Advances in Experimental Medicine and Biology 928

Subash Chandra Gupta
Sahdeo Prasad
Bharat B. Aggarwal Editors

Anti-inflammatory
Nutraceuticals and
Chronic Diseases
Advances in Experimental Medicine and Biology

Volume 928

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N.S. Abel Lajtha, Orangeburg, USA
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Subash Chandra Gupta Sahdeo Prasad
•

Bharat B. Aggarwal
Editors

Anti-inflammatory
Nutraceuticals and Chronic
Diseases

123
Editors
Subash Chandra Gupta Bharat B. Aggarwal
Banaras Hindu University Inflammation Research Institute
Varanasi, Uttar Pradesh San Diego, CA
India USA

Sahdeo Prasad
University of Texas
MD Anderson Cancer Center
Houston, TX
USA

ISSN 0065-2598 ISSN 2214-8019 (electronic)

Advances in Experimental Medicine and Biology
ISBN 978-3-319-41332-7 ISBN 978-3-319-41334-1 (eBook)
DOI 10.1007/978-3-319-41334-1

Library of Congress Control Number: 2016947788

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Contents

1 Curcumin and Its Role in Chronic Diseases . . . . . . . . . . . . . . . . . . . 1

A. Kunwar and K.I. Priyadarsini
2 Berberine and Its Role in Chronic Disease . . . . . . . . . . . . . . . . . . . . 27
Arrigo F.G. Cicero and Alessandra Baggioni
3 Emodin and Its Role in Chronic Diseases . . . . . . . . . . . . . . . . . . . . . 47
B. Anu Monisha, Niraj Kumar and Ashu Bhan Tiku
4 Ursolic Acid and Chronic Disease: An Overview of UA’s Effects
On Prevention and Treatment of Obesity and Cancer . . . . . . . . . . . 75
Anna M. Mancha-Ramirez and Thomas J. Slaga
5 Tocotrienol and Its Role in Chronic Diseases . . . . . . . . . . . . . . . . . . 97
Kok-Yong Chin, Kok-Lun Pang and Ima-Nirwana Soelaiman
6 Indole-3-Carbinol and Its Role in Chronic Diseases . . . . . . . . . . . . . 131
Barbara Licznerska and Wanda Baer-Dubowska
7 Sanguinarine and Its Role in Chronic Diseases . . . . . . . . . . . . . . . . . 155
Pritha Basu and Gopinatha Suresh Kumar
8 Piperine and Its Role in Chronic Diseases . . . . . . . . . . . . . . . . . . . . . 173
Giuseppe Derosa, Pamela Maffioli and Amirhossein Sahebkar
9 Therapeutic Potential and Molecular Targets of Piceatannol
in Chronic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Young-Joon Surh and Hye-Kyung Na
10 Fisetin and Its Role in Chronic Diseases . . . . . . . . . . . . . . . . . . . . . . 213
Harish C. Pal, Ross L. Pearlman and Farrukh Afaq
11 Honokiol, an Active Compound of Magnolia Plant, Inhibits
Growth, and Progression of Cancers of Different Organs . . . . . . . . 245
Ram Prasad and Santosh K. Katiyar

v
vi Contents

12 Celastrol and Its Role in Controlling Chronic Diseases . . . . . . . . . . 267

Shivaprasad H. Venkatesha and Kamal D. Moudgil
13 Boswellic Acids and Their Role in Chronic
Inflammatory Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
H.P.T. Ammon
14 Natural Withanolides in the Treatment of Chronic Diseases . . . . . . 329
Peter T. White, Chitra Subramanian, Hashim F. Motiwala
and Mark S. Cohen
15 Gambogic Acid and Its Role in Chronic Diseases . . . . . . . . . . . . . . . 375
Manoj K. Pandey, Deepkamal Karelia and Shantu G. Amin
16 Embelin and Its Role in Chronic Diseases . . . . . . . . . . . . . . . . . . . . . 397
Hong Lu, Jun Wang, Youxue Wang, Liang Qiao and Yongning Zhou
17 Butein and Its Role in Chronic Diseases . . . . . . . . . . . . . . . . . . . . . . 419
Ziwei Song, Muthu K. Shanmugam, Hanry Yu and Gautam Sethi
18 Garcinol and Its Role in Chronic Diseases . . . . . . . . . . . . . . . . . . . . 435
Amit K. Behera, Mahadeva M. Swamy, Nagashayana Natesh
and Tapas K. Kundu
19 Morin and Its Role in Chronic Diseases . . . . . . . . . . . . . . . . . . . . . . 453
Krishnendu Sinha, Jyotirmoy Ghosh and Parames C. Sil
20 Ellagic Acid and Its Role in Chronic Diseases . . . . . . . . . . . . . . . . . 473
Giuseppe Derosa, Pamela Maffioli and Amirhossein Sahebkar
Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481
About the Editors

Dr. Subash Chandra Gupta is currently Assistant

Professor at Department of Biochemistry, Institute of
Science, Banaras Hindu University, India.

Dr. Sahdeo Prasad is currently at the Department of

Experimental Therapeutics, The University of
Texas MD Anderson Cancer Center, USA.

vii
viii About the Editors

Dr. Bharat Aggarwal is currently Founding Director

of Inflammation Research Institute, San Diego, CA,
USA. He was a Ransom Horne, Jr., Distinguished
Professor of Experimental Therapeutics, Cancer
Medicine and Immunology, The University of
Texas MD Anderson Cancer Center, USA.
Chapter 1
Curcumin and Its Role in Chronic
Diseases

A. Kunwar and K.I. Priyadarsini

Abstract Curcumin, a yellow pigment from the spice turmeric, is used in Indian
and Chinese medicine since ancient times for wide range of diseases. Extensive
scientific research on this molecule performed over the last 3 to 4 decades has
proved its potential as an important pharmacological agent. The antioxidant,
anti-inflammatory, antimicrobial and chemopreventive activities of curcumin have
been extended to explore this molecule against many chronic diseases with
promising results. Further, its multitargeting ability and nontoxic nature to humans
even up to 12 g/day have attracted scientists to explore this as an anticancer agent in
the clinic, which is in different phases of trials. With much more scope to be
investigated and understood, curcumin becomes one of the very few inexpensive
botanical molecules with potent therapeutic abilities.

Keywords Curcumin
Antioxidant
Anti-inflammatory
Anticancer

Turmeric Polyphenol

1.1 Introduction

Curcumin, a natural polyphenol, is one of the most investigated biomolecules from

Mother Nature. Its natural source, Curcuma longa or turmeric is used in Indian
Ayurvedic and Siddha medicines and also in Chinese medicines since thousands of
years [3, 6, 22, 107]. Turmeric is a perennial plant of the ginger family, cultivated in
tropical and subtropical regions of South Asia, and India is one of the largest
producers of turmeric [35]. In Ayurveda, turmeric is used to treat ailments like
arthritis, sprains, open wounds, acnes, stomach upset, flatulence, dysentery, ulcers,

A. Kunwar
K.I. Priyadarsini (&)

Radiation and Photochemistry Division, Bhabha Atomic Research Centre,
Trombay, Mumbai 400085, India
e-mail: [email protected]
A. Kunwar
e-mail: [email protected]

© Springer International Publishing Switzerland 2016 1

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1_1
2 A. Kunwar and K.I. Priyadarsini

jaundice, skin and eye infections. As a dietary agent, turmeric is regularly used as a
spice and also as a coloring agent in Indian cuisine. Both turmeric and its active
principle, curcumin, are permitted like other natural pigments and the food additive,
E number for curcumin is E100. Depending on the origin and soil conditions, the
percentage of curcuminoids in turmeric varies from 2 to 9 % of its dry weight. The
word “curcuminoid” refers to a mixture of four polyphenols, such as curcumin,
demethoxycurcumin and bis-demethoxy curcumin and cyclic curcumin. Out of
these, curcumin is nearly 70 % of the total curcuminoids. In addition to these
curcuminoids, turmeric also contains essential oils primarily composed of mono
and sesquiterpenes, like turmerones, turmerol, etc. The strong yellow color of
turmeric is mainly due to curcuminoids.
Historically, the first scientific report on isolation and chemical characteristics of
curcumin was made in 1815 [115], and its molecular formula and chemical
structure was published in 1910 [74]. Its first laboratory synthesis was demonstrated
in 1913 [68], subsequently in 1964 a method for producing curcumin in high yields
was published [80], and much later, its biosynthesis was understood [35]. Curcumin
is extracted commercially from turmeric by solvent extraction with ethanol fol-
lowed by column chromatography. Using high-performance liquid chromatography
coupled with absorption, fluorescence and mass detectors, curcumin can be detected
in nanomolar quantities in food samples and biological tissues [35, 90].
Apart from the ancient medicinal documents, early research report on therapeutic
use of curcumin appears to date back to 1748; however, the first scientific document
for treating human disease was reported in 1937 [79], wherein at least 67 patients
were treated for chronic cholecystitis, using curcunat, which is equivalent of cur-
cumin. In this study, oral administration for 3 weeks showed symptomatic
improvement in all cases and radiologic improvement by cholecystogram in 18
patients. Interestingly, no ill effects were observed even when the treatment con-
tinued for several months. The efficacy of curcumin was attributed to its ability to
cause the emptying of the gallbladder. Later in 1949, the antibacterial activity of
curcumin [96] was established, since then and till 1970s there were very few reports
on its biological activities [98, 106]. Initial research investigations were focused
mostly on antioxidant and antibacterial activity and the first anticancer report in
human participants was undertaken by Kuttan et al. [67]. However, after the report
by Singh and Aggarwal [108], confirming the anti-inflammatory activity of cur-
cumin by suppressing NF-κB activity, the pace of curcumin research has progressed
systematically. With several encouraging results in rodent models, curcumin
attracted researchers all over the world, to be developed as a potent anticancer drug.
As per Pubmed website, (as of October 2015) there are 8247 articles reported with
the word “curcumin” in the title, including 808 reviews and 141 clinical trials, out
of these more than half have appeared in the last 5 years. It is well accepted by the
scientific community that no other botanical molecule is as efficient and as scien-
tifically celebrated as curcumin.
1 Curcumin and Its Role in Chronic Diseases 3

1.2 Physical, Chemical and Metabolic Reactions

Influencing Curcumin Pharmacology

1.2.1 Physicochemical Properties

Curcumin is a diarylheptanoid, having three important functional moieties. It has

two o-methoxyphenolic groups linked through a heptanoid linker consisting of an
enone moiety and 1,3-diketone group in conjugation (Fig. 1.1). All these groups are
involved in the biological activity of curcumin [35, 42, 88, 89]. Important
physicochemical properties of curcumin are listed in Table 1.1. Like other
β-diketones, curcumin exhibits keto-enol tautomerism, and in solution phase it
mostly exists in the enol form [88]. Curcumin has three acidic protons two from the
phenolic-OH groups (in the range 8.5–10.7) and one from the enolic OH (<8.5).
Curcumin is yellow at neutral and acidic pH with absorption maximum *420–
430 nm and in alkaline solutions, it becomes red in color and the absorption
maximum is shifted to 465 nm. It is practically insoluble in neutral and acidic

H
O O O O
CH3O OCH3 CH3O OCH3

HO OH HO OH

Keto Enol

Fig. 1.1 Chemical structure of curcumin in keto and enol tautomeric forms

Table 1.1 Physico-chemical properties of curcumin

IUPAC name (1E,6E)-1,7-bis (4-hydroxy-3-methoxy phenyl)-
1,6-heptadiene 3,5-dione
Molecular formula C21H20O6
Molecular weight 368.39
Melting point 170–175 °C
Experimental dipole moment in dioxane 3.32 D
Absorption maximum and extinction 425 nm, 55,000 dm3 mol-1 cm-1
coefficient fluorescence maximum in 530 nm
methanol
Solubility Insoluble in water
Soluble in ethanol, methanol, chloroform,
hexane, DMSO
Prototropic equilibrium constant pKa (1), Enolic proton: 7.7–8.5; pKa (2),
(pKa) (three pKas) Phenolic proton: 8.5–10.4; pKa (3), Phenolic
proton: 9.5–10.7
log P value *3.0
Color and odor Yellow at neutral pH, red in alkaline pH; odorless
4 A. Kunwar and K.I. Priyadarsini

water, but is readily soluble in moderately polar solvents like methanol, acetonitrile,
chloroform, dimethylsulfoxide (DMSO), etc. In aqueous solutions its solubility can
be increased by the addition of surfactants, polymers, lipids and proteins. Because
of the presence of serum albumin, clear curcumin solutions in micromolar con-
centration can be prepared in cell culture medium.

1.2.2 Chemical Structural Features Influencing

the Biological Activity of Curcumin

Curcumin has three important functional groups, two o-methoxy phenolic groups,
one enone moiety and an α,β-unsaturated diketone group. Each functional group
has some specific role in crucial biological activity in curcumin. The o-methoxy
phenolic-OH group of curcumin is primarily involved in direct scavenging of
reactive oxygen species (ROS), where it donates an electron or hydrogen atom to
the oxidizing radicals and the resultant curcumin phenoxyl radical acquires stability
through the conjugation and resonating structures [87]. This phenoxyl radical is
regenerated back to curcumin by water-soluble antioxidants like ascorbic acid
making it an excellent chain-breaking antioxidant that has been reported to be at
least ten times better than vitamin E. Curcumin binds to many biomolecules like
proteins, lipids and DNA [42, 88]. The proteins that interact with curcumin include
transcription factors, inflammatory molecules, kinases, tubulins, amyloid-β aggre-
gates, adhesion molecules, growth factors, receptor proteins, protofilaments, prion
proteins, etc. The experimentally reported dipole moment [84] and log P values
(partition coefficient in octanol and water system) of curcumin (Table 1.1) indicate
that the molecule has partial charge transfer character and is moderately polar to be
soluble in lipid-like systems. Because of these properties and flexibility in its
structure, curcumin binds to most of the biomolecules. The hydrophobic interac-
tions and hydrogen-bonding interactions are mainly responsible for the efficient
binding. It is still premature to clarify the role of any single moiety for these
interactions but it appears that the orientation of the enolic group plays a crucial
role.
The α,β-unsaturated β-diketo moiety of curcumin participates in nucleophilic
addition reactions with molecules having functional groups like –SH, –SeH. This
1,4-addition reaction known as Michael addition reaction is of great significance in
curcumin biology, like it reacts with glutathione (GSH) and depletes the GSH in
cells [13]. Similarly through the reaction with the –SeH group, it inhibits thiore-
doxin reductase, an enzyme involved in cellular redox homeostasis [36].
Curcumin undergoes a fast chemical degradation in solution, where products like
ferulic aldehyde, ferulic acid, vanillin, etc. are formed [102, 116]. The degradation
occurs through the β-diketo moiety of curcumin and is increased in presence of light
and decreased when solubilised in aqueous solutions along with lipids, proteins,
surfactants, cyclodextrins, starch etc.
1 Curcumin and Its Role in Chronic Diseases 5

Metabolism of curcumin in mice produced varieties of products. Important

products identified are curcumin glucuronide and curcumin sulfate along with
reduction products like tetrahydrocurcumin, hexahydrocurcumin and octahy-
drocurcumin [11, 37, 56]. The orally administered curcumin undergoes conjugation
whereas the systemically and/or intraperionial (i.p.) administered curcumin
undergoes reduction. These processes occur by phase I and phase II enzymes like
for example, phenol sulfur transferase enzyme is involved in sulfonation and
alcohol dehydrogenase in reduction. Other minor products identified during cur-
cumin metabolism are ferulic acid, dihydroferulic acid, etc. It is still not confirmed
how enzymatic metabolism of curcumin competes with its chemical degradation,
and there is a need to investigate the role of these metabolic and degradation
products in the overall bioactivity of curcumin.

1.2.3 Curcumin–Metal Interactions: Role in Curcumin

Biology

Curcumin binds to many metals and metalloproteins, and the binding is through
covalent interactions, as the diketo group is an excellent metal chelator [89, 90,
117]. Binding of curcumin to metals ions has several biological consequences. Its
binding to Al3+ ions is proposed to be one of the key factors involved in its role in
preventing the pathogenesis of Alzheimer’s disease (AD) [58]. Zn2+-curcumin
complex showed anticancer activity and Au3+ complexes exhibit anti-arthritic
activity [99]. Curcumin–metal chelation can be used to reduce toxicity of heavy
metals like Hg2+, Cd2+, Pb2+ [1, 81]. Several mixed ligand complexes of curcumin
with metals like Cu2+, Ni2+, Mn3+, Pd2+ are also finding applications as antioxi-
dants, superoxide dismutase mimics and anticancer agents [15, 114]. Recently
curcumin derived radiopharmaceuticals are being prepared and explored as new
diagnostic and therapeutic agents. For example, 99mTc4+ and 68Ga3+ complexes of
curcumin reported to bind to amyloid-β (Aβ) fibrils and plaques, are being explored
as novel radiodiagnostic agents for AD [12, 95].

1.2.4 Curcumin Bioavailability

The major issue concerning the development of curcumin based drugs is its
extremely low bioavailability [9, 86]. Due to relatively low intestinal absorption
and rapid metabolism in the liver, the oral bioavailability of curcumin is very low,
while most of it is excreted through the feces within 3–6 h after administration.
Even after oral administration in grams, no significant curcumin was detected in the
plasma, and the highest curcumin levels were found in the intestines and detectable
amounts were observed in the serum, but they fall below detection limit in other
tissues. However, i.p. and intravenous (i.v.) administration has shown better
6 A. Kunwar and K.I. Priyadarsini

Table 1.2 Examples of important formulations used to enhance in vivo bioavailability of

curcumin
Formulation Piperine Curcumin– Cyclodextrin– Pegylated-curcumin Curcumin–
(20 mg/kg) phospholipid curcumin (2.5 mg/kg) phosphatidylcholine
+ curcumin complex (Mervia)
2 g/kg) (100 mg/kg)
Increase in 1.5-fold in 2.2-fold in 1.8-fold in 2-fold in serum 5-fold increase in
bioavailability serum plasma skin plasma

bioavailability than oral administration. The maximum tissue concentrations

recorded after an i.p. injection of 100 mg/kg of curcumin was 73 ± 20, 200 ± 23,
9.1 ± 1.1, 16 ± 3, 8.4 ± 6.0, 78 ± 3 and 2.9 ± 0.4 nmol/g in liver, intestine
mucosa, heart, lungs, muscle, kidneys, and brain respectively [85]. At 10 mg/kg i.
v., maximum serum levels were 0.36 µg/ml, while in another study 2 mg/kg
through tail vain showed plasma concentration of 6.6 µg/ml [9, 86]. Increasing the
dose of curcumin did not result in higher bioabsorption. Being a lipophilic mole-
cule, curcumin is expected to cross blood–brain barrier and reach brain tissue.
However, dietary supplementation did not show significant accumulation in the
brain tissue, but long-term supplementation of mice for nearly 4 months at a dose of
0.5–2 g/kg showed a maximum detectable concentration of 1.5 nmol/g in the brain
tissue [9, 86].
The poor bioavailability of curcumin has thus emerged as one of the major
limitations for its therapeutic applications. To increase the bioavailability of cur-
cumin, researchers have developed several novel formulations. Important among
these are liposomes, nanoemulsions, pegylation, polymers, hydrogels, cyclodex-
trins, piperine-combined, gold and mesoporous silica nanoconjugates and cur-
cumin–iron oxide magnetic nanoparticles [9, 65, 71, 86, 89, 90, 105, 120].
Employing them a significant improvement, not only in the bioavailability of
curcumin but also in its in vivo bioactivity was reported. Most of these formulations
could be dispersed in aqueous buffer medium. There are several reports describing
the preparation and characterization of these nano formulations. Important formu-
lations that showed significant improvement in curcumin bioavailability are given
in Table 1.2.

1.3 Modulation of Cell Signaling Pathways by Curcumin

All the biological activities/functions of a living cell are regulated by a dense

network of signal transduction pathways. The components of signal transduction
pathways are growth factors and their receptors, cytokines and their receptors,
protein kinases, transcription factors and gene expression. Curcumin has been
shown to affect many cellular or molecular pathways in executing its crucial bio-
logical activities. Figure 1.2 gives important signaling molecules involved in bio-
logical activities of curcumin. Some important results are discussed below.
1 Curcumin and Its Role in Chronic Diseases 7

Fig. 1.2 Important biological

activities of curcumin and Curcumin
their associated major
NF-κB
signaling molecules. The text AP-1
in green and red fonts indicate p38
MAPK
upregulation and IL8 IL2
downregulation, respectively TNFα
COX-1
COX-2 ICAM-1
iNOS
IFNγ

Anti-oxidant/ Anti-inflammatory activity Anti-tumor activity

Cyto-protective activity

Therapeutic effect against Cancer, Inflammatory

bowel disease, Neuro-degeneration, Diabetes,
Arthritis, Cardiovascular diseases, Psoriasis
and Microbial infection

1.3.1 Growth Factors/Cytokines and Their Receptors

Growth factors/cytokines are proteins which upon binding to their specific receptors
present on the plasma membrane of the cells stimulate multiple signal transduction
pathways leading to the expression of genes controlling various cellular processes
such as cell cycle, division, apoptosis, movement, inflammatory response to
external stimuli, etc. One of the hallmarks of a cancer cell is the constitutive or
increased expression of growth factors and their receptors inducing signals for the
uncontrolled proliferation or the growth [46]. Recent studies provide evidence that
curcumin targets such growth factors and their receptors to inhibit the growth and
proliferation of cancer cells [4, 6, 49, 110]. For example, curcumin was reported to
inhibit the effects of epidermal growth factor (EGF) and insulin growth factor
(IGF) by downregulating the expression and tyrosine kinase activity of EGF and
IGF receptors in colon cancer cells [93] and MCF-7 breast cancer cells [118].
Similarly curcumin has also been shown to downregulate the expression and
activity of HER-2/erbB2/neu/p185, another member of the EGF receptor super
family closely associated with breast, lung, kidney, and prostate cancers [52].
Further, the overexpression of other growth factors such as platelet-derived
growth factor (PDGF), vascular endothelial growth factor (VEGF) and their
receptors PDGFR and VEGFR, respectively have also been seen in many types of
solid tumors [28, 39]. Park et al. [82] showed that curcumin inhibits
PDGFR-induced proliferation of human hepatic myofibroblasts. In another study
curcumin was shown to block the angiogenesis or the formation of new blood
vessels in various cancer types by inhibiting both VEGF and VEGFR [28].
Some reports indicate that TNF acts as a growth factor for tumor cells.
Similarly other cytokines such as IL6 and IL2 regulate the proliferation of T cells
through the autocrine stimulation and play important roles in mounting the
8 A. Kunwar and K.I. Priyadarsini

inflammatory response. Curcumin has been shown to negatively regulate the

expression of these cytokines both at mRNA and protein levels by inhibiting the
downstream signaling pathways [7, 19, 92]. These effects of curcumin have also
been accounted for its ability to show the antitumor and the anti-inflammatory
activities in various cellular and in vivo model systems.

1.3.2 Protein Kinases

Protein kinases are a class of enzymes, which activate the downstream signaling
molecules through phosphorylation using ATP as a source of inorganic phosphate
(Pi). They are the key transducers such as mitogen activated protein kinases
(MAPKs)/extracellular signal regulated kinases (ERKs), protein kinase A (PKA),
protein kinase B (PKB)/AKT, and protein kinase C (PKC), phosphatidyl inositol 3
phosphate kinase (PI3K), and mammalian target of rapamycin (mTOR) controlling
cell growth, proliferation, cytoprotection, cytokines production and death [3, 4, 49,
110]. Interestingly, curcumin has been shown to modulate the expression and
activity of MAPKs including ERKs, C-Jun, N terminal kinases (JNKs), p38
kinases, and the stress activated protein kinases (SAPK) in suppressing inflam-
mation and cancer [49]. Although inhibitory effect of curcumin on MAPKs has
been documented in several independent studies, there are also some reports, which
indicate that curcumin activates JNKs leading to apoptosis or cell death in tumor
cells [29]. Further, PI3K is the upstream regulator of PKB/AKT and the mTOR
pathways playing a central role in the genesis of cancer. Recent studies have shown
that curcumin inhibits proliferation/growth and induces apoptosis in tumor cells by
inhibiting mTOR pathway and it is mediated through the suppression of PI3K and
PKB/AKT. Additionally, the inhibitory effect of curcumin on mTOR signaling has
also been attributed to its ability to activate adenosine monophosphate kinase
(AMPK1), which senses cellular ATP levels and inhibits mTOR indirectly [122]. In
a separate study, curcumin was shown to block the signals transmitted from EGFR
by inhibiting the PI3K/AKT pathway in colon carcinoma cells [54]. On the con-
trary, curcumin activates the same PI3K and PKB/AKT signaling to induce the
expression of antioxidant genes in pre-cancer cells and thus prevents both tumor
initiation and promotion, a critical stage in carcinogenesis [61, 64]. Further, cur-
cumin also modulates PKC differentially, depending on the cell type and nature of
exogenous/endogenous stimuli. For example it has been shown to inhibit the tumor
promoting action of 12-o-tetradecanoylphorbol-13-acetate (TPA) by inactivating
PKC through oxidizing the vicinal thiols present within catalytic domain [72]. On
the other hand, the chemopreventive activity of curcumin has been linked with its
ability to activate PKC and the downstream p38 MAPK in human monocytes
leading to induction of antioxidant genes [94].
1 Curcumin and Its Role in Chronic Diseases 9

1.3.3 Transcription Factors

Transcription factors are the cellular proteins, which upon activation through sig-
naling cascades bind to the promoter/enhancer regions of their target genes and
regulate their transcription/expression. Curcumin by modulating various signal
transduction pathways affects the activation of a number of transcription factors
such as nuclear factor-κB (NF-κB), activator protein-1 (AP-1), early growth
response-1 (Egr-1), signal transducer and activator of transcription (STAT), per-
oxisome proliferator activated receptor-gamma (PPAR-γ), nuclear factor erythroid
2-related factor 2 (Nrf2), beta (β)-catenin and tumor suppressor p53 [19, 20, 24, 26,
57, 108, 110].
The NF-κB is reported to be constitutively activated in a variety of cancers and
inflammatory disorders. Researchers working on the various aspects of biological
activity have unanimously shown that curcumin is a strong suppressor of NF-κB
activation [104, 108]. The inactivation of NF-κB by curcumin is mediated through
the inhibition of IκBα kinase (IKK) directly and/or by modulating upstream sig-
naling cascade [108]. AP-1 is another transcription factor associated with growth
regulation, cell transformation, inflammation, and innate immune response.
Curcumin suppresses the activation of AP-1 either by inhibiting the upstream
kinases such as JNK, p38 and ERK or by directly interacting with AP-1 DNA
binding motifs present in the promoter regions of the target genes [20]. The tran-
scription factor Egr-1 is one of the immediate early induced gene products in
response to growth factor or carcinogen mediated signaling. Curcumin suppresses
the induction/de novo synthesis of Egr-1 protein in different cell types such as
endothelial cells, fibroblast, and colon cancer cells [26]. The family of STAT pro-
teins (STAT1-STAT7) performs the dual function of signal transducer as well as the
transcription factor. Of the seven STAT proteins, the elevated activity of STAT3 and
STAT5 has been mainly implicated in the angiogenic response and in growth of
various cancer types. Curcumin has been found to inhibit the phosphorylation of
STAT3 and STAT5 by upstream kinases (like janus kinase (JAK) and/or the growth
factor/G-protein-coupled receptor kinases) and their subsequent translocation to
nucleus there by suppressing the tumor cell proliferation and the pro-inflammatory
immune responses [19, 25]. The protein PPAR-γ also plays the dual role of nuclear
receptors of hormones and transcription factor. Curcumin has been shown to induce
the expression of PPAR-γ in various cell types such as colon cancer cells [24] and
hepatocytes leading to suppression of tumor cell proliferation, and the prevention of
liver fibrosis and sepsis [119]. The transcription factor Nrf2 induces the expression
of genes related to cytoprotection, antioxidant enzymes and phase II detoxification
enzymes. Curcumin induces the antioxidant and cytoprotective responses in various
cell types such as epithelial, monocytes, hepatocytes by activating Nrf2. The
mechanism of activation is either through directly interacting with Keap-1 (inhibitor
of Nrf2) or by positively regulating upstream kinases such as PI3K, PKC and MAPK
[53, 94]. The β-catenin and p53 are some of other important transcription factors,
which control the expression of genes involved in cell cycle progression and check
10 A. Kunwar and K.I. Priyadarsini

points. Curcumin has been reported to induce as well as inhibit the transcriptional
activity of β-catenin by differentially modulating the activity of glycogen synthase
kinase-3 (GSK3) [57, 123]. The inhibition of β-catenin activation by curcumin has
been liked with the suppression of tumor cell proliferation and the induction of
apoptosis. Whereas curcumin mediated activation of β-catenin has been attributed
toits anti-AD activity. The p53 protein is a tumor suppressor protein. Depending on
cell type, curcumin regulates the level of p53 in different ways. For example, in
normal thymocytes and myeloid leukemic cells, curcumin downregulates the
expression of p53 and inhibits p53-induced apoptosis [45, 112]. In contrast to such
studies, curcumin has been shown to upregulate p53 in colon, neuroblastoma,
lymphoma, prostate, and human breast cancer cells to induce apoptosis [27, 110].

1.3.4 Gene Expression

Through modulating various signal transduction pathways, curcumin affects the

expression of a number of genes [3, 4, 49, 110]. For example, the antioxidant and
chemopreventive activities of curcumin have been attributed to the upregulated
expression of hemeoxygenase-1 (HO-1), glutathione peroxidase (GPx),
gamma-glutamyl-cysteine ligase (γGCL), and phase II detoxification enzymes like
glutathione S transferase (GST), glutathione reductase (GR), NAD(P)H:
quinonecidoreductase 1 (NOQ1), quinone reductase and epoxide hydrolase [53, 61,
62, 94, 103]. The anti-inflammatory activity of curcumin is associated with its
ability to downregulate the expressions of genes like cyclooxygenase-1 (COX-1),
cyclooxygenase-2 (COX-2), inducible nitrogen oxide synthase (iNOS), and
cytokines such as interleukin-2 (IL-2), interferon-γ (IFNγ), tumor necrosis factor-α
(TNFα), intracellular adhesion molecule-1 (ICAM-1), vascular adhesion
molecule-1 (VCAM-1) and E-selectin [3, 51]. The anticancer effect of curcumin is
mediated through downregulating the expressions of oncogenes (c-Met, c-Myc,
c-Jun, Ras and Mdm2) and anti-apoptotic genes (Bcl2, BclXL, IAP, hTERT, Cyclin
D1, survivin) and upregulating the expressions of tumor suppressor genes (p53, Rb,
PTEN) and apoptotic genes (Bax, FASL and Bim) [49]. Additionally, curcumin has
also been shown to affect the expression of genes like matrix metalloprotease-2,9
(MMP-2,9), macrophage inhibitor protein-1 (MIP-1), vascular endothelial growth
factor (VEGF), endothelial growth factor (EGF), hypoxia-inducible factor-1
(HIF-1), p21, poly-ADP ribose polymerase (PRAP), growth arrest and DNA
damage-inducible (GADD), cytochrome P450 (CYP1A1), and p-glycoprotein
(pgp) associated with varied biological responses such as immunomodulation,
angiogenesis, anti-tumor and drug metabolism [10, 14, 49, 78].
1 Curcumin and Its Role in Chronic Diseases 11

1.4 Biological Activities of Curcumin in Animal Models

Curcumin is one of the most extensively evaluated natural products in rodent

models for therapeutic efficacy against various pathological conditions/diseases.
Among the various activities, the effect of curcumin on carcinogenesis has been
majorly studied in rodents [3, 63, 83, 110]. The transformation of a normal cell to
cancerous cell is a complex process and occurs in three stages namely initiation,
promotion and progression. Interestingly, oral administration of curcumin has been
shown to affect all the three stages of carcinogenesis in chemical and/or genetic
mice models of skin, stomach, duodenum, colon, liver, lung, and breast cancers [63,
83, 110]. Further, several reports are also available on the ability of topically
applied curcumin to inhibit the tumor imitation and tumor promotion of skin car-
cinogenesis [30]. Similar effects of curcumin have been reported in Syrian golden
hamsters, where it prevented chemical induced oral carcinogenesis [70]. In another
interesting set of studies, oral curcumin was shown to inhibit the tumor progression
in a xenograft model of human cancers of prostate, colon, pancreas, bladder and
ovary [3, 110]. There are also reports indicating that curcumin inhibits metastasis of
human breast cancer to the lungs in nude mice [5]. Taken together, curcumin has
shown significant activity of inhibiting carcinogenesis in rodent models.
Curcumin has also been studied in rodent models for its effect on metabolic
enzyme systems and antioxidant activity. For example in F344 mice, curcumin
administered orally at a dosage of 270 mg/kg decreased the levels of metabolizing
enzymes such as cytochrome p450 (CYP) isoforms CYP2B1 and CYP2E1 and
prevented the n-nitrosomethylbenzamine (NMBA) induced oesophageal carcino-
genesis [77]. Since the enzymes CYP2B1 and CYP2E1 are known to metabolize
NMBA into an active carcinogen, the ability of curcumin to suppress their levels is
in line with the hypothesis that curcumin inhibits carcinogenesis at initiation stage.
Additionally, dietary curcumin is shown to play a role in preventing the early stage
of carcinogenesis by inducing phase II enzymes such as GST and epoxy hydrolase
in both mice and rat. Further, curcumin administration (75–300 mg/kg) in rodents is
also associated with the increased expression of antioxidant enzymes like heme
oxygenase (HO-1), glutathione peroxidase (GPx) and γ-glutamate cysteine ligase
(γGCL) in liver, brain, small intestine and kidney tissues [8]. These effects of
curcumin are associated with its ability to prevent nephrotoxicity in rats, reduce
oxidative damage in the heart of diabetes-afflicted mice and brain of transgenic AD
rats. It was also found to inhibit chemically induced hepatic injury and neurode-
generation in rats [8]. There are also studies indicating that curcumin lowered ROS
production in vivo. For example, in mice the orally administered curcumin
(100 mg/kg) prevented lipopolysaccharide induced inflammatory responses in liver
by suppressing the expression of iNOS responsible for nitric oxide (NO) production
[21]. Similarly, oral curcumin was also seen to prevent oxidative damage during
indomethacin-induced gastric lesion by blocking inactivation of gastric peroxidase
as well as directly scavenging peroxide (H2O2) and hydroxyl (
OH) radicals [23].
12 A. Kunwar and K.I. Priyadarsini

Oral curcumin is also effective against carrageenan-induced edema and enhanced

wound-healing in diabetic rats and mice, documenting its anti-inflammatory
activity. Some of the other potential biological effects of curcumin studied in rodent
models through oral administration (50–300 mg/kg) include cardioprotective,
neuroprotective, anti/pro-mutagenic, antifertility, antidiabetic, antifibrotic, anti-
venom, anti-HIV, and anticoagulant activities [22].

1.5 Role of Curcumin in Chronic Diseases

It has been confirmed by several researchers that oxidative stress and oxidative
damage are directly associated with chronic inflammation, which in turn leads to
several chronic diseases, like cancer, metabolic, neurological, pulmonary, intestinal
and cardiovascular diseases. Since curcumin has been identified as an important
remedy against oxidative stress and inflammation, extensive research work has been
undertaken to use curcumin against many chronic diseases. Important examples are
mentioned briefly here [3, 51, 101].
Inflammatory bowel disease (IBD), symptomatic with severe diarrhea, pain,
fatigue and weight loss, involves chronic inflammation of all or part of digestive
tract. Both turmeric extract and polymer loaded curcumin formulations have been
examined in animals and humans for treatment against IBD and many encouraging
results were obtained without any side effects [18, 47, 48, 111]. In mice, oral
administration of curcumin (100 mg/kg per day) for ten consecutive days prior to
the induction of colitis by acetic acid and further continuation for 2 days showed
decreased colon injury and ameliorated macroscopic and microscopic colitis sores.
IBD patients treated with curcumin (550 mg twice daily for 1 month) showed
decreased symptoms and inflammation indices, without any significant side effects.
Rheumatoid arthritis (RA), a long-lasting autoimmune disorder that primarily
affects joints on both sides of the body, is a systemic chronic inflammatory disorder.
To reduce arthritic reaction, it is essential to start early treatment and combination
therapy. As a nonsteroid anti-inflammatory drug and antioxidant, curcumin has
been examined as a potential therapy for RA, accordingly RA patients treated with
curcumin showed significant symptomatic improvement [76, 90]. Similarly cur-
cumin (500 mg) alone and in combination with diclofenac sodium (50 mg)
administered in patients with RA showed best improvement in the overall disease
scores. In a short term, a double-blind crossover study involving 18 young patients
suffering from RA, oral curcumin at a daily dose of 12 g for 2 weeks exerted an
anti-RA activity comparable to that of a standard drug phenylbutazone.
Diabetes mellitus (DM), commonly referred to as diabetes, is a metabolic disease
in which there are high blood sugar levels over a prolonged period. Serious
long-term complications include cardiovascular disease, stroke, chronic kidney
failure, foot ulcers, and damage to the eyes. Effect of curcumin against diabetes was
successfully demonstrated first in one patient in 1972 [109] and since then more
than 200 papers have been published in this related subject [2, 73, 124]. Curcumin
1 Curcumin and Its Role in Chronic Diseases 13

has been shown to reduce blood glucose and glycosylated hemoglobin levels and
prevented weight loss in rodent models. Oral administration of curcumin
(80 mg/kg) showed anti-hyperglycemic effect and improved insulin sensitivity. It
was also effective in improving glucose intolerance. One or two contradicting
effects were also reported, where intragastric administration of curcumin did not
show any effect in blood glucose. Curcumin has been shown to reduce several other
complications associated with diabetes like fatty liver, diabetic neuropathy, diabetic
nephropathy, vascular diseases, musculoskeletal diseases and also islet viability.
Neurodegenerative diseases including Parkinson’s and AD occur due to the
progressive loss of structure or function of neurons, including death of neurons.
Curcumin was considered as a potent drug mainly due to the fact that the epi-
demiological studies indicating reduced risk of AD amongst Indians consuming
turmeric in their diet. In line with this, several experimental studies in mice models
confirmed anti-AD effects of curcumin [44]. It significantly lowered the levels of
oxidized proteins, IL-1β, insoluble and soluble plaque burden in AD transgenic
Tg2576 mice brain. There are several ongoing clinical trials on the effect of cur-
cumin for the prevention of AD. In one such study, daily intake of curcumin 1–
4 g/day over 6 months found a trend towards increased serum Aβ levels which is
considered as a possible clearance of Aβ plaques from the brain, but improvement
in cognitive performance was not observed during the 6 months trial [16]. A few
other studies indicate that curcumin also exhibits antidepressant and neuroprotec-
tive properties [32, 69, 75].
Psoriasis, a chronic skin disease, occurs when the immune system overreacts,
causing inflammation and flaking of skin and is characterized by thick red and scaly
lesions on any part of the body. Since ancient times turmeric has been used as a skin
protectant in the Indian subcontinent. Further experimental evidences strongly
indicate that curcumin is a potential natural product to suppress psoriasis by
inhibiting the keratinocyte proliferation [59]. In a clinical trial, orally administered
curcumin demonstrated therapeutic effects with no adverse reaction in patients with
psoriasis and reduced psoriasis area and severity index [66]. Curcumin skin for-
mulations are being marketed for skin diseases including psoriasis.
In addition to these important diseases, curcumin has been examined for treat-
ment of other chronic diseases like cardiovascular diseases, allergy, asthma,
bronchitis, kidney diseases, obesity, scleroderma, vitiligo, peptic ulcer, pylori
infection and ophthalmic disorders and encouraging results are reported [3, 51]. The
anticancer effects are discussed in detail in the next section.

1.6 Biological Activities of Curcumin in Humans

The remarkable success of curcumin as a therapeutic agent against various chronic

diseases including cancer in preclinical studies has prompted clinicians to undertake
many clinical trials in human subjects [4, 43, 97, 121]. As of October 2015, as
many as 137 clinical trials with curcumin/turmeric extract are listed on a search
14 A. Kunwar and K.I. Priyadarsini

engine (www.clinicaltrials.gov) of which 67 have been completed, 3 terminated, 9

withdrawn and rest are at recruitment (enrolment of human subjects) stage. Table 1.
3 gives the list of clinical trials from different countries and also the purpose. The
maximum number of clinical trials has been reported from USA and the most
common human disease evaluated is the cancer. The trials were primarily under-
taken for safety and efficacy under different disease conditions. Important com-
pleted studies are discussed below.
The phase I clinical trials for safety evaluation of curcumin have indicated that
its intake as high as 12 g/day orally for 3 months is safe. In order to determine the
curcumin’s maximum tolerated dose and safety, a dose-escalation trial has been
recently completed. In this study, curcumin was administered to 24 healthy vol-
unteers at a single oral dose ranging from 0.5 to 12 g and safety was assessed for
72 h after administration. Remarkably, only 7 out of 24 subjects showed minimal
behavioral toxicities like diarrhea, headache, rash, and yellow stool. The pharma-
cokinetics studies in human (sample size *1–30) involving healthy volunteers and
the cancer/IBD/AD patients have revealed that the oral intake of curcumin
(2–12 g/day) results in poor bioabsorption. However, in colorectal cancer
(CRC) patients administered with 3.6 g of curcumin/day for 7 days, although
indicated lower bioavailability in the tumor compared to normal tissue, the
reduction in the levels of a DNA adduct, used as a biomarker, established that a
daily dose of 3.6 g curcumin is pharmacologically efficacious against CRC [38].
Surprisingly, administration of oral curcumin at a similar daily dosage in patients
suffering from hepatic metastases of CRC did not attain the pharmacologically
effective concentration in the liver.
Due to multitargeting effects, curcumin has showed promising chemopreventive
and therapeutic effects against a range of human malignancies such as colorectal
cancer, aberrant crypt foci, familial adenomatous polyposis (FAP), pancreatic
cancer, multiple myeloma, hepatocellular carcinoma, gastric cancer, and colon
cancer in clinical trials. In the first study, topical curcumin applied, either as an
ethanol extract or as an ointment, showed remarkable relief in 62 patients reported
with external cancer lesions. Two phase I trials in CRC patients were conducted,
one in 2001 with 15 patients with 36–180 mg daily for 4 months and another trial
with 15 patients with escalated doses of 0.45–3.6 g daily for 4 months [100]. The
drug was well tolerated by all except three patients with minor side effects along
with improvement in the status of biological parameters indicating efficacy against
CRC. In another study, CRC patients when treated with 0.36 g (in capsule form)
curcumin daily for 10–30 days showed improvement in general health [50].
Curcumin was tested against pancreatic cancer with moderate results. In a single,
blind, randomized, placebo-controlled study in 20 patients with 0.5 g curcumin
with 5 mg piperine for 6 weeks showed reduction in oxidative stress with no
significant effect in pain relief [33]. In another phase II clinical trial on 25 patients
with advanced pancreatic cancer were treated orally with 8 g curcumin daily
showed moderate results [31]. In another open-label phase II trial involving 17
patients with advance pancreatic cancer treated with 8 g of curcumin along with
gemcitabine showed moderate results, while another recent study involving 21
Table 1.3 List of clinical trials with curcumin in patients with different diseases
Pathological conditions/diseases Present No. of Study sites Remarks/conclusions
status of clinical
clinical trials
trials
Cancer (multiple myeloma, pancreatic cancer, FAP, colon cancer, Completed 7 USA Improvement in the histology of
colorectal cancer, leukemia, head and neck cell carcinoma, breast precancerous lesions
cancer, cancer lesions) Reduces markers (M1G DNA adducts) of
colorectal cancer
Decreases the number and size of polyps in
FAP patients
Despite low bioavailability curcumin shows
biological activity against pancreatic, and
multiple myeloma
Reduces cancer lesions
Safe in combination with other anticancer
1 Curcumin and Its Role in Chronic Diseases

drugs
Ongoing 22 USA, Puerto Rico, Canada, UK,
Korea, India, Iran, Israel, Italy,
France, Austria, Belgium
Withdrawn 4 USA
Inflammatory bowel disease (IBD)/H. pylori infection/hepatic injury Completed 5 USA, Israel, Korea Reduces the levels of inflammatory
cytokines and symptoms
Ongoing 6 Israel, France, Itlay
Withdrawn
Cardiovascular diseases and metabolic syndrome Completed 7 France, Iran, Germany, Canada Decreases serum levels of cholesterol and
lipid peroxides, low density lipoprotein
(LDL), ApoB
Increases high density lipoprotein (HDL),
ApoA
Ongoing 5 Thailand, Canada, USA, Singapore
15

Withdrawn 1 USA
(continued)
Table 1.3 (continued)
16

Pathological conditions/diseases Present No. of Study sites Remarks/conclusions

status of clinical
clinical trials
trials
Neurodegenerative diseases, depression, optic neuropathy Completed 7 Puerto Rico, India, China, Thailand, Improves the cognitive functions and overall
USA, Israel, Canada memory in patients with AD
Improves inventory of depressive
symptomatology self-rated version
(IDS-SR30)
Ongoing 9 USA, Israel, Iran, Canada
Withdrawn 2 Israel, USA
Diabetes Completed 4 Iran, USA, Sweden, India Improves β cells function and decreases the
level of serum fatty acids
Decreases serum adipocyte-fatty acid
binding protein (A-FABP) level
Reduces atherogenic risk
Ongoing 4 Thailand, Iran, USA
Withdrawn
Radiotherapy/chemotherapy side effects Completed 4 USA, Israel, India Effective against radiotherapy/chemotherapy
induced dermatitis, mucositis in cancer
patients
Ongoing 3 USA, Iran
Withdrawn
Chronic kidney disease Completed 1 USA Improvement in the levels of inflammatory
cytokines
Ongoing 5 Canada, USA, Finland
Withdrawn
(continued)
A. Kunwar and K.I. Priyadarsini
Table 1.3 (continued)
Pathological conditions/diseases Present No. of Study sites Remarks/conclusions
status of clinical
clinical trials
trials
Allergy, asthma, bronchitis, oral disinfectant, cystic fibrosis Completed 7 USA, Brazil, India Decreases inflammation
Effective anti-inflammatory mouthwash
Ongoing
Withdrawn
Rheumatoid arthritis (RA) Completed 4 Belgium, France, USA, Thailand Improvement in symptoms
Ongoing 2 USA
Withdrawn
Pancreatitis Completed 1 USA Improvement in symptoms and reduction in
oxidative stress
1 Curcumin and Its Role in Chronic Diseases

Ongoing
Withdrawn
Psoriasis Completed 2 USA Decreases phosphorylase kinase activity a
marker of psoriasis disease
Ongoing 1 Italy
Withdrawn
Bioavailability and pharmacokinetics Completed 10 Germany, USA, Austria, Canada, Safe even at 12 g/day
Italy Poor bioavailability in serum and tissues
Ongoing 2 USA, India
Withdrawn 2 Israel, USA
General health benefits Completed 8 USA, France, Korea Dietary supplementation improves the levels
of antioxidant enzymes and reduces
oxidative stress
Improves general health
17

Ongoing 2 France, USA

Withdrawn
18 A. Kunwar and K.I. Priyadarsini

gemcitabine resistant, patients were safe and seem to tolerate well [34, 60]. In a
open-label phase I clinical trial, 14 patients with advanced and metastatic breast
cancer were examined for feasibility and tolerability of curcumin in combination
with docetaxel, based on which it has been recommended that 6 g curcumin/day for
seven consecutive days for 3 weeks would be safe in combination with a standard
dose of docataxel [17].
In a randomized double-blind study, curcumin (100 mg) in combination with
isoflavin (40 mg) for 6 months in 85 patients showed synergism and suppressed
prostrate specific antigen after treatment [55]. Curcumin was also examined against
multiple myeloma and two clinical trials were completed. In one single blinded
crossover pilot study in 26 patients with monoclonal gammopathy of undetermined
significance (MGUS), curcumin showed therapeutic potential against MGUS [41].
In another study, involving 29 patients with asymptomatic, relapsed multiple
myeloma, curcumin was found to be effective either alone or in combination with
bioperine [113]. In a randomized open-label study involving 25 patients with
chronic myeloid leukemia, curcumin (5 g) administered with imatinib (0.8 g)
showed better efficacy in decreasing the nitric oxide levels. This suggested that
curcumin can be used as an adjuvant to imatinib for the treatment of chronic
myeloid leukemia patients [40]. Curcumin has also showed encouraging results in
combination with other cancer drugs like paclitaxel, and cisplatin.
Oral curcumin has also been shown to prevent the H. pylori infection, a pre-
cursor of gastric cancer, prostatic intraepithelial neoplasia, a precursor of prostate
cancer and to reduce the poly numbers and size in patients with FAP. The efficacy
of curcumin against IBD has already been established in clinical trials. Similarly
curcumin could effectively induce the gallbladder emptying and reduce gall stone
formation, a potential risk factor for gallbladder cancer. In another study, oral
curcumin (400 mg three times a day) compared to placebo showed much better
anti-inflammatory response against spermatic cord edema in patients that underwent
surgical repair of hernia and/or hydrocele.
Many other clinical trials against variety of other diseases have also been ini-
tiated and are being continued, interested readers may refer to some of the recent
reviews on curcumin clinical trials [4, 43, 97, 121].

1.7 Conclusions

The natural product, “curcumin”, a constituent of the golden spice, turmeric, a

divine medicinal herb for the Indians, is now one of the highly researched mole-
cules, very often referred by the modern scientists as “Cure-cumin”. Extensive
preclinical studies over the last three to four decades have established the thera-
peutic potential of curcumin against many diseases. Its multitargeting ability and
direct interaction with signaling molecules have added advantage to exploit its use
against several chronic diseases. In the clinic, curcumin has been found to be safe
1 Curcumin and Its Role in Chronic Diseases 19

High absorption curcumin Curcumin drinks Curcumin Gel

Curcumin
Liposomal curcumin
Curcumin mouthwash

Curcumin daily supplements Curcumin tablets

Fig. 1.3 Representative curcumin products in the market

and well tolerated by the patients under study. Its efficacy against diabetes,
inflammatory bowel disease and arthritis are well proven in the clinic. Extensive
clinical trials to develop curcumin as anticancer drug are still continuing, and the
results are moderately encouraging, mainly due to complexity of the disease and
low bioavailability of curcumin. To partly overcome this, new curcumin formula-
tions with improved bioavailability are successfully designed and developed. More
clinical trials with larger participants are warranted to translate the preclinical
research to the clinical cancer medicine. Curcumin- and turmeric-based nutraceu-
tical formulations are becoming very popular and more than hundred varieties of
products in the form of drinks, capsules, creams, gels, food supplements are
available in the market (Fig. 1.3 gives a few representative products). These
products are recommended for both healthy people and also for patients undergoing
treatment of chronic diseases. In view of all these gifted properties; it is not
exaggerating to say that this inexpensive and innocuous dietary agent, a highly
pleiotropic molecule, will be a “Panacea” in near future.

Acknowledgments The authors wish to express sincere thanks to Department of Atomic Energy,
Government of India and acknowledge the contributions of many co-authors and students whose
names appeared in the publications listed from our group.

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Chapter 2
Berberine and Its Role in Chronic Disease

Arrigo F.G. Cicero and Alessandra Baggioni

Abstract Berberine is a quaternary ammonium salt from the protoberberine group

of isoquinoline alkaloids. It is found in such plants as Berberis [e.g. Berberis
aquifolium (Oregon grape), Berberis vulgaris (barberry), Berberis aristata (tree
turmeric)], Hydrastis canadensis (goldenseal), Xanthorhiza simplicissima (yel-
lowroot), Phellodendron amurense[2] (Amur corktree), Coptis chinensis (Chinese
goldthread), Tinospora cordifolia, Argemone mexicana (prickly poppy) and
Eschscholzia californica (Californian poppy). In vitro it exerts significant
anti-inflammatory and antioxidant activities. In animal models berberine has neu-
roprotective and cardiovascular protective effects. In humans, its lipid-lowering and
insulin-resistance improving actions have clearly been demonstrated in numerous
randomized clinical trials. Moreover, preliminary clinical evidence suggest the
ability of berberine to reduce endothelial inflammation improving vascular health,
even in patients already affected by cardiovascular diseases. Altogether the avail-
able evidences suggest a possible application of berberine use in the management of
chronic cardiometabolic disorders.

Keywords Berberine
Antioxidant
Anti-inflammatory
Type 2 diabetes

Cardiovascular disease
Depression

2.1 Introduction

Cardiovascular diseases are yet the most common causes of death and one of the
first causes of disability in industrialized countries and despite the efforts towards
primary prevention of cardiovascular disease, many patients still remain at risk [1].
Lifestyle interventions such as diet and/or physical activity are the most

A.F.G. Cicero (&)
A. Baggioni

Cardiovascular Disease Prevention Research Unit, Department of Medical
and Surgical Sciences, S. Orsola-Malpighi University Hospital,
Via Albertoni 15, 40138 Bologna, Italy
e-mail: [email protected]

© Springer International Publishing Switzerland 2016 27

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1_2
28 A.F.G. Cicero and A. Baggioni

cost-effective approach in delaying or preventing the onset of cardiovascular dis-

ease [2]. Moreover, people without a history of cardiovascular disease who lack
common risk factors have a significantly greater risk of cardiovascular and all-cause
mortality if they do not adhere to a healthy lifestyle [3]. However, lifestyle pro-
grams are often difficult to follow for long periods and some risk parameters, such
as cholesterolemia, are relatively resistant to changes in dietary habits and physical
activity [4]. On the other hand, a relatively large number of dietary supplements and
nutraceuticals have been studied for their supposed or demonstrated ability to
reduce cholesterolemia in humans [5]. The third National Cholesterol Educational
Program suggested to integrate dietary supplements such as soluble fibres, omega-3
polyunsaturated fatty acids (PUFA), plant sterols and soy protein in the diet in order
to achieve an optimal LDL-cholesterolemia [6]. These suggestions have been
supported also by the recent new European guidelines of the management of
dyslipidemias [7] that also cite some other nutraceuticals as potentially useful
lipid-lowering substances. Since cardiovascular disease prevention needs a life
course approach, both the tolerability and safety of dietary
supplements/nutraceuticals used to control plasma cholesterol levels has to be
adequately defined as well as the risk/benefit ratio of their assumption. A relatively
large number of recent reviews already described the mechanism of action and the
efficacy of the different nutraceuticals and botanicals with lipid-lowering effects [8–
10]. In particular, Berberine (BBR) exhibits many different biological activities;
among them, the best characterized are antioxidant, anti-inflammatory,
cholesterol-lowering and anti-hyperglycemic effects.

2.1.1 Physico-Chemical and Pharmacological Properties

of Berberine

Berberine is a quaternary ammonium salt from the group of isoquinoline alkaloids

(2,3–methylenedioxy-9,10-dimethoxyprotoberberine chloride; C20H18NO4+) with a
molar mass of 336.36122 g/mol [11]. It is highly concentrated in the roots, rhi-
zomes and stem bark of various plants including Coptis chinensis, Rhizoma cop-
tidis, Hydrastis canadensis, Berberis aquifolium, Berberis vulgaris, Berberis
aristata, Tinospora cordifolia, Arcangelisia flava and Cortex rhellodendri [12].
Berberine is strongly yellow coloured, which explains the fact that in the past
berberis species were used to dye wool, leather and wood. Under ultraviolet light,
berberine shows a strong yellow fluorescence with a Colour Index of 75,160 [13].
Berberis vulgaris as well as other berberine-containing plants [14] are used
medicinally in virtually all-traditional medical systems, and have a history of usage
in Ayurvedic, Iranian and Chinese medicine dating back at least 3000 years [16].
Ancient Egyptians used barberry fruit with fennel seeds to ward off pestilent fevers
[15]. Indian Ayurvedic physicians used barberry in the treatment of dysentery and
traditional Iranian medicine uses its fruit as a sedative [15, 17]. In northern Europe,
2 Berberine and Its Role in Chronic Disease 29

barberry was used to treat gall bladder and liver problems, while it was used in the
treatment of abnormal uterine bleeds and rheumatism in Russia and Bulgaria [18,
19]. In North America, the Eclectics used barberry for treatment of malaria and as a
general tonic [20]. Also, the American Indians found it effective in improving
appetite and used its dried fruit as a gargle [21, 22].
Medicinal properties for all parts of the plant have been reported, including tonic,
antimicrobial, antiemetic, antipyretic, antipruritic, antioxidant, anti-inflammatory,
hypotensive, antiarrhythmic, sedative, antinociceptive, anticholinergic and chola-
gogue actions, and it has been used in some cases like cholecystitis, cholelithiasis,
jaundice, dysentery, leishmaniasis, malaria and gall stones [23]. Furthermore, ber-
berine has been used for treating diarrhoea and gastrointestinal disorders for a long
time [24, 25]. It has multiple pharmacological effects including; antimicrobial activity
against 54 microorganisms [26], inhibition of intestinal ion secretion and smooth
muscle contraction, inhibition of ventricular tachyarrhythmia, reduction of inflam-
mation, stimulation of bile secretion and bilirubin discharge [27].
Berberine has low bioavailability and poor absorption through the gut wall
(<5 %) and bowel P-glycoprotein contributes to that, actively expelling the alkaloid
from the lumen mucosal cells [28].
In a rat noncompartmental model [29], unbound berberine is transported to bile
through active transportation and it is metabolized by P450 enzyme system in liver,
with phase I demethylation and phase II glucuronidation. Berberine has four main
metabolites identified in rats: berberrubine, thalifendine, demethyleneberberine and
jatrorrhizine, and all of them have glucuronide conjugates [30]. Intestinal bacterial
flora takes role in enterohepatic circulation of berberine and its conjugated
metabolites [28]. On the other hand, very small amount of unchanged berberine is
eliminated in urines [31].
As other alkaloids are present in H. canadensis extracts (i.e. hydrastine and
canadine), berberine may inhibit cytochrome P450 2E1 (CYP2E1) [32] and 1A2
(CYP1A2) [33]. This inhibition is not related to a significant increase in pharma-
cological interactions since the largest part of the available drugs is not metabolized
by these enzymatic systems.

2.1.2 Berberine Modulation of Cell Signalling Pathways

Berberine is a potent antioxidant and anti-inflammatory agent: these properties

could be particularly relevant in the management of type 2 diabetes and cardio-
vascular diseases.
In metabolic disorders, as obesity and type 2 diabetes, increased oxidative stress
is a common feature [34, 35]. It could induce or deteriorate insulin resistance and
diabetes through multiple mechanisms. In the process of oxidative stress, excessive
reactive oxygen species (ROS) are produced, mainly by mitochondria [36, 37].
They could cause damage and apoptosis of pancreatic islet β-cells and reduction of
insulin secretion [38]. ROS also activate c-Jun N-terminal kinase (JNK), protein
30 A.F.G. Cicero and A. Baggioni

kinase C (PKC) and nuclear factor-κB (NF-κB), interfering with the insulin sig-
nalling pathway and causing insulin resistance [39–41]. In addition, oxidative stress
also contributes to the development of chronic complications of diabetes, such as
diabetic nephropathy, retinopathy and neuropathy [37].
Molecular mechanisms of berberine in reducing oxidative stress seem to be
related with multiple cellular pathways (Fig. 2.1).
The NOX family of ROS-generating NADPH oxidases, a family of
membrane-associated enzymatic complexes, is one of the major sources of ROS
production in cells [42]; its activation is often associated to high levels of fatty
acids, cholesterol, glucose or advanced glycation end products (AGEs) [43–45].
Among various NOX isoforms, berberine was reported to suppress the overex-
pression of NOX 2,4 and to decrease ROS production in macrophages and
endothelial cells upon stimulation with inflammatory stimuli [46, 47]. In endothelial
cells, berberine attenuated LDL oxidation induced by ROS and reduces the collapse
of mitochondrial membrane potential, the chromosome condensation, the cyto-
chrome C release and the caspase-3 activation [48]. Circulating endothelial

BERBERINE

Antioxidant activity Antinflammatory activity

SOD NADPH AMPK P38 MAPK IKK -β Rho PPAR -γ

mRNA oxidase mRNA activation activation inhibition inhibition GTPase activation
inhibition

Nrf2 NF -kB AP -1
1 2 Nuclear
3 activation pathway
translocation inhibition

GSH HO1 expression of

SOD activity expression proinflammatory
cytokines

ROS Production
Inflammation
Oxidative stress

Fig. 2.1 Schematic illustration of the molecular mechanisms and pathways of Berberine in
reducing oxidative stress and inflammation. 1 Berberine could inhibit oxidative stress by
upregulation of SOD, and downregulation of NADPH oxidase expression. 2 Berberine
administration induces the activation of the Nrf2 transcription. The effect of berberine on Nrf2
relies on the activation of AMPK, and P38 pathways. 3 Berberine could suppress inflammation by
blocking the MAPK pathways in an AMPK-dependent manner, inhibiting the classic NF-κB
transcription; inhibiting the Rho GTPase pathway, which plays a role in NF-κB regulation, and
attenuating the transcription activity of AP-1, which was possible to be mediated by PPARγ
activation
2 Berberine and Its Role in Chronic Disease 31

microparticles, vesicular structures found in plasma from patients with vascular

diseases so utilized as a surrogate marker of endothelial dysfunction, are oxidative
stress inducers; they promote upregulation of NOX4 expression and ROS pro-
duction. It has been reported that berberine reversed NOX4-derived ROS produc-
tion in human umbilical vein endothelial cells (HUVECs) [46].
NOX could be negatively regulated by adenosine monophosphate-activated
protein kinase (AMPK) activation [49, 50]; in fact AMPK activators, such as
metformin, may exert their cardiovascular protective function through NOX inhi-
bition [51]. AMPK pathway is activated by berberine [52] and it seems to play a
pivotal role in mediating its antioxidant activity [53, 54].
The AMPK is a ubiquitously expressed cellular energy sensor and an essential
component of the adaptive response to cardiomyocyte stress that occurs during
ischemia. AMPK plays also an important role in regulating function of NO syn-
thesis in endothelial cells. In fact, AMPK is an upstream kinase of endothelial nitric
oxide synthase (eNOS) which promotes the phosphorylation of eNOS at Ser1177
site as well as the formation of eNOS and HSP90 complex and NO production [55].
Zhang et al. [56] observed that in HUVECs berberine ameliorated
palmitate-induced endothelial dysfunction by upregulating eNOS and downregu-
lating of NOX4 through the activation of AMPK. In both cultured endothelial cells
and blood vessels isolated from rat aorta berberine enhanced eNOS phosphorylation
and attenuated high glucose-induced generation of ROS, cellular apoptosis, NF-κB
activation and expression of adhesion molecules through AMPK signalling cascade
activation, a key event in preventing oxidative and inflammatory signalling [57].
Besides NADPH oxidase downregulation and NO production, AMPK activation
has been linked to upregulation of the antioxidant enzyme superoxide dismutase
(SOD) [58, 59], which is dismutated to hydrogen peroxide. It was observed an
increased SOD expression in berberine treated diabetic mice [60, 61]. Glutathione
(GSH) is another antioxidant molecule which helps to maintain the balance of redox
state in organisms and acting asco-substrate of glutathione peroxidase (GSH-Px) in
the clearance of peroxides [62]. Berberine treatment promoted a GSH-Px and SOD
hyperactivation in the liver of mice [63], attenuated ROS production and increased
detoxifying enzymes GSH-Px and SOD in NSC34 motor neuron-like cells [64].
Recent studies revealed that berberine suppressed oxidative stress through
induction of the nuclear factor erythroid-2-related factor-2 (Nrf2) pathway [65–67].
Nrf2 is a transcription factor which binds to antioxidative response elements
(ARE) in DNA, leading to transcription of phase II enzymes and cytoprotective
proteins genes such as NAD(P)H quinone oxidoreductase-1 (NQO-1) and heme
oxygenase-1 (HO-1) with a wide range of activities in regulating redox state and
energy metabolism in cells [68]. Now, Nrf2 is recognized as an important mediator
of berberine in reducing oxidative stress, as blocking Nrf2 abolishes the antioxidant
activity of berberine in macrophages and nerve cells [65–67]. The activation of
AMPK, phosphatidylinositol 3-kinase (PI3K)/Akt and p38 kinase cellular pathways
is involved in the effect of berberine on Nrf2, since the block of these pathways
diminishes the stimulating effect of berberine on Nrf2 [65–67].
32 A.F.G. Cicero and A. Baggioni

The anti-inflammatory activity of berberine was observed both in vitro and

in vivo and was noted by the reduction of proinflammatory cytokines as well as
acute phase proteins [69–78].
In cultured metabolically active cells (adipocytes and liver cells), immunocytes
(macrophages and splenocytes) or pancreatic β-cells, berberine treatment reduced the
production of TNF-α, IL-6, IL-1β, matrix metalloprotease 9 (MMP9), cyclooxygenase-2
(COX2), inducible NOS (iNOS), monocyte chemoattractant protein 1 (MCP-1) and
C-reactive protein (CRP) and haptoglobin (HP) [70–100]. In insulin-resistant HepG2
cells, the anti-inflammatory activity of berberine was associated with its
insulin-sensitizing effect. Berberine administration significantly decreased cytokine
production, and reduced serine phosphorylation but increased insulin-mediated tyrosine
phosphorylation of IRS in HepG2 cells treated with palmitate [71].
Berberine could reduce proinflammatory cytokines, acute phase protein and
infiltration of inflammatory cells in animals with diabetes mellitus or insulin
resistance, either induced by streptozocin injection/high-fat diet (HFD) feeding or
spontaneously happened [69, 72, 74–76]. In these animal models, the
anti-inflammatory activity of berberine was observed in different tissues like serum,
liver, adipose tissue, and kidney and was associated with its effect against insulin
resistance or diabetes mellitus [69, 72, 74–76]. Besides evidences from cultured
cells and diabetic animal models, the anti-inflammatory effect of berberine was also
observed in humans: the berberine dose of 1g/day for 3 months significantly
reduced the serum hsCRP and IL-6 level in patients with acute coronary syndrome
following percutaneous coronary intervention [80].
Berberine suppresses inflammation through complex mechanisms. In addition to
antioxidant activity, the AMPK pathway was also crucial for the anti-inflammatory
efficacy of berberine [72]. Blocking AMPK could abolish the inhibitory effect of
berberine on the production of proinflammatory cytokines, like inducible nitric
oxide synthase (iNOS) and COX2 in macrophages [72]. Excessive iNOS in cells
could cause overproduction of NO and had close relationship with the development
of insulin resistance [82]. COX2 is a key enzyme for the synthesis of prostaglandins
[81], which are important mediators for the pathogenesis of diabetes mellitus and
diabetic nephropathy [82].
The anti-inflammatory activity of berberine was also associated with its inhi-
bitory effect on the mitogen-activated protein kinase (MAPK) signalling pathways,
which were activated by inflammatory stimuli [72, 83, 84]. The inhibitory effect of
berberine on MAPKs was dependent on AMPK activation in macrophages [72]. It
seems that conflicting results exist concerning the regulatory effect of berberine on
MAPK signalling. Although some results suggested that berberine suppressed the
inflammation through inhibiting MAPKs [72, 83, 84], others indicated that p38
kinase was activated by berberine which was considered important for berberine’s
efficacy against oxidative stress and inflammation [65–67].
The NF-κB pathway plays a key role in controlling inflammation [85]. In NF-κB
signalling pathway, IκB kinase-β (IKK-β) could be activated by inflammatory
stimuli like TNF-α, as well as nutritional factors like glucose and FFA [86]. The
activation of IKK-β required phosphorylation of the serine residue at position 181
2 Berberine and Its Role in Chronic Disease 33

[87, 88]. In insulin-resistant 3T3-L1 adipocytes [89] and liver/adipose tissues from
obese mice feed with HFD [74], berberine administration greatly reduced phos-
phorylation of ser181 and activation of IKK-β. In addition, the inhibitory effect of
berberine on IKK-β required a cysteine residue at position 179 of IKK-β [89].
Recent studies proved that berberine could reduce renal inflammation in diabetic
rats through inhibiting the Rho GTPase signalling pathway [69]. Rho GTPase is a
member of the superfamily of small GTP binding proteins with multiple biological
functions [90]; it was proven to positively regulate the NF-κB signalling pathway in
diabetic rats [91]. Therefore, in addition to regulation of the classic NF-κB sig-
nalling pathway, berberine could inhibit NF-κB by suppressing Rho GTPase [69,
92]. Furthermore, the inhibitory effect of berberine on Rho GTPase relied on its
antioxidant activity [69].
In addition to NF-κB, transcription factor activator protein 1 (AP-1) also played
a role in the anti-inflammatory activity of berberine [93, 94]. Administration of
berberine to macrophages or epithelial cells greatly attenuated the DNA binding
activity of AP-1 and reduced the production of cytokines like MCP-1 and COX2
[93]. There were reports that the transcription stimulating activity of AP-1 and
NF-κB could be inhibited by activation of peroxisome proliferator-activated
receptor γ (PPARγ) [95–99].

2.1.2.1 Berberine Effects on Glucose Metabolism

In general, there are two distinct pathways to activate glucose uptake in peripheral
tissues; one stimulated by insulin through the IRS-1/PI 3-kinase and the other by
exercise or hypoxia via activation of AMP activated protein kinase (AMPK). In
muscle, which is the major tissue responsible for whole body glucose disposal after
liver, both pathways stimulate the translocation of glucose transporter-4 (GLUT4)
to the cell membrane which accounts for the enhanced glucose uptake [100].
Current data suggest that the berberine effects are complex and may activate
portions of both the insulin and the exercise-induced glucose uptake pathways
[101]. In addition, berberine inhibits intestinal absorption of glucose, which also
contributes to berberine glucose-lowering effect [102].
There is increasing evidence that the most widely expressed GLUT1, initially
thought to be responsible only for basal glucose uptake, can be acutely activated by
cell stressors such as azide [103, 104], osmotic stress [105, 106], methylene blue [107]
and glucose deprivation [108, 109]. In particular, the acute activation of GLUT1 by
hypoxia or azide has been attributed to activation of AMPK [110, 111]. In addition, it
has been recently shown that peptide C activates GLUT1 transport activity in ery-
throcytes, establishing a potential link between GLUT1 activity and diabetes [112].
In cultured human liver cells and rat skeletal muscle, berberine increases insulin
receptor mRNA expression through Protein kinase C-dependent activation of its
promoter [113].
Since berberine was observed to act as an insulin-sensitizing agent in cultured
cells [114], its activity has been compared with that of metformin in different animal
34 A.F.G. Cicero and A. Baggioni

models. In rat models of type 2 diabetes (T2DM), berberine shows to have equal or
better fasting plasma glucose (FPG), insulin-resistance and low-density lipoprotein
cholesterol (LDL-C) lowering activity than metformin by a mechanism involving
retinol binding protein-4 (RBP-4) and (GLUT-4) [115, 116].
Berberine exhibited a high hypoglycemic potential; it has been shown that
berberine activates AMPK with subsequent induction of glycolysis [117]. AMPK,
as an intracellular energy receptor, has attracted more attention and become a new
target for the treatment of diabetes and its cardiovascular complications due to its
regulatory effect on endothelial cell function and energy homeostasis. In H9c2
myoblast cell line treated with insulin to induce insulin resistance, berberine
attenuated the reduction in glucose consumption and glucose uptake at least in part
via stimulation of AMPK activity [118]. berberine enhanced acute insulin-mediated
GLUT4 translocation and glucose transport in insulin-resistant myotubes through
activation of AMPK and PI3K pathway [119] (Fig. 2.2).

Berberine

Pancreas
β-cell

Insulin
receptor (IR)
Peripheral
IR
cells dephosphorylation

Berberine Protein Kinase C

AMPK
Pathway
activation

Fig. 2.2 Main glucose-lowering effects of berberine in the human cells. Berberine administration
could decrease glycemia through the GLP-1 receptor activation in pancreas beta cells, the increase
of Insulin Receptor expression and the AMPK-modulated Glut-4 translocation in peripheral cells
2 Berberine and Its Role in Chronic Disease 35

In a clinical study, the same group observed that berberine significantly lowered
FPG, hemoglobin A1c, triglycerides and insulin levels in patients with T2DM as
well as metformin and rosiglitazone (a combination commonly used for the T2DM
therapy); the percentages of peripheral blood lymphocytes expressing InsR were
significantly elevated after therapy [120].
In a recent meta-analysis of randomized clinical trials, berberine resulted to be
safe and effective in the treatment of patients with T2DM [121].

2.1.2.2 Berberine Effects on Lipid Metabolism and Vascular Health

The cholesterol and triglycerides lowering effect of berberine has been clearly
demonstrated by a recent meta-analysis of randomized clinical trials [122]. The
lipid-lowering activity of berberine, in association with other nutraceuticals, has
been also clearly confirmed in a relatively large number of randomized clinical
trials [123, 124].
The supposed mechanism of action is the increased expression of the liver
receptor for LDL mediated by the inhibition of the Pro-protein-convertase-
subtilisin-kexin-9 (PCSK9) activity [125]. Besides its upregulation effect on the
LDL receptor, berberine could also reduce triglycerides by AMP kinase activation
and MAPK/ERK pathway blocking [126] (Fig. 2.3).
High levels of LDL and their oxidized counterpart, oxidized LDL (oxLDL), in
the blood vessels represent a major risk factor for endothelial dysfunction and
atherosclerosis [127]. Inactivity of LDL receptor (LDLR) or its low-level expres-
sion initiates accumulation of LDL in blood vessels [128]. On the other hand, the
receptor of oxLDL, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1)
identified as the main endothelial receptor for oxLDL also present in macrophages
and smooth muscle cells (SMC), activates a proatherogenic cascade by inducing
endothelial dysfunction, SMC proliferation, apoptosis and the transformation of
macrophages into foam cells and platelet activation via NF-κB activation [129].
LOX-1 contains a lectin-like extracellular C-terminal domain which interacts with
oxLDL, proteolytically cleaved and released as a soluble circulating form (sLOX-1)
that reflects the increased expression of membrane-bound receptors and disease
activities [130].
In human macrophage-derived foam cells treated with oxLDL, berberine inhibits
the expression of LOX-1 [131] as well as the oxLDL uptake of macrophages and
reduces foam cell formation in a dose-dependent manner [132] by activating the
AMPK-SIRT1-PPARγ pathway [133]. Chi and colleagues demonstrated that ber-
berine combined with atorvastatin is more effective in diminishing LOX 1
expression than atorvastatin alone in monocyte-derived macrophages both in vitro
and in rats through modulation of endothelin-1 receptor [134].
Berberine improves also the survival of TNFα-treated endothelial progenitor
cells (EPCs) via the activation of PI3K/AKT/eNOS transcription factor [135]
possibly through AMPK activation. Wu and colleagues showed, both in vitro and
in vivo that berberine reduces the leukocyte-endothelium adhesion and vascular cell
36 A.F.G. Cicero and A. Baggioni

LDL- C decrease

LDL- C receptors

Liver cell
membrane

TG decrease

LDL- C receptors
upregulation

MAPK/ERK inhibition
PCSK9 Inhibition
AMPK activation

BERBERINE

Fig. 2.3 Main lipid-lowering effects of berberine in the human liver cells. Berberine mainly
decreases circulating LDLs by inducing LDLR expression in hepatic cells mediated by inhibition
of PCSK9

adhesion molecule-1 (VCAM-1) expression induced by LPS. Berberine was further

confirmed to inhibit the nuclear translocation and DNA binding activity of
LPS-activated NF-κB signalling pathway [136].

2.1.2.3 Berberine and Central Nervous System Disorders

A large number of preclinical evidence support a possible role of berberine in the

management of Alzheimer’s disease, cerebral ischemia, mental depression,
schizophrenia and anxiety, however the most part of these data have been obtained
in purely experimental models [137]. Of particular interest is the potential antide-
pressant effect of berberine.
Berberine inhibited the immobility period in mice in both forced swim and
tail-suspension test, two animal models of depression, in a dose independent
manner [138, 139]. Among the reported bioactivities of berberine, there is the
inhibition of monoamino oxidase (MAO)-A activity, [140] an enzyme catalyzing
the oxidative deamination of catecholamines, and thus inhibiting degradation of
these neurotransmitters. In fact acute and chronic administration of berberine in
2 Berberine and Its Role in Chronic Disease 37

mice resulted in increased levels of norepinephrine, serotonin and dopamine,

neurotransmitters induced by MAO-A enzyme [140]. In accordance with Kulkarni
and colleagues data, Arora and Chopra [138] showed the protective
antidepressant-like effect of berberine against the reserpine-induced biogenic amine
depletion (a monoamine depletor commonly used to induce depression in animals.
However, at the best of our knowledge, there are no available data on the evaluation
of the potential antidepressant effects of berberine in humans [141].

2.1.2.4 Tolerability and Safety

Highly purified and concentrated berberine is safe, in fact, its Lethal Dose 50
(LD50) in mice is 25 mg/kg in mice [131].
Standard doses of berberine are usually well tolerated and adverse events are rare
and mild. The most studied side effects are those on the gastrointestinal system. In
fact, berberine and its derivatives can produce gastric lesions in animal models
[142]. As shown by the determination of small intestinal transit time measurements
by sorbitol and breath hydrogen test, berberine delays small intestinal transit time,
and this may account for a part of its gastrointestinal side effects (but also of its
antidiarrhoeal one) [143].
The main safety issue of berberine involves the risk of pharmacological inter-
actions. In fact, berberine displaces bilirubin from albumin about tenfold more than
phenylbutazone, thus any herb containing large amounts of berberine should be
avoided in jaundiced infants and pregnant women [144]. Berberine also displaces
warfarin, thiopental and tolbutamide from their protein binding sites, increasing
their plasma levels [145].
Then, berberine can markedly increase blood levels of cyclosporine A because
of CYP3A4 and P-glycoprotein inhibition in liver and gut wall, respectively, and
because of the increase in gastric emptying time, thus causing increased cyclos-
porine A bioavailability and reduced metabolism [146]. In renal transplant recipi-
ents who take cyclosporine 3 mg/kg twice daily, the coadministration of berberine
(0.2 g/day for three times a day for 3 months) increased the mean cyclosporine A
AUC of 34.5 % and its mean half-life of 2.7 h [147].
Even if the main mechanism of berberine pharmacological interaction involves
CYP3A4 and intestinal P-glycoprotein, it also inhibits CYP1A1, potentially inter-
acting with drugs metabolized by this cytochrome isophorm as well. The impact of
this observation in clinical practise has yet to be evaluated since the CYP1A1
metabolized drugs are relatively rare [148].
Overall, the assumption of berberine in dosages of 500–1000 mg/day has to be
considered safe for the most part of subjects and the risk of clinically relevant
pharmacological interaction is limited to cyclosporine and warfarin.
38 A.F.G. Cicero and A. Baggioni

2.2 Conclusion

Berberine is a natural alkaloid with proven antioxidant, anti-inflammatory,

glucose-lowering and lipid-lowering actions, both in animal models and in humans.
Altogether, these effects support the need to study the effects of the long-term
exposition to berberine for the management and prevention of numerous chronic
diseases such as type 2 diabetes and atherosclerosis.

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42 A.F.G. Cicero and A. Baggioni

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2 Berberine and Its Role in Chronic Disease 43

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44 A.F.G. Cicero and A. Baggioni

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2 Berberine and Its Role in Chronic Disease 45

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Chapter 3
Emodin and Its Role in Chronic Diseases

B. Anu Monisha, Niraj Kumar and Ashu Bhan Tiku

Abstract Diseases, such as heart disease, stroke, cancer, respiratory diseases, and
diabetes, are by far the leading cause of mortality in the world, representing 60 % of
all deaths. Although substantial medical advances have been made and many
therapeutic approaches proposed yet traditional medicine and medicinal plants find
an important place in therapy. They have been providing invaluable solutions to the
various health problems. Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is a
natural anthraquinone derivative found in various Chinese medicinal herbs.
Traditionally, it has been used as an active constituent of many herbal laxatives.
However, in the last few years, significant progress has been made in studying the
biological effects of emodin at cellular and molecular levels and it is emerging as an
important therapeutic agent. This review provides an overview of the modulatory
effects of emodin in various diseases and cell signaling pathways, which may have
important implications in its future clinical use.

Keywords Emodin Bioavailability Cancer Anthraquinone Radiosensitizer


Chemosesitizer

3.1 Introduction

Emodin is a member of natural compounds known as anthraquinones. Natural

anthraquinones are found in diverse plant groups from higher plants to fungi and in
some insects. More than half of the natural anthraquinones are found in lower fungi,
mainly Penicillium and Aspergillus species and in lichens. Rest of them are found

B.A. Monisha
Department of Biomedical Science, Bharathidasan University,
Tiruchirappalli, Tamil Nadu 620024, India
N. Kumar
A.B. Tiku (&)
Radiation and Cancer Therapeutics Lab, School of Life Sciences,
Jawaharlal Nehru University, New Delhi 110067, India
e-mail: [email protected]

© Springer International Publishing Switzerland 2016 47

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1_3
48 B.A. Monisha et al.

in higher plants and in isolated instances in insects [24, 131]. Among higher plants,
plants belonging to families Rubiaceae, Rhamnaceae, Fabaceae, Polygonaceae,
Bignoniaceae, Verbenaceae, Scrophulariaceae, and Liliaceae are rich sources of
anthraquinones [134]. Emodin, rhein, chrysophanol, aloe-emodin, and physcion are
the most common naturally occurring anthraquinonesin higher plants [24, 33].
The anthraquinone emodin is identified in 17 plant families distributed world-
wide but is primarily reported in three plant species Fabaceae (Cassia spp.),
Polygonaceae (Rheum, Rumex, and Polygonum spp.), and Rhamnaceae (Rhamnus
and Ventilago spp.) [43]. Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is
present in bark, root, vegetative organ (stem, foliage), reproductive organ (flower,
fruit, seeds, pods), and is produced as secondary metabolite by molds and lichens
[120].
Although emodin was first described more than 75 years ago (reported as
‘frangula-emodin,’ [56], many of its diverse biological properties have been dis-
covered in the last decade (see reviews by Srinivas et al. [120] and Shrimali et al.
[117]). Emodin has also been reported to play a significant ecological role in the life
of many plant species by mediating their interactions with their biotic and abiotic
environment [43].
Emodin is a bioactive anthraquinone and has been an active constituent of many
laxatives and Chinese herbal medicines [71, 73]. It has antitumour, antibacterial,
diuretic, and vasorelaxant effects [39, 57, 175]. It induces growth inhibition in
cancer cells but not in normal cells [98, 122, 162] and modulates cellular redox
status in a dose- and time-dependent manner [58, 120, 163]. The photo-protective
function of emodin against ultraviolet (UV) region of the solar radiation (290–
400 nm) has also been reported [8]. Protective role of emodin against
radiation-induced oxidative and DNA damage in murine splenocytes and in the
concanavalin A (ConA)-induced hyperproliferation was also reported [109, 110]. It
possesses immunosuppressive activities also [85, 142, 145]. Various pharmaco-
logical properties of emodin in both animal and human model systems have been
tabulated (Tables 3.1, 3.2).
Emodin is highly effective in case of pancreatitis, asthma, myocarditis, arthritis,
atherosclerosis, glomerulonephritis, Alzheimer’s, hepatitis, and chronic obstructive
lung disease [117, 156, 157]. It modulates various signaling pathways and produces
many therapeutic effects (Table 3.3).
Besides the beneficial effects, emodin has been reported to cause some toxic side
effects, such as genotoxicity, developmental toxicity, nausea, diarrhea, and renal
failure. Recently, Sevcovicova et al. [108] showed emodin exhibited dual activities;
on one side it was genotoxic inducing primary DNA lesions as well as gene
mutations and on the other it exhibited DNA-protective activity via free radicals
scavenging and reducing activities. Therefore, safety and effectiveness of emodin in
naturopathic treatment is yet to be approved by the U.S. Food and Drug
Administration (FDA). The present review is the compilation of the literature on
effects of emodin in various disease conditions and the underlying molecular
mechanisms.
3 Emodin and Its Role in Chronic Diseases 49

Table 3.1 Therapeutic effects of emodin in animal model systems

Bioactivity Model Mechanism References
Antibacterial, E.coli, Mice, RAW 264.7 Anti-MRSA Alves et al.
antiviral activity cell line (Methicillin-resistant [2], Liu
Staphylococcus aureus) et al. [85],
effect via damaging cell Liu et al.
membrane, affecting [86]
phospholipid
membrane, and reducing
the entry of CVB4 in a
time-dependent manner
Anticancer effects Mice xenografts bearing Promotes cell cycle Ljubimov
LS1034 (in vivo), SW1990 arrest, induces et al. [87],
cells, Mice leukemia apoptosis, inhibits cell Wang et al.
WEHI-3 cells, 4T1 and proliferation, migration, [143],
EO771 breast cancer cells, differentiation, and Kaneshiro
synthetic androgen receptor downregulates androgen et al. [51],
R1881cells, mice, rat, chick receptor Kwak et al.
embryo, zebra fish, Mice Regulates the expression [60], He
bearing gall bladder of NF-κB and et al. [35],
carcinoma SGC-996, NF-κB-regulated Li et al. [71,
GBC-SD cells. angiogenesis-associated 73], Wang
factors, blocks the et al. [140,
phosphorylation of 141], Chang
KDR/Flk-1 and et al. [9],
downstream effector Lin et al.
molecules including [78, 79], Ma
FAK, ERK1/2, p38, et al. [91],
Akt, endothelial nitric Li et al. [68,
oxide synthase and 74, 76], Jia
stimulates phagocytosis et al. [45]
and macrophage
recruitment in vivo
Anti-inflammatory Mouse mammary epithelial Inhibiting receptor Li et al.
activity cells, rat epithelial cells, expression and common [76], Zhu
rats, mice inflammatory pathways, et al. [177],
such as inhibiting Ni et al.
NF-κB activation and [100, 101],
TNF-α production, Sharma and
reducing neutrophil Tiku [109],
infiltration, and cytokine Yang et al.
production [161], Chen
et al. [11],
Han et al.
[32], Li
et al. [69,
70, 75, 77],
Pang et al.
[104]
(continued)
50 B.A. Monisha et al.

Table 3.1 (continued)

Bioactivity Model Mechanism References
Antiallergic activity Mice Acts primarily on Syk to Lu et al.
suppress downstream [88],
signaling events and Nemmar
mast cell activation and et al. [99]
prevents cardiac
inflammation, oxidative
stress, and thrombotic
complications
Anti-hyperlipidaemic C57BL/6 J male mice, rat, Enhances CPT-1 Zhao et al.
activity STZ-induced diabetic mice. expression along with [173], Feng
increased AMPK and et al. [26],
ACC protein expression, Tzeng et al.
protects against diabetic [133], Wu
cardiomyopathy by et al. [148,
phosphorylation and 150]
regulation of
AKT/GSK-3β signaling
pathway
Therapy for hepatic D-galactosamine-sensitized Deactivates MAPKs and Yin et al.
failure mice, Liquid fructose NF-κB signaling [165], Li
feeding rats pathways, and inhibits et al. [75]
TNF-α production
Therapy for bone Mice Suppresses osteoclast Hwang
remodeling and differentiation and the et al. [41],
arthritis bone-resorbing activity Zhu et al.
of mature osteoclasts by [177], Kang
inhibiting et al. [52],
RANKL-induced Kim et al.
NF-κB, c-Fos, and [54]
NFATc1 expression
Treats schizophrenia Rodent Attenuates Mizuno
phosphorylation of et al. [96]
ErbB1 and ErbB2 and
EGF receptor signaling
Cure for gall bladder Guinea pig gall bladder Inhibits voltage Wu et al.
disorder smooth muscle, In vivo and dependent potassium [151], Li
in vitro in SGC996 gall current in gall bladder et al. [74]
bladder carcinoma cell lines smooth muscle cell
model
Table 3.2 Therapeutic effects of emodin in human model systems
Bioactivity Models Mechanism References
Antitumor agent Human HCC cells, MCF-7 cells and Downregulates VEGF, inhibits Integrin Huang et al. [40], Kaneshiro et al. [51],
MDA-MB-231 cells, PC3 prostate Linked Kinase expression through Wang et al. [138, 140, 141], Yan et al.
cancer cells, Human Umbilical Vein AMPKα-mediated reduction of Sp1 and [158], Ma et al. [91]
Endothelial Cells (HUVECs) Human c-Jun proteins He et al. [34], Ok et al. [102], Fu et al.
skin squamous cell carcinoma, HSC5 Downregulates TGF-β signaling pathway, [27], Sun and Bu [127], Li et al. [74],
cells, Prostrate and Lung cancer cells, Blocks Her2/neu binding Jelassi et al. [44], Ma et al. [90], Sui
Parental human ovarian to Hsp90, resulting in et al. [123], Thacker and Karunagaran
adenocarcinoma cell line COC1 and its proteasomal degradation of Her2/neu [129], Masaldan and Iyer [93], Deng
cDDP-resistant derivative Effects ERCCI expression, inhibits the et al. [19], Hwang et al. [42], Zhang
COC1/DDP, human hepatoma cell line expression of ABCG2, decreases the level et al. [169, 172], Tang et al. [128],
SMMC-7721, HL-60 cells, of PRL-3 expression, downregulates Chihara et al. [16], Jun et al. [49]
MCF-7cells, Human NSCLC cell MRP1, and generates ROS
lines, PC9, H1299, H1650, and
3 Emodin and Its Role in Chronic Diseases

H1975, Human breast

(MDA-MB-435s, and MDA-MB-468)
cancer cells, HepG2, BGC, AGS,
HELF
Retina capillary endothelial cells,
HUVECs, LS1034 human colon
cancer cells, LS1034 tumor xenografts
model, Her2 over expressed cancer
cell lines, Human Gastric cancer cell
lines (SGC996, SGC7901, MKN 45,)
gall bladder carcinoma GBC-SD cells,
MCF-7/ADR
(continued)
51
Table 3.2 (continued)
52

Bioactivity Models Mechanism References

Apoptotic agent Human breast cancer cells Bcap-37 Induces apoptosis by intrinsic Shieh et al. [115], Wei et al. [147], Lin
and ZR-75-30, SW1990, Panc-1, mitochondrial and extrinsic death receptor et al. [78, 79], Yaoxian et al. [162], Zu
HPNE cells and ECs, Human pathways, activates caspase and PARP, et al. [178]
Hepatoma cell lines HepG2/C3A increases levels of p53 and Bax, and
(ATCC CRL-1074), PLC/PRF/5 decreases levels of Bcl-2
(ATCC CRL - 8024), and SK-HEP-1 Regulates the expression of NF-κB and
(ATCC HTB-52), SW1990 cells, NF-κB-regulated angiogenesis-associated
HeLa cells factors
Inhibits the expression of cyclin B1 and
Cdc2 to mediate the G2/M arrest in cell
cycle
Cytotoxic agent HeLa cells, Human Multiple Myeloma Generates intracellular ROS and inhibits Wang et al. [144], Muto et al. [98]
cell lines, U266, and IM-9 interleukin-6-induced JAK2/STAT3
pathway selectively
Anti-antherosclerosis Vascular smooth muscle cell (VSMC) Inhibits proliferation of VSMC via Heo et al. [36]
agent caspase dependent apoptosis
Barrier protective Intestinal epithelial cells Attenuates intestinal epithelial barrier Lei et al. [67]
effects dysfunction by inhibiting the HIF-1α and
NF-κB signaling pathways
B.A. Monisha et al.
3 Emodin and Its Role in Chronic Diseases 53

Table 3.3 Therapeutic effects of emodin in chronic diseases and mechanisms of action
Diseases Mechanism References
Allergy Inhibits TNF-α secretion through Lu et al. [88], Kim et al. [53],
the inhibition of PKC or Nemmar et al. [99]
PKC-IKK2 pathways
Cancer Targets PI3K/Akt pathway Su et al. [121], Olsen et al. [103],
Downregulates TGF-β signaling Hsu et al. [37], Yan et al. [158],
pathway and Inhibits β-catenin/Akt Way et al. (2014), Thacker and
Downregulates cytoprotective ERK Karunagaran [129, 130], Li et al.
and Akt cascade [77], Deng et al. [19], Hu et al.
Elevates the levels of Bax, reduces [38], Tang et al. [128], Sun et al.
Bcl-2 and activates caspase-2, -3, [124]
and -9, Suppresses the activation of
P210BCR−ABL downstream
signaling pathways including
CrkL, Akt/mTOR and MEK/ERK
Inhibits TOR signaling pathway
and blocks autophagy
Inhibits ILK expression through
AMPKα-mediated reduction of Sp1
and c-Jun proteins and suppresses
the activation of MAPK signaling
pathways
Inhibits Wnt signaling pathway like
(i) regulating the regulators-p300
and HBP1 (ii) increasing reactive
oxygen species
Cardiovascular Enhances mitochondrial Du et al. [23], Wu et al. [149],
diseases antioxidant components and Song et al. [119], Chen et al.
inducesTNF-α upregulation and [10, 12, 14], Jiang et al. [46]
cardiomyocyte apoptosis
Suppresses pro-inflammatory
cytokines TNF-α and IL-1β due to
inhibition of NF-κB activation
Inhibits IL-23/IL-17 inflammatory
axis, Th17cell proliferation and
viral replication mRNA/protein
Diabetes Regulates PPAR-γ and Xue et al. [155], Wang et al. [142,
AKT/GSK-3β signaling pathway 145], Wu et al. [148, 150],
Arvindekar et al. [3]
Kidney Activates autophagy by modulating Gao et al. [27], Liu et al. [81]
diseases AMPK/mTOR signaling pathways
Liver diseases Increases the mRNA levels of Zhan et al. [167], Dong et al. [20],
PPAR-γ Meng et al. [94], Dong et al. [22],
Inhibits p38 MAPK-NF-κB Lin et al. [81], Dang et al. [18],
pathway leading to suppression of Dong et al. [22], Lee et al. [61],
hepatic IFN-γ, TNF-α, IL-1β, Tzeng et al. [133], Liu et al. [84],
IL-12, IL-6, iNOS, ITGAM, CCL2, Xue et al. [156]
and MIP-2, MIP-2 receptor, and
CXCR2
(continued)
54 B.A. Monisha et al.

Table 3.3 (continued)

Diseases Mechanism References
Enhances CPT-1 expression along
with increase in AMPK and ACC
protein expression and
phosphorylation
Lung diseases Inactivates NF-κB and p38 MAPK Xiao et al. [153], Xue et al. [157],
pathway Sun et al. [125], Yin et al. [164]
Neurological Downregulates PI3K/Akt/GSK-3β Gao et al. [28], Li et al. [72], Yang
disorders signaling pathway, promotes et al. [160], Park et al. [105], Sun
PI3K/Akt-dependent CREB and Liu [126]
phosphorylation and activates class
III PI3K/Beclin-1/B-cell lymphoma
2 pathway
Other diseases Inhibits NF-κB and MAPKs signal Lee et al. [63], Ha et al. [31], Chu
pathways, Blocks et al. [17], Li et al. [68, 74, 76], Lee
HIF-1α/NF-κB-COX-2 signaling et al. [66], Lei et al. [67], Chen
pathways, Interferes with et al. [12], Pang et al. [104]
ROS-ERK1/2/p38 signal pathway
Upregulates PPAR-γ expression
Inhibits AP-1 signaling pathway

3.2 Chemical Structure, Biophysical Properties,

and Bioavailability

The basic chemical structure of anthraquinone is an anthracene ring (tricyclic

aromatic) with two ketone groups in position C9 and C10. The chemical structure
of emodin is depicted in Fig. 3.1 Zhang et al. [171] examined the relationship
between the chemical structure and the activity of emodin and proposed that one
methyl, one hydroxy, and one-carbonyl functional groups are critical for the bio-
logical activities of emodin. The keto forms of emodin are less stable than the enol
form, because the latter is stabilized by p-conjugation in the B ring. These differ-
ences in stability between the enol and keto forms indicate that the contribution of

Fig. 3.1 Structure of emodin, 1,3,8-trihydroxy-6-methyl-anthraquinone

3 Emodin and Its Role in Chronic Diseases 55

the keto forms can be considered negligible for emodin as a free molecule. It is clear
that emodin appears to be a planar molecule and the most stable radical in the gas
phase is the 3-OH species. Despite planarity of radical structures, there is no sig-
nificant electronic delocalisation between adjacent rings [92].
Emodin–DNA interaction mainly involves intercalation of emodin between
bases and with PO2 backbone. Interaction occurs mainly through Ade and Thy
bases and PO2 backbone of double helix. Binding constant for emodin–DNA is
5.59 × 10−3 M−1 [107]. Emodin has low DNA-binding affinity and has low
cytotoxicity against various cancer cells. Addition of pyrazole ring and certain
chemical groups like polymethyleneamine, sugar with anthraquinone chromophore
result in increased binding affinity and cytotoxicity against various cancers. Chain
of varying length, polarity, charge, rigidity, and steric bulk may impart different
DNA binding affinity. Emodin with mono-cationic amino side chain has stronger
cytotoxic potential against cancer cells than di-cationic amino side chain, as indi-
cated by cytotoxicity potency index (IC50) value [154].
Absorption, excretion, tissue distribution, and metabolism of emodin were
studied after a single oral administration of C14-labeled emodin (50 mg/kg) in rat
model [4]. Emodin was quickly absorbed from the gastrointestinal tract.
Radioactivity in the peripheral blood reached a peak 2 h after administration, and
within 24 h subsequently decreased to 30 % of the peak value. In two cannulated
rats, biliary excretion reached a maximum at approximately 6 h and amounted to
49 % dose within 15 h; 70 % of biliary activity was in the form of conjugated
emodin. Urinary excretion amounted to 18 and 22 % dose, in 24 and 72 h,
respectively and most metabolites in pooled urine found were free emodin and
emodic acid. Emodin glycosides are carried unabsorbed to the large intestine
(because of its chemical structure) where metabolism to the active aglycone takes
place by intestinal bacterial flora. The aglycone damages epithelial cells, which
leads directly and indirectly to changes in absorption, secretion, motility and exerts
its laxative effect [97, 135].
In Male Sprague-Dawley rats orally administered PC Polygonum cuspidatum a
widely used Chinese medicine which contains resveratrol and emodin, it was found
that the sulfates/glucuronides of resveratrol and emodin were the major forms in
circulation and in most assayed organs after oral intake [78, 79]. With regard to
tissue distribution emodin was detected as sulfates/glucuronides in lung and kidney,
as free form in liver, but was not detectable in brain and heart [113, 114]. In rats
poor oral bioavailability of emodin is thought to be the result of intestinal and
hepatic glucuronidation [83]. The activity and positional preference of glu-
curonidation of anthraquinones varies with organs, species, substrate concentra-
tions, UGT isoforms, and the substitution at b-positions [148, 150]. Generally, the
conjugated metabolites are recognized as the inactive product of drugs, however,
now there are increasing evidences showing that the conjugated metabolites of
polyphenols demonstrate various bioactivities [25, 111, 112, 116, 159, 166].
Emodin which mainly exists as conjugated metabolite in the circulation and in most
organs, needs to be extensively investigated in future.
56 B.A. Monisha et al.

3.3 Role of Emodin in Various Chronic Disorders

3.3.1 Allergy

Emodin plays an important role in allergic diseases like asthma, rhinitis, and atopic
dermatitis [47, 88]. Asthma is a respiratory disease associated with symptoms like
airway hyper responsiveness, mucus hypersecretion, and bronchial inflammation
[139]. Emodin can be a therapeutic agent in treating allergic airway inflammation. It
inhibited ovalbumin-induced increase in eosinophil counts and hypersecretion of
mucus from goblet cells in air way passage [17]. Emodin also exhibited antiallergic
activities via increasing the stability of the cell membrane and inhibiting extracel-
lular Ca2+ influx [142, 145].
Mast cells play a major role in allergic diseases. Emodin lowered mast
cell-dependent passive anaphylactic reaction in Ig-E sensitized mice and inhibited
degranulation, generation of eicosanoid, and secretion of cytokines in
dose-dependent manner in mast cell [53, 88, 117]. Mast cell degranulation inhi-
bition occurred via attenuation of protein kinase C and IƙB kinase 2 signaling
pathway [53].

3.3.2 Arthritis

Rheumatoid arthritis is a chronic inflammatory disease that causes damage to the

joints and is characterized by inflammation and infiltration of inflammatory cells
into synovial tissue and joint destruction. One of the major transcriptional pathways
involved in joint inflammation is the nuclear factor-κB (NF-κB) pathway. Emodin
exerted anti-inflammatory effects in collagen-induced arthritic mice through inhi-
bition of the NF-κB pathway and has shown therapeutic value for the treatment of
rheumatoid arthritis [41, 177]. Synovial angiogenesis is the main characteristic
feature of rheumatoid arthritis. Emodin decreased expression of various
angiogenesis-related genes like vascular endothelial growth factor (VEGF), HIF-1α,
and cyclooxygenase 2 [31]. Emodin considerably inhibited IL-1β and
lipopolysaccharide (LPS)-stimulated proliferation of RA synoviocytes under
hypoxic condition and reduced the production of pro-inflammatory cytokines
(TNF-α, IL-6 and IL-8), prostaglandin E2, and matrix metalloproteinase (MMP-1,
MMP-13) [31, 117].
Emodin treatment was also helpful in treatment of osteoporosis as it stimulated
osteoblast formation and inhibited osteoclastogenesis. Mice treated with emodin
showed decrease of LPS-induced bone loss and increased bone formation [54].
Emodin is emerging as a potential therapeutic agent to treat arthritis, fractures,
muscle injury, and pain [41, 152, 177].
3 Emodin and Its Role in Chronic Diseases 57

3.3.3 Cancer

Recent studies on emodin are mostly focused on its antitumor properties. These
efforts led to unraveling both the effect of emodin against cancer development and
the underlying molecular mechanisms involved. A number of studies have
demonstrated that emodin inhibited the growth and proliferation of various cancer
cells derived from different tumors, such as cervical, breast, lung, colorectal, and
prostate cancers (Tables 3.1, 3.2). After evaluation with other anthraquinone
derivatives, including emodin 1-O-β-D-glucoside, physcion 1-O-β-D-glucoside,
and physcion, C1 and C3 position of emodin was supposed to be important for its
antitumor function [59]. Meanwhile, emodin displayed over 25-fold differential
cytotoxicity against ras-transformed bronchial epithelial cells to the normal human
bronchial epithelial cells [6]. Emodin also evoked a less or no cytotoxic effect in
several normal cells, together with human fibroblast-like lung WI-38 cells,
HBL-100 cells derived from normal human breast tissue and three primary cultured
rat normal cells [115, 170]. Recently, Sharma and Tiku [109] reported noncytotoxic
effects on murine splenocytes up to 100 µM of emodin. These observations sug-
gested that normal cells might be more resistant to emodin-induced cytotoxicity
than cancer cells. Cells contain various pathways designed to protect them from the
genomic instability or toxicity that can result when their DNA is damaged.
A pivotal role in this response is played by checkpoint proteins that control the
normal passage of cells through the cell cycle. The effect of emodin on cell cycle
has been demonstrated on various cancer cells. In Her2/neu-over expressing
MDA-MB-453 breast cancer cells emodin azide methyl derivative (AMAD) trig-
gered mitochondrial-dependent cell apoptosis involving caspase-8-mediated Bid
cleavage. This derivative-induced G0/G1 arrest by blocking Her2/neu binding to
Hsp90. This was associated with decreasing protein expression of c-Myc, Cyclin
D1, CDK4, and p-Rb [123, 158, 169]. Emodin-induced G2/M phase arrest in
v-ras-transformed cells [6] and the human hepatoma cell line HepG2/C3A cells
[115]. Elevation of p53 and p21 expression might be involved in this G2/M arrest
[115]. In addition to G2/M phase arrest, emodin was reported to block the G1 to S
phase of the cell cycle in human colon carcinoma HCT-15 cells [50] and breast
cancer MDA-MB-453 cells [170]. It downregulated Wnt signaling pathway in
human colorectal cancer cells SW480 and SW620 [129]. Emodin attenuated
radioresistance in the HepG2 cells via upregulation of the apoptotic signals and
downregulation of the proliferative signals [42]. Emodin also inhibited the lung
metastasis of human breast cancer in a mouse xenograft model, and inhibited the
invasion of MDA-MB-231 cells associated with the downregulation of MMP-2,
MMP-9, uPAR, and uPA expression as well as decreased activity of p38 and ERK
[124]. Combination of emodin with curcumin synergistically inhibited survival,
proliferation, and invasion of breast cancer cells and cervical cancer [130].
Apoptosis could be a potential general mechanism of the anti-proliferative and
antineoplastic effects of emodin. A number of studies have demonstrated that
emodin is capable of inducing apoptotic cell death in various cancer cells [120].
58 B.A. Monisha et al.

Several studies revealed that emodin-induced apoptosis was mediated by reactive

oxygen species (ROS) generated from the semiquinone [48, 121], however there
was a report that emodin-induced apoptosis, ROS-independently [13]. It is believed
that the quinoid structure of emodin could be activated to the semiquinone radical
intermediate, which in turn could react with oxygen to generate ROS and induce
oxidative stress [121]. The generation of ROS may contribute to mitochondrial
injury, reduction of mitochondrial transmembrane potential, cytochrome c and
Smac release, and subsequent caspase activation resulting in apoptosis [5].
Several groups examined the role of Bcl-2 family members to further explore the
mitochondria-related pathway involved in emodin-induced apoptosis. Emodin
treatment significantly increased expression level of Bax and Bak, pro-apoptotic
protein, and caused Bax mitochondrial translocation preceding apoptosis [48].
Besides, anti-apoptotic protein Bcl-2 was also involved in emodin-induced apop-
tosis: first, emodin caused a significant decrease in Bcl-2 expression; second,
ectopic expression of Bcl-2 markedly blocked emodin-induced apoptosis [68, 178].
Additionally, PKC-δ and ε may also be involved in emodin-induced apoptosis. It
appeared that PKC was downstream of caspase-3 in the emodin-induced apoptosis
[62]. The inhibitory effects of emodin on tumor-induced metastasis and angio-
genesis in human breast cancer were caused by inhibition of MMPs and VEGFR-2,
which may be associated with the downregulation of Runx2 transcriptional activity
[44, 89, 124].
Emodin also potentiates the anticancer effects of cisplatin on gallbladder and
ovarian cancer cells through ROS-dependent pathway [90, 140]. Emodin
co-treatment was found to downregulate multidrug-resistance-associated protein-1
(MRP-1) and increase the sensitivity of SGC996 cells to cisplatin, carboplatin, or
oxaliplatin [74, 140, 141]. Emodin was also found to be a potential agent for
radio-sensitization of hepatocellular carcinoma (HCC) HepG-2 cells [42, 169].
Alternatively emodin protects cultured human kidney (HEK 293) cells and
murine splenocytes against cisplatin-induced and gamma radiation-induced
oxidative stress, respectively [109, 136]. Theses reported studies showed that
emodin can have differential effects on normal cells and cancerous cells.

3.3.4 Cardiovascular Diseases

Myocarditis is an inflammatory disease of heart that leads to heart failure.

Inflammation and autoimmunity is the major cause of myocarditis. Emodin is a
promising candidate for treatment of myocarditis. The severity of myocarditis was
reduced by treatment with emodin. In a murine acute myocardial infarction model,
emodin reduced the myocardial infarct size [149]. In rat model of experimental
autoimmune myocarditis, it inhibited NF-κB activity there by reducing the level of
pro-inflammatory cytokines TNF-α, IL-1β [119]. Besides it protected against viral
myocarditis by inhibiting IL-23/IL-17 inflammatory axis, Th17 cell proliferation,
and viral replication in mice [46]. Emodin was found to inhibit CVB3 replication
3 Emodin and Its Role in Chronic Diseases 59

(causal agent of viral myocarditis) by inhibiting CVB3 VP1 protein translation. The
fundamental signaling pathways involved in inhibition of CVB3 VP1 protein
translation include Akt-mTORC1-4EBP1, mTORC1-p70S6K, ERK1/2-p90RSK,
and Ca2+-calmodulin (Ca2+-CaM) cascades. Therefore, these pathways might be the
targets for emodin [168].
Atherosclerosis a chronic inflammatory disease characterized by the presence of
atherosclerotic plaque in the arterial intima can lead to hardening and narrowing of
the major arteries. Emodin inhibited NF-κB activation and TNF-α-induced migra-
tion, proliferation and MMP-2, MMP-9 expression in rat aortic smooth muscle cells
(RASMCs) [95]. It also had beneficial effects on stability of atherosclerotic plaque
in Apo-E deficient mice (apo-E has anti-atherosclerosis property) and was found to
inhibit the expression of M-CSF and MMP-9, whereas it enhanced the PPAR-γ
expression [174]. Cardiovascular protective actions of emodin have been attributed
to activation of cardiac natriuretic hormone secretion.
In isolated rabbit atria, emodin heighted atrial natriuretic peptide (ANP) secre-
tion via inhibition of L-type calcium channels and activation of potassium ATP
channels [176]. C-reactive protein (CRP) an inflammatory molecule plays a direct
role in atherogenesis. Emodin was also found to inhibit Hcy-induced CRP gener-
ation in vascular smooth muscles cells via ROS-ERK1/2/p38 signaling pathway and
upregulated PPARγ expression [104]. The above-mentioned results provide a
rationale for the use of emodin in the treatment of cardiovascular homeostasis and
other heart diseases.

3.3.5 Diabetes

Diabetes mellitus is a disease characterized by chronic hyperglycemia, disorders of

lipid, carbohydrates, and microvascular pathology of retina, renal glomerulus, and
peripheral nerves. Emodin injected intraperitoneally for 3 weeks to diabetic mice
(high fat fed diet) and low dose of streptozotocin (STZ) showed significantly
reduced blood glucose, triglycerides, and cholesterol in serum. qRT-PCR results
further confirmed that emodin significantly elevated the mRNA expression level of
PPAR-γ (regulates the mRNA expressions of LPL, FAT/CD36, resistin, and FABPs
(ap2) in liver and adipocyte tissues) [155]. Emodin at a concentration of 3 and
30 µm/L suppressed 11-dehydrocorticosterone-induced lipolysis and adipogenesis,
respectively without affecting corticosterone-induced lipolysis and adipogenesis
[142, 145]. Emodin also reduced the activation of glucocorticoid, which is insulin
antagonizing hormone by inhibiting 11β-hydroxysteroid dehydrogenase type 1
(11β-HSD1) in 3T3-L1 adipocyte cells in a concentration-and time-dependent
manner. Adiponectin is an important insulin-sensitizing adipokine, emodin lead to
assembly of high-molecular weight adiponectin and increased the ratio of
high-molecular adiponectin (possesses insulin-sensitizing activity) to total adipo-
nectin in 3T1-L1 adipocytes [10, 14]. SREBP-1 and SREBP-2 (transcription fac-
tor involved in biosynthesis of cholesterol, fatty acid, and triglyceride) mRNA
60 B.A. Monisha et al.

level was also significantly reduced in the liver and adipose tissue after emodin
treatment [69].
Emodin may also be a potential medication in treating the glucose dependent
structural and functional abnormalities in peritoneal membrane. Emodin regulated
the undesirable effects of concentrated glucose on human peritoneal mesothelial
cells by suppression of protein kinase C (PKC) activation and cyclic AMP response
element binding protein (CREB) phosphorylation [7]. Emodin was found to protect
against diabetic cardiomyopathy by regulating AKT/GSK-3β signaling pathway
[148, 150]. In the presence of high-glucose concentration human umbilical vein
endothelial cells (HUVECs) cultured with 3 µm of emodin showed protection from
endothelial cytotoxicity by suppressing MAPK pathway and inhibiting chemokine
ligand 5 (CCL5) expression [30].
Autoimmune diabetes (AID) is a metabolic disease that progresses through an
intricate relationship of environmental, genetic, and immune factor. Emodin was
able to suppress the chemotactic activity of leukocytes at the insulitis stage
(inflammation of the islets of Langerhans) of AID development [15].

3.3.6 Kidney Diseases

Emodin was also found to have beneficial effects in renal dysfunction. In diabetic
nephropathy it inhibited activation of p38 MAPK pathway and downregulated the
expression of fibronectin. This effect was independent of the blood glucose level
[137]. Emodin was able to protect mice from drug-induced kidney injury. In
cyclosporine-induced kidney nephropathy emodin prevented the overexpression of
Protein Kinase Casein Kinase II (PKCK2) which has a role in apoptosis [118].
In LPS treated NRK-52E cells (Rat kidney epithelial cells) it inhibited
TLR2-mediated NF-κB signaling pathway [70]. Emodin was found to ameliorate
cisplatin-induced apoptosis of rat renal tubular cells in vitro through modulating the
AMPK/mTOR signaling pathways and activating autophagy. Emodin may have
therapeutic potential for the prevention of drug-induced nephrotoxicity [81].

3.3.7 Liver Ailments

Emodin has been found to be beneficial in various liver ailments like

fructose-induced nonalcoholic fatty liver in rat, Con A-induced liver injury, and
LPS-induced fulminant hepatic failure. High carbohydrate/high fat diet fed mice
treated with emodin were protected from hepatosteatosis and metabolic derange-
ment. The mechanism involved were modulation of glutathione homeostasis and
TNF-α inhibition [1]. Con A-induced hepatic injury is a well-characterized murine
model with a pathophysiology similar to that of human viral and autoimmune
hepatitis. Emodin pretreatment protected against Con A-induced liver injury in
3 Emodin and Its Role in Chronic Diseases 61

mice, partially through inhibition of both the infiltration of CD4(+) and F4/80(+)
cells and the activation of the p38 MAPK-NF-κB pathway in CD4(+) T cells and
macrophages [156, 165]. Emodin was capable of improving the lipid accumulation
through the ERS-SREBP1c pathway in fructose-induced nonalcoholic fatty liver
disease [75]. Thus emodin might be applied as a potential candidate for the pre-
vention and intervention of liver diseases. However, in a recent study toxic effects
of emodin were reported in idiosyncratic liver injury model by Tu et al. [132].
Emodin-potentiated liver injury induced by noninjurious dose of LPS. The mech-
anisms underlying this effect are yet to be fully understood.

3.3.8 Lung Diseases

Emodin is considered as a potential pulmonary protective agent against lung tox-

icity induced by particulate air pollution. Particulate air pollution is related with
inflammation, impairment of lung function, and oxidative stress. In lung diseases
emodin reduced oxidative stress and increased the expression and activity of HO-1,
Nrf-2 [99, 157]. Besides acute lung injury is a very well-known fatal disease.
Emodin has been reported to repress LPS-induced pulmonary inflammation, pul-
monary oedema, and can be used to treat acute lung injury (ALI) and acute res-
piratory distress syndrome (ARDS) [153].

3.3.9 Neurological Disorders

Abnormality in cytokine signaling is implicated in the neuropathology and emodin

has been reported to be an effective neuroprotective drug. Subchronic oral
administration of emodin (50 mg/kg) to an epidermal growth factor (EGF)-induced
schizophrenia rat model, resulted in suppressed acoustic startle responses and
abolished prepulse inhibition deficits. Emodin was found to both attenuate EGF
receptor signaling and ameliorate behavioral deficits [96]. Accumulation of
β-amyloid is an important step in pathogenesis of Alzheimer’s disease. Emodin
treatment protected cultured cortical neurons from Aβ25–35-induced toxicity and
inhibited abnormal aggregation of tau protein into paired helical filaments. The
mechanism mediating this neuroprotective effect involve the upregulation of Bcl-2,
the activation ER/PI3K/Akt pathway as well as the inhibition of JNK1/2 phos-
phorylation induced by Aβ25–35 [82, 126].
Chronic emodin (20, 40 and 80 mg/kg) treatments remarkably improved
depression-like behavior in chronic unpredictable mild stress (CUMS) mice.
Abnormal activation of hypothalamic–pituitary–adrenal (HPA) axis is an important
marker of depression. Antidepressant activity of emodin was mediated, at least in
part, by the upregulating Glucocorticoid Respotor (GR) and Brain Derived
Neurotrophic Factor (BDNF) levels in hippocampus [72]. Epilepsy is another
62 B.A. Monisha et al.

chronic brain dysfunction syndrome. COX-2, N-Methyl-D-Aspartate (NMDA)

receptor, and P-glycoprotein have a synergic relationship in the pathogenesis of
epilepsy. Epileptic seizures are tightly associated with upregulated MDR1 gene,
and emodin showed good antagonistic effects on epileptic rats, possibly through
inhibition of NMDA-mediated overexpression of MDR1 and its associated genes
[160]. Neurite outgrowth is an important marker of neuronal differentiation.
Emodin-induced neurite outgrowth in Neuro2a cell by P13K/Akt/GSK3β signaling
pathway [105]. Future studies aiming at precisely understanding the cellular
mechanisms involved in the neuroprotective effects of emodin could open new
avenues for the treatment of various neurodegenerative diseases, and other neuronal
disorders.

3.3.10 Other Diseases

Emodin has been reported to have anti-inflammatory effects in both in vitro and
in vivo system (Tables 3.1, 3.2). Emodin when administered intraperitoneally
reduced LPS-induced mammary gland injury in mastitis [76]. Emodin has also
proved to be a potential candidate in treating serious vascular inflammatory dis-
eases, such as sepsis and septic shock and had anti-inflammatory effects in sepsis
mouse model in vivo [64, 65]. Emodin inhibited inflammation in intestinal
epithelial cells by blocking HIF-1α/NF-κB-COX-2 signaling pathways [67].
Emodin also lessened ocular tissue inflammation and fibrosis by inhibition of
NF-κB pathway after eye injury [11, 55]. Lee et al. [66] reported anti-inflammatory
effects of emodin derivative [6-O-β-D-glucoside emodin (EG)] in vitro in human
umbilical vein endothelial cells (HUVECs) as well as in mice. Recently, Han et al.
[32] reported that emodin attenuated NLRP3 inflammasome activation, leading to
decreased secretion of cleaved IL-1β, and blocking of the inflammasome-induced
pyroptosis. Ocular neovascularization is the origin of blindness related with
ischemic retinal ataxia in conjunction with proliferative diabetic retinopathy (PDR),
retinopathy of prematurity (ROP) and age-related macular degeneration. The
induction of angiogenesis in retina is due to VEGF. Emodin-MgSiO3 nanoparticles
could inhibit the expression of both VEGF gene and protein effectively and can be
an effective therapy for eye-related disorders [106].

3.3.11 Conclusions and Future Perspective

A detailed survey of the literature evaluating the efficacy of emodin in various

disorders, suggests that it can modulate multiple signaling pathways. The primary
signaling pathways affected by emodin are involved in cell proliferation, apoptosis,
differentiation, and have role in inflammation (Fig. 3.2). On one hand it inhibits
antiapoptotic pathways and prosurvival signals, and on the other can reduce
3 Emodin and Its Role in Chronic Diseases 63

PI3K
Inflammosome MAPK

NF-kβ Akt
ERK JNK P38

mTOR GSK3
IL-1β

Fig. 3.2 Common signaling pathways downregulated by emodin

cytotoxic effects through upregulation of autophagy. It can be an effective adjuvant

in cancer therapy by acting as a radiosensitizer making cancer cells more suscep-
tible to radiation, and at the same time protecting the normal cells. In combination
with various chemo therapeutic drugs it increases their efficacy and over comes
drug resistance in various cancers. The primary transcription factor downregulated
by emodin in different disorders is NF-κB that further regulates production/
transcription of cytokines (TNF-α, IL-6), cell adhesion molecules and MMPs that
have important role in various chronic disorders (Fig. 3.3). Recently, emodin has
been found to regulate energy metabolism. It upregulates transcription factor
PPAR-γ that is involved in regulation of adipogenesis and thus can be helpful in
treating metabolic disorders. Thus emodin has potential to be an important drug
molecule. However, the therapeutic potential of emodin is limited by its low
bioavailability. Literature survey on methods to improve the bioavailability of
emodin shows work in this direction has just started and emodin loaded nanopar-
ticles and chemical modification of emodin could be a way forward.
64 B.A. Monisha et al.

Fig. 3.3 Molecules modulated (upregulated ↑/downregulated ↓) by emodin in different chronic

disorders

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Chapter 4
Ursolic Acid and Chronic Disease:
An Overview of UA’s Effects On
Prevention and Treatment of Obesity
and Cancer

Anna M. Mancha-Ramirez and Thomas J. Slaga

Abstract Chronic diseases pose a worldwide problem and are only continuing to
increase in incidence. Two major factors contributing to the increased incidence in
chronic disease are a lack of physical activity and poor diet. As the link between
diet and lifestyle and the increased incidence of chronic disease has been well
established in the literature, novel preventive, and therapeutic methods should be
aimed at naturally derived compounds such as ursolic acid (UA), the focus of this
chapter. As chronic diseases, obesity and cancer share the common thread of
inflammation and dysregulation of many related pathways, the focus here will be on
these two chronic diseases. Significant evidence in the literature supports an
important role for natural compounds such as UA in the prevention and treatment of
chronic diseases like obesity and cancer, and here we have highlighted many of the
ways UA has been shown to be a beneficial and versatile phytochemical.

Keywords Cancer
Obesity
Inflammation
Ursolic acid
Triterpenoids

Phytonutrients

4.1 Introduction

Over the past 50 years, trends in lifestyle and diet have drastically affected the way
human beings experience disease and aging. Tragically, the modern western diet is
poorly balanced and composed of many nutrient-deficient foods. With this in mind,

A.M. Mancha-Ramirez
Department of Cellular and Structural Biology, The University of Texas Health
Science Center San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA
e-mail: [email protected]
T.J. Slaga (&)
Department of Pharmacology, The University of Texas Health Science Center
San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA
e-mail: [email protected]

© Springer International Publishing Switzerland 2016 75

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1_4
76 A.M. Mancha-Ramirez and T.J. Slaga

it is not surprising that chronic disease poses a serious worldwide problem, and one
that has undoubtedly risen from a stark dissonance between today’s modern diet
and that which our ancestors once thrived upon.
The modern western diet is characterized by an excess of salt and refined sugar,
and the readily available low cost, high calorie, nutrient foods that are part of a
typical western diet are believed to be a key component in the increasing obesity
epidemic [77, 116]. Specifically, based on data obtained from the National Health
and Nutrition Examination Survey, some of the most concerning issues with the
typical modern western diet include an increased uptake of refined grains, added
sugars, and saturated fats [81]. Moreover, it has been suggested that some of the
modern agricultural practices and over-processed nature of the typical western diet
has contributed to the nutrient deficiency seen in many components of regularly
consumed food products [116].
As the link between diet and lifestyle and the increased incidence of chronic
disease has been well established in the literature, it makes sense to direct our
attention to phytonutrients as meaningful therapeutic agents for the prevention and
treatment of chronic illnesses such as those discussed in this chapter. Natural
remedies derived from plants play an important role both in the prevention of
chronic disease development and in the treatment of chronic inflammation-driven
diseases. Moreover, plant-derived compounds have been the primary source of
medication for centuries in the Asian subcontinent [3, 7].
Significant evidence in the literature suggests that persistent inflammation is the
primary initiating factor that causes major chronic disease. Unfortunately, novel
approaches towards prevention or treatment of chronic diseases is still moving quite
slowly [1]. It is well known that phytonutrients derived from plants and their
extracts have been widely used in Eastern medicine for thousands of years for a
variety of illnesses included but not limited to hypertension, inflammation, and of
course cancer [58, 74]. Modern epidemiological studies have shown that con-
sumption of nutrient-rich fruit and vegetable-based diets can decrease the risk of
many diseases including metabolic syndrome and cancer [10, 43, 70, 128]. One
such powerful and versatile phytonutrient is ursolic acid (UA), a pentacyclic
triterpene that can be found in apples and rosemary, among other sources [40]. In
this chapter, we will discuss this phytonutrient in great detail including the multi-
tude of ways UA has shown great efficacy in both metabolic disorder maladies as
well as against several types of cancer (Fig. 4.1).
When considering the importance of chronic illness, for perspectives sake, let us
picture a crowded football stadium where every seat is filled; now, imagine that the
people in this stadium represent the population of the United States. According to a
2012 CDC report, approximately half of the fans in our imaginary stadium had one
or more chronic health condition(s), and one in four had two or more chronic health
conditions. Stepping away from our stadium analogy, this translates to approxi-
mately 117 million Americans, half of the adult population, who were living with
chronic illnesses in 2012. Of the ten most common chronic diseases, this chapter will
focus on cancer, obesity, and other illnesses that result from the latter, such as those
associated with metabolic syndrome. Perhaps after using our imagination for the
4 Ursolic Acid and Chronic Disease: An Overview of UA’s Effects … 77

Fig. 4.1 Chemical structure

of ursolic acid (UA) [123]

football stadium exercise, it is not difficult to see why chronic diseases and condi-
tions are among the costliest of all health problems. However, what is difficult to
discern is why we have not managed to diminish this increasing trend of chronic
illness when much of it can be prevented. The unfortunate trend in poor diet choices
coupled with incredibly busy lifestyles, which do not leave any room for regular
exercise, has led to a startling incidence in obesity and subsequently, the rise of
metabolic syndrome in the western world. Metabolic syndrome is a generalized term
for maladies such as hypertension, heart disease, and type II diabetes, all of which
are typically developed as a consequence of an individual having low physical
activity, resulting in obesity. Interestingly, there are several different types of cancer
including colon and breast cancer where risk factor is greatly increased by being
overweight or obese. Consequently, there is growing evidence supporting a strong
link between metabolic syndrome and an increased risk of developing cancer,
making it quite easy to see how these two chronic illnesses are tightly intertwined.

4.1.1 A Brief Overview of Obesity and Metabolic Syndrome

According to the World Health Organization, being overweight is one of the top 10
risk conditions in the world, and one which will affect upwards of one billion people
by 2030 [37]. Perhaps the most devastating aspect of this chronic illness is the fact
that obesity brings about so many different comorbidities that can significantly
compromise a patient’s quality of life; one highly relevant example of this is dia-
betes. Current predictions for the prevalence of diabetes suggest that this disease will
globally increase from 285 million in 2010 to 439 million in 2030 [101]. Not only
has diabetes quickly become an epidemic, but the other characteristic aspects of the
disease such as damage to tissue in the liver, kidney, adipose tissue, pancreas, and
vasculature as a result of pro-inflammation and oxidative stress, generate unique
problems of their own [14]. It is vital to remember that the pathogenesis of obesity is
extremely complex and can be caused by a variety of factors such as a genetic
predisposition, metabolism, physiology, endocrine problems, and behavioral issues
78 A.M. Mancha-Ramirez and T.J. Slaga

[134]. While obesity is traditionally considered fundamental to the development of

type II diabetes, it is now known that both insulin resistance and hyperinsulinemia
are part of this process as well [134]. As current antidiabetic drugs have minimal
efficacy and can often possess safety concerns, the need to identify novel therapies
for this devastating disease is immense. If new antidiabetic agents, ideally derived
from natural compounds, can be identified and utilized, these could hold great
promise for the hundreds of millions of individuals currently seeking better man-
agement of diabetes.

4.1.2 A Brief Overview of Cancer

“Cancer” is considered a rather oversimplified term for upwards of 100 malignant

neoplastic diseases [96]. As cancer is a multifactorial disease with many compli-
cated and intertwined causal pathways, choosing effective targets, and developing
novel therapies can be incredibly difficult. Despite significant time, money, and
effort put forth over the past several decades, the American Cancer Society reported
in 2013 that cancer still remains the second leading cause of death in the United
States. The startling difficulties of developing novel effective treatments coupled
with the fact that it is still one of the leading causes of death in the United States,
has directed a significant portion of research towards cancer chemoprevention.
Chemoprevention utilizes specific natural or synthetic chemical compounds to
inhibit or reverse carcinogenesis and/or to suppress the development of cancer [95].
As many studies have shown that the incidence of cancer could potentially be
decreased by chemoprevention, this strategy has become one that is heavily
investigated. Importantly, the majority of the chemopreventive agents currently
being evaluated are natural products or their derivatives. Many natural compounds,
or phytonutrients, have already been found to exhibit cancer chemopreventive
activities both in vitro and in vivo [49, 86]. Of course, one of the most beneficial
aspects of utilizing phytonutrients for chemoprevention is their nontoxic nature,
which is vastly different from traditional cancer therapies.

4.2 Mechanistic Links Between Obesity, Metabolic

Syndrome Diseases, and Cancer

Although cancer and obesity are two entirely different diseases by definition, they
share many cellular signaling pathways that play a part in their pathogenesis.
Moreover, both cancer and obesity-related diseases share a very important aspect of
pathogenesis, which is inflammation. Chronic inflammation has been linked with
most chronic illnesses including cancer, cardiovascular disease, diabetes, obesity,
and neurologic disease [3]. Significant epidemiological evidence shows an increased
4 Ursolic Acid and Chronic Disease: An Overview of UA’s Effects … 79

risk of acquiring several different types of cancer which is greatly increased by being
overweight or obese [5]. Specifically, a wide variety of cancers, including high-
grade prostate cancer, colorectal cancer, postmenopausal breast cancer, and mela-
noma have been shown to be linked to obesity in epidemiological studies [15, 25, 92,
105]. As you might expect, just as obesity and diabetes have been shown to be
associated with increase cancer incidence, conversely, exercise, and decreased
caloric intake have been shown to be associated with decreased cancer incidence. It
is well known that calorie restriction and exercise aid in the prevention of obesity
and diabetes, but both have also been correlated with decreased cancer incidences in
many epidemiological studies and can directly inhibit tumor formation in certain
cancer models [48, 75, 79]. For example, calorie restriction was able to prevent
tumor formation in various mouse models of cancer including the breast, colon,
brain, prostate, and chemically induced skin cancer [24, 69, 102].
When there is excess dietary intake in an individual, the result is excess fat
storage and improper processing of glucose and lipids [32]. The peptide hormone
insulin is secreted by the b cells of the pancreas and its job is to maintain normal
blood glucose levels by facilitating cellular glucose uptake, regulating carbohy-
drate, lipid, and protein metabolism and promoting cell division and growth [122].
The goal of insulin in healthy humans is to increase glucose uptake and tell the liver
to shut down the process of gluconeogenesis. Conversely, in overweight or obese
humans, this process is derailed by chronic exposure to high levels of glucose and
free fatty acids. Obesity-associated inflammation regulates insulin resistance in
target cells, ultimately resulting in more insulin production in order to reduce blood
glucose and fatty acid levels [51, 122]. The end result of the increased demand for
insulin production is the breakdown of insulin-producing b-cells in the pancreas,
and ultimately type II diabetes (Fig. 4.2).

Fig. 4.2 The role of obesity and chronic inflammation in cytokine and hormone release
80 A.M. Mancha-Ramirez and T.J. Slaga

Excess dietary energy is stored in the adipocytes, and as this process occurs,
cytokines are released along with adipocyte-related hormones which affect systemic
vasculature [28, 106, 115]. Of the cytokines released via the process described
above, some of the most notable are angiogenin, vascular endothelial growth factor,
endostatin, tumor necrosis factor-a, and interleukin (IL)-6 [28, 106, 115].
This release of cytokines that takes place results in the activation of various
signaling pathways including nuclear transcription factor jB (NFjB) [8, 112].
NFjB is a ubiquitous transcription factor that normally resides in the cytoplasm, but
when activated, is translocated to the nucleus, where it induces transcription of
many genes [2]. In terms of obesity and NFjB, the obese state in general is
associated with increased systemic inflammatory cytokine production and chronic
activation of NFjB [55, 71]. In addition to its role in metabolic disorders, NFjB is
also upregulated in a multitude of human cancers and contributes to tumor pro-
motion [19, 47, 62, 72, 109, 110]. The activated form of NFjB is capable of
mediating cancer, atherosclerosis, diabetes, arthritis, and many other inflammatory
diseases [2]. Specifically, the NFjB pathway is one of the most important signaling
pathways involved in immunity, inflammation, proliferation, and anti-apoptotic
defenses. In terms of cancer, inhibition of NFjB leads to downregulation of pro-
teins involved in anti-apoptotic defense mechanisms utilized by cancer cells such as
Bcl-2, Bcl-xL, and c-FLIP; this process thereby promotes apoptotic cell death in
cancer cells [58]. The activation of IKKb plays a major role in inflammation
induced tumor promotion and progression [126]. Experimental studies have shown
the NFjB activity is necessary for the formation of a number of tumor types [38].
Increased NFjB activation may provide a link between obesity, diabetes, and a
range of tumor types [90, 91, 113].
It is well known that AMP-activated protein kinase (AMPK) is a fuel-sensing
enzyme that is activated by changes in the AMP/ATP ratio. The role of this enzyme
is to increase cellular ATP generation and diminish ATP use for less critical pro-
cesses depending upon the situation [93]. In addition to glucose transport, lipid and
protein synthesis, and fuel metabolism, AMPK is also responsible for regulating a
myriad of other physiological processes including but not limited to cellular growth
and proliferation, mitochondrial function, and factors linked to insulin resistance,
including inflammation, oxidative and ER stress, and autophagy [93]. AMPK
restores ATP levels by inhibiting anabolic processes like fatty acid synthesis and
gluconeogenesis and activating catabolic processes like fat oxidation and glucose
uptake [117]. Significant evidence in the literature supports a definite link between
dysregulation of AMPK and insulin resistance in both rodents and humans. In
addition, common antidiabetic drugs, as well as insulin-sensitizing hormones like
adiponectin, function via AMPK [23, 76, 121, 124, 136]. It has also been shown
that AMPK suppresses NFjB activities in different systems through a variety of
mechanisms, and antidiabetic drugs have also shown NFjB-suppressing properties
as a consequence of AMPK activation [9, 94, 133]. As many studies have shown
that calorie restriction can prevent tumor formation in various mouse models of
cancer, the link between obesity and cancer is one that is well represented in the
literature. Calorie restriction models have prevented tumor formation in brain,
4 Ursolic Acid and Chronic Disease: An Overview of UA’s Effects … 81

colon, breast, prostate, and chemically mediated skin cancer [11, 12, 24, 69, 102]. It
has been suggested that the anticancer effects of calorie restriction and exercise may
be mediated by the energy sensor AMPK [67].
Additional mechanistic links between metabolic syndrome and cancer include as
we would expect, many of the cell signaling pathways related to inflammation such
as TNF-a. Additionally, interleukin-6 (IL-6) is also shown to be elevated in obesity
and is positively correlated with BMI [50]. Increased IL-6 levels, as seen in obesity
models, leads to induced JAK-STAT3 signal transduction and stimulates cell
proliferation, differentiation, and metastasis [21, 125]. Data related to insulin
resistance and mechanistic links to cancer suggest that insulin resistance may lead
to a poorer response to cancer treatment and possibly a more aggressive phenotype
in patients with preexisting diabetes [66].

4.3 An Overview of Ursolic Acid

Ursolic acid (UA), 3b-hydroxy-olea-12-en-28-oic acid, is a pentacyclic triterpenoid

that is derived from various different sources such as berries, leaves, flowers, and
fruits of medicinal plants such as Eriobotrya japonica, Calluna vulgaris,
Rosmarinus officinalis, and Eugenia jambolana [64]. The pentacyclic triterpenoids
contain six isoprene units, the basic molecular formula C30H48Ox, and they have
five rings in their skeleton [40]. One of the benefits of UA is that it is widely
distributed in the plant kingdom [84]. Significant evidence in the literature suggests
that consumption of fruits and vegetables rich in triterpenoids have shown bene-
ficial effects against a variety of inflammation-driven diseases including several
different types of cancer such as breast, colon, and pancreas [3, 99] (Table 4.1).
Classified according to the number of isoprene units, the terpenoid compounds
are a large class of natural agents that are synthesized in plants by cyclization of
squalene [123]. Specifically, pentacyclic triterpenoids have been shown in the lit-
erature to have a myriad of functions, however the effective concentrations to
produce different cellular effects varies depending upon the exact phytonutrient in
question [126]. Some of the effects that pentacyclic triterpenoids have been cited as
eliciting include the induction of the anti-inflammatory response, cytoprotective
effects, and apoptotic effects [126]. Triterpenoids are capable of affecting multiple
signaling pathways, and the pentacyclic triterpenoids are particularly studied in the
literature having proven to have many useful clinical properties. The precise
mechanism by which UA derives its various efficacious properties is still unknown,
however it is believed that the presence of a,b-unsaturated carbonyl moieties sig-
nificantly enhances the potency of this specific class of terpenoids [126].
Additionally, pentacyclic triterpenoids have the ability to inhibit multiple targets, a
trait that is suggested to be mediated by the reversible Michael addition of these
phytonutrients to exposed nucleophilic groups of various susceptible signaling
proteins [126]. NF-jB, STAT3, Bcl-2, Bax, ICAM-1, p53, and PKC are all
examples of molecular targets of UA for anticancer and anti-inflammatory activities
82 A.M. Mancha-Ramirez and T.J. Slaga

Table 4.1 Various medicinal plants containing ursolic acid (adapted from [6])
Botanical name Common name
Ocimum sanctum L. Holy Basil (Tulsi)
Eriobotrya japonica Loquat
Vaccinium macrocarpon Cranberry
Harpagophytum procumbens Devil’s Claw
DC
Sambucus nigra L. Elder flowers (European variety)
Mentha piperita L. Peppermint leaves
Eugenia jambolana Java plum
Perilla frutescens Beefsteak plant
Apocynaceae Lavender
Lavandula augustifolia Mill.
Origanum vulgare L. Oregano
Malus domestica Different apples
Melissa officinalis Lemon balm
Rosmarinus officinalis Rosemary
Plantago major Greater plantain
Thymus vulgaris L. Thyme
Crataegus laevigata (Poir) Hawthorn
DC
Coffea arabica Coffee
Calluna vulgaris Heather
Eucalyptus spp. Eucalyptus

[126]. Although the focus here is on chronic illnesses, cancer and obesity, UA has
also been shown to be effective in treatment of chronic diseases related to car-
diovascular conditions, atherosclerosis, and osteoporosis [126].
UA is a phytonutrient with great versatility and is capable of multiple biological
functions; however one potential drawback to its potential use is that despite low
toxicity, it has low water solubility [127]. To overcome this potential drawback and
improve upon its activity and bioavailability, many studies have been carried out in
order to identify structural modifications to achieve this goal. Specifically, UA
derivatives with modifications at the 3-OH and/or 17-COOH positions demonstrate
significant antitumor effects both in vitro and in vivo suggesting that these modi-
fications can enhance the bioavailability of UA [13, 26, 80]. Additionally, structural
studies have also shown that modification of UA with a piperazine group, specif-
ically a piperazine moiety at the C-28 position, can elicit a better inhibitory effect on
cancer cell growth [22, 100]. Overall, of the many reported UA analogs which have
been developed to date, some of the most important modifications include those on
the positions of C-3/C-28, C-11, C-17, and C-28, and modifications on C-2/C-3
positions and ring A [16].
Although there have not been any detailed studies on pure UA and its absorption
and distribution, one study investigating the intestinal uptake of UA (from the
4 Ursolic Acid and Chronic Disease: An Overview of UA’s Effects … 83

ethanolic extract of Sanbucus chinesis) reported that a dose contributing 80 mg/kg

body weight, UA had about 0.6 % oral bioavailability in rats [59]. Moreover, the
half-life was found to be about 4.3 h, and UA was detected in kidney tissue
(3.51 ± 0.57 nmol/mg) after oral administration of 0.2 % UA in the diet to rats
over a period of 11 weeks [59]. There are not any published studies on acute and/or
subacute toxicity of UA alone, but one study looked at a mixture of UA and
oleanolic acid (OA), a similar compound, also a triterpenoid [39, 107]. They found
that a single subcutaneous injection of OA (1.0 g/kg) to mice or rats during a 5-day
period did not cause mortality. Moreover, during multiple administrations of OA
(180 mg/kg, p.o.) for 10 days, no abnormalities were observed in brain, heart, lung,
liver, kidney, thyroid, testes, spleen, or intestines [39, 107]. Several studies aimed at
evaluating UA and/or OA found that these compounds also do not cause irritation
or inflammation when given either topically to the skin or in the diet [31, 39, 53, 59,
64, 107]. UA does not lead to liver damage or elevated liver enzymes which relate
to possible liver damage, and in fact, it is a very potent hepatoprotective component
of several medicinal herbs [29, 60, 68, 104]. UA has a wide range of pharma-
ceutical properties and is a secondary plant metabolite usually present in the stem,
bark, leaves, or fruit peel [123].
UA and its isomer oleanolic acid (OA) have been shown to have many valuable
abilities including inhibition of tumorigenesis, inhibition of tumor promotion, and
inhibition of angiogenesis. Moreover, UA and OA have been shown to possess
antiatherosclerotic, anti-inflammatory, hepatoprotective, gastroprotective, and car-
diovascular treatment abilities [83]. UA is considered to be nutritionally important
in the prevention of several chronic diseases, including diabetes and cancer, but of
course is not limited to only these two illnesses [56, 64]. Used because of its diverse
healing properties in traditional Chinese medicine for centuries, UA has in recent
years been studied in great detail in order to elucidate the exact mechanisms of
action by which it exerts its health beneficial effects, however the precise nature of
these mechanisms have yet to be determined [123]. Studies aimed at predicting the
molecular targets of UA have been carried out which analyze the signaling path-
ways of the predicted targets of UA. Bioinformatic data from these studies
demonstrated that there were 611 possible molecular proteins as targets for inter-
action with UA, and more than 49 functional clusters responding to it as well [36].
Notably, there were 76 pathways that were significantly enriched such as MAPK,
p53, and mTOR pathways [36].
In addition to effects in cancer and obesity, which are the focus of this chapter,
UA has also been shown to have an effect on the liver, on muscle and fat, exhibit
anti-atherogenic and cardioprotective effects, and antimicrobial activity. A very
potent hepatoprotective component of several medicinal herbs, UA is capable of
protecting the liver from chloroform-induced injury and against D-galactosamine-
induced liver injury in rats [29, 60, 68, 104]. Recent studies have also shown that
UA alone or in combination with resveratrol were effective in preventing obesity
and fatty liver disease in mice fed a high-fat diet (Digiovanni and Slaga, unpub-
lished results). UA alone has been shown to elicit an antifibrotic effect in the liver,
but the exact targets for this mechanism have not yet been discerned [36]. Many UA
84 A.M. Mancha-Ramirez and T.J. Slaga

containing plants have anti-bacterial and antifungal activity; in fact, UA has been
shown to have antimicrobial activity against several strains of staphylococci [4,
130]. Finally, UA is a potent inhibitor of components of metabolic syndrome such
as hypertension, triglyceride levels, and accumulation of inflammatory monocytes
and associated atherosclerosis [41, 42, 108, 114].

4.4 Ursolic Acid and Its Effects on Obesity and Metabolic

Syndrome

Among other traditional Chinese medicinal agents such as anthraquinones, several

terpenes including UA have proven to be effective antiobesity agents. It is proposed
that these phytonutrients can aid against obesity by modulating body weight, blood
glucose levels, triglycerides, and total cholesterol [134]. Recently, it has been
demonstrated that UA along with several of its other triterpenoid family members,
can improve insulin signaling by enhancing insulin receptor b subunit phospho-
rylation and Akt in vitro [46, 61, 132]. UA has also been shown to promote glucose
uptake from the bloodstream into peripheral tissues through upregulation of
GLUT4 [20, 33, 119]. One study investigating the ability of UA to promote glucose
uptake found that UA achieved this by enhancing the translocation of GLUT4 to the
plasma membrane in 3T3-L1 adipocytes [46]. A final mechanism by which UA is
suggested to aid in lowering blood glucose levels is by lowering endogenous
glucose production through gluconeogenesis inhibition [14].
Another important target when considering insulin resistance therapies is
PTP1B, a molecule that negatively regulates insulin signaling. Targeting PTP1B is
a very useful approach because it can inhibit the PI3K/Akt signaling pathway to
induce insulin resistance by inhibition of the translocation of GLUT4 to the plasma
membrane [111]. Many studies have evaluated several triterpenoids including UA
for their ability to act as PTP1B inhibitors, and several in vitro findings suggest that
UA can directly inhibit PTP1B and improve insulin sensitivity [61, 78, 88].
Studies utilizing rodents on a high-fat diet showed UA’s ability to normalize
blood glucose levels in animals characterized by diet-induced obesity or diabetes
[73, 89]. One particular study utilized a hypertensive rat model, and showed that UA
was able to lower resting glucose, LDL cholesterol, and triglycerides to near control
levels; it also raised antioxidant enzymes and HDL cholesterol after 6 weeks of
treatment [108]. UA also reversed high-fat diet-induced NFjB signaling and deficits
in metabolic signaling in mice [54, 89]. One study produced findings which suggest
UA may function via a similar mechanism as exercise, due to results showing that
UA stimulates glucose uptake and activates AMPK in insulin resistant cells [34].
Among the many comorbidities associated with metabolic syndrome, in patients
with type II diabetes, hyperglycemia promotes an increase in free radicals and a
decrease in antioxidants leading to lipid peroxidation [14]. It is well established that
free radicals such as reactive oxygen species can have negative effects as they can
4 Ursolic Acid and Chronic Disease: An Overview of UA’s Effects … 85

ultimately lead to cellular dysfunction [30]. Animal studies showed that treatment
with UA was able to decrease liver damage caused by oxidative stress inducing
chemicals such as carbon tetrachloride [65]. Moreover, UA, and other triterpenoids
were also able to increase the activities of the antioxidant enzymes superoxide
dismutase and glutathione peroxidase [68, 118]. Another significant complication
of diabetes is diabetic nephropathy, and therefore UA was tested to determine what
effect it would have on renal function in streptozotocin-induced diabetes. The study
looking at diabetic nephropathy found that UA significantly prevented biochemical
and histopathologic changes in the kidneys associated with diabetes, and lowered
NF-jB activity and renal oxidative stress levels [63]. Finally, phase 2 clinical
studies evaluating the effect of UA on insulin sensitivity and metabolic syndrome
were carried out in 2015 but the results have not yet been published. Despite the
fact that recent studies have demonstrated that UA does exert an antiobesity effect,
the mechanisms of action are still under investigation. Currently, several of the
proposed mechanisms and targets include inhibition of PTP1B, lipolysis stimulation
via cAMP-dependent PKA pathway modulation, modulation of the LKB1/AMPK
pathway, and regulation of adipogenic differentiation [42, 57, 82, 89, 120].

4.5 Ursolic Acid and Its Effects on Cancer

With regards to cancer, UA has been studied in great detail. However, the precise
mechanisms of the many anticancer effects of UA and corresponding molecular
targets for these capabilities are still under investigation. UA has been shown to
have a variety of anticancer capabilities, including the ability to suppress prolif-
eration of several different types of tumor cells, induce apoptosis, and inhibit tumor
promotion, metastasis, and angiogenesis in cancer animal models [98]. Several
mechanisms which have been elucidated for the above mentioned anticancer
abilities of UA thus far and include the ability to suppress multiple cell signaling
pathways such as growth factor receptor activation (EGFR), signaling through
IKK/NF-jB, Akt/mTOR, Cox-2, STAT3, MMP9, and VEGF [97, 126, 131].

4.5.1 Ursolic Acid and Cancer: Notable in vitro Studies

UA has shown great efficacy as an anticancer therapy in many in vitro models,

many of which are accomplished via inhibition of DNA replication, caspase acti-
vation, inactivation of protein tyrosine kinases, induction of Ca2+ release, and
finally via NFjB mediated downregulation of the cellular inhibitor of apoptosis
gene [98]. UA has been reported to induce apoptosis in diverse cell lines via its
immunomodulatory and anti-inflammatory actions [123]. In B16F10 and A375
melanoma cells, the combination of UA with chloroquine was able to synergisti-
cally decreased cell viability [45]. Additionally, studies carried out in mouse skin
86 A.M. Mancha-Ramirez and T.J. Slaga

papilloma and carcinoma cell lines have indicated that the cytotoxic effects of UA
can be enhanced with p-glycoprotein inhibitors [44].
Cranberry-derived UA showed potent anti-proliferative activities against HepG2
liver cancer cells and MCF7 breast cancer cells [35]. UA-mediated apoptosis of
human bladder cancer cells was strongly suppressed by AMPK knockdown [135].
Additionally, UA reduced the viability of human colon cancer cell lines [27].
UA has also shown great promise in breast cancer studies as well, with an ability
to strongly decrease viability of a breast cancer cell line with an associated inhi-
bition of NFjB, as well as, a decrease size of breast cancer xenografts in murine
models [129].
Similar in vitro effects of UA have been seen in prostate cancer models as well
[99]. Notably, it was found that UA was able to suppress NFjB activation induced
by numerous carcinogens, such as TNFa, phorbol ester, okadaic acid, H2O2, and
cigarette smoke [103]. Inhibition of NFjB activation was mediated via IKK kinase
activation, IjBa phosphorylation and degradation, p65 phosphorylation, p65
nuclear translocation, and NFjB dependent reporter gene expression [98]. Results
from multiple myeloma in vitro studies showed that UA was able to inhibit both
constitutive and IL-6-inducible STAT3 activation, which was mediated through the
inhibition of upstream kinases C-SRC, JAK1, JAK2, and ERK1/2 [85]. Moreover,
they also showed that UA could induce expression of the SHP-1 protein, while
knockdown of SHP-1 via RNA interference was able to suppress the induction of
SHP-1 and reverse the inhibition of STAT3 activation. The results from the Pathak
group suggest a critical role of SHP-1 in the mechanism of action of UA, and show
that it can be effective in suppressing important inflammatory networks including
NFjB, STAT3, and AKT [85].

4.5.2 Ursolic Acid and Cancer: Notable in vivo Studies

In addition to the extensive work that has been carried out investigating the efficacy
of UA in in vitro cancer models, significant evidence supporting the versatility and
usefulness of UA has also been amassed in in vivo studies as well. For example, the
effect of UA was evaluated on early stages of skin tumorigenesis utilizing a
two-stage carcinogenesis SENCAR mouse model, and found topical application of
UA alone and/or in combination with calcium D-glutarate during the promotion
stage was able to inhibit tumor multiplicity and tumor incidence [52]. Results from
this study showed that UA is a potent inhibitor of skin tumor promotion and
inflammatory signaling in skin cancer and potentially other epithelial cancers in
humans as well [52]. UA isolated from P. frutescens was also evaluated in a
two-stage skin carcinogenesis mouse model, and was found to significantly inhibit
skin tumor promotion by the tumor promoting agent, TPA [17, 18]. Specifically,
pretreatment with UA resulted in a 42 % inhibition of papilloma formation [17, 18].
Furthermore, when UA was used in combination with resveratrol, a phytochemical
present in grapes, berries, peanuts, and red wine, the combination was found to
4 Ursolic Acid and Chronic Disease: An Overview of UA’s Effects … 87

have a greater inhibitory effect on skin tumor promotion by TPA in a mouse model
than with either agent used independently [17, 18]. Mechanistic studies into the
enhanced inhibitory effects observed in the UA + resveratrol combination showed
an upregulation of tumor suppressor genes such as p21 and PDCD4 as well as a
dramatic increase of p-AMPKa and its downstream target p-Ulk1-ser555 [17, 18].
In a prostate cancer DU145 xenograft nude mouse model, one group analyzed
whether UA could inhibit the growth of prostate cancer and found that UA was able
to significantly suppress the tumor growth in vivo following 6 weeks of treatment
[99]. Also noteworthy in this xenograft prostate cancer study was that at the end-
point of the experiment, there was no significant change in body weight of
UA-treated mice suggesting that UA is a nontoxic compound when administered to
mice. Results from this in vivo prostate cancer mouse model showed a substantial
decrease in VEGF expression and an increase in caspase-3 expression in
UA-treated mice, suggesting that perhaps UA is functioning as an anti-angiogenic
and pro-apoptotic agent against prostate cancer in this model [99]. Additionally,
UA, when administered by gavage, also decreased incidence of chemically induced
preneoplastic lesions in rat colon [27] (Fig. 4.3).
Finally, a phase I trial was carried out in 2015 to evaluate the multiple-dose
safety and antitumor activity of UA liposomes (UAL) in patients with advanced
solid tumors. UA liposome is a new antitumor drug that has significant potential
therapeutic value as it utilizes liposomes to overcome the poor solubility of UA,
increase the therapeutic efficiency, reduce the side effects, and enhance the
bioavailability of the drug. Results from this clinical trial demonstrate that UAL
treatment of patients with advanced solid tumors via multiple-dose and consecutive

Fig. 4.3 An overview of the many oncogenic targets UA has been shown to modulate
88 A.M. Mancha-Ramirez and T.J. Slaga

14-day intravenous infusion every 21 days was safe. Additionally, the prelimi-
nary antitumor activity of UAL was evaluated for the first time in a clinical setting
and their results indicate UAL may be able to potentially improve patient
remission [87].

4.6 Conclusion

As obesity, metabolic syndrome, and cancer have been shown to have similar
mechanistic links, this presents a useful avenue for novel preventive and therapeutic
strategies aimed at fighting these chronic diseases. Dysregulation of important cell
signaling pathways occurs during the constant state of inflammation during obesity,
and this has been linked to an increased overall risk of certain cancers such as breast
and colorectal cancer. Moreover, as discussed earlier, patients with preexisting
conditions brought on by obesity, such as insulin resistance, tend to respond more
poorly to traditional chemotherapeutic agents than otherwise healthy individuals.
As cancer is a multifactorial disease, it is vital to investigate novel therapeutic
compounds that exhibit efficacy against key pathways often dysregulated in cancer
pathogenesis. Due to the unique nature of each individual cancer development,
multi-targeting therapies from natural resources are vital for targeting cancer
development and progression. Triterpenoids such as UA offer unique and novel
drug platforms for development due to the fact that they have been shown to target
critical inflammatory proteins for the prevention and treatment of cancer. As these
phytonutrient have been shown to be nontoxic, it is their safety and ability to affect
various key targets that makes compounds like UA very desirable as a starting point
for future drug development. It is quite possible that triterpenes such as UA could
be used in conjunction with commonly used chemotherapeutic agents to allow for
perhaps a decrease in the chemotherapeutic dose, less adverse effects of the
chemotherapeutic agent, or even synergistic effects between UA and the drug.
While there is significant data in the literature to support an important role for UA
in cancer prevention/therapy, future clinical investigations are needed to further
validate these findings. Here, we have discussed in detail the various ways in which
UA has shown great potential as an anticancer agent in many different types of
cancer; many of these results have significant clinical implications as UA has been
shown to target key cancer pathways.
UA is not only capable of acting as an anticancer compound, but it also has been
shown to have versatile antiobesity properties which we have described in detail.
These effects appear to be partially mediated by inhibition of NFjB and/or acti-
vation of AMPK, but due to the many various effects of UA seen in both cancer and
obesity, the precise mechanisms of action for these effects are still unclear.
Although diet and exercise plus a highly controlled blood glucose level is currently
the recommended treatment for obesity and type II diabetes, one of the major
drawbacks of this standard of care is of course compliance. Where patients may
exhibit poor compliance with a recommended course of diet and exercise,
4 Ursolic Acid and Chronic Disease: An Overview of UA’s Effects … 89

pharmacological agents targeting key disease pathways such as those targeted by

UA could significantly aid in the prevention/of obesity and obesity-related
comorbidities in addition to diet and exercise regimens.
Significant evidence in the literature supports an important role for natural
compounds such as UA in the prevention and treatment of chronic diseases like
obesity and cancer, and here we have highlighted many of the ways UA has been
shown to be a beneficial and versatile phytochemical in both of these diseases.
Although there is extensive data in the literature to support the efficacious nature of
UA in cancer and obesity, there is still significant work to be done in more
mechanistic-oriented research in order to determine the mechanisms by which this
versatile and effective compound exerts its action. Moreover, it is great importance
to undergo further structural studies of UA in order to develop novel derivatives
which may exhibit increased bioavailability while still maintaining the same effi-
cacy and low toxicity seen in the parent compound. As the link between diet and
lifestyle and the increased incidence of chronic disease has been well established in
the literature, it is extremely vital to investigate the use of phytonutrients as
meaningful therapeutic agents for the prevention and treatment of chronic illnesses
such as those discussed in this chapter as well as to elucidate the mechanisms by
which they elicit their action.

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Chapter 5
Tocotrienol and Its Role in Chronic
Diseases

Kok-Yong Chin, Kok-Lun Pang and Ima-Nirwana Soelaiman

Abstract Tocotrienol is a member of vitamin E family and is well-known for its

antioxidant and anti-inflammatory properties. It is also a suppressor of mevalonate
pathway responsible for cholesterol and prenylated protein synthesis. This review
aimed to discuss the health beneficial effects of tocotrienol, specifically in pre-
venting or treating hyperlipidaemia, diabetes mellitus, osteoporosis and cancer with
respect to these properties. Evidence from in vitro, in vivo and human studies has
been examined. It is revealed that tocotrienol shows promising effects in preventing
or treating the health conditions previously mentioned in in vivo and in vitro
models. In some cases, alpha-tocopherol attenuates the biological activity of
tocotrienol. Except for its cholesterol-lowering effects, data on the health-promoting
effects of tocotrienol in human are limited. As a conclusion, the encouraging results
on the health beneficial effects of tocotrienol should motivate researchers to explore
its potential use in human.

Keywords Bone Cancer Cholesterol

Diabetes
Glucose
Osteoporosis




Tocopherol Tocotrienol Vitamin E

K.-Y. Chin
I.-N. Soelaiman (&)

Department of Pharmacology, Universiti Kebangsaan Malaysia Medical Centre,
Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, Bandar Tun Razak, Cheras,
56000 Kuala Lumpur, Malaysia
e-mail: [email protected]
K.-L. Pang
Biomedical Science Programme, School of Diagnostic and Applied Health Sciences,
Faculty of Health Sciences, Universiti Kebangsaan Malaysia,
Jalan Raja Abdul Aziz, 50300 Kuala Lumpur, Malaysia

© Springer International Publishing Switzerland 2016 97

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1_5
98 K.-Y. Chin et al.

5.1 Tocotrienol

Tocotrienol and tocopherol are collectively known as tocochromanols, or com-

monly as vitamin E. They share a similar chemical structure, consisting of a
chromanol ring and a long carbon tail. The difference between tocotrienol and
tocopherol is that the carbon tail of tocotrienol contains three double bonds (an
unsaturated farnesyl tail), whereas the carbon tail of tocopherol contains only single
bonds (a saturated phytyl tail). The difference in the long carbon tail dictates the
difference in biological activity between these two compounds. Similar to toco-
pherol, tocotrienol can be further divided into 4 isomers (alpha, beta, gamma and
delta) based on the position of the methyl side chain on the chromanol ring. Beta-
and gamma-tocotrienols are structural isomers and possess the same number of
methyl group on the chromanol ring. Alpha-tocotrienol has an additional methyl
group whereas delta-tocotrienol has one less methyl group on the chromanol ring
compared to beta- and gamma-tocotrienol [6, 14] (Fig. 5.1).
Tocotrienol and tocopherol isomers are found together in a wide variety of
plants. Tocotrienol is more commonly found in the fruit and seed. Some of the
natural sources that yield the highest amount of tocotrienol include seeds of Bixa
orellana (achiote, annatto) (1.53 mg/g dry weight), Zea mays (maize) (1.42 mg/g
dry weight), Garcinia mangostana (purple mangosteen) (1.33 mg/g dry weight)
and oil of Elaeis guineensis (oil palm) (1.08 mg/g dry weight). Latex of Hevea
brasiliensis (rubber tree) (2.59 mg/g dry weight) also contains high amount of
tocotrienol, but this is not found in other latex-producing plants (summarized by
Müller et al. [98]). Usually alpha-tocopherol is found together with tocotrienol
isomers in varying proportions in natural sources. In palm oil, the proportion of
alpha-tocopherol is around 30 % of the total vitamin E content [111]. In rice bran,
the average percentage of tocotrienol with respect to total vitamin E content is 61 %

Fig. 5.1 Chemical structure

of vitamin E isomers
5 Tocotrienol and Its Role in Chronic Diseases 99

[156]. Vitamin E derived from the seed of Bixa orellana contains 10 %

gamma-tocotrienol and 90 % delta-tocotrienol with no traceable amount of
alpha-tocopherol [42]. Several studies on the tocotrienol content of common food
have been performed [31, 157]. The tocotrienol intake has been found to be very
low (1.9–2.1 mg/person/day) compared to alpha-tocopherol (8–10 mg/day/person)
in a Japanese study. This amount is insufficient compared to the doses used in
experimental studies to achieve the health-promoting benefits of tocotrienol.
Currently, the recommended dietary allowance (RDA) for vitamin E is 15 mg
(22.4 IU) for male and female aged 14 years and above [61]. This estimation is
based on alpha-tocopherol because it is the only fraction retained in the blood.
The RDA for tocotrienol has not been established.
The absorption of tocotrienol is increased with fat-rich food because it is a
lipophilic compound [185]. Tocotrienol and tocopherol are absorbed in the lumen
of the small intestine, whereby they enter the enterocytes via passive diffusion, and
secreted into the lymphatics [43]. They are transported in the form of chylomicron.
The release of vitamin E isomers from the liver to the blood stream is regulated by
alpha-tocopherol transporter protein. The transfer protein has the highest affinity
towards alpha-tocopherol (100 % binding affinity towards RRR-alpha-tocopherol)
and lower binding affinity towards other vitamin E isomers (12 % towards
alpha-tocotrienol, 38 % towards beta-tocopherol, 9 % towards gamma-tocopherol
and 2 % towards delta-tocopherol) [53]. Tocotrienol is distributed by the blood-
stream to various tissues. Significant accumulation of tocotrienol can be found in
the adipose and skin tissue, as demonstrated by rodent experiments [72, 147].
Similar to tocopherols, tocotrienols are also metabolised and excreted in the form of
carboxyethyl-hydroxychromans [81].
Tocotrienol features a variety of biological activities, which enable it to be used
as a candidate agent to prevent chronic diseases. The prominent antioxidant and
anti-inflammatory properties of tocotrienol will be discussed in the following sec-
tions. Tocotrienol is also a known suppressor of the mevalonate pathway involved
in cholesterol synthesis, bone metabolism and carcinogenesis. This will be dis-
cussed in the respective sections.

5.2 Antioxidant Effects of Tocotrienol

Oxidants such as reactive oxygen species (ROS) and reactive nitrogen species
(NOS) are essential for defence against infectious agents, for cell signalling and for
redox homeostasis [170]. They are balanced by endogenous and exogenous
antioxidants in our body, such as antioxidant enzymes, vitamins, thiols and glu-
tathione. Oxidative stress occurs when the production of oxidants overwhelms the
production of antioxidants in our body [152, 153]. Free radical species are capable
of damaging macromolecules such as carbohydrate, protein and DNA in the body
and generate more free radicals, thereby forming a vicious cycle [48]. Many adverse
health conditions, such as neurodegeneration [12], diabetes mellitus [9],
100 K.-Y. Chin et al.

cardiovascular disease [82] and osteoporosis [20], have been linked to oxidative
stress. This highlights a possible role of tocotrienol, a strong antioxidant, in pre-
venting these oxidative stress-related diseases.
Both tocotrienol and tocopherol exert their free radical scavenging activity by
donating phenolic hydrogen at the sixth position of the chromanol ring [113].
Tocotrienol has been shown to be a superior membrane antioxidant compared to its
tocopherol counterpart [161]. This is due to the uniform distribution of tocotrienol
in the membrane and its stronger ability to disorder membrane lipids, thus
enhancing its recycling efficiency [145]. In studies using lipid peroxidation in
microsomes extracted from rat liver, Serbinova et al. showed that the antioxidant
effect of alpha-tocotrienol was 40–60 times stronger than alpha-tocopherol [145].
They attributed the superior antioxidant effect of tocotrienol to its higher recycling
efficacy from chromanoxyl radicals, uniform distribution in the membrane bilayers
and stronger disordering of membrane lipids structure, resulting in a higher efficacy
in its interaction with free radicals [145]. Palozza et al. showed that tocotrienol
isomers inhibited lipid peroxidation, ROS production and heat shock protein
expression in rat liver microsomal membrane and in RAT-1 immortalized fibrob-
lasts challenged with free radicals [121]. The effectiveness of delta-tocotrienol was
found to be greater than gamma-tocotrienol, and similar between gamma- and
alpha-tocotrienol [121]. It was proposed that decreased methylation of the chro-
manol ring, as in delta-tocotrienol, allowed the molecule to be better incorporated
into cell membranes [121]. Kamat et al. showed that palm tocotrienol significantly
reduced the lipid and protein peroxidation products in the microsomal extracts of
the brain and liver from rats [66, 67]. Among the individual isomers, they identified
that gamma-tocotrienol had the highest antioxidant efficacy compared to delta- and
alpha-tocotrienol [66, 67].
The antioxidant properties of tocotrienol were also demonstrated in cell culture
studies. Tan et al. showed that alpha-tocotrienol prevented the decrease of glu-
tathione and mitochondrial depolarization induced by hydrogen peroxide in hepa-
tocytes [166]. The increase of cellular ROS and malondialdehyde, a lipid
peroxidation product, induced by hydrogen peroxide and paracetamol in hepato-
cytes were also suppressed by alpha-tocotrienol. Nizar et al. showed that
gamma-tocotrienol prevented the apoptosis of primary osteoblast induced by
hydrogen peroxide [115]. In a subsequent experiment, they showed that this was
achieved by the preservation of cellular antioxidant defence system, such as
superoxide dismutase, catalase and glutathione peroxidase, and the reduction of
lipid peroxidation product in the treated cells [1].
Tocotrienol also exhibited its antioxidant effect in animal models. Nesaretnam
et al. demonstrated that long-term feeding of rats with diet containing palm oil rich
in tocotrienol significantly reduced the peroxidation potential of hepatic mito-
chondria and microsomes compared to corn oil control [106]. The initial rate of
lipid peroxidation was also found to be slower in the palm oil supplemented group
compared to the corn oil control [106]. Lee et al. showed that rats supplemented
with tocotrienol-rich fraction at 25 or 50 mg/kg body weight for 28 days and forced
to undergo a swimming endurance test showed higher liver superoxide dismutase,
5 Tocotrienol and Its Role in Chronic Diseases 101

catalase and glutathione peroxidase activity but lower liver lipid peroxidation and
reduced liver and muscle protein carbonyl levels compared to the vehicle and
alpha-tocopherol (25 mg/kg body weight) groups [79]. Supplementation of
alpha-tocotrienol (0.04 % weight diet) for one month in rats fed with vitamin
E-deficient diet reduced the level of hydroxyoctadecadienoic acid and isoprostane,
both markers of lipid peroxidation, in plasma and various organs of the rats [186].
The limited examples presented above showcased the antioxidant potential of
tocotrienol ex vivo and in vivo. The role of tocotrienol in suppressing oxidative
stress in relation to specific diseases will be discussed separately in the later
sections.

5.3 Anti-inflammatory Effects of Tocotrienol

Inflammation has been implicated in the pathogenesis of various diseases, such as

cardiovascular disease [80], diabetes mellitus [35], rheumatoid arthritis [30] and
osteoporosis [99]. Prostaglandin and leukotriene generated from arachidonic acid
are important mediators of the inflammatory process. Prostaglandin E2 (PGE2)
synthesized by cyclooxygenase (COX) 1 and 2 are important in activating cytokine
formation, apart from eliciting pain and fever. Leukotriene B4 is another important
chemotactic agent synthesized by 5-lipoxygenase (5-LOX). Nuclear factor
kappa-B, JAK–STAT6/3 (signal transducer and activator of transcription 6/3) and
CCAAT-enhancer binding protein β (C/EBPβ) are transcription factors vital in
inducing the expression of proinflammatory cytokines (reviewed by Jiang [63]).
Wu et al. showed that palm tocotrienol-rich fraction inhibited the release of nitric
oxide and PGE2 in lipopolysaccharide (LPS)-treated human monocytic cells [181].
This was achieved by the suppression of inducible nitric oxide synthase and COX-2
(but not COX-1) expression by tocotrienol-rich fraction [181]. Concurrently, the
production of interleukin-4, interleukin-8 and tumor necrosis factor-alpha was
suppressed by tocotrienol treatment [181]. Using LPS-treated murine peritoneal
macrophages, Ng and Ko demonstrated that tocotrienol-rich fraction inhibited the
production of nitric oxide, PGE2 and proinflammatory cytokines, such as tumor
necrosis factor-alpha, interferon-gamma, interleukin-1 beta and interleukin-6 in
these cells [110]. This contributed to the suppressed protein expression of inducible
nitric oxide synthase, COX (but not COX-1) and nuclear factor kappa-B [110].
They further showed that the anti-inflammatory effects of tocotrienol-rich fraction
were better than alpha-tocopherol and alpha-tocopheryl acetate [110]. Yam et al.
compared the anti-inflammatory effects between alpha-tocopherol, tocotrienol-rich
fraction and individual tocotrienol isomers (alpha-, gamma- and delta-tocotrienol)
in LPS-treated RAW 264.7 murine macrophages [183]. Yam et al. found that
tocotrienol-rich fraction and all three tocotrienol isomers inhibited interleukin-6
and nitric oxide production [183]. Only alpha-tocotrienol could suppress the
production of tumor necrosis factor-alpha. Tocotrienol-rich fraction, alpha- and
delta-tocotrienol lowered the production of PGE2 [183]. The gene expression
102 K.-Y. Chin et al.

of COX-2 was downregulated by tocotrienol-rich fraction and individual isomers

but not by alpha-tocopherol [183]. Wang and Jiang showed that gamma-tocotrienol
inhibited LPS-induced interleukin-6 production in murine RAW267.4 macrophages
by blocking the activation of nuclear factor kappa-B [177]. It also suppressed the
LPS induced upregulation of C/EBPβ needed for IL-6 production and the expres-
sion of its target gene, granulocyte-colony stimulating factor [177]. These effects
could be replicated in bone marrow derived macrophages [177]. Wang et al. further
showed that gamma-tocotrienol blocked the activation of nuclear factor kappa-B by
upregulating its inhibitor, A20, via modulating the synthesis of sphingolipids
involved in inflammatory response [178].
In an vivo study, Qureshi et al. showed that 4-week consumption of diet containing
tocotrienol derived from annatto seeds (100 ppm) in aged and young mice inhibited
the inflammation induced by LPS alone or in combination with interferon-gamma or
interferon-beta [133]. This was evidenced by a reduced nitric oxide and tumour
necrosis factor-alpha level in the treated group [133]. This was achieved by sup-
pression of genes related to proinflammatory cytokines, such as interleukin-1 beta,
interleukin-1 alpha, interleukin-1 RA, interleukin-6, interleukin-12, COX-2, tumor
necrosis factor-alpha etc. [133]. In another experiment, Qureshi et al. compared the
anti-inflammatory effects of alpha-, gamma- and delta-tocotrienol in LPS-treated
BALB/c mice [129]. A dose-dependent decrease of tumor necrosis factor-alpha
synthesis was observed in all tocotrienol-treated groups [129]. The inhibition was
strongest in the delta-tocotrienol group [129]. Using peritoneal macrophages derived
from these mice, it was shown that delta-tocotrienol could suppress the LPS-induced
gene expression of tumor necrosis factor-alpha, interleukin-1 beta, interleukin-6 and
inducible nitric oxide synthase [129]. Heng et al. demonstrated that supplementation
of tocotrienol mixture 400 mg daily for 16 weeks caused a significant reduction of
interleukin-1 and tumor necrosis factor-alpha in a group of subjects (aged 20–
60 years) with metabolic syndrome [50]. Supplementation of tocotrienol 15 mg daily
for 4 weeks significantly decreased the high-sensitivity C-reactive protein in a group
of patients with type-2 diabetes [47].
The role of tocotrienol in suppressing inflammation in individual diseases will be
discussed in detail in later sections.

5.4 Tocotrienol as a Hypocholesterolemic Agent

The mevalonate pathway is responsible for the synthesis of cholesterol and other
isoprenoids. The determining step in this multi-cascade pathway is the synthesis of
3-hydroxy-3-methyglutaryl-CoA (HMG-CoA) from acetyl-CoA via the enzyme
HMG-CoA reductase (HMGR). The expression of HMGR is in turn regulated by
sterol regulatory element-binding proteins (SREBPs), whereby the absence of sterol
isoprenoids in the cells upregulates its expression (reviewed by Buhaescu and
Izzedine [17]). Cholesterol is carried in lipoproteins with specific apolipoproteins
which direct the load to specific tissues. Excretion of cholesterol occurs in the form
5 Tocotrienol and Its Role in Chronic Diseases 103

of bile acid. The rate-determining enzyme in this process is

cholesterol-7-alpha-hydroxylase [65]. Hypercholesterolemia is an important con-
tributing factor to cardiovascular disease and coronary heart disease [159].
Tocotrienol is known as an inhibitor of the mevalonate pathway. The structure of
tocotrienol with its three double bonds is similar to farnesyl, the compound pre-
ceding the formation of squalene in cholesterol synthesis [122]. Tocotrienol pro-
motes the formation of farnesol from farnesyl, thus reducing the formation of
squalene [122]. Gamma- and delta-tocotrienol have been shown to regulate HMGR
in different ways [155]. Delta-tocotrienol stimulates ubiquitination and degradation
of HMGR and blocks processing of SREBPs, while gamma-tocotrienol is more
selective in enhancing HMGR degradation than blocking SREBP processing [155].
In other studies, gamma-tocotrienol has been shown to stimulate apolipoprotein B
degradation by decreasing its translocation into the endoplasmic reticulum lumen
[168]. This also causes a reduction in the number of apolipoprotein B in lipoprotein
particles [168]. Gamma- and delta-tocotrienol have also been demonstrated to
downregulate the expression of genes involved in lipid homeostasis, such as
DGAT2. APOB100, SREBP1/2 and HMGR [190]. These tocotrienols also enhance
efflux of low density lipoprotein (LDL) cholesterol through increasing LDL
receptor expression [190]. Gamma-tocotrienol has been shown to decrease
triglyceride (TG) level in liver cells by decreasing the expression of fatty acid
synthase, SERBP1 and increasing the expression of carnitine palmitoyl transferase
1A [19] (Fig. 5.2).
The effects of tocotrienol on lipid profile have been tested in several animal
models of dyslipidaemia. Different species of animals have been used, including
chicken, swine and rodents (rats, hamsters, guinea pigs). Qureshi et al. found that
chicken supplemented with delta-tocotrienol (50 ppm for 4 weeks) showed reduced
level of total and LDL cholesterol [130]. It also suppressed the lipid elevating effects
of dexamethasone and potentiated the TG-lowering effects of riboflavin [130]. Using
chickens fed with different dosage (50–2000 ppm) of vitamin E isomers and mix-
tures, Yu et al. showed that gamma- and delta-tocotrienol reduced the level of total
and LDL cholesterol most significantly, followed by tocotrienol-rich fraction and
alpha-tocotrienol [188]. Alpha-tocopherol did not exhibit lipid-lowering effects in
their experiment [188]. In another experiment, Qureshi and Peterson demonstrated
that a combination of tocotrienol-rich fraction (50 ppm) and lovastatin (50 ppm) for
4 weeks suppressed HMGR activity more effectively than lovastatin alone in
chickens [125]. The combination was also more effective in reducing serum total and
LDL cholesterol, apolipoprotein B, TG, thromboxane B2 and platelet factor 4 than
individual treatments [125]. Qureshi et al. also showed that alpha-tocopherol
attenuated the inhibition of HMGR by gamma-tocotrienol in chickens [124]. They
concluded that the effective preparation of tocotrienol mixture to maximize its
hypocholesterolemic effects should contain 15–20 % alpha-tocopherol and 60 %
gamma- or delta-tocotrienol [124]. In an experiment using genetically hyperc-
holesterolemic swines, Qureshi et al. showed that 6 weeks treatment of 50 mg
tocotrienol-rich fraction derived from rice bran, gamma-tocotrienol, desmethyl
(d-P21-T3) or didesmethyl (d-P25-T3) tocotrienol individually could cause
104 K.-Y. Chin et al.

Fig. 5.2 The role of tocotrienol in suppressing cholesterol synthesis

significant reduction in serum total and LDL cholesterol, TG, apolipoprotein B,

platelet factor 4, thromboxane B2, glucose and glucagon [126]. These tocotrienols
also lowered HMGR activity but did not affect cholesterol-7-alpha-hydroxylase
activity [126]. After termination of the treatment, the cholesterol-lowering effects of
tocotrienols persisted for 10 weeks [126]. In another study using palm
tocotrienol-rich fraction (50 mg/g diet for 42 days), genetically hypercholes-
terolemic swine responded to the treatment by showing a significant decrease in
serum total and LDL cholesterol, apolipoprotein B, thromboxane B2 and platelet
factor 4 [127]. However, the effects on normal swine were limited to reduction of
total cholesterol and apolipoprotein B [127]. The activity of HMGR in adipose tissue
was reduced with treatment in both normal and hypercholesterolemic swine [127].
In experiment using rats, hamsters or genuine pigs, tocotrienol was shown to
prevent diet or chemical-induced hyperlipidaemia. Burdeos et al. demonstrated that
F344 rats on high fat diet receiving 5 or 10 mg rice bran tocotrienol per day for
3 weeks showed a reduction in TG and phospholipid hydroperoxides (oxidative
stress marker) in the liver and plasma [18]. This reduction could be due to sup-
pressed production and accumulation of TG by the hepatocytes [18]. However, the
cholesterol level did not change in these rats [18]. In a similar experiment by
5 Tocotrienol and Its Role in Chronic Diseases 105

Watkins et al., rats were fed with an atherogenic diet for 6 weeks and the treatment
groups received 50 mg gamma-tocotrienol and 500 mg alpha-tocopherol per kg
diet [179]. The treatment group showed lower plasma cholesterol, LDL, very–
low-density lipoprotein (VLDL), TG, malondialdehyde and fatty acid hydroper-
oxides [179]. Minhajuddin et al. demonstrated that rats on a 3-week atherogenic
diet showed significant reduction in TG, total and LDL cholesterol, malondialde-
hyde and conjugated dienes after supplementation with tocotrienol-rich fraction (8–
48 mg/kg) for 1 week [91]. The activity of HMGR was suppressed with athero-
genic diet, probably due to physiological feedback mechanism [91].
Supplementation of tocotrienol-rich fraction led to further reduction in the activity
of HMGR [91].
Using a chemically induced hypercholesterolemic model, Iqbal et al. showed
that supplementation of rice bran tocotrienol-rich fraction (10 mg/kg/day) signifi-
cantly suppressed the increase in plasma total and LDL cholesterol in rats admin-
istered with 7,12-dimethylbenz[alpha]anthracene [62]. It also suppressed the
activity and protein mass of hepatic HMGR [62]. Salman Khan et al. demonstrated
that Tocomin, a palm tocotrienol mixture, reduced the level of plasma lipoproteins,
cholesterol, apolipoprotein B, small dense LDL and LDL in inflammation-induced
hypercholesterolemia in Syrian hamsters [142]. In the same experiment using
computational modelling, they showed that the inhibition of HMGR was greater
with delta-tocotrienol, followed by gamma-, beta- and alpha-tocotrienol [142].
Raederstorff et al. compared the hypocholesterolemic effects of pure
gamma-tocotrienol and tocotrienol mixture in hamsters [134]. They found that both
gamma-tocotrienol (at 58 and 263 mg/kg) and tocotrienol mixture (at 263 mg/kg)
reduced plasma total and LDL cholesterol but TG and high density lipoprotein
(HDL) cholesterol were not affected [134]. Thus, the hypocholesterolemic effect of
pure gamma-tocotrienol was greater than the tocotrienol mixture [134]. Khor and
Ng showed that presence of alpha-tocopherol might attenuate the hypocholes-
terolemic effect of tocotrienol [74]. In male hamsters fed with diet containing
low-dose alpha-tocopherol (30 ppm), the activity of HMGR was inhibited [74].
However, diet containing high-dose alpha-tocopherol (81 ppm) significantly stim-
ulated the activity of HMGR [74]. On the other hand, in guinea pigs treated with
10 mg tocotrienol (i.p.), showed significant inhibition of HMGR activity but
mixture of 5 mg alpha-tocopherol and 10 mg tocotrienol caused less inhibition
[74]. This observation in turn validated the experiment of Qureshi et al. using
chickens [124]. In a further experiment using guinea pigs injected with palm olein
triglycerides containing either tocotrienol or alpha-tocopherol, Khor et al. indicated
that tocotrienol (1–5 mg) significantly reduced the activity of liver microsomal
HMGR and cholesterol-7-alpha-hydroxylase [73]. The inhibition by
alpha-tocopherol was less effective [73]. Furthermore, the inhibitory effect of
alpha-tocopherol was lost at 5 mg [73]. At a higher dose (50 mg), alpha-tocopherol
significantly elevated the activity of HMGR and reduced the inhibitory effects of
tocotrienol [73].
Effects of tocotrienol supplementation on lipid profile in humans were hetero-
geneous. This could be due to the differences in the composition of the tocotrienol
106 K.-Y. Chin et al.

mixture used, population studied, diet of the subjects, etc. In an earlier study using
palm vitamin E (containing 18 mg tocopherols and 42 mg tocotrienols), Tan et al.
showed that supplementation for 30 days lowered serum total and LDL cholesterol
but the TG and HDL cholesterol were not affected [167]. However, this is not a
randomized controlled trial and the number of subjects was small. Qureshi et al.
showed that palm vitamin E at 200 mg caused a reduction in serum total and LDL
cholesterol, apolipoprotein B, thromboxane, platelet factor 4 and glucose in subjects
during the four week-treatment [128]. A separate hypocholesterolemic group given
200 mg gamma-tocotrienol showed greater reduction in total cholesterol compared
to palm vitamin E group [128]. Daud et al. demonstrated that supplementation of
tocotrienol for 16 weeks (180 mg tocotrienols with 40 mg tocopherols) decreased
the normalized plasma TG and increased HDL in patients undergoing chronic
hemodialysis [36]. These changes might be associated with higher plasma
apolipoprotein and lower cholesteryl-ester transfer protein activity [36]. It should be
noted that the results of this study was not adjusted for medication used by the
subjects. Chin et al. showed that when elderly (>50 years) and young (<50 years)
subjects were supplemented with tocotrienol-rich fraction (160 mg/day, containing
74 % tocotrienols and 26 % tocopherols) for 6 months, the elderly subjects
responded better by showing an increase in HDL cholesterol compared to placebo
group [29]. The products of oxidation, such as protein carbonyl and
advance-glycation end products, were also reduced only in the elderly subjects [29].
Baliarsingh et al. supplemented 19 type 2 diabetic subjects with hyperlipidemia
with tocotrienol-rich fraction (3 mg/kg body weight) for 60 days and total lipids,
total and LDL cholesterol were reduced with treatment [11]. In the study by Qureshi
et al., hypercholesterolemic subjects on American Heart Association step 1 diet
were supplemented with rice bran tocotrienol-rich fraction at different doses (25–
200 mg/kg) [132]. They observed that 100 mg/kg tocotrienol-rich fraction pro-
duced maximum decrease in total and LDL cholesterol, apolipoprotein B and TG
compared to baseline [132]. In another study, Qureshi demonstrated that hyperc-
holesterolemic subjects on American Heart Society step 1 diet supplemented with
rice bran tocotrienol-rich fraction and lovastatin in combination for 25 weeks
showed more significant reduction than individual treatments [131]. The combi-
nation of alpha-tocopherol and lovastatin did not produce similar changes [131].
Tomeo et al. supplemented subjects presented with hyperlipidemia, cere-
brovascular disease and carotid stenosis with 300 mg palm vitamin E (16 mg
alpha-tocopherol and 40 mg tocotrienols in 240 mg palm olein) for 18 months
[169]. Apparent carotid atherosclerotic regression was found in supplemented
subjects while some controls showed progression [169]. Malondialdehyde was
decreased in the treatment group but was increased in the placebo group [169].
However, no changes in total and LDL cholesterol, TG and HDL were found in
both groups [169]. In men with mildly elevated serum lipid level, supplementation
of tocotrienol (4 capsules daily for 6 weeks, each containing 35 mg tocotrienols
and 20 mg alpha-tocopherol) produced no significant effects on serum LDL and
HDL cholesterol, TG, lipoprotein a, lipid peroxide, platelet aggregation velocity
and maximum aggregation, and thromboxane B2 formation compared to men
5 Tocotrienol and Its Role in Chronic Diseases 107

receiving 20 mg alpha-tocopherol only [90]. In the study of O’Byrne et al., subjects

followed a low-fat diet for 4 weeks, then were randomized into treatment groups
receiving placebo, alpha-, gamma-, delta-tocotrienol acetate supplements
(250 mg/d) for 8 weeks while were still maintained on the low-fat diet [120]. It was
found that alpha-tocotrienol increased the in vitro LDL oxidation resistance and
decreased its rate of oxidation [120]. However, tocotrienol treatments did not affect
total and LDL cholesterol, and apolipoprotein B level [120]. In another study,
hypercholesterolemic men and women were randomized to receive three different
brands of tocotrienol from palm or rice bran, or the placebo. The subjects were put
on the American Heart Association Step 1 diet, 21 days before and throughout the
trial period [101]. There were no significant changes in lipid profile between pla-
cebo and treatment after 28 days [101]. Rasool et al. observed that in healthy male
subjects taking placebo or tocotrienol-rich vitamin E at 80, 160 and 320 mg/day for
2 months, arterial compliance, plasma total antioxidant status, serum total and LDL
cholesterol were not different among the groups [136].

5.5 Tocotrienol as Antidiabetic Agent

Diabetes mellitus represents a group of chronic metabolic conditions characterized

by increased blood glucose. Type 1 diabetes results from the destruction of
beta-cell in the pancreas due to autoimmunity, thus causing cessation in insulin
production. Type 2 diabetes is caused by abnormal resistance of the body to the
action of insulin, and the insufficiency of insulin production to overcome this
resistance. It is estimated that 5–10 % of the total diabetes cases belong to type 1
and 90–95 % belong to type 2. Diabetes can affect many organ systems and lead to
serious complications such as damage to the nervous, renal, cardiovascular system
and the eyes [38]. Oxidative stress and inflammation are involved in the devel-
opment and exacerbation of diabetic complications [39].
The antidiabetic effects of tocotrienol have been explored mainly in animal
studies. Diabetes is mostly induced by streptozotocin (STZ), a chemical that can
cause cellular ATP reduction, inhibition of insulin production, DNA alkylation and
death of pancreatic beta cells [75]. Tocotrienol has been shown to improve the
glycemic status and diabetic complications in many instances. Wan Nazaimoon
et al. showed that a diet enriched with tocotrienol-rich fraction (1 g/kg diet) sig-
nificantly reduced the blood glucose level and glycated haemoglobin of
STZ-induced diabetic male rats [176]. However, serum-advanced glycation end
product and malondialdehyde were not affected [176]. Matough et al. showed that
supplementation of tocotrienol-rich fraction at 200 mg/kg body weight for 4 weeks
in STZ-induced diabetic rats reduced the plasma glucose level [85]. The supple-
mentation also increased the erythrocytic superoxide dismutase and glutathione
levels but decreased the level of reduced glutathione and malondialdehyde [85].
Comet assays revealed reduced DNA damage in the supplemented group [85].
108 K.-Y. Chin et al.

Tocotrienol was shown to prevent diabetic nephropathy in several animal

models. Kuhad and Chopra showed that tocotrienol mixture at 25, 50 and
100 mg/kg for 8 weeks dose-dependently reduced diabetic proteinuria, polyuria
and increased serum creatinine and blood urea nitrogen levels and their clearance
[77]. The improvement in renal profile was accompanied by a dose-dependent
decrease in lipid peroxidation product and an increase in non-protein thiol, super-
oxide dismutase and catalase activity in the kidney [77]. Nitrosative stress in the
kidney was also decreased with tocotrienol supplementation as evidenced by a
lower nitric oxide level [77]. Tocotrienol was shown to reduce the level of tumor
necrosis factor-alpha associated with renal hypertrophy and hyperfunction, trans-
forming growth factor-beta associated with renal fibrosis and the expression of
nuclear factor kappa-b p65 subunit indicative of inflammation in the nuclear frac-
tion and apoptosis in the kidney [77]. Siddiqui et al. compared the hypoglycemic
effects of tocotrienol-rich fraction derived from palm oil and rice bran oil in two
separate experiments [151]. Diabetic male rats were treated with either 200 mg/kg
body weight palm oil tocotrienol or rice bran tocotrienol [151]. Both treatments
were found to reduce the fasting blood glucose and percentage of glycosylated
haemoglobin [151]. Improvements in renal profile, indicated by reductions in serum
creatinine and urine protein level, were also seen in both treatment groups [151].
Histopathological examination revealed that both tocotrienol-rich mixtures pre-
vented glomerular hypertrophy, mesangial expansion, tubular atrophy, tubular
basement membrane thickening and proteinaceous cast formation in the lumen
[151]. The improvement could be attributed to the antioxidative properties of
tocotrienol, as evidenced by the decrease in lipid peroxidation products and the
increase in superoxide dismutase and catalase activity in the kidney with tocotrienol
treatment [151]. Overall, efficacy of tocotrienol from palm was higher compared to
rice bran [151]. In the second experiment, Siddiqui et al. fed male Wistar rats with
high fat diet for 5 weeks, then switched them to standard diet and induced diabetes
in them with STZ at the same time [150]. The hypoglycemic effects between
16-week supplementation with palm tocotrienol at 200 mg/kg body weight and rice
bran tocotrienol 400 mg/kg body weight were compared [150]. In agreement with
their previous findings, palm and rice bran tocotrienol prevented the rise of blood
glucose level as early as week 6 [150]. Percentage of glycosylated hemoglobin was
also reduced by both treatments [150]. They also suppressed the increase in TG,
total, LDL and VLDL cholesterol and elevated the HDL cholesterol level [150].
Improvement in renal profile, indicated by decreased urinary creatinine and blood
urea nitrogen and increased creatinine clearance, was seen with both treatments
[150]. These positive changes might be related to improved oxidative status in the
kidney (reduced malondialdehyde level, increased superoxide dismutase, catalase,
glutathione peroxidase and reductase activities) [150]. In addition, the protein
expression of collagen type IV, transforming growth factor-beta and fibronectin
related to renal fibrosis were reduced significantly by palm tocotrienol [150]. This
was accompanied by an improvement in renal histopathology in both treatment
groups [150]. Palm tocotrienol, even though at a lower dosage, exhibited higher
efficacy in preventing diabetic nephropathy than rice bran tocotrienol [150].
5 Tocotrienol and Its Role in Chronic Diseases 109

The presence of cardiovascular risk factors, such as hypertension and hyperc-

holesterolemia, in diabetic patients lead to poorer outcomes compared to those
without [38]. In fact, the risk for cardiovascular death is nearly doubled in diabetic
patients compared with the non-diabetic [21]. As described in the earlier section,
tocotrienol is an effective hypocholesterolemic agent and its effectiveness has been
proven in chemical and diet-induced hypercholesterolemic models. Several animal
studies also demonstrated that the hypocholesterolemic effect of tocotrienol per-
sisted in diabetic model, accompanied by other cardio and vascular protective
effects. Budin et al. fed STZ-induced diabetic rats with palm tocotrienol-rich
fraction 200 mg/kg body weight for 8 weeks and observed positive changes in risk
factors for cardiovascular disease [16]. Adverse changes in fasting blood glucose,
HbA1c and lipid profile were attenuated in the tocotrienol-treated group [16].
Besides, malondialdehyde-4-hydroxynonenal in aorta and plasma, and DNA
damage in lymphocytes were reduced and plasma superoxide dismutase activity
was increased with tocotrienol treatment [16]. Tocotrienol also reduced the
degenerative changes in aortic wall by reducing the proliferation and degeneration
of vascular smooth muscle cells and defects in the aortic walls [16]. Using a
diet-induced diabetic rat model, Patel et al. showed that co-supplementation of
alpha-lipoic acid (1.6 g/kg diet) and palm tocotrienol-rich fraction (0.84 g/kg diet)
for 8 weeks could prevent and reverse the increase in plasma glucose, systolic
blood pressure, interstitial collagen deposition in the left ventricle, diastolic stiffness
and plasma malondialdehyde [123].
Tocotrienol was able to prevent diabetic-associated neuropathy. Kuhad and
Chopra et al. showed that tocotrienol mixture at 25, 50 and 100 mg/kg for 10 weeks
could prevent the increase in plasma glucose in STZ-induced diabetic rats [76].
Transfer latency and percentage of time spend in the target quadrant in a water
maze were decreased in all tocotrienol treated groups compared to the control,
indicating better memory and learning performance [76]. This was attributed to
decreased oxidative stress, indicated by a reduction in lipid peroxide level and an
increase in non-protein thiols, superoxide dismutase and catalase activity, and
decreased nitrosative stress indicated by reduced nitric oxide level in the cerebral
cortex and hippocampus [76]. Inflammation was also prevented by tocotrienol
supplementation, as evidenced by decreased protein expression of interleukin-1b
and p65 subunit of nuclear factor kappa-B in the brain [76]. The reduction in both
oxidative stress and inflammation led to reduced apoptosis of the neurons [76]. In
the subsequent experiment, Kuhad and Chopra supplemented STZ-induced diabetic
rats with 25, 50 and 100 mg/kg tocotrienol mixture and assessed the effects on
hyperalgesia [78]. Tocotrienol mixture prevented the reduction in nociceptive
threshold in the diabetic rats, indicated by increased tolerance for thermal and
mechanical stimuli, and tactile allodynia [78]. They also found that lipid peroxide
and total nitric oxide levels were reduced; while non-protein thiol levels and
activities of superoxide dismutase and catalase were increased in the sciatic nerve
with tocotrienol treatment [78]. Inflammation was also reduced with tocotrienol
treatment, indicated by a reduction in tumor necrosis factor-alpha, transforming
110 K.-Y. Chin et al.

growth factor-1 beta and interleukin-1 beta in the sciatic nerve [78]. Apoptosis
indicated by caspase 3 expression was also decreased in the sciatic nerve [78].
There are limited studies attempted to validate the hypoglycemic effects of
tocotrienol in humans and the results are heterogenous. In a longitudinal study
(median follow-up period 10.2 years) assessing the intake of antioxidants and the
risk of type 2 diabetes in 29,133 Finnish male smokers aged 50–69 years,
Kataja-Tuomola et al. found that all tocotrienol isomers were not associated with
the risk of the disease after multiple adjustment. This could be contributed to the
low levels of tocotrienols in the subjects [71]. In the study of Baliarsingh et al., type
2 diabetic subjects supplemented with tocotrienol-rich fraction (3 mg/kg body
weight) for 60 days showed no changes in fasting and postprandial plasma sugar,
HbA1c and serum creatinine [11]. This could be due to the limited number of
subjects in this study (n = 19) and the low dose of tocotrienol. In a study by
Haghighat et al., type 2 diabetic subjects taking non-insulin medication were ran-
domized to take 15 mg/day tocotrienol mixture or blank canola oil with meals for
4 weeks [47]. It was observed that urinary microalbumin and serum high-sensitivity
C-reactive protein were significantly lowered in the tocotrienol group compared to
the placebo, indicating a better renal profile and a reduced risk for cardiovascular
disease [47].

5.6 Tocotrienol as Antiosteporotic Agent

Osteoporosis is a metabolic bone disease characterized by low bone mass and

deterioration of bone microarchitecture, which subsequently leads to fragility
fracture [68]. Despite the high prevalence in women, osteoporosis affects men as
well, with female to male ratio of 6:1 [64]. Osteoporosis is a result of the imbal-
anced bone remodelling process consisting of bone resorption regulated by osteo-
clast and bone formation regulation by osteoblast. Sex hormone deficiency is the
predominant condition underlying the imbalance in bone remodelling, although
recent studies have switched the focus to the role of oxidative stress and inflam-
mation [2, 84]. Oxidative stress and inflammation inhibit differentiation of osteo-
blast (osteoblastogenesis), but promote differentiation of osteoclast
(osteoclastogenesis) [2, 10, 171]. Hence, an antioxidant and anti-inflammatory
agent like tocotrienol putatively has beneficial effects in preventing bone loss.
The effects of tocotrienol in promoting differentiation of osteoblast and sup-
pressing the differentiation of osteoclast have been demonstrated in cell culture
studies. Ha et al. demonstrated that alpha-tocotrienol but not alpha-tocopherol could
suppress osteoclastogenesis in an osteoblast-bone marrow co-culture [46]. This was
brought about by an inhibition of receptor activator of nuclear factor kappa-b
(RANKL) expression in osteoblasts [46]. In osteoclasts, alpha-tocotrienol sup-
pressed the RANKL-mediated differentiation process by inhibiting c-Fos expres-
sion via suppression of extracellular signal-regulated kinases and nuclear factor
kappa-b activation [46]. Alpha-tocotrienol also prevented the bone resorption
5 Tocotrienol and Its Role in Chronic Diseases 111

activity of mature osteoclasts [46]. Brook et al. compared the suppressive effects of
different tocotrienol isomers (alpha, gamma and delta) on human-derived CD14+
cells [15]. It was found that gamma and delta-tocotrienol were effective in sup-
pressing the formation of osteoclast-like cells and the resorbing activity of these
cells was significantly inhibited. Gamma-tocotrienol was found to be with less toxic
to CD14+ cells, indicating that it is safer compared to delta-tocotrienol [15].
The effectiveness of tocotrienol in preventing bone loss has been demonstrated
in several animal models of osteoporosis. The methods of assessing bone health in
the animals included densitometry which measures bone mineral density; histo-
morphometry which measures bone structure, cells and mineralization activity;
biomechanical test which measures the stiffness and strength of the bone; bone
mineral concentration and serum bone remodelling markers which measure the
bone formation and resorption activity (reviewed in [24, 27]).
Norazlina supplemented ovariectomized rats with palm vitamin E, which was
rich in tocotrienol at either 30 or 60 mg/kg body weight for 8 months and measured
the femoral and vertebral bone mineral density [118]. It was found palm vitamin E
prevented the decline of bone mineral density in both supplemented groups [118].
Muhammad et al. observed that palm tocotrienol at 60 mg/kg body weight for
8 weeks could improve bone structural indices (increased bone volume and tra-
becular number, and decreased trabecular separation) in an oestrogen-deficient
model of osteoporosis induced by ovariectomy [96]. A decrease in osteoclast
number was found in the tocotrienol supplemented group [96]. The effects of
alpha-tocopherol at 60 mg/kg body weight were found to be comparable with palm
tocotrienol [96]. In a subsequent experiment, Muhammed et al. supplemented the
ovariectomized rats with tocotrienol-rich fraction (60 mg/kg for 8 weeks) and
found that it improved bone structural indices (increased bone volume and tra-
becular thickness, and decreased trabecular separation) [97]. Its effects were com-
parable to calcium (15 mg/kg) and oestrogen treatment (64.5 µg/kg) [97].
Aktifanus et al. demonstrated that tocotrienol-rich fraction at 60 mg/kg body weight
for 8 weeks improved bone dynamic indices (decreased single-labelled surface,
increased double-labelled surface, mineral apposition rate and bone formation rate)
in ovariectomized rats [8]. The effects of tocotrienol were better than oestrogen
replacement (64.5 µg/kg body weight) [8]. Using palm tocotrienol at 60 mg/kg
body weight for 8 weeks, Soelaiman et al. demonstrated similar findings and its
effects were superior to calcium supplementation (1 % calcium in drinking water
ad libitum) [154]. Abdul-Majeed et al. supplemented ovariectomized rats with
annatto tocotrienol (60 mg/kg body weight for 8 weeks) or in combination with
lovastatin (11 mg/kg body weight for 8 weeks) [3, 4]. Annatto tocotrienol alone
was found to improve bone structural (increased bone volume, trabecular number
and trabecular thickness, decreased trabecular separation) and cellular indices (in-
creased osteoblast number, osteoid surface and osteoid volume, and decreased
osteoclast number and eroded surface), bone biomechanical strength (increased
load, strain, stress and Young’s modulus) and bone remodelling markers (increased
osteocalcin, a bone formation marker and decreased type-1 C-terminal telopeptide
crosslink, a bone resorption marker) [3, 4]. The combination of annatto tocotrienol
112 K.-Y. Chin et al.

and lovastatin further improved these variables, suggesting an additive or syner-

gistic effect between the two compounds [3, 4]. Deng et al. supplemented
ovariectomized mice with 100 mg/kg emulsified gamma-tocotrienol subcuta-
neously. The supplemented mice showed significant improvements in femoral bone
mineral density, bone structural (increased bone volume, trabecular number, tra-
becular thickness and decreased trabecular separation), dynamic (increased mineral
apposition rate and bone formation rate) and cellular indices (increased osteoblast
number and decreased osteoclast number) [37]. Elevated gene expression of tran-
scription factors runt-2 and osterix in the bone of supplemented groups suggested
increased osteoblast differentiation [37]. This was coupled with increased gene
expression of osteoprotegerin and decreased gene expression of RANKL, indicating
an inhibition of osteoclast formation [37]. Feeding mevalonate (25 mg/kg body
weight) to the supplemented group abolished all the beneficial effects of tocotrienol
treatment, suggesting the involvement of mevalonate pathway in the protective
action of tocotrienol on bone [37].
Using an orchidectomy-induced testosterone deficient model of osteoporosis,
Ima-Nirwana et al. showed that supplementation of palm vitamin E at 30 mg/kg for
8 months prevented the deterioration of femoral and vertebral bone mineral density
and calcium level [58]. Other studies also showed that annatto tocotrienol at
60 mg/kg could prevent deterioration of bone cancellous microarchitecture assessed
with X-ray microtomography (decreased trabecular separation), bone structural
(increased bone volume and trabecular number, decreased trabecular separation),
dynamic (decreased single-labelled surface, increased double-labelled surface) and
cellular (increased osteoblast number, osteoid surface and osteoid volume,
decreased osteoclast number and eroded surface) indices [25, 28]. This was brought
about by increased gene expression of alkaline phosphatase, beta-catenin, collagen
type 1 alpha 1 and osteopontin, indicating increased osteoblast activity [28].
However, no significant changes in bone resorption genes were observed [28].
Annatto tocotrienol was less efficacious compared to supraphysiological testos-
terone enanthate injection (7 mg/kg weekly) in the same experiment [28].
Smoking is a non-modifiable risk factor for osteoporosis. In a model of osteo-
porosis induced by nicotine, Hermizi et al. showed that supplementation of either
tocotrienol-rich fraction or gamma-tocotrienol at 60 mg/kg body weight improved
bone structural (increased bone volume and trabecular thickness), dynamic (re-
duced single-labelled surface and increased mineral apposition rate and bone for-
mation rate) and cellular indices (reduced osteoclast and eroded surface) [51].
Abukhadir et al. indicated that these positive changes were brought about by the
ability of tocotrienol to reverse nicotine-induced suppression of runt-2, osterix and
bone morphogenetic protein-2 [5]. Tocotrienol also inhibited nicotine-induced
elevation of interleukin-1 and interleukin-6 [117, 119]. Nazrun et al. demonstrated
that palm tocotrienol at 100 mg/kg was able to increase trabecular thickness, bone
formation rate, osteoblast number and decrease eroded surface in the osteoporotic
animal [7, 105]. This effect was also brought about by the suppression of
interleukin-1 and interleukin-6 by tootrienol [7]. The effects of tocotrienol in pre-
venting bone loss due to glucocorticoid treatment were also studied. Ima-Nirwana
5 Tocotrienol and Its Role in Chronic Diseases 113

and Fakhrurazi supplemented palm vitamin E in dexamethasone-treated

adrenalectomized male rats and observed no significant changes on bone mineral
density and bone calcium level [56]. In a subsequent attempt, supplementation of
gamma-tocotrienol at 60 mg/kg body weight for 8 weeks increased the fourth
lumbar vertebra bone calcium content in dexamethasone-treated adrenalectomized
male rats [59]. Alpha-tocopherol at the same dose did not exert a similar effect [59].
The beneficial effects of tocotrienol were also shown in healthy male rats sup-
plemented with alpha-tocopherol, delta-tocotrienol or gamma-tocotrienol at
60 mg/kg for 8 weeks [89, 149]. Gamma-tocotrienol supplementation improved all
bone structural, dynamic and cellular indices and biomechanical strength while
alpha-tocopherol and delta-tocotrienol only improved some of the indices, indi-
cating a superior bone-anabolic effect of gamma-tocotrienol over the other vitamin
E isomers [89, 149]. Maniam et al. showed that palm tocotrienol at 100 mg/kg for
4 months reduced malondialdehyde and increased glutathione peroxidase activity
in the bone of healthy male rats. Alpha-tocopherol at the same dose did not exert
similar effects on bone [83].
Despite there are many observational studies on the association between vitamin
E and bone health, most studies measured the intake and blood level of
alpha-tocopherol. Vitamin E (alpha-tocopherol) was associated with increased bone
mineral density or a reduced fracture risk in most studies [26]. However, there has
been no report on the effects of tocotrienol on bone health in humans to date. The
suggested mechanism of tocotrienol in preventing bone loss is summarized in
Fig. 5.3.

Fig. 5.3 The antiosteoporotic mechanism of tocotrienol

114 K.-Y. Chin et al.

5.7 Anticancer Activity

Anticancer activity of vitamin E was discovered and studied extensively since the
first experimental evidence of inhibition of tumorigenesis by vitamin E rich palm
oil [164]. Anticancer property of tocotrienol was found to be independent of its
antioxidant property [32, 162]. Tocotrienol isomer with lower antioxidant proper-
ties like delta- and gamma-tocotrienol usually showed greater potency in killing
cancerous cells. Based on quantitative structure–activity relationship studies,
chromanol structure and phytyl carbon tail were shown to be essential in apoptosis
induction [13]. Decreasing methyl groups on the chromanol head increased
anti-proliferative potency of tocotrienol (delta-tocotrienol > gamma-tocotrienol >
beta-tocotrienol > alpha-tocotrienol) [162]. Shortening the unsaturated phytyl tail
of gamma-tocotrienol by one isoprenyl unit also resulted in greater apoptotic
activity [13]. In silico analysis suggested that esterification of lysine with
gamma-tocotrienol could enhanced its anticancer activity [114]. Tocotrienol
metabolites such as carboxyethyl-hydroxychromans were found to be a less
effective anti-proliferative agent [34].
Besides that, tumor cell uptake and intracellular accumulation of tocotrienol also
determine its anticancer activity. Due to the lipophilic nature of tocotrienols, they
are readily transferred between the membranes and accumulated in cells in a time
and concentration-dependent manner [187]. Studies have shown that serum
physiological tocotrienol level was usually low or absent due to slow absorption,
rapid metabolism and lack of a specific carrier protein [164]. Besides that, toco-
trienol is highly tissue-dependent and it is only found in a few tissues/organs like
the liver and pancreas [52, 55]. Interestingly, in animal models, gamma-tocotrienol
and delta-tocotrienol were found to accumulate in tumors [52, 55]. Different
tocotrienol isomers possess different cellular accumulation tendency, i.e.
delta > gamma > alpha-tocotrienol [52, 87, 88, 116, 141, 144]. This might explain
the poor anticancer activity of alpha-tocotrienol.
Tocotrienol and tocotrienol-rich fraction were demonstrated to induce
anti-proliferation and apoptosis selectively in several cancerous cell lines origi-
nating from the mammary gland [32, 41, 44, 87, 109, 146, 173–175, 189], lung
[70, 86, 184], liver [49, 52, 140, 141, 172], prostate [33, 86, 158], melanoma
[22, 86], colon [40, 182] and leukemic cells [100]. The inhibitory potency of
tocotrienol-rich fraction, tocotrienol isoforms or their succinate synthetic deriva-
tives in different cancerous cells generally follows the order of delta- >gamma- and
beta- >alpha-tocotrienol [22, 33, 40, 52, 140, 172]. Studies also demonstrated that
tocotrienol possessed little or no adverse effect on normal cell viability or growth in
in vitro [49, 70, 87, 88, 141, 158, 184] or animal models [112, 135, 137]. In animal
studies, tocotrienols and tocotrienol-rich fraction were also demonstrated to reduce
tumor load. Oral administration of 0.05 % tocotrienol-rich vitamin E mixture sig-
nificantly reduced liver and lung carcinogenesis in C3H/He mice and 4NQO-treated
ddY mice model [172]. Gamma-tocotrienol or delta-tocotrienol diet (0.1 %) sig-
nificantly delayed tumor growth in C3H/HeN mice inoculated subcutaneously with
5 Tocotrienol and Its Role in Chronic Diseases 115

murine hepatoma MH134 cells [52]. There was no apparent adverse effect and
insignificant body and tissue weight changes in animals fed with 0.1 % of
gamma-tocotrienol or delta-tocotrienol diet [52].
Studies also demonstrated that tocotrienol as a farnesol-mimetic molecule and
HMGR inhibitor possessed great potential to produce synergistic anticancer activity
with other chemotherapeutic agents [86]. Combination of low concentration of
gamma-tocotrienol with several individual statins synergistically inhibited HMGR
activity and Rap1A & Rab6 prenylation [174]. Besides that, combination of
tocotrienol with flavonoids such as genistein, naringenin, hesperetin, tangeretin,
nobiletin, quercetin, apigenin, epigallocatechin gallate or resveratrol demonstrated
synergistic activity in inhibiting human breast cancer MCF7 or/and MDA-MB-435
cells proliferation, which was enhanced further with the addition of tamoxifen [45,
54]. The combination of tocotrienol with docetaxel also resulted in higher pro-
portion of apoptotic cells [22]. Combination of tocotrienols with tamoxifen
increased the arrest of DNA synthesis and triggered an endoplasmic reticulum
(ER)-independent anti-proliferation event in the breast cancer cells [44, 189].
Combination of tocotrienol isomers (delta-tocotrienol and gamma-tocotrienol) was
also found to induce cell death synergistically in DU145 and PC3 [33]. In animal
studies, gamma-tocotrienol also acted synergistically with lovastatin to reduce
tumor load in C57BL/6 mice inoculated with B16 cells [86].
There are limited clinical trials on tocotrienol and cancer conducted to date.
Malaysian Palm Oil Board had conducted two clinical trials on the efficacy of
tocotrienol in breast cancer patients [107]. The first study was conducted to identify
tocopherol and tocotrienol concentrations in malignant and benign adipose tissues
of breast cancer patients after a palm oil diet. The results revealed a higher con-
centration of tocotrienols (alpha-, delta- and gamma-isomers), in the adipose tissues
of patients with benign breast lumps compared with that of patients with malignant
tumors [107]. The second study was a 5-year double-blind, placebo-controlled pilot
trial conducted to compare tamoxifen with or without tocotrienol-rich fraction on
early breast cancer [108]. The risk of dying due to breast cancer decreased by 70 %
and risk of recurrence decreased by 20 % in patients taking the adjuvant tocotrienol
and tamoxifen compared with the patients receiving only tamoxifen [107].
However, there was no significant association between adjuvant tocotrienol therapy
with breast cancer survival rate [108].

5.7.1 Tocotrienol-Induced Caspase Activation and Mode

of Cell Death

Caspases are constitutively present in cells in an inactive precursor form, the pro-
caspases [164]. In receptor-mediated apoptosis, there is early activation of death
receptors (Fas, TNF or TRAIL) which lead to caspase-8 activation. In
mitochondria-mediated apoptosis, there are apoptotic signals from damaged DNA
116 K.-Y. Chin et al.

or loss of mitochondrial membrane potential, which will lead to activation of

caspase-9. Initiator caspases (caspase-8 and -9) activate effector caspases
(caspase-3, -6, and -7) which will then execute the cells by cleaving DNA, PARP
and other structural proteins [164]. Both death receptor- [33, 140, 141] and
mitochondria-mediated [22, 33, 103, 139, 160, 165, 182, 184] pathway of apoptosis
induced by tocotrienol-rich fraction, tocotrienol isomers, ether or succinate syn-
thetic derivatives were reported. Tocotrienol-rich fraction and tocotrienol
isomer-induced apoptosis are selective and greatly dependent on cell types and
treatment conditions. It might involve the upregulation of Bax and tBid protein with
the increased of Bax/Bcl-2 ratio [140].
Besides that, gamma-tocotrienol was also found to induce caspase-12 activation
in +SA cellular apoptosis [173]. Caspase-12 activation is closely associated with
endoplasmic reticulum ER stress-mediated cell death. Further experiments identified
an early involvement of PERK/eIF2a/ATF4 phosphorylation and CHOP-dependent
TRB3-mediated ER stress response pathway in gamma-tocotrienol-induced +SA
cellular apoptosis [173]. Furthermore, gamma-tocotrienol, delta-tocotrienol,
gamma-tocotrienol succinate and delta-tocotrienol succinate were also found to
trigger caspase-independent cell death in DU145, PC3 and LNCap cells whereby the
event of apoptosis could not be abrogated by general caspase inhibitors [33].

5.7.2 Tocotrienol-Induced Upstream Apoptotic Pathway

Modulation

The inhibition of HMGR by tocotrienol not only suppresses cholesterol synthesis but
also inhibits cell proliferation via prenylation of Ras, RhoA or Rap1A signalling
molecules through generation of prenyl intermediates [184]. Farnesyl side chain of
tocotrienol is essential for the post-transcriptional suppression of HMGR.
Gamma-tocotrienol was shown to downregulate HMGR levels in +SA cells via
post-translational protein degradation [173]. However, the gamma-
tocotrienol-induced downregulation of HMGR was not able to block Rap1A &
Rab6 prenylation and trigger +SA cell death [174]. Delta-tocotrienol-induced growth
arrest might be due to HMGR inhibition, causing marked decrease of cyclin
D1/cyclin-dependent kinase CDK4 complex but not cyclin E/CDK2 complex, which
subsequently reduced the phosphorylation of retinoblastoma RB protein and E2F1
expression [41, 103].
6-O-carboxypropyl-alpha-tocotrienol (alpha-T3E) was more potent compared to
alpha-tocotrienol in inducing apoptosis in A549 cells and H28 cells [70, 184]. 6-O-
carboxypropyl-alpha-tocotrienol also harbours the similar farnesyl side chain and
might possess HMG-CoA reductase inhibitory activity. 6-O-carboxypropyl-
alpha-tocotrienol can inhibit RhoA geranyl-geranylation and Ras molecules farne-
sylation, subsequently blocking MEK, ERK and p38 phosphorylation and lead to
cyclin D and Bcl-xL downregulation during G1 arrest and apoptosis [160, 174, 184].
5 Tocotrienol and Its Role in Chronic Diseases 117

Besides that, 6-O-carboxypropyl-alpha-tocotrienol also inactivated Src kinase via

phosphorylation at Tyr527 site and dephosphorylation at Tyr416 site, which
decreased the phosphorylated form of EGFR and its activity [70]. Another study
revealed that gamma-tocotrienol inhibited PI3k/PDK-1/Akt pathway via suppres-
sion of EGFR (ErbB3)-receptor tyrosine phosphorylation [143]. 6-O-
carboxypropyl-alpha-tocotrienol also inhibited Stat-3 activation which might be due
to Src kinase inactivation [70]. 6-O-carboxypropyl-alpha-tocotrienol was shown to
partially inhibit Src kinase. However, this was inadequate to trigger cell death on
A549 cells under hypoxia [69]. Survival and invasion capacity of A549 cells under
hypoxia were suppressed by 6-O-carboxypropyl-alpha-tocotrienol via inhibition of
the Src/HIF-2alpha/PAI-1 and PI3k/PDK-1/Akt signalling pathways [69].
Gamma-tocotrienol was also demonstrated to inhibit PI3k/PDK-1/Akt pathway and
subsequently suppressed the FLIP level [163]. Gamma-tocotrienol also induced +SA
cells apoptosis via TRB3-mediated ER stress [173]. TRB3 inhibited Akt signalling
pathway and this also contributed to the decrease of Akt activity [173].
Combination of gamma-tocotrienol with several individual statins also syner-
gistically induced +SA cells growth arrest and apoptosis which was associated with
inactivation of ERK, p38, JNK MAP kinases and Akt kinases [174, 175].
Downstream Stat-3 inactivation leads to similar downregulation of cyclin D1 and
Bcl-xL [69]. Therefore, HMG-CoA reductase might be involved in upstream reg-
ulation of RhoA/Ras, Src/HIF-2alpha/PAI-1 and PI3k/Akt pathway.
Previous studies demonstrated that tocotrienol-rich fraction, alpha-, gamma- and
delta-tocotrienol inhibited proliferation and induced apoptosis in human breast
cancer MCF-7, MDA-MB-435 and MDA-MB-231 cells, regardless of their
oestrogen receptor status [32, 41, 44, 109, 189]. Delta-tocotrienol was showed to
downregulate cyclin B1 and CDK1 expression in MDA-MB-231 cellular growth
arrest [41]. Combination of gamma-tocotrienol with either epigallocatechin gallate
or resveratrol suppressed MCF-7 cell proliferation via downregulation of cell cycle
regulatory proteins E2F, CDK4 and cyclin D1 [54]. In vitro and in silico studies
revealed that gamma- and delta-tocotrienol specifically bound and activated estrogen
receptor β signalling molecule [32]. Activated estrogen receptor β served as an
upstream signal of apoptosis and suppressed ER alpha-mediated cell survival and
proliferation [32]. Estrogen receptor β upregulated MIC-1, cathepsin D and EGR-1
mRNAs expression which triggered subsequent caspase-regulated cell death [32].
Epidermal growth factor (EGF)-dependent mitogenesis is associated with the
activation of phospholipid-dependent protein kinase C (PKC) and cyclic AMP
(cAMP)-dependent proteins [88]. Tocotrienol-rich fraction, alpha-, gamma-, and
delta-tocotrienol induced apoptosis and inhibit the proliferation of EGF-induced
preneoplastic mammary epithelial CL-S1 cells, neoplastic mammary epithelial-SA
cells and +SA cells [87, 146, 174, 175]. Tocotrienol-rich fraction also reduced
EGFR-independent and cAMP-dependent EGF-induced G-protein mitogenic
signalling in CL-S1 cells [162]. Combination of tocotrienol-rich fraction with
G-protein stimulants (cholera and pertussis toxin) or cAMP agonists (forskolin and
8-Br-cAMP) completely reversed anti-proliferative effects of tocotrienol-rich frac-
tion on CL-S1 cells [162]. Besides that, tocotrienol was shown to inhibit PKC,
118 K.-Y. Chin et al.

adenylate cyclase and cyclic AMP-dependent protein activation which might be

related to its anti-proliferative effects [23, 88, 94]. Delta- and beta-tocotrienol could
inhibit PKC activity leading to transcriptional inhibition of c-myc and hTERT, thus
reducing telomerase activity [40]. In addition, gamma-tocotrienol also induced
downregulation of Id family proteins and EGFR protein with concomitant
activation of JNK MAP kinase pathway [22]. Alpha-tocotrienol but not its acetate
analogue was able to inhibit intrinsic cellular 20S proteasome activity which might
contribute to THP-1 anti-proliferation activity [100]. Tocotrienol-rich fraction,
alpha-, gamma- and delta-tocotrienol might serve as potent DNA synthesis
inhibitors with the rank order of gamma-tocotrienol > delta-tocotrienol >
alpha-tocotrienol [189].

5.7.3 Tocotrienol-Induced Anti-angiogenesis

and Anti-invasion

Anti-angiogenic therapy has recently emerged as a strategy for cancer therapy. As

oxidative stress is an important factor in angiogenesis, it is possible that established
antioxidants such as tocotrienol may minimize the formation of new blood vessels
[60]. An in vitro study revealed that tocotrienol significantly inhibited the prolif-
eration and tube formation of BAEC as well as FGF- or VEGF-induced HUVEC
cells in the order of delta- >beta- >gamma- >alpha-tocotrienol [60, 93–95, 103,
180]. Tocotrienol also suppressed VEGF secretion in colon carcinoma cells DLD-1
and HepG2 cells under normoxic and hypoxic conditions in the order of delta-
>beta- >gamma- >alpha-tocotrienol [148]. In vivo studies revealed that
tocotrienol-rich oil was able to inhibit DLD-1 cells-induced angiogenesis via mouse
dorsal air sac (DAS) assay [93, 103]. Tocotrienol-rich fraction and delta-tocotrienol
were also demonstrated in vivo to possess anti-angiogenic effects as assessed by
chick embryo chorioallantoic membrane (CAM) assay [94, 103, 180].
Tocotrienol-rich fraction supplementation also suppressed serum VEGF level in
BALB/c mice inoculated with mouse mammary cancer 4T1 cells which indicated
its potential anti-angiogenic activity [180].
Western blotting analysis identified that anti-angiogenic activity of
delta-tocotrienol was mediated via apoptosis induction and inactivation of
PI3K/PDK/Akt pathway [93]. The inactivation of PI3K/PDK/Akt pathway then
suppressed the phosphorylation of ERK1/2, eNOS, and GSK3 alpha/beta and
increased the phosphorylated ASK-1 and p38 and p21 which were associated with
apoptosis [103]. Besides that, DNA microarray analysis revealed that delta-
tocotrienol also downregulated VEGF receptor expression and subsequently
blocked intracellular VEGF signalling (phospholipase C-gamma and PKC) in
HUVEC cells [94]. Delta-tocotrienol also managed to suppress IL-8 and angiogenic
factor secretion and downregulate HIF-1 alpha and IL-8 mRNA in hypoxic
conditions [148]. The downregulation of hypoxia-induced HIF-1 alpha expression
5 Tocotrienol and Its Role in Chronic Diseases 119

then inhibited VGEF transcriptional activation [148]. Furthermore, tocotrienol was

demonstrated to inhibit human and mice DNA polymerase λ activity in the order of
delta- >beta- >gamma- >alpha-tocotrienol [95]. Tocotrienol-induced DNA poly-
merase λ inhibition required direct binding of tocotrienol to N-terminal region of
polymerase [95]. Inhibition of DNA polymerase λ might be closely related to
anti-angiogenic activity of tocotrienol.
Tocotrienol-rich fraction and tocotrienols also possessed anti-migration and
anti-invasion properties. Delta-tocotrienol inhibited HUVEC and BAEC cells
migration as shown in scratch assay test [92, 180]. Delta-tocotrienol also demon-
strated profound inhibitory effect on human monocyte U937 cells adhesion to
HAECs via blockage of HAECs surface VCAM-1 mRNA and expression [102].
Both alpha-tocotrienol and gamma-tocotrienol suppressed VCAM-1 expression,
which inhibited human monocyte THP-1 cells adhesion to endothelial HUVEC
cells with the order of alpha-tocotrienol > gamma-tocotrienol [116]. Gamma-
tocotrienol also inhibited melanoma cells invasion via restoration of E-cadherin and
gamma-catenin expression, together with downregulation of Snail, alpha-SMA,
vimentin and twist expression [22].
The anticancer mechanism of tocotrienol is summarized in Fig. 5.4.

Fig. 5.4 The anticancer mechanism of tocotrienol

120 K.-Y. Chin et al.

5.8 Safety of Tocotrienol

A toxicity study conducted by Ima-Nirwana et al. in the mice revealed that palm
tocotrienol at the doses of 500 and 1000 mg/kg body weight (oral) for 14 days
(subacute) and 42 days (subchronic) increased the bleeding the clotting time [57].
After conversion based on body surface area [138], this is equivalent to 40.54 and
81.08 mg/kg in humans. Nakamura et al. observed changes in haematology, blood
serum enzyme levels and organ histology in rats fed with palm tocotrienol
(0.75–3 % weight of diet) for 13 weeks [104]. The no-observed-adverse-effect level
derived from this study was 120 mg/kg body weight for male rats and 130 mg/kg
body weight for female rats [104]. This is equivalent to 19.46 mg/kg in men and
21.08 mg/kg in women. In view of these studies, it is recommended that patients
who are taking anticoagulants such as warfarin, heparin and aspirin to refrain from
using tocotrienol because it may increase haemorrhagic risk. Prolonged use of
tocotrienol at high dose should also be avoided because it may exert adverse effects
on the liver. Taking these into consideration, the use of tocotrienol is relatively safe.
The highest dose used in human recorded in this review is 400 mg [47], equivalent
to 5.71 mg/kg for a 70 kg adults, which is far below the toxic doses.

5.9 Conclusion

The two groups of vitamin E, the tocotrienols and the tocopherols possess distinct
similarities and differences. These differences may be seen even between isomers
within the same group. Extensive in vivo and in vitro studies on vitamin E mixtures
from various natural sources, as well as the pure isomers, have been done. Most of
these studies have shown beneficial effects of the tocotrienol and tocopherol iso-
mers in varying degrees. The source of the naturally occurring vitamin E mixtures
are from palm oil, rice bran or annatto oil. However, in the reported literature, the
vitamin E mixtures differ in composition, some consisting of mixtures of toco-
pherols and tocotrienols, while some are mixtures of tocotrienol isomers only. The
doses used also differ between studies. Even experiments using individual toco-
trienol or tocopherol isomers differ in their effective doses. Thus, it is not easy to
summarize the results, but in general the preclinical literature shows that toco-
trienols are beneficial in metabolic disease conditions associated with elevated free
radicals and inflammatory cytokines, such as dyslipidaemia, diabetes mellitus and
osteoporosis. Some clinical trials on tocotrienols and dyslipidaemia have been
reported, while none on diabetes mellitus or osteoporosis are available so far. Data
from the available clinical trials are inconclusive, and generally the beneficial
effects are slight and not as significantly obvious as the effects seen in the in vivo
and in vitro studies. This begs the question as to whether dose of the tocotrienols
used in the human studies were too low, or the duration of treatment insufficient, or
even whether the sampling methods were adequate. Another cause for the
5 Tocotrienol and Its Role in Chronic Diseases 121

discrepancy could be that the distribution and metabolism of the tocotrienols differ
between humans and the rodent models used. It will be interesting to see whether
the same sort of observations will be seen in clinical trials on the effects of
tocotrienols in diabetics or in osteoporotic patients.
Many well-reported studies on the anticancer effects of tocotrienol were found in
the literature. Most of these studies are in vitro studies using cancer cell lines. Most
of the studies are on breast cancer cells, while some studies are on liver, colon, lung
and leukaemic cell lines. The mechanisms have been elucidated as being apoptotic
and anti-angiogenetic. The anticancer effects of tocotrienol are thought to be sep-
arate from the antioxidant and anti-inflammatory effects. Limited animal studies
have been done using genetically modified mice, and only two reported clinical
trials on the effects of tocotrienols on breast cancer are found in literature. Again the
data from the human studies are scant and inconclusive.
From the above review, tocotrienol presents an exciting possibility as an alter-
native or adjuvant therapy for diseases associated with increased oxidative stress
and inflammation, such as dyslipidaemia and diabetes. Its application in preventing
osteoporosis in humans still awaits results from clinical trial. More coordinated,
well-designed clinical trials are needed to determine the effects on humans, how-
ever, the consistently beneficial effects in in vivo studies indicate that the potential
for human therapeutic use is real. As for the anticancer properties, the potential for
use presents a real challenge to researchers. The apoptotic and anti-angiogenetic
properties seen in the cell culture studies appears very promising and can be the
alternative to the stressful chemotherapy, or at least as an adjuvant to reduce the
side-effects of chemotherapy.

Acknowledgements This work is supported by Universiti Kebangsaan Malaysia via grants

GGPM-2015-036.

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Chapter 6
Indole-3-Carbinol and Its Role
in Chronic Diseases

Barbara Licznerska and Wanda Baer-Dubowska

Abstract Indole-3-carbinol (I3C), a common phytochemical in cruciferous veg-

etables, and its condensation product, 3,3’-diindolylmethane (DIM) exert several
biological activities on cellular and molecular levels, which contribute to their
well-recognized chemoprevention potential. Initially, these compounds were clas-
sified as blocking agents that increase drug-metabolizing enzyme activity. Now it is
widely accepted that I3C and DIM affect multiple signaling pathways and target
molecules controlling cell division, apoptosis, or angiogenesis deregulated in cancer
cells. Although most of the current data support the role of I3C and DIM in
prevention of hormone-dependent cancers, it seems that their application in pre-
vention of the other cancer as well as cardiovascular disease, obesity, and diabetes
reduction is also possible. This chapter summarizes the current experimental data
on the I3C and DIM activity and the results of clinical studies indicating their role
in prevention of chronic diseases.

Keywords Indole-3-carbinol DIM Signaling pathways
Chronic diseases



Animal models Dietary intervention trials

6.1 Introduction

The plant family Cruciferae, particularly members of the genus Brassica, like
cabbage, broccoli, cauliflower, Brussels sprouts, kale, bok choy are rich sources of
sulfur-containing glucosinolates. These secondary products of plant metabolism
include, among others, glucobrassicin and neoglucobrassicin. When plant tissue is
disrupted, an endogenous thioglucosidase (myrosinase) is activated and converts

B. Licznerska
W. Baer-Dubowska (&)

Department of Pharmaceutical Biochemistry, Poznan University of Medical Sciences,
Poznan, Poland
e-mail: [email protected]
B. Licznerska
e-mail: [email protected]

© Springer International Publishing Switzerland 2016 131

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1_6
132 B. Licznerska and W. Baer-Dubowska

glucobrassicin and other indolylic glucosinolates to indoles, principally to

indole-3-carbinol (I3C) [120].
Since the first reports on possible anti-carcinogenic activity of I3C [122]
numerous preclinical studies have confirmed the chemopreventive properties of this
compound by preventing, inhibiting, and reversing the progression of cancer.
Moreover, preliminary clinical trials have shown that I3C is a promising agent
protecting against hormone-dependent as well as hormone-independent human
cancers [47].
Thus, it is not surprising that there are many marked diet supplements containing
I3C. Several mechanistic studies have been performed in order to elucidate the
mechanism of pleiotropic activity of I3C.
This chapter summarizes the current knowledge on the possible interference of
I3C with signaling pathways in vitro and in animal models, as well as its application
in prevention of chronic diseases.

6.2 Physicochemical Properties and Pharmacokinetics

of Indole-3-Carbinol

Among the indoles, generated upon ingestion of cruciferous vegetables, only

I3C (IUPAC: 1H-indol-3-ylmethanol) is commercially available as an off-white
solid. Basic physical and chemical properties of I3C are summarized in Table 6.1.
I3C is chemically unstable in acidic conditions, in vitro in cell cultures and
in vivo in the stomach environment. In such conditions, I3C may rapidly condense
into a series of oligomeric products, of which a dimer, 3,3,-diindolylmethane
(DIM), is considered the most bioactive product (Fig. 6.1) [2, 115]. Several phar-
macokinetics studies, performed mostly in animal models, have been conducted for
I3C and its condensation products [5, 6, 34, 39, 105]. When rainbow trout has been
administered with radiolabeled [5-3H]-indole-3-carbinol, 40 % of total radioactivity
was found in the liver extracts as DIM [34]. Upon oral administration of 250 mg/kg
to mice, the I3C was rapidly absorbed and distributed into variety of tissues and
body fluids (e.g., liver, kidney, lung, heart, brain, and plasma) with highest con-
centrations in liver and kidney, but with rapid clearance (concentrations below the
limit of detection within 1 hour after administration). In the same experiment, DIM
was detected in plasma at 15 min and was still quantifiable after 6 h with a peak at
2 h after I3C dosing [5, 6]. DIM was also found in stomach tissue and contents,

Table 6.1 Physical and Stability Off-white powder

chemical properties of I3C
Molecular weight 147.17386 g/mol
(ALOGPS, www.pubchem.
com; accessed Dec 26, 2015) Melting range 96–99 °C
Storage temperature 2–8 °C
Stability 2–80 °C, considered stable
Water solubility 3.75 mg/ml, mixes with water
6 Indole-3-Carbinol and Its Role in Chronic Diseases 133

Fig. 6.1 Molecular structure and formation of I3C and DIM

intestines, and liver after 1 h following oral administration of I3C to rats [39]. In
human volunteers intervention trial DIM was detected in plasma within 8 h fol-
lowing of 400 mg I3C oral dose [9]. In a phase I clinical trial in women, no I3C was
found in the plasma after administration of a single dose of up to 1200 mg or
multiple-doses at 400 mg provided twice daily for 4 weeks, and DIM was the only
detectable I3C-derived compound in plasma [105]. Fujioka et al. [47] have found
that urinary DIM level after uptake of I3C from Brussels sprouts or cabbage is a
biomarker of glucobrassicin exposure in humans. All these results support the
suggestion that I3C serves as the prodrug rather than the therapeutic agent itself. In
this regard, purified I3C as treatment agent used in in vitro models seems to be
somewhat contradictory, because there is no certainty that any metabolism of DIM
in cells occur. Thus, in this chapter the biological activity and the role in chronic
diseases will refer not only to I3C but also to DIM, its major condensation product
in humans.

6.3 Modulation of Cell Signaling Pathways

by Indole-3-Carbinol

I3C affects multiple signaling pathways and target molecules controlling cell
division, apoptosis or angiogenesis deregulated in cancer cells. Figure 6.2 presents
the overview of the signaling pathways and possible crosstalks influenced by I3C or
DIM. One of the major pathways targeted by I3C is phosphoinositide 3-kinase
134 B. Licznerska and W. Baer-Dubowska

membrane
Wnt

P-glycoprotein GJIC HER2 EGFR E-cadherin

c ell
PI3K β -catenin
Reversal of MDR
GSK-3 β
Akt
MAPK
mTOR AUTOPHAGY

ANGIOGENESIS
NF κ B ERα
AR
nucleus

ERβ AhR
Sp1
Nrf2 BRCA1
miR -21
miR -146 DNA REPAIR

p27

p15 p21 CDK 2 Bcl -2 NQO CYP1B1

COX-2
CDK6
GST CYP1A1/1A2
AKR1C3
APOPTOSIS
METABOLISM
PROLIFERATION
HORMONAL REGULATION
CELL GROWTH INFLAMATION

Fig. 6.2 Signaling pathways and proposed crosstalks ($) affected by I3C/DIM; red-inhibition,
green-induction (AhR-aryl hydrocarbon receptor, AKR-aldo-keto reductase, Akt- protein kinase
B, AR-androgen receptor, BRCA-breast cancer tumor suppressor gene, CDK-cyclin-dependent
kinase, COX-cyclooxygenase, EGFR-epidermal growth factor receptor, ER-estrogen receptor,
GJIC-Gap junctional intracellular communication, GSK-glycogen synthase kinase,
GST-glutathione-S-transferases, HER-receptor tyrosine-protein kinase erbB-2,
MAPK-mitogen-activated protein kinases, MDR-multidrug resistance, NFjB-nuclear factor
kappa-light-chain-enhancer of activated B cells, Nrf2-nuclear transcription factor 2, NQO-NAD
(P)H:quinone oxidoreductases, PI3K-phosphoinositide 3-kinase, Sp1-Sp1 transcription factor)

(PI3K)—protein kinase B (Akt)—mammalian target of rapamycin (mTOR) sig-

naling pathway. This pathway is a cascade of events that plays a key role in the
broad variety of physiological and pathological processes. PI3K/Akt/mTOR sig-
naling pathway is one of the most frequently affected target in all sporadic human
cancers, and it has been estimated that mutations in the individual component of
this pathway account for as much as 30 % of all known human cancers [83, 112].
Akt is a serine/threonine protein kinase functioning downstream of PI3K in
response to mitogen or growth factor stimulation. The inhibition of phosphorylation
and subsequent activation of Akt kinase by I3C or DIM was described in prostate
and breast cancer cells. In addition, I3C abrogated epidermal growth factor (EGF)-
induced activation of Akt in prostate cancer cells. Furthermore, the known down-
stream modulators of the Akt/PI3K cell survival pathway, Bcl-x(L), and BAD
proteins showed decreased expression after I3C treatment [24, 49]. Several genes
that mediate processes involved in carcinogenesis are regulated by transcription
6 Indole-3-Carbinol and Its Role in Chronic Diseases 135

factor NF-jB. It plays a central role in general inflammation as well as immune

responses, although recent evidences suggest that its role in these processes is more
complex [73]. The enhanced activation and expression of NF-jB is linked to
development and progression of human cancers as well as in the acquisition of
drug-resistant phenotype in highly aggressive malignancies [3, 109].
Activation through Akt is important for many tumorigenic functions of NF-jB.
Several studies showed that both indoles inhibited PI3K/Akt/mTOR/NF-jB sig-
naling (reviewed in [4]).
It was also known that there is a crosstalk between Akt and Wnt signaling
pathways through the signal communication between glycogen synthase kinase 3
(GSK-3b) and b-catenin, two of the important molecules in Akt and Wnt pathways,
respectively [109]. It was found that DIM significantly increased the phosphory-
lation of b-catenin, and inhibited b-catenin nuclear translocation [77] suggesting
that DIM could also downregulate the activation of Wnt signaling.
Mitogen-activated protein kinases (MAPK) are involved in cellular response to a
diverse array of stimuli and regulate several cell functions including differentiation,
mitosis, cell survival, and apoptosis [100]. Downregulation of the expression of
MAP2K3, MAP2K4, MAP4K3, and MAPK3 by I3C and DIM was described
suggesting their inhibitory effects on MAPK pathway [76]. It was also reported that
the effects of DIM were mediated by crosstalk between protein kinase A and
MAPK signaling pathways [75].
Gap junctional intracellular communication (GJIC) also involved in regulating
cell proliferation, differentiation and apoptosis is modulated by gap junction
channels via broad variety of endogenous and exogenous agents [90]. I3C was
reported to prevent H2O2-induced inhibition of GJIC by the inactivation of Akt in
rat liver epithelial cells [57].
Several studies have focused on the potential effects of I3C and DIM on the
proliferation and induction of apoptosis in human prostate or breast cancer cell
lines. Cell G1 cycle arrest of breast cancer cells by I3C was related to inhibition of
the expression of cyclin-dependent kinase-6 (CDK6) independently of estrogen
receptor signaling [30]. Moreover, the inhibition of CDK6 expression in human
MCF7 breast cancer cells was further explained by disrupting Sp1 transcription
factor interaction with CDK6 gene promoter [32].
In prostate cancer cell lines, the induction of apoptosis by I3C was
p53-independent [94]. The induction of p21WAF1 expression by DIM was also
independent of p53 status [55]. Pro-apoptotic effect of DIM in HER2/Neu over-
expressing breast cancer cells was connected to inhibition of HER2/Neu activity
[87]. We have also found a decreased expression of HER2/Neu in estrogen inde-
pendent MDA-MB-231 breast cancer cells treated with either I3C or DIM
(Licznerska et al. unpublished data).
I3C has been known to be a negative regulator of estrogens, while DIM a
negative regulator of androgens. I3C inhibited the transcriptional activity of ERa,
the estradiol-activated ERa signaling, and the expression of the estrogen-responsive
genes [89]. Since I3C and DIM could also inhibit the proliferation of ER-negative
breast cancer cells, it is suggested that antitumor activities of indoles could be ER
136 B. Licznerska and W. Baer-Dubowska

independent. DIM was found to be an antagonist of AR, inhibiting

androgen-induced AR translocation to the nucleus [74]. DIM also enhanced
expression of aldo-keto reductase 1C3 (AKR1C3), an enzyme responsible for
inactivation of 5a-dihydrotestosterone and decreasing estrogen levels in mammary
gland and eliminating active androgens from the prostate [92, 101].
Several lines of evidence indicate possible crosstalk between ERa and AhR
signaling pathways [96]. It was shown that I3C triggers AhR-dependent
ERa protein degradation in MCF7 breast cancer cell line disrupting an
ERa-GATA3 transcription factor cross-regulatory loop. This lead to ablation of
ERa expression and loss of ERa-responsive proliferation [86].
Our studies showed that both indoles upregulated AhR and downregulated ERa
expression in non-tumorigenic MCF10A and tumorigenic MCF7 breast epithelial
cells [114]. Upregulation of AhR was also observed in ER-negative MDA-MB-231
cells. This observation is important since increased expression and activation of
AhR result in induction of CYPs involved in estrogen metabolism [113].
Both I3C and DIM elicited an inhibition of cell adhesion, migration and invasion
in the breast cell lines of different ER status [89]. This appeared to be due, in part, to
I3C-induced upregulation of the tumor suppressor gene BRCA1 and other proteins
involved in DNA repair, like RAD51 [105].
Recent studies show that AhR and ERa interact with and modulate the activity
of Nrf2 [52]. This transcription factor plays an essential role in cellular protection
against electrophiles and oxidative stress by upregulating expression of phase II
detoxifying enzymes, including glutathione-S-transferases (GST) and NAD(P)H:
quinone oxidoreductases (NQO) [81]. Significant increase of the Nrf2 transcript
level in MCF7 and MDA-MB-231 breast cancer cell lines was observed after
treatment with I3C. Moreover, increase of Nrf2 expression was correlated with
enhanced expression of NQO1 or GSTP in MCF7 cells and MDA-MB-231 cells
respectively [114].
Recently, a plethora of evidence has demonstrated that epigenetic alterations,
such as DNA methylation, histone modifications, and non-coding miRNAs, con-
sistently contribute to carcinogenesis, and dietary phytochemicals, including glu-
cosinolate derivatives, have the potential to alter a number of these epigenetic
events [46, 119].
In this regard, DIM was reported to decrease promoter methylation of Nrf2
in vitro in TRAMP C1 mouse prostate cell line and in vivo in TRAMP mice
prostate tumors. This effect was at least in part related to decreased expression and
activity of DNA methyltransferases [130]. Moreover, genome-wide promoter
methylation in normal and cancer prostate cells showed broad and complex effects
on DNA methylation profiles reversing many of the cancer associated methylation
alterations, including aberrantly methylated genes that are dysregulated or are
highly involved in cancer progression [129].
Histone modifications by DIM were found in several studies. Decreased histone
deacetylases (HDACs) protein expression were observed in human colon, breast,
6 Indole-3-Carbinol and Its Role in Chronic Diseases 137

and prostate cancer cell lines, human colon cancer xenografts in nude mice and in
mouse prostate cells, influencing apoptotic proteins, like p21, p27, and involved in
inflammation COX-2 [46].
I3C and DIM were found to downregulate miR-21 and miRNA-146, respec-
tively, in Panc-1 pancreatic cancer cells, which was related to induction of
chemosensitivity to gemcitabine in these cells [78, 98]. In another study, I3C
reversed the upregulation of miR-21 caused by lung carcinogen—vinyl carbamate
in mice [88]. DIM upregulated miR-21 in breast cancer MCF7 cells resulting in
reduced proliferation [62], and the let-7 family miRNA leading to inhibition of
self-renewal and clonogenic capacity of prostate cancer cells [69, 70].
To sum up, both I3C and its dimer demonstrate pleiotropic effects on cell sig-
naling and subsequently gene expression regulation. Some of these activities are
summarized in Table 6.2.

Table 6.2 Summary of the major biological effects of indole-3-carbinol (I3C) and its
condensation product—3,3’-diindolylmethane (DIM)
Effecta Target molecules I3C DIM
In In In In
vitro vivo vitro vivo
Induction of phase I CYP1A1, CYP1A2, + + +
enzymes CYP1B1
Induction of phase II GST, NQO + + + +
enzymes
Inhibition of DNA CYP1B1 +
adducts formation
Anti-estrogenic activity ER, AhR + + +
Anti-androgenic activity AR + +
Cell cycle arrest p21, p27, CDK6 + +
Pro-apoptotic Akt, NFjB, GSK3b, JNK + +
Anti-angiogenic activity E-cadherin, a-, b-, and + + +
c-catenins, MMP9
Anti-proliferative activityERb/ERa + + +
DNA repair BRCA1, RAD51 + + +
Reversal of MDR P-glycoprotein + +
Epigenetic modifications DNMTs, HDACs, miRNAs + +
Anti-inflammatory NFjB, COX-2 + + +
activity
AhR aryl hydrocarbon receptor, Akt protein kinase B, AR androgen receptor, BRCA breast cancer
tumor suppressor gene, CDK cyclin-dependent kinase, DNMT DNA methyltransferase,
ER-estrogen receptor, GSK glycogen synthase kinase, GST glutathione-S-transferases, HDAC
histone deacetylases, JNK c-Jun N-terminal kinase, MDR multidrug resistance, MMP matrix
metalloproteinase, NFjB nuclear factor kappa-light-chain-enhancer of activated B cells, NQO
NAD(P)H:quinone oxidoreductases
a
References in the text
138 B. Licznerska and W. Baer-Dubowska

6.4 Role of Indole-3-Carbinol in Chronic Diseases

The term “chronic diseases” appear under different names in different contexts.
According to WHO this term suggests the following shared features:
• The chronic disease epidemics take decades to become fully established—they
have their origins at young age;
• Given their long duration, there are many opportunities for prevention;
• They require a long-term and systematic approach to treatment (WHO Report
2015).
In this context, cardiovascular diseases, chronic respiratory diseases, diabetes
along with cancer are mentioned. However, chronic character of cancer is not so
obvious as in the case of the other illnesses. The idea of considering cancer as
chronic disease emerged recently when it was noted that many cancers, while still
very serious could be manageable chronic diseases with ongoing surveillance
and/or treatment. While this vision has not yet become a reality for most forms of
cancer, the past 10–20 years have brought about a marked acceleration in advance
toward this goal. In this regard some types of metastatic breast cancer have become
manageable over the long term, perhaps most famously with tamoxifen, which can
slow or stop malignant cell growth in many women with estrogen-dependent cancer
by blocking hormone receptors on tumor cells. Moreover, a new class of aromatase
(CYP19) inhibitors that target estrogen production was developed, which seem to
provide better results than tamoxifen [127]. I3C can also inhibit the expression of
aromatase as well as CYP isoforms involved in estrogen metabolism. Therefore,
I3C, as well as its condensation product DIM, appear to be a promising agent for
the prevention or recurrence of human tumors, particularly hormone-dependent
cancers. There are also data suggesting that I3C or DIM might also be useful for
prevention or recurrence of cardiovascular diseases, diabetes, or recurrent respira-
tory papillomatosis, as well as obesity.

6.4.1 The Role of I3C in Cancer Controlling

It is almost 40 years after Lee Wattenberg and William Loub [122] for the first time
reported that I3C inhibited chemically induced breast and forestomach neoplasia in
rodents. Since then the antitumor activity of dietary I3C has been widely studied
and the inhibition of the development of other cancer types, including liver [95],
lung [63], and prostate [110] have been demonstrated.
Breast cancer is the most common and the leading cause of cancer mortality
among women worldwide. The preventive efficacy of I3C on mammary carcinoma
first observed in animal models, was confirmed in many mechanistic studies in cell
cultures and was supported by epidemiological studies with cruciferous vegetables
and their extracts or juices [117, 120].
6 Indole-3-Carbinol and Its Role in Chronic Diseases 139

One of the most important risk factor of breast tumors are estrogens, which are
classified as carcinogenic in humans [58]. These steroid hormones may contribute
to breast cancer development in two ways: (i) acting as promoters by stimulating
cell proliferation, (ii) inducing genotoxicity through the reaction of their active
metabolites with DNA thus acting as tumor initiators.
Experimental studies in vitro in breast epithelial cell lines showed that I3C, DIM,
and cabbage juices induce CYP450 genes CYP1A1, 1A2, 1B1 encoding the key
enzymes of estrogen catabolism. The profile of metabolites was in favor to
2-hydroxyestrogens being noncarcinogenic in comparison to estradiol and
4-hydroxy derivatives [80, 114]. This anti-estrogenic activity of I3C could be
explained by the induction of AhR receptor showed in other studies [114].
Moreover, I3C, DIM, and cabbage juices are capable of upregulating phase II
detoxifying enzymes, including GST and NQO in breast cancer cell lines [114,
120]. Upregulation of GSTs and NQO1 by I3C was correlated with increased levels
of Nrf2, in benign MCF7 and aggressive MDA-MB-231 breast cancer cell lines.
Thus, it may be assumed that I3C protects against estrogen-associated carcino-
genesis by removal of the genotoxic metabolites of estrogens.
Simultaneously, I3C and DIM influenced in situ production of estrogens in
breast epithelial MCF7 cancer cells by reducing the expression of aromatase
(CYP19), the enzyme that synthesizes estrogens by converting C19 androgens into
aromatic C18 estrogenic steroids [80]. Several studies have shown that there is an
overexpression of aromatase gene in breast cancer tissue [82]. Interestingly, the
potential of I3C to reduce estrogenic activity in the breast cancer cells was con-
firmed by other mechanisms, particularly via decreasing the AKR1C3 expression
mentioned in the previous section. In the mammary gland where enzyme converts
androstenedione to testosterone––one of the aromatase substrates [92, 101].
Besides the interference with estrogens metabolism pathways, I3C also affects
DNA repair, the cell cycle progression and apoptosis in breast cancer cell lines. In
this regard it was shown that I3C induces BRCA1 expression and that both I3C and
BRCA1 inhibited estrogen (E2)-stimulated ERa activity in human breast cancer
cells [44]. BRCA1 is DNA repair factor involved in repair of DNA double-strand
breaks. BRCA1gene expression is reduced or completely silenced in a significant
proportion of sporadic breast cancer because of hypermethylation of the gene
promoter [106, 126]. Thus, I3C-induced BRCA1 expression and inhibition of
estrogen-stimulated ERa activity by I3C and BRCA1 showed by some studies
could be one of the antitumor activity of the indole [44].
Several lines of evidence suggest I3C ability to arrest cell cycle in breast cancer
cells. In this regard I3C was reported to inhibit CDK2 activity in breast cancer
MCF7 cell line [48]. Moreover, both I3C and DIM upregulated CDK inhibitors p21
and p27, although in a very high concentration of 200 µM. Activity of this protein
is especially critical during the G1 to S phase transition. Consequently, the ability to
arrest G1 phase by I3C was shown [30]. Other studies suggest the p53 phospho-
rylation by I3C leading to release p53 and inducing the p21 CDK inhibition and G1
cell cycle arrest [18, 86]. Importantly, treatment with I3C and tamoxifen ablated
expression of the phosphorylated retinoblastoma protein (Rb), an endogenous
140 B. Licznerska and W. Baer-Dubowska

substrate for the G1 CDKs, whereas either agent alone only partially inhibited
endogenous Rb phosphorylation [31]. Several studies showed that I3C and its
derivatives are potent inducers of apoptosis in both ER-positive and ER-negative
breast cancer cells[30–32, 50, 80, 103]. Estrogens, particularly estradiol have been
also implicated as a cofactors in human papillomavirus (HPV)-mediated cervical
cancer, both in animal models and in women using oral contraceptives [72].
Interestingly, it was found that estradiol protects cervical cancer cells treated with
DNA-damaging agents such as UVB, mitomycin-C, and cisplatin, from apoptotic
death. I3C was able to overcome the anti-apoptotic effect of estradiol but only in
higher concentrations. Treatment with I3C resulted in loss of the survival protein
Bcl-2. However, the amount of apoptosis versus survival and the level of Bcl-2
depended on the I3C/estradiol ratio [22]. In HPV16-transgenic mice, which develop
cervical cancer after chronic estradiol exposure, apoptotic cells were detected in
cervical epitheliumonly in mice exposed to estradiol and fed on I3C [20].
Experiments in which cervical cancer HeLa and SiHa cell lines were used,
demonstrated that DIM also exerts antitumor effects on these cells through its
anti-proliferative and pro-apoptotic roles, especially for SiHa cells. The molecular
mechanism for these effects may be related to its regulatory effects on MAPK and
PI3K pathway and apoptosis proteins. Thus, DIM may be considered a preventive
and therapeutic agent against cervical cancer [135]. The ability of inhibiting
spontaneous occurrence of endometrial adenocarcinoma and preneoplastic lesions
by I3C was also demonstrated in female Donryu rats. It was suggested that this
effect was due to induction by I3C estradiol 2-hydroxylation [68]. On the other
hand, promotion of endometrial adenocarcinoma in same strains of rats initiated
with N-ethyl-N’-nitro-N-nitrosoguanidine by I3C was described [133]. DIM was
also found to have a potent cytostatic effect in cultured human Ishikawa endome-
trial cancer cells. This effect was related to the stimulation of TGF-a expression and
activation of TGF-a signal transduction pathway [75].
Another hormone-dependent cancer, which might be affected by I3C, is prostate
cancer, one the most prevalent malignancy in men worldwide and the second
leading cause of male death in Western countries [16]. Androgens play a critical
role in prostate cancer cells growth and survival. Androgens bind to the androgen
receptor (AR), a steroid nuclear receptor, which is translocated into the nucleus and
binds to AREs in the promoter regions of target genes to induce cell proliferation
and apoptosis. Approximately 80–90 % of prostate cancers are dependent on
androgen at initial diagnosis, and endocrine therapy of prostate cancer is directed
toward the reduction of plasma androgens and inhibition of AR [37, 54]. It was
demonstrated that both I3C and DIM are able to downregulate AR signaling [74],
but only DIM was shown to be a strong antagonist of AR and inhibitor of its
translocation to the nucleus [30, 74].
Similarly as in the case of breast cancer cells, I3C and its derivatives also affect
cell cycle progression and induce apoptosis. In this regard, cell cycle arrest at G1
checkpoint in different human prostate carcinoma cell lines by I3C and DIM was
described [2]. In LNCaP prostate cancer cells I3C selectively inhibited the
expression of CDK6 protein and transcripts and stimulated the production of the
6 Indole-3-Carbinol and Its Role in Chronic Diseases 141

p16 CDK inhibitor. In vitro protein kinase assays revealed inhibition by I3C CDK2
enzymatic activity and the relatively minor downregulation of CDK4 enzymatic
activity [134].
In PC-3 cell line induction of G1 cell cycle arrest by I3C due to the upregulation
of p21(WAF1) and p27(Kip1) CDK inhibitors, followed by their association with
cyclin D1 and E and downregulation of CDK6 protein kinase levels and activity
was suggested. In addition, I3C inhibited the hyperphosphorylation of the
retinoblastoma (Rb) protein in PC-3 cells. Induction of apoptosis was also observed
in this cell line when treated with I3C. Thus, it was suggested that I3C inhibits the
growth of PC-3 prostate cancer cells by inducing G1 cell cycle arrest leading to
apoptosis, and regulates the expression of apoptosis-related genes [23]. Further
studies showed that I3C-induced apoptosis is partly mediated by the inhibition of
Akt activation, resulting in the alterations in the downstream regulatory molecules
of Akt activation in PC-3 cells [24]. In the case of DIM an inhibition of a crosstalk
between Akt and NF-jB [12], leading to cell cycle arrest and induction of apoptosis
was also described. DIM significantly decreased cellular histone deacetylase
HDAC2 protein level in androgen sensitive LNCaP and androgen insensitive PC-3
cell lines [10]. In all these studies a formulated DIM (BR-DIM) with higher
bioavailability was used and was able to induce apoptosis and inhibit cell growth,
angiogenesis, and invasion of prostate cancer cells [21].
The potential protective activity of I3C and DIM against prostate cancer was
confirmed by microarray analysis, which showed the modulation of the expression
of many genes related to the control of carcinogenesis and cell survival as effect of
indoles treatment of PC-3 cells [76]. It was also demonstrated by several groups that
I3C and DIM may improve the therapeutic effect of conventional chemotherapy of
prostate cancer [44, 71].
Besides the hormone-dependent cancers, both indoles can affect the develop-
ment of some other cancers. In this regard, it was shown that I3C and DIM induced
apoptosis in colorectal cancer cell lines [13, 67, 84]. Interestingly, an effective
inhibition of Akt and inactivation of mTOR was observed as a result of combined
treatment with I3C and genisteinin HT29 colon cancer cells, leading to induction of
apoptosis and autophagy [93].
Anti-carcinogenic activity of I3C was demonstrated in carcinogen-induced lung
cancer in mice [63, 64]. Anti-proliferative effects of I3C and DIM in human
bronchial epithelial cells (HBEC) and A549 adenocarcinomic human alveolar basal
epithelial cells related to marked reductions in the activation of Akt, extracellular
signal-regulated kinase and NF-jB were also described [65].
Moreover, upregulation of several miRNAs induced by chemical carcinogen was
reversed by I3C in mice and rats lung tumors [46, 59].
The signal transducer and activator of transcription 3 (STAT3) is a latent tran-
scription factor required in proliferation and differentiation. The constitutive acti-
vation of STAT3 in human pancreatic carcinoma specimens but not in normal
tissues was shown. Activation of STAT3 was also found in pancreatic tumor cell
lines and was inhibited by I3C although in relatively high concentration (10 µM)
along with induction of apoptosis [79]. Apoptosis in pancreatic cancer cells was
142 B. Licznerska and W. Baer-Dubowska

also induced by DIM as a result of endoplasmic reticulum stress-dependent

upregulation of death receptor 5 [1]. More recent studies showed downregulation of
miRNA-21 and miRNA-221 as a result of I3C or DIM treatment of pancreatic
cancer cells. As upregulation of these miRNAs is characteristic for more aggressive
pancreas cancer, it was suggested that combination of I3C or DIM with conven-
tional chemotherapeutics may increase the chemosensitivity to certain drugs in
resistant pancreatic cancer cells [98, 111].
There are also reports showing the anti-proliferative effect, G1 cell cycle and
induction of apoptosis in thyroid cancer cells by I3C and DIM [116]. However,
earlier reports indicated the enhancement of thyroid gland neoplastic development
by I3C in rat medium-term multi-organ carcinogenesis model [66]. UVB-induced
mouse skin tumors were reduced in mice fed on I3C [29].
Importantly it was also shown that I3C may overcome multiple drug resistance
by downregulation of MDR1-expression in murine melanoma cells and leukemia
cells [7, 8, 28].
These observations further support the possible application of I3C or DIM as
potential adjuvant therapeutics in conventional chemotherapy of several cancers.
Finally, it must be pointed out that although I3C was shown to have
anti-carcinogenic activity in various animal models, at the same time animals
studies have also shown a tumor-promoting activity, when animals were exposed to
I3C after exposure to carcinogens [131, 133]. This aspect has to be clarified in
long-term studies.

6.4.2 Cardiovascular Diseases

Cardiovascular diseases still remain the primary cause of death worldwide. One of
the proposed approaches to reduce the high global incidence is the consumption of
vegetables and fruits containing biologically active components or phytochemical
supplements. Although the most attention was paid to resveratrol, components of
cruciferous vegetables, particularly Brassica oleracea, were also considered a
potential dietary phytochemicals reducing risk of CVDs [97].
In this regard hypocholesterolemic properties of I3C were reported in mice
provided with cholesterol-supplemented diet to which I3C were added. Since
in vitro experiments revealed that I3C and its condensation products effectively
inhibited the enzyme acyl-CoA:cholesterol acyltransferase (ACAT), which is
responsible for the conversion of free cholesterol to the cholesteryl ester, the
hypocholesterolemic effect of I3C in mice was likely mediated by the inhibition of
ACAT [42]. Such mechanism was further confirmed in HepG2 cells. As a result of
treatment with I3C the decreased cholesteryl ester synthesis was associated with
significantly decreased ACAT gene expression and activity [85].
Moreover, antiplatelet and antithrombotic activity of I3C was shown in in vitro
and in vivo studies. I3C significantly inhibited collagen-induced platelet aggrega-
tion in human platelet-rich plasma and suppressed the death of mice with
6 Indole-3-Carbinol and Its Role in Chronic Diseases 143

pulmonary thrombosis induced by intravenous injection of collagen and epi-

nephrine [99].
The protective activity of I3C in heart failure and vascular proliferative disease
was also reported. In this regard, it was shown that I3C can suppress the prolif-
eration of cultured vascular smooth muscle cells and neointima formation in a
carotid injury model via the Akt/GSK3b pathway [51]. Vascular smooth muscle
cells are the principal cell types involved in the pathogenesis of atheriosclerosis and
restenosis after percutaneous coronary intervention [43]. Thus, it was suggested that
I3C may be a part of new therapeutic strategy for vascular proliferative diseases as
well as heart failure. The latter suggestion was supported by the results of the
studies using aortic banding (AB) mouse model, which showed that I3C prevented
and reversed cardiac remodeling induced by AB. This effect was mediated by
AMPK-a and extracelular signal-regulated kinases 1/2 (ERK1/2) [36].
Since AMPK acts as important energy sensor, attenuation of cardiac remodeling in
mice was associated with improved myocardial energy metabolism [36].

6.4.3 Obesity and Diabetes

Chronic inflammatory disease initiated in adipose tissue might lead to

obesity-related insulin resistance and may contribute to an increased risk of diabetes
[132]. It might be assumed that anti-inflammatory phytochemicals may protect
against both diseases. Thus, I3C was also proposed as a potential preventive agent
against obesity and metabolic disorders. In this regard, I3C treatment in
diet-induced obesity (DIO) mice model decreased body weight and fat accumula-
tion and infiltrated macrophages in epididymal adipose tissue. These effects were
associated with improved glucose tolerance and with modulated expression of
adipokines and lipogenic-associated gene products, including acetyl coenzyme A
carboxylase and peroxisome proliferator-activated receptor-c (PPARc) [19]. The
reduced level of inflammatory biomarkers was also confirmed in co-culture of
adipocytes with macrophages treated with I3C [19].
I3C was also capable to normalize tissue expression of genes related to ther-
mogenesis upregulated by high-fat diet, namely uncoupling proteins 1 and 3,
PPARa, PPARc coactivator 1a [26]. The observed improvement of adipogenesis
by I3C could be due to activation of sirtuine SIRT1 [27]. These findings suggest
that I3C has a potential benefit in preventing obesity and metabolic disorders, and
the action for I3C in vivo may involve multiple mechanisms including decreased
adipogenesis and inflammation, along with activated thermogenesis.
Little is known about the possible modulation of different types of diabetes by
I3C. Nevertheless, in recent studies with the genetically modified mice (C57BL/6J
mice) that closely simulated the metabolic abnormalities of the human disease after
the administration of high-fat diet, both I3C and DIM showed a positive modulation
of glucose, insulin, hemoglobin and glycated hemoglobin levels. In the same time a
decreased levels of different mediators of oxidative stress were noticed, including
144 B. Licznerska and W. Baer-Dubowska

thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LOOH) and

conjugated dienes. Simultaneously, in this diabetic mouse model increased levels of
antioxidant enzymes and small molecules (SOD, CAT, GPx, vitamin C, vitamin E,
GSH) were demonstrated. Interestingly, the antioxidant action was comparable to
that of metformin, a standard drug in diabetes 2 treatment [60].

6.5 Biological Activities of Indole-3-Carbinol in Animal

Models

Animal models played a crucial role in discovering the cancer chemopreventive

activity of cruciferous plants. Initially rodent chemical carcinogenesis models were
used to assess anti-carcinogenic activity of minor dietary components, including
I3C. Currently genetically modified animals, mentioned in the previous sections,
allow to assess detailed mechanisms of their biological activity.
In the very first experiments of Wattenberg and his co-workers, benzo[a]pyrene
induced model of lung and forestomach cancer in mice and dimethylbenz[a]an-
thracene induced breast neoplasia in rats were used. In these models I3C when
given prior to carcinogen inhibited the formation of tumors [122]. This effect was
linked to modulation of phase I enzymatic systems, namely cytochrome P450
dependent monooxygenases, involved in carcinogens activation [123, 124]. Later,
several studies using animals carcinogenesis model of liver, colon, and tongue
confirmed the anti-initiating activity of I3C. However, these studies have also
provided evidence for promotional activity of I3C. For example, whereas I3C
pretreatment and co-treatment with liver carcinogen aflatoxin B1 (AFB1) strongly
inhibited AFB1 initiated hepatocarcinogenesis, posttreatment with I3C was strongly
promotional [35].
The modulation of cytochromes P450 is also linked with potential protection
against breast cancer. On the other hand, the same mechanism is probably
responsible for uterine-induced cancer via upregulation of CYP1B1 and increased
precancerous 4-hydroxyestrogen concentration [133]. The increase of carcinogenic
4-hydroxyestrogen following oral administration of I3C were documented also by
the other studies [56]; reviewed in [15]. These observations led to conclusion that
DIM showing higher bioavailability and reducing 4-hydroxyestrogen production
should be recommended as an alternative to I3C in potential chemopreventive
supplementation [15]. More recent studies have confirmed this suggestion that DIM
was more effective in prevention prostate cancer in the transgenic adenocarcinoma
mouse prostate (TRAMP) mice model then I3C [25].
Nevertheless, the more recent studies using “traditional” mouse models or
transgenic animals documented that I3C has been responsible for a decrease of
incidences of carcinogen-induced lung cancer [64, 88, 102], cervical cancer in HPV
gene transgenic mice [61], and UV-induced skin cancer [29].
6 Indole-3-Carbinol and Its Role in Chronic Diseases 145

Moreover, it was shown that in rats bearing the 13762 mammary carcinoma,
addition of I3C to the diet for 6 days prior to antitumor drug ET-743 (trabectidin)
administration almost completely abolished manifestations of hepatotoxicity [41].
These observation further supports the concept that I3C or DIM protecting against
specific cancer may be used in adjuvant therapy to overcome side-effects of con-
ventional therapy.
Specific rodent models like mouse carotid artery injury were developed and used
to assess I3C or DIM protection against cardiovascular diseases, obesity, or dia-
betes. As it was described in previous section, generally the results of these studies
suggest that I3C has a potential benefit in preventing obesity and metabolic dis-
orders, and the action of I3C in vivo may involve multiple mechanisms including
decreased adipogenesis and inflammation, along with activated thermogenesis.

6.6 Biological Activities of Indole-3-Carbinol in Humans

Promising results of the most studies obtained in human cancer cell lines and in
animal models prompted the clinical trials dietary intervention studies to evaluate
the effect of I3C or DIM in risk group of patients or/and volunteers. A major focus
of these trials has been on modulation of hormones metabolism. The urinary
estrogen metabolite ratio of 2-hydroxyesterone to 16a-hydroxyestrone was used in
most of the trials as the surrogate endpoint biomarker.
The validity of this endpoint biomarker was confirmed in the early randomized
clinical trials [14] in which 20 healthy subjects received 400 mg/day of I3C for
3 months. In most of the enrolled subjects I3C increased the 2-hydroxyestrone to
estriol (a precursor of 16a-hydroxyestrone) ratio in sustained manner without
detectible side-effects, although some individuals were resistant to such change. In
another trial women at increased risk for breast cancer were administered with
different doses (range 50–400 mg/day) of I3C for 4 weeks. The results of this study
suggested that the minimum effective dose schedule of 300 mg/day is optimal for
breast cancer prevention, although should be confirmed by long-term breast cancer
prevention trial [128].
In subsequent studies by Bradlow group [91] urine samples were collected from
healthy subjects before and after oral ingestion of 6–7 mg/kg per day for 1 week (7
men) or 2 months (10 women). Analysis of 13 estrogen profiles supported the
hypothesis that I3C induces estrogen 2-hydroxylation resulting in decreased con-
centrations of metabolites known to activate the estrogen receptor and suggested
that I3C may have chemopreventive activity against breast cancer in humans. Later,
phase I trial with women with a high-risk breast cancer were enrolled, subjects
ingested 400 mg I3C daily for 4 weeks followed by a 4 week period of 800 mg I3C
daily [105]. The maximal ratio increase of the urinary 2-hydroxyestrone to
16a-hydroxyestrone was observed with the 400 mg daily dose of I3C, with no
further increase found at 800 mg daily. Beside confirmation of the optimal dose of
146 B. Licznerska and W. Baer-Dubowska

I3C, these studies showed the induction of CYP1A2 which was mirrored by
increase of 2-hydroxyestrone to 16a-hydroxyestrone ratio, and GST.
Cumulative evidence on conversion of I3C to DIM in cell culture, peritoneal and
oral use as well as substantial direct activity seen with DIM led to conclusion that
there is no longer the case for considering I3C to be directly active, and rather DIM
should be considered as a chemopreventive compound of choice [15]. A pilot study
on the effect of BR-DIM on urinary hormone metabolites in postmenopausal
women with a history of early-stage breast cancer showed a significant increase in
levels of 2-hydroxyestrone as result of treatment with only 108 mg DIM/day for
30 days, however, nonsignificant increase (1.46–2.14) of 2-hydroxyestrone to 16a -
hydroxyestrone was noted [33]. In another study cohorts of 3–6 patients
castrate-resistant, non-metastatic prostate cancer received escalating oral doses
twice daily of BR-DIM 75 mg, then 150, 225, and 300 mg. Based on the results of
this trial 225 mg BR-DIM dose twice daily was recommended for phase II trial.
However, modest efficacy of DIM was demonstrated [53].
Cervical intraepithelial neoplasia (CIN) is a precancerous lesion of cervix. When
patients with biopsy proven CIN grade II or III were treated orally with 200, or
400 mg/day of I3C for 12 weeks 50 % of them had complete regression based on
their 12-week biopsy. Moreover, 2-hydroxyestrone to 16a-hydroxyestrone ratio
have changed in a dose-dependent manner [11]. The significant improvement in
confirmed CIN I or II grade was also observed as a result of oral treatment with
2 mg/kg/day of DIM for 12 weeks. Moreover, at median follow-up of 6 months
there was no statistically significant difference in any of the measured outcome
between the DIM and placebo group [40].
Since the incidence of thyroid cancer is 4–5 times higher in women than in men,
estrogens were suggested to contribute the pathogenesis of thyroid proliferative
disease (TPD). In limited (7 patients) phase I clinical trial patients with TPD were
administered with 300 mg of DIM per day for 14 days. DIM was detectable in
thyroid tissue, and the ratio of 2-hydroxyestrone to 16a-hydroxyestrone was
increased. These results suggested that DIM can manifest the anti-estrogenic
activity in situ to modulate TPD [104].
Although major focus of cancer prevention clinical trials of I3C or DIM has been
concentrated on chemoprevention of hormone-dependent cancers, there were also
clinical trials performed in order to evaluate indoles effect on pulmonary cancers. In
this regard in phase I clinical trial patients with recurrent respiratory papillomatosis
(RRP) were treated orally with I3C and had minimum follow-up of 8 months.
Thirty-three percent of the study patients had a cession of their papilloma growth
and had not required surgery since the start of the study [107]. Subsequent
long-term clinical trial performed by the same research group confirmed the pre-
liminary observation indicating that I3C may be a treatment option for RRP [108].
The case of successful use of intralesional and intravenous cidofovir in association
with I3C in 8-year-old girl with pulmonary papillomatosis was also reported [38].
As it was mentioned in the previous sections of this chapter, recently a large
amount of evidence has demonstrated that epigenetic alterations, such as DNA
6 Indole-3-Carbinol and Its Role in Chronic Diseases 147

methylation, histone modifications, and non-coding miRNAs consistently con-

tribute to carcinogenesis, and constituents in the diet, including dietary glucosi-
nolate derivatives, have the potential to alter a number of these epigenetic events
[46]. Different studies on cancer also have shown that miRNAs interact with genes
in many different cellular pathways, displaying a differential gene expression profile
between normal and tumor tissues and between tumor types [17]. Interestingly,
interventions including BR-DIM in prostate cancer patients prior to radical
prostatectomy showed re-expression of miR-34a, which was consistent with
decreased expression of androgen receptor, prostate specific antigen (PSA), and
Notch-1 in tissue specimens [70]. These results suggest that BR-DIM could be
useful for the inactivation of androgen receptor, critically important during the
development and progression of prostate cancer and thus its treatment.
Thus far, seven clinical studies have been registered using I3C and twelve using
DIM (www.clinicaltrials.gov; accessed Dec 26, 2015). Four studies registered for
I3C treatment have been completed for patients with prostate and breast cancers and
one dietary intervention for healthy participants targeting unspecified adult solid
tumors. One trial aiming at I3C effects on estrogen metabolism in obese volunteers
had to be terminated because of slow accrual in the high BMI group. Among twelve
studies registered for DIM, six have been completed for patients with prostate,
breast, and cervical cancers as well as healthy volunteers. Trial aiming at new
therapy of laryngeal papilloma in children was terminated because of lack of suf-
ficient enrollment. Although the results of these trials have not been published yet,
they assure the further extensive prospective studies on chemopreventive and/or
chemotherapeutic potential of I3C and its condensation product.

6.7 Summary and Conclusions

It is well known that in populations which consume higher amounts of cruciferous

vegetables lower incidence rate of cancer occurs or improved biochemical
parameters, such as decreased oxidative stress are noticed [46, 117, 118, 121].
These effects are in part due to the biological activity of I3C and its condensation
products, particularly DIM.
A wide range of cellular pathways are regulated by both indoles. Thus, many
additional targets for indoles could be identified in the future using in vitro cell
cultures and in vivo transgenic animal models and explain a unique
anti-inflammatory and endocrine modulating activity of I3C. Although most of
current data support the role of I3C and DIM in prevention of hormone-dependent
cancers, it seems that their application in prevention of the other cancer as well as
cardiovascular diseases, obesity, and diabetes reduction is also possible.
Experimental in vitro and in vivo studies and clinical trials performed so far,
showed that I3C is a rather safe dietary supplement. However, since the long-term
effects of I3C supplementation in humans are still not clear and due to some
contradictory effects of I3C in animal models, the general use of I3C and DIM
148 B. Licznerska and W. Baer-Dubowska

supplements should be restricted until potential risks and benefits are better char-
acterized. Taking into consideration higher activity of DIM, particularly in BR
form, in comparison with I3C in term of potency and time required to obtain
the effect, this I3C dimer might be a better alternative as chemopreventive sup-
plement. Important aspect of possible clinical application of both indoles is their
drug and radio-sensitization. Emerging new technologies allowing deeper inside in
the mechanism of these glucosinolate derivatives activity should help to better
explore this aspect.

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Chapter 7
Sanguinarine and Its Role in Chronic
Diseases

Pritha Basu and Gopinatha Suresh Kumar

Abstract The use of natural products derived from plants as medicines precedes
even the recorded human history. In the past few years there were renewed interests
in developing natural compounds and understanding their target specificity for drug
development for many devastating human diseases. This has been possible due to
remarkable advancements in the development of sensitive chemistry and biology
tools. Sanguinarine is a benzophenanthridine alkaloid derived from rhizomes of the
plant species Sanguinaria canadensis. The alkaloid can exist in the cationic imi-
nium and neutral alkanolamine forms. Sanguinarine is an excellent DNA and RNA
intercalator where only the iminium ion binds. Both forms of the alkaloid, however,
shows binding to functional proteins like serum albumins, lysozyme and hemo-
globin. The molecule is endowed with remarkable biological activities and large
number of studies on its various activities has been published potentiating its
development as a therapeutic agent particularly for chronic human diseases like
cancer, asthma, etc. In this article, we review the properties of this natural alkaloid,
and its diverse medicinal applications in relation to how it modulates cell death
signaling pathways and induce apoptosis through different ways, its utility as a
therapeutic agent for chronic diseases and its biological effects in animal and human
models. These data may be useful to understand the therapeutic potential of this
important and highly abundant alkaloid that may aid in the development of
sanguinarine-based therapeutic agents with high efficacy and specificity.

Keywords Benzophenanthridine Sanguinarine Nucleic acid binding property



Anticancer Antimicrobial

P. Basu
G.S. Kumar (&)

Biophysical Chemistry Laboratory, Organic and Medicinal Chemistry Division,
CSIR- Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road,
Kolkata 700032, India
e-mail: [email protected]; [email protected]

© Springer International Publishing Switzerland 2016 155

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1_7
156 P. Basu and G.S. Kumar

Fig. 7.1 Molecular structure

of sanguinarine

7.1 Introduction

Sanguinarine[13-methyl[1,3]benzodioxolo[5,6-c]-1,3-dioxolo[4,5-i]phenanthridinium]
(Fig. 7.1) is the well-known member of the relatively small group of quaternary benzo
[c]phenanthridine [QBA] alkaloids. Sanguinaria canadensis, also known as bloodroot,
is a perennial herbaceous flowering plant of the papaveriaceae family. Bloodroot pro-
duces primarily the toxic alkaloid sanguinarine that is largely stored in the rhizome of
the plant. From a medicinal perspective, QBAs in general and sanguinarine in particular,
have many important properties. They display antimicrobial, antifungal and
anti-inflammatory effects in addition to their putative anticancer activity that is widely
studied. The molecular action of sanguinarine in expressing its biological activity has
been a subject of intense debate; it has been shown to interact with many targets, like
nucleic acids, proteins and microtubules, and modify the activities of a wide variety of
enzymes. This review summarizes the current state of knowledge on the properties
of sanguinarine that are important for their potential use in chronic diseases.

7.2 Physicochemical Properties of Sanguinarine

Sanguinarine occurs either as chloride or sulfate crystalline salts and both are
orange-red colored. It is sparingly soluble in aqueous conditions but highly soluble
in many organic solvents. Some of the physical properties of sanguinarine are
summarized in Table 7.1.
One of the remarkable properties of sanguinarine and other QBAs is their ability
to exhibit a pH dependent structural equilibrium between the quaternary iminium
form and the 6-hydroxydihydro derivative or the alkanolamine form (Scheme 7.1).
The reversible, pH dependent equilibrium between the charged iminium (SGI) and
the neutral alkanolamine (SGA) forms in aqueous solution may be represented as
follows [76]

SG þ þ OH $ SGOH

The acid–base structural equilibrium of sanguinarine was characterized first by

Maiti and colleagues primarily by spectroscopy techniques [56] and confirmed
subsequently by others, and was characterized by a pKa value around 8.06 [1, 33,
7 Sanguinarine and Its Role in Chronic Diseases 157

Table 7.1 Physiochemical properties of sanguinarine

Empirical formula C20H14O4N+
Chemical name 13-Methyl-[1,3]-benzodioxolo[5,6-c]-
1,3-dioxolo[4,5-i]phenanthridin-13-ium
Molecular weight (ion) 332.33
Polar surface area 40.8 Å2
Crystal color Orange-red
Solubility Water: slightly soluble <0.3 mg mL−1
Melting point (°C) 278–279 °C
Optical rotation [α]D (solvent) 0 °C (H2O)
Peak position of absorption spectrum (nm) 273, 327, 400, and 470 nm
(In aqueous buffer of pH 6.0)
Molar absorption coefficient (ɛ) (M−1 cm−1) 30,700 at 327 nm
Peak position in fluorescence spectrum Strong, at 570 nm
(nm) (In aqueous buffer of pH 6.0)
pKa 8.06
IC50 or LD50 value (in mice) 19.4 mg kg−1 of body weight

Scheme 7.1 pH dependent structural changes of SGI and SGA forms

36, 39, 42]. These two forms have characteristic absorbance and fluorescence
spectra which are presented in Fig. 7.2a, b. Thus, in aqueous solution at physio-
logical pH both forms of the alkaloid are present. The SGI is unsaturated and
planar; the SGA has a tilted structure and is essentially nonplanar. The aqueous
solubility of the SGI is much higher than that of the SGA. The latter (SGA) form
can penetrate the cell membrane increasing its cellular availability. Inside the cell it
may be converted partially to the SGI form influenced by pH and other factors. Due
to the conjugated aromatic nature of its chromophore sanguinarine has strong
spectral bands in the UV–vis region and also exhibits intense fluorescence
(Fig. 7.2b). The molecule is, therefore, sensitive to light and can cause adverse
phototoxic reactions in the presence of light. The SGA form is known to undergo
photo oxidation at the C6 carbon atom to oxysanguinarine in alkaline solutions
(Scheme 7.2) [75]. The photo oxidation of sanguinarine was found to be acceler-
ated in the presence of Rose Bengal suggesting the role of singlet molecular oxygen
in the reaction mechanism [28].
158 P. Basu and G.S. Kumar

Fig. 7.2 Characteristic

absorption (panel A) and
fluorescence (panel B) spectra
of SGI and SGA forms

The toxicity of sanguinarine has been suggested to be due to its DNA interca-
lation, inhibition of ion pumps and several thiol dependent proteins, and interaction
with cyto skeletal components [24, 86]. Sanguinarine can kill animal cells through a
variety of mechanisms. Epidemic dropsy is a form of edema of extremities that
results from ingesting argemone oil that contains sanguinarine [73]. Application of
sanguinarine to skin may result in tissue damage because of escharotic effect.
Phototoxic effect of sanguinarine against mosquito larvae has also been reported
[67]. Production of singlet oxygen by sanguinarine is known [6, 54]. Phototoxic
action of sanguinarine has also been suggested to be due to the production of H2O2
[6, 78]. Reactive oxygen species (ROS) such as superoxide anions (O2−) or
7 Sanguinarine and Its Role in Chronic Diseases 159

Scheme 7.2 Generation of oxysanguinarine from SGA form in the presence of oxygen by
photochemical process. Reprinted from Suresh Kumar et al. 1997 [75] with permission. Copyright
Elsevier Science S. A

hydrogen peroxide (H2O2) have also been found to be generated in

sanguinarine-treated cells [12, 21, 37, 59]. Recent studies have also suggested that
sanguinarine inhibited the growth cancer cells and induced their apoptosis through
the generation of free radicals [29, 30].
Sanguinarine binds to DNA and RNA in vitro and forms strong intercalation
complexes [15, 33, 34, 36, 57, 61, 71, 76]. It has also been proposed that the SGI
form intercalates to DNA and RNA. The SGA form does not bind to DNA or RNA
but in the presence of large concentration of DNA or RNA the SGA form gets
converted to SGI resulting in the binding of the latter [33, 36, 55]. The crystal
structure of sanguinarine-DNA oligomer complex has been solved recently [25]
(Fig. 7.3) confirming the intercalation model first proposed by Maiti’s group [57].
Interestingly both SGI and SGA forms bind to functional proteins like serum
albumins, hemoglobin and lysozyme [31, 33, 35, 36, 40]. Kundu and coworkers
showed that sanguinarine binds with core histones and induces chromatin aggre-
gation and inhibits important chromatin modifications like acetylation and methy-
lation [70].
160 P. Basu and G.S. Kumar

Fig. 7.3 Sanguinarine molecule intercalated at the interface of two “two molecules” DNA units.
DNA chains color scheme: A = orange, B = yellow, C = green, D = blue. Reprinted from [25]
with permission. Copyright The Royal Society of Chemistry 2011

7.3 Modulation of Cell Signaling Pathways

by Sanguinarine

Sanguinarine has been reported to induce cell cycle arrest and apoptosis in distinct
cancer cells [3, 4, 20, 32, 45, 72, 85]. It modulates multiple signaling pathways
causing inhibition of the initiation of cancer, inducing cell cycle arrest, apoptosis,
and inhibiting metastasis and angiogenesis in a variety of cancer cells [9, 82]
(Fig. 7.4).
One of the first systematic study showing the anticancer effect of sanguinarine
was that of Ahmad et al. [4] who showed that sanguinarine treatment resulted in an
apoptotic death of human epidermoid carcinoma A431 carcinoma cells and the loss
of viability that occurred at much less losses in comparison to normal human
epidermal keratinocytes (NHEKs). The DNA cell cycle analysis revealed that
sanguinarine treatment did not significantly affect the distribution of cells among
the different phases of the cell cycle in A431 cells. It was suggested that the
involvement of the NFĸB pathway may be a mechanism of sanguinarine-mediated
apoptosis [4]. A study by Ding et al. [20] reported that sanguinarine-induced
concentration dependent apoptosis with caspase-3 activation and BCD/oncosis
without caspase-3 activation suggesting two cell death mechanisms. This suggests
7 Sanguinarine and Its Role in Chronic Diseases 161

Fig. 7.4 Schematic diagram showing the multiple cell signaling pathway of action of
sanguinarine in modulating anticancer activity

the effectiveness of sanguinarine against multidrug resistance being a major

obstacle in chemotherapy [20].
Sanguinarine treatment of HaCaT cells was found to inhibit in a dose-dependent
manner the cell proliferation and induce apoptosis; significant cleavage of poly
(ADP-ribose) polymerase occurred in HaCaT cells. A dose-dependent increase in
the level of Bax with a concomitant decrease in Bcl-2 levels and increase in
Bax/Bcl-2 ratio was found. A significant increase in the pro-apoptotic members of
Bcl-2 family proteins, i.e., Bak and Bid was also observed. This was accompanied
by increase in (a) protein expression of cytochrome c and apoptotic
protease-activating factor-1 and (b) activity and protein expression of caspase-3,
caspase-7, caspase-8, and caspase-9. These results showed the involvement of
mitochondrial pathway and Bcl-2 family proteins in sanguinarine-mediated apop-
tosis of immortalized keratiocytes signifying its use in hyper proliferative skin
disorders, including skin cancer [2]. Subsequently, the authors showed that san-
guinarine imparts anti proliferative effects against androgen-responsive (LNCaP)
162 P. Basu and G.S. Kumar

and androgen-unresponsive (DU145) human prostate cancer cells and that this
effect was mediated through deregulation of cell cycle and induction of apoptosis.
The involvement of cyclin kinase inhibitors (cki) and cyclin-dependent kinases
(cdk), i.e., the cki–cyclin–cdk machinery, during cell cycle arrest and apoptosis of
prostate cancer cells by sanguinarine has been suggested [3].
Sanguinarine was found not increase Bax levels in K1735-M2 melanoma cells.
A similar result was obtained in human CEM T-leukemia cells [43]. The interesting
result was that the absence of Bax over-expression occurred with an increase of p53
protein. Matkar et al. [59] demonstrated that sanguinarine-induced death in human
colon cancer cells was p53-independent, although DNA damage was detected in
K1735-M2 cells. The results suggested that mitochondrial depolarization induced
by sanguinarine is associated with nuclear DNA damage, although it may not
exclusively result from pathways activated by the latter event [72].
In vascular smooth muscle cells significant growth inhibition was induced by
sanguinarine as a result of G1-phase cell cycle arrest mediated by induction of
p27KIP1 expression, and resulted in a down-regulation of the expression of cyclins
and cdks. Moreover, sanguinarine-induced inhibition of cell growth appeared to be
linked to activation of Ras/ERK through p27KIP1-mediated G1-phase cell cycle
arrest. Overall, the unexpected effects of sanguinarine treatment in VSMC provided
a theoretical basis for clinical use of therapeutic agents in the treatment of
atherosclerosis [49].
Some researches ascribe the pro-apoptotic properties of sanguinarine to pro-
duction of ROS [29, 30, 43] while others have demonstrated that the effects of
sanguinarine are not accompanied by ROS production [19, 60, 81]. Kim et al. [46]
reported that the mechanism of sanguinarine-induced apoptosis in human breast
cancer MDA-231 cells was due to the generation of ROS. Its ability to directly
interact with glutathione (GSH) was thought to lead to depletion of cellular GSH
and induction of ROS generation [12, 19, 38, 46]. It was found that the quenching
of ROS generation by N-acetyl-L-cysteine (NAC), a scavenger of ROS, reversed
sanguinarine-induced apoptosis effects. It was also found that sanguinarine-induced
rat hepatic stellate T6 cells (HSC-T6 cells) apoptosis was correlated with the
generation of increased ROS, leading to decrease in the mitochondrial membrane
potential (MMP) and the down-regulation of anti-apoptotic protein Bcl-2 [88].
Sanguinarine is a selective inhibitor of mitogen-activated protein kinase phos-
phatase 1 (MKP-1), which is over expressed in many tumor cells [79]. The dis-
ruption of microtubule assembly dynamics [51], the nucleocytoplasmic trafficking
of cyclin D1 and Topisomerase II [32] and the induction of DNA damage [18]
allosteric activator of AMP-activated protein kinase [14] also contributed, at least in
part to the anticancer effects of sanguinarine [52].
Sanguinarine induces a rapid caspase-dependent cell death in human melanoma
cells; partially involving endoplasmic reticulum and mitochondria mediated
responses. This cell death is dependent on the generation of reactive oxygen spe-
cies, and is not prevented by Bcl-XL over expression. The fact that sanguinarine
induces a very rapid cell death with apoptotic features in melanoma cells, together
with the lack of inhibition by over expressing Bcl-XL, highlight the potent
7 Sanguinarine and Its Role in Chronic Diseases 163

anti-melanoma activity of this isoquinoline alkaloid and suggest its potential in the
treatment of skin cancer [9]. Recently Singh et al. [74] identified a number of
differentially expressed proteins which are important in the signaling pathways
modulated by sanguinarine in its action against pancreatic cancer.

7.4 Role of Sanguinarine in Chronic Diseases

Chronic diseases, like heart disease, COPD, stroke, cancer, respiratory diseases like
asthma and metabolic diseases like diabetes are the leading cause of human mor-
tality. Many natural products in general and sanguinarine in particular possess
potent activities that have been useful in the treatment of these chronic ailments,
and these have also been documented in many studies. Anti proliferative activities
have been demonstrated in cells derived from various human carcinomas as
reviewed in the previous section. Various mechanisms by which sanguinarine
induce apoptosis in cancer cells through various pathways have been described. The
therapeutic potential of sanguinarine in cardiovascular disease related to platelet
aggregation has been reported by Jeng et al. [41, 53]. Sanguinarine has promising
antihypertensive and potent antiplatelet effects; it interferes with renin-angiotensin
system and possibly other blood pressure regulating pathways, and also induces
calcium mobilization, thromboxane and cAMP production (Scheme 7.3). Many
studies on in vitro models are reported to this effect and a recent review summarizes
the antihypertensive activity and cardiovascular properties, including hypotensive,
antiplatelet, and positive inotropic effects of sanguinarine [53]. Computational
bioinformatics analysis has identified sanguinarine as a potential candidate drugs
for the treatment of type 2 diabetes [83] but clinical studies are not yet available on
this aspect.

Scheme 7.3 Schematic diagram showing the role of sanguinarine in different chronic diseases
164 P. Basu and G.S. Kumar

7.5 Biological Activities of Sanguinarine in Animal Models

In the previous sections, we reviewed the effect of sanguinarine in exerting cyto-

toxic effects or inhibiting cell proliferation in several normal and cancer cell lines,
including human gingival fibroblasts [58], rat hepatocytes [16], human promyelo-
cytic leukemia HL-60 cells [80], and human osteosarcoma cells [64]. A number of
studies on the effect of sanguinarine have been performed in animal models and the
results suggested a complex scenario; both adverse as well as beneficial effects of
this alkaloid are reported. Niu et al. [62] examined the anti-inflammatory function
of sanguinarine in animal models of acute and chronic inflammation using
lipopolysaccharide (LPS)-induced murine peritoneal macrophages. Sanguinarine
was found to display significant anti-inflammatory effects both in vitro and in vivo.
It was demonstrated that sanguinarine potently inhibited the expression of
inflammatory mediators and inflammation in general. Furthermore, it was shown
that sanguinarine inhibited the activation of mitogen-activated protein kinase
(MAPK), which altered inflammatory mediator synthesis and release in vitro.
Anti-inflammatory effects through inhibition of LPS-induced tumor necrosis
factor-alpha level, interleukin 6 level, and nitric oxide production in serum was
demonstrated for sanguinarine solid lipid nanoparticles in mice endotoxin shock
model induced by lipopolysaccharide [50].
Epidemiological studies have suggested that the use of sanguinarine as an oral
rinse and in toothpaste effectively suppressed dental plaque formation and reduced
gingival inflammation [27, 48, 66] possibly due to its antioxidant, antimicrobial,
and anti-inflammatory effects [26]. However, long-term use of oral products con-
taining sanguinarine has also been reported to lead to an increased incidence of
leukoplakia of the maxillary vestibule [23]. Sanguinarine has been shown to
enhance the skin tumor promoting activity in mouse skin which may have relevance
to its carcinogenic potential [5].
Chan et al. had showed that sanguinarine triggers apoptotic processes in mouse
blastocysts and impairs early post implantation development in vitro and in vivo
[10]. The effect of sanguinarine on early-stage embryo maturation revealed that
short-term exposure to sanguinarine at the oocyte stage causes a long-term dele-
terious impact on subsequent embryonic development leading to retardation of the
oocyte maturation, and deleterious effects on IVF and subsequent embryonic
development [11].
Chelidonium majus extract in general and sanguinarine in particular were
reported to have hERG potassium channel blocking effect. Therefore, in some
situations where cardiac repolarization reserve is weak it may increase the risk of
potentially fatal ventricular arrhythmias in canines [63].
Sanguinarine has been found to modulate the expression of immune related
genes in goldfish and this to some extent was suggested to affect their ability to
resist bacterial pathogens [69].
Sanguinarine was demonstrated to markedly induce the expression of HO-1
which leads to a neuro protective response in mouse hippocampus-derived neuronal
7 Sanguinarine and Its Role in Chronic Diseases 165

HT22 cells from apoptotic cell death induced by glutamate. Sanguinarine signifi-
cantly attenuated the loss of mitochondrial function and membrane integrity asso-
ciated with glutamate-induced neurotoxicity. Sanguinarine protected against
glutamate-induced neurotoxicity through inhibition of HT22 cell apoptosis. JC-1
staining, a well-established measure of mitochondrial damage, which decreased
after treatment with sanguinarine in glutamate-challenged HT22 cells. In addition,
sanguinarine diminished the intracellular accumulation of ROS and Ca2+.
Non-cytotoxic concentration of sanguinarine led to a marked protection against
glutamate-induced oxidative toxicity in mice through activation of the Nrf2-HO-1
pathway. The antioxidant effect of HO-1 was mainly due to reduction in ROS
production and recovery of mitochondrial decay that promoted a decrease in
glutamate-trigged apoptosis in HT22 cells [65].
An antidepressant-like effect of sanguinarine in rats was reported through
inhibition of mitogen-activated protein kinase phosphatase-1 in rates that enable the
use of sanguinarine as anti depressant drug [13]. Radiation protective efficacy of
sanguinarine in mice models have also been reported [87].
Recently, sanguinarine treatment was shown to result in reduction of cell
migration, a dose-dependent inhibition of cell viability and the induction of cell
death by apoptosis in both human (MDA-MB-231 cells) and mouse (A17 cells)
in vitro models of basal-like breast cancer (BLBC). Oral administration of san-
guinarine reduced the development and growth of A17 transplantable tumors in
FVB syngeneic mice. The suppression was correlated with a concurrent up regu-
lation of p27 and down-regulation of cyclin D1 and with the inhibition of STAT3
activation.
The adverse effect, viz., the epidemic dropsy syndrome, is a well-known toxic
effect on human health linked to development of glaucoma due to consumption of
edible oil adulterated with argemone oil that principally contained sanguinarine.
This involves initiation of oxidative stress (ROS formation leading to lipid per-
oxidation, depletion of GSH and decrease of total antioxidant capacity) and death of
red cells via formation of methemoglobin [7, 8, 17]. The detoxification pathway of
sanguinarine in animals and in man may be by conversion to dihydrosanguinarine
which has been reported in rats [22]. On the other hand, in several European Union
nations QBAs and principally sanguinarine are used as feed additives for Pigs.
Sangrovit®, a phytogenic feed additive derived from the rhizomes of Sanguinaria
canadensis, contains high amounts of sanguinarine. This has been shown to
increase feed intake and feed conversion in growing pigs, which results in improved
growth performance and stimulates anti-inflammatory activity [44]. A study to
detect sanguinarine-induced DNA damage has indicated the absence of any DNA
adducts in pigs fed with sanguinarine. The observation that the animals remained
healthy contradicted the cause of epidemic dropsy syndrome to sanguinarine [47].
Also broilers treated with drinking water containing sanguinarine showed reduced
Salmonella Enteritidis count [68].
166 P. Basu and G.S. Kumar

7.6 Biological Activities of Sanguinarine in Humans

Not many studies have been performed in humans on the effect of sanguinarine as
such, but herbal preparations containing sanguinarine have been used in folk
medicines in small doses for the treatment of many disorders like bronchial prob-
lems, asthma, cough and cold remedies, severe throat infections and heart diseases
[26]. Benefits have also been reported for leprosy and tuberculosis treatment,
antimicrobial treatment for the gastrointestinal system, etc. Sanguinarine, as
described in the above section, is traditionally used in tooth pastes and antiseptic
mouth rinses due to its ability to suppress dental plaque gingival inflammation and
periodontal disease through antimicrobial action and ability to inhibit bacterial
adherences [27, 48, 66]. The potential use of sanguinarine in clinical treatment of
allergic asthma has also been suggested from an analysis of gene expression profile
of asthma patients [84]. Sanguinarine is reported to have hERG potassium channel
blocking effect. Therefore, in some situations where cardiac repolarization reserve
is weak, it may increase the risk of potentially fatal ventricular arrhythmias in
canines [63]. The cardiovascular properties of sanguinarine including hypotensive,
antiplatelet and positive inotropic effects have also been described [53]. In a number
of human tumor cells sanguinarine treatment resulted in boosting of intra cellular
ROS, elevation of mitogen-activated protein kinase p38, triggering caspase 3/7
activation, inhibition of mitogen-activated protein kinase phosphatase 1 (MKP-1),
depletion of glutathione and a host of other activities occurred.

7.7 Adverse Effects of Sanguinarine

There are concerns that the use of sanguinarine may cause leukoplakia in the mouth
and oral dysplastic lesions. The use of sanguinarine and blood root for periodontal
disease abacterially elicited inflammation of the gingival and periodontal tissue is
also not based on adequate scientific support. There are unconfirmed reports that
suggested that sanguinarine may cause sedation, faintness, vertigo, and possibly
impair decision-making and increase response time. Bloodroot was reported to be
used to stimulate menstruation and hence it was not advised to be used during
pregnancy [77].

7.8 Conclusions

Plants containing sanguinarine were used extensively in traditional medicine in

many parts of the world since centuries. Subsequent studies by researchers have
found that this molecule has remarkable medicinal relevance. The high medicinal
potency of sanguinarine is reported to cover wide spectrum of ailments. Its ability to
7 Sanguinarine and Its Role in Chronic Diseases 167

inhibit bacterial adherence due to its antimicrobic properties potentiates its clinical
use in periodontitis treatment and mouth washes. Sanguinarine is used as a dietary
supplementation in animal feeds to reduce amino acid degradation, increase feed
intake, and promote growth. Sanguinarine is a putative anticancer agent that is
reported to arrest the cell cycle and induce apoptosis through various mechanisms
that may include binding to DNA and tubilin and inhibiting many enzymes.
Specifically, results from several studies reviewed above have indicated that san-
guinarine inhibits the proliferation of cancer cells of different origins and apoptosis
induction in different malignant cell lines takes place through the activation of cell
surface receptors, intrinsic cytochrome c release from mitochondria pathways, etc.
In spite of a number of studies the precise mechanisms and the diverse pathways by
which the anticancer effect of this compound is manifested are still not fully
understood. The information reviewed and summarized in this chapter reveals that
sanguinarine is a potential cancer drug candidate and detailed studies including
further vivo experiments are indispensable to elucidate whether administration of
sanguinarine as therapeutics would be safe and effective for the treatment of human
diseases.

Acknowledgments GSK acknowledges the Council of Scientific and Industrial Research (CSIR),
Govt. of India for funding the research activities through network projects NWP0036 and
GenCODE (BSC0123). GSK sincerely thanks Dr. M. Maiti, Ex. Scientist of CSIR-Indian Institute
of Chemical Biology, who initiated and introduced him to the research on sanguinarine at the
Biophysical Chemistry Laboratory, and also all the graduate students who have contributed to the
understanding of the chemistry and biochemistry of the alkaloid. PB is a NET-Senior Research
Fellow of the University Grants Commission, Govt. of India.

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Chapter 8
Piperine and Its Role in Chronic Diseases

Giuseppe Derosa, Pamela Maffioli and Amirhossein Sahebkar

Abstract Alkaloids include a family of naturally occurring chemical compounds

containing mostly basic nitrogen atoms. Piperine is an alkaloid present in black
pepper (Piper nigrum), one of the most widely used spices, in long pepper (Piper
longum), and other Piper species fruits belonging to the family of Piperaceae.
Piperine is responsible for the black pepper distinct biting quality. Piperine has
many pharmacological effects and several health benefits, especially against chronic
diseases, such as reduction of insulin-resistance, anti-inflammatory effects, and
improvement of hepatic steatosis. The aim of this chapter is to summarize the
effects of piperine, alone or in combination with other drugs and phytochemicals, in
chronic diseases.

G. Derosa (&)
P. Maffioli

Department of Internal Medicine and Therapeutics, Fondazione IRCCS Policlinico S. Matteo,
University of Pavia, P.le C. Golgi 2, 27100 Pavia, Italy
e-mail: [email protected]
G. Derosa
P. Maffioli
Center for Prevention, Surveillance, Diagnosis and Treatment of Rare Diseases,
Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
G. Derosa
Center for the Study of Endocrine-Metabolic Pathophysiology and Clinical Research,
University of Pavia, Pavia, Italy
G. Derosa
Laboratory of Molecular Medicine, University of Pavia, Pavia, Italy
P. Maffioli
PhD School in Experimental Medicine, University of Pavia, Pavia, Italy
A. Sahebkar
Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
A. Sahebkar (&)
Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical
Sciences, P.O. Box 91779-48564, Mashhad, Iran
e-mail: [email protected]; [email protected]

© Springer International Publishing Switzerland 2016 173

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1_8
174 G. Derosa et al.

Keywords Anti-depressant effects Bioavailability enhancer Insulin-resistance

Piperine

8.1 Introduction

Alkaloids include a family of naturally occurring chemical compounds containing

mostly basic nitrogen atoms. In addition to carbon, hydrogen, and nitrogen, alka-
loids may also contain oxygen, sulfur, or, rarely, other elements including chlorine,
bromine, and phosphorus [1]. Alkaloids also include some related compounds with
neutral, or even weakly acidic properties.
In the recent decades, some alkaloids have been introduced for use in clinical
practice: berberine, for example, has been used for long in oriental medicine to treat
gastrointestinal infections and diarrhea, but also for its beneficial effects on car-
diovascular system. Lately also piperine, formally known as (2E,4E)-5-
(1,3-benzodioxol-5-yl)-1-(1-piperidinyl)-2,4-pentadien-1-one with the following
formula C17H19NO3 (Fig. 8.1), became very used in clinical practice due to its
beneficial properties. Piperine is an alkaloid present in black pepper (Piper nigrum),
one of the most widely used spices, in long pepper (Piper longum), and other Piper
species fruits belonging to the family o Piperaceae. Piperine is responsible of black
pepper distinct biting quality. Black pepper is used not only in human dietaries, but
also for many other purposes such as medicinal, as a preservative, and in
perfumery.

8.1.1 Pharmacological Properties of Piperine

Current literature reveals a wide spectrum of biological activities of piperine as it

stimulates the digestive enzymes of pancreas, helps in inhibiting oxidation reactions
caused by free radicals and enhances the bioavailability of a number of therapeutic
drugs. Moreover, piperine proved to have anti-inflammatory activities in models of

Fig. 8.1 piperine structure

8 Piperine and Its Role in Chronic Diseases 175

many inflammatory autoimmune diseases, such as inflammatory bowel disease,

arthritis, type 1 diabetes as well as cancer [2]. Piperine activates transient receptor
potential vanilloid type 1 receptor, and modulates GABAA receptors [3, 4]. At
similar levels, piperine inhibits both monoamine oxidases (MAOs), for MAO-A and
MAO-B, respectively [5]. Like other natural compounds containing methylene-
dioxyphenyl substituents, piperine affects cytochrome P450 (CYP) isoforms,
inhibiting CYP3A species, and increasing expression of CYP1A and CYP2B in liver
[6]. It also has a biphasic effect on P-glycoprotein activity [7]. Piperine is also
reported to modulate cell signaling pathways such as NF-kB pathway [8].
Piperine is able to modify supplement and drug metabolism, and it also inhibits
drug detoxifying enzymes. This typically increases bioavailability of any compound
which would normally be attacked by these enzymes. This can be good to favor
positive effects of some compound such as curcumin or resveratrol, or it can be bad
by stopping a protective measure against toxic xenobiotics. In fact, piperine inhibits
glucuronidation, a process happening in the liver which attaches a molecule (glu-
curonide) to drugs to signal for their urinary excretion. This process prevents
excessive levels of drugs and supplements in the body, but sometimes inhibits all
uptake and renders some supplements useless. In the scenario of piperine ingestion,
excretion of supplements is hindered and certain drugs and supplements can bypass
this regulatory stage (as not all are subject to it). This is good in some cases, as
piperine is required to give curcumin to the extremities rather than it getting con-
sumed by glucuronidation in the liver. However, in some other cases, it can lead to
elevated levels of certain drugs in the blood. Again, elevated could be good or bad
depending on context; regardless, caution should be taken when approaching this
compound.
Piperine has many potential benefits in clinical practice; a lot of studies were
reported in literature about piperine effects in chronic disease, especially in animals.
In vitro studies showed piperine protective effects against oxidative damage by
inhibiting or quenching free radicals and reactive oxygen species. Piperine treat-
ment also reduced lipid peroxidation in vivo, and beneficially influenced
anti-oxidant molecules and anti-oxidant enzymes in a number of experimental
situations of oxidative stress [9]. The aim of this chapter will be to analyze the
effects of piperine, alone or in combination, in chronic diseases.

8.2 Role of Piperine in Chronic Diseases

8.2.1 Piperine and Oxidative Stress

Piperine suppresses the accumulation of lipid peroxidation products, enhances the

activity of anti-oxidant enzymes and eliminates the accumulation and activation of
polymorphonuclear cell. This was showed by Umar et al. [10] that administered
piperine at a dose of 100 mg/kg and indomethacin at 1 mg/kg body weight once
176 G. Derosa et al.

daily for 21 days in male Wistar rats affected by collagen-induced arthritis (CIA).
Piperine was effective in bringing significant changes on all the biochemical and
inflammatory and parameters studies, in particular piperine significantly reduced the
levels of pro-inflammatory mediators [interleukin-1β (IL-1β), tumor necrosis
factor-α (TNF-α), and prostaglandin-2 (PGE2)] and increased level of
interleukin-10 (IL-10). The protective effects of piperine against arthritis were also
evident from the decrease in arthritis scoring and bone histology. These results
clearly indicate that the protective role of piperine was mediated via its anti-oxidant
effect through the suppression of lipid peroxidation and boosting the anti-oxidant
defense system. Piperine seems to shift the balance of cytokines toward a bone
protecting pattern that acts to both lower levels of TNF-a, IL-1β, and raise the levels
of IL-10. Part of the beneficial anti-inflammatory and cartilage/bone protective
effects of piperine may be mediated through the inhibition of pro-inflammatory
cytokines.

8.2.2 Piperine and Inflammation

Piperine proved to have anti-inflammatory effects on Helicobacter pylori-induced

gastritis in gerbils, through the suppression of inflammatory factors, such as TNF-α,
independent of direct anti-bacterial activities, and may have potential for use in the
chemoprevention of Helicobacted pylori-associated gastric carcinogenesis [11]. In
vitro studies showed that piperine can induce apoptosis and inhibits the expression
of inflammatory cytokines in human cell lines [12, 13].
Piperine also inhibited polyclonal and antigen-specific proliferation of mouse T
lymphocytes, as well as cytokine synthesis and the induction of cytotoxic effector
cells as reported by Doucette et al. [14]. These actions on T lymphocytes were
associated with hypophosphorylation of Akt, extracellular signal-regulated kinase
(ERK), and NF-kB components, in particular IκBα, but not ZAP-70, all molecules
involved in T lymphocyte activation.

8.2.3 Piperine Effects on Hepatic Steatosis

and Insulin-Resistance

Hepatic steatosis is due to an excess of plasma fatty acids, even if de novo lipoge-
nesis is also considered an important contributing factor. Previously published
studies showed that AMP-activated protein kinase (AMPK) is thought to regulate
hepatic lipogenic gene expression by inhibiting transcription factors [15]. The
inhibition of AMPK activates liver X receptor a (LXRa), a major regulator of
lipogenesis. In animal models with high-fat diet (HFD)-induced fatty liver, LXRa is
transcriptionally upregulated and consequently activates lipogenic target genes, thus
8 Piperine and Its Role in Chronic Diseases 177

exacerbating hepatic steatosis [16]. Piperine plays a role in the transcriptional reg-
ulation of LXRa; in particular it antagonized LXRa transcriptional activity by
abolishing the interaction of ligand-bound LXRa with the co-activator
CREB-binding protein. The effects of piperine on hepatic lipid accumulation were
likely regulated via alterations in LXRa-mediated lipogenesis in mice fed a high-fat
diet. Piperine positive effects on insulin-resistance and hepatic steatosis were
reported by Choi et al. [17]. In this study, Authors examined the effect of piperine on
hepatic steatosis and insulin-resistance induced in mice by feeding a high-fat diet
(HFD) for 13 weeks. Administration of piperine (50 mg/kg body weight) to mice
with HFD-induced hepatic steatosis resulted in a significant increase in plasma
adiponectin levels. Also, elevated plasma concentrations of insulin and glucose and
hepatic lipid levels induced by feeding a HFD were reversed in mice when they were
administered piperine. Piperine reversed HFD-induced downregulation of
adiponecitn-AMPK signaling molecules which play an important role in mediating
lipogenesis, fatty acid oxidation, and insulin signaling in the livers of mice. Piperine
significantly decreased the phosphorylation of insulin receptor substrate-1 (IRS-1)
compared with the HFD-fed mice. The positive effects of piperine were confirmed
by Rondanelli et al. [18] in humans. They administered a combination of bioactive
food ingredients (epigallocatechin gallate, capsaicins, piperine, and L-carnitine)
versus placebo, in a randomized, double-blind, 8 weeks trial, involving 86 over-
weight subjects. Consumption of the dietary supplement was associated with a
significantly greater decrease in insulin-resistance, assessed by homeostasis model
assessment, leptin/adiponectin ratio, respiratory quotient, LDL-cholesterol. Leptin,
ghrelin, C-reactive protein decreased, and resting energy expenditure increased
significantly in the supplemented group compared to placebo. These results suggest
that piperine, in combination with epigallocatechin gallate, capsaicins, and
L-carnitine, could be useful for the treatment of obesity-related inflammatory
metabolic dysfunctions.

8.2.4 Piperine and Anti-depressant Effects

Piperine proved to have anti-depressant effects as published by Li et al. [19]: these

Authors investigated the anti-depressant-like effect of piperine in mice exposed to
chronic mild stress (CMS) procedure, administering piperine for 14 days at the
doses of 2.5, 5, and 10 mg/kg. Piperine reversed the CMS-induced changes in
sucrose consumption, plasma corticosterone level and open field activity.
Furthermore, the decreased proliferation of hippocampal progenitor cells was
ameliorated and the level of brain-derived neurotrophic factor (BDNF) in hip-
pocampus of CMS stressed mice was upregulated by piperine treatment in the same
time course.
This was further confirmed by Mao et al. [20] who examined the behavioral and
biochemical effects of piperine in rats exposed to chronic unpredictable mild stress.
The results showed that chronic unpredictable mild stress caused depression-like
178 G. Derosa et al.

behavior in rats, as indicated by the significant decrease in sucrose consumption and

increase in immobility time in the forced swim test. In addition, it was found that
serotonin (5-HT) and BDNF contents in the hippocampus and frontal cortex were
significantly decreased in chronic unpredictable mild stress-treated rats. Treating
the animals with piperine significantly suppressed behavioral and biochemical
changes induced by chronic unpredictable mild stress.
The anti-depressant effects of piperine can be explained throughout upregulation
of the progenitor cell proliferation of hippocampus and cytoprotective activity,
which may be closely related to the elevation of hippocampal BDNF level. The
results indicated that the anti-depressant effects of piperine might be partly related
to its modulating in hypothalamic-pituitary-adrenal activity and thereby the
resulting neurogenesis. Piperine is a monoamine oxidase inhibitor, and thus can
increase the levels of brain monoamines, such as 5-HT or norepinephrine (NE),
involved in upregulation of neurogenesis. Also BDNF plays an important role in
adult neurogenesis: BDNF can promote cell survival in the subventricular zone in
both young and senescent rats [21]. Increase of the 5-HT, NE, and downstream of
BDNF level is, at least part, of the mechanism enhancing neurogenesis,
cytoprotectivity, and anti-depressant effect of piperine, besides its effects on
hypothalamic-pituitary-adrenal axis.

8.2.5 Piperine Analgesic and Anti-pyretic Effects

In literature there are studies about the possible analgesic, and anti-pyretic effects of
piperine as reported by Evan Prince et al. [22]. Authors treated mice with piperine
(20 and 30 mg/kg) intraperitoneally; hot plate reaction test and acetic acid test were
used to determine the analgesic activity of piperine in mice. It was found that
piperine has significant analgesic and anti-pyretic activities without ulcerogenic
effects. The results were comparable with indomethacin which was used as standard
drug for reference. Despite that, further studies are required to elucidate the
mechanism of piperine to confirm these activities.

8.2.6 Piperine as a Bioavailability Enhancer

Because of its properties, piperine increases bioavailability of different agents, listed

in Table 8.1. Mechanism throughout piperine acts as bioavailability enhancer
include: inhibition of a number of enzymes responsible for metabolizing drugs and
nutritional substances; stimulation of the activity of amino acid transporters in the
intestinal lining, inhibition of P-glycoprotein, the protein that removes substances
from cells, decreasing the intestinal production of glucuronic acid, thereby per-
mitting more of the substances to enter the body in active form.
8 Piperine and Its Role in Chronic Diseases 179

Table 8.1 Compounds with Substances

documented bioavailability
enhancement upon piperine Barbiturates Resveratrol
co-administration Pyrazinamide Thiophylline
Beta-carotene Propranolol
Rifampicin Vitamin B-6
Coenzyme Q10 Nalorphine
Selenium Phyllanthin
Curcumin

Among agents whose bioavailability is enhanced by piperine is curcumin.

Curcumin has been extensively studied for its therapeutic properties, such as
anti-oxidant, anti-inflammatory, metabolic, anti-depressant, and neuroprotective
activities [23–32]. However, low oral bioavailability of curcumin has been pro-
posed to limit its approval as a therapeutic agent. Studies have reported that cur-
cumin gets reduced through alcohol dehydrogenase, followed by conjugations like
sulfation and glucuronidation in liver and intestine [24]. Piperine proved to enhance
the bioavailability of curcumin and to potentiate its protective effects against
chronic unpredictable stress-induced cognitive impairment and associated oxidative
damage in mice [33]. Chronic treatment with curcumin (200 and 400 mg/kg, p.o.)
significantly improved behavioral and biochemical alterations induced by chronic
unpredictable stress, restored mitochondrial enzyme complex activities, and atten-
uated increased acetylcholinesterase and serum corticosterone levels. In addition,
co-administration of piperine (20 mg/kg; p.o.) with curcumin (100 and 200 mg/kg,
p.o.) significantly elevated the protective effect as compared to their effects alone.
This was further demonstrated by Banji et al. [34] that treated Wistar rats with
piperine (12 mg/kg) alone, curcumin (40 mg/kg) alone or in combination for a
period of 49 days by the oral route with treatment being initiated a week prior to
D-galactose (60 mg/kg, i.p.). A control group, D-galactose alone and naturally aged
control were also evaluated. The results suggested a superior response to combi-
nation therapy compared to monotherapy as evidenced by improved spatial mem-
ory, reduced oxidative burden, reduced accumulation of lipofuscin, improvement in
signaling, increase in hippocampal volume and protection of hippocampal neurons.
The powerful anti-oxidant nature of both, augmented response of curcumin in the
presence of piperine and enhanced serotoninergic signaling was responsible for
improved cognition and prevention in senescence.
Piperine combined with curcumin also proved to have a better anti-genotoxic
effect compared to single treatment against benzo(a)pyrene (BaP)-induced DNA
damage in lungs and livers of mice [35]. Authors administered curcumin
(100 mg/kg body weight) and piperine (20 mg/kg body weight) separately as well
as in combination orally in corn oil to swiss albino mice for 7 days as pretreatments
and subsequently, 2 h after, BaP was administered orally in corn oil (125 mg/kg
body weight). Pretreatments of curcumin and curcumin plus piperine before
administration of single dose of BaP significantly decreased the levels of 8-oxo-dG
content and percentage of DNA in the comet tail in both the tissues. Moreover, the
180 G. Derosa et al.

genoprotective potential of curcumin plus piperine was significantly higher as

compared to curcumin alone against BaP-induced DNA damage.
The anti-cancer property of curcumin is attributed to its anti-oxidant properties
that inhibit free radicals from mediating peroxidation of membrane lipids or
oxidative DNA damage as both are important initiators of cancer development. The
enhanced anti-genotoxic potential of curcumin plus piperine may be due to increase
bioavailability of curcumin thanks to piperine.
The synergic effect of piperine in combination with curcuminoid was confirmed
also in humans by a meta-analysis conducted by Panahi et al. [36]: in this
meta-analysis Authors evaluated the effectiveness of supplementation with a
bioavailable curcuminoid preparation on measures of oxidative stress and inflam-
mation in patients with metabolic syndrome. Authors concluded that short-term
supplementation with curcuminoid-piperine combination significantly improves
oxidative and inflammatory status in patients with metabolic syndrome. Also in this
case piperine has been shown to overcome several pharmacokinetic drawbacks of
curcuminoids, reducing the activity of glucuronidase enzymes both at the site of
intestinal brush border and liver, resulting in improved absorption of curcuminoids.
Increased intestinal perfusion and enterocyte permeability are additional mecha-
nisms whereby piperine improves the bioavailability of curcuminoids [37].
The same authors also conducted a randomized controlled trial on the effects of a
combination of piperine with curcuminoids on lipid profile, showing that
curcuminoids-piperine combination is an efficacious adjunctive therapy in patients
with metabolic syndrome and can modify serum lipid concentrations beyond what
is achieved with standard of care [38].
This was also reported by Neyrinck et al. [39]: they fed mice with either a
control diet, a high-fat diet or a high-fat diet containing Curcuma longa extract
(0.1 % of curcumin in the high-fat diet) associated with white pepper (0.01 %)
which contains piperine for 4 weeks. The co-supplementation in Curcuma extract
and white pepper decreased high-fat-induced pro-inflammatory cytokines
expression in the subcutaneous adipose tissue, an effect independent of adiposity,
immune cells recruitment, angiogenesis, or modulation of gut bacteria controlling
inflammation.
Piperine also acts as enhancer of quercitin, this was reported by Rinwa et al.
[40]. They evaluated the efficacy of a combination of quercitin with piperine against
chronic unpredictable stress-induced behavioral and biochemical alterations. They
administered quercitin (20, 40, and 80 mg/kg, p.o.), piperine (20 mg/kg, p.o.) and
their combinations daily 30 min before chronic unpredictable stress procedure to
Laca mice for a period of 28 days. Chronic unpredictable stress increased brain
oxidative stress markers and neuro-inflammation (TNF-a) in placebo groups treated
with piracetam (100 mg/kg, i.p.). This was coupled with marked rise in acetyl-
cholinesterase and serum corticosterone levels. Co-administration of piperine with
quercitin significantly elevated their potential to restore these behavioral, bio-
chemical, and molecular changes associated with mouse model of chronic unpre-
dictable stress. Co-administration of quercitin (20 mg/kg) with piperine (20 mg/kg)
significantly lowered TNF-α level which was significant as compared to their
8 Piperine and Its Role in Chronic Diseases 181

effects alone. These results suggest that piperine enhances the neuroprotective
effects of quercitin against chronic unpredictable stress-induced oxidative stress,
neuro-inflammation and memory deficits.
Piperine also enhanced phyllanthin bioavailability as published by Sethiya et al.
[41]. These Authors evaluated the hepato-protective effects of phyllanthin along
with piperine in a mixed micellar lipid formulation (MMLF). Authors compared
phyllanthin (30 mg/kg p.o.), a complex phosphatidylcholine formulation of phyl-
lanthin (CP–PC) (30 mg/kg p.o.), phyllanthin + piperine (CP–P–PC) (30 mg/kg p.
o.), and the reference drug silymarin (100 mg/kg, p.o.) administered daily to rats for
10 days, followed by liver damage by administering a 1:1 (v/v) mixture of CCl4
and olive oil (1 ml/kg, i.p.) for 7 days from day 4 to day 10. The degree of
protection was evaluated by determining the level of marker enzymes (SGOT and
SGPT), bilirubin, and total proteins.
CP–P–PC (30 mg/kg p.o.) showed significant hepato-protective effect by
reducing the levels of serum marker enzymes (SGOT, SGPT, and bilirubin),
whereas, elevated the levels of depleted total protein, lipid peroxidation and
anti-oxidant marker enzyme activities such as, glutathione, superoxide dismutase,
catalase, glutathione peroxidase, and glutathione reductase. The complex MMLF
normalized adverse conditions of rat livers more efficiently than the non-formulated
phyllanthin, confirming, again, the effects of piperine in enhancing low bioavail-
ability of phyllanthin.
Piperine can be used also in combination with resveratrol to enhance its
bioavailability [42]: in this study Authors evaluated if co-supplementation of
piperine with resveratrol affects the bioavailability and efficacy of resveratrol with
regard to cognition and cerebral blood flow. In this randomized, double-blind,
placebo-controlled, within-subjects study, 23 adults were given placebo,
trans-resveratrol (250 mg) and trans-resveratrol with 20 mg piperine on separate
days at least a week apart. The results indicated that when co-supplemented,
piperine, and resveratrol significantly augmented cerebral blood flow during task
performance in comparison with placebo and resveratrol alone. Cognitive function,
mood, and blood pressure were not affected.

8.3 Conclusions

In conclusion, piperine has several important roles that can be useful in clinical
practice, in particular for reduction of insulin-resistance, inflammation, and hepatic
steatosis. However, the most far-reaching attribute of piperine is its inhibitory effect
on enzymatic drug biotransformation reactions in the liver. Piperine strongly
inhibits hepatic and intestinal aryl hydrocarbon hydroxylase and UDP-glucuronyl
transferase, thus enhancing the bioavailability of a number of therapeutic drugs as
well as phytochemicals. Piperine’s bioavailability-enhancing property is also partly
attributed to increased absorption as a result of its effect on the ultrastructure of
182 G. Derosa et al.

intestinal brush border. This promising effect of piperine has been extensively
employed to increase the bioavailability and pharmacological effects of several
drugs and phytopharmaceuticals.

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Chapter 9
Therapeutic Potential and Molecular
Targets of Piceatannol in Chronic Diseases

Young-Joon Surh and Hye-Kyung Na

Abstract Piceatannol (3,3′,4,5′-tetrahydroxy-trans-stilbene; PIC) is a naturally

occurring stilbene present in diverse plant sources. PIC is a hydroxylated analog of
resveratrol and produced from resveratrol by microsomal cytochrome P450 1A11/2
and 1B1 activities. Like resveratrol, PIC has a broad spectrum of health beneficial
effects, many of which are attributable to its antioxidative and anti-inflammatory
activities. PIC exerts anticarcinogenic effects by targeting specific proteins involved
in regulating cancer cell proliferation, survival/death, invasion, metastasis, angio-
genesis, etc. in tumor microenvironment. PIC also has other health promoting and
disease preventing functions, such as anti-obese, antidiabetic, neuroptotective,
cardioprotective, anti-allergic, anti-aging properties. This review outlines the
principal biological activities of PIC and underlying mechanisms with special focus
on intracellular signaling molecules/pathways involved.

Keywords Piceatannol
Astringin
trans-Stilbene
Resveratrol

9.1 Introduction

Piceatannol (3,3′,4,5′-tetrahydroxy-trans-stilbene; PIC) is a naturally occurring

stilbene found in a variety of plant sources including grapes, rhubarb, peanuts,
sugarcane, white tea, and the seeds of passion fruit (Passiflora edulis). Astringin, a
PIC glucoside, is found in red wine. PIC was identified as a selective inhibitor of

Y.-J. Surh (&)

Department of Molecular Medicine and Biopharmaceutical Sciences,
Research Institute of Pharmaceutical Sciences, College of Pharmacy,
Seoul National University, Seoul 151-742, South Korea
e-mail: [email protected]
H.-K. Na (&)
Department of Food and Nutrition, College of Human Ecology,
Sungshin Women’s University, Seoul 142-732, South Korea
e-mail: [email protected]

© Springer International Publishing Switzerland 2016 185

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1_9
186 Y.-J. Surh and H.-K. Na

Anti-aging

Anti-inflammatory
activity Immune Function

Obesity Diabetes
Piceatannol

Cadiovascular Neurodegenerative
Disease disorders

Osteoporosis Cancer

Fig. 9.1 Health beneficial and therapeutic effects of PIC

non-receptor spleen tyrosine kinase (Syk) which plays a critical role in the regulation
of not only immune and inflammatory responses of hematopoietic cells, but also
general physiological functions in a wide variety of non-hematopoietic cells [1].
As a hydroxylated analog and a metabolite of resveratrol (3,5,4′-trihydroxy-
trans-stilbene), PIC has multiple biological functions, including antioxidative,
anti-inflammatory, anticancer, antidiabetic, cardioprotective, neuroprotective, and
immunomodulatory properties (Fig. 9.1). PIC has structural similarity to resveratrol
and both compounds share a spectrum of biological activities [2, 3]. This review
summarizes the updated research findings regarding the cancer chemopreventive/
anticarcinogenic and other health promoting effects of PIC and underlying
mechanisms.

9.2 Chemopreventive/Anticarcinogenic Effects

In an early study, PIC was shown to inhibit the development of 7,12-dimethylbenz

(a)anthracene-induced preneoplastic lesions in a mouse mammary organ culture
model [4]. In addition, the chemotherapeutic effects of PIC have been recently
highlighted [5]. The anticarcinogenic and cancer chemopreventive effects of PIC
are summarized below based on its action mechanisms (Fig. 9.2). The majority of
studies on the anticarcinogenic and chemopreventive effects of PIC were done in
cultured cells, and only few animal as well as human data are available. This may
partly be due to the limited availability of relatively large amount of pure PIC
needed for in vivo studies and resulting high cost.
9 Therapeutic Potential and Molecular Targets of Piceatannol … 187

Cancer cell cycle arrest

Induction of cancer cell death

(apoptosis, autophage, etc.)

DNA strand break in caner cells

Inhibition of cancer cell

invasion and metastasis

Piceatannol Antiangiogenesis

Alleviation of multi-drug resistance

Sensitization to anticancer drug-induced apoptosis

Modulation of tumor microenvironment

Fig. 9.2 Mechanisms of anticarcinogenic and cancer chemopreventive effects of PIC

9.2.1 Cell Cycle Arrest

Treatment of the human colon carcinoma Caco-2 cell line with PIC resulted in the
reduced cell proliferation without influencing the differentiation [6]. Flow cyto-
metric analysis of cell cycle distribution revealed an accumulation of cells in the S
phase. Immunoblot analysis demonstrated that cyclin-dependent kinases (CDK) 2
and 6 as well as cdc2 were expressed at steady-state levels, whereas the expression
of cyclin D1, cyclin B1, and CDK 4 was downregulated. The level of p27KIP1 was
also reduced, whereas that of cyclin E was enhanced. Similar changes were also
observed in studies with HCT-116 colon cancer cells [6].
PIC also inhibits cell cycle progression in the DU145 human prostate cancer cell
line. PIC treatment enhanced the percentage of cells in the G1 phase and decreased
[3H]thymidine incorporation as well as expression of cyclin A, cyclin D1, CDK2,
and CDK4. PIC suppressed CDK4 and CDK2 activities as well, but had no effect
on the levels of p21WAF1/CIP1 or p27KIP1 [7]. PIC inhibited the proliferation of T24
and HT1376 human bladder cancer cells by blocking cell cycle progression in the
G0/G1 phase and induced apoptosis. The G0/G1 phase arrest was attributed to an
increased expression of p21WAF1/CIP1. An enhancement in Fas/APO-1 and
membrane-bound Fas ligand might be responsible for the apoptotic effect induced
by PIC in the aforementioned bladder cancer cells [8].

9.2.2 Induction of Apoptosis

9.2.2.1 Hematologic Tumor Cells

Multiple Myeloma (MM) and Lymphoma

MM is a clonal B cell cancer characterized by proliferation of malignant plasma

cells in the bone marrow. Although the introduction of immunomodulatory drugs
188 Y.-J. Surh and H.-K. Na

like bortezomib or lenalidomide has improved patient survival, MM is still incur-

able, and new treatment options are needed. Treatment of MM cell lines (AMO-1,
U266, and RPMI8226) as well as primary MM cells with PIC reduced proliferation
and stromal cell-derived factor-1 alpha induced migration [9]. PIC induced apop-
tosis of MM cells, as revealed by reduced expression of procaspase 3, increased
cleavage of poly(ADP-ribose) polymerase(PARP)-1 and enhanced release of
cytochrome c (cyt c). The anti-proliferative and proapoptotic activities of PIC in
MM cells appear to be mediated through suppression of Syk [9].
Aberrant activation of Wnt/b-catenin signaling promotes development and pro-
gression of various malignant neoplasms. The Wnt signaling pathway is constitu-
tively activated in MM, which exaggerates cell proliferation. PIC inhibited
Wnt/b-catenin signaling in murine plasmocytoma MPC11 cells as well as human
MM (OPM-2, RPMI-8226, and U-266) cells. PIC induced apoptosis and suppressed
the proliferation of these cells [10]. The combination of PIC with ethacrynic acid or
ciclopirox olamine had a significant additive effect on the vitality of MM cells
compared to single-agent application, while healthy cells remained unaffected [11].
PIC induced apoptosis in BJAB Burkitt-like lymphoma cells as evidenced by
activation of caspase-3 and perturbation of mitochondrial permeability transition [12].
The antileukemic properties of PIC were also assessed using primary, leukemic
lymphoblasts from 21 patients suffering from childhood lymphoblastic leukemia. PIC
was found to be a very efficient inducer of apoptosis in this ex vivo assay as well [12].

Leukemia

PIC and other stilbenes were investigated for their tumor-specific cytotoxicity and
apoptosis-inducing activity, using four human tumor cell lines (squamous cell
carcinoma HSC-2, HSC-3, submandibular gland carcinoma HSG and promyelo-
cytic leukemia HL-60) and three normal human oral cell lines (gingival fibroblast
HGF, pulp cell HPC, periodontal ligament fibroblast HPLF). Among the seven cell
lines, HL-60 and HSC-2 cells were the most sensitive to the cytotoxic action of
these compounds. PIC induced internucleosomal DNA fragmentation and activa-
tion of caspases-3, -8, and -9 in HL-60 cells. [13].
PIC treatment to the human leukemia cell line U937 induced the formation of
apoptotic bodies, DNA fragmentation, and the accumulation of the sub-G1 phase
[14]. The proapoptotic effect of PIC was associated with downregulation of
anti-apoptotic Bcl-2 and cIAP-2, proteolytic activation of caspase-3, and the
degradation/cleavage of PARP. z-DEVD-fmk, a caspase-3-specific inhibitor,
attenuated the proapoptotic activity of PIC in U937 cells [14]. PIC-induced apop-
totic death of human leukemia U937 cells was accompanied by an increase in the
intracellular Ca2+ concentration ([Ca2+]i), inactivation of extracellular signal-
regulated kinase (ERK), activation of p38 mitogen-activated protein kinase
(MAPK), degradation of procaspase-8 and production of t-Bid [15]. PIC treatment
increased the levels of Fas and FasL and their mRNA transcripts. Downregulation
of FADD blocked PIC-induced procaspase-8 degradation and rescued viability of
9 Therapeutic Potential and Molecular Targets of Piceatannol … 189

PIC-treated cells [15]. PIC induced tumor necrosis factor-a (TNF-a)-related

apoptosis-inducing ligand (TRAIL)-mediated leukemia cell death which was
associated with significantly elevated expression of DR5, a death receptor of
TRAIL [16]. Further, PIC-enhanced DR5 promoter activity via Sp1 activation. The
DR5 chimera antibodies significantly suppressed PIC-mediated apoptosis in human
monocytic leukemia cells by inactivating TRAIL signaling [16].

9.2.2.2 Solid Tumor Cells

PIC inhibited the growth of the androgen receptor-negative prostate PC3 cancer cell
line which was associated with accumulation of endogenous ceramide [17]. In
another study, the effects of PIC on proliferation of androgen-dependent LNCaP
and androgen-independent DU145 and PC-3 prostate cancer cells were investigated
[18]. PIC exerted anti-proliferative effects, which was accompanied by cell cycle
blockade in G1/S and S phases in LNCaP and PC-3 cells and induction of apoptosis
in DU145 cells [18]. PIC induced apoptosis of DU145 cells was evidenced by
cleavage of caspase 3 and apoptosis inducing factor AIF and an increase in total cyt
c. The apoptotic changes occurred in concordance with DNA damage, as revealed
by increased phosphorylation of histone H2AX at Ser139. Treatment of
different-stage prostate cancer cells with PIC also resulted in cell type-specific
downregulation of the mammalian target of rapamycin (mTOR), a kinase involved
in growth control of eukaryotic cells [18]. PIC also increased the protein levels of
cleaved caspase-8, -9, -7, and -3 and cleaved PARP in DU145 cells, thereby
inducing apoptosis [19]. PIC induced mitochondrial outer membrane permeability
changes, resulting in cyt c release from the mitochondria to the cytosol. PIC
treatment enhanced the levels of truncated Bid, Bax, Bik, and Bok with a con-
comitant decrease in the levels of Mcl-1 and Bcl-xL [19]. These results indicate that
PIC induces apoptosis via the activation of the death receptor and mitochondrial-
dependent pathways in prostate cancer cells.
PIC induced HCT116 and HT29 colon cancer cell apoptosis by promoting
expression of microRNA-129, and suppressing expression of Bcl-2, a known target
of microRNA-129. Moreover, knock down of microRNA-129 reversed the
reduction of cell viability induced by PIC in these cells [20]. PIC exerted
anti-proliferative activities in prostate cancer cell lines. The anti-tumorigenic effects
of PIC was determined in LNCaP-Luc-xenografts. Two-week pretreatment with
PIC diminished cell colonization, tumor volume, and tumor growth in the xeno-
grafts [21]. Treatment of MDA-MB-231 human breast cancer cells with PIC caused
a rapid release of calcium from the endoplasmic reticulum. This elevation of
intracellular calcium modulates the activity of p53 and the subsequent transcription
of several genes encoding proapoptotic proteins [22]. Nanomolar concentration of
PIC induced c-Myc expression and activated survival signaling in MCF-7 human
breast cancer cells, leading to accelerated cell proliferation [23]. In contrast, PIC at
the concentrations in the micromolar range suppressed c-Myc expression and
induced apoptosis.
190 Y.-J. Surh and H.-K. Na

9.2.3 Induction of Autophage

Environmental conditions or chemical agents can interfere with the function of the
endoplasmic reticulum (ER), and the resultant ER stress can be toxic to the cell if it
is not relieved. Agents that cause ER stress in cancer cells can elicit anticancer
activity. PIC was identified as one of the potent inducers of ER stress. In the
HT1080 reporter cells treated with PIC, there were both splicing of XBP1 and
induction of the ATF4 target gene product CCAAT-enhancer-binding protein
homologous protein (CHOP), leading to ER stress [24]. This was accompanied by a
modest increase in LC3 processing, indicative of a mild autophagic response.
The health beneficial effect of resveratrol may be associated, at least in part, with
its capacity to promote autophagy by activating the NAD+-dependent deacetylase
sirtuin 1 [25]. PIC is a weak activator of sirtuin 1, and its induction of autophage is
independent of this enzyme. Resveratrol and PIC, in combination, induced autop-
hagy and stimulated the deacetylation of cytoplasmic proteins more efficiently than
either compound alone. These findings suggest that both compounds, through
different mechanisms, may synergize in inducing autophagy [25].

9.2.4 DNA Strand Break

Resveratrol is recognized as a naturally occurring antioxidant but it also catalyzes

oxidative DNA degradation in a test tube reaction in the presence of transition metal
ions such as copper. PIC was found to be a more efficient DNA-cleaving agent than
resveratrol in the presence of Cu(II) as assessed by the conversion of supercoiled
pBR322 plasmid into linear forms [26]. In this study, trans-stilbene, which lacks any
hydroxyl group, failed to induce DNA cleavage in the presence of Cu(II). PIC plus
Cu(II) also caused DNA damage in lymphocytes isolated from human peripheral
blood to a greater extent than resveratrol in the presence of Cu(II) as measured by the
Comet assay. Based on these findings, it is hypothesized that anticancer properties of
various plant-derived polyphenols including PIC may involve mobilization of
endogenous copper and the consequent prooxidant action [26].

9.2.5 Anti-metastatic Effects

Cancer invasion and metastasis are two main causes of treatment failure and
cancer-related mortality. Several studies have demonstrated that matrix metallo-
proteinases (MMPs) are critical for tumor invasion and metastasis. PIC inhibited
MMP-2 activity and manifestation of the invasive phenotype of MCF10A human
breast epithelial cells harboring mutated H-ras (H-ras MCF10A cells) more
effectively than resveratrol [27]. PIC attenuated the H-ras-induced phosphorylation
9 Therapeutic Potential and Molecular Targets of Piceatannol … 191

of Akt in a time- and dose-dependent manner, whereas resveratrol, at the same

concentrations, did not exert an inhibitory effect. In vitro kinase assays demon-
strated that PIC significantly inhibited phosphatidylinositol 3-kinase (PI3K) activity
and suppressed phosphatidylinositol (3,4,5)-trisphosphate expression in the H-ras
MCF10A cells. Interestingly, PIC directly binds to PI3K, thereby inhibiting its
catalytic activity. Data from molecular docking suggested that PIC is a more
tight-binding inhibitor of PI3K than resveratrol due to the additional hydrogen bond
between the hydroxyl group and the backbone amide group of Val882 located in
the ATP-binding pocket of PI3K [27].
The anti-invasive and anti-metastatic effects of PIC were investigated in
MDA-MB-231 human breast cancer cells. PIC significantly reduced serum-induced
cell invasion and migration as well as adhesion without affecting the viability of
these cells [28]. Furthermore, PIC markedly inhibited the activity of MMP-9 and its
expression at both protein and mRNA levels. PIC attenuated PI3K and phospho-
rylation of AKT and mTOR, whereas the protein level of the PI3K inhibitor, PTEN
(Phosphatase and Tensin Homolog Deleted on Chromosome Ten) was increased.
Moreover, PIC inhibited transcriptional activity of nuclear factor jB (NF-jB) and its
binding to DNA harboring an MMP-9 promoter consensus sequence. In addition,
PIC diminished NF-jB nuclear translocation by blocking IjBa phosphorylation in
the cytoplasm [28].
In order to evaluate the anti-metastatic property of PIC, the lung metastasis of
MAT-Ly-Lu (MLL) rat prostate cancer cells expressing luciferase were injected
into the tail veins of male nude mice [29]. The oral administration of PIC
(20 mg/kg) significantly inhibited the accumulation of MLL cells in the lung of
these mice [29]. In the cell culture studies, PIC inhibited the basal and epidermal
growth factor (EGF)-induced migration of DU145, MLL, PC3, and TRAMP-C2
prostate cancer cells. In addition, PIC attenuated invasion of DU145 cells in the
absence and presence of EGF [29]. In DU145 cells, PIC reduced secretion and
messenger RNA levels of MMP-9, urokinase-type plasminogen activator (uPA),
and vascular endothelial growth factor (VEGF), while increasing the protein levels
of tissue inhibitor of MMP-2. Additionally, PIC inhibited the phosphorylation of
signal transducer and activator of transcription (STAT) 3. Furthermore, PIC
reduced both basal and EGF-induced interleukin (IL)-6 secretion. PIC also inhibited
IL-6-induced increases in the secretion of uPA and VEGF, STAT3 phosphorylation
and the migration of DU145 cells. [29]. PIC also suppressed the expression of
MMP-9 in DU145 human prostate cancer cells treated with TNF-a. In addition, PIC
reduced the TNF-a-induced invasion of DU145 cells. PIC attenuated MMP-9 gene
expression via the suppression of NF-jB activity. Furthermore, TNF-a-induced Akt
phosphorylation was significantly attenuated in the presence of PIC [30].
PIC suppressed both the proliferation and invasion of cultured AH109A hep-
atoma cells [31]. PIC, at lower concentrations (25–50 lM), induced cell cycle arrest
at G2/M phase, while it caused apoptosis at a higher concentration (100 lM). PIC
suppressed invasive capacity of hepatoma cells by scavenging reactive oxygen
species (ROS). PIC also suppressed the tumor growth and metastasis in
hepatoma-bearing rats [31].
192 Y.-J. Surh and H.-K. Na

9.2.6 Multidrug Resistance (MDR) Modulation

The identification of compounds that overcome the resistance to cancer cell

apoptosis that frequently accompanies MDR is of great therapeutic importance. PIC
effectively inhibited the multidrug resistance-associated protein, MRP1 as assessed
by its ability to suppress the efflux of the fluorescent MRP1 substrate (BCECF)
from human erythrocytes [32].

9.2.7 Modulation of Tumor Microenvironment

Chronic lymphocytic leukemia (CLL) is characterized by the progressive accu-

mulation of clonal B lymphocytes. Proliferation occurs in lymphoid tissues upon
interaction of leukemic cells with a supportive microenvironment [33]. Therefore,
the mobilization of tissue-resident CLL cells into the circulation is a useful thera-
peutic strategy to minimize the reservoir of tumor cells within survival niches. The
exit of normal lymphocytes from lymphoid tissues depends on the presence of
sphingosine-1 phosphate (S1P) and the expression of S1P receptor-1 (S1PR1).
Activated CLL cells displayed reduced expression of S1PR1 and the migratory
response toward S1P. PIC enhanced S1PR1 expression in CLL cells and their
migratory response toward S1P [33].
4T1 mammary carcinoma cells were injected into the mammary fat pad of
syngeneic female BALB/c mice. PIC treatment reduced tumor growth. In tumors
from PIC-treated groups, there was reduced expression of transcription factors
P-NF-jB, P-STAT3, and HIF-1a and multiple proteins involved in regulation of
cell cycle progression (Ki67, cyclin D1, cyclin A, CDK2, and CDK4), angiogenesis
(VEGF-A, VEGFR-2, VE-cadherin, and CD31), and lymphangiogenesis (VEGF-C
and LYVE-1), as well as macrophage infiltration [34]. PIC administration also
significantly increased the number of apoptotic cells and expression of both Bax
and cleaved caspase-3 but reduced anti-apoptotic Bcl-2 expression in tumor tissues.
In addition, PIC reduced the number and the volume of pulmonary tumor nodules
and expression of MMP-9 in the tumor tissues. It also lowered tissue levels of
cytokines/chemokines, including macrophage colony-stimulating factor (M-CSF)
and macrophage chemoattractant protein (MCP-1). PIC inhibited migration of 4T1
mouse mammary cancer cells and monocytes, as well as secretion of MCP-1 and
M-CSF by 4T1 cells. These results indicate that alteration in tumor microenvi-
ronment is an important mechanism by which PIC inhibits tumor proliferation,
angiogenesis, and lymphangiogenesis, leading to suppression of mammary tumor
growth and metastasis [34].
9 Therapeutic Potential and Molecular Targets of Piceatannol … 193

9.2.8 Potentiation of Cytotoxic Effects of Chemotherapeutic

Agents

The potential synergistic effect of PIC on gemcitabine cytotoxicity was investigated

in the human non-small cell lung cancer, A459 cell line. Gemcitabine alone induced
the expression of the proapoptotic proteins Bad and Bak, and pretreatment with PIC
further enhanced Bak expression, leading to an increased number of cells under-
going late apoptosis. PIC can hence potentiate the cytotoxic effects of gemcitabine
by upregulating the proapoptotic protein expression [35].
PIC was found to sensitize the ovarian cancer cells to cisplatin-induced cyto-
toxicity, and this effect was achieved through modulation of several major deter-
minants of chemoresistance [36]. PIC enhanced p53-mediated expression of the
proapoptotic protein NOXA, XIAP degradation via the ubiquitin-proteasome
pathway, and caspase-3 activation. This response was associated with an increase in
Drp1-dependent mitochondrial fission, leading to more effective induction of
apoptosis. In vivo studies using a murine model of ovarian cancer also revealed
apoptotic changes in association with a greater overall reduction in tumor weight
when mice were co-treated with both PIC and cisplatin, in comparison to treatment
with either agent alone [36].
Arabinofuranosyl cytidine (Ara-C) is a chemotherapeutic agent used mainly in
the treatment of hematological malignancies, such as acute myeloid leukemia and
non-Hodgkin lymphoma. It inhibits cancer cell growth by interfering with DNA
synthesis. Incubation of human HL-60 leukemia cells with PIC induced apoptosis
and caused an arrest in the G2/M phase of the cell cycle [37]. PIC depleted intra-
cellular dCTP and dGTP pools, and thereby inhibited the incorporation of [14C]-
labeled cytidine into DNA. PIC significantly abolished all NTP pools, and
sequential treatment with PIC and Ara-C yielded synergistic growth inhibitory
effects [37]. PIC or myricetin alone induced apoptotic cell death in HL-60 cells, but
combined treatment further enhanced the proportion of apoptotic cells [38].

9.2.9 Miscellaneous Effects

It has been reported that glypican-3 (GPC3) is significantly elevated in human

hepatocellular carcinoma compared with benign liver lesions or normal liver. In the
human hepatocellular carcinoma cell lines, GPC3 expression was consistently
observed, and was mainly located in the cell membrane and cytoplasm. PIC
increased the expression of GPC3 [39]. The Cbl family of proteins are evolution-
arily conserved negative regulators of signaling cascades involving receptor and
non-receptor tyrosine kinases. PIC caused the loss of the Cbl family of proteins
[40]. It is likely that the oxidative conversion of the catechol ring of PIC into a
highly reactive o-benzoquinoneis required for the PIC-induced Cbl loss.
194 Y.-J. Surh and H.-K. Na

9.3 Other Pharmacological Effects

9.3.1 Anti-obesity Effects

Obesity is associated with an increased risk of several metabolic diseases, such as

type 2 diabetes, high blood pressure, and cardiovascular disorders as well as cancer.
The differentiation of preadipocytes to adipocytes, a process called adipogenesis, is
essential for expansion of the fat tissue. Dysfunction of adipogenesis is a hallmark
of obesity, and elucidation of underlying mechanisms is essential for identifying
targets for pharmacological and nutritional intervention. CHOP, as an adipocyte
repressor, inhibits the differentiation of preadipocytes. PIC induced CHOP
expression and thereby blocked differentiation of human liposarcoma (LiSa-2)
preadipocytes as characterized by reduced fat droplet formation and VEGF pro-
duction [41]. In another study, PIC inhibited adipogenesis of 3T3-L1 preadipocytes
[42]. In the early phase of adipogenesis, PIC-treated preadipocytes displayed a
delayed cell cycle entry into G2/M phase at 24 h after initiation of adipogenesis.
Furthermore, the PIC-induced expression of mitotic clonal expansion was accom-
panied by reduced activation of insulin signaling. PIC directly binds to insulin
receptor and inhibits insulin receptor kinase activity which may account for its
capability to inhibit adipogenesis [42].
The effects of PIC on obesity-associated complications in Zucker obese rats were
measured after a 6-week supplementation. While PIC tended to improve lipid
handling, it did not mitigate hyperinsulinemia and cardiac hypertrophy. However, it
did increase cardiac expression of ephrin-B1, a membrane protein involved in
maintaining cardiomyocyte architecture. Thus, PIC did not exhibit strong slimming
capacities but could limit several obesity complications [43].

9.3.2 Antidiabetic Effects

As a hydroxylated analog of resveratrol, PIC may have similar or distinct

protective/preventive effects on the metabolic disorders. Resveratrol has been
known to ameliorate diet-induced obesity and insulin resistance [44]. Intragastric
administration of PIC suppressed significantly the increases in the fasting blood
glucose levels in genetically diabetic db/db mice without affecting body weights
and food intake [45]. In addition, PIC significantly suppressed the rises in the blood
glucose levels in db/db mice given glucose after 12-h fasting. These findings
indicate that PIC administration represses the rises in blood glucose levels at early
stages while it can exert a remedial effect on impaired glucose tolerance at late
stages of diabetes in db/db mice [45].
Similar antidiabetic effects of PIC have been observed in C57BL/6Jjcl mice fed
high-fat diet [46]. PIC did not affect high-fat diet-induced body weight gain or
visceral fat gain in these animals, but lowered fasting blood glucose levels. In
9 Therapeutic Potential and Molecular Targets of Piceatannol … 195

agreement with the observation in the previous study by Minakawa et al. [45], a
single intragastric administration of PIC reduced the blood glucose levels in db/db
mice [46]. In skeletal muscle cells, glucose uptake occurs mainly through glucose
transporter 4 (GLUT4) in response to insulin. 5′-Adenosine monophosphate-
activated protein kinase (AMPK) is known as a GLUT4 translocation promoter, and
AMPK activators, hence have a potential to overcome insulin resistance in the
diabetic state. PIC increased glucose uptake in L6 myotubes by promoting GLUT4
translocation to plasma membrane via AMPK activation [45].
The hypoglycemic effect of PIC was examined after intravascular administration
to healthy rats. Intravascularly administered PIC reduced the blood glucose con-
centrations during both fasting and the glucose tolerance test [47]. Furthermore,
PIC increased the insulinogenic index during glucose tolerance tests but had no
influence on insulin sensitivity. Based on these observations, it is likely that PIC
given orally enhances glucose tolerance, and this effect is mediated by intact PIC
itself, not its metabolite, by stimulating early phase secretion of insulin.
PIC effectively mitigated the inhibitory effects of palmitic acid on the
insulin-mediated phosphorylation of insulin receptor substrate-1 in human umbil-
ical vein endothelial cells (HUVECs). The protective effect of PIC against palmitic
acid-induced impairment of insulin signaling was mediated by heme oxygenase-1
(HO-1) activity [48].

9.3.3 Neuroprotective Effects

9.3.3.1 Alzheimer Disease (AD)

b-Amyloid (Ab), a key protein involved in the pathogenesis of AD, is a major

component of senile plaques formed in the brain of AD patients. Apoptotic cell
death caused by ROS has been implicated in the Ab-induced neurotoxicity. PIC
inhibited the formation of Ab fibrils [49]. Notably, the choroid plexus secretes
transthyretin, a protein that has been shown to inhibit Ab aggregation and that may
hence be critical to the maintenance of normal learning capacities in aging. The
misfolding of transthyretin is implicated in amyloid diseases. It is worthwhile
determining whether PIC can inhibit misfolding of this particular protein in exerting
its inhibitory effects on formation and neurotoxicity of Ab fibrils [49].
PIC treatment attenuated the accumulation of ROS and inhibited apoptosis in rat
pheochromocytoma PC12 cells treated with Ab [50]. PIC exerted much stronger
protective effects than did resveratrol [50]. 4-Hydroxynonenal (4-HNE) is a major
lipid peroxidation product produced by oxidative stress, and its level is elevated in
the AD brain. PIC protected PC12 cells from 4-HNE-induced apoptosis as revealed
by attenuation of nuclear condensation, PARP cleavage and downregulation of
anti-apoptotic Bcl-2 expression. PIC also inhibited the phosphorylation of c-Jun
N-terminal kinase (JNK), which is a key regulator of apoptotic cell death [51].
Similar increased cell viability, a decreased apoptosis rate and reduced intracellular
196 Y.-J. Surh and H.-K. Na

ROS accumulation were observed after PIC treatment to PC12 cells [52]. PIC
significantly promoted activation of anti-apoptotic Bad and Akt through phos-
phorylation, while it reduced the Bcl-2/Bax ratio and cleavage of caspase-9,
caspase-3, and PARP.

9.3.3.2 Glutamate-Induced Neurotoxicity

Oxidative cell death is a common mechanism underlying pathogenesis of not only

AD, but also a variety of other neural diseases. The amino acid glutamate at high
concentrations causes depletion of reduced glutathione (GSH) by inhibiting the
glutamate/cystine antiporter system, with a concomitant increase in intracellular
accumulation of ROS. This leads to oxidative stress-induced neuronal cell death.
PIC reduced glutamate-induced ROS formation and cytotoxicity in cultured HT22
neuronal cells [53]. PIC also increased the expression of HO-1 via activation of
nuclear factor-erythroid 2 (NF-E2) p45-related factor 2 (Nrf2), a master transcrip-
tion factor responsible for regulating transcription of many antioxidant/anti-
inflammatory and other cytoptotective genes. Interestingly, the neuroprotective
effect of PIC was partly abolished by either downregulation of HO-1 expression or
blockade of HO-1 activity [53].

9.3.3.3 Ischemic Stroke

Inflammation occurs in the ischemic brain, which consists of various types of cells,
such as neurons, endothelial cells, and immune cells. Macrophage-inducible C-type
lectin (Mincle, CLEC4E) receptor is involved in neuroinflammation in cerebral
ischemia and traumatic brain injury. Mincle, its ligand SAP130, and its downstream
tyrosine kinase Syk were upregulated in the brain after ischemia, and participate in
the pathogenesis of ischemic stroke by initiating inflammation [1]. In mice treated
with PIC intraperitoneally before ischemia and just after reperfusion, the cerebral
ischemic injury was ameliorated. The protective effects of PIC against ischemic
brain injury were attributed to its inhibition of Syk [1]. Subarachnoid hemorrhage
(SAH) accounts for 5–7 % of all strokes. PIC reduced brain edema and ameliorated
neurological deficits after SAH in a rat stroke model [54].

9.3.3.4 Neuroinflammation

Prostaglandin E2 (PGE2), nitric oxide (NO), and proinflammatory cytokines, such

as IL-1b, IL-6, and TNF-a, play pivotal roles in brain injuries. PIC significantly
inhibited the release of NO, PGE2, and proinflammatory cytokines in
LPS-stimulated BV2 microglia [55]. PIC also attenuated the expression of inducible
NO synthase (iNOS) and cyclooxygenase-2 (COX-2) at both transcriptional and
translational levels. PIC exhibits anti-inflammatory properties by suppressing the
9 Therapeutic Potential and Molecular Targets of Piceatannol … 197

transcription of proinflammatory cytokine genes through inhibition of the NF-jB

signaling pathway [55].
Prion diseases are a group of transmissible fatal neurodegenerative disorders of
humans and animals, including bovine spongiform encephalopathy, scrapie, and
Creutzfeldt–Jakob disease. Microglia, the resident macrophages of the central
nervous system, are exquisitely sensitive to pathological tissue alterations. CD36, a
class B scavenger receptor expressed on the surface of microglia, is involved in
microglial activation induced by a neurotoxic prion protein fragment, PrP106–126.
PIC was found to abrogate CD36-mediated iNOS expression induced by neurotoxic
prion peptides in BV2 microglial cells [56].

9.3.4 Cardioprotective Effects

Cardiovascular disease (CVD) is a global health problem. Due to high morbidity

and mortality rates, CVD accounts for an enormous socioeconomic burden.
Consumption of dietary supplements or functional foods for reducing the risk of
CVDs has also gained wide recognition by the general public. PIC is one such
candidate with a potential to prevent CVD-associated disorders, such as hyperc-
holesterolemia, arrhythmia, and atherosclerosis. It also has vasorelaxation and
antioxidant activities [57].
Reperfusion is associated with potentially lethal arrhythmias that occurs within
seconds of the onset of reflow. Presumably due to its additional hydroxyl group, PIC
has more potent free radical scavenging activity than resveratrol in association with
antiarrhythmic and cardioprotective activities in ischemic and ischemic-reperfused
rat hearts [58]. PIC also exerted antiarrhythmic activity in isolated rat hearts sub-
jected to ischemia–reperfusion (I/R) injury [59]. Ischemic tissue injury is caused by
the adhesion of polymorphonuclear neutrophils to the lining of the vascular
endothelium. Administration of albumin nanoparticles loaded with PIC detached the
adherent neutrophils and thereby facilitated their release into the circulation [60].
Atherosclerosis is a major risk factor for CVD, in which inflammation plays a
prominent role in its pathogenesis. Eotaxin, a newly discovered C-C motif chemo-
kine (CCL11), has been shown to increase coronary artery endothelial cell perme-
ability and is involved in endothelial inflammation and vascular smooth muscle cell
migration. Release as well as expression of ecotaxin-1 and its gene promoter activity
were increased in human coronary artery endothelial cells stimulated with IL-13 and
TNF-a, and this was effectively inhibited by PIC as well as resveratrol [61].
PIC pretreatment suppressed cardiac hypertrophy induced by isoproterenol in
mice as assessed by the heart weight/body weight ratio, cross-sectional area, and
expression of hypertrophic markers. The anti-hypertrophic effect of PIC was also
verified in cultured rat neonatal cardiomyocytes, and the underlying mechanism
appears to involve the modulation of the transcription factor GATA binding factor 6
[62].
198 Y.-J. Surh and H.-K. Na

9.3.5 Vascular Endothelial Function/Protection

Glucose-induced oxidative stress is involved in endothelial dysfunction.

Dimethylarginine dimethylaminohydrolase (DDAH) is a regulator of the endothe-
lial NO synthase (eNOS). DDAH activity and expression were decreased in bovine
aortic endothelial cells challenged with 25 mM glucose as compared to control
cells. DDAH inhibition led to intracellular accumulation of asymmetric dimethy-
larginine (ADMA), a natural inhibitor of eNOS. PIC restored the basal DDAH
activity and the ADMA level [63].
Arginase competitively inhibits NOS by use of the common substrate L-arginine.
Arginase II has been reported as a novel therapeutic target for the treatment of
CVDs, such as atherosclerosis. PIC-3′-O-beta-D-glucopyranoside (PG) inhibited
the activity of arginase I and II prepared from mouse liver and kidney lysates,
respectively [64]. Incubation of HUVECs with PG also blocked arginase activity
and increased NO production.
P-selectin glycoproteinligand-1 (PSGL-1) not only functions as an anchor
molecule to capture monocytes and other leukocytes to endothelial cells in ischemic
tissues through its interaction with P-selectin, but also transduces signals to initiate
firm adhesion. Endothelial progenitor cells are derived from monocytes and play an
important role in neovascularization. When transfected with human PSGL-1 gene,
endothelial progenitor cells exhibited higher affinity for activated HUVECs or
recombined P-selectin/intercellular adhesion molecule 1 (ICAM-1) monolayer. The
overexpression of PSGL-1 enhanced expression of beta2-integrin expression on the
surface of endothelial progenitor cells and their adherence affinity, and these effects
were abolished by PIC through suppression of Syk [65].
PIC induced a relaxation in aortic preparations precontracted with phenyle-
phrine. The PIC-induced vascular relaxation in rat aorta appears to be mediated by
an endothelium-dependent NO signaling pathway, at least partially, through the
activation of large conductance Ca2+-activated K+ channels [66].

9.3.6 Antiallergic Effects

Mast cells and basophils are major effector cells in the immunoglobulin E (IgE)-
dependent allergic reactions as well as in the innate immunity. Upon allergen
exposure, these cells are stimulated via the high affinity IgE receptor (FcepsilonRI)
to release several proinflammatory chemical mediators, such as leukotrienes,
immunoregulatory cytokines, and histamine. FcepsilonRI-mediated signaling is
initiated upon tyrosine phosphorylation of this receptor by the Src family kinase
Lyn, which is followed by an activation of Syk. PIC, as a classical Syk kinase
inhibitor, can modulate the FcepsilonRI-mediated signaling in mast cells, and has a
potential for use in the treatment of inflammatory and allergy diseases [67].
9 Therapeutic Potential and Molecular Targets of Piceatannol … 199

Histamine is one of the key chemical mediators that play an important role in
allergic inflammation. PIC 4′-b-glucoside formed in cultured Phytolacca
Americana treated with PIC showed the strong inhibitory effects on histamine
release from rat peritoneal mast cells [68]. Compound 48/80 (8 mg/kg) was used to
induce a systemic fatal allergic reaction in mice. Intraperitoneal administration of
PIC inhibited the compound 48/80-induced systemic anaphylaxis and serum his-
tamine release [69]. PIC treatment also ameliorated local allergic reactions in an
IgE-mediated passive cutaneous anaphylaxis model [70]. The rat basophilic leu-
kemia RBL-2H3 cell line exhibits phenotypic characteristics of mucosal mast cells.
After stimulation with antigens cells release histamine and b-hexosaminidase, a
marker of mast cell degranulation. PIC pretreatment strongly suppressed histamine
release and degranulation in the RBL-2H3 cells stimulated with phorbol
12-myristate 13-acetate (PMA) and the calcium ionophore A23187 [69]. PIC
treatment of the human mast cell line HMC-1 reduced PMA plus A23187-induced
mRNA expression and release of the proinflammatory cytokines (TNF-a and IL-8).

9.3.7 Effects on Immune Cell Functions

Chronic inflammatory diseases are characterized by neutrophil infiltration in

inflamed tissues. Neutrophils are able to release cytotoxic substances and inflam-
matory mediators, which have a potential to maintain persistent inflammation.
Apoptosis is an important process for successful removal of recruited neutrophils.
Neutrophils from healthy volunteers were incubated in vitro with PIC and some
other stilbenoids, including resveratrol. Neutrophils treated with resveratrol and PIC
underwent apoptosis [71]. The ability of PIC to reduce the toxic action of neu-
trophils was verified in another study [72]. It elevated the percentage of early
apoptotic neutrophils, inhibited the activity of protein kinase C, the main regulatory
enzyme in neutrophils, and reduced phosphorylation of protein kinase C isoforms
a, b II, and d on their catalytic region. PIC may hence be useful as a complementary
medicine in states associated with persisting neutrophil activations [72]. The anti-
cancer drug arsenic trioxide is known to be toxic for human mononuclear and
neutrophil cell populations. PIC inhibited the arsenic trioxide-induced phagocytic
ability of neutrophils and degranulation by inhibiting Syk kinase [73].
The effect of PIC on T cell activation, proliferation, and differentiation was
assessed using murine splenic T cells isolated from C57BL/6 mice. PIC treatment
inhibited surface expression of CD4 and CD8 T cell activation markers CD25 and
CD69, reduced production of cytokines IFNc, IL-2, and IL-17, and suppressed
proliferation of activated T cells. Moreover, PIC treatment significantly inhibited
differentiation of CD4+ CD25− CD62L+ naïve CD4 T cells into Th1, Th2, and
Th17 cells, presumably due to inhibition of TcR signaling [74].
200 Y.-J. Surh and H.-K. Na

9.3.8 Modulation of Bone Metabolism

Decreases in new bone formation, followed by estrogen deficiency are implicated in

postmenopausal osteoporosis. PIC was found to stimulate osteoblast maturation and
differentiation required for bone mass increases. These effects were ascribed to
overproduction of bone morphogenetic protein-2 by PIC [75]. Osteoclast formation
occurs when bone marrow macrophages derived from hematopoietic stem cells are
stimulated by osteoclastogenic factors, including receptor activator of nuclear
factor-jB ligand (RANKL). Increased osteoclast formation is responsible for excess
bone resorption, leading to the bone loss seen in osteoporosis, and inflammation-
induced bone destruction [76]. PIC has been reported to inhibit osteoclast function
and formation through upregulation of HO-1 expression. PIC reduced the pro-
duction of microRNA-183 and consequently increased HO-1 expression, leading to
attenuation of osteoclastogenesis [76].
Prostate cancer primarily metastasizes to bone, and the interaction of cancer cells
with bone cells results in a local activation of bone formation and/or bone
resorption. When the cells were grown in the presence of conditioned medium from
osteolytic PC-3 prostate cancer cells, matrix mineralization was largely reduced,
suggesting the suppression of osteoblast differentiation by PC-3-secreted molecules
[77]. Treatment of primary murine osteoblasts with conditioned medium from
osteolytic PC-3 prostate cancer cells led to a marked induction of several cytokine
genes, including Cxcl5, Cxcl12, and Tnfsf11, the latter one encoding for the
osteoclast differentiation factor RANKLl. PIC abrogated transcription of these
genes in osteoblasts stimulated with PC-3-conditioned medium. Similarly, PIC
inhibited the expression of Cxcl5 and Tnfs11 genes in primary human osteoblasts
co-incubated with the PC-3 conditioned medium [77].

9.3.9 Anti-aging Effects

9.3.9.1 Expansion of Life Span

Cellular senescence is characterized by cellular hypertrophy in which cells grow

without division. The genes that regulate this process can be activated or inactivated
by numerous plant polyphenols, including resveratrol [78]. The anti-aging potential
of resveratrol has been subjects of numerous research endeavors [2]. Resveratrol
has been shown to prolong the lifespan of experimental animals, and the main
mechanism involves the activation of Sirtuins (Sir [invertebrates] or Sirt [verte-
brates]). A recent study has demonstrated that both resveratrol and PIC enhance the
expression levels of SIRT1 and its mRNA transcript in THP-1 monocytic cells. An
extract of passion fruit seeds, which is rich in PIC, has also been found to upreg-
ulate sirtuin 1 (SIRT1) mRNA expression [79].
9 Therapeutic Potential and Molecular Targets of Piceatannol … 201

9.3.9.2 Melanogenesis

The effects of passion fruit on melanin inhibition and collagen synthesis were
studied using cultured human melanoma and fibroblast cells [80]. Treatment of
melanoma cells with the seed part of passion fruit resulted in inhibition of
melanogenesis. The production of total soluble collagen in cultured dermal
fibroblast cells was also elevated. PIC is speculated as the major component
responsible for the passion fruit effects on melanogenesis and collagen synthesis
[80]. The skin lightening potential of PIC was investigated in terms of its ability to
inhibit melanogenesis [81]. PIC strongly inhibited tyrosinase activity involved in
melanin biosynthesis and lowered the melanin content in cultured B16F10 mela-
noma cells. The antioxidative property is closely linked to the melanogenic activity.
PIC reduced the ROS accumulation and the ratio of GSH to its oxidized form
GSSG in these cells [81].

9.3.9.3 Photoaging and Skin Inflammation

The use of naturally occurring botanicals with substantial antioxidant activity to

prevent photoaging has received increasing attention. The passion fruit seed extract
and its principal component PIC elevated the GSH levels in human keratinocytes,
while suppressing the ultraviolet (UV)B-induced generation of ROS in these cells
[82]. In addition, the transfer of the medium from the UVB-irradiated keratinocytes
to nonirradiated fibroblasts enhanced MMP-1 activity, and this induction was
reduced when the keratinocytes were pretreated with PIC [82].
In order to identify inhibitors of ultraviolet UV-induced cytotoxicity, more than
50 plant extracts were screened, using cultured normal human epidermal ker-
atinocytes (NHEK). Among them, fruit of rose myrtle (Rhodomyrtus tomentosa)
displayed the most potent effects on UVB-induced cytotoxicity [83]. Both PIC and
the rose myrtle extract reduced the production of UVB-induced cyclobutane
pyrimidine dimers and enhanced the activity of the DNA polymerases in
UVB-irradiated NHEK cells. In addition, the secretion of the inflammatory medi-
ator, PGE2 was decreased [83].
Pretreatment of hairless mouse skin topically with PIC attenuated PMA-induced
expression of COX-2 and iNOS [84]. PIC also diminished nuclear translocation and
DNA binding of NF-jB, a major transcription factor known to regulate these
proinflammatory gene expression, through the blockade of phosphorylation and
subsequent degradation of IjBa. Likewise, the catalytic activity of IKKb and the
phosphorylation of MAPKs in PMA-treated mouse skin were inhibited by topically
applied PIC. In addition, PIC decreased PMA-induced expression of c-Fos and the
DNA binding of AP-1, another transcription factor that plays an important role in
inflammatory signaling [84].
202 Y.-J. Surh and H.-K. Na

9.3.10 Anti-colitic Effects

Inflammatory tissue injury is considered to be implicated in tumor promotion and

progression. For instance, inflammatory bowel diseases (IBD), such as ulcertative
colitis and Crohn’s disease, have been considered to increase the risk of colon
cancer. The potential protective effect of PIC on IBD was investigated in a dextran
sulfate sodium (DSS)-induced murine colitis model that mimics human IBD.
Administration of DSS (2.5 %) in drinking water for 7 days to male ICR mice
resulted in colitis and elevated expression of iNOS and activation of NF-jB [85].
Phosphorylation of ERK and STAT3 was also enhanced after DSS treatment. Oral
administration of PIC (10 mg/kg body weight each) for 7 constitutive days atten-
uated the DSS-induced inflammatory injury, upregulation of iNOS expression, and
activation of NF-jB, STAT3, and ERK [85]. Similarly, PIC administration blunted
the weight loss and clinical signs of intestinal inflammation caused by 5 % DSS in
the BALB/c mice. This was associated with remarkable amelioration of the dis-
ruption of the colonic architecture, significant reduction in colonic myeloperoxidase
activity and decreased the production of inflammatory mediators, such as NO,
PGE2, and proinflammatory cytokines [86].
PIC undergoes extensive phase II hepatic metabolism, which lowers its
bioavailability. To overcome such limitation, a special capsule containing PIC for
colon-targeted delivery was formulated. The anti-colitic effects of PIC in a
colon-targeted capsule were compared with those of PIC in a conventional gelatin
capsule in a trinitrobenzene sulfonic acid-induced rat colitis, another animal model
of human IBD [87]. Colon-targeted PIC elicited greatly enhanced recovery of the
colonic inflammation. When treated to human colon carcinoma HCT116 cells, PIC
inhibited NF-jB signaling while activating the anti-inflammatory transcription
factor, Nrf2. Colon-targeted PIC, but not conventional PIC, modulated production
of the target gene products of these transcription factors in the inflamed colonic
mucosa [87].

9.3.11 Miscellaneous Effects

9.3.11.1 Septic Shock

Interferon regulatory factor 3 (IRF3) mediates the transcriptional induction of

interferon-stimulated genes in response to viral and bacterial infections. PIC
inhibited the LPS-mediated activation of IRF3 and subsequent interferon-stimulated
gene expression [88]. Furthermore, the LPS-mediated induction of tissue factor, a
cell surface protein responsible for initiating the coagulation cascade, was also
inhibited by PIC. The effectiveness of PIC in blocking both the inflammatory
response and the coagulation pathway is likely to be associated with its protection
against LPS-induced septic shock in a murine model [88].
9 Therapeutic Potential and Molecular Targets of Piceatannol … 203

9.3.11.2 Retinal/Ocular Protection

Inflammatory response has a critical role in neuronal damage after retinal I/R injury,
and is regulated tightly by TLR4. The expression of TLR4 was upregulated after
I/R [89]. TLR4 knockout (KO) mice were much less susceptible to the histologic
damage causedby I/R compared to wild-type mice. The phosphorylation level of
NF-jB after I/R in TLR4 KO mice was decreased compared to that in wild-type
mice. The expression of phosphorylated Syk was upregulated after I/R, and this was
blunted in TLR4 KO mice. PIC inhibited the histologic and functional retinal
damage, and reduced the phosphorylation level of NF-jB induced by I/R [89].
Pre- or post-treatment of PIC significantly blocked the LPS-induced ocular
inflammation in rats [90]. Further, PIC also suppressed the expression of COX-2
and iNOS and activation of NF-jB in the ciliary bodies as well as retina, and also in
primary human non-pigmented ciliary epithelial cells treated with LPS. Similarly,
PIC diminished the LPS-induced production of NO and PGE2 in these cells [90].

9.4 Metabolic Formation of PIC

Microsomal preparation of the human cytochrome P450 (CYP)1B1 and recombinant

human CYP1B1 expressed in E. coli catalyze conversion of resveratrol to PIC [91].
Of interest, CYP1B1 is overexpressed in a wide variety of human tumors, and this
observation implies that the anticarcinogenic effects of resveratrol are, at least in
part, mediated by PIC formed by CYP1B1 activity in the cancer cells. In this context,
CYP1B1 in tumors may function to suppress the growth of cancer cells. In addition,
resveratrol was metabolized to PIC by recombinant human CYP1A1 and CYP1A2
as well as by CYP1B1 [92]. Incubation of trans-resveratrol with human microsomes
produced PIC as a major metabolite, and this was markedly diminished by CYP1A2
inhibitors, a-naphthoflavone, and furafylline. Likewise, an antibody raised against
CYP1A2 suppressed the formation of PIC from resveratrol [92].
CYP102A1 from the bacterium Bacillus megaterium was found to metabolize
various drugs through reactions similar to those catalyzed by human CYP enzymes
[93]. Resveratrol is oxidized to PIC by human P4501A2. The formation of PIC from
resveratrol by human and bacterial CYPs is schematically illustrated in Fig. 9.3.

human microsomal
CYP1B1 and CYP1A1/2
bacterial CYP102A1

Resveratrol Piceatannol

Fig. 9.3 Formation of PIC from resveratrol by human microsomal/recombinant CYP1B1 and
CYP1A1/2 activities. The bacterial CYP101A1 also catalyzes the formation of PIC
204 Y.-J. Surh and H.-K. Na

9.5 Absorption and Biotransformation

PIC is rapidly metabolized in the liver and is converted mainly to a glucuronide or

sulfate conjugate. The absorption and metabolism of PIC were investigated fol-
lowing intragastric administration to rats [79, 94]. PIC and isorhapontigenin
(3,4′,5-trihydroxy-3′-methoxystilbene), an O-methyl PIC metabolite detected in the
plasma, upregulated SIRT1 expression in THP-1 human monocytes [79].
The pharmacokinetics of PIC were investigated in male Sprague-Dawley rats
after single intravenous doses of 10 mg/kg body weight. PIC was extensively
glucuronidated, and predominantly eliminated via non-urinary routes. The estimates
of oral bioavailability characterize PIC as a poorly bioavailable compound [95].
Incubation of PIC with human liver microsomes as well as using a panel of 12
recombinant UDP-glucuronosyltransferase isoforms produced distinct glucuronide
conjugates [96]. Similarly, sulfation of PIC was investigated in human liver cytosol
as well as using a panel of recombinant sulfotransferase isoforms. In the presence of
the sulfate donor, 3′-phosphoadenosine-5′-phosphosulfate, mono- and disulfate
conjugates of PIC were produced [97].
Although trans-resveratrol can be biotransformed to PIC by human CYP1A1 and
CYP1A2, it also inhibit reactions catalyzed by these enzymes [98]. Aryl hydro-
carbon receptor (AhR), following ligand-dependent activation, translocates to the
nucleus where it forms a dimer with aryl hydrocarbon receptor nuclear translocator
(ARNT). The AhR/ARNT complex then binds to AhR response elements located in
regulatory regions of some xenobiotic metabolizing enzymes including CYP1A1
and CYP1B1. Resveratrol has been shown to inhibit dioxin-induced AhR activation
and expression of CYP1A1 and CYP1B1 in cultured human mammary epithelial
MCF10A cells [99] and T-47D human breast carcinoma cells [100]. Likewise, PIC
treatment to T-47D breast cancer cells inhibited CYP1A1 and CYP1B1 mRNA
expression induced by a low (1 nM) concentration of dioxin [100]. Moreover, PIC
also inhibited dioxin-induced recruitment of AhR and ARNT to CYP1A1 and
CYP1B1 enhancer regions. These data show that PCI is an inhibitor of
AhR-dependent transcription and suggest that it may contribute to the prolonged
inhibition of AHR-dependent gene expression following resveratrol treatment [100].
PIC, as well as its parent compound trans-resveratrol, decreased the in vitro catalytic
activity of rat CYP1A1 and CYP1A2 by mixed inhibition [101]. PIC was a weak
inhibitor of mouse hepatic microsomal CYP1A2 activity [102].
Secretion of dehydroepiandrosterone, testosterone, and cortisol was markedly
decreased by a nontoxic dose of [103]. In contrast, secretion of rogesterone and
aldosterone was enhanced. This steroid secretion pattern can be explained by the
demonstrated inhibition of CYP17A1, a key enzyme in the androgen biosynthesis
and a target for prostate cancer therapy. Treatment of cells with PIC caused
increased estradiol levels, which was attributed to its inhibition of estrogen sulfate
conjugation catalyzed by SULT1E1 [103].
9 Therapeutic Potential and Molecular Targets of Piceatannol … 205

9.6 Conclusions and Future Perspectives

As a naturally occurring hydroxylated derivative or a metabolite of well-known

stilbene resveratrol, PIC has a spectrum of biological activity similar to resveratrol
although the relative potency varies depending on the experimental system and
dosage. PIC has a catechol moiety and hence can undergo redox cycling to produce
an electrophilic quinone metabolite capable of interacting with cellular nucle-
ophiles. While the number of publications on the health benefits of PIC is growing,
few human studies on this stilbenoid have been performed. A comprehensive
review of PIC concludes that the compound has the health promoting and disease
preventive potential. However, low water-solubility and bioavailability of PIC limit
its pharmaceutical application and also use in functional foods. In this context, it is
noticeable that b-cyclodextrin was found to improve the bioavailability, the solu-
bility and the stability of PIC [104]. Nanoparticle encapsulated PIC may be con-
sidered as an alternative innovative formulation. Derivatives of PIC with enhanced
bioavailability as well as therapeutic efficacy might also be considered for future
human intervention trials.

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Chapter 10
Fisetin and Its Role in Chronic Diseases

Harish C. Pal, Ross L. Pearlman and Farrukh Afaq

Abstract Chronic inflammation is a prolonged and dysregulated immune response

leading to a wide variety of physiological and pathological conditions such as
neurological abnormalities, cardiovascular diseases, diabetes, obesity, pulmonary
diseases, immunological diseases, cancers, and other life-threatening conditions.
Therefore, inhibition of persistent inflammation will reduce the risk of
inflammation-associated chronic diseases. Inflammation-related chronic diseases
require chronic treatment without side effects. Use of traditional medicines and
restricted diet has been utilized by mankind for ages to prevent or treat several
chronic diseases. Bioactive dietary agents or “Nutraceuticals” present in several
fruits, vegetables, legumes, cereals, fibers, and certain spices have shown potential
to inhibit or reverse the inflammatory responses and several chronic diseases related
to chronic inflammation. Due to safe, nontoxic, and preventive benefits, the use of
nutraceuticals as dietary supplements or functional foods has increased in the
Western world. Fisetin (3,3′,4′,7-tetrahydroxyflavone) is a dietary flavonoid found
in various fruits (strawberries, apples, mangoes, persimmons, kiwis, and grapes),
vegetables (tomatoes, onions, and cucumbers), nuts, and wine that has shown
strong anti-inflammatory, anti-oxidant, anti-tumorigenic, anti-invasive,
anti-angiogenic, anti-diabetic, neuroprotective, and cardioprotective effects in cell
culture and in animal models relevant to human diseases. In this chapter, we discuss
the beneficial pharmacological effects of fisetin against different pathological con-
ditions with special emphasis on diseases related to chronic inflammatory
conditions.

H.C. Pal
R.L. Pearlman
F. Afaq (&)

Department of Dermatology, University of Alabama at Birmingham,
Volker Hall, Room 501, 1670 University Blvd., Birmingham, AL 35294, USA
e-mail: [email protected]
F. Afaq
Comprehensive Cancer Center, University of Alabama at Birmingham,
Birmingham, AL, USA

© Springer International Publishing Switzerland 2016 213

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1_10
214 H.C. Pal et al.

Keywords Fisetin Inflammation Chronic diseases Cytokines Transcription
factors
Nutraceuticals
Neurological diseases

Cardiovascular diseases






Pulmonary diseases Diabetes Obesity Allergy Cancer

Abbreviations
AChE Acetylcholinesterase
CAT Catalase
COX Cyclooxygenase
EMT Epithelial-to-mesenchymal transition
EPCR Endothelial cell protein C receptor
ERK Extracellular signal-regulated kinase
GABAA Gamma-aminobutyric acid A
GLUT4 Glucose transporter type 4
GR Glutathione reductase
GSH Glutathione
GSH-Px Glutathione peroxidase
GST Glutathione S-transferase
HDL High density lipoprotein
HMGB1 High mobility group box 1
IL Interleukin
iNOS Inducible nitrogen oxide synthase
LDL Low-density lipoprotein
LOX Lipoxygenase
MAPKs Mitogen-activated protein kinases
MMP Matrix metalloproteinase
MPO Myeloperoxidase
mTOR Mammalian target of rapamycin
NFjB Nuclear factor-kappa B
NO Nitric oxide
Nrf2 Nuclear factor erythroid-2-related factor 2
PECAM-1 Platelet endothelial cell adhesion molecule 1
PGE2 Prostaglandin E2
PI3K Phosphatidylinositol 3-kinase
PPARc Peroxisome proliferator-activated receptor gamma
SCD-1 Stearoyl-CoA desaturase-1
SCEM Small Clot Embolism Model
SOD Superoxide dismutase
SREBP1C Sterol-regulatory-element-binding protein-1c
STAT Signal transducer and activator of transcription
TARC Thymus and activation regulated chemokine
TLR4 Toll-like receptor 4
TSLP Thymic stromal lymphopoietin
VLDL Very-low-density lipoprotein
10 Fisetin and Its Role in Chronic Diseases 215

10.1 Introduction

Inflammation is a powerful and highly complex adaptive component of the body’s

immune response that helps to repair damaged tissue and protects against a variety
of harmful stimuli, such as pathogens, dead cells, or chemical or physical irritants.
In response to harmful stimuli, initiation of the inflammatory reaction, progression
of inflammation, termination of harmful events followed by resolution of inflam-
mation are major coordinated series of events [1, 2]. These inflammatory responses
attract and activate phagocytic cells such as neutrophils, monocytes, and macro-
phages to destroy pathogens, limit tissue damage, and spread of pathogens by
constructing a physical barrier to repair and heal the damaged tissues. Stage of
inflammation is governed by inflammatory mediators, inflammatory cytokines, and
pro-inflammatory transcription factors. Production of these inflammatory regulators
further recruits inflammatory cells to amplify inflammatory condition [3, 4]. The
end point of acute inflammation is usually favorable. However, there is a fine line
between the beneficial and harmful effects of inflammation. Acute inflammation is
generally a short-term immune response that diminishes after healing or elimination
of pathogens. Inadequate immune response or insufficient inflammation may lead to
delayed wound repair and persistent infection of pathogens. However, the beneficial
and harmful outcome of inflammation depends on precisely controlled response.
Uncontrolled acute immune response can result in allergic response or fatal ana-
phylactic shock. On the other hand, prolonged and dysregulated chronic inflam-
mation leads to development of various chronic conditions such as neurological
abnormalities, cardiovascular diseases, diabetes, obesity, pulmonary diseases,
immunological diseases, cancers, and other life-threatening inflammatory diseases
(Fig. 10.1) [5]. Thus, modulating inflammatory response is of preventive and
therapeutic interest, and approaches targeting inflammation are used to treat a wide
variety of illnesses. Although acute inflammatory conditions can be effectively
managed by steroidal anti-inflammatory drugs (SAID) and nonsteroidal
anti-inflammatory drugs (NSAIDs), long-term treatment of chronic inflammatory
conditions by these drugs is associated with severe adverse effects. A growing body
of evidence has demonstrated that long-term use of NSAIDs results in severe
adverse effects in the gastrointestinal tract and also results in liver toxicities [6, 7].
Therefore, inflammation-related chronic diseases require chronic treatment without
side effects [8].
Use of traditional medicines and restricted diet has been utilized by mankind for
ages to prevent or treat several chronic diseases. The term “nutraceuticals” consists
of “nutrition” and “pharmaceutical” and thus is defined as “a food (or part of a food)
that provides medical or health benefits, including the prevention and/or treatment of
a disease” [9]. Bioactive dietary agents (i.e., nutraceuticals) present in several fruits,
vegetables, legumes, cereals, fibers, and certain spices have shown potential to
inhibit or reverse the inflammatory responses and several chronic diseases related to
chronic inflammation [10, 11]. Bioactive foods containing natural anti-inflammatory
agents are gaining attention due to their potential nutritional value, low toxicity, low
216 H.C. Pal et al.

Fig. 10.1 Inflammation-related chronic disease

cost, oral bioavailability, and preventive/therapeutic effects. Nutraceuticals have

demonstrated several health benefits by preventing or delaying the onset of chronic
diseases; therefore, use of nutraceuticals as dietary supplements or functional foods
has also increased in the Western world [8, 12].
Fisetin (3,3′,4′,7-tetrahydroxyflavone) (Fig. 10.2) is one such dietary flavonoid
found in various fruits (strawberries, apples, persimmons, mangoes, kiwis, and
grapes), vegetables (tomatoes, onions, and cucumbers), nuts, and wine (Fig. 10.3).
Concentration of fisetin in these sources ranged from 2 to 160 lg/g of the material
[13]. The highest amount of fisetin has been found to be present in strawberries,
apples, and persimmon. Fisetin average daily intake has been estimated to be
0.4 mg [13, 14]. It is also abundantly present in various acacias trees and shrubs
belonging to Fabaceae family such as Acacia greggii, Acacia berlandieri,
Gleditschia triacanthow, Anacardiaceae family members such as the parrot tree
(Butea fronds), the honey locust (Gleditsia triacanthos). In addition, fisetin can be

Fig. 10.2 Structure of fisetin

10 Fisetin and Its Role in Chronic Diseases 217

Fig. 10.3 Source of fisetin

found in the Quebracho colorado and Rhus cotinus, lac tree (Rhus vemiciflua
Stokes) extract, smoke tree (Cotinus coggygria), Pinopyta species like Callitropsis
nootkatensis (yellow cypress) and other trees and shrubs (Fig. 10.3) [15]. Fisetin is
a potent antioxidant and free radical scavenger. It has shown potential to inhibit cell
proliferation, growth, and survival of various cancer cells via different mechanisms
[16–21]. Its anti-invasive and anti-angiogenic effects were also recently reported
[22–24]. A growing body of evidence has demonstrated that fisetin has potential to
prevent and/or inhibit various chronic inflammation-related conditions [25–30].
Neuroprotective, cardioprotective, and anti-diabetic potentials of fisetin have been
established by employing cell culture studies and animal models relevant to human
diseases [26, 31–37]. More importantly, treatment of these animals with fisetin was
devoid of any sign of measurable toxicity. Using experimental animals, studies
have demonstrated that fisetin was readily absorbed and distributed to the blood
vessels [38]. Moreover, studies have demonstrated that after 40 min of oral
administration, fisetin can be detected within the blood vessels of the brain for 2 h
suggesting that it is well absorbed and bioavailable in the distal organs [38]. Due to
low toxicity and a wide range of beneficial pharmacological effects, fisetin has been
accepted as a nutraceutical and nutritional dietary supplement for neuroprotection.
As the medical sciences progress, we are beginning to understand with greater
detail the mechanisms by which inflammation, cancer, and chronic disease pro-
gress. Despite having our greater understanding, many chronic disease processes
continue to evade and defy modern therapies. For this reason, novel approaches to
the management of chronic disease are necessary. Fisetin is a natural compound that
218 H.C. Pal et al.

Table 10.1 Effects of fisetin on inflammation-related chronic diseases

Chronic Effects of fisetin References
diseases
Neurological Inhibits progression of Parkinson’s, [31, 35–37, 53]
disorders Alzheimer’s, Multiple sclerosis, and
Huntington’s disease
Enhances memory, object recognition, and [34, 48, 58]
learning
Reduces ROS and LPO [26, 52, 62, 63, 67, 68]
Enhances SOD, CAT, GSH, GST, and AChe [63–66].
Reduces TNFa, IL-1b, COX-2, LOX, TXB, [31, 47, 59, 60, 63–66]
iNOS, NO, and MMPs
Downregulates NFjB and MAPK pathways [35, 36,59, 60]
Enhances neuronal differentiation and survival [32, 54, 59, 62]
Enhances serotonin and noradrenalin [61]
Protects from AlCl3 neurotoxicity [63]
Inhibits invasion of glioblastoma [67, 68]
Diabetes Reduces plasma glucose levels and [69, 70]
gluconeogenesis
Enhances glycolysis and glycogen storage [69, 70]
Restores hexokinase, pyruvate kinase, and [69]
lactate dehydrogenase enzyme activities
Inhibits gluconeogenic enzymes [69]
Activates glycogenesis enzymes [69]
Reduces histone acetylation and histone [71].
acetyltransferases
Reduces TNFa, IL6, and NFjB [26, 71]
Reduces vascular permeability [26]
Reduces expression of ICAM-1, VCAM-1, and [26]
E-selectin
Reduces ROS generation [26, 73, 74]
Reduces kidney hypertrophy and albuminuria [35, 36]
Reduces severity of cataracts and delays onset [72]
of cataracts
Reduces neuropathic pain by targeting GABAA [73, 74]
receptors
(continued)
10 Fisetin and Its Role in Chronic Diseases 219

Table 10.1 (continued)

Chronic Effects of fisetin References
diseases
Obesity Reduces high-fat diet-induced weight gain [75]
Inhibits proliferation and differentiation of [75, 76]
adipocytes
Reduces phosphorylation of mTORC1, AKT, [75]
and S6K1
Inhibits cyclin A, cyclin D1, and cdk 4 [76]
expression
Upregulates p27 expression [76]
Reduces total cholesterol, LDL, VLDL, and [77, 78]
HDL
Normalizes bile acid metabolism and reduces [65, 66, 77, 78]
hepatic abundance of CYP7A1
Reduces lipogenesis and inhibits PRAPc, [77, 78]
SREBP1C and SCD-1
Reduces fatty acid synthesis and ATP citrate [77, 78]
lyase
Inhibits high-fat diet-induced expression of [79]
miR-378
Decreases hepatic fat accumulation [79]
Atherosclerosis Inhibits atherosclerosis [88]
Reduces LDL, VLDL, and enhances HDL [88, 89]
Inhibits oxidation of LDLs by macrophages [90]
Inhibits CD36 expression in macrophages [90, 91]
Inhibits ROS generation in endothelial cells [90]
Cancers Reduces cell proliferation, induces cell cycle [16,19, 21, 22, 44, 100,
arrest and apoptosis by intrinsic and extrinsic 103, 104, 107–110, 117,
pathways 118].
Inhibits cell invasion, EMT, and angiogenesis [23, 24, 46, 67, 68, 105,
106, 117]
Inhibits PI3 K/AKT/mTOR, Wnt/b-catenin, [16, 18, 21, 22, 24, 46, 93,
NFjB, MAPKs, AP-1, and other signaling 104, 106, 109, 122–124]
pathways
Protects from UVB-induced DNA damage and [18, 92, 93]
inflammation
Inhibits infiltration of inflammatory cells [18, 92, 93]
Inhibits inflammatory mediators (COX-2, [18, 92, 93, 109]
PGE2, EP receptors, MPO, MMPs, NO, and
iNOS)
Inhibits inflammatory cytokines (TNFa, IL-1b, [18, 92, 93, 122, 123]
IL-6, and IL-8)
(continued)
220 H.C. Pal et al.

Table 10.1 (continued)

Chronic Effects of fisetin References
diseases
Other Reduces allergic reaction and inhibits atopic [25]
inflammatory dermatitis
diseases Inhibits IgE production and allergic [25]
inflammation
Reduces TNFa, IFNc, IL-1b, IL-4, IL-5, IL-6, [29, 125, 126, 128]
IL-8, IL-13, and hexoaminidase production
Inhibits production of COX-2, NO, and iNOS [28, 130]
Inhibits NFjB, MAPKs, Src, and Syk levels in [25, 26, 28, 30, 129, 130]
immune cells

has evoked a great amount of research interest in recent years, demonstrating potent
anti-inflammatory, anti-tumorigenic, and anti-oxidant properties. In this chapter, we
discuss various pharmacological effects of fisetin against different pathological
conditions with special emphasis on diseases related to chronic inflammatory
conditions (Table 10.1).

10.2 Physciochemical Properties of Fisetin

Fisetin is a yellow bioactive pigment with molecular formula C15H10O6 [IUPAC

Name: 2-(3,4-dihydroxyphenyl)-3,7-dihydroxychromen-4-one)] and molecular
weight of 286.2363 g/mol. Fisetin has a density of 1.688 g/ml and melting point of
330 °C. Its topological polar surface area is 107 Å with low lipophilicity
(CLogP = 1.24). It has four hydrogen bond donors, 6 hydrogen bond acceptors, and
one rotatable bond with one covalently bonded unit count. Fisetin is a rare flavone
without 5-hydroxy substitution and has four hydroxyl groups in its structure. Fisetin
is partly soluble in aqueous buffer. Its solubility in ethanol is approximately
5 mg/ml while in DMSO it is highly soluble (approximately 30 mg/ml) at 25 °C
and gives a yellow color.

10.3 Modulation of Cell Signaling Pathways by Fisetin

Studies have demonstrated that fisetin inhibits proliferation of various cancer cells
in vitro and in vivo. Fisetin at lower doses targets Aurora B kinase by inhibiting
kinetochore and centromere localization leading to immature segregation of chro-
mosomes and premature cessation of mitosis without cytokinesis resulting in
10 Fisetin and Its Role in Chronic Diseases 221

aneuploidy [39]. However at higher doses, fisetin inhibited DNA replication

enzymes topoisomerase I and II resulting in chromosomal breakage [40, 41]. In
addition, fisetin inhibited cell cycle progression by targeting cyclins and
cyclin-dependent kinases (cdks) [42, 43]. Fisetin also induced apoptosis in different
cancer cell lines by modulating expression and translocation of Bcl-2 family pro-
teins involved in the intrinsic apoptotic pathway. In addition, fisetin induced
apoptosis via the extrinsic pathway by enhancing expression of cell surface death
receptors and their ligands such as death receptor 5 (DR5), Fas ligand, and tumor
necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in various cancer
cell lines [44]. Higher affinity of fisetin for androgen receptors (AR) than dihy-
drotestosterone suggested that fisetin inhibits AR-mediated transactivation of target
genes in prostate cancer [45]. Moreover, fisetin inhibited cell proliferation and
survival by dual inhibition of PI3 K/AKT and mTOR signaling. At molecular
levels, fisetin inhibited expression of PI3 K, phosphorylation of AKT and expres-
sion and phosphorylation of mTOR. Fisetin also inhibited mTOR kinase activity
and CAP-dependent protein translation by inhibiting mTOR complex formation
[20, 46]. Fisetin exerted its apoptotic effects by suppressing the phosphorylation of
p38, ERK1/2, and AKT [22, 46]. Fisetin treatment also inhibited NFjB DNA
binding activity and activation of NFjB in different cancer cells. In melanoma cells,
fisetin treatment induced apoptosis by targeting PI3 K and Wnt/b-catenin signaling
pathways [21, 22]. Furthermore, it inhibited cell invasion and metastasis by tar-
geting PI3 K, MAPK, and NFjB signaling pathways and downregulated angio-
genesis by reducing MMP and VEGF expression [22–24]. Fisetin also inhibited
inflammatory responses by reducing NFjB signaling, pro-inflammatory mediators
(such as COX-2/PGE2, NO, iNOS, and MPO), and inflammatory cytokines (such as
TNFa, IL-1b, IL-6, and IL-8) in UVB-induced SKH-1 hairless mice [18].
In addition to its antitumor activity, fisetin is a well-known protector of cere-
brovascular and neurodegenerative diseases by inducing neurite outgrowth and
neuronal differentiation via ERK1/2 activation. Fisetin also suppressed
lipopolysaccharides (LPS)-induced neuroinflammation by inhibiting activation of
NFjB and MAPKs pathways and by reducing pro-inflammatory mediators and
inflammatory cytokines in cell culture as well as in brain microglia of neuropro-
tective murine models [31, 35, 36, 47, 48].

10.4 Role of Fisetin in Chronic Diseases

10.4.1 Fisetin and Neurological Diseases

Various neurological pathologies such as stroke, trauma, Alzheimer’s, and

Parkinson’s have been implicated with oxidative stress-induced nerve cell death.
Epidemiological and experimental studies have shown that flavonoids have
222 H.C. Pal et al.

potential to protect the brain due to their ability to modulate intracellular signals
promoting cellular survival [49–51]. Fisetin effectively protected central nervous
system-derived nerve cells (HT-22 cells) and rat primary neurons from glutamate
toxicity, hypoglycemia, and oxidative injuries by altering glutathione
(GSH) metabolism. In addition, fisetin blocked hydrogen peroxide (H2O2)-induced
neuronal death by reactive oxygen species (ROS) scavenging effects [52]. Fisetin
also inhibited in vitro myelin phagocytosis by macrophages responsible for
secretion of inflammatory mediators, suggesting the potential to reduce the risk of a
chronic inflammatory disease of central nervous system called multiple sclerosis
leading to neurological deficits [53]. In addition, fisetin treatment significantly
reduced ROS production without affecting the viability of macrophage cells. Upon
in vitro experimental evaluation of a variety of flavonoids using a well-studied
model system of neuronal differentiation PC-12 cells expressing nerve growth
factor (NGF) derived from rat embryonic origin from the neural crest, Sagara et al.
[54] found that fisetin was the most effective neuroprotective flavonoid. Employing
an extensive mechanistic approach, it was found that fisetin most effectively
induced neurite outgrowth and PC-12 cell differentiation by activation of the
Ras-ERK cascade, particularly by ERK activation [32, 54].
It was also shown that fisetin promotes nerve cell survival by enhancement of
proteasome activity after neurotrophic factor withdrawal, suggesting that it can also
act as neurotrophic factor [55]. Most importantly, Maher et al. [34] found that
fisetin treatment enhances memory in experimental rats by activation of ERK and
induction of cAMP response element-binding protein (CREB) phosphorylation in
rats hippocampal slices. Moreover, intraperitoneal administration of a single dose of
liposomal preparation containing 30 mg/kg dose of fisetin resulted in detection of
8.23 ng fisetin per gram of brain tissue and exerted protective effects demonstrated
by recovery of the cytoarchitecture in ischemic areas of striatum and cortex in rats.
However, a similar dose of fisetin went undetected in the brain when administered
in aqueous preparation [56]. Similarly, intravenous injection of 50 mg/kg fisetin
initiated after 5 min of embolizaion resulted in significant protection in Rabbit
Small Clot Embolism Model (SCEM) [55]. Moreover, oral administration of 5–
25 mg/kg of fisetin resulted in dose-dependent enhancement in long-term memory
in mice [34]. Furthermore, feeding of mice for 10 months with diet containing
fisetin (500 mg/kg of food) resulted in substantial improvement in learning com-
pared to age-matched mice fed on a fisetin-free diet. Moreover, feeding of
fisetin-containing diet to these mice resulted in significantly improved memory in
the morris water maze (MWM) test relative to age-matched mice fed on a
fisetin-free diet [48]. In addition, studies have demonstrated that feeding of 14.8 gm
of dried aqueous strawberry extract (a major source of fisetin) per kilogram of diet
for 8 weeks to 19-months old rats indicated that fisetin-containing strawberry
extract reversed age-related deficits and improved memory in the MWM compared
to control diet fed rats [57]. Feeding of strawberry extract containing diet to rats
demonstrated better protection against spatial deficits caused by irradiation with
1.5 Gy of 1 GeV/n 56Fe particles. The results from this study provided evidence
that strawberry extract fed animals were better able to retain place information (a
10 Fisetin and Its Role in Chronic Diseases 223

hippocampally mediated behavior) compared to control [58]. These studies clearly

demonstrated that fisetin has potential to protect and enhance survival of nerve
cells, induce differentiation and enhance long-term memory.
Pathogenesis of chronic neurodegenerative diseases such as Alzheimer’s,
Parkinson’s, Huntington’s disease, multiple sclerosis, and HIV-associated dementia
is highly associated with neuroinflammation. Inflammation-mediated neurotoxicity
is governed by microglia, which are the primary immune effector cells in the central
nerves system (CNS). Upon stimulation by LPS, interferon c(IFNc) or b-amyloid
activated microglia secrete various pro-inflammatory cytokines (such as TNFa,
PGE2, IL-1, and IL-6) and free radicals like nitric oxide (NO) and superoxide anion.
Secretion of these pro-inflammatory mediators leads to neuroinflammatory diseases.
Fisetin treatment of LPS-stimulated BV-2 microglia cells and primary microglia
cultures greatly reduced secretion of TNFa, PGE2, and NO [59]. LPS-induced
stimulation of TNFa, IL-1b, COX-2, and iNOS were inhibited both at the mRNA
and protein levels after fisetin treatment [47, 59]. Furthermore, fisetin treatment
inhibited the activation of NFjB, a central regulator of inflammation by reducing
IjB degradation and nuclear translation of NFjB/p65 subunit in LPS-stimulated
BV-2 microglia cells. Fisetin treatment also inhibited phosphorylation of p38 in
these cells. Moreover, fisetin protected B35 neuroblastoma cells from toxicity
induced by activated BV-2 microglia cells in coculture [59]. In mice, intraperitoneal
administration of fisetin (10 and 20 mg/kg) improved neuroinflammation by
reducing IL-1b and inhibiting microglial activation. Fisetin treatment to mice
enhanced the level of heme-oxygenase-1(HO-1) expression, an enzyme associated
with endogenous antioxidative activities. In BV-2 microglial cells induction of
HO-1 expression after fisetin treatment was associated with increase in p38 and
AKT phosphorylation [47].
Overexpression of pro-inflammatory markers, COX-2, and MMP-9, from brain
tumor cells and associated microvascular endothelial cells has been linked to
increased disruption of the blood–brain barrier (BBB) and neuroinflammation as
well as enhanced tumor invasion. The production of COX-2, MMP-9, and other
inflammatory markers is regulated by NFjB signaling [60]. Treatment of human
brain microvascular endothelial cells (HBMECs) with fisetin (30 lM) inhibited
capillary-like structure formation in vitro. By employing zymography,
immunoblotting, and qRT-PCR techniques, Tahanian et al. [60] demonstrated that
fisetin treatment inhibited activity, protein expression, and mRNA levels of COX-2
and MMP-9 in HBMECs induced by phorbol 12-myristate 13-acetate
(PMA) exposure. Furthermore, this study also demonstrated that fisetin-inhibited
PMA induced IjB phosphorylation and activation of NFjB.
Huntington’s disease is a fatal neurodegenerative disorder characterized by
disturbed psychiatric, cognitive, and motor functions. It is a late-onset and pro-
gressive disease involving MAPKs, particularly Ras-ERK signaling cascade.
Studies have demonstrated that activation of ERK provides neuroprotection in
Htt-expressing mutant nerve cells, whereas inhibition of ERK promotes nerve cell
death. Ponasterone treatment has been shown to induce death in *45 % cells
within 72 h by inducing Htt (Httex1-103QP-EGFP) mutation in PC12/HttQ103 cells.
224 H.C. Pal et al.

Studies have demonstrated that treatment of PC12/HttQ103 cells with 2.5–10 lM

fisetin increased cell survival by inducing ERK activation in ponasterone-treated
cells without affecting the formation of Httex1-103QP aggregates or the overall
level of Httex1-103QP-EGFP expression [35]. In addition, fisetin treatment reduced
JNK activation in PC12/HttQ103 cells in which Httex1-103QP-induced JNK phos-
phorylation leads to nerve cell death by caspase-3 activation. Furthermore, feeding
of 1–300 lM fisetin-containing diet to Drosophila flies expressing pathogenic
human Htt(w elav:Gal4/w; P{UAS-Httex1p Q93}/+) Httex1p Q93) in neuronal
cells suppressed Huntington’s disease like symptoms by enhanced ERK phos-
phorylation and activation. Feeding of fisetin-containing diet demonstrated the least
neurodegeneration with rescue of *25 % flies with enhanced survival of up to
77 % at 300 lM concentration of fisetin. Moreover, feeding of 0.05 %
fisetin-containing diet to *6 weeks old transgenic R6/2 mouse (a mammalian
model of Huntington’s disease) for 1 week or 7 weeks showed improved perfor-
mance on the rotorod with *30 % increase in life span as compared to wild-type
littermates [35]. Furthermore, fisetin administration (5, 10, and 20 mg/kg, via
gavage, p.o.) to male ICR mice evaluated for despair tests demonstrated
anti-depressant effects. Neurochemical examination showed that fisetin adminis-
tration enhanced serotonin and noradrenalin production in the frontal cortex and
hippocampus and inhibited monoamine oxidase activity [61].
Moreover, a human case study revealed that consumption of a low-fat diet rich in
fisetin and hexacosanol for 6 months resolved the clinical symptoms of Parkinson’s
disease such as cogwheel rigidity, bradykinesia, dystonia, micrographia, hypomi-
mia, constricted arm swing with gait, and retropulsion. However, only little
improvement in tremor or seborrhea was observed [37]. Studies have also
demonstrated that fisetin (20–80 lM) treatment protected hippocampal neuronal
HT22 and osteoblast-like MC3T3-E1 cells from neurotoxin fluoride,
dexamethasone-induced cytotoxicity, and apoptosis by inhibiting ROS production
[62]. Furthermore, aluminum is a potent environmental neurotoxin that affects
cerebral functions by activating astrocytes, microglia, and associated inflammatory
events. Administration of aluminum chloride (AlCl3) has been associated with
increased lipid peroxidation(LPO), reduction of SOD, CAT, GSH, and GST and
compromised acetylcholine esterase (AChE) activity leading to neurodegenerative
disorders and neuroinflammation by production of pro-inflammatory cytokines such
as TNFa, IL-1b, and iNOS. Mice studies have demonstrated that co-treatment of
AlCl3 and fisetin orally at a dosage of 15 mg/kg b.wt. attenuated AlCl3 induced
neurotoxicity [63]. This study demonstrated that pre- and co-treatment of fisetin
reversed AlCl3 impaired recognition memory and discrimination of the object.
Fisetin administration enhanced production of endogenous antioxidants such as
SOD, CAT, GST, and GSH levels in the brain tissues (cortex and hippocampi) of
mice with reduction in LPO. Fisetin treatment also inhibited activation of astrocytic
and microglial as a result of inhibition of AlCl3-induced production of
pro-inflammatory cytokines such as TNFa, IL-1b, and iNOS in cortex and hip-
pocampus of mice. Furthermore, fisetin ameliorated morphological abnormalities
due to neurotoxicity and neuroinflammation induced by AlCl3 [63]. Studies have
10 Fisetin and Its Role in Chronic Diseases 225

demonstrated that flavonoid-rich methanolic extract of Rhus verniciflua containing

fisetin as one of the ten flavonoids possesses neuroprotective and anti-inflammatory
activities. Isolated fisetin from this extract significantly protected HT22 cells from
glutamate-induced neurotoxicity. Fisetin treatment also protected antioxidative
defense system against glutamate-induced oxidative stress by maintaining activities
of different enzymes such as SOD, GSH, GSH-Px, and GR. Fisetin treatment also
inhibited LPS-induced production of NO, iNOS, and COX-2 in BV2 cells [64–66].
Expression of cytosolic phospholipase A2 (cPLA2), COXs, and LOX is
enhanced in hippocampi of Alzheimer’s mice and these are associated with neu-
roinflammation. Studies employing this mouse model showed that oral feeding of
fisetin in diet (0.05 % of feed, i.e., equivalent to 25 mg/kg of daily dose) restored
cPLA2 levels in the hippocampi to levels similar to that of control. Expression of
COXs and 12-LOX were also reduced by fisetin treatment. Feeding of fisetin to
these mice inhibited production of the pro-inflammatory thromboxanes TXB1 and
TXB2, which are increased in this mouse model. Furthermore, levels of the
pro-inflammatory primary metabolites of 5-LOX and 12-LOX were reduced in
fisetin-treated mice. Levels of multiple monohydroxy docosahexaenoic acids that
are the metabolites of decosahexaenoic acid (DHA) generated by either
auto-oxidation of DHA or metabolized by LOX pathways were also strongly
reduced by fisetin treatment in Alzheimer’s mice [31]. Long-term feeding of fisetin
to mice was safe as no significant difference in body weight was observed.
Furthermore, no toxicity was observed in the pathologic evaluation of lungs, liver,
spleen, kidneys, heart, stomach, intestine, or reproductive organs. In acute toxicity
testing, no toxicity was observed at doses up to 2 g/kg, and Ames test was negative
[31].
In a recent important study, Krasieva et al. [38] using label-free two-photon
microscopy of intrinsic fisetin fluorescence, demonstrated that only fisetin (no other
structurally related flavonols such as 3,3′,4′-trihydroxyflavone and quercetin
(3,5,7,3′,4′-pentahydroxyflavone) localized to the nucleoli in living nerve cells
suggesting that the key targets of fisetin reside in the nucleus. Furthermore, fisetin
was rapidly distributed to the blood vessels of the brain followed by a slower
dispersion into the brain parenchyma of living mice after intraperitoneal injection
and oral administration. After 8 min of intraperitoneal injection of 74 mg/kg of
fisetin, it was observed within the blood vessels of the brain and continued until
15 min before diffusing to adjacent parenchyma. More importantly, after oral
administration of 25 mg/kg fisetin, it was readily detectable within the blood ves-
sels of the brain after 40 min and continued until 2 h in blood vessels and par-
enchyma along with more localized areas suggestive of individual neuronal cell
uptake [38].
Fisetin treatment inhibited invasion of glioblastoma GBM8401 cells. Treatment
with fisetin suppressed the expression of multifunctional gene family, ADAM (a
disintegrin and metalloproteinase) involved in myogenesis, neurogenesis,
226 H.C. Pal et al.

tumorigenesis, angiogenesis, and activation of growth factors/cytokines related to

inflammation. The anti-invasive effect of fisetin was associated with induction of
ERK phosphorylation in these cells [67, 68]. Furthermore, studies have demon-
strated that fisetin protected PC-12 cells from enhanced ROS generation induced by
cobalt dichloride. Fisetin treatment increased hypoxiainducible factor 1a (HIF-1a),
its nuclear accumulation and the hypoxia-response element (HRE)-driven tran-
scriptional activation. These effects were the results of fisetin-induced phosphory-
lation of ERK, p38, and AKT proteins in PC-12 cells [68].

10.4.2 Fisetin and Diabetes

Diabetes mellitus is a widespread, chronic illness characterized by persistent

hyperglycemia due to destruction of pancreatic b-islet cells or acquired insulin
resistance of peripheral cells throughout the body. According to the United States
Center for Disease Control, approximately 9.3 % of the United States population
has been diagnosed with diabetes or about 9.3 million people. Fisetin may have a
role to play in diabetes management as a naturopathic option with fewer side effects
than current diabetes therapies. Studies have demonstrated several potential roles
for fisetin in the modulation of diabetes mellitus.
Fisetin has been found to decrease plasma glucose levels in diabetic animal
models by potentiating glycolysis, inhibiting gluconeogenesis, and increasing
glycogen storage. Administration of oral fisetin to diabetic rats over the course of
one month significantly decreased blood glucose levels, increased insulin and
reduced glycosylation of red blood cells [69, 70]. Oral administration of fisetin to
rats demonstrated similar metabolic changes to rats that receive gliclazide, a known
oral hypoglycemic agent. Fisetin achieved these effects via modulation of enzymes
involved in carbohydrate metabolism. In liver and kidney tissues, fisetin supple-
mentation restored the activity of glycolytic pathway enzymes hexokinase, pyruvate
kinase, and lactate dehydrogenase to near-normal levels. In contrast, treatment with
fisetin inhibited the activity of gluconeogenic enzymes glucose-6-phosphatase,
fructose-1,6-bisphosphatase, and glucose-6-phosphate dehydrogenase. Fisetin
treatment also affected intrahepatic glycogen metabolism in diabetic rats via
increased concentration of glycogen, activation of glycogen synthase, and inhibi-
tion of glycogen phosphorylase [69].
Evidence suggests that fisetin may have a role in the regulation of
hyperglycemia-induced inflammatory responses. Innate and secondary immune cells
synthesize and release inflammatory cytokines under hyperglycemic conditions. In
human monocytic THP-1 cells, culture in hyperglycemic environments activates
NFjB, and induces synthesis of IL-6 and TNFa. Treatment with fisetin reduced
10 Fisetin and Its Role in Chronic Diseases 227

expression of these pro-inflammatory cytokines and reduced activation and translo-

cation of NFjB [26, 71]. Fisetin exerted its anti-inflammatory effects via epigenetic
regulation. Changes in expression of inflammatory cytokines were likely due to a
decrease in histone acetylation via inhibition of histone acetyltransferases [71].
In addition to modulation of pro-inflammatory cytokines, fisetin has been found
to decrease hyperglycemic vascular inflammation. Recent studies show that fisetin
can affect several pathophysiologic processes that promote vascular inflammation
including vascular permeability, leukocyte adhesion, and migration, and ROS
generation. Data from in vivo studies suggested that pretreatment with fisetin pre-
vents hyperglycemia-induced increases in vascular permeability of albumin. These
findings were confirmed in murine models. In addition, pretreatment with fisetin
inhibited hyperglycemia-induced overexpression of intercellular adhesion molecule
1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and E-selectin in
endothelial cells; this reduction results in decreased THP-1 adhesion to
hyperglycemia-activated human umbilical vein endothelial cells (HUVECs) [26].
Vascular inflammation in diabetes patients promotes atherosclerosis and thrombo-
sis, and fisetin treatments may be a viable option for reducing the potential for
long-term cardiovascular sequelae.
Fisetin may also play a role in alleviating or reducing common complications
related to diabetes mellitus. There is a well-established link between diabetes
mellitus and abnormalities of lipoprotein metabolism. A recent study demonstrated
that fisetin treatment of rats with streptozotocin-induced diabetes returned serum
LDL and VLDL levels to normal range. Additionally, HDL levels were increased in
fisetin-treated rats compared to controls [70].
Diabetes patients also frequently experience complications due to macromolec-
ular glycosylation and hyperglycemia in multiple organ systems. Fisetin has been
shown to potentiate removal of methylglyoaxal from macromolecules increase
synthesis of glutathione. Activation of glyoxalase 1 by fisetin reduces the number of
proteins glycated by methylgoyoxal. In Akita mice modes, this effect was linked to a
reduction of kidney hypertrophy and albuminuria, both reduced by fisetin treatment
[36]. Ophthalmic complications often include cataracts. A recent study demonstrated
that fisetin treatment of mice with streptozotocin-induced diabetes reduced the
severity of cataracts and delayed onset of late stage cataracts [72]. Moreover,
antinociceptive effects of fisetin against diabetic neuropathic pain targeting
spinal c-aminobutyric acid A (GABAA) receptors in mice with type 1 diabetes have
been reported [73, 74]. Chronic treatment of streptozotocin-induced diabetic rats with
5–45 mg/kg body weight of fisetin administered orally twice per day for two weeks,
delayed development of thermal hyperplasia and mechanical allodynia. Furthermore,
fisetin treatment reduced oxidative stress in tissues of spinal cord, dorsal root gan-
glion, and peripheral nerves. The analgesic effect of fisetin was further potentiated by
combination of fisetin with ROS scavenger phenyl-N-tert-butylnitrone.
228 H.C. Pal et al.

10.4.3 Fisetin and Obesity

In the United States, obesity is one of the greatest public health concerns of the
twenty-first century. Several mechanisms have been suggested by which fisetin
treatment may mitigate the pathogenesis of diet-induced obesity. Preliminary evi-
dence suggests that fisetin supplementation may reduce the risk of developing
obesity by decreasing differentiation and proliferation of adipocytes.
Undifferentiated fibroblasts, or preadipocytes, have been identified as a potential
target of fisetin treatment. Recent studies have demonstrated that differentiation of
3T3-L1 undifferentiated fibroblasts into adipocytes is reduced by fisetin [75, 76].
One study found that fisetin treatment reduced phosphorylation of mTORC1 and
upstream promoters of mTORC1 signaling including AKT and S6K1. In vivo
experiments confirmed these findings; in murine models fed high-fat diets; fisetin
supplementation reduced weight gains and accumulation of white adipose tissue via
suppression of mTORC1 signaling and reduced differentiation of preadipocytes
[75]. Another study suggested that fisetin treatment decreased adipocyte differen-
tiation and proliferation by suppressing mitotic clonal expansion. Fisetin treatment
reduced expression of several key cell cycle promoters including cyclin A, cyclin
D1, and cdk4. In addition, fisetin upregulated the cell cycle inhibitor p27, pro-
moting a sustained G0 phase [76].
Fisetin may play a role in obesity treatment or prevention by modulating
cholesterol homeostasis. In Sprague-Dawley hypercholesterolemic rat models,
treatment with fisetin reduced several critical markers of obesity risk. The blood
lipid profile of rats on a high-fat diet was improved by fisetin treatment; total
cholesterol, LDL, HDL, and hepatic cholesterol levels were all reduced. The
hepatic abundance of CYP7A1 was reduced to near control levels after fisetin
treatment, suggesting a return to normal bile acid metabolism. Another possible
mechanism by which fisetin may regulate obesity pathogenesis is by reducing
hepatic lipogenesis. In Sprague-Dawley rats fed with high-fat diets, fisetin reduced
expression of hepatic mRNA associated with lipogenesis including PPARc,
SREBP1C, and SCD-1 compared to controls. Expression of gene products asso-
ciated with fatty acid synthesis including fatty acid synthase and ATP citrate lyase
were also markedly reduced by fisetin treatment. In addition, fisetin-induced
expression of GLUT4 in 3T3-L1 differentiated adipose cells, decreasing concen-
trations of serum glucose [77, 78]. Another study found that fisetin treatment
inhibited high-fat diet-induced expression of miR-378 and PGC-1B resulting in
decreased hepatic fat accumulation and reversal of metabolic enzyme dysregula-
tion [79]. These data indicate that fisetin treatment may attenuate obesity by
reducing hepatic lipid biosynthesis.
10 Fisetin and Its Role in Chronic Diseases 229

10.4.4 Fisetin and Atherosclerosis

Atherosclerosis is a chronic disease of the arteries and considered a leading cause of

mortality and morbidity associated with cardiovascular disease. Initially,
atherosclerosis was believed to be a disease of lipid accumulation in the arterial
wall; however, a growing body of evidence has demonstrated that dysregulated
lipid metabolism is not the only cause of atherosclerosis, but maladaptive chronic
inflammatory responses also play a critical role in the initiation and progression of
atherosclerosis [80–82]. Accumulation of lipid-laden macrophages in the suben-
dothelial area of the arterial wall is considered a hallmark of atherosclerosis. In
addition, results from recent studies have demonstrated that neutrophils also
modulate the pathogenesis of arthrosclerosis [83, 84]. Moreover, the role of IL-1b,
IL-6, TNFa, P-selectin, and 5-LOX is well documented in the promotion of
atherosclerosis. Experimental and preclinical studies have demonstrated that
anti-inflammatory and immune-modulatory therapies reduced the risk of cardio-
vascular disease and atherosclerosis [85–87].
In vitro studies have suggested that fisetin treatment may be a potent inhibitor of
a key step in atherosclerosis pathogenesis. Uptake of LDLs by macrophages results
in the formation of foam cells, and the accumulation of foam cells promotes
atherosclerotic plaque development. Studies have found that fisetin inhibits oxi-
dation of LDLs by macrophages. Fisetin preserves the antioxidant properties of
a-tocopherol associated with LDLs and prevents oxidation of this critical com-
pound [88, 89]. In addition, fisetin inhibits copper ion-dependent LDL oxidation
and subsequently blocks binding of oxidized-LDLs to the Class-B scavenger
receptor CD36 on macrophages, which has been associated with atherosclerotic
lesions [90, 91]. Inhibition of CD36 receptor expression in macrophages by fisetin
is achieved by decreasing mRNA expression [90]. Although these in vitro findings
are promising, in vivo studies demonstrating these effects have not yet emerged.

10.4.5 Fisetin and Skin Cancer

Basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) are the most
frequently diagnosed non-melanoma skin cancers (NMSCs). Ultraviolet
(UV) irradiation is considered the most important extrinsic factor contributing to
inflammation and skin cancer. Consumption of nontoxic dietary flavonoids to
potentially prevent skin cancers has drawn a great deal of attention. Treatment of
human epidermoid carcinoma A431 cells with fisetin resulted in decease in cell
proliferation. Fisetin treatment enhanced G2/M cell population and induced apop-
tosis through disruption of mitochondrial membrane potential and modulation in
Bcl-2 family proteins. Fisetin treatment also promoted release of cytochrome c and
230 H.C. Pal et al.

Smac/DIABLO proteins from mitochondria to cytosol, inducing activation of

caspases, and PARP cleavage [19]. Studies have demonstrated that the expression
of COX-2, PGE2, MMPs, and other inflammatory mediators increases after UVB
exposure. Fisetin treatment of UVB-irradiated human fibroblasts inhibited the
expression of COX-2, PGE2, and MMPs as well as collagen degradation. Fisetin
treatment also inhibited UVB-induced intracellular ROS and NO production [92].
UVB-induced phosphorylation of MAPKs was inhibited by fisetin treatment. In
addition, fisetin suppressed NFjB activation and translocation of p65 subunit to the
nucleus along with the inhibition of phosphorylation of cAMP response
element-binding protein (CREB) at Ser133 [92].
Oxidative damage and inflammation are key factors in the pathogenesis of skin
cancers. Studies have shown that fisetin treatment to HaCaT cells induced nuclear
factor erythroid-2-related factor 2 (Nrf2)-related HO-1 protein and mRNA
expression. HO-1 is known to inhibit inflammatory responses by inhibiting neu-
trophil trafficking. HO-1 is a member of antioxidant response element (ARE)-
related expression of phase 2 detoxifying genes regulated by Nrf2 and work as a
rate-limiting enzyme in heme catabolism during UV light- or hypoxia-induced
inflammatory responses. Fisetin treatment inhibited TNFa-induced expression of
iNOS and COX-2, production of NO, PGE2, IL-1b, IL-6, and activation of NFjB
in HaCaT cells by inducing nuclear translocation of Nrf2 [93]. Furthermore, topical
application of fisetin inhibited UVB-induced cell proliferation, hyperplasia, and
infiltration of inflammatory cells in SKH-1 hairless mouse skin [18]. Fisetin
treatment also reduced UVB-induced DNA damage evidenced by accelerated
removal of cyclobutane pyrimidine dimers and enhanced expression of p53 and p21
proteins. Moreover, topical application of fisetin resulted in inhibition of
UVB-induced inflammatory mediators (such as COX-2 and PGE2), their receptors
(EP1–EP4), and MPO activity, along with reduction in inflammatory cytokines
(such as TNFa, IL-1b, and IL-6). Fisetin treatment also inhibited UVB-induced
activation of PI3 K/AKT and NFjB signaling pathways [18].
Melanoma, is the least common but most lethal form of skin cancer. Due to its
metastatic potential, melanoma accounts for approximately 80 % of all skin
cancer-related deaths. The incidence of melanoma is increasing worldwide at an
alarming rate. The global incidence rate of melanoma is 12–25 per 100,000 indi-
vidual populations. Incidence rates are highest in Australia and New Zealand
with  60 cases per 100,000 inhabitants per year. The incidence rates in the United
States and Europe are  30 and *20 cases per 100,000 per year, respectively.
According to an estimate, 73,870 new cases of cutaneous melanoma and 9940
deaths due to cutaneous melanoma have been projected to occur in the United
States in 2015 [94, 95]. Exposure to solar UV radiation is still considered one of the
major risk factors for melanoma development. White populations with fair skin are
at higher risk for developing melanoma than pigmented populations. Moreover,
detection of cyclin-dependent kinase inhibitor 2A (CDKN2A) and CDK4 germline
10 Fisetin and Its Role in Chronic Diseases 231

alterations in families have demonstrated a genetic inheritance pattern of melanoma.

In addition, gain of oncogenic functional mutations in BRAF, NRAS, and KIT have
been observed in the majority of melanomas. Furthermore, evidence has demon-
strated the cooperation between these oncogenic mutations and PI3 K/AKT/mTOR,
and PTEN signaling pathways supports melanoma development. In addition, the
cytokine⁄chemokine spectrum of melanoma tumor microenvironment significantly
overlaps with chemoattractant and inflammatory mediators produced by neutrophils
and macrophages at the site of inflammation. Moreover, tumor growth, angiogen-
esis, and metastasis are enhanced by inflammatory tumor microenvironment [96–
98]. An accumulating body of evidence has demonstrated that human melanoma
cells produce various cytokines such as IL-6, IL-8, CXCL1–3 (MGSA-GROa-c),
CCL5 (RANTES), and monocyte chemotactic protein-1 (MCP-1, also known as
CCL2) that are regulated by IL-1b, suggesting that IL-1b may be a potential link
between inflammation and melanoma [99].
Studies have demonstrated that fisetin inhibits melanoma cell growth and
induces apoptosis. Fisetin treatment to human melanoma 451Lu cells induced
G1-phase cell cycle arrest and downregulated cell cycle regulatory cdks (2, −4, and
−6) protein expression. Fisetin treatment resulted in downregulation of Wnt5a
protein expression and its coreceptor (Frizzled/LRP6). Moreover, these treatments
stimulated cytosolic degradation of b-catenin, resulting in decreased nuclear
localization of b-catenin. Furthermore, fisetin treatment downregulated the protein
levels of c-myc, Brn2, and Mitf, which are positively regulated by the
b-catenin/TCF complex. These data demonstrated that fisetin interfered with the
functional cooperation between TCF-2 and b-catenin in melanoma cells [21].
Fisetin also induced apoptosis in melanoma cells through induction of ER stress
and activation of extrinsic and intrinsic apoptotic pathways [100]. Employing silico
modeling and cell-free competition assays, it has been demonstrated that fisetin
inhibits human melanoma cell growth through direct binding to p70S6 K and
mTOR [101]. Moreover, treatment of BRAF-mutant, NRAS-mutant, and wild-type
melanoma cells with fisetin (5–20 lM) resulted in a significant decrease in cell
invasion. The anti-invasive effect of fisetin was also observed in three-dimensional
skin equivalents consisting of human melanoma A375 cells. Furthermore, fisetin
treatment modulated the expression of epithelial-to-mesenchymal transition
(EMT) proteins. The anti-invasive and anti-EMT effects of fisetin were associated
with a decrease in the phosphorylation of MEK1/2 and ERK1/2 and reduction in the
activation of the NFjB signaling pathway [24]. In addition, oral administration of
fisetin (45 mg/kg b.wt.) inhibited melanoma xenograft tumor growth in nude mice
implanted with BRAF mutated melanoma cells. Melanoma growth inhibition and
pro-apoptotic effects of fisetin were observed due to reduction in MAPK and
PI3 K/AKT/mTOR signaling pathways [22]. Moreover, analysis of tumor xeno-
graft tissues revealed that fisetin inhibited EMT progression by reducing the
expression of EMT-related transcription factors such as Snail1, Twist1, ZEB1, and
Slug. Fisetin treatment also inhibited angiogenesis and lung colonization of mela-
noma cells injected intravenously in the tail vein of athymic nude mice [23].
232 H.C. Pal et al.

10.4.6 Fisetin and Prostate Cancer

Prostate cancer is one of the most common cancers and leading causes of death in
men [95, 102]. Consumption of flavonoid-rich diets in East Asian countries such as
China and Japan has been associated with a 60- to 80-fold lower incidence and
reduced mortality of prostate cancer [103]. Fisetin has been found to be effective
against prostate cancer. In vitro studies using fisetin have demonstrated that fisetin
inhibits cell proliferation and induces cell cycle arrest and apoptosis in
androgen-sensitive human prostate LNCaP and CWR22Rʋ1 cells as well as in
androgen receptor (AR)-negative prostate cancer PC-3 cells [103, 104].
Furthermore, fisetin treatment inhibited viability and colony formation of a
P-glycoprotein-overexpressing multi-drug resistant cancer cell line NCI/ADR-RE
[105]. Importantly, fisetin exhibited minimal cytotoxic effects on normal prostate
epithelial cells (PrECs). Fisetin treatment induced cell cycle arrest at G2/M phase in
PC-3 cells; whereas LNCaP cells were arrested at G1 phase of cell cycle. G1-phase
cell cycle arrest in LNCaP cells induced by fisetin treatment was associated with
reduced protein expression of cyclins D1, D2, and E. Fisetin treatment also reduced
the protein expression of cdks 2, 4, and 6 with simultaneous increase in protein
expression of WAF1/p21 and KIP1/p27. Furthermore, fisetin treatment induced
apoptosis in LNCaP cells through induction of pro-apoptotic proteins (Bax, Bak,
Bad, and Bid) and inhibition of anti-apoptotic proteins (Bcl-2 and Bcl-xL), cyto-
chrome c release, activation of caspases (3, 8, and 9) and cleavage of PARP. In
addition, fisetin treatment also reduced protein expression of upstream regulators of
apoptosis such as PI3 K and decreased the phosphorylation of AKT at Ser473 and
Thr308, which are involved in cell proliferation and survival [104]. Fisetin induced
PC3 cell death by induction of autophagy, as observed by an increase in LC3 II
protein expression. Fisetin treatment of PC-3 cells resulted in inhibition of mTOR
kinase activity, basal expression of mTOR and autophosphorylation of mTOR at
Ser2481. It also inhibited formation of mTORC1/2 complexes via downregulation of
Raptor, Rictor, PRAS40, and GbL protein expression. Fisetin treatment also
inhibited the activation of p70-S6 kinase (S6K70) and increased expression of
eukaryotic translation initiation factor 4E-binding protein 1(4EBP1) by its
dephosphorylation from hyperphosphorylated c form to the hypo- or
non-phosphorylated a form. Furthermore, fisetin treatment of PC-3 cells disrupted
assembly of translation complex by increasing eIF4E bound 4EBP1 and simulta-
neous reduction in eIF4G binding to eIF4E [20].
Studies have demonstrated that combination of fisetin with TRAIL resulted in
enhanced apoptosis of TRAIL-resistant androgen-dependent LNCaP cells and the
androgen-independent DU145 and PC-3 cells [44]. In TRAIL-resistant LNCaP
cells, fisetin treatment increased the expression of TRAIL-R1 and reduced NFjB
activity. Moreover, studies have demonstrated that fisetin (5–20 lM) inhibited
adhesion, migration, and invasion of highly metastatic PC-3 cells [46, 105]. Fisetin
treatment significantly reduced protein expression as well as mRNA expression of
MMP-2 and MMP-9 involved in the degradation of ECM to facilitate invasion and
10 Fisetin and Its Role in Chronic Diseases 233

migration of tumor cells. Activation of JNK1/2 was suppressed due to decreased

phosphorylation of JNK1/2 in PC-3 after fisetin treatment; however, phosphory-
lation of ERK1/2 and p38 was not affected in these cells. Furthermore, fisetin
treatment inhibited expression of PI3 K and phosphorylation of AKT and decreased
the protein expression and DNA binding activities of NFjB and AP-1 (c-Fos, and
c-Jun) involved in transcriptional and translational regulation of MMP-2 and
MMP-9 expression, which are required for invasion and migration of prostate
cancer cells [46]. In addition, studies have demonstrated that fisetin promoted an
epithelial phenotype cellular morphology in the two prostate cell lines DU145 and
C4-2 and decreased migration. Fisetin treatment inhibited EMT in prostate cancer
cells by inducing mRNA and protein levels of E-cadherin while downregulating
mRNA and protein levels of vimentin and slug. Moreover, fisetin treatment reduced
EGF-induced YB-1 phosphorylation (required for EMT progression) at Ser102 both
in vitro and in vivo by interacting with the cold shock domain (CSD) domain of
YB-1 [106]. Surface plasmon resonance and computational docking studies sug-
gested that fisetin binds to b-tubulin and stabilizes microtubules by upregulating
microtubule associated proteins (MAP)-2 and -4 [105].

10.4.7 Fisetin and Colon Cancer

In Western countries, colon cancer remains one of the leading causes of

cancer-related deaths. Modification of life style and diet habits, including con-
sumption of vegetables and fruits can reduce the risk of colon cancer. Dietary
flavonoid fisetin inhibited cell growth and clonogenicity of human colon cancer
cells. Fisetin inhibited cell cycle progression in HT-29 cells by G2/M phase arrest.
Fisetin treatment suppressed cdk2 and cdk4 activities resulting in a decrease in the
level of cyclin E and D1 with an increase in p21 levels. Fisetin particularly targeted
cdk4 activity in cell-free system, indicating that cdk4 may be the direct target of
fisetin. In addition, fisetin treatment resulted in reduced phosphorylation (from
hyperphosphorylated to hypophosphorylated) of retinoblastoma (Rb) proteins
[107]. Moreover, cdc2 and cdc25c kinase protein expression and kinase activity of
cdc2 were reduced in HT-29 cells after fisetin treatment. Induction of tumor sup-
pressor gene p53 by fisetin contributes to apoptosis in human colon cancer
HCT-116 cells harboring the wild-type p53 gene [108]. Fisetin-induced apoptosis
was accompanied by reduction in expression of anti-apoptotic Bcl-2 and Bcl-xL
proteins with concomitant increase in pro-apoptotic Bak and Bim proteins.
Fisetin-induced mitochondrial translocation of Bax protein resulted in increased
mitochondrial membrane permeability and release of cytochrome c and
Smac/DIABLO from mitochondria to cytosol. In addition, fisetin treatment resulted
in caspase-3 and PARP cleavage [108, 109]. Moreover, fisetin treatment inhibited
protein expression and activity of COX-2 in HT-29 cells (COX-2 overexpressing
colon cancer cell line), which are known to play a crucial role in colon carcino-
genesis. However, fisetin treatment did not affect COX1 expression in HT-29 cells.
234 H.C. Pal et al.

Fisetin treatment also inhibited activation and translocation of NFjB, which are
required for stimulation of COX-2 expression. PGE2 secretion was also reduced as
a consequence of COX-2 inhibition by fisetin in HT-29 cells. Fisetin did not affect
EP-2 and EP-4 expression, suggesting that fisetin inhibits COX-2/PGE2 signaling
through regulating ligand availability. Moreover, fisetin treatment also inhibited
phosphorylation of EGFR at the Tyr1068 residue in HT-29 cells as a consequence of
PGE2 inhibition, which is a known transactivator of EGFR leading to promotion of
tumor growth and invasion [109]. Furthermore, depletion of Securin expression
(also known as pituitary tumor transforming gene and acts as a marker of inva-
siveness in colon cancers) sensitized human colon cancer cells to fisetin-induced
apoptosis. Phosphorylation of p53 and cleavage of caspase-3 and PARP were
enhanced in HCT116 securin-null cells or in wild-type cells in which securin was
knock down [110]. Studies have also demonstrated that the apoptotic effect of
fisetin on colon cancer cells (COLO205, HCT-116, HT-29, and HCT-15) is
enhanced with N-Acetyl-L-Cysteine treatment, suggesting that fisetin-induced
apoptosis in colon cancer cells is independent of ROS induction [111, 112].

10.4.8 Fisetin and Lung Cancer

Lung cancer is the most deadly cancer in the United States and worldwide. Tobacco
smoking is considered as the most important risk factor for lung cancer develop-
ment. Results from clinical and epidemiologic studies have demonstrated a strong
association between chronic inflammation and lung cancer [113, 114]. A growing
body of evidence has demonstrated that tobacco smoke exposure induces car-
cinogenic inflammatory responses and mutagenic effects in the lungs. Infiltration of
inflammatory cells in the lungs is the initial pathological hallmark of smoking.
These inflammatory macrophages and neutrophils produce pro-inflammatory
mediators and cytokines to further enhance the inflammatory condition and pro-
mote tumor growth [115, 116]. Fisetin treatment significantly inhibited lung cancer
cell proliferation but had minimal toxic effects on normal human embryonic
epithelial cells at physiologically achievable concentration [16, 117]. Fisetin
treatment induced intracellular ROS production, mitochondrial membrane depo-
larization, and apoptosis in NSCLC cells with an increase in Sub-G1 cell popula-
tion. Fisetin treatment resulted in reduced expression of Bcl-2 and enhanced
expression of Bax, as well as activation of caspase-3 and -9 [118]. Relatively
nontoxic concentrations of fisetin inhibited adhesion, invasion, and migration of
A549 cells. Gelatin and casein zymography experiments demonstrated that fisetin
inhibited MMP-2 and u-PA at protein and mRNA levels. In addition, fisetin
decreased ERK1/2 phosphorylation, however it did not affect the phosphorylation
of JNK1/2 and p38. Moreover, fisetin inhibited DNA binding activities of tran-
scription factors NFjB and AP-1 (c-Fos, and c-Jun) in A549 cells [117].
Furthermore, fisetin treatment to lung cancer cells inhibited the expression of
regulatory (p85) and catalytic (p110) subunits of PI3 K and phosphorylation of
10 Fisetin and Its Role in Chronic Diseases 235

AKT both at Ser473 and Thr308 moieties. Fisetin treatment also activated PTEN, a
negative regulator of PI3 K signaling, and increased phosphorylation of AMPKa
kinase thus inhibiting protein translation by mTOR. Fisetin treatment also inhibited
the phosphorylation of mTOR at Ser2448 as a consequence of inhibition of AKT
phosphorylation [16].
In experimental lung carcinogenesis, fisetin inhibited benzo(a)pyrene-induced
lung cancer development in Swiss albino mice [119]. Histological evaluation of
lungs revealed that fisetin treatment significantly reduced the degree of histological
lesions with reduced cell proliferation. Biochemical analysis demonstrated that
fisetin treatment restored enzymatic and nonenzymatic antioxidants. Furthermore,
evaluation of mitochondrial specific enzymes and tumor markers demonstrated that
fisetin treatment inhibited production of isocitrate dehydrogenase, a-ketoglutarate
dehydrogenase, succinate dehydrogenase, malate dehydrogenase and carcinogenic
embryonic tumor antigen in benzo(a)pyrene-induced lung carcinogenesis [120]. In
addition, fisetin treatment resulted in release of cytochrome c and activation of
caspase-3. Furthermore, fisetin treatment inhibited viability of Lewis lung carci-
noma (LLC) and endothelial cells (EAhy 926), with minimum effect on normal NIH
3T3 cells. NIH3T3 cells were five times less sensitive to fisetin than either LLC or
endothelial cells, demonstrating that fisetin specifically targets cancer cells and
endothelial cells involved in tumor angiogenesis [121]. Fisetin treatment to LLC
cells induced apoptosis and accumulation of cells in G2/M phase with concomitant
decrease in G1 phase. Endothelial cells (EAhy 926) were more sensitive to fisetin
treatment with increase in sub-G1cells and decrease in G1, S, and G2/M cells.
Fisetin treatment also inhibited migration and capillary-like structure-forming
abilities of endothelial cells and inhibited tumor growth and angiogenesis in vivo as
demonstrated by reduced expression of PECAM-1. Moreover, antitumor activity of
fisetin was enhanced in combination with cyclophosphamide [121].
Intraperitoneal administration of 1 or 3 mg/kg fisetin in BALB/c mice inhibited
ovalbumin-induced allergic asthma, which is a chronic disease of lung inflamma-
tion, airway hyper-responsiveness and mucus overproduction associated with the
bronchial epithelium, mucus-secreting glands and lung parenchyma [122, 123].
Treatment of fisetin in experimental asthma mouse model resulted in inhibition of
lung inflammation, goblet cell hyperplasia, and airway hyper-responsiveness. These
effects were associated with a decrease in eosinophils and lymphocytes in bron-
choalveolar lavage fluid. In addition, fisetin treatment reduced expression of
eotaxin-1 and thymic stromal lymphopoietin (TSLP) (key initiators of allergic
airway inflammation), IL-4, IL-5, and IL-13 (Th2-associated cytokines) production
in lungs. Fisetin treatment also inhibited mRNA expression of adhesion molecules,
chitinase, IL-17, IL-33, Muc5ac, and iNOS, as wells as eosinophilia and airway
mucus production in lung tissue induced by ovalbumin. In addition, fisetin treat-
ment also inhibited expression of Th2-predominant transcription factor GATA-3
and cytokines in thoracic lymph node cells and splenocytes. Moreover, in
TNFa-stimulated bronchial epithelial cells and OVA-stimulated lung tissues, fisetin
inhibited activation of NFjB by blocking nuclear translocation of subunit p65 and
DNA binding activity [122, 123]. A recent study demonstrated that fisetin inhibits
236 H.C. Pal et al.

LPS-induced acute lung injury by downregulation of TLR4-mediated NFjB sig-

naling pathway in rats [124]. The results of this study demonstrated that
LPS-induced increase of neutrophil, MPO activity and macrophage infiltration in
lung tissues were attenuated by fisetin treatment via inhibition of TLR4 and
NFjB [124].

10.4.9 Fisetin and Other Inflammatory Diseases

Consumption of flavonoid-rich fruits and vegetables has been associated with

reduced risk of other chronic inflammatory conditions. Generally, cross-linking of
the cell-bound specific antigens with mast cell and basophils leads to release of
inflammatory mediators, histamine, leukotrienes and cytokines (such as IL-4, IL-5,
and IL-13) related to IgE production, TH2 differentiation and allergic inflammation.
Pro-inflammatory cytokines derived from activated mast cells play an important
role in the development of acute- and late-phase allergic inflammatory reactions.
Studies have demonstrated that fisetin treatment inhibited allergic inflammation by
reduction in mRNA expression and secretion of IL-4, IL-5, and IL-13 in allergen
stimulated KU812 cells and basophils [125, 126]. In addition, among the 13 fla-
vonoids tested, fisetin was the most potent inhibitor of hexosaminidase secretion
from allergen stimulated RBL-2H3 cells [127]. Furthermore, treatment of fisetin to
PMA plus calcium ionophore A23187 (PMACI) stimulated human mast cells
(HMC-1) suppressed the gene expression and production of inflammatory cytokines
TNFa, IL-1b, IL-4, IL-6, and IL-8 [29, 128]. Moreover, fisetin treatment inhibited
activation of MAPKs by reducing phosphorylation of p38, ERK, and JNK. In
addition, fisetin treatment inhibited PMACI-induced transcriptional activation of
NFjB, NFjB/DNA binding and enhanced phosphorylation and degradation of
IjBa [29, 128]. Further studies on mast cell (HMC-1) by activated T cell membrane
demonstrated that fisetin inhibited mast cell activation by inhibition of cell-to-cell
interactions, reduction in the amount of cell surface antigen CD40 and ICAM-1 and
down regulation of NFjB and MAPKs pathways [129].
Animal studies employing 2,4-Dinitrofluorobenzene (DNFB)-induced allergic
contact dermatitis mouse model demonstrated that Bark of Rhus verniciflua Stokes
containing fisetin as its major constituent and isolated fisetin inhibited TNFa, IL-6
and iNOS production mediated through NFjB signaling pathway [28]. Atopic
dermatitis is a relapsing and pruritic inflammatory skin disease in which infiltration
of inflammatory cells and production of inflammatory cytokines in the skin lesions
is enhanced. Enhanced cutaneous hyper-sensitivity to immunoglobulin E (IgE)-
mediated sensitization promotes development of intense pruritus, edema, erythe-
matous, scaly and lichenified lesions in the skin [25]. During acute response,
production of inflammatory cytokines (such as IL-4, IL-5 and IL-13) and IgEis
increased by infiltratory eosinophil and mast cells (Th2 cells). Fisetin treatment has
been associated with reduced production of these inflammatory mediators from
eosinophils and mast cells. Whereas, in chronic atopic dermatitis, dermal thickening
10 Fisetin and Its Role in Chronic Diseases 237

and tissue remodeling by excessive collagen accumulation due to IFNc and IL-2
(Th1-dominat immune response) is associated with delayed-type hyper-sensitivity.
A recent study by Kim et al. [25] demonstrated that oral administration of fisetin at
20 or 50 mg/kg daily from days 8 to 15, significantly inhibited DNFB-induced
atopic dermatitis-like clinical symptoms such as erythema, edema, oozing, and
excoriation in NC/Nga mice. Fisetin treatment also inhibited DNFB-induced epi-
dermal thickness and infiltration of eosinophils, mast cells, CD4+ T and CD8+ T
cells in ear and dorsal skin. Moreover, fisetin treatment also reduced expression of
Th2 cytokines IL-5, IL-13, TNFa, thymus, and activation regulated chemokine
(TARC) and TSLP mRNA expression produced by dermal leukocytes and ker-
atinocytes. Furthermore, fisetin treatment suppressed production of IFNc and IL-4
by the activated lymph node CD4+ T cells with increased production of IL-10. In
addition, fisetin inhibited activation of NFjB by reducing levels of phosphorylated
p65 [25].
Studies on HUVECs and septic mice have demonstrated that fisetin inhibited
sepsis-related mortality. Fisetin treatment inhibited hyperpermeability and leuko-
cyte migration in septic mice induced by LPS and cecal ligation and puncture
(CLP)-mediated release of high mobility group box 1 (HMGB1) protein. In addi-
tion, fisetin treatment greatly inhibited PMA and CLP-induced expression of
endothelial cell protein C receptor involved in vascular inflammation. Furthermore,
fisetin treatment also inhibited production of TNFa and IL-1b as well as activation
of AKT, NFjB, and ERK1/2 in HUVEC cells induced by HMGB1 [30]. In
addition, in vitro and in vivo studies have demonstrated that fisetin inhibited high
glucose-induced vascular inflammation, vascular permeability, leukocyte adhesion,
and migration, cell adhesion molecule expression, ROS formation and NFjB
activation [26].
A recent study by Kim et al. [130] demonstrated that fisetin suppresses
macrophage-mediated inflammation by blocking Src and Syk, the major NFjB
regulatory protein tyrosine kinases. Fisetin treatment of RAW264.7 cells inhibited
LPS-induced production of NO, transcriptional activation of inflammatory genes
(iNOS, COX-2, and TNFa) and activation of NFjB without any cytotoxic effect on
these cells. Moreover, the autophosphorylation levels of Src and Syk were signif-
icantly suppressed without decreasing total levels of Src and Syk [130].

10.5 Conclusions

Fisetin has demonstrated various health-promoting effects by acting as an

anti-inflammatory, antioxidant, and antitumorigenic agent. Fisetin exhibits benefi-
cial neurologic effects by improving behaviors, learning capabilities, and memory
enhancement in animals. These properties make fisetin a candidate for future
therapies to manage Alzheimer’s, Huntington’s, and other neurological diseases.
Other chronic diseases including diabetes, atherosclerosis, obesity, and lipid dys-
regulation continue to harm patient health and burden healthcare systems with
238 H.C. Pal et al.

exorbitant costs. Similarly, despite great advances in treatment options, effective

treatments for advanced cancers continue to challenge clinicians. For these reasons,
alternative approaches to the management of chronic conditions require innovative
adjuvant and monotherapies to improve patient outcomes. Fisetin has shown
potential to prevent inflammation in in vitro systems and animal models relevant to
chronic inflammation-related life-threatening diseases. However, in-depth clinical
trials are needed to scientifically validate fisetin’s role in inflammation-related
chronic diseases and to translate potential health benefits into clinical application.

Acknowledgments The work highlighted from the author’s laboratory was supported by NIH
Grant R21CA173043.

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Chapter 11
Honokiol, an Active Compound
of Magnolia Plant, Inhibits Growth,
and Progression of Cancers of Different
Organs

Ram Prasad and Santosh K. Katiyar

Abstract Honokiol (C18H18O2) is a biphenolic natural product isolated from the

bark and leaves of Magnolia plant spp. During the last decade or more, honokiol
has been extensively studied for its beneficial effect against several diseases.
Investigations have demonstrated that honokiol possesses anti-carcinogenic,
anti-inflammatory, anti-oxidative, anti-angiogenic as well as inhibitory effect on
malignant transformation of papillomas to carcinomas in vitro and in vivo animal
models without any appreciable toxicity. Honokiol affects multiple signaling
pathways, molecular and cellular targets including nuclear factor-jB (NF-jB),
STAT3, epidermal growth factor receptor (EGFR), cell survival signaling, cell
cycle, cyclooxygenase and other inflammatory mediators, etc. Its chemopreventive
and/or therapeutic effects have been tested against chronic diseases, such as cancers
of different organs. In this chapter, we describe and discuss briefly the effect of
honokiol against cancers of different organs, such as melanoma, non-melanoma,
lung, prostate, breast, head and neck squamous cell carcinoma, urinary bladder
cancer, gastric cancer, and neuroblastoma, etc. and describe its mechanism of action
including various molecular and cellular targets. Although more rigorous in vivo
studies are still needed, however it is expected that therapeutic effects and activities
of honokiol may help in the development and designing of clinical trials against
chronic diseases in human subjects.

R. Prasad
S.K. Katiyar (&)

Department of Dermatology, University of Alabama at Birmingham,
1670, University Boulevard, Volker Hall 557, Birmingham, AL 35294, USA
e-mail: [email protected]
S.K. Katiyar
Comprehensive Cancer Center, University of Alabama at Birmingham,
Birmingham, AL 35294, USA
S.K. Katiyar
Nutrition Obesity Research Center, University of Alabama at Birmingham,
Birmingham, AL 35294, USA
S.K. Katiyar
Birmingham Veterans Affairs Medical Center, Birmingham, AL 35233, USA

© Springer International Publishing Switzerland 2016 245

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1_11
246 R. Prasad and S.K. Katiyar

Keywords Honokiol Ultraviolet radiation Cancer of different organs
Cell



cycle regulation Inflammatory mediators Tumor cell migration

Abbreviations
BCC Basal cell carcinomas
CDK Cyclin dependent kinases
CHS Contact hypersensitivity
COX-2 Cyclooxygenase-2
EGFR Epidermal growth factor receptor
EMT Epithelial-mesenchymal transition
HNSCC Head and neck squamous cell carcinoma
IL Interleukin
iNOS Inducible nitric oxide synthase
MMP Matrix metalloproteinase
NF-jB Nuclear factor-kappa B
NSCLC Non-small cell lung cancer
PCNA Proliferating cell nuclear antigen
PG Prostaglandin
PGE2 Prostaglandin E2
SCC Squamous cell carcinomas
TNF-a Tumor necrosis factor-alpha
UVR Ultraviolet radiation

11.1 Introduction

Honokiol, a biphenolic and bioactive small molecule phytochemical (Fig. 11.1), is

isolated from the bark and leaves of Magnolia plant species (Magnolia officinalis),
and has been widely used in the traditional Japanese medicine Saiboku-to for the
treatment of various ailments due to its anxiolytic, antithrombotic, anti-depressant,
anti-emetic, and antibacterial properties [1]. Since long, the barks and leaves from
the Magnolia plant also have been used in traditional Chinese system of medicine,

Fig. 11.1 Molecular

structure of honokiol, a
phytochemical from
Magnolia spp.
11 Honokiol, an Active Compound of Magnolia Plant … 247

therefore, in the recent past, honokiol achieved a great deal of research interest due
to its diverse biologic and pharmacologic activities that include antibacterial,
anti-inflammatory, anti-fungal, anti-oxidative, and anti-carcinogenic effects [1–8].
Chemically, honokiol is hydrophobic in nature but soluble in organic solvents, such
as acetone. Therefore, to avoid the use of organic solvents in some topical appli-
cations and formulation, which may cause deleterious effects, the research labo-
ratory of Dr. Katiyar has developed a topical formulation by mixing it in a
hydrophilic cream, and this cream-based topical formulation is ready and
easy-to-use for experimental purposes [8].
The protective and therapeutic effect of phytochemicals such as honokiol may be
associated with their antioxidant activity, as overproduction of reactive oxygen and
nitrogen species in the human body is involved in the pathogenesis of many chronic
diseases including cancer. To provide better understanding of the use of honokiol in
prevention and treatment of chronic diseases such lung cancer, prostate cancer, head
and neck cancer, gastric cancer, prostate cancer, urinary bladder cancer and neurob-
lastoma, etc., we are summarizing and explaining the investigations conducted in vitro
and in vivo animal models. It is well documented that many diseases occur or initiated
due to chronic and sustained inflammation in animals as well as in humans, which
includes cancer of several organs and aging processes, etc. Inflammation is a localized
reaction of tissue to infection, irritation, or other injury, e.g., exposure of the skin to
solar ultraviolet (UV) radiation. The key features of inflammation are redness or ery-
thema, warmth, swelling, pain, etc. Inflammation, considered as a necessary response to
clear viral infection, repair tissue insults and suppress tumor initiation and progression.
However, when inflammation is chronic and persists, diseases may develop, including
cancer. Importantly, inflammation involves in all the three stages of tumor develop-
ment: initiation, promotion/progression, and metastasis. During the initiation phase,
inflammation induces the release of a variety of cytokines and chemokines (mostly
pro-inflammatory) that promote the activation of inflammatory cells. These conditions
change the tissue microenvironment, which resulted in the favor of increased cell
survival and proliferation. Clinical and epidemiological evidences suggest a connection
between inflammation and a predisposition for the development of cancer. Here, we
summarize and discuss the therapeutic effects and mechanisms of action of honokiol
against cancers of some specific organs with particular emphasis on ultraviolet
(UV) radiation-induced skin cancer development.

11.2 Effect of UVR Exposure on the Skin: Inflammation

and Immune Suppression

Exposure of the skin to solar UV radiation, specifically UVB (290–320 nm)

spectrum, induces inflammatory mediators and generates oxidative stress, which all
together have been implicated in various skin diseases including the initiation and
progression of non-melanoma and melanoma skin cancers [9–13]. UVB-induced
248 R. Prasad and S.K. Katiyar

inflammatory responses are characterized by the development of edema, erythema,

hyperplastic responses, increases in the expression levels of inducible
cyclooxygenase-2 (COX-2) and production of prostaglandin (PG) metabolites [9].
UV-induced inflammation is considered as an early and important event in tumor
promotion and the growth of skin tumors, and is associated with all the three stages
of tumor development, i.e., initiation, promotion and progression [9]. The pros-
taglandin (PG) metabolites have been implicated in UVB-induced immunosup-
pression as well as implicated in the development of melanoma and non-melanoma
skin cancers. This involvement has been supported by the facts that nonsteroidal
anti-inflammatory drugs exert their effects through COX-2 inhibition and can
reverse the immunosuppressive effects of UV radiation [14, 15]. Among different
PG metabolites, PGE2 is produced abundantly by keratinocytes in UVB-exposed
skin. It is a major and most effective metabolite generated by COX-2 activity and
considered as a potent mediator of inflammatory responses. The role of PGE2 in
UV-induced immunosuppression is supported by the evidence that COX-2-deficient
mice are resistant to UVB-induced suppression of contact hypersensitivity
(CHS) response, whereas the treatment of UVB-exposed COX-2-deficient mice
with PGE2 resulted in suppression of CHS response, thus suggesting the role of
PGE2 in UVB-induced immunosuppression [16]. CHS response is considered as a
prototype of T-cell mediated immune reaction/response. Hence, the regulation of
UVB-induced inflammatory responses has been considered as an important strategy
in reducing the risk of skin cancer. To determine the anti-inflammatory effect of
honokiol, studies have been conducted using in vivo mouse models and subse-
quently its effect on UVB radiation-induced skin tumor development.

11.3 Treatment of Honokiol Inhibits UVB-Induced

Inflammatory Mediators in the Skin

A characteristic response of skin keratinocytes to UVB irradiation is enhanced COX-2

expression and a subsequent increase in the production of PG metabolites in the skin [9,
16, 17]. The exposure of the skin to UV radiation triggers the release of PGs, such as
PGD2, PGF2a, and PGE2, which are produced from arachidonic acid by the action of
COX-2 [17, 18]. Vaid et al. [8] have shown that chronic exposure of the mouse skin to
UVB radiation resulted in greater expression of COX-2 as compared with the skin of
the non-UVB-exposed normal skin. Topical treatment of mice with honokiol, whether
applied before or after UVB-irradiation, resulted in a suppression of COX-2 expression
as compared to the expression in non-honokiol-treated UVB-irradiated mouse skin. The
levels of PG metabolites in the skin with a particular emphasis on PGE2 were also
analyzed. The levels of PGE2 in UVB-irradiated skin were significantly greater as
compared with the skin of the non-UVB-irradiated mouse skin, however, the levels of
11 Honokiol, an Active Compound of Magnolia Plant … 249

Fig. 11.2 This generalized schematic diagram depicts the mechanism of UVR-induced inflam-
mation in the skin. UVR exposure induces inflammatory responses, including overexpression of
COX-2 and PGs production and reactive oxygen species (ROS) generation at early time points
(within few hours) of irradiation. Inflammatory mediators and ROS act as chemotactic factors and
stimulate the infiltration of leukocytes, particularly activated macrophages and neutrophils, at UV
irradiated skin site. Peak time of infiltration is in between 48 and 72 h after UV irradiation of the
skin. Activated infiltrating leukocytes are the major source of inflammatory mediators as well as
ROS. Topical treatment of the skin with honokiol inhibits UVR-induced effects both at 1st stage
(early stage), and 2nd stage (48–72 h after UV) through inhibition of leukocyte infiltration.
Inhibition of UVR-induced inflammatory mediators as well as ROS by honokiol treatment
contributes to the prevention of UVR-induced skin tumor development

PGE2 were significantly lower in the UVB-irradiated mouse skin, which was treated
with honokiol. Skin exposure to UV radiation induces infiltration of inflammatory
leukocytes in the skin, and most prominently are the activated macrophages and
neutrophils. These infiltrating leukocytes (majority of them are CD11b+ cell subset)
have a role in UV-induced immune suppression, and are the major source of inflam-
matory mediators, such as prostaglandins and pro-inflammatory cytokines at the
UV-exposed site, as demonstrated in Fig. 11.2. In addition, these infiltrating CD11b+
leukocytes have been shown to have suppressive effects on immune system. UVB
irradiation also induces production of pro-inflammatory cytokines, such as tumor
necrosis factor-alpha (TNF-a), interleukin (IL)-1b, IL-6, etc., in the skin. Topical
treatment of mouse skin with honokiol resulted in a significant reduction in
UVB-induced production of TNF-a, IL-1b, IL-6 as compared to non-honokiol-treated
UVB-exposed mouse skin.
250 R. Prasad and S.K. Katiyar

11.4 Honokiol Inhibits UVB-Enhanced Expression

of Proliferating Cell Nuclear Antigen (PCNA)
in the Skin

Chronic inflammation caused by UV irradiation promotes cellular proliferation in skin

cells, and it is commonly measured by determining the levels of PCNA in the skin.
Treatment of the skin with honokiol inhibited UVB-induced expression of PCNA in
skin. Uncontrolled cellular proliferation may give rise to tumor growth and its devel-
opment. Based on the above information, it can be suggested that honokiol acts as an
anti-inflammatory agent, which is an initial step of skin carcinogenesis.

11.5 Honokiol Treatment Inhibits UV Radiation-Induced

Skin Tumor Development and Malignant
Progression of Papillomas to Carcinomas in Mice

Non-melanoma skin cancer (NMSC) is the most common cancer in the United States.
The majority of NMSCs is environmentally induced and caused by excessive exposure
to solar UVB radiation which induces inflammation, oxidative stress, suppression of
immune system, and DNA damage. NMSC is composed of two types of skin cancer;
basal cell carcinomas (BCCs), and squamous cell carcinomas (SCCs). The incidence of
SCC has increased approximately 200 % over the past three decades, and represents
about 20 % of NMSC [19, 20]. As topical treatment of honokiol prevents
UVB-induced inflammation and their mediators in the mouse skin, the effect of hon-
okiol was assessed against UVB-induced skin tumor development in SKH-1 hairless
mouse model [8]. To induce tumors, mice were exposed to UVB radiation
(180 mJ/cm2) 3 times a week for 24 weeks. It was observed that topical treatment of
honokiol significantly inhibits UVB-induced initiation and progression of skin tumors
[8]. Honokiol treatment also increased the latency period of skin tumor development.
The tumor multiplicity and tumor size were significantly reduced in the group of
UVB-irradiated mice that were treated with honokiol than in the control group of mice
that were UVB-irradiated but not treated with honokiol. When data were compared in
terms of average tumor volume/tumor bearing mouse between honokiol-treated and
non-honokiol-treated groups, a significant reduction was observed after the treatment of
honokiol. Similar observations were also noted by Chilampalli et al. [21] wherein
chemopreventive effect of honokiol was determined on UVB-induced skin carcino-
genesis. However, these investigators have determined the chemopreventive effect of
topical treatment of honokiol at lower dose (30 lg/200 ml acetone/mouse) as well as
lower dose of UVB exposure (30 mJ/cm2). This study also concluded that honokiol
treatment affords photoprotection in terms of tumor multiplicity. In addition to the
inhibition of skin tumor development in UVB-exposed mouse skin, the malignant
progression of papillomas to carcinomas were also significantly prevented by the
11 Honokiol, an Active Compound of Magnolia Plant … 251

topical treatment of honokiol in mice. Histochemical analysis revealed that most of the
carcinomas were identified as kerato-acanthomas and squamous cell carcinomas [8].

11.5.1 Honokiol Controls Cell Cycle Regulators

in UVB-Induced Skin Tumors

Enhanced expression of cell cycle regulatory proteins such as cyclin-dependent

kinases (CDKs) and cyclins or decreased expression of CDK inhibitors have been
implicated in UV-induced skin carcinogenesis [22, 23]. The cell cycle deregulation
affects skin carcinogenesis under the influence of UV-induced inflammatory
mediators. Regulation of cyclin-CDK complexes plays a key role in cell cycle
progression at different phases in which CDKs are negatively regulated by a group
of functionally related proteins known as CDK inhibitors, such as Kip/Cip family
members [24, 25]. Cip1/p21 is a universal CDK inhibitor, and binds with PCNA to
inhibit PCNA function in DNA replication process [26], while Kip1/p27 is
upregulated in response to anti-proliferative signals. The expression levels of
cyclins (cyclin D1, D2, and E) and CDKs (CDK2, CDK4, and CDK6) were con-
siderably higher in UVB-induced skin tumors compared with non-UVB-irradiated
normal skin from age-matched control mice. However, treatment of the skin with
honokiol resulted in inhibition of UVB-induced expression levels of cyclins (cy-
clins D1, D2, and E) and CDKs in skin tumors compared to skin tumors obtained
from non-honokiol-treated mice. Further, tumor suppressor genes or proteins are
also involved in tumor development. Cip1/p21 also act as tumor suppressor, and
regulates cell cycle progression. Treatment of honokiol enhances the levels of
Cip1/p21 in skin tumors compared with non-honokiol-treated skin tumors. Cell
cycle arrest in tumor cells could lead to the reduction in proliferation potential of
cells as observed by a decrease in the levels of PCNA in UVB-exposed skin and
skin tumors. Thus, the modulation in cell cycle progression and inhibition of cell
proliferation could be one of the possible mechanisms through which honokiol
inhibits UVB-induced skin tumor development [8]. Kip1/p27 is another important
CDK inhibitor that regulates CDK-cyclin activity at G1-S transition of cell cycle
[22]. The level of Kip1/p27 was also upregulated in the skin tumors obtained from
honokiol-treated mice. These observations reflect the molecular mechanism through
which honokiol acts as an anti-inflammatory and anti-skin carcinogenic agent.

11.5.2 Honokiol Inhibits Cell Survival Signals/Pathways

in UVB-Induced Skin Tumors

The risk of UVB radiation-induced skin tumor development increases through

various signaling pathways including the activation of the cell survival kinases,
252 R. Prasad and S.K. Katiyar

such as PI3K/Akt [27, 28]. Studies revealed that the levels of both the catalytic
(p110) and regulatory (p85) subunits of PI3K were enhanced in the UVB-induced
skin tumors as compared with the non-UVB-exposed normal mouse skin; however,
the levels of the p85 and p110 subunits were greatly reduced in the UV-induced
skin tumors from honokiol-treated mice compared to the skin tumors of
non-honokiol-treated mice. Most of the biological effects of PI3K are mediated
through the activation of the downstream target Akt. Akt is a serine/threonine
kinase, which has been identified as an important component of pro-survival sig-
naling pathways [29]. It was observed that the treatment of honokiol resulted in
reduction of UVB-induced phosphorylation of Akt (Ser473) as compared to the skin
tumors of non-honokiol-treated group. Further, cell survival signals have been
associated with cellular proliferation and carcinogenesis [30–32]. The skin tumors
augment UVB radiation-induced activation of PI3K and phospho-Akt as compared
to the non-UVB-exposed mouse skin. The PI3K/Akt signaling pathway regulates
the activity of the transcriptional factor, NF-jB, which in turn known to regulate
several well-known markers of tumor promotion and tumor cell proliferation, e.g.,
COX-2, inducible nitric oxide synthase (iNOS) and PCNA [33, 34]. Thus, the
inhibition of PI3K/p-Akt pathway in skin tumors by honokiol may have a role in
inhibition of UVB-induced skin tumor growth in mouse model.

11.6 Honokiol Inhibits Metastatic Potential

of Melanoma Cells

Melanoma is a leading cause of skin cancer-related deaths due to its propensity to

metastasize at distant organs of the body, and the average survival of patients with
advanced stage melanoma is less than 1 year. The American Cancer Society
indicated that the incidence of melanoma is increasing, and increasing particularly
in children [35]. The oxidative stress plays a significant role in cancer cell pro-
gression and migration. Nox1 is a multi-protein complex that consists of cytosolic
(p47phox, p40phox, and p67phox) and membrane-bound proteins (p22phox, gp91phox)
that when assembled becomes activated, and initiates respiratory bursts or gener-
ation of oxidative stress [36, 37]. A recent study by Prasad et al. [38] reported that
honokiol treatment inhibits the migration capacity of melanoma cells and this effect
was associated with the inhibition of Nox1 expression and NADPH oxidase activity
in melanoma cells. Honokiol not only reduces the NADPH oxidase activity, but
also reduces oxidative bursts in melanoma cells. Treatment of melanoma cells with
honokiol blocks the interaction between membrane-bound and cytosolic-bound
proteins. The p22phox protein is the binding partner of p47phox [39, 40], the subunits
required for oxidase assembly. Failure of this interaction between membrane-bound
and cytosolic-bound proteins would lead to the inactivation of Nox in melanoma
cells, and that would result in the reduction of oxidative stress, which is responsible
for invasive or metastatic phenotype of melanoma cells. The inhibitory effect of
11 Honokiol, an Active Compound of Magnolia Plant … 253

honokiol on migratory potential of melanoma cells was supported by an action of

diphenyleneiodonium chloride, a Nox 1 inhibitor, in melanoma cells. The treatment
of melanoma cells with diphenyleneiodonium chloride also blocked or reduced
melanoma cell migration. Activation of matrix metalloproteinases (MMPs), espe-
cially MMP-2 and MMP-9, plays crucial roles in tissue matrix degradation, and
thus, paves the way for cell migration. Prasad et al. [38] also found that honokiol
treatment reduces the expression of MMP-2 and MMP-9 in Hs294t and SK-Mel28
melanoma cells, thus suggesting a mechanism of action by honokiol.

11.7 Honokiol Promotes Cell Death

of Neuroblastoma Cells

In children, several childhood cancers such as leukemia, neuroblastoma, Wilms

tumor, lymphoma, rhabdomyosarcoma, retinoblastoma, bone cancer including
osteosarcoma and Ewing sarcoma are the leading cause of deaths [35]. Among
various tumor types diagnosed in children, neuroblastomas are the most common
solid cancer and develop from neural crest elements of the sympathetic nervous
system [41]. Based on severity of disease, neuroblastomas are classified into three
risk categories as low, intermediate, and high. Unfortunately, neuroblastomas
diagnosed in children, belongs to high-risk category [42], and it is a severe problem
in pediatric oncology. Children with neuroblastoma achieve a long-term treatment,
and which usually causes other serious complications such hearing loss, cardiac
dysfunction, infertility, and secondary malignancies after therapy [43]. Studies
indicated that honokiol has beneficial effect against neuroblastoma as it can pass
through blood brain barrier and kill neuroblastoma cells without affecting too much
viability of normal brain cells [44]. Autophagy is a self-degradative physiological
process in the body that deals with cell’s destruction and maintains homeostasis or
normal functioning. Studies have shown that honokiol induces autophagy in vari-
ous types of tumor cells including neuroblastoma via diverse mechanisms [45–48].
DNA fragmentation and cell cycle arrest at the sub-G1 phase are two typical
characteristics that indicate that cells are undergoing apoptosis. In human neu-
roglioma H4 cells, honokiol have been reported to cause cell cycle arrest and induce
apoptosis through an activation of p53 [49]. The mammalian target of rapamycin
(mTOR) signaling is a negative regulator of cellular autophagy [50]. Following
phosphorylation, mTOR inhibits activation of downstream protein kinases,
including ULK1 and ATG13, and subsequently suppress cellular autophagy. The
study by Yeh et al. [45] revealed that treatment of neuroblastoma cells with hon-
okiol caused significant downregulation of mTOR phosphorylation which leads to
induction of autophagy of neuroblastoma cells.
254 R. Prasad and S.K. Katiyar

11.8 Honokiol Inhibits the Growth of Head

and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a commonly occurring

malignancy worldwide and account approximately 20,000 deaths in the United
States annually [51, 52]. In addition to several other factors, the over expression of
epidermal growth factor receptor (EGFR) has been commonly reported and
observed in more than 90 % cases of HNSCC. Overexpression of EGFR is asso-
ciated with poor clinical outcomes of HNSCC [53–55]. Therefore, EGFR is con-
sidering as a promising target for the treatment of patients suffering from HNSCC.
Honokiol treatment significantly decreased the cell viability and induced apoptosis
in various HNSCC cell lines, such as derived from tongue, larynx, oral cavity, and
pharynx, and suggests that honokiol possesses broad therapeutic effect on this
malignancy. A recent study published by Singh et al. [56] reported that honokiol
targets EGFR signaling to inhibit HNSCC growth. Treatment of HNSCC cell lines
with honokiol decreases the expression levels of total EGFR as well as p-EGFR and
its downstream target mTOR signaling. An activation of mTOR signaling has been
shown to contribute in tumor growth and progression. To confirm the findings,
authors further verified the inhibition of mTOR and its downstream targets using
rapamycin, an inhibitor of mTOR, and its effect on cell viability. It was found that
treatment of cells with rapamycin results in significant inhibition of cell viability of
HNSCC cells as well as decrease in the levels of mTOR and its downstream targets.
Based on these observations, it appears that honokiol acts as an antagonist and/or
causes increased turnover of EGFR, thus accounting for decreased expression of
EGFR in HNSCC cells. The iNOS is involved in tumorigenesis [57]. Studies
reported that honokiol treatment not only inhibits HNSCC cell proliferation, but
also induced apoptosis in HNSCC cell lines through decreasing the expression level
of iNOS at the protein as well as mRNA levels. NO is an important regulator of
various MMPs, which are over expressed in metastatic cancers and promote cancer
cell migration [58]. Studies also indicated that honokiol treatment reduces meta-
static potential of HNSCC cells by targeting MMPs and inhibiting nuclear
translocation of NF-jB [59]. A proteomic study based on LC-MS/MS analysis
revealed that out of 181 identified proteins, 96 proteins were differentially
expressed in honokiol-treated HN22 cells. Cho et al. [59] list the biological func-
tions of several identified proteins. The endoplasmic reticulum protein 44 (ERp44)
acts as an anchoring protein for ER-resident proteins that lack an ER retention
signal and in many instances, a family of ER oxidoreductases including ERp44
catalyzes oxidative protein folding. The treatment of honokiol significantly reduces
ERp44 expression in HNSCC cell lines. After ERp44 degradation, ER calcium ion
flows into the cytoplasm. ERp44 is a key regulator of protein secretion, calcium
signaling and redox regulation via interaction with IP3R1 in a calcium ion, redox
and pH dependent manner [60]. In pathologic conditions, excessive influx of
cytosolic calcium ion into the mitochondria triggers dysfunction of the mitochon-
drial membrane permeabilization with mitochondrial ROS induction [61]. Studies
11 Honokiol, an Active Compound of Magnolia Plant … 255

have identified that honokiol treatment induced a significant release of cytochrome

c from the mitochondria, followed by an increase in mitochondrial Bax and a
significant decrease in the expression levels of the pro-apoptotic proteins Bid,
Bcl-xL that results in death of HNSCC cells.

11.9 Protective Effect of Honokiol in Breast Cancer

There has been growing emphasis on the importance of epithelial to mesenchymal

transition (EMT), an essential normal physiological process for embryonic development,
tissue remodeling, wound healing, and in cancer progression. An oncogenic EMT not
only derives tumors to gain a mesenchymal phenotype but also facilitate in migration
and invasion potential of cancer cells. The adoption of mesenchymal characteristics not
only promotes separation of cancer cells from primary tumor sites but also provides
favorable microenvironment such as increase in tumor-initiating cell characteristics
including self-renewal, multi potency and resistance to conventional therapeutics [62–
64]. Recent study by Avtanski et al. [65] indicates that honokiol effectively inhibits
EMT in breast cancer cells as evident from morphological changes and molecular
alterations of mesenchymal and epithelial genes. Breast tumors treated with honokiol
also showed reduced expression of mesenchymal markers, and provide convincing
evidence to support the efficacy of honokiol as a potent inhibitor of EMT. An inacti-
vation of serine–threonine kinase liver kinase B1 (LKB1; also known as STK11), a
known tumor suppressor, is correlated with poor prognosis of breast carcinoma [66], and
its knockdown increases motility and invasiveness of cancer cells, through induction in
the expression of many mesenchymal marker proteins indicating its possible role in
EMT [67, 68]. SIRT1 and SIRT3 have been shown to deacetylate LKB1 leading to an
increase in its cytoplasmic localization, binding with STRAD and MO25 and activation
of kinase function. Avtanski et al. [65] have found that honokiol increases the
expressions of SIRT1 and SIRT3, which leads to increase in the cytoplasmic localization
of LKB1 in breast cancer cells. The miRNAs play essential roles in various biological
processes, including cell proliferation, survival, and differentiation. The loss of miR-34a
expression has been reported in many cancer types including breast cancer [69].
Treatment of honokiol enhances miR-34a expression and inhibits mesenchymal markers
while enhancing the expression of epithelial markers in breast cancer cells. These
changes may result in suppression of cell viability of breast cancer cells.

11.10 Growth Inhibitory Effect of Honokiol in Urinary

Bladder Cancer

Urinary bladder cancer is one of the most common urogenital malignant cancer
with an estimated 74,690 new cases and 15,580 deaths occurring in USA during
2014 [70]. Recent studies reported that cancer stem cells might account for
256 R. Prasad and S.K. Katiyar

chemotherapy failure, which are enriched after therapy and have the ability to
generate all types of differentiated cells to repopulate tumors and eventually lead to
metastasis [71–73]. Histone modifications through polycomb repressive complexes
also play an essential role for normal and malignant cell stemness maintenance.
Deregulation of Enhancer of Zeste Homologue 2 (EZH2), an important component
of polycomb repressive complex 2, is frequently detected in a variety of cancer
along with urinary bladder cancer [74–76]. Activation EZH2 specifically represses
the transcription of differentiation-related genes throughout the cell cycle to
maintain the stemness of cells, and this make it a promising therapeutic target for
cancer. Zhang et al. [77] have found that depletion of EZH2 by honokiol treatment
inhibited cell proliferation and clonogenicity of urinary bladder cancer cells.

11.11 Anti-Tumorigenic Activity of Honokiol

in Prostate Cancer

Prostate cancer is the most frequently diagnosed cancer and the second leading
cause of cancer-related death in males after skin cancer in economically developed
countries [70]. The major cause of mortality in prostate cancer is associated with
metastasis. Approximately, 90 % of deaths from solid tumors are caused by
metastasis [78]. Studies have shown that honokiol induces autophagy in cancer
cells [79, 80]. An induction of autophagy with different functional consequences
has been described for a number of structurally divergent naturally occurring
anticancer agents. Studies have implicated that mTOR is a negative regulator of
autophagy. A study published by Hahm et al. [48] reported the suppression of
mTOR and Akt phosphorylation by honokiol treatment in prostate cancer cells
(PC-3). An induction of apoptosis by natural agents is significantly attenuated by
antioxidants because of reduced activation of multi domain Bcl-2 family member
Bax. Hahm et al. [48] also found that treatment of honokiol induced apoptosis in
PC-3 and LNCaP cells, which was associated with induction of Bax and Bak, and
claimed that honokiol may be effective in prevention and therapy of prostate cancer.

11.12 Honokiol Inhibits Metastatic Potential and Tumor

Growth of Gastric Cancer

Gastric cancer is also a leading cause of cancer-related death worldwide, and the
majority of patients exhibit a high incidence of lymph node metastases. Emerging
evidence suggests that EMT leads to increased tumor formation, tissue invasiveness,
11 Honokiol, an Active Compound of Magnolia Plant … 257

and tumor dissemination [81]. Recent studies have shown a close relation between
EMT and gastric cancer progression. A number of signaling pathways, including
developmental transcriptional factors, are involved in regulating the motile-invasion
phenotype of tumor cells [82]. The decreased expression of epithelial marker
E-cadherin in gastric cancer has established the potential role of EMT in gastric
cancer metastasis [83, 84]. Tumor progression locus 2 (Tpl2) is a serine-threonine
kinase, regulates the activation of the mitogen-activated protein kinase, and critically
involved in inflammation, oncogenic events, and tumor progression [85, 86]. Pan
et al. [87] have shown that honokiol treatment regulates Tpl2 mediated mesenchy-
mal markers and significantly down regulates Snail, vimentin, N-cadherin expres-
sion, and upregulates cytokeratin-18 and E-cadherin expression. Tumor growth in
gastric cancer has been associated with Tpl2 [88, 89]. Pan et al. [87] also reported
that honokiol treatment reduces growth of gastric tumor through an inhibition of
Tpl2 expression. Honokiol significantly inhibited tumor angiogenesis as indicated
by reduced microvessel density in tumor mass [87]. Cell cycle regulation is an
important regulatory mechanism to control cell growth, and activation of p53, a
tumor suppressor protein, is involved in the regulation of cell cycle arrest and
apoptosis. The phosphorylation of cell cycle regulatory proteins is involved in
arresting effect of gastric carcinoma cells on the cell cycle at G2/M phase [90, 91].
Yen et al. [92] investigated the anticancer mechanism of honokiol in human gastric
carcinoma using MGC-803 cells and investigated that honokiol induces apoptosis in
gastric carcinoma cells, and the underlying mechanism was mediated through
downregulation of CDC2/cdc25C and upregulation of p53. These studies provide
evidence of anticancer activity of honokiol against human gastric carcinoma.

11.13 Honokiol Inhibits Migratory Potential

of Lung Cancer Cells

Lung cancer is a major cause of cancer-related deaths in the United States as well as
worldwide each year and thus have a tremendous impact on human health and
health care expenditures. Non-small-cell lung cancer (NSCLC) accounts for
approximately 80 % of all types of lung cancer. COX-2 is frequently over express
in lung cancer and associated with excessive production of PGE2, which promotes
tumor cell survival, invasion and metastasis [93–96]. Therefore, COX-2 inhibitors
have shown potential in treatment of lung cancer. Singh and Katiyar [97] reported
the use of honokiol as an inhibitor of COX-2 expression to inhibit migratory
potential of lung cancer cells and supported their findings by the evidence that
treatment of the NSCLC cells with celecoxib, a potent COX-2 inhibitor, resulted in
a reduction in cell migration. The b-catenin signaling is downstream target of
258 R. Prasad and S.K. Katiyar

COX-2 and played important roles in tumor progression as well as cell migration.
b-catenin forms a dynamic link between E-cadherin and cytoskeleton [98, 99], and
this cell-to-cell adhesion may prevent the migration of tumor cells. In contrast, the
breaking of cell-to-cell adhesion due to activation of b-catenin and its nuclear
accumulation may increase the migration potential of tumor cells. Singh and
Katiyar [97] also reported that honokiol induced degradation of b-catenin or
reduces nuclear accumulation, which leads to inhibition of lung cancer cell
migration.

11.14 Honokiol Inhibits Pancreatic Cancer Growth

Pancreatic cancer is one of the most lethal malignancies with increasing incidence in
the United States [33]. Due to its asymptomatic progression, pancreatic cancer is
diagnosed at later stage, when it has already metastasized or locally advanced. The
NF-jB is constitutively activated in a variety of hematologic and solid malignancies,
including pancreatic cancer and controls the expression of an array of genes involved in
cell proliferation and survival through direct and indirect mechanisms [100]. Arora
et al. [101, 102] examined the effect of honokiol against pancreatic cancer and reported
that honokiol showed growth inhibitory potential for pancreatic cancer lines (such as,
Miapaca and PANC-1), which may result of cell cycle arrest and induction of apop-
tosis. Furthermore, to explore the underlying mechanism, these authors have tested the
effect of honokiol on NF-jB signaling in this system. Treatment of honokiol to pan-
creatic cancer cells inhibited transcriptional activity of NF-jB, and decrease protein
expression in the nuclear fraction and suppresses constitutive activation of NF-jB in
pancreatic cancer cells and thus induce cell death.

11.15 Conclusion and Future Prospects

Honokiol, a small molecule phytochemical, has been shown to have significant

chemopreventive and therapeutic effects against cancers of various organs in vitro
and in vivo animal models. Honokiol targets distinct signaling pathways, molecular
and cellular targets and leads to inhibition of growth and progression of tumors of
various organs (Fig. 11.3). Importantly, development of cancers are considered as
chronic disease. Therefore, it has significant potential to serve as a novel agent for
prevention and therapy of chronic diseases, like cancers. This small molecule from
Magnolia spp. may be of significant interest for attenuation of the adverse effects of
environmental factors, such as solar UV radiation, on human skin. The use of
honokiol in combination with already available cancer therapeutic drugs may offer
an enhanced ability to attack other cancer-related targets, reduce toxicity and
resistance to the cancer drugs and may improve the therapeutic efficacy of the
existing cancer drugs.
11 Honokiol, an Active Compound of Magnolia Plant … 259

Fig. 11.3 The schematic diagram reflects the preventative and therapeutic effect of honokiol on
cancers of different organs. Various molecular and cellular targets in cancers of different organs are
affected by the treatment of honokiol in vitro and in vivo models. Inflammation and inflammatory
mediators are the main targets of honokiol in prevention or treatment of cancers. Underline words
indicate the name of organ-specific cancer

Acknowledgments The work reported from Dr. Katiyar’s research laboratory was financially
supported from the funds from National Cancer Institute/NIH (CA183869) and Veterans
Administration Merit Review Award (1I01BX001410). The content of this communication does
not necessarily reflect the views or policies of the funding agencies.

Conflict of interest The author has declared no conflict of interest.

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Chapter 12
Celastrol and Its Role
in Controlling Chronic Diseases

Shivaprasad H. Venkatesha and Kamal D. Moudgil

Abstract Celastrol, a triterpenoid derived from traditional Chinese medicinal

plants, has anti-inflammatory, antioxidant, and anticancer activities. Celastrol has
shown preventive/therapeutic effects in experimental models of several chronic
diseases. These include, chronic inflammatory and autoimmune diseases (e.g.,
rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, inflamma-
tory bowel disease, and psoriasis), neurodegenerative disorders (e.g., Alzheimer’s
disease, Parkinson’s disease, and Amyotrophic lateral sclerosis), atherosclerosis,
obesity, Type 2 diabetes, and cancer. Celastrol modulates intricate cellular path-
ways and networks associated with disease pathology, and it interrupts or redirects
the aberrant cellular and molecular events so as to limit disease progression and
facilitate recovery, where feasible. The major cell signaling pathways modulated by
celastrol include the NF-kB pathway, MAPK pathway, JAK/STAT pathway,
PI3K/Akt/mTOR pathway, and antioxidant defense mechanisms. Furthermore,
celastrol modulates cell proliferation, apoptosis, proteasome activity, heat-shock
protein response, innate and adaptive immune responses, angiogenesis, and bone
remodeling. Current understanding of the mechanisms of action of celastrol and
information about its disease-modulating activities in experimental models have set
the stage for testing celastrol in clinical studies as a therapeutic agent for several
chronic human diseases.

Keywords Celastrol Inflammation Autoimmune diseases Neurodegenerative

diseases
Metabolic disorders

Immune modulation Natural products


Traditional Chinese medicine

S.H. Venkatesha
K.D. Moudgil (&)

Department of Microbiology and Immunology, University of Maryland School
of Medicine, 685 W. Baltimore Street, HSF-1, Suite-380, Baltimore, MD 21201, USA
e-mail: [email protected]
K.D. Moudgil
Division of Rheumatology, Department of Medicine, University of Maryland School
of Medicine, Baltimore, MD 21201, USA

© Springer International Publishing Switzerland 2016 267

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1_12
268 S.H. Venkatesha and K.D. Moudgil

12.1 Introduction

Celastrol is a bioactive component of several traditional Chinese medicinal plants

including, Tripterygium wilfordii (Thunder God Vine), Celastrus orbiculatus,
Celastrus aculeatus, Celastrus reglii, Celastrus scandens, and others that belong to
the Celastraceae family [1–5]. The extracts of the root, bark, and stem of some of
these plants have long been used in China and other Asian countries for the
treatment of a wide range of chronic inflammatory disorders, including rheumatoid
arthritis (RA), systemic lupus erythematosus (SLE), and allergies [1–5]. In this
article, we describe the diverse molecular and cellular pathways modulated by
celastrol, with emphasis on chronic inflammatory, autoimmune, neurodegenerative,
and metabolic diseases [6–12]. Celastrol also possesses anticancer activity [5, 13–
15]. A summary of the anticancer mechanisms employed by celastrol is presented at
the end.

12.2 Physico-Chemical Properties of Celastrol

Celastrol is a pentacyclic triterpene (Fig. 12.1) that belong to a small class of

organic compounds called quinone methides. It has a molecular weight of 450.6
and its molecular formula is C29H38O4. It is a pale brown to orange red crystalline
powder, and its melting point is between 219–230 °C. Celastrol has maximum
UV/visible absorption spectra at 253 and 424 nm. It is sparingly soluble in water,
but is soluble in nonpolar solvents such as dimethylsulfoxide (DMSO) and ethanol.
Celastrol is an electrophilic compound and it can react with nucleophilic thiol
groups of cysteine residues of a variety of proteins to form adducts or induce other

Fig. 12.1 Molecular structure of celastrol. Celastrol is a pentacyclic triterpenoid with a molecular
weight 450.6 and molecular formula C29H38O4. It belongs to a small class of organic compounds
known as quinone methides. Celastrol has an acidic group at one end and a phenolic quinone at the
other end
12 Celastrol and Its Role in Controlling Chronic Diseases 269

modifications within those proteins [6, 16–18]. Apparently, this is one of the
mechanisms by which celastrol can affect biological functions of proteins. Celastrol
is also known as tripterine/tripterin, but the name celastrol is commonly used.

12.3 Celastrol Controls Inflammation and Other

Pathological Processes in Animal Models of Chronic
Diseases

Celastrol has been shown to be beneficial in various chronic disease conditions in

studies in animal models of immune-mediated diseases, neurodegenerative dis-
eases, and others. The preventive/therapeutic potential of celastrol in various in vivo
and in vitro models of these diseases is summarized in Table 12.1. Also mentioned
therein are the cell signaling pathways as well as cellular and molecular targets of
celastrol in various disease processes. The details of these and other mechanisms of
action of celastrol are described below in separate sections. In addition, celastrol has
potent anticancer activity. The mechanisms underlying the anticancer activity of
celastrol are summarized at the end in a separate section.

12.3.1 Inflammatory, Autoimmune, and Allergic Diseases

For RA, using the rat adjuvant arthritis (AA) model, mouse collagen-induced
arthritis (CIA) model and fibroblast-like synoviocytes from RA patients (RA-FLS)
culture model, celastrol has been shown to reduce the severity of clinical and
histopathological features of arthritis, as well as to modulate the production of
pro-inflammatory cytokines and chemokines [8, 9, 19], to reset the T helper 17
(Th17)/T regulatory (Treg) cell balance to facilitate the suppression of arthritis [20],
and to afford protection against bone damage [8, 9, 19] (Table 12.1A). Celastrol
also inhibits RA-FLS invasion and protects against bone and cartilage damage [21].
For multiple sclerosis (MS), celastrol is shown to modulate Th17 responses, to shift
Th1 responses toward Th2, and to increase the production of anti-inflammatory
cytokines in the experimental autoimmune encephalomyelitis (EAE) model of MS
[10, 22]. For SLE (also known as lupus), celastrol treatment decreases transforming
growth factor (TGF)-b production, urine protein excretion, and serum autoantibody
levels in BW F1 and BALB/c mouse models of SLE [23–25]. For ulcerative colitis,
using the mouse dextran sulfate sodium (DSS)-induced colitis model, celastrol has
been shown to modulate oxidative stress, inflammatory cytokines and intestinal
homeostasis [11]. For asthma and other hypersensitivity reactions, celastrol inhibits
histamine and eotaxin production and other mediators involved in hypersensitivity
reactions [26–29]. The main mediators and pathways targeted by celastrol in
above-mentioned diseases are described in Table 12.1A.
270 S.H. Venkatesha and K.D. Moudgil

Table 12.1 Celastrol-induced prevention/treatment of chronic diseases of diverse etiology

Type of disease Experimental model(s) Targets/mechanisms of References
celastrol action
A. Inflammatory and autoimmune diseases
Rheumatoid Rat AA, Rat/Mouse CIA, Modulates pro-inflammatory [8, 9, 19–
Arthritis (RA) Human RA-FLS cytokines, chemokines, and T 21, 88,
helper 17 (Th17)/T regulatory 118, 137–
(Treg) cell balance. Inhibits 140]
RA-FLS invasion and
apoptosis. Decreases
production of antibodies to the
disease-related antigens and
anti-cyclic citrullinated
peptides (aCCP) antibodies.
Also inhibits osteoclast
differentiation, and reduces
cartilage and bone damage
Multiple Rat/mouse experimental Modulates Th17 responses, [10, 22]
sclerosis (MS) autoimmune shifts Th1 response towards
encephalomyelitis (EAE) Th2, decreases TNFa but
increases IL-10. Inhibits
NF-jB expression, nitrites
levels, and expression of
Toll-like receptor (TLR)2
Systemic lupus BW F1 mice, BALB/c mice Decreases transforming [23–25]
erythematosus (chromatin injection) growth factor (TGF)-b, renal
(SLE) (or Lupus) collagen type IV, urine
protein excretion, and serum
autoantibodies
Ulcerative colitis DSS-induced colitis in mice Modulates oxidative stress, [11]
inflammatory cytokines, and
intestinal homeostasis
Psoriasis HACaT keratinocytes Inhibits NF-jB expression [141]
and induces apoptosis through
caspase-3 activation
Hypersensitivity Ovalbumin/allergen-induced Downregulates the expression [26–29]
(e.g., Asthma airway inflammation, of stem cell factor (SCF) in
and skin phorbol myristate fibroblasts, and inhibits the
inflammation) acetate-induced skin production of histamine and
inflammation, and skin eotaxin in mast cells. Inhibits
hypersensitivity to antibody responses and
dinitrochlorobenzene in mice immunoglobulin Fc epsilon
receptor I signaling. Regulates
the balance between isoforms
of MMPs (MMP-2/9) and
TIMPs (TIMP-1/2)
(continued)
12 Celastrol and Its Role in Controlling Chronic Diseases 271

Table 12.1 (continued)

Type of disease Experimental model(s) Targets/mechanisms of References
celastrol action
B. Neurodegenerative diseases
Parkinson’s MPTP mouse model, Modulates pro-inflammatory [12, 31,
disease (PD) SH-SY5Y cells cytokines, prevents the 34]
generation of ROS, lipid
peroxidation, and
mitochondrial membrane
potential. Protects against
cellular injury and apoptotic
cells death
Alzheimer’s Transgenic mouse model Inhibits pro-inflammatory [30, 32,
disease cytokines and expression of 33, 36, 37]
MHC-II molecules. Induces
neuroprotective heat-shock
proteins (Hsps). Lowers
oxidative stress and regulates
BACE-1 expression level via
an NF-jB-dependent
mechanism
Amyotrophic G93A SOD1 transgenic Blocks neuronal cell death, [35]
lateral sclerosis mouse model reduces TNFa, iNOS, CD40,
(ALS) and GFAP immunoreactivity
Gaucher disease GD fibroblasts Modulates molecular [38]
(GD) chaperones and increases
glucocerebrosidase activity
Age-related Human retinal pigment Reduces IL-6 and inhibits [39]
macular epithelial cells (ARPE-19 NF-jB
degeneration cells)
(AMD)
C. Atherosclerotic and metabolic diseases
Atherosclerosis ApoE-deficient mice, Rabbit Inhibits lectin-like oxidized [40, 41,
carotid atherosclerosis low density lipoprotein 142]
model, and human platelets receptor-1 (LOX-1) and
generation of ROS. Reduces
serum level of low density
lipoproteins and VEGF
expression. Inhibits platelet
aggregation by reducing the
expression of P-selectin and
glycoprotein IIb/IIIa on
platelets
(continued)
272 S.H. Venkatesha and K.D. Moudgil

Table 12.1 (continued)

Type of disease Experimental model(s) Targets/mechanisms of References
celastrol action
Obesity Hyperleptinemic Reduces food intake, [42]
diet-induced obese mice increases energy expenditure,
leading to weight loss by
increasing leptin sensitivity
Type 2 diabetes The db/db mouse Acting on the liver, adipose [43]
tissue, and kidney, it Inhibits
NF-kB, reduces insulin
resistance, improves abnormal
lipid metabolism and
oxidative stress, reduces
pro-inflammatory cytokines,
and limits renal injury
AA adjuvant-induced arthritis, CIA collagen-induced arthritis, DSS dextran sulfate sodium, FLS
fibroblasts-like synoviocytes, Hsp heat-shock proteins, MMPs matrix metalloproteases, TIMPs tissue
inhibitor of matrix metalloproteases, MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin, ROS reactive
oxygen species, MHC-II major histocompatibility complex class II, BACE1 beta-site APP-cleaving
enzyme 1, iNOS inducible nitric oxide synthase, GFAP glial fibrillary acidic protein, p-JNK phospho
c-Jun N-terminal kinase, NF-jB nuclear factor kappa B, ROS reactive oxygen species, VEGF vascular
endothelial growth factor

12.3.2 Neurodegenerative Disorders

The effects of celastrol on MS, a neurological disease of autoimmune origin, have

been described above. For other neurological diseases such as Parkinson’s disease,
Alzheimer’s disease and amyotrophic lateral sclerosis (ALS or Lou Gehrig’s dis-
ease), celastrol has been shown to modulate pro-inflammatory cytokine production,
to prevent the generation of reactive oxygen species (ROS), to limit oxidative
damage, to protect against cell death, and to regulate heat-shock proteins (Hsps), as
observed in mouse models and in vitro models of these diseases (Table 12.1B) [12,
30–37]. For Gaucher disease (GD), celastrol modulates molecular chaperones and
increases glucocerebrosidase activity in the GD fibroblasts model [38]. Celastrol is
also known to modulate age-related macular degeneration [39].

12.3.3 Other Diseases

Celastrol can inhibit platelet activation [40], prevent atherosclerotic plaque size in
apo E-deficient mice [41], and decrease ratio of the plaque area and the arterial wall
cross-section area in a rabbit model of carotid atherosclerosis [41], thus revealing
the anti-atherosclerosis effect of this natural triterpene (Table 12.1C). Recently,
celastrol has been reported to be a leptin sensitizer, whose effects are manifest as
12 Celastrol and Its Role in Controlling Chronic Diseases 273

reduced intake of food, increased energy expenditure, and weight loss, and thereby
it may potentially serve as an anti-obesity agent [42]. Celastrol is also effective in
improving insulin resistance and limiting renal injury in a mouse model of type 2
diabetes (T2D) [43]. Furthermore, celastrol can modulate human immunodeficiency
virus (HIV) 1-transactivator of transcription (Tat)-induced inflammatory responses
in astrocytes in vitro by inhibiting the activation of various signaling pathways as
well as the expression of pro-inflammatory chemokines and adhesion molecules
such as intracellular adhesion molecule-1 (ICAM-1)/vascular cell adhesion
molecule-1 (VCAM-1) [44]. Celastrol has also been reported to target defined
functional components of pathogens such as the HIV-Tat [18] to inhibit the tran-
scription and replication of that virus, and the enzyme enoyl-acyl carrier protein
reductase of Plasmodium falciparum [45], which is a drug target for this malaria
parasite. However, because of the limited scope of this article, we have not elab-
orated further on direct effects of celastrol on various pathogens.

12.4 Celastrol Modulates Cell Signaling Pathways

Inflammation involves interplays among a variety of cellular, molecular and bio-

chemical mediators that are activated/induced in response to different
pro-inflammatory stimuli. These mediators comprise diverse pathways (Fig. 12.2)
that are described below. Inflammation is associated with multiple diseases,
including chronic inflammatory diseases, autoimmune diseases, obesity, and can-
cer. Celastrol controls inflammation by targeting one or many of these pathways
depending on the underlying disease process.
The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB)
pathway is a central regulator of inflammation. A broad range of pro-inflammatory
stimuli including cytokines, growth factors, and microbial products activate the
I-kappa B kinase (IKK) complex consisting of IKK1, IKK2 and NF-jB essential
modulator (NEMO). Activated IKK complex phosphorylates I-kappa B (IjB),
which leads to ubiquitination and proteasomal degradation of IjB. The degradation
of IjB in turn activates NF-jB, which then translocates to the nucleus, where it
binds to DNA and regulates the expression of several target genes. NF-kB acti-
vation enhances the production of pro-inflammatory cytokines (e.g., interleukin-1 b
(IL-1b), IL-6 and tumor necrosis factor a (TNFa)) and other inflammatory medi-
ators (e.g., matrix metalloproteinases (MMPs) and inducible nitric oxide synthase
(iNOS)) [46, 47], without much effect on the induction of anti-inflammatory
cytokines (e.g., IL-10 or IL-1 receptor antagonist (IL-1Ra)) [48]. Celastrol inhibits
NF-jB activation and regulates NF-jB-regulated gene expression. It has been
suggested that celastrol targets cysteine 179 in IKK [16] and blocks IKK activity as
well as the degradation and phosphorylation of IjB [16, 21, 49]. This in turn blocks
the activation of NF-jB and its nuclear translocation.
274 S.H. Venkatesha and K.D. Moudgil

Fig. 12.2 Cell signaling pathways and other disease-related processes modulated by celastrol for
the control of chronic diseases. A schematic representation of the cell signaling pathways that are
regulated by celastrol is depicted here. These pathways are activated in response to diverse stimuli
induced by specific ligand-cell surface receptor interaction. This interaction results in activation of
receptor-associated tyrosine kinases, which subsequently activate other cytosolic targets, which
then initiate various signaling pathways. The major pathways regulated by celastrol are described
in the text and shown in the figure. Celastrol is known to target one or more of these pathways
leading to the suppression of inflammation associated with various chronic diseases including,
inflammatory diseases, autoimmune diseases, obesity, and cancer. There is no direct evidence for
celastrol-induced modulation of calmodulin pathway. However, regulation of calcium release and
AMPK activity by celastrol suggests the possibility of regulation of the calmodulin pathway by
celsatrol. Besides cell signaling, celastrol modulates other disease-related processes (e.g.,
angiogenesis, heat-shock protein responses, and proteasome activity) to limit disease progression
and to facilitate recovery, where feasible.

Mitogen activated protein kinase (MAPK) pathway is another important signal

transduction pathway besides NF-jB pathway that is critical for immune-mediated
inflammatory responses [50]. MAPKs are a family of serine/threonine protein
kinases [51]. Three well-defined members of this family are the extracellular
signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, and
they are activated by pro-inflammatory stimuli, including cytokines [50]. ERK
12 Celastrol and Its Role in Controlling Chronic Diseases 275

signal transduction pathway leads to the activation of transcription factors c-Jun,

c-Fos, and activating transcription factor 2 (ATF-2), whereas JNK activation leads to
the activation of c-Jun and ATF-2. Furthermore, the p38 MAPK-mediated processes
involve the participation of transcription factors cAMP response element-binding
protein (CREB) and ATF-2 [50, 52, 53]. Celastrol selectively regulates the MAPK
pathway. It inhibits the phosphorylation of JNK and ERK in various models of
inflammation and arthritis [8, 27, 54]. However, activation of JNK by celastrol has
also been observed in another system [55]. For p38 MAPK, one report stated that
phosphorylation of p38 MAPK is unaffected by celastrol [56], whereas another [57]
indicated that celastrol can activate p38 MAPK; the latter effect has been associated
with the anti-metastatic activity of celastrol against cancer.
The Janus kinase (JAK)-signal transducer and activator of transcription (STAT)
pathway is a common signaling cascade for many cytokines [58, 59]. JAKs are
known to associate with the cytoplasmic domain of cytokine receptors for inter-
feron (IFN)-a/b, IFNc, IL-2, IL-4, IL-6, IL-10, IL-12/23, and others [58]. In gen-
eral, the binding of these cytokines to their respective receptors phosphorylates
JAK, which then leads to the phosphorylation of STATs. Activated STATs
dimerize and translocate to the nucleus, where they bind to promoter regions of
cytokine-responsive genes and thereby activate gene transcription [58]. Different
STATs are involved in the differentiation of naïve T cells into particular T cell
subsets (Th1, Th2, Th17, and Treg). In our studies, we have shown that celastrol
inhibits STAT3 activation and suppresses IL-17 expression as well as Th17 dif-
ferentiation in the rat AA model [8, 20]. In models of cancer, such as multiple
myeloma and hepatocellular carcinoma, constitutive STAT3 activation plays a role
in cell proliferation, survival, and metabolism, and thereby to the disease process.
Celastrol is shown to inhibit STAT3-mediated cell proliferation [60, 61]. It involves
inhibition of activation of upstream JAK1 and JAK2. However, effects of celastrol
on other JAKs and STATs remain to be determined.
Another signaling pathway affected by celastrol is phosphatidylinositol-3-kinase
(PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, which is involved in
immune-mediated diseases and cancer [62–67]. Altered activation of the
PI3K/Akt/mTOR pathway is observed in many human tumors, and it regulates the
proliferation, differentiation, metabolism and survival of cancer cells [68].
Furthermore, there is an association between the accumulation of hypoxia-inducible
factor-1 (HIF-1) and amplified PI3K/Akt/mTOR pathway signaling [69]. HIF-1 is a
transcription factor highly expressed under hypoxic conditions and regulates cell
survival response to hypoxia and cancer [63, 70, 71]. Inhibitors of HIF-1 have been
tried for cancer therapy [72]. Celastrol can inhibit both PI3K activity as well as
HIF-1 [63, 66]. Celastrol inhibited HIF-1 activity in various cancer cell lines by
decreasing the accumulation of HIF-1 and preventing the expression of HIF-1 target
genes. Furthermore, the accumulation of HIF-1 by celastrol is correlated with
inhibition of the phosphorylation of mTOR, ribosomal protein S6 kinase (p70S6K),
eukaryotic initiation factor 4E (eIF4E), and ERK [63]. Contrary to the above,
celastrol is also reported to induce HIF-1 accumulation through the induction of
ROS and Akt/p70S6K signaling, and promote transcription of HIF target genes [70].
276 S.H. Venkatesha and K.D. Moudgil

Therefore, additional studies are needed to clearly understand the role of celastrol in
the regulation of mTOR and HIF-1.
Celastrol possesses antioxidant activity. Oxidative stress is one of the mediators
of inflammation [73]. Oxidative stress builds up with the generation of high levels
of reactive free radicals, such as reactive oxygen species (ROS; e.g., chemically
reactive molecules derived from O2 mainly O2− (superoxide anions),
H2O2 (hydrogen peroxide) and •OH (hydroxyl radicals)) and reactive nitrogen
species (RNS; e.g., radicals derived from nitrogen and oxygen, particularly nitric
oxide (NO)) in the cell [74, 75]. Celastrol is shown to inhibit lipid peroxidation in
rat liver mitochondria by direct radical scavenging [76] as well as neutralizing
oxygen radicals [77]. Celastrol also enhanced the antioxidant defense system and
offered protection against bleomycin-induced pulmonary fibrosis in rats by restor-
ing antioxidant enzymes such as hemoxygenase-1 (HO-1),
glutathione-S-transferase (GSTs) and nicotinamide adenine dinucleotide phosphate
(H) (NADP(H)): quinine oxidoreductase (NQO1) via the NF-E2-related factor-2
(Nrf2) pathway [78]. Similarly, celastrol decreased obesity-induced oxidative stress
by increasing antioxidant enzymes and inhibiting NADH oxidase and ROS [79].
Defense against oxidant system by celastrol has also been attributed to decreased
expression of iNOS and NO production [30, 34], and the blocking of reactive thiols
[17]. In contrast to the above, celastrol has been reported to induce ROS accu-
mulation and to initiate apoptosis through the downregulation on Hsp90 in tumor
cells [80]. Similarly, in osteosarcoma, celastrol caused G2/M phase arrest, and
induced apoptosis and autophagy via the ROS/JNK signaling pathway [81].

12.5 Celastrol has Anti-Angiogenic Activity and Protects

Against Endothelial Barrier Dysfunction

Angiogenesis, the formation of new blood vessels, is a hallmark of cancer [82–84].

However, autoimmune diseases such as RA are also characterized by angiogenesis
in the target organ, the inflamed joints [85, 86]. Accordingly, anti-angiogenic
therapy has been considered for both these categories of disorders [86, 87]. As
discussed above, celastrol treatment inhibits the progression of autoimmune arthritis
in experimental models of AA and CIA [8, 9, 20, 88]. Similarly, celastrol sup-
presses tumor growth, for example, in mouse models and in vitro models of human
prostate cancer [89, 90]. Interestingly, celastrol has been shown to inhibit angio-
genesis, both in vitro and in vivo [83, 91], and to inhibit vascular endothelial
growth factor (VEGF)-induced Akt/mTOR/p70S6 K signaling [83]. Furthermore,
celastrol can inhibit hypoxia-mediated angiogenesis, which involves inhibition of
HIF-1a and its downstream genes such as VEGF [92]. Celastrol’s ability to inhibit
Hsp90 was also implicated in reduced HIF-1a in this process. In another study,
celastrol was shown to inhibit vasculogenesis by decreasing VEGF secretion,
adhesion of endothelial cells to the extracellular matrix (ECM) and their subsequent
12 Celastrol and Its Role in Controlling Chronic Diseases 277

migration, and tubule formation [93]. Inhibition of Akt/endothelial nitric oxide

synthase (eNOS) signaling was implicated in this process. Celastrol has also been
reported to inhibit lipopolysaccharide (LPS)-induced angiogenesis, which involved
suppression of Toll-like receptor-4 (TLR-4)-mediated NF-kB activation [94], and to
inhibit angiogenesis via suppression of VEGFR-1 and VEGFR-2 expression [95].
In RA, vascular endothelial cell physiology is relevant not only for angiogenesis
but also for immune cell interaction and migration through blood vessels, as well as
for maintaining a healthy endothelial barrier. In this regard, celastrol has been
shown to inhibit the expression of cytokine-induced adhesion molecules such as
ICAM-1 and VCAM-1 [96], and to prevent endothelial barrier dysfunction by
inhibiting endogenous peroxynitrite formation in endothelial cells exposed to
pro-inflammatory stimuli [97]. The latter effect involved inhibition of
JAK-2-dependent iNOS and NADPH oxidase type 1 (Nox-1) induction.

12.6 Celastrol-Induced Modulation of Heat-Shock

Response and Its Potential Therapeutic Applications
in Chronic Diseases

Heat-shock proteins (Hsps), also known as “Stress proteins”, can be induced by

heat and other types of stimuli that cause cellular stress [98, 99]. These are highly
conserved proteins evolutionarily. Hsps can be categorized into different families
based on their molecular mass (kD), for example, Hsp110, Hsp90, Hsp70, Hsp60,
Hsp40, and Small Hsps. One of the major functions of Hsps is to protect cells from
damage under different types of stressful conditions [98, 99]. Acting as chaperones,
Hsps bind to cellular proteins, ensure proper folding of cellular proteins, attempt to
repair defective proteins, and protect them against denaturation and other types of
damage. Hsps are also involved in signal transduction and apoptosis. Thus,
heat-shock response is cytoprotective under situations that otherwise would pro-
mote apoptosis of cells. Stress-inducible heat-shock transcription factor-1 (HSF1)
plays an important regulatory role in response to environmental stress and patho-
physiological conditions.
Dysregulation of cellular stress pathways and protein folding can lead to intra-
cellular accumulation of protein aggregates, which is turn can induce tissue
pathology [100, 101]. Many pathophysiological conditions including, neurodegen-
erative disorders (e.g., Alzheimer’s disease, Parkinson’s disease), cardiovascular
diseases, cancer, diabetes, and aging are associated with accumulation of misfolded
and/or aggregated proteins within certain tissues, attributable in part to defective
cellular stress response pathways [17, 32, 100–102]. In addition, inflammation and
oxidant damage also contribute to the pathogenic processes in these disorders.
Accordingly, pharmacological agents that possess anti-inflammatory and antioxidant
activities, and can reset these defective pathways, are increasingly being sought
[17, 32, 102].
278 S.H. Venkatesha and K.D. Moudgil

Celastrol has been shown to have a cytoprotective effect in response to

stress-induced cell death. Celastrol can induce the expression of various Hsps, for
example, Hsp70, Hsp40, and Hsp27, by activation of HSF1 and these Hsps might
contribute to its cytoprotective effect [103]. Celastrol’s antioxidant attributes can
also contribute to its cytoprotective effects. As described under cell signaling, the
antioxidant response involves transcription factors Nrf-2 and Atf4, and celastrol has
been shown to activate both these transcription factors [17]. In one study, celastrol’s
cytoprotective effect was shown to be mediated via induction of Hsp32 (also known
as heme oxygenase-1; HO-1) [104]. This induction of Hsp32 was mediated via
Nrf-2 instead of HSF-1, and Hsp32 in turn inhibited pro-apoptotic JNK. Besides the
induction of Hsps mentioned above, inhibition of Hsp90 has been shown to have a
therapeutic effect in certain neurodegenerative diseases; the latter effect is attribu-
table to selective proteasomal degradation of Hsp90 client proteins [100].
Importantly, celastrol is an inhibitor of Hsp90 [105, 106], and it can modulate
several nuclear transcription factors that are Hsp90 clients, including the aryl
hydrocarbon receptor (AhR) [105, 106]. The involvement of celastrol-induced
inhibition of Hsp90 and its anticancer effect is discussed below.

12.7 Anticancer Effects of Celastrol

Celastrol is known to have anticancer and anti-metastatic activities [15, 82, 83, 107,
108]. In addition, celastrol is shown to enhance the therapeutic efficacy of other
anticancer drugs when used with them, and to potentiate the beneficial effects of
radiotherapy [109, 110]. The major processes involved in these activities and
affected by celastrol include, the inhibition of cellular proliferation, induction of
apoptosis, prevention of malignant tissue invasion, and blockade of angiogenesis
[7, 15, 82, 111]. Celastrol can inhibit cell proliferation and induce apoptosis via
multiple actions. These include, potentiation of TNF-induced apoptosis via sup-
pression of the NF-kB pathway [4]; downregulation of cytokines such as IL-6,
which is an inducer of cell proliferation [112]; activation of caspases [113–115];
inhibition of the expression of anti-apoptotic proteins such as cellular inhibitor of
apoptosis protein 1 and 2 (cIAP1 and cIAP2), cellular FLICE-inhibitory protein
(FLIP), and B-cell lymphoma 2 (Bcl-2) [4, 114]; induction of cell cycle arrest [81,
116]; and downregulation of cell survival proteins coupled with upregulation of
death receptors [117]. Furthermore, celastrol inhibits adhesion, migration and
invasion of tumor cells via reduced expression of specific integrins, as well as
reduced MMP activity [118–120]. In addition, as described above, celastrol can
suppress angiogenesis [63, 83].
Two additional mechanisms contribute to the anticancer effects of celastrol,
namely inhibition of Hsp90 and proteasome inhibition. In regard to Hsp90, celastrol
directly binds to the C-terminal domain of Hsp90 inducing its oligomerization, and
it interferes with specific biological functions through modulation of
Hsp90-associated nuclear transcription factors [106, 121]. In addition, celastrol has
12 Celastrol and Its Role in Controlling Chronic Diseases 279

been shown to inhibit Hsp90 in thiol-related reactions [116], and to downregulate

Hsp90 client proteins via inhibition of enzymes of mitochondrial complexes and
accumulation of ROS [80]. Furthermore, celastrol-induced inhibition of Hsp90
contributes to HIF-1a inhibition and cell cycle arrest [92]. As far as the proteasome
is concerned, celastrol has been shown to inhibit proteasome function by targeting its
chymotrypsin-like activity. This in turn results in the accumulation of ubiquitinated
proteins and some of the known proteasomal substrates such as cell cycle-regulating
proteins and others (IkBa, Bax and p27, etc.) leading to inhibition of cell prolifer-
ation coupled with induction of apoptosis [122, 123]. Accordingly, proteasomal
inhibition has also been exploited in cancer therapy [123, 124]. Inhibition of NF-kB
activation may contribute to the beneficial effect of proteasomal inhibition therapy.
In addition, NF-kB inhibitors combined with standard chemotherapy drugs might be
of benefit in the chronic inflammatory stage of tumor progression [125]. However,
this aspect of cancer therapy needs further evaluation.
Another beneficial effect of celastrol in cancer relates to its ability to remodel
bone by relatively reducing osteoclastic activity, while maintaining or increasing
osteoblastic activity. Metastasis of cancer to the bone may cause osteolysis, which
involves increased bone resorption. In regard to bone remodeling, the receptor
activator of nuclear factor-jB ligand (RANKL) promotes the proliferation and
differentiation of osteoclasts, whereas osteoprotegerin (OPG) secreted by the
osteoblasts is a soluble decoy receptor for RANKL, and it serves as a natural
inhibitor of osteoclast activation. In a study on an osteolytic bone metastasis model,
celastrol suppressed trabecular bone loss; and reduced the number and size of
osteolytic bone lesions, osteoclast number, and bone resorption [126]. In another
study on human osteosarcoma cells, celastrol caused G2/M phase cell cycle arrest,
and induced apoptosis and autophagy [81]. These observations in cancer studies are
supported by the results of our study in the rat AA model [9]. Celastrol protected
against bone and cartilage damage by regulating pro-inflammatory cytokines,
inhibiting RANKL, increasing RANKL/OPG ratio, inhibiting the secretion of
matrix-degrading enzymes such as MMPs, and reducing the number of osteoclasts
without much effect on osteoblasts [9].

12.8 Use of Celastrol-Containing Natural Products

for The Treatment of Chronic Inflammatory
and Autoimmune Diseases in Humans

Celastrol is present in several plants belonging to the celastraceae family. Some of

these plants have been used in traditional Chinese medicine (TCM) for several
decades/centuries as medicinal herbs for the treatment of a wide range of chronic
inflammatory disorders. For example, the extracts of the root, bark and stem of
Tripterygium wilfordii (Thunder God Vine), Celastrus orbiculatus, Celastrus
aculeatus and some other members of the Celastraceae family have been used for
280 S.H. Venkatesha and K.D. Moudgil

the treatment of RA, SLE, and other disorders [1–3, 5, 127]. However, a large part
of this information is based on folklores as well as documented description of the
use of these herbal products in old archived literature. Limited available information
is derived from studies on small numbers of patients and/or scientifically controlled
randomized clinical studies on the use of T. wilfordii in chronic inflammatory and
autoimmune diseases such as RA, juvenile RA, ankylosing spondylitis, and SLE [5,
128–132]. Of these, the most reliable clinical studies have been performed using T.
wilfordii in patients with RA. The efficacy of T. wilfordii extract against RA was
compared with that of two of the mainstream anti-arthritic drugs, namely sul-
phasalazine and methotrexate. Interestingly, T. wilfordii extract reduced the severity
of RA as assessed by well-established criteria, and the efficacy of T. wilfordii was
comparable to, or better than, that of sulphasalazine/ methotrexate [128, 133–136].
Furthermore, the combination of T. wilfordii and methotrexate was better than
methotrexate alone. However, the toxicity profile of this natural product needs
further assessment before it can be approved for therapeutic purposes.

12.9 Conclusions

Celastrol, a natural triterpene, has anti-inflammatory, antioxidant, and anticancer

activities. Besides targeting multiple cell signaling pathways, celastrol modulates
several other pathophysiological processes involved in chronic inflammatory dis-
eases, autoimmune diseases, and cancer [143]. Most of this information on celastrol
is based on in vitro model systems in the laboratory and preclinical studies in
animal models of human diseases. These studies have also offered mechanistic
insights into the use of celastrol-containing herbal extracts from celastraceae family
of plants for the treatment of some of these disorders in the traditional systems of
medicine. This knowledge might encourage the clinical testing of herbal prepara-
tions as has been done for T. wilfordii in RA patients. It is hoped that in the near
future, such natural products might be approved for use in mainstream therapy as
adjuncts for, or in place of, conventional allopathic drugs for RA and some other
chronic diseases. This would be a significant contribution to the therapeutic arsenal
against several chronic debilitating human diseases.

Acknowledgments We thank Dr. Hua Yu, Dr. Brian Astry, Dr. Siddaraju Nanjundaiah, Dr.
Rajesh Rajaiah, and Dr. Li Tong for their contribution to the original studies based on celastrol as
well as for their helpful discussions.

Funding This work was supported by National Institutes of Health (NIH)/National Center for
Complementary and Integrative Health (NCCIH) Grant Number AT004321.
12 Celastrol and Its Role in Controlling Chronic Diseases 281

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Chapter 13
Boswellic Acids and Their Role in Chronic
Inflammatory Diseases

H.P.T. Ammon

Abstract Boswellic acids, which are pentacyclic triterpenes belong to the active
pharmacological compounds of the oleogum resin of different Boswellia species. In
the resin, more than 12 different boswellic acids have been identified but only KBA
and AKBA received significant pharmacological interest. Biological Activity: In an
extract of the resin of Boswellia species multiple factors are responsible for the final
outcome of a therapeutic effect, be it synergistic or antagonistic. Moreover, the
anti-inflammatory actions of BAs are caused by different mechanisms of action.
They include inhibition of leukotriene synthesis and to a less extend prostaglandin
synthesis. Furthermore inhibition of the complement system at the level of con-
version of C3 into C3a and C3b. A major target of BAs is the immune system. Here,
BEs as well as BAs including KBA and AKBA, have been shown to decrease
production of proinflammatory cytokines including IL-1, IL-2, IL-6, IFN-c and
TNF-a which finally are directed to destroy tissues such as cartilage, insulin pro-
ducing cells, bronchial, intestinal and other tissues. NFĸB is considered to be the
target of AKBA. The complex actions of BEs and BAs in inflamed areas may be
completed by some effects that are localized behind the inflammatory process as
such tissue destruction. In this case, in vitro- and animal studies have shown that
BAs and BEs suppress proteolytic activity of cathepsin G, human leucocyte elas-
tase, formation of oxygen radicals and lysosomal enzymes. Pharmacokinetics:
Whereas KBA is absorbed reaching blood levels being close to in vitro IC50, AKBA
which is more active in in vitro studies than KBA, but undergoes much less
absorption than KBA. However, absorption of both is increased more than twice
when taken together with a high-fat meal.Clinical Studies There are a variety of
chronic inflammatory diseases which respond to treatment with extracts from the
resin of Boswellia species. Though, the number of cases is small in related clinical
studies, their results are convincing and supported by the preclinical data. These

H.P.T. Ammon (&)

Department of Pharmacology and Toxicology, Institute of Pharmaceutical Sciences,
University of Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany
e-mail: [email protected]; [email protected]
H.P.T. Ammon
Im Kleeacker 30, 72072 Tuebingen, Germany

© Springer International Publishing Switzerland 2016 291

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1_13
292 H.P.T. Ammon

studies include rheumatoid arthritis, osteoarthritis, chronic colitis, ulcerative colitis,

collagenous colitis, Crohn’s disease and bronchial asthma. It can not be expected
that there is cure from these diseases but at least improvement of symptoms in about
60–70 % of the cases. Side Effects The number and severity of side effects is
extremely low. The most reported complaints are gastrointestinal symptoms.
Allergic reactions are rare. And most authors report, that treatment with BEs is well
tolerated and the registered side effects in BE- and placebo groups are similar.

Keywords Boswellic acids Boswellic extracts Leukotrienes Prostaglandins






Proteolytic enzymes Cytokines Pharmacokinetics Rheumatoid arthritis




Inflammatory bowel diseases Bronchial asthma Diabetes Side effects

Abbreviations
AA Arachidonic Acid
ABA Acetyl-boswellic acid
Ac-OH-LA 3a-Acetoxy-28-hydroxylup-20 (29)
AKBA Acetyl-11-keto-b-Boswellic acid
a-BA a-Boswellic acid
b-BA b-Boswellic acid
BA Boswellic acid
BE Boswellic extract
BS Boswellia serrata
BSA Bovine serum albumin
CD4 CD4 lymphocytes
CD8 CD8 lymphocytes
CIA Collagen-induced arthritis
COX Cyclooxygenase
CRP C-reactive protein
CDAI Crohn’s Disease Activity Index
ConA Concavalin A
ESR Erythrocyte Sedimentation Rate
FCV Forced vital capacity
FEV1 Forced expiratory volume
GSH Reduced glutathione
Hb Hemoglobin
HETE Hydroxyeicosatetraenoic acid
12-HHT 12-Hydroxyheptadecatrienoic acid
HLE Human Leucozyte Elastase
IA2-A Tyrosinephosphatase antibody
IFN-c Interferon-c
IgG Immunoglobulin G
IgM Immunoglobulin M
IL Interleukin
IMG Immune globulin
13 Boswellic Acids and Their Role in Chronic Inflammatory Diseases 293

KBA 11-Keto-b-Boswellic acid

LA Lupenoic acid
LADA Late onset autoimmune diabetes of the adult
5-LO 5-Lipoxigenase
12-LO 12-Lipoxigenase
LPO Lipid peroxidase
LPS Lipopolysaccharide
LTB4 Leukotriene B
LTC4 Leukotriene C4
LTD4 Leukotriene D4
LTE4 Leukotriene E4
MIC Minimal inhibitory concentration
f-MLP n-formylmethionyl-leucyl-phenylalanine
MLD-STZ Multiple low-dose streptozotocin
NADPH Nicotinamide adenine dinucleotide phosphate hydrate
NFjB Nuclear transcription factor jB
NK cells Natural killer cells
NO Nitrogen monoxide
NSAID Nonsteroidal anti-inflammatory drugs
OA Osteoarthritis
PEFR Peak expiratory flow rate
PGE2 Prostaglandin E2
PBMC Peripheral blood mononuclear cells
PEFR Peak expiratory flow rate
PGE1 Prostaglandin E1
PHA Phytohemagglutinin
PMA Phorbol 12-myristate 13-acetate
PMN Polymorph mononuclear neutrophil leucocyte
RA Rheumatoid arthritis
SG Salai guggal
SOD Superoxid dismutase
STZ Streptozotocin
TA Tirucallic acid
Th1 Th1 lymphocytes
Th2 Th2 lymphocytes
TNF-a Tumor necrosis factor alpha
t1/2 Half-life

13.1 Introductory Remarks

Boswellic acids (BAs) belong to the active pharmacological principles of the oleo
gum resin from the trees of different Boswellia species. These trees are plants
typically found in the deserts, where the aborigines since thousands of years,
294 H.P.T. Ammon

scratch the bark and collect fluid, during dry periods. Burning the resin, the fumes
are used for disinfection, improvement of the scent of the air, as well as for cere-
monial and medical purposes. The resins are known as salai guggal (India),
frankincense (pure incense), incense and olibanum.
This chapter summarizes the present knowledge about medical history, phar-
macological active ingredients and therapeutical uses of the resin with special
impact on the anti-inflammatory mechanisms of boswellic acids.

13.2 Medical History

The oldest written document, which mentions frankincense as a drug is the Papyrus
Ebers. In 1873, Moritz Fritz Ebers, professor of Egyptology, received a more than
20 m long papyrus from an Arab businessman [40] describing the medical use of
frankinsence in Egypt. In India, the therapeutic applications of the oleogum resin of
Boswellia serrata, called Salai guggal (SG), are already described in early
Ayurvedic textbooks (Charaka Samhita, 1st–2nd century AD and in Astangahrdaya
Samhita, seventh century AD).
Remedies containing preparations from frankincense (here Boswellia carterii)
were also prescribed by the famous physicians Hippocrates, Celsus, Galenus,
Dioskurides and others for the treatment of tumours, carcinomas, edemas, inflam-
matory diseases including diarrhoea, and diseases of the respiratory tract [40].
Olibanum, the resin of various Boswellia species (Table 13.1), was also known
as a remedy in Europe from ancient times till the beginning of the twentieth
century. Then it disappeared from the list of doctoral prescriptions since scientific
evidence for therapeutical efficacy was missing. Only when Singh and Atal [69] and
Ammon et al. [3] showed that an extract from resin of Boswellia serrata inhibited
inflammation in an animal model resp. formations of leukotrienes in an in vitro
model, the scientific community became interested and the first human pilot studies
were initiated. By now, preparations of the resin are widely used to treat a variety of
chronic inflammatory disorders.

Table 13.1 Some species of Species Growing Product

Boswellia trees producing
incense and the respective B. carteri Birdw. Somalia Olibanum
areas of growth [40] B. sacra Flueck Nubia Olibanum
Saudi Arabia
B. frereana Birdw. Somalia Olibanum
B. bhau-dajiana Birdw. North Somalia Olibanum
B. papyrifera Hochst. Ethiopia Olibanum
B. neglecta S. Moore Somalia Olibanum
B. od0rata Hutch. Tropical Africa Olibanum
B. dalzielli Hutch. Tropical Africa Olibanum
B. serrata Roxb. India Salai Gugal
13 Boswellic Acids and Their Role in Chronic Inflammatory Diseases 295

13.3 Composition of the Resin

The resin of Boswelli species consists of mucus, volatile oil and resin acids
(Table 13.2). However, the quantitative composition of these constituents varies
from species to species.
The resin acids contain pentacyclic and tetracyclic triterpenes. Among the
pentacyclic triterpenes, only some boswellic acids are responsible for many of the
pharmacological effects; but also tirucallic acids (TAs), from the tetracyclic triter-
penic acids, have been shown to be biologically active .
Pentacyclic Triterpenes: Büchele et al. [9] identified 12 different pentacyclic
triterpenes in the samples of Boswellia extracts (BEs) (Table 13.3). From these 12,
the chemical structures of the two most active boswellic acids (BAs) are shown in
Fig. 13.2.
The authors reported significant quantitative differences of pentacyclic triterpe-
nes between various species: A striking difference was observed in the content of
the boswellic acids, i.e. KBA and AKBA (Table 13.3). Recently Beisner et al. [5]
identified a new pentacyclic triterpene from Boswellia serrata, it was just one,
3a-acetyl-20(29)-lupene-24-oic acid and Verhoff et al. [80] observed biological
activity of a novel C(28)-hydroxylated lupeolic acid. Though BAs received most
attention through the group of pentacyclic triterpenes, some other, including lupeol,
exhibit pharmacological activity as well, which are also considered boswellic acids
[49].
Tetracyclic Triterpenes: Among the tetracyclic triterpenes, three TAs have been
identified: 3-oxotirucallic acid, 3-hydroxytirucallic acid and 3-acetoxytirucallic
acid. Other resin compounds with pharmacological activities are: betulinic acid,
epi-lupeol, lupenoic acid, 1-ursene-2-diketone-incensole acetate, isoincensole and
isoincensole acetate and as well as terpenes that can be found in the volatile oil.

13.4 Preclinical Studies

13.4.1 Anti-inflammatory Actions

The first scientific publication with the title “Analgesic effect of the oleogum resin
of Boswellia serrata Roxb”. by Karr and Mennon appeared 1969 [31]. Singh and

Table 13.2 Composition of Boswellia carteri Boswellia serrata Roxb. (%)

oleogum resin of two different Birdw. (%)
Boswellia species [9]
Volatile oil 5–9 7.5–9 to 15
Pure resin ca. 66 55–57
Mucus *12–20 *23
296 H.P.T. Ammon

Fig. 13.1 Boswellia serrata Roxb. Desert of Radschastan, India, taken by the author

Table 13.3 Contents of different pentacyclic triterpenic acids in a frankincense extract

determined by Büchele and Simmet [8]
a-Boswellic acid 13.78 % b-Boswellic acid 19.20 %
Acetyl-a-boswellic acid 3.37 % Acetyl-b-boswellic acid 10.04 %
Lupeoloc acid 2.61 % 11-Keto-b-boswellic acid 6.66 %
Acetyl-lupeoloc acid 1.10 % Aetyl-11-keto-b-boswellic acid 3.81 %
11-Dehydro-a-boswellic acid 0.18 % 9,11-Dehydro-b-boswellic acid 0.83 %
Acetyl-9-11-dehydro-a-boswellic 0.06 % Acetyl-9-11-dehydro-b-boswellic 0.52 %
acid acid
13 Boswellic Acids and Their Role in Chronic Inflammatory Diseases 297

Fig. 13.2 Structures of

11-keto-b-boswellic acid and
acetyl-11-keto-b-boswellic
acid

Atal [69] published a paper entitled: “Pharmacology of an extract of salai guggal

ex-Boswellia serrata, a new non steroidal anti-inflammatory agent”. In this study,
Singh and Atal [69] observed that the oral administration of an alcoholic extract of
the oleogum resin of Boswellia serrata caused inhibition of the carrageenan- and
dextran-induced edema in the paws of rats and mice, suggesting anti-phlogistic
action of a BE. Since such an effect could also be observed in adrenalectomized
rats, the authors concluded that the anti-inflammatory effect of the BE was not due
to the liberation of glucocorticoids. Significant anti-arthritic activity of an acetone
extract from Boswellia carterii in lewis rats was reported by Fan et al. [19].
Moreover, anti-inflammatory, anti-noceptive and antioxidant activities have been
described using extracts from other Boswellia species; in this case, Boswellia
longata [44]. Boswellia extracts were also effective in the treatment of skin
inflammations [71].
A reduction of the carrageenan-induced edema in the paws of mice after oral or
intraperitoneal application of 1 mg/kg b-BA was recently reported by Siemoneit
et al. [68].
Introducing a new model, i.e. papaya latex also causing rat paw inflammation,
Gupta et al. [21] tested a variety of anti-rheumatic agents and BAs and compared
their effects with the actions of carrageenan in relation to rat paw inflammation. It
turned out that in the carrageenan model the inhibitory effects of indomethacin,
piroxicam, ibuprofen and acetylsalicylic acid were more pronounced than those of
BAs, whereas BAs were much more effective in the papaya latex model. The action
of prednisolone was almost similar in both models. This suggests that the
298 H.P.T. Ammon

Fig. 13.3 Arachidonic acid cascade

anti-inflammatory mechanism of BAs is different from the so-called “aspirin-like”

drugs and prednisolone (Fig. 13.3).

13.4.1.1 The Arachidonic Acid Cascade: Cyclooxygenase

and Cyclooxygenase Products

Activation of the arachidonic acid cascade by the membrane bound enzyme

phospholipase A2 (PLA2) is the initial step to start production of mediators of
inflammation. In this cascade (Fig. 13.3) cyclooxygenases and 5-lipoxigenase
produce prostaglandines and leukotrienes, which eventually lead to the typical
inflammatory symptoms. The mechanism of anti-inflammatory action of BEs and
BAs on members of the arachidonic acid cascade has been studied by several
scientists.
Boswellic Extracts: Based on the observations of Singh and Atal [69], Ammon
et al. [3] studied whether or not an alcoholic extract of the BS resin, could affect
prostaglandin synthesis in vitro. For this study, human platelets which contain
cyclooxygenase-1 (COX-1) were used. After in vitro stimulation with the Ca
ionophore A 23187, the extract inhibited 6-keto-PKF-1a synthesis, a product of
COX-1, up to 50 % at a concentration of 100 µg/ml (Fig. 13.4).
In another in vitro model extracts from Boswellia frereana suppressed cytokine
IL-1A-induced prostaglandin E2 synthesis and COX-2. In this case, epi-lupeol was
identified as a principal constituent [6].
13 Boswellic Acids and Their Role in Chronic Inflammatory Diseases 299

Fig. 13.4 Inhibition of 6-keto-PGF1a formation in human platelets by an alcoholic extract of salai
guggal (SG). 100 % = 277 pg 6-keto-PGF1a/107 cells. Number of experiments are given in
parentheses [3]

Boswellic Acids: As discussed above, the resins contain a variety of compounds

which could be responsible for their anti-inflammatory actions. These boswellic
acids are listed in Fig. 13.2.
Among these in a variety of studies most evidence has been gained from
acetyl-11-keto-b-boswellic acid (AKBA) and 11-keto-b-boswellic acid (KBA).
Thus, it was near at hand to study their effect on the production of the arachidonic
acid cascade intermediates. In the assay that used human platelets, AKBA produced
50 % inhibition of 12-hydroxyheptadecatrienoic acid (12-HHT) at a concentration
of 10 lM. Acetyl-boswellic acid (ABA) on the contrary showed no such effect.
Inhibition of COX products by AKBA was also observed by Siemoneit et al. [66,
68], using the human platelets model. BAs, preferably AKBA, inhibited COX-1
product formation with an IC50 * 6 lM. The authors also reported that COX-1 is
more sensitive to inhibition by AKBA than COX-2. In this sense, Siemoneit et al.
[68] described the inhibition of prostaglandin E 2 synthase-1 as the molecular basis
for the anti-inflammatory actions of boswellic acids. This means that BAs, in
particular AKBA, directly interfere with COX-1 and may mediate their
300 H.P.T. Ammon

anti-inflammatory actions not only by suppression of lipoxygenases, as discussed

later, but also by inhibiting cyclooxygenases, preferentially COX-1 [66]. On the
other hand, b-boswellic acid (b-BA) and AKBA stimulated arachidonic acid release
and 12-lipoxigenase activity in human platelets, where AKBA was less potent than
b-BA [47].
Cao et al. [12] observed, that, in addition to AKBA, b-BA, acetyl-a-BA,
acetyl-b-BA and betulinic acid are also inhibitors of COX-1. The IC50 was esti-
mated to be 10 lM.
Other compounds: Verhoff et al. [80] reported that a novel, yet unknown C(28)-
hydroxylated lupenoic acid (LA), that is 3a-acetoxy-28-hydroxylup-20(29)-
en-4b-oic acid (Ac-OH-LA) inhibits the biosynthesis of COX-, 5-LO- and
12-Lipoxigenase (12-LO)-derived eicosanoids from endogenous arachidonic acid
(AA) in activated platelets, neutrophils and monocytes from human blood with
consistent IC50 values of 2.3–6.9 µM. Thus, the data discussed suggests that
cyclooxygenases are targets of a variety of compounds in the resins of Boswellia
species.

13.4.1.2 The Arachidonic Acid Cascade: 5-Lipoxigenase

and 5-Lipoxigenase Products

Until 1991, there was no drug known which could predominantly inhibit the
synthesis of leukotrienes despite the need for such compounds to treat diseases
where leukotrienes have a major impact. It was the publication of Ammon et al. [3]
that showed that BEs not only affected formation of COX products, but to a much
larger extend also inhibited LTB4 synthesis. This publication and a later paper [50],
indicating that certain BAs are responsible for this effect, received large attention by
the scientific community.
Boswellic Extracts: After the stimulation of leukotriene synthesis with the calcium
ionophore A 231876 in polymorphmononuclear neutrophils (PMN)—which con-
tained 5-LO but no COX the same extract that was used for the studies on pros-
taglandin synthesis [3] in a concentration-dependent manner inhibited synthesis of
leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) (a metabolite
of the 5-LO cascade) formation at concentrations between 10 and 80 lg/ml
(Fig. 13.5). In this case, 50 % inhibition occurred at a concentration of 30 lg/ml,
indicating that the inhibitory action of the extract was significantly more pro-
nounced than its effect on COX-1 [3].
Boswellic Acids: Concerning the actions of BAs, Safayhi et al. reported in 1992
BAs to be specific, non-redox inhibitors of 5-LO. In this study, isomers of (a- and
b-) of BAs and their acetyl derivatives were isolated from the oleogum resin of BS.
It was observed that BAs partly decreased the formation of LTB4 in
calcium-ionophore-stimulated PMN in a concentration-dependent manner. AKBA
was most effective with an IC50 value of 1.5 µM.
13 Boswellic Acids and Their Role in Chronic Inflammatory Diseases 301

Fig. 13.5 Concentration-dependent inhibition of LTB4-formation by salai guggal (SG) ethanolic

extract in stimulated rat peritoneal PMNL (a), and the decrease in the inhibition of the sum of
5-LOx products (b) [3]. x-axis represents a and b

Structure–Action Relationships: This observation posed the question whether or

not certain chemical motif in the molecular structure of BAs are required for the
inhibition of leukotriene production. In this sense, Sailer et al. [53] studied the effect
of a variety of derivatives of BAs on leukotriene synthesis in Ca ionophore-
stimulated PMN. From the IC50 values, it was obvious that not all tested com-
pounds inhibited synthesis of leukotrienes and that some of them exhibited only a
partial effect. Taking into account the different chemical structures of the tested
BAs, the data revealed that a hydrophilic function at C-4 position in combination
with an 11-keto group appears to be essential for the inhibition of leukotriene
synthesis by BAs (Table 13.4).
5-LO as target: In intact PMN, signal transduction cascade of events that starts
with the external stimulation of leukocytes, where the participation of calcium ions
Table 13.4 Inhibitory effect R1 R2 IC50 (lM)
of different boswellic acids on
leukotriene AKBA AcO O 2.7 (1.5)
formation/5-lipoxygenase 3-Acetyl-11-OH-BA AcO OH/H Not tested
activity 3-Acetyl-11-MeO-BA AcO MeO/H Partial inhibition
KBA OH O 3.0
b-BA OH 2H Partial inhibition
3-Acetyl-b-BA AcO 2H Partial inhibition
Acetyl-9,11-dehydro-BA AcO – 0.75
9.11-Dehydro-BA OH – Partial inhibition
302 H.P.T. Ammon

is necessary, results in the production of leukotrienes by intracellular 5-LO. In order

to study whether or not BAs might directly interfere with 5-LO, in a cell-free
system from PMN, where this cascade is interrupted, the effect of various deriva-
tives of BAs on 5-LO activity was tested after addition of exogenous arachidonic
acid. It was found that the effects of different BAs were qualitatively similar to those
in intact PMN. However, the IC50 was higher than in intact cells which may be due
to the different environment of 5-LO in a cell-free system. [54].
Mechanism of 5-LO Inhibition: Further studies addressing the mechanism of
direct 5-LO inhibition used the supernatant of PMN. In this study, pentacyclic
triterpenes lacking the 11-keto function and/or carboxyl function on ring A (e.g.
amyrin and ursolic acid) did not or only partially inhibit 5-LO (Table 13.4). These
compounds even caused a concentration-dependent reversal of 5-LO inhibition by
AKBA, whereas the inhibitory actions of 5-LO inhibitors from different chemical
classes were not modified. Thus, it was concluded that AKBA acts directly on the
5-LO enzyme at a site selective for pentacyclic triterpenes which is different from the
arachidonate substrate binding site [51]. Using photoaffinity labelling, it was studied
whether or not 4-azido-5-125 iodo-salicyl-b-alanyl-11-keto-b-boswellic acid, a
photoaffinity analogue inhibiting 5-LO activity as efficiently as a lead compound,
could be displaced from its binding site by AKBA. This was in fact the case. On the
other hand, AA had no such effect. This data also suggests that AKBA in the presence
of calcium binds to a site distinct from the substrate (AA) binding site of 5-LO [54].

13.4.2 Proteolytic Enzymes: Human Leucocyte

Elastase (HLE)

Another factor in the inflammatory process is the release of proteolytic enzymes

from PMN which are involved in the destruction of cartilage. Tausch et al. [78]
observed that BAs suppressed the proteolytic activity of cathepsin G in a com-
petitive reversible manner with an estimated IC50 value of 0.6 lM. The same effect
was observed in humans after oral administration of a BE.
HLE is a serine protease produced and released by PMN. Using pure HLE,
Safayhi et al. [52] screened several pentacyclic triterpenes for inhibitory actions on
HLE. In this study AKBA decreased the activity of HLE with an IC50 value of
roughly 15 lM. Among the pentacyclic triterpenes, tested in concentrations up to
20 lM, substantial inhibition by b-BA, amyrin and ursolic acid was observed.

13.4.3 Oxygen Radicals

Oxygen radicals which are formed in PMN through the action of leukotrienes are
also involved in cartilage destruction in rheumatoid arthritis. Heil et al. [27] studied
13 Boswellic Acids and Their Role in Chronic Inflammatory Diseases 303

the effects of AKBA and of BEs on SOD-quenchable O2-radical formation in intact

PMNs and in a cell-free system. AKBA (IC50 * 10 µM) and extracts
(IC50 * 13 µg/ml) consistently inhibited phorbol-12-myristate-13-acetate (PMA)-
stimulated NADPH oxidase activity in rat peritoneal PMNs and reduced n-
formyl-methionyl-leucyl-phenylazanin (f-MLP) and PMA-induced oxidative burst
in stimulator-sensitive human blood PMN preparations.

13.4.4 The Complement System

The complement system is part of the non-specific humoral defence. It is an

important link between immuno- and inflammatory reactions.
Boswellic Acids: As early as 1987, inhibition of the guinea pig complement
system by a-BA and b-BA in concentration range between 5 and 100 lM has been
reported by Wagner et al. Anticomplementary activities of a mixture of BAs were
also described by Kapil and Moza [30]. They observed that BAs inhibited the
in vitro immunohemolysis of antibody-coated sheep erythrocytes by pooled guinea
pig serum. The reduced immunohemolysis was found to be due to an inhibition of
C3 convertase of the classical complement pathway.
The threshold concentration for inhibition was 100 lg per 0.1 ml diluted buffer
added to the assay. Thus, at least in vitro, BAs can suppress the conversion of C3
into C3a and C3b and therefore its proinflammatory actions of this system.

Summarizing the anti-inflammatory actions of BEs and BAs:

The resin of Boswellia species contains a variety of compounds with
anti-inflammatory properties. Among these are BAs but also some other
constituents have been described. Members of the arachidonic acid cascade
are targets of BEs and BAs as well as proteolytic enzymes, oxygen radicals,
and the complement system.
Most information is available from AKBA and KBA. Among the
cyclooxygenases, COX-1 seems to be most sensible to inhibition by BAs. Of
special interest is the inhibitory action of BAs on 5-LO. 5-LO produces
leukotrienes, which are responsible for plasma exudation, edema production,
chemotaxis and activation of white blood cells which release proteolytic
enzymes and oxygen radicals. 5-LO is more sensitive to BAs than
cyclooxygenases. In addition, BAs directly inhibit proteolytic enzymes and
oxygen radical formation in PMN, and last but not least also the complement
system, which is an important link between immune and inflammatory
reaction. Thus, especially BEs—a multicomponent product as well as BAs—
in contrast to the widely used NSAID drugs—exhibit their anti-inflammatory
action simultaneously affecting a variety of parameters that are involved in
inflammatory processes.
304 H.P.T. Ammon

13.4.5 The Immunosystem

White blood cells play an important role in the defence system of the body.
Granulocytes, monocytes, macrophages and lymphocytes cover the non-specific as
well as the humoral and cellular defence. However, under certain conditions, they
are also closely related to inflammatory autoimmune disorders. Humoral and cel-
lular defence have also been the subject of studies with BEs and BAs.

13.4.5.1 Humoral Defence

Humoral defence is one of the important measures of the body against infectious
diseases. It is related to the activation of B lymphocytes. After contact with anti-
gens, B cells differentiate to plasma cells, which then produce antibodies belonging
to the family of immunoglobulins.

Antibody Titres

Boswellic Acids: Humoral antibody synthesis in mice treated with sheep ery-
throcytes was studied by Sharma et al. [62] by determining the hemagglutinating
antibody titers in the serum. Here, primary antibody production (after a first antigen
injection) and secondary antibody production (after a second injection) were tested.
It was found that a single dose of a mixture of BAs (50–200 mg/kg)—as isolated by
Singh et al. [70] on the day of sensitization produced a dose related reduction
(10.4–32.8 %) in primary hemagglutinating antibody titers on day 4. Significant
reduction in antibody production was obtained with 100 and 200 mg/kg doses. On
the other hand the secondary antibody titers were significantly enhanced at lower
doses, the effect being more prominent at 50 mg/kg. Azathioprine was administered
as a reference compound (200 mg/kg p.o.) following the same schedule and
resulted in only 10.4 % inhibition of primary antibody synthesis and had no effect
on the secondary antibody production.
Usually, the second injection of antigens causes earlier antibody production with
higher affinity and is mainly due to immunoglobulin G (IgG).
Using the technique of complement fixing, a method for analysing antigen and
antibody titers, oral administration of a mixture containing BAs for 5 days around the
time of immunization resulted in a significant decrease in primary and secondary
complement fixing antibody titers at 100 mg/kg [62]. On the contrary, when a BA
mixture (25–100 mg/kg) was given orally for 5 days around immunization a marked
increase (15.38–26.92 %) in antibody production on day +7 was observed. The effect
was more pronounced at a dose of 25 mg/kg than at 50 or 100 mg/kg. The secondary
antibody titers were only marginally increased. Azathioprine treatment (100 mg/kg)
had no significant effects on primary as well as on secondary antibody titers.
13 Boswellic Acids and Their Role in Chronic Inflammatory Diseases 305

In mice, in which treatment with BAs was initiated 7 days prior to immuniza-
tion, BAs (25–100 mg/kg) elicited a dose related increase (37.93–63.79 %) in the
primary humoral response without significantly affecting the expression of the
secondary response. Levamisole (2.5 mg/kg, p.o.), an immunopotentiating agent,
displayed only a 25 % increase in primary and a 6.66 % increase in secondary
antibody titers.

Immunoglobulins

Boswellic Extract As far as low doses of BEs are concerned, the data received
with antibody titers finds their equivalent when immunoglobulins were determined
in the blood of sensitized mice. Previously, it was shown by Khajuria et al. [33] that
oral administration of a biopolymeric fraction (BOS 2000) from B. serrata (1–
10 mg/kg) elicited a dose related increase in the delayed hypersensitivity reaction
(early 24 h and delayed 48 h) in mice. It also stimulated the immunoglobulin M
(IgM) and IgG titers expressed in the form of plaques (PFC) and the complement
fixing antibody titre.
Gupta et al. [26] studied a possible immunological adjuvant effect of BOS 2000 on
specific antibody and the cellular response to ovalbumin in mice. Mice were
immunized s. c. with ovalbumin 100 µg and received BOS 2000, 80 µg on days 1
and 15. Two weeks later, BOS 2000, 80 µg corresponding to about 3.5 mg/kg bw
significantly increased IgG, IgG1 and IgG2a antibody levels in the serum compared
to the ovalbumin group.
At a dose of 80 µg BOS 2000, there was also a significant increase of lym-
phocytes CD4/CD8 and CD80/CD 86 analyzed in spleen cells and with cytokines
(IL-2 and IFN-c) profiles in the spleen cell culture supernatant. From their data, the
authors conclude, that BOS 2000 seems to be a promising balanced Th1- and Th2
lymphocytes directing immunological adjuvant which can enhance the immuno-
genicity of vaccines.
Thus, regarding the humoral defence in the in vivo experiments, it appears that
BAs have a dual effect on antibody titers: at lower doses, there is an increased
formation, whereas at higher doses, BAs may even have inhibitory effects. Whether
or not this data is of relevance for humans remains to be established. And the
question remains: what is a high and what is a low-dose regarding humans.

13.4.5.2 Cellular Defence: Effect on Leucocytes

Cellular defence against infectious microorganisms is mediated by the actions of

white blood cells. This includes proliferation, transformation, and activation of
lymphocytes, as well as induction of infiltration and phagocytosis by macrophages
and neutrophil granulocytes.
306 H.P.T. Ammon

Their functions are organized through the crosstalking among related cells via
cytokines produced and released from various leucocytes. Numerous authors have
dealt with the question whether or not extracts from the Boswellia resin, its volatile
oil, and/or boswellic acids would affect the activity of white blood cells and whether
or not such effects could be attributed to an interaction with the system of cross
talking between white blood cells by cytokines.
Boswellic Extracts and Essential Oils: In their studies on lymphocyte transfor-
mation, Badria et al. [4] used an assay with lymphocytes isolated from venous
human blood. In this study, a methylene chloride extract from the oleogum resin of
Boswellia carterii in the presence of phytohemaglutinin (PHA) or concanavalin A
(CON A) at 1 mg/ml stimulated lymphocyte transformation by 90 %
(EC50 = 0.55 mg/ml). Several compounds from the essential oil were also bio-
logically active. The different BAs and TAs tested, including acetyl-b-BA,
acetyl-a-BA, 3-oxo-TA, AKBA, b-BA, 3-hydroxy-TA and KBA, showed a similar
activity with EC50 values from 0.001 to 0.005 lM.
Mikhaeil et al. [41], who studied the chemical composition of frankincense oil,
also reported that the oil exhibited strong immunostimulant activity (90 %) of
lymphocyte transformation, when assessed in a lymphocyte proliferation assay.
From these studies, it may be concluded, that a variety of components of an extract
from Boswellia resins are effective in stimulating lymphocyte transformation and
thus can contribute to cellular defence.
Boswellic Acids: Sharma et al. [62] reported that, if spleen cells from
non-immunized mice were used, a mixture of various BAs in the range of 1.95–
125.0 lg/ml showed no spontaneous mitogenic activity and the cell viability was
comparable to controls. However, when, the test, was performed in the presence of
mitogen stimulating lipopolysaccharides (LPS, PHA, CON A and alloantigen), a
concentration-dependent inhibition of lymphocyte proliferation by BAs was
observed.
The effect of a mixture of BAs on phagocytosis was also studied by Sharma et al.
[62]. Preincubation of peritoneal macrophages with different concentrations of BAs
(1.95–125 lg/ml) resulted in an enhanced phagocytotic function of adherent
macrophages with a maximal effect occurring at 62.25 lg/ml.
The studies discussed so far have been performed in vitro. They showed stim-
ulating and inhibitory effects of BAs and BEs, respectively, employing different
concentrations. At present, the data is difficult to interpret and transferred to the
human situation.
In an in vivo experiment studying the anti-arthritic activity of boswellic acids
employing bovine serum albumin (BSA) induced arthritis, Sharma et al. [61]
observed that oral administration of a mixture of BAs (25, 50 and 100 mg/kg/day)
reduced the population of leucocytes when BSA was injected into the knee, and
changed the electrophoretic pattern of the synovial fluid proteins. The local injec-
tion of BAs (5, 10 and 20 mg) into the knee 15 min prior to BSA challenge also
reduced infiltration of leucocytes into the knee joint and inhibited the migration
13 Boswellic Acids and Their Role in Chronic Inflammatory Diseases 307

properties of PMN in vitro. As discussed above some BAs are inhibitors of 5-Lo.
So, the mechanism of action of BAs on leucocyte infiltration could be the inhibition
of LTB4—synthesis, i.e. reducing their chemotactic action.

13.4.5.3 The Cross Talk in Cellular Defence

NFjB plays a key role for the function of cytokines in their crosstalk between
immune competent cells. NFjB, which is usually present in the cytosol, is produced
by neutrophil granulocytes. This compound regulates the release of cytokines from
various white blood cells being responsible for proliferation, activation and function
of these cells.

Nuclear Transcription Factor ĸB (NFjB)

As far as possible effects of BEs are concerned, AKBA, as one of their pharma-
cological active compounds, has turned out to be a natural inhibitor of NFjB [16].
Thus, AKBA applied in vivo in mice (100 lmol/kg) for 1 week inhibited the NFjB
activation. Suppression of NFjB and NFjB-regulated gene expression by AKBA is
also reported by Takada et al. [77].
When AKBA was given systemically or locally in a mouse model with psoriasis,
the signalling action of NFjB and subsequent NFjB-dependent cytokine produc-
tion by macrophages was significantly suppressed. This was associated with pro-
found improvement of the psoriasis disease activity score [82]. As far as the
mechanism of action on NFjB is concerned, Syrovets et al. [76] reported that
acetyl-boswellic acids inhibit constitutively activated NFjB signalling by inter-
cepting the IjB-kinase activity in an in vitro model of PC-3 prostate cancer cells.
However, inhibition of NFjB is not only due to an action of BAs in extracts from
Boswellia species. Previously, Moussaieff et al. [45] reported that incensole acetate,
a compound isolated from Boswellia resins, also inhibits NFjB activation. This
action was combined with an anti-inflammatory effect in the inflamed mouse paw
model.

Cytokines

Crosstalk between leucocytes is organized by a group of compounds released from

monocytes, macrophages and T cells, called cytokines. These include interleukines
(IL), interferone-c (IFN-c) and tumornekrosisfactor-a (TNF-a), which regulate
different functions of white blood cells. Since on the one hand cytokines are under
regulation of NFĸB but on the other hand NFĸB is a target of AKBA a compound
of the resin of Boswellia species it was just logical to study whether or not BEs and
BAs would affect production/serum levels of various cytokines.
308 H.P.T. Ammon

Interleukines/IFN-c
Interleukins belong to the group of cytokines. There are pro- and anti-inflammatory
interleukins produced by macrophages and T cells following the recognition of a
pathogen.
Chervier et al. [13] studied the effect of an extract from Boswellic carterii on the
production of TH-1 and TH-2 cytokines by murine splenocytes. The use of an
extract with sesame oil as solvent resulted in a dose-dependent inhibition of IL-2
and IFN-c and a dose-dependent potentiation of IL-4 and IL-10. Gayathri et al. [19]
observed that a crude methanolic extract from BS and 12-ursine-2-diketone, a pure
compounds of BS, inhibited TNF-a, IL-1 B and IL-6 in cultured Peripheral Blood
Mononuclear Cells (PBMC). Observations on TH1/TH2 cytokines also revealed
marked down regulation of IFN-c and IL-12, whereas IL-4 and IL-10—which are
anti-inflammatory interleukins—was upregulated upon treatment with a crude
extract and the pure compound 1-ursene-2-diketone. This indicates that both are
capable of carrying out anti-inflammatory activities at sites where chronic inflam-
mation is present by switching off the proinflammatory cytokines.
Employing an acetone extract from Boswellia carterii Birdw. Fan et al. [18], in
an adjuvant arthritis model in lewis rats 900 mg/kg for 10 consecutive days,
observed significant decrease of arthritic scores. This was associated with sup-
pression of local tissue IL-1B and TNF-a. On the other hand, in a study of Khajuria
et al. [33], the authors demonstrated that oral administration of 1–10 mg/kg of a
polymeric fraction from BS (BOS 2000) increased levels of IL-4, IFN-c and TNF-a
in the serum.

Tumor Necrosis Factor-alpha (TNF-a)

TNF-a is also an important cytokine which activates local restriction of infections.
TNF-a released from macrophages, natural killer (NK) cells, and T cells acti-
vates vascular endothel, increases permeability of vessels for proteins and cells and
entry of IgG and complement into tissues. TNF-a induces fever. Massive liberation
of TNF-a from macrophages can even cause shock.
Inhibition of TNF-a and its signalling has been recognized as a highly successful
strategy for the treatment of chronic inflammatory diseases such as rheumatoid
arthritis. Previously it has been shown by Syrovets et al. [76] that acetyl-a-BA and
AKBA inhibited the generation of TNF-a in concentrations between 1 and 10 lM
in LPS-stimulated human monocytes. AKBA was found to be the most active
compound. The effect was mediated by an indirect inhibition of NFjB and sub-
sequent downregulation of TNF-a expression in human monocytes. In these cells,
Borsches and Grim (personal communication 2000) observed a
concentration-dependent decrease of TNF-a and IL-1B production in a concen-
tration range of 5–20 lM.
13 Boswellic Acids and Their Role in Chronic Inflammatory Diseases 309

The data from this chapter, covering cytokines, suggest that low doses of
Boswellia preparations increase production of proinflammatory cytokines,
whereas high doses are even suppressive. In other words, low doses of BEs
may stimulate cellular defence f.i. in case of infections and high doses may be
useful in defeating the proinflammatory actions of cytokines in autoimmune
inflammatory disorders.

Cytokines in Autoimmune Diabetes

Type 1 diabetes is an autoimmune disease, where a chronic inflammatory process
finally causes death of insulin producing b-cells of pancreatic islets and insulin
deficiency. Autoimmune diseases are—as discussed—associated with overexpres-
sion of proinflammatory cytokines. Among these, in patients with type 1 diabetes
NFjB, TNF-a, IFN-c, IL-1B and IL-2 have been found to be increased in
splenocytes and PBMC [15].
Application of multiple low doses of streptozotocin (MLD-STZ) is a method to
induce autoimmune diabetes in animals similar to type 1 diabetes in humans.
Shehata et al. [64] studied whether or not a BE could prevent hyperglycemia,
inflammation of pancreatic islets and increase of proinflammatory cytokines in the
blood in MLD-STZ treated mice. In this study, treatment with streptozotocin
(STZ) (40 mg/kg i.p. for 5 days) after 10 days produced a permanent increase of
blood glucose, infiltration of lymphocytes into pancreatic islets, apoptosis of
periinsular cells and after 35 days shrinking of islet tissue. This was associated with

Fig. 13.6 Effect of boswellic extract (150 mg/kg i.p. for 10 days) on blood glucose levels of
MLD-STZ-treated mice (mean ± SE; n = 4) [64]
310 H.P.T. Ammon

an increase of proinflammatory cytokines (IL-1A, IL-1B, IL-2, IL-6, IFN-c, TNF-a)

in the blood. In STZ treated mice, simultaneous i.p. injection of 150 mg/kg of BE
over a period of 10 days prevented animals from increase of blood glucose levels
(Fig. 13.6). Histochemical studies showed, that BE avoided lymphocyte infiltration
into pancreatic islets, apoptosis of peri-insular cells and shrinking of islet size.
As far as the cytokines tested are concerned, there was a significant inhibition of
the increase of IL-1A, IL-1B, IL-2, IL-6, IFN-c and TNF-a in the blood. It was
concluded that extracts from the gum resin of Boswellia serrata prevent islet
destruction and consequent hyperglycemia in an animal model of type 1 diabetes,

Fig. 13.7 Effect of Boswellic extract (150 mg/kg i.p. for 10 days) on proinflammatory cytokines
in the serum of MLD-STZ-treated mice. C control (mean ± SE; n = 4) [64]
13 Boswellic Acids and Their Role in Chronic Inflammatory Diseases 311

probably by inhibition of the production/action of cytokines related to induction of

islet inflammation in an autoimmune process (Fig. 13.7).
When these experiments were repeated with KBA or AKBA the same results
were achieved [65]. So far, however, no clinical data is available to support these
animal experiments.

13.5 Antimicrobial and Antiparasitical Effects

Resins in general protect the stems of trees from any microbial attack.
Raja et al. [48] studied the antimicrobial activities of various Boswellic acids
against 112 pathogenic bacterial isolates including ATCC strains. Here, AKBA
exhibited the most potent antibacterial activity showing a Minimal Inhibitory
Concentration (MIC) range of 2–8 lg/ml against the entire Gram-positive bacterial
pathogens tested. It exhibited a concentration-dependent toxicity of Staphylococcus
aureus ATCC 29213 up to 8  MIC. The antibacterial mode of action of AKBA
probably occurred via disruption of the microbial membrane structure.
In another study, methanolic and aqueous extracts from Boswellia dalziellia also
showed a broad spectrum of inhibitory activity against bacteria, both Gram positive,
Gram negative and fungi [2].
Serratol, a diterpene isolated from the gum resin of Boswellia serrata, was
previously tested by Schmidt et al. [57] for its antiprotozoal activity. It was found to
be active against Tripanosoma brucie, Rhode siense (sleeping sickness) and
Plasmodium falciparum (Tropical Malaria).
Essential oils of the gum resin from different Boswellia species including
Boswellia carterii (Somalia), Boswellia papyrifera (Ethiopia), Boswellia serrata
(India) and Boswellia rivae (Ethiopia) were tested by Camardes et al. [11] and
showed antimicrobial activity.

13.6 Clinical Studies

So far, only extracts from the gum resin of Boswellia serrata have been used for
clinical trials. Studies with isolated compounds are still missing. The studies focus
on chronic inflammatory diseases.

13.6.1 Pharmacokinetics of Boswellic Acids

In modern pharmacology, the therapeutic effects of drugs are closely related to their
pharmacokinetic properties. However, as far as extracts from herbal medicine are
concerned, it is impossible to establish pharmacokinetic data since they contain a
312 H.P.T. Ammon

Table 13.5 Antimicrobial and antiparasitical effects of extracts, essential oils, and chemical
constituents from different Boswellia species
Extracts
Methanolic extract (stem Broad spectrum of gram positive and gram negative [2]
bark) bacteria and fungi
B. dalzielli
Methanolic extract Gram positive multiresistent Staphylococcus strains [42]
B. ameero
B. elongata
Boswellic extract Influenza virus A [43]
B. ameero
B. elongata
Methanolic extract Hepatitis virus C [29]
B. carterii
Methanolic extract from Musceocideal [23]
stem bark
Essential oils
Essential oil different B. Gram positive bacteria, gram negative bacteria, antifungal [11]
species
Essential oil B. riva Candida albicans [56]
Chemical constituents
Serratol (diterpene from B. Tripanosoma brucei rhodesiense (sleeping sicknes) [57]
serrata) Plasmodium falsiparum (tropical Malaria)
AKBA Staphylococcus aureus, gram positive bacterial pathogens [48]

variety of compounds which are involved in the final therapeutic actions. Moreover,
their contents vary from species to species. Thus, the only possibility for a rough
standardization is to determine one or more components of an extract where sig-
nificant pharmacological effects can be expected. At present, in case of BEs, these
are KBA and AKBA. Studies with isolated KBA and AKBA in humans do not exist
yet. (Table 13.5)

13.6.1.1 Blood Levels of AKBA and KBA

In an open uncontrolled trial with 12 healthy male volunteers [63], a single oral
dose of a commercial product of an extract out of gum resin from Boswellia serrata
(WokVel™) containing 333 mg was given after standard breakfast. The extract
contained 6.44 % KBA, 2 % AKBA, 18.51 % b-BA, 8.58 % 3-O-acetyl-b-BA,
6.93 % a-BA and 1.85 % 3-O-acetyl-aBA. In this study, only the concentration of
KBA in the plasma was measured (Fig. 13.8). As shown in Table 13.6 maximum
concentration of KBA was found after 4.5 h 2.72 µM. This concentration is in the
range of the IC50 of KBA for the inhibition of leukotriene synthesis in vitro. In this
study, the elimination half life (t1/2b) was 5.97 h. This suggests that when the
above treatment is given every 6 h, the steady state plasma concentration of KBA is
reached after approximately 30 h.
13 Boswellic Acids and Their Role in Chronic Inflammatory Diseases 313

Fig. 13.8 Mean plasma concentration of 11-keto-b-boswellic acid versus time after oral
administration of 333 mg Boswellia serrata extract WokVel™ containing 6.44 % KBA [63]

Table 13.6 Pharmacokinetic Pharmacokinetic parameters Mean SEM

parameters of KBA after oral
administration of 333 mg Cmax (lmol/ml) 2.72  10−3 0.18
Boswellia serrata extract tmax (h) 4.5 0.55
Wok Vel™ containing Kel (per h) 0.17 0.04
6.44 % KBA [63 t½ b (h) 5.97 0.94
AUC0–∞ (lmol/ml h) 27.33  10−3 1.99
Ka (per h) 0.62 0.17
t1/2 a (h) 2.35 0.63
Vd (1) 142.87 22.78
Cl (ml/min) 296.10 24.09

As discussed in Sect. 3, Büchele and Simmet [8] identified 12 different penta-

cyclic triterpenic acids in an extract of Boswellia serrata. Their structures and
contents are shown in Fig. 13.2 and Table 13.3. Using high-performance liquid
chromatography and photodiode array detection, after 10 days of treatment with an
oleogum resin extract of Boswellia serrata (4  786 mg per day), the authors could
most of them (Table 13.3) identify in the plasma of a patient with brain tumor.
After intake of BEs by humans AKBA in the blood is below its IC50 in vitro, but
at least KBA is near to its in vitro value of about 2.4 µM. As far as b-BA is
concerned, which represents the highest percentage in a BE (around 19 % s.
Table 13.3) its concentration in the blood after oral injection of a BE was found to
be there in effective concentrations [68].
Effect of Food Intake: It is very well known that absorption of drugs may be
related to food intake and to the kind of food. In this sense, Sterk et al. [74] studied
the effect of food intake on the bioavailability of BAs from a herbal preparation of
314 H.P.T. Ammon

Boswellia. Twelve healthy subjects fasted 10 h before and 4 h after drug admin-
istration (group A). A second group (B) received a high-fat meal together with the
drug. The volunteers swallowed a single dose of 3 capsules with 282 mg (total of
786 mg) extract, containing 143.4 mg b-BA, 103.71 mg a-BA, 82.71 mg
acetyl-b-BA, 48.12 mg KBA, 28.71 mg AKBA and 26.25 mg acetyl-a-BA. The
time course of the plasma concentrations of the most active boswellic acids, i.e.
KBA and AKBA, was dramatically different between fasted and high-fat meal
volunteers. The calculated pharmacokinetic parameters are shown in Table 13.7.
It is obvious that in the high-fat meal group Cmax of KBA is about 2.7 times and
AKBA 4.8 times higher than in the fasting group. This can be explained by the
lipophilic character of KBA and AKBA. Consequently, BEs should be taken
together with food.

Table 13.7 Pharmacokinetics parameters of 11-keto-b-boswellic acid (KBA) and acetyl-11-

keto-b-bosellic acid (AKBA) after administration of three capsules BSE–018 (786 mg dry extract
of the oleogum resin from Boswellia serrata) as a single oral dose under fasted conditions
(treatment A) and under fed conditions (treatment B) [74]
Parameters Values (geometric mean + range) Treatment B (fed conditions)
Treatment A (fasted conditions) n = 12
n = 12
11-Keto-b-boswellic acid
AUC(0–∞)inf (ng h ml−1) 1.660.72(940.3–3778.1) 3037.15(1481.9–6583.1)
AUC(0–tz) (ng h ml−1) 658.4(137.0–2747.3) 2451.8(1085.0–6125.4)
Cmax (ng ml−1) 83.8 (24.9–243.8) 227.1 (101.0–418.1)
Tmax (h)a 3.5(2.0–4.0) 4.0 (3.0–8.0)
K(h−1) 0.017 0.027
T½ (h) 40.8 25.7
Acetyl-11-keto-b-boswellic acid
AUC(0–∞) (ng h ml−1) 153.6 (59.2–647.9) 748.9(271.4–5316.8)
AUC(0–tz) (ng h ml−1) 47.4 (8.0–232.0) 243.7(53.0–3528.0)
Cmax (ng ml−1) 6.0 (0.9–45.7) 28.8 (13.0–264.5)
a
Tmax (h) 2.0 (0.0–24.0) 3.0 (0.5–60.0)
K (h−1) 0.066 0.046
T½ (h) 10.5 15.0
AUC(O-oo) = area under the concentration time curve, extrapolated to infinity
AUC(0–tz) = area under the concentration time curve
O = last quantifiable sample
Cmax: = maximum plasma concentration
K = overall elimination rate constant by log linear regression
Tmax = time to Cmax
T½ = terminal elimination half-life from K
a
Median (and range)
13 Boswellic Acids and Their Role in Chronic Inflammatory Diseases 315

13.6.1.2 Absorption

Extract preparations from the resin of Boswellia species, in most cases, are
administered by oral route. Compared to the concentrations of BAs, in extract
preparations their concentrations in the blood after oral application are very low.
This holds true, especially for the most active BA, i.e. AKBA. Krüger et al. [37]
explain this phenomenon to be due to poor permeability of AKBA and also
moderate absorption of KBA.

13.6.1.3 Distribution

According to Siemoneit et al. [67] 11-keto-boswellic acids show > 95 % plasma

protein binding. However, due to their lipophilic properties it is possible, that BAs
may accumulate in lipophilic tissues.

13.6.1.4 Metabolism

In rat liver microsomes, human liver microsomes and hepatocytes, Krüger et al.
[36] found that KBA but not AKBA undergoes extensive phase I metabolism.
Oxidation to hydroxylated metabolites is the principal metabolic route. In vitro,
KBA yielded metabolic profiles similar to those obtained in vivo in rat plasma and
liver, whereas no metabolites of AKBA could be identified in vivo. Unexpectedly,
AKBA is not deacetylated to KBA.

13.6.1.5 Topical Administration

Extracts from the gum resin of BS are frequently used topically in ointment
preparations for skin diseases. Singh et al. [71] reported anti-inflammatory activity
of BAs through this route in different acute and chronic models of inflammation
such as arachidonic acid- and croton-oil-induced mouse ear edema,
carrageenan-induced rats paw edema and adjuvant-induced arthritis in rats. The
results of the study revealed that the antiphlogistic effect observed through this
route is in accordance with the study conducted with the systemic application.

13.6.2 Chronic Inflammatory Diseases

From the preclinical studies it is reasonable to conclude that BEs and their active
constituents will be effective in inflammatory disorders, since they inhibit the
responsible related factors, i.e. products of the arachidonic acid cascade, especially
leukotrienes, members of the complement system—and especially factors of the
316 H.P.T. Ammon

immune system which are related to the inflammatory autoimmune system.

However, their actions will not be as immediate as it is the case with nonsteroidal
anti-inflammatory drugs (NSAIDs) or glucocorticoids since it is evident, from
pharmacokinetc studies, that the concentration of the most active compounds, i.e.
KBA and AKBA in the blood does not reach effective levels after a single
administration. This is in accordance with the experience of patients who report that
the therapeutic effect of BEs occurs with a certain delay of 1 or 2 weeks.

13.6.2.1 Rheumatoid Diseases

As early as 1986, Singh and Atal [69] reported that in an arthritic model in rats a
mixture of boswellic acids showed anti-inflammatory activity and Sharma et al. [61]
observed that in another arthritis model a mixture of BAs reduced infiltration of
leucocytes into an arthritic knee. Very recently, Umar [79] studied the effect of an
extract from Boswellia serrata in the collagen-induced arthritis model in rats on
most of the inflammatory parameters discussed. BE was administered at doses of
100 and 200 mg/kg body weight once daily for 21 days. The effects of the treat-
ment in rats were assessed by biochemical parameters (articular elastase, LPO,
GSH, catalase, SOD and NO), inflammatory mediators (IL-1B, IL-6, TNF-a, IL-10,
IFN-c and PGE2), and histopathology in joints. In this study, BE was effective
bringing significant changes to all the tested parameters (articular elastase, LPO,
GSH, catalase, SOD and NO). Oral administration of BE resulted in significantly
reduced levels of inflammatory mediators (IL-1B, IL-6, TNF-a, IFN-c and PGE2),
and increased levels of IL-10. The protective effects of BE against RA were also
evident from the decrease in arthritis scores and bone histology. On the basis of
these studies and traditional experiences, a variety of clinical studies have been
initiated including subjects suffering from rheumatoid arthritis and osteoarthritis.

Rheumatoid Arthritis

Rheumatoid arthritis belongs to the class of autoimmune diseases. The rheumatoid

lesion, which is located in the synovial membrane, includes invasion of macro-
phages and lymphocytes that release cytokines such as interleukines and TNF-a.
Neutrophils which are present in the synovial fluid of the inflamed joints produce
leukotrienes, oxygen radicals and elastase activity, which finally cause synovialitis
and destruction of cartilage.
Boswellic Extracts: Etzel [17] summarized the results of 11 mostly unpublished
studies using extracts from the oleogum resin of Boswellia serrata in patients with
chronic polyarthritis. The criteria were pain, swelling, sensitivity and tolerance. In
five studies, patients were treated intraindividually in 2 placebo-controlled studies.
In a meta-analysis of the above studies, about 50–60 % of the patients responded to
this treatment. Pain and swelling of joints were improved by the commercial
13 Boswellic Acids and Their Role in Chronic Inflammatory Diseases 317

product H15™ (produced by Gufic Ltd. Mumbai, India), when compared to the
placebo group (p < 0.05). Unfortunately, not all of these studies could be
re-examined by the author of this chapter as they were not published. As a con-
sequence, the quality and the outcome of these studies were critiqued by the
German Society of Rheumatology in 1998. The arguments are mainly based on a
study of Sander et al. [55]. In this multicentre controlled trial, the authors studied
the effect of H 15™ versus placebo over a period of 12 weeks in 37 outpatients
with rheumatoid arthritis and chronic polyarthritis being under constant therapy
with steroids and disease-modifying antirheumatic drugs. The patients received 9
tablets of H 15™ (3600 mg) or placebo daily in addition to their previous therapy.
Doses of NSAIDs could be adjusted on demand. Efficacy parameters were the index
for swelling and pain, ESR and CRP. Pain and NSAID doses were documented at
the beginning and at 6 and 12 weeks after initiation. In this study, treatment with
H15™ resulted in no measurable effect. However, this study suffers from the
drawback that the effect of the BE alone in comparison to standard therapy was not
tested. It can be assumed that administration of H15™ to patients who are being
already treated with steroids and patients with basic therapy will not experience an
additional effect.

Osteoarthritis

Osteoarthritis is a common chronic, progressive, skeletal degenerative disorder,

which often affects the knee joint and the shoulder.
Gupta et al. [22] investigated the effect of S-Compound™ (Rahul Pharma,
Jammu Tawi, India), a mixture of various boswellic acids in patients suffering from
chronic osteoarthritis (OA) of the primary type. A total of 50 patients were treated
with either S-Compound™ (30 patients) or with Ibuprofen (20 patients) for 12–
24 weeks. In this study, 60 % of the patients treated with S-Compound™ showed
relief of symptoms in 12 weeks. 33.3 % out of the remaining 40 % recovered in
24 weeks but 2 patients had no relief. Only 30 % of the patients improved with
Ibuprofen.
In a randomized, double-blind, placebo-controlled cross-over study, Kimmatkar
et al. [34] studied efficacy, safety and tolerability of a BE preparation (trade name
WokVel™ capsules) in 30 patients with osteoarthritis in the knee, 15 of them
receiving the drug or placebo for 8 weeks. Each capsule of WokVel™ contained a
standardized extract of Boswellia serrata oleogum resin with a minimum of 65 %
organic acids or a minimum of 40 % total boswellic acids. The patients received
one capsule with 333 mg of the extract three times a day and after a washout phase
the alternative treatment. All patients reported decreased knee pain, increased knee
flexion, an increase in the walking distance and in the ability to climb stairs. The
symptoms returned after withdrawal of the treatment.
In a different double-blind, randomized, placebo-controlled study, Sengupta
et al. [59] evaluated the efficacy and safety of 5-Loxin™ (Laila Natura, New Dehli,
318 H.P.T. Ammon

India) for treatment of OA of the knee. 5-Loxin™ is a novel Boswellia serrata

extract enriched with 30 % AKBA.
Seventy-five OA patients were included in the study. The patients received either
100 mg (n = 25) or 250 mg (n = 25) of 5-Loxin™ daily or a placebo (n = 25) for
90 days. Each patient was evaluated for pain and physical functions using the
standard tools (visual analogue scale, Lequesne’s Functional Index, and Western
Ontario and McMaster Universities Osteoarthritis Index) at the baseline (day 0),
and at days 7, 30, 60 and 90. Additionally, the cartilage degrading enzyme matrix
metalloproteinase-3 was evaluated in the synovial fluid from OA patients.
Measurement of a battery of biochemical parameters in the serum, haematological
parameters and urine analysis were performed to evaluate the safety of 5-Loxin™.
At the end of the study, both doses of 5-Loxin™ conferred clinically and statisti-
cally significant improvements in pain scores and physical function scores.
Interestingly, improvements in pain score and functional ability were recorded not
immediately but first 7 days after the start of treatment. Corroborating the
improvements in pain scores in the treatment groups, the authors also noted sig-
nificant reduction in synovial fluid matrix metalloproteinase-3. In a further study
with 40 patients (20 with the same product and 20 with placebo), Sengupta et al.
[60] reported similar results. Moreover, in an open randomized controlled study,
Sontakke et al. [72] compared the effect of the Boswellia extract preparation
WokVel™ capsules three times daily with a selective COX-2-inhibitor Valdecoxib
(10 mg once daily) over a period of 6 months. Both groups showed comparable
improvement as far as pains, stiffness and physical movement are concerned.

13.6.2.2 Chronic Inflammatory Bowel Diseases

Treatment of the symptoms of bowel disease with preparations containing

olibanum/frankincense has a long tradition (see Chap. 2). Modern science has
attributed these symptoms to different acute and chronic disorders. The latter
include ulcerative colitis, chronic colitis, collagenous colitis and Crohn’s disease.
Moreover, it has been recognized that certain mediators of inflammation and
immunological parameters, i.e. cytokines, play an important role in these disorders.
Thus, it is known that the mucosa of patients with chronic inflammatory bowel
diseases is synthesizing considerable amounts of leukotrienes LTB4, LTD4 and
LTE4, which increase the production of mucus and stimulate contraction of the
smooth muscle of the gastrointestinal tract. As far as the immune system is con-
cerned, autoimmune antibodies have been detected in patients with ulcerative colitis
and Crohn’s disease. In addition, IL-1 and TNF-a have also been shown to be of
importance in intestinal inflammations [73]. Based on these findings, the effect of
oleogum resin preparations from Boswellia serrata were tested in patients with
different chronic inflammatory bowel diseases.
13 Boswellic Acids and Their Role in Chronic Inflammatory Diseases 319

Ulcerative Colitis

Ulcerative colitis is a chronic inflammatory disease with remissions and exacer-

bations affecting primarily the rectal mucosa, the left colon, but in many instances
also the entire colon. It is characterized by rectal bleeding and diarrhoea affecting
mainly, but not exclusively, the youth and early middle age.
Boswellic Extract: In 34 patients, suffering from ulcerative colitis grades II and
III, the effect of an alcoholic extract of Boswellia serrata oleogum resin was studied
according to Singh et al. [70] 350 mg thrice daily for 6 weeks, analyzing stool
properties, histopathology, rectal biopsies via scan microscopy, and blood param-
eters including Hb, serum iron, calcium phosphorus, proteins, total leucocytes, and
eosinophils [23]. Eight patients received sulfasalazine (1 g thrice daily) serving as
controls. All parameters tested improved after treatment with the extract. 82 % out
of treated patients went into remission while the remission rate for sulfasalazine was
75 %.

Chronic Colitis

This disease was characterized by the authors [25] as vague lower abdominal pain,
bleeding per rectum with diarrhoea and palpable tender descending and sigmoid
colon. Its pathophysiology seems to be different from that of ulcerative colitis.
Boswellic Extract: In this study, 30 patients, 17 males and 13 females age 18–
48 years, were included. Twenty patients were given a preparation of the oleogum
resin of Boswellia serrata (S-Compound™) containing KBA 0.63 %, AKBA
0.7 %, acetyl-b-BA and b-BA 1.5 % (900 mg daily divided in three doses for
6 weeks) and ten patients receiving sulfasalazine (3 mg daily divided in three doses
for 6 weeks) served as controls. Out of the 20 patients treated with Boswellia
oleogum resin, 18 patients showed an improvement in one or more of the following
parameters: stool properties, histopathology as well as scanning electron micro-
scopy, Hb, serum iron, calcium, phosphorus, proteins, total leukocytes, and
eosinophils.

Collagenous Colitis

A rarer chronic inflammatory bowel disease is collagenous colitis. It is character-

ized by aqueous diarrhoea, histological thickness of the mucosa and a subepithelial
collagen band.
Boswellic Extract: In a randomized, placebo-controlled, double-blind multicenter
study, quality of life and histology were studied in 25 patients receiving either
400 mg BE (H15™) three times a day or a placebo. After 6 weeks of treatment,
significant improvements were reported for 58.3 % of the Boswellia group and for
30.8 % in the placebo group [38].
320 H.P.T. Ammon

Crohn’s Disease

Crohn’s disease (Ileitis terminalis) is a chronic inflammatory disease of the entire

gastrointestinal tract from the oral cavitity up to the anal area. Most common
locations are the terminal ileum, colon and rectum. Typical symptoms are
abdominal pain, diarrhoea with mucus, purulency and aqueous stools. The
anti-inflammatory treatment at present consists of the use of mesalazine and
sulfasalazine.
Boswellic Extracts: Gerhardt et al. [20] studied the effect of a BE (H15™) on
symptoms in an active state of Crohn’s disease. In a double-blind, verum-controlled
parallel group comparison, 102 patients were randomized. The protocol population
included 44 patients treated with BE H15™ and 39 patients treated with mesala-
zine. As primary parameter, the change of the Crohn’s Disease Activity Index
(CDAI) from the beginning to the end of the therapy was chosen. H15™ was tested
for non-inferiority compared to the standard treatment with mesalazine. In this
study, the CDAI after treatment with H15™ was reduced by 90 and after therapy
with mesalazine by 53 score points in the mean. A difference between both treat-
ments could not be proven to be statistically significant. Thus, the data suggest that
in treatment of Crohn’s disease in an active state the extract from the oleogum resin
of Boswellia serrata is at least as effective as a standard medication under the
conditions of this study.
In a previous study [28] it was investigated whether or not, in a period of
2 years, permanent treatment with Boswellan™ (special BE extract from
Pharmasan Company—Freiburg, Germany) would increase the time of remission.
This was, however, not the case. Obviously, the Boswellia preparations are only
effective in an acute phase of the disease, improving factors related to the CDAI.

13.6.2.3 Respiratory Diseases: Bronchial Asthma

Since ancient times (see Chap. 2) respiratory symptoms are treated with prepara-
tions of olibanum/frankincense. This still holds for bronchitis including cough and
expectoration. A special case is bronchial asthma.
Bronchial asthma is a chronic inflammatory condition characterized by bron-
chial hyper-responsiveness and reversible airways obstruction.
Increased production of leukotrienes both during episodes of asthma and in
patients with stable asthma was shown by Chanarin and Johnston [14]. Bronchial
asthma is also a case of autoimmune diseases.
Boswellia Extracts: In a double-blind, placebo-controlled trial with asthma
patients Gupta et al. [24] tested forty patients with a mean duration of bronchial
asthma of 9.58 ± 6.07 years. They received a preparation of oleogum resin of
Boswellia serrata (S-Compound™) of 300 mg three times daily over a period of
6 weeks. In this study, 70 % of the patients showed improvement of the disease
13 Boswellic Acids and Their Role in Chronic Inflammatory Diseases 321

measured by the disappearance of physical symptoms and by signs such as dysp-

noea, rhonchi, the number of obstructive attacks, increase in respiratory parameters,
including FEV1, FVC and PEFR as well as by a decrease in eosinophilic count and
ESR. In the control group of 40 patients only 27 % of these patients showed
improvements.

13.6.2.4 Autoimmune Diabetes: Tyrosinephosphatase Antibody

Autoimmune Diabetes is associated with an increase of specific markers in the

blood including tyrosinephosphatase antibody (IA2-A), which indicate presence of
insulitis. So far there exists no clinical study whether or not a BE or BA are
effective in human autoimmune diabetes.
Recently in a case report it was shown that a BE decreased blood levels of IA2-A
in a patient with “Late onset Autoimmune Diabetes of the Adult” (LADA).
A female patient 50 years old with a body weight of 72 kg where diabetes was first
diagnosed in November 2012 and who was under insulin therapy (Humalog™,
Humaninsulin basal™) received a BE “Indian Boswellia the Original™, a new
product from Indian Boswellia Laboratory, Agra India, containing 3.6 % KBA and
1.4 % AKBA. The patient received 3 times daily 2 tablets (400 mg BE each) per
oral route over a period of 8.5 weeks.
During this time IA2-A levels in the blood dropped from 25 to 10 K/U/L
indicating improvement of insulitis [58].
The authors are aware, that this is only one case with all its restrictions. But it
may stimulate other attempts to strengthen the hypothesis that BEs or BAs
including KBA and AKBA may be an option for prevention/treatment of autoim-
mune diabetes.

What do the clinical studies teach:

In the clinical studies, that have been performed so far between 6 and
13 weeks and with a limited number of patients, improvement of symptoms
in patients suffering from rheumatoid arthritis, osteoarthritis, chronic
inflammatory bowel diseases and bronchial asthma were reported. This is in
line with preclinical in vitro and in vivo studies showing pharmacological
activity of BEs and some BAs on mediators of inflammation as well as on
factors related to autoimmune disorders. It should, however, be taken into
consideration that the effects of BEs are more likely to be an overall action of
several components of the extract than of single compounds. Nevertheless,
for future clinical studies, extracts should be standardized according to
regional pharmacopoeias, for instance European Pharmacopoea, using KBA
and or AKBA as leading compounds.
322 H.P.T. Ammon

13.6.3 Side Effects

Taking into account the wide use of oleogum resin from different Boswellia species
in ancient times and nowadays, side effects do not appear to be a critical point. This
is also the outcome of the published material. Most of the side effects, if at all, are
related to the gastrointestinal tract. In detail:
Gastrointestinal Tract: In the study using S-Compound™ [22], dyspeptic
symptoms in form of pain in abdomen, distension of abdomen, sour eructation and
loss of appetite were reported in 8 % while 60 % of patients treated with Ibuprofen
had dyspeptic symptoms.
In a further trial with S-Compound™, two from 40 patients complained about
epigastric pain, hyperacidity, and nausea [24]. In a study dealing with ulcerative
colitis [23], 6 out of 34 patients reported retrosternal burning, nausea, fullness of
abdomen, epigastric pain and anorexia.
Böker and Winking [7], studying the effect of H15™, reported that some
patients developed nausea and vomiting. The side effects were reversible after
omission of the treatment. Comparing the effect of H15™ (44 patients) with
mesalazine (39 patients) suffering from Crohn’s disease), Gerhardt et al. [20]
reported no drug related side effects in the H15™ group but 4 patients treated with
mesalazine suffered from headache and gastrointestinal symptoms. In the study of
Gupta et al. [25], out of 20 patients treated with Boswellia gum resin, only 2
patients complained of heartburn and Streffer et al. [75] reported that the prepa-
ration H15™ was well tolerated, only some gastrointestinal complains were
observed. BE (15 patients) was also well tolerated in the study of Kimmatkar et al.
[34] exept for minor gastrointestinal reactions. In the study of Sontakke et al. [72],
one patient out of 33 receiving BE complained about diarrhoea and abdominal
cramps.
Boswellic extracts in higher doses are used in treatment of cerebral edemas
caused by brain tumours. In a prospective, randomized, placebo-controlled
double-blind pilot study [35], where 22 patients with brain tumours received
4200 mg of a BE (H15™) or placebo, diarrhoea grade 1–2 occurred in 6 patients of
the BE group. Other side effects were the same as in the placebo group.
Respiratory Tract: There is a case report from a patient who complained about
asthma symptoms after exposure to fume of incense in the church [46].
Skin: In an other case report Acebo et al. [1] describe a patient suffering from
allergic contact dermatitis using an extract from Boswellia serrata in a naturopathic
cream.
In two patients being treated with H15™ for brain edema, Böker and Winking
[7] described skin irritations. The side effects were reversible after omission of the
treatment.
13 Boswellic Acids and Their Role in Chronic Inflammatory Diseases 323

General Tolerability: No adverse reactions were observed in a double-blind

placebo-controlled study in 20 volunteers receiving topical formulation cream
containing boswellic acids [39]. No side effects were also observed in the phar-
macokinetic study of Sharma et al. [63] and in the study on OA by Sengupta et al.
[59]. And the study of Holtmeier et al. [28] confirmed good tolerance of the
preparation Boswellan™ over a period of 52 weeks.
When Aflapin™ (Laila Impex R & D Center, New Dehli, India) and 5-Loxin™
were used to treat osteoarthritis in comparison to a placebo [59, 60], the safety
parameters were almost unchanged in the treatment group. In a retrospective
analysis in 2000, the laboratory parameters before and after the treatment of patients
suffering from rheumatoid arthritis, ulcerative colitis, Crohn’s disease, neuroder-
mitis, lupus erythematosus, multiple sclerosis, astrocytoma, glioblastoma, bronchial
asthma and psoriasis were tested receiving the Boswellia preparation H15™ over a
period of 6 years.
No significant changes related to the therapy were observed [10].
All together, it appears that extracts from the oleogum resin of Boswellia species
are relatively safe as far as side effects are concerned. This is one of the big
advantages compared to all anti-inflammatory remedies being in use presently.

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J Immunol 1:4755–4763
Chapter 14
Natural Withanolides in the Treatment
of Chronic Diseases

Peter T. White, Chitra Subramanian, Hashim F. Motiwala

and Mark S. Cohen

Abstract Withanolides, and in particular extracts from Withania somnifera, have

been used for over 3,000 years in traditional Ayurvedic and Unani Indian medical
systems as well as within several other Asian countries. Traditionally, the extracts
were ascribed a wide range of pharmacologic properties with corresponding med-
ical uses, including adaptogenic, diuretic, anti-inflammatory, sedative/anxiolytic,
cytotoxic, antitussive, and immunomodulatory. Since the discovery of the archetype
withaferin A in 1965, approximately 900 of these naturally occurring, polyoxy-
genated steroidal lactones with 28-carbon ergostane skeletons have been discovered
across 24 diverse structural types. Subsequently, extensive pharmacologic research
has identified multiple mechanisms of action across key inflammatory pathways. In
this chapter we identify and describe the major withanolides with anti-inflammatory
properties, illustrate their role within essential and supportive inflammatory path-
ways (including NF-κB, JAK/STAT, AP-1, PPARγ, Hsp90 Nrf2, and HIF-1), and
then discuss the clinical application of these withanolides in inflammation-mediated
chronic diseases (including arthritis, autoimmune, cancer, neurodegenerative, and
neurobehavioral). These naturally derived compounds exhibit remarkable biologic
activity across these complex disease processes, while showing minimal adverse
effects. As novel compounds and analogs continue to be discovered, characterized,
and clinically evaluated, the interest in withanolides as a novel therapeutic only
continues to grow.

Keywords Autoimmune Cancer Inflammation Neurodegenerative NF-κB




Withaferin A Withanolide

These authors contributed equally

P.T. White
C. Subramanian
H.F. Motiwala
M.S. Cohen (&)

Department of Surgery, University of Michigan Hospital and Health Systems, 2920K
Taubman Center, SPC 5331, 1500 East Medical Center Drive, Ann Arbor, MI 48109-5331,
USA
e-mail: [email protected]

© Springer International Publishing Switzerland 2016 329

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1_14
330 P.T. White et al.

Abbreviations
5XFAD 5 FAD mutations carried on APP and PS1 transgenes
AChE Acetylcholinesterase
AD Alzheimer’s disease
ADAM10 Adisintegrin and metalloproteinase domain-containing protein 10, or
α-secretase
AP-1 Activator protein 1
APP Amyloid precursor protein
BACE APP cleaving enzyme 1 or β-secretase
BChE Butyrylcholinesterase
Bfl-1/A1 Bcl-2-related protein A1
C/EBPα CCAAT/enhancer-binding proteinα
CCR7 Chemokine (C–C motif) receptor 7
cFLIP C-FADD-like IL-1β-converting enzyme–inhibitory proteins
CNS Central nervous system
COX Cyclooxygenase
CSC Cancer stem cell
EGF Epidermal growth factor
EGFR EGF receptor
ERK Extracellular signal-regulated kinase
FDA Food and Drug Administration
GABA Gamma-aminobutyric acid
GMP Good manufacturing process
HD Huntington’s disease
HIF-1 Hypoxia inducible factor-1
HMGB1 High mobility group box 1
hnRNP-K Heterogeneous nuclear ribonucleoprotein
KHPA Hypothalamic–pituitary–adrenal
Hsp Heat shock protein
HTT Mutant Huntingtin
IAP1 Inhibitor of apoptosis protein-1
IBD Inflammatory bowel disease
ICAM Intercellular adhesion molecule
IFN Interferon
IKK I kappa B kinase
IL Interleukin
iNOS Inducible nitric oxide synthase
JAK Janus kinase
JNK1 c-Jun N-terminal protein kinase
Keap1 Kelch like ECH-associated protein-1
LPS Lipopolysaccharide
14 Natural Withanolides in the Treatment of Chronic Diseases 331

LTB4 Leukotriene B4
MAPK Mitogen-activated protein kinase
MCE Mitotic clonal expansion,
MCP-1 Monocyte chemoattractant protein-1
MMPs Matrix metalloproteinases
MMTV-neu Mouse mammary tumor virus-neu
MPTP 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine
MS Mass spectrometry
mTOR Mechanistic target of rapamycin
MUC-1 Mucin 1
NF-κB Nuclear factor kappa B
NO Nitric oxide
Nrf2 Nuclear factor erythroid 2-related factor 2
OCD Obsessive-compulsive disorder
PBMC Peripheral blood mononuclear cells
PD Parkinson’s disease
PDGF Platelet-derived growth factor
PGE2 Prostaglandin E2
PI3K Phosphatidylinositol-3-kinase
PPARs Peroxisome proliferator-activated receptors
QR Quinone reductase
RA Rheumatoid arthritis
ROS Reactive oxygen species
RTKs Receptor tyrosine kinases
SAR Structural–activity relationship
SFMC Synovial fluid mononuclear cells
SOD Super oxide dismutase
STAT Signal transducer and activator of transcription
TAK1 Transforming growth factor-β-activating kinase
TGF-β Transforming growth factor beta
Th T-helper
TLRs Toll-like receptors
TNBS Trinitrobenzyl sulfonic acid
TNF Tumor necrosis factor
TRAIL Tumor necrosis factor-related apoptosis-inducing ligand
TWIST Twist family BHLH transcription factor
UPLC Ultra-performance liquid chromatography
VEGF Vascular endothelial growth factor
WA Withaferin A
WS Withania somnifera
332 P.T. White et al.

14.1 Introduction

Withanolides are a group of naturally occurring polyoxygenated steroidal lactones

assembled on a C28 ergostane skeleton. The structural skeleton of withanolides
usually varies in the nature and number of oxygenated substituents and the degree
of unsaturation of the rings. Structurally diverse withanolides are typically classified
based on the arrangement of the C-17 side chain into a major C-22/C-26
δ-lactone/lactol group and a minor C-23/C-26 γ-lactone/lactol group with few
exceptions and about 90 % of these compounds possess ketone functionality at C-1
(Figs. 14.1, 14.2) [1–4]. Additionally, withanolides with a C-17 δ-lactone side
chain, as shown in Fig. 14.1, can be further categorized into withanolides with an
unmodified skeleton (e.g., withaferin A and withaperuvin B) and into those with a

(a) Withanolides with a δ-lactone/lactol side chain (unmodified skeleton)

28 Me Me
Me Me
24 HO H HO H
21 H23 25 27 Me
Me
H
O
Me
Me
Me Me
18 22
OH Me Me Me
Me 20 26 O O
12
H O O OH O Me O OH O
O O OH
O 19 11 13 17 Me H O OH Me H
Me H 14 16 H
9 Me H
1 H H
2 10 8 15 OH OMe
5
H H H H
3 4 6 7
OH O
O OH OH OH
OH
Withaferin A Withaperuvin B Cilistol A Withangulatin C

(b) Withanolides with a δ-lactone/lactol side chain (modified skeleton)

O OH
26 Me 21 Me
O 25 Me H O
23
Me O Me Me H
H 24 O
22 O OH O O
22 20 20 26 Me H O
21 H 23 O OH O
Me H O H
O O O
O OH Me HO 24 25
Me H H H Me HO
Me H O
O Me Me
H H OH H OH O
H H H O H H
HO O 27
OAc O
OH
Acnistin I Physalin X Jaborosalactol 18 Physalin C
(Acnistins) (Neophysalins) (Norbornane-type) (Physalins)

Me
Me Me Me
Me 25
O
Me H Me H 24
26
OH O OH O O 23 O
Me O O O 22
Me
21
OH O Me H HO Me OH 20
OH H O
H Me O HO Me OH
H O Me H
OH Me O
H H Me H
O H H
H H
OH Cl
OH
Jaborosalactone 7 Salpichrolide G Jaborosalactone 37 Jaborosalactone 43
(Ring-A aromatic withanolides) (Ring-D aromatic withanolides) (Sativolides) (Spiranoid δ-lactones)

Me O Me
Me 25 26 Me Me
Me 24
23 25
24 O O H
HO H HO 21
22 26
O
Me H 23 O O
O Me Me
20 H H O
21
O O O
H O OH 22 Me
H O Me 20
O O Me H OH H H O
H OH O Me H
Me Me H
H H H H
H H H H
OH O
OH Cl OH
Subtrifloralactone E Tubonolide A Withametelinone Withaphysalin N
(Subtriflora-δ-lactones) (Withajardins) (Withametelins) (Withaphysalins)

Fig. 14.1 Withanolides with a δ-lactone ring

14 Natural Withanolides in the Treatment of Chronic Diseases 333

(a) Withanolides with a γ-lactone/lactol side chain

O
HO OH Me OH
Me Me HO OH
26 O 22
24 25
Me Me 23 Me
Me 28 25
23 26 Me 24 O 24 Me OH
O
Me H H O O O OAc 23 25 26
O Me
Me H H O Me HOH
Me OH H
H H 27
H H H H
O HO O
OH O OH
Ixocarpalactone B Perulactone C Jaborosalactone 15
(Ixocarpalactones) (Perulactones) (Spiranoid γ-lactones)

Me O HO Me
26
Me 25 O
Me O O
OAc 23 24 Me
H H AcO Me 23
H
Me H O
O AcO 26
22 Me
H OH Me H H 24 O
O HO Me OH
O H Me 25
Me H O O H H OAc Me OH O
Me H
OH
OH H H
O H H
OH O
O
Subtrifloralactone K Taccalonolide A Jaborosalactol 24
(Subtriflora-γ-lactones) (Taccalonolide-γ-lactones) (Trechonolides)

(b) Miscellaneous withanolides

Me
O Me
OH
Me H Me Me 20 Me H Me
22 23 20
Me O Me H
OH H O Me O
O OAc 17 H 24
Me H 27 O 17 O
Me H H 25 Me H OH
Me O
H H HO O H H
26
3 O H H
5
OH HO
OH HO OH O
HO
Cilistol U Physanolide A Exodeconolide A
(Withanolides with 3,5-cyclopropane ring) (Withanolide with a 26,27-γ-lactone) (Withanolides with 17-ene group)

Fig. 14.2 Withanolides with a γ-lactone ring and unclassified structural type

modified skeleton (e.g., physalin C and withametelinone) [1, 2, 4]. The occurrence
of unmodified withanolides is more common in nature with approximately 580 of
these naturally occurring withanolides reported in the family Solanaceae alone [1–
3]. Structurally more complex withanolides with a modified skeleton both in the
steroid nucleus and the side chain could possibly result from the biogenetic
transformations of unmodified withanolides [3, 5].
Withaferin A (WA), an archetype of this class was discovered from Withania
somnifera (WS) or Ashwagandha in 1965 [6]. In the past 50 years, approximately
900 withanolides falling into 24 diverse structural types have been discovered [1,
3]. Withanolides are mainly distributed in various genera of Solanaceae, which
includes Acnistus, Aureliana, Brachistus, Browallia, Datura, Deprea, Discopodium,
Dunalia, Exodeconus, Hyoscyamus, Iochroma, Jaborosa, Larnax, Lycium,
Mandragora, Nicandra, Physalis, Salpichroa, Saracha, Solanum, Trechonaetes,
Tubocapsicum, Vassobia, Withania, and Witheringia [1–3, 7–9]. A minor popu-
lation of withanolides has been isolated from other plant sources such as
Dioscoreaceae, Fabaceae, Labiatae, Lamiaceae, Leguminosae, Myrtaceae, and
Taccaceae [1–3] and interestingly from marine sources of Alcyoniidae family
[10–12].
334 P.T. White et al.

Structurally varied withanolides have received significant attention due to their

versatile biological activities demonstrated in vitro and/or in vivo. These activities
have been described as antitumor [7, 13–15], cytotoxic [16–20], apoptotic [21–23],
anti-inflammatory [9, 10, 24–31], immunomodulating [32–34], antimicrobial
[35–37], antistress [34], antioxidant [38], anti-neurodegenerative [39–41],
radiosensitizing [42, 43], and insect antifeedant [44, 45]. Withaferin A, the most
studied withanolide, possesses a wide array of the pharmacological activities
described above and thus carries a great clinical potential for drug development [1,
4, 46–51]. Most notably, the antitumor and associated anti-inflammatory activities
of WA and other withanolides results from targeting multiple signaling pathways
simultaneously, particularly the nuclear factor kappa B (NF-κB), signal transducer
and activator of transcription (STAT), and ubiquitin proteasome pathways (see
Tables 14.1, 14.2) [52–57]. The potent biological activities of withanolides such as
WA and tubocapsenolide A, especially the antitumor and anti-inflammatory prop-
erties have been attributed to the presence of key structural features such as an
α,β-unsaturated ketone in ring A, a 5β,6β-epoxide in ring B, and a lactone side
chain [1, 4, 7, 13, 30, 58–62]. Cysteine residues in the proteins are often implicated
to react with these key electrophilic sites on the withanolide molecule [59, 60, 63,
64]. While other withanolides may possess α,β-unsaturated ketone and/or epoxide
in some respect (e.g., paraminabeolides, capsisteroids, and chantriolides) and are
bioactive, they are generally less potent than those withanolides possessing all three
crucial functional groups.

14.2 Modulation of Inflammatory Cell Signaling Pathways

by Withanolides

Inflammation is a complex immunological process by which our body fights against

infection, cancer, or injury. The initial, acute stage of inflammation is mediated
through the activation of immune cells, the resultant inflammatory cytokines and
intracellular pathways. The initial immune mediators are CD4+ T-cells or T-helper
(Th) cells and are classified as Th-1, Th-2, and Th-17. They play a crucial role in
regulating the cellular and humoral immune responses through recognition of
antigens presented on antigen presenting cells via the major histocompatibility
complex II. Th-1 cells promote the cellular immune response (macrophages) and
primarily produce interferon (IFN)-γ, tumor necrosis factor (TNF)-β, and inter-
leukin (IL)-2, whereas Th-2 cells promote the humoral immune system (antibodies)
and primarily produce IL-4 and IL-10, and Th-17 cells help recruit neutrophils early
in the adaptive response, produce IL-17 cytokine, and are involved in many
autoimmune diseases [82]. The alteration of normal homeostasis of any of the Th
cells through aberrant recognition of self or dysregulated production of cytokines
plays a major role in the formation of chronic inflammatory or autoimmune/
immunomodulatory diseases. Excess cytokine production leads to the over acti-
vation of multiple downstream inflammatory pathways, including Janus kinase
14 Natural Withanolides in the Treatment of Chronic Diseases 335

Table 14.1 Natural and semi-synthetic anti-inflammatory withanolides

Targets
Diseases
Active compounds Structures Plant source inhibited/Inflam- Ref.
involved
mation models
Me

Me H
OAc O-Glc
Me O
OAc O Tacca
Me H O TNF -induced
Chantriolide A plantaginea Inflammation (29)
O H H NF- B
(Dioscoreaceae)*
OH O
Chantriolide A
Glc = β -D-Glucopyranosyl
Me
OH
OH
Me H
Me
Me O H
O OH
OH
Me H

H H superoxide anion
Cilistol G Solanum and elastase
Cilistol G, Capsisteroids Me
Inflammation
Me OAc OH capsicoides release by (9)
A and E Me H
OH and cancer
Me H
OH Me (Solanaceae) human
Me Me O H
Me O H O OH neutrophils
O OH Me H OH
OH
Me H
H H
H H
OH
Capsisteroid A Capsisteroid E
Me
HO H Me
Me
Me O Withania PPAR , Obesity and
O OH O
Me H
Coagulin L coagulans C/EBP , and metabolic (65)
H OH
Glc-O
(Solanaceae) MCE syndrome
Coagulin L
Glc = β-D-Glucopyranosyl
OH OH

Me H Me Me H Me
Me Me
O O
OH H O OH H O
Me H Me H

H H H H
Daturafolisides A and B, Glc-O OH Glc-O OMe
Daturafoliside A Daturafoliside B Datura LPS-induced NO
Baimantuoluoside B, and
Me Me metel(Solanaceae production in Inflammation (27)
12-
Me H Me H ) macrophages
OH O-Glc OH
Deoxywithastramonolide Me O Me O
O H O O H O
Me H Me H

H H H H

OH O OH O
Baimantuoluoside B 12-Deoxywithastramonolide
Glc = β-D-Glucopyranosyl

H
Me
OH
Me Increases
H O O
Me H
Spinal cord
Denosomin Synthetic vimentin, (66)
H H injury
HO
neuroprotective
Denosomin

Me
Me H
Me
H OH
Me Me
OH H O O
2,3-Dihydrowithaferin A Me O
H O O
Me H
O
Me H
Withania
and 4-(2,2-Dimethyl-3- H H Inflammation
H H somnifera(Solana COX-2 (30)
oxocyclopropoxy)-2,3- O
O
O
and cancer
O ceae)
dihydrowithaferin A OH
Me Me
4-(2,2-Dimethyl-3-oxocyclopropoxy)-
2,3-Dihydrowithaferin A
2,3-dihydrowithaferin A
Me

Me H Me
OH
6 ,7 -Epoxy-1-oxo- Me O
O OH O Discopodium
5 ,12 ,17 - Me H LTB4 and COX- Inflammation
H H
penninervium(So (26)
trihydroxywitha-2,24- 2 and cancer
OH O
lanaceae)
dienolide 6α,7α-Epoxy-1-oxo-5α,12α,17α-
trihydroxywitha-2,24-dienolide
Me
HO H Me
Me
Me O
O OH O Physalis
4 -Hydroxywithanolide Me H NF- B and Diabetes and
pruinosa (67)
E H OH MCP-1 obesity
O
(Solanaceae)
OH
4β-Hydroxywithanolide E
336 P.T. White et al.

Formalin-
Me
HO H
induced arthritis
Me Me
Me O
Withania model and cotton
3 -Hydroxy-2,3- O OH O
Me H coagulans pellet granuloma Inflammation (68)
dihydrowithanolide F H OH (Solanaceae) method for sub-
HO
3β-Hydroxy-2,3-dihydrowithanolide F acute
inflammation
Me Me

Me H Me Me H Me
AcO
Me
O O
H O H O
Me H Me H

H H H H LPS-induced
O O Paraminabea
Minabeolides-1, -2, -4, Minabeolide 1 Minabeolide 2 iNOS and COX- Inflammation
acronocephala(A (10)
and -5 Me H Me Me H Me
2 proteins in and cancer
AcO lcyoniidae)
Me
O O macrophages
H O H O
Me H Me H

H H H H
O O
Minabeolide 4 Minabeolide 5
Me Me

Me H Me Me H Me
AcO
OHC
O O
H O H O
Me H Me H

H H H H
O O Paraminabea LPS-induced
Paraminabeolide A Paraminabeolide B Inflammation
Paraminabeolides A–D acronocephala(A iNOS protein in (10)
Me OH Me OH and cancer
AcO H Me H Me lcyoniidae) macrophages
Me
H H
Me H O Me Me H O Me

H H O H H O
O O
Paraminabeolide C Paraminabeolide D

Me H Me H
O O O O
O O IKK , NF- B,
H O H O
O O H
Physalis
Physalins A and O, and Me HO O Me HO O and LPS-induced
Me Me Me alkekengi Inflammation (64)
Isophysalin A H HOH O H HOH O NO production
OH OH (Solanaceae)
Isophysalin A Physalin O
in macrophages
Me H Me H
O O O
O O O
O H O
H
O O
Me HO O Me HO O H
Me Me
H OH O H O
H H
O
Physalin C Physalin B

Me H Me H
O O O O
O O
H O H O
O O
Me HO O Me HO O H
Me Me
H HOH O H H O
O
OH OH
Physalis
Physalin A Physalin N
Physalins C, B, A, N, F alkekengi and
Me H
Me TNF -induced Inflammation
and Withanolides E, F, O O HO H Me
Physalis (59)
O Me NF- B and cancer
H O
G O Me O peruviana
Me HO O H O OH O
Me Me H (Solanaceae)
H H O
O H OH
O
O
Physalin F Withanolide E

Me Me
HO H Me HO H Me
Me Me
Me O Me O
O OH O O H O
Me H Me H

H OH H OH

Withanolide F Withanolide G
14 Natural Withanolides in the Treatment of Chronic Diseases 337

Me

Me H Inflammation
OAc O-Glc
Me O
OH O Tacca and metabolic
Me H O
Plantagiolide I AcO plantaginea PPARs disorders such (29)
H H
Cl
OH O
(Dioscoreaceae)* as diabetes
Plantagiolide I and obesity
Glc = β-D-Glucopyranosyl

Me
H
Me
OH
Me
H O O
OH
Me H

H H
HO
Sominone
Me
Withania Dementia and
Sominone and Induces neurite (39,
Me
H somnifera(Solana Alzheimer's
Withanoside IV OH outgrowth 40)
Me ceae) disease
H O O
OH
OH Me H
HO
O H H
HO
O
OH O O
OH
HO
HO
Withanoside IV

Me
H
Me Me
Me
O O O Tubocapsicum CCR7
O
Me H
Tubocapsanolide A anomalum expression, IKK, Cancer (69)
H H

O
(Solanaceae) and NF- B
OH
TubocapsanolideA

Me
H
Me Me
Me O Tubocapsicum Hsp90-Hsp70
O O
Me H OH (62,
Tubocapsenolide A anomalum chaperone Cancer
H 70)
(Solanaceae) complex
O
OH
Tubocapsenolide A

Me Me
O O
H H
Me Me Me Me
Me Me
H O OH H O OH
O O
Me H OAc Me H

H H H H

O O
OH OH Physalis
Virginol A Virginol B TPA-induced ear
Virginols A–C virginiana(Solan Inflammation (28)
Me edema assay
O aceae)
H
Me Me
Me
H O OH
O
Me H OAc

H H

O
Virginol C
Me
H
Me TNF -induced Inflammation
Me
OH (54)
H O O Withania NF- B and cancer
O
Me H
Viscosalactone B somnifera(Solana
H H
HO
O
ceae) Inflammation
OH COX-2 (30)
Viscosalactone B
and cancer
Me
H
Me Me
AcO
H O O
O
Me H STAT3 and Inflammation
Withacnistin –† (71)
H H STAT5 and cancer
O
OH
Withacnistin

TNF -induced
Angio-
NF- B and
inflammatory
Ubiquitin (55)
diseases and
proteasome
cancer
pathway
NF- B and
338 P.T. White et al.

zymosan-
Inflammation
Me induced (24)
and cancer
H inflammed paw
Me
OH
Me Withania
H O O
model
O
Me H
Withaferin A somnifera(Solana NF- B, AP-1, Inflammation
H H (72)
ceae) Nrf2, and IL-6 and cancer
O
OH
Withaferin A
Inflammation
COX-2 (30)
and cancer
Inflammation (53,
STAT3
and cancer 57)
COX-2, PGE2,
Microglial
STAT1, and (73)
activation
STAT3
LPS-mediated Vascular
(51)
HMGB1 release, inflammatory

ICAMs, and NF- diseases such

B as
atherosclerosis
Inflammation
NF- B and AP-1 (56)
and cancer
Me
H
Me
OAc
Me
H O O
Withaferin A 4,27- O
Me H TNF -induced Inflammation
Semisynthetic (54)
diacetate H H IKK and NF- B and cancer
O
OAc
Withaferin A 4,27-diacetate
Me
HO H Me
Me Ubiquitin
Me Withania Age-related
H O O
O proteasome
Me H
Withanolide D somnifera(Solana macular (52)
H H pathway and NF-
ceae) degeneration
O B network
OH
Withanolide D
Me
H
Me
OH
Me
H O O
O
Me H

H H

OH COX-2 and
OH S
O Withania
OH Inflammation
Withanolide sulfoxide OH somnifera TNF -induced (74)
H H
and cancer
(Solanaceae) NF- B
H Me
O O
O H Me
HO
Me
H
Me
Withanolide sulfoxide

* Reference 28 had the genus Tacca assigned to family Taccaceae, which has been found in older
texts but the APG II system has incorporated this genus into the family Dioscoreaceae. † Obtained
from National Cancer Institute Developmental Therapeutics Program

(JAK)/STAT, NF-κB, phosphatidylinositol-3-kinase (PI3K)/Akt, and mitogen-

activated protein kinase (MAPK). A number of withanolides have demonstrated
significant immunomodulatory effects, including WS extract (primarily aqueous
extract), withanolide A, physalins and coagulins; however, both immuno-
stimulatory and inhibitory actions have been attributed to different withanolides.
WS extract (primarily withanolide A and 2,3 dihydro-3-sulphonile withanone
components) is able to shift the immune response toward Th-1 polarization, activate
cytotoxic natural killer cells [83, 84], and recover depleted T-cells and increase
expression of Th-1 cytokines IL-2 and IFN-γ in models of stress [32, 34, 85, 86].
Conversely, coagulins isolated from Withania coagulans and primarily coagulin-H
have demonstrated immunosuppressive effects similar to prednisolone, inhibiting
stimulated T and B-cell lymphocyte proliferation, and Th-1 cytokine production
14 Natural Withanolides in the Treatment of Chronic Diseases 339

Table 14.2 Anti-inflammatory activity of withanolides in plant extracts

Plant extract Plant Active Effects Therapeutic uses References
part components
Physalis Fruits Withanolides, Antioxidant, Acute renal injury [75]
peruviana polyphenols, anti-inflammatory,
(Solanaceae) and and renoprotective
phytosterols
Calyces – Anti-inflammatory Inflammation [76]
and
immunosuppressive
effect on
macrophages
apoptopic
(downregulates IL-6,
TNF, and MCP-1)
Withania Roots Withanolides Neuroprotective Alzheimer’s [77]
somnifera such as disease
(Solanaceae) withanolide A
and alkaloids
Roots Withanolides, Immunomodulatory, Cancer [78]
alkaloids, and anti-inflammatory,
flavanoids and proapoptopic
(downregulates IL-6,
IL-1β, IL-8, Hsp70,
and STAT-2)
Roots – Antioxidant, Inflammatory [79]
anti-inflammatory, bowel disease
and cytoprotective
Leaves – Neuroprotective Stroke and [80]
against glutamate neurodegenerative
neurotoxicity disorders
Withania Fruits – Antioxidant, Diabetes and [81]
coagulans anti-inflammatory, associated renal
(Solanaceae) antihyperglycemic, complications
immunomodulatory,
and renoprotective
(downregulates
IL-1β, IL-6, TNFα,
IL-4, and IFN-γ)

possibly through IL-2 receptor binding [87, 88]. Similar to coagulins, physalins B,
F, G, and H isolated from Physalis angulata have demonstrated immunosuppres-
sive properties. Physalins B, F, and G showed inhibition of lipopolysaccharide
(LPS)-activated macrophages along with their cytokine (TNF-α, IL-6 and IL-12)
and nitric oxide (NO) production, whereas concanavalin A-induced T-cell prolif-
eration and cytokine production in a mechanism distinct from dexamethasone [89,
90]. Physalin H has demonstrated polarization to Th-2 cells with inhibition of Th-1
cytokines IL-2 and IFN-γ, increased Th-2 cytokines IL-4 and IL-10, and induction
of the heme oxygenase-1 [91].
340 P.T. White et al.

There are strong preclinical and clinical studies that demonstrate that inflam-
mation initially started by immune cell mediators may persist chronically, resulting
in ongoing stimulation of inflammatory mediators and regulatory pathways that
contribute to the pathogenesis of chronic diseases including cardiovascular, neu-
rologic, and pulmonary diseases, as well as cancer, diabetes, and obesity [92–94].
As with acute inflammation, chronic inflammation is mediated through various
signaling factors, which include proinflammatory cytokines such as TNFα, IL-1,
IL-6, IL-8, IL-12, NO, adhesion molecules, and chemokines [95]. Additionally,
transcription factors that regulate the expression of inflammatory mediators such as
NF-κB, activator protein 1 (AP-1), peroxisome proliferator-activated receptor
(PPAR)-γ, STAT3, hypoxia inducible factor-1 (HIF-1), β-catenin/Wnt, hedgehog,
and nuclear factor erythroid 2-related factor 2 (Nrf2) have been linked to chronic
diseases. The DNA-binding capacity of these transcription factors is modified by
several signaling cascades such as JAK/STAT, MAPKs, PI3K/Akt/mechanistic
target of rapamycin (mTOR), and ubiquitin proteasome system [96]. These sig-
naling pathways have a wide range of functions and show complex crosstalk
depending on the cell type and the chronic disease involved. Withanolides have
emerged recently as potential therapeutics for chronic diseases due to their unique
ability to modulate multiple signaling pathways. In this chapter, we will discuss
several different withanolides and their seemingly broad mechanism of action in
modulating key molecular pathways that connect inflammation and chronic
diseases.

14.2.1 Withanolides Modulate the NF-κB Pathway

The NF-κB family of transcription factors plays a prominent role in the immune
system and inflammation. In response to ligation by Toll-like receptors (TLRs) and
IL-1 receptor family members (B-cell and T-cell receptors), NF-κB regulates the
expression of several factors such as inhibitor of apoptosis protein-1 (IAP1),
inducible nitric oxide synthase (iNOS), cytokines, cyclooxygenase (COX)-2,
prostaglandins, growth factors, and effector enzymes [97–99]. NF-κB is activated
by inflammatory cytokines, stress, free radicals, radiation, growth receptors, and
TNFα leading to transcriptional regulation of several genes that are involved in
proliferation, inflammation, cellular survival, apoptosis, angiogenesis, and differ-
entiation [100–103]. The mammalian transcription NF-κB family of proteins
includes RelA (p65), RelB, NF-κB2 (p52), c-Rel, and NF-κB1 (p50). In the absence
of stimuli, inactive forms of NF-κB proteins are present in the cytoplasm due to
their interaction with several inhibitors of kappaB (IκB) proteins. Upon exposure to
stimuli, NF-κB is activated either through canonical or noncanonical pathways by
regulatory IκB kinases (IKK) such as IKKα, IKKβ, and IKKγ. In the most common
classical pathway, IKK phosphorylation leads to IκBα phosphorylation at serine 32
and serine 36, followed by phosphorylation, nuclear localization, DNA binding of
p65–p50 complex, and transcriptional activation of NF-κB responsive genes [104].
14 Natural Withanolides in the Treatment of Chronic Diseases 341

Activation of NF-κB is essential for the survival and expression of inflammatory

mediators. Hence, constitutively active NF-κB is associated with several
inflammation-mediated chronic diseases such as cancer, neurological, and meta-
bolic disorders [98].
A number of naturally occuring withanolides such as chantriolide A, physalins
A, B, C, and O, viscosalactone B, WA, withanolides D, E, F and withaferin A
4,27-diacetate, a diacetyl derivative of WA have been reported to modulate the
regulation of NF-κB [24, 29, 34, 52, 54, 59, 64]. Several studies have examined the
beneficial effect of inhibiting transcriptional activity of NF-κB in chronic inflam-
matory diseases including cancer [24, 55, 56, 72]. In a study led by Aggarwal et al.,
various withanolides isolated from the leaf extract of WS along with their
semi-synthetic acetylated derivatives were tested for their inhibitory effects on
NF-κB activation induced by activators such as cigarette smoke condensate, TNF,
doxorubicin, and IL-1β [54]. Withaferin A and viscosalactone B along with their
4,27-diacetyl derivative inhibited TNF-induced NF-κB activation in human mye-
loid leukemia KBM-5 cells, whereas physagulin D and related glycosidic with-
anolides were inactive. The mechanism through which the above withanolides
blocked NF-κB was through the inhibitory effects of activated IκBα, with subse-
quent phosphorylation of IκBα and p65, followed by prevention of IκBα degra-
dation. Blocking the degradation of IκBα in turn prevents nuclear localization of
p65, and activation of NF-κB-dependent gene products such as Bcl-2-related pro-
tein A1 (Bfl-1/A1), IAP-1, c-FADD-like cFLIP, ICAM1, and COX-2 [54]. To
further understand the SAR, the authors examined TNF-induced NF-κB activity
after treatment of human myeloid leukemia KBM-5 cells with various withanolides
isolated from the leaf extract of WS along with their synthetically modified
acetylated derivatives by electrophoretic mobility shift assay (EMSA). This analysis
pointed toward the importance of the α,β-unsaturated ketone in ring A as a required
structure for the potent inhibition of NF-κB activity [54].
Physalins A, L, G and O, and isophysalin A isolated from Physalis alkekengi
were evaluated for their anti-inflammatory properties and Physalins A, O and
isophysalin A showed significant inhibition of LPS-induced NO production in
macrophages [64]. Based on their structural features characterized by the presence
of either an α,β-unsaturated ketone in ring A (physalin O) or an α,β-unsaturated
ester in lactone side chain featuring an exomethylene group (physalin A and iso-
physalin A), these physalins were able to conjugate with glutathione as identified by
ultra-performance liquid chromatography tandem mass-spectrometry
(UPLC-MS/MS) analysis. Furthermore, peptide mapping and sequencing of alky-
lated IKKβ using micrOTOF-MS revealed alkylation of six cysteine residues on
IKKβ by physalin A indicating IKKβ as a potential target for its anti-inflammatory
mechanism of action. In another related and complementary study, SARs were
performed on a library of withanolides including the physalins. This study revealed
the importance of the oxygenated right-side partial structure (including the lactone
side chain) and the 5β,6β-epoxide, or C5–C6 olefin in the B-ring for the inhibition
of NF-κB activation [59]. Withanolides and physalins with 5β,6β-epoxide inhibited
NF-κB signaling through prevention of IκBα degradation and p65 nuclear
342 P.T. White et al.

localization, whereas those with C5–C6 olefin inhibited NF-κB function by

blocking p65/p50 dimer binding to DNA [59].
Chantriolide A, one of the eight compounds isolated from Taccaplantaginea
exhibited potent inhibition of TNFα-induced NF-κB transcriptional activity in
human hepatocellular carcinoma (HepG2) cells in an NF-κB-luciferase assay [29].
In another study, withanolide D and WA from WS inhibited angiogenesis through
blocking of NF-κB activity by suppressing proteasome-mediated ubiquitin degra-
dation of IκBα in human umbilical vein endothelial cells [52]. Additionally,
4β-hydroxywithanolide E was shown to inhibit inflammatory response in adipo-
cytes via inhibition of NF-κB transcriptional activity [67]. Inhibition of IKKβ
activation by 4β-hydroxy withanolide E through suppression of IKKβ phosphory-
lation was mechanistically distinct from the NF-κB inhibition observed for WA,
where the induction of IKKβ over-phosphorylation was shown to inhibit IKKβ
activation [24]. Moreover in vivo, 4β-hydroxy withanolide E demonstrated an
improvement of impaired glucose tolerance suggesting its potential role for the
treatment/prevention of metabolic disorders including type 2 diabetes [67].
Overexpression of CCR7 in metastatic breast cancer cells has been associated with
lymph node metastasis [69]. In breast cancer cells MDA-MB-231, tubocapsanolide
A inhibited TAK1 to suppress NF-κB-mediated CCR7 expression leading to the
inhibition of lymphatic invasion of breast cancer in vitro and in vivo.
In addition to the inhibition of NF-κB activation, WA and several other with-
anolides have been shown to directly block the expression of LPS- or
TNFα-induced NF-κB-regulated inflammatory genes such as iNOS, COX-1,
COX-2, and NO [10, 13, 14, 25, 26, 64, 74]. Nitric oxide is a small molecule that
regulates MMPs and joints extracellular matrix, and is modulated through iNOS.
COX-1 and COX-2 convert arachidonic acid to prostaglandins, which in turn cause
a significant inflammatory response. COX-1 is constitutively expressed in most cell
types, and is responsible for maintenance of normal physiologic function, whereas
COX-2 is inducible in response to proinflammatory cytokines [26]. Nair and
co-workers were the first to demonstrate the role of withanolides in inhibiting COX
enzymes and provide insight into their anti-inflammatory mechanism [30].
Withaferin A, viscosalactone B, 2,3-dihydrowithaferin A, and 4-
(2,2-dimethyl-3-oxocyclopropoxy)-2,3-dihydrowithaferin A were shown to inhibit
COX-2 enzyme but not COX-1. Interestingly, during this study it was observed that
the presence of a double bond between C-24 and C-25 in the lactone ring was
essential for COX inhibitory activities and a withanolide lacking this unsaturation
in the lactone ring was found to be inactive against both COX-1 and COX-2
enzymes [30].6α,7α-Epoxy-1-oxo-5α,12α,17α-trihydroxywitha-2,24-dienolide
from Discopodiumpenninervium was found to inhibit COX-2 and leukotriene B4
(LTB4) but was inactive against the COX-1 enzyme [26]. Like other withanolides,
withanolide sulfoxide, a sulfoxide dimer of WA was highly selective in inhibiting
COX-2 compared to COX-1[74].
Daturafolisides A and B along with other known withanolides from Datura
metel were shown to exhibit significant reduction in NO production in LPS-induced
RAW 264.7 macrophage cells [27]. Of note, both of these compounds lack the
14 Natural Withanolides in the Treatment of Chronic Diseases 343

α,β-unsaturated ketone in ring A and the 5β,6β-epoxide in ring B, however, they do

possess a δ-lactone side chain. Additionally, withanolides such as paraminabeolides
and minabeolides obtained from a marine source were found to inhibit LPS-induced
iNOS expression in RAW 264.7 macrophages, with minabeolides also effectively
inhibiting COX-2 expression [10].

14.2.2 Withanolide Modulation of the JAK/STAT Pathway

The JAK/STAT pathway is a key-signaling mediator of cytokines and growth

factors such as platelet-derived growth factor (PDGF), epidermal growth factor
(EGF), IL6, as well as oncogenic proteins [105]. Activation of STAT proteins
depends on their binding to cytokines and growth factor receptors on the plasma
membrane followed by tyrosine phosphorylation either directly by receptor tyrosine
kinases (RTKs) or by non RTKs such as JAK or Src [106, 107]. Upon phospho-
rylation, cytoplasmic STAT proteins undergo dimerization via reciprocal
SH2-domain/phosphotyrosine interactions followed by translocation to the nucleus
for DNA binding to STAT-specific response elements leading to transcriptional
activation. There are eight known STAT proteins (STATs 1A, 1B, 2, 3, 4, 5A, 5B,
and 6) that play diverse biochemical roles in several important processes such as
survival, proliferation, apoptosis, invasion, immune response, inflammation, and
angiogenesis [105, 108, 109]. Among the eight isoforms STAT3 and STAT5 are
constitutively activated in several solid tumors, including lung, bladder, breast,
colon, as well as in hematological malignancies [108]. Additionally, STAT3 is also
interconnected with the NF-κB pathway and plays a central role in inflammation
[107].
Several chronic diseases including cancer have been shown to induce aberrant
regulation of STAT3. This transcription factor promotes oncogenic processes such
as invasion, metastasis, and angiogenesis as several genes involved in these
mechanisms such as cyclin D1, c-Myc, vascular endothelial growth factor (VEGF),
mucin 1 (MUC-1), twist family BHLH transcription factor (TWIST) are all regu-
lated by STAT3 [110–114]. Studies have investigated the role of WA from WS in
regulating STAT proteins in different cancer models including colon cancer, breast
cancer, multiple myeloma, and neuroblastoma [53, 57, 115, 116]. In breast cancer,
WA treatment of triple negative MDA-MB-231 and hormonally active MCF-7 cells
effectively decreased the constitutive as well as the IL-6 inducible phosphorylation
of JAK 2 and its downstream target STAT3 thereby inhibiting the transcriptional
activity of STAT3 [115]. In renal carcinoma Caki cells, WA had a similar effect and
also downregulated the expression levels of anti-apoptotic proteins that are regu-
lated by STAT3 like Bcl-2, cyclin D1, survivin, and Bcl-xL, thereby inducing
apoptosis [116]. Docking studies showed that WA not only downregulates the
phosphorylation of STAT3 at the tyrosine Y705, but also prevents dimerization of
STAT3 [57]. In addition to cancer cells, WA also is able to suppress the
phosphorylation of STAT1/3 in murine BV2 microglial cells, leading to a reduction
344 P.T. White et al.

in LPS-induced COX-2 downregulation and PGE2 production [73]. Withacnistin,

an unmodified withanolide blocked both IL-6 as well as EGF-stimulated binding of
STAT3 and STAT5 to gp130 and EGF-receptor (EGFR) in MDA-MB-468 breast
cancer cells. This resulted in subsequent downregulation of STAT3 tyrosine
phosphorylation and decreased nuclear translocation. Further evaluation of
STAT3-DNA binding and transcriptional activity after Withacnistin treatment
revealed blocking of both DNA binding as well as STAT3 reporter activity. This in
turn caused downregulation of STAT3 target genes Bcl-xL and MCL-1 resulting in
apoptosis [71].

14.2.3 Modulation of the AP-1 Pathway by Withanolides

The transcription factor AP-1, which plays a key role in the inflammatory response
is implicated in several diseases such as cancer, psoriasis, inflammatory bowel
disease (IBD), rheumatoid arthritis (RA) and fibrosis [117]. The AP-1 complex
consists of homo and hetero dimers of Jun (JunD, C-Jun, and JunB) and the Fos
(FosB, C-Fos, Fra-1 and Fra-2) family of proteins [118, 119]. Cytokines,
chemokines, hormones, and growth factors as well as external stress factors are
known to activate AP-1 signaling. The AP-1 complex translocates to the nucleus in
response to stress signaling cascades, such MAPKs and c-Jun terminal kinases
[120]. This in turn leads to activation of AP-1 and regulates multiple functions such
as differentiation, transformation, proliferation, and survival [121]. The crude
ethanol extract of WS has been shown to inhibit the nuclear localization of both
AP-1 and NF-κB in LPS-activated peripheral blood mononuclear cells (PBMC) of
both normal and RA patients, as well as synovial fluid mononuclear cells (SFMC)
of RA patients. This in turn led to decreased downstream transcription target genes
such as MMPs, COX-2, and iNOS, all of which are known mediators of RA [122].

14.2.4 Withanolides Can Modulate the PPARγ Pathway

PPARγ was first discovered in adipocyte differentiation and lipid metabolism and is
one of three members in this nuclear receptor family of transcription factors [123].
The other members of the PPARs in mammals are PPARα and PPARβ/δ. The
PPARs activate several genes involved in inflammation, adipogenesis, lipid meta-
bolism, glucose metabolism, cellular differentiation, development, and tumorigen-
esis via binding of the PPAR/retinoid X receptor heterodimer to PPAR-responsive
regulatory elements [124, 125]. PPARγ plays a key role in inflammation through
modulation of proinflammatory transcription factors such as NF-κB and AP-1
[113]. Treatment of 3T3-L1 adipocytes with WA resulted in phosphorylation of
extracellular signal-regulated kinase (ERK), followed by decreased expressions of
PPARγ leading to altered levels of Bcl2 and Bax expression, induction of apoptosis,
14 Natural Withanolides in the Treatment of Chronic Diseases 345

and inhibition of adipogenesis [126]. In addition to WA, other withanolides such as

plantagiolide J and I isolated from Taccaplantaginea [29] and coagulin-L isolated
from Withania coagulans [65] also modulate PPARγ transcriptional activity.

14.2.5 Modulation of the Hsp90 Pathway by Withanolides

Heat shock proteins (Hsp) are ATP-dependent ubiquitously expressed molecular

chaperones that are involved in the folding, assembly, maintenance, and transport
of key regulatory proteins involved in numerous signaling pathways in the cell.
Several environmental and physiological stimuli such as hypoxia, oxidative dam-
age, inflammation, infection, and elevated temperature induce the expression of
these highly conserved molecular chaperone family of proteins as a protein
homeostasis and survival response [70, 114]. The Hsp90 family of proteins
(Hsp90α, Hsp90β, GRP94, and TRAP1) form a large complex with other
co-chaperones such as cdc37, HSP70-HSP90 organizing protein, p27, Hsp32, and
Hsp70. This complex then stabilizes and maintains functional activity of
proteins/kinases in many key signaling pathways, such as PI3K/Akt/mTOR,
p38/MAPK, and NF-κB, all of which play critical roles in inflammation, chronic
inflammatory diseases, and oncogenesis. Through inhibition of Hsp90, and there-
fore inhibition of its oncogenic chaperone clients, cancer cells undergo apoptosis
[124, 125].
Several studies have shown that withanolides such as WA, withalongolides A
and B, tubocapsenolide A, and some of their synthetically modified analoges such
as withalongolide A triacetate and withalongolide B diacetateare are able to target
multiple cancers such as colon, prostate, brain, breast, head and neck, skin, adrenal,
and thyroid both in vitro and in vivo [17, 20, 21, 53, 63, 70, 127–137].
Withanolides such as WA and withalongolide A are known to block Hsp90
chaperone function through blocking the Hsp90/cdc37 complex, and induction of
thiol-mediated oxidative stress [63, 138, 139]. The Hsp90/cdc37 complex facilitates
active conformation of client kinases in particular, such as Akt, cyclin-D1, raf-1,
and cdk4. Blocking this complex leads to dysfunctional or proteasome mediated
degradation of these kinases within multiple oncogenic, pro-survival, and prolif-
erative kinase cascades (p38/MAPK, PI3K/Akt/mToR, NF-κB pathways), which
ultimately leads to cancer cell apoptosis [136, 138]. In addition to targeting the bulk
cancer cell population, WA and withalongolide A triacetate may also target the
cancer stem cell (CSC) population. These CSCs comprise a small fraction of cancer
cells, and are characterized by their tumor initiating and self-renewal capacity. WA
and withalongolide A triacetate block several developmental pathways such as
Wnt/β-catenin, notch, and NF-κB, as well as vimentin and VEGF, all of which are
important in inflammation, self-renewal, and CSC epithelial-to-mesenchymal
transition [115, 140–145].
346 P.T. White et al.

14.2.6 Withanolide Modulation of Nrf2 Pathway

Nuclear factor erythroid 2 related factor 2(Nrf2) is a transcription factor that reg-
ulates genes involved in redox homeostasis, inflammation, energy metabolism and
cellular growth [146]. Under normal homeostatic conditions, Nrf2 is anchored in
the cytoplasm as a complex with Kelch like ECH-associated protein-1 (Keap1),
which facilitates ubiquitin mediated proteasome degradation of Nrf2 and decreased
expression of Nrf2 target genes. However, in response to stimulation by growth
factors, electrophilic stressors, and changes in redox signal, Nrf2 ubiquitination is
disrupted and levels increase rapidly. Nrf2 translocates to the nucleus and upreg-
ulates expression of proteins involved in glutathione and thioredoxin-based
antioxidant defense, drug metabolism and efflux, and proteins associated with
heme and iron metabolism [147]. Nrf2 is engaged in crosstalk with several sig-
naling pathways that play a critical role in the pathogenesis and progression of
chronic diseases, including NF-κB, PI3K, MAPK, glycogen synthase kinase-3β,
and notch [146, 147]. Molecular docking studies have shown that both WA and
withanone interact with the amino acids Ala 69, Gln 75, and Phe 71 of Nrf2 [148].
In another study, WA induced reactive oxygen species (ROS) that activated JNK
and stabilized Nrf2 that resulted in activation of NADPH quinone oxidoreductase
and Tap73 transcriptional function leading to apoptosis of cancer cells [149]. WA
was also shown to inhibit NFκB, AP-1, and Nrf2 in adriamycin-resistant human
myelogenous erythroleukemic K562/Adr cells in a dose-dependent manner [72].
Moreover, compared to other tested natural products such as quercetin, only WA
overcomes attenuated caspase activation and blocking of apoptosis in K562/Adr
cells [72].

14.2.7 Modulation of the HIF-1 Pathway by Withanolides

Under normal oxygen conditions, the HIF-1 α protein is synthesized at a high rate
and rendered transcriptionally inactive due to immediate hydroxylation-dependent
proteasome/ubiquitin degradation by the VHL E3 ligase. However, when hypoxia
is induced through impaired cellular oxygen balance, hydroxylase activity is
downregulated, HIF-α protein is stabilized, and HIF-1 is activated [150].
Transcriptional activation of HIF-1 upregulates several genes that control glycolytic
metabolism, angiogenesis, invasion, metastasis, and cell survival, such as VEGF,
MMPs, stromal cell-derived factor-1, e-cadherin, chemokine receptor 4, EGF, and
transforming growth factor beta (TGF-β) 3 [151–155]. Crosstalk between NF-κB
and HIF pathways has been shown to be associated with several chronic inflam-
matory diseases such as cancer, RA, asthma, and chronic obstructive pulmonary
diseases [156]. In solid tumors, the availability of oxygen within the tumor
decreases as distance from blood vessels increases resulting in the creation of
hypoxic regions [157]. This is known to be responsible in part for therapy resistance
14 Natural Withanolides in the Treatment of Chronic Diseases 347

and metastatic spread [158]. Although, no study thus far directly demonstrates
inhibition of HIF-1 transcriptional activation by withanolides, a few note down-
regulation of migration-promoting HIF-mediated genes such as VEGF, heteroge-
neous nuclear ribonucleoprotein K (hnRNP-K) and MMPs, which lead to restriction
of angiogenesis and metastasis [159].

14.3 Withanolides for Clinical Development

As discussed above, studies show the ability of withanolides to target multiple

interconnected signaling pathways such as PI3K/Akt/mTOR, JAK/STAT, AP-1,
NF-κB, PPARγ, Nrf2 and MAPK. Withanolides target these pathways through
multiple mechanism, such as blocking Hsp90-Cdc37 co-chaperone interaction,
targeting Akt and its downstream pathways, and induction of thiol-mediated
oxidative stress (summarized in Fig. 14.3). Each of these mechanisms and pathway
interactions play important roles in the development of chronic inflammatory dis-
eases. Building on the studies identifying mechanisms of action of withanolides, we
will discuss the clinical importance of withanolides on inflammatory mediated
diseases including chronic inflammatory/autoimmune, cancer, and neurologic.

Fig. 14.3 Schematic diagram representing modulation of various inflammatory pathways by

withanolides
348 P.T. White et al.

14.4 Application of Withanolides

in Inflammatory/Autoimmune Diseases

14.4.1 Osteoarthritis and Rheumatoid Arthritis

In Ayurvedic medicine, withanolides are frequently used to treat both osteoarthritis

(OA) and RA and there are several anti-inflammatory pathways affected by with-
anolides that contribute to chondro protection and treatment. The previously
described NF-κB pathway plays a key role in arthritic inflammation, as do the
downstream effectors NO via iNOS, COX-1, and COX-2 enzymes [160]. NO has
been implicated in chondrocyte apoptosis in RA [161] and WS extracts have
demonstrated reductions in NO in murine macrophage cell lines [122] and human
chondrocytes [162]. Nonsteroidal anti-inflammatory drugs nondiscriminately inhi-
bit COX enzymes, which with chronic use increases the risk of upper GI ulcers.
Withanolides have been demonstrated to exhibit significant selective COX-2
inhibition while sparing COX-1 [13, 26, 163]. Additionally, along a parallel
proinflammatory pathway with arachidonic acid, withanolides inhibited the pro-
duction of LTB4 [26].
Another important mediator of arthritis is the formation of ROS leading to
oxidative stress, and the protective role of Nrf2 pathway in glutathione and
thioredoxin-based antioxidant defense [164]. However, the exact mechanism by
which withanolides act within this pathway is still unclear as studies have
demonstrated that withanolides are both ROS protective through inhibition of lipid
peroxidation [13, 163] and can induce oxidative stress in rabbit chondrocytes [165,
166].

14.4.2 Therapeutic Benefits of Withanolides

for Osteoarthritis and Rheumatoid Arthritis In Vivo

There have been several studies indicating that withanolides have a chrondropro-
tective aspect. WS extract has been shown in animal models to significantly reduce
the effects of collagenase on bovine Achilles tendons with even a suggestion of
collagen stabilization [167]. This has also been demonstrated with inhibition of the
gelatinase activity of collagenase type 2 enzyme in vitro [168]. Several
collagen-induced arthritic rat models have noted significant amelioration of paw
and ankle arthritis, oxidative stress, degradation of cartilage, and improvements in
functional recovery and radiological score [24, 169–171]. Aqueous extracts of WS
have demonstrated a significant dose-dependent reduction in adjuvant-induced hind
foot pad thickness, reductions in immune complement activity [172], as well as
reduced arthritic index, autoantibodies, and CRP levels with results comparable to
methotrexate treatment. These rats similarly demonstrate reductions in oxidative
stress through decreased lipid peroxidation, glutathione-S-transferase activity, and
14 Natural Withanolides in the Treatment of Chronic Diseases 349

an increase in glutathione content and ferric-reducing ability [170]. In a mono-

sodium urate crystal-induced rat model of gout, WS extract demonstrated a sig-
nificant reduction in paw pad volume down to that of normal controls as well as
analgesic and antipyretic effects without any evidence of gastric injury [173].
Despite the conflicting reports of WA increasing oxidative stress and reducing type
II collagen through induction of COX-2 by microRNA-25 [166], the majority of
evidence continues to support the chondroprotective and analgesic properties of
withanolides in arthritis.

14.4.3 Clinical Activity of Withanolides for Osteoarthritis

and Rheumatoid Arthritis

On the basis of the long-standing use of WS extract in the treatment of arthritis in

Ayurvedic medicine and the support from animal models, there have been several
human tissue and blood studies performed in recent years. WS root powder extracts
were given to patients with chronic OA and mild to moderate (Grade 1–2) articular
cartilage and a portion of the cartilage was explanted for analysis. Half of the cartilage
samples had a significant short-term chondroprotective response as demonstrated by
significant decreases in proteoglycan release [168]. Further research on the explanted
cartilage also demonstrated a significant reduction in NO production as an
inflammatory regulator molecule in 50 % of the patient samples [162].
In RA, WS crude ethanol extract significantly inhibited LPS-induced expression
of multiple proinflammatory cytokines from PBMCs and SFMCs taken from RA
and normal patients, including TNF-α, IL-1β, and IL-12 p40. The reduction in
inflammatory cytokines may have resulted from suppression of LPS-activated
NF-κB and AP-1, in addition to inhibition of AP-1 nuclear translocation and
LPS-induced phosphorylation of IκBα [122].
Although there have been several human trials for OA and RA, they were
developed using combinations of Ayurvedic drugs. Combination therapy RA-11
(that includes WS, Boswelliaserrata, Zingiberofficinale, and Curcumalonga) was
given to patients in a randomized, placebo-controlled OA trial. Results demon-
strated a mean reduction in pain (using visual analog scale) and in the modified
WOMAC index (Western Ontario McMaster University OA Index) at 16 and
32 weeks compared to placebo without any significant in adverse events [174].
A pilot prospective study of the combination Ashwagandha and SidhMakardhwaj
in RA demonstrated a significant ACR20 (20 % improvement in tender
joint/swollen join counts and 20 % reduction in 3 of 5 areas, physician global
assessment score, patient global assessment score, pain assessment score, patient
self-assessed disability index score, and ESR) response in 56 % of patients, and a
moderate response in 40 % of patients by EULAR criteria (European League
Against Rheumatism) [175]. Although only part of a combination therapy and the
effects of WS alone cannot be determined, these trials suggest that withanolides
350 P.T. White et al.

may have chondroprotective, anti-inflammatory, and analgesic characteristics in

human joints without significant adverse events.

14.4.4 Applications in Systemic Lupus Erythematous

and Inflammatory Bowel Disease

The pathogenesis of systemic lupus erythematous (SLE) and IBD is rooted in

chronic, aberrant activation of the immune system and inflammatory pathways.
SLE is a complex B-cell mediated autoimmune disease characterized by the gen-
eration of autoantibodies against nuclear antigens (antinuclear antibody) and a type
III hypersensitivity reaction (antibody–antigen complexes) leading to chronic
inflammation and deposition of the antibody–antigen complexes within small
vessels of end organs, such as the kidneys, skin, and brain [176]. In IBD, although
chronic, intermittent inflammation is a cornerstone of disease progression, the
pathogenesis is more complex, involving genetic susceptibilities, perturbation of
mucosal physiology/epithelial barrier, and homeostasis of intestinal innate immune
system [177]. For both SLE and IBD, current therapies are based upon
anti-inflammatory, immunosuppressive, or immune/biologic therapies. Ongoing
therapeutic research is focused on identifying additional immune targets including
modulation of the Th-1, Th-2 and Th-17 responses, inflammatory cytokines, and
downstream inflammatory pathways [178, 179]. As discussed previously, with-
anolides including Withania coagulin, and Physalis angulata have demonstrated
significant immunomodulatory effects on Th cells and inflammatory cytokines
making these compounds an exciting new area of therapeutic development for
inflammatory and autoimmune diseases.

14.4.5 Therapeutic Benefits of Withanolides for SLE

and IBD In Vivo

The recent characterization of the effects of withanolides on immunomodulation has

given rise to several in vivo animal models of SLE and IBD. Aqueous WS extracts
were used to treat a nonautoimmune pristane-induced model of SLE in mice, which
develop SLE-like symptoms, including autoantibody production, proteinuria, and
nephritis. The prophylactic effects of WS extract were characterized by orally
administering WS for one month prior and six months following pristane injections.
WS treatment resulted in significant reductions in ROS formation within intraperi-
toneal macrophages, as well as reductions in inflammatory cytokines (IL-6 and
TNF-α), and decreased inflammation within the kidneys, liver, and lung, however,
no reduction in the humoral response autoantibodies or immune complex deposition
[180, 181].
14 Natural Withanolides in the Treatment of Chronic Diseases 351

Using trinitrobenzylsulfonic acid (TNBS)-induced IBD model in rats, a rectal gel

formulation from aqueous WS extract was administered from the fourth to four-
teenth day, and demonstrated a dose-dependent inhibition of lipid peroxidation up
to 96 %, hydrogen peroxide scavenging up to 82 %, and NO scavenging ability
similar to curcumin. Histopathology demonstrated a significant decrease in colonic
injury with WS treatment along with improvement in macroscopic damage when
compared to untreated controls. WS-treated rats also retained their weight with
improved recovery following induction of the TNBS model [79].
While the animal data for these inflammatory and autoimmune diseases remains
relatively sparse, there has yet to be any major human studies using withanolides in
these diseases. In the meantime, the immunomodulatory effects of withanolides
continue to be characterized and hold therapeutic potential for the treatment of a
number of autoimmune and immune-modulating diseases.

14.5 Application of Withanolides in Cancer Therapy

The discovery that most withanolides have cytotoxic capabilities against a wide
range of cancers has initiated a large breadth of research into the cytotoxic
mechanisms and potential therapeutic benefits of withanolides for cancer treatment
and prevention. As previously described and depicted in Fig. 14.3, withanolides
inhibit multiple aspects of inflammatory pathways. While these inflammatory
pathways have complex interactions between one another, they also interact with
proliferative and oncogenic pathways. Several key mechanisms of cytotoxicity
from withanolides have been described and these include: (a) induction of oxidative
stress, (b) inhibition of proteasome mediated ubiquitin degradation of IκBα (leading
to inhibition of NF-κB and its downstream effectors AP-1 and Nrf-2), (c) inhibition
of transcription factor STAT3, (d) inhibition of Hsp90 (by blocking interaction of
the Cdc37 co-chaperone with Hsp90) with resultant Hsp90 client inhibition in the
PI3K/Akt and MAPK pathways, (e) dysregulation of cytoskeletal and structural
proteins, and (f) angiogenesis inhibition though HIF-1 [1, 137, 182, 183].
Withanolides with significant activity and identified mechanisms are depicted in
Table 14.1, and their cytotoxic characteristics have been demonstrated in multiple
cancer types, including breast, ovarian, colon, head and neck, renal, prostate,
pancreatic, thyroid, glioblastoma, and hematologic cancers such as lymphoma,
leukemia, and multiple myeloma [1, 70, 136, 182–186].

14.5.1 Anticancer Benefits of Withanolides in Animal

Models

The groundwork done in vitro to establish the cytotoxic effect of withanolides

against cancer cell lines and characterize their multiple mechanisms of therapeutic
352 P.T. White et al.

potential led to the translational evaluation of these compounds in several cancer

animal models. Since cancer is a heterogeneous complex disease process, different
cancers utilize different key oncogenic pathways for survival. Because different
aspects of the inflammatory pathways are important in various cancer models,
withanolides demonstrate cytotoxic effects on tumor growth, rate of metastatic
spread, and even prevention. Looking at the STAT3 pathway in a colorectal cancer
in vivo model, WS extract treatment attenuated IL-6 activation of STAT3 and
demonstrated a 1.44 fold decrease in average tumor volume [53].
Following the initial characterization of a novel withanolide tubocapsenolide A to
inhibit Hsp90–Hsp70 complex function in breast cancer through direct thiol oxi-
dation [70], WA was shown to bind to the C-terminal of Hsp90, disrupt the Hsp90–
Cdc37 complex, and inhibit chaperone activity in pancreatic cancer. Pancreatic
cancer xenografts treated daily with withanolides demonstrated a 58 % reduction in
average tumor volume compared to controls [63]. In medullary thyroid cancer, RET,
a known Hsp90 client, is a key proto-oncogene that encodes for a transmembrane
RTK. WA treatment of medullary thyroid xenografts not only inhibited RET
phosphorylation and activation, but also inhibited phospho-Akt and phospho-ERK
protein expression ex vivo. The xenograft tumors also demonstrated significant,
2-week delayed growth kinetics with 80 % of mice responding to WA treatment, as
well as improved survival when compared to controls [187]. Withanolide E has also
been shown to sensitize renal carcinoma cells to tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL)-mediated apoptosis through cFLIP degradation
as a result of inhibited Hsp90 chaperone function. Combination treatment of mouse
renal carcinoma xenografts with withanolide E and a TRAIL death receptor agonist
demonstrated complete and sustained tumor responses in 55 % of treated mice
compared to control or either treatment alone [188].
Cytoskeletal and structural protein inhibition by withanolides has been shown to
lead to cell cycle arrest, decreased epithelial-to-mesenchymal transition (EMT), and
decreased metastasis. In breast cancer, WA binds to and inhibits β-tubulin,
decreasing β-tubulin protein levels in G0–G1 phase cells and severely disrupting
normal spindle morphology, both of which lead to cell cycle arrest [14].
Withaferin A also induces perinuclear vimentin (a mesenchymal protein) accu-
mulation leading to rapid vimentin depolymerization and disrupted morphology. In
both murine mammary carcinoma and human xenograft breast cancer models in
mice, WA and WS treatment showed a dose-dependent inhibition of tumor growth
kinetics, a decrease in number of metastatic lung nodules, and an increase in ser56
phosphorylated vimentin that is indicative of disassembly [189, 190]. Subsequent
studies also showed that WA treatment attenuated TNF-α and TGF-β induced EMT,
increased the epithelial phenotype protein E-cadherin, and inhibited vimentin
expression in addition to inhibited tumor grown kinetics and cell proliferation [23,
143]. Together, these studies characterize the ability of WA to inhibit EMT both
in vitro and in vivo, which may inhibit the metastatic potential of these tumors.
Another area of research in cancer therapy is the inhibition of angiogenesis which
limits the oxygen and nutrient supply to growing tumors as well as their metastatic
capabilities. Withanolides demonstrate significant angiogenesis inhibition [55] and
14 Natural Withanolides in the Treatment of Chronic Diseases 353

as previously described, mechanisms include blocking NF-κB activity along with

inhibition of cyclin D1 expression through suppressing proteasome-mediated
ubiquitin degradation of IκBα [52, 55], and the interplay between NF-κB,
JAK/STAT, HIF-1 and downstream targets VEGF, hnRNP-K, and MMPs [52, 140,
159]. Using both a subcutaneous flank xenograft and lung metastasis murine model
with fibrosarcoma cells, withanone and WA demonstrated significant suppression of
both subcutaneous and lung metastasis tumor growth compared to controls. The
ex vivo tumors then demonstrated decreased expression of hnRNP-K and down-
stream effectors VEGF, Erk p44/42, and MMP2 [159]. In an in vivo assay of
neovascularization using subcutaneously injected Matrigel that contained VEGF and
bFGF, mice treated with 3-azido withaferin A (3-azidoWA) demonstrated a marked
reduction in neovascularization with both preventative and treatment dosing when
compared to untreated controls [50].
Not only have withanolides demonstrated significant cytotoxicity in multiple
cancer models, but they have also demonstrated the capability to prevent cancer
growth and implantation [137]. The induction of the phase II enzyme quinone
reductase (QR) in mouse hepatoma cells has been used as a biological screen for
chemopreventive compounds, and multiple withanolides have demonstrated robust
induction of QR with minimal toxicity [191]. Subsequently in rodent models,
pretreatment with the WS root isolate 1-oxo-5beta, 6beta-epoxy-witha-2-enolide
prevented the formation of cutaneous malignancy and were absent p53 + foci that
were noted in untreated rats that had been exposed to ultraviolet B radiation and
benzoyl peroxide [192]. Additionally, WS root extract inhibited benzo(a)
pyrene-induced forestomach papillomagenesis by 60 % and
7,12-dimethylbenzanthracene-induced skin papillomagenesis by 45 % [193]. It has
been proposed that one mechanism of cutaneous chemoprevention is through
inhibition of carcinogen-induced upregulation of acetyl-CoA carboxylase by sup-
pressing AP-1 activation [194]. In a mouse mammary tumor virus-neu
(MMTV-neu) transgenic model that forms spontaneous tumors, preventative WA
treatment led to a significant 50 % reduction in average mammary tumor weight, a
95 % reduction in average area of invasive carcinoma, and a 73 % reduction in
incidence of lung metastasis [195]. Another important antitumor effect of these
withanolides is that they can also target breast cancer stem cells. Treatment of breast
cancer cells with WA resulted in decreased ability to form mammospheres as well
as significantly decreased aldehyde dehydrogenase activity within the mammary
tumor cells [196]. WS root extract has also been shown to reduce the formation of
spontaneous estrogen receptor negative mammary tumors in the MMTV-neu mice
when mice were fed a diet containing the extract [197].
Overall these in vivo studies also noted that withanolides were well tolerated
without significant reductions in animal weight, necrosis, or fibrosis when com-
pared to placebo [23, 143, 159, 189, 190, 192, 193, 195]. The promising results of
withanolides observed thus far in cancer models have led to ongoing research by
many centers to both identify new withanolides and evaluate existing withanolides
in multiple cancer models. In the last few years, withanolides have also been shown
to demonstrate tumor cytotoxicity using animal xenografts in gliomas [198], B-cell
354 P.T. White et al.

lymphomas [199], ovarian cancer [200, 201], prostate cancer [202, 203], soft tissue
sarcoma [204], cervical carcinoma [205], melanoma, and mesothelioma [206].
These exciting results all point toward the incredible potential of these natural
products for future clinical treatment regimens.

14.5.2 Clinical Applications of Withanolides for Cancer

Despite having improvements in our understanding of the multiple mechanisms of

cytotoxicity with withanolide treatment across an expanding range of cancers, there
has yet to be a significant human clinical placebo-controlled trial brought to com-
pletion and published that evaluates the efficacy of withanolides for the treatment of
cancer. Withanolides are generally regarded as safe and have been used in human
clinical trials for inflammatory and neurologic diseases, and have been evaluated for
the treatment of fatigue for breast cancer patients undergoing chemotherapy [207].
Additional in vivo research on withanolides is ongoing and has overall been quite
promising to identify withanolides as an important cancer therapy, however, further
supporting research is needed to initiate the human clinical trials required to obtain a
better understanding of the clinical treatment effects of withanolides on cancer. To
date, there is no Food and Drug Administration (FDA)-approved good manufac-
turing process (GMP) facility currently purifying and producing purified withanolide
compounds for use in clinical trials, although several facilities in other countries
produce capsules of withanolide plant products and extracts that are not regulated by
the FDA.

14.6 Application of Withanolides in Neurologic Diseases

14.6.1 Neurodegenerative Diseases

Neurodegenerative diseases are characterized by the progressive dysfunction and

loss of neurons in the central nervous system (CNS) and are a major cause of
dementia, cognitive and motor dysfunction. As the pathophysiology of neurode-
generative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD),
and Huntington’s disease (HD) are better understood, the major role of the immune
system and neuroinflammation has become readily apparent [208]. Although the
blood–brain barrier maintains some degree of separation of the CNS from the
systemic immune system to aid in the immune privileged state of the CNS (its
relative inability of nonself antigens to illicit an immune response), the role of the
innate and adaptive immune responses has become a major focus of both study and
intervention [209]. In addition to the formation and deposition of amyloid β plaques
in AD, activation of microglia (macrophage-like CNS cell) and astrocytes,
14 Natural Withanolides in the Treatment of Chronic Diseases 355

increased complement components, cytokines, and TLR pathways, and alterations

in peripheral Th cell responses have also been described [210, 211]. In PD,
mitochondrial dysfunction, oxidative stress, and altered protein handling with Lewy
body deposition as well as inflammation through microglial activation, increased
IL-1β, IL-6, TNF-α, and TLRs, formation of antibodies to neuronal antigens, and
Th cell modulation are important. Finally in HD, mutant Huntingtin
(HTT) expression in neurons and glia leading to microglia proliferation/activation,
increased complement 3, 9 and neuroinflammation are keys to the pathogenesis of
the disease [208, 209].
As previously discussed, withanolides inhibit multiple inflammatory pathways,
and within the CNS astrocytes, Withaferin A has been shown to attenuate
LPS-induced NF-κB, TNF-α, COX-2 and iNOS [212]. In addition to neu-
roinflammatory modulation, several withanolides including WS extract, withano-
sides (particularly withanoside IV and its active metabolite sominone), the synthetic
withanolidedenosomin, withanolide A and coagulin Q have all demonstrated
important effects on stimulating neurite outgrowth and regeneration [66, 210, 211].
In AD, additional key findings demonstrated that withanolide A significantly
downregulates beta-site amyloid precursor protein (APP) cleaving enzyme 1
(BACE1; known as β-secretase, enzyme involved in production of Aβ) while it
upregulates a disintegrin and metalloproteinase domain-containing protein 10
(ADAM10; α-secretase, non-amyloidogenic enzymatic processing of APP).
Additionally, withanolide A increases expression of insulin-degrading enzyme,
which is important in the proteolytic degradation of Aβ [213]. Multiple withano-
lides (WS, WA, and bracteosins) have also demonstrated significant acetyl-
cholinesterase (AChE) or butyrylcholinesterase (BChE) inhibition [214, 215] with
computational docking analysis supporting withanolide A high-affinity binding for
active sites on AChE [216]. These results, combined with the known antioxidant
and anti-inflammatory effects of withanolides have propelled their research forward
in neurodegenerative disorders to further elucidate their mechanism of action.

14.6.2 In Vivo Activities of Withanolides in the Treatment

of Neurodegenerative Diseases

In a systematic review and meta-analysis, Durg et al. identified and analyzed 28

studies evaluating the use of WS in neurobehavioral disorders (including AD, PD,
HD and anxiety/stress) induced by brain oxidation in rodent models [217]. Overall,
WS treatment had a protective effect on brain oxidative stress that corrected
abnormal activity levels of super oxide dismutase (SOD), catalase, glutathione
peroxidase and glutathione, lipid peroxidation, and levels of nitrite and AChE
[217]. In several types of AD mouse models, including Aβ-[25–35] induced
memory deficit and 5XFAD mice (5 FAD mutations carried on APP and PS1
transgenes), withanolides (withanolide A, withanoside IV and its main active
356 P.T. White et al.

metabolite, sominone, and WS extract) significantly ameliorated the impairment of

spatial memory and behavioral deficits [39, 40, 210, 217], while ex vivo brains
showed increased axonal and dendritic protein markers back to control levels [39,
211], increased axonal densities [40], and reduced Aβ levels and plaque depositions
through upregulation of low-density lipoprotein receptor-related protein within the
liver [218].
Parkinson’s disease is characterized by an age-related neurodegenerative pro-
gression that leads to resting tremor, rigidity, and akinesia though loss of
dopaminergic nigro-striatal neurons, particularly within the substantia nigra [219].
The rodent models of Parkinsonism are created through brain injection of
6-Hydroxydopamine [220] or systemic administration of
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [221] or the combination of
manganese ethylene-bis-dithiocarbamate (maneb) and N,N′-dimethyl-4,4′-bipyr-
idinium dichloride (paraquat) [222]. They cause specific degeneration and toxicity
to catecholaminergic and dopaminergic neurons though oxidative stress and for-
mation of hydrogen peroxide, hydroxyl, and superoxide radicals [220–222]. Using
these models, treatment with WS extract resulted in significant improvements in
locomotor control (including rotation, locomotion, muscular coordination and
rearing), amelioration of oxidative parameters (including LPO, glutathione and
associated enzymes, SOD and catalase), correction of ex vivo rodent brain alter-
ations to catecholamine, dopamine, and dopamine metabolites (DOPAC/HVA) in
ex vivo rat brains [222–226]. Some of these effects may be due to mediation of the
otherwise proapototic state through reductions of Bax and Bcl-2 expression and
neuroinflammatory astrocyte activation [227].
Huntington’s disease has autosomal dominant transmission of the mutated HTT
gene that leads to neuron destruction within the basal ganglia, leading to dementia
and the characteristic involuntary writhing movements characteristic of
Huntington’s chorea. Mutant HTT aggregates have been implicated in oxidative
stress through the formation of free radicals, which creates imbalance and plays a
role in subsequent neuroinflammation [228]. Huntington’s disease-like neurobe-
havioral and biochemical changes are induced in rodent models using neurotoxin
3-Nitropropionic acid. Treatment with WS root extract demonstrated significant
dose-dependent attenuation of AChE levels, significantly improved oxidative stress
markers, and improvement in general locomotor, performance and behavioral
changes tested by Rotarod and Morris water [229, 230].
Although the involvement of the immune response and neuroinflammation plays
a significant role in the pathogenesis of neurodegenerative diseases, the specific
connections between these inflammatory pathways and the effects of withanolide
treatment have not been well defined. The role of WS on reducing the oxidative
stress within neurons has been demonstrated in multiple animal models of different
disease processes, as well as the neuro-regenerative role of several withanolides on
axonal and dendritic outgrowths and functional locomotor improvements.
Connecting these effects to neuroinflammation and immunomodulation identifies an
important area of future research.
14 Natural Withanolides in the Treatment of Chronic Diseases 357

14.6.3 Clinical Activity of Withanolides

in Neurodegenerative Diseases

There is an overall paucity of research on the effects of withanolides in patients with

neurodegenerative diseases. WS was a component of one study that evaluated the
efficacy of a traditional Ayurveda treatment in clinically diagnosed PD patients.
Treatment with a combination of eliminative cleansing and a concoction of cow’s
milk with powdered Mucunapruriens, Hyoscyamusreticulatus seeds, WS and
Sidacordifolia roots (analyzed to contain 200 mg L-DOPA per dose) showed
significant improvements in tremor/bradykinesia, stiffness and cramp-like pain
resulting in improved activities of daily living, however, no changes in other
symptoms like dysphonia, dysarthria, wasting, cogwheel rigidity, shuffling gait, or
other locomotor symptoms [231]. However, conclusions regarding the role of WS
cannot be reached due to the mixed nature of the L-DOPA containing herbal
treatment. However, the growing in vitro and animal data continue to support the
use of withanolides in neurodegenerative diseases, and illustrates the potential for
its use in a more controlled clinical trial.

14.6.4 Withanolides in Neurobehavioral/Psychiatric

Diseases

There is a broad range of neurological diseases in which inflammation and partic-

ularly cytokines and microglial activation have been shown to play a pathophysi-
ological role. These include chronic stress [232], anxiety [233], depression [234],
schizophrenia [235, 236], bipolar disorder [237, 238], and obsessive–compulsive
disorder (OCD) [239]. Although the study of how the immune system and inflam-
mation contribute to these diseases is relatively recent, major inflammatory pathways
and cytokines have been identified. One proposed mechanism is through the stress
response, whether through external stimuli or psychological imbalances, which leads
to activation of the hypothalamic-pituitary-adrenal (HPA) axis and short-term ele-
vations in glucocorticoids (mainly cortisol) and a subsequent anti-inflammatory
response. However, prolonged stress and sustained HPA activation causes cortisol
resistance and initiation of pro-inflammatory pathways, such Th cell release of IL-1α
and β, IL-2, IL-6, IL-10, TNF-α cytokines, activation of NF-κB pathway, and
microglial activation [232–238]. In addition to the known anti-inflammatory effects
of withanolides previously discussed, they have also been described as adaptogens
that assist with balance and regulation of the body’s physiologic response to stres-
sors [240]. The adaptogen mechanism of effect has been proposed to occur through
modulation of the HPA axis, inducing stress-activated c-Jun N-terminal protein
kinase (JNK1), inhibiting iNOS expression, and modulation of Hsp70 chaperone
function [241]. Additionally, the inhibitory neurotransmitter gamma-aminobutyric
acid (GABA), which plays an important inhibitory role in neurological disorders, has
358 P.T. White et al.

more recently been shown to have key anti-inflammatory interactions within the
immune system through suppression of T-cells and macrophages, and inhibition of
NF-κB on lymphocytes [242]. The GABA-mimetic activity of WS root extract has
been shown for several decades [243] with a recent study identifying a 27 times
greater affinity of WS for the highly sensitive GABAρ1 receptors compared to
GABAA receptors, though WA and withanolide A were not the active
GABA-mimetics [244]. These GABA-mimetic and adaptogenic effects likely play a
significant role in the long-standing history of using Ashwagandha for the treatment
of anxiety and neurobehavioral disorders, though the vast majority of animal and
human studies have been performed in the last several decades.

14.6.5 In Vivo Activity of Withanolides

for Neurobehavioral/Psychiatric Diseases

Most animal studies evaluating neurologic disorders have focused on the effects of
WS extract using models of either chronic stress or withdrawal to induce anxiety
and depression. Using a chronic, unpredictable, mild footshock to create a chronic
stress model in rats, treatment with aqueous ethanol WS root extract significantly
attenuated chronic, stress-induced abnormalities with improvements in biochemical
imbalances (elevated blood glucose and corticosteroid levels), reduced number and
severity of gastric ulcers, improved behavioral depression and sexual responses,
improved cognitive memory function, and rescued immunosuppression in macro-
phage activity and immunologic pedal edema [245]. In depression and anxiety
rodent models using chronic stress, isolation, sleep deprivation, WS extract treat-
ment demonstrated significant antidepressant effects, and potentiated conventional
antidepressants (tricyclic antidepressant imipramine and selective
serotonin-reuptake inhibitor fluoxetine) measured by reduced immobility time in
the forced open swim test (“behavioral despair/learned helplessness”), and an
anxiolytic effect similar to benzodiazepines (lorazepam and diazepam) in the ele-
vated plus maze, the social interaction test, and the feeding latency test [246–250].
In an OCD rodent model of marble-burying behavior, intraperitoneal injections
of both methanolic and aqueous WS root extracts 30 min prior to evaluation
resulted in significant dose-dependent reductions in marble-burying behavior sim-
ilar to standard fluoxetine alone, and a synergistic effect in combination with
fluoxetine [251]. These in vivo studies demonstrate a consistent amelioration of
stress, anxiety, depressant, and OCD behaviors with corresponding corrections in
biochemical abnormalities, however, the modulation of inflammatory pathways in
these diseases requires further characterization.
14 Natural Withanolides in the Treatment of Chronic Diseases 359

14.6.6 Clinical Activities of Withanolides

in Neurobehavioral/Psychiatric Diseases

Recent clinical trials using WS extract as a neurological treatment have evaluated

effects on psychomotor function in patients with anxiety, bipolar disorder, and
schizophrenia and provide clinical support for centuries of WS use in Ayurvedic
medicine. There have been several randomized controlled trials that demonstrate
significant improvement in anxiety and stress relief [252–257]. Studying ICD-10
anxiety disorders (generalized anxiety disorder, mixed anxiety and depression,
panic disorder, and adjustment disorder with anxiety) assessed by Hamilton
Anxiety Scale, patients were treated with 500 mg WS extract or placebo twice daily
with subsequent clinically guided dose titrations. At 6 weeks, the WS extract
treatment response of 88 % was significantly improved compared to the 50 %
placebo response with no significant difference in overall adverse outcomes or with
abrupt cessation of WS treatment [253]. Subsequent randomized controlled studies
have corroborated their results and demonstrated either significantly decreased
anxiety or improved stress relief using multiple anxiety scales (Hamilton Anxiety
Scale, Beck Anxiety Inventory, Perceived Stress Scale), a range of WS dosing
primarily between 125 and 600 mg/day for extract (one study 12,000 mg/day
whole dried root powder) over the course of 60–84 days [252, 254–257]. None of
the studies reported significant adverse events with WS treatment, with all descri-
bed as mild or comparable to the placebo groups. However, significant limitations
existed in each of the studies that included high rates of patient withdrawal from
study [253], low sample size leading to bias risk and under powering, inconsistent
dosing or lack of withanolide standardization, lack of comparison to standard of
care anti-anxiolytics, and possible methodological flaws [252–257]. The limitations
impair making definitive conclusions regarding the effectiveness of WS in anxiety
disorders, but the body of study identifies WS as a relatively safe therapeutic and
supports the role for conducting additional clinical studies.
In both bipolar disease and schizophrenia, cognitive impairments are consis-
tently associated with poorer functional outcomes. As discussed previously, WS
improves memory and cognitive function when treating neurodegenerative rodent
models, and in healthy adults significantly improves psychomotor function as
demonstrated by simple reaction times, choice discrimination, digit symbol sub-
stitution, digit vigilance, and card sorting tests compared to placebo [258]. In well
maintained bipolar disease patients, a randomized controlled, trial of WS in adjunct
to maintenance bipolar treatment resulted specific improvement in several cognitive
function tests including the digit span backward, Flanker neutral response time, and
social cognition response rating from the Penn Emotional Acuity Test, though no
global cognitive improvement [259]. In schizophrenia, there is currently an ongoing
randomized controlled clinical trial in the United States evaluating the effect of WS
extract on symptom severity and associated stress that also aims to characterize
360 P.T. White et al.

alterations in inflammatory cytokines [260]. The results from this study will help
provide valuable insight into the effect of WS on neuroinflammation and
schizophrenia symptoms.

14.7 Conclusions

Withanolides are an incredibly bio-diverse group of naturally occurring steroidal

lactones. These plant-derived compounds have ongoing use within Ayurvedic
medical practices and are important plant-derived medicinal compounds for a
variety of human diseases and conditions. With WA as the archetype of this novel
class drugs, there are now approximately 900 withanolides identified from a natural
source or synthetically modified. Significant strides have been made in recent years
to advance our understanding of the biochemical, immunomodulatory, and
anti-inflammatory mechanisms of these compounds and plant extracts. Added to
this is emerging in vivo and clinical evidence of safe, efficacious treatment effects
across multiple disease processes ranging from autoimmune/inflammatory disor-
ders, cancer, neurodegenerative diseases, and neurobehavioral/psychiatric diseases.
Ongoing research on withanolides continues to identify new compounds and ana-
logs, both plant derived and synthetically altered, that are more potent or specific
for use in particular diseases. Given their impressive biologic activity in a number
of challenging and complex disease processes and their unique ability to synergize
with many standard drug treatments, these naturally derived drug compounds
undoubtedly will have an important role clinically in well-designed combination
strategies once disease-specific mechanisms of action and synergy are further
validated and characterized. As such, they remain a very hot area of research as a
novel group of medicinal therapeutics.

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NCT01793935
Chapter 15
Gambogic Acid and Its Role in Chronic
Diseases

Manoj K. Pandey, Deepkamal Karelia and Shantu G. Amin

Abstract Kokum, a spice derived from the fruit of the Garcinia hanburyi tree, is
traditionally used in Ayurvedic medicines to facilitate digestion and to treat sores,
dermatitis, diarrhoea, dysentery, and ear infection. One of the major active com-
ponents of kokum is gambogic acid, also known as guttic acid, guttatic acid,
beta-guttilactone, and beta-guttiferin. Gambogic acid’s anti-proliferative,
anti-bacterial; antioxidant and anti-inflammatory effects result from its modulation
of numerous cell-signaling intermediates. This chapter discusses the sources,
chemical components, mechanism of action, and disease targets of the kokum spice.

Keywords Neutraceuticals Dietary agents Gambogic acid Kokum Cancer



Signal transduction pathways

15.1 Introduction

Mother Nature has gifted us a variety of natural agents, including nutraceuticals. One
of the well-known nutraceuticals is Gambogic acid (GA), which is a xanthonoid
derived from the brownish or orange resin from Garcinia hanburyi (Fig. 15.1).
Garcinia hanburyi is a small to medium-sized evergreen tree with smooth gray bark,
and it is native to Cambodia, southern Vietnam, and Thailand. Garcinia indica,
primarily of Indian origin, is known by many names: bindin, biran, bhirand, bhinda,
kokum, katambi, panarpuli, ratamba, amsol, and tamal. In English language, it is
commonly known as mangosteen, wild mangosteen, red mango, Hanbury’s Garcinia,
gambojia, gamboge, and Indian gamboge tree. Germans called this gummi-gutti.
The Garcinia indica seed contains 23–26 % oil, which is used in confectionery,
medicines, and cosmetics. It is used in curries and other dishes as a slightly bitter
spice, a souring agent, and as a substitute for tamarind.

M.K. Pandey (&)
D. Karelia
S.G. Amin

Department of Pharmacology, College of Medicine, Pennsylvania State University, 500
University Dr., Hershey, PA 17033, USA
e-mail: [email protected]

© Springer International Publishing Switzerland 2016 375

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1_15
376 M.K. Pandey et al.

Garcinia hanburyi Fruit and pulp Dried pulp

( False Mangosteen) ( Kokum)

Gambogic Acid

Fig. 15.1 Plant species and fruits by which gambogic acid is derived. Highlighted circles on GA
structure indicate the most common sites for novel derivative generation

In traditional medicine, such as ayurveda, kokum is prescribed for edema,

rheumatism, delayed menstruation, constipation and other bowel complaints, and
intestinal parasites. The extract of Garcinia cambogia is used as an herbal appetite
suppressant and weight-loss supplement.
In last decades, worldwide extensive studies have been performed on Gambogic
acid to understand its full potential as therapeutic agents against variety of diseases
including chronic diseases such as cancers which is summarized in following
sections.

15.2 Physciochemical Properties of GA

GA is also chemically called as Guttic acid, Guttatic acid, beta-Guttilactone, and

beta-Guttiferin. The molecular formula and weight of GA is C38H44O8 and 628.76,
respectively. The appearance of this xanthone is amorphous orange solid. The core
of GA is known as xanthone core and contains a unique 4-oxatricyclo [4.3.1.03,7]
decan-2-one scaffold [1, 2]. Earlier studies regarding its structural activity rela-
tionships (SAR) revealed that the C¼C bond of the a,b-unsaturated ketone in GA is
critical for its antitumor activity, while the HOC(6), C(8)¼O, and C(30)OOH groups
could tolerate a variety of modifications Fig. 15.1 [3, 4]. Along these lines various
modifications have been performed to make GA as a better antitumor agent [5, 6].
15 Gambogic Acid and Its Role in Chronic Diseases 377

15.3 Modulation of Cell Signaling Pathways by GA

Enthusiasm shown by researchers from around the globe clearly suggests that GA
has been one of the “hot” nutraceuticals. GA has shown to be effective on different
chronic diseases (Sect. 4.1 covers different chronic diseases), but its effect on
cancer has been studied the most. Almost a decade ago our group showed that the
anti-inflammatory and anticancer response of GA is associated with its inhibitory
response on Nuclear Factor-Kappa B (NF-jB) [7], since then plethora of studies
suggest that GA regulates several key signaling pathways. GA exhibits
anti-proliferative, antioxidant, and anti-inflammatory effects by modulating cell
signaling pathways, enzymes, and molecular targets, such as epigenetic regulators,
protein kinases, transcription factors, inflammatory biomarkers, and growth regu-
lators. Through microarray analysis, GA modulates many gene products [8, 9]
(Table 15.1).

15.3.1 GA Inhibits Signaling of Nuclear

Factor-Kappa B (NF-jB)

The transcription factor NF-jB is one of the major mediators of inflammation and is
linked with many diseases including cancer, diabetes, arthritis, and neurological
disorders. Therefore, an agent that can suppress NF-jB activation has potential for
clinical use against various chronic illnesses. GA suppression of NF-jB activation
induced by TNF-a, LPS, and various agents [7, 10] leads to the suppression of
NF-jB regulated products, such as cyclooxygenase type 2 (COX-2), inducible
nitric oxide synthase (iNOS), and survival proteins [7, 10, 11]. These actions give it
great potential as a broad-spectrum clinical agent.

15.3.2 GA Inhibits Phosphatidylinositol 3′-Kinase/Protein

Kinase B (PI3K/Akt)

Serine/threonine-specific protein kinase B, commonly designated Akt, is a central

regulator of widely divergent cellular processes, including proliferation, differen-
tiation, migration, survival, and metabolism [12, 13]. Akt is activated by a variety
of stimuli, through growth factor receptors, in a PI3K-dependent manner [12, 13].
Frequently in human cancer, normal signaling along the Akt/PKB/phosphatase, and
tensin homolog (PTEN) pathway is disrupted [14]. Akt plays important roles in
development, progression, and resistance to chemotherapy in cells [12, 13].
Blocking Akt signaling can mediate apoptosis and inhibit the growth of tumor cells
in vitro [14]. GA inhibits Akt activation, which leads to inhibition of tumor cell
proliferation and survival [15–19].
378 M.K. Pandey et al.

Table 15.1 A list of Transcription factor

molecular targets of
Nuclear factor—kappa B #
Gambogic acid
STAT-3 - #
STAT-5 #
Inflammatory cytokines
IL-6 #
Tumor necrosis factor alpha#
Enzymes
Cyclooxygenase-2 #
Inducible nitric oxide synthase#
Matrix metalloproteinase#
Src homology 2 domain-containing typrosine phosphate 2#
Kinases
Focal adhesion kinase#
Janus kinase #
Mitogen-activated protein kinase #
Protein kinase A#
Protein kinase B#
Protein kinase C#
Growth factors
Vascular endothelial growth factor #
Receptors
Chemokine (C-X-C motif) receptor 4 #
Transferrin receptor #
Adhesion molecules
Endothelial leukocyte adhesion molecule-1
Intracellular adhesion molecule-1 #
Anti-apoptotic proteins
B-cell lymphoma protein-2 #
Bcl-xL #
Inhibitory apoptosis protein-1 #
Mcl-1 #
Survivin #
Others
Cyclin D1 #
Heat shock protein 90 #
Heat shock protein 70 "
15 Gambogic Acid and Its Role in Chronic Diseases 379

15.3.3 GA Inhibits Mitogen-Activated Protein

Kinase (MAPK)

MAPKs are evolutionarily conserved enzymes that play a key role in the inflam-
matory stimuli and environmental stresses that lead to the activation of three
independent pathways: p44/42 MAPK extracellular signal-regulated kinases 1 and
2 (ERK1/ERK2), c-Jun N-terminal kinase, and p38 MAPK [20]. In vitro studies of
several cancer cells showed that GA inhibits MAPK pathways [21]. Moreover, this
phytochemical also inhibited ERK in HT-29, HepG2, KBM5, and NCI-H460
cancer cells [22–24].

15.3.4 GA Inhibits Src

The Src family of proteins consists of eight non-receptor tyrosine kinases charac-
terized by a common structure [25]. Src kinases are involved in signal transduction
pathways that are triggered by a variety of surface receptors, including receptors for
tyrosine kinases, integrin, and antigens, as well as receptors coupled with the
G-protein [25, 26]. As a consequence of changes observed in protein expression
and kinase activity in cancer cells, the Src family has been implicated in the
development of cancer [25, 26]. This prompted the design of specific inhibitors, the
most common of which are adenine mimetics, to treat solid tumors and leukemia
clinically [25, 26]. In addition, some of the Src kinases expressed in hematopoietic
cells play pivotal roles in lymphocyte maturation and activation [25]. This finding
encouraged the development of safe and effective Src-specific inhibitors that are
currently in clinical trials as immune-suppressants for the treatment of immuno-
logical disorders [15, 27]. Separate research showing that GA inhibits Src in PC3,
and K562 cells suggests that GA may also have clinical potential against cancers
and immunological disorders in which Src plays a pivotal role [15, 16].

15.3.5 GA Inhibits Signal Transducer and Activator

of Transcription-3 (STAT-3) Pathways

Proteins in the STAT family are among the best studied of the latent cytoplasmic
signal-dependent transcription factors [28–30]. In vitro studies of the MCF-7,
MCF-10A, U266, and MM1.s cell lines suggest that GA modulates the nuclear
translocation and DNA binding of STAT3 and inhibits genes modulated by this
transcription factor [28, 31].
380 M.K. Pandey et al.

15.3.6 GA Inhibits Chemokine X-Receptor 4 (CXCR4)

and Downstream Signaling Pathways

Chemokine receptors belong to class A seven transmembrane G-protein-coupled

receptors and consist of 350 amino acids on average. Primary receptors are defined
as CXCR, CCR, CR, or CX3CR [32]. The function of atypical chemokine receptors
is to modulate immune responses by scavenging, sequestration, buffering as well as
intracellular transport of chemokines from inflammatory sites [33–35]. The receptor
CXCR4 is expressed on almost all of the hematopoietic cells, embryonic pluripo-
tent, and tissue-committed stem cells, allowing them to migrate and invade along
CXCL12 gradients. In malignant cells the chemokine receptor that is most com-
monly found is the receptor CXCR4 [34, 36]. At least 23 different types of tumor
cells from human cancers of epithelial, mesenchymal and hematopoietic origin
express CXCR4 [37]. In cancer, CXCL12 plays a role in the mobilization and
recruitment of these cells to the inflammatory tumor microenvironment,
neo-angiogenic niches, supporting revascularization, tumor growth, and metastasis
[32, 38]. Thus an inhibitor of CXCR4/CXCL12 axis will inhibit tumor metastasis,
GA is one of those inhibitors. Recently, we showed that GA directly interacts with
CXCR4 and inhibits the migration of multiple myeloma cells [17]. We further
demonstrate that GA inhibits CXCR4 regulated pathways and suppresses the bone
loss [17]. Overall, GA has a tremendous potential to be used a therapeutic agent.

15.3.7 GA Inhibits CBP/p300 Histone Aceyltransferase

(HAT) and Histone Deacetylase (HDAC)

The process of histone acetylation and deacetylation in eukaryotic cells alters

chromatin structure and thereby modulates gene expression [39]. HATs and
HDACs are classes of enzymes that effect histone acetylation [40]. These enzymes
can also acetylate and deacetylate several nonhistone substrates, which can have
functional consequences. Altered HAT and HDAC activities can lead to several
diseases, ranging from cancer to neurodegenerative disorders. Therefore, HAT and
HDAC inhibitors are being developed as therapeutic agents. GA inhibits HAT and
HDAC activity in A549 lung cancer cells [41]. These activities of GA demonstrate
its great potential as a therapeutic candidate.

15.3.8 GA Inhibits the Activation of Focal Adhesion

Kinase (FAK)

FAK is a 119- to 121-kDa non-receptor protein kinase widely expressed in various

tissues and cell types [42]. Several studies showed that FAK plays an important role
15 Gambogic Acid and Its Role in Chronic Diseases 381

in integrin signaling [43–45]. Once activated, whether by integrin or non-integrin

stimuli, FAK binds to and activates several other molecules, such as Src, Src
adaptor protein p130Cas, the growth factor receptor-bound protein 2 (Grb2), PI3K,
and paxillin, and thus promotes signaling transduction [44, 46–50]. In a recent
study, FAK was held responsible for uninhibited proliferation, protection from
apoptosis, invasion, migration, adhesion, and spread, as well as tumor angiogenesis
[46, 47]. Our group showed that GA modulates the tyrosine phosphorylation of
FAK and subsequently induce apoptosis by downregulating Src, ERK, and Akt
signaling in prostate cancer PC3 cells [16].

15.3.9 GA Inhibits iNOS

iNOS is expressed in a variety of cell types, particularly inflammatory cells, in

response to diverse pro-inflammatory stimuli [51–53]. iNOS, which may be
induced by bacterial LPS or its derivative lipid A, is expressed by a variety of solid
tumors and generates high levels of nitric oxide inside tumor cells [10]. In vitro
studies showed that GA inhibits LPS- and interferon-gamma-induced iNOS in
RAW246.6 cells [10].

15.3.10 GA Induces the Production of Reactive Oxygen

Species (ROS)

ROS have been linked with various cell signaling pathways [54, 55]. GA, induces
the production of ROS [56].

15.3.11 GA Inhibits COX-2

Overexpression of COX-2 is associated with many cancers and is linked with tumor
cell proliferation and suppression of apoptosis [57, 58]. Therefore, COX-2 inhibi-
tors have great potential in the treatment of cancers and inflammatory conditions, as
evidenced by the U. S. Food and Drug Administration’s approval of celecoxib, a
known COX-2 inhibitor, for the treatment of various inflammatory conditions [59].
GA, too, has been shown to inhibit COX-2 activation induced by TNF-a in KBM5
leukemic cells [7].
382 M.K. Pandey et al.

15.3.12 GA Inhibits Matrix Metalloproteinase 7 & 9

(MMP-7 & 9)

Also known as matrilysin, MMP-7 is a “minimal domain” MMP that exhibits

proteolytic activity against components of the extracellular matrix [60–62]. MMP-7
is frequently overexpressed in human cancer tissues and is associated with cancer
progression [63]. Therefore, MMP-7 inhibitors have great potential in the treatment
of cancer [64]. The studies showed that GA inhibits the expression of MMP-7 in
breast cancer cells like MDA-MB-231 and MDA-MB-435 [65, 66], further sup-
porting the idea that GA may be effective against breast cancer in humans.

15.3.13 GA Inhibits Tubulin

Microtubules are a major component of the cytoskeleton. They are important in

many cellular events and play a crucial role in cell division [67]. As such, micro-
tubules are a highly attractive target for anticancer-drug design. Tubulin-binding
agents, also called anti-microtubule or microtubule-targeted agents, are widely used
chemotherapeutic drugs with a proven clinical efficacy against breast, lung, ovarian,
prostate, and hematologic malignancies, as well as childhood cancers [68, 69].
Research has shown that GA belongs to this class of agents because it inhibits
microtubule assembly and prevents cell division [20].

15.3.14 GA Inhibits Expression of Cyclin D1

The sequential transcriptional activation of cyclins, the regulatory subunits of cell

cycle-specific kinases, is thought to regulate progress through the cell cycle [70].
Thus, cyclins are potential oncogenes, and overexpression of cyclin D1 or ampli-
fication at its genomic locus, 11q13, is commonly seen in breast cancer, head, and
neck cancer, non-small-cell lung cancer, and mantle cell lymphoma [71, 72]. GA
has been shown to inhibit Cyclin D1 in several cancers including leukemia and
multiple myeloma [7, 31].

15.3.15 GA Induces Cleavage of Poly(ADP-Ribose)

Polymerases (PARPs)

PARPs are cell signaling enzymes present in eukaryotes and are involved in poly
(ADP ribosylation) of DNA-binding proteins [73]. Pharmacological degradation of
PARP-1 may enhance the activity of antitumor drugs by inhibiting necrosis and
15 Gambogic Acid and Its Role in Chronic Diseases 383

activating apoptosis [74]. In vitro studies have shown that GA induces PARP
degradation and enhances apoptosis in T98 glioma, HeLa, non-small lung cancer
A549 and NCI-H460, breast cancer, and multiple myeloma cells [7, 11, 17, 22, 24,
31, 66, 75–77].

15.3.16 GA Inhibits Tumor Necrosis Factor-a (TNF-a)

TNF-a is a vital member of the multifunctional superfamily of TNFs and plays

important roles in immunity and cellular remodeling, as well as apoptosis and cell
survival [78]. Because TNF-a is a key player in inflammation and cancer, several
efforts are underway to develop therapeutic TNF-a antagonists. Two such antag-
onists are from the Garcinia species. At a dose of 5 lM, both GA and cambogin
inhibited the release of TNF-a by LPS-activated macrophages [79, 80], suggesting
another mechanism for their antitumor activity.

15.3.17 GA Inhibits the Expression of Bcl-2 Family

Proteins

The bcl-2 gene family consists of at least 25 genes that are proapoptotic or
anti-apoptotic and share at least one of the four characteristic BH domains [81]. The
anti-apoptotic protein bcl-2, which displays sequence homology in all four domains
(i.e., BH1–BH4), promotes cell survival [82]. Increased expression of the bcl-2
protein commonly occurs in human malignancies and is associated with disease
maintenance and progression, resistance to chemotherapy, and poor clinical out-
come. Antisense oligonucleotides targeting bcl-2 have been shown to facilitate
apoptosis in various tumor types [83]. Therefore, bcl-2 inhibitors have great
potential in the treatment of cancer. In vitro and in vivo studies showed that GA
inhibits bcl-2 expression in MGC-803, HL-60, MCF-7, A375M, SMMC-7721,
BGC-823, Jeko-1, and K562 [15, 84–88].

15.3.18 GA Induces BID

Pro-apoptotic BID activates the multi-domain bcl-2 family members bcl-2–

associated X protein (BAX) and bcl-2 homologous antagonist killer (BAK) [89].
Activation of either BAX or BAK produces an allosteric conformational change and
releases cytochrome c [90]. This means that compounds that can induce BID could be
very useful in the treatment of cancer. GA and its derivative GA3 are such inducers
because these agents activate BID and induces apoptosis in cancer cells [91].
384 M.K. Pandey et al.

15.3.19 GA Induces BAD

BAD is proapoptotic and proliferative, suggesting that the cell cycle functions of the
multi-domain bcl-2 family members [89]. BAD antagonizes both the cell cycle and
anti-apoptotic functions of bcl-2 and bcl-xL through BH3 binding [89].
Overexpression of the BH3-only molecule BAD renders the cell unable to arrest in G0
and persistently activates cdk2 [92]. Previous study showed that GA in combination
with nanoparticle Fe3O4 activates BAD and induces apoptosis in LOVO cells [93].

15.3.20 GA Inhibits Cytochrome c

Cytochrome c, an intermediate in apoptosis, is released by the mitochondria in

response to proapoptotic stimuli. The studies have shown that GA, induces the
expression of cytochrome c in colorectal cancer HT-29, bladder cancer T24 and
UMUC3, breast cancer MDA-MB-231, and human hepatocellular carcinoma cells
[94–96].

15.3.21 GA Induces the Activation of Caspase-3

and Caspase-9

Caspases play a central role in mediating various apoptotic responses. In vitro and
in vivo research of GA has shown that it induces the activation of caspase-3 and
caspase-9 in various cancer cells including glioma, osteosarcoma, non-small lung
cancer, leukemia, lymphoma, breast cancer, pancreatic cancer, melanoma and
multiple myeloma and, induces apoptosis [7, 11, 17, 22, 24, 31, 66, 75–77].

15.4 Role of GA in Chronic Diseases

Extensive studies from past one decade have shed light on GA’s potential as
anti-inflammatory and anticancer agents. So far the focus of the studies have been to
identify the molecular targets by which GA exerts its effects, primarily on cancer
cells. However, a very recent study showed that GA could be used as an
anti-psoriatic agent [97]. Importantly, the molecular mechanism by which GA
mediates its effect strongly suggest that it could be used for the prevention and
treatment of many organ and tissue disorders, which are associated with inflam-
mation and oxidative stress. GA alleviates oxidative stress, inflammation in chronic
diseases and regulates inflammatory and pro-inflammatory pathways related with
most chronic diseases.
15 Gambogic Acid and Its Role in Chronic Diseases 385

15.4.1 Cardiovascular Diseases

Cardiovascular Diseases (CVDs), including heart disease, vascular disease and

atherosclerosis, are the most critical current global health threat. Epidemiological
and clinical trials have shown strong consistent relationships between the inflam-
mation markers and risk of cardiovascular diseases [98]. It is widely appreciated
that the key mechanisms in the development of CVDs are inflammation and oxidant
stress, activation of pro-inflammatory cytokines, chronic transmural inflammation,
and C reactive protein (CRP) [99]. Thus cytokines, other bioactive molecules, and
cells that are characteristic of inflammation are believed to be involved in athero-
genesis. An elegant recent study by Liu et al. [100] showed that GA inhibits
pressure overload or isoproterenol infusion-induced cardiac hypertrophy and
fibrosis, through the inhibition of the proteasome and the NF-jB pathway, sug-
gesting that GA treatment may provide a new strategy to treat cardiac hypertrophy
and changes in myocardial NF-jB signaling [100].

15.4.2 Rheumatoid Arthritis

Rheumatoid arthritis (RA) could give rise to a systemic chronic inflammatory

disorder and may impact many organs and tissues but mainly attack flexible
(synovial) joints [101]. It was reported that oxidative stress made an important
contribution to joint destruction in RA [102]. ROS is a significant mediator that
activates a variety of transcription factors including NF-jB and AP-1, thus regu-
lating the expression of over 500 different genes, such as growth factors,
chemokines, cell cycle regulatory molecules, inflammatory cytokines, and
anti-inflammatory molecules [103]. Therefore, transcription factors and genes,
involved in inflammation and antioxidation, are suspected to play a crucial adjective
function in RA. The main treatment of RA is to reduce arthritis reaction, inhibit
disease development and irreversible bone destruction, protect the joints and muscle
function, and ultimately achieve complete remission or low disease activity.
Treatment principles include patient education, early treatment, and combination
therapy [104, 105]. Drug therapy includes nonsteroidal anti-inflammatory drugs
(NSAIDs), slow-acting antirheumatic drugs, immunosuppressive agents, immune
and biological agents, and botanicals. NSAIDs are most common. Our earlier
studies strongly suggest that GA is one of the NSAIDs with anti-inflammatory and
antioxidant actions both in vivo and in vitro and could be used effectively as
anti-RA agent. Recent studies of Cascao et al. [106] support our hypothesis. By
using rat RA model, this group showed that GA inhibits RA by inhibiting the levels
of cytokines and key inflammatory molecules [106].
386 M.K. Pandey et al.

15.4.3 Diabetes and Obesity

Type 2 diabetes is a chronic disease where cells have reduced insulin signaling,
leading to hyperglycemia, and long-term complications, such as heart, kidney, and
liver disease. Recently, more and more studies have shown the critical roles of
oxidative stress and inflammatory reactions in the pathogenesis of diabetes. Studies
have shown that AMP-activated protein kinase (AMPK) plays a key role in
maintaining intracellular and whole-body energy homeostasis. Activation of AMPK
has been shown to ameliorate the symptoms of type 2 diabetes and obesity. In vitro
studies by Zhao et al. [107] demonstrate that GA, activates AMPK by increasing the
phosphorylation of AMPKa and its downstream substrate ACC in various cell lines
[107]. This group also showed that GA induced activation of AMPK was associated
with increased intracellular ROS level. Collectively, these results suggest that GA
may be a novel direct activator of AMPK and could be used as anti-diabetic agent.
However, further studies are required to fully evaluate this function of GA.

15.4.4 Psoriasis

Psoriasis is a chronic inflammatory skin disease characterized by thick, red, and

scaly lesions on any part of the body, which affects approximately 2 % of the
population worldwide [108]. Many cytokines, including interleukin-23(IL-23),
IL-17A, TNF-a, IL-6, IL-1b, and IL-22, are also involved in the pathogenesis of
psoriasis [109]. Along these lines a recent study showed that GA could be used as
an anti-psoriatic agent [97].

15.4.5 Cancer

Inflammation plays key roles in all the ways of tumorigenesis and therapy response
[110]. Activation and interaction between STAT3 and NF-jB are very vital in the
control of cancer cells and inflammatory cells [111]. TNF-a, VEGF, IL-10, MMP-2
and MMP-9, MCP, CD4+ T, AP-1, Akt, PPAR-c, MAP kinases, and mTORC1 are
also important linking factors between inflammation and cancer [111]. It has been
shown that GA suppresses the growth of various cancer cells such as non-small cell
lung cancer [112], human hepatocellular carcinoma [113], oral squamous cell
carcinoma [114], human breast cancer [86], human malignant melanoma [115],
human gastric carcinoma [116], and human leukemia cancer [7] and multiple
myeloma [31, 117]. A variety of mechanisms have been proposed by which GA
inhibits the proliferation of cancer cells and induces apoptosis. These include
inhibition of antiapoptotic proteins Bcl-2 [86, 88] and survivin [118]; induction of
apoptosis-associated proteins p53 [119], bax, and procaspase-3 [115]; activation of
15 Gambogic Acid and Its Role in Chronic Diseases 387

c-jun-NH2-kinase, p38 [20], and GSK-3b [15]; inhibition of topoisomerase II by

binding to its ATPase domain [120], downregulation of the MDM2 oncogene and
subsequent induction of p21 [119]; suppression of LPS induced COX-2 [10]; and
downregulation of human telomerase reverse transcriptase [121]. It has also been
shown that GA directly binds to c-myc [122], transferrin receptors [123], and
CXCR4 [124]. Recently, a proteomic approach revealed that GA suppresses
expression of 14-3-3 protein sigma and stathmin [116]. We have shown earlier that
GA inhibits NF-jB and its regulated gene products in human myeloid leukemia [7];
STAT3 and its regulated gene products in MM [31]. Most recently, we showed that
GA interacts with CXCR4 and inhibits chemotaxis and osteoclastogenesis in MM
[124]. Recently, it is shown that GA is a novel tissue specific proteasome inhibitor,
with potency comparable to bortezomib [25]. In addition, recent studies have shown
that GA is bioavailable, less toxic, effective, and inhibits development of tumors in
animal models, and most importantly it has been approved for phase 2 clinical trial
in solid tumors [4, 121, 125, 126]. Since, GA modulates the expression of proteins
plays important role in survival, migration, invasion and chemoresistance of mul-
tiple myeloma cells (Fig. 15.2), we have been working on the development of GA
as anti-myeloma agent.

MM cells
MCL-1
BCL-2
NF-κB BCL-xL
Gambogic IAPs
acid Cyclin D Survival
IL-6 Anti-apoptosis
MCL-1 Proliferation
JAK/STAT3 Inflammation
BCL-xL
Migration
Raf MEK/ERK Drug resistance
IL-6, VEGF,
TNF-α, PI3K
SDF-1α NF-κB
etc. mTOR
Akt Capspase9
VEGF, TGF

TFs Cytokines
Growth factors
BMSCs

Fig. 15.2 Intimate relationship between multiple myeloma and bone marrow microenvironment.
Bone marrow stromal cells secrete cytokine a growth factors, these growth factors activates several
pathways in multiple myeloma. Gambogic acid targets these pathways
388 M.K. Pandey et al.

15.5 Biological Activities of GA in Animal Models

Besides the extensive in vitro demonstrations of GA’s anti-proliferative effects,

numerous other studies have evaluated its efficacy in various animal models in vivo
(Table 15.2). The in vivo studies have investigated the effects of GA on tumor
angiogenesis and the biomarkers COX-2 and VEGF in prostate carcinoma [16].
One group demonstrated that systemic administration of GA for 3 weeks to athymic
mice bearing non-small lung NCI-H1993 xenografts significantly inhibited tumor
growth [127]. Meanwhile, others have shown that GA can suppress the growth of
cervical carcinoma [128], modulate the growth of colorectal cancer [129], modulate
the growth of prostate cancer in rodents [16], inhibit the growth of human B-cell
lymphoma by inducing proteasome inhibition [130] in nude mice, and inhibits
hepatocellular carcinoma, multiple myeloma, bladder cancer tumor growth, in part
by suppressing angiogenesis, and inducing apoptosis [7, 11, 17, 22, 24, 31, 66, 75–
77]. More recent studies have evaluated GA’s chemosensitizing effects [118]. Our
group evaluated the chemosensitizing effect of GA in combination with paclitaxel,
TNF-a, 5-FU on multiple myeloma [7]. Together, these in vivo animal studies
clearly suggest GA’s anticancer potential when administered either alone or in
combination with currently employed chemotherapeutic agents.

Table 15.2 A list of studies describing antitumor effects of gambogic acid in animals
Tumor Cell line Route Dose (mg/kg) Model References
Lung SPC-A1 i.v. 4, 8 Xenograft [121]
NCI-H1993 i.p. 10, 20 or 30 Xenograft [127]
NCI-H1975 i.v. 8 Xenograft [137]
A549 i.p. 8, 16 and 32 Xenograft [134]
Gastric cancer BGC-823 i.v. 8 Xenograft [85]
Cervical carcinoma HeLa cells i.p. 2 Xenograft [128]
Chronic myeloid KBM5 i.p. 3 Xenograft [24]
leukemia
Liver HepG2 i.v. 1.5 Xenograft [132]
Ovarian SKOV3 1.0 Xenograft [133]
Breast MDA-MB-231 i.v. 4 and 8 Orthotopic [94]
Hepatoma H22 i.v. 2, 4 and 8 Xenograft [125]
H22 p.o. 12.5, 25, 30 and Xenograft [125]
50
SMMC-7721 i.v. 2, 4, and 8 Xenograft [131, 136]
Melanoma B16-F10 i.v. 0.375, 0.75, and Orthotopic [135]
1.5
Colon HT-29 i.v. 5, 10 and 20 Xenograft [129]
15 Gambogic Acid and Its Role in Chronic Diseases 389

15.6 Biological Activities of GA in Humans

There are no clinical studies so far in USA, however in China GA is in Phase II

clinical trial [4, 121, 125, 126]. The plethora of studies clearly suggest GA’s
potential as anticancer agent. It the opportune time to seriously consider this xan-
thone in clinical trial especially in USA.

15.7 Conclusions

The spice derived from kokum, the fruit of Garcinia indica, is used in Indian
cuisines and Ayurvedic medicine. The main component isolated from kokum is
GA, which demonstrates thrust quencher, antioxidant, antimicrobial, antiulceration,
and anticancer properties. Although GA is a potent, biologically active compound,
only a number of studies are carried out in animals and none have been done in
humans. Because of its diverse range of biological activity in vitro, more in vivo
and clinical studies are warranted to establish its true usefulness as a clinical
therapeutic agent in a variety of human diseases.

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15 Gambogic Acid and Its Role in Chronic Diseases 395

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Chapter 16
Embelin and Its Role in Chronic Diseases

Hong Lu, Jun Wang, Youxue Wang, Liang Qiao and Yongning Zhou

Abstract Embelia ribes Burm of Myrsinaceae family has been widely used as an
herb in the traditional medicine of India. Embelin is an active component extracted
from the fruits of Embelia ribes. It has a wide spectrum of biological activities and
is not toxic at low dose. This review focuses on the physical–chemical properties
and bioactivities of Embelin, as well as its effects on chronic diseases such as
tumors, autoimmune inflammatory diseases, parasitic infections, microbial infec-
tions, diabetes, obesity, and cardio-cerebral vascular diseases. The underlying
mechanisms of the effects are also discussed. As a multiple-targeted therapeutic
agent, Embelin has the potential to be used widely for the treatment of a variety of
chronic diseases, including malignant tumors.

Keywords Embelin Anticancer activity Autoimmune inflammatory diseases





Anthelmintic activity Antimicrobial activity Diabetes Cardio-cerebral vascular
diseases

H. Lu
J. Wang
L. Qiao
Y. Zhou

Division of Gastroenterology and Hepatology, The First Hospital of Lanzhou University,
Lanzhou 730000, Gansu Province, China
e-mail: [email protected]
J. Wang
e-mail: [email protected]
Y. Zhou
e-mail: [email protected]
Y. Wang
Department of Physiology, The University of Texas Southwestern Medical Center, Dallas,
TX 75390, USA
e-mail: [email protected]
L. Qiao (&)
Storr Liver Centre, The Westmead Institute for Medical Research, The University of Sydney
at Westmead Hospital, Westmead, NSW 2145, Australia
e-mail: [email protected]

© Springer International Publishing Switzerland 2016 397

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1_16
398 H. Lu et al.

16.1 Introduction

Embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone) also known as embelic acid

or emberine, is a major active constituent of the fruits of Embelia ribes Burm, which
belongs to the Myrsinaceae family. It is also found in other plants, such as
Lysimachia punctata (Primulaceae) [104, 105], and erythrorhiza (Oxalidaceae)
[34].
Embelia ribes Burm, also known as false black pepper in English, Vidanda in
Sanskrit, and Babrang in Hindi, is widely distributed in India, Sri Lanka, Malaysia,
Singapore, and China [45, 124]. It contains benzoquinone derivatives of Embelin
and its dimeric form is named Vilangin [90]. It also contains cyclitol quercitol,
alkaloid christembine, tannins, as well as fatty ingredients including resinoid, fixed
oil, and minute quantities of volatile oils [64, 106, 107]. Embelin is a major
component from all the parts of Embelia Ribes, including fruits, pericarp, root bark,
stem bark, seeds, and leaves [35, 115] with varied quantity ranging from 1.01 to
4.31 % of the weight [105]. The content of Embelin in pericarp, seeds, entirely
crushed fruits, and decorticated seeds along with pericarp was found to be 0.39,
4.18, 3.30, and 3.34 % w/w respectively [35].
Embelia ribes has been widely used as an herb in traditional medicine in India
since time immemorial. The whole plant is used as an anti-inflammatory and
antinociceptive preparation to treat rheumatism, fever, and some skin diseases [89].
The fruits have digestive, carminative, anthelmintic, contraceptive and laxative
effects, and are used to treat tumors, ascites, bronchitis, mental diseases, dyspnoea,
heart diseases, urinary discharges, jaundice, hemicrania, and worms in wounds. The
leaves are astringent, demulcent, and depurative, and are used to treat in pruritus,
sore throat, aphthae ulcers of mouth, indolent ulcers, skin diseases, and leprosy.
Decoction of the roots is used in the treatment of influenza [45, 139].
The active component from Embelia ribes was first isolated more than 4 decades
ago (see structure in Fig. 16.1). It was named Embelin, and was later chemically
synthesized. Embelin has a wide range of medicinal property, including ant fertile
[67], antitumoral [26, 29], anti-inflammatory [60, 137, 138, 144], analgesic [20],
antioxidant [129, 149], hepatoprotective [132], wound healing, antibacterial [32],
and anticonvulsant activities [77, 78].

H OH

HO CH 2 - C 9 H 18 - CH 3

Fig. 16.1 Constitutional formula of Embelin (Molecular formula C17H26O4; Molecular weight
294.39; Melting point 142–143 °C; Appearance Orange solid, Log p (octanol-water) 4.34;
Solubility Insoluble in water, but soluble in organic solvents such as DMSO and Ether) [89]
16 Embelin and Its Role in Chronic Diseases 399

16.2 Physical–Chemical Properties of Embelin

Embelin is a hydroxyl benzoquinone with alkyl substitution and a derivative of

para-benzoquinone. Therefore, it has the basic physical–chemical characteristic of
para-benzoquinone. It shows orange color, and can be used to dye silk and wool
[120]. As a nonpolar molecule, Embelin suffers from erratic bioavailability due to
poor aqueous solubility [96, 98, 128], which limits its clinical application.
Meanwhile, its long alkyl chain (undecyl) provides it with strong lipophilic ability
[69] and high cell membrane permeability [57], which make it more stable and
allow it enter the cells easily.
Sublimation and volatility are other major characteristics of Embelin. It does not
decompose under normal pressure after being heated, and can be distilled with
water vapor, which makes it easy to be extracted and purified.
Embelin is also a phenolic lipid, and is photosensitive. In methanol, it shows a
peak absorbance (k max) at 450 nm [59]. When irradiated with light of the
appropriate wavelength, Embelin can be activated and produces anticancer and
anti-inflammatory effects through antioxidant activity [58, 59].

16.3 Biological Activities of Embelin

Embelin is a phenolic lipid that can be found as a secondary metabolite not only in
plants, but also in fungi, bacteria, and animals both during normal development and
in response to stress conditions such as infection, wounding, and UV radiation
[115]. It is structurally similar to natural coenzyme Q10 (ubiquinones), and it has
antioxidant activity similar to that of coenzyme Q10.
Embelin is also a weak mitochondrial uncoupler that dissociates the electron
transport system from ADP phosphorylation and inhibits ATP synthesis [79]. The
uncoupling effect also leads to increased oxygen consumption. It may explain why
Embelin caused swelling of the mitochondria in rat liver and respiratory inhibition
in germinated cowpea. It can also explain why Embelin prevented angiogenesis
during tumor growth and wound healing by exhausting the low respiratory reserve
of proliferating endothelial cells through uncoupling action [22].
Although Embelin possesses several promising biological activities, preclinical
efforts have been hampered because of water insolubility which leads to poor
bioavailability by oral route. In order to resolve the solubility issue and improve
biological activity, new techniques, such as utilization of the liquisolid compact
systems [95], formulation of Embelin–phospholipid complex (EPC) [98], con-
struction of Embelin-loaded thermosensitive injectable hydrogel system [99, 100],
and synthesis of Embelin analogues [18, 28, 69, 128] have been tried. Polymeric
micelle technique has also been introduced to enhancing the water solubility and
bioavailability [27].
400 H. Lu et al.

Some progresses have been made to improve the solubility and delivery of
Embelin. A micelle system was developed through the conjugation of Embelin and
polyethylene glycol (PEG) through an aspartic acid bridge. The resulting
PEG-Embelin micelles is dual functional and can efficiently deliver both Embelin
itself and other hydrophobic antitumor drugs, such as Paclitaxel, which are
encapsulated in the micelles. This system has been tested with success in cancer cell
line in vitro and animal cancer models in vivo [51, 76]. Diammonium salt of
Embelin exists in some plants and can easily be made from purified Embelin. It was
found to be highly soluble in water and its biological activities were found to be
similar to those of Embelin [28]. It could be an ideal therapeutic agent which can be
administered easily.
Toxicity studies revealed that Embelin is safe to use. It did not show toxic effect
on human fibroblasts at 20 µg/ml for 72 h in vitro [104]. In the acute toxicity study,
no prominent signs of toxicity or death were observed in mice administered orally
with crude hydroalcoholic extract of Embelia ribes at the dose up to 5000 mg/kg
for 24 h [28]. In chronic toxicity study, the administration of Embelin at the dose of
50 mg/kg/day for 14 weeks to Wistar rats did not cause any drastic drop in the
blood counts [132]. In another study, administration of Embelin at 10 mg/kg/day
for 10 weeks in rats did not show toxic side effects on bone marrow, liver, kidney,
and heart [111]. Embelin at doses of 50 mg and 100 mg/kg of body weight/day for
14 days did not cause significant body weight changes, mortality, or apparent signs
of toxic effects in mice [115]. Previous studies had also reported the nontoxic nature
of Embelin on hematopoietic cells when administered for 6 months in mice, rats,
and monkeys [55].
The specifics and details of some biomedicinal activities of Embelin will also be
discussed in the following sections.

16.4 Role of Embelin in Chronic Diseases

16.4.1 Anticancer Activity of Embelin

Anticancer effect of Embelin has been studied in various kinds of cancer, including
breast cancer [29, 133], prostate cancer [94, 106, 107], hepatic cancer [131, 132],
pancreatic cancer [86, 99, 100], colon cancer [24, 25], gastric cancer [141], leu-
kemia [49, 50], and multiple myeloma [47], through in vivo and in vitro experi-
ments. It can effectively inhibit tumor cell proliferation and migration, induce tumor
cell apoptosis, and inhibit tumor invasion, metastasis and angiogenesis.
The molecular mechanisms of the anticancer effect of Embelin have also been
studied extensively. Multiple signaling pathways have been found to be involved.
Embelin was predicted to be a strong inhibitor of XIAP through structure-based
computational screening of a traditional herbal medicine three-dimensional struc-
ture database [92]. It also downregulates the expression of XIAP [27, 50, 86, 141].
16 Embelin and Its Role in Chronic Diseases 401

XIAP belongs to inhibitor of apoptosis (IAP) family [119]. It binds to and inhibits
the initiator caspase-9 and effector caspase-3, caspase-7, and consequently prevents
intrinsic apoptotic pathways [114, 126]. XIAP is overexpressed in various types of
cancer cells [136]. Embelin could induce apoptosis of tumor cell through down-
regulating expression of XIAP and activating caspase cascades [27, 50, 141]. It
could also transform TRAIL-resistant pancreatic cancer cells into TRAIL-sensitive
cells by suppressing the expression of XIAP in vitro [86].
Nuclear factor-jB (NF-jB) plays an important role in tumor cell survival and
proliferation, as well as tumor angiogenesis, invasion, and inflammation [3, 8, 101].
Embelin blocks NF-jB signaling pathway resulting in the suppression of NF-jB
regulated antiapoptotic and metastatic gene products. Embelin inhibits the
expression of ICAM-1, MMP-9, COX-2, cyclin D1, c-Myc, and VEGF [25, 122,
141], all of which are regulated by NF-jB and closely related to proliferation,
invasion, and angiogenesis of tumors. It also inhibits NF-jB-dependent apoptosis
gene products including survivin, XIAP, IAP-1/2, TRAF1, cFLIP and Bcl-2, and
downregulates Bcl-xL [65, 94].
Researches showed Embelin suppressed activation of NF-jB not through
modifying NF-jB proteins itself but through inhibiting IKK (IjB kinase) activa-
tion, IjBa (the inhibitor of NF-jB)-phosphorylation and degradation, and p65
phosphorylation and acetylation, which then inhibited nuclear translocation of
NF-jB and expression of the NF-jB-dependent reporter genes [3, 112, 145]. IKK
could phosphorylate and degrade IjBa [38]. When IjBa was degraded, NF-jB
translocated to the nucleus and activated the transcription of specific genes [41].
These results suggested that Embelin induces apoptosis not only by inhibiting and
downregulating XIAP, but also through suppressing NF-jB signaling. XIAP, as a
downstream target gene, could be activated by NF-jB [63].
Embelin also regulates activity of the PI3 K/AKT pathway by reducing AKT
expression levels, and increases PTEN levels in cancer cell lines in vitro. The
PI3 K/Akt pathway contributes to tumor formation through the anti-apoptotic
activity of Akt. Akt inhibits apoptosis through phosphorylation of GSK3b, which
leads to inactivation of itself [56] and maintains the stabilization of b-catenin [71].
Embelin could suppress b-catenin expression through inhibition of activation of
AKT and GSK-3b, and thus promote apoptosis [94]. Suppression of Akt activation
could also lead to P53 activation and turn on pro-apoptotic signaling pathways for
induction of apoptosis [42]. At the same time, AKT could induce activation of
transcriptional factors, such as NF-jB [72]. Further research indicated that AKT
could activate IKK, and then cause NF-jB activation and cell survival [61]. The
upregulated PTEN can suppress activity of AKT [17].
Embelin upregulates the expression of P53 both in vivo and in vitro [99, 100,
133, 141]. P53 protein, as a key regulator of apoptosis, controls cell cycle pro-
gression and DNA repair, and induces apoptosis by regulating the expression of
proteins like Bcl-2 and Bax, which concomitantly induce Cytochrome C release and
activate Caspase cascades (primarily Caspase 9 and Caspase 3) leading to apoptosis
[130].
402 H. Lu et al.

Embelin inhibits JAK/STAT3 signaling pathways in mouse pancreatic cancer

cells through the downregulation of non-receptor protein tyrosine kinases janus-like
kinase 2(JAK2) and c-Src kinase [47], which then decrease the phosphorylation of
STAT3 in tumor cells [24, 99, 100]. The phosphorylation of STAT3 is mainly
dependent on the activation of JAK although the role of c-Src kinase in STAT3
phosphorylation has been demonstrated too [123]. STAT3 has been shown to
regulate the expression of tumor-associated genes, such as apoptosis inhibitors
(Bcl-xl, Bcl-2, and survivin), cell cycle regulators (cyclin D1), and angiogenesis
inducers (VEGF) [2, 47].
PPARc is a transcriptional factor that belongs to a superfamily of nuclear hor-
mone receptors. It is richly expressed in the normal gastrointestinal epithelium, and
plays a vital role in the regulation of differentiation of gastrointestinal epithelial
cells [150]. It is also abundantly expressed in colon cancer cells, and activation of
PPARc inhibits proliferation and induces apoptosis in some colon cancer cell lines
[25, 73, 127, 148]. Heterozygous loss of PPARc increased the susceptibility to
carcinogen-induced colon cancer and gastric cancer [40]. Embelin effectively
suppressed 1,2-Dimethylhydrazine dihydrochloride (DMH)-induced colon car-
cinogenesis in vivo when PPARc was present, and this effect could be due to
inhibition for NF-jB activity, which is dependent on PPARc [25].
Akt/mTOR/S6K1 signal pathway is closely related to tumorigenesis, metastasis,
and angiogenesis in vitro [9, 82]. Once Akt is stimulated, it can be propagated to
mammalian target of rapamycin (mTOR), a key modulator of protein translation
[75]. S6K1 is a downstream component of mTOR signaling pathway and a key
mTOR effector for tumor cell growth and proliferation [31]. The upregulated
expression of S6K1 gene indicates a poor prognosis in patients with cancer [84].
Embelin was found to inhibit the tumor growth and induce tumor cell apoptosis
through the suppression of Akt/mTOR/S6K1 signaling cascades in human prostate
cancer cells [65].
In rat model of N-nitrosodimethylamine (DENA)-initiated and phenobarbital
(PB)-promoted hepatocarcinogenesis, Embelin (50 mg/kg/day) blocked the induc-
tion of hepatic hyperplastic nodules, prevented body weight loss, and improved the
markers of hepatic function (ALT, AST, ALP, TBIL) and hypoproteinemia [131],
suggesting its preventive effect on tumor formation. In this model of hepatocar-
cinogenesis, a significant decrease in the hepatic glutathione antioxidant defense, an
increase in lipid peroxidation and histological alterations such as dysplasia and
atypical cells with abnormal chromatin pattern were observed. Embelin was able to
improve or even reverse these biochemical and pathological changes possibly
through free radical scavenging and antioxidant activity [132]. Embelin’s radical
scavenging activity has been found to be better than that of a-tocopherol, a
well-known scavenger of peroxyl radical [57].
The immune system plays a crucial role in tumor microenvironment, and
numerous cytokines and growth factors produced by inflammatory cells or immune
cells can affect the regulation of genes that mediate proliferation, prevent apoptosis,
and thereby promote carcinogenesis [81]. NF-kB plays a central role in mediating
the link between inflammation and cancer development, and exerts a
16 Embelin and Its Role in Chronic Diseases 403

procarcinogenic effect principally on immune (myeloid) cells and epithelial cells

[62]. Activated NF-jB regulates the expression of IL-6, TNF-a, IFN-c, IL-1b, IL-8,
and some other cytokines [54, 113].
The level of IL-6 in tumors is directly correlated with increased necrosis, pro-
liferation, and vascular invasion, and the level of circulating IL-6 is highly corre-
lated with poor survival. Moreover, exogenous and endogenous IL-6 can effectively
activate JAK/STAT pathway [24, 74]. IL-6 associates with inflammatory cells and
immune suppressive cells including Th1, Th2, Th17, regulatory T cell (Treg), and
myeloid-derived suppressor cells (MDSCs) [93, 99, 100]. Embelin suppresses
immune suppressive IL-17A+ cells, GM-CSF+ cells, MDSCs and Treg by inhibiting
IL-6 secretion [99, 100]. It decreases the levels of IL-1b, IL-17a, and IL-23a, and
inhibits the infiltration of CD4+ T cells and macrophages in colonic tissues [24].
IL-17-producing effector T helper (Th17) cells are crucial for inflammation and
may have a potential role in carcinogenesis [142, 147]. The roles of IL-6 and IL-23
in the maturation of Th17 cells have been identified [10].
TNF-a can stimulate accumulation of inflammatory cells via induction of
expression of adhesion molecules such as ICAM-1 in neighboring endothelial cells,
and give rise to production of secondary cytokines [60], resulting in expansion of
inflammatory reactions. TNF-a can also activate NF-jB by degrading IjBa [3]. So,
the relationship between TNF-a and tumor is beyond doubt. TNF-a is synthetized
as a membrane anchored protein (pro-TNF-a), and the soluble component of
pro-TNF-a is then released into the extracellular space by the action of a protease
called converting enzyme (TACE) [91]. Embelin could decrease the level of TNF-a
through blocking the expression of TACE in human breast cancer cells [29].
Embelin could inhibit osteoclastogenesis, which is related to bone loss induced
by RANKL and tumor cells, such as multiple myeloma and breast cancer cells
in vitro, through the suppression of NF-jB activation [112]. Thereby, Embelin
could be used for patients with secondary bone lesions associated with various
cancers.
In summary, Embelin has antitumor effect on a variety of cancers, and it exe-
cutes its antitumor effect through multiple pathways (Fig. 16.2). The use of
multi-target drugs has been increasingly accepted because of clearer realization that
cancer is caused by dysregulation of multiple pathways [83]. Sorafenib and
Sunitinib are examples of multi-target drugs that modulate several tyrosine
kinases/signal transduction pathways. Embelin has a great potential for the treat-
ment and prevention of cancer.

16.4.2 Embelin and Autoimmune Inflammatory Diseases

Rheumatoid arthritis, inflammatory bowel disease (IBD), psoriasis, multiple scle-

rosis (MS), and autoimmune encephalomyelitis (AE) are all included in the cate-
gory of autoimmune inflammatory diseases, and possess common clinical
characteristics, namely unknown etiology, immune correlation, and the lack of
404 H. Lu et al.

Fig. 16.2 Schematic diagrams showing antitumor mechanism of Embelin. EB Embelin, XIAP
X-chromosome linked inhibitor of apoptosis protein, NF-jB Nuclear factor-jB, IjBa the inhibitor
of NF-jB, IKK IjB kinase, AKT protein kinase B, GSK3b glycogen synthase kinase-3b, JAK
janus-like kinase, PPARc peroxisome proliferator-activated receptor gamma, mTOR mammalian
target of rapamycin, S6K1 ribosomal S6 kinase 1, STAT3 signal transducer and activator of
transcription 3

effective therapeutic agents with few side effects. Embelia ribes have been shown to
relieve the symptoms of fever and skin diseases for many years. The effects of
Embelin on ulcerative colitis, psoriasis, autoimmune encephalomyelitis, rheumatoid
arthritis and the underlying mechanisms have also been studied.
Embelin was found to be effective in treating acetic acid-induced ulcerative
colitis in rats. It relieved symptoms such as body weight loss, diarrhea, and gross
intestinal bleeding. It decreased wet weight of the colon tissue [7] and alleviated
colonic shortening and splenomegaly [68], indicating the improvement of the
inflammation of colon. It decreased the colonic myeloperoxidase (MPO) activity
and lipid peroxides (LPO), as well as serum lactate dehydrogenase (LDH). It also
16 Embelin and Its Role in Chronic Diseases 405

increased the reduced glutathione (GSH) in this rat ulcerative colitis model [7, 137,
138]. Wet weight of the colon tissue is considered as a reliable indicator of the
extent of the inflammation in colitis. MPO exists abundantly in the neutrophils, and
the levels of colonic MPO reflect the infiltration of activated neutrophils in colon
which in turn reflect the severity of the colitis [21]. LPO is the product of membrane
lipid peroxidation, and it can cause further lipid peroxidation and generate more
reactive metabolites through self propagation chain reaction. LPO exhausts cellular
antioxidants and accelerates the development of inflammation and ulceration. GSH
can block free radical damage, enhance the antioxidant activity, facilitate the
transport of amino acids, and play a critical role in detoxification [44]. The ele-
vation of LDH in serum indicates enhanced production of lactic acid [80] and
increased release of LDH by damaged cells.
Pro-inflammatory cytokines (TNF-a, IFN-c, IL-1b, IL-6 and IL-12), and
anti-inflammatory cytokines (IL-4, IL-10, IL-11) play a central role during the
occurrence and development of IBD [46, 121]. TNF-a can disrupt the epithelial
barrier, induce the apoptosis of epithelial cells, and stimulate the secretions of
chemokines from intestinal epithelial cells. It can also activate immune system of
the bowel through recruiting and activating neutrophils and macrophages [15].
IL-1b can increase the infiltration of inflammatory cells into the intestine. Embelin
was found to ameliorated colitis by suppressing the level of TNF-a, IL-1b, and IL-6
in the colonic tissues of mice with dextran sulfate sodium (DSS)-induced colitis
[68].
Epidermal keratinocytes are known to participate in immune and inflammatory
reactions by producing a variety of cytokines including TNF-a, which plays a
crucial role in autoimmune diseases of skin, such as psoriasis. TNF-a released from
keratinocytes promotes the accumulation of inflammatory cells. Embelin could
inhibit topical edema, and decrease skin thickness, tissue weight, and MPO activity
in phorbol ester (TPA)-induced inflammation in mouse ear, which serves as a
model of psoriasis. The possible mechanism might be the blockage of the pro-
duction of pro-inflammatory cytokines (TNF-a, IL-1b) by keratinocytes [60].
Experimental Autoimmune Encephalomyelitis (EAE) is an autoimmune disorder
of the CNS that serves as an animal model for human MS [66]. Dendritic cells
(DCs) play a definitive role in the induction and maintenance of self-tolerance, and
the absence of these cells can lead to autoimmune inflammatory diseases, such as
MS. Transforming growth factor-beta (TGF-b) signaling in DCs was found to be a
prerequisite for the control of autoimmune encephalomyelitis [146], and it could
program DCs into a tolerogenic state [16], and thereby limit the inflammatory
response and promote the recovery from EAE [52]. Embelin was shown to sig-
nificantly increase TGF-b/b-catenin signaling, decrease STAT3 phosphorylation in
DCs, maintain DCs in their tolerogenic state, and block the expression of
pro-inflammatory factors (IFN-c, IL-12, IL-6, and IL-23), which lead to the
improvement of the EAE clinical score and alleviation of CNS inflammation and
demyelination [144].
The effect of Embelin on rheumatoid arthritis was also affirmed. Embelin was
found to reduce inflammation and bone erosion in a mouse model of inflammatory
406 H. Lu et al.

arthritis possibly through the induction of apoptosis [30] and suppression of

osteoclastogenesis [112].
To summarize, Embelin has anti-inflammatory and immunomodulatory effects,
and can be potential treatment for autoimmune inflammatory diseases.

16.4.3 Anthelmintic Activity of Embelin

Embelia ribes Burm has been used as an anthelminthic remedy in India for a long
time. The fruits of Embelia ribes are widely used to expel the adult stage beef
tapeworm and other intestinal parasites [23, 39]. The ethanolic extract from the
seeds of Embelia ribes (10–200 lg/mL) exhibited potent anthelmintic activity
against roundworm Rhabditis pseudoelongata in vitro [43]. Crude hydroalcoholic
extract from the fruits showed significant anthelmintic activities against the dwarf
tapeworm (Hymenolepis nana) in vivo and against hookworm larva and Necator
americanus in vitro [28]. Diammonium salt of Embelin showed anthelmintic
activity against intestinal cestodes (Hymenolepis diminuta and Hymenolepis
microstoma), trematode (Echinostoma caproni), nematode (Heligmosomoides
polygyrus), and hookworm larva in vitro. The in vivo anthelmintic activity of
diammonium salt of Embelin against Hymenolepis diminuta and Hymenolepis nana
was also confirmed. However, the anthelmintic activity against Hymenolepis
microstoma, Echinostoma caproni, or Heligmosomoides polygyrus was not con-
firmed [6, 28]. In addition, Embelin was moderately active against Leishmania
amazonensis and Leishmania braziliensis lysing 70 % of the parasites at
100 µg/ml, and potently active against Trypanosoma cruzi trypomastigotes with
100 % lysis at 100 µg/ml in vitro [34].

16.4.4 Antimicrobial Activity of Embelin

The crude extracts from Embelia ribes have displayed moderate antibacterial
activity against Salmonella typhi, Staphylococcus aureus, Enterobacter aerogenes,
and Klebsiella pneumoniae [5, 109, 135].
Embelin was found to suppress Escherichia coli and methicillin-sensitive and
methicillin-resistant strains of Staphylococcus aureus with minimal inhibitory
concentration(MIC) values of 50, 250, and 62 lg/ml respectively [34]. At high
concentration (100 lg/disk), it significantly inhibited the growth of Staphylococcus
aureus, Streptococcus pyogenes, Shigella flexneri, Shigella sonnei, and
Pseudomonas aeruginosa; and was moderately effective against Salmonella typhi,
Shigella boydii, and Proteus mirabilis [19]. Further study indicated Embelin
showed bactericidal activity against Gram positive organisms, and bacteriostatic
activity against Gram negative organisms [108].
16 Embelin and Its Role in Chronic Diseases 407

In combination, Embelin and oxacillin showed synergistic antimicrobial effects

against 4 (ATCC 29213, ATCC 43300, EMRSA15, and TRSA2) out of 9
Staphylococcus aureus strains (fractional inhibitory concentration sum (RFIC)
indices 0.203–0.477). Embelin and tetracycline combination were bactericidal
against 3 (ATCC 43300, MRSA4, and TRSA2) out of 9 strains (RFIC 0.400–
0.496). Both combinations showed an additive effect in most of the strains with no
occurrence of antagonism. Addition of Embelin completely reversed the resistances
to Oxacillin and tetracycline in ATCC 43300, MRSA1, MdRSA2, and MdRSA3,
which are multi-drug-resistant strains [115].
Streptococcus mutans plays a significant role in dental infection by effectively
utilizing sugars and synthesizing large amounts of exopolysaccharides, which are
indispensable in adhesion of bacteria and accumulation of biofilm. The organized
structure and mechanisms of biofilm are responsible for the emergence of drug
resistant bacteria [70]. Embelin could block the biofilm formation of Streptococcus
mutans, and is expected to become novel drug candidates for treating oral infections
caused by S. mutans [32].
The crude extracts from Embelia ribes have displayed moderate antifungal
activity against Candida albican, Candida tropicalis, Candida parapsilosis, and
Aspergillus fumigatus [110, 134]. Embelin exhibited good inhibitory activity
against Candida tropicalis [110, 134], and Candida albican [87] with MIC50
values of 700 and 120 mg/L, respectively, by means of EUCAST (European
Committee for Antifungal Susceptibility Tests). The MIC50 values were below
700 mg/L for Candida albican, Candida tropicalis, Candida parapsilosis, Candida
albidus, and Aspergillus Flavus by NCCLS (The National Committee for Clinical
Laboratory Standards, USA) method [134]. Embelin also showed antifungal
activity against dermatophytic fungi Epidermophyton floccosum, Microsporum
canis, Microsporum gypseum, Trichophyton mentagrophytes, and Trichophyton
rubrum with MICs ranging from 50 to 100 µg/ml using agar dilution method [34].
The methanol extract of Embelia ribes was proven to be active on hepatitis C
virus (HCV) protease (PR) with  90 % inhibition at 100 µg/ml, and the active
component Embelin was found to be a potent HCV-PR inhibitors with an IC50
value of 21 µM [37, 102].
In summary, Embelia ribes and Embelin have antibacterial, antifungal, and
antiviral activities in vitro. Animal researches are needed to study the in vivo effect
of Embelin before clinical application can be considered.

16.4.5 Embelin and Its Antidiabetic Properties

Ethanolic extract of Embelia ribes improved hyperglycaemia, and decreased serum

insulin, lactate dehydrogenase, creatinine in Wistar rats with streptozotocin
(40 mg/kg, intravenously)-induced diabetes. It also decreased systolic blood pres-
sure and heart rate, as well as total cholesterol and triglycerides in these diabetic rats
[14].
408 H. Lu et al.

In rats with alloxan-induced diabetic, oral administration of Embelin (25 and

50 mg/kg) for 21 days significantly decreased fasting blood glucose, improved
body weights, and restored biochemical parameters such as TGL, TC, TB, CR,
LDH, ALP, VLDL, TP, and albumin. It also significantly improved and almost
normalized the histological changes of liver, kidney, and pancreas in these diabetic
rats. However, the antidiabetic activity of Embelin (50 mg/kg) was found to be
inferior to that of glibenclamide (10 mg/kg) [77, 78, 88].
The underlying mechanism of the antidiabetic effect of Embelia ribes and
Embelin might be the protection of b-cells against reactive oxygen
species-mediated damage by enhancing cellular antioxidant defense. It is indicated
the antioxidant defense system is weakened in STZ-induced diabetic rats [14, 88],
and the islet b-cells are susceptible to damage caused by oxygen free radicals.
Increases of lipid peroxides (LPO), superoxide dismutase (SOD) and catalase
(CAT) indicate an oxidative stress condition, and glutathione (GSH) is one of the
primary defenses that counteract the oxidative stress. Extract of Embelia ribes at the
dose of 100 mg/kg and 200 mg/kg increased SOD, CAT and GSH levels in pan-
creatic tissue of diabetic rats [11, 13, 14]. Embelin was found to decrease the
malondialdehyde (MDA), restore depleted GSH as well as antioxidant enzymes
SOD and CAT in liver and pancreatic tissues of STZ-induced diabetic rats [14, 36,
88], suggesting that antidiabetic activity of Embelin is at least partially due to the
improvement of the oxidation defense mechanism, which protects islet b-cell
against oxidative damage.
It is documented that cytokines TNF-a and IL-6 are associated with insulin
resistance [85]. TNF-a could induce serine phosphorylation of insulin receptor
substrate-1 (IRS-1), interfere with normal tyrosine phosphorylation and insulin
signaling, which causes insulin resistance [48]. The high level of IL-6 is also related
with decreased insulin sensitivity in skeletal muscles and liver, and IL-6 could
block IRS phosphorylation, which ultimately lead to decreased gluconeogenesis
and increased glycogenolysis [33]. Embelin decreased the levels of cytokines
TNF-a and IL-6 in STZ-induced diabetic rats [88], and therefore improved insulin
resistance and normalized the metabolism of glucose and lipid.
PPARc is the molecular target to treat T2DM, and its expression is downreg-
ulated in multiple tissues in insulin resistance and diabetes [103]. Embelin induced
PPARc expression in the epididymal adipose tissue of rats with HFD+STZ-induced
diabetes. It also caused plasma membrane translocation and activation of glucose
transporter protein 4 (GLUT4), and relative increases in the expressions of PI3K
and p-Akt, implying that Embelin increased insulin-mediated PI3K/p-Akt signaling
dependent glucose uptake in adipose tissue through the translocation and subse-
quent activation of GLUT4 [36]. GLUT4, along with GLUT1, plays an important
role in tissue glucose uptake and body glucose homeostasis regulation [143].
Embelin has also been used to treat obesity, which is a risk factor of T2DM. It
prevents body weight gain, visceral fat accumulation, and blood pressure increase.
It also decreased serum levels of leptin in the murine model of high fat diet (HFD)-
induced obesity [12].
16 Embelin and Its Role in Chronic Diseases 409

Embelin also exhibited nephroprotective and anti-polyuric effects in rats with

lithium-induced nephrogenic diabetes insipidus (NDI). It increased body weight,
and decreased plasma and urine creatinine, blood urea nitrogen levels, and urine
protein levels. Embelin, as a potent antioxidant, seemed to protect kidneys through
promoting antioxidant status as evidenced by the increased level of GSH in kidneys
[117].

16.4.6 Embelin and Cardio-Cerebral Vascular Diseases

Aqueous extract of Embelin Ribes exhibited cardioprotective effect, and it ame-

liorated myocardial injury through enhancing the antioxidant defense in isopro-
terenol (ISO)-induced myocardial infarction in rats [11, 13]. Recently, Embelin was
also found to have cardioprotective effect [118, 149]. It significantly decreased the
serum levels of cardiac enzymes such as CK-MB, LDH, and AST in rats with
ISO-induced myocardial infarction. It also decreased serum levels of lipids and
lipoproteins, and improved histopathological changes such as myocardial degen-
eration and necrosis. The underlying mechanism of this cardioprotective activity of
Embelin might be the recovery of myocardial mitochondrial respiratory enzyme
activities, the enhancement of antioxidant defense status, and the inhibition of
mitochondria dependent apoptotic damage [118].
It is known that inflammation is one of the most vital factors which results in
ventricular dysfunction following myocardial ischemia–reperfusion injury [53,
116]. Increased expression of inflammatory cytokines is highly correlated with the
deterioration of myocardial function [140]. It could trigger the systematic inflam-
matory response syndrome (SIRS), and lead to myocardial dysfunction and multiple
organ failure (MOF). In a rabbit model of myocardial ischemia–reperfusion injury
following cardiac arrest (CA), Embelin downregulated the expression of
pro-inflammatory cytokines TNF-a, IL-1b, and IL-6 through inhibition of NF-jB in
myocardial tissues. It blocked apoptosis and necrosis of myocardium, and improved
cardiac function and hemodynamics following resuscitation [149]. This observation
indicates that Embelin may protect the heart against myocardial ischemia–reper-
fusion injury via its anti-inflammatory abilities.
The fruits of Embelia ribes are traditionally used as a brain tonic to treat mental
disorders in India. The ethanolic extract of Embelia ribes fruits was also proven to
have potent neuroprotective effects [4]. The protective effect of Embelin on global
ischemia/reperfusion-induced brain injury was validated using an experimental rat
model. Pretreatment with Embelin (25 and 50 mg/kg) normalized locomotor
activity and hanging latency time and decreased beam walking latency. The
treatment also reduced lipid peroxidation, increased the total thiol content and
glutathione-S-transferase activity in brain, and decreased the area of infraction. The
antioxidant activity of Embelin might be the foundation of its neuroprotective effect
[137, 138].
410 H. Lu et al.

It has been proved that Embelin was effective in the treatment of seizure, epi-
lepsy, and anxiety in animals. It has anticonvulsant activity against both grand mal
and petit mal epilepsy. In animal experiment, Embelin inhibited seizures induced by
maximal electroshock and pentylenetetrazole in a dose-dependent manner. The
anticonvulsant activity of Embelin was comparable to that of phenytoin and dia-
zepam [77, 78]. Embelin also showed a dose-dependent anxiolytic effect in animal
experiment [1].

16.5 Conclusion

Embelin, as a bioactive ingredient of Embelia ribes Burm, possesses higher

medicinal value. It can suppress the growth, invasion, and metastasis of malignant
tumors; inhibit inflammatory; and ameliorate infections of bacteria, viruses, and
parasites. It is also antidiabetic, neuroprotective and cardioprotective. It has the
potential in the treatment of malignant tumor, autoimmune diseases, infections,
diabetes, and obesity, as well as cardio-cerebral vascular diseases. Further in vivo
animal studies and well-designed clinical trials are needed to validate the clinical
application.

Acknowledgments This work was supported by the grant “The Model for Prevention and
Control of Malignant Tumor in Area with High Incidence from Wuwei Gansu Province” (National
Science and Technology Program, Grant Code: 2012GS620101 to YN Zhou).

Conflict of interest The authors declare no conflict of interest.

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Chapter 17
Butein and Its Role in Chronic Diseases

Ziwei Song, Muthu K. Shanmugam, Hanry Yu and Gautam Sethi

Abstract Natural compounds isolated from various plant sources have been used
for therapeutic purpose for centuries. These compounds have been routinely used
for the management of various chronic ailments and have gained considerable
attention because of their significant efficacy and comparatively low side effects.
Butein, a chacolnoid compound that has been isolated from various medicinal
plants has exhibited a wide range of beneficial pharmacological effects, such as
anti-inflammatory, anticancer, antioxidant, and anti-angiogenic in diverse disease
models. This article briefly summarizes the past published literature related to the
therapeutic and protective effects of butein, as demonstrated in various models of
human chronic diseases. Further analysis of its important cellular targets, toxicity,
and pharmacokinetic profile may further significantly expand its therapeutic
application.

Keywords Butein
Anticancer
Inflammation
Angiogenesis
Antioxidant

Z. Song
H. Yu
Department of Physiology, Yong Loo Lin School of Medicine, MD9-04-11,
2 Medical Drive, Singapore 117597, Singapore
M.K. Shanmugam
G. Sethi (&)
Department of Pharmacology, Yong Loo Lin School of Medicine,
National University of Singapore, Singapore 117600, Singapore
e-mail: [email protected]
H. Yu
Institute of Biotechnology and Nanotechnology, A*STAR, The Nanos,
#04-01, 31 Biopolis Way, Singapore 138669, Singapore
H. Yu
Singapore-MIT Alliance for Research and Technology, 1 CREATE Way,
#10-01 CREATE Tower, Singapore 138602, Singapore
H. Yu
Mechanobiology Institute, National University of Singapore, T-Lab, #05-01,
5A Engineering Drive 1, Singapore 117411, Singapore

© Springer International Publishing Switzerland 2016 419

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1_17
420 Z. Song et al.

17.1 Introduction

Plant-derived natural compounds and their derivatives have been used as thera-
peutic agents over the centuries. However, their exact chemical structures, functions
as well as molecular targets are not completely known as of yet. Their high ther-
apeutic efficiency and low side effects have made themselves ideal alternatives of
synthetic drugs [4, 56]. Thus extensive qualitative and quantitative tests have been
done to evaluate and to extend their therapeutic potential as drug candidates.
Butein, a plant polyphenol (2′,3,4,4′-2′,4′,3,4- or 3,4,2′,4′-tetrahydroxychalcone),
with the chemical structure shown in Fig. 17.1, has been reported to exhibit several
important pharmacological effects, such as anti-inflammatory, anti-cancer,
anti-oxidant, and anti-angiogenic [5, 33, 37, 50, 58]. It was first found in
Toxicodendronvernicifluum (formerly called RhusVerniciflua Stokes, RVS), a tree
widely used as a local food additive and therapeutic supplement in South East Asia
[62]. It can also be isolated from the heartwood of Dalbergiaodorifera, the seed of
Cyclopiasubternata and the stems of Semecarpusanacardium, as well as many
other plants [41]. The various plant sources of butein are shown in Table 17.1 [60].
The first dietary butein supplement Inh-AR, launched by Megabol, is a sport
pro-hormone supplement to control estrogen production by inhibiting aromatase
[1]. Its activities in aromatase inhibition and estrogen regulation have also made
butein a promising candidate for breast cancer treatment. Besides cancer [2, 30, 73],
it also has exhibited promising effects in the treatment of many other chronic
diseases, including inflammation [63], glaucoma [14], cardiovascular diseases [9],
and metabolic complications [48]. Although molecular mechanisms underlying its
various reported pharmacological effects remain to be elucidated, butein has been
found to modulate several important cellular signaling pathways involved in eti-
ology of chronic diseases. Meanwhile, it exhibits low toxicity against normal cells,
thereby indicating its potential both as a chemopreventive and chemotherapeutic
agent [28, 71]. In this chapter, we have briefly summarized various published
reports related to the pharmacological properties and biological activities of butein,
and emphasized on its therapeutic effects against various chronic diseases.

Fig. 17.1 The chemical

structure of butein
17 Butein and Its Role in Chronic Diseases 421

Table 17.1 Plant sources of butein [60]

Family Plant Part
Adoxaceae Viburnum propinquum Hemsl. Leaves
Anacardiaceae Cotinuscoggygria Scop. Heartwood
Searsiaverniciflua Stokes Syn. Stem bark
Toxicodendronvernicifluum (Stokes) FA Barkley Stem bark
Semecarpusanacardium L. Stem bark
Asparagaceae Sansevierialiberica Ger. and Labr. Rhizomes
Asteraceae Bidensbipinnata L. Flowers
Bidenspilosa L. Whole plant
Bidenstripartita L. Whole plant
Coreopsis douglasii (DC.) HM Hall Flowers
Coreopsis gigantean (Kellogg) HM Hall Flowers
Coreopsis maritime (Nutt.) Hook. f. Flowers
Coreopsis petrophiloides BL Rob. & Greenm. Flowers
Coreopsis lanceolata L. Flowers
Cosmos sulfurous Cav. Flowers
Dahlia variabilisDesf. Flowers
Dahlia coccinea Cav. Petals
VernoniaanthelminticaWilld Seeds
Fabaceae Acacia pycnathaBenth. Heartwood
Adenantherapavonina L. Wood
Bauhinia purpurea L. Seeds
Butea frondosaRoxb. Flowers
Butea monosperma (Lam.) Taub. Flowers
Caragana intermedia Kuang & HC Fu Syn. Whole plant
CaraganakorshinskiiKom.
Cyclopiasubternata Vogel Syn. Seeds
Cyclopiafalcata (Harv.) Kies
Dalbergiaodorifera TC Chen Heartwood
DipteryxlacuniferaDucke Fruits
Millettianitida var. hirsutissima Z. Wei Stems
Millettiaspeciosa Champ. Roots
Sophoraalopecuroides L. Whole plant
Viciafaba L. Fruits
Pinaceae AbiespindrowRoyle ex D. Don Stems
Rubiaceae Hydnophytumformicarum Jack. Tubers
Schisandraceae Schisandrapropinqua (Wall.) Baill Whole plant
Solanaceae Solanum lycopersicum Lam. Fruits
422 Z. Song et al.

17.2 Physiochemical Properties of Butein

As a flavonoid compound, butein exhibits the various common structure features

and biological activities shared by the flavonoid family. Flavonoids are known to
provide health benefits, such as disease prevention, antioxidant activities, and
chemoprevention [63, 66]. These compounds can be classified into different groups
based upon their specific structural features, and each group has different biological
activities and efficiency. Butein belongs to the chaloconoid subgroup of flavonoid
family, with two aromatic carbon rings linked together by the a,b-unsaturated
carbonyl group and a double bond [7]. Chemical and physical properties of butein
are summarized below in Table 17.2.
There are several studies on the structural-activity relationship of butein, providing
new insights into the specific chemical structural features that exhibit diverse biological
activities. Studies show that the position of hydroxyl groups on aromatic carbon rings is
crucial for the bioactivities of chalcone compounds [53], and that the double bond at
C2–C3 as well as the 4-oxo functional group contributes to the inflammatory activities
of flavonoids [22]. In addition, a study to compare butein and luteolin (two flavonoid
compounds without and with C ring structures, respectively) proves that, butein is more
effective in the induction of heme oxygencase-1 (HO-1) and the suppression of NF-jB
activation, thus suppressing LPS-induced inflammatory responses in the macrophage
RAW264.7 cells. This report clearly indicates that although both flavonoids exhibit
anti-inflammatory activities, but the C ring structure of butein contributes to its sig-
nificantly higher efficacy [63].

Table 17.2 Chemical and Molecular formula C15H12O5

physical properties of butein
Molecular weight 272.25278 g/mol
Exact mass 272.068473 g/mol
Monoisotopic mass 272.068473 g/mol
XLogP3 2.8
Hydrogen bond donor count 4
Hydrogen bond acceptor count 5
Rotatable bond count 3
Topological polar surface area 98 A^2
Heavy atom count 20
Formal charge 0
Complexity 367
Isotope atom count 0
Defined atom stereocenter count 0
Undefined atom stereocenter count 0
Defined bond stereocenter count 1
Undefined bond stereocenter count 0
Covalently bonded unit count 1
Source: [51]
17 Butein and Its Role in Chronic Diseases 423

17.3 Role of Butein Against Chronic Diseases

As a multi-targeted compound with diverse biological effects, butein has been reported
to target several important cell signaling pathways. Although the underlying mecha-
nisms and molecular targets remain to be elucidated, published results indicate that
butein exhibit therapeutic effects on several chronic diseases including inflammation,
cancer, cardiovascular diseases, and metabolic complications. This section briefly
summarizes its reported therapeutic effects against various chronic ailments.

17.3.1 Inflammatory Diseases

Inflammation is body’s primary protective response against harmful foreign stimuli.

The primary purpose of inflammation is beneficial: to clear out damaged tissues and
to initiate the healing process. However, aberrant inflammatory response can lead to
some severe complications such as cancers [41], cardiovascular diseases and
rheumatoid arthritis [10, 40]. It is known that the activation of nuclear factor-jB
(NF-jB) regulates the production of many pro-inflammatory mediators. NF-jB can
be activated by extracellular stimuli and later translocate from cytoplasm to nucleus
[63]. As stated in the previous section, butein suppresses lipopolysaccharide (LPS)-
induced inflammatory responses in the macrophage RAW264.7 cells by
dose-dependently inhibiting various enzymes and pro-inflammatory mediators,
including nitrite [26], tumor necrosis factor-a (TNF-a) [26, 37], COX-1 [59],
COX-2 [37], inducible nitric oxide synthase (iNOS) [63], PGE-2 [26], and nitric
oxide (NO) [26, 43] production. It is hypothesized that these anti-inflammatory
effects are mediated by suppression of deregulated activation of NF-jB and JNK1/2
signaling pathways [26]. It also induces the expression of HO-1, an enzyme that
inhibits NF-jB activation in macrophages [63].
Besides suppressing NF-jB in macrophage RAW264.7 cells, butein also inhibits
the activation of NF-jB in human mast cells leading to the decreased production of
TNF-a, IL-6, and IL-8 [58], and in adipocytes by blocking IKKb, a NF-jB
upstream kinase [68]. And it also abrogates ß-glucuronidase and lysozyme in rat
neutrophils [5]. Moreover, it also exhibits anti-inflammatory effects on lung
epithelial A549 cells by inhibiting TNF-a-induced ROS generation, NF-jB acti-
vation, MAPK phosphorylation and Akt phosphorylation, and by decreasing
TNF-a-induced monocyte cell adhesion to lung epithelial cells [58]. In addition, it
also reduces IL-8 secretion and MMP-7 expression in intestinal epithelial HT-29
cells, and suppresses the E-selectin expression in human umbilical vein endothelial
cells [38]. It also exhibits significant activity against bowel inflammatory diseases
by decreasing the expression of IL-6, IL-1b, interferon (IFN)-c, and MMP-9 in
IL-10 deficient mice [41].
424 Z. Song et al.

17.3.2 Cancer

As stated above, butein can interfere with tumor progression by suppressing inflam-
matory reactions [52]. It is also well recognized as a promising anticancer agent and
functions by inhibiting cellular growth, migration as well as by promoting apoptosis. Its
anticancer effects have been reported against various tumor cells, including breast
cancer [67], colon cancer [74], leukemia [31], melanoma [15], osteosarcoma cells [24],
prostate cancer [30], hepatocellular carcinoma [57], lung cancer [42], and neuroblas-
toma cells [8]. Its anticancer effects are achieved by modulating various critical cellular
signaling pathways and enzymes involved in tumorigenesis and apoptosis. It is well
established that PI3K/Akt/mTOR is one of the major signaling pathways regulating cell
survival and motility [17]. And its activation plays a key role in tumor cell growth and
invasion [6, 17]. Butein can significantly suppress PI3K/Akt phosphorylation in human
prostate cancer cells [30] and cervical cancer cells [2]. It is also found to downregulate
MMP-9 and uPA expression by inhibiting Akt/mTOR/p70S6K, thus acting as anti-
metastatic agent [46].
Various studies also show that butein induces reactive oxygen species
(ROS) production, which is also critical for cell apoptosis, cell senescence and cell
division [65]. It has been reported that it induces G2/M phase cell cycle arrest in
hepatoma cells by regulating ROS level, JNK expression, ATM, and Chk activity
[50]. It also induces ROS generation, ERK1/2, and p38MAPK suppression in
triple-negative breast cancer MDA-MB-231 cells [72]. Similarly, it causes apop-
tosis of neutroblastoma cells by increasing ROS level and modulating various
enzymes [8]. And it also shows antiproliferative and apoptotic activities in HeLa
human cervical cancer cells, by inhibiting the expression of PI3K, Akt and mTOR
phosphorylation, and by inducing the ROS generation [2]. These effects were
further confirmed in vivo in a nude mouse model, where it significantly suppressed
cervical tumor growth with limited side effects by inhibiting PI3K/Akt/mTOR
activation and increasing ROS generation [2]. It also increases caspase-3, -8, -9
activities in neutroblastoma cells and caspase-3 activity in HeLa cells, the
expression of which is critical for apoptosis [31]. Moreover, Bcl-2 downregulation
is also observed in butein-induced apoptosis of neutroblastoma cells [8]. It is also
shown that butein increases Bax while suppressing Bcl-2 expression in Hela cells.
Bax and Bcl-2 are both critical enzymes for apoptosis, and the Bax/Bcl-2 ratio
contributes to the butein-induced apoptosis [2].
Another critical molecule involved in the anticancer activity of butein is the
signal transducer and activator of transcription 3 (STAT3), a transcription factor.
STAT3 can regulate the NF-jB signaling pathway, and thus plays a major role in
the inflammation-related tumorigenesis [21, 76]. It is activated by IL6/JAK sig-
naling pathway [29]. The IL6/JAK/STAT3 mechanism has been found to partici-
pate in the progression of various malignant tumors, such as prostate cancer, breast
cancer, and myeloma [41]. Butein inhibits the IL6/JAK/STAT3 pathway by sup-
pressing c-Src and JAK1/JAK2 activation, and by inhibiting Bcl-xL, Bcl-2, and
cyclin D1 expression [57]. Moreover, it blocks the ERK1/2 and NF-jB signaling
17 Butein and Its Role in Chronic Diseases 425

pathways in human bladder cancer cells, which inhibits the cell proliferation and tumor
invasion [78]. It also negatively regulates NK-jB activation by modulating critical
molecules such as TNFa1, TRADD, TRAF2, NIK, TAK1/TAB17.1, and IKK-b [55].
Besides STAT3, matrix metalloproteinase (MMP) is also critical in tumor cell apop-
tosis, tumor progression, and invasion. It has been found that butein suppresses the
activation of MMP-9 in prostate cancer cells [18, 35], and the activation of MMP-2 and
MMP-9 in Hela cells [2]. Butein is also noted to inhibit both MMP-9 activity and
STAT3 activation in Colo 205 cells [41], which indicates its possible therapeutic
potential for treating bowel inflammation-induced colon cancer.
There are several critical enzymes and molecules regulated by butein in various
tumor cell lines. For example, glutathione-s -transferase (GST), an enzyme that
reduces H2O2-induced oxidative stress, is significantly increased after butein
treatment in human caucasian neuroblastoma IMR-32 cells [77]. And it also sup-
presses some other enzymes critical for tumorigenesis, such as histone deacetylase
enzymes [54], aromatase [67] and recombinant human aldo-keto reductase family
[61]. And butein can inhibit DNA, RNA, and protein synthesis by reducing the
cellular uptake of thymidine, uridine, and leucine of cancer cells [74]. Moreover,
butein treatment suppresses colony formation in UACC-812 human breast cancer
cells co-cultured with fibroblasts [34]. And it induces apoptosis in U937 human
leukemia cells by modulating caspase 3-dependent pathways [23].

17.3.3 Cardiovascular Diseases

Cardiovascular diseases are one of the leading causes of mortality worldwide [49].
They are also closely associated with various other complications, such as obesity
and diabetes [20]. Butein has been found to exhibit cardiovascular protection effects
by improving blood circulation, in which platelets aggregation and thrombosis play
a key role. It has been reported that butein also exhibits anti-thrombin effects by
inhibiting the blood coagulation drawn from New Zealand white rabbits [43]. It also
inhibits ADP-induced platelet aggregation [43] and adrenalin-induced secondary
aggregation in human plasma [45]. Besides its anti-platelet effects, butein can also
suppress the viability and motility of vascular smooth muscle cells, both of which
can lead to atherosclerosis by thickening the blood vessels [9]. This activity is
further confirmed in vivo in animal models of phenylephrine-induced contracted rat
aorta [75] and balloon-injured rat carotid arteries [8].

17.3.4 Diabetes and Metabolic Complications

Diabetes is a group of metabolic diseases caused by either insufficient supply of

insulin (Type I diabetes mellitus) or aberrant cellular response to insulin (Type II
diabetes mellitus). Butein can significantly suppress diabetes induced by
426 Z. Song et al.

streptozotocin [44]. It also has been found to have protective effects on functional
pancreatic beta-cells and rat islets. Type I diabetes is mainly caused by beta-cell
destruction, in which various inflammatory cytokines are involved, including IL-1b,
TNF-a, IFN-c, etc. [25]. Moreover, it protects the cellular activity of beta cells by
inhibiting cytokine-mediated cell death, NO production, iNOS expression, and
NF-jB translocation [25], thus indicating its potential for Type I diabetes mellitus
prevention.

17.3.5 Liver Diseases

It has been reported that butein is also active in liver fibrosis prevention, by
increasing albumin production [16] and inhibiting hepatic stellate cell proliferation
[69]. In vivo data shows that butein can reduce hydroxyproline and malondialde-
hyde levels in liver fibrosis mouse model induced by CCl4 [36]. It can also decrease
the toxicity caused by ethanol in hepatic stellate cells and hepatoma HepG2 cells
[64], indicating its potential to prevent alcohol-related liver complications, such as
cirrhosis, fatty liver, and alcoholic hepatitis. The effects to decrease alcohol-induced
toxicity are achieved by suppressing ROS production, MMP-1 and -2 inhibitors in
HSCs. It has been also found that butein inhibits the JNK/p38 MAPK signaling
pathway and upregulates NF-jB [64]. Moreover, butein inhibits rat hepatocyte
apoptosis by decreasing PARP cleavage, DNA fragmentation, and caspase acti-
vation [60].

17.3.6 Other Protective and Therapeutic Effects

Butein has been found to have therapeutic effects against the initiation and pro-
gression of several other major human diseases. For example, it has anti-HIV effects
by causing around 58 % inhibition of HIV-1 protease [70]. It can also suppress
ICAM-1 expression and increase leukocyte function-associated antigen-1 positive
cells in nephritic glomeruli [19]. And it can decreases the protein level in urine and
cholesterol level in plasma, thus indicating its therapeutic potential for nephropathy,
a kidney disease which can be caused by either IgA deposition in glomerulus or
chemotherapy agents [60]. Moreover, it is potential drug candidate for tuberculosis,
as it can inhibit myocolic acids production and HadB activity, by blocking the
activity of Mycobacteriumbovis BCG, a myobacteria that causes tuberculosis [3]. It
also shows protective effects in several neuronal cell lines. It largely reduces the
toxicity induced by glutamate in mouse HT22 neuronal cells [11, 12]. And it can be
a potential pain killer by inhibiting SNARE-complex, a critical enzyme involved in
the synaptic vesicle transmission [27].
17 Butein and Its Role in Chronic Diseases 427

17.4 Preclinical Studies

Although the therapeutic effects of butein have been demonstrated in various

in vitro models of different cell lines as mentioned above, yet it still need to be
further confirmed in vivo in order to have practical application. And several studies
have reassured its bioactivities in animals. It is revealed that the tumorigenesis of
xenograft cervical tumors can be inhibited in nude mice with minor side effects, by
intraperitoneal injection of butein [2]. Similarly, extracts from RhusVerniciflua
Stokes (RVS) containing butein has been shown to decrease lung cancer tumor size
in a xenograft mouse model. It inhibits angiogenesis and suppresses tumor growth,
by inhibiting VEGF activity which indicates its potential for treating angiogenesis
dependent cancers [13]. Moreover, injection of butein significantly reduces the
colonic inflammation in IL-10-/- mice with drug-induced colitis [41]. Also, butein
has shown to prevent drug-induced acute renal failure in rats, indicating its potential
for nephropathy treatment [60]. Additionally, it also has exhibited protective effects
in rat models with spinal cord injury [47] and CCl4-induced liver fibrosis [36],
which is, again, consistent with respective in vitro results. There are few preclinical
trials to analyze the possible therapeutic potential of butein in humans. Extracts
from RVS with butein significantly reduced the tumor size in an 82 years old
patient after 5 months of treatment [39]. Gastric cancer has always been one of the
leading diseases, and the effect of conventional therapies such as surgical resection
is limited for elderly patients. So RVS extracts containing butein can be an ideal
alternative treatment. Also, this finding indicates that the therapeutic effects of
butein can also be elevated and optimized in a combination with other useful
flavonoid compounds. Extracts from RVS with butein significantly reduced the
tumor size in a 82-years-old patient after 5 months of treatment [39]. Gastric cancer
has always been one of the leading diseases, and the effect of conventional therapies
such as surgical resection is limited for elderly patients. So RVS extracts containing
butein can be an ideal alternative treatment. Also, this finding also indicates that the
therapeutic effects of butein can also be elevated and optimized in a combination
with other useful flavonoid compounds.

17.5 Conclusions

Herbal medicine can be traced back to hundreds of years ago, and our ancestors use
different plants with different bioactivities in different combinations. However, the
ancient application of herbal medicine is solely based on experience, without
qualitative or quantitative tests. With the development of drug testing techniques,
the chemical structures and activities of these therapeutic compounds have been
extensively studied, by using high throughput screening and various in vitro as well
as in vivo models. Butein is a multi-target compound that can exhibit a wide array
of beneficial effects by modulating a wide variety of cellular signal transduction
428 Z. Song et al.

TNF-α

PI3K/Akt/mTOR

ROS

Butein

c-Src

ß-glucuronidase

Fig. 17.2 Major signaling pathways and enzymes regulated by butein, # indicates inhibition or
downregulation; " indicates activation or upregulation. The molecules and pathways in the inner
circle are all upregulated or activated, while those in the outer two circles are downregulated or
inhibited by butein

cascades (Fig. 17.2). Its therapeutic effects have been validated in various cell lines
as well as mice models. However, neither Food and Drug Administration (FDA) in
US nor European Food Safety Authority (EFSA) has approved it as a therapeutic
drug. The only formulation containing butein available at present is Inh-AR, a
dietary supplement. There are many structure-activity studies on butein to reveal the
relationship between its chemical structural features and reported pharmacological
effects [22, 53, 63]. These studies provides in-depth understanding of its observed
biological effects and may also aid to synthesize similar compounds in future that
can achieve the same, or even better, therapeutic effects with better pharmacokinetic
profile. Although butein has many beneficial effects and low side effects, its toxicity
still needs to be elucidated. It is shown that the RhusVerniciflua Stokes, one of the
17 Butein and Its Role in Chronic Diseases 429

original sources of butein, can cause severe skin complications [32], thereby
indicating that butein may also have potential adverse effects. However, there is
currently lack of relevant toxicity studies with this therapeutic agent. Overall, butein
appears to be a promising drug candidate because of its various beneficial effects
and therapeutic actions against major human chronic diseases. However, additional
clinical trials are needed to validate its safety and efficacy so that it can be rapidly
applied for human use in near future.

Acknowledgments This work was supported by National University Health System Bench to
Bedside grant to GS.

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Chapter 18
Garcinol and Its Role in Chronic Diseases

Amit K. Behera, Mahadeva M. Swamy, Nagashayana Natesh

and Tapas K. Kundu

Abstract The various bioactive compounds isolated from leaves and fruits of
Garcinia sps plants, have beencharacterized and experimentally demonstrated to be
anti-oxidant, anti-inflammatory and anti-cancer in nature.Garcinol, a polyiso-
prenylated benzophenone, obtained from plant Garcinia indica has been found to be
aneffective inhibitor of several key regulatory pathways (e.g., NF-kB, STAT3 etc.)
in cancer cells, thereby being ableto control malignant growth of solid tumours
in vivo. Despite its high potential as an anti-neoplastic modulator ofseveral cancer
types such as head and neck cancer, breast cancer, hepatocellular carcinoma,
prostate cancer,colon cancer etc., it is still in preclinical stage due to lack of sys-
tematic and conclusive evaluation ofpharmacological parameters. While it is
promising anti-cancer effects are being positively ascertained fortherapeutic
development, studies on its effectiveness in ameliorating other chronic diseases
such ascardiovascular diseases, diabetes, allergy, neurodegenerative diseases etc.,
though seem favourable, are veryrecent and require in depth scientific investigation.

Keywords STAT3
Epigenetic enzymes
Inflammation
Tumorigenesis

18.1 Introduction

Garcinol (camboginol) is a natural compound originally extracted from dried fruit

rind of Garcinia indica (Clusiaceae) (Fig. 18.1). The plant Garcinia indica (also
known as kokum) has been utilized in culinary field, cosmetics, and confectionary
industries and most importantly in Ayurvedic medicine. The plant has been
immensely valued for its medicinal utility. In traditional Indian Ayurvedic

A.K. Behera
M.M. Swamy
T.K. Kundu (&)

Transcription and Disease Laboratory, Molecular Biology and Genetics Unit,
Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur P.O.,
Bangalore 560064, India
e-mail: [email protected]
N. Natesh
Central Government Health Scheme Dispensary, No. 3, Basavanagudi, Bangalore, India

© Springer International Publishing Switzerland 2016 435

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1_18
436 A.K. Behera et al.

Fig. 18.1 a Image of Garcinia indica tree. b Ripe fruits of Garcinia indica. c Chemical structure
of garcinol

practices, leaves and fruits of this plant have been used for treatment of inflam-
matory ailments and digestive disorders [1]. As per Ayurvedic classical texts, it has
been mentioned as cardiac tonic, anti-helminthic and used in intestinal disorders,
hemorrhoids, tumors, etc. [2]. Many groups have put efforts to identify and char-
acterize the biological function and medicinal value of different phytochemicals
obtained from this plant. Different studies have independently demonstrated that
garcinol possesses antibacterial, antioxidant, gastroprotective, and most impor-
tantly, antineoplastic properties. Recent investigations have revealed that garcinol
treatment significantly affects multiple biological pathways, suggesting its potent
therapeutic role in multitude of human disorders.
The diseases which are persistent and long lasting, such as cancer, cardiovascular
diseases, diabetes, arthritis, neurodegenerative diseases, allergies, etc., are included in
the group of chronic diseases. With current advancement in medical science, complete
cure is not possible for the pathological conditions seen in chronic diseases. However,
with medical intervention controlling measures could be taken so as to alleviate the
pathological severity or retard the progression of the disease. As a result the inevitable
outcome of mortality in case of such diseases could be delayed. Several of the chronic
diseases could be prevented on the grounds of healthy lifestyle. Thus, self-awareness
and personal management of illness also could help to avoid complications and
increase survival. Nevertheless, researchers have been putting constant efforts to find
cures for such diseases. In the field of drug development for human diseases, naturally
available compounds and small molecules derived from medicinal plant sources have
been under investigation in last few decades. On a similar basis, there have been
several preclinical studies carried out with garcinol testifying and underscoring its
value with therapeutic promises in manifold human disorders including chronic
diseases, which will be discussed in subsequent sections.

18.2 Physicochemical Properties of Garcinol

Biochemical Properties: According to one investigation, the role of functional

groups namely 13,14-dihydroxy, present on garcinol, seems to be important in
18 Garcinol and Its Role in Chronic Diseases 437

regulating its anticancer activity in context of oral cancer. In this study it has been
revealed that 13,14-dimethoxy derivative of garcinol has very little effect in regulating
cell cycle and apoptosis of SCC15 cell line compared to unmethylated garcinol. The
mechanism behind this difference has been attributed to the ability of garcinol to bind
and inhibit 5-lipoxygenase (5-Lox), where hydroxyl groups at C13 and C14 seem to
be critical in interaction with the catalytic domain of 5-Lox [3]. Importance of C-13
and C-14 has been further shown by another study, where substitution of C-13 and
C-14 of isogarcinol (an intramolecular cyclization product of garcinol) could enhance
the specificity of the molecule in context of inhibition of histone acetyltransferase
(HAT) activity [4]. It has been further discussed in Sect. 18.7.
Pharmacokinetic Properties: According to the calculation by Guide to
Pharmacology (IUPHAR/BPS) online tool, Garcinol has four hydrogen bond
acceptors and three hydrogen bond donors. It has 10 rotatable bonds. Its 2-D
structure would have topological polar surface area of 111.9 Å2. It displays an
octanol–water partition coefficient: log P of 10.07. The number of Lipinski’s rules
broken is 2, making it pharmacologically less amenable to be an orally active drug,
which is a theoretical prediction.
There have been very few studies toward understanding of bioavailability of gar-
cinol. According to one investigation, effect of garcinol seems to be more effective
in the absence of serum in in vitro cell culture system. The addition of 10 % FBS
(fetal bovine serum) to medium leads to approximately 10-fold decrease in IC50
value of garcinol for HCT116 cell growth. Addition of BSA (bovine serum albu-
min) protein to media showed similar effect, indicating interaction of garcinol with
serum proteins. However, no such effect was observed with cambogin (found in
Garcinia cambogia, having similar but not identical structure with that of garcinol).
Cellular uptake assessment studies showed that intracellular garcinol level in HT-29
and HCT116 cells was 2–5-fold more than that of cambogin after incubation in
serum-free Hank’s balanced salt solution for 1 h. However, in the presence of FBS
the intracellular level of garcinol was dramatically reduced under similar experi-
mental conditions [5].
Pharmacokinetic properties of garcinol with respect to its absorption, metabo-
lism, tissue distribution, excretion and physiological toxicity, etc. are needed to be
investigated in animal model before considering for clinical trial toward therapeutic
advancement, despite its promising anticancer properties.

18.3 Modulation of Cell Signaling Pathways by Garcinol

Garcinol has been demonstrated to inhibit HATs such as p300 and PCAF both
in vitro and in vivo; as a result it could suppress HAT-dependent transcription from
chromatin. It was further shown by microarray analysis that inhibition of HAT with
treatment of garcinol could suppress expression of majority of genes tested at global
level in HeLa cells. Several of downregulated genes were revealed to be
438 A.K. Behera et al.

proto-oncogenes, justifying anticancer property of garcinol [6]. Garcinol treatment

has been shown to cause upregulation of several tumor suppressor miRNAs, of
which miR-200c was found to target and downregulate Notch1 in pancreatic CSCs
[7]. The administration of garcinol has been shown to cause significant reduction in
expression level of cyclooxygenase-2 (COX-2), cyclin D1, and vascular endothelial
growth factor, which is proposed to occur via inhibition of the extracellular
signal-regulated protein kinase 1/2, PI3K/Akt and Wnt/b-catenin signaling path-
ways [8, 9] (Fig. 18.2).
Garcinol has been shown by several groups to target and inhibit STAT3 signaling
pathway. The first report on this aspect has shown that garcinol treatment could
reduce STAT3 expression as well as the levels of phosphorylated STAT3 in breast,
prostate, and pancreatic cancer cell lines [10]. This finding has been confirmed by
other groups and it has been further demonstrated that garcinol could inhibit
acetylation and dimerization of STAT3, thereby negatively affecting its nuclear
localization and DNA-binding ability thus transcriptional property of STAT3 in cell
[11] (Fig. 18.3). In breast cancer, it inhibited Wnt-beta-catenin pathway which acts
as a pro-survival signaling pathway in aggressive cancers. Beta-catenin level was

NF-kB
STAT3
Wnt-Β-catenin Proliferative
Gli1 pathway
Cyclin D1
Notch 1
MET E-cadherin FAK
Epigenetic
p300/CBP
pathway
DR4, DR5
Vimentin
Cytosolic
Apoptotic
Garcinol ZEB 1 EMT
cytochrome c
pathway ZEB 2
Bax, tBid
Cleaved caspase 3 Bcl2 Anti-apoptotic
XIAP pathway
FLIP
MMP7
Metastatic
VEGF
pathway
NF-kB
iNOS Inflammatory
NO pathway
COX2

Fig. 18.2 Different cellular pathways positively and negatively regulated by garcinol in cell. DR4
death receptor 4, Bax Bcl2-associated X Protein, tBid truncated BH3 interacting-domain death
agonist, NF-kB nuclear factor kappa light chain enhancer of activated B cells, STAT3 signal
transducer and activator of transcription 3, FAK focal adhesion kinase, CBP CREB-binding
protein, ZEB1 zinc finger E-box-binding homeobox 1, Bcl2 B-cell lymphoma 2, XIAP X-linked
inhibitor of apoptosis protein, FLIP FLICE-like inhibitory protein, MMP7 matrix
metalloproteinase-7, VEGF vascular endothelial growth factor, iNOS inducible nitric oxide
synthase, NO nitric oxide, COX-2 cyclooxygenase-2
18 Garcinol and Its Role in Chronic Diseases 439

decreased at both cytoplasmic and nuclear level with increase in phosphorylated

form of beta-catenin (which is targeted for degradation) upon treatment with garcinol
in MDA-MB-231 and BT-549 cells. There was concomitant reduction in expression
of cyclin D1 which is a downstream target of beta-catenin [12].
Pro-apoptotic role of garcinol has been demonstrated by several groups in var-
ious cancer cell lines. Multiple reports demonstrate that garcinol treatment could
lead to downregulation of NF-kB pathway, which appears to be the underlying
mechanism in the induction of apoptosis [13, 14]. However, there are other reports
which show modulation of a different pathway to activate apoptosis. According to
one such study, it can lead to loss of membrane potential of mitochondria and
release of cytochrome c to cytoplasm, which in turn triggers intrinsic apoptotic
pathway with activation of caspase 9 and 3 in human leukemia HL60 cells [15]. Yet
another study finds that it could upregulate the expression of death receptors: DR4
and DR5 could sensitize the colon cancer cells, e.g., HCT116 and HT29 cells
toward TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis.
According to this report, garcinol could induce activation of caspase 8, which

Nuclear localization,
Phosphorylation Phosphorylation
IKK IkBα NF-kB

Gene regulation
JAK2 p300
Phosphorylation

Acetylation

Garcinol

STAT3 STAT3

STAT3 STAT3

STAT3 STAT3

Nuclear localization and gene regulation

Fig. 18.3 Scheme to show the mechanism through which garcinol suppresses NF-kB and STAT3
pathways. Garcinol inhibits IKK activity, resulting in reduced degradation and increased
accumulation of IkBa, which in turn phosphorylates NF-kB and leads to degradation of NF-kB.
Garcinol inhibits both phosphorylation and acetylation of STAT3 by JAK2 and p300, respectively,
which reduces the nuclear localization of STAT3. Garcinol also directly binds to STAT3 and
impairs the dimerization and thus DNA-binding and transcriptional regulation ability of STAT3
440 A.K. Behera et al.

subsequently helps in activation of intrinsic apoptotic pathway [16]. This study

highlights significance of combinatorial therapy where garcinol could play an
important role to potentiate TRAIL-induced apoptosis in TRAIL-resistant cancer
cells. Garcinol has been found to inhibit double-strand break (DSB) repair including
nonhomologous end joining (NHEJ) in HeLa cervical cancer cells as well as A549
lung cancer cells and enhance the incidence of senescence induced by ionizing
radiation (IR), exhibiting radiosensitization properties [17].

18.4 Role of Garcinol in Chronic Diseases

Cancer: Garcinol has been revealed by many groups to be anticarcinogenic and

pro-apoptotic in several cancers, such as prostate cancer, colorectal cancer, breast
cancer, pancreatic cancer, head and neck cancer, liver cancer, etc. In a hamster chick
pouch model of oral cancer induced by 7,12-dimethylbenz[a]anthracene (DMBA),
treatment with garcinol suppressed 5-Lox pathway, thereby leukotriene B4 (LTB4)
biosynthesis led to reduction in cell proliferation, cancer lesions, and the growth of
visible tumor [18]. Parallel studies in head and neck cancer have led to the under-
standing that it could inhibit STAT3 and NF-kB signaling pathway in head and neck
cancer [19]. Researchers have been able to demonstrate that it could also suppress
activation of the kinases involved in subsequent activation of STAT3, e.g., c-Src,
JAK1, and JAK2. Moreover, it could inhibit activation of positive regulators of
NF-kB such as transforming growth factor-b-activated kinase 1 (TAK1) and IKK
(IkB kinase) and prevent phosphorylation and degradation of negative regulator of
NF-kB such as IkBa (nuclear factor of kappa light polypeptide gene enhancer in
B-cell inhibitor, alpha). By blocking activation of pro-survival and anti-apoptotic
pathways (such as STAT3 and NF-kB pathways), garcinol was demonstrated to be
able to inhibit growth of HNSCC in nude mice. Apart from inhibiting growth of
tumor cells in xenograft of MDA-MB-231 cells in SCID mice, it also inhibits
epithelial to mesenchymal transition (EMT) process which is necessary for metas-
tasis of cancer cells. Garcinol treatment has been found to decrease the expression of
the mesenchymal markers such as vimentin, ZEB1, and ZEB2 in aggressive breast
cancer cell lines MDA-MB-231 and BT-549, and can lead to increased expression of
miR-200 and let-7 family of miRNAs along with E-cadherin favoring the process of
mesenchymal to epithelial transition (MET) [12].
A study involving pancreatic cancer stem-like cells shows that garcinol could
significantly suppress stem-like properties of PANC-1 side population (exhibiting
cancer stem-like cell properties) as well as metastatic potential by downregulating
the expressions of Notch1, ABCG2, Mcl-1, Gli-1, and EZH2. Moreover, it could
upregulate tumor suppressor miR-200c which was revealed to target and down-
regulate Notch 1 [7]. It exhibited enhanced bioactivity with high level of synergism
with curcumin in reducing proliferation and inducing apoptosis in pancreatic cancer
(PaCa) cells in vitro [20]. Garcinol induces apoptosis in hepatocellular carcinoma
18 Garcinol and Its Role in Chronic Diseases 441

Hep3B cells (deficient for p53), where it has been demonstrated to activate death
receptor and mitochondrial apoptotic pathway with significant activation of caspase
9 and 3. It could lead to accumulation of reactive oxygen species (ROS), endo-
plasmic reticulum (ER) stress modulator GADD153 (growth arrest and DNA
damage inducible gene 153), increased Bax2/Bcl-2 ratio, elevated tBid (truncated
Bid), and caspase 8 in cancer cell. This study highlights the therapeutic application
of garcinol in the context of p53-independent apoptosis [21]. Garcinol also sup-
pressed the growth of prostate cancer cell lines such as LNCaP, C4-2B, and PC3
in vitro and could induce apoptotic cell death. This chemopreventive nature of
garcinol was demonstrated to occur via inhibition of NF-kB pathway [13]. Study
with colorectal cancer cell line HT-29 shows that garcinol can lead to inhibition of
tyrosine phosphorylation of focal adhesion kinase (FAK), which is a mediator of
integrin-regulated intracellular signaling in adherent cells, thus impairing cellular
proliferation and migration. It also inhibited expression of MMP7 (matrix metal-
loproteinase 7) in HT-29 cells, thereby leading to inhibition of invasiveness of the
cancerous cells. Researchers were also able to demonstrate that it could lead to
inhibition of growth promoting c-Src, MAPK/ERK, and PI3K/Akt signaling
pathways and can cause release of cytochrome c from mitochondria to cytosol and
induction of apoptosis in HT-29 colorectal cells [22].
Diabetes: Diabetes is a pathological condition characterized by prolonged and
high levels of sugar in blood of the patient. Either inadequate production of insulin
or inability of cells of body to respond to insulin for proper metabolism of sugar or
both could lead to diabetes. Diabetic condition is known to cause damage in small
blood vessels to the eye, kidney, and nerves causing diabetic retinopathy, diabetic
nephropathy, and diabetic neuropathy, respectively, with damage to respective
organs. Investigations have revealed marked hyperacetylation of histones in several
organs of diabetic rats such as retina. One study has successfully targeted histone
acetylation in retinal Müller glia cells grown in a diabetes-like concentration of
glucose, which would mimic pathogenesis of diabetic retinopathy. In this report the
researchers show that garcinol treatment could lead to reduction in histone acety-
lation, consequently suppressing expression of the pro-inflammatory factors
implicated in diabetic retinopathy [23]. With a similar perspective, curcumin analog
C66 has been found to prevent diabetic nephropathy in mice via inhibition of HAT
activity of p300/CBP, which seems to be one of important mechanisms modulated
by curcumin and its analogs to exert antidiabetic effects [24–26], further empha-
sizing the significance of garcinol in the prospects of treatment of diabetic com-
plications as a modulator of HATs. Another line of research indicates that garcinol
could also target the process of adipogenesis and reduce accumulation of lipids in
3T3-L1 cells [27]. With further research anti-adipogenic effect of garcinol could be
exploited to manage obesity, which is known to increase the risk of type 2 diabetes.
Cardiovascular Diseases: Many cardiovascular diseases result in cardiac fibrosis
which in turn leads to thickening and stiffening of cardiac muscle in the progression
to heart failure. It has been found that excessive expression of collagen-1 (which
normally provides structural support to heart) in cardiac fibroblasts in fibrotic
442 A.K. Behera et al.

condition could be inhibited by treatment with garcinol which would target

acetyltransferase p300/CBP, required for basal expression of Collagen1 [28]. Thus
garcinol might be useful as a part of chemoprevention therapy in retarding cardiac
fibrosis in cardiovascular diseases. Similar line of research with other HAT inhi-
bitors further corroborates and favors the idea of suppression of HAT in the context
of cardiovascular diseases. For instance, anacardic acid, a reported inhibitor of
p300/PCAF [29], was found to reverse cardiac hypertrophy induced by ethanol in
fetal mice [30]. Investigation on the mechanism revealed that anacardic acid could
suppress the over-expression of NKX2.5, Cx43, and b-MHC genes (involved in
fetal heart development) induced by alcohol via inhibition of HATs. Yet another
study demonstrates that phenylephrine (PE)-induced cardiac hypertrophy is medi-
ated by HAT activity of p300/CBP, inhibition of which by anacardic acid could
prevent hypertrophic stimulation in primary neonatal cardiomyocytes [31]. Thus
utilization of HAT inhibitors such as garcinol with proper and validated combi-
nation with other chemical modulators such as anacardic acid could be considered
for further research to develop therapeutics to ameliorate cardiovascular diseases.
Neurodegenerative Diseases: Garcinol could enhance neuronal survival by
modulating ERK pathway and promote neurite growth in epidermal growth factor
(EGF)-responsive neural precursor cell [32]. Antioxidant nature of garcinol has
been experimentally demonstrated by many groups. Garcinol can scavenge
hydroxyl-free radicals and can relieve oxidative stress in the cell; a concept which
has been tested to verify whether it can confer neuroprotection against damaging
effects of free radicals. Indeed garcinol was found to reduce inducible nitric oxide
synthase (iNOS) level (induced by lipopolysaccharide) in primary astrocytes and
could enhance survival of neurons in neuron–astrocyte co-culture system [33]. This
finding raises the possibility of garcinol being considered in development of ther-
apeutics for neurodegenerative diseases such as Alzheimers’s, Parkinson’s to
attenuate oxidative stress-induced neurotoxicity.
Allergy: Beneficiary role of garcinol has been investigated in certain allergic
conditions such as asthma. Asthma is a chronic inflammatory disease of respiratory
tract which can be induced by alcohol, aspirin, and exercise or triggered by the
presence of allergens in workplace such as wood dust, spray paint, latex, iso-
cyanates, smoke, and air pollutants. Symptoms of asthma include shortness of
breath, coughing, wheezing associated with reversible airflow obstruction, and
bronchospasm. Environmental factors play a very important role in determining
nature of allergy. Thus, alteration in epigenetic mechanisms from genetic interac-
tion with environment could determine severity of pathophysiological outcome. It
has been observed that acetylation marks on H3 and H4, site specifically H3K9,
H3K14, H3K27, H3K18, and H4K16, were significantly increased on Notch1
promoter in case of asthmatic lung CD4+ T cells compared to control group,
concomitant to increased activity of p300 and PCAF and decreased activity of
HDAC1 and HDAC2. In an attempt to reverse this condition researchers have used
garcinol as an inhibitor of HAT and successfully reduced the acetylation marks on
Notch1 promoter, thereby achieving reduction in expression level of Notch1 in
18 Garcinol and Its Role in Chronic Diseases 443

asthmatic T cells. Further investigation on asthmatic parameters with garcinol

intervention showed that it could significantly suppress the levels of inflammatory
cytokines such as IL4, IL5, and IL13 in asthmatic lung T cells [34]. This study
indicates that with further investigation garcinol might prove to have applications in
treatment of asthma.

18.5 Biological Activities of Garcinol in Animal Models

In situ studies of colon carcinogenesis induced by azoxymethane (AOM) in male

F344 rats showed that dietary garcinol administration at doses of 0.01 and 0.05 %
could suppress expression of iNOS and COX-2 as well as reduce O(2)(-) and NO
generation and ultimately could achieve 26 and 40 % reduction in frequency of
aberrant crypt foci (ACF) development, respectively. Significant reduction in pro-
liferating cell nuclear antigen (PCNA) index in ACF was also observed with gar-
cinol treatment. Garcinol administration in such cases was also seen to enhance
activities of liver glutathione S-transferase (GST) and quinone reductase (QR),
which play an important role in detoxification process of body [35]. In a similar
vein, another study demonstrates chemopreventive properties of garcinol in
colitis-associated colon cancer, where garcinol treatment could successfully reverse
dextran sulfate sodium (DSS)-induced colitis/inflammation-related colon tumori-
genesis in male ICR mice [8]. In context of hepatocellular carcinoma (HCC),
researchers have shown that treatment with garcinol could successfully inhibit
growth of HCC in athymic nude mice through suppression of STAT3 signaling
[11]. Garcinol was found to inhibit autophagy (0–6 h duration of treatment) via
activation of PI3K/Akt and mTOR pathway and induce apoptosis in PC-3 human
prostate cancer cells. Additionally, garcinol exhibited antitumor activity in xeno-
graft mouse model of prostate cancer, where it could achieve 80 % tumor reduction
[36]. Studies performed with animal models of cancer with garcinol have been
summarized in Table 18.1.

18.6 Biological Activities of Garcinol in Humans

Garcinol is still in preclinical stage. There have been numerous in vitro studies with
cell lines of human origin as well as in animal models for different human diseases,
to assess efficacy of garcinol toward development of chemoprevention modalities.
However, there has not been any systematic study on human as a whole organism.
A commercially available medicinal product called GarCitrin® (an extract obtained
from Garcinia cambogia) contains 5 % of garcinol as constituent along with
minimum of 50 % of hydroxycitric acid (HCA). The major purpose of using
GarCitrin® relies on activity of HCA to block biosynthesis of fatty acids and
lipogenesis thus aiding in weight management in an individual [37]. Although it is
444 A.K. Behera et al.

Table 18.1 Physico-chemical properties of garcinol

Molecular weight: 602.36
Chemical formula: C38H50O6
Composition: C (75.71 %), H (8.36 %), and O (15.93 %)
IUPAC name: (1S,5R,7R)-3-[(3,4-dihydroxyphenyl)(hydroxy)methylidene]-6,6-dimethyl-1-
[(2S)-5-methyl-2-(prop-1-en-2-yl)hex-4-en-1-yl]-5,7-bis(3-methylbut-2-en-1-l)bicycle [3.3.1]
nonane-2,4,9-trione
Structure: isoprenylated benzophenone
Density: 1.1 ± 0.1 g/cm3
Melting point: 122 °C
Optical rotation: [a] 22 D—143°
Solubility: Up to 100 mM in DMSO and up to 100 mM in ethanol
ACD/Labs Percepta platform based predication
Boiling point: 710.8 ± 60.0 °C at 760 mmHg
Vapor pressure: 0.0 ± 2.4 mmHg at 25 °C
Enthalpy of vaporization: 109.1 ± 3.0 kJ/mol
Molar refractivity: 175.6 ± 0.3 cm3
Polarizability: 69.6 ± 0.5 10–24 cm3
Surface tension: 43.8 ± 3.0 dyne/cm
Molar volume: 541.1 ± 3.0 cm3

hypothesized that combination of garcinol with HCA would enhance bioavailability

of HCA, however, there have not been any scientific investigation to determine
exact effect of garcinol in this context.

18.7 Derivatives and Analogs of Garcinol

High cytotoxicity of garcinol has prompted several groups to explore the avail-
ability of bioactive compounds with analogous chemical structures as well as
synthesizing derivatives on garcinol scaffold with better biological utility.
According to one such study, mono-substituted isogarcinol (IG: an intramolecular
cyclization product of garcinol) derivatives, LTK-13 (14-isopropoxy IG), LTK-14
(14-methoxy IG), and LTK-19 (13,14 disulfoxy IG) were specific inhibitors of
p300-HAT compared to parental compound, which inhibits both p300 and
PCAF HAT activity. Further investigation with the derivative LTK14, which is
specific inhibitor of p300 and non-toxic to cells, showed that it could suppress HIV
multiplication in T lymphocytes via HAT inhibition [4]. Another study, in context
of cancer, showed that two oxidative derivatives of garcinol namely, garcim-1 and
garcim-2, possessed greater inhibitory potential on growth of colon cancer cells [5].
Compounds from different species of Garcinia genus having similar structures
also have been isolated and found to be exhibiting similar biological activities,
18 Garcinol and Its Role in Chronic Diseases 445

Table 18.2 Summary of in vivo cancer chemopreventive studies with garcinol

Types of Cell Animal Pathway/genes targeted by References
cancer considered for garcinol
xenograft
HNSCC CAL27 Mouse Inhibition of NF-kB Li et al.
pathway [39]
Prostate PC-3 Mouse Activation of Apoptosis, Wang
inhibition of autophagy, et al. [36]
Activation of p-mTOR and
p-PI3 Kinase/Akt pathway
Hepato-cellular PLC/PRF5 Mouse Inhibition of STAT3 Sethi et al.
signaling [11]
Colon In situ Mouse Inhibition of PI3K/Akt and Tsai et al.
(induction by Wnt/beta-catenin pathway, [8]
DSS) inhibition of inflammation
HNSCC CAL27 Mouse Inhibition of NF-kB and Li et al.
STAT3 signaling pathway [19]
Oral In situ Hamster Inhibition of 5-lipoxygenase Chen et al.
(induced by pathway [18]
DMBA)
Breast MDA-MB-231 Mouse Suppression of NF-kB and Ahmad
Wnt-beta-catenin pathway et al. [12]
Breast MDA-MB-231 Mouse Inhibition of STAT3 Ahmad
signaling et al. [10]
Tongue In situ Rat Suppression of Yoshida
(induced by cyclooxygenase et al. [9]
4-NQO) (COX) expression
Colon In situ Rat Inhibition of iNOS and Tanaka
(induced by COX expression and et al. [35]
azoxymethane) suppression of free radical
[O(2)(-) and NO]
generation.
In situ: chemicals used to induce carcinogenesis at the relevant sites or organs in the animal
4NQO 4-nitroquinoline 1-oxide, DMBA 7,12-dimethylbenz[a]anthracene, DSS dextran sulfate
sodium

underscoring the pharmaceutical importance of the molecular scaffold of garcinol

(Fig. 18.1) . Few of such compounds showing therapeutic values have been listed
in Table 18.2. Among polyphenol analogs of garcinol, chalcones have been
emphasized for their structural similarity and biological activity. Structurally,
chalcones possess aromatic rings and a,b-unsaturated ketone system, but not iso-
prenyl group present in garcinol. Biologically, similar to garcinol, chalcones are
potent antioxidants, and anti-inflammatory in nature. Chalcones have been shown to
target NF-kB and Akt pathway in cells and exhibit antitumor activity in several
human cancer cell lines [38].
Table 18.3 Structural bioactive analogs and polyphenol precursors of garcinol isolated from Garcinia sps
446

Name of Garcinia sp. Name of the Chemical structure Biological activity References
compound isolated
Garcinia indica, Garcinia Isogarcinol (a) Anticancer, pro-apoptotic (a) Pieme et al.
ovalifolia, Garcinia (b) Immuno-suppressant [40]
mangostana OH (b) Fu et al. [41]
HO O O H

H
O O

Garcinia assigu Gracinol 13-O- Cancer chemopreventive Ito et al. [42]

methyl ether
OMe
HO O O H

H
O OH

Garcinia achachairu Guttiferone A (a) Neuroprotective (a)

Rusby (b) Anticancer Nuñez-Figueredo
(c) Gastroprotective et al. [43]
OH
H
(b) Pardo-Andreu
HO O O
et al. [44]
(c) Niero et al.
O OH [45]

Garcinia yunnanensis Oblongifolin (a) Anticancer, pro-apoptotic (a) Feng et al.

(b) Anti-metastatic [46]
OH (c) Antiallergic (b) Wang et al.
HO O O
[47]
(c) Lu et al. [48]
O OH
A.K. Behera et al.

(continued)
Table 18.3 (continued)
18

Name of Garcinia sp. Name of the Chemical structure Biological activity References
compound isolated
Garcinia xanthochymus Xanthochymol Anticancer Matsumoto et al.
Pro-apoptotic [49],
OH Einbond et al.
HO O O H
[50]

H
O OH

Garcinia brasiliensis 7-Epiclusianone Anticancer effect on glioblastoma Sales et al. [51]

O O H
Garcinol and Its Role in Chronic Diseases

O OH

Garcinia multiflora Garcimultiflorone OH Pro-apoptotic Liu et al. [52]

D
OH
HO O O

O OH

Garcinia hanburyi Gambogic acida Anticancer and pro-apoptotic (a) Wang et al.
effect on [53]
OH (a) Lung cancer (b) Shi et al. [54]
O
O O O O (b) Chronic myelogenous (c) Wen et al. [55]
leukemia
(c) Colorectal cancer
447

OH O
(continued)
Table 18.3 (continued)
448

Name of Garcinia sp. Name of the Chemical structure Biological activity References
compound isolated
Garcinia kola Kolaviron OH O Antidiabetic and hypoglycemic Iwu et al. [56],
OH Oyenihi et al. [57]
OH
HO O
H
O
O
HO O
OH

OH
Garcinia mangostana Alpha-Mangostin (a) Anti-inflammatory, (a) Janhom and
cytoprotective and anti-apoptotic Dharmasaroja
OH O
in SH-SY5Y cells [58]
O
(b) Anticancer (b) Hafeez et al.
HO O OH
(c) Anti-invasive [59]
(c) Wang et al.
[60]
Garcinia nujiangensis Nujiangexanthone Antiasthmatic effect Lu et al. [61]
A OH O
HO

O O OH
OH

a
Approved by the Chinese Food and Drug Administration for the treatment of lung cancer and is in Phase II clinical trials [62]
A.K. Behera et al.
18 Garcinol and Its Role in Chronic Diseases 449

18.8 Conclusions

Among several natural compounds isolated from fruit rind of Garcinia indica,
garcinol has been found to be the major component responsible for medicinal utility
of the plant. Initial studies showed potent antioxidant and anti-inflammatory
properties of garcinol. Further, in vitro and in vivo studies in animal models have
demonstrated its role as an effective anticancer agent in multitude of cancers, such
as prostate, colon, head and neck, pancreatic, and breast cancers. Therapeutic
potential of garcinol has been found not to be limited to cancers alone. Several
scientific researches indicate that it could exhibit benefits in many other chronic
diseases such as cardiovascular disorders, diabetes, neurodegenerative diseases, and
certain allergies such as asthma. However, majority of investigations carried out
with garcinol have explored and emphasized on its promising antineoplastic
properties. Researchers have identified and elucidated the molecular targets of
garcinol in different cancer cells and explained the basis of antitumor activity of
garcinol. It could suppress the growth in cancer cells via inhibition of several of
pro-survival pathways such as NF-kB, STAT3, and Akt/PI3K pathways and
simultaneously can induce apoptotic cell death. Additionally, it inhibits epithelial to
mesenchymal transition and metastasis of cancer cells and favors mesenchymal to
epithelial transition. Despite several animal studies justifying therapeutic role of
garcinol in last decade, it is still in preclinical stage of drug development due to lack
of proper pharmacokinetic studies. Further investigations are required to determine
toxicological aspects of garcinol and assess the parameters encompassing its
bio-metabolism such as absorption, systemic and/or localized distribution, and
excretion at the level of whole organism. It is important to understand the bio-
logically effective dosage or concentrations of garcinol for a specific disease, route
of administration, and temporal scheme of administration for better bioavailability
in whole organism, for ultimate development toward clinical applications. Proper
pharmacological assessment of garcinol could shed light into its relative propensity
of being effective as a future drug and encourage researchers to look for structural
analogs and synthesize derivatives of garcinol to improve on the limitations and
make it more effective and amenable for clinical therapy.

Acknowledgments TKK was supported by grants from Department of Biotechnology, Govt. of

India (Chromatin and Disease: Programme support (grant number: Grant BT/01/CEIB/10/III/01).
TKK is a recipient of the Sir J.C. Bose National Fellowship, Department of Science and
Technology, Government of India. AKB is a Senior Research Fellow of the Council of Scientific
and Industrial Research, Government of India. We acknowledge contribution of Prof. Gautam
Sethi, from NUS, Singapore for in vivo experiments on hepatocellular carcinoma with garcinol as
part of collaborative research with us. GS was supported by grants from National Medical
Research Council of Singapore [Grant R-184-000-211-213].
450 A.K. Behera et al.

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1646
Chapter 19
Morin and Its Role in Chronic Diseases

Krishnendu Sinha, Jyotirmoy Ghosh and Parames C. Sil

Abstract Chronic diseases can be referred to the long-term medical conditions

which are mostly progressive in nature, i.e., it deteriorates over time. Diabetes,
arthritis, heart disease, stroke, cancer, and chronic respiratory problems (e.g., COPD)
are not a few examples of chronic diseases and chronic diseases are the leading
causes of death and disability all over the world. Chronic diseases and conditions are
among the most common, costly, and preventable of all health problems. Affordable
cost, presence mostly in the consumables, and minimal side effects make the natu-
rally occurring compounds interesting and attractive for pharmacological study in
recent years. Plants produce diverse types of low molecular weight products mainly
for the defense purpose. Among them, the group of secondary metabolites related to
a polyphenolic group has been named flavonoids and are of great interest due to their
incredible pharmacological properties. In these regard, due to its potent
anti-inflammatory, anti-apoptotic and many important pharmacological properties
(relevant to chronic diseases, e.g., urate transporter inhibitor related to gout, mod-
ulator of immunosystem related to chronic hypersensitivity, etc.), morin [morin
hydrate:2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopy ran-4-one; 3,5,7,
20,40 pentahydroxyflavone], widely found among the Moraceae family, considered
as one of the most important key bioflavonols. However, little is known about the
molecular mechanisms of its action on such conditions. In this chapter, we have
summarized most of the findings, if not all, available till date.

K. Sinha
P.C. Sil (&)

Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M,
Kolkata 700054, West Bengal, India
e-mail: [email protected]; [email protected]
K. Sinha
Department of Zoology, Jhargram Raj College, Government of West Bengal,
Jhargram 721507, West Bengal, India
J. Ghosh
Chemistry Department, Banwarilal Bhalotia College Asansol,
Ushagram, Asansol 713303, West Bengal, India

© Springer International Publishing Switzerland 2016 453

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1_19
454 K. Sinha et al.

Keywords Morin Chronic diseases Anti-inflammatory Antioxidant Cellular

signaling

19.1 Introduction

Affordable cost, presence mostly in the consumables, and minimal side effects make
the naturally occurring compounds interesting and attractive for pharmacological
study in recent years. Plants produce diverse types of low molecular weight products
mainly for the defense purpose. Among them, the group of secondary metabolites
related to a polyphenolic group has been named flavonoids and are of great interest
due to their incredible pharmacological properties [14, 24]. These compounds are
consumed regularly as a part of the diet, present in flowers, tea, red wine, fruits, nuts,
herbs, vegetables, seeds, spices, stems, etc. and are known to possess a number of
biological and pharmacological activities like anti-hepatotoxic, anti-inflammatory,
antiulcer, antioxidant, etc., and also have the ability to inhibit various enzyme
activities [12, 27]. Among these, morin [morin hydrate:2-(2,4-dihydroxyphenyl)-
3,5,7-trihydroxy-4H-1-benzopy ran-4-one; 3,5,7,20,40 pentahydroxyflavone]
(Fig. 19.1) is belonging to the group of flavonols (a class of flavonoids having
3-hydroxyflavon backbone) found in the branches of white mulberry (Morus alba L),
osage orange (Maclura pomifera), almond (Psidium guajava), fig (Chlorophora
tinctoria), mill (Prunus dulcis), old fustic (Maclura tinctoria), and other family
members of Moraceae along with sweet chestnut (Castanea sativa, family Fagaceae)
[4, 35]. This compound exhibits different types of pharmacological activities, like free
radical scavenging, anti-inflammatory, xanthine oxidase inhibitor property, protec-
tive effect on DNA from damage caused by free radicals, prevention of low-density
lipoprotein oxidation, anticancer activity, etc. [14, 35]. Further studies both in in vitro
and in vivo indicate that it possesses numerous add on health benefits. However, no
article has yet been published that comprises and explains its botanical origin and
pharmacological activities in spite of the current research progress made on

Fig. 19.1 Molecular

structure of morin [Reprinted
from Biochim. Biophys. Acta
[General Subjects] 1850:769–
783; with the permission from
Elsevier; License Number:
3764180672838]
19 Morin and Its Role in Chronic Diseases 455

pharmacological/biological activities of morin. So, in this chapter, we would like to

compile the known pharmacological activities of morin on different chronic diseases.
Morin is thought to be a major bioactive molecule which could be used for the
prevention of hepatotoxicity. Also, the protective effects of this molecule against
oxidative stress and inflammation were investigated in some earlier studies [14]. In
addition, a number of other beneficial pharmacological effects including the pre-
vention of low-density lipoprotein oxidation, immunomodulation (the downregula-
tion of immune responses), inhibition of xanthine oxidase, and anticancer activity
were also reported [11, 14, 25, 34, 35, 46]. All these properties justify its validity as an
agent in the pharmacological treatment of several chronic diseases. Chronic diseases
are long-term medical conditions that are generally progressive [27]. Diseases like
inflammatory bowel disease, COPD, diabetes, myocardial infraction (MI), several
neurological diseases, chronic environmental pollutant-mediated toxicity, cancer, etc.
can be considered as chronic diseases [27]. Reports from Centers for Disease Control
and Prevention showed up to 2012, about 117 million people in US had one or more
chronic health conditions and every one of four adults had two or more chronic health
conditions (http://www.cdc.gov/chronicdisease/overview/). They also reported 7 of
the top 10 causes of death in 2010 were chronic diseases, whereas 2 of these chronic
diseases—heart disease and cancer—together accounted for nearly 48 % of all
deaths. Obesity is another serious health concern. During 2009–2010, more than
one-third of adults, or about 78 million people, were obese. Other very crucial chronic
disease conditions are arthritis, diabetes-related kidney failure, etc. where among 53
million adults with a clinical diagnosis of arthritis, more than 22 million have trouble
with their daily activities due to arthritis (http://www.cdc.gov/chronicdisease/
overview/). However, the possibility of preventing or controlling cancer using fla-
vonoids from fruits has created a considerable amount of interest as high intake of
vegetables and fruits and is reported to be associated with low incidence of cancer [30,
38]. Results from a number of studies suggest that phytochemicals can safely mod-
ulate the biology of the cancer cells and induce cancer cell death. Some properties of
this molecule have also been reported that may regulate the inflammatory responses
and halt carcinogenesis and cancer progression [20]. However, little is known about
the molecular mechanisms of the anticancer effects of this unique molecule [30].

19.2 Physciochemical Properties of Morin

Morin has a molecular weight of 302.2357 g/mol, exact mass of 302.042653 g/mol,
monoisotopic mass of 302.042653 g/mol, molecular formula of C15H10O7, XLogP3
of 1.5, hydrogen bond donor count of 5, hydrogen bond acceptor count of 7,
rotatable bond count of 1, topological polar surface area of 127 A2, heavy atom
456 K. Sinha et al.

count of 22, formal charge of 0, complexity of 488, isotope atom count of 0, defined
atom stereocenter count of 0, undefined atom stereocenter count of 0, defined bond
stereo center count of 0, undefined bond stereo center count of 0, a covalently
bonded unit count of 1, and experimental melting point 303.5 °C [5]. Morin is a
naturally occurring molecule (can also be synthesized) and it inhibits or retard the
oxidation of a substance to which it is added. It counteracts the harmful and
damaging effects of oxidation in animal tissues. The structure [Fig. 19.1] of morin
hydrate shows that it is an isomeric form of quercetin. Quercetin and morin both
have OH in position 3, a carbonyl group in position 4, and a resorcinol moiety.
However, there is a difference in the hydroxylation pattern on B-ring. In morin, the
hydroxylation is at the meta-position (in Morin), whereas it is in the ortho position
in quercetin. For all its groups and an ortho hydroxylation pattern on B-ring,
quercetin is regarded to have the highest antioxidant potential of the flavonoids but
morin hydrate has also been demonstrated to have higher effectiveness of certain
oxidative processes. Morin is soluble in methanol (50 mg ml−1) and generously
soluble in alcohol whereas faintly soluble in ether and acetic acid. It is also soluble
in aqueous alkaline solutions and gives intense yellow color. This color changes to
brown upon water exposure. It is also soluble in water (0.25 mg/ml, 20 °C;
0.94 mg/ml, 100 °C). In respect to pharmacokinetic study, Hou et al. investigated
and compared the pharmacokinetics of morin and its isomer quercetin in rats. Parent
forms and their glucuronides and sulfates in serum were studied. They found that
after oral dosing of both the parent forms, morin, and its glucuronides, and sulfates
were present in the blood stream and a nonlinear pharmacokinetics was observed
for morin. On the other hand, negligible bioavailability of quercetin is presented as
its glucuronides and sulfates were only detected in the blood and the metabolites
showed linear pharmacokinetics at the two doses studied. The total AUC of parent
form with conjugated metabolites showed that the extent of absorption of morin
was threefold compared to that of quercetin. Hou et al. [18] proved that the fates of
the flavonols were markedly affected by difference in hydroxylation pattern on
B-ring. Xie et al. showed how morin interacted with human serum albumin (HSA).
The interaction has been investigated using fluorescence, Fourier transform,
infrared spectroscopic approaches, and UV absorption [45]. Under the physiolog-
ical condition, there is a specific binding site on HSA for morin, and the binding
affinity was found to be 1.13 ± 0.11  10−5 L Mol−1. The intrinsic fluorescence
of morin was noticeably boosted in the presence of HSA due to excited-state proton
transfer. The level of protonation of the hydroxyl groups played a significant role
during the morin–HSA binding process and it was evident from the fact that with
the increase of the buffer pH from 6.4 to 8.4, binding ability of morin to protein
decreased. The interaction between morin and HSA induced an apparent decline of
the protein a-helix and b-sheet structures [45].
19 Morin and Its Role in Chronic Diseases 457

19.3 Role of Morin in Chronic Diseases: Modulation

of Cell Signaling Pathways in Animal and Human
Model

Morin proves itself effective against many chronic diseases. An increasing number of
studies showed that morin significantly modulate different cell signaling pathways
related to chronic pathophysiological conditions, including gastrointestinal com-
plications, diabetes, cardiovascular disease, cancer, arthritis, neurodegenerative
disease, and several inflammatory diseases. As various oxidative stress-related dis-
orders are led by inflammation, antioxidant and anti-inflammatory activities of morin
play a critical role in the therapeutic process of these disorders. Different studies
prove that morin is effective against several chronic diseases like chronic neurode-
generative disease, chronic cardiovascular pathophysiology, chronic gastrointestinal
pathophysiology, chronic hypersensitivity and immunological disorders, cancer and
several oxidative stress-related chronic pathophysiological disorders, arthritis, dia-
betes, and related pathophysiology. Morin alters the levels of phosphorylated Akt
kinase, Erk1/2, AIF release, cytosolic Bax, and regulated the NF-jB’s nuclear
translocation, and acts against the imbalances in the levels/activities of different
enzymes (e.g., elastase), inflammatory mediators, proinflammatory cytokines,
inflammatory enzymes, glycoproteins, reactive oxygen species, lysosomal acid
hydrolases, and transcription factors (e.g., NF-jB, p65, and AP1) to produce the
desirable effects. Also, different others pathways are involved in morin’s protective
actions and these are described extensively in the following sections.

19.3.1 Morin Against Chronic Neurodegenerative Disease

Excessive glutamate receptors activation (i.e., excitotoxicity) leads to acute and

chronic neurological disorders including stroke. The neuroprotective role of two
natural polyphenol antioxidants, mangiferin and morin, has been previously
reported in a model of ischemic brain damage using an in vitro model of excitotoxic
neuronal death involving N-Methyl-D-aspartate (NMDA) receptor over activation
[15]. Campos-Esperza et al. [7] showed the intricate molecular details of the neu-
roprotection exert by morin. They found that morin significantly reduced the
reactive oxygen species formation while activate the antioxidant enzyme system,
and reinstate the altered mitochondrial membrane potential to its basal state. They
also observed that morin could inhibit glutamate-induced calpains activation, nor-
malized the levels of phosphorylated Akt kinase and Erk1/2. It also inhibited AIF
release from mitochondria, restored the normalized level of cytosolic Bax, and
regulated the NF-jB’s nuclear translocation. These changes ultimately lead to
reduction of apoptotic neuronal death induced by glutamate. These results, based on
excellent antioxidant and anti-apoptotic properties of morin, support the clinical
application of morin as a trial neuroprotector in the pathology involving excitotoxic
458 K. Sinha et al.

neuronal death [7]. Zhang et al. investigated the neuroprotective effects of morin on
1-methyl-4-phenylpyridinium ion (MPP+)-induced apoptosis in neuronal differen-
tiated PC12 cells as well as in a 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine
(MPTP) mouse model of Parkinson disease (PD) where they found that MPP+, in
PC12 cells, induced ROS formation and apoptosis, whereas morin significantly
attenuated the MPP+-induced loss of cell viability, apoptosis, and inhibit ROS
formation. In mice model, morin significantly attenuated the MPTP-induced
nigrostriatal lesions, dopaminergic neuronal death, striatal dopamine depletion, and
permanent behavioral deficits. The results clearly suggest the neuroprotective
actions of this unique molecule both in vitro and in vivo and indicate the possibility
of being a novel therapeutic agent for the treatment of PD and other neurodegen-
erative diseases [49].
Ammonia is considered as a potent neurotoxin. It has been intensely associated
in the pathogenesis of hepatic encephalopathy. Subash and Subramaniam evaluated
the chronotherapeutic effect of morin, on ammonium chloride (AC)-induced
hyperammonemia in a rat model. Morin significantly ameliorate AC-induced
pathophysiological changes in respect to the circulating levels of urea, ammonia,
hydroperoxides (HP), thiobarbituric acid reactive substances (TBARS), liver
markers [aspartate transaminase (AST), alanine transaminase (ALT), and alka-
linephosphatase (ALP)], superoxide dismutase (SOD), glutathione peroxidase
(GPx), catalase (CAT), reduced glutathione (GSH), and vitamins A, C, and E. The
authors speculated that the chronotherapeutic effect of morin in hyperammonemic
rats might be due to temporal variations of lipid peroxidation and of antioxidants,
urea cycle enzymes, etc., temporal variations of metabolic enzymes involved in the
degradation of morin and the temporal variation in its bioavailability [36].

19.3.2 Morin Against Arthritis

In Ghanaian traditional medicine, leaf extracts of Ficus exasperata P. Beauv.

(Moraceae) have been and are being commonly used for the treatment of various
pathological states including inflammatory disorders. The main active ingredient in
all the extracts is morin. A recent study was conducted to evaluate the antiarthritic
effect of an ethanolic extract of F. exasperata (FEE) in an arthritis model (using
rats) in which the disease was induced by the Freund’s adjuvant [3]. For this study,
dexamethasone and methotrexate were used as positive controls. Like these positive
controls, FEE also showed significant dose-dependent antiarthritic properties in
adjuvant-induced arthritis. In addition, like antirheumatic drug methotrexate and the
steroidal anti-inflammatory agent dexamethasone, FEE was found to significantly
reduce the arthritic edema in the lateral paw of the animals and prevented the spread
of the edema from the lateral to the contralateral paws, suggesting the protective
role of the extract in inhibiting systemic spread. In rat brain homogenates, the
extract has been reported to exhibit reducing activity, scavenge DPPH radical, and
prevent lipid peroxidation. Detection of phenol in the extract suggests that the
19 Morin and Its Role in Chronic Diseases 459

ethanolic extract of these leaves probably exerts antiarthritic activity after oral
administration and it also has antioxidant properties; combination of the both may
contribute to its beneficial activity [3]. Zeng et al. [48] investigated the effect of
morin on type II collagen-induced arthritis (CIA) in rats and also explored the
underlying molecular mechanisms of synovial angiogenesis. Morin significantly
attenuated arthritic development which is specified by reduction of paw swelling
and arthritis scores. Morin also noticeably reduced the serum levels of proinflam-
matory cytokines, e.g., interleukin (IL)-6 (IL6), tumor necrosis factor-a (TNFa),
etc. Along with that, it increased the level of anti-inflammatory cytokine
interleukin-10 and improved pathological changes of joints as evident from his-
tology. Morin also distinctly inhibited expression of vascular endothelial growth
factor (VEGF), basic fibroblast growth factor, and CD31 in synovial membrane
tissues. It also reduced the serum levels of VEGF in CIA rats. In in vitro study,
morin also significantly inhibited VEGF-induced human umbilical vein endothelial
cells migration and tube formation. These results nicely showed that morin had
antirheumatoid potential which it exerts probably by inhibiting synovial [48].
Sultana and Rasool proved that the combination therapy of morin along with a
NSAID was very effective in suppressing the pathogenesis of rheumatoid arthritis
(RA), against adjuvant-induced arthritis (an experimental model for RA) in rats.
They found that imbalances in the levels/activities of elastase, inflammatory
mediators (TNFa, IL1b, MCP1, VEGF, and PGE2), paw edema, glycoproteins
(hexose and hexosamine), urinary constituents (hydroxyproline and glycosamino-
glycans), reactive oxygen species (LPO and NO), lysosomal acid hydrolases (acid
phosphatase, N-acetylglucosaminidase, b-galactosidase, and cathepsin D),
proinflammatory cytokines (TNFa, IL1b, IL17, IL6, and MCP1), inflammatory
enzymes (iNOS and COX2), RANKL, and transcription factors (NF-jB, p65, and
AP1) were regulated back effectively to near control level by morin and indo-
methacin, which were elevated in case of RA. Their findings were supported by
histopathological and radiological analysis, whereas body weight, bone collagen,
and the antioxidant status [superoxide dismutase (SOD), catalase (CAT), glu-
tathione peroxidase (GPx), glutathione, and ceruloplasmin)] were found to be
decreased in RA and that was restored back by the combinatorial therapy [37]. Gout
is a general systemic joint disorder where hyperuricemias is a hallmark of gout [3].
A serum uric acid level above 9 mg dL−1 is considered as gouty arthritis [6].
Pathological manifestation of gout follows over production or decreased excretion
of uric acid (purine metabolic end product) [26]. Urate–anion transporter (URAT1)
in the brush border membrane of the proximal tubule in kidney is the main
transporter involved in the maintenance of serum uric acid level by reabsorbing the
urate from the lumen to the cytosol in kidney tubules [9, 14]. Different drugs
including xanthine oxidase inhibitors (e.g., allopurinol), inhibitors of urate reab-
sorption at proximal renal tubule-like probenecid, benzbromarone, etc. are being
used in the treatment of gout. However, some undesirable side effects like hepa-
totoxicity are associated with the benzbromarone and other agents having
hypouricemic activity [14]. Certain natural herbs were reported to have the xanthine
oxidase inhibitor activity along with other types of mechanisms and those are
460 K. Sinha et al.

helpful in the treatment of gouty arthritis and hyperuricemia-related disorders.

Morin hydrate is one of them having the xanthine oxidase inhibitor activity and the
other type of mechanism which can reduce the rheumatic disorders [14]. The urate
reabsorption inhibitory effect of morin at the brush border of proximal renal tubule
membrane vesicles explains the above-said action of morin on the kidney [46].
Wijeratne et al. [43] using oxonate-induced hyperuricemic rat model showed the
uricosuric activity of morin hydrate. Above-mentioned information suggest that
morin has been used in the treatment of rheumatic disorders.

19.3.3 Morin Against Chronic Cardiovascular

Pathophysiologies

Cardiovascular diseases (CVD) are the major cause of chief mortality worldwide due
to its complicated nature. Among CVD disorder, MI is a major one. If there is
imbalance between the coronary supply and its myocardial demand, then myocardial
infarction takes place and it causes necrosis of the myocardial tissue. Morin shows
cardiovascular protection in isoproterenol (ISO)-induced myocardial infarction in
rats and this protection is attributed to its free radical scavenging activity by the
polyphenolic group [2]. It also shows significant beneficial effect on lipid profiles,
blood pressure, and serum glucose levels from the high-fat diet-induced hyperten-
sive rats. ISO, which is a synthetic catecholamine, has been documented to produce
severe stress in the myocardium, resulting in myocardial infarction, if it is admin-
istered in supramaximal doses. Cardiac necrosis due to the administration of ISO
includes increased oxygen consumption, calcium overload and accumulation,
increased myocardial cAMP levels, alterations of membrane permeability, and
increase in lipid peroxides level [2]. Al-Numair et al. reported that morin pretreat-
ment (20, 40, and 80 mg/kg, respectively) daily for a period of 30 days decreases
significantly the activities of cardiac marker enzymes such as lactate dehydrogenase,
creatine kinase, aspartate transaminase and creatine kinase-MB, membrane bound
enzymes (such as calcium-dependent adenosine triphosphatase), sodium
potassium-dependent adenosine triphosphatase, and magnesium-dependent adeno-
sine triphosphatase, in serum. Calcium-dependent adenosine triphosphatase and
magnesium-dependent adenosine triphosphatase were increased, whereas the
activity of sodium potassium-dependent adenosine triphosphatase was found to
decrease in the heart. At the same time it also showed a significant decrease in
glycoprotein (hexosamine, fucose, hexose, and sialic acid) levels in serum and heart.
ISO administration disrupts the redox balance and produces myocardial infarction
via free radical-mediated b-adreno-receptor mechanism. However, positive alter-
ations of these biochemical parameters were successfully achieved with morin
pretreatment daily for a period of 30 days. From their studies, the authors concluded
that morin has a protective role in ISO-induced MI in rats and the observed effects
might be due to the free radical scavenging, antioxidant, and membrane-stabilizing
19 Morin and Its Role in Chronic Diseases 461

properties of morin [2]. These beneficial effects of morin are due to its multitude of
biological function such as antioxidant, anti-inflammatory, and free radical scav-
enging activity. Another group of investigators also conducted a study to prove the
cardio-protective benefits of this flavonoid in ISO-induced myocardial infarcted rats.
A significant increase in the levels of cardiac markers was detected in ISO-induced
myocardial infarcted rats although morin pretreated animals could regulate the
abnormalities in electrocardiograph and biomarkers. Results also showed an
increased lipid peroxidation product in ISO-induced myocardial infarcted rats. The
animals, pretreated with morin, however, showed reduction of lipid peroxidation.
Histopathological studies supported all these observations as pretreatment with
morin-inhibited myocardial damage. Combining, the results of the above-mentioned
studies proved that the pretreatment of morin exhibits protective effect and are
rational to understand its beneficial effects on cardio-protection against myocardial
injury. The results also indicate the cardio-protective ability of morin [31]. However,
till now, the molecular mechanism of its protective actions is not crystal clear, and it
requires more studies to prove its inherent molecular mechanisms.
The protective effect of morin against deoxycorticosterone acetate (DOCA)-
induced hypertension was recently investigated in male Wistar rats. DOAC salt
hypertensive rats showed considerably increased systolic and diastolic blood pres-
sure in association with considerably increased systolic and diastolic blood, AST,
ALP, ALT, GGT, urea, uric acid, and creatinine levels in the plasma. However,
morin effectively lowered all the enzymes’ level up to that of the control. The study
indicates antihypertensive effect of morin [32]. In vitro studies have established that
oxidized low-density lipoprotein (ox-LDL) has increased atherogenicity compared
to native LDL. Upon oxidative modification of LDL, alteration in its structure allows
LDL to be taken up by scavenger receptors on smooth muscle, macrophage, and
endothelial cells. This lead to the formation of lipid-laden foam cells which is the
hallmark of primary atherosclerotic lesions. Naderi et al. found that morin signifi-
cantly inhibits in vitro LDL oxidation. Thus, they proved that morin would probably
be helpful to prevent atherosclerosis [28]. In a study, Wu et al. demonstrated that
morin hydrate significantly reduced the tissue necrosis in post-ischemic and reper-
fused rabbit hearts by >50 %. They showed that, besides scavenging oxyradicals,
morin hydrate discreetly inhibits a free radical generating enzyme xanthine oxidase,
from the ischemic endothelium. However, they also speculate that morin hydrate
may chelate some metal ions which help in further oxyradical formation and thus
inhibiting the process of oxidative stress generation [44].

19.3.4 Morin Against Chronic Gastrointestinal

Pathophysiology

Galvez et al. reported that morin possesses intestinal anti-inflammatory activity in

the chronic stages of trinitrobenzenesulphonic acid model of rat colitis. They
showed that the morin administration enabled tissue recovery following a colonic
462 K. Sinha et al.

insult with trinitrobenzenesulphonic acid. It also reduced myeloperoxidase activity,

colonic leukotriene B4, and interleukin-1b levels, improved colonic oxidative
stress, and inhibited colonic nitric oxide synthase activity [11]. In another study it
has been shown that morin significantly ameliorate nonsteroidal anti-inflammatory
drug (NSAID)-induced gastropathy in a rat model [35] (Fig. 19.2a, b). The gas-
troprotective action of morin is primarily attributed to its potent antioxidant nature.
Morin also significantly inhibits indomethacin (IND)-induced inflammatory
responses and gastric damage. IND induces ROS production which indirectly leads
to NF-jB activation. A simultaneous proinflammatory response gets initiated
leading to neutrophil infiltration. Activated NF-jB increases the redox burden by
inhibiting catalase production and inducing iNOS production. On the other hand,
increased TNF-a and IL-1b activate NF-jB. This produces ROS and creates a
positive feedback loop. However, morin effectively inhibits ROS production,
scavenges free radicals, and chelates noxious Fe2+. Besides, morin pretreatment
effectively inhibits IND-mediated NF-jB activation by inhibiting IKK activation.
Thus, it reduces proinflammatory cytokine production, related to apoptosis and
gastric damage. Morin also prevents downregulation of catalase and thus helps to
restore the cellular redox homeostasis (Fig. 19.3). In conclusion, this study provides
evidence that morin has significant potential as a therapeutic intervention for
IND-induced gastric mucosal injury. From the study it can be said that future
detailed pharmacokinetic and pharmacodynamic studies are expected to establish
morin as a gastroprotective agent [35]. The main inflammatory parameters in the
intestinal inflammation are free radicals, nitric oxide, leuckotrienes, etc. and are
responsible for the production of inflammatory mediators in the intestinal inflam-
matory conditions [11]. Morin hydrate has been shown to inhibit nitric oxide
synthase activity and the leukotriene-b4 synthesis [17]. Moreover, because of its
inhibitory effect on the myeloperoxidase activity, the intestinal inflammation mar-
ker of neutrophil infiltration activity was evidently increased [16]. Inhibition of
IL-1b, one of the proinflammatory cytokines responsible for the induction of
inducible nitric oxide synthase (iNOS) activity in enterocytes, is another important
anti-inflammatory activity attributed to the morin hydrate [33]. Inflammatory bowel
disease (IBD) is a chronic phase of inflammatory disorder and is known to be
associated with two closely related conditions (Crohn’s disease and Ulcerative
colitis) in the intestine. Synthesis and upregulation of proinflammatory mediators
(such as reactive oxygen species, cytokines and platelet-activating factors, etc.) are
the main aetiological events that occurred in the development of IBD. 5-amino
salicylic acid and local or systemic gluco-corticosteroids are the drugs which are
nowadays used for the management of IBD to exert their benefit through various
mechanisms [40]. Experimentally, rats were imparted colitis by single injection of
colonic instillation of the hapten trinitrobenzene sulphonic acid dissolved in ethanol
and the colitic animals were treated with morin hydrate which showed the beneficial
effect on 4th week following colitis insult [11]. Myeloperoxidase, IL-b4,
interleukin-1b (IL-1b) synthesis, glutathione (GSH) and malondialdehyde
(MDA) levels, and nitric oxide synthase (NOS) activity are different biochemical
mediators reported to be involved in the colonic inflammation [14]. Inhibition of the
19 Morin and Its Role in Chronic Diseases 463
 464 K. Sinha et al.

b Fig. 19.2 Effect of morin on gastric mucosa in IND-induced gastric injury. Control: vehicle
treatment alone; IND: IND treatment alone; morin + IND: treatment with morin IND; Morin:
morin treatment alone. a Open stomach showing injured mucosa. Note that the injury (in the form
of reddish black ulcers of different sizes) is highest in the IND (red arrows) and morin almost
completely prevented the ulceration. b Sections of gastric mucosa were stained with hematoxylin–
eosin. The IND group showed marked changes with outward mucosal damage (red arrow),
presence of inflammatory exudates (green arrows), extensive vasocongestion (black arrows), and
damaged submucosa (blue arrow) [Reprinted from Biochim. Biophys. Acta [General Subjects]
1850:769–783; with the permission from Elsevier; License Number: 3764180672838]

Fig. 19.3 Graphical representation of molecular mechanisms of gastroprotective action of morin

against IND-induced gastropathy in rats (‘Solid arrows’ indicating stimulatory interaction; ‘blunt
arrow’ indicates inhibitory interaction; ‘broken arrows’ indicate plausible mechanisms) [Reprinted
from Biochim. Biophys. Acta [General Subjects] 1850:769–783; with the permission from
Elsevier; License Number: 3764180672838]

synthesis of most important cytokine, IL-1b, decreased in the NOS and free radicals
involved in the inflammatory cascade are believed to be the factors involved in the
anti-inflammatory activity of morin.
19 Morin and Its Role in Chronic Diseases 465

19.3.5 Morin Against Diabetes and Related Pathophysiology

Noor et al. [29] showed that morin hydrate inhibits amyloid formation by human
islet amyloid polypeptide (IAPP, amylin) and disaggregates preformed IAPP
amyloid fibers that were evident from transmission electron microscopy (TEM) and
right-angle light scattering. IAPP is responsible for type-2 diabetes-related islet
amyloid formation and is also evident in islet cell transplants leading to graft
failure. Human IAPP have fewer inhibitors and it is extremely amyloidogenic.
However, due to the specific substitution pattern on the B-ring, morin hydrate
signifies a novel type of IAPP amyloid inhibitor [29]. Vanitha et al. [41] found that
morin significantly reduced the blood glucose and enhanced the serum insulin
levels in experimentally induced type I diabetic rats. Morin dose dependently
reduced glucose-6-phosphatase, fructose-1,6-bisphosphatase, hexokinase, and
glucose-6-phosphate dehydrogenase activities in liver. It significantly protected
pancreatic islets’ overall morphology as well as preserve insulin-positive cells in
diabetic rats. From the study it is evident that morin might be advantageous in the
treatment of diabetes and regulation of carbohydrate metabolic enzyme activities. It
is of prime importance in the pathophysiological conditions [41]. Abuohashish et al.
[1] found the putative advantageous effect of morin on diabetic osteopenia in rats
which they suggested might be due to both the anti-inflammatory and antioxidant
properties. They found that morin is particularly effective in bone metabolic dis-
order which is a major chronic problem in age-related disease. In case of diabetic
rats significant bone loss was observed at the level of bone turnover parameters
including osteocalcin (OC), bone alkaline phosphatase (bALP), and collagen type 1
cross-linked C-telopeptide (CTX). However, the study showed that morin treatment
obviously attenuated these elevations in those parameters [1]. A significant
impairment in trabecular bone microarchitecture, density, and other morphometric
parameters have been detected by performing bone micro-CT scan of diabetic rats,
and those were efficiently ameliorated by morin treatment. Besides, in diabetic rats,
serum levels of glucose, TBARS, IL-1b, IL-6, and TNF-a were significantly ele-
vated, while that of insulin and GSH was decreased. These changes were bring back
to normal after 5 weeks morin treatment, suggesting the protective effect of morin
against diabetic-induced osteopenia [1].

19.3.6 Morin Against Chronic Hypersensitivity

and Immunological Disorders

Kim et al. showed that morin suppressed IgE-mediated allergic responses in a

mouse model. It inhibited production of TNF-a, IL-4, and degranulation of antigen
(Ag)-stimulated mast cells. They also found that morin inhibited the phosphory-
lation of Syk which plays a very important role in the Syk activation. Morin also
inhibits activation of linker for activation of T cells (LAT) in rat basophilic
466 K. Sinha et al.

leukemia (RBL)-2H3 cells and bone marrow-derived mast cells (BMMCs) along
with the inhibition of p38, Akt and the MAP kinases, ERK1/2, JNK, and Fyn
kinase. With further investigations, their results suggest that the morin suppresses
the IgE-mediated allergic response by principally inhibiting Fyn kinase in mast cells
[23]. Findings of Fang et al. [10] indicated that morin might have the ability to
regulate immune response through modulating the cytokine profiles exhibited in
chronic immunotoxic pathophysiology. They have shown that morin and its
derivatives (sulfates/glucuronides) were effective on LPS-activated RAW 264.7
cells (macrophages) in reducing NO, TNF-a, and IL-12 production. Moreover,
phagocytic activities of the peripheral blood cells were significantly lowered in the
morin-treated cells in respect to control. These lowering in the NO production and
reduced macrophage phagocytic activities corresponded to LPS-resistant state is
very important to treat various chronic autoimmune diseases [10].

19.3.7 Morin Against Cancer and Several Oxidative

Stress-Related Chronic Pathophysiological Disorders

Recently, attention has been given on the anti-cancer activity of morin in various
kinds of cancers. For example, it exerted protective effect on chemically produced
rat tongue carcinogenesis [20] and inhibited phorbol ester-induced transformation
of rat hepatocytes.[19]. Besides, by inhibiting the lipoxygenase pathway, this
molecule could inhibit the peroxisome-proliferated activator receptor-induced ker-
atinocyte differentiation [39]. The inhibitory activity of this molecule was reported
during the release of inflammatory cytokines (such as IL-8, IL-6, and TNF) from
mast cells [21]. In an in vivo study, morin hydrate showed anticancer activity in
cancer models (like inhibit the growth of COLO205 cells in nude mice) [8]. The
transcription factor NF-jB is known to be involved in various kinds of cell pro-
liferation, cell survival, tumorigenesis, and inflammation. Induction of NF-jB
activation pathway induced by TNF, ceramide, lipopolysaccharide, IL-1, phorbol
12-myristate 13-acetate, and H2O2 was reported to be suppressed by morin. The
process involved the inhibition of IjB (inhibitory subunit of NF-jB) kinase that
leads to suppression of phosphorylation and degradation of IjBa and consequently
nuclear translocation of p65 occurs. NF-jB-dependent reporter gene expression
[activated by TNF receptor (TNFR), TNF, TNFR-associated factor 2,
TNFR1-associated death domain, NF-jB-inducing kinase, IǩB kinase, and the p65
subunit of NF-jB] was also reported to be inhibited by morin hydrate. Besides, it
could also downregulate the NF-jB-related products that are involved in the cell
survival (i.e., inhibitor of apoptosis proteins 1 & 2, survivin, X-chromosome-linked
IAP, and BcL-xL), invasion (matrix metalloproteinase-9), and proliferation (cyclin
D1 and cyclooxygenase-2) [14].
Zeng et al. in their study demonstrated that morin hydrate acts as a broad-
spectrum antioxidant as it scavenges both xanthine oxidase/hypoxanthine-generated
19 Morin and Its Role in Chronic Diseases 467

oxyradicals and also nonenzymatic, nitrogen-derived radicals. They showed the

effectiveness of morin hydrate on rabbit corneal endothelial cells against oxyradicals
and nitric oxide-induced damage. This way morin hydrate might be helpful in
preventing free radical-induced damage in chronic corneal problems and also in its
effective preservation [47]. In a study, Hsiang et al. [19] found that morin, in a
dose-dependent manner, reticent (terephthalic acid) TPA-induced cellular transfor-
mation in Chang liver cells. TPA-induced AP-1 activity, while through the inhibition
of p38 kinase, morin inhibited the AP-1. Moreover, morin induced the S-phase
which suggests that a cell cycle checkpoint was activated by morin to block DNA
synthesis as a protective measure against TPA-induced genotoxicity. Their study
established this molecule as a potent anti-hepatocellular transformation agent that
inhibited cellular transformation [19]. Kim et al. proved that morin has excellent
antioxidant and anti-inflammatory nature for which it can be considered as an
excellent agent against the chronic diseases. They proved that morin extensively
modulate reactive species (RS)-induced NF-jB activation through its RS scavenging
activity. Their findings indicate that morin neutralizes RS in vitro, inhibits t-
BHP-induced RS generation, and represses redox-sensitive transcription factor
NF-jB activation through reduced DNA binding activity, nuclear translocation of
p65/p50, and IjBa phosphorylation. More precisely, it can be said from their study
that, in endothelial cells, suppression of the NF-jB cascade by morin was modulated
through the ERK and p38 MAPKs pathways and morin’s antioxidant effect, as a
consequence, extended the expression level of NF-jB-dependent proinflammatory
genes, thereby reducing COX-2, iNOS, and 5-LOX [22]. During the metabolic
process, a limited range of various metals for the enzymatic and nonenzymatic
process in organic and inorganic forms are required. However, various heavy metals,
like mercury, (known as a wide-spread environmental pollutant) can cause severe
alterations in humans and animals [13, 14]. Morin hydrate has been shown to protect
various organs from mercuric chloride-induced toxicity. Moreover, it exerts pro-
tective effects on the alterations of serum markers like LDH, AST, and ACP levels
which are increased in renal nephritis and renal infarction [42]. Kidney is a multi-
purpose organ in the body. It is in control for excretion of metabolic wastes from the
body and for the regulation of homeostasis (e.g., acid base balance, electrolyte
balance, water balance, calcium level, blood pressure) in the body. However, when
the metabolites are more toxic than its precursor, they can lead to severe toxication
which ultimately remains responsible for kidney damage [14]. Humans are exposed
to these heavy metals in many ways in daily life. Mercury is one of them causing
kidney damage. Morin, in such cases, could be used as an effective antioxidant.
Morin possesses a variety of biological functions against oxidative stress-induced
damage, such as cardiovascular cells, glomerular mesangial cells, hepatocytes, oligo
dendrocytes, and in neurons [42, 44].
468 K. Sinha et al.

19.4 Conclusions

From the diverse studies reviewed in this article, morin emerged as a useful natural
flavone in the management of different chronic pathophysiological conditions.
Despite these direct pharmacological activities, morin could help to detect param-
eters related to severe chronic diseases. It could also be used as an excellent and
novel pathological detection tool. Recently, fluorescence-based, time saving, stain
with morin hydrate has been developed and it stains phospho-proteins in
one-dimensional SDS-PAGE where Al3+ was used as a ‘‘fixed bridge.’. With the
help of this novel quantification method based on kinetic measurement of the
fluorescence decrease of Al3+–morin complex it can be used to determine the
bisphosphonate content in plasma samples. This method would upkeep research on
the development of drug delivery systems for the related bisphosphonates.
However, despite the in vivo and in vitro studies, trying to elucidate the mecha-
nisms of action morin, in future, more studies are required to clearly understand the
precise molecular mechanism with great depth. Detailed preclinical studies and its
clinical experimentations are also extremely needed to provide a basis for potential
expediency of this gift of nature, morin, in the treatment and mitigation of chronic
diseases.

References

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19 Morin and Its Role in Chronic Diseases 471

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Chapter 20
Ellagic Acid and Its Role in Chronic
Diseases

Giuseppe Derosa, Pamela Maffioli and Amirhossein Sahebkar

Abstract Ellagic acid is a natural anti-oxidant phenol found in numerous fruits and
vegetables, in particular pomegranate, persimmon, raspberry, black raspberry,
strawberry, peach, plumes, nuts (walnuts, almonds), and wine. The anti-proliferative
and anti-oxidant properties of ellagic acid have prompted research into its potential
health benefits. The aim of this chapter will be to summarize potential benefits of
ellagic acid supplementation in chronic diseases.

Keywords Ellagic acid
Cancer
Chronic inflammation
Diabetes

G. Derosa (&)
P. Maffioli

Department of Internal Medicine and Therapeutics, University of Pavia,
Fondazione IRCCS Policlinico S. Matteo, P.Le C. Golgi, 2, 27100 Pavia, Italy
e-mail: [email protected]
G. Derosa
Center for Prevention, Surveillance, Diagnosis and Treatment of Rare Diseases,
Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
G. Derosa
Center for the Study of Endocrine-Metabolic Pathophysiology and Clinical Research,
University of Pavia, Pavia, Italy
G. Derosa
Laboratory of Molecular Medicine, University of Pavia, Pavia, Italy
P. Maffioli
PhD School in Experimental Medicine, University of Pavia, Pavia, Italy
A. Sahebkar
Biotechnology Research Center, Mashhad University of Medical Sciences,
Mashhad, Iran
A. Sahebkar (&)
Department of Medical Biotechnology, School of Medicine,
Mashhad University of Medical Sciences, P.O. Box: 91779-48564, Mashhad, Iran
e-mail: [email protected]; [email protected]

© Springer International Publishing Switzerland 2016 473

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1_20
474 G. Derosa et al.

20.1 Introduction

A diet rich in polyphenols, which are found in fruits and vegetables, is strongly
associated with a reduced risk for developing chronic diseases such as cancer and
cardiovascular disease [1]. Ellagic acid is a natural phenol anti-oxidant found in
numerous fruits and vegetables. Ellagic acid is a dimeric derivative of gallic acid
and rarely occurs free in diet crops, but usually occurs in food products conjugated
with glycoside moiety (glucose, xylose) or forms part of ellagitannins (polymeric
molecules) [2, 3]. These compounds usually occur in fruits (pomegranates, per-
simmon, raspberries, black raspberries, strawberries, peach, plumes), nuts (walnuts,
almonds), vegetables, and wine. Ellagitannins are the bioactive polyphenols present
in pomegranate; however, they are not absorbed intact by the human gut, but they
can be hydrolyzed to ellagic acid by colonic gastrointestinal flora [3, 4]. The
anti-proliferative and anti-oxidant properties of ellagic acid have prompted research
into its potential health benefits. The aim of this chapter will be to summarize
potential benefits of ellagic acid supplementation.

20.2 Physiochemical Properties of Ellagic Acid

Ellagic acid (2,3,7,8-tetrahydroxy-chromeno[5,4,3-cde]chromene-5,10-dione,

C14H6O8) (Fig. 20.1) was first discovered in 1831. It is a highly thermostable
molecule, with a melting point of 350 °C, with a molecular weight of
302.197 g/mol, slightly soluble in water, alcohol, and ether, but soluble in caustic
potash [5]. Ellagic acid is a weak acid, which is ionized at physiological pH [5]. It
structurally presents four rings representing the lipophilic domain, four phenolic
groups, and two lactones, which form hydrogen-bond sides and act as electron
acceptors, respectively, and that represent the hydrophilic domain.

Fig. 20.1 Ellagic acid

structure
20 Ellagic Acid and Its Role in Chronic Diseases 475

20.3 Role of Ellagic Acid in Chronic Diseases

20.3.1 Ellagic Acid and Inflammation

Ellagic acid proved to have anti-inflammatory effects in acute or chronic model of

ulcerative colitis [6]. In the study by Marin et al., the acute model of ulcerative colitis
was represented by female Balb/C mice treated with dextran sulfate sodium
(DSS) (5 %) for 7 days, while concomitantly receiving a dietary supplement of
ellagic acid (2 %). In the chronic ulcerative colitis model, instead, female C57BL/6
mice received 4-week-long cycles of DSS (1 and 2 %) interspersed with week-long
recovery periods along with a diet supplemented with ellagic acid (0.5 %). In acute
model, ellagic acid slightly ameliorated disease severity as observed both macro-
scopically and through the reduction of inflammatory mediators including
interleukin-6, tumor necrosis factor-α (TNF-α), and interferon-γ. In the chronic
model, ellagic acid significantly inhibited the progression of the disease, reducing
intestinal inflammation and decreasing histological scores. Daily treatment with
dietary ellagic acid significantly reduced cyclooxygenase-2 (COX-2) and inducible
nitric oxide synthase (iNOS) expression in colon tissue in a chronic model of
DSS-induced colitis. This action of ellagic acid is noteworthy, because COX-2 and
iNOS activation produce excessive inflammatory mediators which may be detri-
mental to the integrity of the colon and contribute to the development of intestinal
damage. These data are consistent with those of other studies conducted by Rosillo
et al. [7]. Similarly, these authors showed that ellagic acid administration reduced the
expression of both COX-2 and iNOS in a model of Cronh’s disease induced by
administration of trinitrobenzenesulfonic acid (TNBS) in rats. Myeloperoxidase
activity and pro-inflammatory cytokines, such as TNF-α production, were correlated
with the development of colonic inflammation and dietary ellagic acid, as well as
pomegranate enriched with or without ellagic acid, was able to diminish both
parameters. Ellagic acid probably acts on NF-κB family. NF-κB activation actively
contributes to the development and maintenance of intestinal inflammation, pro-
moting the expression of various pro-inflammatory cytokines including
interleukins-1, -2, -6, -8, -12, and TNF-α. NF-κB activation also mediates the
transcription of pro-inflammatory genes such as COX-2 and iNOS. In the study by
Rosillo et al. [7], the nuclear protein expression of NF-κB p65, involved in ulcerative
colitis and Crohn’s disease, was drastically decreased upon dietary treatment with
ellagic acid and ellagic acid-enriched pomegranate extract.

20.3.2 Role of Ellagic Acid in Cancer

The anti-cancer effect of ellagic acid has been studied in many human cancer cell
lines including those of skin, esophageal, and colon cancer where it exhibited
anti-proliferative activity, with the ability to cause cell cycle arrest and induce
476 G. Derosa et al.

apoptosis [8]. Ellagic acid takes part in various DNA maintenance reactions pre-
venting genomic instability which otherwise leads to cancer [9]. Ellagic acid has an
anti-proliferative effect and induces apoptosis via a mitochondrial pathway in
Caco-2 cells, an in vitro model for colon cancer, without interfering with the normal
colon cells [3]. In particular, a study exploring the activity of ellagic acid
demonstrated that it could induce apoptosis in 1,2-dimethyl hydrazine (DMH)-
induced colon carcinoma and participate in a wide range of DNA maintenance
reactions that prevent genomic instability [8]. Ellagic acid prevents PI3K/Akt
activation that, in turn, results in the modulation of its downstream Bcl-2 family
proteins [8] involved in the activation of the intrinsic apoptotic pathway [3]. Bax
expression and caspase-3 activation were noted after ellagic acid supplementation
leading to elevation of cytochrome c levels and finally cell death [3].

20.3.3 Ellagic Acid and Liver Protection

Ellagic acid possesses anti-oxidant, anti-hepatotoxic, anti-steatosic, anti-cholestatic,

anti-fibrogenic, anti-hepatocarcinogenic, and anti-viral properties that improve the
hepatic architectural and functions against toxic and pathological conditions.
Hepatotoxicity refers to liver dysfunction or liver damage associated with exposure
to drugs or xenobiotics. Ellagic acid increases anti-oxidant response through the
transcriptional activation of nuclear erythroid 2-related factor 2 (Nrf2), and indi-
rectly having a scavenging activity against a variety of reactive oxygen species.
Moreover, ellagic acid inhibits alcohol-induced liver cell damage increasing the
anti-oxidant levels, scavenging free radicals, and stabilizing cell membranes as
reported by Devipriya et al. [10]. They showed that in female albino Wistar rats,
weighing treated with ellagic acid against alcohol-induced damage, there was an
inhibition of alcohol-induced toxicity by improving body weight, restoring
anti-oxidant status, modulating micronutrients, and attenuating the lipid levels in
the circulation. Administration of ellagic acid effectively reduced the level of lipids
in the circulation, preventing lipid peroxidation [11]. This was confirmed by Yu
et al. [12] which reported that ellagic acid supplementation reduced the elevations
of plasma cholesterol in hyperlipidemic rabbits. It can be speculated that ellagic
acid might have decreased the activity of 3-hydroxy-3-methylglutaryl coenzyme A
(HMG CoA) reductase, or enhanced the rate of lipid degradative process and
increased the hepatic bile acids and fecal neutral sterol and thus decreased the level
of other lipids.
Ellagic acid has revealed potential activities against HBV infection as reported
by Pathak et al. [13] which identified that ellagic acid inhibits the HBx-induced
transcriptional activation for replication of the virus. Recently, the anti-viral activity
of ellagic acid against HCV was described by Reddy et al. [14], and Ajala et al.
[15]; these authors demonstrated that ellagic acid inhibits NS3/4A protease activity
in vitro, suggesting an interaction between ellagic acid with the unconventional
Zn-binding site present in the core region of the enzyme, blocking its activity.
20 Ellagic Acid and Its Role in Chronic Diseases 477

20.3.4 Ellagic Acid and Advanced Glycation End Products

It has been reported that the gradual build-up of advanced glycation end products
(AGEs) in body tissues is a major contributor to many progressive diseases
including diabetic complications [16] and Alzheimer’s disease [17, 18].
AGE-modified plasma proteins could bind to AGE receptors (RAGE) on the sur-
face of the cell, activate cell signaling, and lead to the production of reactive oxygen
species and inflammatory factors. Moreover, proteins on the extracellular matrix
crosslink with other matrix components, leading to a loss in their function.
As mentioned above, pomegranate fruit, which is a rich source of phenolics, in
particular ellagitannins, showed anti-oxidant and anti-inflammatory effects. Taking
into account the effects on AGE, pomegranate fruit juice and extracts have been
shown to exert neuro-protective effects by ameliorating symptoms of Alzheimer’s
disease potentially caused by the abnormal accumulation of AGEs in the brain [19].
This finding was further supported by Rojanathammanee et al. [20], who
determined whether a dietary intervention with ellagic acid could attenuate
microgliosis in a rat model of Alzheimer’s disease. Three months of pomegranate
feeding lowered TNF-α concentrations in brain, and lowered nuclear factor of
activated T-cell (NFAT) transcriptional activity compared with controls.
Immunocytochemistry showed that pomegranate, but not control-fed mice, had
attenuated microgliosis and amyloid β plaque deposition, involved in Alzheimer’s
disease.

20.3.5 Ellagic Acid and Diabetes

Ellagic acid seems to play an anti-diabetic activity through the action on β-cells of
pancreas, stimulating insulin secretion and decreasing glucose intolerance. This
effect of ellagic acid was reported by Fatima et al. [21]. Treatment with Emblica
officinalis extract, rich in ellagic acid, has been reported to result in a significant
decrease in the fasting blood glucose in a dose- and time-dependent manner in
diabetic rats. Ellagic acid significantly increased serum insulin in diabetic rats in a
dose-dependent manner. Insulin-to-glucose ratio was also increased by Emblica
officinalis treatment. Immunostaining of pancreas showed that Emblica officinalis
extract (250 mg/kg) increased β-cell size, but a higher dose of 500 mg/kg increased
β-cells number in diabetic rats. Moreover, Emblica officinalis extract significantly
increased plasma total anti-oxidants and liver GSH and TBARS. Elevation in
glucose tolerance by ellagic acid suggested that ellagic acid probably works by
stimulating insulin secretion from pancreatic β-cells. These results infer that ellagic
acid may exert anti-diabetic activity through the action on β-cells of pancreas
resulting in an increase in β-cell size and number, increasing anti-oxidant status,
decreasing blood glucose, increasing serum insulin, and β-cell morphology, and
morphometry [21].
478 G. Derosa et al.

20.4 Conclusions

Ellagic acid seems to be a very promising agent for the treatment of chronic
diseases, especially ulcerative colitis, Cronh’s disease, Alzheimer’s disease, and
diabetes. Ellagic acid also seems to have hepatoprotective and anti-cancer proper-
ties. The anti-diabetic potential of ellagic acid makes it a potentially interesting
alternative to traditional glucose-lowering medications and a strong candidate for
diabetic drug research. However, detailed toxicity evaluations need to be performed
before any therapeutic application in humans could be suggested. Proof-of-concept
safety and efficacy trials are also warranted to verify the interesting preclinical
findings. Further studies are needed to see if the very good results observed in
animal will be confirmed in humans.

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Author Index

A M
Afaq, Farrukh, 213 Maffioli, Pamela, 173, 473
Amin, Shantu G., 375 Mancha-Ramirez, Anna M., 75
Ammon, H.P.T., 291 Monisha, B. Anu, 47
Motiwala, Hashim F., 329
B Moudgil, Kamal D., 267
Baer-Dubowska, Wanda, 131
Baggioni, Alessandra, 27 N
Basu, Pritha, 155 Na, Hye-Kyung, 185
Behera, Amit K., 435 Natesh, Nagashayana, 435

C P
Chin, Kok-Yong, 97 Pal, Harish C., 213
Cicero, Arrigo F.G., 27 Pandey, Manoj K., 375
Cohen, Mark S., 329 Pang, Kok-Lun, 97
Pearlman, Ross L., 213
D Prasad, Ram, 245
Derosa, Giuseppe, 173, 473 Priyadarsini, K.I., 1

G Q
Ghosh, Jyotirmoy, 453 Qiao, Liang, 397

K S
Karelia, Deepkamal, 375 Sahebkar, Amirhossein, 173, 473
Katiyar, Santosh K., 245 Sethi, Gautam, 419
Kumar, Gopinatha Shanmugam, Muthu K., 419
Suresh, 155 Sil, Parames C., 453
Kumar, Niraj, 47 Sinha, Krishnendu, 453
Kundu, Tapas K., 435 Slaga, Thomas J., 75
Kunwar, A., 1 Soelaiman, Ima-Nirwana, 97
Song, Ziwei, 419
L Subramanian, Chitra, 329
Licznerska, Barbara, 131 Surh, Young-Joon, 185
Lu, Hong, 397 Swamy, Mahadeva M., 435

© Springer International Publishing Switzerland 2016 481

S.C. Gupta et al. (eds.), Anti-inflammatory Nutraceuticals and Chronic Diseases,
Advances in Experimental Medicine and Biology 928,
DOI 10.1007/978-3-319-41334-1
482 Author Index

T Wang, Youxue, 397

Tiku, Ashu Bhan, 47 White, Peter T., 329

V Y
Venkatesha, Shivaprasad H., 267 Yu, Hanry, 419

W Z
Wang, Jun, 397 Zhou, Yongning, 397