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Cite this: Nat. Prod. Rep., 2011, 28, 953

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Bufadienolides and their antitumor activity
Huimin Gao,*a Ruxandra Popescu,b Brigitte Koppb and Zhimin Wanga
Received 30th August 2010
DOI: 10.1039/c0np00032a
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Covering: 1998 to July 2010

Since 1998, a great number of bufadienolides have been reported from plants, animals and plant cell
suspensions, as well as from fungi and bacteria cultures. This review summarizes both new
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bufadienolides and those obtained from new sources, together with data regarding the antitumor
activity of bufadienolide derivatives.

1 Introduction position C-17b. Since the isolation of the first bufadienolide,

2 Plant sources of bufadienolides scillaren A, from Egyptian squill in 1933,1 this family of
2.1 Crassulaceae compounds has attracted the attention of scientists worldwide,
2.2 Hyacinthaceae leading to the isolation of numerous bufadienolides with various
2.3 Ranunculaceae chemical structures and diverse biological activities. Two thor-
2.4 Leguminoseae ough reviews, covering the chemical characterization of bufa-
2.5 Sterculiaceae dienolides derived from plant and animal sources from 1967 to
3 Animal sources of bufadienolides 1997, were published in 1998 by Krenn and Steyn, respectively.2,3
3.1 Bufonidae In recent years, several reviews have focused on various aspects
3.2 Colubridae related to the isolation, characterization and biological activities
3.3 Mammalian bufadienolides of cardiotonic steroids and their analogues,4–7 among which
4 Biotransformation of bufadienolides bufadienolides only occupied a small subsection, and in which
4.1 Plant cell suspension culture the chemical description of bufadienolides was outdated. A
4.2 Microbial transformation review article describing natural and synthetic 2H-pyran-2-ones
4.3 Metabolites of bufadienolides in vivo provided an overview of the natural bufadienolides reported up
4.4 Analytical determination of bufadienolides in biolog- to 2005.8
ical samples Considerable progress has been made regarding the cellular
5 Antitumor activities pharmacology of bufadienolides and the potential of this class of
5.1 Antitumor activities in vitro compounds as chemotherapeutic agents.7,9–11 The ecological
5.2 Antitumor activities in vivo value of particular bufadienolides has also been discussed.12–18
5.3 Toxicity of potent antitumor bufadienolides For example, several bufadienolides are sequestered and stored
6 Concluding remarks for defensive purposes by fireflies12,13 and the Asian snake.14,15
7 Acknowledgements Previous publications reported the isolation of bufadienolides
8 References from animal and plant species. Vegetal species containing bufa-
dienolides mainly belong to the following genera and families:
Cotyledon and Kalanchoe (Crassulaceae), Urginea /Scilla and
Bowiea (Hyacinthaceae), Holmeria and Moraea (Iridaceae),
1 Introduction Bersama and Melianthus (Melianthaceae), Helleborus (Ranun-
culaceae), as well as Thesium (Santalaceae). Animal sources of
Bufadienolides are an important group of polyhydroxy C-24 bufadienolides include fireflies Photinus spp. (Lampyridae),
steroids and their glycosides. They are characterised by the snakes Rhabdophis spp. (Colubridae) and toads Bufo spp.
presence of a six-membered lactone (a-pyrone) ring located at (Bufonidae).2,3 Currently, compounds belonging to the class of
bufadienolides continue to be isolated from plant and animal
a
Institute of Chinese Materia Medica, China Academy of Chinese Medical species. Moreover, biotransformation, a new and effective
Sciences, Beijing, 100700, China. E-mail: [email protected]; Fax: +86
approach for the biosynthesis of biologically active compounds,
(10)84014128; Tel: +86 (10)84014128
b
Department of Pharmacognosy, University of Vienna, A-1090 Vienna, has led to the production of bufadienolide derivatives based on
Austria parent compounds such as bufalin, cinobufagin, resibufogenin

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and bufotalin. The present review summarizes new isolated Crassulaceae family, and Melianthus comosus and Bersama
bufadienolides and new sources of known bufadienolides from abyssinica from the Melianthaceae family.22
1998 to 2010, together with their reported antitumor activity. In addition, three a-pyrone ring-opening derivatives of bufa-
dienolides, kalanhybrin A 8, B 9 and C 10, were isolated as active
components from the cytotoxic fractions of the methanol
2 Plant sources of bufadienolides extracts of Kalanchoe hybrida, together with the known bufa-
2.1 Crassulaceae dienolides 5 and 6 and daigredorigenin-3-acetate 11.23,24
Bufadienolides were also detected in the extract of Kalanchoe
Two new insecticidal bufadienolides, bryophyllin C 1 and methyl crenata25 and the juice of Bryophyllum pinnatum26 on the basis of
daigremonate 7, have been isolated from the leaves of Kalanchoe TLC, DAD-UV spectra, and HPLC-MS data. In addition to the
pinnata19 and K. daigremontiana  tubiflora,20 respectively, along known free bufadienolide 2, hellebrigenin 12, hellebrigenin-3-
with the known compounds bryophyllin A 2, bersaldegenin- acetate 13 and bryophyllin B 14, three new glucosylated bufa-
1,3,5-orthoacetate 3, daigremontianin 4, bersaldegenin-1-acetate dienolides, namely kalanchosides A 15, B 16 and C 17, together
Published on 17 March 2011 on http://pubs.rsc.org | doi:10.1039/C0NP00032A

5, and bersaldegenin-3-acetate 6. In line with the structure– with the known glucoside thesiuside 18, were isolated from the
activity relationship, the 1,3,5-orthoacetate and a-pyrone moiety aerial parts of Kalanchoe gracilis.27
were shown to be the important structural elements for the
strong insecticidal activity and cytotoxicity.21Although bufadie-
2.2 Hyacinthaceae
nolides occur in a wide variety of genera of several families, such
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bufadienolide orthoesters were only found in six species of three The Hycinthaceae family consists of bulbous plants with thick
genera in two families: Kalanchoe daigremontiana (syn. Bryo- and sometimes contractile roots. Two genera (Urginea and
phyllum daigremontianum), K. tubiflora, and their hybrid Bowiea) of this family are known to be a source of bufadieno-
K. daigremontiana  tubiflora as well as K. pinnata from the lides, most of which are glycosides with one to three sugars in

Huimin Gao obtained her Ph.D. Brigitte Kopp is a Professor at

degree in 2006 from the Institute the Department of Pharmacog-
of Chinese Materia Medica, nosy, University of Vienna. Her
China Academy of Chinese research interests are focused on
Medical Sciences. She continued the development of methods for
at the same institute from 2006, quality assurance of herbal
working first as an assistant medicine products, phyto-
researcher and then as an asso- pharmaceuticals and food
ciate researcher (from 2008 supplements, and the isolation
onwards) She undertook post- and structure elucidation of
doctoral research (2008 and natural compounds with immu-
2009) at the Department of noregulatory, spasmolytic,
Pharmacognosy, University of choleretic or antiinflammatory
Huimin Gao Vienna, under the guidance of Brigitte Kopp activities.
Professor Brigitte Kopp. Her
research interests are focused on
the isolation and structural elucidation of biologically active
natural products.

Ruxandra Popescu received her Zhimin Wang is a Professor at

Ph.D. degree in Natural the Institute of Chinese Materia
Sciences in 2010 under the Medica, China Academy of
supervision of Professor Brigitte Chinese Medical Sciences. His
Kopp, at the Department of group is actively involved in the
Pharmacognosy, University of isolation, structure determina-
Vienna. Her research focused on tion, and synthetic optimization
cancer drug discovery from of natural products.
natural products.

Ruxandra Popescu Zhimin Wang

954 | Nat. Prod. Rep., 2011, 28, 953–969 This journal is ª The Royal Society of Chemistry 2011
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a chain linked to the 3-hydroxyl of the genin, although branched Urgineoideae of the Hyacinthaceae, in which both Drimia and
chain glycosides and sugar attachment to other positions than Urginea genera have been placed, is phytochemically character-
3b-OH has also been found. ized by the presence of bufadienolides.30 The botanical classifi-
Based on the taxonomic proximity of the genera Scilla and cation of Drimia and Urginea is ambiguous. Urginea has at times
Urginea, and the knowledge that bulbs of Urginea maritima been included in the genus Drimia, which has resulted in various
(Scilla maritima) contained bufadienolides, Scilla maderensis was synonyms,31 as for example D. robusta (syn. D. alta), D. altissima
suspected to contain similar constituents. TLC screening of (syn. U. altissima) and D. depressa (syn. U. capitata and
different extracts also supported the presence of substances such U. depressa).
as proscillaridin and scillaren A.28 Phytochemical screening of The bulbs or leaves of Drimia robusta have been widely used in
Scilla natalensis and Ledebouria ovatifolia extracts revealed that South Africa for medicinal purposes,32 e.g. as a diuretic for
S. natalensis contained both saponins and bufadienolides, while cleaning the bladder and treating diseases of the uterus. The
L. ovatifolia contained only bufadienolides.29 The subfamily active principles were identified as two 6b-acetoxy bufadienolide

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derivatives: 6b-acetoxy-3b,8b,12b,14b-tetrahydroxybufa- 3-epi-O-methylscilliphaeosidin 22, 3-oxo-11a,14b-dihydrox-

4,20,22-trienolide (12b-hydroxyscillirosidin) 1931 and 6b-ace- ybufa-4,20,22-trienolide 23, 12a-scilliphaeosidin 2438 and 16b-
toxy-3b,8b,14b-trihydroxy-12-oxobufa-4,20,22-trienolide 20.33 acetoxy-3b,14b-dihydroxy-19-formylbufa-4,20,22-trienolide 25
The former was previously identified in Urginea sanguinea34 and (scillicyanosidin).39 Accordingly, seven 4,20,22-bufatrienolide
U. maritima;35 however, no spectroscopic or physical data were glycosides, 26–32, were identified in the bulbs of U. maritima38,40
provided. An investigation by Pohl31 gave a detailed structural and U. altissima31 as 6b-acetoxy scillarenin 3-O-b-D-glucopyr-
elucidation and stereochemistry on the basis of MS and NMR anoside 26, 6b-acetoxy scillarenin 3-O-b-D-glucopyranosyl-(1/
spectra. 4)-a-L-rhamnopyranoside 27, scillirubrosidin 3-O-a-L-rhamno-
The bulb sap of Urginea epigea, like many other South African pyranosyl-(1/4)-a-L-rhamnopyranoside 28, scillirosidin 3-O-b-
Urginea species, usually induces skin irritation upon handling, D-glucopyranosyl-(1/4)-a-L-rhamnopyranoside 29, scillarenin
indicating the presence of bufadienolides. A new bufadienolide 3-O-b-D-glucopyranosyl-(1/4)-30 -O-acetyl-a-L-rhamnopyrano-
with a double bond at C-3 position, 14b-hydroxybufa-3,5,20,22- side 30, scillarenin 3-O-b-D-glucopyranosyl-(1/4)-20 -O-acetyl-
tetraenolide 21, was isolated from its methanol extract.33 This a-L-rhamnopyranoside 31,38 and 14b-hydroxybufa-4,20,
class of compounds has been previously identified in the 22-trienolide 3b-O-{a-L-rhamnopyranosyl-[(1/4)-b-D-gluco-
composition of Scilla maritima36 and Bersama abyssinica.37 pyranosyl]-(1/3)-a-L-rhamnopyranoside} (urginin) 32.31 These
Moreover, this is the first reported natural occurrence of compounds were accompanied by a 3,20,22-bufatrienolide
a bufadienolide with a 3,5-diene system, although they have been glycoside, 11a-hydroxyscilliglaucoside 33, and a bufadienolide
previously synthesized by removing the sugar group from scilli- glycoside, 11a-acetylgamabufotalin 3-O-(4-O-b-D-glucopyr-
glaucoside. anosyl)-a-L-rhamnoside 34.40 In addition, two unusual glyco-
Several new free 4,20,22-bufatrienolides from the bulb of sides, lydenburgenin 35 and riparianin 36, along with the known
Urginea maritima and U. lydenburgensis were assigned as compound rubellin 37, in which the aglycone was doubly linked

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to the sugar moiety, were identified as main active compounds of is consistent with chemotaxonomic trends for the Urgineoideae
Urginea lydenburgensis,39 U. riparia and Drimia macrocentra.41 family.
The sugar linkage of compounds 35–37 is similar to that of the
previously isolated compounds orbicuside A 38 and tyledoside A
2.3 Ranunculaceae
39 from the Ranunculaceae family,42,43 which possess multi-
linked positions. This type of multi-linked bufadienolide glyco- Helleborus is the only genus belonging to the family of Ranun-
side can be synthesized using the procedure developed by Dixon culaceae known to contain bufadienolides and their analogues.
and coworkers.44 Three bufadienolide derivatives of hellebrigenin with the steroid
Investigation into bufadienolides by Crouch led to the isola- aglycone of hellebrin were previously isolated from this genus,3
tion of two additional compounds, 3b,16b-dihydroxy-5b-bufa- and much effort has been devoted to species such as H. torqua-
20,22-dienolide 40 and 16b-hydroxy-5b-bufa-20,22-dienolide 3b- tus,46 H. orientalis,47 H. caucasicus,48 H. bocconei subsp. siculus49
O-b-D-galactoside 41, from the bulbs of the poisonous plant D. and H. thibetanus.50 Three novel bufadienolides, hellebortin A
depressa from South Africa.45 The isolation of these compounds 42, B 43 and C 44, have been isolated by bioassay- and
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radioimmunoassay-directed HPLC analyses of the methanol detected, although they have been described from Sterculiaceae
extract of the seeds of H. torquatus, of which 42 and 44 are the species.55
first isolated 19-norbufadienolides.46 Fresh rhizomes of H. ori-
entalis provided the bufadienolide glycoside 45, which showed 3 Animal sources of bufadienolides
a potent cytotoxic effect. The rhizomes also contained the known
compound 46 and deglucohellebrin 47,47 which were originally The animal sources of bufadienolides include species belonging
isolated from Urginea altissima51 and Helleborus odorus,52 and to the families Bufonidae (toads), Lampyridae (fireflies) and
were recently also identified in the underground parts of H. Colubridae (snakes) as well as mammalian tissues.2,3 Rapid
caucasicus, an endemic plant of Caucasian flora.48 Phytochemical development of the preparative and analytical technology
investigation of the rhizomes of the Chinese species H. thibetanus allowed the identification of new bufadienolides in toad and
led to the isolation and characterization of two new bufadieno- snake species, whereas no new bufadienolides from the Lampy-
lides, tigencaoside A 48 and tigencaoside B 49, as well as the ridae family were reported during this period.
known compounds 12 and 47.50
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3.1 Bufonidae
2.4 Leguminoseae Toad species are one of the richest sources of bufadienolides. The
compounds occur in the free form, and also conjugated at the C-3
The Leguminoseae is the latest family in which the presence of
position with sulfates, dicarboxylic esters and amino acid
natural bufadienolides was reported. A novel bufadienolide,
dicarboxylic acid esters. An overview of well-known bufadieno-
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hellebrigenin-3-O-a-L-rhamnopyranosyl-(1/4)-O-b-D-galacto-
lide-containing Bufo species was included in a comprehensive
pyranoside 50,53 was isolated from the seeds of Mimosa pudica,
review.2 Recent investigations indicated that bufadienolides are
used in traditional Indian medicine as an effective emetic. The
present in several diverse anurans56–60 of the Bufonidae family,
stems of Millettia ovalifolia are poisonous, and indicated the
except Melanophryniscus species.61,62
presence of a bufadienolide, 4,5-dehydro-14b-hydroxy-
A hemisuberate of hellebrigenin 52 was isolated from the skin
scilladienolide 3-O-b-D-glucopyranoside 51.54
of Bufo bufo gargarizans,63 and three unusual bufadienolide
conjugates, 11a,19-dihydroxytelocinobufagin-3-(12-hydroxy-
2.5 Sterculiaceae
dodecanoic acid ester) 53, 11a,19-dihydroxytelocinobufagin-3-
Species belonging to the Sterculiaceae family have been tradi- (14-hydroxy-7-tetradecenoic acid ester) 54 and 11a,
tionally used in South African medicine. Phytochemical and 19-dihydroxytelocinobufagin-3-(14-hydroxytetradecanoic acid
pharmacological studies of five species, namely Cola greenwayi, ester) 55, were separated from the ovaries of the cane toad,
C. natalensis, Dombeya burgessiae, D. cymosa and Hermannia B. marinus. The conjugates that possessed hydroxylated mono-
depressa, suggested the probable presence of bufadienolides in carboxylic acids differed from the previously described esters.64
the leaves of D. burgessiae and D. cymosa. No cardenolides were The commercial toad venom ‘chansu’ provided five new bufa-

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dienolides with inhibitory activity towards cancer cell growth, acetoxybufarenogin 65 and 11a,12b-dihydroxybufalin 66.68 Six
19-oxobufalin 56, 1b-hydroxybufalin 57, 3b-for- new bufadienolides, 67–72, and two new C23 steroids 73 and 74,
myloxyresibufogenin 58, 19-oxodesacetylcinobufagin 59 and 6a- together with three known compounds 75–77, were isolated and
hydroxycinobufagin 60, together with the known compounds characterized from the venom of B. bufo gargarizans by spec-
5b-hydroxybufotalin 61, 19-hydroxyresibufogenin (resibufagi- troscopic analysis and single-crystal X-ray diffraction.69
nol) 62 and 12b-hydroxyresibufogenin 63.65 Compounds 61–63 Additionally, a rare series of 20,21-epoxybufenolides, namely
were originally obtained by semisynthesis from bufotalin, resi- 20S,21-epoxyresibufogenin 78, 20R,21-epoxyresibufogenin 79,
bufagin and resibufogenin.66 Investigations of ‘chansu’ by several 3-oxo-20S,21-epoxyresibufogenin 80, 3-O-formyl-20S,21-
different research groups led to the extraction of the unconju- epoxyresibufogenin 81 and 3-O-formyl-20R,21-epoxyr-
gated bufadienolides 5b,12b-dihydroxycinobufagin 64,67 16b- esibufogenin 82, were isolated from the Chinese drug ‘chansu’.70
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The structure of 20,21-epoxyresibufogenin has been previously 3.2 Colubridae

obtained from this drug; however, the stereochemistry of the
The Asian snake Rhabdophis tigrinus has specialized defensive
20,21-epoxy group was not determined.71 The compounds
glands located on its neck, which contain steroidal toxins of the
20S,21R-epoxymarinobufagin 83, marinobufagin 84, bufalin 85,
bufadienolide class.2 A recent investigation reported the presence
telocinobufagin 86 and hellebrigenin 12 were extracted from the
of seventeen bufadienolides in the nuchal gland fluid of
secretion of the Brazilian toad Rhinella schneideri. Furthermore,
R. tigrinus, among which six compounds, 98–103, were new
chemically modified derivatives such as 3b-acetoxymar-
natural products. Further observations have confirmed that the
inobufagin 87, 3b-acetoxybufalin 88, 3b-acetoxytelocinobufagin
defensive steroids are not synthesized by R. tigrinus itself.
89 and 3b-acetoxy-20S,21R-epoxymarinobufagin 90, were
Instead, the snake is dependent on a diet of toads from which it
prepared from corresponding natural bufadienolides.59
can sequester these compounds.14 In the absence of direct dietary
In addition to classical separation procedures, on-line struc-
sources of bufadienolides, the young R. tigrinus are chemically
tural characterization technologies, such as LC–DAD-MS/MS
protected from predators by maternal provisioning.15
and LC–DAD-SPE-NMR, have also been employed for the
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identification of natural or biologically modified bufadienolides.

3.3 Mammalian bufadienolides
Several investigations were published on the identification of
bufadienolides in crude toad venom72–75 and its preparations76–78 The presence of bufalin and its derivatives in adrenal glands,
as well as in toad skin,79 utilizing the comparison of the retention hypothalamus, plasma, urine and bile of human, porcine and
time with reference substances and on-line UV spectra, as well as canine origin has been recorded previously.3,80,81 Biochemical,
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typical fragmentation rules obtained by LC–DAD-MS/MS. The immunological and spectroscopic methods led to the isolation of
detailed APCI-MS fragmentation rules of bufadienolides compounds such as proscillaridin A 104, identified in human
summarized by Ye provided a good basis for the detection of plasma,82–84 and marinobufagin 84, purified from human urine
chemical constituents in the crude toad venom and the identifi- after acute myocardial infarction.85 Enhanced marinobufagin
cation of 35 bufadienolides, including four new constituents. production occurred in volume-expanded rats86 and in patho-
However, with the exception of a-bufalin 91, the structures of logical states associated with fluid retention, including
three other compounds were not finally determined due to preeclampsia,87,88 hypertension in the end-stage renal disease,89
limited MS information.72 A similar investigation was conducted and salt-sensitive hypertension in Dahl rats.90 Another digitalis-
for systematic screening of novel bufadienolides in the toad skin like factor was isolated from the peritoneal dialysate of volume-
of Bufo bufo gargariizans by UPLC–ESI-TOF-MS. In addition expanded patients with chronic kidney failure and its structure
to 19 known bufadienolides, 20 putative novel compounds, was not determined.91 Recently, telocinobufagin 86 was identi-
including 8 stereoisomers, were characterized.79 HPLC and LC– fied in the plasma of patients with renal failure.92 Although
DAD-MS/MS analyses of the toad venom collected from a growing body of evidence strongly supports the hypothesis that
various Bufo species, namely B. marinus, B. melanosticus, B. endogenous bufadienolides were synthesized in, and released
viridis, and B. bufo gargarizans, indicated the occurrence of five from, the adrenal gland,93,94 the biosynthetic pathway remains
new buadienolide sulfates 92–96 and a free bufadienolide 97 in unknown. Moreover, while plant-derived bufadienolides origi-
the venom of B. melanosticus,74 These structures were charac- nate from pregnenolone, it has been shown that the production
terized in more detail by further LC–DAD-MSn and LC–SPE- of mammalian bufadienolides does not involve cholesterol side-
NMR analyses.75 chain cleavage.95

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4 Biotransformation of bufadienolides 113, desacetylcinobufagin 16-O-b-D-glucoside 114 and 3-epi-

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desacetylcinobufagin 16-O-b-D-glucoside 115,98,99 among which

Bufadienolides show potent cardiotonic and antitumor activity, compounds 111–115 were new bufadienolide derivatives. From
and interest in developing bufadienolides as anticancer agents the supernatant of the culture of P. grandiflorum and cinobufa-
has grown progressively. However, due to the narrow thera- gin, compound 108 was isolated as a major product with the yield
peutic index, the risk of cardiac toxicity has to be taken into of 37.8%, while cinobufotalin 116 and desacetylcinobufagin 117
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account. Therefore, there is a demand for analogues with were obtained as minor products. The two plant suspension
enhanced therapeutic activity and decreased toxicity.7 Biotrans- cultures exhibited similar transformation patterns of cinobufa-
formation uses organisms to generate chemical modifications, gin, such as 5-hydroxylation and 16-O-deacetylation. Moreover,
and thus is an important tool for the development of new active glucosylation was identified as a characteristic modification
compounds.96 Biotransformation has been successfully applied occurring in the suspension culture of C. roseus with cinobufagin.
to several natural bufadienolides, including bufalin 85, cinobu- Bufalin in a cell suspension culture of P. grandiflorus provided
fagin 105, resibufogenin 106 and bufotalin 107, increasing the two new products, 3-epi-telocinobufagin 118 and 3-epi-bufalin 3-
diversity of this class of compounds. O-b-D-glucoside 119.101 In the culture of P. grandiflorum with
resibufogenin, new biotransformed products, 1b-hydroxy-resi-
4.1 Plant cell suspension culture bufogenin 120 and 3-epi-marinobufagin 121, were identified
Preliminary screening experiments indicated Catharanthus roseus together with the known compounds 3-epi-resibufogenin 122 and
and Platycodon grandiflorum as the optimal cell suspension marinobufagin 84.102 In addition, resibufogenin could be iso-
culture systems for cinobufagin, leading to a great variety of merized by plant cell cultures of Ginkgo biloba.103
compounds.97–100 Incubation of cinobufagin with C. roseus
yielded eight products, namely desacetylcinobufotalin 108, 3-epi-
4.2 Microbial transformation
desacetylcinobufagin 109, 3-epi-desacetylcinobufotalin 110,
1b-hydroxyldesacetylcinobufagin 111,97 cinobufagin 3-O-b-D- More than 25 strains of filamentous fungi and 4 strains of
glucoside 112, 3-oxo-desacetylcinobufagin 16-O-b-D-glucoside bacteria were screened for biotransformation of bufadienolides

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including bufalin 85, cinobufagin 105, resibufogenin 106 and lead to the formation of new bufadienolide derivatives. Cino-
bufotalin 107. As a result, several microbes were considered bufagin can suffer metabolic changes in rat bile, generating
representative for the biotransformation potential, including compounds such as 1a-hydroxy-3-epi-desacetylcinobufagin 189,
Alternaria alternata AS 3.4578,104,106,107,119 Mucor spino- 1b-hydroxy-3-epi-desacetylcinobufagin 190, 1a,5a-dihydroxy-3-
sus,105,112,113 Aspergillus niger,105 Cunninghamella blakesleana,113 epi-desacetylcinobufagin 191, and 2a,5b-dihydroxy-3-epi-desa-
Mucor polymorphosporus,115,118 Mucor subtilissimus, Pseudo- cetylcinobufagin 192, and the known 3-epi-desacetylcinobufagin
monas aeruginosa,116 Fusarium solani AS 3.1829,108 Penicillium 109, 3-epi-desacetylcinobufotalin 110, desacetylcinobufagin 117,
aurantigriseum AS 3.4512109 and Nocardia sp. NRRL 5646.117 12b-hydroxy-3-epi-desacetylcinobufagin 128, as well as 3-oxo-
Fungi and bacteria cultures provided 76 biotransformed prod- desacetylcinobufagin 131. In addition to the desacetylation at C-
ucts with slight structural differences, among which 40 16, hydroxylation at C-1 and C-5 preferentially occurred in rat
compounds were new bufadienolides. bile.123 After oral administration of ‘chansu’, four metabolites
According to the bioconverted products shown in Table 1, were detected in rat plasma, of which two were identified as 3-epi-
cinobufagin, resibufogenin and bufalin were biotransformed to bufalin 91 and 3-epi-resibufogenin 122.124
12b-hydroxylated products, suggesting that bufadienolides
follow the same transformation pathway in the cited microbes.
Interestingly, a chemical method was recently reported for the 4.4 Analytical determination of bufadienolides in biological
12b-hydroxylation of steroids; however, in comparison with the samples
mentioned biocatalytic means, the method yielded a lower Bufadienolides have a narrow therapeutic index in clinical prac-
number of products and it was accompanied by the formation of tice with the occurrence of severe cardiac toxicity. Thus, the
by-products.121 Additionally, 7b-hydroxylation was also readily importance of detecting and measuring bufadienolides in body
observed for bufalin as a parallel and competing metabolic route fluids and tissues for therapeutic drug monitoring led to the
with 12b-hydroxylation, but not for cinobufagin and resibufo- development of HPLC-UV methods applied to rat serum,125,126 rat
genin in the same cultures, which might result from the close plasma127,128 and human liver.129 However, HPLC-UV analysis
spatial positions of C-7 and the epoxy ring for the latter two cannot always be applied for the identification of bufadienolides
compounds. Again, for cinobufagin and bufalin, selective dehy- in biological matrices. The search for sensitive detection methods
drogenation at C-3 and deacetylation at C-16 in microbial prompted the establishment of the fluoroimmunoassay method
cultures were considered to be similar to the metabolic patterns for the determination of bufalin; however, the lack of commer-
in rat liver microsomal fraction122 and rat bile, respectively.123 cially available assay kits limits its usage.130 LC–MS/MS analysis
is a more sensitive and applicable tool that has been used for the
identification of bufadienolides in dog131 and rat plasma,132 and
4.3 Metabolites of bufadienolides in vivo
gastrointestinal tract model in vitro.133 Recently, a novel, rapid
In addition to biotransformations occurring in cultures of plant and specific UPLC–ESI-MS/MS method has been established for
cells, bacteria or fungi, the in vivo metabolic pathway can also the simultaneous determination of bufalin, cinobufagin and

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Table 1 Products of microbial biotransformation

Product of biotransformation Parent compound Microbe

12b-Hydroxyldesacetylcinobufagin 123a Cinobufagin Alternaria alternata104

Aspergillus niger105
Mucor spinosus105
3-Oxo-12b-hydroxylcinobufagin 124a Cinobufagin Alternaria alternata104,106
3-Oxo-12b-hydroxyldesacetylcinobufagin 125a Cinobufagin Alternaria alternata104,106
12-Oxo-cinobufagin 126a Cinobufagin Alternaria alternata104
3-Oxo-12a-hydroxylcinobufagin 127a Cinobufagin Alternaria alternata104
3-epi-12b-Hydroxyldesacetylcinobufagin 128a 3-epi-Desacetylcinobufagin Alternaria alternata106
12-Oxo-desacetylcinobufagin 129a Cinobufagin Alternaria alternata107
3-Oxo-cinobufagin 130 Cinobufagin Fusarium solani AS 3.1829108
Penicillium aurantigriseum109,116
Mucor spinosus105
3-Oxo-deacetylcinobufagin 131 Cinobufagin Aspergillus niger105
Published on 17 March 2011 on http://pubs.rsc.org | doi:10.1039/C0NP00032A

Mucor spinosus105
Penicillium aurantigriseum109,116
3-epi-Deacetylcinobufagin 109 Cinobufagin Penicillium aurantigriseum109
3-epi-Cinobufagin 132 Cinobufagin Penicillium aurantigriseum109
12b-Hydroxylcinobufagin 133 Cinobufagin Alternaria alternata104,106
Aspergillus niger105
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Mucor spinosus105
1b-Hydroxylcinobufagin 134 Cinobufagin Mucor spinosus105
1b,12b-Dihydroxylcinobufagin 135 Cinobufagin Mucor spinosus105
7b,12b-Dihydroxylcinobufagin 136a Cinobufagin Cunninghamella elegans110
Syncephalastrum racemosum111,120
5,12b-Dihydroxylcinobufagin 64a Cinobufagin Syncephalastrum racemosum111,120
4b,11a-Dihydroxylcinobufagin 137a Cinobufagin Syncephalastrum racemosum111,120
4b,12a-Dihydroxylcinobufagin 138a Cinobufagin Syncephalastrum racemosum111,120
Cinobufotalin 116 Cinobufagin Syncephalastrum racemosum111
Desacetylcinobufagin 117 Cinobufagin Aspergillus niger105
Mucor spinosus105
Penicillium aurantigriseum109
3-epi-12b-Hydroxylcinobufagin 139 Cinobufagin Mucor spinosus105
5b-Hydroxylcinobufagin 140 Cinobufagin Mucor spinosus105
3-Acetylcinobufagin 141 Cinobufagin Nocardia sp.117
Bufalin 85 Cinobufagin Aspergillus niger105
12b-Hydroxylbufalin 142 Cinobufagin105 Alternaria alternata106
Aspergillus niger105
Bufalin106,113 Mucor spinosus112
Cunninghamella blakesleana113
7b-Hydroxylbufalin 143a Cinobufagin105 Alternaria alternata106
Aspergillus niger105
Bufalin106,112,113 Mucor spinosus112
Cunninghamella blakesleana113
11b-Hydroxylbufalin 144a Bufalin Mucor spinosus112
16a-Hydroxylbufalin 145a Bufalin Mucor spinosus112
7b,16a-Dihydroxylbufalin 146a Bufalin Mucor spinosus112
1b,7b-Dihydroxylbufalin 147a Bufalin Mucor spinosus112
1b,12b-Dihydroxylbufalin 148a Bufalin Mucor spinosus112
3-epi-7b-Hydroxylbufalin 149a Bufalin Mucor spinosus112
12a-Hydroxylbufalin 150a Bufalin Cunninghamella blakesleana113
7b,12a-Dihydroxylbufalin 151a Bufalin Cunninghamella blakesleana113
7b,15a-Dihydroxylbufalin 152a Bufalin Mucor spinosus113
5b,7b-Dihydroxylbufalin 153a Bufalin Mucor spinosus113
3-Oxo-bufalin 154 Bufalin Penicillium aurantigriseum109
3-epi-Bufalin 91 Bufalin Fusarium solani AS 3.1829108
Penicillium aurantigriseum109
1b-Hydroxylbufalin 57 Bufalin Mucor spinosus112
3-Oxo-12b-hydroxylbufalin 155 Bufalin Alternaria alternata106
3-Oxo-7b-hydroxylbufalin 156 Bufalin Alternaria alternata106
15a-Hydroxylbufalin 157 Bufalin Mucor spinosus112
15b-Hydroxylbufalin 158 Bufalin Mucor spinosus112
Telocinobufagin (5-hydroxylbufalin) 86 Bufalin Mucor spinosus112
3-Oxo-12b-hydroxylresibufogenin 159a Resibufogenin Alternaria alternata106
Pseudomonas aeruginosa114
3-epi-7b-Hydroxylresibufogenin 160a Resibufogenin Mucor polymorphosporus115,118
5,7b-Dihydroxylresibufogenin 161a Resibufogenin Mucor polymorphosporus115,118
7a-Hydroxylresibufogenin 162a Resibufogenin Mucor polymorphosporus115,118
3-epi-12b-hydroxylresibufogenin 163a Resibufogenin Mucor polymorphosporus115,118
5,12b-Dihydroxylresibufogenin 164a Resibufogenin Mucor polymorphosporus115,118
3-epi-12a-Hydroxylresibufogenin 165a Resibufogenin Mucor polymorphosporus115,118
5,12a-Dihydroxylresibufogenin 166a Resibufogenin Mucor polymorphosporus115,118

964 | Nat. Prod. Rep., 2011, 28, 953–969 This journal is ª The Royal Society of Chemistry 2011
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Table 1 (Contd. )

Product of biotransformation Parent compound Microbe

a
1b,12a-Dihydroxylresibufogenin 167 Resibufogenin Mucor polymorphosporus115,118
3-Oxo-12a-hydroxylresibufogenin 168a Resibufogenin Mucor polymorphosporus115,118
12-Oxo-resibufogenin 169a Resibufogenin Mucor polymorphosporus115
3-epi-16a-Hydroxylresibufogenin 170a Resibufogenin Mucor polymorphosporus115
7b,16a-Dihydroxylresibufogenin 171a Resibufogenin Mucor polymorphosporus115,118
12b,16a-Dihydroxylresibufogenin 172a Resibufogenin Mucor polymorphosporus115,118
1b,16a-Dihydroxylresibufogenin 173a Resibufogenin Mucor polymorphosporus115,118
12a,16a-dihydroxylresibufogenin 174a Resibufogenin Mucor polymorphosporus115,118
7b-Hydroxylresibufogenin 175 Resibufogenin Mucor polymorphosporus115,118
Marinobufagin (5-hydroxylresibufogenin) 84 Resibufogenin Mucor polymorphosporus115,118
12b-Hydroxylresibufogenin 63 Resibufogenin Alternaria alternata106
Mucor polymorphosporus115
Published on 17 March 2011 on http://pubs.rsc.org | doi:10.1039/C0NP00032A

Mucor subtilissimus114
12a-Hydroxylresibufogenin 176 Resibufogenin Mucor polymorphosporus115,118
16a-Hydroxylresibufogenin 177 Resibufogenin Mucor polymorphosporus115,118
3-Oxo-16a-hydroxylresibufogenin 178 Resibufogenin Mucor polymorphosporus115,118
3-Oxo-D4-resibufogenin 179 Resibufogenin Mucor polymorphosporus115
3-epi-Resibufogenin 122 Resibufogenin Fusarium solani AS 3.1829108
Penicillium aurantigriseum109
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3-Oxo-resibufogenin 180 Resibufogenin Penicillium aurantigriseum109

Pseudomonas aeruginosa116
3-Acetyl-15b-hydroxylbufotalin 181 Resibufogenin Nocardia sp117
3-Acetyl-resibufogenin 182 Resibufogenin Nocardia sp117
12b-Hydroxylbufotalin 183a Bufotalin Alternaria alternata119
3-Keto-12b-hydroxylbufotalin 184a Bufotalin Alternaria alternata119
3-Keto-bufotalin 185 Bufotalin Alternaria alternata119
3-epi-Marinobufagin 121 Marinobufagin Comamonas testosteroni17
3-Oxo-marinobufagin 186 Marinobufagin Comamonas testosteroni17
D1,4-3-Oxo-resibufogenin 187 Marinobufagin Comamonas testosteroni17
D1,4-3-Oxo-bufalin 188 Marinobufagin Comamonas testosteroni17
a
Reported as a new bufadienolide.

resibufogenin in rat plasma. The three analytes could be deter- SF-268 (glioma-derived) cell lines. Among them, compound 6
mined within 3.0 min, meeting the requirements for high- and 11 displayed almost complete inhibition of MCF-7 and NCI-
throughput determination of biosamples.134 H460 cell growth at a concentration of 4 mg/mL. Compound 5
showed weaker cytotoxicity towards MCF-7 cell line and
inhibitory activity against the NCI-H460 cell line.24 Compound
5 Antitumor activities
36, riparianin, displayed moderate activity against the MCF-7,
Bufadienolides possess a wide range of bioactivities, including TK10 (renal) and UACC62 (melanoma) cell lines, with mean
cardiotonic, renal sodium excretion, blood pressure stimu- TGI (total growth inhibition) values of <3.5, <7 and <10.5 ppm
lating,135–137 biphasic effects on NO production,138 immuno- respectively.41 Bufadienolide monoglycosides 45–47 showed
regulatory139–141 and antineoplastic. Of all the activities described particular sensitivity against HSC-2 (squamous cell carcinoma)
for bufadienolides, the antitumor activity is to date one of the and A375 (skin) cancer cell lines, and relative resistance to
most interesting research subjects. Here, we provide a general HepG2 (liver) cells. Although the bufadienolide glucoside 46
overview of the antitumor activity of a number of bufadienolides indicated cytotoxic activity on both tumor cells and normal
obtained from plants, animals and by biotransformation. human pulp cells (HPC), the bufadienolide rhamnosides 45 and
47 provided higher tumor specificity and therefore only weak
cytotoxicity against HPC. These results suggested that the
5.1 Antitumor activities in vitro
monosaccharides attached at the bufadienolide aglycone
Bufadienolides obtained from plants, animals and by biotrans- contributed to the mediation of the tumor specificity.47 The
formation have been extensively evaluated for their cytotoxic bufadienolide derivatives 56–60 were found to be active against
activity towards a variety of cancer cells, including human the KB (nasopharynx) and HL-60 (leukemia) cancer cell lines,
leukemia, hepatoma, lung carcinoma, and gastro-intestinal and with increased sensitivity towards the latter.65 20S,21-Epoxy-
breast cancers. All the bufadienolides showed potent cytotoxic bufadienolide, a cytokine interleukin (IL)-6 inhibitor, also
activity. As already indicated in the literature, these data showed cytotoxic effect on HL-60 cells.142,143
contribute to structure–activity relationship studies aimed at the Products resulting from the biotransformation of cinobufagin
design of novel bufadienolides of pharmaceutical interest. and resibufogenin by P. grandiflorum exhibited potent cytotoxic
Interestingly, marked differences characterize the potencies of activity against Bel-7402 (liver), HeLa (cervical), BGC-823
these structurally similar compounds. For instance, compounds (gastric), MCF-7 and HL-60 human cancer cell lines. 3-epi-
5, 6 and 8–11 from the family Crassulaceae were evaluated for Desacetylcinobufagin 109, 3-epi-desacetylcinobufotalin 110 and
their cytotoxicity towards MCF-7 (breast), NCI-H460 (lung) and 1b-hydroxyldesacetylcinobufagin 111 showed higher cytotoxic

This journal is ª The Royal Society of Chemistry 2011 Nat. Prod. Rep., 2011, 28, 953–969 | 965
 View Online

activity against HL-60 cells than the parent compound, with IC50 study was carried out on the inhibitory effect of resibufogenin on
values of 4.90, 0.02 and 3.10 mM, respectively. It is suggested that transplantation tumor growth of HeLa human cancer cells in
1b-hydroxylation and 3-epimerization could improve the cyto- nude mice.154 A dose of 15 mg/kg of resibufogenin displayed
toxicity of cinobufagin derivatives.97 In contrast, minimal remarkable inhibitory activity, although accompanied by acute
structural modifications of bufadienolides such as substitution neurotoxicity. An ethanol extract of Chinese toad venom has
with 3b-hydroxy, 3-ketone or 16-deacetyl groups achieved by been shown to inhibit the growth of transplated H22 liver cancer
microbial transformation decreased the cytotoxic effect.109 The cells in nude mice at doses of 0.5–5 mg/kg and to cause hepato-
metabolites of cinobufagin were less cytotoxic than cinobufa- toxicity at 5 mg/kg.155
gin.112 Similarly, mono-hydroxylation at different positions of
the structure of cinobufagin could significantly alter the cyto-
toxic activity towards human hepatoma and leukemia cells.105 A 5.3 Toxicity of potent antitumor bufadienolides
detailed structure–activity relationship of natural bufadienolides
as well as their chemical and biotransformed derivatives was Although bufadienolides open new perspectives for anticancer
therapy, the cardiotoxicity of the compounds should be taken
Published on 17 March 2011 on http://pubs.rsc.org | doi:10.1039/C0NP00032A

described by Kamano144,145 and Ye.100 In addition, the essential

structural requirements for increasing the inhibitory activities into account. Cardiotoxicity has been attributed mainly to the
have been identified by Zhang and coauthors.117 inhibitory effect on the Na+/K+-ATPase and the alteration of
Despite the cytotoxic potency showed by bufadienolides intracellular calcium stores; the possibility of bufadienolides to
against different tumour cells, the mechanism of action under- act on sites other than the Na+/K+-ATPase has also been
raised.156 Several publications referred to intoxication caused by
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lying the growth inhibitory effect remains unknown in most

cases. Bufalin has been shown to induce cell differentation and bufadienolides or by formulations containing bufadieno-
apoptosis, events that were interlinked and regulated by distinct lides.135,157 The isolation of new bufadienolides has been carried
protein kinase C isozymes.146 In leukemic cells, bufalin has been out in an attempt to find compounds with potent anticancer and
shown to activate the activating protein (AP)-1 via the mitogen- low cardiotoxicity. Daniel and coauthors reported selective
activated protein kinase (MAPK) cascade that included c-Jun N- antitumour activity induced by hellebrin derivatives that lacked
terminal protein kinase,147 while the involvement of Tiam1 has cardioactive groups, towards malignant T lymphoblasts in
been reported in both cervical cancer and leukemia cells.148 contrast to normal peripheral blood mononuclear cells.158 This
Bufalin-induced apoptosis in endometric stromal cells was study suggested the possibility of obtaining potent anticancer
characterized by cell cycle arrest in the G0/G1 phase, down- bufadienolides with low or even no cardiotoxic effect.
regulation of the pro-survival proteins Bcl-2 and Bcl-XL, up-
regulation of the pro-apoptotic protein Bax and cleavage of
caspase-9.149 The phosphatidylinositol 3-kinase (PI3K)/Akt 6 Concluding remarks
signaling pathway was suggested to be involved in the onset of The review has highlighted the abundance of natural bufadie-
programmed cell death in bufalin-treated gastric cancer cells.150 nolides, and their products obtained by biotransformation,
This substance induced cell death by apoptosis in lung adeno- reported in the last twelve years. During the period, the narrow
carcinoma cells, which was linked to the production of reactive therapeutic index of this class of compounds encouraged a search
oxygen species.151 In adrogen-dependent and -independent for potent bioactive compounds with low or no cardiotoxicity.
prostate cancer cells, bufalin and cinobufagin increased intra- Biotransformation, an effective tool for the structural modifi-
cellular calcium levels and caused apoptosis via the mitochon- cation of natural products, led to the discovery of new bufadie-
drial pathway with translocation of Bax, release of cytochrome c nolides with unique structural diversity and potent
and activation of caspases.152 pharmacological activity. The strong antitumor activity reported
In a recent review, Mijatovic discussed the potential of for the compounds discussed herein, both in vitro and in vivo,
cardiotonic steroids to exert antineoplastic activity due to their should prompt efforts to understand the underlying mechanism
interaction with the sodium pump. The binding site of cardio- of action, and further emphasizes the possibility of developing
tonic steroids resides mainly on the a subunit of the Na+/K+- bufadienolides as antitumor agents.
ATP-ase. Since the sodium pump was shown to play a role in
cancer progression and the a subunit was found to be over-
expressed in malignant cells, inhibition of the a subunit of the
7 Acknowledgements
Na+/K+-ATP-ase by bufadienolides provides potential for their
development as anticancer agents.7 This work was supported by NSFC (30801512), the Special
Program for New Drug Innovation of the Ministry of Science
and Technology (2009ZX09301-005 and 2009ZX09308-003) and
5.2 Antitumor activities in vivo
financed within the project ‘‘Quality assurance of Herbal
The cytotoxicity of bufadienolides in vitro has been widely Medicinal Products (HMPs) from TCM’’, aided by the Austrian
studied; however, few reports have evidenced their activity Federal Ministry of Science and Research (BMWF) and the
in vivo. Bufalin exhibited a significant anti-tumor effect with no Federal Ministry for Health, Family, and Youth (B.K.). The first
marked toxicity in the orthotopic transplantation tumor model author is thankful to the BMWF for providing a postdoctoral
of human hepatocellular carcinoma in nude mice. The mecha- scholarship within the program ‘‘Technology Grants East/
nism of apoptosis induction in transplanted tumor cells indicated Central/South Asia’’, in the frame of the Eurasia–Pacific Uninet
the up-regulation of the expression of the gene Bax.153 A similar administered by OeAD-GmbH.

966 | Nat. Prod. Rep., 2011, 28, 953–969 This journal is ª The Royal Society of Chemistry 2011
 View Online

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