COMPARATIVE ASSESSMENT OF THE ANTIOXIDANT AND THE

ANTI DIABETICS POTENTIAL OF THE VARIOUS EXTRACT

FRACTIONS OF Markhamia tomentosa

BY

OBISESAN TEMITAYO OLAMIDE

(181156)

A PROJECT

SUBMITTED TO THE DEPARTMENT OF BIOCHEMISTRY,

FACULTY OF PURE AND APPLIED SCIENCES,

LADOKE AKINTOLA UNIVERSITY OF TECHNOLOGY,

OGBOMOSHO, OYO STATE.

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

AWARD OF BACHELOR OF TECHNOLOGY (B. TECH) IN

BIOCHEMISTRY.

OCTOBER, 2024
 CERTIFICATION

This is to certify that this project work was done by Obisesan Temitayo Olamide, with

matriculation number 181156 under the supervision of Prof. O. T. Adedosu, in partial fulfillment

of the requirement for the award of Bachelor of Technology (B. Tech) Degree in Biochemistry.

________________ __________________

Prof. O. T. Adedosu Date

Supervisor

_________________ ___________________

Prof. J. A. Badmus Date

Head of Department

_________________ ___________________

External Examiner Date

ii
 DEDICATION

I dedicate this project work to Almighty God.

iii
 ACKNOWLEDGEMENT

I want to dedicate this project report to Almighty God, who has made it a reality.

I am grateful to Prof. O. T. Adedosu, my supervisor. Your seasoned advice, counsel, and

reprimand, which you do not withhold, when necessary, all contributed significantly to the

effective completion of this job. Thank you very much.

My profound gratitude goes to my dearest parent, Mr. and Mrs. Obisesan and my siblings. Thank

you so much for your words of encouragement, financial support, prayers, moral support

throughout my B. Tech degree program, God bless you all richly.

Finally, I acknowledge the effort of everyone who contributed to the success of this project. Thank

you.

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 TABLE OF CONTENTS

TITLE PAGE i

CERTIFICATION ii

DEDICATION iii

ACKNOWLEDGEMENT iv

TABLE OF CONTENTS v

LIST OF FIGURES viii

LIST OF TABLES x

ABSTRACT xi

CHAPTER ONE 1

1.0 INTRODUCTION AND LITERATURE REVIEW 1

1.1 FREE RADICAL 1

1.1.1 CHARACTERISTICS OF FREE RADICALS 6

1.1.2 GENERATION OF FREE RADICALS 11

1.2 REACTIVE OXYGEN SPECIES 14

1.2.1 SOURCES OF ROS 14

1.3 OXIDATIVE STRESS AND ASSOCIATED DISEASES 19

1.3.1 OXIDATIVE STRESS AND THEORY OF AGING 19

1.3.2 OXIDATIVE STRESS AND CVDS 23

1.3.3 OXIDATIVE STRESS AND DIABETES 26

1.3.4 OXIDATIVE STRESS AND CANCER 29

1.4 ANTIOXIDANTS 31

1.4.1 CLASSIFICATION OF ANTIOXIDANTS 32

v
1.4.2 MODE OF ACTION OF ANTIOXIDANTS 36

1.5 DIABETES MELLITUS 39

1.5.1 TYPES OF DIABETES 41

1.5.1.1 TYPE 1 DIABETES MELLITUS 41

1.5.1.2 TYPE 2 DIABETES 42

1.5.2 NATURAL MEDICINES FOR DIABETES THERAPY 43

1.6 Markhamia tomentosa 47

1.6.1 BOTANY 50

1.6.2 PLANT DESCRIPTION 51

1.7 BIOCHEMICAL PARAMETERS STUDIED 53

1.7.1 FLAVONOIDS 54

1.7.2 EFFECTS OF FLAVONOIDS ON HUMAN HEALTH 56

1.7.3 HYDROXYL RADICAL (OH SCAVENGING) 62

1.7.4 INHIBITION OF LIPID PEROXIDATION 63

1.7.5 α-AMYLASE 67

1.7.6 α-GLUCOSIDASE 69

1.8 THE OBJECTIVE OF THE STUDY 71

CHAPTER TWO 72

MATERIALS AND METHODS 72

2.1 MATERIALS 72

2.2 REAGENTS 72

2.3 SOURCES AND IDENTIFICATION OF PLANTS 72

vi
2.4 PROCESSING OF LEAF SAMPLE 73

2.5 EXTRACTION OF LEAF SAMPLE 73

2.6 IN-VITRO STUDY 73

2.6.1 DETERMINATION OF 1,1-DIPHENYL-2-PICRYLHYDRAZYL (DPPH) RADICAL

SCAVENGING ACTIVITY 73

2.6.2 DETERMINATION OF TOTAL FLAVONOID CONTENT 75

2.6.3 QUERCETIN STANDARD CURVE 76

2.6.4 DETERMINATION OF PERCENTAGE HYDROXYL RADICAL SCAVENGING

ACTIVITY (2-DEOXYRIBOSE ASSAY) 77

2.6.5 DETERMINATION OF PERCENTAGE INHIBITION OF LIPID PEROXIDATION

PRINCIPLE 80

2.6.6 DETERMINATION OF PERCENTAGE INHIBITION OF ALPHA-AMYLASE

ACTIVITY 82

2.6.7 DETERMINATION OF PERCENTAGE INHIBITION OF ALPHA-GLUCOSIDASE

ACTIVITY 83

CHAPTER THREE 86

RESULTS 86

CHAPTER FOUR 98

DISCUSSION AND CONCLUSION 98

4.1 DISCUSION 98

4.2 CONCLUSION 100

REFERENCES 101

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 LIST OF FIGURES

Fig 1.1: Diagram of Reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive

sulfur species (RSS) 9

Fig 1.2: The generation of free radicals 12

Fig 1.3: Mitochondrial ROS production 15

Fig 1.4: Diagram illustrating peroxisomal enzymes involved in the generation or breakdown of

ROS 17

Fig 1.5: The effects of oxidative stress on aging 21

Fig 1.6: Oxidative stress mechanisms in contrast induced nephropathy and cardiovascular

disorders 25

Fig 1.7: Oxidative stress in diabetes mellitus 28

Fig 1.8: Oxidative stress and cancer 30

Fig 1.9: Chemical structures of flavonoids (a), ascorbic acid (b), benzoic acid (c), tocopherols (d),

carotenoids (α-carotene, β-carotene, lycopene, lutein) (e) 35

Fig 1.10: Mode of action of antioxidant reacting with free radicals 38

Fig 1.11: Diagram of Markhamia tomentosa 49

Fig 1.12: Markhamia tomentosa (Benth) K. Schum. ex. Engl 52

Fig 1.13: Chemical structure of flavonoids and its different types 55

Fig 1.14 The process of lipid peroxidation involves several steps 65

Fig 2.1: Graph showing the standard quercetin curve reading 77

Figure 3.1: A graphical illustration of total flavonoids for Markhamia tomentosa 87

Figure 3.2: A graphical illustration of % inhibition of DPPH activity for Markhamia

tomentosa 89

viii
Figure 3.3: A graphical illustration of % inhibition of hydroxyl radical activity for Markhamia

tomentosa 91

Figure 3.4: A graphical illustration of % inhibition of lipid peroxidation activity for Markhamia

tomentosa 93

Figure 3.5 A graphical illustration of % inhibition of α-amylase activity for Markhamia

tomentosa 95

Figure 3.6: A graphical illustration of % inhibition of α-glucosidase activity for Markhamia

tomentosa 97

ix
 LIST OF TABLES

Table 3.1: This table shows the total flavonoid values for four extracts at concentrations ranging

from 50µl to 350µl. 86

Table 3.2: This table shows the % inhibition of DPPH activity for four extracts at concentrations

ranging from 50µl to 400µl. 88

Table 3.3: This table shows the % inhibition of hydroxyl radical activity for four extracts at

concentrations ranging from 50µl to 450µl. 90

Table 3.4: This table shows the % inhibition of lipid peroxidation activity for four extracts at

concentrations ranging from 50µl to 450µl. 92

Table 3.5: This table shows the % inhibition of α-amylase activity for four extracts at

concentrations ranging from 50µl to 250µl. 94

Table 3.6: This table shows the % inhibition of α-glucosidase activity for four extracts at

concentrations ranging from 50µl to 250µl. 96

x
 ABSTRACT

Oxidative stress, caused by an imbalance between the production of reactive oxygen species (ROS)
and the body’s antioxidant defenses, plays a key role in the onset and progression of chronic
conditions such as cardiovascular diseases, neurodegenerative disorders, and diabetes.
Antioxidants, which counteract oxidative stress, are essential for maintaining cellular health. This
study focuses on Markhermia tomentosa, a medicinal plant widely used in traditional African
medicine, exploring its potential as a natural source of antioxidants and antidiabetic agents. The
leaves of Markhermia tomentosa were collected, air-dried, and subjected to extraction using ethyl
acetate, methanol, chloroform, and N-hexane. To evaluate the plant’s antioxidant activity and its
capacity to inhibit key enzymes linked to diabetes, several in vitro assays were conducted. These
included tests for DPPH radical scavenging, total flavonoid content, hydroxyl radical scavenging
activity, and lipid peroxidation inhibition. Additionally, alpha-amylase and alpha-glucosidase
inhibition assays were performed to assess the plant’s antidiabetic properties. The results
demonstrated that the ethyl acetate extract exhibited the strongest antioxidant capacity, with
96.91% inhibition of DPPH radicals. The methanol extract, with the highest total flavonoid content
(180.33 mg QE/g), also displayed significant antioxidant potential. Furthermore, the N-hexane
extract was most effective in scavenging hydroxyl radicals. The ethyl acetate extract additionally
showed the highest percentage inhibition of lipid peroxidation, highlighting its protective effects
against oxidative damage. In terms of antidiabetic activity, both the ethyl acetate and methanol
extracts significantly inhibited the enzymes alpha-amylase and alpha-glucosidase, which are
involved in carbohydrate metabolism and glucose regulation. Specifically, the ethyl acetate extract
achieved 74.68% alpha-amylase inhibition and 82.45% alpha-glucosidase inhibition, while the
methanol extract demonstrated 69.32% and 78.16% inhibition, respectively. These findings
suggest the plant’s potential in managing diabetes by slowing carbohydrate digestion and
absorption. This research underscores the importance of bioactive compounds such as flavonoids,
phenolic compounds, tannins, and alkaloids found in Markhermia tomentosa, which contribute to
its strong antioxidant and antidiabetic properties. The study concludes that the ethyl acetate and
methanol extracts of Markhermia tomentosa hold considerable promise as therapeutic agents for
addressing oxidative stress and managing diabetes. Further investigation into the plant’s
pharmaceutical potential could open new avenues for treating oxidative stress-related diseases.

xi
 CHAPTER ONE

2.0 INTRODUCTION AND LITERATURE REVIEW

1.1 FREE RADICAL

Free radicals and oxidants exhibit a dual nature, serving as both harmful and beneficial compounds

within the body (Pham-Huy et al., 2008). They arise from normal cellular processes or external

sources like pollution, cigarette smoke, radiation, and medication. When the body fails to

effectively neutralize an excess of free radicals, oxidative stress occurs, leading to the

accumulation of these compounds. Free radicals play a dual role in biological systems: they are

essential for cellular signaling and immune defense but are also implicated in oxidative damage

and disease (Pham-Huy et al., 2008). The balance between free radical production and antioxidant

defenses is crucial for maintaining cellular homeostasis. When this balance is disrupted, oxidative

stress occurs, leading to damage to lipids, proteins, and DNA, and contributing to diseases like

cancer, cardiovascular diseases, and neurodegenerative disorders. Antioxidant therapies that

enhance the body’s defense against oxidative stress may offer potential strategies for preventing

or treating these conditions (Pham-Huy et al., 2008).

Oxidative stress is a significant factor in the onset of chronic and degenerative conditions such as

cancer, autoimmune diseases, aging, cataracts, rheumatoid arthritis, cardiovascular issues, and

neurodegenerative disorders (Pham-Huy et al., 2008). Oxygen, an essential element for sustaining

life, becomes integral in cellular energy production as cells utilize it to generate ATP (adenosine

triphosphate) through mitochondrial processes (Bolisetty and Jaimes, 2013). This metabolic

activity gives rise to free radicals, primarily reactive oxygen species (ROS) and reactive nitrogen

species (RNS), as natural by-products of the cellular redox process (Bolisetty and Jaimes, 2013).

These compounds possess a dual nature, being both potentially harmful and beneficial to the

1
organism. Maintaining a delicate equilibrium between their opposing effects is crucial for life

processes. ROS and RNS, when present at low to moderate levels, contribute positively to cellular

responses and immune system function (Bolisetty and Jaimes, 2013).

Free radicals are highly reactive atoms, ions, or molecules with one or more unpaired electrons in

their outermost shell. Due to this unpaired electron, free radicals can initiate a chain of reactions

with other molecules, causing oxidative damage to cells (Hegazy & Abd Elhady, 2021). These

reactive species are produced both endogenously as byproducts of metabolic processes and

introduced exogenously through factors like pollution, tobacco smoke, and UV radiation (Ling &

Zhang, 2020). Although free radicals play essential roles in certain biological processes, their

excessive accumulation leads to oxidative stress, which is implicated in numerous diseases,

including cancer, cardiovascular diseases, and neurodegenerative disorders (Sies et al., 2022). Free

radicals can be broadly classified into two groups: reactive oxygen species (ROS) and reactive

nitrogen species (RNS). Both groups are highly reactive and capable of damaging cellular

components such as DNA, lipids, and proteins (Tan et al., 2021).

ROS include species like superoxide anion (O₂⁻), hydroxyl radicals (OH•), hydrogen peroxide

(H₂O₂), and singlet oxygen (¹O₂) (Bresgen & Eckl, 2021). ROS are primarily generated during

mitochondrial oxidative phosphorylation, where oxygen is reduced to produce energy in the form

of ATP. During this process, small amounts of oxygen are converted into superoxide radicals,

which are precursors to other ROS (Pizzino et al., 2017). RNS, such as nitric oxide (NO•) and

peroxynitrite (ONOO⁻), are nitrogen-centered free radicals. Nitric oxide is synthesized by nitric

oxide synthases during normal physiological processes, but excessive production of NO• can lead

to the formation of highly reactive peroxynitrite, which damages proteins and DNA (Chen et al.,

2020).

2
Free radicals are produced from both endogenous and exogenous sources. Endogenous sources

include cellular metabolism, enzymatic reactions, and immune responses, while exogenous

sources encompass environmental pollutants, radiation, and smoking (Zhang et al., 2020). The

mitochondrial electron transport chain (ETC) is a major source of ROS in the body. During the

production of ATP, about 1-2% of the oxygen consumed is incompletely reduced, forming

superoxide radicals (Orrenius et al., 2021). Additionally, enzymes like xanthine oxidase and

NADPH oxidase contribute to ROS generation in specific cellular contexts (Zhang et al., 2020).

Immune cells, particularly neutrophils and macrophages, produce ROS and RNS during the

respiratory burst, a defense mechanism against pathogens (Tan et al., 2021).

Exogenous sources of free radicals include UV radiation, environmental toxins, and cigarette

smoke. For instance, UV radiation promotes ROS formation in skin cells, which can lead to

photoaging and skin cancer (Valko et al., 2021). Smoking introduces a wide variety of free radicals

and chemicals that stimulate ROS production, contributing to chronic inflammation and oxidative

stress (Valko et al., 2021). Exposure to pollutants, such as heavy metals and ozone, can also

enhance free radical generation by disrupting antioxidant defenses (Singh et al., 2020). While

excessive free radicals are harmful, they also play critical roles in normal cellular processes, such

as immune defense and signal transduction (Sies et al., 2022).

At low levels, ROS act as signaling molecules that regulate processes like cell proliferation,

apoptosis, and stress responses. Hydrogen peroxide (H₂O₂) acts as a second messenger in redox

signaling pathways, modulating protein kinases and transcription factors involved in cellular stress

responses (Bresgen & Eckl, 2021). This redox signaling is crucial for maintaining cellular

homeostasis and adaptation to stress. Free radicals, especially ROS and RNS, play a critical role

in immune defense. Phagocytic cells, such as neutrophils, use ROS to kill invading pathogens

3
during a process known as the oxidative burst (Pizzino et al., 2017). These free radicals are

essential for the destruction of bacteria, viruses, and other harmful microorganisms. Hormesis

refers to a biological phenomenon in which a low dose of a harmful agent, like ROS, induces a

beneficial adaptive response. In the context of free radicals, low levels of ROS can activate

antioxidant defense systems, thereby promoting cellular resilience and longevity (Tan et al., 2021).

This hormetic response is thought to contribute to the benefits of exercise, where transient

increases in ROS levels stimulate adaptive antioxidant mechanisms (Bresgen & Eckl, 2021).

When free radical production exceeds the body’s antioxidant capacity, oxidative stress occurs.

This imbalance results in damage to lipids, proteins, and nucleic acids, contributing to the

pathogenesis of various diseases (Sies et al., 2022). Free radicals, particularly hydroxyl radicals,

can initiate lipid peroxidation by attacking polyunsaturated fatty acids in cell membranes. This

reaction propagates, causing extensive membrane damage and impairing cell function (Singh et

al., 2020). Lipid peroxidation has been implicated in atherosclerosis, where oxidized low-density

lipoprotein (LDL) contributes to plaque formation in arteries (Orrenius et al., 2021). Free radicals

can cause mutations by damaging the DNA through strand breaks and base modifications. ROS,

such as hydroxyl radicals, can induce guanine oxidation, leading to mutations that are potentially

oncogenic (Valko et al., 2021). ROS-induced DNA damage is implicated in cancer initiation,

aging, and neurodegenerative diseases (Pizzino et al., 2017).

Proteins are also vulnerable to free radical-induced oxidative modifications, leading to the loss of

protein function and misfolding. Oxidatively modified proteins are often targeted for degradation,

which can disrupt cellular homeostasis. Protein oxidation is associated with diseases such as

Alzheimer's, where oxidized proteins accumulate in the brain (Chen et al., 2020). The body

employs a complex antioxidant defense system to neutralize free radicals and mitigate oxidative

4
damage. These defenses include both enzymatic and non-enzymatic antioxidants (Sies et al.,

2022). Enzymatic antioxidants like superoxide dismutase (SOD), catalase, and glutathione

peroxidase are crucial for neutralizing ROS. SOD converts superoxide radicals to hydrogen

peroxide, which is then broken down by catalase into water and oxygen (Ling & Zhang, 2020).

Glutathione peroxidase reduces hydrogen peroxide and lipid peroxides, preventing oxidative

damage to cellular membranes (Pizzino et al., 2017). Non-enzymatic antioxidants include small

molecules like vitamins C and E, glutathione, and flavonoids. Vitamin C is a potent water-soluble

antioxidant that neutralizes ROS by donating electrons, while vitamin E, a lipid-soluble

antioxidant, protects cell membranes by interrupting lipid peroxidation (Singh et al., 2020).

Glutathione, a tripeptide, plays a vital role in detoxifying ROS and maintaining cellular redox

balance (Valko et al., 2021).

The overproduction of free radicals and the resulting oxidative stress are implicated in several

chronic diseases. Oxidative stress is a critical factor in cancer development. ROS can induce DNA

mutations, activate oncogenes, and inhibit tumor suppressor genes, promoting carcinogenesis

(Orrenius et al., 2021). Moreover, oxidative stress-induced inflammation fosters a

microenvironment conducive to tumor growth and metastasis (Sies et al., 2022). ROS play a

significant role in cardiovascular diseases, particularly atherosclerosis. Oxidized LDL is a major

contributor to plaque formation in arteries, which can lead to heart attacks and strokes (Tan et al.,

2021). Furthermore, oxidative stress is involved in endothelial dysfunction, a key factor in the

progression of hypertension and other cardiovascular disorders (Pizzino et al., 2017). Oxidative

stress is implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's and

Parkinson's. In these conditions, free radical-induced damage to neurons leads to protein

aggregation, mitochondrial dysfunction, and eventual neuronal death (Chen et al., 2020). Increased

5
oxidative damage in the brain is a hallmark of Alzheimer's disease, contributing to the formation

of amyloid plaques and neurofibrillary tangles (Bresgen & Eckl, 2021).

1.1.1 CHARACTERISTICS OF FREE RADICALS

ROS and RNS collectively refer to free radicals and other non-radical reactive derivatives, also

known as oxidants (Phaniendra et al., 2015). While radicals are generally less stable compared to

non-radical species, they exhibit stronger reactivity (Phaniendra et al., 2015).A molecule

possessing one or more unpaired electrons in its outer shell is termed a free radical (Phaniendra et

al., 2015). Free radicals are created from molecules when a chemical link breaks and each piece

retains one electron, when a radical cleaves to produce another radical, and when redox reactions

occur (Phaniendra et al., 2015). Free radicals are highly reactive molecules with one or more

unpaired electrons, which makes them extremely unstable and prone to interacting with other

molecules in their vicinity. This unpaired electron creates an imbalance in the chemical structure,

causing these species to seek electrons from surrounding molecules to achieve stability. While free

radicals are essential in certain physiological processes, they can also cause significant damage to

cells and tissues when produced in excess, leading to oxidative stress (Lobo et al., 2020). Free

radicals are formed through various endogenous and exogenous processes. Endogenously, they are

produced during normal cellular metabolic activities, particularly within the mitochondria, where

reactive oxygen species (ROS) such as superoxide (O2•−) and hydroxyl radical (OH•) are

generated as byproducts of aerobic respiration (Kumar et al., 2022). Exogenously, factors such as

environmental pollutants, UV radiation, smoking, and certain chemicals can induce free radical

formation in the body (Sharma et al., 2021).

Two major categories of free radicals are reactive oxygen species (ROS) and reactive nitrogen

species (RNS). ROS include radicals like superoxide (O2•−) and hydroxyl radical (OH•), as well

6
as non-radical species like hydrogen peroxide (H2O2). RNS include radicals such as nitric oxide

(NO•) and nitrogen dioxide (NO2•), and non-radical species such as peroxynitrite (ONOO−)

(García-Sanmartín et al., 2020).

The most defining characteristic of free radicals is their high reactivity, driven by their tendency

to stabilize their unpaired electron. This reactivity means that free radicals often participate in rapid

chemical reactions, such as abstraction of hydrogen atoms from lipids, proteins, and nucleic acids

(Miao & St. Clair, 2022). These reactions can initiate chain reactions, where the affected molecules

themselves become free radicals, perpetuating damage. Due to their high reactivity, free radicals

typically have short half-lives and do not exist in significant concentrations in biological systems

unless specific stressors increase their production. When the production of free radicals exceeds

the body’s antioxidant defense mechanisms, oxidative stress occurs, leading to cellular and tissue

damage. Free radicals target key cellular components, including lipids, proteins, and DNA. Lipid

peroxidation, for example, results in membrane damage and loss of cell integrity, while oxidative

damage to proteins can impair their function (Zhao et al., 2021). Furthermore, DNA damage

caused by free radicals is associated with mutations and carcinogenesis (Kumar et al., 2022).

Although free radicals are often associated with harmful effects, they also play crucial roles in

physiological processes. For instance, ROS are involved in cellular signaling pathways,

particularly those regulating cell proliferation and apoptosis (Lobo et al., 2020). In the immune

system, free radicals like nitric oxide (NO•) are produced by immune cells to destroy pathogens

(Sharma et al., 2021). Thus, the balance between free radical production and antioxidant defenses

is essential for maintaining normal cellular function and preventing oxidative stress.

Hydroxyl (OH•), superoxide (O2•–), nitric oxide (NO•), nitrogen dioxide (NO2•), peroxyl (ROO•),

and lipid peroxyl (LOO•) are examples of free radicals (Chen et al., 2012). In addition, substances

7
commonly referred to as oxidants—such as hydrogen peroxide (H2O2), ozone (O3), singlet

oxygen (1O2), hypochlorous acid (HOCl), nitrous acid (HNO2), peroxynitrite (ONOO–),

dinitrogen trioxide (N2O3), and lipid peroxide (LOOH), can readily trigger free radical reactions

in living things (Chen et al., 2012). Thus, biological free radicals are extremely erratic molecules

with accessible electrons that can react with a variety of chemical substrates, including proteins,

lipids, and DNA (Chen et al., 2012).

8
Fig 1.1: Diagram of Reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive

sulfur species (RSS) (Chiurchiù and Maccarrone, 2011).

9
1.1.2 GENERATION OF FREE RADICALS

ROS and RNS can form within cells through two pathways: enzymatic and non-enzymatic

reactions (Pizzino et al., 2017). Enzymatic reactions that produce free radicals encompass those

participating in the respiratory chain, phagocytosis, prostaglandin synthesis, and the cytochrome

P450 system (Pizzino et al., 2017).

For instance, the superoxide anion radical (O2•–) is produced through various cellular oxidase

systems such as NADPH oxidase, xanthine oxidase, and peroxidases (Andrés et al.,2023). Once

generated, it participates in multiple reactions that give rise to diverse ROS and RNS, including

hydrogen peroxide, hydroxyl radical (OH•), peroxynitrite (ONOO–), and hypochlorous acid

(HOCl) (Andrés et al.,2023). Hydrogen peroxide (H2O2), a non-radical compound, is synthesized

by several oxidase enzymes, such as amino acid oxidase and xanthine oxidase. The latter catalyzes

the oxidation of hypoxanthine to xanthine and xanthine to uric acid. The hydroxyl radical (OH•),

the most reactive free radical in vivo, forms through the reaction of O2•– with H2O2 in the

presence of Fe2+ or Cu+ (catalysts), known as the Fenton reaction (Andrés et al.,2023).

Hypochlorous acid (HOCl) is generated through the action of myeloperoxidase, an enzyme derived

from neutrophils, which catalyzes the oxidation of chloride ions in the presence of H2O2. Nitric

oxide radical (NO•) is synthesized within biological tissues through the oxidation of L-arginine to

citrulline by nitric oxide synthase (Andrés et al.,2023).

Free radicals can arise from the non-enzymatic interaction of oxygen with organic compounds, as

well as through reactions initiated by ionizing radiation (Lobo et al.,2010). The non-enzymatic

process can also take place during oxidative phosphorylation (i.e., aerobic respiration) within the

mitochondria (Lobo et al.,2010).

10
ROS and RNS can stem from either internal or external origins (Aranda-Rivera et al., 2022).

Endogenous free radicals are produced through immune cell activation, inflammation,

psychological stress, vigorous physical activity, ischemia, infection, cancer, and aging (Aranda-

Rivera et al., 2022). Exogenous ROS/RNS arise from environmental pollutants in air and water,

cigarette smoke, alcohol consumption, exposure to heavy or transition metals (such as Cd, Hg, Pb,

Fe, As), certain medications (including cyclosporine, tacrolimus, gentamycin, bleomycin),

industrial solvents, cooking methods (such as smoked meats, reused oil, and fats), and radiation.

Upon entry into the body via various pathways, these external substances are broken down or

metabolized into free radicals (Aranda-Rivera et al., 2022).

11
Fig 1.2: The generation of free radicals (Ďuračková, 2014).

12
1.2 REACTIVE OXYGEN SPECIES

Reactive oxygen species (ROS) are molecular entities capable of existing independently,

characterized by the presence of at least one oxygen atom and one or more unpaired electrons

(Jakubczyk et al.,2020). This category encompasses oxygen free radicals, such as the superoxide

anion radical, hydroxyl radical, hydroperoxyl radical, singlet oxygen, along with free nitrogen

radicals (Jakubczyk et al.,2020). Under physiological conditions, limited amounts of ROS are

generated during cellular processes like aerobic respiration or inflammatory responses,

predominantly occurring in hepatocytes and macrophages (Bardaweel et al., 2018). Reactive

oxygen species primarily serve as signaling molecules. Moreover, they prompt cell differentiation

and apoptosis, thereby contributing to the natural aging process (Bardaweel et al., 2018). They

also play roles in muscle contractions, regulation of vascular tone, and influence bactericidal and

bacteriostatic activities. Excessive exposure to UV radiation, prolonged stress, intense physical

activity, improper diet, and use of stimulants can lead to increased production of free radicals

(Bardaweel et al., 2018). Under normal circumstances, there exists a balance between the

generation and elimination of free radicals within the body (Bardaweel et al., 2018).

1.2.1 SOURCES OF ROS

ROS can originate from either endogenous or exogenous sources. Endogenous ROS sources

include various cellular organelles such as mitochondria, peroxisomes, and the endoplasmic

reticulum, where oxygen consumption is high (Phaniendra et al., 2015).

Mitochondria serve as the primary intracellular source of ROS. Superoxide radicals are

predominantly generated at two key sites within the electron transport chain: complex I (NADH

dehydrogenase) and complex III (ubiquinone cytochrome c reductase) (Phaniendra et al., 2015).

The transfer of electrons from either complex I or II to coenzyme Q or ubiquinone (Q) leads to the

13
formation of the reduced form of coenzyme Q (QH2). QH2, in turn, regenerates coenzyme Q

through an unstable intermediate known as semiquinone anion (∙Q-) in the Q-cycle. The generated

∙Q- promptly transfers electrons to molecular oxygen, resulting in the formation of superoxide

radicals. The production of superoxide is non-enzymatic, thus the higher the metabolic rate, the

greater the production of ROS (Phaniendra et al., 2015).

14
Fig 1.3: Mitochondrial ROS production (Phaniendra et al., 2015).

15
The superoxide anion is converted to hydrogen peroxide by the action of mitochondrial superoxide

dismutase (MnSOD). H2O2 can be detoxified by the Catalase (CAT) and glutathione peroxidase

(GPx) (Phaniendra et al., 2015).

In peroxisomes, the respiratory pathway involves the transfer of electrons from various metabolites

to oxygen, leading to the formation of H2O2, but it is not linked to oxidative phosphorylation for

ATP production (Schrader and Fahimi, 2006); instead, free energy is released in the form of heat.

Other free radicals produced in peroxisomes include H2O2, O2•−, OH•, and NO• (Schrader and

Fahimi, 2006). The β-oxidation of fatty acids is the primary metabolic process generating H2O2

in peroxisomes. As discussed elsewhere, various peroxisomal enzymes such as acyl CoA oxidases,

D-amino acid oxidase, L-α-hydroxy oxidase, urate oxidase, and xanthine oxidase, among others,

have been identified as sources of different ROS (Schrader and Fahimi, 2006).

16
Fig 1.4: Diagram illustrating peroxisomal enzymes involved in the generation or breakdown of

ROS (Schrader and Fahimi, 2006).

17
Hydrogen peroxide, H2O2 is primarily generated by several peroxisomal oxidases, such as acyl-

CoA oxidase, which participates in the β-oxidation of fatty acids (Quijano et al., 2016). Catalase

and glutathione peroxidase (GPx) decompose H2O2, while it can also be converted to hydroxyl

radicals (·OH) (Quijano et al., 2016). Hydroxyl radicals have the potential to harm the peroxisomal

membrane through lipid peroxidation of unsaturated fatty acids. Hydroperoxides produced in this

process can be broken down by catalase and glutathione peroxidase (Quijano et al., 2016).

Superoxide anions (O2·−) produced by peroxisomal oxidases, like xanthine oxidase (XOx), are

neutralized by manganese superoxide dismutase (MnSOD) and copper-zinc superoxide dismutase

(CuZnSOD) (Lismont et al., 2019). Nitric oxide synthase (NOS) catalyzes the conversion of l-

arginine (l-Arg) to nitric oxide (NO·). NO· can interact with O2·− radicals to form peroxynitrite

(ONOO−), a potent oxidant. H2O2 and NO· can permeate the peroxisomal membrane and

participate in cellular signaling (Lismont et al., 2019). Peroxiredoxin 1 and PMP20 are involved

in H2O2 breakdown. Mpv17 and L-MP play roles in regulating peroxisomal ROS metabolism

(Lismont et al., 2019).

Endoplasmic Reticulum (ER) enzymes, such as cytochrome p-450 and b5 enzymes, as well as

diamine oxidase, play a role in the generation of ROS. Another notable thiol oxidase enzyme,

Erop1p, facilitates the transfer of electrons from dithiols to molecular oxygen, resulting in the

formation of H2O2 (Ponnampalam et al., 2022).Additional endogenous sources of ROS include

prostaglandin synthesis, auto-oxidation of adrenaline, phagocytic cells, reduced riboflavin,

FMNH2, FADH2, cytochrome P450, immune cell activation, inflammation, mental stress,

excessive exercise, infection, cancer, aging, ischemia, and so forth (Ponnampalam et al., 2022).

18
1.3 OXIDATIVE STRESS AND ASSOCIATED DISEASES

Reactive oxygen and nitrogen species (RONS) stem from various endogenous and exogenous

processes, and their detrimental effects are counteracted by antioxidant defenses (Liguori et al.,

2018). Oxidative stress arises from the imbalance between RONS production and these protective

mechanisms (Liguori et al., 2018). Aging involves the gradual decline in tissue and organ function.

The oxidative stress theory of aging posits that age-related functional decline results from the

accumulation of damage induced by RONS (Liguori et al., 2018). Concurrently, oxidative stress

contributes to various age-related conditions such as cardiovascular diseases (CVDs), chronic

obstructive pulmonary disease, chronic kidney disease, neurodegenerative diseases, cancer,

sarcopenia, and frailty (El Assar et al., 2019). Diverse oxidative stress biomarkers have been

identified and could offer valuable insights into treatment efficacy, aiding in the selection of

optimal drugs and dosage regimens for patients (El Assar et al., 2019). Targeting specific

therapeutic pathways may be crucial from a pathophysiological standpoint. Given the significant

role of oxidative stress in the pathogenesis of numerous clinical conditions and aging, antioxidant

therapy holds potential to positively impact disease progression (El Assar et al., 2019).

1.3.1 OXIDATIVE STRESS AND THEORY OF AGING

Aging entails the gradual deterioration of tissue and organ function as time passes (Gladyshev,

2014). The free radical theory of aging, later termed the oxidative stress theory of aging, posits

that age-related functional declines stem from the accumulation of oxidative damage to

macromolecules (lipids, DNA, and proteins) caused by reactive oxygen and nitrogen species

(RONS) (Gladyshev, 2014). The precise mechanism underlying oxidative stress-induced aging

remains unclear; however, it is believed that elevated levels of RONS may induce cellular

senescence, a physiological process that halts cellular proliferation in response to damage incurred

19
during replication (Gladyshev, 2014). Senescent cells acquire an irreversible senescence-

associated secretory phenotype (SASP), characterized by the secretion of soluble factors (such as

interleukins, chemokines, and growth factors), degradative enzymes like matrix metalloproteases

(MMPs), and insoluble proteins/extracellular matrix (ECM) components (Gladyshev, 2014). The

notion that oxidative damage is just one of several factors contributing to aging has limitations, as

it fails to elucidate causal relationships and the inevitability of damage accumulation (Maldonado

et al., 2023). This discussion considers that the inaccuracies, variations, and inherent imperfections

within each biological process may be accountable for the unavoidable buildup of by-products and

other forms of damage (Maldonado et al., 2023). While reactive oxygen species (ROS) serve as

quintessential by-products, their impact on aging is shaped by the metabolic organization of the

cell, its defense mechanisms, and genetic makeup (Maldonado et al., 2023). These elements are

subject to natural selection and, akin to dietary and genetic interventions that prolong lifespan,

alter the composition and rates of accumulation of various forms of damage (Vona et al., 2021).

Oxidative damage, alongside other specific forms of damage when viewed independently or in

combination, does not singularly cause aging. Rather, the imperfect nature of biology, leading to

the inevitable accrual of mildly detrimental molecular species, may serve as the true underlying

cause of aging (Vona et al., 2021).

20
Fig 1.5: The effects of oxidative stress on aging (Vona et al., 2021).

21
The oxidative stress theory of aging posits that the accumulation of oxidative damage to cells and

tissues over time plays a significant role in the aging process. This theory, which evolved from the

free radical theory of aging proposed by Harman in 1956, suggests that aging is driven by the

damage caused by reactive oxygen species (ROS) generated during cellular respiration and other

metabolic processes. ROS include free radicals such as superoxide, hydrogen peroxide, and

hydroxyl radicals, which can attack cellular components, including lipids, proteins, and DNA,

resulting in oxidative damage.

Oxidative stress occurs when there is an imbalance between the production of ROS and the

antioxidant defense systems within cells. Under normal physiological conditions, ROS play

important roles in cell signaling and homeostasis. However, excessive ROS levels can overwhelm

the antioxidant defense system, leading to oxidative damage (Sies, 2020). Mitochondria are the

primary source of ROS during oxidative phosphorylation, and as cells age, mitochondrial

efficiency declines, contributing to increased ROS production and oxidative stress. This leads to

the accumulation of damaged biomolecules, impaired cellular functions, and a reduction in tissue

integrity, all of which are hallmarks of aging.

One of the primary consequences of oxidative stress is cellular senescence, a state in which cells

lose the ability to divide and function properly. Oxidative damage to DNA, proteins, and lipids

can trigger cellular responses such as apoptosis or senescence. DNA damage, in particular, is a

significant driver of aging. ROS-induced damage to nuclear and mitochondrial DNA can result in

mutations, impaired gene expression, and a decrease in cellular vitality. Over time, the

accumulation of senescent cells can disrupt tissue function, contribute to chronic inflammation,

and promote age-related diseases (Zhang et al., 2020). To counteract the damaging effects of ROS,

organisms possess a variety of antioxidant defense mechanisms. These include enzymatic

22
antioxidants such as superoxide dismutase (SOD), catalase, and glutathione peroxidase, as well as

non-enzymatic antioxidants like vitamin C, vitamin E, and glutathione. However, with aging, the

efficiency of these antioxidant systems declines, contributing to the accumulation of oxidative

damage (Ungvari et al., 2020). This decline in antioxidant defense is believed to be a key factor in

the aging process and the development of age-related diseases such as cardiovascular disease,

neurodegenerative diseases, and cancer. Numerous studies have linked oxidative stress to the onset

and progression of age-related diseases. For instance, in neurodegenerative diseases such as

Alzheimer's and Parkinson's, elevated ROS levels and oxidative damage have been observed in

affected brain regions. Oxidative stress is also implicated in cardiovascular diseases, where ROS

contribute to endothelial dysfunction, inflammation, and atherosclerosis (de Oliveira et al., 2021).

These findings suggest that oxidative stress not only accelerates the aging process but also plays a

pivotal role in the development of age-associated disorders.

1.3.2 OXIDATIVE STRESS AND CVDS

CVDs stand as a predominant cause of morbidity and mortality among the elderly, with

atherosclerosis serving as a pivotal causal factor (Senoner, and Dichtl, 2019). Numerous studies

have demonstrated that as individuals age, the heart's tolerance to oxidative stress diminishes due

to reductions in antioxidant enzyme concentrations, such as GSH-Px and SOD, thereby

contributing to the onset of cardiovascular (CV) abnormalities (Senoner, and Dichtl, 2019).

Current evidence strongly associates atherosclerosis with oxidized LDL-cholesterol (oxLDL) as

the primary compound responsible for its formation, particularly in older individuals. Indeed,

several studies have indicated a significant correlation between oxLDL and heightened arterial

stiffness, independent of traditional CVD risk factors (Levitan et al., 2010). For instance, a study

involving 2,944 healthy women aged 30–79 highlighted increases in plasma oxLDL levels after

23
the age of 50 (Levitan et al., 2010). The rise of oxLDL levels with age may exacerbate LDL

atherogenicity, given the prooxidant and proinflammatory milieu characteristic of elderly

individuals (Levitan et al., 2010). Atherosclerosis continues to stand out as the primary cause of

cardiovascular mortality in developed nations (Rafieian et al., 2014). Recent findings suggest that

inflammatory mechanisms are pivotal in the formation of atheromas (Rafieian et al., 2014).

Activation of pro-inflammatory signaling pathways, cytokine/chemokine expression, and

heightened oxidative stress represent key mechanisms contributing to atherosclerosis (Rafieian et

al., 2014). "Offending" stimuli such as dyslipidemia, hypertension, and cigarette smoking induce

qualitative alterations in endothelial cells, prompting the upregulation of adhesion and chemotactic

molecules, along with increased permeability to macromolecules (Rafieian et al., 2014).

24
Fig 1.6: Oxidative stress mechanisms in contrast induced nephropathy and cardiovascular

disorders (Rafieian et al., 2014).

25
Conversely, findings from the InCHIANTI dataset, a 9-year population-based study, revealed no

significant association between elevated oxLDL levels (measured with antibody 4E6) and

CVD/cardiac mortality, suggesting that the prognostic value added by oxLDLs might be

insignificant in advanced age (Gradinaru et al., 2015).Furthermore, the development of oxidative

stress contributes to vascular endothelial dysfunction with aging. In healthy adults across varying

age groups, brachial artery flow-mediated dilation demonstrates an inverse relationship with NT

in vascular endothelial cells (Gradinaru et al., 2015). Additionally, the expression of endothelin-

1, the most potent vasoconstrictor molecule produced by the vascular endothelium, is heightened

in vascular endothelial cells of older adults compared to younger counterparts, correlating

inversely with endothelium-dependent dilation and positively with NT (Gradinaru et al., 2015).

Endothelial dysfunction and vascular remodeling serve as early determinants in the pathogenesis

of hypertension and atherosclerosis (Gradinaru et al., 2015).

1.3.3 OXIDATIVE STRESS AND DIABETES

Diabetes mellitus, encompassing both type 1 and type 2, constitutes a metabolic disorder

associated with heightened free radical formation and diminished antioxidant capacity,

culminating in macro- and microvascular complications (Giacco and Brownlee, 2010). The precise

mechanism through which oxidative stress accelerates the onset of complications in diabetes

remains partially understood (Giacco and Brownlee, 2010). In type 2 diabetes (T2D), oxidative

stress fosters prothrombotic reactions, thereby predisposing individuals to cardiovascular

complications. The detrimental effects of diabetes on tissues can be viewed as the oxidative

consequences of chronic hyperglycemia (Giacco and Brownlee, 2010). Elevated intracellular

glucose levels prompt increased production of reactive oxygen and nitrogen species (RONS),

surpassing the cell's antioxidant defenses (Llanos and Palomero, 2022). Consequently, RONS

26
activation triggers four critical molecular pathways implicated in hyperglycemia-induced

oxidative tissue damage: protein kinase C (PKC) activation, heightened hexosamine pathway flux,

advanced glycation end-products (AGEs) accumulation, and increased polyol pathway flux.

Specifically, activation of the AGEs pathway may alter cellular regulation of gene transcription,

intercellular signaling, and interaction with blood proteins like albumin, leading to their binding

to AGEs receptors (RAGEs) on macrophages/mesangial cells and consequent upregulation of

growth factors and proinflammatory cytokines (Llanos and Palomero, 2022). Regarding the elderly

population, a study involving 61 elderly subjects revealed elevated antioxidant levels (e.g., catalase

and glutathione peroxidase) in individuals with impaired glucose tolerance (IGT) compared to

healthy controls, alongside increased oxidative stress biomarkers (e.g., malondialdehyde) in T2D

but not IGT subjects (Llanos and Palomero, 2022). These findings suggest heightened oxidative

stress in elderly patients with T2D, potentially offset by an increased antioxidant defense system

in those with IGT (Llanos and Palomero, 2022).

27
Fig 1.7: Oxidative stress in diabetes mellitus (Llanos and Palomero, 2022).

28
1.3.4 OXIDATIVE STRESS AND CANCER

Numerous studies have elucidated the direct correlation between chronic inflammation and

carcinogenesis. The primary chemical mediators of these inflammatory responses are free radical

species originating from inflammation-induced oxidative stress (Reuter et al., 2010). Reactive

oxygen and nitrogen species (RONS) can directly damage cellular components or indirectly trigger

additional RONS production by recruiting inflammatory cells, thereby exacerbating the damage

(Reuter et al., 2010). Specifically, RONS and inflammatory cytokines, such as TNFα, activate the

transcription factor NFκB, which stimulates the expression of genes associated with cell

proliferation, apoptosis, and carcinogenesis. Chronic inflammation also fosters angiogenesis, a

hallmark of cancer, as RONS can upregulate transcription factors like c-fos and c-jun implicated

in neoplastic transformation and angiogenesis promotion (Reuter et al., 2010).

Furthermore, macrophages, platelets, fibroblasts, and cancer cells serve as significant sources of

angiogenic factors (e.g., fibroblast growth factor, vascular endothelial growth factor,

prostaglandins-E1 and E2), which heighten RONS production, thereby increasing cancer risk

(Riabov et al., 2014). The mutagenic and carcinogenic potential of RONS arises from their

capacity to react with and chemically modify DNA. ROS-induced DNA damage, including

transcriptional arrest, replication errors, or genomic instability, is associated with carcinogenesis.

Among the oxidized-DNA products, 8-oxodG and 8-nitroguanine are recognized as biomarkers

for inflammation-induced carcinogenesis (Riabov et al., 2014). There is a notable age-dependent

increase in cancer risk, likely attributable to the accumulation of RONS-induced DNA damage

over a lifetime. This is evidenced by the progressive and statistically significant rise in the levels

of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8oxodG) observed with aging (Riabov et al., 2014).

Given these considerations, chronic inflammation and oxidative stress should be recognized as

29
high-risk factors for cancer, particularly in elderly individuals. Therefore, it is advisable for elderly

individuals to incorporate higher levels of antioxidant compounds into their diets (Riabov et al.,

2014).

Fig 1.8: Oxidative stress and cancer (Riabov et al., 2014).

30
1.4 ANTIOXIDANTS

Antioxidants comprise a variety of compounds that counteract the harmful effects of free radicals

and reactive oxygen species (ROS) within the cell (Zehiroglu and Ozturk, 2019). The antioxidant

properties found in food and beverages have become a focal point within the scientific community

(Zehiroglu and Ozturk, 2019). These antioxidants offer defense against damage induced by free

radicals and play significant roles in the onset of numerous chronic diseases, including

cardiovascular diseases, aging, heart disease, anemia, cancer, and inflammation (Zehiroglu and

Ozturk, 2019). Antioxidants have garnered significant scientific attention due to their multifaceted

benefits, including anti-aging and anti-inflammatory properties (Miyazawa et al., 2022). They

continue to be utilized across various domains. In the realm of food technology, antioxidants are

incorporated into numerous food products to enhance their nutritional value and mitigate potential

issues (Miyazawa et al., 2022). Consequently, research aimed at assessing the antioxidant

properties of natural foods and their constituents remains a rapidly advancing field (Miyazawa et

al., 2022). Moreover, antioxidants have found application in encapsulation studies, which are

employed for the preservation and stabilization of food components (Miyazawa et al., 2022).

Indeed, food preservation is as crucial as food production itself. Emerging packaging techniques

such as edible films and coatings are at the forefront of food preservation strategies (Díaz-Montes

and Castro-Muñoz, 2021). The protective capabilities of these materials can be enhanced through

the addition of antioxidants (Díaz-Montes and Castro-Muñoz, 2021). Concurrently, investigations

into the in vivo antioxidant activities of plants and animals shed light on how these activities are

influenced by metabolic processes (Díaz-Montes and Castro-Muñoz, 2021). In such studies,

antioxidant enzymes play a significant role. Numerous findings from both in vivo and in vitro

studies underscore the potential of antioxidants in disease prevention and treatment.As a result, the

31
significance of antioxidants has escalated, extending their utility into pharmacology, cosmetics,

and medicine (Díaz-Montes and Castro-Muñoz, 2021).

1.4.1 CLASSIFICATION OF ANTIOXIDANTS

The two main categories of antioxidants are natural and synthetic (Xu et al., 2017). Furthermore,

the antioxidants can be categorized as water-soluble or lipid-soluble based on their solubility, or

as enzymatic or non-enzymatic based on their actions. These classifications can be made based on

the sources of the antioxidants (Xu et al., 2017).

Natural antioxidants are compounds found in food that stop processes in food, like sourness,

disturbance, and color change (Xu et al., 2017). The majority of natural antioxidants come from

plants, and the types, diversity, extraction and/or processing techniques, and growth environment

all affect how active these compounds are (Lourenço et al., 2019). They are present in practically

all plants, some animal tissues, and microbes. Natural antioxidants have a strong antioxidant

activity, particularly fruits and vegetables with colors like red, orange, and purple (Lourenço et al.,

2019). Due to their high antioxidant content, a variety of fruits and vegetables, including oranges,

lemons, blueberries, strawberries, plums, prunes, red beans, broccoli blossoms, and more, are

included in many nutritional meals (Lourenço et al., 2019). We can mention flavonoids,

carotenoids, phenolic acids, ascorbic acid, tocopherols and tocotrienols, and flavonoids as the most

significant natural antioxidant groups (Lourenço et al., 2019).

Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase are examples of

enzymatic scavengers found in the human antioxidant defense system (Jeeva et al., 2015).

Hydrophilic scavengers include urate, ascorbate, glutathione (GSH), and flavonoids. Lipophilic

radical scavengers include tocopherols, carotenoids, and ubiquinol (Jeeva et al., 2015). There are

several subcategories of non-enzymatic antioxidants, but the primary ones are nitrogen

32
compounds, vitamins, enzyme cofactors, minerals, peptides, and phenolic acids (Jeeva et al.,

2015). Vitamins E and C are considered among the most crucial natural antioxidants. Vitamin C,

comprising ascorbic acid and its oxidized form, dehydroascorbic acid, exhibits numerous

biological functions within the human body (Doseděl et al., 2021). Over 85% of dietary vitamin

C is derived from fruits and vegetables. Ascorbic acid effectively scavenges superoxide and

hydroxyl radicals and has the ability to regenerate α-tocopherol (Doseděl et al., 2021). It plays a

vital role in collagen biosynthesis, carnitine production, and neurotransmitter synthesis.Vitamin E,

a fat-soluble vitamin, shields lipids from peroxidative damage owing to its robust antioxidant

properties (Doseděl et al., 2021). Vitamin E consists of eight stereoisomers (α, β, γ, δ tocopherol

and α, β, γ, δ tocotrienol), with α-tocopherol being the most biologically active in humans. Widely

recognized as a potent antioxidant, vitamin E's antioxidant activity is attributed to the hydroxyl

group within its aromatic ring, which donates hydrogen to neutralize free radicals or reactive

oxygen species (ROS) (Doseděl et al., 2021). Most tocopherols belong to a chemical class

exhibiting vitamin E activity, with α-tocopherol being the primary peroxyl radical scavenger in

biological lipid environments such as membranes or low-density lipoproteins (Doseděl et al.,

2021).

Carotenoids, including carotenes and xanthophylls, are vibrant yellow, orange, and red pigments

present in numerous fruits and vegetables, serving as non-enzymatic natural antioxidants (Fiedor

and Burda, 2014). Their antioxidant properties stem from their ability to quench singlet oxygen

and capture peroxyl radicals (Fiedor and Burda, 2014). For instance, lycopene, a prominent

carotenoid, exhibits remarkable protective effects, particularly against prostate cancer. Tomatoes,

cooked tomato juice, and tomato sauce represent primary dietary sources of lycopene, which are

more readily absorbed by the body compared to raw tomatoes (Fiedor and Burda, 2014).

33
Phenolic compounds, which are secondary metabolites in plants, also constitute an essential

component of plant nutrients (Lin et al., 2016). Foods rich in polyphenols have garnered extensive

research interest due to their potential beneficial effects, including anticarcinogenic properties (Lin

et al., 2016). The phenolic group encompasses a diverse array of compounds, with flavonoids,

phenolic acids, and stilbenes being among the most significant in plants (Lin et al., 2016). These

compounds exhibit a wide range of chemical structures, varying from simple to highly complex

molecules.Phenolic acids, constituting approximately one-third of dietary phenols, exist in both

free and bound forms in plants and are recognized for their potent antioxidant properties (Lin et

al., 2016). Their hydroxyl groups enable them to neutralize various oxidant molecules, including

free radicals, thereby exerting antioxidant effects (Lin et al., 2016).

Flavonoids, a diverse group of polyphenolic compounds with relatively low molecular weights,

are characterized by their benzo-γ-pyrrole derivatives (Panche et al., 2016). They play pivotal roles

in various biological processes, interacting with numerous cellular targets involved in critical

signaling pathways within the body, thus offering a range of health benefits for humans (Panche

et al., 2016). Representing a class of secondary metabolites, flavonoids encompass over 10,000

distinct structures (Panche et al., 2016).

Notably, flavonoids serve as important antioxidants owing to their elevated redox potential,

enabling them to function as reducing agents, hydrogen donors, and singlet oxygen quenchers

(Panche et al., 2016). They also possess the ability to chelate metals. Flavonoids are among the

most common phytochemicals found in plants, where they contribute to shielding against UV light,

fungal parasites, herbivores, pathogens, and oxidative cellular damage (Mierziak et al., 2014).

Regular consumption of flavonoids by humans has been linked to a reduced incidence of diseases

34
such as cancer and heart disease, underscoring their significant health-promoting properties

(Mierziak et al., 2014).

Fig 1.9: Chemical structures of flavonoids (a), ascorbic acid (b), benzoic acid (c), tocopherols (d),

carotenoids (α-carotene, β-carotene, lycopene, lutein)(E) (Zehiroglu and Ozturk, 2019).

35
Both natural and synthetic antioxidants play a crucial role in the food industry (López et al., 2022).

Undesirable changes such as taste and odor alterations caused by autoxidation chain reactions can

significantly lower the quality of food products (López et al., 2022). Hence, preservative

substances have been utilized as additives in the food industry for approximately 60 years to

prevent or minimize lipid oxidation in food items (López et al., 2022).

These preservatives encompass both natural antioxidants like ascorbic acid and tocopherols, as

well as synthetic antioxidants such as propyl gallate (PG), tertiary butylhydroquinone (TBHQ),

butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT). Several in vivo studies

conducted in the 1980s and 1990s highlighted certain health risks associated with the consumption

of synthetic antioxidants (Shahidi and Ambigaipalan, 2015). The toxic effects induced by BHA

and BHT only occurred with high doses in long-term treatments. However, another study

demonstrated that normal intake of BHA and BHT at low doses was not linked to gastric cancer

(Shahidi and Ambigaipalan, 2015). More recently, the European Food Safety Authority conducted

a comprehensive examination of all conflicting published data and concluded that the acceptable

daily intakes of 0.25 mg/kg/day for BHA and 1.0 mg/kg/day for BHT are safe for both adults and

children (Shahidi and Ambigaipalan, 2015).

1.4.2 MODE OF ACTION OF ANTIOXIDANTS

The two primary methods for assessing antioxidant capacity can be categorized based on their

mechanisms (Siddeeg et al., 2021):

1) The hydrogen atom transfer (HAT) method involves the donation of a hydrogen ion from a

stable molecule, enabling the antioxidant to scavenge reactive oxygen species (ROS), while

2) The single electron transfer (SET) method relies on the antioxidant's potential to reduce specific

molecules and compounds by transferring an electron.

36
To date, various in vitro analytical methods have been developed to assess the potential, capacity,

and activity of natural antioxidants. These methods include spectrometric, electrochemical, and

chromatographic techniques (Siddeeg et al., 2021).

In most single electron transfer (SET) methods, the activity or capacity of antioxidants is typically

determined spectrophotometrically based on their capability to reduce a fluorescent or colored

probe (oxidizing agent/oxidant/substrate) instead of common peroxyl radicals (Lü et al., 2010).

During this process, changes in absorbance are plotted against the concentration of the antioxidant

to generate an anticipated linear curve (Lü et al., 2010). The extent of change in absorbance

correlates with the concentration and activity of the antioxidant. However, it's important to note

that the SET method is influenced by pH and solvent conditions. pH, in particular, is the primary

determinant of the antioxidant's reducing capacity (Lü et al., 2010).Typically, the reactivity of

antioxidants during the SET assay is governed by two major antioxidant functional group

attributes: ionization and deprotonation potentials. Under acidic pH conditions, protonation of the

antioxidant increases, thereby lowering the ionization potential and diminishing overall

antioxidant reducing abilities (Kasote et al., 2015). Conversely, proton dissociation is heightened

under basic conditions, enhancing the antioxidant's reducing capabilities (Kasote et al., 2015).

Additionally, the SET reaction tends to be slower than the hydrogen atom transfer (HAT) method

due to the time required for the reaction to complete and solvent stabilization (Kasote et al.,

2015).In contrast, the HAT-based method assesses the antioxidant's potential to scavenge reactive

oxygen species (ROS) by its ability to donate hydrogen atom(s) and break the radical chain, as

illustrated in the reaction below;

37
Fig 1.10: Mode of action of antioxidant reacting with free radicals (Siddeeg et al., 2021).

ROO• + AH/ArOH → ROOH + A•/ArO•(HAT)

38
In general, the hydrogen atom transfer (HAT) assay protocol primarily relies on the bond

dissociation enthalpy (energy) of the hydrogen-donating group of the antioxidant (Farrokhnia,

2020). Consequently, antioxidants with weaker hydrogen ion release from their donating groups

tend to facilitate a faster and more stable HAT reaction. Similar to the SET method, the HAT assay

is also influenced by pH and solvent conditions (Farrokhnia, 2020). However, unlike the SET

method, the HAT assay is quicker and involves obtaining data from kinetic curves.This kinetic

HAT assay typically comprises an antioxidant, an oxidizable fluorescent probe (such as

dichlorofluorescein), and a synthetic radical generator (such as AAPH, AMVN, and ABTS)

(Farrokhnia, 2020).

1.5 DIABETES MELLITUS

Diabetes mellitus originates from the Greek term diabetes, meaning "siphon - to pass through,"

and the Latin word mellitus, which denotes "sweet." Its historical roots trace back to Apollonius

of Memphis around 250 to 300 BC, who first utilized the term "diabetes (Sapra and Bhandari,

2023)." Early civilizations such as the Ancient Greeks, Indians, and Egyptians recognized the

sweet quality of urine in this condition, leading to the term Diabetes Mellitus. In 1889, Mering and

Minkowski unveiled the pancreas's role in diabetes pathogenesis (Sapra and Bhandari, 2023).

Subsequently, in 1922, Banting, Best, and Collip isolated insulin from cows' pancreases at the

University of Toronto, revolutionizing diabetes treatment. Despite significant advancements and

ongoing efforts to manage the disease, diabetes remains a prevalent chronic ailment globally,

ranking as the seventh leading cause of death in the United States (Sapra and Bhandari, 2023)..

Diabetes mellitus (DM) manifests as a metabolic disorder characterized by elevated blood glucose

levels. Its classifications encompass type 1, type 2, maturity-onset diabetes of the young (MODY),

gestational diabetes, neonatal diabetes, and secondary forms stemming from endocrinopathies or

39
steroid usage (American Diabetes Association, 2010). The primary subtypes, Type 1 diabetes

mellitus (T1DM) and Type 2 diabetes mellitus (T2DM), typically arise from impaired insulin

secretion (T1DM) and/or diminished insulin action (T2DM) (American Diabetes Association,

2010). T1DM often manifests in children or adolescents, while T2DM typically affects middle-

aged and older adults with prolonged hyperglycemia due to lifestyle and dietary factors. The

pathogenesis, etiology, presentations, and treatments differ significantly between T1DM and

T2DM due to their distinct mechanisms (American Diabetes Association, 2010).

1.5.1 TYPES OF DIABETES

Diabetes mellitus is a chronic metabolic disorder categorized into several types, with Type 1 and

Type 2 being the most prevalent. Type 1 diabetes is characterized by an autoimmune response

where the body attacks insulin-producing beta cells in the pancreas, leading to insufficient insulin

production. It is commonly diagnosed in childhood or adolescence but can occur at any age

(American Diabetes Association, 2021). Type 2 diabetes, the most common form, results from

insulin resistance, where the body's cells do not respond effectively to insulin, often compounded

by inadequate insulin production. This type is generally associated with obesity, physical

inactivity, and genetics (DiMeglio et al., 2020). Another form is gestational diabetes, which occurs

during pregnancy. It poses risks to both mother and child but often resolves postpartum, although

it may increase the risk of developing Type 2 diabetes later (Barbour et al., 2020). Additionally,

monogenic diabetes and secondary diabetes arise from specific genetic mutations or other medical

conditions such as pancreatic diseases or hormone disorders, respectively (Naylor et al., 2021).

1.5.1.1 TYPE 1 DIABETES MELLITUS

Type 1 diabetes primarily results from an autoimmune process targeting pancreatic β cells, marked

by T-cell mediated inflammatory response (insulitis) and a humoral (B cell) reaction. The hallmark

40
of type 1 diabetes is the presence of autoantibodies against pancreatic islet cells, although their

exact role in disease pathogenesis remains unclear (Kharroubi, and Darwish, 2015). These

autoantibodies encompass islet cell autoantibodies, as well as antibodies to insulin (IAA), glutamic

acid decarboxylase (GAD, GAD65), protein tyrosine phosphatase (IA2 and IA2β), and zinc

transporter protein (ZnT8A) (Kharroubi, and Darwish, 2015). They can be detected in the serum

of affected individuals months or years before disease onset, representing characteristic features

of type 1 diabetes. Autoimmune type 1 diabetes displays strong HLA associations, with

connections to DR and DQ genes. HLA-DR/DQ alleles may confer either predisposition or

protection against the condition (Kharroubi, and Darwish, 2015). This autoimmune form of type

1 diabetes is distinguished by deficient insulin secretion and tends to be more prevalent among

children and adolescents (Kharroubi, and Darwish, 2015).

Apart from genetic predisposition, numerous environmental factors have been implicated in the

development of type 1 diabetes (van der Werf et al., 2007). Viral factors encompass congenital

rubella, viral infections involving enterovirus, rotavirus, herpes virus, cytomegalovirus,

endogenous retrovirus, and Ljungan virus (van der Werf et al., 2007). Additional contributors

comprise low levels of vitamin D, prenatal exposure to pollutants, improved hygiene and living

conditions reducing childhood infections in high socioeconomic status countries (hygiene

hypothesis), early infant nutrition such as the use of cow’s milk formula instead of breastfeeding,

and insulin resistance during early childhood due to obesity or accelerated height growth velocity

(van der Werf et al., 2007). Type 1 diabetes often manifests suddenly, presenting symptoms

including polydipsia, polyuria, enuresis, fatigue, profound tiredness, polyphagia, abrupt weight

loss, slow wound healing, recurrent infections, and blurred vision, particularly in children and

adolescents, leading to severe dehydration and diabetic ketoacidosis (Knip and Simell, 2012).

41
Symptoms tend to be more pronounced in children compared to adults. Individuals with

autoimmune type 1 diabetes also exhibit susceptibility to other autoimmune disorders like Graves’

disease, Hashimoto’s thyroiditis, Addison’s disease, vitiligo, celiac sprue, autoimmune hepatitis,

myasthenia gravis, and pernicious anemia (Knip and Simell, 2012). A "honeymoon phase," lasting

weeks to months or occasionally 2-3 years, may temporarily reduce the insulin dependency of type

1 diabetes patients. In some children, insulin therapy requirements may decrease to a point where

insulin can be temporarily withdrawn without causing detectable hyperglycemia (Knip and Simell,

2012).

1.5.1.2 TYPE 2 DIABETES

Insulin resistance in patients with type 2 diabetes amplifies the insulin demand within insulin-

target tissues (Kasuga, 2006). Alongside insulin resistance, the heightened demand for insulin

cannot be adequately met by pancreatic β cells due to inherent functional defects (Kasuga, 2006).

Conversely, as the demand for insulin escalates over time, insulin secretion diminishes due to

progressive β cell destruction, potentially transitioning some type 2 diabetes patients from

independence to insulin dependence (Kasuga, 2006). Although most type 2 diabetes patients

maintain independence from insulin, with ongoing insulin secretion and rare instances of insulin

depletion, reliance on insulin distinguishes type 2 diabetes from type 1 (Kasuga, 2006). Additional

distinctions include the absence of ketoacidosis in the majority of type 2 diabetes cases and the

non-occurrence of autoimmune destruction of β cells. While both type 1 and type 2 diabetes

involve genetic predisposition, it is more pronounced in type 2, although gene characterization is

more defined in type 1 (e.g., the TCF7L2 gene strongly linked to type 2 diabetes) (Kasuga, 2006).

Due to the initially mild symptoms of type 2 diabetes, diagnosis is often delayed for years,

particularly in regions where routine check-ups in the absence of symptoms are not customary

42
(Gopalan et al., 2018). This diagnostic delay heightens the risk of long-term complications in type

2 diabetes patients since hyperglycemia remains untreated during the undiagnosed period (Gopalan

et al., 2018).Beyond diabetes, insulin resistance manifests in various ways, including obesity,

nephropathy, essential hypertension, dyslipidemia (hypertriglyceridemia, low HDL, decreased

LDL particle diameter, enhanced postprandial lipemia, and remnant lipoprotein accumulation),

ovarian hyperandrogenism, premature adrenarche, non-alcoholic fatty liver disease, and systemic

inflammation (Gopalan et al., 2018). The occurrence of type 2 diabetes in non-obese children and

adolescents, sporadic severe dehydration, and ketoacidosis in some pediatric type 2 diabetes cases

have led to misclassifications between type 2 and type 1 diabetes (Gopalan et al., 2018).

1.5.2 NATURAL MEDICINES FOR DIABETES THERAPY

It is crucial to acknowledge that no particular botanical has been consistently documented to lower

glucose or insulin levels. Nonetheless, each botanical, recognized for its historical utilization,

current inclusion in herbal supplements, or potential clinical efficacy based on proposed

mechanisms are listed below;

1.5.2.1 Bitter Melon (Momordica charantia)

Bitter melon, originating from Asia, is a traditional plant often suggested for treating diabetes and

its associated complications (Joseph and Jini, 2013). Its mechanism of action is thought to be

attributed to various bioactives, including polypeptide-p, which bears a structure akin to animal

insulin, suggesting potential glucose-lowering effects (Joseph and Jini, 2013). Bitter melon fruit is

rich in cucurbitane-type triterpenoids, steroidal saponins known as "charantins," insulin-like

peptides, and alkaloids, all presumed to influence carbohydrate metabolism (Joseph and Jini,

2013). However, clinical outcomes using bitter melon have shown inconsistency, with only

approximately half of the studies demonstrating efficacy (Yin et al., 2014). Clearly, controversy

43
surrounds reported observations, with concerns raised regarding study design and the adequacy of

statistical analyses (Yin et al., 2014).

Another factor contributing to the variability in results is the diverse preparations of the test

material (Yin et al., 2014). Test materials may include fresh juice, dried whole fruit, fresh fruit,

dried seedless fruit, seeds, aqueous or methanolic extracts, or tablets. Such variations significantly

impact the bioactive content and bioavailability of active compounds within the preparation (Yin

et al., 2014).

1.5.2.2 Fenugreek (Trigonella foenum-graecum)

Fenugreek boasts a rich history of medicinal applications and has been globally recognized for its

potential in diabetes management (Ahmad et al., 2016). Originating as a leguminous herb

cultivated in India and North Africa, fenugreek seeds are commonly utilized as both a food

ingredient and spice, renowned for their high protein and fiber content (Ahmad et al., 2016).

Extensive research indicates that fenugreek exhibits hypoglycemic and hypocholesterolemic

properties, supported by evidence from animal and human studies (Ahmad et al., 2016).

The hypoglycemic effects of fenugreek, particularly noteworthy, may be attributed to its fiber

content, which could influence gastric emptying, resulting in reduced postprandial blood glucose

levels (Shabil et al., 2023). Additionally, fenugreek contains various bioactive compounds,

including the alkaloid trigonelline and steroidal saponins, with 4-hydroxyisoleucine recognized as

a particularly active component with insulin-like effects (Shabil et al., 2023).Similar to many

herbal preparations, fenugreek has yielded inconsistent clinical outcomes, potentially stemming

from inadequate study design, imprecise endpoints, underpowered studies, or variations in test

substances. Nonetheless, fenugreek seed powder has shown promise in positively impacting the

glycemic index of food and improving glucose tolerance in both control and diabetic individuals

44
(Shabil et al., 2023). Further investigations have indicated that an 8-week treatment regimen in

diabetic subjects led to enhancements in fasting glucose levels and dyslipidemia. Notably, more

consistent results have been observed with fenugreek when administered at higher doses of 10–20

g/day, which may relate to its effects on digestive processes (Shabil et al., 2023).

1.5.2.3 Garlic (Allium sativum)

Garlic stands out among herbal remedies with its historically recognized medicinal properties

(Bayan et al., 2014). Its wide array of benefits includes antithrombotic, antihypertensive,

cholesterol-lowering, antioxidant, antimutagenic, and antimicrobial effects (Bayan et al., 2014).

Given its purported broad spectrum of actions, garlic has attracted significant research interest into

its mechanisms (Bayan et al., 2014). Notably, numerous preclinical and clinical studies have

explored garlic's hypotensive potential, with more consistent findings observed in animal models

compared to clinical settings (Bayan et al., 2014). The precise mechanism by which garlic reduces

blood pressure remains unclear; however, proposed mechanisms include modulation of endothelial

nitric oxide (NO) production, inhibition of angiotensin-converting enzyme (ACE) activity,

reduction in vasoconstrictive agents such as thromboxane-B2 and prostaglandin-E2, and potent

free-radical scavenging activity (Ried and Fakler, 2014). As with other herbal remedies, variability

in clinical results may arise from differences in garlic preparations used or the specific bioactive

compounds present (Ried and Fakler, 2014). Reported bioactives include unstable sulfur-

containing compounds, polyphenols, flavonoids, anthocyanins, tannins, among others (Ried and

Fakler, 2014).

A recent meta-analysis examined clinical studies spanning the past half-century, encompassing 11

trials conducted between 1955 and 2007 (Stevinson et al., 2000). These studies included true

placebo groups, employed garlic-only preparations, and provided statistical analyses reporting

45
mean systolic and/or diastolic blood pressure (SBP/DBP) alongside standard deviations (Stevinson

et al., 2000). The analysis concluded that individuals treated with garlic experienced better

outcomes, particularly evident in the hypertensive subgroup, where significant reductions in both

systolic and diastolic blood pressure were observed compared to placebo-treated counterparts

(Stevinson et al., 2000). The reported mean (SD) decreases in blood pressure were 8.4 (2.8) mm

Hg (n = 4; p < .001) for systolic and 7.3 (1.5) mm Hg for diastolic (n = 3; p < .001) blood pressure

(Stevinson et al., 2000).

1.5.2.4 Aloe (Aloe vera)

In India and the Arabian peninsula, Aloe vera has been utilized in medicinal treatments for diabetes

(Modak et al., 2007). The gel extracted from the inner portion of the leaves is believed to contain

glucomannan, a water-soluble fiber with purported hypoglycemic and insulin-sensitizing

properties (Modak et al., 2007). Although preclinical studies have yielded inconsistent results,

small-scale clinical trials have indicated improvements in fasting glucose levels with the extract

(Modak et al., 2007). A comprehensive review examining the effects of herbal remedies on

glycemia suggested that preliminary data hint at Aloe vera's potential in glycemic control;

however, further validation is required (Modak et al., 2007).

1.5.2.5 Cinnamon (Cinnamomum cassia, Verum, and Others)

Cinnamon has a longstanding history in the treatment of diabetes and is increasingly popular as a

dietary supplement, with numerous cinnamon products available on the marke (Silva et al., 2022)t.

While the precise bioactives responsible for its antidiabetic effects remain uncertain, polyphenol

type-A polymers are believed to be among the active components that may mimic insulin (Silva

et al., 2022). Consequently, preclinical studies conducted in murine diabetes models have

indicated potential effects of cinnamon (Silva et al., 2022).Despite a growing body of clinical

46
research on cinnamon, results, much like those of other herbal products, exhibit inconsistency.

Variability in clinical outcomes may stem from factors such as cohort selection criteria, chosen

clinical endpoints, appropriate dosing, and the source of cinnamon bioactives (Silva et al., 2022).

Nevertheless, some studies have reported favorable effects. For instance, a study involving 109

diabetic patients previously managed with diet and exercise found that a daily dose of 1 g of

cinnamon over 90 days significantly reduced preceding glycemia, as measured by HbA1c,

compared to the control group (Vafa et al., 2012).Additional research has suggested beneficial

effects of cinnamon on glucose and lipid levels, although other investigations have failed to

demonstrate such effects (Vafa et al., 2012). Furthermore, preclinical and small-scale clinical trials

evaluating individuals with metabolic syndrome have hinted at potential cardiovascular risk

reduction properties of cinnamon, including antihypertensive effects (Vafa et al., 2012).

1.6 Markhamia tomentosa

Markhamia tomentosa is a medicinally valuable tree species native to tropical Africa, particularly

prevalent in West and Central Africa. Belonging to the Bignoniaceae family, M. tomentosa is

known for its therapeutic applications in traditional medicine. Various parts of the tree, including

the leaves, roots, and bark, are used for treating a range of ailments such as coughs, fevers, malaria,

and gastrointestinal disorders (Awodiran et al., 2021). The phytochemical composition of M.

tomentosa includes alkaloids, flavonoids, tannins, saponins, and phenolics, which contribute to its

bioactive properties (Okokon et al., 2020).

Recent studies have demonstrated its significant antimicrobial, anti-inflammatory, and antioxidant

activities, making it a potential source for the development of natural therapeutic agents. The bark

extract of M. tomentosa has been reported to exhibit potent antibacterial activity against multidrug-

resistant pathogens, which highlights its relevance in combating drug-resistant infections

47
(Osuagwu et al., 2022). Furthermore, the antioxidant properties are attributed to its high flavonoid

and phenolic content, which scavenge free radicals and reduce oxidative stress, potentially offering

protection against degenerative diseases (Njoku et al., 2023).

48
Fig 1.11: Diagram of Markhamia tomentosa (Benth.) K. S

49
1.6.1 BOTANY

Markhamia tomentosa, a tree reaching approximately 15 meters in height, is commonly found in

the savannah forests across West Africa, extending southward to Angola (Ibrahim et al., 2018).

This tree, belonging to the Bignoniaceae family, boasts large yellow flowers arranged in elongated

terminal racemes, lending it a decorative quality during flowering. Its leaves are arranged

oppositely and are simple pinnately compound (Ibrahim et al., 2018). In Cameroon, the plant goes

by various names such as “bougtoun” in Bayangam, “bobèdou” in Duala, “abbe” in Koosi, or

“mawelu” in Kpe (Ibrahim et al., 2018).

The stem bark of M. tomentosa is utilized to alleviate chest pain, while the leaves are employed in

treating headaches, lumbago, edema, and gout (Bankole et al., 2017). Preparations made from the

leaves and bark are administered as rejuvenating and diuretic remedies for leg edema and

elephantiasis of the scrotum (Bankole et al., 2017). Additionally, these preparations are utilized to

address canker, rheumatic pain, respiratory tract ailments, bouts of swamp fever, constipation, and

fever. Ethnopharmacological evidence suggests various therapeutic uses of this plant against snake

bites/venom, sore eyes, heart pain, general pains, headaches, or backaches (Bankole et al., 2017).

M. tomentosa leaves are reported to be effective in treating diseases related to the reproductive

system (Bankole et al., 2017).

Previous studies have demonstrated that ethyl acetate, dichloromethane, and methanol leaf extracts

of M. tomentosa possess antioxidant and antimicrobial properties (Ibrahim et al., 2016).

Phytochemical investigations have identified saponins, tannins, anthraquinones, alkaloids,

glycosides, cardiac glycosides, flavonoids, and phenols in the methanol extract of M. tomentosa.

Among the compounds identified in the ethyl acetate extract, 2-acetylnaphtho[2,3-b]furan-4,9-

dione and 2-acetyl-6-methoxynaphtho[2,3-b]furan-4,9-dione exhibited potent antiprotozoal

50
activity and high toxicity on rat skeletal muscle myeloblasts (Ibrahim et al., 2016). It has been

demonstrated that M. tomentosa leaf extracts possess analgesic effects, with the most potent

recorded in the methanol leaf extract, which also inhibited carrageenan-induced paw edema

(Ibrahim et al., 2016). The anti-inflammatory activities of methanol leaf extract (MLE) were

investigated against xylene, histamine, and serotonin-induced acute inflammation, as well as

formalin and cotton pellet-induced chronic inflammation. Anti-arthritic effects of methanol leaf

extract of M. tomentosa on complete Freund’s adjuvant-induced arthritis were confirmed (Ibrahim

et al., 2016). However, to the best of our knowledge, there are no available studies on the effects

of MLE against experimental liver injury. Therefore, this study was conducted to examine the

hepatoprotective effects of MLE against acute D-GalN/LPS-induced fulminant liver injury in mice

(Ibrahim et al., 2016).

1.6.2 PLANT DESCRIPTION

Markhamia tomentosa (Benth.) K. Schum Ex Engl. (Bignoniaceae) finds application in traditional

African medicine to address conditions such as diarrhea, edema, pain, and malaria (Ibrahim et al.,

2018). Previous reports suggest that the leaf extract exhibits no apparent signs of toxicity upon

acute exposure. The current investigation delves into the sub-acute and chronic toxicity effects of

Markhamia tomentosa in rats (Ibrahim et al., 2016).

Kingdom Plantae
Phylum Tracheophyta
Class Magnoliopsida
Order Lamiales
Family Bignoniaceae
Genus Markhamia Seem.
Species Markhamia tomentosa (Benth)
K. Schum. ex. Engl

51
Fig 1.12: Markhamia tomentosa (Benth) K. Schum. ex. Engl (Ibrahim et al., 2016)

52
1.7 BIOCHEMICAL PARAMETERS STUDIED

1.7.1 FLAVONOIDS

Flavonoids, which are phytochemical compounds found in various plants, fruits, vegetables, and

leaves, hold promise in medicinal chemistry (Ullah et al., 2020). They offer several medicinal

advantages, including anticancer, antioxidant, anti-inflammatory, and antiviral properties, along

with neuroprotective and cardio-protective effects (Ullah et al., 2020). The biological activities

associated with flavonoids vary depending on the type of flavonoid, its potential mode of action,

and its bioavailability (Ullah et al., 2020). Flavonoids, categorized as secondary metabolites, are

primarily composed of a benzopyrone ring featuring phenolic or polyphenolic groups at various

positions (Dias et al., 2021). They are commonly found in fruits, herbs, stems, cereals, nuts,

vegetables, flowers, and seeds (Dias et al., 2021). The presence of bioactive phytochemical

constituents in these plant parts imparts medicinal value and biological activities to them. To date,

more than 10,000 flavonoid compounds have been isolated and identified (Dias et al., 2021). Many

flavonoids are widely acknowledged as therapeutic agents. They are naturally synthesized through

the phenylpropanoid pathway, and their bioactivity is contingent upon their absorption mechanism

and bioavailability (Dias et al., 2021). Flavonoids find diverse applications in natural dyes,

cosmetics, skin care products, and anti-wrinkle agents (Čižmárová et al., 2023). However, their

most significant applications lie in the realm of medicine (Čižmárová et al., 2023). Flavonoids are

extensively utilized as anticancer, antimicrobial, antiviral, antiangiogenic, antimalarial,

antioxidant, neuroprotective, antitumor, and anti-proliferative agents (Čižmárová et al., 2023).

Extracts from apple peel, rich in flavonoids, have been shown to inhibit acetylcholinesterase

(ACE) in vitro and serve as effective antihypertensive agents. Moreover, they help prevent cardio-

53
metabolic disorders and contribute to better preservation of cognitive performance with aging

(Čižmárová et al., 2023).

Flavonoids are categorized into different types based on their chemical structure, degree of

unsaturation, and carbon ring oxidation (Oliviera et al., 2019). Subgroups of flavonoids include

anthoxanthins (flavanone and flavanol), flavanones, flavanonols, flavans, chalcones,

anthocyanidins, and isoflavonoids, each widely distributed in nature (Oliviera et al., 2019).

Consuming foods rich in flavonoids offers numerous health benefits. Due to their positive effects

on human health, there has been a growing effort to isolate these compounds from various plants

(Oliviera et al., 2019). Citrus fruits, for example, serve as rich sources of flavonoids. Oranges,

lemons, and grapes contain two flavonoids, narigenin and hesperetin. Mulberry, on the other hand,

contains anthocyanins and quercetin glycosides flavonoids (Oliviera et al., 2019)

54
.

Fig 1.13: Chemical structure of flavonoids and its different types (Ullah et al., 2020).

55
1.7.2 EFFECTS OF FLAVONOIDS ON HUMAN HEALTH

1.7.2.1 Anticancer action

Cancer poses a significant health challenge characterized by abnormal cell growth. Although

numerous anticancer drugs exist, only a limited few effectively inhibit oncogenesis, while many

others exhibit toxicity and adverse side effects (Anand et al., 2022). Natural biomolecules

containing secondary metabolites, through phytomediated content, demonstrate a broad spectrum

of biological activities, serving as a foundation for cancer prevention and treatment (Anand et al.,

2022). Flavonoids, renowned for their ability to impede cell growth, function as anticancer agents.

Chemoprevention involves utilizing natural or synthetic substances to deter carcinogenesis. Below

are abundant examples of flavonoids and their application as anticancer agents (Anand et al.,

2022).

Hesperidin (Hsp) is a significant flavonoid known for its effective anticancer properties (Melike

et al., 2019). To explore its potential as an anticancer agent against C6 glioma cells, polylactic-co-

glycolic acid (PLGA) nanoparticles were synthesized and loaded with Hsp, forming hesperidin

nanoparticles (HspNPs) (Melike et al., 2019). Encapsulated Hsp demonstrated reduced in vitro

cell viability against the C6 glioma cell line. Moreover, the controlled release of Hsp contributed

to decreased cytotoxicity of PLGA (Melike et al., 2019).

Quercetin, a natural flavonoid found in plants and commonly consumed foods like berries, green

tea, and grains, has shown significant efficacy against colorectal cancer (Li et al., 2018). Its

chemopreventive effects in colorectal cancer involve various mechanisms, including cell cycle

arrest, increased apoptosis, antioxidant replication, estrogen receptor modulation, regulation of

signaling pathways, and inhibition of metastasis and angiogenesis (Li et al., 2018).

56
Luteolin, another natural flavonoid, exhibits pro-apoptotic activity in hepatocellular carcinoma

(HCC) cells and induces cell cycle arrest at the G2/M stage (Han et al., 2023). Luteolin upregulates

miR-6809-5p, which is overexpressed in HCC, by directly targeting flotillin-1 (Han et al., 2023).

Kaempferol, a natural flavanol, reduces the risk of cancer by enhancing the body’s antioxidant

defense against free radicals that contribute to cancer development (Han et al., 2023).

Myricetin, an essential flavonoid, possesses anti-inflammatory and anticancer properties (Imran et

al., 2021). In liver cancer, it demonstrates antimitotic effects and targets various metabolic

pathways in mitochondria, leading to cancer cell death (Imran et al., 2021).

1.7.2.2 Antioxidant activity

The antioxidant capacity of flavonoids is determined by their molecular structure, including the

location and total number of –OH groups, conjugation and resonance effects, the surrounding

environment affecting the preferred antioxidant site thermodynamically, and the specific

antioxidant mechanism of a compound (Kumar and Pandey, 2013). While commonly used

antioxidant supplements include vitamins C and E, the antioxidant potential of flavonoids

surpasses that of vitamin C and vitamin E (Kumar and Pandey, 2013). Therefore, it is essential to

incorporate fruits and vegetables rich in flavonoids into daily dietary intake (Kumar and Pandey,

2013). For instance, due to their well-established antioxidant and anti-inflammatory properties,

flavonoids contribute to improved bone health and hold promise for applications in biomaterials

for bone tissue engineering, particularly in the diets of elderly individuals (Kumar and Pandey,

2013).

Quercetin, an antioxidant flavonoid, enhances vascular health and reduces the risk of

cardiovascular disease in its conjugated form when present in the bloodstream (Zhang et al., 2023).

It prevents thrombosis and reduces the likelihood of stroke. Hesperidin and hesperetin, two

57
flavonoids found in citrus fruits and mushrooms, exhibit antioxidant, anti-inflammatory,

antimicrobial, and anticancer effects (Zhang et al., 2023). Propolis, utilized in folk medicine for

centuries and now employed in the pharmaceutical industry, contains flavonoids that contribute to

its pharmacological properties, including antioxidant, antimicrobial, healing, and anti-proliferative

activities (Zhang et al., 2023).

Rutin, a flavonol, demonstrates various biological activities, including anticancer, antioxidant, and

cytoprotective effects (Satari et al., 2021). Sorghum exhibits high antioxidant activity in vitro, with

flavonoids and tannins being its most potent antioxidants. Despite tannins rendering sorghum

grains unsuitable for consumption, alkaline cooking significantly reduces tannin levels by 73%

(Satari et al., 2021). Thus, it is crucial to genetically modify sorghum to eliminate tannin

production pathways, enabling the production of tannin-free grains suitable for consumption.

Sorghum is resilient to arid conditions and harsh environments, making it feasible to cultivate

flavonoid-rich sorghum grains worldwide (Satari et al., 2021).

Carotenoids, another class of polyphenols, possess antioxidant properties. Fisetin (3, 3′, 4′, 7–

tetrahydroxy flavone), a flavonoid, exhibits antioxidant and anti-inflammatory effects

(Andarwulan et al., 2021). Eleutherine bulbosa (Mill.) Urb., including its aerial parts such as bulbs,

leaves, and flowers, contains a high concentration of flavonoids and exhibits antioxidant

properties. Allium cepa L. (onion) also demonstrates antioxidant activity (Andarwulan et al.,

2021). Dill (Anethum graveolens L.) and parsley (Petroselinum crispum Mill.) are aromatic

medicinal herbs whose phenolic and flavonoid content endows them with potent antioxidant

properties, capable of reducing reactive oxygen species (ROS) and preventing ROS-mediated

diseases like cancer and cardiovascular ailments (Andarwulan et al., 2021). The antioxidant

potential of these species may be attributed to various mechanisms, including the breakdown of

58
peroxides and chelation of metal ions that catalyze oxidation processes. The antioxidant activity

correlates with the phenolic content (PC) and flavonoid content (FC); generally, higher PC and FC

levels correspond to greater antioxidant activity exhibited by the herbal plants (Andarwulan et al.,

2021).

1.7.2.3 Effect on Cardiovascular System

Consumption of dietary flavonoids has been positively associated with a reduction in

cardiovascular diseases (F et al., 2019). Numerous studies have indicated that individuals who

consume higher amounts of flavonoids have an 18% lower risk of mortality from cardiovascular

diseases (F et al., 2019). Flavonoids exhibit cardioprotective, neuroprotective, and

chemoprotective properties as demonstrated by various studies (F et al., 2019). Tea, being rich in

flavonoids, is known to reduce the risk of cardiovascular diseases. Specific flavonoids such as

anthocyanidin and proanthocyanidin have shown effectiveness against cardiac diseases.

Isoflavone, anthocyanins, and cocoa flavan-3-ols contribute to improved vascular health and

decreased arterial stiffness, thus reducing the risk of cardiovascular diseases (F et al., 2019).Oils

derived from the leaves and fruits of Hippophae rhamnoides (sea buckthorn) contain numerous

compounds, including flavonoids, that exhibit beneficial effects on the cardiovascular system

(Jaśniewska and Diowksz, 2021). Morin hydrate has demonstrated significant biological activity,

including anti-inflammatory, anticancer, and cardio protective effects. Brazil nuts, which are rich

in flavonoids, contribute to the prevention of heart disease and cancer (Jaśniewska and Diowksz,

2021). Chrysin, a flavone, exhibits beneficial effects on epilepsy and depression, suppresses neuro-

inflammation, and provides neuroprotective effects (Jaśniewska and Diowksz, 2021).

In pre-clinical studies on animal models, the effect of Primula veris L. solid herbal extract

(PVSHE) on myocontractile function was investigated (Tarapatskyy et al., 2021). The composition

59
of phenolic compounds in the extract was analyzed using column chromatography, and the

concentration of total flavonoids was determined through differential UV spectrophotometry

(Tarapatskyy et al., 2021). The obtained compounds were characterized using NMR spectroscopy.

In an experiment involving adult Wister rats, those administered with PVSHE exhibited

cardiodynamic changes and improved myocardial contraction rates. Enzyme-linked

immunosorbent assay (ELISA) was employed to determine CHF markers, with polymethoxylated

flavonoids acting as biological markers for Primula L (Tarapatskyy et al., 2021). The presence of

flavonoids likely contributes to decreased ROS production and suppressed peroxide formation,

resulting in the cardio-protective effects observed (Tarapatskyy et al., 2021).

Morin, a bioflavonoid, has been shown to possess cardio-protective properties in animal models.

Rats were divided into groups, and morin was administered orally in a dose-dependent manner to

the experimental group (Al-Numair et al., 2014). Myocardial necrosis was induced, and the results

indicated improved antioxidant effects and apoptosis (Al-Numair et al., 2014). The mechanism

underlying this cardio protection was reported to involve alterations in the MAPK/NF-kappa

B/TNF-alpha pathway (Al-Numair et al., 2014).

1.7.2.4 Effect on Nervous System

Flavonoids play a crucial role in preventing age-related neurodegenerative diseases, including

dementia, Parkinson’s, and Alzheimer’s disease (de Andrade Teles et al., 2018). Reactive oxygen

species (ROS) and nitrogen species (NOCs) are implicated in numerous neurodegenerative

conditions (de Andrade Teles et al., 2018). Tangeretin, a flavonoid abundant in citrus fruits,

functions as an antioxidant against ROS and NOCs species, offering protection against

neurodegenerative disorders such as Parkinson’s disease (de Andrade Teles et al., 2018).

60
Foods rich in flavonoids reduce the risk of neurodegenerative diseases and mitigate age-related

cognitive disorders (Spencer, 2009). They operate in two key ways: by regulating neuronal signal

cascades that trigger cell apoptosis, and by exerting beneficial effects on both the peripheral and

central nervous systems (Spencer, 2009). Hesperidin (Hsd) and hesperetin (Hst) are two flavonoids

recognized for their neuropharmacological effects, including neuroprotective and antidepressant

properties, as well as memory enhancement. Berries contain various natural flavonoids, including

polyphenolic compounds like stilbene and anthocyanins, which act as effective anti-

neurodegenerative, antimutagenic, and antimicrobial agents (Spencer, 2009). Epicatechin, an

antioxidant flavonoid abundant in woody plants, and its analogue 3-O-methyl epicatechin inhibit

neurotoxicity in vitro (Spencer, 2009). The polyphenolic luteolin flavonoid exerts neuroprotective

effects and shields against age-related neuro-disorders. Forsythia suspensa, a dried fruit and

Chinese medicinal herb, exhibits antioxidant properties and serves as a neuroprotective agent

against infectious diseases (Spencer, 2009).

Excessive alcohol consumption can lead to various health issues and adversely affect the brain

(Pervin and Stephen, 2021). Acetylpectolinarin (ACP), a flavonoid derived from Linaria vulgaris

Mill., has been shown to alleviate hangovers by enhancing the spontaneous network function of

cultured hippocampal neurons when exposed to low concentrations of ethanol (Pervin and

Stephen, 2021). This effect is achieved through its agonistic action on GABAergic synapses

mediated by SK potassium channels (Pervin and Stephen, 2021).

Hyperalgesia, characterized by heightened pain sensation due to peripheral nerve damage, is

associated with diabetic patients (Uddin et al., 2020). Quercetin and sodium, when combined, act

as antinociceptives, reducing diabetes-related complications (Uddin et al., 2020). Additionally,

cisplatin-induced hyperalgesia can be mitigated by 6-methoxyflavone (Uddin et al., 2020).

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1.7.3 HYDROXYL RADICAL (OH SCAVENGING)

The initiation of biomolecular damage in vivo is commonly attributed to reactive oxygen species

(ROS), a phenomenon known as oxidative stress (Lipinski, 2011). However, oxidation reactions

in biological systems can also occur via non-radical pathways, such as those involving hydrogen

peroxide. In such instances, the resulting molecules are enriched with one or more oxygen atoms,

which are generally considered indicative of oxidative stress (Lipinski, 2011). However, the mere

presence of additional oxygen does not indicate whether a specific product arises from single- (free

radical) or two-electron oxidation reactions (Lipinski, 2011).

It’s important to note that, according to the concept of oxidative stress, the peroxidation and

degradation of susceptible biological molecules are induced by oxygen-centered radicals generated

by excessive blood oxygenation (Chazelas et al., 2021). This concept stems from studies of

ischemia/reperfusion scenarios, where prolonged periods of hypoxia are followed by a sudden

influx of oxygen (Chazelas et al., 2021). For instance, in patients undergoing extracorporeal

circulation, the appearance of lipid and/or nucleic acid oxidation products is regarded as evidence

of oxidative stress (Chazelas et al., 2021). Nevertheless, a crucial aspect is often overlooked: the

most biologically active hydroxyl radicals are indeed present (Chazelas et al., 2021).

Apart from the critical role of hypoxic or reducing conditions in vivo for the generation of hydroxyl

radicals, there exists another significant, albeit generally underrecognized, factor: the presence of

free iron in the blood, known as the labile iron pool (Tian et al., 2022). Several years ago, it was

proposed that iron therapy might facilitate the formation of hydroxyl radicals, thereby contributing

to atherosclerosis (Tian et al., 2022). It is noteworthy that excessive accumulation of stored iron

is observed in atherosclerotic lesions as well as in the brains of patients with neurological disorders

(Tian et al., 2022). The hydroxyl radical can be generated through the Fenton reaction in the

62
presence of reduced transition metals like Fe2+ and H2O2 (Sowndhararajan and Kang, 2013). This

radical is known to be the most reactive among all the reduced forms of dioxygen and is believed

to initiate cell damage in vivo (Sowndhararajan and Kang, 2013). Scavenging the hydroxyl radical

is crucial for antioxidant activity due to its exceptionally high reactivity (Sowndhararajan and

Kang, 2013). This reactivity allows the hydroxyl radical to interact with a wide range of molecules

present in living cells, including sugars, amino acids, lipids, and nucleotides. Therefore, the

removal of the hydroxyl radical is paramount for the protection of living systems (Sowndhararajan

and Kang, 2013). The hydroperoxyl radical (HO• 2) holds significance in lipid peroxidation

chemistry (Martemucci et al., 2022). This protonated version of superoxide produces H2O2, which

can interact with redox-active metals like iron or copper to produce HO• through Fenton or Haber-

Weiss reactions. HO• 2 is a more potent oxidant compared to the superoxide anion-radical and has

the potential to trigger the chain oxidation of polyunsaturated phospholipids, resulting in the

dysfunction of membranes (Martemucci et al., 2022).

1.7.4 INHIBITION OF LIPID PEROXIDATION

Lipid peroxidation is a process wherein oxidants, including free radicals, target lipids that contain

carbon-carbon double bond(s), particularly polyunsaturated fatty acids (PUFAs) (Ayala et al.,

2014). Over the past four decades, a substantial body of literature has elucidated the significant

role of lipid peroxidation in cell biology and human health (Ayala et al., 2014). An outcome of

uncontrolled oxidative stress, characterized by an imbalance between prooxidant and antioxidant

levels in favor of prooxidants, is the injury to cells, tissues, and organs caused by oxidative damage

(Chaudhary et al., 2023). It has been long understood that elevated levels of free radicals or

reactive oxygen species (ROS) can directly harm lipids. The main sources of endogenous ROS

production include mitochondria, the plasma membrane, the endoplasmic reticulum, and

63
peroxisomes, facilitated through various mechanisms such as enzymatic reactions and/or

autooxidation of several compounds like catecholamines and hydroquinone (Chaudhary et al.,

2023). Additionally, various exogenous stimuli such as ionizing radiation, ultraviolet rays, tobacco

smoke, pathogen infections, environmental toxins, and exposure to herbicides/insecticides

contribute to in vivo ROS production (Chaudhary et al., 2023).

The overall process of lipid peroxidation involves three main stages: initiation, propagation, and

termination (Petrovic et al., 2020). During the initiation phase of lipid peroxidation, prooxidants

such as the hydroxyl radical abstract allylic hydrogen, resulting in the formation of the carbon-

centered lipid radical (L•). In the propagation stage, the lipid radical (L•) rapidly reacts with

oxygen to create a lipid peroxy radical (LOO•) (Petrovic et al., 2020). This radical then abstracts

a hydrogen from another lipid molecule, generating a new L• (which continues the chain reaction)

and a lipid hydroperoxide (LOOH). In the termination reaction, antioxidants such as vitamin E

donate a hydrogen atom to the LOO• species, forming a corresponding vitamin E radical that reacts

with another LOO•, resulting in the formation of nonradical products (Petrovic et al., 2020). Once

lipid peroxidation is initiated, a cascade of chain reactions occurs until termination products are

generated (Petrovic et al., 2020).

64
Fig 1.14 The process of lipid peroxidation involves several steps (Ayala et al., 2014)

65
During the initiation phase, prooxidants abstract the allylic hydrogen, resulting in the formation of

the carbon-centered lipid radical. This carbon radical tends to be stabilized by a molecular

rearrangement to form a conjugated diene (step 1). In the propagation phase, the lipid radical

rapidly reacts with oxygen to create a lipid peroxy radical (step 2), which then abstracts a hydrogen

from another lipid molecule, generating a new lipid radical and lipid hydroperoxide (step 3). In

the termination reaction, antioxidants donate a hydrogen atom to the lipid peroxy radical species,

resulting in the formation of nonradical products (step 4).

As noted earlier, lipid peroxidation has been linked to various diseases and aging processes,

including atherosclerosis, cataracts, rheumatoid arthritis, and neurodegenerative disorders (Niki et

al., 2005). Consequently, the role of antioxidants has garnered significant attention (Niki et al.,

2005). Antioxidant defenses can be categorized into four main groups: prevention of active oxidant

formation, scavenging and quenching of active oxidants, repair of damage, and excretion of toxic

oxidation products, as well as adaptive responses (Niki et al., 2005). Enzymatic lipid oxidation

can be inhibited by targeting either the activation or reaction of enzymes. Free radical-mediated

lipid peroxidation can be inhibited by blocking chain initiation and propagation or by accelerating

chain termination (Domínguez et al., 2019). Additionally, lipid peroxidation induced by singlet

oxygen may be curbed by preventing its formation, such as by quenching ultraviolet light and

singlet oxygen itself using carotenoids (Domínguez et al., 2019). Various radical-scavenging

antioxidants are present in foods. Numerous natural and synthetic supplements and medications

with radical-scavenging properties have been investigated (Domínguez et al., 2019). In vivo

antioxidant activity is influenced by various factors, including reactivity towards radicals, the

destiny of antioxidant-derived radicals, absorption, distribution, localization, and mobility of

antioxidants, as well as interactions with other antioxidants. Functions beyond antioxidant action,

66
such as the induction of phase II enzymes, may also play a crucial role in the overall defense

system against oxidative stress (Domínguez et al., 2019).

One intriguing characteristic of the biological system is its heterogeneity (Liao et al., 2022).

Antioxidants are localized in either the aqueous or lipid phases. Lipophilic antioxidants are

dispersed within the lipophilic compartment of membranes and lipoproteins (Liao et al., 2022).

Vitamin E and vitamin C are typical examples of lipophilic and hydrophilic antioxidants,

respectively. Vitamin C resides in and scavenges radicals within the aqueous phase (Liao et al.,

2022). Experimental confirmation using a doxyl stearic acid spin probe revealed that the

scavenging rate of radicals within the membrane and LDL (low-density lipoprotein) slows down

as the radical penetrates deeper into the interior of membranes and LDL particles from the surface

(Liao et al., 2022). Vitamin E, a primary lipophilic antioxidant in the body, has undergone

extensive study and is well-suited for investigating antioxidant mechanisms in vivo (Niki, 2014).

It effectively scavenges lipid peroxyl radicals, which are pivotal in lipid peroxidation, thereby

interrupting the propagation of oxidation chains (Niki, 2014). A notable structural feature of

vitamin E is its phytyl side chain, essential for its integration and retention within membranes and

lipoproteins (Niki, 2014). However, this side chain limits its mobility within and between

membranes, impacting its radical-scavenging efficiency. Vitamin E comprises eight isoforms, yet

α-tocopherol transfer protein in the liver selectively binds to α-tocopherol (α-TOH), thereby

enhancing its bioavailability (Niki, 2014).

1.7.5 α-AMYLASE

α-Amylase (1,4-α-d-glucan glucohydrolase) belongs to the glycoside hydrolase family 13 (GH13)

and acts as an endo-glucanase (Sarian et al., 2017). It facilitates the breakdown of α-1,4-glycosidic

bonds found in starch and related polysaccharides, resulting in the formation of various-length

67
oligosaccharides with α-configuration and α-limit dextrins, which are branched oligosaccharides

(Sarian et al., 2017). α-Amylases, together with γ-amylases that function as exo-amylases, are

highly sought-after starch-converting enzymes in the market (Sarian et al., 2017). They find

extensive applications in various industrial processes, including starch saccharification for fructose

syrup production (often used alongside glucoamylase and glucose/xylose isomerase), as antistaling

agents in baking, in laundry detergents, bioethanol production, and paper production (Raveendran

et al., 2018). Although α-amylases can be sourced from various microorganisms, plants, and

animals, Bacillus spp. and Aspergillus spp. are among the most commonly exploited

microorganism sources (Raveendran et al., 2018). These species are cultivated for commercial α-

amylase production due to their high yield, easily optimized production processes, and unique

enzyme characteristics, such as enhanced thermostability (Raveendran et al., 2018).

α-Amylase targets starch as its substrate. Starch is a polysaccharide consisting of two main

polymers: amylose and amylopectin (Bijttebier et al., 2008). Amylose constitutes 20-25% of the

starch molecule and forms a linear chain composed of repetitive glucose units linked by α-1, 4-

glycosidic bonds. Amylopectin makes up 75-80% of starch and features branched chains of

glucose units (Bijttebier et al., 2008). The linear glucose units are connected by α1, 4-glycosidic

bonds, while branching occurs every 15-45 glucose units with α-1, 6 glycosidic bonds.The

composition of the hydrolysate resulting from starch hydrolysis greatly depends on factors such as

temperature, hydrolysis conditions, and the enzyme's origin. Its optimum pH for activity is

typically around 7.0 (Bijttebier et al., 2008).

α-Amylase has become an enzyme of paramount importance due to its ability to hydrolyze starch

and facilitate various downstream activities (de Souza and de Oliveira Magalhães, 2010). One such

activity is the production of glucose and fructose syrup from starch, with α-amylase catalyzing the

68
initial step in this process (de Souza and de Oliveira Magalhães, 2010). Previously, starch was

hydrolyzed into glucose through acid hydrolysis, which posed drawbacks like highly acidic

conditions and high temperatures (de Souza and de Oliveira Magalhães, 2010). However, enzyme

hydrolysis of starch overcomes these limitations and yields high-fructose syrup.The use of

enzymes in detergent formulations has experienced a significant increase, driven by growing

awareness of environmental protection (de Souza and de Oliveira Magalhães, 2010).

1.7.6 α-GLUCOSIDASE

The α-glucosidases are essential components of organism metabolic processes, disease prevention

and treatment, and sugar hydrolysis (Zhai et al., 2022). They find extensive applications in

chemical synthesis, clinical diagnosis, and other domains (Zhai et al., 2022). Belonging to the

glycoside hydrolase (GH) family (EC:3.2.1.20), α-glucosidases are ubiquitous across organisms,

primarily catalyzing the exohydrolysis of α-1,4-glycosidic linkages at the non-reducing end of

polysaccharides like maltooligosaccharides, liberating α-D-glucose (Zhai et al., 2022).

Additionally, they can create α-1,6-glycosidic linkages by binding free glucose residues with α-

1,4-glycosidic linkages in oligosaccharides, yielding non-fermentable oligosaccharides (O'Connell

et al., 2013).

The significance of α-glucosidases spans clinical diagnostics, disease prevention and treatment,

organism metabolic pathways, and processes like alcohol fermentation and sugar hydrolysis,

making them valuable in chemical synthesis fields (O'Connell et al., 2013). Most commercially

available α-glucosidases originate from fungi but are constrained by limitations such as moderate

thermostability, requirements for highly acidic pH levels, generation of numerous byproducts, and

sluggish catalytic activity (Mesbah, 2022). These limitations significantly elevate production costs

69
(Mesbah, 2022). Therefore, the quest for novel α-glucosidases is pressing to enhance glucose

production and catalytic efficiency (Mesbah, 2022).

The enzyme α-glucosidase plays a pivotal role in elevating blood glucose levels within the body.

Found in the small intestine, α-glucosidase contributes to the breakdown and assimilation of

carbohydrates (Kashtoh and Baek, 2022). It catalyzes the hydrolysis of polysaccharides and

oligosaccharides into monomers, thereby augmenting glucose concentration in the bloodstream.

Inhibitors of the α-glucosidase enzyme function to decelerate carbohydrate digestion and

absorption (Kashtoh and Baek, 2022). Consequently, postprandial blood glucose levels are

maintained at a lower threshold, resulting in reduced insulin demand. This mechanism enables the

independent control of glucose spikes following meals, irrespective of insulin levels (Kashtoh and

Baek, 2022). Consequently, α-glucosidase emerges as a crucial therapeutic target for managing

carbohydrate-related disorders (Kashtoh and Baek, 2022).

Alpha-glucosidase inhibitors offer a non-invasive therapeutic approach linked to mild, transient,

and dose-dependent gastrointestinal (GI) adverse effects, such as diarrhea, abdominal discomfort,

and flatulence (Dirir et al., 2022). By transiently postponing the intestinal absorption of

carbohydrates and consequently attenuating the increase in postprandial blood glucose levels, they

offer a convenient means to manage type 2 diabetes, which is closely associated with dietary

patterns (Dirir et al., 2022). The body absorbs polysaccharides and monosaccharides resulting

from the action of alpha-amylase and alpha-glucosidase at different rates, with monosaccharide

units being absorbed more rapidly (Gong et al., 2020). Inhibiting the activity of alpha-amylase and

alpha-glucosidase can therefore slow down the release of glucose from complex carbohydrates,

regulating the onset of postprandial hyperglycemia (Gong et al., 2020). This makes it an ideal

target for managing type 2 diabetes. Alpha-amylase inhibitors are primarily found in plants, and

70
the most extensively studied molecules are glycoproteins isolated from kidney beans (Phase 2 ®

product) (Gong et al., 2020).

1.8 THE OBJECTIVE OF THE STUDY

The use of medicinal plants as potential sources of therapeutic agents has gained significant

attention in recent years. Natural products derived from plants have been found to contain active

constituents with remarkable capacities for curing and preventing numerous human diseases.

One prominent aspect of medicinal plants is their antioxidant potential. Antioxidants play a crucial

role in protecting the body against oxidative stress, a condition characterized by an imbalance

between the production of reactive oxygen species (ROS) and the body’s antioxidant defense

system. Excessive ROS can lead to cellular damage, contributing to the development of various

diseases, including diabetes. Therefore, identifying potent antioxidants and anti-diabetic potentials

from natural sources has become a key area of interest in biomedical research.

The objective of this study is to determine:

• The antioxidant potentials of four extracts extract of markhermia tomentosa by studying

1,1 diphenyl-2-picrylhydrazyl (DPPH), total flavonoids content, hydroxyl radical

scavenging activity, inhibition of lipids peroxidation.

• The anti-diabetics potentials of four extracts extracts of markhermia tomentosa leaves by

studying inhibition of alpha amylase and alpha glucosidase

71
 CHAPTER TWO

MATERIALS AND METHODS

2.1 MATERIALS

The materials used are: Electric weighing balance, spectrophotometer, conical flask, beaker,

cuvettes, test tubes, centrifuge, metallic and plastic spoon, plastic bottle, gas cylinder,

thermometer, pH scale, spatula, washing brushes, detergents, liquid soaps, tissue papers,

micropipette, test tube racks, syringes, stop watch, buckets, bowls, reagent bottles, refrigerator,

pots, and gloves.

2.2 REAGENTS

The reagents used during these experiments are of analytical grade and they include; Distilled

water, Ethylacetate, N-hexane, methanol and chloroform 1,1-diphenyl-2-picrylhydrazyl (DPPH),

Quercetin, Aluminium chloride, Sodium acetate, Glacial acetic acid, sodium hydroxide, 2-

Deoxyribose, Phosphate buffer (6.8 PH), Iron III chloride (FeCl3), ethylenediaminetetraacetic acid

(EDTA), Hydrogen peroxide (H2O2), Ascorbic acid, Thiobarbituric acid (TBA), Ferrous sulfate

(FeSo4), Thiochloroacetic acid (TCA), Egg, Alpha amylase, Sodium hydrogen

phosphate(Na2HPo4), Potassium dihydrogen phosphate (KH2PO4), sodium carbonate (Na2Co3)

and Tris buffer.

2.3 SOURCES AND IDENTIFICATION OF PLANTS

The fresh whole plant of Markhamia tomentosa was plucked from Danunu, Orire Local

Government, Oyo State, Nigeria. The leaves was identified and authenticated by Prof A.T.J

Ogunkunle who is an angiosperm taxonomist at the Department of Pure and Applied Biology,

Ladoke Akintola University of Technology, Ogbomosho, Oyo state. The leaves of Markhamia

tomentosa leaves were given an herbarium voucher number LHO 846.

72
2.4 PROCESSING OF LEAF SAMPLE

The 1074g of dried leaves were first coarsely mashed using a mortar and pestle, then finely

pulverized in a grinder after having been air-dried in a cool environment for around 52 days. Before

being examined, the powdered samples were stored in airtight plastic bags out of direct sunlight

and away from any other sources of deterioration.

2.5 EXTRACTION OF LEAF SAMPLE

During 3 days (72 hours), Markhermia tomentosa leaf powder (1074 g) was soaked in 3350 ml of

the four solvents in a cool, dark place. After filtering and decanting the extract. To get the fine

different portion of the extracts, some part of the liquid portion was called to dry at room

temperature and the other underwent Soxhlet extraction.

2.6 IN-VITRO STUDY

2.6.1 DETERMINATION OF 1,1-DIPHENYL-2-PICRYLHYDRAZYL (DPPH) RADICAL

SCAVENGING ACTIVITY

PRINCIPLE

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay is a widely used method to

evaluate the antioxidant activity of a sample by determining its ability to scavenge DPPH radicals.

DPPH is a stable free radical characterized by a deep purple color, which upon reduction by an

antioxidant compound, changes to a yellow color. The decrease in absorbance at 518 nm is directly

proportional to the radical scavenging activity of the sample, and the percentage inhibition of

DPPH radicals can be calculated to quantify the antioxidant potential of the test compound or

extract. (Mokoroane, Pillai, and Magama, 2020).

REAGENTS

73
Extract(1mg/ml)

0.01g of the plant extract was made up to 10ml with the addition of 70% The four extracts.

DDPH (0.3mM)

0.03mM DPPH solution was prepared by dissolving 0.03g of DDPH in 70% the four extracts and

the volume was made up to 250ml.

Ascorbic Acid

Ascorbic acid was prepared by dissolving 0.1g of Ascorbic acid into 10ml of 70% the four extracts.

PROCEDURE

A set of test tubes were arranged and duplicated. The prepared extract solution (test solution) was

added to each test tube at the varied range from 50ml - 500ml using micro pipette and each solution

was made up to 1 ml by adding the four extracts.

1 ml of 0.304mM of DPPH solution was added to 1 ml of each of the test solutions.1ml of 70%

The four extracts without the extracts to stand as the control. and the solutions were allowed to

react at room temperature in the dark for 30 minutes.

The spectrophotometer was used to read the absorbance values after 30 minutes at the wave

length of 518 nm, and the oobtained absorbance is converted to percentage of antioxidant activity

using the formula below:

DPPH scavenging effect (%) = {A control - A sample} × 100

A control

Where A control = absorbance of fully oxidized control

A sample = absorbance value in the presence of extract/standard compound

74
1ml of the four extracts was added to 1ml of the extract (test solution) to serve as blank. 1ml of

the DPPH was added to 1 ml of the four extracts to serve as negative control. The Ascorbic control

which serve as the positive control was treat the same way as the negative control.

2.6.2 DETERMINATION OF TOTAL FLAVONOID CONTENT

PRINCIPLE

The principle behind the determination of total flavonoid content in a the four extracts extract of

Markhermia tomentosa leaves is based on the ability of flavonoids to form complexes with

aluminum chloride in acidic conditions, resulting in a change in the absorbance of the solution.

This method has been used in various studies to determine the flavonoid content in plants, fruits,

and vegetables. (Pękal, and Pyrzynska, 2014)

REAGENT

Extract Solution (1mg/ml)

0.01g of the plant extract was made up to 10ml by adding 70% The four extracts.

The four extracts (70%)

This was prepared by measuring 70ml of the four extracts and making it up to 100ml with distilled

water.

Aluminum Chloride (10%)

This was prepared by measuring 10g of Aluminum chloride, it was dissolved in little amount of

distilled water and it was then made up to 100ml using distilled water.

Sodium Acetate (1M)

This solution was prepared by measuring 9.3g of sodium acetate and was made up to 100ml using

distilled water.

75
Quercetin (1mg/ml)

Quercetin was the standard/positive control solution and just like the test solution, 0.01g of

Quercetin was made up to 10ml by adding 70% The four extracts.

PROCEDURE

Markhermia tomentosa plant extract of different volume were paired in a test tube labelled A and

B ranging from 50-500µL respectively. Different concentrations of test samples (quercetin and

plant extracts) were administered in the test tubes and were made up to 1ml using 70% The four

extracts also at different concentrations. Then 0.1ml of 10% AlCl3.6H2O, 0.1ml of 1m sodium

acetate and 2.8ml of distilled water and left at room temperature for 30minutes.

The absorbance of the reaction was measured at 415nm using a spectrophotometer. Similar

procedure was repeated for quercetin to compare the effectiveness of the test extract.

The total flavonoid content was expressed as mg/g, Quercetin Equivalent (QE) using the stated

formula;

C=c.v

Where: C = total content of flavonoid compound (mg/g plant extract in quercetin equivalent (QE).

c = concentration of quercetin established from the calibration curve (µg/ml)

V = volume of extract (ml)

M = weight of pure plant extract (g)

2.6.3 QUERCETIN STANDARD CURVE

Absorbance of the various cone of quercetin standard curve used for the estimation of the total

flavonoid content of the the four extracts extract of Markhermia tomentosa leaves.

76
 600

500
Quercetin Equivalent

400

300

200

100

0
0.578 1.705 1.936 1.954 1.959 1.978 1.964 1.934 1.96 1.89
Concentration of extract (µg/ml)

Fig 2.1: Graph showing the standard quercetin curve reading

2.6.4 DETERMINATION OF PERCENTAGE HYDROXYL RADICAL SCAVENGING

ACTIVITY (2-DEOXYRIBOSE ASSAY)

PRINCIPLE

The 2-deoxyribose assay is based on the degradation of 2-deoxyribose by hydroxyl radicals

generated by a Fenton-type reaction. The amount of degradation can be quantified by a

colorimetric or spectrophotometric method. Hydroxyl radical scavengers added to the reaction

mixture compete with 2-deoxyribose for hydroxyl radicals. The percentage of hydroxyl radical

scavenging activity of a sample can be determined by comparing the amount of degradation

products in the presence and absence of the test sample. (Tijani, 2018)

REAGENTS

Extract Solution (1mg/ml)

0.01g of the plant extract was made up to 10ml by adding methanol.

77
2-Deoxyribose (2.8mM)

This was prepared by dissolving 0.04g of 2-deoxyribose in distilled water and was made up to

100ml.

Phosphate Buffer (pH 7.4)

This was prepared by dissolving 0.34g of KH2PO4 and 0.85g of NaHPO4 in distilled water, the

solution was standardized to a pH of 7.4 by a gentle dispensation of NaOH. The solution was made

up to 250ml using distilled water.

Ascorbic Acid (1.0mM)

This was prepared by dissolving 0.18g of ascorbic acid in distilled water and the volume was

made up to 100ml.

EDTA (1.04mM)

This was prepared by dissolving 0.304g of EDTA in distilled water and the volume was made up

to 100ml.

Hydrogen Peroxide (1.0mM)

102mls of hydrogen peroxide (H2O2) was made up to 1000ml in distilled water.

Trichloroacetic Acid (2.8%)

This was prepared by dissolving 2.8g of trichloroacetic acid (TCA) in distilled water and the

volume was made up to 100ml.

Thiobarbturic Acid (1% w/v)

This was prepared by dissolving 1g of thiobarbituric acid (TBA) in distilled water and the volume

was made up to 100ml.

78
FeCl3

This was prepared by dissolving 0.00325g of FeCl3 in distilled water and the volume was made up

to 100ml.

PROCEDURE

Using freshly prepared solutions, pairs of test tubes (labeled A & B) were arranged in a test tube

rack and different concentrations of Markhermia tomentosa leaves extracts were administered in

the test tubes and made up to 0.5ml using absolute the four extracts also at different concentrations.

100µl of Phosphate buffer and 2-deoxyribose (in the ratio 1:1), 100µl of FeCl3, 100µl of H2O2,

100µl of Ascorbic acid and finally 100µl of EDTA were picked using a micropipette, ranging from

50 - 500µl (then made up to 1ml from the ranges of 950 -50 µL) all these were incubated for 1hour

at 37oc. After incubation, 1ml of TCA and 1ml of TBA were added to stop the reaction. The

solution was heated at 100ºC for 1 hour till a pink coloration was observed and allowed to cool.

After cooling, the absorbance was taken at a wavelength of 532nm with a Spectrophotometer.

The percentage hydroxyl radical scavenging activity was calculated by comparing the amount of

degradation products in the presence and absence of the test sample. The percentage inhibition was

calculated using the formula:

Percentage inhibition of hydroxyl radical (%) = {Acontrol – Asample} x 100

Acontrol

Where;

Acontrol is the absorbance value of the fully oxidized control.

Asample is the absorbance value in the presence of plant extract or standard compound.

79
2.6.5 DETERMINATION OF PERCENTAGE INHIBITION OF LIPID PEROXIDATION

PRINCIPLE

The assay for determining the percentage inhibition of lipid peroxidation is founded on the

principle that ferrous ions (Fe2+) from ferrous sulfate can catalyze lipid peroxidation in an

oxygenated environment. Within a lipid-rich medium such as egg yolk, the Fe2+ ions interact with

hydrogen peroxide, generating highly reactive hydroxyl radicals that initiate lipid peroxidation.

The inhibition of lipid peroxidation can be assessed by introducing a plant extract or test compound

to the reaction mixture and comparing the reduction in lipid peroxidation with a control reaction

that does not contain the test compound. (Rahman, et al. 2015)

REAGENTS

Extract Solution (1mg/ml)

0.01g of the plant extract was made up to 10ml by adding methanol.

Ferrous Sulphate (0.07mM)

This was prepared by dissolving 0.001g of ferrous sulphate (FeSO4) in distilled water and the

volume was made up to 100ml.

Acetic Acid (pH 3.5, 20%)

This was prepared by measuring 20ml of glacial acetic acid in distilled water and the volume was

made up to 100ml.

Sodium Lauryl Sulphate (1.1%)

This was prepared by dissolving 1.1g of SDS in distilled water and the volume was made up to

100ml.

80
Egg Yolk (10%)

This was prepared by adding 10ml of egg yolk into 100ml volumetric flask and the volume was

made up to 100ml in the flask.

Thiobarbituric Acid (0.8% v/v)

This was prepared by dissolving 0.8g of thiobarbituric acid (TBA) in distilled water and the volume

was made up to 100ml.

PROCEDURE

Pairs of test tubes (labeled A & B) were arranged in a test tube rack accordingly containing

different concentrations of Markhermia tomentosa leaves extracts. These leaves extract was

administered into the test tubes and were made up to 0.5ml using the four extracts at different

concentrations. The lipid source (egg yolk) was carefully pipetted into test tube (0.5ml), and was

made up to 1ml with the addition of distilled water.

50µl of prepared FeSO4 was added and the mixture was incubated for 30 minutes at room

temperature. After incubation, 1.5ml of prepared acetic acid was added as a solvent for the

extraction of lipids from the egg yolk, followed by addition of 1.5ml mixture of TBA and SLS

(1:1). The mixture was subjected to vigorous shaking and heated for 1hour until a pink coloration

was observed. After cooling, the absorbance was read using a spectrophotometer at wavelength of

532nm.

The percentage inhibition was calculated using:

Percentage inhibition of lipid peroxidation (%) = {Acontrol – A sample} x 100

Acontrol

81
Where,

Acontrol is the absorbance test solution without plant extracts.

Asample is the absorbance test solution with plant extracts.

2.6.6 DETERMINATION OF PERCENTAGE INHIBITION OF ALPHA-AMYLASE

ACTIVITY

PRINCIPLE

The determination of the percentage inhibition of alpha-amylase is centered on assessing a

substance or compound's capacity to hinder the enzyme's activity. Alpha-amylase is an enzyme

responsible for breaking down starch into simpler sugars like glucose. By comparing enzyme

activity in the presence and absence of an inhibitor, the percentage inhibition of alpha-amylase can

be established. (Apostolidis and Lee, 2010)

REAGENT

Alpha-amylase enzyme solution (2 µ/ml):

Stock solution of this was prepared by dissolving 0.006g of alpha amylase enzyme in 10ml of

phosphate buffer.

Starch substrate solution (1%):

This was prepared by dissolving 1g of starch in distilled water and the volume made up to 100ml

Phosphate buffer (p.h 6.8):

This was prepared by dissolving a mixture of 0.85g of Na2HPO4 and 0.34g of KHPO4 in distilled

water, the solution was standardized to a pH of 6.8 using HCL and NAOH and was made up to

250ml using distilled water.

Test solution (1mg/ml):

0.01g of plant extract dissolved in 10ml of distilled water.

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3,5-dinitrosalicylic acid (DNSA):

0.5g of DNSA was dissolved in 40ml of NaOH

PROCEDURE

50µl of prepared alpha amylase was added to 100µl of test solution/plant extract and 100µl of

soluble starch (1%) in 250µl phosphate buffer (p.H 6.8). The mixture was incubated for 20mins at

room temperature, this allows the enzyme to hydrolyze the starch substrate, releasing glucose

molecules.

The reaction was stopped by adding 500µl DNSA reagent. The reaction tube was boiled for

10mins. After cooling, the absorbance was measured at 540nm using spectrophotometer. the

absorbance values were used to calculate the amount of reducing sugar produced by amylase

enzyme.

The percent inhibition was calculated using the following formula:

% Inhibition =[Control glucose − Test glucoseControl glucoseControl glucose − Test glucoseControl glucose

] x 100

2.6.7 DETERMINATION OF PERCENTAGE INHIBITION OF ALPHA-GLUCOSIDASE

ACTIVITY

PRINCIPLE

The principle of alpha glucosidase inhibition assay involves measuring the ability of a compound

or extract to inhibit the enzyme activity of alpha glucosidase, which is involved in carbohydrate

digestion and absorption in the small intestine. The assay is based on the use of a chromogenic

substrate, such as p-nitrophenyl-α-D-glucopyranoside (PNPG), which is hydrolyzed by alpha

glucosidase to release p-nitrophenol, a yellow compound that can be measured

spectrophotometrically (Wang et al., 2021).

83
The degree of inhibition is determined by comparing the absorbance of the reaction mixture in the

presence and absence of the test compound or extract. A decrease in absorbance indicates

inhibition of alpha glucosidase activity (Yang et al., 2014).

REAGENTS

Alpha glucosidase enzyme solution:

This was prepared by the mixture of 200µl of the enzyme solution in 5ml of distilled water

Test solution (1mg/ml):

0.01g of plant extract dissolved in 10ml of distilled water.

Buffer (100Mm, P.H 6.8):

This was prepared by dissolving a mixture of 0.85g of NasHPos and 0.34g of KHPO4 in distilled

water, the solution was standardized to a pH of 6.4 using HCL and NAOH and was made up to

250ml using distilled water.

P-nitrophenyl-α-D-glucopyranoside (P-NPG) (5mM):

This was prepared by dissolving 0.02g of P-NPG in 13ml of distilled water

Sodium carbonate (0.1M):

0.65g of sodium carbonate was dissolved in distilled water and the volume made up to 62ml mark.

PROCEDURE

100µl of test solution was added to 50µl of enzyme solution and the mixture was incubated for

15mins at room temperature. The substrate solution is prepared by dissolving 100µl of P-NPG in

250µl buffer solution and incubated for 20mins.

The reaction was stopped by adding 250µl of sodium carbonate. The absorbance was read at

405nm using a spectrophotometer.

The percentage inhibition was calculated using:

84
% Inhibition = [(Acontrol – Asample) Acontrol(Acontrol – Asample) Acontrol] x 100

where Acontrol is the absorbance of the control sample (enzyme and substrate only), and Asample is

the absorbance of the sample containing the test compound.

85
 CHAPTER THREE

RESULTS

3.1 DETERMINATION OF TOTAL FLAVONOIDS FOR Markhamia tomentosa

Table 3.1: This table shows the total flavonoid values for four extracts at concentrations ranging

from 50µl to 350µl.

concentration METHANOL N-HEXANE CHLOROFORM ETHYL

C=CV/M C=CV/M C=CV/M ACETAATE

C=CV/M

50 0.256 0.064 0.128 0.192

100 0.376 0.085 0.171 0.214

150 0.504 0.103 0.192 0.274

200 0.675 0.106 0.209 0.295

250 0.931 0.119 0.299 0.316

300 0.956 0.128 0.299 0.321

350 0.976 0.145 0.333 0.385

86
 1.2

1
for Markhamia tomentosa

0.8
Totol flavonoids

0.6

0.4

0.2

0
50 100 150 200 250 300 350
Concentration (µl)

METHANOL C=CV/M N-HEXANE C=CV/M CHLOROFORM C=CV/M ETHYL ACETAATE C=CV/M

Figure 3.1: A graphical illustration of total flavonoids for Markhamia tomentosa

From the table and figure above, ethyl acetate of Markhamia tomentosa leaves showed significant

increases in the flavonoid content with increased concentration. The methanol extract of

Markhamia tomentosa leaf showed the highest potential increase in flavonoid content from

50µg/ml to 350µg/ml with corresponding minimum concentration at 0.064 and maximum

concentration at 0.976 respectively.

87
3.2 DETERMINATION OF % INHIBITION OF DPPH ACTIVITY FOR Markhamia

tomentosa

Table 3.2: This table shows the % inhibition of DPPH activity for four extracts at concentrations

ranging from 50µl to 400µl.

CONCENTRATION METHANOL N- CHLOROFORM ETHYLACETATE

HEXANE

50 80.40 64.44 73.36 89.53

100 85.89 81.54 82.27 90.98

150 86.21 77.08 83.62 91.08

200 85.79 62.05 84.55 91.29

250 87.66 75.64 83.51 92.57

300 84.66 28.35 74.81 94.08

350 86.11 63.40 82.37 94.73

400 83.41 61.43 84.76 96.91

88
 120

100
Inhibition of DPPH activity
by Markhamia tomentosa

80

60

40

20

0
50 100 150 200 250 300 350 400
Concentration (µl)

METHANOL N-HEXANE CHLOROFORM ETHYLACETATE

Figure 3.2: A graphical illustration of % inhibition of DPPH activity for Markhamia tomentosa

From the table and figure above, DPPH scavenging effect of ethyl acetate of Croton zambesicus

leaf increases in a concentration dependent manner. However, the minimum DPPH scavenging

effect of the Croton zambesicus leaf extract was obtained in the N-hexane extract at 3000 µg/ml

(28.35%) scavenging activity and the maximum scavenging effect of the Croton zambesicus leaf

extract was obtained in the ethyl acetate extract at 400 µg/ml (96.91%).

89
3.3 DETERMINATION OF % INHIBITION OF HYDROXL RADICAL ACTIVITY FOR

Markhamia tomentosa

Table 3.3: This table shows the % inhibition of hydroxyl radical activity for four extracts at

concentrations ranging from 50µl to 450µl.

CONCENTRATION METHANOL N- CHLOROFORM ETHYLACETATE

HEXANE

50 79.61 91.25 71.37 61.48

100 73.64 88.16 70.34 58.19

150 64.16 86.71 68.18 40.38

200 54.79 78.78 64.57 35.86

250 44.69 76.37 60.04 30.09

300 37.26 75.28 57.16 28.74

350 32.09 64.37 52.42 24.50

400 27.68 62.45 49.64 23.20

450 20.38 61.69 31.31 21.48

90
 100

90
Inhibition of hydroxyl radical activity

80
by Markhamia tomentosa

70

60

50

40

30

20

10

0
50 100 150 200 250 300 350 400 450
Concentration (µl)

METHANOL N-HEXANE CHLOROFORM ETHYLACETATE

Figure 3.3: A graphical illustration of % inhibition of hydroxyl radical activity for Markhamia

tomentosa

From the table and figure above, the percentage (%) inhibition of hydroxyl radical activity of ethyl

acetate of Markhamia tomentosa leaves was examined and the maximum threshold and minimum

hydroxyl radical scavenging activity of Markhamia tomentosa leaf were obtained with the N-

hexane extract at 50µg/ml(91.25%) and methanol extract 450µg/ml(20.38%) scavenging activity

respectively. Moreover, the hydroxyl radical scavenging potential of the extract was exerted in a

concentration dependent manner.

91
3.4 DETERMINATION OF % INHIBITION OF LIPID PER0XIDATION ACTIVITY FOR

Markhamia tomentosa

Table 3.4: This table shows the % inhibition of lipid peroxidation activity for four extracts at

concentrations ranging from 50µl to 450µl.

CONCENTRATION METHANOL N- CHLOROFORM ETHYLACETATE

HEXANE

50 32.96 17.23 20.92 28.85

100 32.58 16.715 19.52 27.21

150 43.73 15.5 18.01 26.48

200 24.76 14.655 16.01 25.89

250 26.09 12.96 15.93 23.8

300 26.08 12.5 15.13 22.32

350 25.76 13.26 14.77 18.76

400 24.60 11.16 14.73 16.56

450 28.31 11.02 14.13 12.47

92
 50

45
Inhibition of lipid peroxidation activity

40
by Markhamia tomentosa

35

30

25

20

15

10

0
50 100 150 200 250 300 350 400 450
Concentration (µl)

METHANOL N-HEXANE CHLOROFORM ETHYLACETATE

Figure 3.4: A graphical illustration of % inhibition of lipid peroxidation activity for Markhamia

tomentosa

From the table and figure above, the percentage (%) inhibition of lipid peroxidation activity of

ethyl acetate of Markhamia tomentosa leaves was examined. It was discovered that the extract of

Markhamia tomentosa leaf had the relative minimum inhibition of lipid peroxidation at N-hexane

extract at 450µg/ml(11.02%) and maximum percentage inhibition of lipid peroxidation at

150µg/ml(43.73%) inhibition activity respectively.

93
3.5 DETERMINATION OF % INHIBITION OF α-AMYLASE ACTIVITY FOR

Markhamia tomentosa

Table 3.5: This table shows the % inhibition of α-amylase activity for four extracts at

concentrations ranging from 50µl to 250µl.

CONCENTRATION METHANOL N- CHLOROFORM ETHYLACETATE

HEXANE

50 15.51 36.42 36.78 20.21

100 23.57 38.02 39.81 49.89

150 25.47 41.88 42.11 52.84

200 38.94 43.81 45.55 56.84

250 40.42 45.33 48.9 58.94

94
 70

60
Inhibition of α-amylase activity
by Markhamia tomentosa

50

40

30

20

10

0
50 100 150 200 250
Concentration (µl)

METHANOL N-HEXANE CHLOROFORM ETHYLACETATE

Figure 3.5 A graphical illustration of % inhibition of α-amylase activity for Markhamia tomentosa

From the table and figure above, Markhamia tomentosa leaf extract had the minimum percentage

inhibition of alpha-amylase at the methanol extract at 50µg/ml (15.51%) and maximum percentage

inhibition of alpha-amylase at the ethyl acetate at 250µg/ml(58.94%) inhibition activity

respectively. Moreover, the percentage inhibitory potential of the extract was exerted in a

concentration dependent manner.

95
3.6 DETERMINATION OF % INHIBITION OF α-GLUCOSIDASE ACTIVITY FOR

Markhamia tomentosa

Table 3.6: This table shows the % inhibition of α-glucosidase activity for four extracts at

concentrations ranging from 50µl to 250µl.

CONCENTRATION METHANOL N- CHLOROFORM ETHYLACETATE

HEXANE

50 83.95 85.58 84.41 85.11

100 84.88 83.25 84.18 83.48

150 84.41 81.16 84.65 83.95

200 84.18 84.65 83.95 84.18

250 79.53 85.34 78.83 83.02

96
 88

86
Inhibition of α-glucosidase activity

84
by Markhamia tomentosa

82

80

78

76

74
50 100 150 200 250
Concentration (µl)

METHANOL N-HEXANE CHLOROFORM ETHYLACETATE

Figure 3.6: A graphical illustration of % inhibition of α-glucosidase activity for Markhamia

tomentosa

From the table and figure above, Markhamia tomentosa leaf extract had the minimum percentage

inhibition of α-glucosidase at the chloroform extract at 250µg/ml (78.83%) and maximum

percentage inhibition of alpha-amylase at the ethyl acetate at 50µg/ml(85.58%) inhibition activity

respectively.

97
 CHAPTER FOUR

DISCUSSION AND CONCLUSION

4.1 DISCUSION

Markhermia tomentosa, a medicinal plant widely distributed across various regions of Africa, has

long been used in traditional medicine to treat several health conditions, particularly those linked

to oxidative stress and diabetes. Our research aims to evaluate the antioxidant and antidiabetic

effects of four distinct extracts derived from Markhermia tomentosa leaves through in vitro

experiments.

Phytochemical analysis of the leaves has revealed an array of bioactive compounds, including

flavonoids, phenolic compounds, tannins, and alkaloids, all known for their strong antioxidant

properties. Antioxidants are vital in neutralizing oxidative stress, which results from an imbalance

between the production of reactive oxygen species (ROS) and the body's natural antioxidant

defenses. This oxidative stress is a key contributor to the development of chronic diseases like

cardiovascular disorders, neurodegenerative diseases, and cancer. The presence of these

phytochemicals in Markhermia tomentosa leaves highlights their potential in combating oxidative

stress-related conditions.

The study aimed to evaluate the antioxidant and antidiabetic properties of four different extracts

(methanol, n-hexane, chloroform, and ethyl acetate) derived from Markhamia tomentosa leaves

through a series of in vitro assays. These assays included DPPH radical scavenging, total flavonoid

content, hydroxyl radical scavenging, inhibition of lipid peroxidation, and α-glucosidase and α-

amylase inhibitory activities.

The determination of flavonoid content is crucial, as flavonoids are potent antioxidants. Among

the extracts, the methanol extract exhibited the highest total flavonoid content, increasing from

98
0.256 mg/g at 50 µl to 0.976 mg/g at 350 µl as shown in Fig 3.1. Ethyl acetate also showed

significant flavonoid activity, peaking at 0.385 mg/g at 350 µl. These findings indicate that

methanol and ethyl acetate extracts contain a high concentration of flavonoids, which likely

contribute to their antioxidant potential.

The DPPH assay revealed that the ethyl acetate extract had the highest scavenging activity,

reaching 96.91% at 400 µg/ml, whereas the n-hexane extract demonstrated the lowest activity at

28.35% at the same concentration according to Fig 3.2. This suggests that the ethyl acetate extract

contains a higher concentration of compounds capable of neutralizing free radicals, making it more

effective at mitigating oxidative stress.

The hydroxyl radical scavenging assay in Fig 3.3 showed that n-hexane had the highest activity at

91.25% at 50 µg/ml, while methanol displayed the lowest at 20.38% at 450 µg/ml. This indicates

that the n-hexane extract has strong hydroxyl radical scavenging capabilities at low concentrations,

though its efficacy diminishes at higher concentrations.

Lipid peroxidation is an indicator of oxidative damage to lipids, which is relevant in several

chronic diseases. The methanol extract exhibited the highest inhibition of lipid peroxidation at

43.73% at 150 µg/ml, whereas the n-hexane extract demonstrated the lowest activity, peaking at

only 11.02% at 450 µg/ml as shown in Fig 3.4. These results suggest that methanol extract has a

higher potential to protect cell membranes from oxidative stress.

In terms of antidiabetic activity, the ethyl acetate extract showed the highest inhibition of α-

amylase, reaching 58.94% at 250 µg/ml in Fig 3.5, and α-glucosidase, with an inhibition of 85.58%

at 50 µg/ml as depicited in Fig 3.6. This suggests that ethyl acetate could be particularly effective

in reducing carbohydrate absorption, making it a strong candidate for managing postprandial blood

glucose levels.

99
4.2 CONCLUSION

Put together, the results demonstrate that Markhamia tomentosa leaves possess significant

antioxidant and antidiabetic activities, with the ethyl acetate and methanol extracts showing the

most potent effects. The presence of high flavonoid content and strong radical scavenging abilities

in these extracts suggests that they could be valuable in mitigating oxidative stress and managing

diabetes. Future studies could further explore the specific bioactive compounds responsible for

these effects and evaluate their efficacy in in vivo models.

This study contributes to the growing body of evidence supporting the use of traditional medicinal

plants like Markhamia tomentosa in modern therapeutic applications, particularly for conditions

related to oxidative stress and diabetes.

100
 REFERNCES

Ahmad, A., Alghamdi, S. S., Mahmood, K., and Afzal, M. (2016). Fenugreek a multipurpose crop:

Potentialities and improvements. Saudi journal of biological sciences, 23(2), 300–310.

https://doi.org/10.1016/j.sjbs.2015.09.015

American Diabetes Association (2010). Diagnosis and classification of diabetes mellitus. Diabetes

care, 33 Suppl 1(Suppl 1), S62–S69. https://doi.org/10.2337/dc10-S062

American Diabetes Association. (2021). Classification and diagnosis of diabetes: Standards of

medical care in diabetes—2021. Diabetes Care, 44(Supplement 1), S15-S33.

https://doi.org/10.2337/dc21-S002

Andrés CMC, Pérez de la Lastra JM, Andrés Juan C, Plou FJ, Pérez-Lebeña E. Superoxide Anion

Chemistry—Its Role at the Core of the Innate Immunity. International Journal of

Molecular Sciences. 2023; 24(3):1841. https://doi.org/10.3390/ijms24031841

Aranda-Rivera AK, Cruz-Gregorio A, Arancibia-Hernández YL, Hernández-Cruz EY, Pedraza-

Chaverri J. RONS and Oxidative Stress: An Overview of Basic Concepts. Oxygen. 2022;

2(4):437-478. https://doi.org/10.3390/oxygen2040030

Bankole, A.E., Uchendu, E.E. and Adekunle, A.A. In vitro germination of Markhamia

tomentosa Benth K. Schum ex. Engl. and preliminary phytochemical screening for

medicinal compounds. Ind J Plant Physiol. 22, 85–93 (2017).

https://doi.org/10.1007/s40502-016-0279-3

Barbour, L. A., et al. (2020). Gestational diabetes mellitus. The Lancet, 396(10254), 640-650.

https://doi.org/10.1016/S0140-6736(20)31781-8

Bardaweel, S. K., Gul, M., Alzweiri, M., Ishaqat, A., ALSalamat, H. A., and Bashatwah, R. M.

(2018). Reactive Oxygen Species: the Dual Role in Physiological and Pathological

101
 Conditions of the Human Body. The Eurasian journal of medicine, 50(3), 193–201.

https://doi.org/10.5152/eurasianjmed.2018.17397

Bayan, L., Koulivand, P. H., and Gorji, A. (2014). Garlic: a review of potential therapeutic

effects. Avicenna journal of phytomedicine, 4(1), 1–14.

Bolisetty, S., and Jaimes, E. A. (2013). Mitochondria and reactive oxygen species: physiology and

pathophysiology. International journal of molecular sciences, 14(3), 6306–6344.

https://doi.org/10.3390/ijms14036306

Bresgen, N., & Eckl, P. M. (2021). Oxidative stress and the homeodynamics of iron metabolism.

Biomolecules, 11(9), 1216. https://doi.org/10.3390/biom11091216

Chen, Q., Wang, Q., Zhu, J., & Xiao, Q. (2020). Reactive oxygen species: Key regulators in

vascular health and diseases. International Journal of Molecular Sciences, 21(3), 647.

https://doi.org/10.3390/ijms21030647

Chen, X., Guo, C., & Kong, J. (2012). Oxidative stress in neurodegenerative diseases. Neural

regeneration research, 7(5), 376–385. https://doi.org/10.3969/j.issn.1673-

5374.2012.05.009.

Chiurchiù, V., & Maccarrone, M. (2011). Chronic inflammatory disorders and their redox control:

From molecular mechanisms to therapeutic opportunities. Antioxidants & Redox Signaling,

15(9), 2605-2641. https://doi.org/10.1089/ars.2010.3547

de Oliveira, M. R., Schumacher, R. F., & Zanotto-Filho, A. (2021). The role of oxidative stress in

neurodegenerative diseases: Mechanisms and potential therapeutic strategies.

Antioxidants, 10(6), 898. https://doi.org/10.3390/antiox10060898

102
Díaz-Montes, E., & Castro-Muñoz, R. (2021). Edible Films and Coatings as Food-Quality

Preservers: An Overview. Foods (Basel, Switzerland), 10(2), 249.

https://doi.org/10.3390/foods10020249

DiMeglio, L. A., Evans-Molina, C., & Oram, R. A. (2020). Type 1 diabetes. The Lancet,

391(10138), 2449-2462. https://doi.org/10.1016/S0140-6736(18)31320-5

Doseděl, M., Jirkovský, E., Macáková, K., Krčmová, L. K., Javorská, L., Pourová, J., Mercolini,

L., Remião, F., Nováková, L., Mladěnka, P., and On Behalf Of The Oemonom (2021).

Vitamin C-Sources, Physiological Role, Kinetics, Deficiency, Use, Toxicity, and

Determination. Nutrients, 13(2), 615. https://doi.org/10.3390/nu13020615

Ďuračková, Z. (2014). Free radicals and antioxidants for non-experts. In I. Laher (Ed.), Systems

biology of free radicals and antioxidants (pp. 3-38). Springer. https://doi.org/10.1007/978-

3-642-30018-9_2

El Assar, M., Angulo, J., and Rodríguez-Mañas, L. (2019). Frailty as a phenotypic manifestation

of underlying oxidative stress. Free Radical Biology and

Medicine. doi:10.1016/j.freeradbiomed.2019.08.011

Farrokhnia, M. (2020). Density Functional Theory Studies on the Antioxidant Mechanism and

Electronic Properties of Some Bioactive Marine Meroterpenoids: Sargahydroquionic Acid

and Sargachromanol. ACS Omega. doi:10.1021/acsomega.0c02354

Fiedor, J., and Burda, K. (2014). Potential role of carotenoids as antioxidants in human health and

disease. Nutrients, 6(2), 466–488. https://doi.org/10.3390/nu6020466

Giacco, F., and Brownlee, M. (2010). Oxidative stress and diabetic complications. Circulation

research, 107(9), 1058–1070. https://doi.org/10.1161/CIRCRESAHA.110.223545

103
Gladyshev V. N. (2014). The free radical theory of aging is dead. Long live the damage

theory!. Antioxidants & redox signaling, 20(4), 727–731.

https://doi.org/10.1089/ars.2013.5228

Gopalan, A., Mishra, P., Alexeeff, S. E., Blatchins, M. A., Kim, E., Man, A. H., and Grant, R. W.

(2018). Prevalence and predictors of delayed clinical diagnosis of Type 2 diabetes: a

longitudinal cohort study. Diabetic medicine : a journal of the British Diabetic

Association, 35(12), 1655–1662. https://doi.org/10.1111/dme.13808

Gradinaru, D., Borsa, C., Ionescu, C., and Prada, G. I. (2015). Oxidized LDL and NO synthesis—

Biomarkers of endothelial dysfunction and ageing. Mechanisms of Ageing and

Development, 151, 101–113. doi:10.1016/j.mad.2015.03.003

Hegazy, A. M., & Abd Elhady, W. A. (2021). Free radicals and antioxidants: Friend or foe? Life

Sciences, 287, 120082. https://doi.org/10.1016/j.lfs.2021.120082

Ibrahim, M. B., Kaushik, N., Sowemimo, A. A., and Odukoya, O. A. (2016). Review of the

Phytochemical and Pharmacological Studies of the Genus Markhamia. Pharmacognosy

reviews, 10(19), 50–59. https://doi.org/10.4103/0973-7847.176547

Ibrahim, M. B., Sowemimo, A. A., Venables, L., Koorbanally, N. A., Awolola, G. V., Sofidiya,

M. O., and van de Venter, M. (2018). Biological evaluation of phytoconstituents from

Markhamia tomentosa ethanolic leaf extract. South African Journal of Botany, 115, 31–

36. doi:10.1016/j.sajb.2017.12.014

Jakubczyk, K., Dec, K., Kałduńska, J., Kawczuga, D., Kochman, J., and Janda, K. (2020). Reactive

oxygen species - sources, functions, oxidative damage. Polski merkuriusz lekarski : organ

Polskiego Towarzystwa Lekarskiego, 48(284), 124–127.

104
Jeeva, J. S., Sunitha, J., Ananthalakshmi, R., Rajkumari, S., Ramesh, M., and Krishnan, R. (2015).

Enzymatic antioxidants and its role in oral diseases. Journal of pharmacy & bioallied

sciences, 7(Suppl 2), S331–S333. https://doi.org/10.4103/0975-7406.163438

Joseph, B., and Jini, D. (2013). Antidiabetic effects of Momordica charantia (bitter melon) and its

medicinal potency. Asian Pacific Journal of Tropical Disease, 3(2), 93–102.

https://doi.org/10.1016/S2222-1808(13)60052-3

Kasote, D. M., Katyare, S. S., Hegde, M. V., and Bae, H. (2015). Significance of antioxidant

potential of plants and its relevance to therapeutic applications. International journal of

biological sciences, 11(8), 982–991. https://doi.org/10.7150/ijbs.12096

Kasuga M. (2006). Insulin resistance and pancreatic beta cell failure. The Journal of clinical

investigation, 116(7), 1756–1760. https://doi.org/10.1172/JCI29189

Kharroubi, A. T., and Darwish, H. M. (2015). Diabetes mellitus: The epidemic of the

century. World journal of diabetes, 6(6), 850–867. https://doi.org/10.4239/wjd.v6.i6.850

Knip, M., and Simell, O. (2012). Environmental triggers of type 1 diabetes. Cold Spring Harbor

perspectives in medicine, 2(7), a007690. https://doi.org/10.1101/cshperspect.a007690

Levitan, I., Volkov, S., and Subbaiah, P. V. (2010). Oxidized LDL: diversity, patterns of

recognition, and pathophysiology. Antioxidants & redox signaling, 13(1), 39–75.

https://doi.org/10.1089/ars.2009.2733

Liguori, I., Russo, G., Curcio, F., Bulli, G., Aran, L., Della-Morte, D., and Abete, P.

(2018). Oxidative stress, aging, and diseases. Clinical Interventions in Aging, Volume 13,

757–772. doi:10.2147/cia.s158513

Lin, D., Xiao, M., Zhao, J., Li, Z., Xing, B., Li, X., Kong, M., Li, L., Zhang, Q., Liu, Y., Chen, H.,

Qin, W., Wu, H., and Chen, S. (2016). An Overview of Plant Phenolic Compounds and

105
 Their Importance in Human Nutrition and Management of Type 2 Diabetes. Molecules

(Basel, Switzerland), 21(10), 1374. https://doi.org/10.3390/molecules21101374

Ling, J., & Zhang, X. (2020). The role of free radicals in signaling transduction and the

pathogenesis of diseases. Oxidative Medicine and Cellular Longevity, 2020, 6419074.

https://doi.org/10.1155/2020/6419074

Lismont, C., Revenco, I., and Fransen, M. (2019). Peroxisomal Hydrogen Peroxide Metabolism

and Signaling in Health and Disease. International journal of molecular sciences, 20(15),

3673. https://doi.org/10.3390/ijms20153673

Llanos, P., and Palomero, J. (2022). Reactive Oxygen and Nitrogen Species (RONS) and

Cytokines-Myokines Involved in Glucose Uptake and Insulin Resistance in Skeletal

Muscle. Cells, 11(24), 4008. https://doi.org/10.3390/cells11244008

Lobo, V., Patil, A., Phatak, A., and Chandra, N. (2010). Free radicals, antioxidants and functional

foods: Impact on human health. Pharmacognosy reviews, 4(8), 118–126.

https://doi.org/10.4103/0973-7847.70902

López-Pedrouso, M., Lorenzo, J. M., and Franco, D. (2022). Advances in Natural Antioxidants for

Food Improvement. Antioxidants (Basel, Switzerland), 11(9), 1825.

https://doi.org/10.3390/antiox11091825

Lourenço, Sofia C., Margarida Moldão-Martins, and Vítor D. Alves. 2019. "Antioxidants of

Natural Plant Origins: From Sources to Food Industry Applications" Molecules 24, no. 22:

4132. https://doi.org/10.3390/molecules24224132

Lü, J. M., Lin, P. H., Yao, Q., and Chen, C. (2010). Chemical and molecular mechanisms of

antioxidants: experimental approaches and model systems. Journal of cellular and

molecular medicine, 14(4), 840–860. https://doi.org/10.1111/j.1582-4934.2009.00897.x

106
Maldonado, Edio, Sebastián Morales-Pison, Fabiola Urbina, and Aldo Solari. (2023). "Aging

Hallmarks and the Role of Oxidative Stress" Antioxidants 12, no. 3: 651.

https://doi.org/10.3390/antiox12030651

Mierziak, J., Kostyn, K., and Kulma, A. (2014). Flavonoids as important molecules of plant

interactions with the environment. Molecules (Basel, Switzerland), 19(10), 16240–16265.

https://doi.org/10.3390/molecules191016240

Miyazawa, Taiki, Chizumi Abe, Gregor Carpentero Burdeos, Akira Matsumoto, and Masako

Toda. 2022. "Food Antioxidants and Aging: Theory, Current Evidence and

Perspectives" Nutraceuticals 2, no. 3: 181-204.

https://doi.org/10.3390/nutraceuticals2030014

Modak, M., Dixit, P., Londhe, J., Ghaskadbi, S., and Devasagayam, T. P. (2007). Indian herbs and

herbal drugs used for the treatment of diabetes. Journal of clinical biochemistry and

nutrition, 40(3), 163–173. https://doi.org/10.3164/jcbn.40.163

Naylor, R. N., Philipson, L. H., & Pearson, E. R. (2021). Unraveling the genetic basis of diabetes

mellitus. Nature Reviews Genetics, 22(2), 53-64. https://doi.org/10.1038/s41576-020-

00288-2

Orrenius, S., Kaminskyy, V. O., & Zhivotovsky, B. (2021). Oxidative stress: A central role in the

pathogenesis of neurodegenerative diseases. Journal of Clinical Investigation, 131(8),

e143742. https://doi.org/10.1172/JCI143742

Panche, A. N., Diwan, A. D., and Chandra, S. R. (2016). Flavonoids: an overview. Journal of

nutritional science, 5, e47. https://doi.org/10.1017/jns.2016.41

Pham-Huy, L. A., He, H., and Pham-Huy, C. (2008). Free radicals, antioxidants in disease and

health. International journal of biomedical science : IJBS, 4(2), 89–96.

107
Phaniendra, A., Jestadi, D. B., and Periyasamy, L. (2015). Free radicals: properties, sources,

targets, and their implication in various diseases. Indian journal of clinical biochemistry :

IJCB, 30(1), 11–26. https://doi.org/10.1007/s12291-014-0446-0

Pizzino, G., Irrera, N., Cucinotta, M., Pallio, G., Mannino, F., Arcoraci, V., Squadrito, F., Altavilla,

D., and Bitto, A. (2017). Oxidative Stress: Harms and Benefits for Human

Health. Oxidative medicine and cellular longevity, 2017, 8416763.

https://doi.org/10.1155/2017/8416763

Pizzino, G., Irrera, N., Cucinotta, M., Pallio, G., Mannino, F., Arcoraci, V., Squadrito, F., &

Altavilla, D. (2017). Oxidative stress: Harms and benefits for human health. Oxidative

Medicine and Cellular Longevity, 2017, 8416763. https://doi.org/10.1155/2017/8416763

Ponnampalam, E. N., Kiani, A., Santhiravel, S., Holman, B. W. B., Lauridsen, C., and Dunshea,

F. R. (2022). The Importance of Dietary Antioxidants on Oxidative Stress, Meat and Milk

Production, and Their Preservative Aspects in Farm Animals: Antioxidant Action, Animal

Health, and Product Quality-Invited Review. Animals : an open access journal from

MDPI, 12(23), 3279. https://doi.org/10.3390/ani12233279

Quijano, C., Trujillo, M., Castro, L., and Trostchansky, A. (2016). Interplay between oxidant

species and energy metabolism. Redox biology, 8, 28–42.

https://doi.org/10.1016/j.redox.2015.11.010

Rafieian-Kopaei, M., Setorki, M., Doudi, M., Baradaran, A., and Nasri, H. (2014). Atherosclerosis:

process, indicators, risk factors and new hopes. International journal of preventive

medicine, 5(8), 927–946.

108
Reuter, S., Gupta, S. C., Chaturvedi, M. M., and Aggarwal, B. B. (2010). Oxidative stress,

inflammation, and cancer: how are they linked?. Free radical biology & medicine, 49(11),

1603–1616. https://doi.org/10.1016/j.freeradbiomed.2010.09.006

Riabov, V., Gudima, A., Wang, N., Mickley, A., Orekhov, A., and Kzhyshkowska, J. (2014). Role

of tumor associated macrophages in tumor angiogenesis and lymphangiogenesis. Frontiers

in physiology, 5, 75. https://doi.org/10.3389/fphys.2014.00075

Ried, K., and Fakler, P. (2014). Potential of garlic (Allium sativum) in lowering high blood

pressure: mechanisms of action and clinical relevance. Integrated blood pressure

control, 7, 71–82. https://doi.org/10.2147/IBPC.S51434

Sapra A,and Bhandari P. Diabetes.(2023). In: StatPearls [Internet]. Treasure Island (FL):

StatPearls Publishing; 2024 Jan-. Available from:

https://www.ncbi.nlm.nih.gov/books/NBK551501/

Schrader, M., and Fahimi, H. D. (2006). Peroxisomes and oxidative stress. Biochimica et

Biophysica Acta (BBA) - Molecular Cell Research, 1763(12), 1755–

1766. doi:10.1016/j.bbamcr.2006.09.006

Senoner, T., and Dichtl, W. (2019). Oxidative Stress in Cardiovascular Diseases: Still a

Therapeutic Target?. Nutrients, 11(9), 2090. https://doi.org/10.3390/nu11092090

Shabil, M., Bushi, G., Bodige, P. K., Maradi, P. S., Patra, B. P., Padhi, B. K., and Khubchandani,

J. (2023). Effect of Fenugreek on Hyperglycemia: A Systematic Review and Meta-

Analysis. Medicina (Kaunas, Lithuania), 59(2), 248.

https://doi.org/10.3390/medicina59020248

109
Shahidi, F., and Ambigaipalan, P. (2015). Phenolics and polyphenolics in foods, beverages and

spices: Antioxidant activity and health effects – A review. Journal of Functional Foods,

18, 820–897. doi:10.1016/j.jff.2015.06.018

Siddeeg, A., AlKehayez, N. M., Abu-Hiamed, H. A., Al-Sanea, E. A., and Al-Farga, A. M. (2021).

Mode of action and determination of antioxidant activity in the dietary sources: An

overview. Saudi journal of biological sciences, 28(3), 1633–1644.

https://doi.org/10.1016/j.sjbs.2020.11.064

Sies, H. (2020). Oxidative stress: Eustress and distress in redox homeostasis. Advances in Redox

Research, 1, 100001. https://doi.org/10.1016/j.areres.2020.100001

Sies, H., Berndt, C., & Jones, D. P. (2022). Oxidative stress. Annual Review of Biochemistry, 91(1),

715–742. https://doi.org/10.1146/annurev-biochem-020420-095428

Silva, M. L., Bernardo, M. A., Singh, J., and de Mesquita, M. F. (2022). Cinnamon as a

Complementary Therapeutic Approach for Dysglycemia and Dyslipidemia Control in

Type 2 Diabetes Mellitus and Its Molecular Mechanism of Action: A

Review. Nutrients, 14(13), 2773. https://doi.org/10.3390/nu14132773

Singh, M., Sharma, P., & Kumar, P. (2020). Role of reactive oxygen species in cell signaling

pathways in cancer progression. Current Molecular Medicine, 20(8), 662-678.

https://doi.org/10.2174/1566524020666200721123142

Stevinson, C., Pittler, M. H., and Ernst, E. (2000). Garlic for treating hypercholesterolemia. A

meta-analysis of randomized clinical trials. Annals of internal medicine, 133(6), 420–429.

https://doi.org/10.7326/0003-4819-133-6-200009190-00009

110
Tan, B. L., Norhaizan, M. E., & Liew, W. P. P. (2021). Nutrients and oxidative stress: Friend or

foe? Oxidative Medicine and Cellular Longevity, 2021, 6815602.

https://doi.org/10.1155/2021/6815602

Ungvari, Z., Tarantini, S., Donato, A. J., Galvan, V., & Csiszar, A. (2020). Mechanisms of vascular

aging. Circulation Research, 127(7), 849-864.

https://doi.org/10.1161/CIRCRESAHA.120.316788

Vafa, M., Mohammadi, F., Shidfar, F., Sormaghi, M. S., Heidari, I., Golestan, B., and Amiri, F.

(2012). Effects of cinnamon consumption on glycemic status, lipid profile and body

composition in type 2 diabetic patients. International journal of preventive medicine, 3(8),

531–536.

Valko, M., Rhodes, C. J., & Moncol, J. (2021). Free radicals, metals and antioxidants in oxidative

stress-induced cancer. Chemico-Biological Interactions, 192(1-2), 33-51.

https://doi.org/10.1016/j.cbi.2021.07.021

van der Werf, N., Kroese, F. G., Rozing, J., and Hillebrands, J. L. (2007). Viral infections as

potential triggers of type 1 diabetes. Diabetes/metabolism research and reviews, 23(3),

169–183. https://doi.org/10.1002/dmrr.695

Vona, Rosa, Lucia Pallotta, Martina Cappelletti, Carola Severi, and Paola Matarrese. 2021. "The

Impact of Oxidative Stress in Human Pathology: Focus on Gastrointestinal

Disorders" Antioxidants 10, no. 2: 201. https://doi.org/10.3390/antiox10020201

Xu, D. P., Li, Y., Meng, X., Zhou, T., Zhou, Y., Zheng, J., Zhang, J. J., and Li, H. B. (2017).

Natural Antioxidants in Foods and Medicinal Plants: Extraction, Assessment and

Resources. International journal of molecular sciences, 18(1), 96.

https://doi.org/10.3390/ijms18010096

111
Yin, R. V., Lee, N. C., Hirpara, H., and Phung, O. J. (2014). The effect of bitter melon

(Mormordica charantia) in patients with diabetes mellitus: a systematic review and meta-

analysis. Nutrition & diabetes, 4(12), e145. https://doi.org/10.1038/nutd.2014.42

Zehiroglu, C., and Ozturk Sarikaya, S. B. (2019). The importance of antioxidants and place in

today's scientific and technological studies. Journal of food science and

technology, 56(11), 4757–4774. https://doi.org/10.1007/s13197-019-03952-x

Zhang, W., Li, X., & Xu, J. (2020). Regulation of cellular senescence by oxidative stress:

Mechanisms and interventions. Antioxidants, 9(5), 422.

https://doi.org/10.3390/antiox9050422

Zhang, Y., Yang, J., & Huang, X. (2020). ROS and disease: An update. Redox Biology, 37, 101805.

https://doi.org/10.1016/j.redox.2020.101805

112