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IN VITRO ANTIOXIDANT AND PHYTOCHEMICAL ACTIVITY OF ETHYL ACETATE
AND n-HEXANE FRACTION OF Ficus exasperata VAHL LEAVES
BY
FAJANA ELIZABETH DAMILOLA
19/2844
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF

BACHELOR OF SCIENCE (B.Sc.) IN BIOCHEMISTRY
SUBMITTED TO THE
DEPARTMENT OF BIOCHEMISTRY
SCHOOL OF BASIC MEDICAL SCIENCES
BENJAMIN S. CARSON (SNR.) COLLEGE OF HEALTH AND MEDICAL SCIENCES
BABCOCK UNIVERSITY
ILISHAN-REMO
OGUN STATE
SUPERVISOR
DR. ESIABA, IJEOMA
MAY, 2022
CERTIFICATION
This is to certify that this research project titled “IN VITRO ANTIOXIDANT AND
PHYTOCHEMICAL ACTIVITY OF ETHYL ACETATE AND n-HEXANE FRACTION
OF Ficus exasperata VAHL LEAVES” was done and complied by FAJANA ELIZABETH
DAMILOLA with matriculation number 19/2844 from the Department of Biochemistry,
Benjamin S. Carson (Snr.) College of Health and Medical Sciences, Babcock University, Ilishan-
Remo, Ogun State, under the supervision of DR. ESIABA, I.
FAJANA ELIZABETH DAMILOLA _______________________
(Student) Signature/Date
DR. ESIABA IJEOMA _____________________
(Supervisor) Signature/Date
ii
DEDICATION
This work is dedicated to God Almighty for his love and grace in my life. This work is also
dedicated to Mr. Femi Fajana and Mrs. Silifatu Fajana, for their financial and emotional support.
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ACKNOWLEDGEMENTS
Fore mostly, I would like give all the credit to God Almighty, His guidance, love, mercy and
providence, wisdom and understanding to see me through to the end of this project. I want to
express my gratitude to my beloved parents, Mr. Femi Fajana and Mrs. Silifatu FajanaI would
take this opportunity to express my gratitude to my most illustrious supervisor, Dr. Esiaba
Ijeoma, for her guidance, assistance and dedication to the success and completion of this project.
My heartfelt gratitude goes to the Department of Biochemistry, which is led by the Head of
Department Dr. Kayode Abolanle, for providing an opportunity and facilities to perform this
work successfully.
Furthermore, I would like appreciate the laboratory technician, Mr. Oredele Kunle, for taking
time out of his busy schedule to guarantee that the tests carried out during my research went
successfully and efficiently.
My specific gratitude to Aboyade-Cole Adedoyin, a member of my project team, for her
assistance and wonderful teamwork during this project, as well as my other team members, Salau
Ridwan and Nwaukwa Chinedum for their assistance and support.
Mummy Fola Oyebade, deserves special appreciation for her regular check-ups, prayers and
encouragement during this project.
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TABLE OF CONTENTS
Content
Pag
CERTIFICATION ii
DEDICATION iii
ACKNOWLEDGEMENTS iv
LIST OF FIGURES viii
ABSTRACT ix
CHAPTER ONE: INTRODUCTION 1
1.1 Background study 1

1.2 Statement problem and justification 4
1.3 Objectives of study 5
1.3.1 General objective 5
1.3.2 Specific objective 5
1.4 Significance of the study 5
1.5 Scope of the study 6
CHAPTER TWO: LITERATURE REVIEW 7
2.1 Antioxidants 7
2.2 Types of antioxidants 9
2.3 Mechanism of action of antioxidants 12
2.4 Levels of antioxidant action 13
2.5 Characteristics of antioxidants 14
2.6 Effects of Antioxidants 14
2.7 Chain Reactions of Free Radicals 16
2.8 Phytochemicals 17
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2.8.1 Classes of Major Phytochemicals, Food Sources, and Nutritional Benefits 17
2.8.2 Polyphenols 18
2.8.3 Flavonoids 19
2.9 Ficus exasperata Vahl (sandpaper) 22
2.9.1 Botanical description, habitat and distribution 22
2.9.2 Taxonomy of Ficus exasperata Vahl 22
2.9.3 Cultural and Ethnomedicinal uses 23
CHAPTER THREE: MATERIAL AND METHODS 25
3.1 Materials and Reagents 25

3.2 Location of the Study 25
3.3 Collection and Authentification of Plant Material 25
3.4 Preparation of Plant Extract 26
3.5 In-Vitro Anti-Oxidant Assays 26
3.5.1 Determination of In vitro DPPH (2, 2- diphenyl-1-picrylhydrazyl) Radical Scavenging
Activity 26
3.5.2 In vitro FRAP (Ferric Reducing Antioxidant Power) Assay 28
3.5.3 Determination of Total Antioxidant Capacity (Phosphomolybdenum Method) 28
3.5.4 Determination of In vitro ABTS (2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)
Radical Scavenging Activity 29
3.5.6 Determination of Total Phenolic Content 30
3.5.7 Determination Total flavonoid content (TFC) 30
3.6 Statistical Analysis 31
CHAPTER FOUR: RESULT 32
4.1 Free radical scavenging effect on the DPPH Assay 32

4.2 Free radical scavenging ability by the use of ABTS radical cation assay 34
4.3 Total antioxidant capacity of F. exasperata leaves fractions (ethyl acetate and n-hexane
fractions) 36
4.4 Ferric reducing antioxidant power (FRAP) of F.exaperata Vahl leaves ethyl acetate and n-
hexane fraction 38
4.5 Total flavonoid content of the ethyl acetate and n-hexane fraction of F. exasperata leaves40
4.6: Total phenolic content of the ethyl acetate and n-hexane fraction of F. exasperata leaves 42
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CHAPTER FIVE: DISCUSSION, CONCLUSION AND RECOMMENDATION 44
5.1 Discussion 44
5.2 Conclusion 48
5.3 Recommendation 49
REFERENCES 50
APPENDICES 66
vii
LIST OF FIGURES
Figure Page
Figure 2.1 Classification and sub-classification of antioxidants found in natural sources 12
Figure 2.2 Chemical structure of flavonoid polyphenols 21
Figure 2.3 Ficus exasperata Vahl leaves 24
Figure 4.1 DPPH radical scavenging activity of the ethyl acetate and hexane fraction of Ficus
exasperata Vahl leaves 33
Figure 4.2 ABTS radical scavenging activity of the ethyl acetate and hexane fraction of Ficus
Figure 4.3 Total antioxidant capacity of the ethyl acetate and n-hexane fractions of Ficus
Figure 4.4 Ferric reducing antioxidant power (FRAP) of the ethyl acetate and n-hexane fraction
F.exasperata Vahl leaves 39
Figure 4.5 Total flavonoid content of the ethyl acetate and n-hexane fraction of F. exasperata
Vahl leaves 41
Figure 4.6 Total phenolic content of the ethyl acetate and n-hexane fraction of F. exasperata Vahl
leaves 43
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ABSTRACT
When biochemical molecules like proteins, lipids, and nucleic acid are attacked, they produce
reactive oxygen species (ROS), which are involved in the pathogenesis of oxidative stress-
related disorders like diabetes, nephrotoxicity, cancer, and cardiovascular disorders, etc.
Antioxidants are utilized to protect cells and tissues from the harmful effects of reactive oxygen
species (ROS). Free radicals are known to be quenched by natural antioxidants. Flavonoids and
phenols have been shown to have antioxidant properties and are abundant in most therapeutic
plants. These phytochemicals are currently being investigated as a potential alternative and
supplementary medicine. Ficus exasperata Vahl leaves are used in traditional medicine for the
treatment of hypertension, rheumatism, arthritis, intestinal pains, etc. In this study, the in vitro
antioxidant and phytochemical activities of the ethyl acetate and n-hexane fraction of the F.
exasperata Vahl leaves were determined using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical
scavenging, ferric reducing antioxidant power (FRAP), ABTS (2, 2'-azino-bis (3
ethylbenzothiazoline-6-sulfonic acid) radical scavenging and total antioxidant capacity assays
respectively. The total phenolic content of the fractions was determined using the Folin-
Ciocalteu method and the flavonoid content was determined using the method of Ordonez et al.
(2006). The results obtained showed both fractions significantly (p<0.05) exhibited antioxidants
activities at different concentrations tested. The ethyl acetate fraction exhibited higher
antioxidant activity and had higher phenolic and flavonoid content than the n-hexane fraction.
Therefore, the result of this research revealed that both fractions contain secondary metabolites
linked to antioxidant activity. For this reason, the current study suggests that they be further
investigated to improve their potential applicability in the treatment of oxidative stress-related
illnesses.
Keywords: Ficus exasperata Vahl, Antioxidants, phytochemicals, ethyl acetate, n-hexane
Word count: 263
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CHAPTER ONE
INTRODUCTION
1.1 Background study
The imbalance between free radical generation and antioxidant defences in the body is referred
to as oxidative stress (Kivrak, Yurt, Kaplan, Alkan, & Altun, 2017). Free radicals are defined as
molecules that have one or more unpaired electrons, making them highly reactive molecules
capable of attacking any stable molecule such as proteins, carbohydrates, and lipids (Kumar &
Salam, 2018). Reactive oxygen species (ROS) are the most common and widely known free
radicals. They include superoxide (O2 ‐), hydroxyl (HO-), hydrogen peroxide (H2O2), and nitric
oxide (NO-).
Most metabolic activities in the body produce reactive oxygen species (ROS), which can harm
essential biomolecules such as proteins, nucleic acids, and lipids if they are not scavenged by
antioxidants (Sharma, Gupta, & Prabhakar, 2019). Free radicals are recognized to have a role in
the aging process as well as the pathogenesis of stress-related diseases such as diabetes,
nephrotoxicity, hepatotoxicity, cancer, cardiovascular disease, inflammation, and neurological
disorders (Neha, Haider, Pathak, & Yar, 2019).
An antioxidant is a chemical that gives an electron to a free radical, turning it into a harmless
molecule. Catalase, superoxide dismutase, Vitamin C, and glutathione peroxidase are examples
of natural antioxidants that have been reported to be capable of scavenging ROS (Bratovcic,
2020). Antioxidant defence systems protect human cells against ROS attack, however, some
cells have been proven to be vulnerable to ROS at low concentrations of antioxidant enzymes
(Truong, Jun, & Jeong, 2017). To measure the susceptibility of tissues to oxidative damage, the
1
cellular antioxidant level is utilized. During oxidative stress, this amount usually changes
(Birnie-Gauvin, Costantini, Cooke, & Willmore, 2017).
Plants that are part of our everyday diet naturally contain a wide range of antioxidants. Vitamin
C, E, and carotenoids are examples of dietary antioxidants (Huang, 2018). Consumption of
antioxidant-rich foods and fruits plays an important role in enhancing the body's natural
resilience to oxidative stress (Sztretye et al., 2019). Plants also contain phenols, flavonoids, and
alkaloids, which are non-nutrient antioxidants. These polyphenols have been examined and
reported as free radical quenchers (Renuka, Devi, & Subramanian, 2018).
Butyrate hydroxyanisole, butylated hydroxytolune, and propyl gallate are all commercially
marketed antioxidant medicines (Kebede & Admassu, 2019). However, research has revealed
that these synthetic antioxidants can be harmful (Atta, Mohamed, & Abdelgawad, 2017). As a
result, several limitations on their use have been imposed. Researchers are now concentrating
their efforts on antioxidants derived from plants, among other things (Salehi et al., 2018).
Plants have supplied a novel source of medicine and have tremendously benefited mankind in
maintaining their health since the beginning of time. It is believed that 80% of the world's
population uses herbal remedies to meet their health demands (Stenvinkel, Painer, Johnson, &
Natterson‐Horowitz, 2019). Herbal medication is popular because it is more culturally
appropriate, more readily available, and has fewer negative side effects (Saeed et al., 2017).
Over 1200 plants have been traditionally used for their antioxidant properties around the world
(Hosseinpour-Jaghdani, Shomali, Gholipour-Shahraki, Rahimi-Madiseh, & Rafieian-Kopaei,
2017). According to ethnopharmacological studies, medicinal plants play a critical role in the
treatment of oxidative stress-related illnesses (Widodo, Sismindari, Asmara, & Rohman, 2019).
2
Plant extracts contain phytochemicals such as flavonoids, tannins, phenols, and alkaloids, which
are well-known antioxidants and are currently being investigated as alternative and
supplementary treatments for oxidative stress-related illnesses (Forni et al., 2019). Plant-based
antioxidants are relatively safe, cost-effective, and beneficial in disease treatment, according to
several efficacy studies conducted on herbal plants.
The genus Ficus is well-known for its antioxidant capabilities, due to its high content of phenols
and flavonoids (Ashraf et al., 2021). The F.exasperata Vahl plant is used as in traditional
medicine for prevention, treatment or/and management of diarrhoea, liver disease, skin
infections, stomach disorders, helminthiasis, lactation disorders, epilepsy, tuberculosis, sterility,
and diabetes mellitus (Grace & Kayode, 2018). In East Africa, F. exasperata Vahl is another
extensively distributed evergreen herb. It is used to treat rheumatism, snakebite, stomach
discomfort, gastrointestinal pain, gynecological issues, malaria, high blood pressure,
rheumatism, arthritis, intestinal pains and colics, epilepsy, bleeding and wounds and diabetes
mellitus by traditional healers (Bekoe, Kitcher, Gyima, Schwinger & Frempong, 2018). There
has been evidence that the fractions of F.exasperata Vahl leaves contain important therapeutic
compounds for stress-related diseases.
Given this context, the goal of this study is to determine the in vitro antioxidant activity of F.
exasperata Vahl leaves ethyl acetate and n-hexane fractions and also to look into and provide
preliminary information on the ethyl acetate and n-hexane fractions of F. exasperata Vahl
leaves as potential bio-resources for making conveniently accessible herbal formulations that are
more successful in treating and managing oxidative stress-related illnesses.
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1.2 Statement problem and justification
Atherosclerosis, diabetes mellitus, hypertension, ischemia complications, malignancies,
cardiovascular diseases, eye diseases, lung, pancreas, and renal disorders, cancer, as well as
ageing and diseases of the reproductive system have all been linked to oxidative stress (Islam &
Shekhar, 2015).
In developing and underdeveloped countries, stress-related disorders have become an epidemic.
The majority of traditional therapy strategies aim to alleviate the clinical signs of these illnesses
as well as their consequences. However, investigations have revealed that they increase toxicity,
causing harm to sensitive organs such as the liver and brain, and they are also suspected of being
mutagenic (Jnaid, Yacoub & Al-Biski, 2016).
In light of this, supplementary medications for oxidative stress-related illnesses have grown in
popularity, and plant-based antioxidant therapy is now commonly used in most developing
nations (Nuhu et al., 2021).
Since antioxidants are crucial in reducing oxidative stress-related diseases, many plant extracts
and secondary metabolites are being studied for their antioxidant activities (Stagos, 2019).
Antioxidants derived from plants are vital in inhibiting the activation of oxidation-induced
signaling pathways in our bodies (Namadina, Haruna, & Sanusi, 2020). As a result, determining
the antioxidant activity of fractions of F. exasperata Vahl leaf is a crucial step toward better
understanding how it might be used to treat various stress-related illnesses.
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1.3 Objectives of study
1.3.1 General objective
The general objective of this study is to determine in vitro antioxidant and phytochemical
activities of the ethyl acetate and n-hexane fraction of Ficus exasperata Vahl leaves.
1.3.2 Specific objective
i. To evaluate in vitro DPPH radicals scavenging effect of the ethyl acetate and n-hexane
fraction of Ficus exasperata Vahl leaves.
ii. To determine in vitro ferric reducing power of the ethyl acetate and n-hexane fraction of
Ficus exasperata Vahl leaves.
iii. To evaluate the total phenolic and total flavonoid contents of the ethyl acetate and n-
hexane fraction of Ficus exasperata Vahl leaves.
iv. To evaluate in vitro ABTS radical scavenging activity of the ethyl acetate and n-hexane
fraction of Ficus exasperata Vahl leaves.
v. To determine the total antioxidant capacity of the ethyl acetate and n-hexane fraction of
Ficus exasperata Vahl leaves.
vi. To compare the antioxidant and phytochemical values between the ethyl acetate and n-
hexane fraction of Ficus exasperata Vahl leaves.
1.4 Significance of the study
The leaves of Ficus exasperata Vahl contain bioactive constituents that belong to important
phytochemical groups. Some of the phytochemicals found in the leaves have antioxidant
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properties, these bioactive components could be used to develop drugs that are affordable and
less toxic for the treatment, management and prevention of diseases related to oxidative stress
1.5 Scope of the study
This study is based on the in vitro evaluation of the antioxidant and phytochemical (total
phenolic and total flavonoid content) activity of ethyl acetate and n-hexane fraction of Ficus
exasperata Vahl leaves.
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CHAPTER TWO
LITERATURE REVIEW
2.1 Antioxidants
Antioxidants are substances that can neutralize or reduce the harmful effects of oxidants in the
body. Antioxidants work by donating an electron to an oxidant chemical, inhibiting the oxidant
compound's activity (Zulaikhah, 2017). The binding of antioxidants to free radicals terminates
the oxidative chain reaction which is a chemical reaction that can produce free radicals that lead
to chain reactions that can damage the cells of organisms. Some examples of antioxidants are
ascorbic acid, thiols, and polyphenols (Rao, 2016). A chemical reaction in which electrons are
transferred from one molecule to an oxidizing agent is known as oxidation. Free radicals are
created during oxidation reactions. These highly reactive free radicals have one or more unpaired
electrons in their outermost shell. The chain reaction begins when they are generated.
Antioxidants react with free radicals, stopping the chain reaction and inhibiting additional
oxidation reactions by oxidizing themselves (Mamta, Misra, Dhillon, Brar, & Verma, 2013).
Free radicals are atomic entities that have unpaired electrons in their atomic orbitals and can
exist independently. As a result, these radicals are extremely reactive and can either remove or
give electrons to other molecules, serving as a reductant or oxidant, respectively (Engwa, 2018).
In living organisms, oxygen is required for energy production via the electron transport chain, a
system that releases energy (ATP) to allow the cell to carry out its usual physiological functions.
This is due to its high redox potential, which makes it an excellent oxidizing agent capable of
taking electrons from reduced substrates with ease. Because of the contradicting effects of
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oxygen in living beings, the antioxidant system evolved to defend against excessive oxidation
and counteract reactive oxygen species (ROS). The mitochondria are the most important
generator of reactive oxygen species (ROS) (Ifeanyi, 2018).
At, low concentration ROS have beneficial effects and are involved in some physiological
functions such as immune function (defence against pathogenic organism), cellular pathways,
and redox regulation (Di Meo, Reed, Venditti, & Victor, 2016). Also according to Ahmad
(2018), it aids in the toning of smooth muscles, which in turn regulate the normal working of
blood vessels and internal organs. But when the concentrations of ROS and RNS are too high, it
results in the generation of oxidative stress and nitrosative stress respectively, causing potential
damage to biomolecules (Phaniendra, Jestadi, & Periyasamy, 2014).
Endogenous sources of free radicals (ROS and RNS) include mitochondria, peroxisomes,
endoplasmic reticulum, phagocytic cells, neutrophils, NADPH/xanthine oxidase, neuroendocrine
cells, macrophages, and others, and exogenous or external sources like pollution, alcohol,
tobacco smoke, heavy metals, transition metals, industrial solvents, pesticides, certain
xenobiotics like halothane, paracetamol, and radiation (Rahal et al., 2014).) Free radicals harm
important biomolecules such as nucleic acids, lipids, and proteins, by altering their normal redox
status leading to increased oxidative stress.
Excess oxidants lead to a decrease in antioxidants, resulting in an oxidation–reduction imbalance
in organisms. Oxidative stress is caused by a deficiency in the antioxidant system, which is
characterized by high quantities of reactive species (oxygen, hydroxyl free radical, and so on).
Mitochondria are involved in the creation of important biological compounds as well as the
delivery of ATP to cells via oxidative phosphorylation. In the oxidative phosphorylation process,
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enzymes catalyze a variety of redox reactions. Reactive oxygen species (ROS) are produced
when oxidative phosphorylation is inefficient, which can lead to mitochondrial malfunction. The
principal and possible sources of free radicals are determined to be mitochondrial redox
metabolism, phospholipid metabolism, and proteolytic processes (Singh, Kukreti, Saso, &
Kukreti, 2019).
Oxidative stress may be defined as an imbalance or disturbance in the production of free radicals
and antioxidants in favour of oxidants. Cell damage can occur at the molecular level due to an
imbalance between free radicals and antioxidants and since these oxidants are generated at a
different rate as a normal product of aerobic metabolism, complex biochemical mechanisms are
required to regulate this process (Pruchniak, Aražna & Demkow, 2015).
Antioxidants are chemicals that bind with free radicals to nullify their effect. They bind with free
radicals by giving up their electrons. Resulting in the termination of oxidative chain reactions
and the free radicals are no longer able to attack the cell. The antioxidants themselves attain a
free e radical state after donating their electron but can accommodate the change in electrons
without becoming reactive and harmful.
2.2 Types of antioxidants
Antioxidants are classified in several ways;
i. They can be classed as enzymatic or non-enzymatic antioxidants based on their action.
Enzymatic antioxidants that can break down and get rid of free radicals in a multistep
process in the presence of cofactors such as copper (Cu), zinc (Zn), manganese (Mn),
selenium (Se), and iron can convert dangerous oxidative products to HO and
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subsequently to water (Fe). Superoxide dismutase (SOD), catalase (CAT), and
glutathione peroxidase are antioxidant enzymes (GPx). Reduced glutathione (GSH),
vitamin C, E, β-Carotene, flavonoids, isoflavones, flavones, anthocyanins, catechins,
phenolic compounds, and isocatechins, as well as lipoic acid, are non-enzyme
antioxidants found in plants and fruits (Zulaikhah, 2017).
ii. Non-enzymatic antioxidants such as vitamin C, vitamin E, plant polyphenols, carotenoid
and glutathione work by disrupting free radical chain reactions.
iii. Antioxidants are classed as either water-soluble or lipid-soluble based on their solubility.
Vitamin C is a water-soluble vitamin that can be found in cellular fluids including cytosol
and cytoplasmic matrix.
iv. Antioxidants are classified as small or large molecule anti-oxidants based on their size. In
a mechanism known as radical scavenging, small molecule antioxidants neutralize ROS
and move them away. The principal antioxidants in this category are vitamin C, vitamin
E, carotenoids, and glutathione (GSH). Enzymes (SOD, CAT, and GPx) and sacrificial
proteins (albumin) are large molecule antioxidants that absorb ROS and prevent them
from damaging other important proteins.
v. Antioxidants are classified as natural or synthetic antioxidants based on their occurrence
(Sharifi-Rad et al., 2020).
vi. Antioxidants are classified in the following ways based on their kinematics:
a) Antioxidants that can break chains by interacting with weak O–H or N–H bonds in
peroxyl radicals, such as phenol, naphthol, hydroquinone, aromatic amines, and
aminophenols.
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b) Quinines, nitrones, and iminoquinones are antioxidants that can break chains by
interacting with alkyl radicals.
c) Aromatic amines, nitroxyl radicals, and variable valence metal compounds are
antioxidants that terminate cyclic chains.
d) Antioxidants that decompose hydroperoxide, such as sulfide, phosphide, and
thiophosphate.
e) Diamines, hydroxyl acids, and bifunctional chemicals are examples of metal-
deactivating antioxidants.
f) Several antioxidants, including phenol sulfide, work together to form a synergistic
reaction in which the phenolic group reacts with peroxyl radicals and the sulfide
group reacts with hydroperoxide.
vii. Synthetic antioxidants: These are phenolic compounds that function as scavengers for
free radicals, halting the chain reaction. Example includes;
a) Butylated hydroxyl anisole (BHA).
b) Butylated hydroxytoluene (BHT).
c) Propyl gallate (PG) and metal chelating agent (EDTA).
d) Tertiary butyl hydroquinone (TBHQ).
e) Nordihydro guaiaretic acid (NDGA) (Azat Aziz, Shehab Diab, & Abdulrazak
Mohammed, 2019).
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Figure 2.1 Classification and sub-classification of antioxidants found in natural sources. Source:
(Anwar, Hussain, & Mustafa, 2018)
2.3 Mechanism of action of antioxidants
Antioxidants are thought to have two main modes of action. The first is a chain-breaking
mechanism in which the principal antioxidant gives an electron to the system's free radical. The
second method includes quenching chain-initiating catalyst to remove ROS/reactive nitrogen
species initiators (secondary antioxidants). Antioxidants influence biological systems through a
variety of processes, including electron donation, metal ion chelation, co-antioxidants, and gene
expression modulation (Gulcin, 2020).
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2.4 Levels of antioxidant action
Antioxidants in defense systems work on multiple levels, including prevention, radical
scavenging, repair, and de novo, as well as the fourth line of defense, adaptation (Lobo et al.,
2010).
Preventive antioxidants, which inhibit the generation of free radicals, are the first line of
protection. Although the exact method and site of radical generation in vivo are yet unknown,
metal-induced hydro-peroxide and hydrogen peroxide decompositions must be one of the major
causes. Some antioxidants degrade hydro-peroxides and hydrogen peroxide to alcohols and
water, respectively, without generating free radicals, while some proteins sequester metal ions to
prevent such reactions (Lobo et al., 2010).
Lipid hydro-peroxides are decomposed to corresponding alcohols by glutathione peroxidase,
glutathione-s-transferase, phospholipid hydro-peroxide glutathione peroxidase (PHGPX), and
peroxidase. The ability of PHGPX to decrease hydro-peroxides of phospholipids embedded in
bio-membranes makes it unique. Hydrogen peroxide is converted to water by glutathione
peroxidase and catalase enzymes (Lobo et al., 2010).
Antioxidants, which scavenge active radicals and hence reduce chain initiation and/or break
chain propagation reactions, are the second line of defense. Endogenous antioxidants that
scavenge radicals include hydrophilic and lipophilic antioxidants. Hydrophilic radical-
scavenging antioxidants include vitamin C, uric acid, bilirubin, albumin, and thiols, while
lipophilic radical-scavenging antioxidants include vitamin E and ubiquinol. Vitamin E is widely
13
considered to be the most powerful lipophilic antioxidant for scavenging free radicals (Lobo et
al., 2010).
Repair and de novo antioxidants are the third lines of protection. Proteinases, proteases, and
peptidases are proteolytic enzymes that recognize, break down, and remove oxidatively changed
proteins from the cytosol and mitochondria of mammalian cells, preventing the buildup of
oxidized proteins (Lobo et al., 2010).
The overall defense mechanism against oxidative damage includes DNA repair processes. There
are a variety of enzymes that repair damaged DNA, including glycosylases and nucleases.
Another key role is adaptation, in which the signal for free radical production and reactions
causes the creation and delivery of the appropriate antioxidant to the correct location (Lobo et
al., 2010).
2.5 Characteristics of antioxidants
Monohydroxy or polyhydroxy phenol compounds with varying ring replacements are the most
common antioxidants utilized in meals today. The activation energy of these molecules to give
hydrogen is minimal. As a result of the stability of the delocalized radical electron, the ensuing
antioxidant radical does not initiate another free radical. The donation of hydrogen from
antioxidants and metal chelating agents can delay or prevent the propagation and start of free
radical chain reactions. Because of its stability, the generated antioxidant-free radical is not
prone to fast oxidation.
Antioxidant free radicals can also create a stable complex molecule with lipid-free radicals,
preventing part of their damage (Hamid, Aiyelaagbe, Usman, Ameen & Lawal, 2010).
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2.6 Effects of Antioxidants
Free radicals are continually on the lookout for healthy cells to assault their fragile outer
membranes, resulting in cellular death and degeneration. Today's experts believe that free
radicals are responsible for disease, infection, stress, and aging. Free radicals can also harm
sports performance by slowing or stopping muscle development and diminishing aerobic
capacity. Furthermore, free radicals have been shown to create abnormalities in both normal
RNA and life-sustaining DNA, which is the genetic material of cells (Warner et al., 2004).
In the outer shell of normal body molecules, there are two electrons (a paired group). A free
radical is a molecule that has only one (unpaired) electron in its outer shell. When oxygen in the
bloodstream is combined with any of a diverse group of chemicals, such as those commonly
found in polluted air, in primary and/or second-hand cigarette smoke, free radicals form
naturally, and damage is accelerated by the normal radiation found in sunlight and by increasing
exercise, particularly running and other aerobic activities. This is simple to comprehend because
aerobic activity can raise oxygen use by ten to twenty times typical levels. Free radical
generation skyrockets as more oxygen enter the bloodstream (Hamid, Aiyelaagbe, Usman,
Ameen & Lawal, 2010).
Free radicals' direct muscle-destroying effects persist for several hours after activity has ended.
Free radical damage can be avoided by including antioxidants in one's diet or using anti-oxidant
supplements. A good anti-oxidant supplement, the complex has several different types of anti-
oxidants that seek for and kill free radicals at many different cellular places, giving it an
advantage over diet sources. Vitamin E, for example, protects only the cell's outer fatty layers. It
will not stabilize DNA, which is one of the anti-oxidant Vitamin C's key actions.
15
Anti-oxidant synergy refers to the process by which different anti-oxidants spread through the
bloodstream to protect cells at different locations. When a specific anti-oxidant comes into
contact with a free radical in the bloodstream at its designated activity site, it naturally binds to it
and converts it to harmless water and oxygen. As a result, cellular damage decreases as anti-
oxidant levels rise due to the administration of higher doses of a wider range of anti-oxidants,
and performance and health improve (Hamid, Aiyelaagbe, Usman, Ameen & Lawal, 2010).
2.7 Chain Reactions of Free Radicals
Initiation stage: It refers to the first step in the process of forming a radical species. This is a
homolytic cleavage event in the vast majority of cases. Following are the equations that represent
the reaction:
a. I→I ̇
b. I ̇ +RH→ R ̇+ I-H
Propagation stage: The 'chain' part of chain reactions is described by this term. Once a reactive
free radical has been formed, it can combine with stable molecules to produce other reactive free
radicals. These new free radicals produce other free radicals, and so forth. Hydrogen abstraction
or radical addition to double bonds are frequently used in propagation phases. The following
equations are used to show this:
a. R ̇ + O2 → ROO ̇
b. ROO ̇ + RH → ROOH + R ̇
c. RO ̇ + RH → ROH + R ̇
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Termination stage: When two free radical species react to generate a stable, non-radical adduct,
this is called adduct formation. The equations that represent this are as follows:
a. R ̇ + R ̇ → R – R
b. R ̇ + ROO ̇ → ROOR
c. ROO ̇ + ROO ̇ → ROOR + O2
Antioxidants + O2 → oxidized antioxidants (Losada-Barreiro & Bravo-Díaz, 2017).
2.8 Phytochemicals
Phytochemicals are bioactive components found naturally in plants, and their concentrations and
secretions differ from one plant to the next. Terpenoids, polyphenols, phenolic components,
alkaloids, carotenoids, phytosterols, saponins, and fibers are the most common classes. They
contribute to human health in a variety of ways, including antioxidant characteristics, cell
differentiation effects, improved detoxification enzyme activity, DNA metabolism effects, DNA
repair maintenance, cancer cell death, cell proliferation reduction, and so on (Thakur, Singh,
Khedkar, 2020). Phytochemicals can have a wide range of physiological effects, including acting
as antioxidants, mimicking biological hormones, and preventing disease development (Hayes,
2005).
2.8.1 Classes of Major Phytochemicals, Food Sources, and Nutritional Benefits
All plant products, including fruits, vegetables, legumes, grains, herbs, and spices, contain
phytochemicals. No one plant material contains all of the essential phytochemicals that humans
require. As a result, it is recommended that a wide variety of plant materials, such as fruits,
vegetables, cereals, herbs, and spices, be ingested to maximize the benefits of the phytochemical
17
complex. Phytochemicals are found in almost all plant materials consumed by humans. Cabbage,
lettuce, tomatoes, carrots, watermelon, mangoes, pawpaw, grapes, oranges, apples, cashew apple
and nut, mustard, pear, oats, sweet potatoes, whole wheat, beans, ginger, onions, red pepper
spinach, sesame seed, and garlic are all good sources of phytochemicals. (Obeta, 2015).
Phytochemicals function in synergy, according to Birt (2006), and their combined effects are
stronger than the sum of their individual effects when served together. The hundreds of
phytochemicals identified so far are organized into groups
2.8.2 Polyphenols
Xanthophylls, carotenes, and vitamins are the most common natural antioxidants found in plant
materials (vitamin E and C). These natural antioxidants, particularly polyphenols and
carotenoids, have a variety of biological effects, including anti-inflammatory, antibacterial,
antiviral, anti-aging, and anticancer properties (Xu, et al., 2017).
Given the benefits of using dietary components with low toxicity, a large supply of materials,
and a low cost, nutritional therapy is a significant technique for preventing and/or treating a
variety of diseases, as well as contributing to individual well-being. The revelation of the
intriguing potential advantages of modifying one's eating choices has thrown light on the role of
naturally occurring plant chemicals in health promotion and maintenance.
Many human diseases and maladies have been linked to the protective benefits of these
phytochemicals, and research into nutraceuticals, functional meals, and natural health products
has become a popular issue in recent years.
18
They are a type of phytochemical with strong antioxidant properties. Their antioxidant
capabilities are determined by their chemical and physical properties, which influence
metabolism through their molecular structures (Ajila et al., 2011). Phenolic acids, flavonoids,
gingerol, curcumin, and other compounds are among them (Amit & Priyadarsini 2011).
Dietary phenols are split into two groups: benzoic derivatives such as gallic acid and cinnamic
derivatives such as caffeic acids. Gallic acid is the most well-known polyphenol that works well
in a polar medium because it has a stronger ability to deprotonate hydroxyl free radicals. Gallic
acid is a strong scavenger, capable of deactivating a wide range of ROS and RNS primarily
through electron transport across the cellular physiological pH (Mareno et al., 2014). The
inactivation of lipid-free radicals or the prevention of the breakdown of hydroperoxides into free
radicals is the mechanism by which phenolic compounds operate as antioxidants (Jin et al., 2010;
Kibiti and Afolayan, 2015).
Flavonoid is a type of polyphenolic substance found in vegetables, fruits, grains, seeds, leaves,
flowers, bark, and other plants. Gingerol is extracted from the rhizomes of ginger, while
curcumin (diferuloylmethane) is the major bioactive component of turmeric and is recognized to
have strong antioxidant action.
2.8.3 Flavonoids
Flavonoids are the biggest class of phenolic chemicals, with a three-ring basic structure (C6-C3-
C6). Flavan-3-ols, anthocyanins, flavones, isoflavones, flavanones, and flavonols are the six
primary classes defined by their substitution pattern in the B- and C-rings (J. Harborne and
Baxter, 2000). Proanthocyanidins are another name for flavonoid polymers. Flavonoids are
19
secondary metabolites found in plants that have a role in pigmentation, antioxidants,
antimicrobials, antistressors, and UV irradiation protection (Vaya and Aviram, 2001). So far,
more than 4000 flavonoids have been identified in plant components ingested by humans, with
around 650 flavones and 1030 flavanols identified (Ghasemzadeh et al., 2010).
Flavonoids may be found in practically all plant-based foods and beverages, but their
concentration varies depending on the ripeness of the fruits, variety, and processing. Su et al.
(2005) found that most flavonoids increase the effectiveness of vitamin C (ascorbic acid) and act
as antioxidants. Flavonoids' antioxidant activities are linked to their ability to scavenge free
radicals, metal-reducing activity, and metal chelation, all of which are implicated in the
formation of reactive hydroxyl radicals. The polyphenolic chemical structure of the C6-C3-C6
ring system is responsible for these properties. The antioxidant activity is influenced by the
quantity and location of hydroxyl groups in the structure (Rodríguez-Arce & Saldías, 2021).
Examples of dietary sources of flavonoids include Apple skin, Berries, Broccoli, Celery, Onions,
citrus fruits, citrus peels, etc. Due to their ability to control critical cellular enzyme activity as
well as their anti-oxidative, anti-inflammatory, anti-mutagenic, and anti-carcinogenic capabilities
(Panche, Diwan, & Chandra, 2016). By scavenging free radicals and/or chelating metal ions,
flavonoids' functional hydroxyl groups mediate their antioxidant actions (Kumar, Mishra, &
Pandey, 2013).
Because it gives hydrogen and an electron to hydroxyl, peroxyl, and peroxynitrite radicals, the β-
ring hydroxyl configuration is the most important factor in scavenging ROS and RNS (Reactive
Nitrogen Species), stabilizing them and giving rise to a rather stable flavonoid radical (Kumar
and Pandey, 2013).
20
Mechanisms of antioxidant action may include;
i. Inhibition of enzymes or chelation of trace elements involved in a free radical creation to
reduce ROS production.
ii. Scavenging ROS
iii. Antioxidant defence regulation or protection (Manandhar, 2018).
The majority of the mechanisms outlined above are involved in flavonoid activity. Some of the
actions mediated by them could be the consequence of a combination of radical scavenging
activity and enzyme functions interacting (Lewandowska et al., 2015). Microsomal mono-
oxygenase, glutathione S-transferase, mitochondrial succinoxidase, NADH oxidase, and other
ROS-producing enzymes are all inhibited by flavonoids
21
Figure 2.2 Chemical structure of flavonoid polyphenols. Soure: (Collins, Saleh, & Kalisch,
2022).
2.9 Ficus exasperata Vahl (sandpaper)
2.9.1 Botanical description, habitat and distribution
Ficus exasperata (linn) is a member of the Moraceae family, which consists of over 800 species
of shrubs, trees, and other plants found in tropical and moderate temperate climates. They are
commonly referred to as "fig plants or fig trees" around the world (Ohunayo et al., 2020). Ficus
exasperata Vahl is a species of evergreen forest tree that grows up to 20 meters tall. The leaves
are distichous, alternating, ovate to elliptic, sub-coriaceous to coriaceous, apex acuminate, base
acute to obtuse, upper surface scabrous with a highly rough surface, giving them the name
22
sandpaper tree. Lateral veins; 3-5 pairs, the basal pair branching and reaching the lamina's border
at or above the middle. Stipules are 0.2-0.5m long, strigose, and caducous, and the petiole is 0.5-
4cm long. Solitary or in pairs, figs can be found in the leaf axils and on aged wood. Fresh figs
are subglobose, 1-2.5cm in diameter, hispidulous, with a 0.5-1m long peduncle and 1mm long
basal bracts dispersed on the peduncle. It produces figs in pairs in the leaf axils. It is widely
spread in Zambia, Mozambique, northern Angola, Ethiopia, Senegal, and other Central African
nations.
2.9.2 Taxonomy of Ficus exasperata Vahl
Kingdom: Plantae
Sub-kingdom: Viridaeplantae
Phylum: Tracheophyta
Subphylum: Euphyllophytina
Infraphylum: Radiatopses
Class: Magnoliopsida
Subclass: Dilleniidae
Superorder: Urticanae
Order: Urticales
Family: Moraceae
23
Tribe: Ficeae
Genus: Ficus
Specific epithet: exasperata– Vahl.
Botanical name: Ficus exasperata Vahl (Ahmed, Karim, Mueen Ahmed, & Abedin, 2012).
2.9.3 Cultural and Ethnomedicinal uses
The leaves are commonly used as an abrasive to polish hard surfaces like furniture and utensils.
Ficus exasperata Vahl is a beneficial medicinal plant of biological value that is used to cure and
manage a variety of ailments in Africa. The leaves are employed in the treatment of malaria,
haemorrhoids, and diarrhoea in Cameroonian folk medicine when the water extract is taken
orally. The viscid sap is used to cure wounds and stomach aches in the Ivory Coast. Studies on
F. exasperata stem barks, leaves, and root extracts have revealed the plant's remarkable
antibacterial capabilities. The hydro-alcoholic extract of the plant's leaves has been shown to
have anti-inflammatory, anti-nociceptive, and antipyretic properties. The hydro-alcoholic extract
of stem-bark showed antiulcerogenic, antioxidant, hypotensive, lipid-lowering, and
hypoglycaemic properties. Phytochemicals such as alkaloids, flavonoids, and tannins are
abundant in the plant. This means the plant could be used to treat/or manage oxidative stress
related diseases.
24
Figure 2.3 Ficus exasperata Vahl leaves. Source: (Gloria Tit & Oluwafemi, 2019)
25
CHAPTER THREE
MATERIALS AND METHODS
3.1 Materials and Reagents
Materials and reagents used in this study include: F. exasperata Vahl leaves, electric blender, hot
air oven, rotary evaporator, beaker, conical flask, funnel, filter paper, Petri dishes, water bath,
test tubes, test tube racks, weighing balance, separating funnel, n-hexane, ethyl acetate,
spectrophotometer, spatula, 70% methanol, distilled water, DPPH reagent, FRAP reagent which
contains 500mL of acetate buffer (300 mM pH 3.6), 50mL of 2,4,6-tri (2-pyridyl)-s-triazine
(TPTZ) (10mM), FeCl3. 6H2O (20mM), (200mg/ml) gallic acid, phosphate buffer, 0.6M sulfuric
acid, (28mM) sodium phosphate and 4mM ammonium molybdate.
3.2 Location of the Study
This study was carried out at the Department of Biochemistry, School of Basic medical sciences,
Babcock University, Ilishan Remo, Ogun State, Nigeria.
3.3 Collection and Authentification of Plant Material
The fresh leaves of Ficus exasperata were plucked from a Ficus exasperata tree on a plantain
farm close to the new market square, Ilishan Remo, Ogun State and was identified and
authenticated at the University of Lagos Herbarium and a voucher number of LUH: 8387 was
obtained.
26
3.4 Preparation of Plant Extract
The leaves of Ficus exasperata Vahl were washed and cleaned thoroughly with distilled water to
remove all debris; it was then air-dried for about one month and pulverized into a fine powder
using an electric blender.
Methanolic extract of F. exasperate: Pulverized F. exasperate leaves (450 g) were macerated
with 3.6L of 70% methanol. The mixture was shaken intermittently and was allowed to stay for
72hours at room temperature. After 72hours, it was filtered using whatmann No. 1 filter paper.
The filtrate was concentrated using a rotary evaporator at 40°C and allowed to dry.
After concentration, the methanol extract was reconstituted in distilled water and also partitioned
using, n-hexane and ethyl acetate respectively to obtain its various fractions. The various solvent
fractions were then evaporated to dryness using a rotary evaporator at 40ºC.
3.5 In-Vitro Anti-Oxidant Assays
3.5.1 Determination of In vitro DPPH (2, 2- diphenyl-1-picrylhydrazyl) Radical Scavenging
Activity
Using the DPPH reagent, the DPPH radical scavenging activity of methanol extracts of Ficus
exasperata leaves was measured and compared, as described by McCune and Johns (2002). The
approach relies on the stable free radical DPPH, which has a deep violet color that can be
detected using spectrophotometry. This is a simple and rapid approach for determining
antioxidant scavenging potential. In methanol, DPPH in its oxidized form produces a rich violet
color. When an antioxidant gives an electron to DPPH, it is reduced, and its color changes from
deep violet to yellow. At 517 nm, DPPH solutions had a significant absorption, resulting in deep
27
violet color. The ability of the test materials to scavenge DPPH free radicals indicates their free
radical scavenging capacity or antioxidants potential, which demonstrates their effectiveness,
prevention, interception, and repair mechanisms against injury in a biological system.
Preparation of DPPH Solution (0.1 M)
The DPPH solution (0.1M) was produced by dissolving 0.39 mg of DPPH in methanol in a
volumetric flask with a final volume of 1000 µL. As a result, the purple-colored DPPH free
radical solution was created and stored at 20°C for later use.
Procedure
1mL DPPH solution was added to the sample and standard solutions of various concentrations
(20, 40, 60,100, and 200µg/ml) and incubated for 30 minutes in the dark at room temperature. 1
mL methanol and 1 mL DPPH solution were mixed to provide a control. Finally, using a
spectrophotometer set at 517 nm, the absorbance of the solutions was determined. The
experiment was carried out in triplicate. As the standard, gallic acid was utilized. The percentage
of inhibition of radical scavenging activity was measured. Triplicate measurements were made.
The percentage antioxidant potential was calculated using the formula:
AC-AO
I%= ×100%
AC
Where AC = absorbance of the control (1 mL methanol + 1 mL DPPH solution), AO =
absorbance of the sample solution, and I% = percentage of inhibition.
28
3.5.2 In vitro FRAP (Ferric Reducing Antioxidant Power) Assay
The overall antioxidant activity was measured using the FRAP method, which measures the
reduction of ferric tripyridyl triazine complex to ferrous colored form in the presence of
antioxidant compounds (Hajimahmoodi et al., 2013). This method was initially reported by
Benzie & Strain (Benzie & Strain, 1996).
Procedure
The ferric reducing antioxidant power reagent was prepared by mixing acetate buffer 300mM,
10ml TPTZ (2, 4, 6- tripyridyl-S- triazine 10mmol/l) in HCl 40mM and 20mM FeCl 3.6H2 O in
the proportion 10:1:1 at 37 ºC. A sample (fractions) and standard solution (Gallic acid) of
different concentrations (20, 40, 60, 100, and 200 µg/ml) were introduced into five distinct pairs
of test tubes and to each 2ml of the freshly prepared FRAP reagent was added and was mixed
thoroughly. An intense blue color complex was formed indicating that the ferric tripyridyl
triazine (Fe3+ TPTZ) complex has been reduced to a ferrous (Fe 2+) complex and the absorbance
was measured at 593nm with the aid of a spectrophotometer, the absorbance was recorded
against the reagent blank (3.995ml FRAP reagent + 5µl distilled water) after 30minutes
incubation at 37 ºC, using Gallic acid as standard. The experiment was carried out in triplicate.
3.5.3 Determination of Total Antioxidant Capacity (Phosphomolybdenum Method)
Using a phosphomolybdenum assay with gallic acid as a reference standard, the total antioxidant
capacity of the methanol extract of F. exasperata leaves was calculated. According to the
approach provided by Prieto, Pineda, and Aguilar (1999), phosphomolybdenum is a quantitative
29
method for investigating the reduction rate among antioxidant, oxidant, and molybdenum ligand
(Raju, Vasudevan, Anbazhagan, & Venkataraman, 2003).
Procedure
The sample (fractions) and standard (gallic acid) solution of different concentrations (20, 40, 60,
100, and 200 µg/ml) were introduced into five distinct pairs of test tubes and to each 1mL of the
reagent (0.6M sulfuric acid, 28mM sodium phosphate, and 4mM ammonium molybdate) was
added. The tube was capped and incubated in a boiling water bath at 95°C for 90 minutes. In a
UV spectrophotometer, the absorbance of the solution was measured at 695nm against a blank
after cooling to room temperature. 1mL of the reagent solution and 0.1mL of distilled water
made up the blank solution. It was incubated at the same temperature as the other samples. The
experiment was carried out in triplicate. The averages of three different samples were calculated.
The positive reference standard was gallic acid.
3.5.4 Determination of In vitro ABTS (2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic
acid) Radical Scavenging Activity
The ABTS radical cation decolorization assay was used to determine the free radical scavenging
activity of the sample solution (Re, Pellegrini, Proteggente, Pannala, Yang & Rice-Evans, 1999).
The reaction between 7 mM ABTS in water and 2.45 mM potassium persulfate (1:1) produced
an ABTS cation (+) radical. Before usage, it was kept in the dark at room temperature for 12-16
hours. After that, the ABTS+ solution was diluted with methanol to attain an absorbance of 0.700
at 734 nm. After mixing the sample and standard solutions of different concentrations (20, 40,
60, 100,and 200 µg/ml) with 3.995 ml of diluted ABTS+ solution, the absorbance was measured
30
30 minutes later. In each test, a suitable solvent blank was used. The experiment was carried out
in triplicate. Using the formula, the percent inhibition of absorbance at 734 nm was obtained.
ABTS·+ scavenging effect (%) = [ AB− AA

AB ]
×100
Where AB is the absorbance of ABTS radical + methanol; AA is the absorbance of ABTS
radical + sample extract/standard. Gallic acid was used as a standard substance.
3.5.6 Determination of Total Phenolic Content
The total phenolic content of then-hexane and ethyl acetate extracts was calculated using the
Singleton et al (1999) approach, 2.5 ml Folin-Ciocalteu reagent (1:10 v/v) and 2 ml sodium
carbonate (7.5 percent w/v) were mixed with each extract (concentration 20 and 200 µg/ml) the
standard (gallic acid) solution (20, 40, 60, 100 and 200 µg/ml).
Color development took place after the liquid was vortexed for 15 seconds and incubated at
400C for 30 minutes. A calibration curve was developed using gallic acid as the standard. Using
a spectra UV/visible spectrophotometer, absorption was measured at 750nm.
3.5.7 Determination Total flavonoid content (TFC)
Ordonez et al (2006) approach was used to calculate the total flavonoid content of the samples.
The sample solution (20 and 200 µg/ml) and standard solutions (20, 40, 60, 100, and 200 µg/ml)
were combined with 0.5 ml of 2 % Aluminum chloride (AlCl 3) produced in methanol. To
establish the presence of flavonoids, the following combination was incubated at room
temperature for 60 minutes for color development (yellow). Using a UV/visible
spectrophotometer, absorbance at 420nm was measured.
31
3.6 Statistical Analysis
Triplicates of the experiments were carried out. The data was presented as mean and standard
deviation (SD). To see if there were any significant differences between the ethyl acetate and n-
hexane fractions, multiple unpaired t-tests were used. When p < 0.05, a difference was
considered statistically significant.
32
CHAPTER FOUR
RESULTS
4.1 Free radical scavenging effect on the DPPH Assay
The DPPH radical scavenging activity of ethyl acetate and n-hexane fraction of Ficus exasperata
Vahl leaves is shown in figure 4.1. At 60 µg/ml, 100 µg/ml and 200 µg/ml of the fractions,
percentage (%) inhibition of the n-hexane fraction was lower than in the ethyl acetate fraction. A
significant difference (p<0.05) was not observed between the fractions (ethyl acetate and n-
hexane fraction) at 20 µg/ml concentration.
33
DPPH
45
40
35
30
% Inibition of DPPH
25 HE
EA
20
15
10
0
20 40 60 100 200
Concentration of Fractions (µg/ml)
Figure 4.1 DPPH radical scavenging activity of the ethyl acetate and hexane fraction of Ficus
exasperata Vahl leaves
HE: n-hexane fraction
EA: ethyl acetate fraction
DPPH: 1, 1-diphenyl-2-picrylhydrazyl) radical scavenging power
*: significant difference at p<0.05
34
4.2 Free radical scavenging ability by the use of ABTS radical cation assay
The ABTS radical scavenging activity of ethyl acetate and n-hexane fraction of Ficus exasperata
Vahl leaves is shown in figure 4.2. At all concentrations of the fractions, the percentage (%)
inhibition of the ethyl acetate fraction was significantly higher than the n-hexane fraction.
35
ABTS
120
100
80
% Inibition of ABTS
HE
60
EA
40
20
0
20 40 60 100 200
Figure 4.2 ABTS radical scavenging activity of the ethyl acetate and hexane fraction of Ficus
ABTS: 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid
*: significant difference p<0.05
36
4.3 Total antioxidant capacity of F. exasperata leaves fractions (ethyl acetate and n-hexane
fractions)
The total antioxidant capacity of the ethyl acetate and n-hexane fractions of F. exasperata Vahl
leaf is shown in figure 4.3. The n-hexane fraction at all concentrations had significantly lower
antioxidant capacity than the ethyl acetate fraction.
37
TAC
400
350
300
250
HE
200
µgGAE/mg
EA
150
100
50
0
20 40 60 100 200
Figure 4.3 Total antioxidant capacity of the ethyl acetate and n-hexane fractions of Ficus
38
4.4 Ferric reducing antioxidant power (FRAP) of F.exaperata Vahl leaves ethyl acetate and
n-hexane fraction
The Ferric reducing antioxidant power (FRAP) of ethyl acetate and n-hexane fraction of
F.exaperata Vahl leaves is shown in figure 4.4. Statistically, there was a significant difference
(p<0.05) between the FRAP reducing antioxidant power of the ethyl acetate and n-hexane
fraction at 20 µg/ml and 200 µg/ml concentration. But at 40 µg/ml, 60 µg/ml and 100 µg/ml
concentration, there was no significant difference (p<0.05).
39
FRAP
80
70
60
50
mMFe2+ equivalent/g of sample
HE
40
EA
30
20
10
0
20 40 60 100 200
Figure 4.4 Ferric reducing antioxidant power (FRAP) of the ethyl acetate and n-hexane fraction
F.exasperata Vahl leaves
40
4.5 Total flavonoid content of the ethyl acetate and n-hexane fraction of F. exasperata
leaves
The total flavonoid content of the ethyl acetate and n-hexane fraction of F. exasperata Vahl
leaves is shown in figure 4.5. Statistically, at the concentration of 20µg/ml, there was no
significant difference (p<0.05) between the total flavonoid content of the two fractions (ethyl
acetate and n-hexane Fraction) of the F. exasperata Vahl leaf. But at the 200 µg/ml
concentration of the fraction, the total flavonoid content of the ethyl acetate fraction was
significantly higher than the n-hexane Fraction.
41
TFC
70
60
50
40
µgQE/mg
HE
EA
30
20
10
0
20 200
Figure 4.5 Total flavonoid content of the ethyl acetate and n-hexane fraction of F. exasperata
Vahl leaves
42
4.6: Total phenolic content of the ethyl acetate and n-hexane fraction of F. exasperata
leaves
The total phenolic content of the ethyl acetate and n-hexane fraction of F. exasperata leaves is
shown in figure 4.6. At 200µg/ml, the ethyl acetate fraction had a significantly higher phenolic
content than the hexane fraction.
43
TPC
30
25
Phenolic content (µgGAE/mg)
20
HE
15
EA
10
0
20 200
Figure 4.6 Total phenolic content of the ethyl acetate and n-hexane fraction of F. exasperata
Vahl leaves
44
CHAPTER FIVE
DISCUSSION, CONCLUSION AND RECOMMENDATION
5.1 Discussion
Clinical and medical practitioners are becoming increasingly interested in the hunt for natural
antioxidants. This is because reactive oxygen species (ROS) and oxidative stress have been
related to the aetiology of a variety of diseases, including diabetes, cancer, cardiovascular
disease, kidney, lung, and liver damage, among others. Free radicals have also been linked to the
pathophysiology of inflammatory diseases such as rheumatoid arthritis, according to research
(Liu, Zhou, Ziegler, Dimitrion, & Zuo, 2017).
On the other hand, synthetic antioxidants have been associated with toxicity, high cost, and
adverse effects (Kibiti and Afolayan, 2015). Plants, on the other hand, may provide better
antioxidant options. Plant leaves, stems, flowers, fruits, and roots have long been known to have
therapeutic potential, and as a result, they have been extensively researched as potential remedies
for a number of stress-related disorders.
The yield and antioxidant activity of a plant extract are dependent on the extraction solvent used,
according to Gong et al. (2012). Due to polarity differences, different solvents are used to isolate
antioxidant molecules. Methanol, ethanol, dichloromethane (DCM), water, and acetone are some
of the most commonly used extraction solvents. Due to their polarity, ethanol, methanol, and
water are ideal solvents for extracting polar molecules such as phenolic compounds and
flavonoids. For the extraction of non-polar substances, non-polar solvents such as ether, acetone,
and dichloromethane are preferred (Lefebvre, Destandau, & Lesellier, 2021). Methanol was used
45
as a solvent to extract antioxidant components from Ficus exasperata Vahl leaves in this
research. The separating funnel method was also used to fractionalize it into ethyl acetate and n-
hexane fractions.
One of the most commonly known approaches to determining the antioxidant activity of
medicinal plants is to measure the decrease of the DPPH radical. Plant extracts' ability to
contribute electrons to the DPPH radical is tightly linked to their ability to inhibit the radical
(Daniel and Dluya, 2016). In most solvents, such as methanol, ethanol, and water, the DPPH
radical is stable. As a result, the radical is often made in an ethanol or methanol solution
(Venkatesan, Choi, & Kim, 2019). The DPPH radical was prepared in methanol for this
experiment. Because it does not require specific equipment or methodologies, the in vitro DPPH
scavenging assay was chosen for this investigation because it is accurate, simple, and reliable. At
a wavelength of 517 nm, the extracts' in-vitro radical scavenging activities against DPPH were
measured.
The DPPH radical can be converted to a non-reactive state by binding hydrogen atoms or
receiving an electron (A. Kumar, Behl, & Chadha, 2020). The colour of the solution changes
from purple to yellow when DPPH is converted to DPPH-H (diphenylhydrazine) in the presence
of an antioxidant agent. The level of discolouration increases when the number of DPPH radicals
in the solution is reduced. The DPPH changes colour, indicating that the extract has radical-
inhibiting activities (Gebrehiwot et al., 2020). Phenolics and flavonoids have been reported to
reduce the DPPH radicals through their hydrogen donating ability (Shafiee et al., 2020). It was
observed that the scavenging activities of the ethyl acetate fraction of F. exasperata Vahl leaves
were increased with the increasing concentrations compared to the n-hexane fraction (Figure
4.1). The involvement of free radicals, especially their increased production leads to the
46
development of cardiovascular diseases and cancer. Thus, the consumption of F. exasperata
Vahl leaves can be beneficial in preventing oxidative stress related to numerous chronic diseases.
Scavenging of ABTS radical cations is useful in determining the antioxidant capacity of both
lipophilic and hydrophilic antioxidants. Furthermore, phenolic compounds are either hydrophilic
or lipophilic depending on their structure, which explains their great capacity to scavenge ABTS
radicals. The radical scavenging potential of a range of substances, including plant extracts, has
been assessed using this approach. The blue-green ABTS radical solution will be reduced to a
colourless neutral state by a molecule with the property of electron donation (Re et al., 1999;
Wangcharoen & Morasuk, 2007; Camacho-Luis et al., 2008; Krishna & Nair, 2010; Aiyegoro
and Okoh, 2010; Kekuda et al., 2015). In the present study, both fractions (ethyl acetate and n-
hexane fraction) were able to scavenge ABTS radical in a concentration-dependent manner. But
the scavenging ability of the ABTS radicals by the ethyl acetate fraction was found to be higher
than that of the n-hexane fraction. This shows that Ficus exasperata Vahl leaves ethyl acetate
fraction present a higher ability to scavenge the ABTS radical than that of the n-hexane fraction.
DPPH is scavenged mostly by lipophilic molecules, whereas ABTS represents both hydrophilic
and lipophilic scavenging action (MacDonald-Wicks, Wood, & Garg, 2006).
It was observed that increasing the concentrations of fractions (ethyl acetate and n-hexane
fraction) enhanced their ferric reducing capacity. More Fe 3+ were reduced to Fe2+ as a result of
antioxidant components donating more electrons (Ganu et al., 2010; Mitra et al., 2014). High
absorbance indicated that the extract's potential to reduce was increasing (Dehpour et al., 2009).
The ferric reducing power activities of the two fractions indicate that they are reductones (ethyl
acetate and n-hexane fractions). Reductones are engaged in a variety of antioxidant mechanisms,
including chain initiation prevention, metal ion binding, peroxide breakdown, and radical
47
scavenging (Yuliet, Sukandar, & Adnyana, 2020). The phytochemicals in the fractions either
directly bind the metal ions or, by occupying their coordination sites, indirectly decrease their
chelating re-activities (Edeoga et al., 2005; Njoku and Obi, 2009). The result obtained from this
assay suggests these fractions of Ficus exasperata Vahl leaves may play a protective role against
oxidative damage by sequestering Fe2+ ions.
The total antioxidant capacity (TAC) is determined by extracts reducing molybdenum (VI) to
molybdenum (V) and forming a green phosphate/molybdenum (V) complex at an acidic pH. The
sample's high absorbance values indicated that it possessed significant antioxidant activity. The
ethyl acetate fraction of the F. exasperata Vahl leaves had significant total antioxidant activity
compared to the n-hexane fraction and the effect increased with increasing concentration (Figure
4.3). The difference in the number of antioxidants in the fractions may be attributed to the
differences in the amount and kind of existing antioxidant compounds in them such as
carotenoids, phenol and ascorbic acid (Ghosh et al., 2012). The antioxidant activity shown by the
F. exasperata Vahl leaves fractions may be due to the presence of phenolic compounds and
flavonoids.
One of the most important in vitro antioxidants assays is the determination of the total flavonoid
and phenolic content of extracts. These two polyphenols are thought to be the most bioactive
molecules in plant extracts for diverse antioxidant activities (Altemimi, Lakhssassi, Baharlouei,
Watson, & Lightfoot, 2017). According to Heim et al. (2002) and Fukumoto et al. (2000),
increases in the number of hydroxyl groups in polyphenols and decreases in glycosylated groups
are directly connected to increased scavenging activity.
48
As a result, plant extracts' phenolic and flavonoid content have a direct role in their antioxidant
properties. Furthermore, other researchers, such as Arnous et al. (2002) and Lee et al. (2003),
have proposed that the antioxidant activity of plant extract is due to the synergy of various
phytochemicals in plants. Due to their hydroxyl groups, which are attached to their aromatic ring
structures (Mohamed et al., 2010) and help to quench the radicals either by donating their
electrons and thus neutralizing them or by electron delocalization over all three-ring systems
achieved by ortho-dihydroxyl of the B-ring and 4-oxo group of the ring C of the flavonoid,
which actively reduces radicals, flavonoids and phenols naturally exhibit strong scavenging
ability (Jin et al., 2010). The activity of proanthocyanidins, a flavonoid family, is mediated via
ferric ion inactivation, chelation, or suppression of the superoxide-driven Fenton's reaction
(Kibiti and Afolayan, 2015). One of the most common sources of reactive oxygen species in
cells and tissues is the Fenton reaction.
The phytochemical analysis of F.exasperata Vahl leaves fractions revealed that the fractions
(ethyl acetate and n-hexane fraction) contained flavonoids and phenols, which help protect cells
against oxidative stress. From the result, it was observed that the ethyl acetate fraction had higher
phenolic and flavonoid content than the n-hexane fraction. This is because in general,
polyphenols and flavonoids are polar, so it would be easy to dissolve in the fractions with a polar
or semi-polar solvent. The results show that both fractions examined have strong in
vitro antioxidant activity. This is why these herbs have long been used in African traditional
medicine to treat and control oxidative stress-related ailments like rheumatism, diabetes, stomach
discomfort, wound healing, and oral antiseptic.
49
5.2 Conclusion
From this study, it is concluded that:
1. The Ficus exasperata Vahl leaves possess antioxidant and phytochemicals activities.
2. The ethyl acetate fraction of the leaves of Ficus exasperata Vahl has higher antioxidant
properties than the n-hexane fraction.
3. The ethyl acetate fraction of the leaves of Ficus exasperata Vahl has higher phenolic and
flavonoid content than the n-hexane fraction.
5.3 Recommendation
Further studies are recommended for the comparison of ethyl acetate and n-hexane fraction of
Ficus exasperata Vahl leaves for the prevention, management and/or treatment of oxidative
stress-related diseases such as cancer, asthma, inflammatory disorders, Alzheimer’s disease,
Parkinson’s disease, etc.
50
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APPENDICES
Appendix A
Table 1 Free radical scavenging effect on the DPPH Assay
SAMPLE CONCENTRATIO %INHIBITION MEAN±STDEV

N OF FRACTIONS
USED
(µg/mL)
1(unit) 2(unit) 3(unit)
ETHYL 200 40.88625 42.16537 41.79991 41.617±0.538

ACETATE
100 36.13522 35.08451 35.63271 35.617±0.429
60 22.38465 21.33394 21.92782 21.882±0.43
67
40 10.96391 11.32937 11.42074 11.238±0.197
20 7.354957 5.481955 6.304249 6.38±0.767
n-HEXANE 200 32.38922 33.34856 32.80037 32.846±0.393
100 32.4349 31.61261 31.42988 31.826±0.437
60 18.7757 18.73001 18.27318 18.593±0.227
40 12.38008 12.74555 12.88259 12.669±0.212
20 6.441297 9.821836 6.441297 7.568±1.594
Table 2 Free radical scavenging ability by the use of ABTS radical cation assay
SAMPLE CONCENTRATION %INHIBITION MEAN±STDEV

OF FRACTIONS
USED
(µg/mL)
1(unit) 2 (unit) 3(unit)
ETHYL 200 98.2069 96.96552 97.37931 97.517± 0.516

ACETATE
100 72.68966 72.82759 73.10345 72.874± 0.172
60 64.55172 64.41379 63.58621 64.184± 0.426
40 52.27586 53.93103 52.96552 53.057± 0.679
20 45.10345 44.13793 44.27586 44.506± 0.426
n-HEXANE 200 61.93103 59.86207 59.72414 60.506±1.009

68
100 43.58621 43.17241 42.34483 43.034±0.516
60 42.2069 36.68966 36.41379 38.437±2.668
40 36 36.96552 36.41379 36.46±0.396
20 33.37931 33.65517 33.93103 33.655±0.225
Table 3 Ferric reducing antioxidant power (FRAP) Assay
SAMPLE CONCENTRATION mMFe2+ EQUIVALENT/g OF MEAN±STDEV

OF FRACTIONS SAMPLE)
USED
(µg/mL)
1(unit) 2 (unit) 3(unit)
ETHYL 200 70.39744 70.26923 69.88462 70.184±0.218

ACETATE
100 53.47436 52.96154 52.83333 53.09±0.277
60 46.16667 49.37179 46.42308 47.321±1.454
40 44.62821 43.60256 43.98718 44.073±0.423
20 40.52564 40.78205 40.26923 40.526±0.209
69
n-HEXANE 200 64.37179 63.98718 63.08974 63.816±0.537
100 54.11538 53.98718 55.78205 54.628±0.818
60 46.16667 48.85897 47.70513 47.577±1.103
40 43.21795 42.57692 43.08974 42.962±0.277
20 37.0641 37.44872 37.32051 37.278±0.16
Table 4: Total Antioxidant Content
SAMPLE CONCENTRATION GALLIC ACID EQUIVALENT MEAN±STDEV

OF FRACTIONS
USED µgGAE/mg
(µg/mL)
1 (unit) 2(unit) 3(unit)
ETHYL 200 333.9565 334.8261 337 335.261±1.28

ACETATE
100 197 210.4783 203.087 203.522±5.511
60 125.6957 133.087 137.4348 132.073±4.846
40 119.1739 117.8696 117.4348 118.159±0.739
20 55.69565 68.30435 63.95652 62.652±5.229
70
n-HEXANE 200 94.82609 101.3478 99.6087 98.594±2.757
100 45.69565 46.13043 44.82609 45.551±0.542
60 19.17391 18.73913 21.34783 19.754±1.141
40 12.21739 11.78261 10.47826 11.493±0.739
20 6.130435 10.04348 7.869565 8.014±1.601
Table 5: Total Flavonoid Content
SAMPLE CONCENTRATION QUERCETIN EQUIVALENT MEAN±STDEV

OF FRACTIONS
USED (µgQUE/mg)
(µg/mL)
ETHYL 200 59.24324 62.21622 59.78378 60.414±1.293

ACETATE
20 2.756757 11.94595 9.513514 8.072±3.887
n-HEXANE 200 43.2973 35.45946 39.51351 39.423±3.2
20 4.918919 3.027027 3.837838 3.928±0.775
71
Table 6: Total Phenolic Content
SAMPLE CONCENTRATION GALLIC ACID EQUIVALENT MEAN±STDEV

OF FRACTIONS
USED (µgGAE/mg)
(µg/mL)
ETHYL 200 27.78512 27.61983 27.70248 27.702±0.067

ACETATE
20 8.61157 6.876033 7.041322 7.51±0.782
n-HEXANE 200 15.63636 17.3719 16.71074 16.573±0.715
20 6.049587 6.628099 6.46281 6.38±0.243
72

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IN VITRO ANTIOXIDANT AND PHYTOCHEMICAL ACTIVITY OF ETHYL ACETATE

AND n-HEXANE FRACTION OF Ficus exasperata VAHL LEAVES

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF

SCHOOL OF BASIC MEDICAL SCIENCES

BENJAMIN S. CARSON (SNR.) COLLEGE OF HEALTH AND MEDICAL SCIENCES

DR. ESIABA, IJEOMA

FAJANA ELIZABETH DAMILOLA _______________________

DR. ESIABA IJEOMA _____________________

successfully and efficiently.

My specific gratitude to Aboyade-Cole Adedoyin, a member of my project team, for her

Ridwan and Nwaukwa Chinedum for their assistance and support.

encouragement during this project.

LIST OF FIGURES viii

CHAPTER ONE: INTRODUCTION 1

1.1 Background study 1

3.1 Materials and Reagents 25

4.1 Free radical scavenging effect on the DPPH Assay 32

Figure 2.1 Classification and sub-classification of antioxidants found in natural sources 12

Figure 2.2 Chemical structure of flavonoid polyphenols 21

Figure 2.3 Ficus exasperata Vahl leaves 24

exasperata Vahl leaves 33

exasperata Vahl leaves 35

exasperata Vahl leaves 37

F.exasperata Vahl leaves 39

Keywords: Ficus exasperata Vahl, Antioxidants, phytochemicals, ethyl acetate, n-hexane

Word count: 263

1.1 Background study

nephrotoxicity, hepatotoxicity, cancer, cardiovascular disease, inflammation, and neurological

disorders (Neha, Haider, Pathak, & Yar, 2019).

(Birnie-Gauvin, Costantini, Cooke, & Willmore, 2017).

C, E, and carotenoids are examples of dietary antioxidants (Huang, 2018). Consumption of

reported as free radical quenchers (Renuka, Devi, & Subramanian, 2018).

Natterson‐Horowitz, 2019). Herbal medication is popular because it is more culturally

(Hosseinpour-Jaghdani, Shomali, Gholipour-Shahraki, Rahimi-Madiseh, & Rafieian-Kopaei,

several efficacy studies conducted on herbal plants.

infections, stomach disorders, helminthiasis, lactation disorders, epilepsy, tuberculosis, sterility,

extensively distributed evergreen herb. It is used to treat rheumatism, snakebite, stomach

discomfort, gastrointestinal pain, gynecological issues, malaria, high blood pressure,

compounds for stress-related diseases.

more successful in treating and managing oxidative stress-related illnesses.

Atherosclerosis, diabetes mellitus, hypertension, ischemia complications, malignancies,

In developing and underdeveloped countries, stress-related disorders have become an epidemic.

mutagenic (Jnaid, Yacoub & Al-Biski, 2016).

nations (Nuhu et al., 2021).

understanding how it might be used to treat various stress-related illnesses.

1.3.1 General objective

1.3.2 Specific objective

fraction of Ficus exasperata Vahl leaves.

Ficus exasperata Vahl leaves.

hexane fraction of Ficus exasperata Vahl leaves.

fraction of Ficus exasperata Vahl leaves.

Ficus exasperata Vahl leaves.

hexane fraction of Ficus exasperata Vahl leaves.

1.4 Significance of the study

1.5 Scope of the study

exasperata Vahl leaves.

generator of reactive oxygen species (ROS) (Ifeanyi, 2018).

damage to biomolecules (Phaniendra, Jestadi, & Periyasamy, 2014).

endoplasmic reticulum, phagocytic cells, neutrophils, NADPH/xanthine oxidase, neuroendocrine