NAME:ADEDEJI BOLUWATIFE

MATRIC NO:H/ST/22/2272

TOPIC:HISTOARCHITECTURAL EVALUATION OF
ETHANOLIC AND ETHYL ACETATE EXTRACT OF FICUS
CAPENSIS ON IRON INDUCED OXIDATIVE STRESS IN RAT
BRAIN.

CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of the study

Neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, and stroke,

represent a significant and growing public health concern worldwide. These conditions are

characterized by the progressive degeneration of neurons, leading to cognitive and motor

impairments, and are associated with substantial morbidity and mortality. Oxidative stress,

which arises from an imbalance between the production of reactive oxygen species (ROS)

and the ability of cells to detoxify them, has been implicated as a common pathological

feature in the development and progression of neurodegenerative diseases (Uttara et al.,

2009).

One of the key mechanisms through which oxidative stress exerts its detrimental effects on

neuronal cells is through the process of lipid peroxidation. Lipid peroxidation involves the

oxidative degradation of polyunsaturated fatty acids, resulting in the generation of reactive

aldehydes, such as malondialdehyde (MDA), and other toxic by-products that can damage

1
cellular membranes and disrupt normal cellular function (Halliwell & Chirico, 1993). The

brain is particularly vulnerable to lipid peroxidation due to its high content of polyunsaturated

fatty acids and its high oxygen consumption, which makes it more susceptible to oxidative

damage (Uttara et al., 2009).

Iron (Fe2+), an essential transition metal in the brain, plays a crucial role in various

physiological processes, including neurotransmitter synthesis and myelin production.

However, excessive levels of iron can lead to the generation of ROS through Fenton

chemistry, resulting in oxidative stress and neuronal damage. The accumulation of iron in the

brain has been implicated in the pathogenesis of several neurodegenerative disorders, and

iron-induced oxidative stress has been linked to the progression of these conditions (Zecca et

al., 2004).

Given the central role of oxidative stress and lipid peroxidation in neurodegenerative

diseases, there is growing interest in identifying natural compounds with neuroprotective

properties that can mitigate these processes and potentially serve as therapeutic agents.

Natural products derived from medicinal plants have gained attention as potential sources of

neuroprotective compounds due to their diverse chemical composition and the presence of

bioactive phytochemicals with antioxidant and anti-inflammatory properties (Farooqui et al.,

2012).

Medicinal plants have been used right from ancient times as they serve as very good sources

of drugs due to their high efficacy, ready availability at reduced cost, and adverse effects

(Herve et al., 2008). The use of herbs in the prevention and management of various diseases

is increasingly gaining acceptance not only in rural communities but also in urban cities

(Otitoju et al., 2014). This popularity is not necessarily based on any proven scientific

evidence but often, on the acclaimed medicinal benefits of such herbs (Ach, 2017).

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Nevertheless, many of these herbs contain phytochemical constituents like saponins, phenols,

and flavonoids with useful bioactivities. Such phytochemicals in plants like Euphorbia hirta

and Cassia glauca account for their potential antioxidant and other bioactivities which can be

employed in the management of oxidative stress and chronic human diseases like cancer,

hypertension, and diabetes mellitus that are closely associated with it (Chaitanya et al., 2010).

Among the group of plants endowed with enormous potential for the management of various

diseases is the Ficus species. Ficus religiosa has been employed locally in the treatment of

asthma, haematuria, cough, and diarrhea among other diseases (Damanpreet & Rajesh, 2009).

Research has shown that F. religiosa extract has anticholinesterase activity and methanol

extract of the fruit exhibits anticonvulsant activity (Damanpreet & Rajesh, 2009). F. Capensis

has been used both as an antispasmodic and ant-sickling drug (Sirisha et al., 2010). In

Nigeria, F. Capensis has been used by the Igede people as a treatment for dysentery and in

wound dressing (Igoli et al., 2005). It is also used in circumcision, leprosy, and epilepsy

(Joshua, 2006). The need to affirm that F. Capensis may well be useful in the management

and prevention of diabetes mellitus and other chronic diseases related to oxidative stress is

the major motivating factor for this research. F. Capensis commonly known as the fig tree is a

medicinal plant found in terrestrial zones mostly along rivers (Joshua, 2006). It belongs to the

family Moroaceae. F. Capensis is a spreading deciduous or evergreen tree with a thick bole

and spreading roots (Juurlink, 2001). It produces fruits throughout the year and the leaves are

broad and green. In comparison to other plants, the flowers are relatively inconspicuous. It

produces both male and female flowers in a special hollow, pear-shaped inflorescence called

syconia. Many people refer to this inflorescence as a fig (Adebayo-Tayo & Odeniyi, 2012).

The fig has an ostiole or small opening at its tip, which is the site of entrance by a tiny

stringless wasp called the fig wasp that serves as its sole pollinator (Njoku-oji et al., 2015).

3
Oxidative stress is attributed to the etiology of several diseases such as diabetes, cancer, and

cardiovascular disorders. However, reactive oxygen species may have beneficial functions in

cellular signalling and combating microorganisms in cells. Enhanced production of reactive

oxygen species or depletion of the antioxidant defence system can cause an imbalance

between oxidants and antioxidants (Ilaiyaraja et al., 2011). Overexpression of ROS causes

damage to lipids, proteins, and DNA structure (Bloomer et al., 2013). To prevent oxidative

consequences, cells are surrounded by a defense mechanism that consists of antioxidant

enzymes such as superoxide dismutase, catalase, and glutathione peroxidase (GSH-Px;

Ilaiyaraja et al., 2011).

Reactive oxygen species damage the cell in many ways, e.g., by oxidation of proteins or

nucleic acids (mutagenic effect) or by oxidation of unsaturated fatty acids in cell membranes

(Frijhoff et al., 2015). Reactive oxygen species are detoxified endogenously by various

mechanisms, enzymatically by superoxide dismutase or glutathione peroxidase and by

endogenous low-molecular-weight antioxidants, e.g., vitamins C and E. In healthy cells, there

is a balance between the formation and detoxification of Reactive oxygen species (“redox

homeostasis”). If a state of increased Reactive oxygen species formation and or diminished

Reactive oxygen species detoxification is present, it is called “oxidative stress” (Sies, 2015).

Oxidative stress occurs in inflammation, atherosclerosis, and ischemia-reperfusion injury,

contributes to the process of tumorigenesis, and is held responsible for the aging process

(Forman & Zhang, 2021). In addition, oxidative stress is an important feature of

neurodegenerative and neurological diseases such as Alzheimer’s, Parkinson’s, and epilepsy

(Fabisiak & Patel, 2022).

1.2 Statement of the Problem

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The effects of high-protein diets have been of great interest in the last decade.

Supplementation with high-protein diets is often used to improve physical status causing an

effective reduction in body weight and fat deposition. Some studies have shown the

beneficial effects of lipid diets on rodent brains such as protecting against cerebral ischemia

and reducing apoptosis in the ischemic cortex. Nevertheless, little is known regarding the

effects of a lipid diet on brain oxidative stress markers.

1.3 Justification of the Study

As a neuroprotective agent, an administered substance of interest should be able to reverse

some of the pathological damage or prevent further damage to the neuronal cells. A lot of

studies have found that active compounds present in natural products have high contents of

antioxidant and anti-inflammatory properties. These properties can protect the neurons

against neurodegenerative conditions thereby preserving or enhancing cognitive function

(Essa et al., 2012; Hamid et al. 2018). The findings of this study have the potential to

contribute to the development of novel neuroprotective interventions for neurodegenerative

disorders associated with oxidative stress.

1.4 Aim and Objectives of the Study

1.4.1 Aim of the study

The aim of the study is to evaluate the histoarchitectural evaluation of effects of ethanolic and

ethyl acetate extract of Ficus capensis on iron induced oxidative stress in the rat brain.

1.4.2 Objectives of the study

Objectives of the study are to:

i. Evaluate the phytochemical screening of Ficus capensis.

ii. Carry out characterization of the plant using GC-MS.

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iii. Carry out inflammatory marker test.

iv. Carry out histopathological examination of the brain.

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 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Neuroprotection

Neuroprotection is a term used to refer to strategies and relative mechanisms that shield the

central nervous system from neuronal injuries caused by chronic (e.g., Alzheimer’s and

Parkinson’s diseases) or acute (e.g., stroke) neurodegenerative diseases (Juurlink, 2001).

These acute or chronic diseases result from breakdown and deterioration of neurons of the

central nervous system and often result in the deterioration of the cognitive as well as the

intellectual faculties of the sufferers. The onset of neurodegenerative diseases symptoms is

usually gradual as well as progressive and includes loss of memory, primarily short-term,

difficulty in learning, motor coordination, and many other functional loses (Joshua, 2006).

Aging, defined as a complex physiological process involving both morphological and

biochemical changes that progressively unfold as we get older (Souza et al., 2013), has been

found to be closely associated with neurodegenerative diseases. Aging stands out as a major

risk factor among the other etiological factors of neurodegenerative diseases, including

hypertension, genetic and/or environmental factors, and infections. With increasing age,

aggregation of proteins, inflammation, oxidative stress, and loss of neurotransmitters, which

are common to the pathology of neurodegenerative diseases, also occur more often

(Damanpreet & Rajesh, 2009). Nature remains to be a veritable source of medicine to

mankind. Many important drugs such as vincristine, artemisinin, and gentamicin, which are

still in use today, are obtained from natural sources or are designed on structural fingerprints

of naturally occurring molecules (Otitoju et al., 2014). Furthermore, traditional medicine

remains a vital alternative source of medicine all over the world today with some estimates

7
suggesting to accounts for about 80% of the primary healthcare system in some developing

countries (e.g., Nigeria, Ghana, China, and India (Adebayo-Tayo & Odeniyi, 2012)). The

increasing incidence of resistance (especially to antibiotics and antimalarials), undesirable

side effects, high cost, and lack of efficacy after prolonged usage of the existing drugs in use

has led to a renewed interest in the development of new drug candidates from natural sources

(Everitt et al., 2007. For example, drugs such as amantadine, memantine, donepezil,

selegiline, galantamine, and rivastigmine that are clinically available for the management of

neurodegenerative diseases are only able to provide symptomatic relief and slow the

progression of the diseases (Frijhoff et al., 2015). These drugs, such as the synthetic

donepezil, are also associated with side effects (Everitt et al., 2007). Hence, a great deal of

research focus has been given in recent years to herbs and other natural products used in

ethnomedicine around the world for age-related central nervous system diseases. Numerous

natural products, but primarily plants extracts, have been reported to be used in traditional

medicine for neuroprotective, memory enhancing, and anti-ageing purposes. Examples of

such plants include Ginkgo biloba, Panax ginseng, Curcuma longa, Bacopa monnieri, and

Salvia officinalis (Njoku-oji et al., 2015). These plants have been studied to confirm the

traditional claim with special attention given in understanding the mechanism by which they

elicit the neuroprotective effects. This review is designed to give a brief description of a

number of neurodegenerative diseases, the most important pathological events associated

with the diseases, and an overview of therapeutic options with some popular plant derived

neuroprotective agents.

2.1.1 Neurodegeneration diseases

Neurodegenerative disease is a term applied to a variety of conditions that result from a

chronic breakdown and deterioration of neurons, particularly those of the central nervous

system (CNS). These neurons may accumulate aggregated proteins which cause dysfunction

8
(Houghton and Howes, 2005). Alzheimer’s disease, Parkinson’s disease, multiple sclerosis,

amyotrophic lateral sclerosis and spongiform encephalopathy are some of the common forms

of neurodegenerative diseases (Chiba et al., 2007). These diseases are commonly found in

elderly people and in advanced industrialized societies where life expectancy is long. They

are a major cause of morbidity, mortality and impose severe strains on the social welfare

systems, and as a result are gaining increased recognition by the World Health Organisation

(WHO) (Houghton and Howes, 2005). Neurodegenerative diseases are characterized by a

gradual onset of progressive symptoms including loss of memory and tremor, difficulty in

learning or retaining information, inability to handle complex tasks, impaired spatial

orientation and abilities, language deficits and behavioural changes. These symptoms have

been recognized as a feature of increasing age for a long time and are acknowledged in many

traditional medical systems. However, it is only recently that they have been recognized and

received attention from mainstream medicine as distinctive diseases (Houghton & Howes,

2005).

2.1.1.1 Alzheimer’s Disease

The AD is the most prevalent and devastating disorder of the NDs. It is an incurable disease

of cognition and behavioural impairment that affects social and occupational activities and is

also a leading cause of institutionalization in the elderly (Otitoju et al., 2014). Clinically, AD

is characterized by a progressive and irreversible memory deficits, cognitive deterioration,

and personality changes, with a mean duration of about 8.5 years between onset of clinical

symptoms and death (Ach, 2017). Memory impairments are first to appear in the early stage

of the disease, after which motor and sensory functions are affected as the disease progresses.

The onset of AD is usually above 65 years of age, with risk from this age doubling every 5

years. Hence, it has been suggested that the risk for AD for persons living into their eighties

rises to 20–40% depending on a variety of factors such as population dynamics and

9
geography (Uttara et al., 2009). As the world population continues to age in parallel with

economic development, the number of people with NDs and the associated dementia also

continues to increase (Halliwell & Chirico, 1993). This increase has in turn prompted an

enormous increment in research interest and efforts on the discovery of new therapeutic

agents for primary, auxiliary, or tertiary prevention of these diseases (Zecca et al., 2004). The

pathological hallmark of AD is the accumulation of protein aggregates to form two major

lesions, namely, neurofibrillary tangles (NFTs) and senile plaques. Senile plaques are

composed of fibrillar amyloid 𝛽 (A𝛽) peptides produced by cleavage of the A𝛽 precursor

protein (APP), whereas NFTs consist of hyperphosphorylated microtubule associated tau

protein (Farooqui et al., 2012). The mechanisms by which A𝛽 peptide aggregates act to cause

AD are thought to include induction of oxidative damage as well as inflammation and

neurotoxicity (Damanpreet & Rajesh, 2009). It is now known that the dysfunction of the

central cholinergic system which plays a key role in the retrieval and storage of memory in

the CNS is responsible for the cognitive deficit associated with AD (Herve et al., 2008).

Reports of substantial neocortical deficits in choline acetyltransferase (ChAT), the enzyme

responsible for the synthesis of acetylcholine (ACh), discoveries of reduced choline uptake,

ACh release, and loss of cholinergic perikarya from the nucleus basalis of Meynert, along

with the emerging roles of ACh in learning and memory, led to the “cholinergic hypothesis of

AD” (Igoli et al., 2005).

2.1.1.2. Parkinson’s Disease.

The PD is the second most common ageing-related neurodegenerative diseases that can

greatly impair quality of life with significant consequences in terms of cost of patient care

(Juurlink, 2001). Primarily a movement disorder, as opposed to AD which is mainly a

cognitive disease, PD affects approximately 1% of the human population over the age of 60

(Souza et al., 2013). Its classical signs include resting tremors, bradykinesia, extrapyramidal

10
rigidity, and loss of postural reflexes such as disturbance in walking or equilibrium. The PD

involves loss of dopaminergic neurons of the pars compacta region of the substantia nigra and

their terminals in the corpus striatum (Ilaiyaraja et al., 2011). Since neurodegeneration is not

restricted to the basal ganglia, PD is also linked with nonmotor disorder like dementia. The

association between PD and oxidative damage of neuronal cells has been well established.

For example, the breakdown of dopamine (DA) by autooxidation has been shown to be

linked to semiquinone metabolism and the generation of superoxide anion, hydrogen

peroxide (H2O2), and monoamine oxidase (MAO) expression (Njoku-oji et al., 2015).

2.1.1.3 Other Neurodegenerative Diseases

Amyotrophic lateral sclerosis is thought to be caused by the mutation of the gene coding for

the enzyme superoxide dismutase (SOD) and also by the misfolding of the same enzyme. The

ALS is incurable and has generally a median survival of three years from onset to death. Its

symptoms include tripping or stumbling when running, foot and wrist drop, slurred speech,

and depression (Zecca et al., 2004). Huntington disease (HD) is another incurable ND. It has

an adult onset with autosomal dominant inherited disorder characterized by progressive brain

degeneration, causing rapid deterioration and eventually death. Symptoms of the diseases

include involuntary movement, dementia, and behavioral changes (Halliwell & Chirico,

1993). Prion diseases refer to a group of rare NDs caused by the aggregation of misfolded

prion proteins. Prion proteins are known to be infectious and are presumed to cause some

type of NDs referred to as spongiform encephalopathy: for example, Creutzfeldt-Jacob

disease and kuru in humans, Scrapie in sheep, and bovine spongiform encephalopathy in pigs

collectively referred to as prion diseases. A major feature of these diseases is that they are

transmissible (Chaitanya et al., 2010). Cerebrovascular diseases such as stroke cause acute

degeneration of the CNS unlike the previously discussed chronic NDs. About 85% of stroke

cases are of ischemic origin and have a slightly different etiology from the chronic ND.

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Interruption of blood supplies to the brain leads to a cascade of events that causes irreversible

neuronal damage. Stroke is said to be the second leading cause of death in industrialized

countries (Everitt et al., 2007) and has been reported to lead to dementia in 25% of patients

within three months after a stroke (Adebayo-Tayo & Odeniyi, 2012). Interestingly, the

deposition of both A𝛽 and APP in the cortical and subcortical brain areas of nondementia

patients following stroke has been reported (Ach, 2017).

2.2 Ficus capensis

Ficus Capensis commonly known as bush fig, fig of heaven, is a fast-growing, deciduous, or

evergreen tree (Damanpreet & Rajesh, 2009). It usually grows to about 5- 12 metres (16-39

ft) in height but may attain a height of 35-40 metres (115-131 ft). The large, alternate, and

spirally- -arranged leaves are ovate to elliptic with irregularly serrated margins. Fresh foliage

is conspicuous red colour and papery. The bark of young trees is smooth and pale greyish

white in colour, in contrast to the flaky, yellow back of F. sycomorus; with increasing age, the

bark becomes darker and rough. The figs are carried on short or long drooping spurs (or

fascicles) which may emerge from surface roots, the trunk or especially from lower main

branches. The figs are 2 to 4 cm in diameter and acquire a rosy, speckled exterior when ripe.

The fig seeds are dispersed after passing through the intestinal tracts of birds, bats and

primates. The tree is found from Cape Verde and Senegambia, across tropical West Africa to

Cameroon and the Central Africa Republic, then eastwards to Eritrea, Northern Somalia and

Yemen, and southwards through all tropical eastern and southern African countries. The tree

is of variable height depending on location. In Senegal, the plant can be 4-6 m tall, while in

Nigeria, it can be 6-9 m tall or up to 20 m or more. The trunk is 1.2 m or over in girth, with

spherical crown, often low branching. The tree is of open and wooded Saudano-Guinean

savanna and secondary jungle, by watercourses from Senegal to West Cameroon, in lowlands

to high altitudes, and widespread in tropical Africa (Chaitanya et al., 2010). In Nigeria, the

12
tree is cultivated in all parts of the country but is more abundant in the Middle Belt (North-

Central) of Nigeria. It is commonly called Ogbaikolo among the Igala’s, Opoto in Yoruba

(Joshua, 2006); in Nsukka area of Enugu State, the plant is known as Akokoro, in Hausa as

Uwaryara (Joshua, 2006). It is known as Rimabichehi by the Fulanis and Obada in Edo State

(Joshua, 2006).

2.3 Ethnomedicinal uses of Ficus capensis

Almost all the parts of F. Capensis plant have been found useful. Some parts are used to treat

pregnancy-related ailments most especially cases of threatened abortion (Otitoju et al., 2014).

The bark decoction is used in Senegal in baths for newborns, children with rickets and

feverish children. The bark pulped up with Xylopia fruit is given in enemas for oederna

(Frijhoff et al., 2015). The latex is used for treating wounds, toothache, eye problems, general

body pain, lung and throat problems, gonorrhoea and as an anti-emetic. Root preparations of

Ficus Capensis are used for the treatment of cough, sore throat, diarrhea, stomach pain in

babies, chest pain, infertility, uterine pain, gonorrhoea, oedema, and as an emmenagogue and

emetic (Adebayo-Tayo & Odeniyi, 2012). Bark decoctions or infusions are used against pain,

rheumatism, diarrhoea, stomach problems, oedema in children, infertility and as a

galactagogue; bark macerations are drunk for treatment of fever and cough, and the powdered

bark is applied on skin rashes and mouth sores (Adebayo-Tayo & Odeniyi, 2012). The leaves

are chewed as a remedy for peptic ulcer, leaf maceration is drunk against chest problems, leaf

infusions are drunk to treat tonsillitis and stomach pain. Sap squeezed from leaves is applied

onto wounds, leaf decoctions are used as a disinfectant wash and in the treatment of

ophthalmia, the sap of young shoots is taken against gonorrhoea, preparations are used to

treat infertility, tuberculosis, abscesses and sores, and as a lactogenic, purgative and

aphrodisiac. The plant has been used extensively for the management of leprosy, epilepsy,

rickets, infertility, gonorrhoea, oedema, respiratory disorders and as emollient (Herve et al.,

13
2008). In Nigeria, F. Capensis has been used by the Igede people Benue state as a treatment

for dysentery and in wound dressing (Ilaiyaraja et al., 2011). Gill (1992), reported the use of

the plant leaves in treating dysentery, oedema, epilepsy and rickets in infants among some

tribes in Edo-Delta areas, the Igala people of Kogi State believe F. Capensis to possess

immune-boosting properties, hence, forming part of most of their traditional remedies for

several ailments (Souza et al., 2013).

2.4 Phytochemical Constituent of Ficus capensis

The results of the phytochemical screening by (Uzoekwe & Mohammed, 2015) showed the

presence of tannins, terpenoids, alkaloids, flavonoids, cardiac glycosides and reducing sugars,

with steroids and anthraquinones absent in the water extract of the leaves and bark of Ficus

Capensis. Saponin was present in the bark but absent in the leaves. Terpenoids, flavonoids,

steroids, cardiac glycosides and reducing sugars were present in ethanol extract of the leaves

and bark, while anthraquinones were absent. Alkaloids were present in the ethanol extracts of

the bark but absent in leaves. Adebayo & Adeniyi (2012), reported the presence of tannins in

the bark. Saponins were highest in the leaves, reduced in the stem and least in the bark.

Alkaloids and phenolics were highest in the bark while their quantity was least in the leaf.

Terpenoids and flavonoids were highest in the leaf samples. Owolabi et al. (2009) equally

reported the presence of saponins, cardiac glycosides, tannins and flavonoids with traces of

alkaloids and anthracene derivatives in the stem bark. The qualitative and quantitative

phytochemical analyses of aqueous leaf extract revealed the presence of reducing sugar,

saponins, tannins, flavonoids, soluble carbohydrates, alkaloids, steroids, hydrogen cyanide,

glycosides, terpenoids, fats and oil (Sirisha et al., 2010). The following compounds were

found from n-hexane and ethyl acetate fractions of ethanol extract of F. Capensis leaves: 4, 4,

24-trimelhyl-cholesta-8-en-3-B-ol, mixture of camp sterol, stigmasterol and β-sitosterol,

stigmasterol, 3-B-O’glucoside and 4, 5, 7-trihydroxy flavan-3-ol, xanthotoxin, and β-amyrin

14
(Njoku-oji et al., 2015). Francois et al. (2010) reported the presence of carvacrol (65.78%), α-

caryophyllene (29.81), caryophyllene oxide (25.70 %), linalool (3.97%), 3-tetradecanone

(2.90%), geranyl acetone (1.20%), 3,7,11- trimethyl-3-hydroxy-6,10-dodecadiene-1-ylacetate

(1.53%), hexahydro farnesyl acetone (1.21%), α-caryophyllene (0.81%), 2-methyl-3-hexyne

(0.69%) and scytalone (0.69%).

2.5 Proximate Analysis and Mineral Contents

Ficus Capensis leaves were found to have high quantities of calcium, magnesium and

phosphorus. Iron, zinc, copper and manganese were present but not in very high

concentration. Sodium and potassium were absent. Manganese was absent in F. Capensis

bark while calcium and magnesium were present in the highest concentrations (Uzoekwe &

Mohammed). The result of proximate analysis of the leaves of F. Capensis reported by

(Uzoekwe & Mohammed) was moisture content 25.80%, proteins17.47%, crude fat1.80%,

ash11.00%, crude fibre 41.00% and carbohydrates 2.93% from samples collected in Edo

state, Nigeria. Results obtained by Isah et al. (2013) were moisture content 33.55.60%,

protein-11.83%, crude fat-1.01%, ash-11.40% and crude fibre 15.95%. Achi, (2017) reported

lipid content-1.83%, fiber-4.77%, moisture content-104.53%, carbohydrate-73.77%, ash-

6.65% and protein 6.31% for samples collected in Anambra state, Nigeria. F. Capensis bark

yielded 10.00% - moisture, 3.73%-protein, 2.00%-crude fat, 10.95% ash, 20.50%-crude fibre,

and 52.82%- carbohydrate (Uzoekwe & Mohammed, 2015). Ojokuku & Okunowo, (2010)

reported their findings to be moisture- 9.8%, protein-3.63%, crude fat-1.92%, ash-15.60%,

crude fibre 16.38%, carbohydrate-52.66% for samples collected in Lagos state, Nigeria. Isah,

(2013) reported moisture-80.48%, protein -1.31%, crude fat -0.22, ash- 1.34% and crude fibre

-6.00%. The differences in their proximate content may be due to the different geographical

locations of collection.

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2.6 Fe2+

Iron’s aqueous solution chemistry is based on two oxidation states, Fe 2+ and Fe3+, although

some iron-binding proteins generate high-valent Fe(IV) or Fe(V) intermediates during their

catalytic cycles. Both Fe2+ and Fe3+ forms are thermodynamically stable and kinetically

reactive. Living organisms continuously require interconversion of molecules at specific

moments in reactions where neither inert nor very unstable metallic species would be

adequate substitutes. The interconversion of Fe2+ and Fe3+ species facilitates many electrons

transfer and acid–base reactions necessary in biology. However, organisms would not have

incorporated iron into their biological systems were it not for iron’s abundance. Iron is the

second most abundant metal (after aluminium) and the fourth most abundant element in the

Earth’s crust (Herve et al., 2008). Furthermore, when life first began about 3,500 million

years ago, the Earth had a reductive atmosphere. In the reduced Fe 2+ state, soluble iron

existed at high concentrations in the sea and was thus available to the first living organisms.

After the “Great Oxygen Event” (about 2,500 million of years ago), the atmosphere became

oxidative due to the continuous oxygen emission by photosynthetic organisms (Damanpreet

& Rajesh, 2009). Today, iron in water mainly exists as insoluble Fe 3+. The mass extinction

forced the evolution of organisms that could master a new challenge: Fe3+ insolubility.

Iron’s rich coordination chemistry creates an enormous range in Fe 3+/Fe2+ redox potential that

spans almost across the entire biological range. The standard Fe 3+/Fe2+ redox potential of

10.77 V is a reference with limited biological significance since this value refers to a 1 M

concentration of all species at atmospheric pressure. From a biological perspective, this value

changes depending not only on iron concentration but also on the presence of molecules with

different Fe2+ or Fe2+ affinities. In the presence of biomolecules with very high Fe 3+ affinity,

the thermodynamic tendency of Fe3+ reduction to Fe2+ decreases and therefore, the redox

potential decreases below 0.77 V. In fact, the Fe 3+/Fe2+ redox potential can reach even

16
negative values. For example, the lowest (most negative) Fe 3+/Fe2+ redox potential (20.70 V)

is found in the tris-catecholate siderophore Enterobacter, the biomolecule with the highest

Fe3+- affinity reported thus far (Igoli et al., 2005). In contrast, in the presence of “soft” bases

that stabilize Fe2+ such as N-heterocyclic ligands phenanthroline (phen) or 2,20 -bipiridyl

(bipy), the Fe3+/Fe2+ redox potential values lie at 1 V (Uttara et al., 2009). Based on these

chemical properties, organisms elected iron as the main metal to carry out different and

crucial functions. However, they had to overcome other difficulties generated by iron’s

chemical behavior in the oxidizing atmosphere created after the “oxygen crisis”: Fe 3+

insolubility and the production of Reactive Oxygen Species (ROS).

2.7 High Oxidation States of Iron

High-valent iron commonly denotes compounds and intermediates in which iron is found in a

formal oxidation state >3. High-valent iron–oxo species have been proposed and in some

cases identified. These species play key roles as intermediates in the catalytic oxidation of

organic substrates by heme, nonheme mononuclear iron, and nonheme diiron proteins

(Njoku-oji et al., 2015). In the case of heme-containing proteins, the cytochrome P450 is by

far the most studied enzyme with a high-valent iron-oxo unit as the active intermediate.

Cytochrome P450 belongs to a family of heme-thiolate enzymes that activate O 2 for the

oxidation of nonactivated hydrocarbons (Souza et al., 2013). Their active site is known in

detail from a number of X-ray crystal structures. They have in common an iron–

protoporphyrin IX center coordinated to a cysteine thiolate (Juurlink, 2001). Species Feiv= O

porphyrin have been proposed as active oxidants in a number of oxidation reactions such as

alkane hydroxylation, olefin epoxidation, and alcohol oxidation. High valent iron-oxo units

have also been identified as powerful oxidant intermediates in a variety of important

biochemical transformations related to nonheme proteins. The first characterization of a Fe iv=

O intermediate of a nonheme iron enzyme was provided by studies of several RKG-

17
dependent oxygenases that catalyze either hydroxylation or halogenation of their substrates

(Igoli et al., 2005). In the coordination sphere, iron is coordinated by three protein ligands:

two His and one Asp or Glu. This His2Asp (or Glu) motif is known as the “facial triad”

because they occupy one face of an octahedron, leaving three remaining sites on the opposite

face for substrate coordination. Although the Feiv =O species is the oxidant most commonly

postulated for these enzymes, an Fev= O oxidant has been proposed for the Rieske

dioxygenases that catalyze CIS dihydroxylation of arene double bonds (Halliwell & Chirico,

1993). Feiv=O intermediates have also been proposed to participate in the mechanism of some

nonheme diiron proteins, of them methane monooxygenase (MMO) probably the most

studied. MMO is an enzyme capable of oxidizing the CAH bond in methane and other

alkanes (Zecca et al., 2004). Its active site consists of an asymmetric dinuclear iron center in

which two hexacoordinated Fe2+ are bridged by two carboxylate groups (from Glu and an

acetate anion) and a hydroxyl group (Otitoju et al., 2014). The mechanism proposed for

MMO begins with a two electron reduction of O 2 via the Fe2+-Fe2+ form of the enzyme to

produce a peroxo-Fe3+-Fe3+ state. This peroxo- form is oxidized to a reactive species that has

been characterized as a bis-oxo-Fe(IV) (Chaitanya et al., 2010).

2.8 Antioxidants

There are many types of antioxidants; they can be classified by their mechanism of action.

Preventative antioxidants include peroxide decomposers and metal ion decomposers, while

chain-breaking antioxidants intercept chain-carrying radicals. Many chain-breaking

antioxidants donate a hydrogen atom to the chain-carrying radical thereby stopping the

oxidation process. This results in an antioxidant radical. However, this radical is much less

reactive than the original chain-carrying radical. But even this much more domesticated

radical must be removed. Tocopherol (TOH) is a typical donor antioxidant in this class; it

protects against lipid peroxidation (Adebayo-Tayo & Odeniyi, 2012).

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