CHAPTER ONE

INTRODUCTION

1.1 Background of the Study

Since ancient times, people have taken advantage of the biological effects
of plants as traditional remedies for certain diseases. Approximately 80% of the
world’s population uses medicinal plants as traditional treatment, mainly in
developing countries (Lee et al. 2019). Empirical knowledge in some regions
have identified plants useful for the treatment of certain diseases, but the use of
a given plant as a traditional remedy also depends on popular beliefs in that
region (Adeniyi et al. 2018). The interest of the scientific class in the study of
compounds of plant origin is increasing worldwide, especially in developing
countries where the use of herbal medicines is widely used for their basic health
needs (Yadav, 2018).

The food and pharmaceutical industries have become increasingly

interested recently in analyzing some medicinal plants, mainly their phenolic
profile. Phenolic compounds are secondary metabolites produced in plants,
which protect them against biotic and abiotic stress; some also have beneficial
effects for the organisms that consume them (Li et al. 2018). Their chemical
structure consists of one or more aromatic rings linked to at least one hydroxyl
group, and they are divided according to this structure into phenolic acids,
simple phenols, flavonoids, coumarins, ligans, and tannins. Several of these
compounds have a high antioxidant capacity, believed to assist in the prevention
of certain chronic diseases (Gallegos-Zurita, 2016; Chutipaijit and
Sutjaritvorakul, 2018; Xu et al. 2019; Valduga et al. 2019). A wide range of
publications in the scientific literature have reported on the extraction processes
used to determine the phenolic compounds and antioxidant capacity of such
plants, but comparisons between them are difficult because of differences in
their methods for obtaining of the raw material, their processing, the different
types and mixtures of solvents used, and expression of units of antioxidant
capacity and content of phenolic compounds (Kuri-García et al. 2017).

A free radical is defined as any atom or molecule possessing unpaired

electrons. The reactive oxygen species are oxygen derived free radicals such as
superoxide anion (O2), hydroxyl (OH•), hydroperoxyl (OOH•), peroxyl (ROO•)
and alkoxyl (RO•) radicals and non free radicals such as hydrogen peroxide
(H2O2), hypochorous acid (HOCl), ozone (O3) and singlet oxygen (O2)
(Halliwell and Gutteridge, 1999). It can be formed in living organisms by both
endogenously (respiration, peroxisomes stimulation of polymorphonuclear
leucocytes and macrophages) and exogenously (ionizing radiation, tobacco
smoke, pollutants, pesticides and organic solvents) (Irshad and Chaudhuri,
2002). These free radicals are produced by our body and to stabilize the body's
natural function, but the excess amount could cause the cell and tissue damage
(Sen et al. 2010). It can also cause oxidative damage to proteins, lipids and
DNA and chronic diseases such as cancer, diabetes, aging and other
degenerative diseases in humans (Aiyegoro and Okoh, 2010).

An antioxidant can be broadly defined as any substance that delays or

inhibits oxidative damage to a target molecule (Yamagishi and Matsui, 2011).
The characteristic feature of an antioxidant is ability to scavenge the free
radicals due to their redox hydrogen donators and singlet oxygen quencher (Wu
et al. 2011). The free radicals can be scavenged by the natural (plants) and
synthetic (butylated hydroxyl toluene, butylated hydroxyl anisol and tetra butyl
hydro quinone) antioxidants (Mbaebe et al. 2012). But the usages of these
synthetic antioxidants are now replaced because the natural antioxidants could
be considered as safer without any side effects (Meenaksh et al. 2011). In recent
decades, many researchers are interested in medicinal plants for evaluation of
antioxidant phytochemicals such as phenols, flavonoids and tannins which have
received more attention for their potential role in prevention of human diseases
(Upadhyay et al. 2010).
 Traditional medicine practices in ancient human civilizations worldwide
have demonstrated that plants are one of the most promising sources of effective
medicinal agents. Therefore, scientific studies have been carried out on the
antimicrobial activities of plant extracts against different types of
microorganisms, which have resulted in the development of alternative plant-
based antimicrobial drugs.

Antibacterial is an agent that kills microorganisms or stops their growth

(Merriam, 2019). Antibacterial medicines can be grouped according to the
microorganisms they act primarily against. For example, antibiotics are used
against bacteria. They can also be classified according to their function. Agents
that kill microbes are microbicides, while those that merely inhibit their growth
are called bacteriostatic agents. The use of Antibacterial medicines to treat
infection is known as Antibacterial chemotherapy, while the use of
antimicrobial medicines to prevent infection is known as antimicrobial
prophylaxis (Merriam, 2019).

Diospyros mespiliformis is a tall, evergreen tree 15-50 m high, with

dense, rounded and buttressed stem. Bark grey-black or black, smooth in young
trees rough with small regular scales in older trees, pinkish when slashed.
Young branchlets are green, tomentellous with pinkish white hairs, glabrescent
later. Crown is very branchy with dense foliage. Some common names of
Diospyros mespiliformis are in Yoruba: “Agbalumo” Hausa: “Kanya” and Igbo:
“Udara”, Kilba: “Wachira”. Diospyros mespiliformis, commonly known as
Jackal Berry or African Ebony, has various traditional uses across different
regions. Some of the traditional uses of Diospyros mespiliformis include:
Treatment of fevers, Wound dressings, Antidote for poisonous substances,
Treatment of malaria, syphilis, and leprosy and Antidiabetic properties

Fruits are usually globose, fleshy, greenish and pubescent when young,
yellowish to orange yellow and glabrous when ripe, bell shaped, with persistent
style and enlarged calyx and contain 4-6 seeds. Seeds are dark brown, bean-
shaped shiny and glabrous. Flowers are pentamerous, white and fragment. Male
flowers are sessile hairy and clustered on axillary peduncles. Female flowers are
solitary, shortly pediculate and axillary with 5-lobed calyx. The leaves are
simple, alternate, leathery and dark green with small hairs on the underside of
old leaves . The fruit is a fleshy berry, with an enlarged calyx yellow to orange
when ripe (Thompson, 2007).

In spite of the wide spread ethno-botanical studies of plants, this work is

to determine in-vitro the antioxidant and antibacterial activities of Diospyros
mespiliformis.

1.2 Statement of the Problem

Free radicals activities and the problem of antibiotics resistance in

biological systems has been a major challenge especially in underdeveloped and
developing countries of the world. A new effective and eco-friendly substance
which can inhibit oxidation and microbial activities with little or no effect to the
hosts is needed, hence the project work.

1.3 Aim and Objectives

The aim of the Study is to determine the antioxidant and the antibacterial
activities of Diospryros mespliliformis ethanol leaves extract.

Specific objectives of the study are to:

i. Determine the phytochemical constituents of Diospryros

mespliliformis ethanol leaves extract.
ii. Determine the in-vitro antioxidant activities of Diospryros
mespliliformis ethanol leaves extract
iii. Determine the in-vitro antibacterial activities of Diospryros
mespliliformis ethanol leaves extract
1.4 Significance of the Study

The study will provide basis for the identification and recognition of
plants abandoned due to lack of knowledge of their biological and medicinal
importance, such as the case of Diospryros mespliliformis, in underdeveloped
and developing countries especially Nigeria, and also a means of finding
alternatives that are effective and eco-friendly.

1.5 Scope of the Study

This Study is limited to the determination of antioxidant and antibacterial

activities of Diospryros mespliliformis ethanol leaves extract in-vitro, to obtain
a new effective and eco-friendly substance to combat the challenges faced by
conventional modern-day medicine.
 CHAPTER TWO

LITERATURE REVIEW

2.1 Medicinal Plants

A medicinal plant is a plant that is used with the intention of maintaining

health, to be administered for a specific condition, or both, whether in modern
medicine or in traditional medicine (Smith-Hall et al., 2012; Ahn, 2017). The
Food and Agriculture Organization estimated in 2002 that over 50,000
medicinal plants are used across the world (Schippmann et al., 2002).

2.1.1 Future of Medicinal Plants

The future of medicinal plants is promising, considering the vast number

of medicinal plants that are yet to be selected and investigated for their
phytochemical compositions. Medicinal plants have provided an avenue for
understanding the scaffold for synthetic drug design and development. In
addition, the future of medicinal plants will influence the medical practice,
considering the occurrence of new pathogens and diseases, which calls for
alternative or complementary medicine (El-Ghani, 2016). The rising tide in
antibiotic resistance by microorganism has raised significant concern in the
medical field and, thus, an urgent demand for the discovery of safe, natural
compounds in this postgenomic era. The use of medicinal plants as
nutraceuticals and functional foods is on the increase, as means of ensuring
preventive medicine and finding a solution to this global concern of evolution of
drug-resistant microorganisms (Dhama et al. 2014).

2.1.2 Phytochemical Constituents of Medicinal Plants

Medicinal plants contain bioactive organic chemical compounds often

referred to as phytochemicals, which play a defensive role against major chronic
diseases in both host-metabolic or genetic dysfunctional disease and infectious
disease, and found in grains, vegetables, fruits, and other plant products (Nayak
et al. 2015; Esposito et al. 2016). Phytochemicals perform intermediary
metabolic activities, and they function as primary metabolites such as fats and
sugars found in all plants, while secondary metabolites are found in a smaller
range of plants and provide specialized functions. Secondary metabolites are
biomolecules produced for sustainability or adaptation of plants in the
environment as a result of many factors, such as protection from drought,
pollination, and predation, but are not required for immediate survival of the
plants (Kennedy and Wightman, 2011).

Screening of chemical composition of medicinal plants revealed that they

contain different bioactive compounds which include saponins, tannins, and
alkaloids (Ezike et al. 2016). The main functional classes of phytochemicals
with therapeutic potential include antioxidants, anticancer agents, immunity-
potentiating agents, detoxifying agents, and neuropharmacological agents
(Kennedy and Wightman, 2011). Each of the functional classes of the
phytochemicals consists of a wide range of compounds at varying potency and
sometimes with multiple functions (Esposito et al. 2016). Plants produce unique
arrays of phytochemicals that frequently belong to existing biochemical motifs
(Singh et al. 2015). Among the bioactive compounds, triterpenoids provide anti-
inflammatory activity and tannins possess astringent, anti-inflammatory, and
antimicrobial activity properties (Akinpelu et al. 2015). Saponins often have
medicinal features as blood cleansers, expectorants, and antibiotics.

2.1.3 Preparation of Medicinal Plants

Medicinal plants are often tough and fibrous, requiring some form of
preparation to make them convenient to administer. According to the Institute
for Traditional Medicine, common methods for the preparation of herbal
medicines include decoction, powdering, and extraction with alcohol, in each
case yielding a mixture of substances. Decoction involves crushing and then
boiling the plant material in water to produce a liquid extract. Powdering
involves drying the plant material and then crushing it to yield a powder that
can be compressed into tablets. Alcohol extraction involves soaking the plant
material in cold wine or distilled spirit to form a tincture (Dharmananda, 1997).

Traditional poultices were made by boiling medicinal plants, wrapping

them in a cloth, and applying the resulting parcel externally to the affected part
of the body (Mount, 2015). When modern medicine has identified a drug in a
medicinal plant, commercial quantities of the drug may either be synthesized or
extracted from plant material, yielding a pure chemical (Atanasov et al., 2015).
Extraction can be practical when the compound in question is complex
(Pezzuto, 1997).

2.2 Phytochemicals

Chemical compounds produced as a result of metabolic reaction during

plant growth are known as phytochemicals. Harborne (1973) and Okwu (2004),
refer to such metabolic chemicals as “secondary metabolites” which include
alkaloids, flavonoids, coumarins, tannins, terpenes, terpenoids, phenols, gums,
polysaccharides, and glycosides. According to (Sodipo et al., 2000), most
phytochemicals serve as natural antibiotics, which assist the body in fighting
microbial invasion and infections.

2.2.1 Alkaloids

Originally, the name alkaloids (which means alkali-like) was given to all
organic bases isolated from plants. But today, alkaloids are defined as naturally
organic bases which contain a pyridine and it can be further defined as natural
plant compound having a basic character and containing at least one Nitrogen
atom in heterocyclic ring. Alkaloids are poisonous, but are used medically in a
very small quantity, they are usually colorless, crystalline and nonvolatile solid
which are insoluble in water, but are soluble in ethanol, ether, chloroform etc.
Some alkaloids are liquid which are soluble in water e.g. coniine and nicotine
and few are colored e.g. berberine is yellow. Most alkaloids have a bitter taste
and are optically active (laevonotarory) are generally tertiary nitrogen
compound and contain one or two nitrogen atom usually in the tertiary state in a
ring system and most of them contains oxygen (Okwu, 2001).

Alkaloids are usually found in in the seeds, roots, leaves, tender pods
bark of various plants and generally occur as salts of various plant acids e.g.
acetic, oxalate, citric, malic, tartaric acid, etc.

2.2.2 Flavonoids

Flavonoids are an important class of natural products; particularly, they

belong to a class of plant secondary metabolites having a polyphenolic
structure, widely found in fruits, vegetables and certain beverages. They have
miscellaneous favorable biochemical and antioxidant effects associated with
various diseases such as cancer, Alzheimer's disease (AD), atherosclerosis, etc.
(Burak and Imen, 1999; Ovando et al., 2009; Lee et al., 2009). Flavonoids are
associated with a broad spectrum of health-promoting effects and are an
indispensable component in a variety of nutraceutical, pharmaceutical,
medicinal and cosmetic applications. This is because of their antioxidative, anti-
inflammatory, anti-mutagenic and anti-carcinogenic properties coupled with
their capacity to modulate key cellular enzyme functions. They are also known
to be potent inhibitors for several enzymes, such as xanthine oxidase (XO),
cyclo-oxygenase (COX), lipoxygenase and phosphoinositide 3-kinase
(Metodiewa et al., 1997; Hayashi et al., 1988; Walker et al., 2000). In nature,
flavonoid compounds are products extracted from plants and they are found in
several parts of the plant. Flavonoids are used by vegetables for their growth
and defence against plaques (Havsteen, 2002).

Flavonoids play a variety of biological activities in plants, animals and

bacteria. In plants, flavonoids have long been known to be synthesized in
particular sites and are responsible for the colour and aroma of flowers, and in
fruits to attract pollinators and consequently fruit dispersion to help in seed and
spore germination, and the growth and development of seedlings (Griesbach,
2005). Flavonoids protect plants from different biotic and abiotic stresses and
act as unique UV filters (Takahashi and Ohnishi, 2004), function as signal
molecules, allopathic compounds, phytoalexins, detoxifying agents and
antimicrobial defensive compounds.

2.2.3 Tannins

Tannins are very widely distributed in the plant kingdom. There are two
types of these bioactive compounds known, such as condensed and
hydrolyzable tannins. The first group consists of large flavonoid polymers
which are characterized by the binding properties to microbial proteins. This
property is responsible for the antibacterial activity of tannins (Bernhoft 2010;
Nagesh et al., 2012), which protect the plant against fungi and insects. These
compounds are detected in all parts of plants, but the most are found in wood,
bark, fruits and galls (Altemimi et al., 2017; Shrestha et al., 2014; Zhang et al.,
2015). Like the other phenolic compounds, tannins have antioxidant effects but
they also show antimicrobial, antiprotozoal, anti-inflammatory, antidiabetic,
anticarcinogenic, hepatoprotective, cardioprotective and antiallergic properties
(Macáková et al., 2014). Condensed tannins were detected in poplar leaves (Li
et al., 2011; Rubert-Nason et al., 2013), black locust wood (Fan et al., 2010 ),
willow wood (Brereton et al., 2017), willow cortex (Heiska et al., 2007;
Juntheikki and Julkunen-Tiitto 2000) and in poplar bark (Li et al., 2011).

2.2.4 Glycosides

Anthraquinone glycosides are found in medicinal plants such as rhubarb,

cascara, and Alexandrian senna (Wang et al., 2013; Chan and Lin, 2009). Plant-
based laxatives made from such plants include senna (Hietala et al., 1987),
rhubarb (Akolkar and Praful, 2012) and Aloe (Elumalai et al., 2012). The
cardiac glycosides are powerful drugs from medicinal plants including foxglove
and lily of the valley. They include digoxin and digitoxin which support the
beating of the heart, and act as diuretics (USDA, 2017). Senna alexandrina,
containing anthraquinone glycosides, has been used as a laxative for millennia
(Hietala et al., 1987).
 The foxglove, Digitalis purpurea, contains digoxin, a cardiac glycoside.
The plant was used on heart conditions long before the glycoside was identified
(USDA, 2017; Texas and University, 2017).

2.2.5 Phenolic compounds

Phenolic compounds are a main class of secondary metabolites in plants

and are divided into phenolic acids and polyphenols. These compounds are
found combined with mono‐ and polysaccharides, linked to one or more
phenolic group, or can occur as derivatives, such as ester or methyl esters
(Harborne et al. 1999). Among the several classes of phenolic compounds, the
phenolic acids, flavonoids, and tannins are regarded as the main dietary
phenolic compounds (King and Young, 1999). Many studies have shown a
strong and positive correlation between the phenolic compound contents and the
antioxidant potential of fruits and vegetables (Reddy et al. 2010; Someya et al.
2002; Sarawong et al. 2014). This antioxidant mechanism, present in the plants,
has an important role in the reduction of lipid oxidation in (plant and animal)
tissues, because when incorporated in the human diet, not only it conserves the
quality of the food, but it also reduces the risk of developing some diseases.
Studies have shown that a diet rich in fruits and vegetables contributes to the
delay of the aging process and to the decrease of the inflammation and oxidative
stress risk, related with chronic diseases (e.g., cardiovascular diseases,
arteriosclerosis, cancer, diabetes, cataract, disorders of the cognitive function,
and neurological diseases) (Eliassen et al. 2012; Projer et al. 2013; Tanaka et al.
2012).

2.3 Phytochemical Screening

The active ingredients of medicinal and aromatic plants extracts are in the
roots, stems, bark, leaves, or flowers (Kennedy and Wightman, 2011). Most
times, the actions of these active ingredients on the human CNS could be
associated with ecological roles or biochemical identities in other plants and
higher animals (Ukamaka et al. 2015). The significant steps to utilize these
biologically active compounds from plant resources involve the extraction,
primary screening, isolation, purification, and characterization as well.

Pharmaceutically, the extraction involves the separation of constituents of

plant and microorganism tissues using selective buffer solution or solvents
according to standard protocols (Udochukwu et al. 2015). The crucial
parameters that can influence the quality of an extract from the plant include
parts used as a sample (leaf, bark, or root), solvent and its concentration used
for extraction, and extraction method, while the effect of chemical constituents
obtained from the extract depends on the nature of the plant material, its source,
the extent of processing, and level of moisture, as well as particle size
(Oramadike and Ogunbanwo, 2017). Factors that can influence the secondary
metabolite composition and quantity in an extract include type and time of
extraction, nature and concentration of the solvent, processing temperature, and
polarity of analytes (Oramadike and Ogunbanwo, 2017).

2.3.1 Antioxidants

The chemical compounds which can delay the start or slow the rate of
lipid oxidation reaction in food systems are called Antioxidants. Biological
antioxidants include enzymatic antioxidants (e.g., Superoxide dismutase,
catalase and glutathione peroxidase) and nonenzymatic antioxidants such as
oxidative enzyme (e.g., cycloxygenase). The leaves, seeds, fruits, and wood
barks extract of P. dulce have potential activity against free radicals has proved.
The whole plant has active free radical scavenging potential against synthetic
radicals of DPPH, NO, superoxide, and hydroxyl ions (Katekhaye and Kale,
2012; Nagmoti et al., 2012; Sukantha et al., 2011). The leaf extracts of P. dulce
exhibited antioxidant and antifungal activity against human pathogenic bacteria
(Suman. 2017).Detailed investigations into the active components responsible
for the observed antimicrobial activity may open new avenues for drug
development and control of antibiotic-resistant pathogenesis. Thus, earlier
studies suggest that P. dulce possess antioxidant, antibacterial, as well as
antifungal activities (Sukantha et al., 2011)

2.3.2 Antimicrobials

An antimicrobial is an agent that kills microorganisms or stops their

growth. Antimicrobial medicines can be grouped according to the
microorganisms they act primarily against. For example, antibiotics are used
against bacteria, and antifungals are used against fungi. They can also be
classified according to their function. Agents that kill microbes
are microbicides, while those that merely inhibit their growth are
called bacteriostatic agents. The use of antimicrobial medicines to treat
infection is known as antimicrobial chemotherapy, while the use of
antimicrobial medicines to prevent infection is known as antimicrobial
prophylaxis

2.4 Plant Description

Diospyros mespiliformis is a tall, evergreen tree 15-50 m high, with dense,

rounded and buttressed stem. Bark grey-black or black, smooth in young trees
rough with small regular scales in older trees, pinkish when slashed. Young
branchlets are green, tomentellous with pinkish white hairs, glabrescent later.
Crown is very branchy with dense foliage. Fruits are usually globose, fleshy,
greenish and pubescent when young, yellowish to orange yellow and glabrous
when ripe, bell shaped, with persistent style and enlarged calyx and contain 4-6
seeds. Seeds are dark brown, bean-shaped shiny and glabrous. Flowers are
pentamerous, white and fragment. Male flowers are sessile hairy and clustered
on axillary peduncles. Female flowers are solitary, shortly pediculate and
axillary with 5-lobed calyx. The leaves are simple, alternate, leathery and dark
green with small hairs on the underside of old leaves. The fruit is a fleshy
berry, with an enlarged calyx yellow to orange when ripe (Thompson, 2007).

2.4.1 Taxonomical Classification

Kingdom: Plantae

Subkingdom: Tracheobionta

Superdivision: Spermatophyta

Division: Magnoliophyta

Class: Magnoliopsida

Subclass: Dilleniidae

Order: Ebenales

Family: Ebanaceae

Genus: Diospyros. L

Specie: mespiliformis Hochst. ex A. DC. (USDA, NRCS, 2017).

2.4.2 Ethnomedicinal Uses Leaves:

Used as astringent, febrifuge, hemostatic, mildly laxative, stimulant .and

vermifuge. Infusion of the leaves is used in treatment of fevers, pneumonia,
syphilis, leprosy and yaws (Chivandi and Erlwanger, 2011). The leaves are also
used for treatment of headache, arthritis and skin infections. The leaves and
fruits are chewed or applied as infusion for treating gingivitis, toothache, and
for wound dressing to prevent infection Root and Bark: an infusion of the bark
is used to treat stomach ache (El-Kamali, 2011). Bark and roots for infections
such as malaria, pneumonia, syphilis, leprosy, dermatomycoses, as an
anthelmintic and to facilitate child birth. Barks and roots are used as psycho-
pharmacological drug and to treat tumor. Roasted and pulverized roots are taken
to treat jaundice; Bark preparations are administered to treat cough, bronchial
diseases, tuberculosis, syphilis and leprosy, and applied externally to wounds,
ulcers, bruises and furuncles. The bark is also used in veterinary medicine as
vermifuge (Abba et al., 2016). Fruits and Seeds: Fruits decoction or infusions
are taken to treat dysentery, diarrhea, and menorrhagia. Fruit ash is applied to
fungal skin infections and fruit powder to ulcers, whereas seed decoctions are
administered against headache. Twigs are chewed to clean teeth. Its seeds are
also known to have nutraceutical value in managing high cholesterol, reducing
risk of type-2 diabetes, and for weight control (Mohammed et al., 2016).

2.5.3 Phytochemical Constituents of Diospyros mespiliformis

Phytochemical plumbagin is isolated from rook-bark. Tannins, saponins and

substances similar to scopolamine are present. It also has high fluoride content.
Studies on Diospyros mespiliformis has revealed the presence of triterpenes, α-
amyrinbaurenol, trihydroxy-triterpenoid acid, α-amyrin, βsitosterol, lupeol,
betulin, behenic acid, and naphtoquinones like; Diospyrin, Isodiospyrin,
Diosquinone, plumbagin (Abba et al., 2016). Preliminary phytochemical
screening also revealed the presence of several secondary metabolites namely;
anthraquinones, tannins, triterpenes, saponins, steroids (Shagal et al., 2011)

2.6. Pharmacological Uses

2.6.1 Antioxidant activity

Mohammed et al., (2016) investigated the in-vitro antioxidant using DPPH

assay, total phenolic, and total flavonoid content of ethanol, ethanol and
petroleum ether leaf extracts of Diospyros mespiliformis. The ethanol extract of
D. mespiliformis were found to contain higher amount of phenolic, and
flavonoid compounds.

2.6.2 Anti-proliferative effect

Abba et al., (2016) reported anti-proliferative property of Diospyros

mespiliformis stem bark extracts against radicles of a Guinea corn (Sorghum
bicolour) which relate to its use as anticancer agent.

2.6.3 Antimicrobial activity

Shagal et al., (2011) investigated the antimicrobial activity of aqueous and
ethanol extracts of leaves, stembark and root of Diospyros mespiliformis.
Activity of the extracts was tested against clinical isolates of Salmonella typhi,
Escherichia coli, Staphylococcus aureus, Streptococcus spp, Shigella sp and
Klebsiella pneumonia. Staphylococcus aureus was sensitive to ethanol extract of
the leaves. The ethanolic and water extract of Diospyros mespiliformis showed
significant antimicrobial activity against the tested microorganisms.

Antibacterial activity of ethanol and water extracts of Diospyros mespiliformis

was carried out by Dangoggo et al., (2016). The test organisms included clinical
isolates of Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus
pyogenes, Salmonella typhi, Pseudomonas aeruginosa, Escherichia coli and
Shigella spp. Antibacterial activity of crude extract of the leave of ethanol of
Diospyros mespiliformis produced zone of inhibition of 12mm, 14 mm on E.
coli at 90 mg/ml and 120 mg/ml concentration respectively and 12mm and 13
mm on P. aeruginosa at 90 and120 mg/ml respectively. There was no zone of
inhibition on Salmonella typhi. For the water extract, it produced zone of
inhibition of 10mm,11mm,12mm,13mm on S. aureus at
30mg/ml,60mg/ml,90mg/ml and 120mg/ml concentrations respectively and
11mm,13 mm on P. aeruginosa and also 11mm,14 mm on E. coli at
90mg/ml,120mg/ml, on Shigella spp 10mm,11mm at 90mg/ml and120mg/ml.
The ethanol extract of Diospyros mespiliformis leaf showed significant activity
on E. coli, S. aureus, Shigella spp and P. aeruginosa. The leaf and bark extract
of Diospyros mespiliformis showed significant antifungal activity against
Aspergillus niger, Aspergillus flavus and Microsporum gypseum at various
concentration.

2.6.4 Antiplasmodial activity

In-vivo Antiplasmodial Activity of Aqueous, N-Butanol and Ethylacetate

Fractions of Leaf and Stem Bark Ethanol Extracts of Diospyros mespiliformis
was tested on Plasmodium berghei Infected Mice by Oguche, (2012). The
results indicated that the leaf and stem bark ethanol extracts of Diospyros
mespiliformis are significantly effective against Plasmodium berghei could be
used in the management of malaria.

2.6.5 Analgesic activity

The ethanol extract of Diospyros mespiliformis was evaluated for its folkloric
usage in the relief of pain and fever. Antipyretic, analgesic and anti-
inflammatory effect of the extracts were evaluated in rats and mice. Studies
were carried out on yeast induced pyrexia in rats; acetic acid induced writhing
in mice, formalin test and egg albumin-induced anti-inflammatory activity inn
rats. The extract (50 and 100mg/kg i.p) gave a potent antipyretic effect for
100mg/kg and significant activity (p<0.05) against all the analgesic and anti-
inflammatory models used Adzu et al., (2015) extracted and carried out a
bioassay-guided fractionation of the stem bark of Diospyros mespiliformis with
solvents of varying polarity. Lupeol was isolated from the stem bark. This
compound alone or synergistically might be responsible for the beneficial effect
of the plant in treatment of pain related ailments.

2.6.6 Hypoglycemic activity

Mohammed et al., (2009) carried out α-glucosidase enzyme inhibition activity

of isolated bioactive compounds from Diospyros mespiliformis. The compounds
were identified as lupane type triterpenes; lupeol, betulinic acid, betulin and
lupenone. Lupeol, botulin and lupenone showed potent α-glucosidase inhibitory
activity.

2.6.7 Neuropharmacological activity

The aqueous extract of Diospyros mespiliformis stem bark in mice (100 and 200
mg/kg p.p.) produced a significant prolongation of pentobarbital-induced
sleeping time and reduced the spontaneous motor activity and exploratory
behavior. The extract prolonged onset of the phases of seizure activity but did
not protect mice against lethality induced by pentylenetetrazole (Dangoggo et
al., 2016)
 CHAPTER THREE

MATERIALS AND METHODS

3.1 Materials

Round bottom flask, Pencil, Test tube and test tube rack, Ruler, Conical flask,
Foil paper, Hand gloves, Spatula, Graduated cylinder, Cotton wool, Beakers,
Petri dishes, Cork borer, Masking tape etc.

3.1.1 Equipment

Centrifuge, Uv-vis spectrophotometer (Jenway,7351), wooden mortar and

pestle, sieve, Incubator, Autoclave machine, Oven, Weighing balance etc.

3.1.2 Chemicals/Reagents

Ethanol, Mayer's reagent, Conc. H 2SO4, Ferric chloride solution, 1,1-Diphenyl-

2-picrylhydrazyl (DPPH), Sodium hydroxide, Acetic anhydride, Molisch’s
reagent, Hydrochloric acid, ABTS, Distilled water etc.

3.2 METHODS

3.2.1 Sample Collection and Identification

Plant leaves will be collected from Wuro-patuji in Mubi South Local

Government Area, Adamawa State. It will be identified and authenticated by a
Botanist in the Department of Biological Sciences, Adamawa State University,
Mubi.

3.2.2 Sample Preparation

The leaves will be washed and rinsed with water and dried in the shade for a
week. The dried samples will be grinded with a wooden motar and pestle and
sieved using a clean kitchen sieve to obtain a fine powder and will be stored in a
tight container until required for use.
3.2.3 Sample Extraction

Two hundred grams of dried powder of Diospyros mespiliformis leaves will be

weighed and soaked in 1000ml of 70% ethanol and left for 72 hours. Thereafter
it will be decanted. The extract will be filtered using Whatman No. 1 filter paper
and then pass through a sterilized filter paper to avoid any contamination and
evaporated to dryness with a water bath at temperature of 50°F.The dried
extract will be then weighed and stored in a sterilized screw capped bottle in a
refrigerator until prior to the analysis.

3.2.4 Phytochemical Analysis

Phytochemical analysis will be carried out qualitatively to determine: Tannins,

Quinones, Alkaloids, Flavonoids, Phenolic compound, Carbohydrates,
Glycosides and, Amino acids and Proteins using typical standard methods with
slight modifications.

3.2.4.1 Test of Tannins (Ferric Chloride Test)

Plant extract (1 mL) will be treated with ferric chloride (FeCl3). Formation of
blue green color indicates the presence of tannins (Ugochukwu et al., 2013).

3.2.4.2 Test of Quinones

Plant extract (1 mL) will be taken and 5 mL distilled water will be added, and
then turbidity will be observe.

3.2.4.3 Test for Alkaloids (Mayer's Test)

Plant extract (1 mL) will be taken and few drops of Mayer’s reagent will be
added.A creamy white precipate indicates the presence of alkaloids (Trease and
Evans, 2002).

3.2.4.4 Test for Flavonoids (Alkaline reagent Test)

Plant extract (1 mL) will be taken and treated with 3-5 drops of 20% NaOH
solution. The formation of intense yellow color, which becomes colorless on
addition of 0.5 mL dilute HCl indicates the presence of flavonoids (Saxena et
al., 2013).

3.2.4.5 Test for Phenols (Ferric Chloride Test)

Plant extract (1 mL) will be taken and 5-6 drops of aqueous ferric chloride
solution will be added and the formation of deep blue or black color will
observe (Tiwari et al., 2011).

3.2.5 Determination of Antioxidant Activity

The antioxidant activities of plant extract will be assessed using four common
assays, namely: 1,1- diphenyl-2-picrylhydrazyl (DPPH) radical scavenging
method, ABTs radical scavenging activity.

3.2.5.1 DPPH (1,1-Diphenyl-2-picrylhydrazyl) Scavenging Activity

The DPPH Scavenging activity of extracts will be determined

spectrophotometrically according to Clarke et al. (2013), with some slight
modification (Abdulkadir et al., 2015). Briefly, a 160 μl of DPPH solution (0.6
mg/100 ml ethanol) will be mixed with 40 μl of the extract prepared in several
concentrations. The mixture will be kept in dark at room temperature. After 30
min of incubation, the absorbance will be measured at 517 nm. The antiradical
activity will be expressed as IC50 the values, i.e., concentration of the studied
extracts, which causes a 50% decrease in the absorbance at 517 nm as compared
to the control. The percent inhibition will be calculated according to the
following equation:

DPPH scavenging (%) = (A of control – A of sample)/ A of control) × 100

3.2.5.2 ABTS radical scavenging activity

ABTS scavenging capacity will be determined according to the method

described by (Benslama et al. 2019). The ABTS+ radical will be generated by
the mixture of ABTS solution (7 mM) with potassium persulfate (K2S2O8)
solution (2.45 mM). The mixture will be incubated in the dark at room
temperature for 16-24 h. The standard solution of ABTS will be diluted by the
addition of ethanol to have an absorbance of 0.700 (±0.02) at 734 nm. An
aliquot of 40 µL of extract will be mixed with 160 µL of ABTS and absorbance
will be recorded after 30 minutes. The antiradical activity will be expressed as
IC50 the values, i.e., concentration of the studied extracts, which causes a 50%
decrease in the absorbance at 432 nm as compared to the control. The percent
inhibition will be calculated according to the following equation:

ABTs radical-scavenging (%) = (A of control – A of sample)/ A of control) ×

100.

3.2.5.3 Ferric-Reducing Antioxidant Power Assay (FRAP)

The FRAP assay will be carried out according to Benzie and Strain (1996) with
slight modification (Abdulkadir et al., 2016). The FRAP reagents will be
prepared using 10 mmol TPTZ (2, 4, 6-tripyridyl-s-triazine) solution in 40
mmol HCl, 20 mmol iron (III) chloride aqueous solution and acetate buffer (pH
3.6) with the ratio 1:1:10 (v/v), respectively. The FRAP reagents will be freshly
prepared before starting the experiment and warmed at 37°C in a water bath for
30 minutes before use. Fifty microliters of sample will be added into 1.5 mL of
the FRAP reagent. The absorbance of the reaction mixture will be measured
using spectrophotometer at 593 nm after 30 minutes of incubation. A volume of
2000 μM of iron (II) sulfate solution will be used as a standard and further
diluted to 1000, 500, 250, 125, 62.5 and 31.25 μM. The results will be
expressed as μmol Fe(II)/g dry weight of the plant material. Three replications
will be maintained and the mean values will be calculated.
3.2.6 Determination of Antibacterial Activity

Standardized method for disc diffusion Microorganisms to be used in this study

include Salmonella typhi, Escherichia coli, Staphylococcus aureus,
pseudomonas aeruginosa, shigella represent pathogenic species commonly
associated obtained from the Microbiology Laboratory of Adamawa State
University, Mubi, Nigeria. All the bacterial strains will be sub-cultured from the
original culture, store at 70°C and maintain on Muller-Hinton (MH) agar plates
at 4°C, and grown at 37°C when required.

3.2.6.1 Sterilization Procedure

In other to avoid any type of contamination and mass contamination by the test
organisms, the antibacterial screening will be performed in a way that all types
of precautions will be highly maintained. Petri dishes and all other glass wares
were sterilized by autoclaving in the oven at a certain temperature for 30
minutes.

3.2.6.2 Antibacterial activity Assay

Four concentrations of the extract (0.05 mg/mL, 0.10 mg/mL, 0.20 mg/mL, and
0.40 mg/mL) will be prepared and assessed using disc diffusion method by
(Prasannabalaji et al. 2012). Stock culture of test bacteria will be grown in
nutrient broth medium at 37°C for 22 h. The culture suspensions will be
prepared and adjusted to approximately 1.5 x 108 cfu of bacteria/mL (0.5
McFarland turbidimetry). 100 mL of the inoculum will be spread over plates
containing sterile nutrient agar. Circular discs of 6 mm diameter impregnated
with 50 mL of each concentrations of extract will be placed on the surface of
the media. The plates will be incubated at 37°C for 24 h. Each concentration of
extracts will be carried out in triplicate. After incubation, the diameter of the
zone of bacterial growth inhibition around each disc will be measured and
recorded in millimeter. Control antibiotic (tetracycline 100µg/mL) will be used
as a positive control and distilled water will be used as a blank.
3.2.6.3 Minimum Inhibitory Concentration (MIC)

0.85% normal saline will be added in sterile plate. A stock concentration of 100
mg/mL for crude extracts in ethanol will be pipetted into the first row of the
plate and a serial dilution will be prepared. To each well 30 µL of 3.3x strength
isosensitised broth and 10µL 5x105 cfu/mL of bacteria will be added.
Tetracycline (100g/mL) will be used as positive control and ethanol will be used
as solvent control. The plates will be incubated at 37°C for 24 h. After that, 10
µL of resazurin indicator will be added in each well. The plates will be
incubated at 37°C for 3 h and recorded for the result. From result, blue color
will be recorded as positive. The lowest concentration at which color did not
change from blue to pink will be recorded as the MIC value. The average of
three from four values will be calculated to MIC value (Sarker et al. 2007).

3.3 Statistical Analysis

Studies will be performed in triplicates and data will be expressed as mean ±

SEM, analyzed via one-way ANOVA. Analystat version 1.6.50 will be used for
the analysis and statistical significance will be set at P < 0.05.