CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of the Study

Traditional Atrican medicine is a system of healing practices including herbalism,

spiritualism and 103 divination, where people depend mostly on the indigenous flora for
curing discases (Mokgobi, 2014; Truter, 2007). Africa is endowed with enormous
biodiversity and natural resources and it is estimated to constitute around forty thousand
species of plants, among them 5,000 species are used medicinally (Mahomoodally, 2013).
Recently, the uses of medicinal plants continue to thrive in Africa; where it was reported that
75- 80% of the population relies entirely on traditional medicine for curing diseases and to
maintain a good health (Khalid et al., 2012; Mahomoodally, 2013). The continuum usage of
medicinal plants has paved the way for researchers to explore their curative properties and to
discover bioactive compounds. Herein, the traditional knowledge had represented an essential
starting point for the ethnopharmacological approach in drug discovery and development
(Heinrich, 2010). Many medicinal plants in Africa that are widely used in traditional
medicines have not been explored in detail for their chemical constituents and potential
pharmacological activities (Dirar et al., 2019a). Guiera senegalensis J.F. Gmel., (Syn. G.
glandulosa Sm.), belonging to the family Combretaceae, is one of such plant having
widespread use in Africa. It is a shrub of 1-3 meters high and it grows throughout the Sahel
region from Mauritania through Northern Nigeria to Sudan (Burkill, 1985). It is a typical
species of semi-desert areas and dry localities of infrequent rainfall regions (Bosisio et al.,
1997), and it is an important nectar plant in the Savanna region (Dukku 2010). It is reported
to have many local names and different common names among tribes within the same
country, G, senegalensis is widely traded in the market of most African traditional medicine,
and the whole plant is used by traditional healers for the treatment of many diseases (Eloff et
al., 2008)

However, the dried bitter leaves are the most important part of the plant and commonly sold
in African markets, and it is widely acknowledged as a "cure-all' medicine ([Link] al.,
2013). Apart from its valuable traditional uses, G. senegalensis is reported to have cultural
belicfs; where the branches are put on graves, while the roots are fixed on vehicles to prevent
large storms and tightened around the waist for a safe jourey (Stoate ct al., 2001). The
variation in G. senegalensis as a marketed species and quantity depends on the local and

1
international demand. For example, in Sudan, G. senegalensis is widely sold in local shops
called "Atareen" as dried leaves. Previously, Khalid et al. (2007) reported the volumc of the
most popular exported

Sudanese medicinal plants and their revenue. However, the survey reported that G.
senegalensis is not a common species exported to the international market, and its global
trade figure data is scarce.

1.2 Statement of the Problem

The problem addressed in this study is the lack of comprehensive scientific understanding of
the phytochemical composition, antioxidant activity, potential of Guiera senegalensis leaves
extracts. Despite its traditional use in various medicinal practices, there is limited scientific
data regarding the bioactive compounds present in the plant and their therapeutic properties.
Therefore, the study aims to investigate the phytochemical constituents of Guiera
senegalensis leaves extracts and evaluate their antioxidant and anti-diabetic activities

1.3 Aim and Objectives of the Study

The aim of this study is to investigate the phytochemical composition, antioxidant activity,
and potential of Guiera senegalensis leaves extracts

Objectives:

i. To determine the phytochemical constituents present in the leaves extracts of Guiera

senegalensis.
ii. To evaluate the antioxidant potential of Guiera senegalensis leaves extracts.
iii. To determine the total phenolic and flavonoid content of Guiera senegalensis leaves
extracts.

1.4 Significance of the Study

The study on phytochemical investigation, antioxidant, and potential of Guiera Senegalensis

leaves extracts holds significant importance for several reasons:

Medicinal Potential: Guiera senegalensis is a plant species with a long history of Traditional
use in various indigenous systems of medicine. By conducting a Comprehensive
phytochemical analysis, this study can identify the bioactive Compounds present in the plant,
thus providing insights into its medicinal potential.

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This information can be utilized for the development of new natural therapeutics or
Pharmaceutical products

Antioxidant Activity: Oxidative stress caused by an imbalance between the production of

reactive oxygen species (ROS) and the body’s antioxidant defence mechanisms is implicated
in the development of various chronic diseases. Assessing the antioxidant Activity of Guiera
senegalensis extracts can help determine its ability to scavenge free Radicals and protect
against oxidative damage. This knowledge can contribute to the Development of natural
antioxidant supplements or functional foods.

Natural Product Research: In the era of drug discovery, natural products have served as a rich
source of bioactive compounds, Guiera senegalensis, being a plant with Medicinal
importance, can contribute to the pool of natural products. Studying its phytochemical
composition and biological activities can aid in the discovery of novel Compounds that can
be further developed into therapeutic agents.

In conclusion, the study on phytochemical investigation, antioxidant, and anti-diabetic

Potential of Guiera senegalensis leaves cxtracts addresses the knowledge gap regarding the
medicinal properties of this plant, the findings of this research can have Implications for drug
development, functional food formulation, and the management Of oxidative stress-related
disorders and diabetes.

1.5 Scope and limitation of the Study

Phytochemical Investigation: This involves the identification and analysis of the Chemical
constituents present in Guiera senegalensis leaves extracts

Phytochemicals are naturally occurring compounds derived from plants that may have
potential health benefits.

Antioxidant Potential: Guiera senegalensis leaves extracts are evaluated for their Antioxidant
activity. Antioxidants help protect the body against oxidative stress. Which is caused by an
imbalance between the production of reactive oxygen Species and the body’s antioxidant
defences.

Lack of Clinical Studies: While preliminary research and in vitro studies can provide valuable
insights, it is important to note that the topic’s limitations may include a lack of clinical

3
studies. Clinical trials involving human subjects are Necessary to determine the safety and
efficacy of Guiera senegalensis leaves Extracts for antioxidant and anti-diabetic purposes.

Variability of Results: The phytochemical composition and biological activities Of plant

extracts can vary depending on various factors, including the Geographical location, plant
growth conditions, extraction methods, and

Analytical techniques used, consequently, different studies may yield different Results,
making it challenging to draw definitive conclusions.

Limited Understanding of Mechanisms: While the topic explores the potential Antioxidant
and anti-diabetic activities of Guiera senegalensis leaves extracts, the underlying mechanisms
of action may not be fully understood. Further Research is needed to elucidate the specific
pathways and molecular targets Involved.

Safety Considerations: Although Guiera senegalensis is traditionally used in

Some cultures for medicinal purposes, it is essential to consider potential safety Concerns and
side effects associated with the use of plant extracts. Toxicological Studies and
comprehensive safety evaluations are necessary to establish the Safety profile of these
extracts.

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 CHAPTER TWO
2.0 LITERATURE REVIEW

2.1 Guiera Senegalensis

Guiera senegalensis is a semi-ever green shrub that grows up to 3-5 m tall with
spindly bole or many branched from base. All parts are covered with black glandular dot,
bark fibrous more or less smooth to fine scaly gray to brown. Flowers are bisexual, regular
petals, and creamy white to yellowish densely short hair. The Plant is widely distributed in
West Africa. In Burkina Faso, the gall is used to treat fowl pox and have antiseptic and
antifungal activities (Sanago et al., 2012). Preliminary tests on Guiera senegalensis suggests
its usefulness in traditional medicine. In Sudan, nearly 80, 000 people in Ghubaysh village
use the plant as medicine. Random survey was conducted on selected group of people, where
66 were male and 60 female with age range between 20-50 years. The study showed that
boiled and soaked leaves are used for treating many diseases, while dried roots are used for
wound treatment. Common ailment treated by leaves extract are jaundice (51.5 %) and 48.5
% showed it can be used to treat diabetes mellitus, hypertension, cough, arthritis, diarrhea,
malaria. Guiera senegalensis is also used in cosmetics, animal feed and fuel (Nabaa et al.,
2016).

Nabaa et al. (2016) reported that Guiera senegalensis water extracts of the leaves has no
inhibition activity against all the tested fungal strains; Stemphyllum solani, Aspergillus flavus,
Trichoderma viride, Penicilliun sp, Fusaruim verticillatum, Cladosporium cladosporioide
and Fusarium solani. Similarly, the toxicity test using brine shrimp suggest that the leaves of
the plant are apparently not toxic. Phytochemical tests of G. senegalensis leaf extract
indicated the presence of alkaloid, flavonoid, terpenoid, tannin, carbohydrate, protein, steroid
and saponin.

A study on the antimicrobial and phytochemical analysis on fraction of Guiera

senegalensis from Alasan Tumbuwal Nigeria was carried out by Simon and Aminu (2016).
The leaf sample was extracted using maceration technique with petroleum ether (PE),
dichloromethane (DCM) and methanol. The crude extract of pet-ether proved to be active
against Escherichia coli and Pseudomonas aeruginosa with inhibition zones of about 14 mm
and 21 mm respectively.

5
DCM extract was only active against Salmonella typhi while methanol extract showed no
activity against the entire organism under test. Column chromatography fraction of the crude
extract from PE and DCM gave 17 and 19 fraction respectively. On the basis their TLC
profile, the fractions of PE and DCM were grouped into three.. Second fraction of pet-ether
(PE2) showed activity on the entire test organism except Salmonella typhi. On the other hand,
first fraction (D1) from DCM extract was active against Escherichia coli, Pseudomonas
aeruginosa and Klebsiella pneumoniae. D2 was active on all the microbes whereas D3 was
active against Pseudomonas aeruginosa and Salmonella typhi. The phytochemical tests
revealed the presence of steroid, saponins, flavonoid and alkaloid (Simon and Aminu, 2015).

Report by Abdulmalik et al. (2019) showed that the methanolic extract of Guiera
senegalensis revealed the presence of anthraquinones, alkaloids, flavonoids, cardiac
glycoside, saponins, steroids/terpenoids, phenolic compounds and tannins but glycosides
were not detected. The antioxidant activities of Senna occidentalis and Guiera senegalensis
leaf extracts with DPPH were found to be 43.468-72.564 % and 47.74866.154 %
respectively. The 50 % that is inhibitory concentration (IC 50) of Guiera senegalensis were
found to be 58.55 µg/ml, while that of Senna occidentalis was 77.565 µg/ml. (Abdulmalik et
al., 2019). Several factors are known to affect the therapeutic efficacy of medicinal plants
which include but not limited to the age of plant, time of collection, extraction method,
geographical location and the general climatic conditions of area. Consequently, there is need
to carry out the phytochemical screening, antioxidant and antimicrobial peoperties
determination of Guiera senegalensis methanol leaf extract from Gombe state, Nigeria.

2.1.1 Family Combretaceae

'The Combretaceae are a family of flowering plant in the order myrtales. Family include
about 530 species of tree, shrub and lianas in ca 10 genera (Christenhusz and Byn, g 016).
The family include the lead wood tree, combretum imberbe. Three genera conocarpus,
languncuilaria and lumnitzera, row in mangrove habitat (mangals). The combretacea are wide
spread in the subtropics and tropic, some member of this family produce useful construction
timber, such as indigbo from terminaliainvarensis. The commonly cultivated quisqualisindica
is now placed in genus combretum

2.1.2 Genus Guiera

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Guiera is a lowering plant genus in the family Combretaceae. Family Combretaceae trees or
shrubs, sometime climbing plants (Andrews, 1950). Comprising about 20 genera and 500
species t rese species distributed around thte globe and central of diversity in

Africa and Asia. G. senegalensis belongs to this family is widely distributed in west and
central Africa on eached soils, follows; as underwood in low savanna forests, mostly in sandy
soils and on very dry sites; indicative of overgrazing (Heywood et al.,2007).

Guiera senegalensis (Combretaceae), locally known as Sabara" or Barbarta" in Hausa

Language of Northern Nigeria. Guiera senegalensis is widely distributed in the savannah
region of west and central Africa in Senegal, Gambia, Mali, Niger, Burkina Faso, Guinea-
Bissau, Guinea, Nigeria, Chad, Mauritania and Sudan (Sombie et al., 2011). Is a small shrub
upto 3 m high, with scattered black dots; branches pubescent. Leaves grey-green, single or
opposite, oblong-elliptic, mucronate at the apex, rounded or slightly cordate at the base.
About 2 to 4 cm long by 1 to 2 cm wide, sofly tomentose on both surfaces, with scattered
black glands beneath lateral nerves fine.

2.1.3 Botanical Description

Scientific Classifcation

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Myrtales

Family: Combretaceae

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 Genus: Guiera

Species: senegalensis

2.1.4 Morphology

Guiera senegalensis, very well known in its native area, generally occurs as a shrub that can
grow to a height of 3 to 5 m according to habitat. Its stem presents numerous <nots that send
out branches. The ash-grey stem and pranches have fibrous or pubescent bark and bear
opposing, short petiolatec oval leaves, sometimes mucronate, sometimes even cordate at their
base, about 2 to 4 cm long by 1 to 2 cm wide. These grey-green leaves, darker on their upper
surface, display black spots on their lower surface and are slightly downy on poth sides.
These features lend the plant an overall silver green colour that is conspicuous in brush land
(Anka et al., 2020). Flowering occurs almost throughout the year, when it is leafy. Often
blooms twice a year, during the dry season and the rainy season. Each flower has a calcinal
tube ovoid, welded to the ovary. This tube iS topped by a bellflower blade with 5 teeth
screened black and persistent points to fruiting. The ligulform corolla is composed of 5 petals
also riddled with black spots. The stamens are 10 on wo rows of 5, all inserted on the calyx.
The ovary has a single box containing 4 to 6 eggs (Anka et al., 2020). The fruit is an achene
around 3 cm long brown or green ash, spindle-shaped, hairy with sides and the remains of the
calyx (Alshafei et al., 2016).

2.2 Habitat and Repartition

t is a plant that grows primarily in Sudanian Sahel area, on soils sandy leached or exhausted,
fallow and dry stations. Plant pioneer disseminated by cattle in the fallow land, it is also
indicative of overgrazing. It is found from Senegal to Cameroon to Sudan. Widespread and
common, locally gregarious and very abundant (Alshafei et al., 2016).

2.3 Medicinal Uses of the Plant

G. senegalensis has been reported to have numerous raditional medicinal uses, many
ethnobotanical studies have been conducted by many authors confirmed that G. senegalensis
has a good reputation as medicinal traditional medicinal plant, It is reported that, it is used to
treat dysentery and/or diarhea (Maydell and john, 1986). Many authors reported that G.

8
snenegalensis used in traditional medicine to treat Malaria and /or fever (Jigam et al., 2011),
Abdominal (igametal., 2011). It is also reported as antileprosy pains, Epilepsy (Somboro et
al., 2011), depressant (Maria et al., 2012), cough and/or cold, (Maydell and john, 1986),
nakebite (Shatima et al., 2012), Eczema (Somboro et al., 2011), syphilis (Jigam et al., 2011),
hypertension and hypotension (Salihu and Usman, 015), diabetes (Shatima et al., 2012),
Impotence (Somboro et al., 2011) , Breast cancer and Jaundice (Fadimu et al., 2014).

Method of Preparation / Region / Ethnic

S/N Traditional Use Part Used Administration Group

Leaves, Decoction or infusion; Hausa, Fulani

1 Treatment of malaria stem bark taken orally (Nigeria, Niger)

Infusion or powdered
Management of leaves mixed with
2 diarrhea and dysentery Leaves water Senegal, Mali

Wound healing and Leaves, Crushed fresh leaves

3 treatment of ulcers bark applied topically Sahel regions

Respiratory problems
(e.g. cough, Leaves, Boiled and inhaled as Burkina Faso,
4 bronchitis) roots steam or drunk Nigeria

West African
savanna
5 Fever reduction Leaves Decoction taken orally communities

Hausa, rural
Stem, Nigerian
6 Toothache relief twigs Used as chewing sticks communities

7 Gonorrhea and STDs Roots Decoction taken orally Niger, Mali

Anti-inflammatory Leaves, Poultice applied to Traditional healers

8 and pain relief bark affected area across Sahel

9 Hypertension Leaves Infusion or boiled Northern Nigeria

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 Method of Preparation / Region / Ethnic
S/N Traditional Use Part Used Administration Group

extract consumed orally

Traditional
Liver and kidney Leaves, medicine
10 disorders roots Decoction taken orally practitioners

2.4 Pharmacological Activities Review

G. senegalensis ras various pharmacological properties that rave been reported by

researchers, It showed positive effects from the methanolic foots extracts as anti-tuberculosis
(Adedayo, 2015) and antidiarrhoeal (Williams et al., 2009), also it exhibited significant result
as ulcer protective (Aneiagu et al., 2005). 'The aqueous and ethanolic leaves extract at lower
doses is not harmful to the liver and therefore can be exploited as is served in the treatments
of some illnesses (Oshobu et al., 2014). Methanolic extract it of leaves revealed positive
result as anti-plasmodia, analgesic and devoid of anti-infammatory activites (Jigam et al.,
2011). It is also reported that the gall senegalensis exhibited an interesting antifiungal activity
against 9 strains tested (Sombie er al., 2013), while the leaves extract showed that it has no
inhibition activity against all of the tested fungal strains ( Alshafei et al.,2016), while
Guieranone which isolated from the leaves of senegalensis exhibited potent activity against
the fungus Cladosporium cucmeinun (silva et al., 2003). The extract of the leaves of G.
senegalensis was found to detoxify (in vitro) venom from two snake species (Abubakar et al.,
2000). 'Total alkaloid extract and beta carboline alkaloids presented an interesting
antiplasmodial activity associated with a low cytotoxicity (fiot et al., 2006). The extract (500
g) effectively inhibited the formation of peroxides in sunflower oil and showed the highest
antioxidant activity (Mariot er al., 2006). The chloroform extract of roots of & senegalensis
exhibited a pronounced anti- malarial activity while two alkaloids isolated from the active
extract, Harman and tetrahydroharman, showed antimalarial activity and displayed low
toxicity (Ancolio et al., 2002). The gall of G. senegalensis by using hydro acetic extract
showed the most potent antioxidant activity and anti-lipid peroxidation activity (Sombie et
al., 2013). It i also reported that pallotannin and tow condensed tannins exhibit interesting
antioxidant properties (Bouchet et al., 1998). The extract from galls of Guiera senegalensis
possess eflèctive antiacetyl cholinesterase, antilipid peroxidation in rat's brain homogenate

10
and erythrocytes hemolysis inhibitory activities Sombie et al., 2011). Leaf decoction of G.
senegalensis was reported to possess an antitussive activity (Sanogo et al., (1998).

2.5 Antimicrobial

The substances that can inhibit pathogens and have itle toxicity to host cells are considered
candidates for developing new antimicrobial drugs. On the other hand Ndiseriminate Se of
commercial antimicrobial drugs in the treatment of infectious diseases has resulted in
multiple-drug resistance to many human Pathogenic Microorganisms. This situation has
necessitated a more radical approach in the search for New antimicrobial substances from
various sources which could be used as novel Antimicrobial chemotherapeutic agents
(Navarro et al., 1996) and the resultant difficulty In the treatment of infectious diseases
(Tenover, 2006) and also the fact that the number S scientists hto are developing new
antibacterial agents has dwindled, even as bacteria Evolve ever more clever mechanisms of
resistance to antibiotics (Krause, 1992). The Search for new antibiotics includes arious
sources utch as thte synthetic compounds, Ioactive agents from aquatic microorganisms, and
natural products including medicina Plants.

2.6 Phytochemicals

The secondary metabolites of plant have been mentioned as a phytochemicals.

Phytochemicals are the naturally occurring, biologically active compounds found in Plants
which have capabilities of disease inhibiting (Abdurraham et al., 1992). In this Way
prevention from many diseases and food spoilage is possible (Chetia et al., 2014), (Kokeva et
al., 2000). Before the introduction of orthodox medicines medicinal plants were used.
Flowers, eaves, stems, seeds, roots, bark and fruit are constituents of the Herbal medicines.
Component of phytochemicals represent medicinal value of the natural Plants
(Akinmoladunet al., 2007). (Pascaline et al., 2011). Phytochemicals performs physiological
actions on human body. Important phytochemicals are tannins, alkaloids, phenolic
compounds and flavonoids (Kolaket al., 2009).

Phytochemicals are non-nutritive compounds found in plant and which may Nave protective
or disease preventive properties. These phytochemicals are Mostly secondary metabolites of
which over 10,000 have been isolated. In Many cases, these substances serve as plant defense
mechanism against Predation by microorganism, insects and herbivores. However, it has been
demonstrated that these chemical substances can also protect human against diseases. They
include the following:
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S/N Chemical Class Bioactive Compound(s) Known Biological Activity

Quercetin, Kaempferol, Antioxidant, anti-inflammatory,

1 Flavonoids Luteolin antimicrobial

Condensed tannins, Antidiarrheal, astringent,

2 Tannins Hydrolyzable tannins antimicrobial

Guieranone A, Guieranone Antiplasmodial (antimalarial),

3 Alkaloids B cytotoxic

Antifungal, anti-inflammatory,
4 Saponins Triterpenoid saponins immune-stimulant

Monoterpenes,
5 Terpenoids Sesquiterpenes Antibacterial, anti-inflammatory

Phenolic
6 compounds Gallic acid, Caffeic acid Antioxidant, hepatoprotective

Anti-inflammatory, cholesterol-
7 Steroids β-sitosterol lowering

1,8-cineole, α-pinene, Antimicrobial, insecticidal,

8 Essential oils camphene analgesic

Antifungal, anti-inflammatory,
9 Coumarins Scopoletin vasodilator

Antimicrobial, laxative,
10 Anthraquinones Emodin anticancer

2.6.1 Alkaloids

They are natural plant compounds with a basic character and usually contain one or more
nitrogen atom in a heterocyclic ring. They are usually colourless, crystalline, non-volatile
solids which are insoluble in water but soluble in ethanol, ether, chloroform and other organic
solvents. Only very few liquids are soluble in water. Most alkaloids have a bitter taste and are

12
optically active. Most alkaloids are physiologically active while some are extremely
poisonous The first medically useful example of an alkaloid was morphine isolated in 1805
fom the opium Papaver somniferum. Many alkaloids are commonly found to have
antimicrobial properties. The mechanism of action of highly aromatic planar quaternary
alkaloids such as berberine and har ane is attributed to their ability to intercalate with DNA
(Knek et al., 1997).

2.6.2 Flavonoids

Flavonoids are a class of water soluble plant pigments. They are a group of polyphenolic
compounds possessing 15 carbon atoms; two benzene rings joined by a linear three carbon
chain. Since the flavonoids are known to be synthesized by plants in response to microbial
infection, it should not be surprising that they have been found in vitro to be effective
antimicrobial substances against a wide array of microorganisms. Their activity is probably
due to their ability to complex with extracellular and soluble proteins and to complex with
bacterial cell wall (Ajali, 2004). More lipophilic flavonoids may also disrupt microbial
membranes. Human studies suggest that flavonoids may reduce the risk of cardiovascular
disease and stroke (Knek et al., 1997).

✅ 1. Flavonoids (e.g., Quercetin, Kaempferol, Luteolin)

Biological Activities: Antioxidant, anti-inflammatory, antimicrobial, antimalarial.

Relevance to Traditional Use:
Malaria and fever: These flavonoids scavenge free radicals and reduce oxidative stress
associated with malaria.
Wound healing: Promote tissue regeneration and reduce inflammation.
Cough and respiratory issues: Anti-inflammatory effect soothes mucosal irritation.
2.6.3 Saponins

Saponins are glycosides with distinctive foaming characteristics. They are natural detergents
found in certain plants. They are found in many plants especially certain desert plants. They
got their name from the soapwort plant (Saponaria) the root of which was used historically as
a soap. Saponins have detergent or surfactant properties because they contain both water
soluble and fat soluble component. S aponins are amphipathic compounds, possessing both
hydrophilic and lipophilic portions. They are, therefore, surface active and can be used as
emulsifiers. Molecular weight is of the prder 180-2000 Daltons. At concentrations below 200-

13
500 ppm saponins exist as monomers; above 200-500ppm, they aggregate as micelles with a
molecular weight of approximately 100,000 Dalton. Some saponins are sweet while others
are bitter (Menkhetti, 2005). The antifungal and antibacterial properties of saponins are
important in cosmetic application in addition to their emollient effects. Saponins have both
current and potential applications in animal and human nutrition, in pig and poultry raising
facilities and in dog and cat foods. Saponins have ammonia binding activity when added to
the diet, can bind to ammonia and certain other odoriferous components in the excreta and
prevent them from being released into the air. It is however interesting hat human do not
suffer severe poisoning from saponins (Stewart, 2005).

✅ 4. Saponins (Triterpenoid Saponins)

Biological Activities: Immune-modulating, antifungal, anti-inflammatory.

Relevance to Traditional Use:
Cough and bronchitis: Saponins may aid expectoration (loosening mucus).
General infections: Enhance immune response and fight microbes.
2.6.4 Tannins

Tannins is a general descriptive name for a group of polymeric phenolic substances capable
of tanning leather or precipitating gelatin from solution, property known as astringency. Their
molecular weight range from 500 to 3000 kD and are found in almost every plant part: bark,
wood, leaves, fruits and roots. Tannins are divided into two groups, hydrolysable and
condensed tannins. Hydrolysable tannins are based on Gallic acid, usually as multiple esters
with D-glucose; while the more numerous condensed tannins often called proanthocyanidins
are derived from flavonoid monomers. Tannins may be formed by condensation of flavan
derivatives which have been transported to woody tissues of plants. AIternatively; tannins
may be formed by polymerization of quinone units. This group of compounds has received a
great deal of attention in recent years, since it was suggested that the consumption of tannin-
containing beverages, especially green teas and red wines, can cure or prevent a variety of
illness (Herbert, 1989). Many human physiological activities, such as stimulation of
phagocytic cells, host mediated tumor activity, and a wide range of anti-infective activities
have been assigned to tannins. One of their molecular actions is to complex with proteins
through so-called nonspecific forces such as hydrogen bonding and hydrophobic effects, as
well as by covalent bond formation. Thus, their mode of antimicrobial action may be related

14
to their ability to inactivate microbial adhesions, enzymes, cell envelope transport proteins etc
(Stewart, 2005).

2.6.5 Phenolics and Polyphenols

Some of the simplest bioactive phytochemicals consists of a single phenolic ring. Cinnamic
and caffeic acids are common representatives of a wide group of phenylpropanederived
compounds which are in the highest oxidation state: The common herbs, Tarragon and
Thyme, both contain caffeic acid, which is effective against viruses (Wild, 1994), bacteria
(Bratner and Grein, 1994) and fungi (Duke, 1985). Catochol and pyrogallol both are
hydroxylated phenolsshown to be toxic to microorganisms. Catochol has two -OH groups,
and pyrogallol has three. The site(s) and number of hydroxyl groups on the phenol group are
thought to be related to their relative toxicity to microorganisms, with evidence that increased
hydroxylation results in increased toxicity (Geissman, 1963). In addition, some authors have
found that more highly oxidized phenols are inhibitors. The mechanisms thought to be
responsible for phenolic toxicity to microorganisms include enzyme inhibition by the
oxidized compounds, possibly through more non specific interactions with the proteins
(Mason and Wesserman, 1987). Phenolic compounds possessing a C3 side chain at lower
level of oxidation and containing no oxygen are classified as essential oils and often cited as
antimicrobial as well Eugenol is vell characterized representative found in clove oil. Eugenol
is considered bacteriostatic against both fungi and bacteria (Duke, 1985).

✅ 6. Phenolic Compounds (Gallic Acid, Caffeic Acid)

Biological Activities: Potent antioxidant, hepatoprotective, antimicrobial.

Relevance to Traditional Use:
Liver disorders: Protect liver cells from oxidative damage.
Hypertension: Antioxidants improve vascular function.
Cough and cold: Antimicrobial action targets upper respiratory tract pathogens.
2.7 Bioavailability of Herbal Drugs

The bioavailability of the active constituents of the herb is another area of Considerable
importance. Before a compound can act systemically it must Dass from the gastrointistinal
tract into the blood stream. This is an area in which surprisingly little is known for herbal
constituents. Compound, such as Perberine and hydrastine in the popular botanical goldensea
Hydrastic Canadensis L), are essentially not absorbed following oral consumption. Studies

15
showing systemic effect in animal have all involved parenteral Administration of these
alkaloids. Yet goldenseal remains one of the best-Selling herbs, is widely promoted, and is
accepted by a misinformed public as Nonspecific immunostimulant (Tyler, 1999). Cinnabar
has been for a long Ime in traditional medicine. The toxic effects of inorganic mercury are
well recognized, but because of its insolubility it has been assumed that this Compound
would not be significantly absorbed from the gastrointestinal tract. However, investigation of
(Yeoh et al., 1986) on the oral absorption of Cinnabar in mice found a significant increase in
mercury concentration in the Liver and kidney. Concomitant use of cinnabar and drugs
containing Bromides, sulphates, sulphides, nitrates and iodine may enhance its toxicity by
increasing the gastrointestinal absorption (Shaw Et al., 1995).

2.8 Antimicrobial Compounds in Guiera senegalensisis

Essential oils extracted from herbs are generally recognized as containing the Active
antimicrobial compounds. Herbs have been recognized to possess a Broad spectrum of active
constituents that exhibit antimicrobial (AM) activity. These active compounds are produced
as secondary metabolites associate with the volatile essential oil (E0) fraction of these plants
(Ajali, 2004). A wide Range of AM agents derived from Eos have the potential to be us for
Packaging systems which is one of the promising forms of active Systems aimed at protecting
food products from microbial contamination. Many studies have evaluated the AM activity of
synthetic AM and/or natura AM agents incorporated into packaging materials and have
demonstrated Effective AM activity by controlling the growth of microorganisms (Ajali,
2004). Allicin and allyl isothocyanate are sulfur-containing compounds. Allicin, Isolated from
garlic oil, inhibits the growth of both Gram-negative and Gram-Positive bacteria. Sulfur-
containing compounds are also present in onions, Leeks, and chives. The essential oil fraction
is particularly high in cloves, and Eugenol comprises 95% of the fraction. The presence of
these compounds in Cinnamon and cloves, when added to bakery items, function as mold
inhibitors In addition to adding flavor and aroma to baked products (Ross Et al., 2001).

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 CHAPTER THREE
3.0 MATERIALS AND METHOD
3.1 Introduction

Guiera senegalensis is a plant species widely used in traditional medicine for its various
health benefits. The leaves of G. senegalensis have been reported to possess antioxidant
properties, which can help protect against oxidative stress and related diseases. This chapter
aims to investigate the phytochemical composition and antioxidant potential of G.
senegalensis leaves extracts.

3.2 Sample Collection

Guiera senegalensis samples will be collected from muda lawal market bauchi state and will
be transported in cleaned polyethene bag and sacks to the biochemistry laboratory of
Abubakar Tatari Ali polytechnic Bauchi State. It will be identified and authenticated by a
biochemist at biochemistry laboratory of Bauchi State polytechnic. It will be air-dried at
room temperature.

3.3 Sample Preparation

Drying: Dry the collected leaves to remove excess moisture.

Grinding: Grind the dried leaves into a fine powder to increase the surface area for extraction.

Storage: Store the powdered leaves in airtight containers to maintain their quality and prevent
contamination.

The powder plant samples will be weighed (500 g) and soaked in methanol for a week with
occasional shaking to facilitate the extraction of phyto-constituents. It will be decanted and
filtered using No.1 whatman filter paper and then concentrated under reduced pressure with
rotatory evaporator until a semi solid residue will be obtained.

3.4 Materials

Solvents: Use suitable solvents such as methanol, ethanol, or water for extraction.

Equipment: Use equipment such as rotary evaporators, freeze dryers, or oven dryers for
extract preparation.

Reagents: Use reagents such as DPPH, ABTS, or Folin-Ciocalteu reagent for antioxidant
assays.
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3.5 Determination of Phytochemical Constituents

The qualitative phytochemical tests will be carried out according to well established
protocols. Wagners test will be used for the presence of alkaloids (Musa et al., 2018).
Similarly, froth test was used for Saponins (Musa et al., 2018). The formation of blue black
precipitate using 1% FeCl3 will be used as a test for tannins (Trease and Evans, 2002). Test
for flavonoids will be carried out according to the method reported by Kwaji et al. (2018).
Salkowski’s method will be used for the presence of terpenoids (Sonali et al., 2015),
anthraquinones (Musa et al., 2018) and Glycosides (Sonali et al., 2015).

Application

Pharmaceuticals: Guiera senegalensis leaves extracts may be used as a potential source of

natural antioxidants for pharmaceutical applications.

Food industry: The extracts may be used as natural preservatives or additives in food
products.

Cosmetics: The extracts may be used in cosmetic products due to their antioxidant and anti-
inflammatory properties.

Principle

Extraction: The principle of extraction is based on the solubility of phytochemicals in

solvents.

Antioxidant assays: The principle of antioxidant assays is based on the ability of

phytochemicals to scavenge free radicals or reduce oxidative stress.

Procedure
 Weigh 100g of the powdered leaves and extract with 500ml of methanol using a
Soxhlet apparatus for 4 hours.
 Repeat the extraction process three times to ensure maximum yield.
 Concentrate the extract using a rotary evaporator at 40°C.

3.5.1 Determination of Total Alkaloids

Crude extract (10 mg) will be weighed and 20 ml of methanol: ammonia (68:2) will be
added. After 24 hrs, the ammonia solution will be decanted. The procedure will be repeated
thrice to obtain maximal extracts. The extracts will be concentrated and then treated with 1m

18
HCl and kept over-night. The acidic solution will be extracted with 20 ml of CHCl3 thrice.
The organic layers will be then pooled and evaporated to dryness after which it will be
basified with NaOH solution to pH-12 and then extracted with 20ml CHCl3 again. The acid
layer will be evaporated and dried over absorbent cotton. The fraction will be weighed and
expressed as mg/100 g Atropine sulfate.

3.5.2 Determination of total flavonoids

The method is based on the formation of the flavonoid - aluminium complex which has an
absorption maximum (max ) at 415nm. 100 μl of the sample extracts in methanol (10 mg/ml)
will be mixed with 100 μl of 20 % AlCl3 in methanol and a drop of acetic acid, and then
diluted with methanol to 5 ml. The absorption at 415 nm will be read after 40 min. Blank
samples will be prepared from 100 ml of sample extracts and a drop of acetic acid, and then
diluted to 5ml with methanol. The absorption of standard quercetin solution (0.5 mg/ml) in
methanol was measured under the same conditions. All determinations were carried out in
triplicate.

3.5.3 Determination of total saponin content

About 2 mg of extract will be weighed in a 250 ml beaker and 100 ml of isobutyl alcohol will
be added. The mixture will be agitated using orbital shaker for 5 hours to ensure uniform
mixture. It will be then filtered with Whatman No.1 filter paper into a 100 ml beaker
containing 20 ml of 40 % saturated solution of MgCO3. The mixture obtained will be filtered
again using Whatman No.1 filter paper. The filtrate (1 ml) will be taken in a 150 ml
volumetric flask using pipette and 2 ml of 5 % iron (III) chloride (FeCl3) solution will be
added and made up to the mark with distilled water. It will be allowed to stand for 30 min for
color to develop. The absorbance will be read against the blank at 380 mm.

3.5.4 Determination of total phenols

A 0.5g sample will be homogenized in 10x volume of 80 % ethanol and centrifuged at 6000
rpm for 20 min. The procedure will be repeated twice. The supernatants will be combined and
evaporated to dryness. The residue will be then dissolved in 20 ml of distilled water. Several
aliquots will be pipetted out and the volume in each test tube will be made up to 3 ml with
distilled water. Folin-ciocalteau reagent (0.5 ml) will be added and equal volume of NaCO3.
The tubes will be placed in a boiling water bath for exactly one minute. The test tubes will be
cooled and the absorbances will be read at 600 nm in a spectrophotometer against a blank. A

19
standard gallic acid solution (0.2- 1 ml) with concentration range of 2.0 – 10 µg/mL will be
also treated as above and the standard gallic acid graph will be plotted (Oladummoe et al.,
2016).

3.5.5 Determination of steroids

0.1ml and 0.2 ml of triple acid extract will be taken and a set of standards (0.5 to 2.5 ml) will
be taken and made up to 5 ml with ferric chloride diluting reagent. A blank will be prepared
simultaneously by taking 5.0 ml diluting reagent. Then add 4.0 ml of concentrated sulphuric
acid to each tube. After 30 minutes’ incubation, intensity of the colour developed will be read
at 540 nm. Standard calibration curve will be obtained from various diluted concentrations of
cholesterol expressed as mg/100 g.

3.5.6 Determination of tannin

Weigh 0.5 g of the powdered sample and transfer to 250ml conical flask. Add 75 ml of water.

% AA= 100 {[(AbS sample AbS blank)/ AbS control x100

Blank= methanol (1.0 ml) +sample solution (2.0 ml)
Negative control= DPPH (1.0 ml, 0.25ml) + Methanol (2.0 ml)
Positive control= Ascorbic acid was use as standard
The scavenging reaction between (DPPH) and an antioxidant (H-A) will be written as;
Heat the flask gently and boil for 30 mins. Centrifuge at 2000 rpm for 20 mins and collect the
supernatant in 100 ml volumetric flask and make up the volume. Transfer 1ml of the sample
extract to 100 ml volumetric flask containing 75ml water. Add 5ml of Folin-Denis reagent, 10
ml of sodium carbonate solution and dilute to 100ml with water. Shake well. Read the
absorbance at 700 nm after 30 mins.

3.6 Evaluating the Antioxidant Potential of Guiera senegalensis

To evaluate the antioxidant potential of Guiera senegalensis, the following methods can be
employed:

Extraction of Phytochemicals

Solvent Extraction: Use solvents like methanol, ethanol, or water to extract phytochemicals
from Guiera senegalensis leaves.

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Optimization: Optimize extraction conditions, such as solvent ratio, temperature, and time, to
maximize yield.

In Vitro Antioxidant Assays

DPPH Assay: Measure the ability of extracts to scavenge DPPH (2,2-diphenyl-1-

picrylhydrazyl) free radicals.

ABTS Assay: Measure the ability of extracts to scavenge ABTS (2,2'-azino-bis(3-

ethylbenzothiazoline-6-sulfonic acid) free radicals.

FRAP Assay: Measure the ability of extracts to reduce ferric ions (Fe3+) to ferrous ions
(Fe2+).

Hydrogen Peroxide Scavenging Assay: Measure the ability of extracts to scavenge hydrogen
peroxide (H2O2).

Total Phenolic and Flavonoid Content

Folin-Ciocalteu Assay: Measure the total phenolic content of extracts using the Folin-
Ciocalteu reagent.

Aluminum Chloride Assay: Measure the total flavonoid content of extracts using aluminum
chloride.

Chromatographic Analysis

HPLC (High-Performance Liquid Chromatography): Separate and identify individual

phytochemicals in extracts.

GC-MS (Gas Chromatography-Mass Spectrometry): Identify and quantify volatile

compounds in extracts.

Cell-Based Antioxidant Assays

Cell Viability Assays: Evaluate the protective effects of extracts against oxidative stress-
induced cell damage.

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Intracellular ROS Assays: Measure the ability of extracts to reduce intracellular reactive
oxygen species (ROS) levels.

3.7 Phytochemical Analysis

 Determine the total phenolic content using Folin-Ciocalteu reagent:

 Prepare 1mg/ml of the extract in methanol.
 Add 1ml of Folin-Ciocalteu reagent to 1ml of the extract.
 Measure the absorbance at 765nm after 30 minutes.
 Calculate the total phenolic content using a standard curve.

3.8 Statistical Analysis

Calculate the mean and standard deviation of the antioxidant activity and phytochemical
content.

Use ANOVA or t-test to compare the differences between the extract concentrations and
antioxidant activities.

Calculate the IC50 value (concentration required to inhibit 50% of radical activity) using
regression analysis.

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