PHYTOCHEMICALS AND ANTIBACTERIAL POTENTIALS OF AQUEOUS AND

ETHANOLIC EXTRACTS OF Garcinia Kola on Salmonella Enterica AND Escherichia

Coli

BY

UMAR AHMED IBRAHIM

ST/BST/M/HND/22/005

A PROJECT PROPOSAL SUBMITTED TO THE DEPARTMENT OF BIOLOGICAL

SCIENCE TECHNOLOGY, FEDERAL POLYTECHNIC MUBI, ADAMAWA STATE.

IN PARTIAL FULFILLMENT FOR THE AWARD OF HIGHER NATIONAL

DIPLOMA IN SCIENCE LABORATORY TECHNOLOGY BIOLOGY FEDERAL
POLYTECHNIC

APRIL, 2024

1
 CHAPTER ONE

INTRODUCTION

1.1 Background of the study

Garcinia kola, commonly referred to as bitter kola, stands as a tropical tree indigenous to the

regions of West and Central Africa. Its seeds have long been employed in traditional medicine

due to their diverse medicinal properties, prompting scientific interest in the phytochemicals

within Garcinia kola and their potential antibacterial efficacy (Ojewole, 2007).

Phytochemicals, naturally occurring compounds found in plants, exhibit diverse biological

activities that extend beyond the plant's essential growth and development functions. Instead,

they often serve a protective role against pests, diseases, and environmental stressors. Within the

realm of Garcinia kola, researchers have concentrated their efforts on characterizing and

investigating the phytochemical composition present in its seeds, particularly in relation to

potential antibacterial effects (Olayinka and Ajayi (2016)).

Extraction methods play a pivotal role in obtaining bioactive compounds from plant materials,

with aqueous and ethanolic extracts being commonly utilized Sarker and Nahar, (2012). The

choice of solvent, whether water or ethanol, significantly influences the types and concentrations

of phytochemicals extracted. Comparative studies on different extraction methods aim to identify

the most potent extracts (Mazza, 1998).

To assess the antibacterial potential of these extracts, researchers typically conduct experiments

against specific bacterial strains. In this context, Salmonella enterica and Escherichia coli,

prevalent bacteria associated with foodborne illnesses, become the focal points. Researchers

2
rigorously evaluate the inhibitory effects of Garcinia kola extracts on the growth or viability of

these bacteria through meticulous experimentation (Iwu, Duncan, Okunji, 2002; Olayinka and

Ajayi, 2016).

The antibacterial properties inherent in plant extracts are often attributed to specific

phytochemicals, including alkaloids, flavonoids, tannins, and saponins. These bioactive

compounds may disrupt bacterial cell membranes, inhibit crucial enzymes, or interfere with

bacterial cell processes, collectively resulting in the inhibition of bacterial growth (Cowan, 1999;

Cushnie and Lamb, 2005).

Exploration into the antibacterial potentials of Garcinia kola extracts against Salmonella

enterica and Escherichia coli bears immense significance for multiple reasons. Firstly, it offers

valuable insights into the potential application of these plant extracts as natural antibacterial

agents, with potential implications in food preservation and the development of alternative

antimicrobial agents (Abdulazeez and Mann, 2018). Moreover, such research contributes

substantially to our comprehension of the bioactive compounds inherent in traditional medicinal

plants, paving the way for the exploration and development of novel pharmaceuticals or

nutraceuticals (Olayinka and Ajayi, 2016).

1.2 Statement of the Problem:

To date, the literature provides limited insight into the detailed phytochemical composition of

Garcinia kola and its direct impact on the growth and viability of Salmonella enterica and

Escherichia coli (Omodamiro, Adeniyi and Oyedapo, 2021). Understanding the specific

compounds responsible for antibacterial properties and elucidating their mechanisms of action

3
against these bacterial strains is crucial for the development of potential natural antibacterial

agents (Olusola, Ayo and Olusola, 2017).

Therefore, the primary aim of this study is to determine the phytochemical profile of Garcinia

kola extracts and assess their antibacterial activities against Salmonella enterica and Escherichia

coli. By addressing this gap in knowledge, the research endeavors to contribute valuable

information to the fields of pharmacognosy and antimicrobial research, potentially paving the

way for the development of novel therapeutic agents (Uzochukwu, Okoye, Nwodo, Enejoh and

Ifemeje, 2022).

1.3 Aim of the study

To determine the phytochemicals and antibacterial activities of extract of Garcinia kola on

Salmonella enterica and Escherichia coli

1.4 Specific objectives of the study

The specific objectives of the study are to;

i. screen for phytochemical components of Garcinia kola

ii. determine the antibacterial activity of Garcinia kola extract on Salmonella enterica

and Escherichia coli

1.5 Significance of the Study

This study holds multifaceted significance across various domains, making substantial

contributions to scientific knowledge, healthcare practices, and industrial applications.

4
The investigation deepens our comprehension of the phytochemical components of Garcinia

kola extracts by identifying and quantifying specific compounds, thereby advancing plant

chemistry knowledge.

The study carries noteworthy implications in the quest for natural antibacterial agents. By

evaluating the antibacterial activity of Garcinia kola extracts against Salmonella enterica and

Escherichia coli, the research explores their potential as alternatives to conventional antibacterial

agents. The outcomes may pave the way for novel therapeutic agents, aligning with the growing

interest in natural remedies for health and wellness.

From a pharmaceutical and medicinal perspective, the study's findings may guide the

formulation of new products. The antibacterial potential of Garcinia kola extracts could be

leveraged to develop pharmaceuticals for medical applications, addressing the ongoing demand

for innovative therapeutic solutions. Additionally, the study's outcomes may benefit the

nutraceutical industry, where natural plant extracts are increasingly sought after for potential

health benefits.

Considering implications for food safety, the antibacterial properties of Garcinia kola extracts

may find application in food preservation. If proven effective against bacteria such as

Salmonella and Escherichia coli, these extracts could be explored as natural and safe

preservatives, aligning with consumer demand for clean-label and natural food products.

1.6 Scope and Limitations

The scope of the study involves a comprehensive analysis of the phytochemical composition and

antibacterial activities of Garcinia kola extracts. It encompasses the identification and

5
quantification of specific compounds, including alkaloids, flavonoids, tannins, and saponins. The

antibacterial aspect involves in vitro assays against Salmonella enterica and Escherichia coli.

6
 CHAPTER THREE

MATERIALS AND METHODS

3.1 The Study Area

Mubi metropolis is a geo-political area comprising of two local government areas; Mubi North

and Mubi South. The metropolis will be located between latitudes 10° 05' and 10° 30'N of the

equator and between longitude 13° 12' and 13° 19'E of the Greenwich meridian. The two Local

government areas occupy a land area of 192,307 Km 2 and support a total population 260,009

people (National Population Census 2006). The area shares international boundary with

Cameroon Republic to the east and local boundary with Maiha L.G.A in the South, Hong L.G.A

in the West, Michika L.G.A in the East. The major ethnic groups in Mubi includes; Gude, Fali,

Kilba, Higgi, Marghi and Nzanyi (Adebayo, 2004).

3.2 Cleansing and Sterilization of Glassware’s

All the glass wares to be used for the projects will be will behed appropriately with detergents,

air-dried and sterilized in hot air oven at 160oC for 1 hour before used.

3.3 Collection of Plant Materials

The seeds of Garcinia kola will be purchased in Mubi main market. The seeds will be identified

the Botany Unit, Department of Biological Sciences Technology, Federal Polytechnic Mubi and

also by comparing their morphological and anatomical characteristics with the standard

description

7
3.2 Extraction Procedure:

3.2.1 Aqueous Extract:

The Garcinia kola seeds will be thoroughly will behed under running water to remove any dirt or

foreign particles. After drying at room temperature, the seeds will be ground into a fine powder

using a mechanical grinder. The powdered seeds (200g) will be then macerated in 500ml of

distilled water for 24 hours, allowing the water to extract the phytochemicals. The mixture will

be then filtered using a fine mesh filter, and the filtrate will be concentrated using a rotary

evaporator under reduced pressure.

3.2.2 Ethanolic Extract:

A separate batch of 200g of powdered seeds will be soaked in 500ml of ethanol for 72 hours.

This process will allows the ethanol to extract different phytochemicals that may not be water-

soluble. The ethanolic extract will be then filtered and concentrated under reduced pressure using

a rotary evaporator.

3.3 Phytochemical Analysis:

The qualitative phytochemical screening for alkaloids, flavonoids, tannins, saponins, glycosides,

and phenolic compounds will be carried out by standard procedure (Sofowora, 1993; Egwaikhide

et al., 2007) as follows;

3.3.1 Test for tannins

8
0.5 g each portion will be stirred with about 10 ml of distilled water and then filtered. Few drops

of 1% ferric chloride solution will be added to 2 ml of the filtrate occurrence of a blue-black,

green or blue-green precipitate indicates the presence of tannins.

3.3.2. Test for steroids

To 0.2 g of each portion, 2 ml of acetic acid will be added, the solution will be cooled well in ice

followed by the addition of conc. H 2SO4 carefully. Colour development from violet to blue or

bluish-green indicated the presence of a steroidal ring i.e. aglycone portion of cardiac glycoside

3.3.3 Test for terpenoids

A little of each portion will be dissolved in ethanol. To it 1 ml of acetic anhydride will be added

followed by the addition of conc. H2SO4. A change in colour from pink to violet showed the

presence of terpenoids.

3.3.4 Test for saponins

One gram of each portion will be boiled with 5 ml of distilled water, filtered. To the filtrate,

about 3 ml of distilled water will be further added and shaken vigorously for about 5 minutes.

Frothing which persisted on warming will be taken as evidence for the presence of saponins.

3.3.5 Test for flavonoids

Each sample (0.30 g) weighed into a beaker will be extracted with 30 cm3 of distilled water for 2

hours and filtered with Whatman filter paper number 42 (125 mm). To 10 cm3 of the aqueous

filtrate of each wood extract will be added 5 cm3 of 1.0 M dilute ammonia solution followed by

the addition of 5 cm3 of concentrated tetraoxosulphate (VI) acid. Appearance of yellow

colouration which disappeared on standing shows the presence of flavonoids.

9
3.3.6 Test for alkaloids

Extraction of component from 2 grams of each powder sample will be carried out using 5%

tetraoxosulphate (VI) acid (H2SO4) (20 cm3) in 50% ethanol by boiling for 2 minutes and filtered

through Whatman filter paper number 42 (125 mm). The filtrate will be made alkaline using 5

cm3 of 28% ammonia solution (NH3) in a separating funnel. Equal volume of chloroform (5.0

cm3) will be used in further solution extraction in which chloroform solution will be extracted

with two 5 cm3 portions of 1.0 M dilute tetraoxosulphate (VI) acid. This final acid extract will be

then used to carry out the following test: 0.5 cm3 of Dragendorff’s reagent (Bismuth potassium

iodide solution) will be mixed with 2 cm3 of acid extract and precipitated orange colour infers

the presence of alkaloid.

3.6.7 Test for glycoside

To 2.00 g of each sample will be added 20 cm3 of water, heated for 5 minutes on a water bath

and filtered through Gem filter paper (12.5 cm). The following tests will be carried out with the

filtrate:

(a) 0.2 cm3 of Fehling’s solutions A and B will be mixed with 5 cm3 of the filtrate until it

became alkaline (tested with litmus paper). A brick-red colouration on heating showed a positive

result.

(b) Instead of water, 15 cm3 of 1.0 M sulphuric acid will be used to repeat the above test and

the quantity of precipitate obtained compared with that of (a) above. High precipitate content

indicates the presence of glycoside while low content shows the absence of glycoside

3.4 Antibacterial Assay:

10
3.4.1 Bacterial isolate

Standard strains of Salmonella enterica and Escherichia coli will be obtained from Microbiology

Laboratory, Federal Polytechnic Mubi, Adamawa State. These bacterial strains will be sub-

cultured on nutrient agar plates and maintained under suitable conditions to ensure their viability.

3.4.2 Preparation of Inoculum

A standardized inoculum will be prepared by adjusting the turbidity of the bacterial cultures to

match the 0.5 McFarland standard. This ensures that the number of bacteria used in the

antibacterial assay is consistent.

3.5 Antibacterial activity of Garcinia kola

The dried aqueous and ethanolic extracts of Garcinia kola will be reconstituted in distilled water

and dimethyl sulfoxide (DMSO) respectively to obtain final concentrations of 200mg/ml for this

test. The susceptibility test will be done using agar well diffusion method. 0.1ml aliquot of each

test organism suspension will be transferred onto dried nutrient agar plates in duplicate and will

be spread evenly with a sterile bent glass rod. After drying, three (3) wells will be bored (using

6mm diameter cork borer) into the dried nutrient agar plates and 0.5ml each of the extracts

(aqueous or ethanolic) will be aseptically introduced into two wells. Distil water and or DMSO

will be introduced into the third well as control. The plates will be then incubated at 37°C for

24h after which zones of inhibition will be measured in centimeters and recorded appropriately

(Tula et al., 2014)

11
 REFERENCES

Abdulazeez, A. T., and Mann, A. (2018). Antibacterial potential of aqueous and ethanolic
extracts of Garcinia kola seeds against Salmonella enterica and Escherichia coli. Journal
of Medicinal Plants Research, 12(14), 165-173.

Adebayo, A.A. (2004). Mubi region: a geographical synthesis. Paraclete publishing, Yola. 17-25

Cowan, M. M. (1999). Plant products as antimicrobial agents. Clinical Microbiology Reviews,

12(4), 564-582.

Cushnie, T. P., and Lamb, A. J. (2005). Antimicrobial activity of flavonoids. International

Journal of Antimicrobial Agents, 26(5), 343-356.

Egwaikhide, P. A., Okeniyi, S. O., and Gimba, C. E. (2007). The medicinal plants and extraction
of phytochemicals. Journal of Advances in Biological Research, 1(5-

Iwu, M. M., Duncan, A. R., and Okunji, C. O. (2002). New Antimicrobials of Plant Origin. In
Perspectives on New Crops and New Uses (pp. 457-462). ASHS Press.

Mazza, G. (1998). Anthocyanins in grapes and grape products. Critical Reviews in Food Science
and Nutrition, 38(4), 291-328.

Ojewole, J. A. (2007). Laboratory evaluations of the hypoglycemic effect of Garcinia kola

(Heckel) seed in rats. Phytotherapy Research, 21(10), 978-981.

Olayinka, A. A., and Ajayi, O. S. (2016). Antibacterial and phytochemical evaluation of

Garcinia kola seed extract. International Journal of Current Microbiology and Applied
Sciences, 5(2), 433-443.

Olusola, A. O., Ayo, R. G., and Olusola, B. A. (2017). Evaluation of antibacterial and
antioxidant properties of Garcinia kola seeds. International Journal of Biological
Chemistry, 11(3), 116-123.

Omodamiro, O. D., Adeniyi, B. A., and Oyedapo, O. O. (2021). Phytochemical profiling and
antibacterial activities of Garcinia kola (Heckel) seeds extracts. Journal of Herbs, Spices
and Medicinal Plants, 27(2), 158-174.

Sarker, S. D., and Nahar, L. (2012). Natural medicine: The genus Garcinia. CRC Press, United
States.

12
Sofowora A (1993). Standardization of Herbal Medicinal Plants and Traditional Medicine in
Africa. Spectrum Books Limited, LagosNigeria. pp. 51-61.

Tula, M. Y., Danchal, T. B., Iruolaje, F. O., and Onyeje, G. A. (2014). Studies on Phytochemical
Constituents and Antibacterial Potentials of Extracts of Balanites aegyptiaca
(Del.) Parts on Antibiotic Resistant Bacterial Isolates. European Journal of
Medicinal Plants, 4(7), 854-864.

Uzochukwu, S. V., Okoye, F. B., Nwodo, U. U., Enejoh, O. A., and Ifemeje, J. C. (2022).
Phytochemical composition and antibacterial activity of Garcinia kola seeds against
Salmonella enterica and Escherichia coli. Journal of Natural Products and Plant
Resources, 12(1), 12-21.

13