PHYTOCHEMICAL ANALYSIS OF GARCINIA KOLA AND IT'S

INVITRO EFFECTS ON KLEBSIELLA PNEUMONIAE STRAINS

ISOLATED FROM ST VERONICA HOSPITAL BIAKA
UNIVERSITY.

By

TAMBE VICTORIN

HS23BPT003

A Research Project Submitted to The Department of Pharmaceutical

Studies, School of Health Sciences of Biaka University Institute of Buea
For the Partial Fulfilment of The Requirements for An Award of The
Bachelor in Pharmacy Technology.

Supervisor:

Madam: Aguh edith

 Chapter one
1.1 Background

Klebsiella pneumoniae is a common pathogen responsible for various infections, including

pneumonia, urinary tract infections, and septicemia. The prevalence of antimicrobial resistance

in K. pneumoniae is a growing concern in healthcare settings worldwide. Natural products, such

as plant extracts, have gained attention as potential sources of novel antimicrobial agents. One

plant of interest in this context is Garcinia kola, also known as bitter kola. Garcinia kola is a

tropical tree native to West Africa and has been traditionally used for its medicinal properties.

Studies have shown that extracts from Garcinia kola possess antimicrobial activity against a

range of pathogens, including K. pneumoniae (Okwu et al., 2008). Further research on the

antimicrobial potential of Garcinia kola compounds could lead to the development of new

therapeutic agents to combat drug-resistant infections.

Traditional medicines produced from plants have become more important as alternative

medicines in treating a broad spectrum of ailments, and researchers are continuing to pay

attention to the use of plants materials in the treatment of many afflictions (Mahmoud and Abba

et Al.,)( Dogara and Labaran, et Al., ). The majority of the developing world believes that this

plants based product are safer and more cost-effective (A Dogara and S.W. Hamad et Al.,). With

the emergence of new diseases and microorganism resistance, the usage of these plants products

in developed, developing, and underdeveloped countries (S. Kayfi and M.D. Abdulrahman et

Al.,) Ethnopharmacology and medication discovery employing plants-based products are still in

critical healthcare delivery worldwide. Garcinia kola (Bitter kola) is regarded as a miracle plant

because every component has medicinal use. The use of herbs has a long history in health care

delivery in Africa. According to World Health Organization (W.H.O 2012), the evolving public
health threat of antimicrobial resistance is driven by both appropriate uses of anti-infective

medicines. The scientific evaluation of traditional drugs of plant seed origin and screening of

more effective and safe antimicrobial agents has continued to gain medicinal importance stated

by (Biswas et Al., 2012). The medicinal values of many of these seed cannot be over emphasized

in the light of oral traditions and folklores from the distance past that have continued to extol the

healing virtues of these seeds and their extracts according to (Amabeoku and Kinyua et Al.,

2010). Medicinal plants can be regard as the richest bioresource of drug of modern medicine,

folks medicine and chemical entities or templates for synthetic drugs by (Joshua and Takudzwa

et Al., 2013). The discovery of medically important metabolites in common and abundant plant

would minimize over exploitation of well known, rare medicinal plants. Plant seeds contain

bioactive components such as flavonoids, glycosides, saponins and tannins according to (Tiwari

et al., 2011), which possess medicinal properties that are harnessed for the treatment of different

diseases stated by (Prohp et al., 2009).

Dietary plant seeds with proven antioxidant properties may function as a direct anti-radical

chain breaker of free radical propagation, interaction with transition metals and inhibition of

Reactive Oxygen Species (ROS) generating enzymes postulated by (Hassan et al.,2007). The use

of plants for medicinal purposes continues to this day, usually in the form of traditional

medicine, which is now recognized by the World Health Organization as a building block for

primary health care. Well-known examples of drugs with plant origins includes aspirin, atropine,

digoxin, ephedrine, morphine, quinine, reserpine, vincristine and vinblastine, as well as several

plant steroidal sapogenins which serve as semi-synthetic precursors to the steroidal drugs

according to (Lim 2012)

Garcinia kola belongs to the Garcinia genus of the Clusiaceae family and Malpighiales order. It

contains more than 180 members all over the globe. It is found all over Asia and in tropical

African countries. It has been traditionally used for various health benefits, including its

antimicrobial properties. Phytochemical analysis of Garcinia kola has revealed the presence of

bioactive compounds such as flavonoids, alkaloids, saponins, and tannins, which are known to

have antimicrobial properties, anti oxidants properties just to name a few (Abulrahman et Al.,

2022).

This study therefore focused on the bioactive potentials of the extract from the plant on some

microorganisms.

1.2. Problem statement

The emergency of antibiotics resistance strains especially on klebsellia pneumonia has impose a

significant threat or challenge in the health care sector of Cameroon and in the general

population of Cameroon this as lead to an increase in the health care cost as well as an increase

in the mortality rate of those affected with the pathogen in Cameroon thus the aim of this study is

to analysis the phytochemicals constituent of Garcinia kola and it's invitro effects on klebsiella

pneumoniae strains which could be an alternative means of solving the problem of antibacterial

resistance in Cameroon.

1.3. Significance of study

1. Addressing Antimicrobial Resistance: The research topic is significant as it addresses the

urgent global issue of antimicrobial resistance, particularly in the context of Klebsiella

pneumoniae strains. By exploring the antimicrobial properties of Garcinia kola, this study may

provide insights into novel treatment options that could help combat antibiotic-resistant
infections caused by Klebsiella pneumoniae. Natural Product Discovery: Investigating the

phytochemical composition of Garcinia kola and its effects on Klebsiella pneumoniae strains can

lead to the discovery of new bioactive compounds with antimicrobial properties. Natural

products have been a valuable source of novel antimicrobial agents, and this research could

contribute to expanding the arsenal of natural-based treatments for bacterial infections.

Promoting Sustainable Healthcare Practices: Utilizing plant-derived compounds like those found

in Garcinia kola for their antimicrobial effects aligns with the growing interest in sustainable and

eco-friendly healthcare practices. By exploring the natural products as alternatives to

conventional antibiotics, this research may support the development of sustainable treatment

options that are less likely to contribute to the development of further antimicrobial resistance.

1.4. Research question

What is the minimum inhibitory concentration and maximum bactericidal concentration of

Garcinia kola extracts on klebsiella pneumoniae strains ?

1.5. General objective

To evaluate the phytochemical constituents of Garcinia kola and it's invitro effects on klebsiella

pneumoniae strains.

1.5.1. Specific objectives

To investigate the phytochemical constituents present in Garcinia kola (bitter kola) .

2) Determine the minimum inhibitory concentration (MIC) and maximum bactericidal

concentration (MBC) of Garcinia kola extracts on klebsiella pneumoniae strains .

3). Evaluate the combine effects Garcinia kola extracts with conventional antibiotics commonly

used to treat klebsiella pneumoniae infections.

1.6. Rationale

The rationale for conducting a phytochemical analysis of Garcinia kola and studying its in vitro

effects on Klebsiella pneumoniae strains lies in the potential of natural products to serve as

sources of novel antimicrobial agents. Given the increasing prevalence of antimicrobial

resistance in K. pneumoniae and the limited efficacy of conventional antibiotics, there is a

critical need to explore alternative treatment options. Phytochemical analysis of Garcinia kola

can help identify the bioactive compounds present in the plant that may exhibit antimicrobial

properties.(Okwu, D. E., Iroabuchi, F., & Okoronkwo, M. (2008). Evaluation of the

phytochemical composition of Garcinia kola seed.

1.7. Limitation of the study

This research work focuses on in vitro experiment and it may not fully represent real world

conditions.The study did not explore long term effects

1.8. Definition of terms

Antibiotics resistance: Minimum Inhibitory Concentration (MIC): The minimum inhibitory

concentration is the lowest concentration of an antimicrobial agent (such as an antibiotic or

disinfectant) that inhibits the visible growth of a microorganism in a standardized test. It is used

to determine the effectiveness of an antimicrobial agent against a specific microorganism.

2. Phytochemicals: Phytochemicals are naturally occurring compounds found in plants that have

biological activity and potential health benefits. These compounds are not essential nutrients but

may have antioxidant, anti-inflammatory, antimicrobial, or other beneficial effects on human

health.

3. Maximum Bactericidal Concentration (MBC): The maximum bactericidal concentration is

the highest concentration of an antimicrobial agent that kills a specific microorganism. It is

determined by performing a test similar to the MIC test but with higher concentrations of the

antimicrobial agent to assess its bactericidal activity.

4. Antibiotic Resistance: Antibiotic resistance occurs when bacteria develop the ability to

survive exposure to antibiotics that would normally kill them or inhibit their growth. This can

happen through genetic mutations or the acquisition of resistance genes, leading to the reduced

effectiveness of antibiotics in treating bacterial infections.

 CHAPTER TWO

2.1 Overview of klebsiella pneumoniae.

K. pnuemoniae is a bacterium that normally reside in the human intestines without causing

diseases, but can lead to a range of illnesses when it infiltrate other areas of the body such as

pneumonia, urinary tract infections, and septicemia, Thus Understanding their pathogenesis,

epidemiology, method of transmission and classification of these pathogens will provide us with

an inside view of these pathogen.

2.1.1 Botanical Classification of Staphylococcus aureus

Kingdom: Bacteria

Phylum: Proteobacteria

Class: Gammaproteobacteria

Order: Enterobacterales

Family: Enterobacteriaceae

Genus: Klebsiella

Specie: K. pneumonaie

2.1.2 Morphological description of S. aureus

Klebsiella is a genus of Gram-negative, non-motile, encapsulated, rod-shaped bacteria under the

microscope with size ranging from 1 – 2 nanometer in length and 0.5 – 0.8 nanometer in width

(Sahil, 2018). When treated with Indian ink, it appears as a clear halo against a dark background.
While on MacConkey Agar medium, K. pneumoniae colonies appears pink to purple without a

green metallic sheen (Sahil, 2018).

2.1.3 Virulence factor, pathogenesis and mode of transmission of K.

pneumoniae
K. pneumonia produces several types of toxins such as siderophores, lipopolysaccharides

(endotoxins), and various enzymes, this toxins damages host cells which greatly contribute to

pathogenicity of the bacterium (Katty et al., 202). The bacterium invade the host cell by

attaching in to the host using fimbriae (Katty et al., 2022). Lipopolysaccharides (Endotoxins) s

are components of the outer membrane of Gram-negative bacteria like Klebsiella pneumoniae.

When the bacterium is killed or undergoes cell lysis, these endotoxins are released which can

trigger a strong immune response in the host, leading to the release of inflammatory mediators

such as cytokines, excessive cytokine release can cause widespread inflammation, tissue damage,

and potentially lead to septic shock (Rosen et al., 2015). Klebsiella pneumoniae as the ability to

produces siderophores, which are molecules that scavenge iron from the host environment. Iron

is essential for bacterial growth, so the production of siderophores allows the bacterium to obtain

iron and thrive in host tissues. High levels of iron can lead to oxidative stress and damage to host

cells (Pan et al., 2008). Klebsiella pneumoniae produces enzymes such as proteases, lipases, and

hemolysins, which can break down host tissues and cells. For example, proteases can degrade

proteins in host tissues, disrupting cell structure and function. Lipases can break down lipids in

host cell membranes, compromising their integrity. Hemolysins can damage red blood cells and

disrupt cellular membranes (Piperaki et al., 2017).

2.2 Overview of Garcinia kola.

Garcinia kola (G. kola) is one of the medicinal plants, which has been used in African ethno

medicine because of its purgative, antiparasitic, antimicrobial properties.(Iwu M. et al.,).

Garcinia kola is botanical name for bitter kola. It is flowering plant found in subtropical or

tropical moist lowland forests of Nigeria, Senegal, Sierra Leone, Liberia, Ghana, Gabon, Ivory

Coast, Democratic Republic of the Congo, Cameroon and Benin Republic . The name bitter kola

came from the bitter astringent and resinous taste, which is followed by a slight sweetness from

chewing the kola. In Nigeria, G. kola is called aku-ilu, adu or ugolo in Igbo, orogbo in Yoruba,

and namijingworo in Hausa. It is highly valued in Africa and used for hospitality during cultural

and social ceremonies.

2.2.1 Botanical description of Garcinia kola .

Garcinia kola, commonly known as bitter kola, is an evergreen plant that grow mostly in West

and Central Africa and is highly valued for its medicinal properties, it has a reddish – yellow,

globose with 2 -4 brown, oblong seeds which are embedded in a freshy orange pulp (Okoye,

2014)

2.2.2 Classification of G. kola

Kingdom: Plantae:

Phylum: Angiosperms

Class: Magnoliopsida

Order: Malpighiales

Family: Clusiaceae
Genus: Garcinia

Species: Garcinia kola

2.2.3 physicochemical composition of Curcuma longa

Although G. kola seeds are more valued for their medicinal properties rather than as foodstuff,

the kernels are still commonly consumed, which justifies concerns about their nutritional value

(Okoye et al. 2014). There are wide discrepancies among the published results on the species

primary metabolites content. Generally, the studies agree on relatively high amounts of moisture

in the seeds (about 70%), suggesting their vulnerability to mould infestation and possible

storage/post-harvest processing difficulties. Present saccharides, also described as nitrogen-free

extracts (NFE), form the largest part of the seed proximate composition (around 65%), while the

content of minerals is very low (1.5% on average). The mean value for crude protein was found

to be 3.5%, with lysine (2.4 g/kg), leucine (1.9 g/kg) and valine (1.7 g/kg) being the predominant

essential amino acids (AA) and glutamic acid (6.8 g/kg) with arginine (5.5 g/kg) as the highest

abundant nonessential AA in both kernels and seeds’ hulls (Eleyinmi et al. 2006). The crude fat

generally varies about 6.2% with oleic acid (C 18:1; 38 mg/kg), linoleic acid (C 18:2; 36 mg/kg)

and palmitic acid (C 16:0; 32 mg/kg) being the dominant fatty acids in both seeds and hulls

(Eleyinmi et al. 2006). The crude fibre content was determined at 9.4% on average. Before

consumption, people generally prefer to peel the seeds, discarding the hulls as worthless waste.

However, due to their high protein content (9.92 g/100 g), these husks may represent a valuable

fodder source for domestic animals, whose diet is usually based only on natural pastures of poor

quality and thus quite low in protein content (Eleyinmi et al. 2006). If grinded into a powder, the

hulls can be incorporated into enriched feeding mixtures. They were reported to contain
 relatively high amounts of vitamin C (23.1–69 mg/100 g), potassium (25–722 mg/kg) and

phosphorus (3.3–720 mg/kg) (Okwu 2005; Onyekwelu et al. 2015). They are also low in anti-

nutrients such as phytate and oxalate, and are thus considered safe for consumption without any

reports on harmful overdosing (Onyekwelu et al. 2015; Konziase 2015).

Bitter kola (Garcinia kola) is known to contain various phytochemicals such as flavonoids,

tannins, alkaloids, saponins, and phenolic compounds(Adedapo et al. (2009) Various classes of

secondary metabolites have been isolated from different plant parts of G. kola. Of these, perhaps

the most studied are flavonoids and their related structures. Benzophenon,benzofurans and

benzopyran analogues, vitamin E derivatives, xanthones and phytosterols have also been isolated

from G. kola in the past. Many of the present constituents, namely, kolaviron, garcinianin,

kolanone, gakolanone, garcionic acid, garcinal, garcifuran A and B, and garcipyran A, appear to

be exclusive for G. kola

(Pretorius et al.,(2003). Some of the structures are shown in figure one below

X anthones
Fig. 1 Chemical structures of secondary metabolites found in bitter kola (Garcinia kola)

2.2.4 pharmacological properties of G. kola

Garcinia kola is the specific species of plant within the genus Garcinia. It is known for its bitter-

tasting seeds and medicinal properties.( Nworu CS et al.,2010). Garcinia kola has been

traditionally used in African medicine for various therapeutic purposes. The seeds of Garcinia

kola contain bioactive compounds such as kolaviron, which have been studied for their potential

health benefits. Some of the medicinal uses of Garcinia kola include:

Anticancer effect:

Garcinol is attracting a scientific interest mainly due to its ability to inhibit histone

acetyltransferase (HAT), a novel drug target in cancer research. As a HAT inhibitor, garcinol

was found effective at hindering the process of non-homologous end joining in the DNA repair

mechanism, ultimately causing apoptosis of the cancer cells (Oike et al. 2012; Schobert and

Biersack 2019). Other suggested mechanisms of garcinol’s anticancer effect is interference with

NF-jB, iNOS, COX-2 (especially in the inflammatory-induced cancers, such as colorectal

cancer), VEGF, and signal transducer and activator of transcription 3 (STAT-3) pathway (Liu et

al. 2015; Schobert and Biersack 2019). In vivo and in vitro anti-cancer properties of garcinol

have been quite recently and exhaustively reviewed by Aggarwal et al. (2020) and Schobert and

Biersack (2019).

Anti-inflammatory activity:

Garcinol disposed an anti-inflammatory activity in various animal models of induced

inflammation. Majority of studies agree on a mechanism that appears to be related to the

interference with NF-jB, iNOS, ERK, COX-2, p38 mitogen-activated protein kinases (MAPK),

lipoxygenase (5-LOX), TNF-a, interleukin (e.g. IL-2, IL-6, IL-23), nuclear factor of activated T-

cells (NF-AT) (Liu et al. 2015; Schobert and Biersack 2019). Some authors also suggested that

anti-inflammatory effect of garcinol is associated with HAT suppression (Ferriero et al. 2018).

Neurodegenerative disorders and drug withdrawal

Garcinol was found to be an inhibitor of monoamine oxidase B (MAO-B), and as such, it might

be helpful in Parkinson’s disease treatment by retarding dopamine depletion (Mazumder et al.

2018). Additionally, it was discovered that garcinol attenuated the sideeffects and increased

bioavailability of L-DOPA, a dopamine precursor commonly used in the treatment of

Parkinson’s disease symptoms (Mazumder et al. 2016; Ryu et al. 2018). Garcinol also decreased

mortality and seizure scores in mice, presumably by suppressing brain-derived neurotrophic

factor (BDNF) and by having effect on neurotransmitter systems, including those involving

glutamate and GABAA (Hao et al. 2016). Garcinol was also observed to decrease inflammation

of microglia in rats via down regulation of NF-jB pathway and inhibiting COX-2, iNOS, and IL

expression (Wang et al. 2017). A relatively unusual effect of garcinol has been discovered—in

rats exposed to cocaine, garcinol inhibited restoration via reconsolidation-based modes following

cocaine reactivation. The effect of garcinol on reactivated memories were long-lasting,

suggesting a potential in control of drug abstinence and addiction (Fuchs and McLaughlin 2017).

Antiviral and antimicrobial activity

One of the early studies involved investigation on antiviral activity of garcinol against HIV,

where again it was found to be potentially exerting its effect via inhibition of histone

acetyltransferase (HAT) of the HIV infected cells (Mantelingu et al. 2007). Similarly as in the
case of kolaviron, garcinol showed some degree of activity also against influenza virus

(Hatakeyama et al. 2014). It appears that garcinol exerts its antiviral activity against influenza

through regulation of the viral polymerase function (Schobert and Biersack 2019). Garcinol has

demonstrated antibacterial, anti-yeast and antiprotozoal activity which was in some cases equal

or better than conventional treatment. Again, mechanism of its antimicrobial effect might be

related to the HAT inhibitory activity.

Antiparasitic activity

Although G. kola is commonly used in folk medicine to treat malaria, there are relatively few

studies on its antimalarial effect. KV showed anti-malarial activities by suppressing Plasmodium

bergheii in infected laboratory mice (Oluwatosin et al. 2014; Tshibangu et al. 2016). Of all KV

components, GB1 exhibited the almost the same in vitro antimalarial effectivity on P. falciparum

as quinine. In the in vivo test, it was observed that GB1 significantly increased the average life

span of Plasmodium-infected mice (Konziase 2015). Recently, KV was also showed to be

effective against Trypanosoma infections (e.g. T. congolense) both in vitro and in vivo. It has

been suggested that KV may exert its antitrypanosomal activity by interfering with trypanothione

reductase, an enzyme responsible for homeostasis maintenance (Timothy et al. 2021).

Anti-snake venom activity

Anti-snake venom activity forms a relatively narrow area of KV research. As far as we know,

only one study addressed this issue. Quite recently Okafor and Onyike (2020) suggested that the

KV may produce inhibitory effect against hydrolytic enzymes of Naja nigricollis venom, namely

phospholipase A2 (PLA2), protease, hyaluronidase and l-amino acid oxidase, and thus also

neutralize their myotoxic, oedemic, haemolytic and procoagulant effects. However, KV was
assayed at quite high doses (venom:KV 1:5 w/w) and reasonable inhibition was only observed in

the case of PLA2. It is questionable whether these high doses of KV are clinically relevant.

Effect on reproduction and infertility

Garcinia kola is relatively widely used in traditional medicine as an aphrodisiac. Corresponding

with this fact, studies have been focused on examining the effect of present substances on

reproductive properties. KV was found to prevent testicular damage and decline of sex hormones

upon administration of various toxic agents. Administration of these agents resulted in increased

levels of antioxidant/detoxifying enzymes (catalase, superoxide dismutase, glutathione S-

transferase) and markers of oxidation (e.g. elevated hydrogen peroxide and malondialdehyde).

Additionally, the rats that had been treated with KV also showed improved semen characteristics

(e.g. sperm count). It was also found out, that KV has lowered the negative effect of EGEE on

activities of 3b-hydroxysteroid dehydrogenase (3b-HSD) and 17b-hydroxysteroid dehydrogenase

(17b-HSD), enzymes that are associated with production of steroidal hormones (e.g.

testosterone) (Adedara and Farombi 2013).

Diabetes

Investigations were made to figure out if KV can act as a potential source of diabetes treatment.

The compound showed a hypoglycaemic effect in alloxaninduced diabetic rabbits and

streptozotocin-induced diabetic rats (Iwu et al. 1990b; Adaramoye and Adeyemi 2006;

Adaramoye 2012). Though there is no generally accepted mechanism of action yet, KV was

suggested to produce its antidiabetic effect via inhibition of a-glucosidase and a-amylase

activities (Iwu et al. 1990b; Salau et al. 2020). Recent study has also suggested that KV may play
a regenerative role in pancreatic islets in streptozotocin-induced diabetic rats (Oyenihi et al.

2021).

2.2.5 Mechanism of G.kola against klebsiella pneumoniae.

Klebsiella pneumoniae is a significant human pathogen that is increasingly becoming resistant to

multiple antibiotics, posing a challenge for the treatment of infections caused by this bacterium.

In this context, the search for alternative antimicrobial agents, especially those derived from

natural sources, has gained attention. Garcinia kola, a plant with a long history of traditional

medicinal use, has shown promise in combating bacterial infections, including those caused by

Klebsiella pneumoniae. Understanding the mechanisms by which Garcinia kola acts against this

pathogen is crucial for harnessing its therapeutic potential. Garcinia kola, The mechanism of

action of Garcinia kola against Klebsiella pneumoniae can be attributed to its bioactive

compounds, particularly kolaviron. Kolaviron is a complex mixture of bioflavonoids, including

biflavonoids and prenylated xanthones, which have been shown to possess antimicrobial activity.

Studies have indicated that kolaviron extracted from Garcinia kola can disrupt the cell membrane

of bacteria, leading to cell lysis and death ( Omojasola et al. (2015). The mechanism of action of

kolaviron against Klebsiella pneumoniae in terms of inhibition of cell wall synthesis can be

explained by its interaction with enzymes involved in peptidoglycan biosynthesis. Peptidoglycan

biosynthesis is a complex process that involves the sequential action of several enzymes,

including glycosyltransferases and transpeptidases. These enzymes are essential for the synthesis

and cross-linking of peptidoglycan, which is crucial for maintaining the integrity and stability of

the bacterial cell wall. Kolaviron may interfere with the activity of these enzymes by binding to

their active sites or by disrupting their function through allosteric inhibition. By inhibiting key

enzymes involved in peptidoglycan biosynthesis, kolaviron can disrupt the synthesis and
assembly of the bacterial cell wall, leading to cell lysis and death. This mechanism of action is

similar to that of β-lactam antibiotics, which also target enzymes involved in cell wall synthesis

(Adeyemi et al. 2015)

2.3 The evaluation of phytochemical constituent in Garcinia kola

A qualitative analysis of aqueous Garcinia kola nut extract was done by Joel et al 2023 to

determine various secondary metabolite in G. kola the results obtained showed a positive test of

alkaloid upon the addition o f 1.0 mL of Picric Acid was added to about 2.0 mL of the extract. A

yellow precipitate was observed which indicated the presence of alkaloid, 1.0 mL of the filtrate

was diluted in 1.0 mL of water and shaken vigorously. Persistence foam indicated the presence

of saponins, the extract (1.0 mL) was diluted in 2.0 mL of 10% NaOH. A yellow precipitate was

formed and following the addition of dilute HCl, the yellow colour turned colourless. This

indicated the presence of flavonoids, 1.0 mL of 10% Ferric chloride was added to the extract (1.0

mL). The formation of a greenish brown precipitate indicated the presence of phenols, 5.0 mL of

the extract was added to 2.0 mL of 1.0% HCl. Deposition of a red precipitate showed the

presence of tannins and Salkowski test was used to detect the presence of terpenoids. 5ml of the

G. kola was mixed 2ml of chloroform and 3ml of cocentrated sulpharic acid was carefully added

to form a layer. A reddish - brown coloration of the inter face formed indicated the presence of

terpenoids.

2.4The invitro effect of G. kola on K. pneumonia

According to research conducted by Ibrahim et al. (2020), Garcinia kola, a plant native to

Western and Central Africa, has been found to have significant medicinal importance. The study

investigated the antibacterial activity of aqueous and methanol extracts from G. kola seeds
against several bacterial isolates, including Klebsiella pneumoniae, Shigella species, and

Salmonella typhi. The methanol extract demonstrated greater antibacterial activity compared to

the aqueous extract, showing effects against K. pneumoniae and Shigella species, but not against

S. typhi. The minimum inhibitory concentration (MIC) of the aqueous extract on all test

organisms was 1.40 mg/ml, while the MIC of the methanol extract was 1.25 mg/ml for K.

pneumoniae and Shigella species. Additionally, the methanol extract exhibited bactericidal

effects on K. pneumoniae and Shigella species at a concentration of 2.5 mg/ml. studies

conducted by Arekenase at al., 2012 found the zone of inhibitions from 17 to 23mm for ethanolic

extract of G. kola and 20 to 27mm for aqueous (using hot water) extract at a concentration

ranging from0.625 to 2.50mg/ml.

 CHAPTER THREE

RESEARCH METHODOLOGY

3.1 RESEARCH DESIGN

The research follows an experimental design where the aim is to evaluate the intro effect of

Garcina kolo on K. pneumonia and to conduct a qualitative phytochemical analysis on the plant.

The study involves both qualitative and quantitative assessments.

3.2 Study area

This research was carried out in the Biaka university Teaching laboratory which is found in

Buea, Fako division in the South West Region.

3.3 Bacterial species

The test organisms for the study will be an isolates of K. pneumonia species.

3.4 Research Materials and Equipment

Mortar and Pestle, Conical flask, Beaker, Measuring cylinders, Water bath, Filter papers, Petri

dishes, Auto clave, Capillary tube, Inoculation wire loop, Funnels, Syringes, Culture plates

mannitol salt, potato glucose agar and sabouraud destrose agar), weighing balance, foil paper,

conical flask, Oven, Refrigerator, Condenser. Chemical reagent used includes Fehling solutions

(A and B), Methanol, distilled water, Ferric chloride 3.5% (3.5 mL of FeCl 3 in 96.5 mL of

solvents), Dilute tetraoxosulphate (IV) acid (H2SO4), Sodium hydroxide (NaOH), Zinc chips,
Meyer’s reagent, Dragendoff’s reagent, Ammonia, Chloroform (CHCl 3), Nutrient agar, Ethyl

acetate, Acetone, Aqueous hydrochloric acid (1.0% HCl), Ethanol, Benzene and methanol.

3.5 Samples collection

Samples of Klebsiella pneumoniae bacteria will be collected from stool of an infected at St

Veronica Hospital, Biaka, Buea, Cameroon.The collected samples will be then immediately

close in an air tight container to avoid air contamination, and will be brought brought to the

Biaka teaching lab. Garcinia kola will be purchase from the Muea market.

3.6 Collection and Preparation of Plant Extract

The outer layer of the Garcinia kola will be carefully peeled with a knife, rinsed with potable

water, sliced and dried in the oven at 50 °C for 24 hours. The dried samples will be then milled

using a Blender into powder and allowed to pass through a sieve with a nominal mesh size of 0.2

mm in diameter. 250g of sieved powder will be dissolved in 500ml of ethanol overnight (24hrs)

for extraction, after 24hrs of extraction it was heated on a water bath to evaporate all the ethanol

and kept for further analysis.

3.7 Isolation of K. pnomonia

3.7.1 Preparation of MacConkey agar

10g of MacConkey agar will be weighed using an electronic balance and dissolved in 90ml of

distilled water in a conical flask and then heated using a water bath to completely dissolve.

MacConkey agar will be then sterilizes in an autoclave at a pressure of 121 degrees for 15mins to

ensure that the media is free from microorganisms. The container or conical flask will be

removed from the autoclave and allow cool at temperature of 50 degree and the sterilize medium
will be then immediately poured into 8 petri dishes of 100mm and bubbles where remove by

flaming. Following solidification of the agar , place the petri dishes in a plastic sleeve and store

at 4 degree until needed.

3.7.2 Culture and antimicrobial susceptibility testing:

The following steps outline the culturing of K. pneumonia on culture media which has been

solidified in the petri – dish.

All materials and equipment used for culturing were Sterilize with a Bunsen banner. The isolated

sample collected was dissolved in 2mls sterile distilled water in the test tube to ensure the even

distribution of the bacteria on the surface of the plate and to prevent clumping of the bacterial. A

sterile swap was gently deep into the dissolve dissolved sample in the test tube and excess liquid

was press against the walls of the test tube and it was inoculated on the patri dish near lame to

prevent any contamination. The strike plate was closed and incubated for 48hrs at a temperature

of 37-degree census. After the incubation period the plates will be observed for the presence a

large, mucoid, and red with diffusing red pigment colonies which indicated the successful

growth of K. pneumonia. Wells will be created on the fresh agar plates using a sterile cork borer.

A sterile loop will be used to transfer a small amount of the bacterial from the agar plater into

fresh agar plate. Each well was filled with 100μl of the ethanolic kola extract at the desired

concentration. The plates were incubated at 37°C for 24 hours. The diameter of the zones of

inhibition was measured using a ruler.

3.8 Phytochemical Analysis of turmeric, neem and tulsi leaves extract

Chemical tests for the identification of bioactive chemical constituents in the turmeric extract

were carried out using a standard procedure as shown below;

3.8.1 Test for phenols and Tannins
2 ml of the extract was mixed with 3ml of 2% solution of FeCl3. A black coloration indicated

the presence of phenol and tannins.

3.8.2 Test for terpenoids (Salkowski’s Test)

2 ml of the extract was mixed with 3 ml of chloroform. Then 3 ml of concentrated sulfuric acid

was added carefully and shaken gently. A reddish brown coloration formed at the interphase

showed positive results for the presence of terpenoids.

3.8.3 Test for glycosides

2 ml of the extract was mixed with 3ml of glacial acetic acid containing 3 drops of 2% FeCl3. To

another tube containing 3 ml of concentrated sulfuric acid, the mixture was poured into it. A

brown ring at the interphase indicated the presence of glycosides.

3.8.4 Test for flavonoids (Shinoda test)

2 ml of the extract was mixed with 5 pieces of magnesium ribbons and concentrated

hydrochloric acid was added drop wise noting the color change. Pink or orange coloration

indicated the presence of flavonoids.

3.8.5 Test for saponins

4 ml of the solvent extract was placed in a test tube and shaken vigorously noting down the

observations. The formation of stable foam indicated the presence of saponins

3.9 Data Analysis:

The data obtained from the antimicrobial susceptibility testing was analyzed using descriptive

statistics and the different phytochemical constituents where evaluated qualitatively by visually
observation of their changes. The mean and standard deviation of the zone of inhibition were

calculated for each concentration of the ethanolic extract of the kola. A comparative analysis will

be conducted to determine the differences in antimicrobial activity between the different

concentrations and bacterial strains.

3.10 Ethical Considerations:

Prior approval for this research was obtained from the relevant ethics committee. All laboratory

experiments were conducted following standard biosecurity and safety protocols to ensure the

safe handling of microorganisms.