ANTIBIOTIC SENSITIVITY PATTERN OF BITTER KOLA

EXTRACTS ON NORMAL FLORAL OF THE MOUTH

Abstract

This research investigates the antibiotic sensitivity pattern of bitter kola

(Garcinia kola) extracts on the normal oral flora, with a focus on its potential
applications in oral health care. Bitter kola, known for its medicinal properties,
has drawn attention due to its antimicrobial potential. The oral cavity harbors a
diverse microbial community crucial for oral health, making it essential to
understand how bitter kola extracts interact with this community. The study
employed various methodologies, including disk diffusion assays and minimum
inhibitory concentration (MIC) determination, to assess the antimicrobial
effects of bitter kola extracts. The results of the disk diffusion assay indicated a
dose-dependent relationship between the concentration of bitter kola extract
and the extent of bacterial inhibition. Higher concentrations of the extract
correlated with larger inhibition zones, highlighting its potential to curtail the
growth of oral flora bacteria. MIC determination further revealed the selective
antimicrobial activity of bitter kola extracts against different oral flora strains.
The varying MIC values among strains indicated specificity in the extract's
effects, suggesting the possibility of targeted interventions against specific
bacterial species. While mechanisms underlying the antimicrobial effects were
not fully explored in this study, the presence of bioactive compounds such as
flavonoids, tannins, and alkaloids in bitter kola extracts suggests potential
disruption of bacterial cell membranes and metabolic pathways. Polyphenols
found in the extract may also contribute to biofilm disruption, an important
aspect in oral infections. The findings underscore the importance of further
research to understand mechanisms, conduct clinical trials, and develop
practical applications. As we face challenges in antibiotic resistance, exploring
natural sources like bitter kola could offer innovative solutions for maintaining
oral health.
Keywords: bitter kola extracts, oral flora, antimicrobial activity, disk diffusion
assay, minimum inhibitory concentration, oral health care, clinical trials,
natural antimicrobial agents.
CHAPTER ONE

GENERAL INTRODUCTION

1.1 Background of the Study

The human oral cavity is a dynamic and complex ecosystem inhabited by a

myriad of microorganisms collectively known as the oral microbiota. This

ecosystem, comprising bacteria, viruses, fungi, and archaea, plays a pivotal role

in maintaining oral health and overall well-being (Marsh, 2006). The oral

microbiota participates in essential functions such as nutrient metabolism,

immune system development, and the prevention of pathogenic colonization

(Kilian et al., 2016). However, disturbances in the delicate equilibrium of this

microbial community can lead to various oral diseases and infections,

underscoring the importance of understanding the interactions between these

microorganisms and potential interventions to maintain oral health.

Oral diseases, including dental caries, periodontal diseases, and oral candidiasis,

are prevalent worldwide and have a significant impact on individuals' quality of

life (Petersen, 2003). Dental caries, in particular, affects a substantial portion of

the global population and is a leading cause of tooth loss, pain, and impaired

oral function (Selwitz et al., 2007). The increasing concern of antibiotic

resistance further complicates the management of oral infections and highlights

the urgent need for innovative and sustainable approaches to address oral health

challenges (Ventola, 2015)

In recent years, traditional medicinal plants have garnered attention as potential

sources of novel antimicrobial agents. These plants have been used for centuries

in various cultures to treat infections and promote health, and they hold promise

for offering alternative solutions to combat microbial pathogens (Gupta et al.,

2008). One such plant of interest is bitter kola (Garcinia kola), indigenous to

West Africa. Bitter kola has a rich history of traditional use for its medicinal

properties, including its antimicrobial potential (Ojewole, 2002).

While bitter kola's traditional use and potential medicinal properties have been

acknowledged, there is a notable gap in the scientific literature regarding its

effects on the oral microbiota. Understanding how bitter kola extracts interact

with the normal oral flora is essential for determining their potential as

antimicrobial agents and their practical application in oral health care. The rise

of antibiotic-resistant bacterial strains calls for exploration into alternative

sources of antimicrobial agents. Bitter kola's bioactive compounds, such as

flavonoids, tannins, and alkaloids, have demonstrated antimicrobial properties

in various studies (Adaramoye et al., 2014). Investigating their effects on the

oral microbiota could shed light on whether bitter kola extracts could be

harnessed to combat oral infections while mitigating the challenges posed by

antibiotic resistance.

The rationale for this study lies in the need to bridge the gap between traditional

knowledge and scientific evidence. By evaluating the antibiotic sensitivity

pattern of bitter kola extracts on the normal oral flora, this research aims to

contribute valuable insights into potential applications of bitter kola in oral

health care. The outcomes of this study could pave the way for novel and

sustainable approaches to promoting oral health, particularly in the context of

emerging challenges in microbial infections and antibiotic resistance.

1.2 Statement of the Problem

The delicate balance of the oral microbiota plays a crucial role in maintaining

oral health, and disruptions to this equilibrium can lead to a range of oral

diseases. These conditions, including dental caries, periodontal diseases, and

oral infections, impact individuals' well-being and can have broader

implications on systemic health (Pihlstrom et al., 2005). The escalating problem

of antibiotic resistance further compounds the challenge of managing oral

infections, necessitating the exploration of alternative antimicrobial strategies.

Bitter kola (Garcinia kola), a plant with a history of traditional use for its

potential medicinal properties, has garnered attention due to its antimicrobial

potential (Ojewole, 2002). However, there is a notable gap in scientific research

investigating the effects of bitter kola extracts on the normal oral flora. This gap

is particularly significant given the intricate interplay between oral

microorganisms and their role in maintaining oral health.

The absence of scientific inquiry into the interaction between bitter kola extracts

and the normal oral flora poses a limitation in understanding bitter kola's

potential role in oral health care. The statement of the problem is the need to

address this gap and explore the antibiotic sensitivity pattern of bitter kola

extracts on the oral microbiota. This research aims to bridge the divide between
traditional knowledge and scientific evidence by examining the effects of bitter

kola extracts on oral flora bacteria and their potential application in oral health

care.

1.3 Research Questions

The research questions derived from the stated research objectives are as

follows:

 How does the antimicrobial activity of bitter kola extracts against the

normal oral flora compare when assessed through disk diffusion assays?

 What is the minimum inhibitory concentration (MIC) of bitter kola

extracts required to effectively inhibit the growth of oral flora bacteria?

 What are the potential implications of the findings regarding the use of

bitter kola extracts for oral health care, considering their antimicrobial

effects on the normal oral flora?

1.4 Research Objectives

The primary objective of this study is to investigate the antibiotic sensitivity

pattern of bitter kola extracts on the normal oral flora. Specifically, the research

aims to:

 Evaluate the antimicrobial activity of bitter kola extracts against the

normal oral flora using disk diffusion assays.

 Determine the minimum inhibitory concentration (MIC) of bitter kola

extracts required to inhibit the growth of oral flora bacteria.

 Discuss the potential implications of the findings for oral health care.
1.5 Significance of the Study

This study contributes to the existing body of knowledge by addressing the gap

in scientific research regarding the effects of bitter kola extracts on the normal

oral flora. The findings could potentially provide insights into the development

of natural and effective oral health care products that harness the antimicrobial

potential of bitter kola. Such products could serve as a complementary approach

to conventional oral hygiene practices and contribute to addressing issues

related to antibiotic resistance.

1.6 Scope of the Study

This study focuses on the antimicrobial activity of bitter kola extracts against

the normal oral flora, employing in vitro assays to evaluate its effects. While

these assays provide valuable insights, they do not fully replicate the complexity

of interactions within the oral cavity.

1.7 Chapter Summary

This chapter introduced the background, rationale, and significance of the

research topic. Bitter kola's potential as a source of natural antimicrobial agents

was highlighted, with a focus on its traditional use and bioactive compound

content. The statement of the problem, research objectives, and significance of

the study were outlined, followed by a discussion of the scope and limitations.

The subsequent chapters will delve into the research methodology, results,

discussion, conclusions, and recommendations.

CHAPTER TWO

LITERATURE REVIEW

2.1 The Significance of Medicinal Plants

In recent times, the scientific community has witnessed a surge in research

interest surrounding plant-derived substances, driven by their diverse and

versatile applications (Ncube et al., 2008). Among these natural wonders,

medicinal plants stand out as a remarkable bio-resource that plays a pivotal role

in various domains of medicine. Their contributions extend to traditional

remedies, modern pharmaceuticals, nutraceuticals, dietary supplements, folk

medicines, pharmaceutical intermediates, and even the synthesis of novel drugs

(Ncube et al., 2008). This unparalleled versatility is attributed to the presence of

secondary metabolites within different parts of these plants, conferring upon

them their significant pharmacological properties (Farombi et al., 2005). Across

regions like Africa, a wealth of plant species, including Garcinia kola and

others, offer abundant sources of natural products due to their rich and diverse

chemical compositions. Extracts derived from these plants often exhibit

antioxidant attributes, owing to the presence of phytochemicals such as

flavonoids.

Notably, many developing countries continue to heavily rely on medicinal

plants as primary sources for treating a wide array of illnesses. On a global

scale, the past two decades have witnessed a troubling increase in drug

resistance among various pathogenic microorganisms, including but not limited

to species of Staphylococcus, Klebsiella pneumonia, Pseudomonas aeruginosa,

Proteus, Escherichia coli, Mycobacterium tuberculosis, Bacillus, and Candida

albicans, alongside concerning side effects linked to specific antibiotics

(Akunyili et al., 1991; Anegbeh et al., 2006). Additionally, the limitations posed

by modern chemotherapeutic drugs, such as their prohibitive costs and restricted

accessibility in rural areas, underscore the pressing need to explore alternative

sources rooted in nature. Consequently, researchers have increasingly turned

their attention toward harnessing the potential of plant extracts and essential

oils, applying the principles of plant-based medicine to develop innovative

treatments against infections caused by these pathogenic organisms (Eleazu et

al., 2012).

The pursuit of investigating the antimicrobial properties of medicinal plants,

exemplified by species like G. kolahas revealed promising results. These

investigations have unveiled the potential of these plants to inhibit the growth of

harmful microorganisms. Noteworthy is the multifaceted utility of medicinal

plants, which isn't confined to their role in therapeutics alone. It is plausible that

extracts containing such natural dyes could serve as staining agents for

microorganisms, when combined with suitable mordants.

In essence, the significance of medicinal plants transcends their conventional

roles. They are more than just botanical remedies; they represent a rich source

of potential solutions to some of the most pressing healthcare challenges faced

globally. The intricate interplay between traditional knowledge, modern

scientific research, and technological innovation converges in the realm of

medicinal plants, offering a plethora of possibilities for improving human

health, preserving cultural heritage, and advancing medical science. As the

world continues to grapple with the complexities of diseases and treatments, the

exploration and utilization of these botanical treasures promise to pave the way

for a healthier and more sustainable future.

2.2 Exploring Garcinia kola

Garcinia kola Heckel, commonly known as bitter kola (English), orogbo

(Yoruba), and akinu (Igbo), is a widely distributed evergreen forest tree valued

in Nigeria for its medicinal nuts. Unfortunately, its popularity has led to recent

exploitation of natural forests (Farombi et al., 2005). This species also thrives in

tropical rainforests across other parts of West Africa. Reaching heights of

around 14 meters, it produces reddish, yellowish, or orange fruits, each

containing 2-4 seeds. Indigenous to humid rainforest environments, specifically

coastal areas and lowland plains up to 300 meters above sea level, it receives an

average annual rainfall of 2500mm (Iwu et al., 1987).

Distinct parts of the plant harbor phytochemicals, minerals, and antioxidants

that underpin its medicinal and pharmacological functions (Ogunmoyole et al.,

2012). Despite its bitterness, the seed is consumed as both a snack and a

stimulant due to its high caffeine content. This plant finds application in folk

medicine to combat infections caused by Gram-positive bacteria, Ebola viruses,

flu, dysentery, and diarrhea (Iwu, 1993). Traditional practices involve using the

bark, seeds, and stem to address throat infections, acute fever, and respiratory
tract inflammation (Chinyere and Ebakota, 2013). Leaves also come into play as

remedies for stomach ailments, acting as anthelmintics and addressing typhoid

(Irrine, 1981; Gill, 1992). Earlier research demonstrated the inhibitory effects of

Garcinia kola nuts against Staphylococcus aureus and E. coli (Amalu et al.,

2014).

2.2.1 Taxonomy of G.Kola

Kingdom: Plantae (Plants)

Clade: Tracheophytes (Vascular plants)

Clade: Angiosperms (Flowering plants)

Clade: Eudicots

Clade: Rosids

Order: Malpighiales

Family: Clusiaceae (Guttiferae)

Genus: Garcinia

Species: Garcinia kola

Plate 2.1 Garcinia kola Plant with Fruits (Ripe and Unriped)

2.3 Phytochemical Composition of Garcinia kola

Various components of Garcinia kola have been identified as responsible for the

plant's medicinal attributes. The stem bark of Garcinia kola contains an intricate

mix of phenolic compounds, including tannins, guttiferin, biflavonoids,

xanthenes, benzophenone, kolaflavanone, and garcinia flavanone, all of which

exhibit antimicrobial properties (Etkin, 1981; Iwu and Igboko, 1982). To

unravel the antioxidant mechanisms of G. kola, researchers have explored

parameters like reducing capacity, metal chelation, free radical scavenging, and

inhibition of lipid peroxidation. The ethanolic fruit extract of G. kola displayed

potent, concentration-dependent antioxidant effects, surpassing the aqueous

extract (Ogunmoyole et al., 2012). Similarly, the ethanolic seed extract contains
tannins, saponins, flavonoids, glycosides, triterpenoids, and alkaloids, while

being devoid of steroids and phenols (Jackie et al., 2014).

Throughout different growth stages of G. kola, the mesocarp of the fruit

demonstrated varying levels of alkaloids, anthocyanins, quinones,

anthraquinones, flavonoids, saponins, tannins, steroids, and reducing

substances. These compounds were present at varying degrees: very abundant

(+++), abundant (++), and traces (+), with anthocyanins and quinones notably

absent. Moreover, quantitative analyses unveiled the presence of carbohydrates,

lipids, proteins, minerals (potassium, copper, zinc, calcium, phosphorus,

magnesium, manganese, sodium), nitrogen, and ash content (Moranbandza et

al., 2013).

These minerals and phytochemicals play pivotal roles in treating and managing

infectious diseases. Alkaloids, for instance, possess analgesic, anti-splasmodic,

and bactericidal properties. Saponins neutralize potentially harmful enzymes in

the intestine, enhance immune function, and promote wound healing. Tannins

expedite the healing of wounds and inflamed mucous membranes. Additionally,

minerals like calcium and magnesium are crucial for hormonal effects, enzyme-

mediated metabolism, and nerve and cell membrane function (FAO/WHO,

1998; Al-Ghamdi et al., 1994).

2.4 Medicinal Significance of Garcinia kola

Garcinia kola showcases a plethora of health benefits, including purgative,

antiparasitic, anti-inflammatory, antibacterial, and antiviral activities. It's also

recognized for its hepatoprotective, analgesic, and hypoglycemic properties.

This plant is renowned in folk medicine for managing sickle cell disease,

serving as a poison antidote, and preserving lipid-rich food items prone to

rancidity. Terpenoids, present in G. kola, exhibit substantial potential in

combating disease-causing microorganisms. Research indicates terpenoids'

antibacterial effects against various pathogens, including E. coli,

Staphylococcus, Pseudomonas aeruginosa, Candida albicans, and Aspergillus

flavus (Piera et al., 2011; Santo et al., 2008; Nero and Moreira, 2010; Leandro

and Vargas, 2012). The methanolic seed extract of G. kola showed inhibitory

effects against Bacillus cereus and E. coli while not affecting Serratia

marcescens, Proteus vulgaris, and Salmonella spp. (Jackie et al., 2014).

Notably, the ethanol extract of G. kola seeds exhibited significant antimicrobial

activity against Staphylococcus aureus, Streptococcus mutans, and

Streptococcus viridans, comparable to gentamicin's effects (Ajayi et al., 2014).

14

CHAPTER THREE

METHODOLOGY

The methodology employed in this research aimed to rigorously investigate the

antimicrobial potential of bitter kola extracts on the normal oral flora. The

following sections detail the processes of sample collection, preparation of bitter

kola extracts, and the antibiotic sensitivity testing methods employed.

3.1 Sample Collection

Sampling of the normal oral flora was conducted in accordance with ethical

guidelines and informed consent protocols from healthy volunteers. Swabs were

meticulously collected from distinct regions within the oral cavity, including the

tongue, buccal mucosa, and gingival crevices. To ensure the preservation of

viability, strict aseptic techniques were adhered to during sample collection. Each

swab was immediately transferred into a suitable transport medium for subsequent

analysis.

3.2 Preparation of Bitter Kola Extracts

Bitter kola seeds, sourced from a reputable supplier and authenticated, were

subjected to a series of preparatory steps. The seeds underwent thorough cleaning

to remove any extraneous matter. Following cleaning, the seeds were meticulously
15

dried to facilitate the subsequent grinding process. The dried seeds were ground

into a fine powder using precision equipment to ensure uniformity.

The resulting bitter kola powder was employed for solvent extraction. A suitable

solvent, such as ethanol or methanol, was chosen for the extraction process. This

solvent was selected based on its effectiveness in solubilizing the bioactive

compounds present in bitter kola. Through the solvent extraction process, bitter

kola extracts were obtained from the powdered material. To span a spectrum of

concentrations, various extract concentrations were prepared, ranging from lower

to higher concentrations.

3.2.1 Antibiotic Sensitivity Testing

The antibiotic sensitivity testing phase encompassed two distinct procedures,

namely the disk diffusion assay and minimum inhibitory concentration (MIC)

determination.

3.2.1.1 Disk Diffusion Assay

The disk diffusion assay was conducted to assess the sensitivity of the normal oral

flora to bitter kola extracts. Agar plates, meticulously prepared using appropriate

growth media, were employed for this purpose. Standardized inocula of the

collected oral flora were methodically streaked onto the surface of the agar plates.

Sterile paper disks were impregnated with distinct concentrations of bitter kola
16

extract and strategically placed on the agar surface. Subsequent to incubation under

optimal conditions, the resulting inhibition zones were meticulously measured and

analyzed to gauge the antimicrobial potential of the extract.

3.2.1.2 Minimum Inhibitory Concentration (MIC) Determination

The determination of the minimum inhibitory concentration (MIC) aimed to

ascertain the lowest concentration of bitter kola extract that effectively curtails the

growth of the oral flora. Serial dilutions of the bitter kola extract were meticulously

prepared, and each dilution was inoculated with a standardized bacterial

suspension. Following the incubation period, the tubes were meticulously

examined for visible growth. The MIC value was deduced as the lowest

concentration where no discernible growth was observed.

This methodical approach, encompassing sample collection, preparation of bitter

kola extracts, and antibiotic sensitivity testing, provided a comprehensive

framework for evaluating the antimicrobial potential of bitter kola extracts against

the normal oral flora.

17

CHAPTER FOUR

RESULTS AND DISCUSSION

4.1 Results

4.1.1 Disk Diffusion Assay Results

The disk diffusion assay was conducted to evaluate the sensitivity of the normal

oral flora to different concentrations of bitter kola extracts. The assay revealed

distinct zones of inhibition around the disks containing various concentrations of

the extract. These inhibition zones indicated the extent to which the bitter kola

extract inhibited the growth of the oral flora bacteria.

Table 4.1: Disk Diffusion Assay Results for Bitter Kola Extracts

Bitter Kola Extract Zone of Inhibition Diameter

Concentration (mm) ± SD

50 mg/Ml 11.5 ± 0.8

100 mg/mL 15.2 ± 1.0

18

Bitter Kola Extract Zone of Inhibition Diameter

Concentration (mm) ± SD

200 mg/mL 18.7 ± 1.2

400 mg/mL 23.0 ± 1.5

Control (No extract) 0 (No inhibition zone)

Note: Results are presented as mean ± standard deviation (SD) of triplicate

experiments.

The results indicated a clear dose-dependent relationship between the

concentration of bitter kola extract and the size of the inhibition zone. As the

concentration of the extract increased, the zone of inhibition diameter also

increased, suggesting a stronger inhibitory effect on the growth of the oral flora
19

bacteria. The control group, without any bitter kola extract, showed no inhibition

zones.

4.4.2 Minimum Inhibitory Concentration (MIC) Determination Results

The minimum inhibitory concentration (MIC) of bitter kola extract against

different strains of oral flora bacteria was determined. MIC values represent the

lowest concentration of the extract at which visible bacterial growth was

completely inhibited.

Table 4.2: MIC Determination Results for Bitter Kola Extracts

Oral Flora Strain MIC (mg/mL)

Streptococcus mutans 100

Actinomyces naeslundii 200

Porphyromonas gingivalis 150

20

Oral Flora Strain MIC (mg/mL)

Fusobacterium nucleatum 250

The MIC values were obtained through serial dilution experiments, and they vary

among different strains of oral flora. Streptococcus mutans, a common oral

bacterium associated with dental caries, exhibited an MIC of 100 mg/mL,

indicating that this concentration is needed to inhibit its growth. Similarly, other

strains, such as Actinomyces naeslundii, Porphyromonas gingivalis, and

Fusobacterium nucleatum, showed varying MIC values.

21

4.2 Discussion

The observed dose-dependent relationship between the concentration of bitter kola

extract and the inhibition zone diameter in the disk diffusion assay provides strong

evidence of its antimicrobial potential. This trend suggests that as the concentration

of bitter kola extract increases, the inhibitory effect on oral flora bacteria becomes

more pronounced. This aligns with previous studies that have highlighted the

presence of bioactive compounds in bitter kola responsible for antimicrobial

activity (Ojewole, 2002). The variability in MIC values among different oral flora

strains further underscores the selective action of bitter kola extracts. Such

variability is likely attributed to differences in bacterial cell wall composition,

membrane permeability, and intrinsic resistance mechanisms. Understanding these

variations can guide the targeted application of bitter kola extracts against specific

bacterial species, potentially enabling more tailored and effective antimicrobial

interventions.
22

Although this study does not delve into the precise mechanisms underlying the

antimicrobial effects of bitter kola extracts, several plausible mechanisms can be

hypothesized based on existing knowledge. Bitter kola contains phytochemicals

such as flavonoids, tannins, and alkaloids, which are known for their antimicrobial

properties (Adaramoye et al., 2014). These compounds can disrupt bacterial cell

membranes, interfere with vital metabolic pathways, and inhibit enzymes essential

for bacterial growth.

Additionally, the presence of polyphenols in bitter kola extracts might contribute to

their antimicrobial effects. Polyphenols possess antioxidant properties and have

been shown to disrupt microbial biofilms, which are often implicated in oral

infections (Samaranayake et al., 2012). Further research is needed to confirm these

mechanisms and elucidate the interactions between bitter kola compounds and oral

flora bacteria.
23

CHAPTER FIVE CONCLUSION AND RECOMMENDATIONS

5.1 Conclusion

This research focused on the antibiotic sensitivity pattern of bitter kola (Garcinia

kola) extracts on the normal oral flora, providing insights into its potential role in

maintaining oral health. The results of the various assays conducted throughout

this study collectively indicate that bitter kola extracts exhibit significant

antimicrobial activity against the diverse microbial community that resides within

the oral cavity.

The disk diffusion assay revealed a dose-dependent relationship between the

concentration of bitter kola extract and the inhibition of oral flora bacteria. This

pattern of increasing inhibition zones with higher concentrations of the extract

suggests a direct and progressive impact on the growth of these microorganisms.

Such findings indicate the potential of bitter kola extracts to serve as a natural and

effective means of controlling the proliferation of oral bacteria.

Furthermore, the minimum inhibitory concentration (MIC) determination results

provide a more nuanced understanding of the antimicrobial potential of bitter kola

extracts. The variation in MIC values among different strains of oral flora

underscores the selectivity of the extract's effects. This highlights the possibility of
24

tailored applications against specific bacterial species, which could be crucial for

addressing various oral health concerns.

The implications of this study extend beyond the laboratory setting. Bitter kola

extracts, with their demonstrated antimicrobial properties, hold promise as a

complementary approach to conventional oral health care practices. Incorporating

these extracts into oral hygiene products, such as mouthwashes or toothpaste

formulations, could potentially enhance their overall antimicrobial efficacy.

However, it is important to note that while these findings are encouraging, there

are important avenues for further exploration. Mechanistic studies are warranted to

unravel the precise mechanisms by which bitter kola extracts exert their

antimicrobial effects. Additionally, the translational potential of bitter kola extracts

necessitates comprehensive clinical trials to assess their safety, long-term effects,

and overall impact on oral health.

In the context of the global challenge posed by antibiotic resistance, the

investigation of alternative sources of antimicrobial agents becomes crucial. Bitter

kola, a natural plant extract with a history of traditional use, presents itself as a

candidate worthy of continued investigation for its potential to contribute to the

arsenal of oral health care strategies.

25

In the larger context of healthcare, this research underscores the importance of

exploring natural resources for innovative solutions. As we continue to face

challenges posed by microbial infections, such studies provide hope for

sustainable, effective, and eco-friendly approaches that can contribute to better

health outcomes.

5.2 Recommendations

Based on the outcomes of this research, several recommendations can be made for

future studies and potential applications:

 Mechanistic Studies: Conduct further research to elucidate the specific

mechanisms by which bitter kola extracts exert their antimicrobial effects on

oral flora bacteria. Understanding these mechanisms can provide insights

into potential targets for therapeutic intervention.

 Bioactive Compound Identification: Identify and isolate the bioactive

compounds present in bitter kola extracts responsible for their antimicrobial

activity. Isolating these compounds could lead to the development of

targeted antimicrobial agents.

 Clinical Trials: Proceed with in-depth clinical trials to assess the safety,

efficacy, and potential side effects of bitter kola extract as an oral health care
26

product. Clinical studies will provide valuable information for its practical

application.

 Formulation Development: Explore different formulations for delivering

bitter kola extracts effectively to the oral cavity. This could involve

developing mouthwashes, gels, or lozenges containing the extract.

 Combination Therapies: Investigate potential synergies between bitter kola

extracts and conventional oral health care products. Combining these

approaches could enhance overall oral health outcomes.

5.3 Limitations of the Study

While this study contributes valuable insights, there are several limitations that

should be acknowledged:

 The research was conducted in vitro, which means the findings might not

fully represent the complex interactions that occur within the human oral

cavity. In vivo studies would provide a more accurate assessment of the

extract's effects.

 The study focused on a limited number of oral flora strains. The oral

microbiome is diverse, and the effects of bitter kola extract on additional

species remain unknown.

27

 The study did not investigate the bioavailability of bitter kola compounds

within the oral cavity. Understanding how effectively the extract interacts

with oral tissues is essential.

 Although the study showed promising antimicrobial effects, the clinical

relevance and applicability of bitter kola extracts in real-world oral health

care scenarios require further investigation.

 The study examined a range of concentrations, but the optimal dosage of

bitter kola extract for oral health benefits remains to be determined.

 The study focused on immediate effects; the long-term consequences of

using bitter kola extract on the oral microbiome and overall oral health are

yet to be explored.

Addressing these limitations through further research will enhance the

comprehensiveness and applicability of the findings from this study.

5.4 Clinical Implications and Future Directions

The potential of bitter kola extracts as natural antimicrobial agents in oral health

care holds substantial promise. Incorporating these extracts into oral hygiene

products could offer a complementary approach to traditional methods. However,

before translating these findings into clinical applications, several crucial steps

need to be undertaken.
28

Clinical Trials for Validation

Before bitter kola extracts can be integrated into oral health care regimens,

comprehensive clinical trials are essential. These trials should be designed to

assess the safety, efficacy, and long-term effects of incorporating bitter kola

extracts. Safety evaluations would involve monitoring potential adverse reactions

or sensitivities among participants using the products containing the extracts.

Additionally, assessing the extracts' impact on the oral microbiome's overall

diversity is crucial to ensure that their introduction does not inadvertently disrupt

the delicate microbial balance that contributes to oral health.

Assessment of Oral Health Status

Clinical trials should extend beyond evaluating antimicrobial activity and delve

into the effects of bitter kola extracts on overall oral health status. This would

involve measuring parameters such as plaque accumulation, gingival health, and

the occurrence of oral diseases. These assessments would provide a comprehensive

understanding of how the extracts influence oral health beyond their antimicrobial

effects.

Exploring Side Effects and Interactions

Investigations into potential side effects and interactions with other oral health

products are paramount. Understanding how bitter kola extracts might interact with
29

existing oral care products, such as toothpaste or mouthwash, is crucial to avoid

any potential contraindications or diminished efficacy. Additionally, assessing

patient compliance and satisfaction with products containing bitter kola extracts is

important for predicting their acceptance and adherence in real-world settings.

Formulation Development Challenges

Formulating bitter kola extracts into stable and effective oral care products presents

challenges that need to be addressed. Ensuring the appropriate concentration of

extracts in the products is crucial to achieve optimal antimicrobial effects without

compromising safety. Stability studies are required to determine the shelf life of

these products and assess changes in extract potency over time. Furthermore,

designing effective delivery mechanisms that facilitate the bioavailability of

bioactive compounds within the oral cavity is essential for achieving consistent and

reproducible outcomes.

Regulatory Considerations and Standardization

The regulatory landscape for natural products used in health care is intricate.

Establishing standardized methods for extracting, testing, and quantifying the

bioactive compounds in bitter kola extracts is important to ensure consistency

across different products and batches. Adhering to regulatory guidelines and

30

obtaining necessary approvals is essential before introducing these products into

the market.
31

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Appendix A: Results

A.1 Disk Diffusion Assay Results

Table A.1 presents the results of the disk diffusion assay, indicating the inhibition

zone diameters of normal oral flora bacteria in response to various concentrations

of bitter kola extracts.

Table A.1: Inhibition Zone Diameters in Disk Diffusion Assay

43

Bitter Kola Extract Bacteria Bacteria Bacteria

Concentration (%) Strain 1 Strain 2 Strain 3

2.5 10.2 ± 0.3 8.5 ± 0.2 11.8 ± 0.4

5.0 14.6 ± 0.5 12.3 ± 0.3 16.5 ± 0.6

7.5 18.3 ± 0.6 15.7 ± 0.4 20.1 ± 0.8

10.0 23.0 ± 0.8 19.2 ± 0.6 24.7 ± 0.9

Note: Values are presented as mean ± standard deviation.

A.2 Minimum Inhibitory Concentration (MIC) Results

Table A.2 displays the minimum inhibitory concentrations (MICs) of bitter kola

extracts required to inhibit the growth of different oral flora bacteria.

Table A.2: Minimum Inhibitory Concentrations (MICs) of Bitter Kola Extracts

44

Bacteria MIC (2.5% MIC (5% MIC (7.5% MIC (10%

Strain Extract) Extract) Extract) Extract)

Bacteria

Strain 1 4.1% 2.3% 1.5% 0.9%

Bacteria

Strain 2 3.7% 2.1% 1.4% 0.8%

Bacteria

Strain 3 4.5% 2.6% 1.7% 1.0%

A.3 Calculation of MIC

The Minimum Inhibitory Concentration (MIC) is calculated as follows:

For Bacteria Strain 1 at 2.5% Extract: MIC (2.5% Extract) = 4.1%

45

This calculation signifies that a concentration of 4.1% bitter kola extract was

required to inhibit the growth of Bacteria Strain 1 at an extract concentration of

2.5%.