CHAPTER ONE

1.1 Background of the Study

Garcinia kola seed known in commerce as bitter kola (Iwu et al., 1982), has a

bitter astringent and resinous taste, somewhat resembling that of raw coffee,

followed by a slight sweetness. Bitter kola is a highly valued ingredient in

African ethno medicine because of its varied and numerous uses which are

social and medicinal, thus making the plant an essential ingredient in folk

medicine. Medicinal plants such as G. Kola are believed to be an important

source of new classical substances with potential therapeutic benefits (Eisner,

2000).

Cola nut seed, an evergreen originally endemic to West Africa was

introduced to many tropical countries, Jamaica being one of them. It belongs

to the Sterculiaceae family having more than 125 species (Odebunm et

al.,2008) and is locally known as bissy, cola or kola nuts (Duke, 2009). The

cola nut fruit is shaped like a capsule and is comprised of fleshy, irregularly

shaped seeds which are pink, red or white when fresh, and become brown and

hard once they are dried. The seeds are called nuts because of their bitter and

astringent taste (Adam et al., 2011). The plant’s rich history of traditional use

paved the way for cytotoxic and anti-microbial screens (Lowe et al., 2001) in

addition to identifying its key phytochemicals.

Plants are important in our everyday existence, the use of plants in traditional

medical practice has a long drawn history, and remains the main stay of

primary health care in most of the third world. Traditional medicines are used

by about 50% of the world population, in both developing and developed

countries where modern medicines are predominantly used (Mythilpriya et al.,

2007). The study of plants used in folklore remedies in treatment of microbial

infections have attracted the attention of many scientists as possible

alternatives to the existing drugs to which many infectious micro-organisms

have become resistant.

1.2 Statement of the Problem

Presently, there are global problems of multiple antibiotics resistance

as well as emergence of new and emergence of previously eradicated diseases

leading to a pressing need for new and more potent anti-microbial compounds

of natural origin to complement the existing synthetic antimicrobial anti-

sickling drugs. This leads to our study on Garcinia kola and cola nut.

1.3 Significance of the study

It is hoped that the findings of this study would be useful as a guide and in the

development of new drugs for resistant bacteria and other microbe/pathogens.

1.4 Scope of the study

This work covers the antibacterial activity of this plant seed Garcinia kola and

Cola nut selected clinical isolate. The work will investigate whether this plant

herb will inhibit growth on the test organisms.

1.5 Objective of the study

The main objective of the research work is to test the antibacterial activity of

the extract on the selected pathogenic clinical isolate.

 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 MEDICINAL PLANTS AS HERB

Historically, plant derived medicines, which have made large contributions to

human health and well-being provide source of inspiration for novel drug and

had served as models for western drugs (Farnsworth, 2009). It is estimated that

over 70% of modern pharmaceutical products are based on herbs. For instance,

artemisinin from Artemisia annua, used in the manufacture of Artesunate and

other artemisinin-based drugs, which serves as potent anti-malaria drug, is a

popular drug in the markets (Brisibe, et al., 2008). Plant derived medicines have

many benefits such as; low toxicity status/ relative safety, accessibility and

affordability. Plants parts have been a source of herbal medicine which has

been shown to be effective to about 80% of population as primary health care

(Akinyemi, et al., 2000). One of such plants is Garcinia kola, a member of the

Guttiferae species found throughout West and Central Africa. Every part of

Garcinia kola (bitter kola) is an important component in traditional herbal

medicine worldwide (Dalziel, 1937). Considering the enormous relevance of

Garcinia kola in folkloric medicine. Cola nut is not left aside as it uses are

numerous to mention in traditional medicine and herbal word at large.

2.2 GARCINIA KOLA

Garcinia kola (bitter kola) is a dicotyledonous plant belonging to the family

of plants called Guttiferae. It is a perennial crop growing in the forest,

distributed throughout West and Central Africa (Iwu, 1993). G. kola is also

found distributed in the forest zone of Sierra Leone, Ghana, Cameroon and

other West African countries. In Nigeria, it is common in the South Western

States and Edo State (Otor, et al., 2001). It is a medium sized evergreen tree,

about 15-17m tall and with a fairly narrow crown. The leaves are simple, 6-

14cm long and 2-6cm across, shiny on both surfaces and spotted with resin

glands. The small flowers are covered with short, red hairs (Iwu, 1993).

The fruit is a drupe of 5-10cm in diameter and weighs 30-50g. It is usually

smooth and contains a yellow-red pulp. The fruit changes color during

maturation from green to orange, and each fruit contains 1-4 seeds (Juliana,

et al., 2006).

SCIENTIFIC CLASSIFICATION

Kingdom: Plantae

Order: Malpighiales
Family: Clusiaceae

Genus: Garcinia

Species: G. kola

Binomial name: Garcinia kola

2.1.2 COMMON NAMES OF GARCINIA KOLA

Garcinia kola has been referred to as a “wonder plant” because

every part of it has been found to be of medicinal importance

(Dalziel, 2007). It is also called bitter cola, male kola due to the reported

aphrodisiac properties. It is commonly called “Orogbo” in Yoruba language,

Aku ilu‟ in Igbo language and “Namijin goro” in Hausa language (Dalziel,

2007).

2.2.1 MEDICINAL VALUES AND TRADITIONAL USES OF GARCINIA

KOLA.

Garcinia kola is chewed extensively in Southern Nigeria as a masticatory and

it is readily served to visitors, especially among the Igbo tribe in Eastern

Nigeria, as a sign of peace and acceptance of visitors. The root of the plant is

used as favorite bitter chew-sticks in West Africa (Otor, et al., 2001). The
stem bark is used in folklore remedies as a purgative among the natives of

Eastern Nigeria and the latex is externally applied to fresh wounds to prevent

sepsis, thereby assisting in wound healing. It is also popular among the people

of Nigeria for nervous alertness and induction of insomnia. Garcinia kola is

highly valued for medicinal use. This plant has been referred to as a “wonder

plant” because every part of it has been found to be of medicinal importance

(Dalziel, 2007). The seeds are chewed as an aphrodisiac or used to cure cough,

dysentery, chest colds, liver disorders, diarrhoea, laryngitis, bronchitis, and

gonorrhea (Adesina, et al., 2005). The seed is used to prevent and relieve cold;

it can also be used to treat headache, stomach ache and gastritis (Ayensu,

2008). It has also been reported for the treatment of jaundice, high fever, and as

purgative (Iwu , 2009). In Sierra Leone, the roots and bark are taken as a tonic

for sexual dysfunction in men. The bark is also added into palm wine to

improve its potency (Iwu, et al., 2009). Traditional medicine practitioners in

Nigeria, particularly in the Ogoni area use a decoction of Garcinia kola stem

bark for the treatment of dysmenorrhoea, fever, inflammation and burns

(Adesina, et al., 2005). "Bitter kola is anti-poison and helps to detoxify the

system, it has the ability to repel evil men and spirits, it could sound

superstitious but it works (Iwu, 2009).

Physiological Properties of Garcinia kola

Garcinia kola seed i s believed to contain a wide spectrum of organic

compounds (which several researches have confirmed) such as flavonoids

which confer on it some antimicrobial (Iwu et al., 2009) actions against gram

negative and gram positive microorganism. The biological activities of

flavonoids include action against allergies, inflammation, free radicals and

hepatoxins (Terashima et al., 2002). Several other works confirmed its

hypolipidermic (Oluyemi et al., 2007), antiulcerogence effect (Mechendran et

al., 2002), though its effect on the reproductive system have been

investigated with no significant difference/change in sperm count

(Ajibade et al., 2011). However, Akpantah et al., (2003) and Akinloye et

al., (1999) findings varies as due to the presence of biflavonoid and xanthane

in Garcinia kola seed, the compounds are potent anti-oxidants. Therefore,

administration of Gacinia kola seeds extract caused an increase in

testosterone production in spraque-dawley rats, due to the anti-oxidant

properties of its constituents. Garcinia kola has been proven to exhibit

pharmacological uses in treating coughs, throat infections, bronchitis,

hepatitis and liver disorders (Farombi et al., 2005). It is served as bitter

stimulant and as snake repellent when they are placed round the compound

(Nair, 2000). Garcinia kola is used in the treatment of liver disease and

diarrhea (Iwu et al., 2000). Some other medicinal uses of this plant include

purgative. This plant has also shown bronchodilator effect (Orie and Ekon,

2003), antihepatoxic effect (Braid, 2001) and anti-oxidant activity

(Adaramoye, 2005). In Nigeria, a cold water extract of the roots and bark with

salt are administered to cases of ukwala (bronchial asthma or cough) or

agboo (vomiting) (Iwu et al., 2000). According to Adesuyi et al (2012), the

medicinal properties of bitter kola can be classified under: - Purgative

Antiparasitic

2.2.3 Chemical Constituents of Garcinia kola

The phytochemical compounds isolated from G. kola include tannins, saponins,

alkaloids, cardiac glycosides (Ebana, et al., 2001). Other phytochemical

compounds isolated from G. kola seeds are biflavonoids such as

kolaflavone and 2-hydroxybi-flavonols. Two new chromanols, garcioic and

garcinal, together with tocotrienol were reported isolated from G. kola

(Terashima, et al., 2002). Morabandza, et al., (2013) had also determined

the chemical composition of Garcinia kola Heckel (Clusiaceae) mesocarp.

2.2.4 Anti-microbial properties of Garcinia kola

Adegboye, et al., (2008) had investigated the in vitro antimicrobial activities of

crude extract of Garcinia kola against some bacterial isolates comprising of

both Gram-positive and Gram- negative organisms. In another study, the

antimicrobial interaction between Garcinia kola seed (GKS) and gatifloxacin

(GAT), a fourth generation fluoroquinolone was evaluated by a modification of

the checkerboard technique using Bacillus subtilis and Staphylococcus aureus

as the test organisms (Ofokansi, et al., 2008). The antimicrobial activity of five

solvent extracts of Garcinia kola seeds had also been investigated against 30

clinical strains of H. pylori and a standard control strain, NCTC 11638, using

standard microbiological techniques (Collise et al., 2011). In vitro anti-

Vibrio activities of methanol and aqueous extracts of Garcinia kola seeds

against 50 Vibrio isolates obtained from wastewater final effluents in the

Eastern Cape Province, South Africa were also investigated (Penduka, et al.,

2011). The bioactivity of G. kola seeds was also assessed on Streptococcus

pyogenes, Staphylococcus aureus, Plesiomonas shigelloides and Salmonella

typhimurium (Christinah and Roland, 2012). Trichomonacidal effects of G.

kola nuts were also investigated (Gabriel and Emmanuel, 2011). Esimone et

al., 2002 also investigated the effect of Garcinia kola seed extract (100 mg/kg)

on the pharmacokinetic and antibacterial effects of ciprofloxacin hydrochloride

(40 mg/kg).

Anti-diabetic Properties of Garcinia kola

The hypoglycaemic and hypolipidaemic effects of fractions from kolaviron

(KV) (a Garcinia kola seed extract) were investigated in normal and

streptozotocin (STZ)-diabetic rats (Adaramoye and Adeyemi, 2006).

Garcinia kola seed Powder (GKP) had also been shown to have antidiabetic,

antilipidemic and anti-atherogenic properties with a tremendous potential to

protect against coronary heart disease (Udenze, et al., 2012). Significant

hypoglycaemic and hypolipidemic activity of Garcinia kola in alloxan-

induced diabetic Wistar rats had been reported (Nwangwa, 2012).

Hepatoprotective and anti-oxidant activities

The hepatoprotective effect of Garcinia kola seed extract against paracetamol

induced hepatptoxicity had been investigated in rats (Alade and Ani, 1990). The
protective effects of Garcinia kola against a dose of Carbon-Tetrachloride

(CC14)-induced liver damage in experimental rats were also investigated

(Mathew and Blessing, 2007). Antioxidant p o t e n t i a l s of five fractions

(ME1–ME5) of methanolic extract of Garcinia kola seeds was also studied

(Tebekeme, 2009).

Garcinia kola Effects on Fertility

Garcinia kola seed meal had been shown to improve semen characteristics and

sexual drive (libido) in matured rabbit bucks (Iwuji and Herbert, 2012). In

another study, long- term ingestion of Garcinia kola seed diet had been shown

to cause a significant reduction in sperm count, sperm motility, and ultimately

infertility in the male wistar rat (Mesembe, et al., 2013). Garcinia kola seed at

200mg/kg body weight altered oestrous cycle and partly inhibits ovulation in

female rats (Akpantah, et al., 2005).

Other studies on Garcinia kola

Anti-ulcer potential and proton pump inhibitory activity of kolaviron (KV)

isolated from Garcinia kola Heckel had been evaluated using different ulcer

models and was suggested to emerge as a potent anti-ulcer compound

(Onasanwo, et al., 2011). Garcinia kola (Heckel) seed extract evaluated in

albino Wistar rats possess significant anti-pyretic activity, which justified its

ethnomedicinal use (Kakjing, et al., 2014). Garcinia kola 0.5% aqueous

solution eye drop significantly reduces intraocular pressure (IOP) as compared

to baseline (Adebukunola, et al., 2010). Comparative study on the efficacy

of Garcinia kola in reducing some heavy metal accumulation in liver of

Wistar rats was also carried out. Garcinia kola has the highest

hepatoprotective effect to Cd followed by Hg and least protection against Pb

toxicity in rats and its administration was beneficial in reducing heavy metal

accumulation in the liver (Nwokochaa, et al., 2011). Administration of

Garcinia kola for a period of six weeks in rabbits elicited no

observable histo-pathological effects on the histology of the liver (Charity, et

al., 2012). Garcinia kola had been shown to enhance erythropoiesis in rabbits

and rats and as well has no long term significant toxicological implication

(Unigwe and Nwakpu, 2009). In another study, Garcinia kola significantly

reduced tissue damage induced by lipopolysaccharide (LPS) (Okoko and

Ndoni, 2009). Daily administration of Garcinia kola (G. kola) in growing

Wistar rats for a period of 70 days showed a depressive effect on appetite

and water intake with resultant poor feed utilization efficiency and mass gain

of rats in a dose- dependent manner. Plasma alanine aminotransferase (ALT)

and aspartate aminotransferase (AST) activities were elevated (P<0.05) but

histological examinations of liver, heart and lungs of experimental rats revealed

no alterations. The extract enhanced sexual interest (libido) of the male rats

but did not necessarily improve their fertility rate (Otor, et al., 2001).

2.3 COLA NUT

Cola is a genus of trees native to the tropical forests of Africa, classified in the

family Malvaceae, subfamily Sterculioideae (or treated in the separate

family Sterculiaceae). Species in this genus are sometimes referred to as kola

tree or kola nut for the caffeine-containing fruit produced by the trees that is

often used as a flavoring ingredient in beverages. The genus is related to

the South American genus Theobroma, or cocoa. They are evergreen trees,

growing up to 20 m tall (about 60 feet), with glossy ovoid leaves up to 30 cm

long and star-shaped fruit (Mitchell and Ahmad 2006)

SCIENTIFIC CLASSIFICATION

Kingdom: Plantae

Order: Malvales

Family: Malvaceae

Genus: Cola

Species: C. acuminata

2.3.1 Use in Ethnomedicine

Chewing of the fresh Cola acuminata nut is a well-known practise of the

Western and sub-Saharan native populations of Africa particularly in Northern

Nigeria, Ivory Coast, Sierralione Liberia and Sudan (Russell, 2005), whereas

the dried nuts are used as beverages and as pharmaceutical agents in Europe

and North America. Bissy has a bitter taste and high caffeine content
(Benjamin et al., 2001). It is used traditionally as a caffeine stimulant and it is

chewed in many West African cultures individually or in a group setting and is

often used ceremonially, be it at weddings, child naming, funeral, the

presentation of tribal chiefs or guests (Sonibare et al., 2009) . Chewing kolanut

can ease hunger cramps, stimulates digestion and is also used for euphoric

qualities. Its effects are comparable to other xanthine containing herbs like

cocoa and tea (Endrini et al., 2011); nonetheless, the effects are distinctively

different as it produces a stronger state of euphoria and well-being (Benjamin

et al., 2001). It is thought to enhance alertness and physical energy, elevate

mood, increase tactile sensitivity, and suppress appetite. It may also increase

body temperature, blood pressure, and respiratory rate (Natural Standard,

2012). It has been used as a common additive to American and European soft

drinks (Natural Standard, 2012). The plant produces fruit pods containing seeds

that are used to treat poisoning, digestive disorders (Warner, 2007) and asthma

(Odebunmi et al., 2009). Small doses are used to treat migraine, motion and

morning sickness. In addition, it has been used to relieve inflammation

disorders such as rheumatism and gout, and has been administered to treat

pneumonia and typhoid fever when great nervous irritability was present. Kola

nut is also used to treat diarrhoea and has been used as a diuretic (Mitchell,

2003).

2.3.2 Pharmacological Effects of C. acuminata

Anti-Cancer Potential of C. acuminata Extract

Being the leading cause of death in Jamaica and the third leading cause of

death worldwide, cancer is a disease of major concern and the search for

compounds that can combat these deadly diseases is ongoing. Presently, 60%

of chemotherapeutic drugs are from natural origin (Cragg and Newman, 2005),

however more often than not they are associated with insufferable side effects

which make the hunt for other molecular entities or plant extracts an ongoing

one. Because of its abundant enthomedicinal practices, the bissy but plant

seemed ideal to be screened for anti-cancer potential even though the plant

has no traditional use in that arena. Research from our lab showed that of the

five solvents used (hexane, dichloromethane, acetone, methanol and water), the

hexane bissy nut extract was most effective as it (160 µg/ml) killed 100% of

breast (BC) and prostate (DU-145 and PC) cancer cell lines (Lowe et al.,

2012). Further, research has shown that extracts from the bissy nut were

cytotoxic against MCF-7 cell line (Fontenot et al., 2007) while Edrini and

others (Endrini et al., 2011) showed that reduction in cell viability of the cell

line was through apoptosis (at 60 µg/ml) in which case less cells were present

in the S and G2 phase of the cell cycle when compared to the positive control.

Bissy nut was also shown to reduce the viability of LNCaP and DU145 with

growth inhibition (GI50) of 15 ppm and 3.6 ppm respectively (Fontenot et al.,

2007). A significant increase in mRNA of anti-apoptotic genes; Bcl2 and Bax

were observed in addition to an increase in protein and mRNA levels of

androgen regulated genes. These results show that the bissy nut plant may
provide a key for modulating breast and prostate cancers in an AR-dependent

manner. Further research should be ex- plored against other cell lines, in

addition to mechanistic and in vivo studies. Additionally, the plant’s

chemopreventive properties from other angles can also be explored, such as

testing its ability to inhibit the ac- tivities of CYP1 family of enzymes

implicated in cancer initiation. The Rho proteins family of proteins is also

known as the cellular switches and their modulation can be explored in cancer

prevention and treatment. Such studies would determine the anticancer

potential of this extract and pave the way for nutraceutical and pharmaceutical

treatment options.

Other Biological Activity Properties of C. acuminata

The effects of fresh kola nut extracts on female Swiss Webster mice on post

natal development and their off spring was investigated and it was found that

mice whose mothers were exposed to the kola nut extract showed a decline in

the rate of post natal body weight gain but they experienced eye opening and

hair gain relatively faster than their respective controls (Ajarem and Ahmad,

2004) implying kola nuts affects the utero development and the effects seem

perma- nent. Kola nut was also shown to exhibit a depressive effect on biphasic

locomotor activity in male mice models at high concentrations (10 mg/kg)

(Ajarem, 2000) . Results obtained from limited human trials infer that kola nut

may have some weight loss properties, positive chronotropic and weak diuretic

properties. Animal studies show analeptic and lipolytic (fat-burning) properties

(Natural Standard, 2012) . More recent research shows that it stains the

cytoplasm of various rat tissues which shows that it can be a suitable alternative

for histological staining and so be more environmentally friendly and cost

effective than synthetic dyes (Shehu et al., 2012).

2.3.2 PHYTOCHEMISTRY OF COLA ACUMINATA

Kola nuts are rich in xanthine alkaloids such as theobromine (up to 0.1%),

caffeine (0.6% - 3.0%) and kolatin (Adeyeye et al., 2012) . The plant contains

9.73% moisture, 2.72% ash, 3.02% fat, 19.14% protein, 7.3% crude fiber and

58.09% carbohydrate (Adeyeye et al., 2012) . The plant is a source of other

xanthine alkaloids, tannins, proanthocyanidins and anthocyanins such as “kola

red”. Specimens of African kola yielded more caffeine (2.24 per cent) than

Jamaican kola (1.93 per cent) (Mitchell, 2003). The plant accumulates

approximately 38% amino acids (Adeyeye et al., 2012) and appreciable levels of

appreciable levels of (+)-catechin, (−)-epicatechin, procyanidin B1 (epicatechin-

(4beta-->8)-catechin), and procyanidin B2 (epicatechin-(4beta-->8)-epicatechin)

(Adeyeye et al., 2012). It also has higher levels of alkaloids, tannins and

saponins than Cola nitida (Adeyeye et al., 2012). The phenolics and

anthocyanins are likely to provide antioxidant activity (Adeyeye et al., 2012)

and some of the active ingredients have been identified as non-steroidal

compounds that are bioactive against prostate and breast cancer cells (Fontenot
et al., 2007) and so these compounds could be responsible for the observed

bioactivity against breast and prostate cancer cell lines. The tannic acid in the

kola nuts helps to precipitate a number of toxins in the gut preventing their

absorption (Lowe et al., 2012) hence its traditional use as an antidote. Given

the potential anticancer and microbial properties, high throughput assays can

be used to screen the known phytochemicals in hopes of identifying the

individual ones or combinations responsible for the observed bioactivities.

Further screens can be conducted on these to assess their potential

chemopreventive properties

2.4 ANTI-MICROBIAL PROPERTIES OF C. ACUMINATA

Microbial infections are of major importance to the medical arena since they are

responsible for 90% of infections found in health care services. The occurrence

of MDR bacterial strains seems to be the major cause of treatment failure

(Koyama, 2006). Not only do they warrant concern in the medical arena but to

food preservation as well. Presently there is growing global demand for

consumers to reduce chemical preservation that can be unfavorable to human

health (Cho, et al., 2005). Subsequently the search for plant metabolites such as

polyphenols, glycosides and alkaloids amongst others are currently undergoing

detailed investigations in response to consumer requirements for fresher and

more natural additive-free products (Nychas, 2005). Research shows that

methanolic and aqueous extracts of Cola acuminata seeds displayed

antimicrobial activity against; Bacillus subtilis, Staphylococcus aureus,

Escherichia coli, Klebsiella pneumonae, Pseudomonas aeruginosa and Candida

albicans ranging from 8 - 24 mm and results compared well with Gentamicin

and Nystatin (Adam et al., 2011). Bissy nuts are also employed in the treatment

of malaria, fever, ringworm, scabies, gonorrhea and dysentery (Odugbemi,

2006). These results concur with the use of the plant in traditional medicine and

implicate its promise as an antimicrobial agent. Further research can be

conducted to identify key phytochemicals be them individual or a combination

responsible for the observed bioactivities. Research can be extended to in vivo

and clinical trials to confirm the anti-microbial properties and once again pave

the way for nutraceuticals and or pharmaceuticals that can be used to treat

certain infections especially those caused by current strains now resistant to

existing anti-microbial treatments.

2.4.5 Some Major Groups of Antimicrobial Phytochemicals from Plants.

Phytochemicals are non nutritive compounds found in plant and which may

have 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;

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 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 harane is

attributed to their ability to intercalate with DNA.

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.,2007).

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. Saponins 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 order 180-2000

Daltons. At concentrations below 200-500 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. 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 that human do not suffer

severe poisoning from saponins.

Tannins

Tannins is a general descriptive name for a group of polymeric phenolic

substances capable of tanning leather or precipitating gelatin from solution, a

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. Alternatively; 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 to

their ability to inactivate microbial adhesions, enzymes, cell envelope transport

proteins etc.

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 phenylpropane derived 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 phenols shown 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, 2003). 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, 2007). 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 a well characterized

representative found in clove oil. Eugenol is considered bacteriostatic against

both fungi and bacteria.

CHAPTER THREE

MATERIALS AND METHODS

3.1 Plant collection and identification

The seed of cola nut and bitter cola were purchased from Eke market Oko,

Orumba North L.G.A Anambra state.

Preparation of sample:

Drying and grinding: The seed of cola nut and bitter cola were washed

and dried in sunlight and then grinded to form the powder. These

powdered samples will then be stored in airtight bottles.

Preparation of aqueous extract:

Two hundred gram portion of the powered sample was extracted with 80ml of

water in 250cm3 conical flask, and were allowed to stay overnite, the extracts

were separated using sterile muslin cloth and filtered through a sterile filter paper

Preparation of crude ethanolic extracts:

 50g of powered cola nut and bitter cola seed will be weighed into the soxhelt

extractor respectively. It was done under 3-4 hours using 350mls of ethanol.

The ethanol extract will be concentrated into dryness by evaporation of the

solvent in stream bath and the weight was noted. The sample will be labelled

and will be stored in a sterile container and refrigerator at 40c until used.

Determination of antibacterial activity

Test organisms

The micro organisms used will be the gram positive bacteria Staphylococcus

aerus, Streptococcus spp, Bacillus spp and the gram negative bacteria

Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi. They will be

obtained from Glanson laboratory at Awka and from National Agency for Food

and Drug Administration and Control (NAFDAC) at Agulu town, all in

Anambra State, Nigeria.

Antibacterial assay

The antibacterial activity will be performed by agar disc diffusion method. The

bacteria strains will be grown in nutrient broth. Muller Hinton agar will be the

media used to study the bacteria susceptibility. The broth cultures were grown

for 24 hours and serially diluted in the same broth (sterilized at 121 0C for 15

minutes) to 10-3. The 24hrs broth culture contains approximately 8.0 X10 7

Cfu/ml, 1.7 X 107 Cfu/ml, 2.8 X 107Cfu/ml, 3.2 X10 Cfu/ml, 2.6 X106 Cfu/ml

and 5.0 X 107 Cfu/ml for Escherichia coli, Staphlococcus aureus, Salmonella
typhi and Streptococcus spp and pseudomonas spp respectively, as determined

by plate count method. Sterile swab stick will be used to inoculate the media by

dipping it in the diluted culture and spreading all the surfaces of the agar plate.

Sterile paper disc about 10mm diameter will be soaked with the extract and

allowed to dry for some minutes. This will be placed on the surface of

inoculated agar plates. The plates will then be incubated for 24hours at 37 oC.

After incubation, the diameter zone of inhibition will be measured and recorded

using meter rule in (mm). The extract with the highest zone of inhibition will be

chosen for subsequent experimental work.

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