CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND OF THE STUDY

Plants have contributed immensely to health care across the globe. This is due to the

recognition of the value of traditional medical systems, particularly in African origin, and the

identification of medicinal plant from indigenous and traditional knowledge, which have

significant healing power.

Among all plant families, members of the Lamiaceae have been used for centuries as

alternative medicine. Ocimum gratissimum L (Lamiaceae), is used in the treatment of

different diseases, for example upper respiratory tract infections, diarrhoea, headache, fever,

ophthalmic, skin disease and pneumonia (Koche et al, 2012). The Ocimum oil is active

against several species of bacteria (Staphylococcus aureus, Listeria monocytogenes,

Escherichia coli, Shigella, Salmonella and Proteus) and fungi (Trichophyton rubrum, T.

mentagrophytes. Cryptococcus neoformans, Penicillum islandi cum, and Candida albicans

(Lopez et al., 2005). Various species of Ocimum gratissimum for example O. viride Linn, O.

suave Linn, O. basilicum Linn and O. canum Sims have been highlighted for their numerous

medical uses (Mshana et al., 2000) and benefits.

Reports have it that ocimum have numerous properties, such as the tannins and sweet

smelling volatile oil known to have antibacterial agent (Elujoba 2000). The volatile oil also

stops spasm, the hyperactivity of the gastrointestinal tract, by combining with the

antibacterial activity and thus lowers the amount of contraction of the muscle of the stomach

and gastrointestinal tracts, stopping the diarrhoea (Elujoba, 2000) that are usually adverse for

most other pathogenic bacteria. It can be isolated from soil, silage and other environmental

sources.Several species and varieties of plants of the genus Ocimum have been reported to

yield oil of diverse nature,commonly known as basilic oils.

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Janine de Aquino Lemos et al.,(2005) reported some chemical compounds and active

ingredients found in these plants such as; eugenol,linaol,methyl cinnamate,camphor and

thymol. It has been demonstrated that the eugenol isolated from ocimum gratissimum

presented antimicrobial,insecticidal,antihelminthic,nematicidal activities or fungistatic

properties. Due to this favorable reasons,search for plant products with antimicrobial

properties intensified in recent years (Carmona et al., 2013; Prasannabalaji et al.,2012). It is

therefore very necessary that the search for newer antibiotic sources be a continued process.

For many years, medicine had depended exclusively on leaves, flowers and barks of plants;

only recently have synthetic drugs come into use and in many instances, these carbon copies

of chemicals identified in plants.

According to WHO, a medicinal plant is any plant which in one or more of its organs,

contains substances that can be used for therapeutic purposes or which are precursors for the

synthesis of useful drugs (Junaid et al .,2006). At present nearly 30% or more of the modern

pharamacological drugs are derived directly or indirectly from plants and in home-opathic or

ayurvedic medicines, medicinal plants, their parts and extracts dominate the scenes. The

commercial value of various innumerable drugs and pharmaceuticals derived from tropical

forests systems on worldwide basis is projected at 20 billion dollars a year(Sharif and

Banik,2006).

Although usage of these herbal medicines increases their quality, safety and efficiency are

more serious concerns in industrialized and developing countries. Bacterial infections are one

of the prominent causes of health problems physical, disabilities and mortalities around the

world. Plant that have been used in medicine as antimicrobial agents since ancient times

could provide a promising solution for drug resistance species. Thypoid fever is a common

and sometimes fatal infection of both adults and children that causes bacteremia and

inflammatory destruction of the intestine and other organ. Chloramphenicol has been the

2
treatment of choice for thypoid fever for over 40years, but the widespread emergence of

multidrug resistance. Salmonella typhi (resistance to ampicillin sulfamethoxazole) has

necessitated the research for other therapeutic options. Thypoid fever has become rare in

industrialized countries, yet it remains a major cause of entire disease in children in

developing countries. Globally, it is estimated that typhoid account for 16million cases each

year, resulting in over 600,000 deaths. Thypoid fever therefore continues to be a public

problem in Africa.

It has been reported that a substantial percentage (38%) of prescription contained one or more

of the neutral products of plant origin as the therapeutic agent. Many of these indigenous

medicinal plants are used as species and food plants; they are also sometimes added to foods

for medicinal purposes (Okwu; 2001). The plant Kingdom has proven to be the most useful in

the treatment of diseases and they provide an important source of all the World’s

pharmaceuticals. They, in all fact of life, have served a valuable starting material for drug

development (Edeoga et al., 2005).

The use of herbal medicine has always been part of human culture, as some plants possess

important therapeutic properties, which can be used to cure human and other animal diseases

(Rios and Receo, 2005).

1.2 AIMS AND OBJECTIVES

This study was designed to evaluate the antibacterial efficacy of Ocimum gratissimum on

bacterial strain E.coli activities. Also to determine the antimicrobial activity of the leaves

against the clinical isolate.

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 CHAPTER TWO

LITERATURE REVIEW

2.1 Introduction

Research on herbs, spices and medicinal plants originated with our ancestors thousands of

years ago it's now a popular subject that appeals to life scientist due to the problems of drugs

resistance and cost of drugs. Scientist in Africa and other developing countries are conducting

researches into local plants which are used in traditional medicine (Umar M, et al.,2016).

The use of synthetic and chemically based drugs in the treatment of various bacterial diseases

leads to a long-term complication to the recipients, since most of the chemically synthetic

drugs possess serious side effects that might make their disadvantages to outweigh their

advantages, because some chemical constituents can be carcinogenic, cytolytic or cytotoxic

when administered in large doses.

According to (Umar et al., 2016),the use of ethnomedicinal or natural plants as substitutes of

chemically synthetic drugs is imperative in order to prevent negative side effects and toxicity

of the orthodox drugs with the natural means of treatment.Majority of chemically synthesized

drugs have serious adverse effects to the recipients, which may lead to temporary or

permanent disability and incapacitations. Also, gastrointestinal disorders, dysentery,

diarrhoea and candidiasis are very serious infections that can lead to frequent morbidity and

mortality in tropical countries like Nigeria. These disorders are serious diseases that can

affect many people at various stages of their lives causing distress and discomfort (Umar M,

et al.,2016). Sometimes, the disorder can even lead to hospitalization. Majority of the

etiologic agents of gastroenteritis were found to be resistant to variety orthodox drugs, as

such complementary and alternative therapy is the only future to the success of pharmacology

(Azaizeh H and Fulder S, 2003).Treatment of diseases has always been associated with the

administration of drugs gotten from plants, animals and mineral sources.

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The use of plants or herbs extract in the treatment of human ailments is a very ancient art.

Investigation of African medicinal plants for antimicrobial activities ranks highest among

biological test carried in many plants and their extracts (Moss MO and Adams MR., 2002).

Herbal medicines tend to look primitive and unscientific when compared to synthetic

(conventional) drugs, which are thought to be more reliable than those made from plants.

Herbal medicine is still the mainstay of about 75-80% of the world population, mainly in

developing countries for primary health care as documented by Kamboj VP and Edeoga HO.,

(2000). A lot of research has been carried out on the herb, O. gratissimum. Though, literature

search has not revealed any study on the effect of O. gratissimum on the histology of the lung

of the albino rat, some works closely related to it has been documented. This plant is used by

herbalists to treat a variety of maladies, from bacterial infections and diabetes to pain and

liver damage. Several studies have been performed that lend credence to herbalist use of this

plant for treating diarrhoea and other gastrointestinal infections (Ezekwesili et al., 2004).

Use of natural means of treating infectious diseases will be the future of pharmacology in the

development of effective drugs with low or no toxicity to the recipient. Scent leaf has been

used traditionally for the treatment of gastrointestinal disorders, dysentery, diarrhoea and

candidiasis caused by various gastrointestinal inhabiting microorganisms. Direct oral

administration of raw scent leaf juice has been used for long in many tribes in order to treat

gastrointestinal disorders, dysentery, diarrhoea and candidiasis of varying degrees.

Traditional medicine is more accessible to most population in the world than orthodox

medicine. In fact, it is reported that 60-80% of the population of every country of the

developing countries has to rely on traditional or indigenous forms of medicine (Neergheen-

Bhujun 2013). Therefore this research was designed to determine the effect of scent leaf (O.

gratissimum) and to identify the common phytochemicals constituents of scent leaf that may

be inhibitory to gastrointestinal pathogens as well as the in vivo toxicity level of the extract.

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The research findings can further be used by the clinics, pharmaceutical industries and other

medical sectors in tackling the menace of the aforementioned disorders.

Current studies on the formation of reactive oxygen species (ROS) has shown that normal

body metabolism cannot function well without these species (Nelson and Cox, 2005). They

are useful to the body when moderately produced or strictly regulated (Manoj et al., 2009).

The highly reactive free radicals continuously produced within healthy human cells include

hydroxyl radicals, superoxide radicals as well as excited or single state oxygen radicals.

To sustain life, there are some enzymes in the human system like catalase, superoxide

dismutase (SOD), glutathione systems that help to regulate and control the escalation of these

radicals in the body, a situation known as oxidative stress. Complementary to these enzymes

are some plant species that have been found useful in controlling or regulating the

overproduction of these free radicals in the body (Manoj et al., 2009).

All plant and animal cells contain antioxidants that prevent damage due to the action of

reactive oxygen species (ROS). These highly reactive oxygen species when left uncontrolled

are capable of causing cell death or may form DNA adducts that could cause cancer-

promoting mutations (Robin, 2002). To prevent uncontrolled propagation of these free

radicals, cells normally contain a dozen or more antioxidant control systems that regulate the

many necessary and desirable free radicals presents. Those mechanisms include endogenous

enzymes like catalase, superoxide dismutase, thioredocxin and glutathione peroxidase (Aloh

and Ozougwu, 2010). When functioning properly, antioxidant system suppresses and control

excessive free radicals production, allowing control oxidative energy metabolism to proceed

normally without cellular or molecular damage. Oxidative stress occurs where there is an

imbalance between the production of reactive oxygen and the biological system’s ability to

readily detoxify the reactive intermediate or easily repair the resulting damage. All forms of

life maintain a reducing environment within their cells by enzymes through a constant input

6
of metabolic energy. Disturbances in this normal redox state could lead to the production of

peroxides and free radicals that damage all compounds of the cell, including proteins, Lipids

and DNA (Rimbach et al., 1999).

Many phytochemicals have been identified as components of food and more are still being

discovered (Ajali, 2004). Some of the phytochemicals of greater importance are plant

steroids, flavonoids, tannins, glucosides, saponins and alkaloids. This study is to investigate

the possible secondary plant metabolites present in the ethanol leave extract of Ocimum

gratissimum with a view of evaluating the acute toxicity of the leave extract and its

antioxidant status in vitro.

Nigeria is covered with a large number of plant species, some of which have been used for

centuries in folkloric medicines to diagnose, prevent and treat various ailments, but the

scientific investigations and information on the therapeutic potentials of medicinal plants are

limited. This lack of scientific knowledge has restricted the use of traditional herbs as

remedies to be used in conjunction with or as an alternative to orthodox medical treatment

(El-Mahmood , et al.,2010). Therefore, this research study aimed to qualitatively analyze the

phytochemical of scent leaf (ocimum gratissium), and to determine the antimicrobial effect of

the leaf extracts on some selected clinical bacterial isolates, with view to determine the

alternative or complementary therapy (using non synthetic drugs) against diseases caused by

the test organisms.

2.2 Uses of medicinal plants

Plants are very important sources of drugs used for centuries in the treatment of various

microbial infections. Most of the plant-based drugs when used correctly, pose less or no toxic

effect to the recipients compared to the chemically synthetic drugs. This necessitates the use

of natural plants in the complementary medicine in order to showcase the long term adverse

effects of some synthetic drugs. Herbal drugs have increasingly been used worldwide during

7
the last few decades as evidenced by rapidly growing global and national markets of herbal

drugs.

According to World Health Organization estimates, the demand for medicinal plants is about

US $14 billion a year and by the year 2050 it would be about a trillion US dollars. Now

people rely more on herbal drugs because of high price and harmful side effects of synthetic

drugs, and this trend is growing not only in developing countries but in developed countries

too (Tor-anyiin, T.A, Shaato R., 2003). A number of plants have been indicated to possess

antimicrobial properties from traditional uses.

Medicinal plants have therefore been described as one in which one or more of its organs

contain substance that can be used for therapeutic purposes (Rios JL, Recio MC., 2005). It

has been estimated that about one in four of all prescribed drugs, and almost 7,000 different

medicaments contain compounds of plants origin or their derivatives with their commercial

value being put at about$40 billion annually (Carter, A.P, Cleons WM.,2000). Studies

indicated that about 33% of drugs produced in the developed countries are derived from

plants.

2.3 Scent Leaf (Ocimum gratissium)

Ocimum gratissimum is popularly known as scent leaf. It is a full developed flowering plant

with root, stem and leaves systems. The plant Ocimum gratissimum is one of those plants

widely known and used for both medicinal and nutritional purposes. It is a perennial plant

that is widely distributed in the tropics of Africa and Asia. It belongs to the Family Labiatae

and it is the most abundant of the genus Ocimum. The common names of the plant are Basil

Fever plant or Tea bush and vernacular names include Daidoya tagida (Hausa), Nichonwu

(Igbo), Tanmotswangiwawagi (Nupe)and Efinrin (Yoruba) (Abdullahi et al., 2003).

It is woody at the base and has an average height of 1-3 meters. The leaves are broad and

narrowly ovate, usually 5-13cm long and 3-9cm wide. It is a scented shrub with limegreen

8
leaves (USDA, 2008). The plant is consumed by the Igbos as a leafy vegetables and the

nutritional importance of this plant center on its usefulness as a seasoning because of its

aromatic flavor. It is also used by the Igbos in the management of the baby’s cord. It is

believed to keep the baby’s cord and wound surface sterile.

2.4 Morphology of ocimum gratissium

O. gratissimum is a shrub up to 1.9m in height with stems that are branched. The leaves

measure up to 10 x 5 cm, and are ovate to ovate-lanceolate, sub-acuminate to acuminate at

apex, cuneate and decurrent at base with a coarsely crenate, serrate margin, pubscent and

dotted on both the sides. The leaves show the presence of covering and glandular trichomes.

Stomata are rare or absent on the upper surface while they are present on the lower surface.

Ordinary trichomes are few, while the long ones up to 6-celled are present on the margins

mostly; the short ones which are 2 celled, are mostly found on the lamina. Petioles are up to 6

cm long and racemes up to 18 cm long. The peduncles are densely pubescent. Calyx is upto

5mm long, campanulate and 5-7 mm long, greenish-white to greenish-yellow in colour.

Nutlets are mucilaginous when they are wet (Bhat KG., 2003)

On the 2 surfaces of the leaf epidermal cells are typical of irregular contours, and diacytic

stomata, secretory glands most abundant in the leaf, are also present in simple pluricellular

hairs on the leaf veins. The cross section shows the epidermis monoestratificada (beam), a

layer of parenchyma fenced in sub-epidermal position, followed by parenchymal pond, and

finally the epidermis monoestratificada lower.

The vitamin compositions of scent leaves is 32.26 mg/100 and 31.46 mg/100 . Since vitamin

C (ascorbic acid) promotes the health of teeth and gums, lungs and bronchia, and joints, aids

the purification of blood (Ogunlesi et al., 2010), its (ascorbic acid) content suggests that its

consumption and use in herbal medicine can prevent common cold and other diseases like

prostate cancer (Igile et al., 2013). The following minerals were present calcium, magnesium,

9
potassium, sodium, iron, copper and zinc, with zinc being in the highest concentration

(46.80± 0.03 mg/kg and 58.00±0.12 mg/kg) followed by copper (9.45±0.11 mg/kg and 16.60

± 0.03 mg/kg) in O. gratissimum respectively.

2.5 History of Bacteria (Prokaryotes)

Bacteria all are prokaryotes,“before nucleus”smallest, simplest, oldest cells on earth with size

1-5µm (vs eucaryotes 10-100µ long) if human hair were enlarged to size of tree trunk a

bacterial cell would be size of cockroach. 1st cells – survived for 2 Billion Years as only

kinds of cells on earth even after much more efficient cells appeared (eukaryotes) they

remained successful and abundant. Bacteria are the most common organism on the earth

which dominate the biosphere in terms of numbers, I.e more bacterial cells in a handful of

dirt than all people who have every lived, 1 tsp rich soil contains 1 Billion bacterial cells;

40,000 species, one species of bacteria (Prochlorococcus sp.). outnumbers all other organisms

in the open oceans eg. as much as 70% of bacteria alive on earth live on and below the ocean

floor (Umaret et al.,2016)

Bacteria rarely occur in isolated colonies of a single specieswhile individual bacterial cells

are too small to see without a microscope, bacteria are often found in microbial communities

that are visible to the naked eye. Bacteria may also be classified as gram-positive or gram-

negative. Gram-positive bacteria have a thick cell wall while gram-negative bacteria do not.

Gram staining, bacterial culture with antibiotic sensitivity determination, and other tests like

genetic analysis are used to identify bacterial strains and help determine the appropriate

course of treatment. Cell Wall almost all bacteria have a rigid cell wall surrounding the cell

membrane bacterial cell walls have a unique structure and chemistry unlike the cell walls in

any other kingdom,chemistry of cell wall varies from species to species in most bacteria the

cell wall is made of bacterial starch ( peptidoglycan) the cell wall protects the bacteria and

allows them to live in “extreme” environments the rigid cell wall produces the three basic

10
shapes of bacterial cells which are cocci (spheres), rods (bacilli), spirals (curves and

corkscrew shapes). About half of all bacterial species are motile and can move up to

50µm/sec [~100x’s body length/sec]. They can move by flagella most use one or more

flagella, flagellum is whip-like rod that rotates like propeller to move bacteria along, bacterial

flagellum is the only rotary motor known in the living world can spin at 6,000 rpm allows

bacteria to move ~10x’s their length/second. New bacterial cells can only grow slightly in

size when we say bacteria “grow” we usually mean they are reproducing, ie. increasing in

numbers bacterial growth = bacterial reproduction one of the reasons bacteria are so

successful is that most reproduce very rapidly most bacterial reproduction is by asexual

fission asexual reproduction is much faster than sexual reproduction.The Life Cycle of

bacteria is measured as the time between when the cell is first formed and when it divides by

fission time between divisions = generation time (Azaizeh H et al.,2003).

All life requires food for survival, in most organisms food must provide two main resources

which are building blocks (nutrients) and an energy source. As does all life bacteria require

sources of Carbon, Hydrogen, Oxygen and Nitrogen, Phosphorus, etc as well as several other

elements as a rule bacteria require fewer and often simpler essential nutrients than higher life

forms eg. humans require about a dozen essential organic molecules; vitamins, some amino

acids and some fatty acids, many bacteria can make all the organic molecules they need from

simple atoms and inorganic molecules; they require NO essential organic molecules also,

bacteria, as a group, rely on an extremely diverse group of nutrients compared to us.bacteria

can use 1000’s of different kinds of nutrients: avirtually every natural and human made

chemical can be eaten by at least some bacterial species including minerals in rock, acids,

hydrogen sulfide, sulphur, etc. (Ezekwesili et al., 2014) some bacteria can even break down

pesticides, herbicides, petroleum, asphalt, DDT, concrete, computer chips, paints, even

plastic and glass eg. the plastic we throw in the oceans harbors 1000’s of species of bacteria

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forming a uniquely identifiable plastisphere, bacteria can extract phosphorus from virtually

any phosphorus containing compound, organic or inorganic because bacteria can “eat” almost

anything, they are very effective decomposers and recyclers. Bacteria are of major

importance in the world’s biogeochemical cycles eg. nitrogen cycle, carbon cycle, etc.

Because bacteria are relatively simple and very adaptable they can survive, and even thrive,

under many conditions that we would consider quite harsh eg. ideal conditions for humans:

nice balmy temperaturesnot too hot, not too dry, plenty of food and drink, not overcrowded,

etc. But bacteria, as a group, have the widest tolerances for temp, pH, salt, etc than any other

kingdom of organisms their rates of growth and reproduction are greatly affected by various

environmental factors: temperature preferences, pH tolerance, salt concentrations, oxygen

requirements, light (for photosynthetic bacteria), each species has its own preferences and

tolerance limits for these factors (Neergheen-Bhujun.,2013).

2.6 Bacterial Infections

Food Poisoning, It is estimated that between 24 and 81 million cases of food borne diarrhea

disease occur each year in the United States, costing between $5 billion and $17 billion in

medical care and lost productivity.

More than 90 percent of the cases of food poisoning each year are caused by Staphylococcus

aureus, Salmonella, Clostridium perfringens, Campylobacter, Listeria monocytogenes, Vibrio

parahaemolyticus, Bacillus cereus, and Entero-pathogenic Escherichia coli. These bacteria

are commonly found on many raw foods. Normally a large number of food-poisoning

bacteria must be present to cause illness. Poor personal hygiene, improper cleaning of storage

and preparation areas and unclean utensils cause contamination of raw and cooked foods.

Researchers have recently reported (2010) that the 1400 or so different kinds of bacteria on

our fingers can leave a unique “microbeprint” on surfaces such as computer keyboards and

12
mouse, samples up to 2 weeks old could be linked to a specific use (Moss Mo and Adams

MR.,2002).

Some Examples of Bacterial Diseases:

Tuberculosis (Mycobacterium tuberculosis)

Gonorrhoea (Neisseria gonorrhea)

Anthrax (Bacillus anthracis)

Syphilis (Treponema pallidum)

Borellia ~relapsing fever, lyme disease

Leptospira ~ leptospirosis from urine of dogs, cats, pigs

Helicobacter ~ ulcers

Legionella ~ legionnaires disease

Bordetella ~whooping cough

Salmonella ~ typhoid fever

Shigella ~ bacillary dysentery

Yersinia ~ plague

Vibrio cholera ~ cholera

Clostridium: botulism, gas gangrene, tetanus

Staphylococcus aureus

Streptococcus pneumonia.

2.7 Treatment Of Bacteria Infections

Antibiotics also known as (antibacterial) are medications that fight bacterial infections. They

work by disrupting the processes necessary for bacterial cell growth and proliferation. The

first antibiotic was penicillin. Penicillin-based antibiotics, such as ampicillin, amoxicillin, and

penicillin G, are still available to treat a variety of infections and have been around for a long

time.Several types of modern antibiotics are available, and they are usually only available

13
with a prescription in most countries. Topical antibiotics are available in over-the-counter

(OTC) creams and ointments. It's important to take antibiotics exactly as prescribed. Failure

to do so could make a bacterial infection worse. Antibiotics don't treat viruses, but they're

sometimes prescribed in viral illnesses to help prevent a "secondary bacterial infection."

Secondary infections occur when someone is in a weakened or compromised state due to an

existing illness. Doctors may prescribe bacterial supplements to improve physical health

(A.Gurib-Fakim et al.,2006).

2.8 Influence of Traditional Medicine In The Treatment Of Bacterial.

Traditional medicine is the sum total of knowledge, skills and practices based on the theories,

beliefs, and experiences indigenous to different cultures that are used to maintain health, as

well as to prevent, diagnose, improve, or treat physical and mental illnesses. Traditional

medicine that has been adopted by other populations (outside its indigenous culture) is often

termed complementary or alternative medicine (CAM)(A. Gurib-Fakim et al.,2006).

The World Health Organization (WHO) reported that80% of the emerging world’s

population relies on traditional medicine for therapy. During the past decades, the developed

world has also witnessed an ascending trend in the utilization of CAM, particularly herbal

remedies (V. Chintamunnee and M. F. Mahomoodally 2012). Herbal medicines include

herbs, herbal materials, herbal preparations, and finished herbal products that contain parts of

plants or other plant materials as active ingredients. While 90% of the population in Ethiopia

use herbal remedies for their primary healthcare, surveys carried out in developed countries

like Germany and Canada tend to show that at least 70% of their population have tried CAM

at least once. It is likely that the profound knowledge of herbal remedies in traditional

cultures, developed through trial and error over many centuries, along with the most

important cures was carefully passed on verbally from one generation to another. Indeed,

modern allopathic medicine has its roots in this ancient medicine, and it is likely that many

14
important new remedies will be developed and commercialized in the future from the African

biodiversity, as it has been till now, by following the leads provided by traditional knowledge

and experiences ( S. Shohawon et al., 2013).

The extensive use of traditional medicine in Africa, composed mainly of medicinal plants,

has been argued to be linked to cultural and economic reasons. This is why the WHO

encourages African member states to promote and integrate traditional medical practices in

their health system. Plants typically contain mixtures of different photochemicals,also known

as secondary metabolites that may act individually, additively, or in synergy to improve

health. Indeed, medicinal plants, unlike pharmacological drugs, commonly have several

chemicals working together catalytically and synergistically to produce a combined effect

that surpasses the total activity of the individual constituents.

15
 CHAPTER THREE

3.1 Sources of Materials

Fresh leaves of Ocimum gratissimum L was collected from one of the resident building close

to profile hotel,Osere Area,Ilorin in the month of July 2021.

3.2 Preparation of Ocimum Gratissimum

The fresh leaves were harvested and properly rinsed in running water and finally rinsed in

sterile distilled water. The leaves were blended fresh using electric blender. The soluble

ingredients were then extracted by solubilization using ethanol as solvent.

The ethanol extract of the active ingredient of the leaves were carried out using the method as

described by (Koche et al.,2012). 25g of the grinded fresh leaves were soxhlet extracted using

250 ml of 95% ethanol. The extraction lasted for 6 hours. The volatile oil obtained was

concentrated by evaporation using water bath at 100°C for 1 hour. The crude extract was

diluted with 30% dimethylsulphoxide (DMSO) to obtain concentration of 250, 200, 150, 100,

and 50mg/ml.

3.3 Test Microorganisms

The strain used in this work was E. coli obtained from a diagnostic laboratory in Ilorin

metropolis.The bacteria was maintained by weekly transfers in tryptic soy broth (TSB) and

distributed in 5ml volume in screw-capped tubes. Cells were grown at 37°C for 48 hours and

cultures were kept at 40°C.

3.4 Antibacterial Test

The antibacterial tests of the plant extracts were tested on the test isolate using the agar-gel

diffusion inhibition test. In the agar-gel diffusion inhibition test as described by Koche et al.,

(2012), 0.2 ml of a 24 hours broth culture containing 1 X 106 cells/ml of organism was

aseptically introduced and evenly spread using bent sterile glass rod on the surface of gelled

16
sterile Mueller-Hinton agar plates.Fixed volumes (0.1 ml) of the extract were then introduced

into the wells in the plates. A control well was in the center with 0.01 ml of the extracting

solvent. The plates were allowed on the bench for 40 minutes for pre-diffusion of the extract

to occur (Esimone et al., 1998) and then incubated at 37°C for 24 hours. The resulting zones

of inhibition were measured.

3.5 Maximum Inhibitory Concentration (MIC)

The MIC of the potent extracts was determined according to the macro broth dilution

technique (Baron and Finegold 1990). Standardized suspensions of the test organism was

inoculated into a series of sterile tubes of nutrient broth containing two-fold dilutions of leaf

extracts and incubated at 37°C for 24 hours. The MICs were read as the least concentration

that inhibited the growth of the test organisms.

3.6 Minimum Bactericidial Concentration (MBC)

The MBCs were determined by first selecting tubes that showed no growth during MIC

determination; a loopful from each tube was subcultured onto extract free agar plates,

incubated for further 24 hours at 37°C. The least concentration, at which no growth was

observed, was noted as the MBC.

17