Tuesday, July 7, 2009

Antibacterial activity of Ocimum gratissimum on Some Selected Pathogenic Bacteria

CHAPTER ONE

INTRODUCTION

1.1 Background of Study

During the last century, the practice of herbalism became mainstream throughout the world. In spite of great advances observed in modern medicine, plants still make an important contribution to health care. This is due to the recognition of the value of traditional medical systems, particularly of Asian origin, and the identification of medicinal plants from indigenous pharmacopoeias, which have significant healing power. The use of medicinal plants as herbal remedies to prevent and cure several ailments differ from community to community (Sherif and Banik,2006; Kubmarawa et al., 2007). Medicinal plants have been used for centuries before the advent of orthodox medicine. Leaves, flowers, stems, roots, seeds, fruit, and bark can all be constituents of herbal medicines. The medicinal values of these plants lie in their component phytochemicals, which produce definite physiological actions on the human body. The most important of these phytochemicals are alkaloids, tannins, flavonoids and phenolic compounds (Afolabi et al., 2007; Doughari and Manzara, 2008).

Medicinal plants are distributed worldwide, but they are most abundant in tropical countries (Calixto, 2000; Lewis, 2001). In Brazil alone, about 80,000 species of higher plants were described which offer enormous prospects for discovering new compounds with therapeutic property (Nakaruma et al., 1999).

Ocimum gratissimum (African Basil) belongs to the family Labiatae. It is widely distributed in tropical and warm temperate regions. Ocimum gratissimum is called 'efinrin' by the yorubas of the southwestern part of Nigeria, 'nchanwu' by the Igbos and 'Dai'doya' by the Hausas. It has been reported to contain the terpenoids, eugenol and thymol, saponins and alkaloides (Gill, 1988). Aromatic oil from the leaves consist of thymol (32-65%). Ocimum gratissimum Linn (Labiatae) is grown for the essential oils in its leaves and stems. Eugenol, thymol, citral, geraniol and linalool have been extracted from the oil (Sulistiarini, 1999). Essential oils from the plant have been reported to possess an interesting spectrum of antifungal properties (Dubey et al., 2000). The antinociceptive property of the essential oil of the plant has been reported (Rabelo et al., 2003). The whole plant and the essential oil are used in traditional medicine especially in Africa and India. The essential oil is also an important insect repellant. Ocimum gratissimum is germicidal and has found wide use in toothpastes and mouth washes as well as some topical ointments (Nakamura et al., 1999; Holets et al., 2003; Pessoa et al., 2003). It is used as an excellent gargle for sore throats and tonsillitis. It is also used as an expectorant and a cough suppressant. The plant extract is used against gastrointestinal helminths of animals and man (Fakae, 2000; Chitwood, 2003). In addition, Ocimum gratissimum carminative properties make it a good choice for stomach upset. It is used as an emetic and for hemorrhoids. The plant is also used for the treatment of rheumatism, paralysis, epilepsy, high fever, diarrhea, sunstroke, influenza, gonorrhea and mental illness (Dhawan et al., 1977; Oliver, 1980; Abdulrahman, 1992; Osifo, 1992; Sofowora, 1993; Sulistiarini, 1999). In addition, the plant is used as a spice and condiment in the southern part of Nigeria. The plant is commonly used in folk medicine to treat different diseases such as upper respiratory tract infections diarrhoea, headache, ophthalmic, skin diseases, pneumonia, cough fever and conjunctivitis (Onajobi, 1986). It is used by the Igbos in southern Nigeria in the management of baby's cord . It is believed to keep the baby's cord and wound surfaces sterile (Iwu, 1986) . Ocimum gratissimum has been reported to be active against several species of bacteria and fungi (Nwosu and Okafor, 1995; Nakaruma et al., 1999).

Much has been documented on the antimicrobial properties of the leaf extract of this plant, this work is therefore aimed at evaluation of the antimicrobial property of the stem extract of thes plant.


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 

1.2 Statement of the Problem

The problem of microbial resistance to antibiotics is growing and the future of antimicrobial drugs is still uncertain. Therefore, actions must be taken to reduce this problem. To control drug resistance among microorganisms, alternative source of antibiotic should be sourced for. According to the world health organization medicinal plants would be the best source to obtain a variety of drugs. About 80% of individuals from developed countries use traditional medicine, which has compounds derived from medicinal plants (Boyd et al. 1994). Therefore, such plants should be investigated to better understand their antimicrobial property.

1.3 Main Objective

To determine the antimicrobial activity of Ocimum gratissimum on some selected pathogenic bacteria.

1.4 Specific Objectives

  1. Determination of the phytochemical constituent of the extract
  2. Determination of the sensitivity of the clinical isolates to the extract
  3. Determination of the Minimum Inhibitory Concentration (MIC) of the extract.
  4. Determination of the Minimum Bactericidal Concentration (MBC) of the extract
  5. Determination of the effect of pH on the activity of the extract

on Staphylococcus aureus, Bacillus cereus, Proteus mirabilis, Salmonella paratyphi and Pseudomonas aeruginosa.


 


 


 


 

CHAPTER TWO

LITERATURE REVIEW

2.1 Plants as source of Antimicrobials

Historically, plants have provided a source of inspiration for novel drug compounds, as plant derived medicines have made large contributions to human health and well-being. Their role is two fold in the development of new drugs: (1) they may become the base for the development of a medicine, a natural blueprint for the development of new drugs, or; (2) a phytomedicine to be used for the treatment of disease. There are numerous illustrations of plant derived drugs. Some selected examples, including those classified as antiinfective, are presented below.

The isoquinoline alkaloid emetine obtained from the underground part of Cephaelis ipecacuanha, and related species, has been used for many years as and amoebicidal drug as well as for the treatment of abscesses due to the spread of Entamoeba histolytica infections. Another important drug of plant origin with a long history of use, is quinine. This alkaloid occurs naturally in the bark of Cinchona tree. Apart from its continued usefulness in the treatment of malaria, it can be also used to relieve nocturnal leg cramps. Currently, the widely prescribed drugs are analogs of quinine such as chloroquine. Some strains of malaria parasites have become resistant to the quinines, therefore antimalarial drugs with novel mode of action are required (Boyd et al., 1994)

Similarly, higher plants have made important contributions in the areas beyond antiinfectives, such as cancer therapies. Early examples include the antileukaemic alkaloids, vinblatine and vincristine, which were both obtained from the Madagascan periwinkle (Catharanthus roseus syn. Vinca roseus). Other cancer therapeutic agents include taxol, homoharringtonine and several derivatives of camptothein. For instance, a well-known benzylisoquinoline alkaliod, papaverine, has been shown to have a potent inhibitory effect on the replication of several viruses including cytomegalovirus, measles and HIV (Turano et al. 1989). Most recently, three new atropisomeric naphthylisoquinoline alkaloid dimers, michellamines A, B, and C were isolated from a newly described species tropical liana Ancistrocladus korupensis from the rainforest of Cameroon. The three compounds showed potential anti-HIV with michellamine B being the most potent and abundant member of the series. These compounds were capable of complete inhibition of the cytopathic effects of HIV-1 and HIV-2 on human lymphoblastoid target cell in vitro (Boyd et al., 1994).


 

2.2 The Development of Phytomedicines and the Ethnomedicinal Approach

The first generation of plant drugs were usually simple botanicals employed in more or less their crude form. Several effective medicines used in their natural state such as cinchona, opium, belladonna and aloe were selected as therapeutic agents based on empirical evidence of their clinical application by traditional societies from different parts of the world. Following the industrial revolution, a second generation of plant based drugs emerged based on scientific processing of the plant extracts to isolate "their active constituents." The second-generation phytopharmaceutical agents were pure molecules and some of the compounds were even more pharmacologically active than their synthetic counterparts. Notable examples were quinine from Cinchona, reserpine from Rauvolfia, and more recently taxol from Taxus species. These compounds differed from the synthetic therapeutic agents only in their origin. They followed the same method of development and evaluation as other pharmaceutical agents (Stewart et al., 2008).

The sequence for development of pharmaceuticals usually begins with the identification of active lead molecules, detailed biological assays, and formulation of dosage forms in that order, and followed by several phases of clinical studies designed to established safety, efficacy and pharmacokinetic profile of the new drug. Possible interaction with food and other medications may be discerned from the clinical trials.

In the development of "Third Generation" phytotherapeutic agents a top to bottom approach is usually adopted. This consists of first conducting a clinical evaluation of the treatment modalities and therapy as administered by traditional doctors or as used by the community as folk medicine. This evaluation is then followed by acute and chronic toxicity studies in animals. Studies should, when applicable, include cytotoxicity studies. It is only if the substance has an acceptable safety index would it be necessary to conduct detailed pharmacological/ biochemical studies (Boyd et al., 1994).

Formulation and trial production of the dosage forms are structured to mimic the traditional use of the herb. The stability of the finished product is given careful attention during the formulation of the final dosage form. This is a unique blend of the empiricism of the earlier first generation botanicals with the experimental research used to prove the efficacy and safety of second generation isolated pure compounds. Several pharmaceuticals companies are engaged in the development of natural product drugs through the isolation of the so-called active molecules from plant extracts.


 


 

2.3 The Genus Ocimum

The genus Ocimum belongs to the family Lamiaceae and is comprised of more than 50 species of herbs and shrubs distributed in tropical and subtropical regions of Asia, Africa and the Americas. Most members of this family such as Hyptis, Thymus, Origanum, Salvia and Mentha species are considered economically useful because of their basic natural characteristics as essential oil producers. These essential oils are composed primarily of monoterpenes and sesquiterpenes and have been the subject of extensive studies due to their economic importance(Lawrence, 1993).

The individual species within the genus Ocimum have been observed to show significant variation in the aromatic character as well as morphological features. Such observations have been attributed to the abundant cross pollination that occurs within this genus resulting in considerable degrees of variation in the genotypes, hence diversity in growth characteristics, leaf size, flower colour, physical appearance and aroma (Stewart et al., 2008).

Consequently, high diversity of species, subspecies, varieties and chemotypes are evident in this genus, each having distinct aromatic characters, morphological features and chemical composition in the essential oil distillates. Such difference in the essential oil compositions in Ocimum
basilicum from different geographical localities led to the classification of basil into chemotypes on the basis of the prevalent chemical components (Stewart et al., 2008) or components having composition greater than 20 percent (Grayer et al., 1996). Four main chemotypes and numerous other sub-chemotypes were established on the basis of the structural features of the main constituents as belonging to either the phenylpropanoid group (methyl chavicol, eugenol, methyleugenol and methyl cinnamate) or the terpenic group (linalool and geraniol), which are derived from the shikimic acid and the mevalonic acid biosynthetic pathways respectively. Other latter studies on the basils from other geographical regions have added new chemotypes to that list based on the established classification scheme (Lawrence, 1992; Grayer, 1996). Some of such chemotypic entries include terpenen-4-ol type from O. canum and thymol type from Ocimum gratissimum (Sanda et al., 1998; Yusuf et al. 1998; Keita et al. 2000); geranyl acetate type from Ocimum minimum (Ozcan and Chalchat, 2002); citral and camphor types from Ocimum
americanum (Mondello et al., 2002); and p-cymene type from O. suave (Keita et al. 2000). A report on the chemical constituents in O. canum from Rwanda indicated the oil to be composed of 60-90 percent linalool (Stewart et al., 2008).

2.4 The Plant Ocimum gratissimum

2.4.1 Taxonomy

Kingdom: Plantae

Phylum: Tracheaphyta

Division: Magnoliopsida

Subdivision: Spermatophyta

Class: Magnoliopsida

Subclass: Asteridae

Order: Lamiales

Family: Lamiaceace

Sub-family: Nepetoideae

Genus: Ocimum

Specie: gratissimum

Common Names

English: African Basil

Hausa: Dai'ta doya

Yoruba: Efirin

Igbo: Nchanwu


 

2.4.2 Botanical
Description

Ocimum gratissimum is a herbaceous perenniel herb, woody at the base,stem is 1-3 meter long. Leaves are broadly to narrowly ovate, usually 5-13cm long and 3-9 cm wide. Both surfaces are capiously glandular punctate, upper surface is glabarate to sparsely puberulent. Lower surface on veins, margins is serrate, apex is acuminate, the base is cuneate, petioles is 1-6 cm long. Calyx is 3-5 mm long, enlarging upto calyx. The tube is usually 2-2.5 mm at anthesis, sparsely hispidulous or puberculent and dotted with sessile oil globules, glabrous within, the upper lobe is ovate, median lobes of lower lip are shorter than lateral ones; corolla is greenish white to greenish yellow, 4-7mm long. Nutlets: subglobus, 1.5-2mm in diameter, slightly ngose (Wegner et al., 1999).

2.4.3 Ecology and distribution

In its native area, Ocimum
gratissimum occurs from sea-level up to 1500 m altitude in coastal scrub, along lake shores, in savanna vegetation, in submontane forest, and disturbed land. In South-East Asia it is not frequently found in open locations like roadsides and clearings, but more often cultivated as a hedge plant, up to about 300 m altitude (Sulistiarini, 1999). Ocimum
gratissimum is found throughout the tropics and subtropics, both wild and cultivated. Its greatest variability occurs in tropical Africa (from where it possibly originates) and India. In South-East Asia it is cultivated mainly as a home garden crop, only in Vietnam is it grown on a commercial scale as well (Sulistiarini, 1999).


 

2.4.4 Propagation and Reproductive Biology

Ocimum
gratissimum is propagated by seed or cuttings. In a growth trial in Colombia, germination of Ocimum gratissimum was very poor (<10%); cuttings took 28 days to take root. Flowering started after 136 days and continued until 195 days. Seed matured after 259 days. Flowering and seed set were much poorer than in Ocimum
basilicum L. or Ocimum minimum L. In South-East Asia flowers can be found year-round. In northern India, oil content of young plants was low (2.3%) until the seed setting stage, then remained constant at 2.8% until the seed maturation stage (Sulistiarini, 1999). It prefers most and fertile soil during growth but will tolerate drought after flowering (Sulistiarini, 1999).

2.4.5 Properties of Ocimum gratissimum

Essential oils: The fresh aboveground parts of O. gratissimum contain 0.8-1.2% essential oil. The chemical composition of the oil is variable and at least 6 chemotypes have been reported, characterized by the main component of the essential oil: eugenol, thymol, citral, ethyl cinnamate, geraniol and linalool. An overview of the occurrence of the various types and possible implications for the taxonomy is lacking. The eugenol type is the most important economically; the thymol type was formerly important, but most thymol is now produced synthetically, while natural thymol is mostly obtained from Thymus vulgaris L. or Trachyspermum ammi (L.) Sprague ex Turrill. The other types are of little economic importance. The eugenol-type oil is a brownish-yellow to pale yellow liquid with a powerful, warm-spicy and aromatic odour, reminiscent of clove oil, but with a sweet-woody, almost floral top note. The dry-out is more bitter than that of clove oil. Analysis of a sample of an essential oil of the eugenol type from Vietnam indicated that the main component was eugenol (71%) with small amounts of D-germacrene and (Z)-beta-ocimene. In a sample from southern China the eugenol content was as much as 95%. Samples from Madagascar had eugenol contents of 40-90%, with very variable other components. Medicine: The thymol-type oil is a dark yellow to orange-yellow or brownish liquid with a medicinal-spicy, warm and somewhat herb-like odour. Its flavour is warm, slightly astringent and burning, and has a sweet medicinal aftertaste. Analysis of several samples of essential oils from Ocimum gratissimum from Central and West Africa rich in thymol indicated that their main constituents were thymol, gamma-terpinene, p-cymene and eugenol. The concrete obtained by solvent extraction is much richer in thymol than the distilled oil. A geraniol-rich type, found in the United States, contained mainly geraniol (84-88%) with small amounts of gamma-muurolene, neral, beta-caryophyllene and limonene. The citral type, reported from Iran, Pakistan and India is rich in citral (67%) and geraniol (26%) (Sulistiarini, 1999).


 


Figure 1: Ocimum gratissimum

2.4.6 Uses of Ocimum gratissimum

2.4.6.1 Medicinal uses

The whole plant and the essential oil have many applications in traditional medicine, especially in Africa and India. In folk medicine, it is used in the treatment of upper respiratory tract infection, diarrhoea, headache, ophthalmia, skin disease, pneumonia, cough, fever, conjunctivitis, gonorrhoeal, mental illness, high fever and influenza (Abdulrahman, 1992; Correa, 1932; Dhawan et al., 1977; Oliver, 1980; Osifo, 1992; Sofowora, 1993; Sulisfiarin, 1999).

Ocimum gratissimum leaf or the whole herb are popular treatments for diarrhoea (Dalziel, 1956). The plant is rich in volatile oils which contain upto 70% thymol; whose antimicrobial avtivity is well known. Infact, the antimicrobial activity of the water-saturated oil had been shown to be proportional to the thymol content in preparations where Ocimum gratissimum is used as cold infusion (El-said et al., 1969). Therefore , the antimicrobial effect of the extracted thymol is probably suffient explanation for the antidiarrhoeal effect. However, in certain other preparations, Ocimum gratissimum when boiled with water to form decoction will contain little of the steam-volatile thymol. Such aqeous decoctions were shown to be devoid of antimicrobail activity, but they do relax the guinea pig ileum and rat jejunum invitro (Sofowora, 1982).

The current antifungal therapies used such as amphotericin B, and Fluconazole have certain limitations due to side effects and emergencxe of resistant strains. Ocimum gratissimum has been reported earlier to have invitro antifungal activity against some dermatophytes. Lexa et al., (2005) was able to demonstrate the antifungal property of Ocimum gratissimum. The chloroformic fraction of the extract inhibited 23 isolates (92%)
of Cryptococcus neoformans at a concentration of 62.5µg/ml. Silva et al., 2005, on the other hand was able to demonstrate the effect of the essential oil of Ocimum gratissimum invitro against human pathoigenic dermatophytes. In there experiment, hexane fraction of the extract at a concentration of 125µg/ml. Eugol inhibited the growth of 80% of dermatophytes at the same concentration (Lexa et al., 2005).

The oil of Ocimum gratissimum has been formulated into creams for clinical trials where favourable results were reportes for certain dermatologyal disorders of microbial complications (Sofowora, 1993). Mixtures for internal administration has also been formulated to utilize the antithelmintic property of the oil. The oil has been incorporated into tooth pastes on experimental basis for oral hygeine (Sofowora, 1982).

Recent studies on Ocimum gratissimum proved the plant extract can be a source of medication for people living with Human Immunodeficiency Virus, (HIV) and Acquired Immune Deficiency Virus, AIDS (Elujoba, 2000).

Ocimum gratissimum has been shown to possess antioxidant activity.Afolabi et al., 2007 was able to establish the antioxidant property with regards th the phytochemical property.


 

2.4.3.2 Non medicinal uses

In Indonesia (Sumatra) a tea is made from the leaves, while in Thailand the leaves are applied as a flavouring. In Indonesia the eugenol-type of Ocimum gratissimum is used in the ceremonial washing of corpses and is planted in graveyards. In India O. gratissimum, named 'ram tulsi', is widely used in religious ceremonies and rituals (Sulistiarini, 1999). It is also used as insect repellent (Sofowora, 1982).

2.5 Phytochemical

Phytochemicals are naturally occurring, biologically active chemical compounds in plants. The prefix "Phyto" is from a Greek word meaning plant. In plants, phytochemicals act as a natural defense system for host plants and provide colour, aroma and flavour. More than 4000 of these compounds have been discovered to date and it is expected that scientists will discover many more. Phytochemicals are protective and disease-preventing particularly for some forms of cancer and heart diseases. The most important action of these chemicals with respect to human beings is somewhat similar in that they function as antioxidants that react with the free oxygen molecules or free radicals in our bodies.They are not essential nutrients and are not required by the human body for sustaining life (Burrello, 2005).


 

2.5.1 Types of phytochemicals

The thousands of phytochemicals that have been discovered are grouped based on function and sometimes source. These groupings include the widely studied, flavanoids, phyto-oestrogens, phytosterols and carotenoids. These classes and others can be further divided into subclasses(Burrello, 2005).

The polyphenols include a large subgroup of chemicals called flavonoids. Flavonoids are plant chemicals found in a broad range of fruits, grains, and vegetables. They are being studied to find out whether they can prevent chronic diseases such as cancer and heart disease. The flavonoids found in soybeans, soy products, garbanzo beans, chickpeas, and licorice may mimic the actions of the female hormone estrogen. Estrogen-like substances from these plant sources are called phytoestrogens (Setchell, 1999). They may play a role in the development of some hormone-dependent cancers such as breast and prostate cancer (Gruenwald et al., 2004).

Other polyphenols (including some flavonoids) act as antioxidants. These are thought to rid the body of harmful molecules known as free radicals, which can damage a cell's DNA and may trigger some forms of cancer and other diseases. These compounds are commonly found in vegetables such as broccoli, brussels sprouts, cabbage, and cauliflower and in teas . Grapes, egg plant, red cabbage, and radishes all contain anthocyanidins – flavonoids that act as antioxidants and may protect against some cancers and heart disease. Quercetin, another flavonoid with antioxidant properties, is found in apples, onions, teas, and red wine. Ellagic acid, found in raspberries, blackberries, cranberries, strawberries, and walnuts, also is said to have anti-cancer effects (Kushi et al., 2006).

Carotenoids, which give carrots, yams, cantaloupe, squash, and apricots their orange color, are also promoted as anti-cancer agents. Tomatoes, red peppers,Sent leaf (Ocimum gratissimum) and pink grapefruit contain lycopene, which proponents claim is a powerful antioxidant. The phytochemicals lutein and zeaxanthin, found in spinach, kale, and turnip greens, may reduce the risk of some cancers (Gruenwald et al., 2004).

Another group of phytochemicals, called allyl sulfides, are found in garlic and onions. These compounds may stimulate enzymes that help the body get rid of harmful chemicals. They may also help strengthen the immune system (Craig,1999).

Phyto-oestrogens are naturally occurring plant compounds that structurally resemble mammalian oestrogen . they copy or counteract the effect of oestrogen in the body.

consumption of isoflavone, a phyto-oestrogen, is associated with cancer prevention, improved cardiovascular health and improved bone health. Isoflavones contained in soy are believed to be helpful in the treatment of menopausal symptoms while isoflavones isolated from the roots of the kudzu plant may be used to treatalcoholism by lowering a person's tolerance for alcohol and thus reduce the pleasure response to drinking
(Burrello, 2005).

Phytosterols are plant sterols that occur in most plant species but appear to be most abundant in the seeds of green and yellow vegetables. They are important in the human diet because they help to reduce the amount of dietary cholesterol absorbed by the body by blocking uptake in the intestine. They also facilitate cholesterol excretion from the body. This is especially important as cholesterol is a well established risk factor for heart disease (Burrello, 2005).

2.6 An Overview of Test Organisms

2.6.1 Pseudomonas aeruginosa

Pseudomonas aeruginosa is member of the Gamma Proteobacteria class of Bacteria. It is a Gram-negative, aerobic rod belonging to the bacterial family Pseudomonadaceae. It measures 0.5 to 0.8 µm by 1.5 to 3.0 µm. Almost all strains are motile by means of a single polar flagellum. Its optimum temperature for growth is 37oC, and it is able to grow at temperatures as high as 42oC. It is tolerant to a wide variety of physical conditions, including temperature. It is resistant to high concentrations of salts and dyes, weak antiseptics, and many commonly used antibiotics(Kenneth,2008).


Pseudomonas aeruginosa has a predilection for growth in moist environments, which is probably a reflection of its natural existence in soil and water (Kenneth, 2008). Pseudomonas aeruginosa isolates may produce three colony types. Natural isolates from soil or water typically produce a small, rough colony. Clinical samples, in general, yield one or another of two smooth colony types. One type has a fried-egg appearance which is large, smooth, with flat edges and an elevated appearance. Another type, frequently obtained from respiratory and urinary tract secretions, has a mucoid appearance, which is attributed to the production of alginate slime. The smooth and mucoid colonies are presumed to play a role in colonization and virulence (Kenneth, 2008). Pseudomonas aeruginosa strains produce two types of soluble pigments, the fluorescent pigment pyoverdin and the blue pigment pyocyanin. The latter is produced abundantly in media of low-iron content and functions in iron metabolism in the bacterium. Pyocyanin (from "pyocyaneus") refers to "blue pus", which is a characteristic of suppurative infections caused by Pseudomonas aeruginosa (Kenneth, 2008).

Pseudomonas bacteremia resembles other bacteremias, producing fever, tiredness, muscle pains, joint pains, and chills. Bone infections are marked by swelling, redness, and pain at the infected site and possibly fever. Pseudomonas meningitis causes fever, headache, irritability, and clouded consciousness. Ear infection is associated with pain, ear drainage, facial paralysis, and reduced hearing. Pseudomonas infections of the eye cause ulcers that may spread to cover the entire eye, pain, reduced vision, swelling of the eyelids, and pus accumulation within the eye. Pseudomonas aeruginosa pneumonia is marked by chills, fever, productive cough, difficult breathing, and blue-tinted skin (Belinda, 2006). Patients with cystic fibrosis with pseudomonas lung infections experience coughing, decreased appetite, weight loss, tiredness, wheezing, rapid breathing, fever, blue-tinted skin, and abdominal enlargement. Skin infections can cause a range of symptoms from a mild rash to large bleeding ulcers. Symptoms of Pseudomonas folliculitis include a red itchy rash, headache, dizziness, earache, sore eyes, nose, and throat, breast tenderness, and stomach pain. Pseudomonas wound infections may secrete a blue-green colored fluid and have a fruity smell. Burn wound infections usually occur one to two weeks after the burn and cause discoloration of the burn scab, destruction of the tissue below the scab, early scab loss, bleeding, swelling, and a blue-green drainage (Belinda, 2006).

2.6.2 Bacillus cereus

Bacillus cereus are Gram-positive, facultative aerobic sporeforming rod. They can be differentiated from other Bacillus species by their cell position and biochemical tests. The optimum growth temperatures range from 30 to 50°C, although some psychrotrophic strains can grow down to 4 to 5°C. They can grow at pH values of between 4.3 and 9.3, and can grow at water activity values down to 0.912. The organism produces heat resistant spores and these may germinate if cooling is too slow (Hocking et al., 1997).

Bacillus cereus causes two types of food-borne illnesses. One type is characterized by nausea and vomiting and abdominal cramps and has an incubation period of 1 to 6 hours. It resembles Staphylococcus aureus (staph) food poisoning in its symptoms and incubation period. This is the "short-incubation" or emetic form of the disease. The second type is manifested primarily by abdominal cramps and diarrhea following an incubation period of 8 to 16 hours. Diarrhea may be a small volume or profuse and watery. This type is referred to as the "long-incubation" or diarrheal form of the disease, and it resembles food poisoning caused by Clostridium perfringens. In either type, the illness usually lasts less than 24 hours after onset. In a few patients symptoms may last longer (Kenneth, 2008). The short-incubation form is caused by a preformed, heat-stable emetic toxin, ETE. The mechanism and site of action of this toxin are unknown, although the small molecule forms ion channels and holes in membranes. The long-incubation form of illness is mediated by the heat-labile diarrheagenic enterotoxin Nhe and/or hemolytic enterotoxin HBL, which cause intestinal fluid secretion, probably by several mechanisms, including pore formation and activation of adenylate cyclase enzymes(Kenneth, 2008).


 

2.6.3 Proteus mirabilis

Proteus mirabilis a Gram-negative, facultatively anaerobic
bacterium. It shows swarming motility, and urease activity. It can produce hydrogen sulfide gas, and forms clear films on growth media. It is commonly found in the intestinal tract of humans. P. mirabilis is not pathogenic in guinea pigs or chickens
( Gus, 2006).

Proteus species possess an extracytoplasmic outer membrane, a feature shared with other gram-negative bacteria. In addition, the outer membrane contains a lipid bilayer, lipoproteins, polysaccharides, and lipopolysaccharides ( Frenod et al., 2006).

Infection depends on the interaction between the infecting organism and the host defense mechanisms. Various components of the membrane interplay with the host to determine virulence. Inoculum size is important and has a positive correlation with the risk of infection. Certain virulence factors have been identified in bacteria. The first step in the infectious process is adherence of the microbe to host tissue. Fimbriae facilitate adherence and thus enhance the capacity of the organism to produce disease. Escherichia coli, Proteus mirabilis, and other gram-negative bacteria contain fimbriae, which are tiny projections on the surface of the bacterium. Specific chemicals located on the tips of pili enable organisms to attach to selected host tissue sites (an example is, urinary tract endothelium). The presence of these fimbriae has been demonstrated to be important for the attachment of Proteus mirabilis to host tissue. The attachment of Proteus species to uroepithelial cells initiates several events in the mucosal endothelial cells, including secretion of interleukin 6 and interleukin 8. Proteus organisms also induce apoptosis and epithelial cell desquamation. Bacterial production of urease has also been shown to increase the risk of pyelonephritis in experimental animals. Urease production, together with the presence of bacterial motility and fimbriae, may favor the production of upper urinary tract infections (UTIs) by organisms such as Proteus ( Gus, 2006).

Enterobacteriaceae (of which Proteus is a member) and Pseudomonas species are the microorganisms most commonly responsible for gram-negative bacteremia. When these organisms invade the bloodstream, endotoxin, a component of gram-negative bacterial cell walls, apparently triggers a cascade of host inflammatory responses and leads to major detrimental effects. Because Proteus and Pseudomonas organisms are gram-negative bacilli, they can cause gram-negative endotoxin-induced sepsis, resulting in systemic inflammatory response syndrome (SIRS). SIRS has a mortality rate of 20-50% (Hocking et al., 1997).

The presence of the sepsis syndrome associated with a UTI should raise the possibility of urinary tract obstruction. This is especially true of patients who reside in long-term care facilities, who have long-term indwelling urethral catheters, or who have a known history of urethral anatomic abnormalities. The ability of Proteus organisms to produce urease and to alkalinize the urine by hydrolyzing urea to ammonia makes it effective in producing an environment in which it can survive. This leads to precipitation of organic and inorganic compounds, which leads to struvite stone formation. Struvite stones are composed of a combination of magnesium ammonium phosphate (struvite) and calcium carbonate-apatite ( Gus, 2006).

Struvite stone formation can be sustained only when ammonia production is increased and the urine pH is elevated to decrease the solubility of phosphate. Both of these requirements can occur only when urine is infected with a urease-producing organism such as Proteus. Urease metabolizes urea into ammonia and carbon dioxide: Urea ® 2NH3 + CO2. The ammonia/ammonium buffer pair has a pK of 9.0, resulting in the combination of highly alkaline urine rich in ammonia. Symptoms attributable to struvite stones are uncommon. More often, women present with UTI, flank pain, or hematuria and are found to have a persistently alkaline urine pH (>7.0) ( Gus, 2006).


 

2.6.4 Staphylococcus aureus

Bacteria of the genus Staphylococcus are gram-positive cocci that are microscopically observed as individual organisms, in pairs, and in irregular, grapelike clusters. The term Staphylococcus is derived from the Greek term staphyle, meaning "a bunch of grapes." Staphylococci are nonmotile, non–spore-forming, and catalase-positive bacteria. The cell wall contains peptidoglycan and teichoic acid. The organisms are resistant to temperatures as high as 50°C, to high salt concentrations, and to drying. Colonies are usually large (6-8 mm in diameter), smooth, and translucent. The colonies of most strains are pigmented, ranging from cream-yellow to orange (Robert et al., 2009 ).

The ability to clot plasma continues to be the most widely used and generally accepted criterion for the identification of Staphylococcus aureus. One such factor, bound coagulase, also known as clumping factor, reacts with fibrinogen to cause organisms to aggregate. Another factor, extracellular staphylocoagulase, reacts with prothrombin to form staphylothrombin, which can convert fibrinogen to fibrin. Approximately 97% of human S aureus isolates possess both of these forms of coagulase (Robert et al., 2009 ).

The postulated sequence of events that leads to infection is initiated with carriage of the organism. The organism is then disseminated via hand carriage to body sites where infection may occur (either through overt breaks in dermal surfaces, such as vascular catheterization or operative incisions, or through less evident breakdown in barrier function, such as eczema or shaving-associated microtrauma) (Robert et al., 2009 ).

The hallmark of staphylococcal infection is the abscess, which consists of a fibrin wall surrounded by inflamed tissues enclosing a central core of pus containing organisms and leukocytes. From this focus of infection, the organisms may be disseminated hematogenously, even from the smallest abscess. The ability to elaborate proteolytic enzymes facilitates the process. This may result in pneumonia, bone and joint infection, and infection of the heart valves. In immunocompromised hosts (eg, patients with cancer who are neutropenic and have a central venous line), 20-30% develop serious complications or fatal sepsis following catheter-related S aureus bacteremia (Robert et al., 2009 ).

Persistent deep-seated infections have now been linked to small-colony variants of the organism. This population is more resistant to antibiotics and grows slowly. These organisms have been described in patients with cystic fibrosis and may contribute to the persistence of S aureus in these patients (Robert et al., 2009).

The organism may also elaborate toxins that can cause specific diseases or syndromes. Enterotoxin-producing strains of S aureus cause one of the most common food-borne illnesses. The most common presentation is acute onset of vomiting and watery diarrhea 2-6 hours after ingestion. The symptoms are usually self-limited. The cause is the proliferation of toxin-producing organisms in uncooked or partially cooked food that an individual carrying the staphylococci has contaminated (Robert et al., 2009 ).

A rare but well-described disorder in neonates and young children is staphylococcal scalded skin syndrome (Ritter disease). The organism produces an exfoliative toxin produced by strains belonging to phage group II. Initial features include fever, erythema, and blisters, which eventually rupture and leave a red base. Gentle shearing forces on intact skin cause the upper epidermis to slip at a plane of cleavage in the skin, which is known as the Nikolsky sign. How the exfoliative toxins produce epidermal splitting has not been fully elucidated (Robert et al., 2009 ).

The most feared manifestation of S aureus toxin production is toxic shock syndrome (TSS). Although first described in children, it was most frequently associated with women using tampons during menstruation. Since the early 1990s, at least half of the cases have not been associated with menstruation. The syndrome is associated with strains that produce the exotoxin TSST-1, but strains that produce enterotoxins B and C may cause 50% of cases of nonmenstrual TSS. These toxins are superantigens, T-cell mitogens that bind directly to invariant regions of major histocompatibility complex class II molecules, causing an expansion of clonal T cells, followed by a massive release of cytokines. This cytokine release mediates the TSS; the resultant pathophysiology mimics that of endotoxic shock (Robert et al., 2009 ).

In a recent worldwide trend, the proportion of infections caused by CA-MRSA has increased. Initially noted in tertiary care centers, these infections are now increasingly common in the community. Resistance to methicillin confers resistance to all penicillinase-resistant penicillins and cephalosporins (Robert et al., 2009 ).


 

2.6.5 Salmonella spp

These are gram negative rod shaped bacteria. Salmonella organism are found in virtually all animals, birds (including poultry), reptiles, rodents, domestic animals, and humans(Murray et al., 1998). Some serotypes of Salmonella are virtually species specific(Hook, 1990).

Salmonella spp. Cause many types of infections, from mild self-liumiting to life-threatening typhoid fever. The most commom form of Salmonella disease is self-limiting gastroenteritis with fever lasting less than 2 days and diarrhoea lasting for seven days. Typhoid fever, the best-studied enteric fever, is characterized by fever, headache, diarrhoea, and abdominal pain and can produce fatal respiratory, hepatic, spleen, and/or neurologic damage. Bacterimia, meningitis, respiratory disease, cardiac disease, osteomyelitis, and other local infections caused by Salmonella spp. Have been reported. S. typhi and S. paratyphi A and B cause gastroenteritis, bacteremia, and enteric fever (Hook, 1990).


 


 


 

CHAPTER THREE

MATERIALS AND METHODS

3.1 Sample collection/Identification

The stem of Ocimum gratissimum was used for this study. The stems were collectedFrom

University of Jos Senoir Staff Quarters, Bauchi road, Jos. It was then identified at the haberium

of the Federal College of Forestry, Jos. The stems (after washing with distilled water) were wet-

milled using laboratory mortal and pestle and then allowed to air-dry to constant weight under

shade. The stem was further micronized using an electric blender. The powders were stored in

airtight bottles until required.

3.2 Extraction and Determination of Phytoconstituents

Hundred grammes (100 g) of the powdered plant material was soaked in 500 ml each of distilled water and 99.9% ethanol separately in 1000 ml sterile conical flasks at room temperature for 72 hours. The content was filtered with Whatman No. 1 filter paper. The filtrates were gently evaporated to dryness and then packed in separate clean dry bottles and stored at room temperature until required. The extracts were screened for the presence of carbohydrates, tannins, alkaloids, saponins, phenolics, anthraquinones and cardiac glycosides as described by Trease and Evans (1984) and Sofwora (1986).


 

3.3 Test Organisms

Bacillus cereus, Staphylococcus aureus, Proteus mirabilis, Salmonella paratyhi

and Pseudomonas aeriginosa were obtained from the federal College of Vetenary

and Medical Laboratory Technology, vom.


 

3.4 Preparation of Stock Solutions of the Extract

The stock solutions of the two extracts (aqeuous and ethanolic) were prepared by

dissolving 4g of each extract in 10ml of sterile distilled water to give a

concentration of 400mg/ml. The stocks were labelled appropriately and

refrigerated at 4oC until required for use.


 

3.5 Preparation of the Test bacterial Isolates

Fresh plates of the test bacteria were made from the isolate cultures obtained on agar slants. Colonies of Fresh cultures of the different bacterial isolates were picked and suspended in 5 ml nutrient broth in well-labeled sterile bijou bottles. They were incubated for 24 h at 37ºC.


 

3.6 Determination of Antimicrobial Activity of Extracts

The agar well dilution method as described by Lino and Deogracious (2006) was used. Standardized inoculum (0.5 McFarland turbidity standard equivalent to 5 x 108cfu/ml) NCCLS, 1990) of each test bacterium was spread onto sterile nutrient agar plates so as to achieve even growth. The plates were allowed to dry and a sterile cork borer (3.0 mm diameter) was used to bore wells in the agar plates. The extracts (ethanolic and aqeuous) were prepared by double dilution method. This was done by adding 5ml of the stock solution to a testtube containing 5ml of sterile distilled water to achieve a concentration of 200mg/ml. This concentration (200mg/ml) was further diluted to concentrations of 100mg/ml and 50mg/ml. Subsequently, 0.1 ml of each concentration of the extracts was introduced in wells earlier bored in the nutrient agar plate cultures. Chloramphenicol (100 mg/ml) (Joabez Corporation) was used as a positive control and 20% nutrient agar as negative control. The plates were then incubated at 37oC for 24 h. Antimicrobial activity of the extracts was determined after incubation period by measurement of zones of inhibition. The sensitivity test was done in replicate and mean zone of inhibition was taken.


 

3.7 Effect of pH on activity on the plant extract


 

This was carried out as described by Emeruwa (1982). Two grammes of the extract powder was dissolved in 5 ml of distilled water in a test tube and the contents filtered. One millimeter each of the filtrate was pipetted into two separate test tubes. To the first test tube few drops of 1N HCl was added dropwise until pH 3 was attained, and to the second test tube few drops of 1N NaOH was also added dropwise until pH 10 was attained. All the extracts were allowed to soak for 30 min after which they were neutralized (pH 7) using the appropriate solvents. Another test tube containing extracts only (untreated) was left for the same period (30 min) to serve as control. Antimicrobial activity of both the treated and untreated extracts were then determined as described in 3.6.


 

3.8 Determination of Minimum Inhibitory Concentration (MIC)

The MIC of the potent extracts was determined according to the broth dilution technique
(Junaid et al., 2006). Standardized suspensions of the test organisms (1.0 x 108 cfu/ml) were inoculated into a series of sterile tubes of nutrient broth containing two-fold dilutions (400mg/ml, 200mg/ml, 100mg/ml, 50mg/ml, 25mg/ml, 12.5 and 6.25mg/ml) of leaf extracts, and incubated at 37°C for 24 h. The MICs were read as the least concentration that inhibited the growth of the test organisms.


 

3.9 Determination of Minimum Bactericidal Concentration (MBC)

To obtain the MBC, two loopfuls of broth culture were taken from the tubes that showed no growth in the above test tubes used for the MIC determination and were inoculated on to agar plates. The plates were incubated at 37oC for 24 h and observed for growth. The least concentration that did not show any growth after incubation was regarded as the MBC.


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 

CHAPTER FOUR

RESULTS

The result of the preliminary phytochemical screening of the ethanolic and aqueous stem extract of Ocimum gratissimum showed no differences in the phytochemical constituents. Saponins, alkaloids,`phenol, renin, balsam, terpenes and steroids, Cardiac glucoside, tannins and cardiac glycosides were found to be present (Table1).

With respect to the antimicrobial sensitivity of the test organisms to aqueous plant extract, all the test organisms showed zones of inhibition to the highiest concentration used (400mg.ml) with Proteus mirabilis showing a zone of inhibition of 17mm while Bacillus cereus, Salmonella paratyphi and Pseudomonas aeruginosa zones of inhibition of 13mm, 12mm and 11mm respectively (Plates 1,2 and 3). Staphylococcus aureus showed no zone of inhibition to all the concentrations. No zones of inhibition were reported at lower concentrations with the exception of Bacillus cereus showing zones of inhibition of 11mm and 10mm to concentrations of 200mg/ml and 100mg/ml respectively and Proteus mirabilis that showed a zone of inhibition of 10mm at a concentration of 200mg/ml (Table 2).

For the ethanolic extract, Staphylococcus aureus and Salmonella paratyphi showed the higest zones of inhibition (18mm) at 400mg/ml while Bacillus cereus,Proteus mirabilis and Pseudomonas aeruginosa were 14mm, 11mm, and 9mm respectively.Bacillus cereus, Proteus mirabilis, Pseudomonas aeruginosa and Salmonella paratyphi showed zones of inhibition of 13mm,10mm, 9mm and 7mm respectively at a concentration of 200mg/ml. Only B. cereus showed zone of inhibition of 11mm at a concentration of 100mg/ml. No zones of inhibition were observed for all the test organisms at the least concentration (50mg/ml). The control (100 mg/ml of chloramphenicol) showed a higher zone of inhibition compared with the extract (Table 3).

Table 1. Phytochemical screening of the stem extract of Ocimum gratissimum


 

Phytoconstituent 

Aqueous

Ethanolic

Alkaloid

+ 

+ 

Flavonoid

- 

- 

Tannin

+ 

+ 

Saponin

+ 

+ 

Cardiac glucoside

+ 

+ 

Terpenes and Steroids

+ 

+ 

Balsam

+ 

+ 

Renin

+ 

+ 

Phenol

+ 

+ 


 

  • = Not present

+ = Present


 


 


 


 


 


 


 


 

Table 2. Antimicrobial Sensitivity of the Aqueous extract of Ocimum gratissimum


  

Zones of Inhibition (mm)

   

Concentrations(mg/ml) 

S. aureus 

B. cereus 

P. aeruginosa 

P. mirabilis 

S. paratyphi 

400 

- 

13 

11 

17 

12 

200 

- 

11 

- 

10 

- 

100 

- 

9 

- 

- 

- 

50 

- 

- 

- 

- 

- 

Reference standard drug

35 

30 

33 

26 

27 

Reference standard drug = 100 mg/ml Chloramphenicol

- = No zone of inhibition


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 

Table 3. Antimicrobial Sensitivity of the Ethanolic extract of Ocimum gratissimum


  

Zones of Inhibition 

   

Concentrations (mg/ml) 

S. aureus 

B. cereus 

P. aeruginosa 

P. mirabilis 

S. paratyphi 

400 

18 

14 

9 

11 

18 

200 

- 

13 

9 

10 

7 

100 

- 

9 

- 

- 

- 

50 

- 

- 

- 

- 

- 

Reference standard drug

35 

30 

26 

26 

27 


 

Reference standard drug = 100 mg/ml Chloramphenicol                        

- = No zone of inhibition


 


 


 


 

Slight increase in the activity of all the extracts were observed when the were subjected to varying pH conditions (pH 3 and pH 10) at a concentration of 400mg/ml. For the ethanolic extract,Bacillus cereus showed zones of inhibition of 18mm and 16mm while Salmonella paratyphi showed zones of inhibitions of 20mm and 19mm at pH 3 and 10 respectively . For the aqueous extract, Bacillus cereus showed zones of inhibition of 15mm at both pH of 3 and 10 while Salmonella paratyphi showed zones of inhibitions of 16mm and 14mm at pH 3 and 10 respectively (Table 4).

For MIC, the growth of all the isolates (with the exception of S. aureus)
were inhibited at a higher concentrations of the aqueous and ethanolic extract (400mg/ml and 200mg/ml). Non of the test organisms were inhibited at lower concentrations. The MBC for Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Salmonella
parattyphi and Proteus mirabilis ranged from 200mg/ml to 400mg/ml for the ethanolic extract while that of the aqueous extract is 400mg/ml for Bacillus cereus and Proteus mirabilis. Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella parattyphi had no MBC for the aqueous extract (Table 5 ).


 


 


 


 


 


 


 


 


 


 


 


 


 

Table 4. Effect of pH on the Antimicrobial activity of the extract (400mg/ml) of Ocimum gratissimum


   

Zones of Inhibition (mm)

   


 

Oraganisms 

Ethanolic

extractS 

 


 

Aqueous

extract


 

 


 

*Ethanolic extract 

** Aqueous extract 

 

pH 3 

pH 10

pH 3 

pH 10 

  

S. paratyphi

20 

19 

16 

14 

17 

12 

B. cereus

18 

16 

15 

15 

15 

13 


 

*Untreated extract pH 4.5, **Untreated pH 4.8


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 

Table 5. MIC and MBC of Ethanolic and Aqueous extract of Ocimum gratissimum


 


 

Test organism


 

Concentration of Extract mg/ml MIC MBC  


 


 

400 


 

200 


 


 

100 


 

50 


 

25 


 


 

12.5 


 

6.25  


 


 


 


 


 

E 


 


 

A 


 

E 


 

A 


 

E 


 

A 


 


 

E 


 

A 


 

E 


 

A 


 


 

E 


 

A  


 

E


 

A


 


 

E 


 

A  


 

E 


 

A 

S. aureus 

-

+

+

+

+

+

+

+

+

+

+

+

+

+

400

-

400

-

B. cereus 

- 

-

-

-

+

-

+

+

+

+

+

+

+

+

200

200

400

-

P. aeruginosa

-

-

-

+

+

+

+

+

+

+

+

+

+

+

200

400

400

-

S. paratyphi

-

-

-

+

+

+

+

+

+

+

+

+

+

+

200

400

400

-

P. mirabilis

-

-

-

-

+

+

+

+

+

+

+

+

+

+

200

200

-

400

Key

  • = no growth

+ = growth

E = Ethanolic extrac

A = Aqueous extract


 

CHAPTER FIVE

DISCUSSION

Discussion

Preliminary phytochemical investigations of the stem extract of Ocimum gratissimum revealed the presence of some phytochemical compounds. These phytochemicals have earlier been reported in the leaf extract of this plant (Afolabi et al., 2007). These secondary metabolites are linked to antimicrobial activity of the plant material. Drugs contained in medicinal plants are called active principles and these active principles are divided into a number of groups. Carbohydrates present in plants are mostly in the form of pentoses, sucrose and soluble sugars which are intermediate plant constituents and whose pharmacognostic significance in stem form the fact that they combine with a numerous variety of compounds to form glycosides. The presence of glycoside moieties like saponins, anthracene and cardiac glycolsides, some of which are known to structurally resemble sex hormones (oestrogens, gestrogens and androgens) are known to protect against gastric infections caused by enteric pathogens (Salmonella paratyphi) thus justifying the use of this plant in traditional medicinal practice (El-Mahmood et al., 2008).

All the extracts from the different solvents demonstrated antimicrobial activity with ethanol showing the highest activity against the test organisms with exception of Proteus mirabilis and Pseudomonas aeruginosa. Aqueous extract demonstrated higher activity against these two organisms. Variation in activity among different extracting solvents has earlier been reported (Falodun et al., 2006). When plant materials are ground in water or the plant cells are damaged, some phenolases and hydrolases are often released and these enzymes might have modulatory effect on the activity of the active compounds in the extract or there may be incomplete extraction of the active principles thus explaining the low activity. Traditionally, however crude plant extracts are prepared with water as infusions, decoctions and poultices, therefore it is very unlikely that the herbalist is able to extract all these compounds, which are responsible for the activity observed in ethanolic extract. Generally, ethanol showed broader and greater spectra of activity against the tested organisms (El-Mahmood et al., 2008).

The results obtained also showed that the activity of all the extracts were concentration dependent. Similar results have been reported by several researchers. Highest activity was demonstrated by the standard reference antibiotic chloramphenicol (control). This is because the antibiotic is in pure state and has refined processes that have established it as a standard antibiotic (Prescott et al., 2002). The activity of the extract against the test organisms indicates that it can be a potential source of a broad spectrum antibiotic (Junaid, 2006).

Acid and alkaline treatment was carried out in order to slightly simulate the stomach and duodenal conditions and also to predict the condition under which the drug would be more effective, if it is to be formulated for commercial purposes and since the plant is also used in the treatment of constipation and diarrhea traditionally.

Demonstration of high MIC and MBC values is an indication that the phytoconstituents of the plant have little therapeutic properties and therefore justifies its uncommon usage in traditional traditional medicinal uses though the therapeutic properties may be increased if used in a refined form.

The resistance of Staphylococcus aureus to the aqueous extract justifies the work of Adebolui et al., 2005 where S. aureus was resistant to the hot and cold aqueous leaf extract of Ocimum gratissimum. Furthermore, Adebolu et al. (2005)
and Lexa et al. (2006) reported the activity of essential oil of the plant on Staphylococcus, Bacillus spp, Proteus mirabilis and Pseudomonas aeruginosa where higher zones of inhibition were observed at a lower concebtration of 75µg/ml (the highiest concentration used) . Lima et al. (1993) tested 13 essential oils obtained from plants against dermatophytes. Ocimum gratissimum was found to be the most active in inhibiting 80% of the dermatophyte strains tested and producing zones greater than 10 mm diameter. Similarly, Nwosu and Okafor (1995) reported the antifungal activities of extracts of ten medicinal plants collected from Southeastern Nigeria against seven pathogenic fungi. According to the report, O. gratissimum inhibited the growth of Trichophyton rubrum and T. Mentagrophytes. In this present study, a lower activity of the stem extract may also be due to the high volatibility of the oil, leading to the escape or evaporation of the oil during the evaporation and for the cold-water extract, it may be due to insufficient release of the oil during extraction. The antimicrobial substance present in the oil that is probably responsible for this observation is likely to be eugenol. This component has been demonstrated to have both antibacterial (Nakaruma et al., 1999) and antihelmintic activities (Pessoa et al., 2002).

The organisms used for the purpose of this investigation are associated with various forms of infections; Staphyhlococcus aureus , Bacillus cereus (food poisoning), P.
mirabilis (wound infections and UTIs), Pseudomonas aeruginosa (opportunistic pathogen) and Salmonella paratyphi (paratyphoid fever) (Prescott et al. 2002). Results of this investigation therefore have shown that Ocimum gratissimum is a potential source of antibiotic substances for use against these test organisms.

5.2 Conclusion and Recommendation

In conclusion, Salmonella typhi and Staphylococcus aureus were sensitive to the ethanolic extract while only Proteus mirabilis was sensitive to the aqueous extract. It is recommended that the antibacteria property of the stem of Ocimum gratissimum be tested against more pathogenic microbes,different solvents such as methanol should be used for the extraction of the bioactive components, and the stem extracts should also be tested invivo to authenticate effectiveness of the stem extract for the treatment of diseases asssociated with the test organisms.


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 

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APPENDIX 1

Procedure for Preliminary Phytochemical Screening of Plant Extract

Test for Alkaloids

To 2.0ml of the extract, few drops of dragendorf reagent was added and observed for orange colouration.

Test for Flavanoids

2.0ml of 5% NaOH was added to the extract and observed for yellow colouration.

Test for Tannins

1.0ml of the extract was diluted with 4.0ml of distilled water in a ratio of 4:1. This was followed by the addition of few drops of FeCL3 solution. The mixture was observed for blue or green colour formation

Test for Saponins

To 2.0ml of the extract, 5ml of distilled water and shaked vigorously for 2 minute which few drops of olive oil eas added. Formation of an emulsion indicated the presence of saponins.

Test for Cardiac Glycoside

To 1.0ml of the extract, 1.0ml of Barfoed's reagent was added. The solution was heated on hot plate for 2 minute. The formation of brick red precipitate indicates the presence of cardiac glycoside

Test for Phenols

chloroform of FeCl3 was added to 2.0ml of the extract. Deep bluish green colouration indicated the presence of phenols .


 


 


 

APPENDIX II

Preparation of Turbidity Standard

  1. 1% v/v solution of sulphuric acid was prepared by adding 1ml of concentrated sulphuric acid to 99 ml of water.

  1. 1%w/v of Barium chloride was prepared by dissolving 0.5g of dihydrate barium chloride (BaCl2.2H2O) in 50 ml of distilled water.

  1. 0.6 ml of the barium chloride solution was added to 99.4 ml of sulphuric acid solution and mixed.
  2. A small volume of the turbid solution was transfered to a capped tube.


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 

Plates


 


 



 

Plate 1: Sensitivity of Bacillus cereus to stem extract of Ocimum gratissimum


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 



 


 

Plate 2: Sensitivity of Proteus mirabilis to stem extract of Ocimum gratissimum


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 



 


 


 

Plate 3: Sensitivity of Pseudomomas aeruginosa to stem extract of Ocimum gratissimum


 


 

Research: Olaniyi, Reuben Olayinka

Student project for the award of BSc in microbiology

Email:olayinkaolaniyi60@gmail.com


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 

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