MINIMUM INHIBITORY CONCENTRATION, MINIMUM BACTERICIDAL CONCENTRATION OF PSIDIUM GUAJAVA AND OCIMUM GRATISSIMUM ON BACTERIA

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ABSTRACT

The antibacterial activities of the crude extracts from the leaves of Psidium guajava and Ocimum gratissimum were investigated on both gram positive and gram negative bacteria which were isolated from the soil, stream water, cow dung and the skin of humans. These organisms were cultured on different media. Gram staining and biochemical tests were carried out on the organisms for identification. Organisms like E. coli, Pseudomonas, Streptococcus, Staphylococcus aureus, Salmonella, Shigella and Bacillus were identified. The plants were extracted after two weeks of air drying and grinding using the grinder. Two solvents, ethanol and water were used for extraction and about 30g each of the leaves were macerated 200ml of the solvents for 48hrs, which were then evaporated to dryness using the water bath to obtain the crude extract which weighed about 4g each of the extract in ethanol and 2.5g each of the extract in water. The sensitivity, minimum inhibitory concentration and the minimum bactericidal concentration of the extracts were tested on the isolates and the organisms showed susceptibility to the extracts except Pseudomonas. The phytochemical screening of the crude extracts from the plants were also investigated and constituents like tannins, saponins alkaloids and others were seen at different concentrations on different leave extract. The result all showed that these plants can be used in the formulation of oral antibacterial drugs and support the reason why some countries use them as traditional herbal medicine. In conclusion the leave extracts were effective and had a wide range of activity against both gram positive and gram negative bacteria.




TABLE OF CONTENTS

Title page                                                                 i

Certification                                                                                             ii

Dedication                                                                        iii

Acknowledgement                                    iv

 Table of contents                                                           v

List of tables                                                                               ix

Abstract                  x                                                                                                                       

CHAPTER ONE                                                           

INTRODUCTION                                                                       

1.0  BACKGROUND OF THE STUDY                      1

1.1Brief description of Psidium guajava (Guava)                                        2

1.2 Brief description of Ocimum gatissimum (Scent leave)                                                         2

1.3 Justification for the work                                                                                                        3

1.4 Aims and objectives                                                                                                                 3

CHAPTER TWO

LITERATURE REVIEW                                                           

2.0   Overview                                                                         4

2.1    Psidium guajava                                                                     4

2.1.1 Chemical composition                                                5

2.1.2 The leaves                                         6

2.1.3 The roots                                                  7

2.1.4 The seeds                                                                  7

2.1.5 Antibacterial activity:                    7

2.1.6 Anti-diarrheal                                          8

2.1.7 Anti-inflammatory effect:                            8

2.1.8 Antispasmodic:                                               9

2.1.9 CNS activity                                                       9

2.1.10 Conjunctivitis:                                                10

2.1.11 Coughs                                                                   10

2.1.12 Diabetes:                                                            10

2.1.13 Skin use                                10

2.1.14 Vaginal disorders                                                 11

2.2  Ocimum gratisimum                                                                12

2.2.1 Chemical composition                                          12

2.2.2      Antibacterial  activity                         12                            

2.2.3      The oil of Ocimum gratissimum                                 13

2.3     Description of test organism                                               14

2.3.1 Salmonella sp;                                          14

2.3.2. Shigella sp                                       14

2.3.3    Escherichia coli;                                          15

2.3.4 Bacillus cereus;                                                    15


CHAPTER THREE

3.0 MATERIALS AND METHOD                                               

3.1 Sources of leaves                                                          16

3.2 Sources of isolation of organisms                                      16

3.3 Isolation of the organisms                                         16

3.4 Preparation of media and culturing of                16           

3.5 Identification of organisms                            17

3.6 Biochemical identification of the organisms                           17

3.6.1 Catalase test                                      17

3.6.2 Indole test                                                  17

3.6.3 Oxidase test                                          17

3.6.4 Coagulase test                                    17

3.3.5 Motility test                                               17

3.6.6 Citrate test                             18

3.7 Preparation of the extracts                                                      18

3.8 Phytochemical screening                                    18

3.8.1 Flavonoids:                                         19

3.8.2 Tannins:                                                        19

3.8.3 Test for alkaloids:                                               19

3.8.4 Test for sterols and steroids:          20

3.8.5 Test for the carborhydrate:                                                      20

3.8.6 Starch                                     20                                                                 

 3.9 Preparation of the inoculum                                        21

3.10 Bacterial sensitivity testing using agar well diffusion method                21

3.11 Determination of the minimum inhibitory concentration of the plant extracts             21

3.12 Determination of the minimum bactericidal concentration (MBC)                22


CHAPTER FOUR

4.0 RESULTS      

4.1 Result from the gram staining reaction                                                      25

4.2 Result from biochemical tests                              25

4.3 Result of the phytochemical screening P. guajava and O. gratissimum                                                 26

4.4 Antibacterial sensitivity test                     27

4.4.1 P. guajava                                                                             27

4.4.2 O. gratissimum                                        28

4.5 Determination of the minimum inhibitory concentrations using tube dilution method                     28

4.5.1 MIC of P. guajava in ethanol             28

4.5.2 MIC of P. guajava in water                                 29

4.5.3 MIC of O. gratissimum in ethanol                                            30

4.5.4 MIC of O. gratissimum in water                                               30 

4.6 Determination of MBC                                                  31


CHAPTER FIVE

5.1 Discussions                                                                        

5.2 Conclusion                                                                                33

REFERENCES                      34




 

LIST OF TABLES

Table 4.1 Gram staining reaction                                        25

Table 4.2 Biochemical test of different isolates                          25 

Table 4.3 Phytochemical screening of P. guajava and O. gratissimum                                    26

Table 4.4 Antibacterial sensitivity test with P. guajava                            27

Table 4.5 Antibacterial sensitivity test with O. gratissimum                   28

Table 4.6 MIC of P. guajava in ethanol                                           28

Table 4.7 MIC of P. guajava in water                                         29

Table 4.8 MIC of O. gratissimum in ethanol                                 30

Table 4.9 MIC of O. gratissimum in water                                    30

Table 4.10 MBC of P. guajava and O. gratissimum in ethanol and water                           


 

 

 

 

 

CHAPTER ONE

INTRODUCTION


1.0   BACKGROUND OF THE STUDY

The use of plants and herbs extract in the treatment of human ailments is a very ancient art, a practice that has been passed on for generations and scientists in Africa and other developing countries are conducting research into local plants abundant in the continent for their possible use in traditional medicine (Nneamaka, 1991).

Research into traditional plants and herbs received further boost due to increasing resistance to many orthodox medicine and thus a research for new organic molecules of plants with antimicrobial properties (Sofowara, 1993). Bacteria are responsible for high mortality rates in numerous developing countries with as many as 50,000 people dying as a consequence of infection. Plants remedies are increasingly being recognized by scientists as a very important cost alternative to industrially produced antibiotics which are not available to all who need them because of their high price. So present work expresses that plants have great potential as antimicrobial compounds against microorganisms (Rajendra et al., 2012).

Amongst the various forms of treatment for diarrhea, the use of traditional plant remedies is common and widespread. The world health organization (WHO) has categorized more than 20000 plant species with medicinal properties providing treatment for such complaints as pneumonia, ulcers, diarrhea, bronchitis, colds and diseases of the respiratory tracts. One method among the many ways in which plants are used in popular remedies is to extract and consume essential plant oils. Essential oils are complex chemical mixtures, typically composed of more than a hundred compounds by and large are responsible for plant aromas. They are obtained from different parts of the plants. Flowers, leaves, seeds, barks, and tubers of many plants have medicinal properties (Goncalves et al., 2008).

Increased developments of resistance to current antibiotics have strengthened scientific research for discovery of new drugs. However, new leads/hits in drugs discovery are been developed from natural sources due to growing scientific link between the folkloric medicinal use of some of these natural products especially of plant origin, to biological activity. Hence plants continue to provide a good source for new drugs (Kunle and Egharevba, 2009; Kunle et al., 2013, Begum et al., 2002)


1.1 Brief description of Psidium guajava (Guava)

Guava is a small tropical tree that grows up to 35 feet tall; it is grown for its fruits in tropics. It is a member of the Myrtaceae family, with about 133 genera and more than 3800 species. The leaves and bark of Psidium guajava free have a long history of medicinal uses that are still employed today (Nwinyi et al., 2008). The leaves and bark of the guava plant have been used to treat diarrhea, other gastro intestinal disorders, tooth aches, colds and swelling in areas such as Tahiti (Weiner, 1971), India (Dutta et al.,2000), Africa (Rabe and Vanstaden, 1997;Tona et al., 2000; Len et al., 2002). Guava is used for skin disorders such as astringent for acne, rashes and ringworm (Coe and Anderson, 1999). (Duta et al., 2000) tested Psidium guajava against dermatophytes, Trichophyton tonsurans, Trichophyton rubrum, Microsporium fulvrum and Candida albicans.


1.2 Brief description of Ocimum gatissimum (Scent leave)

Ocimum gatissimum is called ‘efinrin’ by the Yorubas of the south western part of Nigeria, ‘nchanwu’ by the Igbos and ‘Daidoya’ by the Hausas. It has been reported to contain the terpenoids, eugenol, thymol saponins and alkaloids (Gill, 1988). Ocimum gratissimum is germicidal and has been found widely used in tooth pastes and mouth washes as well as some topical ointments (Nakamu et al., 1999; Holets et al., 2003; Pessoa et al., 2003.

Ocimum gratissimum leaf or the whole herb are popular treatments for diarrhea (Dalziel, 1956). In fact, the water saturated oil has been shown to be proportional to the thymol content in preparations where Ocimum gratissimum is used as cold infusion (El-said et al., 1969)


1.3 Justification for the work.

This work is been embarked upon due to increased resistance by bacteria to many already synthesized drugs due to changes or modification in the active site of those drugs and the abuse of orthodox drugs.

Due to the high price of the orthodox medicines in the market, the antimicrobial effect of these plant extracts on bacteria will help to reduce the overdependence on orthodox or synthetic drugs which greater population of the society cannot afford due to economic/financial restriction.


1.4 Aims and Objectives.

1. To determine the active components in Ocimium gratisimum and Psidium guajava

2. To isolate and identify some bacteria like E. coli, Staphylococcus aureus, Salmonella and Shigella.

3. To determine the Minimum inhibitory concentration and the minimum bactericidal concentration of Ocimum gratissimum on the isolates.

4. To determine the Minimum inhibitory concentration and Minimum bactericidal concentration of Psidium guajava on the isolates.   



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