EVALUATION OF ANTIBACTERIAL PROPERTY OF DOGOYARO (AZADIRACHTA INDICA) LEAF EXTRACT ON PATHOGENIC ORGANISMS

ABSTRACT

Medicinal plants are traditional plants/herbs that have been used in healthcare centers to treat various types of illness. Examples of medicinal plants are Dogoyaro leaf, paw-paw leaf, e.t.c. Medicinal plants have a long history of use and their use is widespread in both developing and developed countries. The study evaluated the antibacterial effect of Dogoyaro (Azadirachta indica) leaf on pathogenic organisms. The fresh leaves of Dogoyaro (Azadirachta indica) leaves were collected at National Root Crop Research Institute Umudike in Ikwuano, Abia State, Nigeria. The plant was identified and authenticated at the Herbarium of the Department of Plant sciences and Biotechnology, Michael Okpara university of Agriculture, Umudike, Abia State. The antibacterial activity of Dogoyaro (Azadirachta indica) leaf extracts on some pathogens was investigated with different solvents (Ethanol and aqueous) against test organisms (Staphylococcus aureuas, Esherichia coli and Pseudomonas aeruginosa) using agar well diffusion method. The different extracts were prepared at different concentration (200mg/ml, 100 mg/ml, 50mg/ml, 25 mg/ml, 12.5 mg/ml and 6.25mg/ml). Amongst the different Dogoyaro (Azadirachta indica) leaf extracts studied, the ethanolic extracts have more significant inhibitory effect than the aqueous extracts. The diameter zone of inhibition (mm) produced by ethanolic and aqueous leaf extract of the Dogoyaro (Azadirachta indica) indicated that the aqueous extracts of Dogoyaro (Azadirachta indica) showed minimal antibacterial activity against the isolates at 200 mg/ml, 100 mg/ml, 50 mg/ml and 25 mg/ml concentrations as against the ethanolic extracts. The results also show that as the concentrations of the extract increases there is a corresponding increase in the zones of inhibition and comparing the activity, ethanolic extract of neem leaf exhibited higher antimicrobial activity against the test bacteria than aqueous extract. This difference may be due to the inhibitory ability of the ethanol even without extract, whereas the ability of the aqueous extract to exhibit this inhibition may be primarily due to its penetrative ability. From this study, it was observed that ethanol extracts exhibited better MIC and MBC on the test organisms. The present work has shown that Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were susceptible to extracts of Dogoyaro (Azadirachta indica) which means the plant has antibacterial property. The results of this study suggest that the leaf of Dogoyaro (Azadirachta indica) can be used as an antibacterial agent against infections caused by Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.

TABLE OF CONTENTS

Title Page                                                                                                                                 i

Certification                                                                                                                           iii

Dedication                                                                                                                              iv

Acknowledgements                                                                                                                v

Table of Contents                                                                                                                   vi

List of Tables                                                                                                                          ix

Abstract                                                                                                                                  x

CHAPTER ONE

1.0       Introduction                                                                                                                1

1.1       Aims and Objectives                                                                                                  5

CHAPTER TWO

2.0       Literature Review                                                                                                       6

1.1       Azadirachta Indica                                                                                                     6

2.2       Medicinal Uses                                                                                                           7

2.3       Phytochemicals                                                                                                          8

2.4       Antimicrobial Activity                                                                                               9

2.4.1    Antibacterial and Anti-Fungal Activities                                                                   10

2.5       Antibacterial Effects of Crude Extract of Azadirachta Indica against                        

Escherichia coli, Salmonella sp and Staphylococcus aureus                                     10

2.6       Test Organisms                                                                                                           12

2.6.1    Escherichia coli                                                                                                          12

2.6.2    Staphylococcus aureus                                                                                               14

2.6.3    Pseudomonas aeruginosa                                                                                           14

CHAPTER THREE

3.0       Materials and Methods                                                                                               16

3.1       Study Area                                                                                                                  16

3.2       Materials                                                                                                                      16

3.3       Sample Collection                                                                                                      16

3.4       Test Organisms                                                                                                           17

3.5       Confirmation of Test Isolate                                                                                      17

3.5.1    Morphological appearance                                                                                         17

3.5.2    Gram Staining                                                                                                            17

3.5.3    Motility test                                                                                                                18

3.5.4    Biochemical tests                                                                                                        18

3.6       Preparation of Crude Extracts                                                                                    20

3.7       Preparation of Concentration of Plant Extract                                                           21

3.8       Antimicrobial Susceptibility Testing                                                                         21

3.9       Determination of Minimum Inhibitory Concentration (MIC)                                     22

3.10     Minimum Bactericidal Concentration (MBC)                                                           22

CHAPTER FOUR

4.0       Result                                                                                                                          23

CHAPTER FIVE

5.0       Discussion, Conclusion and Recommendation                                                          31

5.1       Discussion                                                                                                                   31

5.2       Conclusion                                                                                                                  33

5.3       Recommendation                                                                                                        33

References                                                                                                                  34

LIST OF TABLES

CHAPTER ONE

1.0   INTRODUCTION

Plants have formed the basis of traditional systems of medicine that have been in existence since time immemorial and continued to provide mankind with new remedies (Jonathan et al., 2007). A medicinal plant is one which contains substances that can be used for therapeutic purposes. For a long time, plants have been valuable and indispensible sources of natural products for the health of human beings and they have a great potential for producing new drugs (Nascimento et al., 2000). Plants act generally to stimulate and supplement the body’s forces; they are the natural food for human beings (Akinnibosun and Itedjer 2012). For thousands of years before the advent of modern medicine, herbs and substances derived from plants have been the mainstay of traditional medicine around the world (Ortuno et al., 2006). According to World Health Organization, medicinal plants would be the best source to obtain varieties of drugs. About 80% of individuals from developed countries have used traditional medicine, which has compounds derived from plants. Therefore, such plants should be investigated for better understanding of their properties, safety and efficiency (Chintamunnee and Mohomoodally 2012).  Mouhssen (2013), because of an increasing interest in plants as source of agents to fight microbial diseases and the alarming incidence of antibiotic resistance of pathogenic microbes in particular, there is still constant need for discovering new and effective therapeutic agents. The control of infectious diseases is badly endangered by the rise in the number of Microorganism that are resistant to antimicrobial agents. This is because infectious caused by resistant Microorganisms often fail to respond to conventional treatment, resulting in prolonged illness and greater risk of death. Antibiotics resistance is a type of drug resistance where Microorganisms are able to survive exposure to an antibiotic. The primary causes of antibiotic resistance are genetic mutation in bacteria (Fernando and Jose 2013).  Inappropriate and irrational use of antimicrobial medicines provides favourable conditions for resistant Microorganisms to emerge, spread and persist. The greater the duration of exposure to the antibiotic, the greater the risk of the development of resistance, irrespective of the need for the antibiotic as resistance toward antibiotics becomes more common, a greater need for alternative treatment arises. However despite a push for new antibiotic therapies there has been a continued decline in the number of newly approved drugs (Mankkam et al., 2015). Antibiotics resistance therefore posses a significant problem. Nevertheless efforts are in place to solve the problem. Nowadays, bacteria are considered as the main cause of morbidity and mortality in many developing countries, especially amongst children. Among the bacterial pathogens, Escherichia coli, Salmonella specie, and Staphylococcus aureus are most prevalent. In the recent decades despite developments of different types of antibiotics by pharmacological industries, resistance to antibiotics has been increased significantly in many bacterial pathogens. In general, bacteria have several genetic mechanisms to transmit and acquire resistance to antibiotics. The antibacterial resistance in the bacterial populations is rapidly spreading; this is a serious threat to successful treatment of infectious diseases. On the other hand, the side effects associated with the commercial antibiotics are frequently reported. In this regard, there is an urgent and continuous need for finding and investigating novel antimicrobial compounds. Most of the antibiotics have been developed from microorganisms. Moreover, the plant materials remain an important resource for finding the novel antimicrobial compounds. There are many published reports on the antibacterial effectiveness of the traditional herbs against the gram-positive and gram-negative bacteria. Microbial cells are negatively affected by plant-derived substances via various mechanisms of actions as these substances attack the phospholipid bilayer of the cell membrane and disrupt enzymatic systems (Ahmad et al., 2006). The medicinal herbs have the bacteriostatic effects on the enzymatic activity associated with energy production, or they can cause denaturation of proteins, modifying cell wall permeability, or causing the loss of macromolecules. Therefore, it is difficult for the microorganisms to develop resistance against these medicinal herbs.

Medicinal plants have a long history of use and their use is widespread in both developing and developed countries. According to reports of the World Health Organization, 80% of the world’s population relies mainly on traditional therapies which involve the use of plant extracts or their active substances. Microorganisms have developed resistance against many antibiotics due to the indiscriminate use of antimicrobial drugs. Furthermore, antibiotics are sometimes associated with side effects (Cunha, 2001), whereas there are some advantages of using antimicrobial compounds of medicinal plants, such as fewer side effects, better patient tolerance, relatively less expensive, acceptance due to long history of use and being renewable in nature (Vermani and Garg, 2002). It is known that more than 400, 000 spp. of tropical flowering plants have medicinal properties and this has made traditional medicine cheaper than modern medicine. Some plant decoctions are of great value in the treatment of diarrhoea or gastrointestinal disorder, urinary tract infections, skin infections, infertility, wound and cutaneous abscesses (Ergene et al., 2006). The tree, Azadirachta indica of the family Maliaceae; popularly known as neem tree or dogon yaro (Hausa) is an evergreen tree, native to the Southeast Asia and found in most tropical countries. It has been in use since ancient times, to treat a number of human ailments and also as household pesticide (Chattopadhyay and Bandyopadhyay, 2005). Extracts from the bark, leaves, fruits and roots have been used to control leprosy, intestinal helminthosis and respiratory disorders. Every part of the neem tree has been used as traditional medicine for house-hold remedy against various human ailments from antiquity. The tree is still regarded as ‘Village dispensary’. It is a plant known over 2000 years as one of the most versatile medicinal plants having a wide spectrum of activity. Enterobacteriaceae, the enteric bacteria are facultative anaerobic Gram-negative rods that live in the intestinal tract of animals in health and diseases. The Enterobacteriaceae are among the most important bacteria medically. A number of genera within the family are human and animal intestinal pathogens (for example, Salmonella, Shigella and Yersinia). Several others are normal colonist of the human gastrointestinal tract (for example, Escherichia coli, Enterobacter, Klebsiella), but these bacteria, as well, may occasionally be associated with diseases in humans and animals.

Azadirachta indica also known as neem, is called dogoyaro, atuyabasi ogwuakom or maina in Yoruba, Igbo and Hausa dialects of Nigeria respectively and is widely distributed across Nigeria. It is famed to be the most extensively researched plant so far and its products as popular. (Pritima and Selvaraj, 2008). A. indica belongs to the family Meliaceae. “Azad dhirakat” is a Persian word meaning an “Excellent or Noble Tree”. It is native to East India and Myanmar (Burma), Southeast Asia, West Africa and abundant in all regions of Nigeria irrespective of the local geography of the region (Pritima and Selvaraj, 2008).

Every part of the tree, including leaf, twig, bark, flower, fruit, root and seed, is used in one form or another and reputed to have hypoglycemic activity (Biswas et al., 2002; Ebong et al., 2008), antidiabetic activities and other pharmacological and biological properties. It's a fast growing evergreen tree measuring up to 1.5 meters in diameter with hard fissured or scaly bark. The leaves are alternate, pinnate with dentate margins; young leaves are reddish-purple in color. Tiny cream colored flowers, give raise to green oblong fruits with seeds. Fruits, when ripe turn yellow in color. The leaves and fruits (raw) are bitter but edible and widely eaten in Nigeria as “agbo”, an herbal cocktail preparation for malaria and in India as part of food or for medicinal reasons. It has been reported that Mahatma Gandhi encouraged scientific investigations of neem tree as part of his revitalization program of the Indian tradition because he loved the plant so much, and also to increase commercial interest in the plant.

The Chemical constituents in contain many bioactive compounds including alkaloids, flavonoids, tri-terpenoids, phenolic compounds, carotenoids, steroids and ketones. The main active principle present in neem is azadirachtin, a natural insecticide. Azadirachtin is a mixture of seven isomeric compounds labeled as azadirachtin A to G and azadirachtin E is more effective. Other compounds found in the plant include salannin, volatile oils, meliantriol and nimbin. Neem leaf is effective in treating various diseases as eczema, ringworm, acne, anti-inflammatory activities, antihyperglycemic and also used to treat chronic wounds, diabetic foot and gangrene. It is also believed to remove toxins from the body, neutralize free radicals and acts as a blood purifier. Recently it has been used as an anticancer preparation and it has hepato-renal protective activity and hypolipidemic effects. Boiled neem leaf water makes an excellent antiseptic to clean wounds, soothes swellings and eases skin problems.

•                AIMS AND OBJECTIVES

The aim of this study is to evaluate the antibacterial effect of Dogoyaro (Azadirachta indica) leaf on pathogenic organisms

The objectives are;

•       To determine the antibacterial effects of Dogoyaro (Azadirachta indica) leaf extract against some selected pathogens

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