PHYTOCHEMISTRY AND EVALUATION OF SOME PLANT EXTRACTS ON STREPTOCOCCUS MUTANS AND CANDIDA ALBICANS

ABSTRACT

The upsurge in the prevalence of side effects of many synthetic antimicrobial agents and incidence of multidrug resistant bacteria has spurred scientists on the research for plant based antimicrobial of therapeutic potentials. Azadirachta indica and Psidium guajava present such potential of high medicinal value. These plants are used in Nigeria traditionally as condiments and for treatment of various ailments such dental plague . The phytochemical composition of Azadirachta indica and Psidium guajava showed the presence of Tannin, Alkaloid, Saponin and Flavonoid. Aqueous and ethanol extracts of Azadirachta indica and Psidium guajava leaves were screened for antibacterial activity against Candida albican and Streptococcus mutans. Both extracts were found to exhibit selective inhibition against the isolates. The diameter zones of inhibition exhibited by the extracts were between 2 ± 0.01 – 10 ± 0.10 mm. The Minimum Inhibitory Concentration (MIC) determined by the agar diffusion method was between 10.00 and 2.50 mg/ml-1. Ethanol extracts showed more `inhibitory effect compared to the aqueous extracts. Results obtained show that the extracts of Azadirachta indica and Psidium guajava possess some level of antibacterial activities against Steptococcus mutans and Candida albicans.

TABLE OF CONTENTS

Title Page                                                                                                                    i

Certification                                                                                                                ii

Dedication                                                                                                                   iii

Acknowledgements                                                                                                    iv

Table of Contents                                                                                                       v

Lists of Tables                                                                                                            vii

Abstract                                                                                                                       viii

CHAPTER ONE

1.0        Introduction                                                                                              1

1.1       Aims and Objectives                                                                                       4

1.2       Objectives                                                                                                       4

CHAPTER TWO

2.0       Literature Review                                                                                           5         

2.1       Dogoyaro/Neem (Azadirachta indica)                                                            5

2.2       Phytochemicals of Azadirachta indica                                                           8

2.3       Antimicrobial Activity                                                                                   9

2.4       Antibacterial and Anti-Fungal Activities                                                       10

2.5       Guava (Psidium guajavaLinn.)                                                                      10

2.6       Streptococcus mutans                                                                                     14

2.7       Candida albicans                                                                                            15

CHAPTER THREE

3.0       Materials and Methods                                                                                   17

3.1       Collection of Samples                                                                                    17

3.2       Media Used                                                                                                     17

3.3       Sterilization                                                                                                    17

3.4       Collection of Test Organisms                                                                         17

3.5       Isolation and Identification of Bacteria and Fungi                                         17

3.6       Preparations of Plant Extracts                                                                        18

3.7       Phytochemical Analysis                                                                                 18

3.7.1    Test for alkaloid                                                                                             18

3.7.2    Test for flavonoid                                                                                           19

3.7.3    Determination of saponins                                                                              19

3.7.4    Test for tannin                                                                                                20

3.8       Antibiotic Sensitivity Testing                                                                         20       

3.8.1    Agar diffusion method                                                                                   20

3.9       Determination of Minimum Inhibitory Concentration (MIC)                         20

CHAPTER FOUR

4.0       Results                                                                                                            22

CHAPTER FIVE

5.0       Discussion Conclusion Recommendation                                                      27

5.1       Discussion                                                                                                       27

5.2       Conclusion                                                                                                      28

5.3       Recommendation                                                                                           28

References                                                                                                      29

LIST OF TABLES

Table                              Title                                                          Page

1:                     Antibacterial activity of the aqueous of Azadirachta

Indica and Psidium guajava against Streptococcus

mutans and Candida albicans                                                            23

2:                     Antibacterial activity of the ethanol extract of Azadirachta

Indica and Psidium guajava against Streptococcus mutans

and Candida albicans                                                                         24

3:                     Phytochemical composition of Azadirachta

Indica and Psidium guajava                                                               25

4:                     Minimum inhibitory concentration (MIC) of the aqueous and

ethanol extracts of Azadirachta indica and Psidium guajava                       26

CHAPTER ONE

1.0        INTRODUCTION

The microorganisms present in the mouth consist of a diverse and populous collection of bacteria, fungi, and transient viruses. Bacteria make up the largest number of varieties (Akinyele et al.,2014). More than 350 cultivable bacterial species have been identified in the oral cavity and molecular analyses suggest that an equal number of non-cultivable flora are also present (Marcotte and Lavoie, 1998). Bacterial accumulation on oral surfaces is a major factor in the development of most of the common dental diseases such as dental caries, plaque and periodontal disease (Vohra et al.,2012).

Dental plaque is a complex biofilm found on the tooth surface which forms the major reason of the development of dental caries. The accumulation and development of plaque depends upon the outcome of the interactions between the adhesiveness of plaque to the tooth surface and the physical shear forces which serve to dislodge and remove the plaque (Vohraet al.,2012). Tooth brushing with toothpaste is the most widely practiced form of oral hygiene in most countries (Pannutiet al.,2003) and the success of any toothpaste, in part, lies on its ability to eliminate pathogenic oral microflora. A wide range of chemicals, mainly antimicrobial agents, have been added to toothpastes in order to produce a direct inhibitory effect on plaque formation (Fine etal., 2006). Fluoridated toothpastes have been proven to protect teeth against attack from bacteria (Vohraet al.,2012).

Nigerians basically employ two methods to remove debris (plaque) from the mouth which are either by use of tooth brush and paste or by use of parts of various plants native to West Africa, referred to as “African chewing sticks” (Bankoleet al.,2012). Meanwhile, in absence of toothpaste, a large proportion of persons living in rural areas and some who are also in the urban areas make use of the natural toothbrush known as “chewing sticks. Ojoet al.,(2007) defined chewing sticks as parts of higher plants which are cut into suitable lengths and used for the maintenance of oral hygiene. They are important Non Timber Forest Product (NTFP) widely used for dental cleaning in the tropical West Africa (Akande and Hayashi, 1998). Plants from which chewing sticks are derived are abundant and diverse in Nigeria rural communities. Almost the entire rural population in Nigeria use chewing sticks for orodental hygiene (Bankoleet al.,2012).

This is due to the fact that the Nigerian chewing sticks, are readily available, cheap and efficacious (Kareem et al.,2012). Chewing sticks are recommended for oral hygiene by the World Health Organization, and some of them, or their extracts, are also used in ethno-medical treatment of oral infections (Ndukwe, et al.,2005). Several studies have demonstrated the antiplaque and antibacterial activities of extracts of these Nigerian Chewing Sticks (NCS) against oral bacteria, such as Streptococcus mutans and Streptococcus mitiswhich are organisms commonly implicated in dental caries and orodental infections. The plants used as chewing sticks are carefully selected for certain properties such as foaminess, hardness, or bitterness and certain species are more popular than the others, in which a great number of these plant species have related medicinal properties that may be antibacterial and are as important today as they were thousands of years ago (Hullins, 2010).

 In addition, Hoodaet al.,(2010) reported that extracts of chewing sticks can be incorporated into tooth pastes or used as mouth washes. The choice of chewing sticks to be used in most cases depends on its cleansing action of the teeth; the therapeutic value, or preferred taste or flavour. The sticks (which may be stem or root with bark removed or retained) are cut to convenient lengths and washed thoroughly with fresh water to get rid of any dirt (Ogundiyaet al.,2006). The usefulness of chewing sticks in oral hygiene maintenance has been considered comparatively effective as tooth brush (Van-Palentsteinet al.,2002).

Oral diseases impact our quality of life and may lead to systemic and threatening diseases. The relationship between the high incidence of oral diseases and microorganisms is well known. Because of the increased microbial resistance to antibiotics, toxic and harmful effects of few common antimicrobial agents, there is a continuous need for alternative therapies which areaffordable, non toxic and effective, such plants (Van-Palentsteinet al.,2002).

Alternative medicines are commonly included in therapeutic and diagnostic disciplines outside the conventional health system(Bankoleet al.,2012). In developing countries, access to dental healthcare is restricted and expensive and thus it is limited to emergency dental care procedures (Bankoleet al.,2012).

Dental caries is o most common infection of all oral diseases. It is proved that cariogenic microorganisms, especially Streptococcus mutans plays an essential role in the pathogenesis of dental caries. It is involved in the initiation of almost all carious lesions in enamel (Bankoleet al.,2012).

The main cause of dental caries is attributing to oral biofilm, also known as dental plaque, a film of microorganisms sticking to the tooth surface (Bankole et al.,2012). Streptococcus mutanshas the ability to metabolize dietary sucrose and synthesize glucan by cell surface and extracellular glucosyl transferase. This glucan is an insoluble sticky or slimy gel relatively inert and resistant to bacterial hydrolytic enzymes which causes plaque to adhere tenaciously to tooth surface (Akinyeleet al.,2014). Streptococcus mutans organisms in the plaque produce organic acids such as lactic acid that gradually destroy the enamel to form a cavity to Streptococcus mutans, Lactobacillus acidophilus probably also play role in acid production in the plaque and involved in root caries and periodontal diseases Enterococcus faecalis (Akinyeleet al.,2014).

Candida albicans, are some other microbial species that knowingly cause several oral diseases, such as dental caries, endodontic infections, periodontal diseases and oral Candidiasis. The most effective method to eradicate C.albicans is the use of sodium hypochlorite and 2% chlorhexidine(Van-Palentstein et al.,2002).

 1.1   AIMS AND OBJECTIVES

The aim of this research is to study the antimicrobial effect of Azadirachta indica (Dogoyaro /Neem) and Psidium guajava L. (Guava) on Streptococcus mutans and Candida albican.

1.2       OBJECTIVES

1. To determine the phytochemical constituents of Azadirachta indica (Dogoyaro/Neem) and Psidium guajava L.  (Guava) .

2. To determine the antimicrobial activity of Azadirachta indica (Dogoyaro/Neem) and Psidium guajava L.  (Guava)on Streptococcus mutans and Candida albican.

3. To determine the minimal inhibitory concentration of Azadirachta indica (Dogoyaro/Neem) and Psidium guajava L. (Guava)on Streptococcus mutans and Candida albican.

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