ANTIMICROBIAL ACTIVITIES OF ESSENTIAL OIL FROM CYMBOPOGON CITRATUS (LEMON GRASS) AGAINST ENTERIC ORGANISMS

ABSTRACT

Plants produce certain bioactive compounds which are naturally toxic to microorganisms and so have been investigated as therapeutic agents. The study was carried out to determine the bioactive compounds contained in the essential oil of Cymbopogon citratus and to evaluate in-vitro the antimicrobial effect of the essential oil on five bacterial species. The essential oil was evaluated quanlitatively for phytochemical analysis and quantitative analysis to determine the chemical composition using Gas Chromatography-Mass Spectrophotometry (GC-MS). The result obtained for Phytochemical Screening revealed 24 compounds such as Limonene, Alpha citral, Aromadendrene, Terpineolcis beta, d-Camphene, Sabinene, Myrene, Caorylic aldehyde, Is-alpha pinene, Cucumber alcohol, Cephrol, Neral, Neryl alcohol, Geranaldehyde, Geraniol acetate Artemiseol. The in-vitro evaluation of the essential oil against five bacterial Pathogens of gastrointestinal tract (enterics) namely Salmonella spp, Eschericheria coli, Proteus spp, Klebsiella spp and Shigella spp showed significant inhibitory activities in each bacteria species, (P=0.50) with varying zones of inhibition in each species which includes Shigellaspp ≥ 14.75mm, Proteus spp≥17.25mm, Klebsiella spp ≥18.25, Samonella spp ≥15.75mm and Escherichia coli ≥16.75mm with the conc. oil. The Minimum Inhibitory Concentration of Cymbopogon citratus oil of all enteric bacterial pathogen is ≥ 0.25ml and the Minimum bacteriocidal concentration of Cymbopogon citratus oil ranged from 0.25 to 1.0ml for all test bacterial isolate.  Gram negative organisms possess peptidoglycan layer and membrane made up of lipoproteins and polysaccharide which make them possess a complex cell wall thus making them resistant to antibiotics, this study of in-vitro testing of antimicrobial activity indicates promising results for the use of essential oil from Cymbopogon citratus against diseases caused by Salmonella spp, Shigella spp, E.coli, Klebsiella spp and Proteus spp.

TABLE OF CONTENTS

Title page                                                                                                                                i

Certification                                                                                                                           ii

Dedication                                                                                                                              iii

Acknowledgement                                                                                                                  iv

Table of Contents                                                                                                                   v

List of Table’s                                                                                                                        viii

Appendices                                                                                                                             ix

Abstract                                                                                                                                  x

CHAPTER ONE

1.1 INTRODUCTION                                                                                                            1

1.2 Aims and Objectives                                                                                                        5

CHAPTER TWO

2.0 LITERATURE REVIEW                                                                                                 6       

2.1 Botany description of cymbopogon citratus                                                                     6

2.1.1 Taxonomical classification                                                                                           6

2.1.2 Common names                                                                                                             7

2.2  Propagation                                                                                                                 7

2.3  Agro-climatic requirements                                                                                       8

2.4 Oil content of cymbopogon citratus                                                                                 8

2.5 Storage and packaging of essential oil                                                                             8 

2.6 Uses of some plans                                                                                                           9

2.7 Economic importance                                                                                                       9

2.8 Health benefit of C. citratus                                                                                             9

2.9 Photochemistry of C. citratus                                                                                           10

2.10 Anti-bacterial potential                                                                                                  12

2.10.1 Anti-fungal activity                                                                                                     12

2.10.2 Anti-oxidant properties                                                                                               13

2.10.3 Cytotoxicity and mutagenicity                                                                                    13

2.10.4 Anti-malarial activity                                                                                                  14

2.10.5 Hypoglycemic and hypolipidemic effects                                                                   14

CHAPTER THREE

3.0 MATERIALS AND METHOD                                                                                        15

3.1 Plant materials                                                                                                                  15

3.2 Extraction of essential oil                                                                                                 15

3.3 Analysis of essential oil from Cymbopogon citratus using Gas Chromatography – Mass Spectrophotometry (GC-MC)                                                                                                        15

3.4 Media preparation                                                                                                                                                16

3.4.1 Salmonella Shigella Agar (SSA)                                                                                                                       16

3.4.2 MacConkey agar (MCA)                                                                                                                                   17

3.4.3 Blood Agar                                                                                                                                                        17

3.5 Microorganism collection and maintenance                                                                    17

3.6 Isolation and identification of bacteria                                                                             17

3.7 Biochemical tests                                                                                                              18

3.7.1 Catalase                                                                                                                         18

3.7.2 Indole test                                                                                                                      18

3.7.3 Citrate utilization test                                                                                                    18

3.8 Bioassay procedure                                                                                                          19

3.8.1 Preparation of disc                                                                                                         19

3.8.2 Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)                                                                                                                       20

2.9 Data analysis                                                                                                                     21

CHAPTER FOUR

4.1 RESULTS                                                                                                                         22

CHAPTER FIVE

5.0 DISCUSSION, CONCLUSION AND RECOMMENDATION                                      28

5.1 discussion                                                                                                                         28

5.2 Conclusion                                                                                                                        30

5.3 Recommendation                                                                                                             30

References

Appendix

LIST OF TABLES

Table    Title                                                                             Page

1.         Bioactive compounds from C.citratus                                                                      10

4.1       Bioactive compounds analysed from essential oil                                                    22

4.2       Identification and Characterisation of bacterial isolates                                           23

4.3       Mean zones of inhibition (Disc diffusion assay)                                                       24

4.4       MIC of C.citratus oil (tube dilution assay)                                                                25

4.5       Minimum Bacteriocidal Concentration                                                                    26                                                                                                                                                                                

APPENDICES

Appendix 1. Chromatogram intensity

Appendix 2. Compounds analyzed from essential oil of C. Citrates

Appendix 3. SPSS output result of one way Analysis of Variance

CHAPTER ONE

1.0    INTRODUCTION

Essential oils are botanical extracts of various plant materials and do not only originate from flowers, but from herbs, trees and various other plant materials, (Husmu et al, 2010). An essential oil is a concentrated hydrophobic liquid containing volatile aroma compounds from plants. Essential oils are also known as “oil of” the plant from which they were extracted, such as oil of Cymbopogon citrates (lemon grass). Oil is essential in the sense that it contains the essence of the plants fragrance.

It is estimated that the global number of plants is the order 300,000 and about 10% of these contains essential oils and could be used as a source for their production (Husmu et al, 2010). In industries, essential oils are typically extracted from fresh or partially dried leaves, using various methods of extraction (Husmu et al, 2010).

The use of medicinal plant extracts is part of a competitive market, which includes pharmaceuticals; food, cosmetics and perfumery market mainly use their active substance. Cymbopogon Citratus popularly known as Citronella grass or lemon grass (Negrelle et al, 2007). This species belong to the Poaceae family, which comprises approximately 500 genus and 8,000 herb species (Negrelle et al, 2007). Cymbopogon citratus is a perennial grass plant widely distributed worldwide and most especially in tropical and subtropical countries (Francisco et al, 2011). Several reports have linked it origin to Asia (Indochina, Indonesia and Malaysia), Africa and the America. The plant can grow up to 6 inch high and its bulb like stem consists of terrete and glabrous linearly venated sheathed leaves with narrow base and acute apex. The leaf height is about 100cm length and 2cm in width. When squeezed, the leaves usually produce yellow or amber color, aromatic, essential oil (Adejuwon and Esther, 2007).

The use of whole herbs and extractives has remained the main approach of folk medicine. Practitioners in the treatment of ailments and debilitating diseases (Olorunnisola et al, 2014) usually claimed that such whole herbs and extractives are efficacious against several ailments and diseases without recourse to scientific proofs. Increased cases of opportunistic diseases emanating from side effects associated with synthetic drugs continue to necessitate incremental efforts in searching for effective biological substitutes with little or no side effect. Therefore, efforts are being directed towards elucidating potential resources such as ethno-medicinal plant (Patil, 2010). New, robust and less cumbersome extraction techniques assisted by recent development in biotechnology have enhance investigation of natural compounds faster with more precision than before leading to isolation of bioactive compound with intense health benefit (Wang and Welle, 2006). According to folk medicine, several plant possess ethno-medicinal benefits as Cymbopogon citratus also known as lemon grass.

The common methods to extract essential oil from medicinal plant, including Cymbopogon citratus, are hydrodistillation (HD), steam distillation, steam and water distillation, maceration, empyreumatic (or destructive) distillation and expression. It is proved through a number of studies (Dhobi et al, 2009), that the quality of primarily influenced by their extraction procedures. In contrast, these common methods can induce thermal degradation, hydrolysis and water solubililization of some fragrance constituents (Dhobi et al, 2009). In addition, the oil obtained through solvent aided extraction contains residues that pollute the foods fragrances to which they are added. As a means to overcome this sort of drawback, an advance and improved method such as microwave-assisted extraction (C0sta et al, 2006), Ohmic-assisted hydrodistillation (Chanthal et al, 2012), subcritical water extraction (Carlson et al, 2001) and ultrasound-assisted extraction (Paviani et al, 2006) have been applied to shorten extraction time, improve the extraction yield and reduce the operational costs.

Recently, microwave-assisted hydro distillation (MAHD) procedures for isolating essential oils have become attractive for use in laboratories and industry due to its effective heating, fast energy transfer and environmental friendly extraction techniques. Its acceptance as potential and powerful alternative for conventional extraction techniques has been proved through several research (Ashgari et al, 2010). However, there are no reports of the simultaneous comparison of MAHD and HD extraction procedures on extraction of essential oil from Cymbopogon Citratus (Lemon grass) Therefore, the aim of the present study was to investigate the applicability of microwave-assisted hydrodistillation (MAHD) technique as an alternative to conventional hydrodistillation (HD) in isolation of Cymbopogon Citratus extracts based on the extraction yield and constituents of oils obtained under optimized condition (Ramitha et al, 2014). The effect of operational parameter such as extraction time and water to raw materials ratio were evaluated to identify its optimum condition for extraction and this applicability was appreciated by using the result of subsequent GC/MS analysis by comparing their main constituents and oxygenated compounds (Ramitha et al, 2014).

The demand of essential oil in current industry has increased due to its bioactive compound that shows various therapeutic effects. Microwave-assisted hydrodistillation (MAHD) is an advance hydrodistillation (HD) technique, in which a microwave oven is used as the heating source. MAHD extraction of essential oil from Lemongrass (Cymbopogon Citratus) was studied. The effect of different parameters, such as water to plant material ratio (6:1, 8:1, 10:1), microwave power (200 W,250W) and extraction time (30min, 60min,90min,120min) on the extraction yield and its major constituents were investigated and the results were compared with those of the conventional HD. These essential oils were further analyzed by Gas Chromatography/Mass Spectrometric (GC-MS) to evaluate the effect of extraction method on the content of its main constituents which were neral, geranial and myrcene and some minor compound such as linalool, geranic acid and citronellol. The optimum parameter were found at water to plant material ratio of 8:1, microwave power of 250W and 90 minutes of extraction and the yield obtained under this condition was 1.46%. GC-MS analysis has proved that the use of microwave irradiation did not adversely influence the composition of essential oils as the main constituents found through both methods were almost similar in terms of quality and quantity. The results obtained indicate that MAHD method provided a good alternative for the extraction of essential oil from Lemongrass (Cymbopogon Citratus) (Ramitha et al, 2014).

1.1 AIMS AND OBJECTIVES

The broad aim of this research is to investigate the antimicrobial activity of essential oil from Cymbopogon citratus against selected enteric bacteria species. The objectives of this study includes

1. To isolate essential oil from Cymbopogon citratus using steam distillation method.
2. To characterize and identify isolated bioactive compounds from essential oil of Cymbopogon citratus leaves.
3. To test for the antimicrobial activity of essential oil of Cymbopogon citratus leaves on enteric bacterial species using disc diffusion method. Example Salmonella spp, Escherichia coli, Klebsiella spp, Proteus spp and Shigella spp
4. To characterize and document inhibitory patterns of the essential oil on the test bacterial isolates

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