ANTIMICROBIAL ACTIVITY OF ESSENTIAL OIL FROM CITRUS PARADISI (GRAPE) AGAINST ISOLATES FROM THE RESPIRATORY TRACT

ABSTRACT

The antimicrobial activity of essential oil of citrus paradise feel has been demonstrated against a range of organism (Streptococcus pneumoniae and Klebsiella pneumoniae). The oil was obtained from the peels of grape fruits by hydro-distillation procedure and analyzed using gas chromatography coupled by mass spectrometer. 18 compounds were identified namely, cyclohexene, 1-methyl-4-(1-methylethenyl), Bicyclo (3,1,1) hept-3-en-2-01,4,66,-trimethyl, limonene oxide, bicycle (4,1,0) heptanes,-3-cycloprophyl, -7- hydroxymethyl, trans,  2-cy- clohecene-1-01,2-methyl-5-(1-methylethonyl)-cis, 2-cycloheven-1-one,2 7-oxabicyclo(4.1.0)hept-ane, 1-methyl -4- (2-methyloxivanyl), 1,2-cyclohexanediol, 1-methyl -4- (1-methyl-ethenyl),2,7-octadiene-1,6-diol,2,6-dimethyl, Limonene oxide,cis, Z,Z,Z-4-6.9-Nonadecatriene, Bicyclo(3.1.1) heptanes-2,3-diol, 2,6,6,-trimethyl.Hexadeianoic acid,  Z,Z- 2,5-pertadecadien-l-ol-  2- Butyl-3-methyl -5- (2-methylprop-2-enyl) cyclohexanone  Alpha-Farnesene, Oleic acid and octadecanoic acid.The oil extracts exerted different degree of inhibitions on the test organism or isolates was dependent on the dissolution concentrations used. Klebsiellla pneumoniae had the highest zone diameter of inhibition 2.0mm while the Streptococcus pneumonia had 10.5mm. The undiluted had 13.5mm for Klebsiella pneumonia and 11.5m for Streptococcus pneumiae. The MIC value of the essential oil against Klebsiella pneumoniae was at 12.5% concentration. However, the essential oil exhibited a bactericidal effect against the organism Streptococcus pneumoniae and Klebsiella pneumoniae at 50% concentration. This study finally has shown that grape  peels from Nigeria contains some antibiotic principles which may be explored for use in the treatment of certain diseases (respiratory tract infections).

TABLE OF CONTENTS

Title Page                                                                                                                    i

Certification                                                                                                               ii

Dedication                                                                                                                  iv

Acknowledgement                                                                                                      v

Table of Contents                                                                                                       vi

List of Tables                                                                                                              viii

Abstract                                                                                                                      ix

CHAPTER ONE

1.0   INTRODUCTION                                                                                              1

1. 1 Aim                                                                                                                      2                                                         

1.2 Objectives                                                                                                             3

CHAPTER TWO

2.0 Literature Review                                                                                                 4

2.1 General Concept                                                                                                   4

2.2 ESSENTIAL OIL COMPOSITION                                                                     5

2.3 Factors That Influence the Determination of the Antimicrobial Activity                        6

2.3.1The Assay Technique                                                                                         7

2.3.2 The Test Microorganism                                                                                   11

2.3.3 Culture Conditions                                                                                            12

2.4 Mechanism of Action of Plant Essential Oil (Eos)                                              12

2.4.1 Cell Wall and Membrane Disturbance                                                              13

2.4.2 ATP Production                                                                                                 14

2.4.3 Protein Synthesis                                                                                               15

2.4.4 PH   Disturbance                                                                                                16

2.4.5 Intracytoplasmic Changes                                                                                 17

2.4.6 DNA                                                                                                                   17

2.4.7 Quorum  Sensing                                                                                               18

2.5 Citrus Paradisi and importance of EO                                                                 20

CHAPTER THREE

MATERIALS AND METHODS

3.1 Plant Collection and Identification                                                                       23

3.2 Essential Oil Extraction from Grape Fruit                                                           23

3.3  Isolation of test organism                                                                                    23

3.4 Characterization/ Identification                                                                            24           

3.4.1 Gram Staining                                                                                                   24

3.4.2 Catalase Test                                                                                                      25

3.4.3 Bile Solubility Test                                                                                            25

3.4.4 Citrate Test                                                                                                        25

3.4.5 Voges Proskauer Test                                                                                        26

3.5 Antimicrobial Susceptibility Using Disc Diffusion Method                                26

3.5.1 Preparation of Disc                                                                                            26

3.5.2 Sensitivity Assay                                                                                               26

3.5.3 Minimum inhibitory Concentration (MIC) and Minimum

3.5.4 Minimum Inhibitory Concentration                                                                 

3.5.5 Minimum Bactericidal Concentration                                                               27

3.6   Analysis of Plant Extract (Citrus Paradisi) Using Gas

       Chromatography-Mass Spectrometry (GC-MS)                                                 27

CHAPTER FOUR

4.1 RESULTS                                                                                                             29   

CHAPTER FIVE

DISCUSSION, CONCLUSION AND RECOMMENDATION

5.1Discussion                                                                                                              36

5.1Conclusion                                                                                                             37

5.2Recommendation                                                                                                  37

References

Appendix

LIST OF TABLES

Tables     Title                                                                Pages 

1                      Characterization and Identification of Bacterial Isolates                        31

2                      Zone diameter of inhibition (mm)                                                      32

3                      Minimum Inhibitory Concentration (MIC) (mm)                              33

4                      Minimum Bactericidal Concentration (MBC) (mm)                         34

5                      Names of Compounds (Essential oil)                                                 35

CHAPTER ONE

1.0   INTRODUCTION

The increasing problems of antibiotics drug resistance by pathogenic organisms, in the past decades and recently have led to the  continues exploration of natural plant products for new antibiotic agents. (Ayoola, et al; 2008, Belmekki, et al; 2013 Dahpour, et al; 2012). Many of these products are produced in plants as secondary metabolites and often used in plants for defense against microbial attack (Shittu, et al; 2008).

Since ancient times, the antimicrobial impact of essential oils and their components isolated from aromatic and medicinal plants has been recognized. During the last decades these properties have been confirmed. The control of food spoilage and pathogenic microorganisms is achieved mainly by chemical control but the use of synthetic chemicals is limited due to a number of undesirable aspect which include carcinogenicity, teratogenicity, acute toxicity and the requirement of extended degradation period with the subsequent development of environmental pollution problem. The awareness of modern consumer’s profile that demand the absence of synthetic chemicals in food preservation accompanied by the requirement for extended shelf life in the majority of food products in conjunction with the scientific community and agro-industries and pharmaceutical industries to search for natural compounds that may satisfy consumers request. Essential oil may be included in this group. Essential oil area class of such volatile compound produced with inherent antimicrobial properties (Uysal, et al; 2011). Essential oils (EO’S) obtained from plants are a complex mixture of some compounds such as hydrocarbon, alcohol esters, aldehydes and have been reported to exhibit inhibitory activity against a wide spectrum food spoilage microorganism (Uysal, et al; 2011).

Citrus is one of the most consumed fruit in the world and contain a high amount of useful by-products which includes essential oil (Shieber, et al; 2001). It is mostly consumed fresh or used as raw materials for juice and wine (Shieber, et al; 2001)

Grape fruit (Citrus paradisi) belong to the citrus genus, a texa of flowering plants in the family Rutaceae. (Uysal,et al;2011). Grape is one of the citrus fruit cultivated and consumed in Nigeria (Odubanyo,et al; 2002). The yield of grape fruit and oranges juice is about half of the fruit weight thereby generating a very high amount of waste annually (Bocco, et al; 1998, Balili, et al; 1996).

Essential oil and plant products have had a tremendous application in food, cosmetics and folk medicine and are continuously investigated for their antimicrobial activities (Girish, et al; 2008).

Antimicrobial effects of essential oils from citrus paradise peel from other country has been reported e.g. Turkey (Uysae, et al; 2011). The essential oil composition of peels of citrus reticulate L, C , paradisi and C. medica from Nigeria has been examined. (Gbolade,et al; 2011). However there has not been any information in the effect of essential oils of Citrus Paradisi peel of Nigeria origin on a range of disease causing organisms.

1. 1:  AIM

This study aimed at determining the antibacterial activity of essential oils from C. paradisi peel against isolates from the respiratory tract.

1.2 OBJECTIVES

i.               To extract and identify the essential oils present in C. Paradisi  peel

ii.              To isolate, characterized and identify the organisms associated with the respiratory tract.

iii.            To determine the antibacterial potential of the essential oils against the isolates

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