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
- To isolate essential oil from Cymbopogon citratus using steam
distillation method.
- To characterize and identify isolated
bioactive compounds from essential oil of Cymbopogon citratus leaves.
- 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
- To characterize and document
inhibitory patterns of the essential oil on the test bacterial isolates
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