ABSTRACT
The present study was carried out to find out the antimicrobial activity of ethanolic, methanolic, extract of lemon fruit parts like Juice, Leaves Stem and Roots. Antimicrobial analysis was done by using disc diffusion method against bacterial. Methanolic extract of lemon stem exhibited the minimum zone of inhibition against Staphylococcus aureus 13mm, whereas Ethanol extract of lemon Stem extract exhibited least zone of inhibition 22mm against Staphylococcus aureus. Ethanolic extract of lemon Leaves showed maximum zone of inhibition 14mm against Staphylococcus aureus whereas Methanol extract showed least zone of inhibition 12mm. MIC value was determined by using micro test tubes dilution method. The least concentration was obtained 0.625 mg/ml for ethanolic extract on Staphylococcus aureus and 1.250 mg/ml on Escherichia coli. The MBC value also determined and phytochemical analysis showed the presence of Tannins, Flavonoids, Saponins, Steroids, Terpenoid and Phenol.
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 Antibacterial Actions of Essential Oils
2
1.2 Mechanism of Action Of Lemon Extract against Pathogenic Microorganisms 4
1.3 Aims and Objectives 5
1.4
Objectives 5
CHAPTER TWO
2.0 Literature Review 6
CHAPTER THREE
3.0 Materials and Methods
12
3.1 Sample
Collection 12
3.2. Test Microorganisms 12
3.3media
Used 13
3.4 Preparation of Media 13
3.4
Sterilization 13
3.5 Preparation of Extract 13
3.6 Identification and
Characterization Of Isolates 13
3.6.1
Gram Staining 13
3.7 Biochemical Cultural
Characteristics 14
3.7.1 Catalase test 14
3.7.2
Coagulase Test 14
3.7.3 Citrate Test 14
3.7.3 Motility Test 15
3.7.4 Indole Test 15
3.7.5 Triple Sugar Iron Test 15
3.7.6 Sugar fermentation 16
3.8
Qualitative Phytochemical Analysis 16
3.8.1 Test for saponins. 16
3.8.2 Test for Tannins 16
3.8.3 Test for Flavonoids 16
3.8.4 Test for Alkaloids 17
3.8.5
Determination of Total Phenols 17
3.8.5 Test for Steroids 17
3.8.6 Test for Terpenoid 18
3.9 Antimicrobial Sensitivity by Disc
Diffusion Methods 18
3.9.1 Minimum inhibitory concentration (MIC) 18
3.9.2 Minimum
bacteriocidal concentration (MBC): 19
CHAPTER FOUR
4.0 RESULTS 20
CHAPTER FIVE
5.0
Discussion, Conclusion And Recommendation 27
5.2
Conclusion 28
5.3
Recommendation 29
References 30
LIST OF TABLES
Table Title Page
1: Qualitative phytochemical constituent of
citrus
lemon
leaf, stem, juice and root extracts 21
2: Biochemical characterization of
isolated microorganisms. 22
3 Antimicrobial activity of extract
against Gram positive isolates. 23
4: Antimicrobial activity of extract
against Gram negative isolates. 24
5: Minimum inhibitory concentration values
of
different extracts of citrus lemon. 25
6: Minimum bactericidal concentration of
values
of
different extract of citrus lemon 26
CHAPTER ONE
1.0
INTRODUCTION
Medicinal plants have the ability to inhibit
the growth of wide range of pathogenic microorganisms due to presence of
essential oils. The antimicrobial impact of essential oils and its various
components extracted from medicinal plants has been well documented (Hammer et al., 2002; Hood et al., 2003; Duschatzky et
al., 2005). Essential oils have been extracted from complex mixture of
volatile molecules produced by the secondary metabolism of medicinal plants.
Hammer et al., (1999) reported that
the essential oils extracted from medicinal plants contain approximately 20-60
components of quite different concentrations.
Essential oils are natural,
volatile liquid, complex compounds characterized by a strong odor, rarely
colored, soluble in lipid and organic solvents. It could be synthesized by all
plant organs, i.e. buds, flowers, leaves, stems, twigs, see trichomes (Bozin et al., 2006). Essential oils generally
have 2-3 major components at fairly high concentrations (20-70%) compared to
other components present in trace amount. For example, Carvacrol (30%) and
thymol (27%) are the main components of the Origanum compactum essential
oil (Betts, 2001).
The major components include two
groups of distinct bio-synthetical origin, which may determine the biological
activity against the pathogenic microorganisms (Pichersky et al., 2006). The majority of essential oils are composed of
terpenes and terpenoids and other aromatic and aliphatic constituents, all
characterized by low molecular weight. Terpenes are the major group of plant
natural products characterized by an extensive variety of structural types and
the most valuable compounds (Degenhardt et
al., 2009). The terpene compounds are hydrocarbons of general formula (C5H8)n
formed from isoprene units. These compounds could be acyclic, fruits, roots,
wood or bark, and are stored in secretary cells, cavities, canals, epidermic
cells or glandular monocyclic, bicyclic or tricyclic (Abed, 2007). On the basis
of diversity in their chemical structure, they could be classified into several
groups as monoterpenes (C10), sesquiterpenes (C15), and
diterpenes (C20). The majority of the components of essential oils
are monoterpenes represent approximately 90% of the essential oils. These are
generally volatile in nature with pleasant odor (Bakkali et al., 2008).
The chemical profile of essential
oils varies in the number of molecules, stereochemical properties of molecules,
and also depends on the type of extraction. The extraction products may vary in
quality, quantity and in composition according to climate, soil composition,
plant organ, age and vegetative cycle stage (Masotti et al., 2003; Angioni et al.,
2006). Essential oils or some of their constituents are indeed effective
against a large variety of organisms including bacteria and viruses (Duschatzky
et al., 2005), fungi (Hammer et al., 2002) and protozoa (Monzote et al., 2006).
1.1
ANTIBACTERIAL ACTIONS OF ESSENTIAL OILS
Conner (1993) found that cinnamon,
clove, pimento, thyme, oregano, and rosemary plants had strong inhibitory
effect against several bacterial pathogens. It has been also reported that
essential oils extracted from some medicinal plants had the antibacterial
effects against all the five tested food borne pathogens due to presence of
phenolic compounds such as carvacrol, eugenol and thymol (Kim et al., 1995). However, Ramos-Nino et al., (1996) found that benzoic acids,
benzaldehydes and cinnamic acid were able to inhibit the growth of Listeria
monocytogenes. Similarly, Ouattara et
al., (1997) observed the antibacterial activity of selected spices on the
meat spoilage bacteria.
Arora and Kaur (1999) analyzed the
antimicrobial activity of garlic, ginger, clove, black pepper and green chilli
on the human pathogenic bacteria viz. Bacillus sphaericus, Enterobacter
aerogenes, Escherichia coli, Pseudomonas aeruginosa,
Staphylococcus aureus, Streptococcus epidermidis, Salmomella
typhi and Shiguella flexneri and stated that amongst all the tested
spices, aqueous garlic extracts was sensitive against all the bacterial
pathogens. Similarly, effect of clove extracts on the production of verotoxin
by enterohemorrhagic Escherichia coli O157:H7 was investigated by
Sakagami et al., (2000) and it was
evident from the study that the verotoxin production was inhibited by clove
extract. However, Elgayyar et al., (2001)
examined the effectiveness of cardamom, anise, basil, coriander, rosemary,
parsley, dill and angelica essential oil for controlling the growth and
survival of pathogenic and saprophytic microorganisms. The results of their
study showed that essential oils extracted oregano, basil and coriander plants
have inhibitory effect against Pseudomonas aeruginosa, S. aureus and Yersinia
enterocolitica.
Sakandamis et al., (2002) observed the effect of oregano essential oils on the
behaviour of Salmonella typhimurium in sterile and naturally
contaminated beef fillets stored under aerobic and modified atmospheres. They
have concluded that the addition of oregano essential oils checked the
reduction in initial population of the tested bacterial pathogens. However,
Hood et al., (2003) reported that the
bacterial growth may be inhibited by the ample application of essential oils or
their use at high concentrations and their mode of action results in decline of
the bacterial cells. Similarly, Sokovic et
al., (2009) observed the antibacterial activity of essential oils extracted
from thyme and mint leaves against the Staphylococcus aureus, Salmonella
typhimurium and Vibrio parahaemolyticus. The result showed that all
the plants have antibacterial activity against the tested pathogens but the
effect of thyme leaves extract was more pronounced compared to other plants.
Moreover, Shan et al., (2011) showed
cinnamon, oregano, clove, pomegranate peel, and grape seed were found effective
against S. enterica at room temperature, but the clove extracts possess
highest antibacterial activity.
1.2
MECHANISM OF ACTION OF LEMON EXTRACT
AGAINST PATHOGENIC MICROORGANISMS
Antimicrobial actions of essential
oils lead to the leaking of cell membrane and increased the membrane
permeability (Lambert et al., 2001;
Oussalah et al., 2006). The
permeabilization of the cell membranes is directly associated with loss of ions
and reduction in membrane potential, collapse of the proton pump and depletion
of the ATP pool (Di Pasqua et al., 2006;
Turina et al., 2006). The disturbed
cell structure may affect others cellular structures in a cascade type of
action (Carson et al., 2002).
Essential oils pass through the cell wall and cytoplasmic membrane may disrupt
the structural arrangement of different polysaccharides, fatty acids and
phospholipids layers (Burt, 2004; Longbottom et al., 2004). It may also coagulate in the cytoplasm and damage
lipids and proteins layers (Burt, 2004).
Cytotoxic effects of essential oils
were analyzed in-vitro experiments against most of pathogenic gram
positive and gram negative bacteria not only confined to human or animal
pathogens parasites but also found effective in for the preservation of
agricultural/marine products (Arnal-Schnebelen et al., 2004). The antimicrobial effect of essential oil components
such as thymol, menthol and linalyl acetate might be due to a perturbation of
the lipid fractions of bacterial plasma-membranes, which might be affected the membrane permeability and leakage
of intracellular materials (Trombetta et
al., 2005). The other action of
essential oils on the cell membrane is the inhibition of toxin secretion. Ultee
and Smid (2001) reported that the exposure of B. cereus to carvacrol
resulted on inhibition of diarrheal toxin production and use of oregano
completely abolish the enterotoxin production of S. aureus. However,
Ultee et al., (2000) reported that
the secretion of toxins may be prevented by modifications in the bacterial
membrane due to the attachment of the essential oil which might control the
trans-membrane transport process across the plasma membrane and limit the
release of toxins to the external environment (de Souza et al., 2010).
The disruption of the cell membrane
by essential oils may help in various vital processes such as energy conversion
processes, nutrient processing, synthesis of structural macromolecules, and
secretion of many growth regulators (Oussalah et al., 2006). Moreover, Turina et
al., (2006) emphasized that effect of specific ions due on plasma membrane
has strong effect on the protons motive force, intracellular ATP content and
overall activity of microbial cells such as turgor pressure, solutes transport
and metabolism regulation process.
1.3
AIMS AND OBJECTIVES
The
aim of this study is to evaluate the potential of different parts of lemon extract
on pathogenic microorganism strains by using routine antibacterial assay
techniques.
1.4 Objectives
1. To
determine the phytochemical component of the different parts of lemon extract.
2. To
determine the antimicrobial effect of the different part extracts of lemon
against pathogenic microorganisms.
3. To
determine the minimal inhibitory concentration and minimal bactericidal
concentration of the different parts of the lemon extracts.
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