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Product Code: 00007106

No of Pages: 47

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 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.


Title Page                                                                                                                                i

Certification                                                                                                                            ii

Dedication                                                                                                                              iii

Acknowledgements                                                                                                                iv

Table of contents                                                                                                                    v

Lists of Tables                                                                                                                         vii

Abstract                                                                                                                                  viii



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



2.0     Literature Review                                                                                                          6



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



4.0        RESULTS                                                                                                                  20



5.0 Discussion, Conclusion And Recommendation                                                               27

5.2 Conclusion                                                                                                                        28

5.3 Recommendation                                                                                                              29

       References                                                                                                                       30












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


     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).


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.


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.


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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