INVITRO ANTIMICROBIAL ACTIVITY OF ESSENTIAL OILS

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International Standard Serial Number (ISSN): 2319-8141 International Journal of Universal Pharmacy and Bio Sciences 3(3): May-June 2014

INTERNATIONAL JOURNAL OF UNIVERSAL PHARMACY AND BIO SCIENCES Bio

Sciences

IMPACT FACTOR 1.89*** ICV 5.13*** Research Article ……!!!

INVITRO ANTIMICROBIAL ACTIVITY OF ESSENTIAL OILS N.Uma Maheswari* and R.Renuga P.G And Research Department of Microbiology, Sengamala Thayaar Educational Trust Women’s College, Mannargudi, Thiruvarur (Dt)-614001Tamilnadu, India. KEYWORDS: Antimicrobial activity, Mustard oil, Gingelly oil,

ABSTRACT In the present study deals with the antimicrobial activity of essential oils against pathogens. Pathogens were isolated from the soil and identified

Eucalyptus oil, Well diffusion method. For Correspondence: N.Uma Maheswari* Address: P.G And Research Department of

by staining, biochemical test. The gram negative, gram positive bacteria are such as E. coli, Staphylococcus aureus, Pseudomonas aeruginosa were isolated. Antibacterial activity of three essential oils such as Mustard oil, Eucalyptus oil analysed against the pathogens by well diffusion method. Compared to Eucalyptus oil, Gingelly oil, Mustard oil

Microbiology, Sengamala Thayaar Educational

have showed maximum zone of inhibition against Staphylococcus

Trust Women’s College,

aureus 36mm followed by E. coli, Pseudomonas aeruginosa, A.niger

Mannargudi, Thiruvarur

followed by Rhizopus, A.flavus. The antimicrobial activity of essential

(Dt)-614001Tamilnadu, India.

oil is useful for controlling human pathogens. In addition to their use for food and cosmetics, the potential of essential oils for the treatment acne and cancer merits further exploration in the future.

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INTRODUCTION: An antimicrobial is a substance that kills or inhibits the growth of microorganisms, such as bacteria, fungi or protozoan. Antimicrobial drugs either kill microbes (Microbiocidal) or prevent the growth of microbes (Microbiostatic). Disinfectant are antimicrobial substances no living objects or outside the body. The history of antimicrobial begins with the observations of Pasteur and Joubert, who discovered that one type of bacteria could prevent the growth of another. The antimicrobial activity of plant oils and extracts has been recognized for many years. Volatile or essential oils are volatile stream. They differ entirely in both chemical and physical properties from fixed oils. They are secreted in oil cells, in secretion ducts, cavities or in glandular hairs. Herbs and spices are antimicrobially active themselves (1,4) and are distinguished by having particular essential oils. As essential oils are essential additives to food or drug products, the biological activities of those oils should be fully explored. The antimicrobial activity of most essential oils produced world wide is frequently reported: including antibacterial (8﴿, antifungal ﴾9﴿, amoebicidal (7), etc. Antimicrobial activity of essential oils against human, animal, plant﴾14﴿ and food-borne (16) pathogens have been investigated. In vitro activity of common constituents of essential oils (10,15) have also been recorded. The antibacterial activities of spices and their essential oils have been known for a long time, and a number of researches on the antibacterial effect of spices, essential oils and their derivatives have been reported. Plant essential oils are the potentially useful source of antimicrobial compounds. Numerous studies have been published on the antimicrobial activities of plant compounds against many different types of microorganisms, including foodborne pathogens (6). Mustard oil is used for many purposes. Mustard seed can be added to food to make it more spicy. It has inherent pungent flavor which is used for cooking food, body oil for massage, hair oil and is thought to reduce skin dryness, and improve blood circulation, muscular development and skin texture, the oil is also thought to be antibacterial and may even repel insects. (11). Eucalyptus oil Eucalyptus oil should not be confused with the term “eucalyptol”, another name for cineole. The cineole based oil is used as component in pharmaceutical preparations to relieve the symptoms of influenza and colds, in products like cough sweets, lozenges, ointments and inhalants. Eucalyptus oil has antibacterial effects on pathogenic bacteria in the respiratory tract. Inhaled eucalyptus oil vapor is a decongestant and treatment for bronchitis. (13)

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Sesame oil Sesame oil is a source of vitamin E. Vitamin E is an antioxidant and has been correlated with lowering cholesterol levels. Sesame oil is also contains magnesium, copper, calcium, iron, zinc and vitamin B6. Copper provides relief for rheumatoid arthritis. (12) Characteristics of microorganisms: E.coli are gram negative non sporing bacilli, most strains are motile and most strains are fimbriate. Staphylococcus aureus are gram positive spherical cocci approximately 1cm in diametric, arranged characteristically in grape like clusters. Pseudomonas aeruginosa are gram negative. They grow readily on ordinary culture media.

It grows well at 37°c-42°c. Aspergillus niger is grow on

sabouraud agar medium with in a temperature being 25°c for 7 days. Aspergillus flavus is grow on sabouraud agar medium within a temperature being 25° for 7 days. Mycelium of two kinds one submerged in the substratum and other aerial constituting the filaments or stolons. MATERIALS AND METHODS Sample collection: Soil sample was collected from Mannargudi, Thiruvarur Dt, Tamil Nadu, India. The collected sample was transferred to the sterile plastic bags and sealed. Collection of essential oils Available essential oils were collected from Medical shop in Mannargudi, Thiruvarur Dt, Tamil Nadu, India. Essential oils are Eucalyptus oil, Mustard oil, Gingelly oil. Preparation of Ethanol extract concentration of essential oils The collected essential oil were concentrated under vaccum 50oC dried and weighed Ethanol extracts taken at different concentration 25%, 50%, 75% and 100% against test pathogens. Antimicrobial activity Well diffusion technique (Bauer et al., 1966) The bacterial and fungal culture are used for well diffusion method. One well of 5mm size made in the help of sterile cork borer under aseptic condition in laminar air flow chamber. The wells were loaded with 5% of the essential oil (5gm in 100ml of solvent). The plates were incubated at 37ºc for 24 hours. The plates were observed after 24 hours for clearing zone around the well. The zone of inhibition was calculated by measuring the diameter of the inhibition zone around the well, the zone of diameter was measured in 5mm. the reading were taken in all the replicates the averaged values were tabulated. The sabouraud agar plates were prepared and the fungal spores were spreaded are the Sabouraud agar plates. One well of 5mm, size made in the agar plates with the help of sterile cork borer under aseptic condition in laminar air flow chamber. The well were loaded with 0.2ml of essential oil. The Full Text Available On www.ijupbs.com

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plates were incubated at 28ºc for 48 hours. The plates were observed after 48 hours for clearing zone around the well. The zone of inhibition was calculated by measuring the diameter of the inhibition zone around the well, the zone of diameter was measured. RESULTS AND DISCUSSION The present study has been under taken to find out the effectiveness of the Ethanol extracts of essential oils against some human bacterial and fungal pathogens. Isolation of organisms Nutrient agar and PDA plates were prepared .Then soil samples was inoculated into agar plates The bacterial colonies

were identified by grams staining and biochemical test where as fungi by

lactophenol cotton blue mounting technique . Antimicrobial activity The antimicrobial potency of Ethanol extracts of essential oils were tested against

some human

pathogenic bacteria and fungi assessed for the presence or absence of zone of inhibition. The results were relative to antimicrobial activity by measuring the diameter of the zone of inhibition. Antibacterial activity of Essential oil Ethanol extracts of mustard oil Ethanol extracts of mustard oil was effectively controls the Staphylococcus aureus (9.5±2.3mm). Minimum zone of inhibition was noticed

in

Staphylococcus

aureus

100% 25%

(2.3±3.2mm). Ethanol extracts of eucalyptus oil Ethanol extracts of eucalyptus oil was showed minimum inhibition against S. aureus (2.3±3.2mm) at 25% concentration and maximum activity (3.2±6.2mm) at 100% level. Minimum activity was noticed in E.coli (3.2±4.3mm) at 25% concentration. The Ethanol extracts of eucalyptus oil against pseudomonas aeruginosa showed minimum activity (2.5±8.6mm) at 25% concentration and maximum activity (3.0±6.4mm) at 100%. Ethanol extracts of gingelly oil Ethanol extracts of gingelly oil showed minimum activity on Staphylococcus aureus (2.3±4.1mm) at 25% and maximum activity (3.8±5.2mm) at 100% level. The Ethanol extracts of gingelly oil against E.coli showed minimum activity (2.1±5.2mm) at 25% concentration and maximum activity of (3.5±4.1mm) at 100% level. Ethanol extracts of gingelly oil against Pseudomonas aeruginosa showed minimum activity (2.3±4.5mm) at 25% concentration and maximum activity (4.2±4.4mm) at 100% level. Ethanol extracts of mustard oil was showed maximum zone of inhibition against Staphylococcus aureus, Pseudomonas aeruginosa, E.coli. (Table -2).

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Antifungal activity Ethanol extracts of mustard oil was found to be significantly showed maximum shown of inhibition at 100% (11.5±7.3mm) for Aspergillus niger, Aspergillus flavus (8.3±6.1mm) and Rhizopus (9.2±6.1mm). Ethanol extracts of gingelly oil Ethanol extracts of gingelly oil was showed maximum activity at 100% (4.5±2.3mm) and minimum activity (2.3±3.0mm) against Aspergillus flavus. Aspergillus niger Ethanol extracts of gingelly oil was showed maximum activity at 100% (5.4±6.5mm) and minimum activity at 25% (2.3±1.0mm). The Ethanol extracts of gingelly oil was showed maximum activity at 100% (6.1±7.5mm) and minimum activity at 25% (3.5±2.3mm) against Rhizopus. Ethanol extracts of eucalyptus oil The Ethanol extracts of eucalyptus oil was showed a activity at 100% (4.5±1.5mm) and minimum activity at 25%(2.5±3.0mm) against Aspergillus niger. The Ethanol extracts showed maximum activity at 100% (5.7±6.1mm) and minimum activity of 25%(3.2±3.3mm) for Aspergillus flavus. The Ethanol extracts showed maximum activity at 100% (6.8±5.4mm) and minimum activity of 25% (3.4±2.2mm) for Rhizopus. Ethanol extracts of mustard oil 100% (11.5±7.3mm) (8.3±6.1mm) (9.2±6.1mm) was showed maximum zone of inhibition against Aspergillus niger, Aspergillus flavus and Rhizopus. Compared to Mustard oil , Eucalyptus oil , Gingelly oil have shown maximum zone of inhibition against Staphylococcus aureus

followed by E.coli, Pseudomonas aeruginosa .

Aspergillus niger , followed by Aspergillus flavus , Rhizopus . Table 1: S.No 1 2 3 4 5 6 7 8 9 10 11

Morphological And Biochemical Characteristics Of Bacterial Pathogens. Morphological and biochemical characteristics Gram’s staining Motility Shape Indole Test Methyl Red Test VogesProskauer Test Citrate Test Urease Test Trible Sugar Iron Test Catalase Test Oxidase Test

S.aureus

Escherichia coli

P.aeuroginosa

+ Cocci + + + K/A -

+ Rod + + K/K + -

+ Rod + + + K/K -

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Table 2 : Antibacterial Activity Of Ethanol Extracts Of Essential Oils. Zone of inhibition(mm) S.No 1

Essential oils Mustard oil

2

Eucalyptus oil

3

Gingelly oil

25% 5.5±2.0 6.2±1.5 4.5±2.1

50% 8.5±4.1 7.4±3.2 6.6±2.4

75% 5.8±2.31 8.8±5.12 9.0±3.16

100% 9.5±8.9 6.5±6.4 6.7±2.1

2.3±3.21 3.2±4.3 2.5±8.6 2.3±4.1 2.1±5.2 2.3±4.5

4.2±3.41 3.5±2.3 2.7±4.2 2.3±2.3 2.5±3.1 3.5±5.2

2.3±3.2 3.7±2.5 2.8±6.2 3.5±3.2 2.8±2.5 2.3±3.5

3.2±6.2 4.0±5.1 3.0±6.4 3.8±5.2 3.5±4.1 4.2±4.4

Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa. Values are triplicates and represented as mean ± standard deviation Table 3 : Antifungal Activity Of Ethanol Extracts Of Essential Oils. S.No

Essential oils

1

Mustard oil

2

Eucalyptus oil

3

Gingelly oil

Zone of inhibition(mm) 25% 50% 6.3±2.2 7.4±2.3 5.3±3.5 6.2±4.3 4.2±4.8 5.6±4.1

75% 9.2±1.2 6.2±2.3 7.2±3.8

100% 11.5±7.3 8.3±6.1 9.2±6.1

2.5±3.03 3.2±3.3 3.4±2.2 2.3±1.0 2.3±3.0 3.5±2.3

3.2±4.1 4.3±2.3 5.2±2.2 4.1±3.2 4.2±2.3 5.1±2.2

4.5±1.5 5.7±6.1 6.8±5.4 5.4±6.5 4.5±2.3 6.1±7.5

2.7±0.2 3.1±3.0 3.5±4.1 3.4±5.1 3.7±5.5 4.1±6.5

Aspergillus niger Aspergillus flavus Rhizopus Values are triplicates and represented as mean ± standard deviation CONCLUSIONS: In the present study deals with the antimicrobial activity of essential oils against pathogens. In this study, the pathogens were isolated from the soil and identified by staining, biochemical test. The gram negative gram positive bacteria are such as E.coli, Staphylococcus aureus, Pseudomonas aeruginosa were isolated . Antibacterial activity of three essential oils such as Mustard oil , Eucalyptus oil analysed against the pathogens. Compared to Eucalyptus oil, Gingelly oil, Mustard oil have showed maximum zone of inhibition against Staphylococcus aureus followed by E.coli, Pseudomonas aeruginosa.

Aspergillus niger,

followed by Rhizopus, Aspergillus flavus. The antimicrobial activity of essential oil is useful for controlling human pathogens. In addition to their use for food and cosmetics, the potential of

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essential oils for the treatment of acne and cancer merits further exploration in the future. Further pharmacological and toxicological studies will be required to establish the therapeutic use of this oil. REFERENCES: 1. Akgul A & Kivanc M (1988a). Inhibitory effects of Turkish spices and oregano components on some food borne fungi, Int, J.Food Micro. 6;263-268. 2. Akgul A & Kivanc M (1988b). Inhibitory effects of six Turkish thyme-like spices on some common food borne bacteria. Die Nahrung. 32;201-203. 3. Akgul A & Kivanc M (1988c). Sensitivity of four food borne moulds to essential oils from Turkish spices, herbs and citrus peel.J.Sci Food Agric. 47:129-132. 4. Akgul A (1989). New spice from Turkey:Laser trilobum(L).Borkh.I. Inhibitory effect on food contaminating microorganisms, Acta Alimentaria. 18:65-69. 5. Bauer, A.W., Kirby, W.M., Truck, H., and Shreeies, J.C., (1996). Antibiotic Susceptibility testing by standardized single well diffusion method, AM.J.Clin,Pathol. 45;496. 6. Biavati B., ozcan,M. and Piccaglia,R (2004). Composition and antimicrobial properties of Satureja aneifolia Ten and Thymbra Sintenesii Bornm. Et Aznav.subp. is aurica P.H.Davis essential oils. Annals microbial. 54:393-401. 7. De Blasiv, Debrot S, Menoud PA, Gendre L & Schowing J (1990). Amoebicidal effect of essential oils in vitro J.Toxicol clip.Exp 10:361-373. 8. Deans SG & Ritchie GA (1987). Antibacterial propertiesof plant essential oils. International Journal of food Microbiology 5:165-180 9. Garg SC & Dengre SI (1998). Antifungal efficacy of some essential oils. Pharmazie 43:141143. 10. Hinou JB Harvala CE & Hinou EB (1989). Antimicrobial activity screening of 32 common constituents of essential oils. Pharmazie 44.302-303. 11. Hulan H.W, Kramer JK, Mahadevan S.Sauer FD (1976). “Relationship between eurcic acid and Myocardial changes in male rats” Lipids. 1(1) 9-15. 12. Kamal –Eldin, Afaf Appelqvist,Lars-Ake (1195). The effects of extraction methods on sesame oil stability journal of the Americanoil chemist’s society. 72 (8); 967-969. 13. Lawless J., (1995). The illustrate Encyclopedia of Essential oils, Element Books ISBN 185-230-661-0. 14. Panizzi L, Flamini G, Cioni PL & Morelli I (1993). Composition and antimicrobial properties of essentialoils of four Mediterranean Lamiaceae. J.ethnopharmacol. 39:167-170 15. Shapiro S, Meier A & Guggenheim B (1994). The antimicrobial activity of essential oils and essential oil components towards oral bacteria. Oral Microbiol Immunol. 9:202-208. Full Text Available On www.ijupbs.com

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