Abstract
The present study dealt with the hydro distillation of Nigella sativa seeds and GC-MS analysis. The total composition of the oil was 86.7%. The seed volatile oil was tested against 19 microbes (Gram positive, Gram negative and fungi), which showed that Gram negative bacteria Haemophilus influenza, Klebsiella pnemoniae, and Proteus vulgaris were highly sensitive against the antimicrobial agent, whereas the fungi such as Trichoderma vibriae,. Pencillium rubrum; and Dermatophyte: Trichophyton mentagrophytes; had no response at the 20µl concentration.
Nigella sativa; Volatile oil; Antimicrobial activity; Antifungal activity
BIOLOGICAL AND APPLIED SCIENCES
GC-MS analysis of Nigella sativa seeds and antimicrobial activity of its volatile oil
Saptha Jyothi Gerige* * Author for correspondence: gsapthajyothi@gmail.com ; Mahesh Kumar Yadav Gerige; Muralidhara Rao; Ramanjaneyulu
Departament of Biotecnology; Sri Krishnadeveraya University; Anantapura - A. P. - India
ABSTRACT
The present study dealt with the hydro distillation of Nigella sativa seeds and GC-MS analysis. The total composition of the oil was 86.7%. The seed volatile oil was tested against 19 microbes (Gram positive, Gram negative and fungi), which showed that Gram negative bacteria Haemophilus influenza, Klebsiella pnemoniae, and Proteus vulgaris were highly sensitive against the antimicrobial agent, whereas the fungi such as Trichoderma vibriae,. Pencillium rubrum; and Dermatophyte: Trichophyton mentagrophytes; had no response at the 20µl concentration.
Key words: Nigella sativa, Volatile oil, Antimicrobial activity, Antifungal activity
INTRODUCTION
The role of the volatile oils is predominant in many industries, particularly in the pharmaceutical, clinical and food industries. The oil and its constituents are well documented as antimicrobial agents (Knobloch et al., 1989, Pepeljnjak et al., 2003). The volatile oils are complex mixtures of the compounds which mainly having monoterpines, sesquiterpines hydrocarbons with general formula (C5H8)n (Svoboda and Hampson, 1999). These oils act in monitoring the blood circulation, nerve growth, liver, and cholesterol etc (Vardharajan, 1985). It also possesses antimicrobial activity. Some Plants like Nigella sativa, Cuminum Cyminum, Papaver somniferum, etc. have been considered as a protective agent against carcinogenesis (Aruna and Sivaramakrishnan, 1996, Hailat, 1995). The oil from N.sativa is used in preliminary clinical medicine for cough and bronchial asthma (Vardharajan, 1985). The seeds are considered as carminative stimulant, diuretic, emenagogue, galactagogue and are used in the treatment of mild cases of puerperal fever (Vardharajan, 1985, Hailat, 1995).
N.sativa is an annual herb and is widely spread in southern Europe and western Asia, abundantly in Mediterranean region (Vardharajan, 1985). In India it is found in Punjab, Bihar, Assam and Himachal Pradesh (Vardharajan, 1985). The seeds are trigonous black rugulose tubercular and used as a flavouring agent and for the medicinal purposes (Vardharajan, 1985, Hailat, 1995).
In view of its medicinal value the N.sativa was chosen for the extraction of essential oil with an aim to establish its antimicrobial activity against Gram-positive and Gram-negative bacteria and fungi.
MATERIALS AND METHODS
The seeds of N.sativa were procured from local market and were fine powered and hydro-distilled at 100ºC in a Clevenger apparatus (Haborne, 1984). The volatile oil was collected, dried over anhydrous sodium sulphate, stored in brown bottles and finally kept in refrigerator for further GC-MS analysis.
GC-MS Analysis
A Shimadzu 17A GC coupled with Shimadzu QP5050 A (quadruple) Mass Spectrometer (Shimadzu, Japan), equipped with EI and a fused silica column DB-5 (30m x 0.25 mm i.d.) of 0.25µm film thickness was needed. The oven temperature at 50ºC for 5 minutes and then programmed from 50-280ºC for 40 minutes. Helium flow rate of 2ml/min, with the split ratio of 1:30 mode was used for sample injection of 1µl and ionization voltage of MS-analysis was run by EI technique at 70ev. The volatile oil constituents were identified by matching their MS and retention index data with those of the standards ethnic spectra and by matching their fragmentation pattern in Mass Spectra with those of WILEY 139.LIB and NIST 12.LIB (3) The retention indices were calculated by Kovats's procedure (Masada, 1976, Adams, 1989).
Antimicrobial susceptibility test
The antimicrobial activity was determined by the disc diffusion method using the Kirby-Bauer method (Bauer and Kirby, 1966). The discs of 6 mm diameter were prepared with Whatman No 1 filter paper. The volatile oil of concentration 20µg for the test was applied to the discs. Inoculum was prepared with the fresh cultures of bacterial strains, which were grown in tryptic-soy agar for 18h at 37±1ºC with physiological saline, 3 x 106 cells ml-1 . Inoculum density was compared with Mac-Farlands standard solution of BaSO4 (0.1ml of 1% BaCl2 + 9.9ml of 1% H2SO4). The cultures were cultivated on Sabouraud dextrose agar with addition of 50mg/l Chloramphenicol (sigma, Germany) for 5 days for the yeasts and 10 days for fungi and dermatophytes at 25±5ml of Muller Hington agar for the bacterial strains, and the same amount of inoculum was cultured Sabouraud agar for the fungi. Then the agar was inoculated with the culture and incubated at room temperature for 25 minutes. The discs were arranged on the surface of the inoculated agar plates and pressed gently to adhere to the surface of the agar. The plates were incubated for 24-48h at 35-37ºC. After incubation, the diameter of the zone of inhibition was measured.
RESULTS AND DISCUSSION
The GC-MS analysis of the N. sativa volatile oil showed 31 compounds (Table-1), which included two new chemical compounds viz. 2(1H)-Naphthalenone (C11H18O) and Uvdin (C15H24O3).
The percentage of total compounds was 86.7%.
The results of the antimicrobial activity of the N. sativa volatile oil were presented in Table-2 and compared with the standard and accordingly, the efficacy of volatile oil was far better than the standard. Further, from the data found in Table-2, the order of sensitivity of microorganisms was Gram-negative bacteria followed by Gram-positive bacteria, Yeast and Dermatophyte, but at 20mg of volatile oil, the Trichophyton mentagrophytes, tricoderma vibriae, penicillium rubrum were found to be resistant to the volatile oil. The presence of biological active compounds such as α-thujene, 2(1H)-naphthalenone, α-pinene, α-phellandrene, limonene, thymoquinone, myristicin etc in N.sativa volatile oil contributed the antimicrobial activity of volatile oil.
CONCLUSIONS
The presence of rich biological active compounds in N.sativa volatile oil, which contributed antimicrobial properties, has highlighted the plant as good medicinal plant.
ACKNOWLEDGEMENTS
We are thankful to G.V. Lakshmi, G.Ramachandra, Natural products research division, # 2 - 674, ADONI, 518302, A.P, India for their help in oil extraction and analysis.
Received: September 26, 2006; Revised: July 20, 2007; Accepted: July 28, 2008.
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Publication Dates
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Publication in this collection
12 Jan 2010 -
Date of issue
Oct 2009
History
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Reviewed
20 July 2007 -
Received
26 Sept 2006 -
Accepted
28 July 2008
