GC-MS Analysis of Nigella Sativa Seeds and Antimicrobial Activity of Its Volatile Oil

1189
Vol.52, n. 5: pp. 1189-1192, September-October 2009 BRAZILIAN ARCHIVES OF

ISSN 1516-8913 Printed in Brazil
BIOLOGY AND TECHNOLOGY
A N I N T E R N A T I O N A L J O U R N A L
GC-MS Analysis of Nigella sativa Seeds and Antimicrobial

Activity of its Volatile oil
Saptha Jyothi Gerige*, Mahesh Kumar Yadav Gerige, Muralidhara Rao and
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 from N.sativa is used in preliminary clinical

medicine for cough and bronchial asthma
The role of the volatile oils is predominant in (Vardharajan, 1985). The seeds are considered as
many industries, particularly in the carminative stimulant, diuretic, emenagogue,
pharmaceutical, clinical and food industries. The galactagogue and are used in the treatment of mild
oil and its constituents are well documented as cases of puerperal fever (Vardharajan, 1985,
antimicrobial agents (Knobloch et al., 1989, Hailat, 1995).
Pepeljnjak et al., 2003). The volatile oils are N.sativa is an annual herb and is widely spread in
complex mixtures of the compounds which mainly southern Europe and western Asia, abundantly in
having monoterpines, sesquiterpines hydrocarbons Mediterranean region (Vardharajan, 1985). In
with general formula (C5H8)n (Svoboda and India it is found in Punjab, Bihar, Assam and
Hampson, 1999). These oils act in monitoring the Himachal Pradesh (Vardharajan, 1985). The seeds
blood circulation, nerve growth, liver, and are trigonous black rugulose tubercular and used
cholesterol etc (Vardharajan, 1985). It also as a flavouring agent and for the medicinal
possesses antimicrobial activity. Some Plants like purposes (Vardharajan, 1985, Hailat, 1995).
Nigella sativa, Cuminum Cyminum, Papaver In view of its medicinal value the N.sativa was
somniferum, etc. have been considered as a chosen for the extraction of essential oil with an
protective agent against carcinogenesis (Aruna and aim to establish its antimicrobial activity against
Sivaramakrishnan, 1996, Hailat, 1995). The oil
*
Author for correspondence: gsapthajyothi@gmail.com
Braz. Arch. Biol. Technol. v.52 n.5: pp. 1189-1192, Sept/Oct 2009
1190 Gerige, S. J. et al.
Gram-positive and Gram-negative bacteria and 18h at 37±10C with physiological saline, 3 x 106
fungi. 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
MATERIALS AND METHODS were cultivated on Sabouraud dextrose agar with
addition of 50mg/l Chloramphenicol (sigma,
The seeds of N.sativa were procured from local Germany) for 5 days for the yeasts and 10 days for
market and were fine powered and hydro-distilled fungi and dermatophytes at 25±5ml of Muller
at 1000C in a Clevenger apparatus (Haborne, Hington agar for the bacterial strains, and the same
1984). The volatile oil was collected, dried over amount of inoculum was cultured Sabouraud agar
anhydrous sodium sulphate, stored in brown for the fungi. Then the agar was inoculated with
bottles and finally kept in refrigerator for further the culture and incubated at room temperature for
GC-MS analysis. 25 minutes. The discs were arranged on the
surface of the inoculated agar plates and pressed
GC-MS Analysis gently to adhere to the surface of the agar. The
A Shimadzu 17A GC coupled with Shimadzu plates were incubated for 24-48h at 35-370C.
QP5050 A (quadruple) Mass Spectrometer After incubation, the diameter of the zone of
(Shimadzu, Japan), equipped with EI and a fused inhibition was measured.
silica column DB-5 (30m x 0.25 mm i.d.) of
0.25µm film thickness was needed. The oven
temperature at 500C for 5 minutes and then RESULTS AND DISCUSSION
programmed from 50-2800C for 40 minutes.
Helium flow rate of 2ml/min, with the split ratio of The GC-MS analysis of the N. sativa volatile oil
1:30 mode was used for sample injection of 1µl showed 31 compounds (Table-1), which included
and ionization voltage of MS-analysis was run by two new chemical compounds viz. 2(1H)-
EI technique at 70ev. The volatile oil constituents Naphthalenone (C11H18O) and Uvdin (C15H24O3).
were identified by matching their MS and The percentage of total compounds was 86.7%.
retention index data with those of the standards The results of the antimicrobial activity of the N.
ethnic spectra and by matching their fragmentation sativa volatile oil were presented in Table-2 and
pattern in Mass Spectra with those of WILEY compared with the standard and accordingly, the
139.LIB and NIST 12.LIB (3) The retention efficacy of volatile oil was far better than the
indices were calculated by Kovats’s procedure standard. Further, from the data found in Table-2,
(Masada, 1976, Adams, 1989). the order of sensitivity of microorganisms was
Gram-negative bacteria followed by Gram-
Antimicrobial susceptibility test positive bacteria, Yeast and Dermatophyte, but at
The antimicrobial activity was determined by the 20mg of volatile oil, the Trichophyton
disc diffusion method using the Kirby-Bauer mentagrophytes, tricoderma vibriae, penicillium
method (Bauer and Kirby, 1966). The discs of 6 rubrum were found to be resistant to the volatile
mm diameter were prepared with Whatman No 1 oil. The presence of biological active compounds
filter paper. The volatile oil of concentration 20µg such as α-thujene, 2(1H)-naphthalenone, α-
for the test was applied to the discs. Inoculum was pinene, α-phellandrene, limonene, thymoquinone,
prepared with the fresh cultures of bacterial myristicin etc in N.sativa volatile oil contributed
strains, which were grown in tryptic-soy agar for the antimicrobial activity of volatile oil.
GC-MS Analysis of Nigella sativa Seeds and Antimicrobial 1191
Table 1 - Chemical composition of Nigella sativa volatile oil constituents.

Compound Percentage Compound Percentage
α-Thujene 2.4 p-Cymene-8-ol 0.4
3-Methyl Nonane 0.6 Nerol 1.3
α-Pinene 1.2 Estragole 1.9
Sabinene 1.4 Dihydrocarvone 0.3
β-Pinene 1.3 Carvone 2.0
Myrcene 0.6 Thymoquinone 11.8
n-Decane 0.4 Anisaldehyde 1.7
α-Phellandrene 0.6 Trans-Anethole 27.1
p-Cymene 9.0 Carvacrol 3.7
Limonene 4.3 α-Longipinene 0.3
1-Methyl-3-propyl benzene 0.7 n-Tetradane 0.2
γ-Terpinene 0.5 Longifolene 5.7
1-Ethyl-2,3-dimethyl benzene 0.2 Uvidine 1.3
2(1H)-Naphthalenone 2.6 Myristicin 1.4
Fenchone 1.1 n-Hexadecane 0.2
Terpinen-4-ol 0.7 Apiole 1.0
Total 27.6 60.3
Table:2 - Antimicrobial activity of the Nigella sativa volatile oil by Disc diffusion method.
Inhibition Zone in mm
Microorganisms
µg)
Oil disc (20µ Standard
Gram-Positive Staphylococcus aureus MTCC 737 18 25

Bacterial species Streptococcus pneumoniae MFBF 14 nt
Bacillus subtilus MTCC 121 10 nt
Micrococcus luteus MTCC 1541 11 20
Gram-Negative
Bacterial species Pseudomonas aeruginosa MTCC 1688 14 11
E.Coli MTCC 1687 14 17
Vibrio cholerae MFBF 16 nt
Salmonella typhi MFBF 10 nt
Proteus vulgaris MTCC 1771 12 12
Haemophilus influenzae MFBF 32 nt
Neisseria gonorrhoeae MFBF 18 nt
Klebsiella pneumoniae MFBF 16 16
Yeast Candida albicans MTCC 184 08 12
Fungi Aspergillus niger MTCC 1344 10 13

Aspergillus flavus MFBF 09 14
Tricoderma vibriae MFBF 0 16
Penicillium rubrum MFBF 0 16
Chaetomium globosum MFBF 07 12
Dermatophyte Trichophyton mentagrophytes MFBF 00 12
nt=not tested., Clindamycin 2mg/ml for S.aureus, Gentamicin 2mg/ml for Ps. aeruginosa, Proteus vulgaris; Tetracycline
3mg/mlE.Coli and B.subtilus Clotrimazole 5mg/ml for C. albicans; Nystain 10mg/ml for A.niger and T.mentagrophytes.
MFBF: number of strains from the collection of microorganisms of the Dept. of Microbiology and biotechnology, Anantapur.
MTCC: Microbial type culture collection centre.
1192 Gerige, S. J. et al.
CONCLUSIONS NAHA, 25-28 september, St. Louis Missouri, USA

105-127.
The presence of rich biological active compounds Vardharajan, S. (1985), The wealth of India-A
Dictionary of Indian raw materials and industrial
in N.sativa volatile oil, which contributed
products, Vol. 1A, Publication and information
antimicrobial properties, has highlighted the plant directorate, CSIR, NewDelhi.
as good medicinal plant. Aruna. and Sivaramakrishnan, V. M. (1996),
Anticarcinogenic effects of the essential oils from
Cumin, Poppy and Basil. Phytotherapy Research, 10
ACKNOWLEDGEMENTS (7), 577-580.
Hailat, N.; Batinheh, Z.; Lafi, S.; Raweily, E.; Aqel, M.;
We are thankful to G.V. Lakshmi, Al-Katib, M. and Hanash, S. (1995), Effect of Nigella
G.Ramachandra, Natural products research sativa volatile oil on Jarkat T cell leukemia
polypeptides. Int. J. Pharmacog, 33 (1), 16-20.
division, # 2 - 674, ADONI, 518302, A.P, India for
Haborne, J. B. (1984), Phytochemicals methods, 2nd ed,
their help in oil extraction and analysis. Academic press London.
Masada, Y. (1976), Analysis of Essential Oils by Gas
chromatography and mass Spectrometry, John Wiley
REFERENCES and Sons, New York.
Adams, R. P. (1989), Identification of essential oils by
Knobloch, K.; Pauli, A.; Iberl, B.; Weigand, H. and Ion Trap Mass spectroscopy. Academic Press,
Weis, N. (1989), Antibacterial and antifungal London.
properties of essential oil components. J. Essent. Oil Bauer, A. W.; Kirby, M. (1966), Antibiotic
Res., 1:119-128. Susceptibility testing by standard disc method. Am.
Pepeljnjak, S.; Kosalec, I.; Kolodera, Z. and Kustrak, J. Clin. Patho. 10, 45; 493-496.
D. (2003), Natural Antimycotics from Croatian National Committee for Clinical Laboratory Standards
Plants, in Plant-derived Antimycotics. Current Trends Approved standard M.2-A6. (1997), Performance
and Future Prospects (Eds. M. Rai, D.Mares), Standards for antimicrobial disc susceptibility testing
Harworth Press, New York, pp 41-84. 6th edition, NCCLS, Waynae.
Svoboda, K. P.; Hampson, J. B. (1999), Bioactivity of
essential oils of selected temperature aromatic plants:
antibacterial, antioxidant, anti-inflammatory and Received: September 26, 2006;
other related pharmacological activities. Procceedings Revised: July 20, 2007;
Accepted: July 28, 2008.

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1189

Vol.52, n. 5: pp. 1189-1192, September-October 2009 BRAZILIAN ARCHIVES OF

GC-MS Analysis of Nigella sativa Seeds and Antimicrobial

INTRODUCTION from N.sativa is used in preliminary clinical

Table 1 - Chemical composition of Nigella sativa volatile oil constituents.

Gram-Positive Staphylococcus aureus MTCC 737 18 25

Yeast Candida albicans MTCC 184 08 12

Fungi Aspergillus niger MTCC 1344 10 13

CONCLUSIONS NAHA, 25-28 september, St. Louis Missouri, USA

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