International Journal of Research in Pharmaceutical and Biomedical Sciences

ISSN: 2229-3701

______________________________________________________________________Research Paper

Chemical compsition and antimicrobial activity of essential oil of tubers of Cyperus rotundus Linn. collected from Dehradun (Uttarakhand)
Anupam Bisht1*, G.R.S. Bisht2, Mamta Singh1, Richa Gupta1 and Vinod Singh1
1Department

of Pharmaceutical Sciences, Sardar Bhagwan Singh P.G Institute of

Biomedical Sciences & Research, Balawala, Dehradun, Uttarakhand, India.

2Department

of Microboilogy,Sardar Bhagwan Singh P.G Institute of Biomedical Sciences &

Research, Balawala, Dehradun, Uttarakhand, India.

___________________________________________________________________________
ABSTRACT Cyperus rotundus a member of family Cyperaceae is a perennial,stoloniferous herb.The essential oil (0.2%) was extracted by hydrodistillation from the tubers of Cyperus rotundus collected from Balawala(Dehradun),Uttarakhand. The hydrodistilled oil was subjected to GC-MS analysis.Sixteen compounds were identified constituting 67.546% of the total oil. The major components were oxo-isolongifolene (16.268%),-gurjunene(10.219%) and (z)-Valerenyl acetate (8.888%) and -salinene(4.480%). The oil was screened for its antimicrobial potential. The oil was found to be effective against various bacterial and fungal strains viz. Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, Candida parapsilosis, Aspergillus flavus, Aspergillus fumigatus and Fusarium oxysporum in different concentrations. Key Words: Cyperus rotundus,tubers,essential oil,GC-MS,Antimicrobial activity. INTRODUCTION Essential oils are secondary metabolites of various parts of plants13.Essential oil extracted from plants have unique therapeutic benefits. These are utilized in various forms such as ointment, lubricant, lotions, perfumes, perfumed water, fragrent baths, massage and incense3.The antimicrobial action of essential oils is one of the most extensively studied aspects of plant medicine. Antimicrobial action of essential oil is due to the presence of active compounds that can inhibit microbial growth.11 Cyperus rotundus Linn.(Nut grass) is commonly known as Nagarmotha belonging to the family cyperaceae.Cyperus rotundus is originated in india as a weed and it is now widely distributed in the tropics and subtropics1.Cyperus rotundus plant is a perennial glabrous herb with elongate slender stolon beraing hard black fragrant tubers and triquetrous aerial stems, leaves numerous, narrowly linear, finely acuminate. The bulbous root (tubers) have many medicinal uses. The tubers are refrignant, demulcent and tonic10. ________________________________________ *Address for correspondence: E-mail: anupam_sajwan@yahoo.com The decoction of the roots and tubers are excellent antidote to all poisons. Romans used the tubers as emmenagogue in uterine complaints5.Tubers are anthelmintic, diaphoretic, astringent and stimulant. The tubers have anti-inflammatory and antipyretic activity1. Decoction of dried root is taken orally for diarrohea8. water extract is taken orally as an anthelminitic and as an emmenagogue. Tubers and rhizomes, crushed and boiled in goats milk are taken orally for colic, to treat diarrhea,to treat vomiting in children and to relieve flatulence in children2. Dried tuberous roots, known as source of aromatics, used in perfumes and agarbatties4. In the earlier report the essential oil of rhizome of Cyperus rotundus collected from south India,contained -copaene (11.4%-12.1%), cyperene (8.4-11.7%), valerenal (8.79.8%),caryophyllene oxide (7.8-9.7%) andtranspino carveol (5.2-7.4%)6.Lawal reported cyperone (7.9-11.0%), myrtenol (7.9-7.1%) caryophyllene oxide (5.4%),-pinene (5.3-11.3%), -pinene (10.8%) and -Selinene (6.6%) in oil samples collected from two different locations of 661

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International Journal of Research in Pharmaceutical and Biomedical Sciences

ISSN: 2229-3701

South Africa9. The essential oil of Cyperus rotundus from Germany contain cyprotene,copaene,cyperene,-selinene,rotundene, cadalene, rootkatene13.The Tunisian species oil was characterized with high content of sesquterpene with cyperene (30.9%).7 Although work have been carried out on the chemical composition of the oils of Cyperus rotundus5, 6,7,8,9. In this study, the composition of essential oil obtained from Cyperus rotundus growing in dehradun,a new location is compared with the previous study. MATERIALS AND METHODS The fresh tuber roots of Cyperus rotundus Linn.were collected from village balawala, Dehradun (Uttarakhand).Identification of plant specimen was confirmed from Forest Research Institute, Dehradun.The fresh tubers(500 g.) were hydrodistilled for 6-7 hrs using Clavenger apparatus(lighter than water).The oil so obtained was extracted with ether,dried over anhydrous Na2SO4 and ethereal layer was finally evaporated at room temperature. Physical properties of oil was studied. Oil was light yellow in colour and was having characteristic smell.It gets volatilized when exposed to atmosphere and was soluble in ether. The other physical parameters of oil like specific gravity, refractive index were also observed. The extracted essential oil was subjected to GC-MS analysis (Table 1). The GC-MS analysis of oil was carried out on a PERKIN ELMER CLARUS-500 model coupled with CLARUS-500 Mass spectrometer (it is equivalent to DB-5) with a RTX-5 column (60m X 0.32mm X 0.25m). Injection volume was 0.1l in the split mode (split flow 50ml/minute) Helium as a carrier gas at a flow rate of 1ml/min , inlet temperature were 210C.Oven temperature was held at 60C to 220C for 10 min followed by linear temperature programming at the rate of 3C temperature rise per minute .The column was coupled directly to the Perkin Elmere Clarus mass spectrometer operated in the electron ionization mode at 70ev, ion source temperature 150C.Mass spectral identification were made by matching the mass against the NIST library software and the Retention time comparison with the publisher data of Wiley. The essential oil of Cyperus rotundus was screened for its antibacterial activity against various bacterial strains viz. Bacillus subtilis MTCC 121, Micrococcus luteus MTCC 106, Staphylococcus aureus MTCC 2940, Escherichia coli MTCC 2939, Pseudomonas aeruginosa MTCC 424, Klebsiella sp. MTCC 109. The bacterial cultures were provided by Department of Microbiology, of the institute. The antibacterial activity was determined by agar dics diffusion method. Nutrient agar media was used for antibacterial activity.0.1 ml of suspension of bacterial strains were swabbed over the agar plates.

Petriplates were allowed to dry. With the help of sterilized forceps sterile paper disc containing different concentration of essential oil (10l/disc) were placed on agar surface of the petri plates containing nutrient agar medium. In the centre of the plate control antibiotic chloramphenicol (10l of 30g/ml stock solution) was placed as positive control and tween 20 as negative control. Plates were incubated at 37C for 24 hrs and observed for zone of inhibition. The essential oil was further subjected to the antifungal activity and summarized results are shown in table 3.The fungal strains taken for the study were Candida albicans MTCC 1673,Candida parapsilosis MTCC 1744,, Aspegillus flavus MTCC 1973, Aspergillus fumigatus MTCC 3070, Fusarium oxysporum MTCC 2480. 0.1 ml of cell suspension of of Candida spp. and the spore suspension of three molds viz Aspergillus flavus,Aspergillus fumigatus and Fusarium oxysporum spreaded over the Sabouraud Dextrose agar plates. Petriplates were allowed to dry. With the help of sterilized forceps sterile paper disc were placed over agar surface at equidistant and an aliquot of 10l of different concentration i.e 100%,50%,25%,12.5% and 6.25% of each were soaked on to the sterile filter paper disc for determination of Minimal Inhibitory Concentration(MIC).The petriplates were incubated at 27C1oC for 72 hrs and inhibition zone diameter were recorded after observation. Nystatin was used as standard antifungal antibiotic(10l of 100g/ml stock solution). RESULTS AND DISCUSSION Steam distillation of Cyperus rotundus tubers yield 0.2% of yellow oil with characteristic odor. The specific gravity and refractive index of the oil was found to be 0.8426,1.464 respectively. The analysis of the essential oil of Cyperus rotundus by GC-MS allowed the identification of sixteen compounds representing 67.546 % of the total composition of oil(Table1).The major components were 5-oxoisolongifolene (16.268%), -gurjunene (10.219%), (z)-Valerenyl acetate (8.888%),Salinene(4.480%).The other components were valerenic acid(3.669%) and -cadinene(3.401%). At 100% concentration the oil shown good activity against Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Pseudomonas aeruginosa and less effective against Micrococcus luteus and Klebsiella sp. At low concentration the oil was effective against S.aureus(Table2). Oil shown good antifungal activity against Candida parapsilosis and Aspergillus fumigatus. The oil inhibit spore formation of fusarium oxysporum and Aspergillus flavus (Table 3).

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International Journal of Research in Pharmaceutical and Biomedical Sciences

ISSN: 2229-3701

CONCLUSION Major components of essential oil of Cyperus rotundus tubers collected from Dehradun were 5oxo-isolongifolene(16.268%),-gurjunene (10.219%) ,(z)-Valerenyl acetate (8.888%),Salinene (4.480%). The other components were valerenic acid (3.669%) and cadinene(3.401%).The oil composition of Cyperus rotundus obtained from Dehradun region was different from south region which contain copaene (11.4%-12.1%), cyperene (8.4-11.7%), valerenal (8.7-9.8%), caryophyllene oxide (7.89.7%) as main constituents.Comparing the results with those previously reported in literature. There is differences regarding the major constituents of oil of C.rotundus. The oil was effective against E.coli,S.aureus and shows good antimicrobial activity against B.subtilis in comparison to chlorampenicol.Oil is inhibitory

against Aspergillus flavus ,Fusarium oxysporum and inhibit spore formation in Fusarium fumigatus. The compositional difference between C.rotundus found in Dehradun due to climatic and environmental conditions.This shows the existence of more chemical diversity within the C.rotundus species.It was concluded that essential oil of Cyperus rotundus growing in Dehradun region contain different constituents as reported earlier.It suggested the different chemotype of Cyperus rotundus. ACKNOWLDGEMENTS Authors thanks the management of S.B.S.PGI.Balawala, Dehradun, for laboratory facilities, Professor F.C. Garg Head of the Department of Microbiology of the institute for providing microbial cultures.

Table 1: Percentage composition of Cyperus rotundus Linn. oil sample

S. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Identified compounds 5-oxo-isolongifolene -gurjunene (z)-Valerenyl acetate -Salinene Valerenic acid -Cadinene 9H-Cycloiso longifoline,8-oxo (+)--Ylangene Iso spathulenol Isolongifolene,7,8-Dehydro-8A-Hydroxy Iso cyclo seychellene 9-Aristolene-1,alpha-ol -elemene (-)Caryophyllene oxide -cardinene Cis -copane-8-ol Percentage 16.268 10.219 8.888 4.480 3.669 3.401 3.241 2.901 2.614 2.400 2.397 2.164 1.327 1.325 1.210 1.042

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International Journal of Research in Pharmaceutical and Biomedical Sciences

ISSN: 2229-3701

Table 2: Minimal Inhibitory Concentration of essential oil from Cyperus rotundus Linn. active against some bacterial cultures
S. No. Test Organism Concentration showing inhibition zone diameter(mm) 100% 50% 25% 12.5% 6.25% Chloramphenicol 30g/disc 1 2 3 4 5 Bacillus subtilis Micrococcus luteus Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa Klebsiella sp. 20 12 18 18 15 12 16 10 10 08 30 22 30 30 20 Control Tween 20 -

12

30

Table 3: Antifungal activity of essential oil of Cyperus rotundous Linn.

S. No. Test Organism Concentration showing inhibition zone diameter(mm) 100% 50% 25% 12.5% 6.25% Nystatin 100g/disc 1 2 3 4 5 Canida albicans Candida parapsilosis Aspergillus flavus Aspergillus fumigatus Fusarium oxysporum 12 20 * 18 * 08 * 13 * * 08 * 22 14 15 20 20 Control Tween 20 -

(-) = No inhibition of fungal growth (*) = Inhibition of spore formation

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International Journal of Research in Pharmaceutical and Biomedical Sciences

ISSN: 2229-3701

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