International Journal of Current Biotechnology

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1 Vijisaral Elezabeth. D and Subramanian Arumugam, GC MS Analysis of Ethanol Extract of Cyperus rotundus Leaves Int.J.Curr.Biotechnol., 2014, 2(1): International Journal of Current Biotechnology ISSN: Journal Homepage : GC MS Analysis of Ethanol Extract of Cyperus rotundus Leaves Vijisaral Elezabeth. D and Subramanian Arumugam* PG & Research Department of Chemistry, Nehru Memorial College, Puthanampatti , Tiruchirappalli, Tamilnadu, India. A R T I C L E I N F O A B S T R A C T Article History: Received 10 January 2014 Received in revised form 15 January 2014 Accepted 22 January 2014 Available online 25 January 2014 Key words: Cyperus rotundus, Bio-active components, GC-MS, Hexadecanoic acid. Introduction In recent years the use of plants in the management and treatment of diseases has gained considerable importance. Plant and fruits are considered as one of the main source of biologically active compounds. The plants of the genus Cyperus are used in traditional Ayurvedic medicine in Chinese and Native American as astringent, diaphoretic, diuretic, analgesic, antispasmodic, aromatic, carminative, antitussive, emmenagogue, litholytic, sedative, stimulant, stomachic, vermifuge, tonic and antibacterial. It is also useful for dietary management of psychotic diseases and metabolic disorders (Anonymous, 1950). C.rotundus L., (Family-Cyperaceae), also known as purple nutsedge or nutgrass, is a common perennial weed with slender, scaly creeping rhizomes, bulbous at the base and arising singly from the tubers which are about 1-3 cm long. The tubers are externally blackish in colour and reddish white inside, with a characteristic odour. The stems grow to about 25 cm tall and the leaves are linear, dark green and grooved on the upper surface. Inflorescences are small, with 2-4 bracts, consisting of tiny flowers with a red-brown husk. The nut is three-angled, oblong-ovate, yellow in colour and black when ripe. C.rotundus is indigenous to India, but *Corresponding author. address: nmcaaru@gmail.com. Mobile no: The investigation was carried out to determine the possible bioactive components of leaves of Cyperus rotundus using GC-MS analysis. The chemical compositions of the ethanol extract of leaves of Cyperus rotundus was investigated using perkin-elmer Gas chromatography- Mass Spectrometry, while the mass spectra of the compounds found in the extract was matched with the National Institute of Standard and Technology (NIST) library. Thirty four compounds were identified; n-hexadecanoic acid (21.99%) was found to be major components followed by 9, 12, 15-Octadecatrienoic acid, ethyl ester (Z, Z, Z) (11.78%), Hexadecanoic acid, ethyl ester (10.12%), Ethanone, 1-(2-hydroxy-5-methylphenyl) (9.9%), 3, 7, 11, 15-Tetramethyl-2- hexadecan-1-ol (6.73%), Phytol (5.53%), 4H-Pyron-4-one, 2, 3-dihydro- 3, 5-dihyroxy-6-methyl (3.80%), Caryophyllene oxide (2.51%) and D-Allose (2.40%). is now found in tropical, subtropical and temperate regions (Uddin et al., 2006). The ethanolic extract of C. rotundus in the dose of 128.1± 11.6 mg/kg was found to protect 50% dogs against apomorphine induced vomiting and it is also produced relaxation of rabbit ileum and spasmolytic effect against contractions induced by acetylcholine, barium chloride and 5-hydroxitriptamine, showing a direct relaxant action on the smooth muscle (Sing et al., 1970). The present investigation was carried out to determine the possible bioactive chemical components from Cyperus rotundus leaves by GC-MS. Materials and Methods Collection of Plant Material The leaves of Cyperus rotundus were collected from Kumarai Village in Cuddalore District of Tamilnadu, India during January to December 2012 and authenticated by the Director of the Rapinat Herbarium and Centre for Molecular Systematics, St.Joseph s college (campus), Trichirappalli, Tamilnadu, India. Fresh leaves were cleaned with running tap water and dried under the shade. Then the dried plant leaves were ground to fine powder mechanically and preserved in airtight containers for further analysis. GC-MS Analysis The GC-MS analysis of leaves was carried on extract prepared by successive extraction method in Soxhlet. The previously dried powdered leaves (100g) were extracted 19 Int.J.Curr.Biotechnol. Volume 2; Issue 1; Jan, 2014

2 Table 1: Components detected in the plant of ethanol extract of Cyperus rotundus Leaves Molecular %Peak S.No Compound Name MW RT Peak Area Formula Area 1. 3-Methyl-3-butenoic acid C5H8O Cyclopenten-1-one, 2-hydroxy- C5H6O ,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one C6H8O l-pantoyl lactone C 6 H 10 O Hydrouracil, 1-methyl- C5H8N2O H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- C6H8O Benzofuran, 2-methyl- C9H8O Ethanone, 1-(2-hydroxy-5-methylphenyl)- C9H10O Phenol, 2,6-dimethoxy- C8H10O Caryophyllene C 15 H {2-[3-Methyl-3-(5-methyl-furan-2-yl)-butyl]-oxiran-2- yl}-ethanone C14H20O Caryophyllene C15H (2,6,6-Trimethylcyclohexa-1,3-dienyl)but-3-en-2-one C13H18O D.Allose C6H12O Ethanone, 1-(3,4-dimethoxyphenyl)- C10H12O Caryophyllene oxide C15H24O Oxabicyclo[9.1.0]dodeca-3,7-diene, 1,5,5,8- tetramethyl-, [1R-(1R*,3E,7E,11R*)]- C 15 H 24 O Cycloocta-1,3,6-triene, 2,3,5,5,8,8-hexamethyl- C14H ,10-Dimethyl-2,6-dimethylenebicyclo[7.2.0]undecan- 5 -ol C 15 H 24 O Kauran-18-al, 17-(acetyloxy)-, (4 )- C22H34O Isopropenyl-4,7-dimethyl-1-oxaspiro[2.5]octane C12H20O Tetradecanoic acid C14H28O Ethyl-1-thio- -d-glucopyranoside C 8 H 16 O 5 S ,7,11,15-Tetramethyl-2-hexadecen-1-ol C 20 H 40 O n-hexadecanoic acid C16H32O Hexadecanoic acid, ethyl ester C18H36O Nonadecanoic acid, ethyl ester C 21 H 42 O Phytol C20H40O ,12-Octadecadienoic acid, ethyl ester C20H36O ,12,15-Octadecatrienoic acid, ethyl ester, (Z,Z,Z)- C 20 H 34 O Octadecanoic acid C 18 H 36 O Z-2-Octadecen-1-ol C18H36O Z-9-Pentadecenol C15H30O Eicosanoic acid, ethyl ester C22H44O MW: Molecular Weight, RT: Retention Time for 6-8 hours in a Soxhlet apparatus with ethanol successively. GC-MS analysis of this extract was performed with GC clarus 500 Perkin Elmer system and Gas chromatograph interfaced to a Mass spectrometer (GC-MS) equipped with a Elite 5 fused silica capillary column (30 m x 250 µm 1D x 1 um df, composed of 5% Phenyl 95% Dimethyl siloxane). For GC-MS detection, and electron ionization system with ionizing energy of 70 ev was used. Helium gas (99.999%) was used as the carrier gas at constant flow rate 1 ml / min and an injection volume of 2 ul was employed (Split ratio of 1:10); Injector temperature C; ion-source temperature C. The oven temperature was programmed from C (isothermal for 2 min) with an increase of 6 0 C/min, to C, then 6 0 C/min to C, ending with a 10 min isothermal at C. Mass spectra were taken at 70 ev, a scan interval of 0.5 seconds and fragments from 45 to 450 Da. Total GC running time was 36 minutes. The relative % amount of each component was calculated by comparing its average peak area to the total areas, software adopted to handle mass spectra and chromatograms was a turbomass. Interpretation on mass spectrum of GC-MS was done using the database of US National Institute of Standard and Technology (NIST) having more than 65,000 patterns. The mass spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library. The name, molecular weight and structure of the components of the test materials were ascertained. Results The components present in the ethanol extract of plant of Cyperus rotundus leaves were identified by GC-MS analysis (Figure 1). The active principles with their retention time (RT), molecular formula, molecular weight (MW), peak area and concentration of peak area (%) in the ethanol extract of plant of Cyperus rotundus leaves are presented in the Table 1. Thirty four compounds were detected in ethanol extract of the plant. The results revealed that, n-hexadecanoic acid (21.99%) was found Volume 2; Issue 1; Jan, 2014 Int.J.Curr.Biotechnol. 20

3 Table 2: Activity of Phyto-Components identified in the ethanol extracts of the plant of Cyperus rotundus Leaves S. No Name of the compound Molecular formula Nature of compound 1. Phytol C20H40O Diterpene **Activity Antimicrobial, Anticancer, Dieruretic Methyl-3-butenoic acid C5H8O2 Butenoic acid Antibaterial 3. Z-9-Pentadecenol C15H30O Alcoholic Antimicrobial 4. 9,12-Octadecadienoic acid, ethyl ester C 20 H 36 O 2 Linolenic acid Ester Anti androgenic, Hypocholesteolemic, Nematicide, 5-Alpha reductase inhibitor, Antiacen, Antiandrogenic, Cancer preventive, Dermatitigenic. 5. Octadecanoic acid C18H36O2 Stearic acid No activity reported. Insectifuge, Cancer preventive, 6. 9,12,15-Octadecatrienoic Nematicide, Hypatoprotective, C20H34O2 Linolenic acid acid, ethyl ester, (Z,Z,Z)- Antihistaminic, Antiandrogenic, Antieczemic, Antiacen, 5-Alpha reductase inhibitor, Antiarthritic, Anticoronary. 7. n-hexadecanoic acid C16H32O2 Palmitric acid Antioxidant, Nematicide, Pesticide, Lubricant, Antiandrogenic, Hemolytic, 5-Alpha reductase inhibitor. 8. 3,7,11,15-Tetramethyl-2- hexadecen-1-ol C20H40O Terpene alcohol Antimicrobial Antioxidant, 9. Hexadecanoic acid, ethyl Nematicide, Pesticide, ester C18H36O2 Ester compound Lubricant, Antiandrogenic, Hemolytic, 5-Alpha reductase inhibitor Caryophyllene C15H24 Sesquiterpene Anti-inflammatory actvity 11. Caryophyllene oxide C15H24O Sesquiterpene Antitumor, Anesthetic, Antibacterial, Analagesic, Antioxidant H-Pyran-4-one, 2,3- dihydro-3,5-dihydroxy-6- methyl C6H8O4 Flavanoid Antimicrobial, Antiproliferative. 13. Phenol, 2,6-dimethoxy- C8H10O3 phenol Antifungal 14. Benzofuran, 2-methyl- C 9 H 8 O Coumaran Antihelminthic, Antidiarrhoeal. 21 Int.J.Curr.Biotechnol. Volume 2; Issue 1; Jan, 2014

4 to be major components followed by 9, 12, 15- Octadecatrienoic acid, ethyl ester (Z, Z, Z) (11.78%), Hexadecanoic acid, ethyl ester (10.12%), Ethanone, 1-(2- hydroxy-5-methylphenyl) (9.9%), 3, 7, 11, 15-Teramethyl- 2-hexadecan-1-ol (6.73%), Phytol (5.53%), 4H-Pyron-4- one, 2, 3-dihydro-3, 5-dihyroxy-6-methyl (3.80%), Caryophyllene oxide (2.51%) and D-Allose (2.40%). Table 2 lists the major phytocomponents and their biological activities obtained through the GC MS study Cyperus rotundus leaves. Discussion In the present study, the GC-MS analysis of the ethanol extract of Cyperus rotundus leaves showed the presence of fourteen bioactive compounds were represented in Table 2. In terms of percentage amounts n-hexadecanoic acid was predominant in the extract. This compound have all shown to have hypochloesterolemic activity, antioxidant and lubricant activity (Praveen Kumar et al., 2010). Plant products including phenols and flavonoids in the plant extracts have been reported to be radical scavengers and inhibitors of Lipid Peroxidation (Xie et al., 1993 and Formica et al., 1995). The antioxidant properties of phenolic acids and flavonoids are due to their redox properties, ability to chelate metals and quenching of singlet oxygen (Rice-Evans et al., 1996). Terpenoids are an important part of volatiles from plants. Caryophyllene oxide, an oxygenated terpenoid, well known as preservative in food, drugs and cosmetics, has been tested in vitro for antibacterial, antifungal activity (Guillen et al., 1996 and Yang et al., 1999.). It is also suggested as potential anticarcinogenic agent exhibit cytotoxic activity against several solid tumor cell lines (Kubo et al., 1996). The caryophyllene oxide in medicinal plants is reported to be potentially useful for diuretic activity (Ratnasooriya et al., 2004). The results show that, reactive oxygen species promoting substances such as phytol constitute a promising novel class of pharmaceuticals for the treatment of rheumatoid arthritis and possibly other chronic inflammatory diseases (Ogunlesi et al., 2009). 9-12, Octadecadienoic acid (Z, Z) have the property of anti-inflammatory and anti-arthritic as reported by the earlier worker (Lalitha Rani et al., 2009). á-caryophyllene is a sesquiterpene that has antiinflammatory activity(rigo, Fernanda, et al., 2007). The presence of various above bioactive compounds justifies the use of the plant leaves for various ailments by traditional practitioners. Acknowledgement The authors would wish to acknowledge the management and Principal of Nehru Memorial College for providing research facilities and encouragements. References Formica JV, Regelson W, Review of the biology of Quercetin and relatd bioflavonoids. Food Chem Toxicol, 33, 1995, Guillen, MD., Cabo, N., Burillo S. Journal of Sci Food Agric., 1996, 70, 359. Yang, D., Michel, L., Chaumont, J and Millet-Clerc, J. Mycopathologia., 1999, 148, 79D82. Lalitha Rani, S., Mohan, V.R., Regini, G.S. and Kalidass, C. GC-MS analysis of ethanolic extract of Pothos scandens leaf. J. Herb. Medi. Toxicology. 2009, 3, Ogunlesi M, Okiei W and Osibote AE, Analysis of the essential oil from the dried leaves of Euphorbia hirta Linn. (Euphorbiaceae), a potential medication for asthma. African.J. Biotech. 8: Praveen Kumar P, Kumaravel S, Lalitha C, Screening of antioxidant activity, total phenolics and GC-MS study of Vitex negundo, African Journal of Biochemistry Research. 2010; 4(7): Ratnasooriya, WD., Pieris, KP., Samaratunga, U and Jayakody, JR. Ethnopharmacol Journal., 2004, 91(2-3), Rice-Evans CA, Miller Nj, Paganga G, Structureantioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine, 20, 1996, Rigo, Fernanda, D.C., Juliano, F., Maria, C., Luiz, P. and Jobo, B.C. Anti-inflammatory effects of compounds alpha-humulene and (-) trans-caryophyllene isolated from Fig 1: GC-MS Chromatogram of the ethanol extracts of plant of Cyperus rotundus Leaves Volume 2; Issue 1; Jan, 2014 Int.J.Curr.Biotechnol. 22

5 the essential oil of Cordia Verbenacea. Euro. J.Pharma. 2007, 569, Singh N, Kulshrestha VK, Gupta MB and Bhargava K P. A pharmacological study of Cyperus rotundus, Indian J Med, Res, 1970,58, Uddin SJ, Mondal K, Shilpi JA, Rahnan MT. Antidiarrhoeal activity of Cyperus rotundus. Fitoterapia 2006; 77 (2): Xie B, Shi H, Chen Q, Ho CT, Antioxidant properties of fractions and polyphenol constituents from green, long and black teas. Life Sci, 17, 1993, Int.J.Curr.Biotechnol. Volume 2; Issue 1; Jan, 2014