PHYTOCHEMICAL ANALYSIS OF LEPIDIUM SATIVUM USING UV-VIS AND GC-MS

Jency Malar .M.S 1 , J. Shifa Vanmathi 2 and K.Chairman 3 . 1. Research scholar, Department of Zoology, Manonmaniam Sundaranar University, Abishekapatti. 2. Guest faculty, PG and Research Department of Zoology, St.John’s College, Palayamkottai, Tirunelveli.Manonmaniam Sundaranar University, Abishekapatti.. 3. Guest faculty, UG and PG Department of Microbiology, Kamarajar Govt. Arts College, Surandai, Manonmaniam Sundaranar University, Abishekapatti. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

The present study was carried out to characterize the bioactive constituents present in seed and whole plant extracts of Lepidium sativum using UV-VIS and GC-MS. The crude extracts were scanned in the wavelength ranging from 200 to 800 nm by using Perkin Elmer spectrophotometer and the characteristic peaks were detected. For GC-MS analysisabout 25g of powdered plant material was uniformly packed into a thimble and extracted with 150ml of ethanol as solvent using this plant extract was prepared. Helium gas (99.999%) was used as the carrier gas at constant flow rate 1ml/min and an injection volume of 2μl was employed (split ratio of 10:1); Injector temperature 80°C; Ion-source temperature 250°C. The oven temperature was programmed from 110°C (isothermal for 2 min.), with an increase of 10°C/min, to 200°C, then 5°C/min to 250°C, ending with a 9min isothermal at 280°C. Mass spectra were taken at 70 eV; a scan interval of 0.5seconds and fragments from 45 to 450 Da. The UV-VIS profile showed different peaks ranging from 280 and 290 nm with absorbance values of 0.26 and 3.98 respectively.The spectra for phenolic compounds (tannins) and flavonoids typically lie in the range of 230-290 nm.The results of the GC-MS analysis provide different peaks determining the presence of 28 phytochemical compounds in seed extract and the major phyto constituents were (Peak area 16.23%),o-ethyl S-2-Dimethylaminoethyl Ethylphos, (14.37%)Oleoyl chloride and (12.50%) cis-9-Hexadecenal (8.97%). Phytochemical compoundspresent in whole plant extract was 79 and the major phyto constituents were Eugenol (7.69 % ); Hexadecanoic Acid, Ethyl Ester (7.50%) and Stigmast-5-EN-3-OL, (3.BETA.)-(7.14 %) were reported by GC-MS analysis. The results revealed the major compounds are fatty acid esters and alkaloids which showed antioxidant, antimicrobial and anticancer activities.

ISSN: 2320-5407
Int. J. Adv. Res. 6(9), 813-825 814 Introduction:-Herbal medicines are in great demand in both developed and the developing countries in primary healthcare because of their great efficacy and little or no side effects. In India, the indigenous system of medicine namely Ayurvedic, Siddha and Unani have been in existence for several centuries. These traditional systems of medicine together with homoeopathy and folklore medicine continue to play a significant role largely in the health care system of the population (Yadavet al., 2011,). The tribals and rural population of India are highly dependent on medicinal plant therapy for meeting their health care needs. This attracted the attention of several botanist and plant scientists of several medicinal plants and there was a spurt of scientific literature. (Cerutti, 1991).
Plantsare the best sources for chemical ingredients or phytochemical agents for cure of different disease. Medicinal plants are an inexhaustible source of molecules with very different biological and pharmacological activities(KshitijChauhanet al, 2012).Lepidiumsativum Linn (Brassicaceae) commonly known as Asaliyo, is an erect, glabrous annual herb cultivated as a salad plant throughout India, Europe and United States. The seeds are used in chronic enlargement of liver and spleen, as carminative adjunct to purgatives, in skin diseases, dysentery, diarrhoea, asthma and in liver complaints (shuklaet al., 2015).
Lepidiumsativum, Family Brassicaceae, is a fast-growing, edible plant botanically related to watercress and mustard and known to share their peppery, tangy flavour and aroma (Prajapatiet al., 2014). In some regions, garden cress is known as garden pepper cress, pepper grass or pepperwort. Cress is one of the easiest vegetables to grow as it can grow just about anywhere. The plant isused as an antiasthmatic; anti-scorbutic; aperient; diuretic; galactogogue ; poultice and stimulant (Cassidy, 2002).
The total glucosinolates of the seeds of Lepidium sativum revealed the presence of two glucosinolates, glucotropaeolin and gluconasturin. On the other hand, four glucosinolates were isolated from the fresh herb and were identified as 2ethyl butyl glucosinolate, methyl glucosinolate (glucocapparin), butylglucosinolate in addition to the glucotropaeolin which was isolated also from the seeds. The glucosinolates were identified by (UV, MS). The individual corresponding isothiocyanates (aglucone) which was obtained by enzymatic hydrolysis of the individual glucosinolates were identified using GC / MS technique (Radwanet al., 2007).

Collection of plant materials
Fresh materials of Lepidium sativum was grown in the laboratory of Sri Paramakalyani College, Alwarkurichi, Tamilnadu, India. The plant was dried in shade and was pulverized using mortor and pestle separately and stored in a closed vessel for further use.

Preparation of plant extracts -Solvent extraction
Crude plant extract was prepared by Soxhlet extraction method. About 25g of powdered plant material was uniformly packed into a thimble and extracted with 150ml of ethanol as solvent. The process of extraction was continued for 24htill the solvent in siphon tube of an extractor become colourless. After that the extract was taken in a beaker and kept on hot plate and heated at 30-40ºC till the solvent got evaporated. Dried extract was kept in refrigerator at 4ºC for their future use in phytochemical analysis (Martins et al., 2001).

Ultraviolet-visible spectroscopy analysis:
The extracts of L.sativum were examined under visible and UV light for proximate analysis. For UV-VIS spectrophotometer analysis, the extracts were centrifuged at 3000 rpm for 10 min and filtered through Whatmann No. 1filter paper by using high pressure vacuum pump. The sample is diluted to 1:10 with the ethanol. The extracts were scanned in the wavelength ranging from 200-800 nm using Spectrophotometer and the characteristic peaks were detected. The peak values of the UV-VIS were recorded. Each and every analysis was repeated twice for the spectrum confirmation (AH and Aysel 2003).

Gas Chromatography Mass Spectrum (GC-MS) Analysis:
GC-MS analysis of the extract was performed using a Thermo GC -Trace ultra Ver: 5.0 system and Gas chromatograph interfaced to a Mass spectrometer (GC-MS)(Perkin-Elmer GC Clarus 500 system) equipped with TR 5 -MS capillary standard non-polar column (30mmX0.25mm 1D X 1 μMdf). For GC-MS detection, an electron ionization system with ionizing energy of 70 eV was used. Helium gas (99.999%) was used as the carrier gas at 815 constant flow rate 1ml/min and an injection volume of 2μl was employed (split ratio of 10:1); Injector temperature 80°C; Ion-source temperature 250°C. The oven temperature was programmed from 110°C (isothermal for 2 min.), with an increase of 10°C/min, to 200°C, then 5°C/min to 250°C, ending with a 9min isothermal at 280°C. Mass spectra were taken at 70 eV; a scan interval of 0.5seconds and fragments from 45 to 450 Da. Relative quantities of the chemical compounds present in each of the extracts of L. sativumwas expressed as percentage based on peak area produced in the chromatogram. (  The phenolic contents of methanolic extract of Lepidium sativum was determined by UV spectrophotometric method. The total contentof phenolic compounds were found to be 46.0 mg GAE/100 g in methanolic extract of Lepidium sativum. The flavonoids content was determined by UV spectrophotometric method. The total content of flavonoids was found to be 4.28 mg QE/100 g in methanolic extract of Lepidium sativumrespectively (RizwanAhamadet al., 2015).
In recent years GC-MS studies have been increasingly applied for the analysis of medicinal plants as this technique has proved to be a valuable method for the analysis of non-polar components and volatile essential oil, fatty acids, lipids and alkaloids.
The GC-MS chromatogram shows the peak area separation of the components. The above mentioned isolated compounds from the ethanol extract of Lepidium sativium seem to possess the reported biological activity and further study of these phytoconstituents may prove the medicinal importance in future.