PRELIMINARY PHYTOCHEMICAL SCREENING AND GC-MS ANALYSIS OF METHANOLIC LEAF EXTRACT OF DRYPETES SEPIARIA (WIGHT & ARN.) PAX. & HOFFIM. FROM SILAMBUR SACRED GROVE, TAMILNADU

Drypetes sepiaria (Wight & Arn.) Pax. & Hoffim a medium sized tree member of Euphorbiaceae was investigated to determine the phytochemical constituents present in various extracts of the leaves through GC-MS analysis. Powdered leaf plant materials were subjected to successive extraction with organic solvents such as methanol by Soxhlet extraction method. In the present study, GC-MS analysis revealed that a total of 23 different compounds identified by using methanol extract and all the identified compounds were medicinally valuable for the treatment of various human ailments. In addition, all the phytochemical compounds were needed for further investigations on toxicological aspects for the development of new lead of therapeutic interest.


INTRODUCTION
Plants have been a rich source for drug discovery (Mishra and Tiwari, 2011). Plants and plant parts have been provide a good source of pharmaceutical active compounds, such as phenolic compounds, nitrogen compounds, vitamins, terpenoids, saponin and some other secondary metabolites, which are rich in valuable bioactivities of antioxidant, anti-inflammatory, antitumor, antimutagenic, anti-carcinogenic (Maridass et al., 2008). The genus Drypetes (Putranjivaceae (Euphorbiaceae) comprises nearly 160 species which has been used in the folk medicine of many cultures for many years (Nganga et al., 2008). Even though the species was different, they used to treat similar disorders. Among the members of the genus Drypetes earlier phytochemical studies on some species including D. parrifolia, D. laciniata, D. inaequalis, D. armoracia, D. gossweileri, D. molunduana, D. roxburghii have yielded flavonoids, chalcone glycosides, saponins, tripterpenoids, phenolics, alkaloids, etc.
Drypetes sepiaria (Wight & Arn.) Pax. & Hoffim. an ever green tree locally known as Kalvirai (Tamil) commonly grown in foothills and shrub jungles and some sacred groves of Tamil Nadu. Drypetes sepiaria is traditionally used to treat pain and inflammation and seeds are used as a wild edible food and their root paste can be used as an antidote for scorpion bite. Decoction of leaves and seeds is also noted for reducing rheumatic inflammation (Arinathan et al., 2007;Bharath Kumar and Suryanarayana, 2011). As per earlier literature, there is no scientific investigations found in D. sepiaria on phytoconstituents present. In ethnomedicinal point of view as described above, the GC-MS analysis was carried out with methanolic leaf extract of D. sepiaria to investigate the chemical constituents present in it.

Collection of plant materials and preparation of the extract
The fresh leaves of D. sepiaria was collected from the sacred grove of Silambur (Lat, 11.35 °N; Long, 79.31°E), Ariyalur District, Tamil Nadu, India. The specimen was botanically identified and confirmed by Rapinat Herbarium, St. Joseph's College, Tiruchirappalli. The preserved plant specimens were submitted to the Department of Botany, Annamalai University, Annamalainagar, Tamil Nadu for further reference. The leaves were chopped into small pieces, shade-dried and coarsely powdered by using a pulverizor. The powdered leaf were then subjected to successive extraction with organic solvents such as hexane chloroform and ethanol by Soxhlet method (Catherine et al., 1997). The extracts were then collected and distilled off on a water bath at atmospheric pressure and the last trace of the solvents was removed and stored at 4˚C. They were used for GC-MS analysis.

Preparation of extract
The powdered leaf of D. sepiaria (500 g) was extracted with methanol (95%) and double distilled water separately in a soxhlet extractor. The extract was evaporated to dryness at 60°C under reduced pressure in a rotary evaporator and kept in refrigerator at 4°C till used. The extracts were dissolved in dimethylsulphoxide to make the final concentrations at the time study.

Preliminary phytochemical screening
A small portion of the dry extracts were subjected to preliminary phytochemical screening to detect the presence of various phytoconstituents present in the leaves of D. sepiaria (Harborne, 1973;Evans, 2003).

Gas chromatography-mass spectrometry (GC-MS) analysis
GC-MS analysis was performed with GC-MS Clarus 500 Perkin Elmer Equipment. Compounds were separated on Elite-5 capillary column

RESULT AND DISCUSSION
Preliminary phytochemical analysis showed (Table 34) the presence of alkaloids, flavonoids, saponins, and phenols showed in petroleum ether leaf extract and presence of flavonoids, saponins, phenols, steroids in methanolic solvent. Steroids, flavonoids, saponins and phenol Hexane leaf extract showed terpenoids and glycosides only. In chloroform leaf extract showed steroids, tannins, and saponins only.  (Manjamalai et al., 2010).

Identification of compounds
All the compounds were identified from methanol extracts based on direct comparison of the retention times and their mass spectra with the spectra of known compounds stored in the spectral database, NIST (Version year 2005).
Phenolic compounds have antimicrobial properties. Phenol and phenolic compounds have been extensively used in disinfections. Thus the reported antimicrobial properties of both plants may be attributed to phenolic compounds. Plants with tannins are used for healing of wounds, varicose ulcers and burns (Nafiu et al., 2011). Among the identified phytochemicals, n-hexadecanoic acid has the property of antioxidant activity and it justifies with the earlier work in Alstonea venenata (Sutha, 2012).

CONCLUSION
The present investigation through the present study revealed that the species D. sepiaria is a reliable source of bioactive compounds like fatty acid esters, alcohols, hydrocarbons, alkanes, amines, terpenes, and sugars that justify the traditional usage of this species by the local healers in Tamil Nadu, India, for various ailments. As GC-MS is the first step towards understanding the nature of active principles. Further investigation in this species is suggested for the development of novel drugs.