Isolation and Characterization of Bio- Actives from Roots of Hemidesmus indicus with Special Reference to Anti-Oxidant Activity

Abstract

Hemidesmus indicus, Asclepiadaceae is a well known and a potent herb used in the traditional system of medicine. Its medicinal properties have reported since ancient times. It is a very rich source of terpenoids, flavanoid, phenolic, tannins, etc. Literature shows that the plant has very good antioxidant potential; also it shows antimicrobial, anticancer, anti diabetic and many more pharmacological activities. There are many constituents which are reported from Hemidesmus indicus, but still further detailed investigations are required for the complete identification of the chemical profile of the plant with special reference to its antioxidant and hepatoprotective activity. So, this research work focuses on isolation of phytoconstituents, more specifically targeted phenolics from the bioactive fraction of methanolic extract of this plant. Identification of possible bioactive compounds by various spectroscopic methods like IR, Mass, and Nuclear Magnetic Resonance spectroscopy and to check for its antioxidant potential formed a major basis of the present study. Standardization of the plant, Hemidesmus indicus was done by morphological and microscopical study of roots and powder materials, phytochemical screening for the presence of active constituents, and evaluation of physicochemical parameter like ash values, extractive values and preliminary phytochemical screening. The extract of Hemidesmus indicus was prepared by soxhlet using petroleum ether, ethyl acetate and methanol as the solvent, this successive extraction was carried out for a period of 6 hrs. The methanolic extract was thus obtained was used for the extraction of phenolic compounds. This crude extract was then loaded in the column and the isolation was carried out by vacuum assisted column chromatography using gradient elution till single pure compounds HI001, HI003, HI004, HI005 were isolated. FTIR and MASS spectrum of HI001 was recorded for the identification and characterization and the probable structure was evaluated. Other pure compounds were isolated in very less quantity so subjected only to the FTIR study and probable structures of isolated compounds were predicted by their spectral data. The in vitro antioxidant potential of isolated compounds was evaluated. In conclusion the data suggested that compound HI001, HI004 and HI005 of plant have very good antioxidant potential when evaluated using DPPH free radical scavenging activity. IC50 value of HI001 for free radical scavenging activity using DPPH was found 6μg/ml while IC50 value of HI004 and HI005 was found to be 12.5μg/ml. Result of the entire study showed that FTIR data of the isolated compounds (HI004 and HI005) suggested the presence of phenolics hydroxyl groups and the probable structure predicted was similar to that of proanthocyanidins, indicated potential role of this compounds in the observed potent anti oxidant activity. Mass spectra and FTIR spectra of HI001 showed presence of diglycoside structure with ester linkage. This compound showed positive Liebermann, Burchardt test suggestive of presence of steroidal nucleus. Further scope of this study includes more specific structural characterization of the isolated compounds using different spectroscopic analysis and to check for the invitro and invivo hepatoprotective activity of the isolated compounds in different animal models.