Bioactivity guided fractionation of whole plant of medicago sativa with isolation and analysis of phytoconstituents therefrom

Abstract

quotBackground: Medicago sativa Linn. (Fabaceae) is used to treat cardiovascular diseases, cancer, diabetes and menopausal symptoms in women. Hypertension is one of major killer worldwide and ACE inhibitors play an important role in treatment of hypertension. Many antihypertensive synthetic drugs are available in the market but have certain side effects. There is a need of safe, effective and economical alternative of synthetic drugs. newlineAim: The purpose of present study was to investigate antihypertensive activity of M. sativa and to identify and isolate bioactive phytoconstituent(s). newlineMaterials and Methods: Leaf, root and stem extracts were prepared by successive extraction using solvents of different polarities. Extracts were subjected to ACE-inhibitory activity by direct spectrophotometric assay. Extracts were also subjected to antioxidant evaluation by DPPH free radical scavenging model. A mobile phase was developed by TLC for most bioactive extract and its HPTLC fingerprinting was done. Isolation and characterization of fractions from methanol extract of M. sativa was done using flash chromatography and GC-MS, respectively. Isolation of Lupeol was performed using column chromatography. newlineResults and Discussion: Among all other extracts, methanol extract of M. sativa root showed the maximum and dose dependent in vitro ACE inhibitory activity with IC50 value of 34.43±0.31µg/ml concentration. Antioxidant activity of methanol extracts of root, leaf and stem, revealed with IC50 value 31.86and#956;g/ml, 17.23and#956;g/ml and 13.67and#956;g/ml. Methanol extract of M. sativa root showed distinct spots in TLC system using mobile phase Chloroform: Methanol: Ethyl acetate (1:5:5). HPTLC fingerprinting of this extract showed 8 peaks at 254nm and 6 peaks at 366nm. Further, 30 different fractions were isolated by flash chromatography. GC-MS of fractions revealed the presence of total 28 phytoconstituents in that extract. Two of those phytoconstituents, Didodecyl phthalate and Lupeol, possess therapeutic potential in cardiovascular disease which can be c