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Kundan Singh Bora*, Anupam Sharma


Background: Medicago sativa Linn. (Family - Leguminosae) has been used traditionally for the treatment of a variety of CNS disorders. Our previous work inferred that methanol extract of M. sativa aerial parts exhibited significant anti-anxiety activity on elevated plus-maze (EPM) model of anxiety. Objective: The current study has been designed to evaluate the anxiolytic activity of different fractions of bioactive methanol extract of the aerial parts of M. sativa. An attempt has been made to isolate the bioactive (anxiolytic) principle by resorting to bioactivity directed fractionation and column chromatographic technique. Materials and Methods: The bioactive methanol extract was fractionated by solvent partitioning, and each fraction was evaluated for the anxiolytic activity using the EPM model in mice. Further, bioactive fraction was subjected to column chromatography, and evaluated for the anxiolytic activity. Finally, the anxiolytic activity of isolated compound was further confirmed using light/dark test and hole-board test model of anxiety, and characterization and quantification of isolated compound was done by spectroscopic techniques and TLC densitometric method respectively. Results: Anti-anxiety activity guided fractionation of sub-fraction F4 of ethyl acetate fraction of methanol extract led to isolation of 4',5,7-trihydroxyflavone (apigenin) which showed significant (p<0.05) anxiolytic activity by increasing the average time spent, and number of entries in open arms in EPM model of anxiety at a dose of 2 mg/kg, p.o., in mice with respect to the standard drug diazepam. Further, the anti-anxiety activity of isolated compound was confirmed using light/dark test and hole-board test. Conclusions: On the basis of findings of the present study, it is concluded that the isolated compound - apigenin is responsible for anxiolytic activity of M. sativa.

Keywords: Medicago sativa; Anxiolytic activity; Apigenin; Elevated plus-maze.

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