The present study aimed towards assessing the isolated lupeol derivative <em>in vitro</em> for testosterone biogenesis enhancement by the action on isolated rat leydig cells. Studies were carried out to separate a novel bioactive molecule from an Indian medicinal plant, <em>Hygrophila spinosa</em>, used as an aphrodisiac and spermatogenic for years. Unsaponifiable bioactive fraction, prepared from seeds of the plant, was subjected to isolation of phytoconstituents by column and flash chromatography. The isolated compound was characterized by spectral studies as lupeol methyl ether. Later, it was subjected to <em>in vitro</em> studies using isolated leydig cells from rat for its action. Different amounts of isolated compound were incubated with leydig cells for 3 hours and concentration of testosterone after incubation was considered as a parameter. Dehydroepiandrosterone was used as positive control in the studies. The results indicated that the isolated compound was active and enhanced the testosterone biogenesis, as evident from increased amount of testosterone found in a medium treated with compound. This was the first report included isolation, chemical characterization and biological activity of Lupeol methyl ether from <em>Hygrophila spinosa</em> T. Ander.
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