Autotoxic compounds from fresh alfalfa leaf extracts: Identification and biological activity

Many investigators have attempted to identify the allelochemicals in alfalf a (Medicago sativa), that cause autotoxicity. The autotoxic compounds from fresh alfalfa leaves were separated and quantified, and their biological ac tivity was determined. Chemical separation procedures involved an 80% metha nol extract of fresh alfalfa leaves, treatment with activated charcoal, mic rocrystalline cellulose thin-layer chromatography (MCTLC), and finally sepa ration by Sephadex LH-20 column chromatography. The various fractions were examined further by high-performance liquid chromatography (HPLC). Prelimin ary identification by HPLC analysis resulted in peaks with retention times close to those of chlorogenic (m/z = 354) and salicylic acid (m/z = 138) st andards, and these compounds were confirmed with CC-MS. Several other peaks remain unidentified. Chlorogenic acid occurs in relatively large amounts ( 0.39 mg/g) in alfalfa aqueous extracts as compared to salicylic acid (0.03 mg/g), and bioassays suggest that chlorogenic acid is involved in alfalfa a utotoxicity.