Study on the Allelopathy of Alfalfa (Medicago sativa L.): II. Isolation and identification of allelopathic substances in alfalfa

Allelopathic substances in alfalfa plants (Medicago sativa L.) were purified from methanol extracts of alfalfa shoots. The methanol extracts were fractionated into an acidic fraction, and the sample was divided into 10 fractions by thinlayer chromatography (TLC). Growth of radish seedlings was inhibited by the substances obtained from Rf values of 0.5–0.6 and 0.6–0.7. These fractions were eluted with distilled water, and the eluates were analyzed by high-performance liquid chromatography (HPLC). Six peaks were detected on the chromatogram. Growth of radish seedlings was inhibited on three peaks. Of these peaks, two were identified using HPLC, a mass-spectrometer, IR, and ¹NMR as follows: peak b, ferulic acid and peak f, salicylic acid. The growth of alfalfa and radish seedlings was inhibited at lower concentrations of these substances.