A method of isoflavones isolation from red clover as standards for analyses

Abstract

Five compounds having an isoflavone structure were isolated from the tops of red clover (Trifolium pratense). On the basis of spectral (UV, MS) and chromatographic (TLC, HPLC) analyses the compounds were identified as biochanin A, formononetin, pratensein, genistein and daidzein. Biochanin A and formononetin - two main clover estrogens - were obtained in crystalline forms in the amounts of 50 mg and 15 mg, respectively (per 250 g of D. W.). Homogenous fractions of pratensein, genistein, and daidzein were also obtained. The obtained standards were used as markers for peak identification in HPLC. The presented method of isoflavone isolation is simple and quick. It allows for the simple isolation of a mixture of crude isoflavone aglycones which may be used in qualitative and semi-quantitative (TLC) analyses. The individual standards are obtained through column chromatography and may be used for quantitative analyses.