Studies on Phytoestrogenic and Nonphytoestrogenic Compounds in Trifolium incarnatum L. and Other Clover Species Using Pressurized Liquid Extraction and High Performance Column Liquid Chromatography with Photodiode-Array and Fluorescence Detection

Abstract

HPLC coupled with photodiode array (PDA) and fluorescence (FL) detectors has been used for the identification and determination of phytoestrogenic (isoflavones and coumestrol) and nonphytoestrogenic (flavones) compounds in hydrolyzed and nonhydrolyzed extracts obtained from aerial parts of Trifolium incarnatum L. and related clover species. The effective isolation technique of pressurized liquid extraction (PLE) was used. Various types of extraction solvents, i.e., ethanol, n-propanol, n-butanol, and water solutions (75%, v/v) of methanol or ethanol at changeable or constant temperatures were tested, taking into account the chemical character of isolated compounds. Higher PLE efficiency in relation to isoflavone aglycones was found for ethanol. Predominant isoflavone glycosides determined in clover samples were sissotrin and ononin, reaching levels above 1.4% dry weight (wt) in T. medium. The presence of flavone compounds (apigenin and luteolin) in aerial parts of T. incarnatum, occurring in amounts exceeding 0.4% dry wt, documented chemotaxonomic distinction of this species from other clovers examined. Additionally, within the group of phytoestrogenic isoflavones, only biochanin A and formononetin derivatives were identified in above-ground parts of T. incarnatum. The application of simultaneous PDA and FL detection enabled unambiguous confirmation of the lack of a strong phytoestrogen, coumestrol, in all hydrolyzed and nonhydrolyzed clover extracts.