Characterisation of red clover (Trifolium pratense L.) isoflavones under different extraction conditions

Red clover (Trifolium pratense L.) is a widely used forage crop rich in isoflavones, bioactive compounds with diverse healthprotective effects. In legumes, isoflavones occur in various chemical forms, but their health benefits are most pronounced in their free forms—aglycones. Although different extraction parametres (temperature, pH, sample-tosolvent ratio) can influence the efficiency of isoflavone extraction, this study specifically examined the effect of different HCl concentrations (2M, 4M, and 6M), with and without evaporation of the extract to dryness, on the hydrolysis of glycoside isoflavones into aglycones. The leaves and the whole plant of red clover were used as samples. High- Performance Liquid Chromatography (HPLC) was used for the identification and quantification of the isoflavones. The results revealed significant variations in isoflavone content among different plant parts. Red clover leaves had the highest total isoflavone content, with formononetin (3.739 mg/g DM) and biochanin A (2.484 mg/g DM) as the dominant compounds, whereas whole-plant extracts contained a higher content of daidzein (0.440 mg/g DM) and genistein (0.170 mg/g DM) compared to the leaves. Regarding the extraction method, the highest total isoflavone concentration (6.556 mg/g DM) was obtained from leaf extracts that were evaporated to dryness after hydrolysis with 2M HCl.