Determination of isoflavones in the aerial part of red clover by HPLC-diode array detection

Red clover (Trifolium pratense L.) is a biennial plant which has been used as feeding material for ruminants, but also as a health food for humans due to its estrogenic, antispasmodic, and expectorant properties. Red clover contains a large number of flavones, the four most important being daidzein and genistein and their precursors formononetin and biochanin A, respectively. The purpose of the current project was to quantify these four isoflavones in the aerial part of red clover in samples collected at the flowering, vegetative, and fruiting stages, and accordingly, to determine which of the three growth stages of red clover, contains the highest isoflavone amount. Thus a method based on reversed-phase high performance liquid chromatography (HPLC) using diode array detection has been developed and validated. The linearity, precision, accuracy, and sensitivity of the method allow for the fast and reliable determination of the aforementioned substances from the aerial part of red clover. Analysis of the plant at different growth stages showed that the highest amount of isoflavones was detected during the vegetative stage.