Quantification of isoflavones aglycones in the late harvest aerial parts of Trifolium pratense L. using different hydrolysis methods

Abstract

Background. Red clover (Trifolium pratense L.) is a perennial legume that has received considerable amount of interest as an alternative valuable source of isoflavones endowed with a variety of health protective effects. Isoflavones found in the red clover possess estrogenic and antiproliferative effects. Conjugated isoflavones like daidzin and genistin are inactive but become active in aglycone (daidzein and genistein) form, when the glucose molecule is removed from the structure. To obtain the highest amounts of aglycones, the best method of hydrolysis must be determined. Aim. The aim was to obtain the highest possible amounts of isoflavones genistein and daidzein from Trifolium pratense L. blossoms using different methods of hydrolysis. Methods. Ultrasound-assisted extraction was performed using 1.0 g of dried and milled flower heads and 30 mL of 70 % ethanol. Ultrasound power 250 W, temperature 40°C, extraction time 10- and 30-min. Alkali hydrolysis was implemented using 25% NaOH. The pH was changed to 10.5 and then the extracts were sonicated. After hydrolysis, the samples were neutralized to pH 5.7 using 25% acetic acid. Acidic hydrolysis was performed after sonication. 37% HCl was added to the whole medium ratio 1:12 (v/v) and the flask was heated under reflux condenser for 1 h. Hydrolysed extracts pH was changed to ∼2.5 by adding aqueous solution of 2 M NaOH while stirring. Thermal hydrolysis was performed after sonication placing extract under reflux for 1 h. Maceration results was used as control. Extraction samples for quantification of aglycones were investigated using HPLC. Results. Ultrasound-assisted extraction without hydrolysis yielded lower levels of aglycones than simple maceration but using alkaline and thermal hydrolysis larger amounts of aglycones were obtained. The highest amounts of isoflavones were obtained throughout thermal hydrolysis: 1066.96 μg/g daidzein and 150.8 μg/g genis [...].