Determination of isoflavone content by hplc-uv method and in vitro antioxidant activity of red clover (trifolium pratense l.)

Abstract

In this study, the methanol and acid hydrolyzed methanol extracts prepared from the aerial parts of two varieties of Trifolium pratense (red clover) collected from different parts of Turkey, namely T. pratense L. var. pratense and T. pratense L. var. sativum Schreb., have been analyzed to determine the concentrations of four isoflavones, daidzein (D), genistein (G), formononetin (F) and biochanin A (B). A RP-HPLC-UV method for the determination of these isoflavones in red clover was developed. Chromatographic separation was carried out by using a gradient system consisting of 0.3 % acetic acid and methanol on a reversed phase column, Supelcosil™ LC-18 (15 cm x 4.6 mm id, 5μm). The highest content of total D, G, F, and B were found as 0.0447, 0.0115, 0.0251 and 0.0039 % in T. pratense var. pratense, respectively. Antioxidant capacity of the methanol extracts of aerial parts of plant samples was also studied. Studied extracts were found to possess some antioxidant activities, as determined by the DPPH free radical scavenging test, thiobarbituric acid and trolox-equivalent antioxidant capacity assays. The phenolic contents of the samples were determined using Folin-Ciocalteu's reagent.