Effects of wavelengths and standard products on the analysis of isoflavone content in red clover

Red clover (Trifolium pratense) isoflavones exhibit many medicinal and health food functions and are widely used as phytoestrogens in clinical practice. Accurate quantification of isoflavones is of great importance for their further study and application. The four major isoflavones are biochanin A, formononetin, genistein, and daidzein, which are usually used as standard products for quantitative determination of isoflavones. However, scientific selection of standard products and their effects on the determination results have not been reported. In this study, the effects of different wavelengths on isoflavone content in red clover were studied using these four chemical constituents as standard products. The experimental results showed highly significant differentiation of content under different wavelengths and by the use of different standards (P < 0.05). Based on the results, the four isoflavones could be divided into two groups, the FD group (formononetin and daidzein) and the BG group (biochanin A and genistein). The inter-group differences were highly significant (P < 0.05), whereas the intra-group differences were small. The differences may be primarily caused by the compatibility of compounds with red clover UV absorption, and were not closely related to the content of compounds. The UV absorption curve for biochanin A and genistein was close to the slope of the red clover extract at 275 nm. Therefore, it is suggested that these two compounds can be used as standards to determine isoflavone content at 275 nm. In addition, this study showed that the difference in the content of isoflavones was so small that the four standards were all used in semi-quantitative or qualitative comparisons.