The Antioxidant Study Of Isoflavones And Isoflavone Metabolites

Isoflavones mainly composed of genistein and daidzein are a class of phenolic compounds produced in high concentration in leguminous plants, such as soybeans. After being absorbed, these dietary isoflavones are subjected to degradation by gut microflora to diverse metabolites, including dihydrodaidzein (DHD), equol, O-desmethylangolensin (O-Dma) and dihydrogenistein (DHG) etc.Isoflavones are of various biological activities, such as antioxidant activities, preventing cancer, lowing the ratio of cardiovascular disease, and so on. It is generally believed that many of the beneficial effects of isoflavones are at least partially related to the antioxidant activity. Oxidative stress, might play a pivotal role in the pathogenesis of chronic inflammatory diseases. Excess in reactive oxygen species, such as superoxide anion radical or hydrogen peroxide, has harmful effects on DNA which may lead to cell apoptosis. Therefore, accurate detection of antioxidant activity of isoflavones and their metabolites may provide an important guide for both SAR (Structure Activity Relationship) study and drug research and development.Although the antioxidant activity of isoflavones daidzein, genistein and several synthesized isoflavone metabolites has been reported in several studies, the results are inconsistent. The objective of the present study was to find out the pivotal factors that influence an accurate detection of hydroxyl radical, superoxide anion and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity. In hydroxyl radical assay, it was first found that methanol and ethanol showed almost the same hydroxyl radical scavenging capacity, which was much higher than that of acetone at the concentration of 0.4% (v/v) in the reaction system. In contrast, acetonitrile did not show any hydroxyl radical scavenging ability at the same concentration. The reaction system of hydroxyl radical scavenging assay is not stable enough. Meanwhile, both isoflavones and their metabolites are of low water solubility. Therefore, it is not fittable to determine the antioxidant activity of isoflavones and their metabolites by using hydroxiyl radical scavenging assay. We here showed for the first time that organic solvents, including methanol, ethanol and acetone, were of strong superoxide radical-scavenging activity at the concentrations down to 0.1% (v/v), however, no such kind of activity was observed with acetonitrile at the concentrations even up to 2.0% (v/v). Even though the superoxide radical-scavenging is of high sensitivity, while is not suitable to be used for determination of acidic compounds. Therefore, the superoxide radical-scavenging assay is of restriction. Both DPPH radical and the reaction system of DPPH assay are more stable in comparison with that of hydroxyl radical and superoxide radical. In DPPH assay, we found for the first time that the detected DPPH scavenging activity was much improved by extending the reaction time. The determination sensitivity was much increased by extending the reaction time from 30min to 72h. In DPPH assay, we found that at all the four tested concentrations, equol was the most effective DPPH scavenger, the DPPH scavenging activity of genistein was higher than that of daidzein. In addition, when the concentration of the detected compound was more than 1.33mmol/L, the DPPH scavenging capacity of all the four isoflavone metabolites, including DHD, DHG, O-Dma and equol were higher than that of isoflavone themselves. The improved DPPH assay was regarded as the most suitable method for determination of antioxidant activity of isoflavones and isoflavone metabolites.