Improved in vitro assays of superoxide anion and 1,1-diphenyl- 2-picrylhydrazyl (DPPH) radical-scavenging activity of isoflavones and isoflavone metabolites

Free radical-scavenging activity of isoflavones and some isoflavone metabolites have been described previously, but the results are inconsistent. The objective of the present study was to find out the pivotal factors that influence an accurate detection of both superoxide anion and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity. We here showed for the first time that organic solvents, including methanol, ethanol and acetone, were of strong superoxide radical-scavenging activity at concentrations down to 0.1% (v/v), however, no such activity was observed with acetonitrile at concentrations up to 2.0% (v/v). In DPPH assay, we found that the DPPH radical-scavenging ratio increased together with the extended reaction time. Based on our findings, improved in vitro assays for the detection of radical-scavenging activity of both isoflavones (daidzein and genistein) and isoflavone metabolites, including dihydrodaidzein (DHD), dihydrogenistein (DHG), and O-desmethylangolensin (O-Dma), were established.