Determination of antioxidant activity by DPPH· scavenging assay

In 96-well plates, each 100 μl flavonoid sample was appropriately diluted by the addition of 50% ethanol. A DPPH radical solution (concentration: 600 μM), prepared by dissolving the DPPH radical in 100% ethanol in advance, was added in aliquots of 100 μl per well (finally making an volume of 200 μl per well with 75% ethanol concentration). After stirring on a shaking table till sufficient mixing had occurred, the plate was allowed to stand in a dark place for 30 min. The absorbance OD₅₁₇ at the DPPH absorption wavelength (517 nm) for each well was measured using a multiplate reader (SpectraMax M5, Molecular Devices, CA, USA) [22]. The DPPH radical scavenging activity (%) was calculated by standardizing the difference between the absorbance OD₅₁₇ of a measurement sample and the absorbance OD₅₁₇ of a control (without DPPH) with the absorbance OD₅₁₇ of the control [23, 24].