This assay was performed by a method of Brand-Williams et al. (1995) with some modifications. Briefly, 250 μL of the crude extract was mixed with 4.75 mL of the DPPH solution (1.2 × 10−4 M), which had the initial absorbance (Ainitial) at 515 nm of 1.1. The mixture was held in darkness for 15 min and determined for its absorbance at 515 nm. The measured value was designated as the final absorbance (Afinal), which was subtracted from the Ainitial to yield the difference of absorbance (Adifference) as follows:
The Adifference of the sample was used in calculation for the DPPH radical scavenging activity by comparison with a calibration curve, between Adifference and μM Trolox, made with a series of Trolox solutions (82–417 μM), and the activity was expressed as mmol Trolox equivalent per 100 g flour