2.6.1. Determination of DPPH-Free Radical Scavenging Activity (FRSA)

The DPPH-FRSA assay, based on both electron transfer and hydrogen atom transfer reactions, was determined according to the method of Molyneux [26] with slight modifications [24]. The FRSA of each freeze-dried extract at various concentrations was tested by measuring their ability to quench DPPH. The DPPH, a stable free radical, was reduced, causing the purple color of the DPPH solution to change to bright yellow in the presence of antioxidants that possess hydrogen-donating or chain-breaking properties, and the intensity of this change can be monitored spectrophotometrically. An aliquot of 250 µL of each extract sample (concentration range 3.33–20 µg/mL) or BHT was added to 500 µL of 100 µM DPPH solution. BHT was used as the reference sample at the same concentration of the extracts and a mixture without sample, or BHT, was used as the control. The Abs was measured at 517 nm after incubation (at room temperature in the dark) for a period of 30 min. The FRSA was calculated as a percentage of DPPH decoloration using the following equation:

The results were expressed as EC₅₀ value (µg/mL), which is defined as the sample concentration that can quench 50% of the DPPH free radicals. A lower EC₅₀ value was indicative of a higher antioxidant activity.