4.5.1. DPPH (2,2-Diphenyl-1-picrylhydrazyl) Assay

In this method, the in vitro free radical-scavenging potential of BBS and BBS-NiONPs at different concentrations (50, 100 and 200 µg/mL) was determined using a microplate reader [39]. Reagent solution was prepared by adding 2.4 mg of DPPH to 25 mL of methanol. The procedure involved the addition of 180 µL of reagent solution into 20 µL of the test sample to make 200 µL of the final reaction mixture. The mixture was subjected to a shaker followed by incubation for 1 h. Ascorbic acid was used as a reference antioxidant. DPPH solution without sample was taken as control and methanol was used as a blank solution. Finally, the absorbance of the control and tested samples was measured at 517 nm using a microplate reader to find radical scavenging activity using the following formula below:

where, AC and AN refer to the absorbance of the control and NPs at 517 nm.