4.5.3. Ferric Reducing Power Assay

In the ferric reducing power test, is measured the reduction in the bright red color potassium ferricyanide to the intense blue color ferric ferrocyanide (Prussian blue), which occurs by means of antioxidants in acidic environments, with an increase in absorbance at 700 nm [84].

The ability of all the newly synthesized compounds to reduce iron(III) to iron(II) was determined by the modified Oyaizu method [92–94]. The sample solutions were prepared in DMSO at a concentration of 500 μM. Two milliliters of each tested compound solution were mixed with 2 mL of 0.2 M phosphate buffer (pH 6.6) and 2 mL of 1% K3[Fe(CN)6] solution. After 20 min incubation at 50 ◦C, 2 mL of 10% trichloroacetic acid solution was added, and the mixture was centrifuged for 15 min at 4500 rpm. Finally, 2 mL of the upper layer was mixed with 2 mL of deionized water and 0.4 mL of 0.1% FeCl3 solution, and after 5 min the absorbance was recorded at 700 nm with an Analytik Jena Specord 40 UV/VIS spectrophotometer. All analyses were performed on three replicates, and the results were averaged and compared with those of antioxidant standards used, i.e., AA, BHA, and BHT.
