*4.4. Evaluation of Antioxidant and Radical Scavenging Activities of POE*

The antioxidant and radical scavenging activities of POE were studied using ferric reducing/antioxidant potency (FRAP) and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical [8]. Briefly, for determination of the antioxidant activity of POE by FRAP, 200 μL of Ferrozine™ reagent (10 mM Ferrozine™ in 40 mM HCl: 20 mM FeCl3: 0.3 M acetate buffer pH 3.6 1:1:10 ratio) was added to scalar volumes of POE. After 4 min incubation at 37 ◦C, absorbance was measured at 595 nm at room temperature using a microplate reader.

For determination of the radical scavenging activities of POE, 100 μL of 95% methanol was added to scalar volumes of POE and mixed with 100 μL of freshly prepared DPPH solution (0.15 mg/mL in methanol). After a 30 min incubation in the dark at room temperature, absorbance was read at 490 nm with a microplate reader. Ascorbic acid (0.1 mg/mL) was used as a reference to determine the values of both the antioxidant and radical scavenging activity. These activities are expressed as mg of ascorbic acid equivalents (AAE) per mL.
