3.9. Total Antioxidant Activity by 2,2-Diphenyl-1-Picril-Hidrazil (DPPH) Assay

The antioxidant activity was determined according to the modified method of Kelebek et al. [41] using 2,2-diphenyl-1-picril-hidrazil (DPPH) as a free radical. Briefly, five different aqueous dilution samples of the orange juices (5%, 10%, 15%, 20%, and 30%, v/v) were firstly prepared. A set of standard DPPH solutions (0–95 µM) in methanol were also prepared. These standard solutions were used to make the calibration curve. An aliquot of 75 µL of diluted orange juice solution was added to 2.93 mL of a 60 µM DPPH solution in methanol, and left to react for 45 min in a dark at room temperature. Finally, the absorbance of each sample was measured at 515 nm using a 6405 UV/Vis spectrophotometer (Jenway, Staffordshire, UK), and using methanol as blank solution. The DPPH concentration in mg/mL (C_DPPH) in the reaction medium was calculated from the calibration curve with Equation (1). All tests were performed in duplicate.

Effective concentration (EC₅₀) was then calculated using the remaining DPPH percentage (% DPPH_remain), as shown in Equation (2). Antioxidant values were expressed as antiradicalar power (ARP), which is the inverse of EC50 [42] and represents the antioxidant efficiency.