2.4.2. Superoxide Radicals Generated by Xanthine/Xanthine Oxidase (X/XO) System

Another antioxidant test was performed in order to evaluate the antioxidant activity of urolithin A using a more physiological system of superoxide radical generation [25]. Here, 22.8 μM nitroblue tetrazolium (NBT), 90 μM xanthine, and 16 mM Na2CO3 were mixed in phosphate buffer (pH = 6.9). Then, 240 μL of this cocktail was added to the well. Next, 30 μL of urolithin A and 30 μL of xanthine oxidase (168 U/L) were added to start the reaction. Before measurement, the plate was incubated for 2 min at 37 ◦C. The superoxide radicals' (O2 −) scavenging activity was assessed spectrophotometrically at 560 nm. The inhibitory activity of XO was also assayed spectrophotometrically at 295 nm. Gallic acid was used as a reference antioxidant compound.
