2.9. In Vitro Antioxidant Ability

The in vitro antioxidative ability was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay method [17] with slight modifications. In brief, 200 µmol/L of DPPH solution was prepared in 75% ethanol before use and kept away from light. The samples were then dissolved in distilled water to 2 and 4 mg/mL CoQ10 equivalent concentrations. Next, 100 µL was taken from each sample and mixed with 100 µL of DPPH ethanol solution in 96-well plates. The final sample concentrations were 1 and 2 mg/mL CoQ10 equivalent. The mixed solution was incubated for 30 min at 40 °C in the dark and light absorption was measured at 517 nm using Tecan Infinite 200 PRO (TECAN, Switzerland).

A mixture of 100 μL distilled water and 100 μL DPPH solution was used as the “blank 1,” a mixture of 100 μL distilled water and 100 μL 75% ethanol was used as “blank 2,” and a mixture of 100 μL of each sample solution and 100 μL of 75% ethanol was used as a “sample blank.” DPPH radical scavenging activity was evaluated using the following formula.

Radical scavenging activity (%) = ((Absorbance of blank 1 − Absorbance of blank 2) − (Absorbance of sample − Absorbance of sample blank))/(Absorbance of blank 1 − Absorbance of blank 2) × 100.