Antioxidant Potential of O. sinensis Melanin

The ability of O. sinensis melanin to scavenge free radicals was measured using the DPPH method, and the procedure was adapted from Surendirakumar et al. [23 (link)]. Briefly, different concentrations of O. sinensis melanin were prepared with DMSO, and 0.05 mg/mL DPPH (ethanol solution) was mixed at a volume ratio of 1:1. After incubation in the dark at 24 °C for 30 min, a spectrophotometer (SpectraMax i3x, Molecular Devices, Shanghai, China) was used to read the absorbance at 517 nm. Trolox was used as a control. The antioxidant activity of O. sinensis melanin was also examined using the ABTS method as described by Khemakhem et al. [41 (link)]. Approximately 7.4 mM ABTS storage solution and 2.6 mM K₂S₂O₈ storage solution were prepared, mixed with equal volumes, and reacted at 4 °C for 12–15 h (protected from light), and then diluted with ethanol to OD734 = 0.7 to obtain the ABTS working solution. The samples and ABTS working solution were mixed at a volume ratio of 1:4, kept in the dark at 24 °C for 30 min to react, and a spectrophotometer (SpectraMax i3x, Molecular Devices, Shanghai, China) was used to read the absorbance at 714 nm.
The ability to scavenge the DPPH (or ABTS) radical was calculated using the following equation:
where A₀: DPPH (or ABTS)+ DMSO; A₁: DPPH (or ABTS)+ melanin; and A₂: melanin + ethanol.