2.5.1. Determination of O2− by Nitro-Blue Tetrazolium (NBT) Reduction Test

The reduction of NBT to insoluble blue formazan was used as a probe for O₂⁻ generation inside the viable cells according to the method in [23] with slight modification. Supernatants of SHSY5Y cells described in Section 2.4.1 were eliminated and replaced by a medium with different concentrations (0.25, 0.5, 1, 2, and 4 mg/mL) of tested plant extracts, then 15 µL of 1% NBT solution was added. Untreated cells (negative control: considered as moderate absorbance) and cells treated with 200 μM H₂O₂ for 1 h (positive control: considered as massive absorbance). Following 3 h incubation, the supernatants were removed, and the formation of formazan product was solubilized in 100 µL DMSO. After mixing the contents in each well, the absorption of colored formazan solution was read at 620 nm using a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).