2.2. MTT Assay for Cell Viability Analysis

SH-SY5Y cells were seeded in 96-well plates (CoStar, USA) separately at a density of 4,000 cells per well. Triphala extract power (AL1675; Dabur India Ltd., Alwar, India) was prepared and diluted in the same way as our previous study [23, 27]. Firstly, to determine the optimal concentrations of Triphala and H₂O₂, higher concentrations (0.08, 0.4, 2, 10, 50, and 250 μg/mL) and lower concentrations (0.014, 0.041, 0.12, 0.37, 1.11, 3.33, and 10 μg/mL) of Triphala were applied to treat SH-SY5Y cells for 48 h. Meanwhile, cells were treated with 300, 400, 500, 600, 700, and 800 μmol/L of hydrogen peroxide (H₂O₂) for 20 h, separately. Then, cell viability was measured using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay (Boster Biological Technology Company, Wuhan, Hubei, China). Briefly, each well was incubated with 10 μL of MTT solution for 4 h at 37°C, added with 110 μL formazan solvent, and the absorption at 490 nm was measured using a microplate reader (Beijing Pulang New Technology Co., Beijing, China).

Next, the neuroprotective effect of Triphala on H₂O₂ toxicity was estimated by preincubating cells with increasing concentrations (0.014, 0.041, 0.12, 0.37, 1.11, 3.33, and 10 μg/mL) of Triphala for 24 h and then challenging the cells with 400 μmol/L H₂O₂ for an additional 20 h. Cells treated without either H₂O₂ or Triphala were considered as blank control, while the H₂O₂-induced injury cell model, in which only H₂O₂ was added, was regarded as model control. After the drug intervention, cell viability was determined using the MTT assay as described above and the percentage of cell viability was calculated as follows: viability (%) = (OD_experiment − OD_blank)/(OD_model − OD_blank) × 100%. The percentage of the protective effects was calculated; thus, protective effects (%) = 1 − viability (%).