3.5. Neuroprotective Assay

The MTT assay was used to assess the protective effects of compounds 1–3 on SH-SY5Y human neuroblastoma cells against H₂O₂-induced oxidative stress [27]. The SH-SY5Y cells (1.2 ×10⁴ cells/mL) were cultured at 37 °C in a 5% CO₂ and 95% air atmosphere in 96-well plates for 48 h. Subsequently, the cells were treated with 2-fold serial dilutions of compounds (100, 50, 25, 12.5, and 0 μM) for 3 h, followed by the addition of 1000 μM H₂O₂. After six hours, 20 μM MTT (5 mg/mL in PBS) was introduced to each well and incubated for an additional four hours. Subsequently, the medium was discarded, and DMSO was employed to dissolve the formazan. Cell viability was quantified as a percentage of the control group (100%) by measuring absorbance at 490 nm with a Tecan microplate reader. Statistical analysis and group comparisons were conducted using GraphPad Prism software.