4.4. Malondialdehyde (MDA) Determination

To quantify the MDA [56], the powder of frozen leaves (100 mg) was homogenized with 1.5 mL of 0.1% (w/v) trichloroacetic acid (TCA) and centrifuged for 10 min at 10,000× g, 4 °C. Two aliquots containing 250 µL of supernatant were collected. One aliquot was mixed with 1 mL of 20% (w/v) TCA, and to the other one with 1 mL of 20% (w/v) trichloroacetic acid and 0.5% (w/v) thiobarbituric acid (TCA + TBA). Both aliquots were placed in a hot bath (95 °C) for 30 min, cooled, and centrifuged at 10,000× g (10 min at 4 °C). Specific absorbance was measured at 532 nm and the non-specific absorbance at 600 nm. MDA was calculated by subtracting the non-specific absorbance from the specific absorbance and using the extinction coefficient of 155 mM⁻¹ cm⁻¹ [56]. Data are presented as mean ± standard deviation (n = 9, per condition).