2.8.4. Estimation of Malondialdehyde (MDA) and Hydrogen Peroxide (H2O2) Contents

The method [41] was used to determine the amount of MDA in fresh pepper leaves. New leaf samples (0.5 g) were extracted with 5% TCA and centrifuged for 10 min at 4000× *g*. 2 mL of the extract was combined with 2 mL of 0.6% thiobarbituric acid (TBA) solution and placed in a water bath for 10 min. After cooling, the generated color's absorbance was measured at 532, 600, and 450 nm. The following equation was used to calculate the MDA content:

$$6.45 \times (\text{A}\_{532} - \text{A}\_{600}) - 0.56 \times \text{A}\_{450}$$

Fresh pepper leaves were tested for H2O2 content [46]. Fresh pepper leaves (0.5 g) were added to 4 mL cold acetone, then 3 mL of the extract was mixed with 1 mL of 0.1% titanium dioxide in 20 percent (*v*:*v*) H2SO4, then the mixture was centrifuged at 6000× *g* for 15 min. The yellow color generated at 415 nm was detected.
