2.2.1. Lipid peroxidation evaluation
For the estimation of lipid peroxidation, the malondialdehyde
(MDA) content in the leaves was determined as described [21].
This adapted method was carried out with some modifications:
300 mg of leaves was ground in a mortar under
liquid nitrogen and homogenised in a solution containing
0.1% (v/v) trichloroacetic acid (TCA) and 20% (m/v) insoluble
polyvinylpolypyrrolidone. The homogenate was centrifuged
at 10,000 g for 10 min, and then, 250 μL of the supernatant
was added to a solution composed of 1 mL of 0.5% (v/v)
2-thiobarbituric acid plus 20% (v/v) trichloroacetic acid. The
mixture was incubated in a dry bath at 95 °C for 30 min. Then,
the homogenate was maintained in an ice bath for 1 min and
centrifuged at 10,000 g for 10 min to separate the residues
formed during the heating process. The concentration of MDA
was determined in triplicate at 535 nm using an absorptivity
of 155 mM−1 cm−1
, following a correction for nonspecific
turbidity determined by the absorbance at 600 nm.