*2.4. Assessment of Nonenzymatic Antioxidant Activities (Flavonols, Tocopherols, and Total Phenolics)*

#### Methanolic Extract Preparation

For this test, 0.5 g of dry leaf sample was dissolved in 4 mL of methanol (80%) and then centrifuged at 7000× *g* for 15 min. The methanolic extract was used for the tests. The total phenolics were measured according to the protocol of Conde [35]. According to this protocol, a 0.1-m methanolic extra was added to 2.5 mL of 10% Folin–Ciocalteu reagent. Then, for neutralization of the obtained mixture of sodium bicarbonate, 7% was added. The final mixture was transferred to a spectrometer machine to measure the total phenolics at an absorbance of 765 nm. The content of flavonol was determined according to the Akkol method [36]. A 0.5-mL methanolic extract was homogenized with 0.4 mL of aluminum chloride (2%) and 1.5 mL of sodium acetate (5%). After preparation of the supernatant, it was kept at room temperature for 2.5 h. The flavonoid content was determined in the supernatant at an absorbance of 445 nm. The content of tocopherol was determined according to the protocol of Kayden [37]. For this purpose, 3 mL ethanol was mixed with 0.1 g of leaf samples, and the soluble solution was then centrifuged at 7000× *g* for 15 min. The obtained mixture was added to 0.1 mL ethanol extract, 0.2 mL bathophenanthroline at a concentration of 0.2%, 0.001 M of 0.2 mL ferric chloride, and 1 mM 0.2 mL phosphoric acid. The content of tocopherol was recorded by measuring the absorbance of the supernatant at 534 nm.

#### *2.5. Assay of Hydrogen Peroxide (H2O2), Malondialdehyde (MDA), Superoxide Radical (O2* •−*), Soluble Proteins (SP), and Electrolyte Leakage (EL)*

Malondialdehyde is representative of lipid peroxidation, which was measured by the protocol described by Siddiqui [38]. In this experiment, 0.1% trichloroacetic acid (TCA) was used for the homogenization of fresh leaves, after which the sample was centrifuged at 8000× *g* for 25 min. The obtained amount of supernatant was mixed with TCA solution in the range of 20%, which contained 0.5% thiobarbituric acid. In the next process, the

soluble solution was kept at 98 ◦C for 25 min. Then, the soluble solution was kept at room temperature. The final soluble solution was centrifuged a second time at 2000× *g* for 15 min at 5 ◦C. Finally, to estimate malondialdehyde, the absorbance was determined at 532 nm.

The levels of H2O2 were determined using the protocol reported by Patterson [39]. For this study, samples (leaves) in the specified amount of 0.5 g were mixed in a mortar and pestle by adding 10 mL cold acetone. The mixture was centrifuged at 4000× *g* for 25 min. In the next step, titanium chloride at a concentration of 20% in 2 mL of concentrated HCl and 2 mL of ammonia at the specified level of 17 M were added to the supernatant (1 mL). The supernatant was extracted with acetone, which was conducted by the addition of 2 N H2SO4 in 10 mL for proper absorbance. To remove immiscible inputs, the mixture was centrifuged again. The absorbance of the supernatant was recorded at 410 nm. The levels of H2O2 were determined based on a standard curve, which was created based on the known levels of H2O2 and formulated as μmole g−<sup>1</sup> FM. The soluble protein (SP) levels were assigned according to the protocol of Bradford [40] and measured based on the effect of Coomassie Brilliant Blue (G25) on changes in protein levels. The final data were obtained using a spectrometer machine. The amount of superoxide radical (O2 •−) was determined according to the method of Li [41]. According to this protocol, 200 mg leaf tissue samples were mixed with phosphate buffer at pH 7.8 in the amount of 65 mM and then centrifuged at 4000× *g* for 20 min. The supernatant was incubated in 10 mM of hydroxylamine hydrochloride and 65 mM of phosphate buffer (pH = 7.8) for 15 min at 27 ◦C. In the next step, 7 mM α-naphthylamine plus 17 mM sulfanilamide was added to the mixture, preserved for 25 min and then recorded at an absorbance of 530 nm at 25 ◦C. Finally, to determine the final rate of O2 •−, nitrogen dioxide radicals (NO2) were applied to generate a standard curve. Electrolyte leakage (EL) was calculated based on the protocol of Valentovic [42]. According to this protocol, 0.3 g of leaf samples were mixed with 15 mL of deionized water. Then, the mixture was kept at the optimum temperature (25 ◦C) for 2.5 h. In this stage, EC1 was recorded as the primary electrical conductivity of the mixture. To obtain EC2 as the secondary electrical conductivity, the samples were transferred to one autoclave and kept at 120 ◦C for 17 min. At the end of the test, EL was determined based on the following formula:

$$\text{EL (\%)}=\text{EC}\_1/\text{EC}\_2 \times 100\tag{1}$$
