2.3.8. Proline Content

Proline content was determined using the Bates's method [26]. Leaf tissue (0.5 g) was homogenized in 10 mL of 3% aqueous sulfosalicylic acid for 10 min, followed by filtration. Two milliliters of the filtrate were mixed with 2 mL of acid ninhydrin and 2 mL of glacial acetic acid, then placed in a boiling water bath for 1 h at 90 ◦C, and the reaction was completed in an ice bath. The developed color was extracted in 4 mL of toluene, and the intensity of the reaction mixture was determined spectrophotometrically (JENWAY 6300, Staffordshire, UK) at the wavelength of 520 nm. Proline concentration was determined from a standard curve and calculated on a fresh weight basis as follows: μmoles proline/g of fresh weight = [(μg proline/mL × mL toluene)/115.5 μg/μmole]/[(g sample)/5].

#### 2.3.9. DPPH Free Radical Scavenging Assay

The antioxidant activity was assessed by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging method [27,28]. We added 2.4 mL of 0.1 mM DPPH to 1.6 mL of methanolic leaf extract, vortexed the mixture, and incubated it at room temperature in the dark. The absorbance of the samples was measured after 30 min at 517 nm using a spectrophotometer (JENWAY 6300, Staffordshire, UK). The percentage of DPPH scavenging activity was calculated as % inhibition of DPPH = (A517 control –A517 sample/A517 control) × 100.

#### 2.3.10. Extraction and Determination of Antioxidant Enzymes Activity

We used about 0.5 g of fresh young mango leaves to determine the concentration of superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), and polyphenol oxidase (PPO) enzymes. Crude enzyme extracts were prepared using ground fresh leaves samples in liquid nitrogen that were homogenized in 10 mL of phosphate buffer pH 6.8 (0.1 M) and then centrifuged at 2 ◦C for 20 min at 20,000 rpm in a refrigerated centrifuge. The clear supernatant was the crude enzyme extract [29].

• *Superoxide Dismutase (SOD) Activity*

SOD (EC 1.15.1.1) activity was determined by measuring the inhibition of the autooxidation of pyrogallol by employing a method described by Marklund and Marklund [30]. We used a 10 mL reaction mixture consisting of 3.6 mL of distilled water, 0.1 mL of enzyme extract, 5.5 mL of 50 mM phosphate buffer (pH 7.8), and 0.8 mL of 3 mM pyrogallol (dissolved in 10 mM HCl). The pyrogallol reduction rate was measured at 325 nm with a UV–VIS spectrophotometer (PD 303, Apel Co., Ltd., Saitama, Japan). One unit of enzyme activity is the amount of the enzyme leading to 50% inhibition of the auto-oxidation rate of pyrogallol at 25 ◦C [31].

• *Catalase (CAT) Activity*

Catalase (E.C.1.11.1.6) activity was assayed based on Chen et al. [32]. The reaction mixture with a final volume of 10 mL, containing 40 μL of enzyme extract, was added to 9.96 mL of H2O2 phosphate buffer pH 7.0 (0.16 mL of 30% H2O2 to 100 mL of 50 mM phosphate buffer). Catalase activity was measured as the change in H2O2 absorbance in 60 s against a buffer blank at 250 nm using a UV–VIS spectrophotometer (PD 303, Apel Co., Ltd., Saitama, Japan). The blank sample was made by using buffer instead of the enzyme extract. One unit of enzyme activity is the amount of the enzyme reducing 50% of H2O2 in 60 s at 25 ◦C [31].

• *Peroxidase (POX) Activity*

Peroxidase (EC 1.11.1.7) activity was assayed in a reaction mixture containing 5.8 mL of 50 mM phosphate buffer pH 7.0, 0.2 mL of the enzyme extract, and 2 mL of 20 mM H2O2. The change in optical density was determined spectrophotometrically (PD 303, Apel Co., Ltd., Saitama, Japan) within 60 s at 470 nm at 25 ◦C after adding 2 mL of 20 mM pyrogallol [33]. One unit of enzyme activity is the amount of the enzyme catalyzing one micromole of H2O2 per minute at 25 ◦C [31].

• *Polyphenol Oxidase (PPO) Activity*

Polyphenol oxidase (EC 1.10.3.1) activity was determined using 125 μmol of phosphate buffer (pH 6.8), 100 μmol of pyrogallols, and 2 mL of enzyme extract. After incubating the mixture for 5 min at 25 ◦C, the reaction was stopped by adding 1 mL of 5% H2SO4. The developed color was read spectrophotometrically at 430 nm (PD 303, Apel Co., Ltd., Saitama, Japan) [34].
