*4.9. Determination of Total Phenolic and Flavonoid Contents*

Total phenolic content (TPC) of tomato was measured by dissolving 5 mg of air-dried leaf powder in 10 mL methanol according to Slinkard and Singleton [80] using Folin– Ciocalteu reagent protocol. Total flavonoid content (TFC) of tomato leaves was evaluated using the aluminum chloride colorimetry method described by Chavan et al. [81]. A standard calibration curve was constructed using quercetin in different concentrations (0.05–1 mg/mL). Tomato extract or quercetin (2 mL) was mixed with 500 μL of 10% aluminum chloride solution and 500 μL of 0.1 mM sodium nitrate solution. The absorbance of the reaction mixture was measured after incubation at room temperature for 30 min at wavelength 430 nm using a UV–VIS spectrometer (Jenway, Tokyo, Japan). Soluble protein content (PC) was estimated in both control and treated plants following Bradford [82] using Coomassie Brilliant Blue G-250 dye and the absorbance was recorded at 595 nm using bovine serum albumin as standard.

#### *4.10. Assay of Antioxidant Enzymes*

Antioxidant enzymes were extracted by homogenizing 1 gm fresh tomato leaf tissue in chilled 50 mM phosphate buffer (pH 7.0) supplemented with 1% polyvinyl pyrolidine and 1 mM EDTA using prechilled pestle and mortar. After centrifuging at 18,000× *g* for 30 min at 40 ◦C, the supernatant was used for enzyme assay. Determination of the activity of superoxide dismutase (SOD, EC 1.15.1.1) and NBT photochemical reductions was recorded at 560 nm using the Bayer and Fridovich [83] method in a 1.5 mL assay mixture containing sodium phosphate buffer (50 mM, pH 7.5), 100 μL EDTA, L-methionine, 75 μM NBT, riboflavin, and 100 μL enzyme extract.

The catalase assay (CAT, EC1.11.1.6) activity method of Luck [84] was used and monitored the change in absorbance at 240 nm for 2 min. For the calculation, an extinction coefficient of 39.4 mM−<sup>1</sup> cm−<sup>1</sup> was used. Ascorbate peroxidase (APX, EC 1.11.1.11) activity was tested by monitoring absorption change at 290 nm for 3 min in a 1 mL reaction mixture containing potassium phosphate buffer (pH 7.0), 0.5 mM ascorbic acid, hydrogen peroxide, and enzyme extract. The calculation of the extinction coefficient of 2.8 mM−<sup>1</sup> cm−<sup>1</sup> was used [85].
