Antioxidant Enzyme Extraction and Assay from Tomato Leaves

Plant extracts were prepared from tomato leaves after 45 days and antioxidant enzymes were assayed as described by Ahmad et al. (2015) (link). Briefly, fresh tomato leaves were ground using liquid nitrogen and the ground leaf samples were stored at –80 ^◦C. The ground leaf samples (approximately ∼1 g) were homogenized on ice using 10 mL of 50 mM phosphate buffer (pH 7.8) and then incubated for 10 min at 4 ^◦C. Subsequently, the homogenate was filtered using Advantech Qualitative Filter Papers (110 mm) and centrifuged at 4,000 × g for 15 min at 4 ^◦C. The supernatant was used for the determination of enzyme activities. The activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT) (EC 1.11.1.6), and peroxidase (POD, EC 1.11.1.7), were measured using assay kits (kit Numbers. A001-1, A007-1, A084-3, respectively; Nanjing Jiancheng Bioengineering Institute, China), following the manufacturer’s instructions (http://elder.njjcbio.com/index_en.php) (Liang et al., 2017 (link)). This experiment was conducted in triplicate.