A015 1

T-AOC activity was measured according to previous report [28 (link)]. Total antioxidant capacity assay kit (A015–1, Nanjing Jiancheng Bioengineering Institute (Nanjing, China)) was used according manufacturer’s instructions. T-AOC activity was expressed in U/mL and U/gprot in serum and tissues respectively.

Hepatopancreas were homogenized with PBS (1:9) and centrifuged immediately at 4000 × g, 4°C for 10 min. The obtained supernatants were used to measure biochemical parameters with relevant kits. Specifically, total protein was measured with BCA Protein Assay Kit (A045-3, Jiancheng Bioengineering Institute, Nanjing, China). Total antioxidant capacity (T-AOC), catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH), and malondialdehyde (MDA) in supernatants were analyzed with A015–1, A007-1-1, A001–1, A006-1-1, and A003-1 kits (Jiancheng Bioengineering Institute, Nanjing, China), respectively. In addition, total cholesterol (T-CHO), lysozyme (LZM), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in plasma were detected using A111-1-1, A050-1-1, C010-1-1, and C009-1-1 kits (Jiancheng Bioengineering Institute, Nanjing, China), respectively. The phenoloxidase (PO) determination was as referenced by Lin et al. (2012) (link) with kits (G0146W, Suzhou Grace Biotechnology Co., Ltd., China).

Approximately 200 mg of fresh stem from each sample was homogenized with 1,800 μL of PBS (pH 7.4, 0.1 M) with a glass homogenizer, then centrifuged at 3,500 × g for 10–12 min. The supernatant was used in determining total proteins, total amino acids, and antioxidant capacity.
The Coomassie brilliant blue method was used in determining total protein content with commercial total protein assay kit (A045-2) made by Nanjing Jiancheng Bioengineering Institute, China, and absorbance was measured at 595 nm. Total amino acid content was determined also with a commercially available test kit (A026-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Copper ions were reacted with various amino acids for the production of blue complexes. Absorbance at a certain wavelength stands was directly proportional to total amino acid content. Absorbance was measured at 650 nm.
Various antioxidative compounds can reduce Fe³⁺ to Fe²⁺, and Fe²⁺ can react with phenanthroline to produce a stable complex, which can be quantified at a certain wavelength. Total antioxidant capability was determined with the commercial total antioxidative capability assay kit (A015-1) of Nanjing Jiancheng Bioengineering Institute, China, and absorbance was measured at 520 nm (Hussain et al., 2016 (link); Dai et al., 2018 (link); Yang et al., 2018 (link)).