Antioxidant enzyme assay kits

All reagents and PRMI 1640 media were obtained from Sigma–Aldrich Ltd. (Beijing, China) or J&K Scientific Ltd. (Beijing, China). Antioxidant enzyme assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The BCA protein assay kit was obtained from Beyotime Institute of Biotechnology (Jiangsu, China). Polyvinylidine fluoride membranes and Centricon YM-3 were purchased from the Millipore Corp. (USA). Rabbit anti-cyt c (AF0146) and rabbit anti-VDAC1 (DF6140) were obtained from Affinity Biosciences (OH, USA). Fetal bovine serum was purchased from Hangzhou Sijiqing Biomaterials Co., Ltd. (Hangzhou, China).
¹H NMR and ¹³C NMR spectra were recorded using a Varian Mercury spectrometer operating at 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR. HR-MS spectra were obtained on an Orbitrap Elite (Thermo Scientific) mass spectrometer. UV/Vis spectra were recorded at 25 °C with a Perkin-Elmer Lambda 25 spectrophotometer that was equipped with temperature-controlled cell holders (USA). Fluorescence intensity and fluorescence spectra were measured at 25 °C with an Infinite M200 Pro Multimode Reader (Switzerland) and a Shimadzu RF-5301 spectrofluorimeter (Japan), respectively.

The jejunal and ileal mucosal tissue samples from piglets were powdered in liquid nitrogen, collected immediately in PBS solution, left to stand for 3 h, and then centrifuged at 3,000 × g at 4 °C for 10 min. Digestive enzymes and antioxidant enzyme assay kits were purchased from Nanjing Jiancheng Company. The digestive enzymes measured were alkaline phosphatase, maltase and sucrase, and the antioxidant enzymes measured were total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-PX), malondialdehyde (MDA) and superoxide dismutase (SOD). The BCA kit (Beyoncé) was used to determine the protein concentration to calibrate the enzyme activities. Detailed procedures and calculation formulae refer to the corresponding kit instructions.