Sephadex g 25

Purple wheat (Triticum aestivum L.) of the cultivar Shannong Purple Wheat One were harvested in August, 2018 (base on the GB 1351–2008) at maturity from Shangdong, China. Purple wheat bran protein (PWBP) was prepared from purple wheat bran. Alcalase (2 × 105 U/g), flavourzyme (3 × 104 U/g), papain (8 × 105 U/g), neutral enzyme (3 × 104 U/g), and trypsin (2.5 × 105 U/g) were purchased from Beijing Solarbio Science &Technology Co., Ltd. (Beijing, China) DETA-Sepharose FF and Sephadex G-25 were purchased from Beijing Solarbio Science &Technology Co., Ltd. (Beijing, China).

Chinese pecans were purchased from Lin’an Tongda Food Co., Ltd. (Hangzhou, China). Compound protease (120 U/mg) and glutathione (GSH) were obtained from Chinese Shanghaiyuanye Bio-Technology Co., Ltd. (Shanghai, China). 1,1-diphenyl-2-picrylhydrazyl (DPPH) was bought from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Sephadex G-25 was obtained from Beijing Solarbio Biotechnology Co., Ltd. (Beijing, China). H₂O₂ was obtained from Sigma-Aldrich (Shanghai, China) Trading Co., Ltd. (Shanghai, China). Caco-2 cells were acquired from the Chinese Academy of Sciences (Kunming, China). Cell Counting Kit-8 (CCK-8), ROS assay kit, Catalase assay kit, and SOD assay kit with WST-8 were obtained from Beyotime Biotechnology Co., Ltd. (Shanghai, China). All the reagents were analytical grade.

The procedure for the synthesis of cyclic single-stranded T was adapted from the protocols reported in our previous research. Generally, 24 nmoles of CTS and 24 nmoles of Tt were lyophilized and re-dissolved in 300 μL of MES buffered solutions (2-(N-mophorlino)ethanesulfonic acid, 250 mM, pH = 7.6), respectively, and cooled to 0 °C on ice. Then the solutions were mixed and incubated on ice for another 30 min. After that, 600 μL of cyanogen bromide solution (5M in dry acetonitrile) was added to the reaction mixture and vortexed for 30 min on ice, which was followed by adding 12 mL of LiClO₄ solution (2%w/vin acetone). The mixture was incubated on dry ice for 1 h and then centrifuged at 14,000 rpm and 4 °C for 10 min. After disposing of the supernatant, the pellet was dried and re-dissolved in 120 μL of autoclaved H₂O and separated by 12% denaturing PAGE gels. The desired band was sliced and collected, and the target product DNA was extracted and desalted by Sephadex G25 (Solarbio Science, Beijing, China). The quantification was conducted on Implen Nanophotometer N60 (Germany).