Gsh kit

DCFH-DA and an Annexin V-FITC/PI apoptosis kit were purchased from Beijing 4A Biotech Co., Ltd. (Beijing, China). DPPH, ABTS, TPTZ, MTT, and LPS were purchased from Sigma-Aldrich (Shanghai, China). SOD, CAT, and GSH kits were purchased from Nanjing Jiancheng (Nanjing, China). FBS, DMEM, streptomycin, and penicillin were purchased from Gibco (Grand Island, NY, USA). ELISA kits for human TNF-α, IL-6, and IL-1β were obtained from (Lianke) Biotech (Hangzhou, China).

The cerebral cortex of the right hemisphere was collected 24 hours after pMCAO. The weight of the brain tissue was determined, then the tissues were immersed in normal saline nine times, and mechanical homogenization was performed in an ice box. The tissue homogenate at a concentration of 0.5%‐10% was then prepared and analyzed using SOD, CAT, GSH‐Px, MDA, and GSH kits following the manufacturers' instructions (A001, A007, A005, A003, and A006; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The activity of SOD was determined by xanthine oxidase assay, based on its reduction ability to superoxide anion radical. Ammonium molybdate could terminate CAT to decompose hydrogen peroxide, and the remaining hydrogen peroxide reacted with ammonium molybdate to produce yellow complex. We measured the change of yellow complex to explore the activity of CAT. The activity of GSH‐Px was represented by the speed of its catalyzing GSH. The levels of MDA were determined via measuring the red products, which were formed by the condensation of MDA with thiobarbituric acid. The levels of GSH were determined by measuring 5‐thio‐2‐nitrobenzoic acid, a kind of yellow substance, which was produced in the process of GSH oxidized by 5,5'‐dithiobis‐2‐nitrobenzoic acid.

Standard compounds geniposide, gardenoside, genipin-1-gentiobioside, shanzhiside, chlorogenicacid, crocin-1, crocin-2, crocetin, geniposidic acid, and genipin with over 98% purity were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Cell Counting Kit-8 purchased from EnoGene technology Co., Ltd. (Nanjing, China). Annexin V-FITC/PI apoptosis detection kit purchased from BestBio technology Co., Ltd. (Shanghai, China). DAPI staining solution purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). TNF-α,IL-6, and NO enzyme-linked immuno sorbent assay (ELISA) kits (R&D, USA) were purchased from Changchun Bestgene Biotechnology Co., Ltd. (Changchun, China). ALT, AST, ALP, MDA, SOD, and GSH kits were obtained from Nanjing JianCheng Institute of Biological Engineering (Nanjing, China). Acetonitrile and Formic acid (Fisher, Fair Lawn, NJ, USA) were of HPLC grade.

Glutathione (GSH) levels in the cell extracts were determined by using a GSH kit (Jiancheng Bioengineering Co. Ltd., Nanjing, China) according to the manufacturer’s instruction [49 (link)]. Briefly, a mixture of 0.1 mL of cell extract and 0.9 mL of 5% trichloroacetic acid was centrifuged at 2300 g for 15 min at 4 °C. Then, 0.5 mL of the supernatant was added into 1.5 mL of 0.01% 5,5-dithio -bis-2-nitrobenzoic acid (DTNB), and the reaction was monitored at 412 nm. The amount of GSH was expressed in terms of μmol/mg protein.

The level of GSH was measured following the method of Beutler with certain modification (18 (link)). The determination of GSH was based on the ability of the -SH group to reduce 5,5′-dithiobis(2-nitrobenzoic acid) and form a yellow anionic product whose optical density was measured at 412 nm. The levels of GSH were determined using the GSH kit from Nanjing Jiancheng Bioengineering Institute.