2 6 dichloroindophenol

The following reagents were acquired from Sigma Aldrich Chemical Co. (St. Louis, MO, USA): Folin–Ciocalteu reagent, 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,6-Dichloroindophenol, L-ascorbic acid, gallic acid, quercetin, α-amylase (EC 3.2.1.1), pepsin from porcine gastric mucosa (EC 3.4.23.1), pancreatin from porcine pancreas (EC 232.468.9), and bile extract porcine (EC 232.369.0).

In this study, the following reagents were used: gallic acid standard (GAE), Folin–Ciocalteu's Phenol reagent, sodium carbonate, 2,6-dichloroindoPhenol, D-glucose, L(+)-Ascorbic acid, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were obtained from Sigma Aldrich (German). Phenol (99.5%, Riedel-de Haën, Germany), sulphuric acid (95–98%, Panreac Quimica SA, Spain), oxalic acid (99%, Panreac Quimica SA, Spain), methanol (99.5%, Panreac Quimica SA, Spain), potassium ferricyanide (98%, Avonchem, United Kingdom), trichloroacetic acid (99.5%, Sigma-Aldrich, Germany), Hexan (≥97%, Riedel-de Haën, Germany), and ferric chloride (98%, SRL, India).

Folin & Ciocalteu's phenol reagent, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), 2,4,6-tripyridyl-1,3,5-triazine (TPTZ), 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), gallic acid monohydrate, 2,6-dichloroindophenol, (+)-catechin hydrate, and vitamin C were purchased from Sigma-Aldrich (Shanghai, China). The hesperidin was purchased by Chengdu Institute of Biology, Chinese Academy of Sciences (Chengdu, China). The synephrine was purchased from National Institute for the Control of Pharmaceutical and Biological Products (NICPBP, Beijing, China). All other reagents of HPLC-grade and analytical grade were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China).

Peaches (Prunus persica cv. Yanhong) were purchased from an orchard situated in Yaodu District, Linfen City, Shanxi Province. The fruits were picked at commercial mature stage. Peaches with uniform shape, size, and color, as well as no insect pest and mechanical damage, were selected and quickly transported to the laboratory of Shanxi Normal University in open cartons.
CO gas with a purity of 99.99% was purchased from Beijing Huaneng Special Gases Co., Ltd. (Beijing, China). Oxalic acid, gallic acid, rutin, potassium ferricyanide, trichloroacetic acid, and sodium hydroxide (analytical grade) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Analytical-grade 1,1-diphenyl-2-picrylhydrazyl radical, 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl, and 2,6-dichloroindophenol were purchased from Sigma (USA). Other reagents of analytical grade were purchased from Alfa Aesar Company (Tianjin, China).

Fresh satsuma mandarins were obtained from Berry Good farms (Tifton, GA, USA). Maltodextrin (MD, dextrose equivalent = 9–13) was obtained from Now Foods (Bloomingdale, IL, USA). Acetic acid, ascorbic acid, 2,6-Dichloroindophenol, metaphosphoric acid, and sodium bicarbonate were purchased from Sigma Aldrich (St. Louis, MO, USA).

The in vitro inhibition of hDHODH was measured using an N-terminally truncated recombinant hDHODH enzyme as described previously [36 (link)]. Briefly, the hDHODH concentration was adjusted in a way that an average slope of approximately 0.2 AU/min served as the positive control (e.g., without inhibitor). The standard assay mixture contained 60 µM 2,6-dichloroindophenol (Sigma, St. Louis, MO, USA), 50 µM decylubiquinone (Sigma), and 100 µM dihydroorotate (Sigma). The hDHODH enzyme with or without at least six different concentrations of the compounds was added, and measurements were performed in 50 mM TrisHCl, 150 mM potassium chloride (KCl) (Merck), and 0.1% Triton X-100 (Sigma) at pH 8.0 and at 30 °C [37 (link)]. The reaction was started by adding dihydroorotate and measuring the absorption at 600 nm for 2 min. For the determination of the IC50 values, each data point was recorded in triplicate. DHODH inhibitors Cmp 1, Cmp 2, Cmp 3, Cmp 4, and Cmp 5 were provided by Immunic Therapeutics. Brequinar, uridine, 2′-deoxyuridine, cytidine, and 2′-deoxycytidine were purchased from Sigma. UCK2 inhibitor, UCK2-IN-20874830 was obtained from ChemDiv (San Diego, CA, USA).