S phenol reagent

ACS-grade solvents (acetone, methanol, and 95% ethanol), HPLC-grade solvents (water, methanol, and acetonitrile), glass wool, Gibco fetal bovine serum, glacial acetic acid and hydrochloric acid were acquired from the Fisher Scientific Co., LLC (Suwanee, GA, USA). Sephadex LH-20, Amberlite XAD-16, Folin & Ciocalteu’s phenol reagent, gallic acid (>99%), (+)-catechin hydrate (>99%), chlorogenic acid (≥98%), protocatechuic acid (≥97%), vanillic acid (≥97%), fluorescein 3′,6′-dihydroxyspiro[isobenzofuran-1[3H]9′[9[H]xanthen]-3-one), Trolox [(±)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, 97%], TPTZ (2,4,6-tripyridyl-S-triazine, ≥99.0%), quercetin-3-O-rutinoside hydrate (≥94%), iron(II) sulfate heptahydrate (≥99.0%), iron(III) chloride hexahydrate (97%), 2′,7′-dichlorofluorescin diacetate (DCFH-DA, ≥97%), and 2,2′-azobis [2-amidinopropane] dihydrochloride (ABAP) were purchased from the Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Cyanidin-3-O-glucoside chloride was obtained from the Indofine Chemical Company, Inc. (Hillsborough, NJ, USA). Advanced DMEM, phosphate-buffered saline (PBS), Hanks’ balanced salt solution (HBSS), penicillin-streptomycin, and trypsin-EDTA were purchased from Life Technologies (Grand Island, NY, USA). Human colorectal adenocarcinoma (Caco-2) cells were procured from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Total phenols were estimated by the slightly modified Folin-Ciocalteu method [27 (link)–28 ]. Briefly, 0.05 mL of each diluted solution of extracts was added to 0.1 mL of 2 mol/L Folin-Ciocalteu’s phenol reagent (Sigma, St. Louis, MO, USA). Then 0.2 mL of sodium carbonate solution (120 g/L) was added in the reagent after 30 s to 8 min and made up to 2.5 mL. The tube was laid for 5 min in a water bath at 50±0.5°C and then put in a cold water bath. The absorbance at 760 nm was measured. 0.05 mL of methanol was used instead of extracts as a control sample. The amounts were expressed in milligrams of gallic acid equivalents (GAE) per gram of dry matter. The standard curve was prepared with aqueous solutions of pure gallic acid.

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).