Epicatechin

The liquid chromatography standards: phloridzin, phloretin, chlorogenic acid, ferulic acid, and caffeic acid were purchased from Sigma Aldrich (Oakville, ON, Canada); catechin, epicatechin, quercetin, quercetin-3-O-galactoside and quercitin-3-O-glucoside from ChromaDex, Inc. (Santa Ana, CA, USA); quercitin-3-O-rhamnoside, quercitin-3-O-galactoside and anthocyanin standards from Indofine Chemical Company (Hillsborough, NJ, USA). The analytic solvents, chemicals, and enzymes were obtained from Sigma Aldrich (Oakville, ON, Canada). Mouse embryo 3T3-L1 cell line (ATCC, CL-173) was obtained from Cedarlane (Burlington, ON, Canada). Phenazine methosulfate (PMS) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) were purchased from Sigma–Aldrich Canada Ltd. (Oakville, ON, Canada). Dulbecco's modified Eagle's medium (DMEM), penicillin-streptomycin solution, fetal bovine serum, bovine calf serum, trypsin-EDTA solution, dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), insulin solution and all other cell biology grade chemicals were purchased from Sigma–Aldrich (Oakville, ON, Canada). Lipolysis colorimetric assay kit was purchased from Biovison Inc. (Milpitas, CA, USA) and adipogenesis assay kit from Cayman Chemical Co. (Ann Arbor, MI, USA).

Analysis was performed on (Thermo Scientific UltiMate 3000 UHPLC) system equipped with UV detector. Zorbax Eclipse Plus C18 column (100 × 4.6 mm, 3.5 μm) was used. The mobile phase consisted of acetonitrile (A) and 0.1% formic acid (B). The linear gradient profile was as follows: 10–20% (A) for 45 min, 20–30% (A) for 15 min, 30–40% (A) for 10 min, and 40–10% for 10 min. The flow rate of mobile phase was set at 0.8 mL/min, the temperature was 40 °C. The detection wavelength was 240, 254, 270, and 520 nm. The identification of each compound was carried out by the comparison of both retention time and UV-VIS spectra of reference compound. Epicatechin and cyanidin-3-O-glucoside, analytical standards were obtained from ChromaDex. Quercetin (purity ≥ 95%) and chlorogenic acid (purity 96.6%) were purchased from Sigma-Aldrich and Dr. Ehrenstorfer GmbH respectively.

Reference compounds procyanidin A2 (PAC-A2), procyanidin B2 (PAC-B2), catechin, and epicatechin were purchased from Chromadex (Irvine, CA, USA). 4-Dimethylaminocinnamaldehyde (DMAC), Folin–Ciocalteu reagent, Sephadex LH-20, and Supelco LC-18 12 ml solid-phase extraction (SPE) cartridges were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). All organic solvents were high pressure liquid chromatography (HPLC) grade and obtained from VWR International (Suwanee, GA, USA).

Inhibition and kinetic reactions were carried out in Black costar 96-well plates bought from Thermo Fischer Scientific (Pittsburgh, PA, USA). α-naphthoflavone, furafylline, sulphaphenazole, and miconazole were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vivid^® Blue screening kits and vivid^® substrates, 7-benzyl-oxymethyloxy-3-cyanocoumarin, (BOMCC-cat. no. P2975) and 7-ethoxy-methloxy-3-cyanocoumarin (EOMCC-cat. no. P3024) were obtained from Life Technologies, (Grand Island, NY, USA). CYP2C19 (cat. no. P2864), CYP2C9 (cat. no. P2861), and CYP1A2 (cat. no. P2863) blue screening kit included baculosome (respective isozymes and NADPH-P450 reductase); a regeneration system (glucose-6-phosphate, glucose-6-phosphate dehydrogenase) and NADP⁺ were used for the study. Standards for epicatechin, catechin, chlorogenic acid, caffeic acid, and p-coumaric acid for identification and quantification of constituents of herbal extracts were purchased from Chromadex (Wesel, Germany). Ultra-Pure double distilled and deionized water was obtained from Milli-Q plus water purification system (Millipore, Bedford, MA, USA). All reagents were of analytical grade.

All the chemicals used were analytical or reagent grade and were not purified further. Folin-Ciocalteu reagent, gallic acid, sodium carbonate, ascorbic acid, l-cysteine, sodium phosphate monobasic monohydrate (NaH₂PO₄·H₂O), disodium hydrogen phosphate (Na₂HPO₄), caffeine, and theobromine were obtained from Sigma Aldrich (St. Louis, MO, USA). (+)-Catechin hydrate (≥99%; ASB-000003310), (−)-epicatechin (≥99%; ASB-00005127), procyanidin B2 (≥90%; ASB-00016231) were purchased from ChromaDex Inc. (Irvine, CA, USA). Acetonitrile, methanol, (HPLC-grade), ethanol, n-hexane, citric acid and formic acid were acquired from Merck (Merck, Germany). Deionized water (18 MΩ/cm) from an Aqua Solution system (Aqua solution, Inc. Jasper, Georgia, USA) was used for the preparation of all solutions.