Methanol and acetonitrile of hplc grade

Roswell Park Memorial Institute (RPMI) 1640 medium, penicillin-streptomycin (Pen Strep), fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Phorbol 12-myristate 13-acetate (PMA), LPS (Escherichia coli 055:B5), RIPA buffer, DMSO were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 1× Halt Protease and Phosphatase Inhibitor Cocktail was purchased from Pierce (Rockford, IL, USA). Human TNF-α and IL-1β enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (Minneapolis, MN, USA). Alamar blue reagent for cell viability assay was purchased from Life Technologies (Grand Island, NY, USA). Primary antibodies specific to COX-2, p-p38, p38, p-ERK1/2, ERK1/2, p-JNK1/2, JNK1/2, p-IκBᾳ, IκBᾳ, p-IKKᾳ/β, p-NFκBp65 and β-actin were purchased from Cell Signaling Technology (Beverly, MA) and, in addition, anti-rabbit secondary antibody conjugated to horseradish peroxidase was obtained from Cell Signaling Technology (Beverly, MA). Methanol and acetonitrile of HPLC grade were purchased from Fisher Scientific (Loughborough, UK). Phyllanthin, hypoPhyllanthin, niranthin gallic acid, ellagic acid, corilagin and geraniin (purity > 98%) were purchased from ChromaDex (CA, USA). Dexamethasone was obtained from CCM Duopharma Biotech Bhd (Selangor, Malaysia).

EPR was purchased from Anguo Chinese Chemicals Herbal Medicine Factory (Anguo, China) and authenticated by Professor Jincai Lu (School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, China); hypoxanthine, niacinamide, phenylalanine, betaine and N,N-dimethylglycine were supplied by Sigma Corporation (St. Louis, MO); corilagin, brevifolin carboxylic acid, brevifolin, ellagic acid, 3,3′-di-O-methyl ellagic acid-4′-O-β-d-xylopyranoside, 3,3′-di-O-methyl ellagic acid-4′-O-β-d-glucopyranoside, 3,3′-di-O-methyl ellagic acid, quercetin, yuexiandajisu C, helioscopinolide E and (–)-(1S)-15-hydroxy-18-carboxycembrene were isolated from EPR and fully characterized based on chemical and spectroscopic analysis (UV, IR, NMR and MS) in our laboratory. The purity of each compound isolated was more than 98% determined by HPLC analysis. Methanol and acetonitrile of HPLC grade were purchased from Fisher Scientific (Pittsburgh, PA), and formic acid of HPLC grade was obtained from Concord Tech. Co. Ltd. (Tianjin, China). Distilled water was purified using a Milli-Q system (Millipore, Bedford, MA).

Ephedra (Ephedrae Herba, Batch number 201611) was purchased from Nei Monggol Chifeng Rongxingtang Drug Co., Ltd. (Shenyang, China) and identified by Associate Professor YanNian Wang, a traditional Chinese medicine (TCM) identifying expert from Department of Traditional Chinese Medicine Processing, Shenyang Pharmaceutical University. The contents of EH, PEH, and MEH were quantitatively determined by HPLC and were 9.5, 8.4, and 1.1 mg/g in ephedra, respectively. EH (Batch number 171241-201607; purity, 99.7%), MEH (Batch number 171247-201601; purity, 99.9%), PEH (Batch number 171237-201608; purity, 100%), and internal standard (matrine; purity > 98.5%) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Methanol and acetonitrile of HPLC grade were supplied by Fisher Scientific (Pittsburgh, PA, USA). Ether and isopropyl alcohol (analytical grade) was purchased from Kermel Chemical Reagent Co. Ltd. (Tianjin, China). Distilled water was obtained from Wahaha Co. Ltd. (Hangzhou, China).