Epigallocatechin 3 gallate

The following chemicals were commercially obtained: KH₂PO₄ (Merck KGaA, Darmstadt, Germany); Na₂HPO₄, citric acid, NaN₃, sodium carboxymethyl cellulose (CMC, molecular weight ≈ 250 kDa), mucin from bovine submaxillary glands (type I-S) with a molecular weight between 0.4 and 4.0 MDa [22 (link), 23 ], (-)-epicatechin (EC), (-)-epigallocatechin-3-gallate (EGCG), methyl gallate, saccharin (Sigma-Aldrich, Steinheim, Germany); quercetin-3-O-rutinoside (rutin) (Carl Roth, Karlsruhe, Germany); (-)-epicatechin-3-gallate (ECG), (-)-epigallocatechin (EGC) (Extrasynthese, Genay, France); acetonitrile (J.T. Baker, Deventer, Netherlands); D₂O, DMSO-d₆, and 3-(trimethylsilyl)propionic acid sodium salt (TMSP) (Euriso-top, Gif-sur-Yvette, France). All chemicals were of analytical grade or better and were used without further purification. Ultrapure water was prepared with a Milli-Q Gradient A10 system (Millipore, Schwalbach, Germany).

Madin-Darby canine kidney (MDCK) cells, human cervical cancer HeLa cells and human lung epithelial A549 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured at 37 °C/5% CO₂ in Minimum Essential Medium (MEM; Invitrogen, Carlsbad, CA, USA), Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen) or Roswell Park MEMorial Institute 1640 Medium (RPMI; Hyclone, Logan, UT, USA), respectively, supplemented with 10% FBS (Invitrogen). Influenza viruses A/Puerto Rico/8/34 (PR8), A/Hong Kong/8/68 (HK), and B/Lee/40 (Lee) were obtained from the ATCC. The two type A influenza strains were amplified in 10-day-old embryonated chicken eggs, while the type B strain was inoculated into MDCK cells in the presence of 2 μg/mL TPCK-treated trypsin at 37 °C for 3 days. The NA inhibitor, oseltamivir carboxylate (OSV-C; ≥98%), was purchased from United States Biological (Swampscott, MA, USA). The cell entry blocker (-)-epigallocatechin-3-gallate (EGCG, ≥95%) and the polymerase inhibitor ribavirin (RBV) were from Sigma-Aldrich (St. Louis, MO, USA). Stocks were prepared in water (for EGCG) or DMSO (for OSV-C and RBV); the final concentration of each was 50 mM.

All reagents used for analytical determinations, (water, acetonitrile, methanol, formic acid and hydrochloric acid) were from VWR International (Fontenay-sous-Bois, France).
Hyperoside (purity > 98%), kaempferol-3-O-glucoside (purity > 99%), procyanidin B1 (purity ≥ 80%), procyanidin B2 (purity ≥ 90%), and catechin (purity ≥ 99%) were purchased from Extrasynthese (Genay, France); rutin (purity > 97%), quercetin-3-O-glucoside (purity ≥ 98%), quercetin-3-O-glucuronide (purity > 98%), epicatechin (purity > 90%), epicatechin-3-gallate (purity > 98%), epigallocatechin-3-gallate (purity > 95%), Gallic acid, sodium carbonate, and Folin–Ciocalteu’s reagent were from Sigma-Aldrich (St. Louis, MO, USA); caftaric acid (purity ≥ 95%) and procyanidin C1 (purity ≥ 80%) were from Phytolab GMBH & Co. (Vestenbergsgreuth, Germany); and procyanidin B3 (purity > 98%) was from Carbosynth Limited (Compton, Berkshire, UK).

Epigallocatechin-3-gallate (EGCG) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and the stock solution was prepared in DMSO. Purified LPS from Escherichia coli was obtained from List Biological Laboratory Inc. (Campbell, CA, USA) and dissolved in endotoxin-free water. Monosodium urate (MSU) and ATP were purchased from Invivogen (Carlsbad, CA, USA). Nigericin and H₂DCFDA were purchased from Sigma-Aldrich. A caspase inhibitor, Z-YVAD-FMK, was obtained from Calbiochem (Darmstadt, Germany). An antibody for mouse caspase-1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). An antibody for interleukin-1β (IL-1β) was from R&D Systems (Minneapolis, MN, USA). All other reagents, if not specified, were purchased from Sigma-Aldrich.