Harpagoside

Gallic acid, catechin, chlorogenic acid, p-OH benzoic acid, vanillic acid, epicatechin, syringic acid, 3-OH benzoic acid, 3-OH-4-MeO benzaldehyde, p-coumaric acid, rutin, sinapinic acid, t-ferullic acid, naringin, 2,3-diMeO benzoic acid, benzoic acid, o-coumaric acid, quercetin, harpagoside, t-cinnamic acid, naringenin, and carvacrol were purchased from Sigma-Aldrich (Milan, Italy). Methanol (HPLC-grade) and formic acid (99%) were obtained from Carlo Erba Reagenti (Milan, Italy).

High purity chemicals: sodium carbonate, sodium acetate trihydrate, and anhydrous aluminum chloride were acquired from Sigma-Aldrich (Germany). Folin-Ciocâlteu reagent was purchased from Merck (Germany). The standard chemicals; chlorogenic acid, p-coumaric acid, caffeic acid, rutin, apigenin, quercetin, isoquercitrin, hyperoside, kaempferol, quercetol, myricetol, fisetin, gallic acid, aucubin, catalpol, and harpagoside were sourced from Sigma-Aldrich (Germany), ferulic acid, sinapic acid, gentisic acid, patuletin, luteolin from Roth (Germany), caftaric acid from Dalton (United States), harpagide, and 8-O-acetyl-harpagide from PhytoLab GmbH & Co. (Germany). HPLC grade solvents (methanol, acetonitrile, ammonium acetate, and silver nitrate) were purchased from Sigma-Aldrich. Distilled, deionised water was produced by a Direct Q-5 Millipore (Millipore SA, Molsheim, France) water system.

Standards of phytochemicals (gallic acid, catechin, harpagoside, cyanidin chloride and ascorbic acid), phenolic acids (gallic acid, 3,5-dihydroxybenzoic acid, protocatechuic acid, chlorogenic acid, gentisic acid, 4-hydroxybenzoic acid, caffeic acid, vanillic acid, syringic acid, 3-hydroxybenzoic acid, 4-courmaric acid, sinapic acid, ferulic acid, 3-courmaric acid, 2-courmaric acid, salicylic acid and trans-cinnamic acid), 1,1-diphenyl-2-picrylhydrazyl, vanillin, aluminum chloride, sodium hydroxide, ferric ammonium sulphate and leucocyanidin were obtained from Sigma-Aldrich Fine Chemicals (St. Louis, MO, USA). Deuterium-labeled standards of 4-hydroxybenzoic acid (2,3,5,6-D4) and salicylic acid (3,4,5,6-D4) were obtained from Cambridge Isotope Laboratories (Andover, MA, USA). Folin–Ciocalteu’s phenol reagent, sodium carbonate, sulphuric acid, butanol, hydrochloric acid, formic acid and methanol were purchased from MERCK (Darmstadt, Germany).

LPS (E. coli 0111: B4), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sulfanilamide, N-(1-naphthyl)-ethylenediamine dihydrochloride, dimethyl sulfoxide (DMSO), harpagoside, cinnamic acid, and TAK-242 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Harpagide was purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China). Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS), and an antibiotic-antimycotic solution were purchased from Invitrogen Co. (Grand Island, NY, USA). A sodium nitrite solution was purchased from Fluka (Buchs, Switzerland). Phosphoric acid was purchased from Junsei (Tokyo, Japan). Gene-specific primers were synthesized by Bioneer (Daejeon, Korea). AMV reverse transcriptase, a dNTP mixture, random primers, RNasin, and Taq polymerase were purchased from Promega (Madison, WI, USA). Goat anti-mouse IgG-HRP, goat anti-rabbit IgG-HRP, goat anti-goat IgG-HRP, and antibodies specific for iNOS, COX-2, IL-1β, NF-κB p50, NF-κB p65, IKK-α/β, p-IKK-α/β, ERK1/2, p-ERK1/2, IRF3, STAT1, p-STAT1, JAK1, and β-actin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Antibodies against TNF-α, IκB-α, p-IκB-α, SAPK/JNK, p-SAPK/JNK, p38, p-p38, p-JAK1, p-IRF3, INF-β, TLR4, and Myd88 were purchased from Cell Signaling Technology (Beverly, MA, USA).