4 hydroxybenzoic acid

Chemical reagents (acetonitrile, L-ascorbic acid, methanol, acetic acid, and formic acid) were purchased from Sigma-Merck, Poznań, Poland. For the qualitative and quantitative content of polyphenols the following standards were used: gallic acid, caffeic acid, isovanilic acid, chlorogenic acid, neochlorogenic acid, p-coumaric acid, 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, orientin (luteolin-8-glucoside), quercetin, quercetin-3-O-glucoside, procyanidin B2, luteolin, kaempferol (Sigma-Aldrich, Poznań, Poland), cyanidin-3-O-glucoside, cyanidin-3-O-galactoside, cyanidin-3-O-arabinoside (Polyphenols Laboratories AS, Sandnes, Norway), cyanidin-3-O-xyloside (Toronto Research Chemicals, North York, ON, Canada), quercetin-3-O-galactoside, quercetin-3-O-vicianoside, quercetin-3-O-rutinoside, (+)-catechin and (−)-epicatechin (Extrasynthese, Genay, France). All other chemicals were of analytical grade.

All standards and reagents were LC-MS grade and were used without any further treatments. Authentic standards of 2,5-Dihydroxybenzoic acid (gentisic acid), 3,4-dihydroxybenzoic acid (protocatechuic acid), 4-hydroxybenzoic acid, cinnamic acid, epicatechin, eriodictyol, ferulic acid, gallic acid, myricetin, p-coumaric acid, pinoresinol, quercetin, resveratrol, rosmarinic acid, salicylic acid, syringic acid, taxifolin, and vanillic acid were purchased from Sigma-Aldrich (Stenheim, Germany). Apigenin, caffeic acid, catechin, ethyl vanillin (internal standard, IS), galangin, genistein, naringenin, tyrosol, and vanillin were acquired from Alfa Aesar (Karlsruche, Germany), whereas hydroxytyrosol and luteolin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
2-Propanol (LC–MS grade) and methanol (MeOH) (LC–MS grade) were purchased from Fisher Scientific (Geel, Belgium) and Merck (Darmstadt, Germany), respectively. Formic acid 99%, ammonium acetate and sodium hydroxide monohydrate for trace analysis ≥ 99.9995% were all obtained from Fluka (Buchs, Switzerland). Ultrapure water (18.2 MΩ resistivity) was provided by a Millipore Direct-Q UV purification System (Millipore, Bedford, MA, USA). The wine samples were filtered using regenerated cellulose syringe filters (RC filters, pore size 0.2 μm, diameter 15 mm) purchased from Phenomenex (Torrance, CA, USA).

Reagents: Folin-Ciocalteu, sodium persulfate, gallic acid (GA), BHT, 2,2′-azinobis-3-ethylbenzothiazoline-6-sulphonic acid, diammonium salt (ABTS), Trolox, ferrous chloride, and 2,2-diphenyl-1-picrylhydrazyl (DPPH), were purchased from Sigma-Aldrich (Athens, Greece); 2,4,6-Tris (2-pyridyl)-s-triazine (TPTZ) was purchased from Fluka Chemica (Athens, Greece). Dimethylsulfoxide (DMSO), (Merck KGaA, Darmstaadt, Germany) was used as a solvent. Water was obtained from a Milli-Q water purification system (TGI Pure Water Systems, Topway Global, Greenville, SC, USA). Lichrosolv hypergrade for LC-MS acetonitrile was supplied by Merck (Darmstadt, Germany). Water (18.2 MΩ) was from a Milli-Q water system (Millipore, Bedford, MA, USA). Acetic acid was from LGC Standards (Middlesex, UK). Eighteen standard phenolic compounds were used (gallic acid, protocatechuic acid, HydroxyTyrosol, 4-hydroxybenzoic acid, chlorogenic acid, gentisic acid, caffeic acid, vanillic acid, syringic acid, p-coumaric acid, rutin, ferulic acid, trans-m-hydroxycinnamic acid, o-coumaric acid, salicylic acid, luteolin, eriodictyol and cinnamic acid) purchased from Sigma Aldrich.

Most of the chemicals used for extraction and characterization were analytical grade and purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). The standards for antioxidant assays included gallic acid, quercetin, catechin and L-ascorbic acid, which were purchased from Sigma-Aldrich (St. Louis, MO, USA). For antioxidant assays, Folin and Ciocalteu’s phenol, aluminium chloride hexahydrate, vanillin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric (III) chloride anhydrous, 2,4,6-tripyridyl-s-triazine (TPTZ), potassium persulfate and 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulphonate) (ABTS) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and sodium carbonate anhydrous (chem-supply; Gillman, SA, Australia), sodium acetate hydrated (Ajax Finechem, Scoreby, VIC, Australia) and sulfuric acid 98% were purchased from RCI Labscan Limited, (Bangkok, Thailand). The HPLC grade methanol was purchased from Fisher Chemical Company (San Jose, CA, USA). The mobile phases for LC-ESI-QTOF/MS and HPLC were mainly comprised of 50% acetic acid solution (Sigma-Aldrich, St. Louis, MO, USA) and acetonitrile (LiChrosolv, Darmstadt, Germany). The reference standards including protocatechuic acid, catechin, 4-hydroxybenzoic acid, chlorogenic acid, quercetin 3-O-glucoside, kaempferol 3-O-glucoside and quercetin were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Softwood kraft lignin (KL) with a 20% moisture content was received from FPInnovations. [2-(Methacryloyloxy)ethyl] trimethylammonium chloride solution (METAC, 80 wt % in H₂O), potassium persulfate (K₂S₂O₈), 4-hydroxybenzoic acid, sulfuric acid (H₂SO₄, 98%), hydrochloric acid, sodium hydroxide (NaOH, 97%) and deuterium oxide (D₂O) were purchased from Sigma-Aldrich (Markham, ON, Canada). All chemical reagents were used as received. Ethanol (95 vol %) and anionic polyvinyl sulfate (PVSK) were purchased from Fisher Scientific and Wako Pure Chem. Ltd., Japan.

For the online ABTS with HPLC-DAD and liquid chromatography-mass spectroscopy (LC-MS), the following standards were used: naringenin, gallic acid, catechin hydrate, caffeic acid, chlorogenic acid, ferulic acid, rutin, luteolin, sinapic acid, trans-cinnamic acid, 4-hydroxybenzoic acid, phenylisothiocyanate, quercetin, ellagic acid, tyramine, pyrogallol, and vanillic acid, which were purchased from Sigma Aldrich (Sydney, Australia). Epicatechin and kaempferol were obtained from Extrasynthese (Genay, France).

Amino acid standard kit, caffeic acid, cinnamic acid, chlorogenic acid, p-coumaric acid, gallic acid, gentisic acid, trans-ferulic acid, salicylic acid, sinapic acid, syringic acid, vanillic acid and 4-hydroxy benzoic acid were purchased from Sigma Aldrich, Bengaluru, India. β-Mercaptoethanol, PMSF, Tris-HCl, ammonium acetate, phenol, bovine serum albumin, glucose, fructose and sucrose were purchased from SRL, Mumbai, India. Serum glutamate pyruvate transaminase (GPT), serum glutamate oxaloacetate transaminase (GOT), alkaline phosphatase (ALP), serum urea, triglycerides, total cholesterol, high-density lipoprotein cholesterol and total protein were determined using commercially available kits (SPAN Diagnostics Ltd., Surat, India).