Ferulic acid

Sodium potassium l(+)-tartrate tetrahydrate was obtained from VWR Chemicals (Vienna, Austria). Potassium iodide was obtained from BDH Prolabo Chemicals (Leuven, Belgium). Iodine, sodium hydroxide (1 and 0.1 M), sodium thiosulfate (0.1 M) and sodium hydroxide pellets were obtained from Gram-mol (Zagreb, Croatia). Sulphuric acid (96%), starch (p.a., soluble) and phenolphthalein were obtained from Kemika (Zagreb, Croatia). Bromothymol blue was obtained from Merck (Darmstadt, Germany). (+)-Catechin, (-)-epicatechin, 2,5-dihydroxybenzoic acid (2,5-DHBA), 3,4-dihydroxybenzoic acid (3,4-DHBA), 3-hydroxytyrosol, caffeic acid, ellagic acid, quercetin, naringenin, luteolin-7-O-glucoside, pinobanksin, p-coumaric acid and syringic acid were obtained from Sigma-Aldrich, Merck (St. Louis, MO, USA). Gallic acid was obtained from Alfa Aesar (Thermo Fischer Scientific, Tewksbury, MA, USA). Ferulic acid and resveratrol were obtained from Extrasynthese (Genay, France). Honeywell research chemicals (Charlotte, NC, USA) supplied ethanol (HPLC grade) and acetonitrile (LC-MS grade). Sigma-Aldrich supplied formic acid (LC-MS grade), ultrapure water (LC-MS grade) and ethanol (96%).

As samples for TLC, O. regalis extract and standards were used. They were applied to Silica gel 60 F264 plates (Merckmillipore.com), according to standard procedures [11 ]. As solvents ethyl acetate - formic acid - acetic acid - water (100:11:11:27) for flavones or toluene - ethyl acetate - formic acid (50:40:10) for polyphenolcarboxylic acids were used. UV detection was performed at 365 nm.
The following standards were used: ferulic acid, apigenin (Extrasynthese, Genay, France), chlorogenic acid (Carl Roth, Rothenfels, Germany), rosmarinic acid and rutoside (Sigma Aldrich, St. Louis, Missouri, USA).

Rosmarinic acid and the 7-O-glucoside derivatives of apigenin, luteolin, eriodyctiol, quinic acid, caffeic acid, 5-O-caffeoylquinic acid, 4-O-hydroxybenzoic acid, salvianolic acid B, caffeic acid, ferulic acid, and 4-O-coumaric acid were obtained from Extrasynthese (Genay Cedex, France). Trolox, sulforhodamine B (SRB), DPPH (2,2-diphenyl-1-picrylhydrazyl) radical, acetic acid, ellipticine, trypan blue, trichloroacetic acid (TCA), Tris, lipopolysaccharide (LPS), carnosol, nisin, ascorbic acid, and butylated hydroxyanisole (BHA) were obtained from Sigma Chemical Co (St Louis, MO, USA). Fetal bovine serum (FBS), L-glutamine, trypsin-ethylenediaminetetraacetic acid (EDTA), penicillin/streptomycin solution (100 U/mL and 100 mg/mL, respectively), Roswell Park Memorial Institute (RPMI) 1640 Medium and Dulbecco’s Modified Eagle Medium (DMEM) were from Hyclone (Logan, Utah, UT, USA). The Griess reagent system was purchased from Promega Corporation (Madison, WI, USA). Mueller-Hinton agar was from VWR (Prolabo Chemicals, USA); formic acid and ethanol was from Panreac (Barcelona, Spain). n-Hexane, methanol, and acetonitrile were purchased from Lab-Scan (Lisbon, Portugal). Purified water was obtained from a Direct-Q^® water purification system (Merck Life Science, Darmstadt, Germany).

Methanol of analytical grade purity was from Lab-Scan (Lisbon, Portugal). The culture media Brain Heart Infusion Agar (BHIA), Mueller Hinton Broth (MHB), Mueller Hinton Agar (MHA), and all the antibiotics were obtained from Oxoid (Hampshire, UK). The saline solution was prepared with NaCl from Merck (Darmstadt, Germany). The dye resazurin and crystal violet were purchased from Sigma-Aldrich (St Louis, Mosby, USA). All the organic solvents were HPLC grade. Ultrapure water from purified system (Isopad Isomantle, Gemini BV, Pr Beatrixlaan 301, 7312 DG Apeldoorn, Netherlands) was used. External standards caffeic acid, catechin, chlorogenic acid, ferulic acid, gallic acid, (-)-epicatechin, naringin, p-hydroxybenzoic acid, quercitrin, and rutin were purchased from Extrasynthese (Lyon Nord, Genay Cedex, France). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), non-essential amino acids, penicillin, streptomycin, and trypsin–EDTA were obtained from Invitrogen Corporation (Life Technologies, SA, Madrid, Spain).

The following chemicals were used: methylglyoxal (40% in water), 2,2–diphenyl–1–picrylhydrazyl,2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid, trolox, metformin hydrochloride, 98–100% formic acid, methanol (HPLC grade), acetonitrile (HPLC gradient grade and LC–MS grade), and water (LC–MS grade) were purchased from Merck–Sigma–Aldrich (Sigma–Aldrich Sp. z o.o., Poznań, Poland); NaCl, KCl, Na₂HPO₄ and KH₂PO₄ (reagent grade) were obtained from Chempur (Piekary Śląskie, Poland); quercetin, quercitrin, astragalin, hyperoside, rutin, nicotiflorin, narcissin, taxifolin, gallic acid, p-coumaric acid, ferulic acid, caffeic acid, and chlorogenic acid were from Extrasynthese (Genay Cedex, France); galegine sulfate was purchased from SelectLab (Münster, Germany). Water was glass-distilled and deionized.
The stock solutions of standards (1 mg/mL) were prepared by dissolving 5 mg of a reference compound in 5 mL of methanol. Working standard solutions in the range of 10–400 μg/mL were made by mixing with 50% aq. (aqueous) methanol (v/v), filtered through hydrophilic Millex Syringe Filters (Durapore 0.22 μm; Millipore, Burlington, MA, USA) and stored at −20 °C.

HPLC-grade methanol, acetonitrile, and formic acid were obtained from Wako Pure Chemical Industries (Osaka, Japan). Phenolic compound standards for rutin, catechin, chlorogenic acid, benzoic acid, ferulic acid, and quercetin were procured from Extrasynthese (Genay, France). Cyanidin 3-O-rutinoside and cyanidin-3-O-glucoside were purchased from Fujicco Co., Ltd. (Kobe, Japan). Other routine reagents and chemicals were procured from Sigma–Aldrich (St. Louis, MO, USA).

Methanol and ethanol of analytical grade (Fisher Chemical, Loughborough, UK) were used as extraction solvents. Folin-Ciocalteu, gallic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2-diphenyl-1-picrylhydrazyl (DPPH), sodium carbonate, potassium persulfate, 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), acetic acid, hydrochloric acid, ferric chloride, acetonitrile, and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St. Louise, MO, USA). All the organic solvents were HPLC grade. Ultrapure water from a purified system (Isopad Isomantle, Gemini BV, Pr. Beatrixlaan 301, 7312 DG Apeldoorn, The Netherlands) was used. External standards (purity ≥ 99%) caffeic acid, catechin, chlorogenic acid, diosmetin, ferulic acid, glycitin, isorhamnetin, (-)-epicatechin, luteolin-7-O-glucoside, luteolin-4′-O-glucoside, myricetin, naringin, p-hydroxybenzoic acid, quercetin, and rutin were purchased from Extrasynthese (Lyon Nord, Genay Cedex, France). Hispidin (purity ≥ 98%) was purchased from Sigma-Aldrich.