Myricetin

Gallic (3), ellagic (38), protocatechuic (45), neochlorogenic (48), caffeic (49), chlorogenic (50), syringic acids (53), myricitrin (59), isoquercitrin (60), hyperoside (64), kaempferol 3-O-glucoside (67), quercitrin (68), isorhamnetin 3-O-glucoside (72), quercetin (77), myricetin (78), kaempferol (80), (+)-catechin (82), and prunin (87) were obtained from Extrasynthese (Genay, France). All reagents and solvents were of analytical grade.

Wine alcohol 65% v/v was obtained from winery Ouro Verde-Miolo Wine Group (Casa Nova, BA, Brazil). Folin-Ciocalteu reagent, ethanol, potassium persulfate, phosphoric acid, and potassium phosphate monobasic were purchased from Merck (Darmstadt, Germany). Trolox (6-hydroxy-2,5,7,8-tetramethylchromate-2-carboxylic acid) and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and 2,2-azino-bis (3-ethylbenzthiazoline-6 sulfonic acid) (ABTS) were supplied by Sigma-Aldrich (St. Louis, MO, USA). Methanol was from J.T. Baker (Phillipsburg, NJ, USA). Ultrapure water was obtained with a Marte Científica purification system (São Paulo, SP, Brazil). External standards of gallic acid, syringic acid, benzoic acid, p-coumaric acid, o-coumaric acid, cinnamic acid, procyanidin B₁, catechin, procyanidin B₂, and caffeic acid came from Sigma-Aldrich (St. Louis, MO, USA). Epigallocatechin gallate, epicatechin, epicatechin gallate, procyanidin A₂, quercetin 3-glucoside, rutin, kaempferol 3-glucoside, myricetin, and isorhamnetin 3-glucoside, quercetin-3-O-glucopyranoside came from Extrasynthese (Genay, France). The trans-Resveratrol was obtained from Cayman Chemical Company (Michigan, EUA, Ann Arbor, MI, USA).

All chemicals used were of high-performance liquid chromatography (HPLC) analytical grade. Acetonitrile was purchased from from J.T. Baker (Deventer, The Netherlands). Formic acid and orthophosphoric acid (85% w/w) were purchased from Fluka (Buchs, Switzerland) and ethanol from Kemika (Zagreb, Croatia). Phenolic standard compounds of analytical standard grade used for identification and quantification were: delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, peonidin-3-O-glucoside, malvidin-3-O-glucoside, epigallocatechin, procyanidin B1, procyanidin B2, rutin (quercetin 3-O-rutinoside) and myricetin from Extrasynthese (Lyon, France); (–)-epicatechin, (+)-catechin and (–)-and epicatechin-gallate from Sigma-Aldrich (St. Louis, MO, USA); kaempferol, quercetin and isorhamnetin from Fluka (Steinheim, Germany) and quercetin-3-O-glucoside from Sigma (St. Louis, MO, USA).

In total, 1 × 10⁵ clone 1C116 or 2C21 cells, were cultured with each material (concentration ranges are described below) or vehicle control (dimethyl sulfoxide: DMSO) and 20 ng/mL PMA in 96-well U-bottom tissue culture plates in 200 μL of CCM for 24 h. The materials were 0–100 μg/mL flavonoids (quercetin, isoquercitrin, hyperoside, galangin, kaempferol, morin, myricetin, hesperetin, hesperidin, acacetin, linarin, isorhoifolin, (all purchased from Extrasynthese, Lyon, France)), 0–1000 μM IPP (Sigma-Aldrich), 0–2000 μM risedronate, 0–33.5 μM methylamine (Sigma-Aldrich), 0–33.5 μM ethylamine (Sigma-Aldrich) and 1 μg/mL purified anti-human CD3 mAb (OKT3: Thermo Fisher Scientific). After the incubation, the culture supernatant was corrected, and the concentration of IL-2 was analyzed using an ELISA kit (BD Biosciense).

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.

All chemicals and reagents were either analytical-reagent or HPLC grade. Ultrapure deionized water, with a resistivity of 18.2 MΩ cm, was obtained from a Milli-Q^® Integral water purification system with a Q-pod purchased from Millipore (Bedford, MA, USA). Acetic acid, acetonitrile, and 2-propanol were purchased from VWR International S.r.l. (Milan, Italy); hydrochloric acid and sodium hydroxide were purchased from Carlo Erba (Milan, Italy). The methanol HPLC gradient grade was obtained from Merck (Darmstadt, Germany). Standard solutions of vanillic acid, trans-ferulic acid, syringic acid, myricetin, naringenin, pinocembrin, luteolin, and hesperidin were purchased from Extrasynthese (Genay Cedex, France). Chlorogenic acid was purchased from HWI Analytik GmbH (Rülzheim, Germany). Gallic acid, caffeic acid, apigenin, quercetin, kaempferol, catechin, epicatechin, indometacine, histamine, and anti-α-tubulin antibody were purchased from Sigma-Aldrich S.r.l. (Milan, Italy). The IFN-γ was obtained from Pepro Tech EC (London, England). The anti-ICAM-1 antibody (sc-51632) was purchased from Santa Cruz Biotechnology (Milan, Italy), anti-COX-2 (35-8200) from Thermo Fisher Scientific (Milan, Italy), and -α- tubulin (T6074) from Sigma-Aldrich (Milan, Italy).

The following materials were purchased from the suppliers indicated in parentheses: chlorogenic acid (MP Biomedicals); gossypetin (Indofine Chemical Company); butein, eriodictyol, formononetin, herbacetin, isorhamnetin, kaempferol, myricetin, and quercetin (Extrasynthèse); daidzein and genistein (LC Laboratories); vestitol (Plantech); apigenin, biochanin A, coniferyl alcohol, o-coumaric acid, m-coumaric acid, coumestrol, 5-hydroxyferulic acid, luteolin, naringenin, phloretic acid, sinapic acid, and umbelliferone (Merck); p-coumaric acid, erythronic acid, isoferulic acid, and succinic anhydride (TCI); caffeic acid, trans-cinnamic acid, 3,4-dimethoxycinnamic acid, ferulic acid, p-methoxycinnamic acid, phenylalanine, tetronic acid, and L-tyrosine (FUJIFILM Wako Pure Chemical). Umbellic acid was prepared from umbelliferone by a treatment with 1 M NaOH at 90°C for 1 h. Isoliquiritigenin was obtained from our laboratory stock (Shimamura et al., 2007 (link)).