Hesperetin

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).

All solvents used for solid-phase extraction and HPLC-DAD analysis were LC-MS grade from either Sigma-Aldrich (Madrid, Spain) or VWR Chemicals (Barcelona, Spain). Luteolin, diosmetin, chrysoeriol, eriodictyol, hesperetin, homoeriodictyol, and homohesperetin were provided by Extrasynthese (Genay, France).

Acetonitrile (99.9%) was of high-performance liquid chromatography (HPLC) grade from Fisher Scientific (Lisbon, Portugal). Phenolic compound standards (caffeic acid, ferulic acid, hesperetin, naringenin, quercetin-3-O-glucoside and quercetin-3-O-rutinoside) were from Extrasynthèse (Genay, France). Formic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other general laboratory reagents were from Panreac Química S.L.U. (Barcelona, Spain). Water was treated in a Milli-Q water purification system (TGI Pure Water Systems, Greenville, SC, USA).

Acetonitrile 99.9% was of HPLC grade from Fisher Scientific (Lisbon, Portugal). Phenolic compound standards (caffeic acid ≥ 99%, p-coumaric acid ≥ 90%; hesperetin ≥ 99%, luteolin-7-O-glucoside ≥ 99%, quercetin-3-O-glucoside ≥ 99%, rosmarinic acid ≥ 99% HPLC purity) were from Extrasynthese (Genay, France). Trypan blue, lipopolysaccharide (LPS), dexamethasone, acetic acid, formic acid, ellipticine, sulforhodamine B (SRB), trichloroacetic acid (TCA) and Tris were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium was purchased from HyClone. RAW264.7 cells were acquired from ECACC (‘‘European Collection of Animal Cell Culture”) (Salisburg, UK), Griess reagent system kit was purchased from Promega, Fetal bovine serum (FBS), L-glutamine, Hank’s balanced salt solution (HBSS), trypsin-EDTA (ethylenediaminetetraacetic acid), penicillin/streptomycin solution (100 U/mL and 100 mg/mL, respectively), RPMI-1640 and DMEM media were from Hyclone (Logan, UT, USA). Water was treated in Milli-Q water purification system (TGI Pure Water Systems, Greenville, SC, USA).

All the solvents and chemicals used in this study were analytical grade. Methanol, ethanol, acetonitrile LC-MS grade, formic acid LC-MS grade, 85% phosphoric acid, and water LC-MS grade were purchased from Merck^® (Darmstadt, Germany). Gallic acid, quinic acid, caffeic acid, ferulic acid, protocatechuic acid, neochlorogenic acid (3-O-caffeoylquinic acid), chlorogenic acid (5-O-caffeoylquinic acid), cynarin (1,3-dicaffeoylquinic acid), isochlorogenic acid, kaempferol-3-O-glucoside, quercetin-3-O-glucoside, ferrous sulfate, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH^●), (±)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tris(2-pyridyl) -1,3,5-triazine (TPTZ), 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), Folin–Ciocalteu reagent, sodium carbonate, ferric chloride, AlCl₃, NaNO₂, and CuSO₄∙5H₂O were purchased from Merck^®, Sigma-Aldrich^®, or Fluka™ (Milan, Italy). Flavonoid standards of apigenin, apigenin-7-O-glucoside, hesperidin, hesperetin, luteolin-7-O-glucoside, naringin, and naringenin were purchased from Extrasynthese (Genay, France). Ultrapure water (18 MΩ·cm) was obtained with a Milli-Q Advantage A10 System apparatus (Millipore, Milan, Italy).