Isoquercetin

Standards of (+)-catechin, malvidin-3-glucoside, caffeic acid, quercetin, iso-quercetin, and gallic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The purity grade of all standards was greater than 99%.
Porcine amylase, porcine pepsin, porcine pancreatin, and fetal bovine serum were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Formic acid (HPLC-grade), acetonitrile (HPLC-grade), ethanol, Dulbecco’s Modified Eagle Medium (DMEM)culture medium, methyl thiazolyl tetrazolium, EDTA, penicillin, streptomycin, DPPH, acetic acid, TPTZ, FeCl₃, ABTS, and potassium persulfate were purchased from a commercial reagent company (Omic Inc., Beijing, China). In addition to HPLC-grade reagents, all of the other reagents were of analytical grade. Ultrapure water was obtained from a Milli-Q water purification system (Millipore Corp., Bedford, MA, USA).

Lyophilised and powdered samples (about 250 mg) were extracted with 2 mL of methanol using sonication (10 min at 25 ± 2 °C) and centrifuged for 10 min at 15,000 rpm. The supernatant was filtered through 0.22 μm filter (Millex^®GP, Millipore, Merck, Darmstadt, Germany). RP-HPLC analyses was carried out according to Simlat et al.^{34 (link)} using a Merck–Hitachi liquid chromatograph (LaChrom Elite, Hitachi, Tokyo, Japan) equipped with a DAD detector L-2455 and Purospher^®RP-18e (250 × 4 mm/5 mm) column (Merck, Darmstadt, Germany). Quantification was carried out at λ = 254 nm for phenolic acids and λ = 370 nm for flavonoids. Identification was performed through a comparison of the retention times of the peaks with authentic reference compounds and co-chromatography with standards. Quantification consisted of measurement of the peak area with reference to the standard curve derived from five concentrations (0.03125–0.5 mg ml⁻¹). Commercially available standards of the phenolic acids: chlorogenic, neochlorogenic (Sigma–Aldrich, St Louis, MO, USA), isochlorogenic A (ChromaDex, Irvine, CA, USA), and rosmarinic (Sigma–Aldrich, St Louis, MO, USA), as well as the flavonoids isoquercetin, and quercitrin (Sigma–Aldrich, St Louis, MO, USA), were used to generate the calibration curves. The analysis was repeated three times.

Analytical standards (Zeaxanthin, Lutein, β-carotene, Gallic acid, Protocatechuic acid, 4-Hydroxy-benzoic acid, Vanillic acid, Chlorogenic acid, Caffeic acid, p-coumaric acid, Ferulic acid, Sinapic acid, Rutin, Kaempferol, IsoQuercetin, Astragalin, Myricetin, Quercetin, α-tocopherol, γ-tocopherol, δ-tocopherol, 5-methyltetrahydrofolate, tetrahydrofolate, 5-formyltetrahydrofolic acid, l-ascorbic acid) were obtained from Sigma Aldrich (Poznan, Poland). 10-formylfolic acid and pteroyltri-γ-L-glutamic acid were obtained from Schirck’s Laboratories (Jona, Switzerland). α-amylase obtained from Sigma Aldrich (Poznan, Poland) and γ-glutamyl hydrolase obtained from rat blood plasma (Europa Bioproducts Ltd., Cambridge, UK). Other reagents (acetonitrile, triethylamine, ethyl acetate, pyrogallol, ethanol, potassium hydroxide, orthophosphoric acid, methanol, nitric acid, lanthanum chloride, potassium dihydrogen phosphate) were purchased from Merck (Warsaw, Poland).

Ethanol and sodium carbonate were purchased from Avantor Performance Materials Poland (Gliwice, Poland). Gallic, m-coumaric, o-coumaric, p-coumaric, caffeic, 5-O-caffeoylqunic, ferulic, isoferulic, vanillic, salicylic, 3-hydroxybenzoic, 4-hydroxybenzoic, protocatechuic, syringic, rosmarinic acid, quercitrin, apigenin 7-O-glucoside, eriocitrin, taxifolin, 3-O-methylquercetin, isorhamnetin, isorhamnetin-3-O-glucoside, luteolin, kaempferol, rutin, hyperoside, isoquercetin, sakuranetin, aluminum chloride, and LC–MS grade acetonitrile were purchased from Sigma-Aldrich Chemical Co. (St. Louis, Mo, USA). Folin–Ciocalteu reagent was supplied by Chempur (Piekary Śląskie, Poland). Quercetin was purchased from Fluka (Buchs, Switzerland). Catechin, naringenin 7-O-glucoside, eriodictyol, luteolin-7-O-glucoside, naringenin 7-O-glucoside, luteolin 3,7-diglucoside, nicotiflorin, narcissoside, gentisic, sinapic acid, and myricetin were provided by ChromaDex (Irvine, CA, USA). Astragalin, apigenin, naringenin, kaempferol-3-rutinoside, and tiliroside were supplied by Roth (Karlsruhe, Germany). LC–MS grade water was prepared using a Millipore Direct-Q3 purification system (Bedford, MA, USA).

Standards of the phenolic compounds 2-OH cinnamic acid, 4-OH benzaldehyde, apigenin, caffeic acid, catechin, chlorogenic acid, emodin, epicatechin, gallic aicd, genistein, glycitein, hesperidin, homoorientin, isoquercetin, isovitexin, isorhamnetin, kaempferol, luteolin, n-feruloyl octopamine, naringenin, neochlorogenic acid, mauritianin, miquelianin, orientin, p-coumaric acid, pinocembrin, quercetin, quercitrin, rhamnetin, rutin, salicylic acid, taxifolin, umbelliferone, vitexin, and the internal standard probenecid and verapamil hydrochloride were purchased from Sigma–Aldrich (St. Louis, MO, USA). Methanol (LC-MS grade, ≥99.9%) was obtained from Riedel de Haën (Seelze, Germany). Formic acid (LC-MS grade, 99%) was purchased from VWR (Leuven, Belgium). Pure water was attained from a Milli-Q purification system (Millipore, Bedford, MA, USA).

Ultra-pure water (H₂O) was obtained by a Milli-Q Direct 8 system (Millipore, Milan, Italy), methanol, acetonitrile (ACN) and formic acid (HCOOH) LC-MS grade were purchased from Sigma Aldrich (Milan, Italy). For the quantitative and qualitative analysis of flavonoids two columns were employed respectively: a Kinetex C18 150 × 4.6 mm (100 Å), packed with 2.6 µm particles, and a Kinetex C18 150 × 2.1 mm, 2.6 µm column (Phenomenex, Bologna, Italy). Both columns were protected with C18 precolumns (Phenomenex).
Flavonoids standards (diosmetin 6,8 di C-glucoside, neohesperidin, eriocitrin, isoquercetin, narirutin, diosmetin, hesperetin) and polymetoxyflavones (tangeretin) were purchased from Sigma Aldrich. For cell culture unless stated otherwise, all reagents and compounds were purchased from Sigma Chemicals Company (Milan, Italy).