Taxifolin

Human hepatocellular carcinoma cells HepG2 and HuH7 and kidney epithelial cells Vero were obtained from National Control Laboratory for Biologicals (National Institute of Health Islamabad). All cell lines were grown in DMEM (Gibco by Life Technologies) supplemented with 10% Fetal Bovine serum (FBS) (Gibco by Life Technologies) and 1% penicillin-streptomycin with 5% CO₂ at 37 °C. Taxifolin (Sigma Aldrich) dissolved in Dimethyl sulfoxide (0.1% (v/v)) was employed for the treatment of HepG2 and Huh7 cells at a confluency of ∼70%. Cells were treated with Taxifolin at 0.1 µM, 0.125 µM, 0.15 µM, 0.20 µM and 0.22 µM for 24 h.

CaCl₂, NaCl, NaOH, calcein, Triton X-100, Sephadex G-50, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), dimethylsulfoxide (DMSO), and plant polyphenols: chalcones (4′-hydroxychalcone (≥99%, high-performance liquid chromatography (HPLC)), cardamonin (≥98%, HPLC), isoliquiritigenin (≥98%, HPLC)), dihydrochalcones (phloretin (≥99%, HPLC)), stilbenes (resveratrol (≥99%, HPLC), piceatannol (≥98%, HPLC)), isoflavones (daidzein (≥98%, HPLC), biochanin A (≥95%, HPLC), genistein (≥98%, HPLC), genistin (≥95%, HPLC)), flavanones (liquiritigenin (≥97%, HPLC), naringenin (≥95%, HPLC)), flavan-3-ols (catechin (≥98%, HPLC)), flavononols (taxifolin (≥90%, HPLC)), flavonols (quercetin, myricetin, and rutin), and lignans (honokiol (≥98%, HPLC)) were purchased from Sigma-Aldrich Company Ltd. (Gillingham, United Kingdom).
Lipids synthetic 1,2-dioleoyl-sn-glycero-3-phospho-(1-rac-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1-rac-glycerol) (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol (CHOL) were obtained from Avanti Polar Lipids (Avanti Polar Lipids, Inc., Alabaster, AL, USA).

Neochlorogenic acid, gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, p-hydroxybenzoic acid, 1,3-dicaffeoylquinic acid, p-coumaric acid, rutin, quercetin 3-β-galactoside, ferulic acid, taxifolin, 3,4-dicaffeoylquinic acid, trans-m-coumaric acid, quercetin 3-α-L-rhamnoside, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, rosmarinic acid, myrcetin, luteolin, quercetin, trans-cinnamic acid, apigenin, kaempferol, fenofibrate, dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), hematoxylin, eosin, and trizma base were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), streptomycin, and penicillin were purchased from Invitrogen (Carlsbad, CA, USA). Other reagents were commercially available and of special grade.

Methanol (LC-MS grade) and sodium hydroxide (>99%) were purchased from Merck (Darmstadt, Germany). Ammonium acetate (≥99% for HPLC) and formic acid (LC-MS Ultra) were purchased from Fluka (Buchs, Switzerland). Isopropanol was obtained from Fisher Scientific (Geel, Belgium). Ultrapure water was provided by a Milli-Q purification apparatus (Millipore Direct-Q UV, Bedford, MA, USA). Regarding the standards that were used, syringic acid 95% was purchased from Extrasynthèse (Genay, France). Gallic acid 98%, ferulic acid 98%, epicatechin 97%, p-coumaric (4-hydroxycinnamic acid) 98%, homovanillic acid 97%, quercetin 98%, oleuropein 98%, pinoresinol 95%, caffeic acid 99%, taxifolin 98%, vanillic acid 97% and syringaldehyde 98% (internal standard) were obtained from Sigma-Aldrich (Steinheim, Germany). Hydroxytyrosol 98% and luteolin 98% were acquired from Santa Cruz Biotechnologies. Vanillin 99%, apigenin (4,5,7-trihydroxyflavone) 97% and tyrosol (2-(4-hydroxyphenyl) ethanol) 98% were purchased from Alfa Aesar (Karlsruhe, Germany). Stock standard solutions of individual compounds (1000 mg/L) were solubilized in methanol and stored at −20 °C in dark brown glass bottles. Intermediate standard working solutions containing the analytes were prepared by appropriate dilution of the stock solutions with methanol:water (80:20, v/v) over the concentration range 0.1–8 mg/L.

Formic acid, dimethylformamide (DMF), acetonitrile, taxifolin, and ammonium acetate were purchased from Sigma‐Aldrich (Poole, UK). The (‒)‐epicatechin reference standard was purchased from Toronto Research Chemicals (Canada). The reference standards 5‐(3′,4′‐dihydroxyphenyl)‐γ‐valerolactone (3,4DHVL) and 5‐(3′‐methoxy‐4′‐hydroxyphenyl)‐γ‐valerolactone (4H3MVL) were synthesized in‐house as described by Chang et al.^[15] The phase‐2 conjugates, specifically 5‐(3′‐hydroxyphenyl)‐γ‐valerolactone‐4’‐glucuronide (3HGV4‐glucuronide), 5‐(4′‐hydroxyphenyl)‐γ‐valerolactone‐3’‐glucuronide (4HVL3‐glucuronide), 5‐(3′‐hydroxyphenyl)‐γ‐valerolactone‐4’‐sulfate (3HGV4‐sulfate), and 5‐(4′‐hydroxyphenyl)‐γ‐valerolactone‐3’‐sulfate (3HGV4‐sulfate) were synthesized in‐house as described in the Supporting Information.

Dimethyl sulfoxide (DMSO), elastase from human leukocytes, N-succinyl-Ala-Ala-Ala-p-nitroanilide, collagenase from Clostridium histolyticum (Type I), collagen-fluorescein, oleanolic acid, hyaluronidase from bovine testes (Type I-S), hyaluronic acid sodium salt, 4-(dimethylamino)benzaldehyde (DMAB), 1,10-phenanthroline (1,10-Ph), taxifolin, quercetin and other chemicals were from Sigma-Aldrich (Prague, Czech Republic). Methanol (HiPerSolv CHROMANORM for HPLC, LC-MS grade) was from VWR International s.r.o. (Stříbrná Skalice, Czech Republic). All solutions were prepared using reverse-osmosis deionized water (Ultrapur, Watrex, Prague, Czech Republic). Nitrogen and helium (99.999% for all) were obtained from Linde Gas (Prague, Czech Republic).

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).