Eriocitrin

Ultra-pure water (18 mΩ) was obtained from a Milli-Q water purification system (Millipore Co., Ltd., Milford, MA, USA). High-performance liquid chromatography (HPLC)-grade methanol, acetic acid, and 56 analytical standards, including catechinic, scopolin, chlorogenic acid, epicatechinic, vanillic acid, caffeic acid, purerarin, syringic acid, daidzin, glycitin, scopoletin, eriocitrin, umbelliferone, p-coumaric acid, dihydroquercetin, sinapic acid, genistin, liquiritin, ferulic acid, salicylic acid, rutin, isoferulic acid, m-coumaric acid, naringin, hesperidin, resveratrol, xanthotoxol, silydianin, sinapyl alcohol, o-coumaric acid, liquiritigenin, kaempferol, 2’-hydroxygenistein, eriodictyol, daidzein, psoralen, glycitein, quercetin, didymin, bergaptol, naringenin, luteolin, cinnamic_acid, hesperetin, genistein, bergapten, diosmetin, isoliquiritigenin, coumestrol, sinensetin, formononetin, medicarpin, imperatorin, biochanin A, tangeretin, and rotenone (displayed in Table S2), were purchased from Sigma-Aldrich Co., Ltd. The stock solutions of these authentic standards were 10.0 mg of each standard dissolved in 10 mL methanol. Then, the stock solutions were diluted to various concentrations before analysis. All stock solutions were sealed with Parafilm^® and stored in a −20 °C freezer.

All organic solvents were HPLC grade and purchased from Mallinckrodt Baker (St. Louis, MO, USA). The water used for the mobile phase preparation was purified by a Milli-Q purification system (Millipore, São Paulo, Brazil). The formic acid (≥95%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade. Flavonoid and organic acids standards, hesperidin (hesperetin 7-O-rutinoside), eriocitrin (eriodictyol 7-O-rutinoside), narirutin (naringenin 7-O-rutinoside), naringin (naringenin 7-O-neohesperidoside), rutin (quercetin 3-O-rutinoside), naringenin (4′,5,7-trihydroxyflavanone), quercetin (3,5,7,3′, 4′-pentahydroxyflavone), caffeic acid (3,4-dihydroxycinnamic acid) and gallic acid (3,4,5-trihydroxybenzoic acid) (≥97.0%) were all HPLC grade and purchased from Sigma-Aldrich. The standard and stock solutions were stored at −20 °C during the analyses. The mobile phases were prepared in a volume/volume ratio.

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).

Eriocitrin (≥98.0%, HPLC), dimethyl sulfoxide, gelatin, glutamine, acridine orange, ethidium bromide, and medium 199 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS), trypsin-EDTA, and fetal bovine serum (FBS) were acquired from Moregate (Brisbane, Australia). MCDB-104 was purchased from Wako Pure Chemicals Industries (Osaka, Japan). Atelocollagen bovine dermis (type I collagen) was purchased from Koken (Tokyo, Japan). Epidermal growth factor (EGF) was purchased from BD Bioscience (Bedford, MA, USA) and human basic fibroblast growth factor (bFGF) (recombination) was purchased from Australia Biologicals (San Ramon, CA, USA). Phospho- or total forms of c-Raf, MEK 1/2, ERK 1/2, VEGFR2, PI3K, AKT, mTOR, caspase-9, caspase-3, PARP, and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA). MMP-2 and MMP-9 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Lipid emulsion and fertilized chicken eggs were acquired from Dongkook Pharmacy Co. in Seoul, Korea and Pulmuone Farm in Bonghwa, Korea, respectively. Other chemicals were all bought from Sigma-Aldrich (St. Louis, MO, 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).

Flavonoids were extracted and purified from pulp tissue as follows. Briefly, pulp tissue (0.5 g) was extracted with 5 mL of Milli-Q water centrifuged at 3000 g for 30 min at 4 °C. Next, the extract was subsequently filtered through a 0.45-micrometer PVDF filter (13 mm diameter, Análisis Vínicos, Tomelloso, Ciudad Real, Spain). Flavonoid content was determined by HPLC-DAD. Separation of flavonoids was carried out with a XBridgeTM BEH C18 (4.6 × 150-millimeter, 2.-micrometer Column XP), maintained at 40 °C, and a secondary gradient elution with water 0.1% TFA (solvent A) and acetonitrile 0.1% TFA (solvent B) at a flow rate of 1 mL/min as follows. From 0 to 5 min 100% A, 5 to 30 min 95% A and 5% B, 30 to 33 min 50% A and 50% B and from 33 to 37 min 100% B. Wavelength was registered from 220 to 550 nm and flavonoids were quantified at 280 nm. Identification and quantification of the different flavonoids was achieved by comparison with the retention times and peak areas of authentic standards of hesperidin, narirutin, naringin, eriocitrin, dydimin and rutin (Sigma-Aldrich, Barcelona, Spain). Concentrations are expressed as mg/100 g of fresh weight. Samples were extracted twice and the results are the mean of two replicates (mean ± SD).

N,N-dimethylformamide (anhydrous, 99.8%), 2,2-diphenyl-1-picrylhydrazyl (DPPH), ascorbic acid (≥99.0%) , flavonoids analytical standard with purity ≥97.0% (neoeriocitrin, eriocitrin, narirutin, naringin, hesperidin, neohesperidin), limonoid aglycones analytical standard with purity ≥95.0% (limonin, nomilin), and the HPLC-grade solvents were purchased from Sigma Chemical Co. (St. Louis, MO, USA). N,N-dimethyl-L-proline (≥95.0%) was purchased from Extrasynthese (Genay, France). Analytical-grade water (resistivity ≥18 MΩ cm) and all other solvents and reagents of analytical grade were obtained from Carlo Erba Reagents (Milan, Italy).