Eriodictyol

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).

Analytical standards of apigetrin (apigenin 7-glucoside), chrysin, prunetin, rhamnazin, morin, isoquercetin, vitexin, isovitexin, quercitrin, kaempferitrin, taxifolin, luteolin, 3-O-methylquercetin, kaempferol, hyperoside, rutin, isorhamnetin, isokaempferide, rhamnetin, sakuranetin, luteoloside, eriodictyol-7-O-glucopyranoside, narirutin, naringin, LC grade acetonitryl, DMSO, DPPH radical, ampicillin, bovine serum, ferric chloride, hyaluronidase from bovine testes type I -S, sodium phosphate buffer pH 7, oleanolic acid, 2,4,6-tris(2-pyridyl)-s-triazine TPTZ, and hyaluronic acid were obtained from Sigma-Aldrich Fine Chemicals (St. Louis, MI, USA). Naringenin, astragalin, apigenin, tiliroside, and nicotiflorin were purchased from Roth (Karlsruhe, Germany). Dihydromyricetin was supplied by LGC (LGC Group, Teddington, UK), and laricitrin by Extrasynthese (Lyon, France). Quercetin was obtained from Fluka (Buchs, Switzerland). Catechin, luteolin-7-O-glucoside, luteolin 3,7-diglucoside, Naringenin 7-O-glucoside, eriodictyol, and myricetin were supplied by ChromaDex (Irvine, CA, USA). LC-MS grade water was prepared using a Millipore Direct-Q3 purification system (Bedford, MA, USA). Ethanol was obtained from Avantor Performance Materials (Gliwice, Poland).

To obtain N-terminally His-tagged Sb06g029550 for enzymatic assays, cDNA was subcloned into the NheI–XhoI sites of a Gateway Nova pET-53-DEST vector (Merck KGaA). The plasmid was transformed into E. coli strain BL21 Star (DE3) pLysS (Life Technologies). Expression was induced by incubating the culture at 18° for 48 hr and then at 30° for 5 hr. Then, 2 mg of protein was purified from the cell pellet (5 g) with an IMAC system (Takara). The reaction mixtures (500 μl) for assay contained 0.1 M potassium phosphate buffer (pH 7.0), 1 mg of NADPH, and 200 μg of a substrate [naringenin, eriodictyol, dihydroquercetin (ChromaDex, Inc.), or (±)-taxifolin (dihydrokaempferol) (ChromaDex, Inc.)]. The recombinant protein (250 μg) was added, and the mixture was incubated for 12 hr at 30°. The reaction was terminated by extraction with ethyl acetate (500 μl). Following evaporation of 200-μl extracts, residues were dissolved in 2 N HCl in 50% methanol, incubated for 1 hr at 80°, dried, and dissolved in 200 μl of 0.1% formic acid in water. The reaction mixtures were acid-treated to convert the direct reaction products, flavan-3, 4-diol, and flavan-4-ol, to their respective anthocyanidin or 3-deoxyanthocyanidins, and analyzed by LC-MS/MS (Sawada et al. 2009 (link)).