Tricin

Metabolites were analyzed using HPLC-ESI-TOF MS as previously described [28 (link)]. Briefly, their separation was conducted on a HPX-87H column with 8% cross-linkage (150-mm length, 7.8-mm inside diameter, and 9-μm particle size; Bio-Rad, Richmond, CA) using an Agilent Technologies 1100 Series HPLC system. Metabolites were eluted isocratically with a mobile-phase composition of 0.1% formic acid in water at a flow rate of 0.5 ml/min. Drying and nebulizing gases were set to 13 liters/min and 30 lb/in², respectively, and a drying-gas temperature of 330°C was used throughout. ESI was conducted in the negative ion mode and using a capillary voltage of −3,500 V. Luteolin, chrysoeriol (ChromaDex, Inc., Irvine, CA), tricin (ChromaDex, Inc., Irvine, CA), and selgin were quantified via 8-point calibration curves of authentic standard compounds for which the R² coefficients were ≥ 0.99. Stock solutions of metabolites used for enzymatic assays and standard curves were quantified spectrophometrically using published molar absorption coefficients: S-adenosylmethionine (ε = 15,400 L.mol^-1.cm^-1 at 254 nm) [29 (link)], Luteolin (ε = 14,790 L.mol^-1.cm^-1 at 350 nm) [30 (link)], chrysoeriol (ε = 15,400 L.mol^-1.cm^-1 at 347 nm) [30 (link)], and tricin (ε = 41,000 L.mol^-1.cm^-1 at 349 nm) [31 (link)].

Acetonitrile and methanol of HPLC grade were purchased from Sigma-Aldrich (St. Louis, MO, USA). Quercetin 3-methyl ether, eriodictyol, ampelopsin (dihydromyricetin), taxifolin (dihydroquercetin), aromadendrin (dihydrokaempferol), tamarixetin, quercetin 3,3'-dimethyl ether, isokaempferide, chrysoeriol, homoeriodictyol, and isoferulic acid were purchased from BioBioPha Co., Ltd (Kunming, China), and oroxylin A was purchased from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China). Laricitrin, syringetin, and tricetin were from Extrasynthese (Lyon, France), and tricin was from ChromaDex (Irvine, CA, USA). Quercetin, isorhamnetin, kaempferol, luteolin, baicalein, eriocitrin, SAM, and formic acid for HPLC were obtained from Aladdin (Shanghai, China). Myricetin, apigenin, and 7,8-dihydroxyflavone hydrate were purchased from TCI (Shanghai, China). Caffeic acid, naringenin, hesperetin, neoeriocitrin, and ferulic acid were also purchased from Sigma-Aldrich.

Mobile phases of acetonitrile and methanol for HPLC (high-performance liquid chromatography) and phenolic acids, including caffeic acid, ferulic acid, naringenin, neoeriocitrin, and hesperetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dihydroflavonols (dihydroquercetin dihydromyricetin and dihydrokaempferol), eriodictyol, homoeriodictyol, quercetin 3,3’-dimethyl ether, quercetin 3-methyl ether, chrysoeriol, and isokaempferide were obtained from BioBioPha Co., Ltd. (Kunming, China). Eriocitrin, baicalein, luteolin, quercetin, kaempferol, isorhamnetin, and SAM were from Aladdin (Shanghai, China). Apigenin, myricetin, and 7,8-dihydroxyflavone hydrate were from TCI (Shanghai, China) and tricin was purchased from ChromaDex (Irvine, CA, USA). Tricetin and syringetin were purchased from Extrasynthese (Lyon, France) and baicalein 7-methyl ether was purchased from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China). RPMI-1640 medium, fetal bovine serum (FBS), HEPES, and trypsin-EDTA were purchased from Gibco (Waltham, MA, USA). Cell Counting Kit-8 was purchased from Dojindo Technology (Shanghai, China).