Petunidin chloride

To detect major anthocyanidins derived from anthocyanins, HPLC analysis was performed according to the previous report with minor modifications [22 (link)]. Briefly, anthocyanin pigments were extracted with a solvent mixture of acetone:methanol:water:formic acid (40:40:20:0.1, v/v/v/v). Extracts were filtered through Sep-Pak C18 cartridge (Waters Scientific, Ontario, CA). For hydrolysis of anthocyanins to aglycones, 3 mL of 2N HCl in 50% (v/v) aqueous methanol was added to the sample powder. After incubation at 100°C for 1 h, the extracts were injected onto the Eclipse ZOBRAX XDB-C18 Rapid Resolution Threaded Column (4.6 × 150 mm, 5 μm; Agilent Technologies) on an UltiMate 3000 HPLC system (Thermo Scientific), using delphinidin chloride, cyanidin chloride, peonidin chloride, malvidin chloride (Sigma-Aldrich), and petunidin chloride (EXTRASYNTHESE) as standards. Quantification of anthocyanin aglycone was determined at the wavelength of 520 nm. For detection of leucoanthocyanidins and anthocyanidins from in vitro assay, the samples were separated in the Inno C18 column (4.6 mm × 250 mm, 5 μm; Innopia, Korea) on an UltiMate 3000 HPLC system (Thermo scientific) with detection at 280 nm.

HPLC-grade methanol and formic acid for the extraction of the phenolics were purchased from Sigma-Aldrich (Steinheim, Germany). For the mobile phases, we used HPLC–MS grade acetonitrile and formic acid from Fluka Chemie (Buch, Switzerland). The following standards were used for the quantification of phenolic compounds: Fluka Chemie (Buch, Switzerland): quercetin-3-glucoside (≥90%), p-coumaric acid (≥98.0%), kaempferol-3-rutinoside (≥95.0%), and kaempferol-3-glucoside (≥90.0%), delphinidin-3-O-glucoside chloride (≥95.0%), peonidin chloride (≥97%), and pelargonidin chloride (≥97%); Sigma-Aldrich: quercetin-3-rutinoside (≥90.0%), ferulic acid (≥99.0%), luteolin 7-O-β-D-glucoside (≥98%), 3-caffeoylquinic acid (≥95%) and 5-caffeoylquinic acid (≥95.0%); Extrasynthese (Genay, France): isorhamnetin-3-glucoside (≥95.0%), cyanidin 3-O-galactoside chloride (≥97%) and petunidin chloride (≥95%). The water for phenolic compounds extraction and mobile phases was double distilled and purified with a Mili-Q Millipore system (Merck Millipore, Billerica, MA, USA).