Malvidin chloride

Anthocyanin and stilbene content analysis was performed by the method HPLC-MS as described [45 (link),47 (link),48 (link)]. Briefly, for anthocyanins 100 mg fresh cells tissue were subsequently homogenized using a mortar and a pestle in 1 mL of 1% (v/v) hydrochloric acid in methanol. Then, shredded tissue was extracted for 1 d at 4 °C. For stilbenes 100 mg of the dried shredded cells tissue were extracted for 2 h at 60 °C in 3 mL of methanol. Then, anthocyanin and stilbene extracts were filtered through a 0.25-um nylon membrane for further analysis. Next, samples were separated on Shim-pack GIST C18 column (150 mm, 2.1-nm i.d., 3-_m part size; Shimadzu, Japan) the on HPLC LC-20AD XR analytical system (Shimadzu, Japan), equipped with an SPD-M20A photodiode array detector. Liquid chromatography-high-resolution mass spectrometry for qualification of all components was performed using a 1260 Infinity analytical system (Agilent Technologies, Santa Clara, CA, USA) as described [24 (link),44 (link)].
The commercial standard cyanidin chloride, petunidin chloride, delphinidin chloride, malvidin chloride, t-resveratrol, t-piceid, t-piceatannol, ε-viniferin were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used as the control.

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.