4.7. Extraction and Determination Anthocyanin, Catechin, and Epicatechin

A liquid nitrogen grinding sample (0.2 g) was extracted in 1 mL of acidified methanol (1% HCl, v/v) at 4 °C in the dark for 12 h with manual shaking three times. The extract liquor was centrifuged at 1000 rpm at 4 °C for 2 min, the supernatant was collected, and the precipitate was re-extracted in an equal volume of extracting solution. Each extract was performed in triplicate. The combined-supernatant solutions were evaporated to concentrate using a rotary evaporator (RE52AA, Yarong, Shanghai, China) at 30 °C. Then the concentrate was re-dissolved in 0.8 mL extracting solution and filtered through a 0.22 μm microporous membrane (Jinteng, Tianjin, China) for the following analysis.

Quantification of the total anthocyanin was performed according to previously reported protocols [2,19,58], with minor modification. Absorption of the extracts at wavelengths of 530 nm and 657 nm was measured at 25 °C using Multimode Microplate Readers (Spectra Max M 2, Hercules, CA, USA). The concentration of the total anthocyanin was calculated according to the following formula: (A₅₃₀ − 0.25 × A₆₅₇)/FW. A₅₃₀ and A₆₅₇ are the absorptions at the 530 nm and 657 nm wavelengths, respectively. FW is the fresh weight (in grams) of the plant tissues used for the extraction.

The contents of delphinidin chloride, cyanidin-3,5-di-O-glucoside, catechin, and epicatechin were all detected on Waters HPLC 1525 system (Milford, MA, USA), equipped with a Waters 2996 photodiode array detector. Chromatographic separation was performed with a Waters Xbridge C18 column (4.6 mm × 250 mm, 5 μm particle size) a column temperature of 30 °C and 20 μL injection volume at a flow rate of 1.0 mL/min. The mobile phase consisted of 0.5% trifluoroacetic acid in water (A) and 100% acetonitrile (B). The gradient profile as follows (all concentrations are v/v): 0–3 min, 0% to 86% A; 3–10 min, 86% to 85% A; 10–30 min, 85% to 70% A; 30–45 min, 70% to 95% A; 45–46 min, 95% to 100% A. The spectra were scanned from 210 nm to 600 nm. The detection of anthocyanin, catechin, and epicatechin was carried out at 520 nm, 280 nm and 280 nm, respectively. All reagents were of HPLC grade. Standards of delphinidin chloride and cyanidin-3,5-di-O-glucoside were purchased from Sigma-Aldrich (St. Louis, MO, USA). Standards catechin and epicatechin were purchased from Yuanye Shanghai.