*2.2. HPLC Analysis of Anthocyanidins*

Extraction and HPLC analysis of anthocyanidins in PCC were carried out according to the Agricultural Industry Standard of the People's Republic of China (NY/T 2640-2014, Determination of anthocyanidins in plant origin products-High performance liquid chromatography). Basically, accurately weighed 0.200 g powdered samples were placed in a 15 mL plastic tube, and 5.00 mL of extracting solution consisting of ethanol:water:hydrochloric acid = 2:1:1 (volume) was added to extract anthocyanidins. The extraction mixture was sonicated for 30 min at room temperature, then hydrolyzed under boiled water for one hour. Then, the cooled extraction mixture was centrifuged using a HITACHI high-speed refrigerated centrifuge (Katsuta, Japan) at 8000 rpm for 10 min. The supernatant was accurately fixed to 5.00 mL volume and filtered through a 0.45 μm polyvinylidene fluoride syringe filter before HPLC analysis.

The quantification of anthocyanidins was carried out on a reversed-phase HPLC system (LC-20AD, Shimadzu, Tokyo, Japan) coupled with a photodiode array (PDA) detector (SPD-M20A, Shimadzu, Tokyo, Japan). The column used was a Waters C18 (3.9 × 150 mm, 5 μm) kept at 35 ◦C. The gradient elution was carried out with a binary solvent system consisting of ultrapure water (A) and acetonitrile (B), both containing 1% formic acid, at a constant flow rate of 0.8 mL/min. The injection volume was 20 μL. Anthocyanidin compounds were detected at the wavelength of 530 nm. Individual anthocyanidins were quantified via comparison of the peak areas with those of the known standards. The anthocyanidins standards (delphinidin, cyanidin, petunidin, pelargonidin, peonidin, and malvidin) were purchased from Sigma-Aldrich (Darmstadt, Germany).
