Cyanidin 3 o glucoside chloride

Approximately 1 g of peach peel powder was suspended in 5 mL 0.05% HCl in methanol, extracted at 4°C for 12 h, and followed by a centrifugation to collect the supernatant. The residue was extracted for another two times. The supernatants were combined and filtered using a 0.22 μm Millipore membrane, then evaporated in a rotary evaporator at 30°C. The residual was resuspended with 1 ml methanol, filtered with a 0.22 μm Millipore membrane, then analyzed by HPLC using an ZORBAX SB-C18 analytical column (4.6 × 250 mm, 5 μm, Agilent Technologies, Santa Clara, CA, United States). A quantitative analysis of phenolic compounds were analyzed by an Agilent 1260 Liquid Chromatograph equipped with a diode array detector using solvent A (formic acid : water, 5:95, v/v) and solvent B (methanol) with the following gradient: 0–2 min, 5%; 2–7 min, 5–15%; 7–20 min, 15–20%; 20–25 min, 20–27%; 25–32 min, 27%; 32–41 min, 27–35%; and 41.01–43 min, 5% (Cheng et al., 2014 (link)). The post-run-time was 5 min. The flow rate was 0.8 ml min^-1 at 30°C. The wavelength was set at 520 nm. Cyanidin 3-O-glucoside chloride purchased from Sigma (CAS:7084) was used as a standard.

Qualitative analysis of the extract was performed by high-performance liquid chromatography (HPLC) using an HP 1100/1200 instrument (Agilent Technologies, Palo Alto, CA, USA), equipped with an autosampler (100 µL sample loop), and a diode array detector [26 (link)]. About 225 μg of raw extract was loaded in 100 µL of HPLC starting conditions (6% of eluent A, composed by water/acetonitrile/formic acid, 87:3:10 v/v/v) on a C12 column (Synergi 4 µm Max-RP 80 A, 250 × 4.6 mm ID, Phenomenex, Torrance, California, USA), preceded by a Synergi guard column (4 × 3.00 mm, Phenomenex) with the same stationary phase, at room temperature. The flow rate was set at 0.5 mL/min with a multistep gradient of eluent B (water/acetonitrile/formic acid, 40:50:10 v/v/v in A from 6% to 90%, according to the scheme provided in Table 1. Elution was followed by recording the absorbance between 190 and 700 nm. As a reference, a standard pool composed by 60 µL of a 2.5 ng/µL solution of malvidin-3-O-glucoside chloride, 60 µL of a 5 ng/µL solution of cyanidin-3-O-glucoside chloride, and 60 µL of a 2.5 ng/µL solution of peonidin-3-O-glucoside chloride (Sigma-Aldrich) was run on the same gradient.