Quercetine

Extracts were filtered prior to the analysis by 0.22 μm PVDF syringe filter (PVDF2025A, Hyundai micro, Seoul, Korea) and then analyzed by HPLC-Diode array detector (DAD) using an Agilent 1260 liquid chromatograph (Agilent technologies, Santa Clara, CA, USA) with quaternary pump and auto sampler. Separation was performed using Symmetry C18 column (250 × 4.6 mm, 5 μm, Agilent Zorbax SB-C18, Santa Clara, USA) at 25 °C. The mobile phase was composed of solvent A (99.9% v/v, acetonitrile, Sigma-Aldrich, St. Louis, USA) and solvent B (0.1% v/v, acetic acid, Dajung, Namyangju). The system gradient was run with the following gradient elution program (0 min, 0% A/100% B; 5 min, 15% A/85% B; 50 min, 50% A/50% B, 60 min, 100% A/0% B, 70 min, 0% A/100% B). The flow rate was kept constant throughout the analysis at 0.3 mL/min and the injection volume was 20 µL. The DAD detector was scanned for 240–340 nm for monitoring of the different groups of phenolic compounds. Phenolic standards, ascorbic acid, gallic acid, epigallocatechin gallate, chlorogenic acid, chatechin, caffeic acid, vanillic acid, syringic acid, coumaric acid, vanillin, sinapic acid, ferulic acid, benzoic acid, quercetine, and eugenol were purchased from Sigma-Aldrich (St. Louis, USA). Identification of phenolic compounds was performed by comparing their retention time and spectra with those of external standards.

Folin−Ciocalteu reagent was purchased from Kemika (Zagreb, Croatia). Methanol (MS grade), employed as extraction solvent was supplied by Scharlaub (Chemie S.A., Barcelona, Spain), ultrapure water was produced in the laboratory with a Milli-Q gradient system (Millipore, Bedford, MA, USA) and the formic acid was supplied by Merck (Darmstadt, Germany). Catechin (CAS: 154–23–4, ≥99.0%), epiCatechin (CAS: 490–46–0; ≥98%), phloretin (CAS: 60–82–2, ≥99.0%), phloridzin (CAS: 60–81–1, ≥98%), procyanidin B1 (CAS: 20315-25-7, ≥90%), procyanidin B2 (CAS: 29106-49-8, ≥90%), procyanidin A2 (CAS: 41743-41-3, ≥99.0%), quercetine (CAS: 117-39-5, ≥95%), quercetin 3-β-D-glucoside (CAS: 482-35-9, ≥90%), quercetin-3-rutinoside (CAS: 207671-50-9, ≥94%), myricetin (CAS: 529-44-2, ≥96.0%), chlorogenic acid (CAS: 327–97–9, ≥95%), 4-hydroxycinnamic acid (CAS: 7400-08-0, ≥98%), 2-6-dimethoxybenzoic acid (CAS: 1466-76-8, ≥98%) and patulin (CAS: 149-29-1, ≥98.0%) were obtained from Sigma–Aldrich (Chemie Check GmbH, Steinheim, Germany).