Epicatechin

Methanol and water (LC-MS grade) were purchased from HiPerSolv CHROMANORM, VWR Chemicals BDH (Amsterdam, The Netherlands). Formic acid 98–100% was purchased from Merck (Darmstadt, Germany). For the determination of phenolic compounds, apigenin 98%, caffeic acid 98%, catechin 97%, cinnamiccinnamic acid 97%, chrysin 98%, diosmin 97%, epicatechin, epigallocatechin 97%, ferulic acid 98%, epicatechin gallate 98%, hesperidin 98% (internal standard), kaempferol 98%, luteolin 98%, myritecin 97%, myricitrin 97%, naringin 98%, p-coumaric acid 98%, quercetin 98%, quercitrin 99%, rosmarinic acid 98%, protocatechuic acid 97%, rutin 98%, sinapic acid 98%, syringaldehyde 97%, syringic acid 98%, taxifolin 98%, vanillic acid 98%, and vanillin 98% were used and were purchased from Sigma-Aldrich (Stenheim, Germany). Stock standard solutions of all the analytical standard compounds were prepared with LC-MS-grade methanol at 1000 mg/L and were afterward stored in dark brown glass bottles at −20 °C.

HPLC standards (chlorogenic acid and (−)-epicatechin) and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). DMSO and organic solvents (chloroform, isopropanol, methanol, and ethanol) were obtained from Sigma-Aldrich, Samchun Chemical (Seoul, Republic of Korea), and Daejung Chemical (Siheung, Republic of Korea). Inorganic salts were from Sigma-Aldrich, and silica gel was from Merck (Rahway, NJ, USA). DMEM and FBS were supplied by Hyclone (Logan, UT, USA), and Antibiotic-Antimycotic was from Gibco (Grand Island, NY, USA).

Samples were sent to E & J Gallo (Modesto, CA, USA) to conduct compositional analyses [29 (link)]. For whole BSG, 100 g dry BSG was added to 500 mL of 50% ethanol and adjusted to pH 2.0. The mixture was agitated for 12 h at 25 °C then centrifuged at 5770× g for 10 min (Avanti J-E Centrifuge, Beckman Coulter, Brea, CA, USA). The supernatant was filtered through a 0.45 µm cellulose acetate filter with a glass microfiber prefilter layer (Whatman Puradisc, Cytiva Life Sciences, Marlborough, MA, USA) and stored at 5 °C until further analysis. For BSGE, the supernatant from Section 2.1 (before diluting to 5%) was directly analyzed.
Acetonitrile (HPLC grade), phosphoric acid, and additional consumables were purchased from VWR International (Radnor, PA, USA). Deionized water was obtained by an ELGA LabWater Purelab Flex System (Woodridge, IL, USA). The following HPLC standards were purchased from Sigma-Aldrich (St. Louis, MO, USA): gallic acid (CAS 149-91-7, ≥97.5%), protocatechuic acid (CAS 99-50-3, ≥97%), syringic acid (CAS 530-57-4, ≥95%), caffeic acid (CAS 331-39-5, ≥98%), p-coumaric acid (CAS 501-98-4, ≥98%), ferulic acid (CAS 537-98-4, ≥99%), astilbin (CAS 29838-67-3, ≥98%), (+)-catechin hydrate (CAS 225937-10-0, ≥98%), (−)-epicatechin (CAS 490-46-0, ≥98%), and quercetin dihydrate (CAS 6151-25-3, ≥99%). Oenin chloride (7228-78-6, ≥95%) was purchased from Indofine Chemical Company (Hillsborough, NJ, USA).
This method was adapted from a previous study [30 (link)]. BSG and BSGE samples were centrifuged at max speed of 21,100× g for 10 min at 10 °C (Sorvall Legend Micro 21R Refrigerated Centrifuge, Thermo Scientific, Waltham, MA, USA) prior to analysis via HPLC coupled with a diode array detector (DAD). The system included an Agilent 1260 Infinity Micro Degasser (Model G1379B), High Performance Autosampler (Model G1367C), Thermostatted Column Compartment (Model G1316B), DAD (Model G1315C), a Zorbax C18 precolumn (2.1 × 12.5 mm, 1.8 mm) coupled to a Zorbax Eclipse C18 analytical column (2.1 × 100 mm, 1.8 mm) (Agilent Technologies, Santa Clara, CA, USA). Two mobile phases were used: phase A was water/phosphoric acid at 99.5:0.5 v/v and phase B was acetonitrile/phosphoric acid at 99.5:0.5 v/v. The flow rate was set to 0.2 mL/min and the column temperature to 40 °C. The binary gradient of the mobile phases started at 90%, then 81% at 30 min, 67% from 30.75–37.5 min, 5% at 40.5 min, then 90% from 43.5–46 min. The injection volume was 4 μL for each sample. Calibration curves were based on 6 points covering a range from 1.0 mg/L to 200 mg/L for all compounds of interest besides catechin with a range of 1.0 mg/L to 1000 mg/L. All compounds were quantified by their corresponding standards except hydroxycinnamic acid, which was expressed in caffeic acid equivalents, and polymeric tannins, expressed in catechin equivalents. After every twentieth sample, an additional continuous calibration verification (CCV) sample was spiked with a known concentration of (+)-catechin and quercetin. Recovery rates were calculated from the difference between unspiked and spiked samples divided by the spiked concentrations. Recovery rates were 97–103% for CCV samples.