3 coumaric acid

2,7-dichlorofluorescein diacetate (DCFH-DA), 2,2′-Azo-bis-aminopropane (ABAP) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hanks' Balanced Salt Solution (HBSS), fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM), and other cell culture reagents were purchased from Gibco (Invitrogen, Barcelona, Spain). Methyl syringate, epicatechin, syringic acid, catechin, 4-coumaric acid, gallic acid, quercetin, apigenin, luteolin, kaempferol, naringenin, formononetin, genistein, 3-coumaric acid, daidzein, pelargonidin, 2-coumaric acid, biochanin A and cyanidin were purchased from Sigma. Ultrapure water was obtained using a Milli-Q system (Millipore, Bedford, MA, USA) and methanol, acetonitrile and 2-propanol were purchased from Teknokroma (Barcelona, Spain). All other chemicals were of analytical grade.

HPLC-DAD analysis was performed by using a Thermo-Finnigan Spectra-System HPLC system (Thermo-Finnigan, Waltham, USA) equipped with a P2000 binary gradient pump system, a SCM 1000 degasser, an AS 100 automatic injector, an UV6000LP DAD and ChromQuest software for data processing. Separation was achieved on a C18 RP Lichrosphere 250 × 4.6 mm, 5-µm (Merck, Milan, Italy) column equipped with a C18 RP Lichrosphere 5-µm guard column (Merck, Milan, Italy). The mobile phase was composed of trifluoroacetic acid/ultrapure water (0.1:99.9, v/v) (A) and methanol (B). The flow rate was 1 mL/min, and the injection volume was 20 µL. The elution program was as follows: 95% A as the initial condition, maintained for 2 minutes; 80% A for 8 min; 25% A for 57 min; 0% A for 13 min; and 95% A for 5 min. DAD spectra were recorded in full scan mode over a wavelength range of 200 to 400 nm. Identification was achieved by comparing the retention times and spectra with authentic standards (Fig. 1). Each compound was quantified as mg/Kg sample by means of calibration with external standards: gallic acid, protocatechuic acid, procyanidin B1, gallocatechin gallate, 3-coumaric acid and rutin purchased from Sigma-Aldrich (Milan, Italy) and 2coumaric acid purchased from Extrasynthese (Genay Cedex, France).