Extract characterization, reagents, and apparatuses
The reference substances, (+)-catechin (purity > 99 %), (−)-epicatechin (purity > 99 %), (−)-epicatechin-3-gallate (purity > 97.5 %), procyanidin dimer B1 (purity > 80 %), and procyanidin dimer B2 (purity > 90 %), were from Extrasynthèse (Genay, France). Gallic acid (99 % purity) was from Sigma-Aldrich (St. Louis, Missouri, USA). Procyanidin dimers B3-B7 were purified from natural extracts by high-speed counter current chromatography (HSCCC) according to Köhler et al.  and Esatbeyoglu et al. , and were characterized by nuclear magnetic resonance (NMR). Purified procyanidin extract from cocoa (Breko GmbH, Bremen, Germany) was used as a complex reference sample for the identification of procyanidin trimer C1 [EC-(4α-8)-EC-(4α-8)-EC], tetramer [EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC], pentamer [EC-(4α-8)-EC-(4a-8)-EC-(4α-8)-EC-(4α-8)-EC], and hexamer [EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC].
Grape seed extract constituents were assessed by the reversed phase (RP) HPLC method of Prodanov et al. , with adaptation for a 3 μm particle size stationary phase to increase the separation efficiency . Two HPLC apparatuses were used: (i) HPLC/PAD/FL, a Varian 920-LC Galaxie (Varian Instruments, Walnut Creek, California, USA) that was coupled to a photodiode array (PDA) and fluorescence (FL) detectors in series and (ii) an Agilent series 1100 HPLC/PAD/MS (Palo Alto, California, USA) that was coupled to a quadrupole mass spectrometer (MS) (Hewlett-Packard series 1100 MSD) with an electrospray interface (ESI). Separation was performed on an ACE-3-C18-AR (200 mm × 4.6 mm, 3 μm particle size) column from Advanced Chromatography Technologies (Aberdeen, UK) and a guard-column (20 × 4.6 mm) of the same material. The mobile phase was a linear gradient of solvent A (water:acetic acid, 98:2) to solvent B (acetonitrile:acetic acid, 98:2) as follows: from 0 to 20 % of B in 80 min, from 20 to 28 % of B in 35 min, from 28 to 100 % of B in 5 min, isocratically 100 % of B for 10 min, from 100 to 0 % of B in 5 min, and equilibration of the column for the next analysis for 10 min. The total run time was 140 min. The flow rate of the mobile phase was 0.6 mL/min. Chromatograms were acquired by measurement of absorption at 280 nm (by the PDA) and fluorescence at 316 nm (273 nm excitation). For ESI, the drying gas was N2 (10 L/min at 340 °C), the nebulizing pressure was 40 psi, and the capillary tension was 4000 V. Mass spectra were obtained by scanning negative ions from m/z less than 200 at 100 V, m/z 200–1000 at 200 V, and m/z 1000–2500 at 250 V. Mass spectra were recorded from m/z 100 to 2500.
Gallic acid, (+)-catechin, (−)-epicatechin, (−)-epicatechin-3-O-gallate, and procyanidin dimers B1 and B2 were identified by co-elution and comparison with the masses and UV spectra and retention times of commercial references. Procyanidin dimers B3, B4, B5, and B7 were identified by comparison of the UV absorbance spectra, fluorescence following excitation at 273 nm, molecular ions, and retention times of reference substances isolated by HSCCC from natural extracts [20, 21] and characterized by NMR. Procyanidin trimer C1 [EC-(4α-8)-EC-(4α-8)-EC], tetramer [EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC], pentamer [EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC], and hexamer [EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC] were identified by comparison of the UV absorbance spectra, fluorescence following excitation at 273 nm, molecular ions, and retention times of the corresponding complex reference sample from a purified procyanidin extract from cocoa . For the procyanidin pentamer [EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC], both the single- and the double-charged ions were present, but for the procyanidin hexamer [EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC], only the double-charged ion was present in the mass spectra. Procyanidin trimers, C2 [C-(4α-8)-C-(4α-8)-C] and [EC-(4α-8)-EC-(4α-8)-C], were identified based on mass, UV absorbance spectra, fluorescence following excitation at 273 nm, retention time, and order of elution . The galloylated procyanidin dimers B1-3-G and B2-3′-G were identified by mass, UV absorbance spectra, retention times, and order of elution .
Gallic acid, (+)-catechin, (−)-epicatechin and (−)-epicatechin-3-gallate were quantified by calibration curves of standards. All procyanidin nongalloylated dimers to hexamers were referred to the calibration curve of the procyanidin dimer B1 from the fluorescence chromatograms. The quantities of galloylated procyanidin dimers B1-3-G and B2-3′-G were calculated based on a calibration curve for the (−)-epicatechin-3-gallate from the 280 nm absorbance chromatograms.
Forty-six non-smokers healthy volunteers (20 males and 26 females, mean age: 34 years, age range: 17 to 60 years) were invited to participate. Individuals who reported consuming antioxidant supplements or omega-3 polyunsaturated fatty acids and those with addiction to alcohol or drugs were excluded. None of the participants received any pharmacological treatment during urine collection. Fruits and vitamin supplements were discontinued 24 h prior to collection of the first urine sample. All subjects were instructed to maintain their usual diets and to not participate in sports activities during the two day study period. The subjects consumed the biscuits with the 100 % of compliance. All subjects provided written informed consent and the study protocol (IB 2030/13 PI) was approved by the Ethics Investigation Committee of the Balearic Islands (Spain).
The first day, each volunteer ate eight traditional biscuits (Quely from Quely S.A., Mallorca Islands, Spain) during the day (4 in breakfast and 4 in dinner) with no plant-derived phenol supplementation. The biscuits, of 9 g of weight, are made with wheat, sunflower oil, yeast, olive oil and salt. An overnight urine sample, starting prior to sleep and ending with the first morning urine while still in a fasting state, was obtained the next day.
The second day each volunteer ate eight traditional biscuits supplemented with 0.6 % (wt/wt) of red grape (Vitis vinfera L.) seed extract (Quely Cor from Quely S.A.,) during the day, corresponding to about 250 mg of phenols, the phenols content of the biscuits was measured by Folin’s method and expressed as gallic acid. This represent a total polyphenol intake about 2 g/d. The red grape seed extract (exGrape®) was purchased from La Gardonnenque-Groupe Grap’Sud® (Cruviers-Lascours, France). The second urine sample was collected on the third morning as described above.
At 2 h after collection of each non filtered urine sample, the redox potential was measured at 25 °C using a Crison potentiometer (Crison Instruments S.A., Barcelona, Spain), with a platinum electrode as the working electrode and a saturated calomel electrode as the reference electrode. Results are shown as means ± standard errors and compared using Student’s paired t-test.