The aim of this paper is to study how grape ripeness and ethanol concentration affect the extraction of color and phenolic compounds from skins and seeds during the maceration/fermentation process. Simulated maceration assays were carried out with the grapes at three stages of berry development (vitis vinifera cv. Tempranillo) and different percentages of ethanol in the maceration media. Both ripeness and ethanol content have a considerable effect on the extraction of color and phenolic compounds. Of these two factors, ripeness increases the extractability most. The presence of ethanol in the medium facilitates anthocyanin and especially proanthocyanidin extraction, but it also decreases copigmentation phenomena, which can decrease the color intensity. The higher the ethanol concentration is in the maceration media, the higher the astringency of proanthocyanidins.

Procyanindin extract (PE) is a mixture of polyphenols, mainly procyanidins, obtained from grape seed with putative antiinflammatory activity. We evaluated the PE effect on RAW 264.7 macrophages stimulated with lipopolysaccharide plus interferon-gamma that show a rapid enhanced production of prostaglandin E2 (PGE2) and nitric oxide (NO). Our results demonstrated that PE significantly inhibited the overproduction of NO, dose and time dependently. PE caused a marked inhibition of PGE2 synthesis when administered during activation. Moreover, PE pretreatment diminished iNOS mRNA and protein amount dose dependently (10-65 microg/mL). PE (65 microg/mL) pretreatment inhibited NFkappaB (p65) translocation to nucleus by nearly 40%. Trimeric and longer oligomeric-rich procyanidin fractions from PE (5-30 microg/mL) inhibited iNOS expression but not the monomeric forms catechin and epicatechin. Thus, we show that the degree of polymerization is important in determining procyanidin effects. PE was considerably a more effective inhibitor of NO biosynthesis (IC50 = 50 microg/mL) in comparison to other antiinflammatories, such as aspirin (3 mM), indomethacin (20 microM), and dexamethasone (9 nM). In conclusion, PE modulates inflammatory response in activated macrophages by the inhibition of NO and PGE2 production, suppression of iNOS expression, and NFkB translocation. These results demonstrate an immunomodulatory role of grape seed procyanidins and thus a potential health-benefit in inflammatory conditions that exert an overproduction of NO and PGE2.

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Among procyanidins (PC), monomers, such as catechin and epicatechin, have been widely studied, whereas dimer and trimer oligomers have received much less attention, despite their abundance in our diet. Recent studies have showed that as dimers and trimers could be important in determining the biological effects of procyanidin-rich food, understanding their bioavailability and metabolism is fundamental. The purpose of the present work is to study the stability of PC under digestion conditions, the metabolism and the bioavailability by using a combination of in vitro and in vivo models. Simultaneously, the matrix effect of a carbohydrate-rich food on the digestibility and bioavailability of PC is investigated. The results show a high level of stability of PC under gastric and duodenal digestion conditions. However, the pharmacokinetic study revealed limited absorption. Free forms of dimers and trimers have been detected in rat plasma, reaching the maximum concentration 1 h after oral intake of a grape seed extract.Procyanidins: Dimers: Trimers: Digestion: Bioavailability Procyanidins (PC) are found in most plants and in a wide range of foods, such as red wine, cocoa, tea and fruits, and thus they are a part of the human diet. PC belong to the group of flavonoids, and are phenolic compounds mainly formed of (þ)-catechin and (2 )-epicatechin units with C 4 -C 8 and/or C 4 -C 6 bonds. PC are formed from the condensation of monomeric units, between two and five units for oligomers and over five units for polymers (1) .Grape seed procyanidin extracts (GSPE) have shown several bioactivities. They improve antioxidant cell defences (2) and the plasma lipid profile (3,4) , limit adipogenesis (5) and function as insulinomimetic (6) and anti-inflammatory (7) agents. A preliminary study of an in vitro model by the authors has also determined that dimeric and trimeric oligomers are the most powerful PC molecules that mimic the complete GSPE (8) . Thus, to explain these health effects of PC and understand the mechanism by which PC act at the cellular level in vivo, it is essential to determine PC stability during the digestive process as well as their bioavailability.Despite it being clear that monomers are absorbed in human subjects and animals, there are controversies about the bioavailability of oligomeric forms. Different studies, following the ingestion of chocolate (9 -12) , black and green tea (13 -16) , red wine (17,18) and grape seed extract (1) , have shown that during digestion, the oligomers are fragmented into monomeric units of catechin and epicatechin. These are then absorbed, appearing in plasma and urine primarily as glucuronidated, methylated and sulphated metabolites. A study by Rios et al. (19) with six healthy subjects who consumed a rich PC cocoa beverage studied the depolymerisation of PC in the stomach and proved that they were remarkably stable in the stomach environment. Another study by Sano et al. (20) was the first work to detect procyanidin B1 in human serum after oral intake of a GSPE, the ma...

Phenolics from grapes and wines can play a role against oxidation and development of atherosclerosis. Stilbenes have been shown to protect lipoproteins from oxidative damage and to have cancer chemopreventive activity. We describe a method for the direct determination of stilbenes in several red wines using high-performance liquid chromatography with UV detection. In a survey of 12 commercial wines from the south of Brazil (Rio Grande del Sul), levels of delta-viniferin are reported for the first time in different varieties of red wines. Brazilian red wine contains trans-astringin, trans-piceid, trans-resveratrol, cis-resveratrol (in high quantity: 5 times more than the trans form), epsilon-viniferin, and a compound isolated for the first time in wine, trans-delta-viniferin. Isolation and identification of delta-viniferin was achieved by NMR after extraction and fractionation of red wine phenolics. delta-Viniferin contributes, as well as cis-resveratrol and trans-piceid, to a significant proportion of stilbenes in wine dietary intake, particularly with Merlot varieties containing an average level of 10 mg/L for delta-viniferin, 15 mg/L for cis-resveratrol, and 13 mg/L for trans-piceid. The total stilbene intake from wine origin was estimated for the Brazilian population as 5.3 mg/day per person (on the basis of a regular wine consumption of 160 mL/day). delta-Viniferin can contribute to around 20% of total stilbenes in wine (average of 6.4 mg/L in red Brazilian wines). It would be important in the future to investigate the origins of the differences in wine stilbene levels in relation to the vine varieties, and the bioavailability of the newly extracted stilbene delta-viniferin in plasma after consumption of different types of wines.

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In the analysis of biological samples, such as plasma or serum, the quantity of sample available is a critical parameter in most cases. A good approach is the use of the microelution SPE (μSPE) plates as sample pre-treatment technique in which the loaded sample volume is low. An off-line μSPE and ultra-performance LC-ESI-MS/MS (UPLC-ESI-MS/MS) method was developed and validated to determine procyanidins and anthocyanins in spiked plasma samples. The sample pre-treatment μSPE allowed the simultaneous determination of procyanidins and anthocyanins from plasma by using a small sample volume (350 μL) and without an evaporation step previous to the chromatographic analysis. Moreover, the use of UPLC technique allowed to determine the studied compounds at low concentration levels in a short analysis time (12.5 min approximately). Then, the developed method was applied to determine the studied compounds, procyanidins and anthocyanins, and their metabolites in rat plasma samples. Previously, the rats had consumed 5000 mg/kg of a grape pomace extract and the plasma was extracted 4 h after administration. The procyanidins catechin and epicatechin glucuronide, methyl catechin and epicatechin glucuronide, and methyl catechin and epicatechin sulphate were detected at μM concentration level, and the parent anthocyanins at nM.

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