Vitisin A content in Chilean wines from Vitis vinifera Cv. Cabernet Sauvignon and contribution to the color of aged red wines

Vitisin A was prepared from malvidin 3-glucoside and pyruvic acid in model wine medium, isolated by countercurrent chromatography, and purified by preparative high-performance liquid chromatography (HPLC). The synthesized compound was used as a reference standard to quantify vitisin A in Chilean wines from Vitis vinifera cv. Cabernet Sauvignon, including a vertical row of wines from the same vineyard over 16 years. Maximum vitisin A content was reached within the first year of storage. Importantly, up to half of the initial amount of vitisin A in young wines was still present in 15 year old wines. Although vitisin A was found to be much more stable as compared to other monomeric C-4 underivatized anthocyanins, it also slowly degrades after reaching its peak concentration. The "color activity concept" was applied to vitisin A, malvidin 3-glucoside, malvidin 3-(6' '-acetylglucoside), and polymeric pigments isolated by countercurrent chromatography in order to estimate their contribution toward the overall color expression of wines. It was found that vitisin A is only a minor contributor to the visually perceived color of aged red wines (color contribution approximately 5%). The major contributor is the polymeric fraction (color contribution approximately 70-90%).     

Effect of copigments and grape cultivar on the color of red wines fermented after the addition of copigments

The prefermentation addition of copigments led to significantly different red wines according to the copigment structure (flavonol or hydroxycinnamic acid) and the grape cultivar [Tempranillo (= Cencibel) or Cabernet Sauvignon]. The flavonol rutin enhanced copigmentation and anthocyanin extraction, improving the red color, but the hydroxycinnamic acids (especially caffeic acid) had converse results. The above effects were higher in Cabernet Sauvignon wines, particularly if rutin or p-coumaric acid was used. These wines showed the highest copigmentation as they contained more anthocyanins and flavonols, whereas the coumaroylated anthocyanins of Tempranillo wines could have prevented the action of the added copigments. After 21 months, the main pyranoanthocyanins found were the malvidin-3-glucoside 4-vinylphenol and the malvidin-3-glucoside 4-vinylcatechol (pinotin A) adducts. The results suggested that the former adduct was primarily generated following enzymatic decarboxylation of p-coumaric acid during fermentation, whereas pinotin A was formed through a pure chemical reaction, which depended on the concentration of free caffeic acid during aging.     

Isolation and structural characterization of new anthocyanin-derived yellow pigments in aged red wines

Two newly formed yellow pigments that revealed unique spectral features were detected and isolated from an aged Port red wine by TSK Toyopearl HW-40(s) gel chromatography and characterized by UV-visible spectrophotometry, 1H NMR and 13C NMR, and mass spectrometry (LC-ESI/MS). The UV-vis spectra of these pigments showed maximum absorption at 478 nm that is significantly hypsochromically shifted from those of original grape anthocyanins and other pyranoanthocyanins, exhibiting a more yellow hue in acidic solution. The structures of these pigments correspond to methyl-linked pyranomalvidin 3-glucoside and its respective coumaroyl glucoside derivative. They were shown to arise from the reaction between acetoacetic acid and genuine grape anthocyanins. Isolation and NMR identification using 1D and 2D NMR techniques are reported for the first time for this new family of anthocyanin-derived yellow pigments occurring in red wines.     

Antiradical properties of red wine portisins

The free radical chemistry of two polyphenolic pigments from red wine, belonging to the family of portisins, has been investigated after reaction with O(2)(?-) radicals using electron paramagnetic resonance (EPR) spectroscopy. Two portisins derived from malvidin-3-glucoside and cyanidin-3-glucoside were used for this study. Stable free radicals were detected with both portisins and correspond to unpaired electrons localized on the B as well as F rings of the portisins. Interpretations were confirmed by comparison with the spectra of free radicals formed by oxidation of the model compounds cyanidin-3-glucoside, malvidin-3-glucoside, and catechin. These results concur with previous work reporting the higher antiradical properties of these pigments compared to their anthocyanin precursors.     

Occurrence of anthocyanin-derived pigments in red wines

Several anthocyanin-derived pigments that showed UV-visible spectra different from those of the original grape anthocyanins were detected by HPLC-DAD analysis in 1-year-old bottled Port wines from the Douro region. Among these, three malvidin 3-glucoside derived pigments were detected in large amounts, representing approximately 60% of the total anthocyanidin monoglucosides content. These pigments were isolated, purified, and identified by LSI-MS and NMR ((1)H, DQF-COSY, ROESY, HSQC, and HMBC) techniques. The major pigment is malvidin 3-glucoside pyruvic adduct, previously characterized, and the other two corresponded to its respective acetyl and coumaroyl glucoside derivatives. The latter is reported for the first time in red wines.     

Antioxidant capacities and phenolics levels of French wines from different varieties and vintages.

Phenolics from grapes and wines can play a role against oxidation and development of atherosclerosis. Levels of phenolics, major catechins [(+)-catechin, (-)-epicatechin, procyanidin dimers B1, B2, B3, and B4], phenolic acids (gallic acid and caffeic acid), caftaric acid, malvidin-3-glucoside, peonidin-3-glucoside, and cyanidin-3-glucoside were quantified by HPLC with UV detection for 54 French varietal commercial wines taken from southern France to study the antioxidant capacity and the daily dietary intake of these compounds for the French population. The highest antioxidant capacity was obtained with red wines and ranged from 12.8 mmol/L (Grenache) to 25.2 mmol/L (Pinot Noir). For white wines, Chardonnay enriched in phenolics by special wine-making was found to have an antioxidant capacity of 13.8 mmol/L, comparable to red wine values. For red wines classified by vintages (1996-1999) antioxidant capacities were approximately 20 mmol/L and then decreased to 13.4 mmol/L for vintages 1995-1991. Sweet white wines have 1.7 times more antioxidant capacity (3.2 mmol/L) than dry white wines (1.91 mmol/L). On the basis of a still significant French wine consumption of 180 mL/day/person, the current daily intake of catechins (monomers and dimers B1, B2, B3, and B4) averaged 5 (dry white wine), 4.36 (sweet white wines), 7.70 (rosé wines), 31.98 (red wines), and 66.94 (dry white wine enriched in phenolic) mg/day/resident for the French population. Red wine, and particularly Pinot Noir, Egiodola, Syrah, Cabernet Sauvignon, and Merlot varieties, or Chardonnay enriched in phenolics during wine-making for white varieties contribute to a very significant catechin dietary intake.     

Enzymatic synthesis of [3'-O-methyl-(3)H]malvidin-3-glucoside from petunidin-3-glucoside

Malvidin-3-glucoside has been labeled by enzymatic synthesis in a single-step experiment. Catechol-O-methyl transferase catalyzed the B-ring methylation of petunidin-3-glucoside, and S-Adenosyl-L-[methyl-(3)H] methionine was the methyl donor. Solid phase extraction and preparative high-performance liquid chromatography were necessary to separate [3'-O-methyl-(3)H]malvidin-3-glucoside from an isomer and the starting material. The specific activity was 2.2 Ci mmol(-1), and the yield of incorporation was 1.1%. A possible application of the labeled material is the study of anthocyanin reactions in complex mixtures such as red wine where products are difficult to isolate and analyze.     

Effect of red grapes co-winemaking in polyphenols and color of wines

The red grapes co-winemaking effect on phenolic fraction and wine color has been studied for the first time, where Monastrell was comacerated and cofermentated with Cabernet Sauvignon and Merlot. Changes in the relative abundance of anthocyanins were observed as well as hyperchromic shifts at 530 and 620 nm; these effects remain constant after aging. Co-winemaking also favored copigmentation, giving way to more stable anthocyanins and facilitating their polymerization. With regard to color evolution, the mixture of Monastrell with Merlot grapes was more appropriate than with Cabernet Sauvignon for aging wines in oak barrels. The extent of copigmentation was more important in young wines than in aged wines. This is mainly due to the self-anthocyanin monomer reactions in the case of young wines, whereas in aged wines copigmentation is mainly due to the reaction between the anthocyanins and other polyphenolic cofactors. Discriminant analysis showed the possibility of differentiating wines according to the aging time and the type of wine, with color parameters (color intensity, OD 620 nm, and OD 520 nm) being the most important discrimination variables in the first case and petunidin-3-glucoside and peonidin-3-glucoside contents in the second case.     

Characterization of anthocyanins and pyranoanthocyanins from blood orange [Citrus sinensis (L.) Osbeck] juice

Anthocyanins from blood orange [Citrus sinensis (L.) Osbeck] juices were isolated and purified by means of high-speed countercurrent chromatography and preparative high-performance liquid chromatography. Structures of the pigments were then elucidated by electrospray ionization multiple mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. The major anthocyanins of the juice were characterized as cyanidin 3-glucoside and cyanidin 3-(6"-malonylglucoside). Furthermore, six minor anthocyanins were detected and identified as cyanidin 3,5-diglucoside, delphinidin 3-glucoside, cyanidin 3-sophoroside, delphinidin 3-(6"-malonylglucoside), peonidin 3-(6"-malonylglucoside), and cyanidin 3-(6"-dioxalylglucoside). The occurrence of the latter compound in blood oranges is reported here for the first time, together with full NMR spectroscopic data. Further investigations revealed the presence of four anthocyanin-derived pigments, which are formed through a direct reaction between anthocyanins and hydroxycinnamic acids during prolonged storage of the juice. These novel pyranoanthocyanins were identified as the 4-vinylphenol, 4-vinylcatechol, 4-vinylguaiacol, and 4-vinylsyringol adducts of cyanidin 3-glucoside through comparison of their mass spectrometric and chromatographic properties with those of synthesized reference compounds.     

Evolution and stability of anthocyanin-derived pigments during Port wine aging.

For three years, the evolution of the three major anthocyanidin monoglucosides (malvidin 3-glucoside, malvidin 3-acetylglucoside, and malvidin 3-coumaroylglucoside) and their anthocyanin-pyruvic acid adducts was monitored in Port wines stored in oak barrels. The degradation reactions of all pigments followed first-order kinetics in all the wines studied. The degradation rate constants of the anthocyanin-pyruvic acid adducts were much lower than those of the anthocyanidin monoglucosides. The results of both anthocyanins and pyruvic acid adducts show that acylation on the sugar moiety of all the pigments decreased their stability in wine. The levels of malvidin 3-glucoside-pyruvic acid adduct and its acylated forms increased right after wine fortification with wine spirit before starting to decrease around 100 days. The initial formation of anthocyanin-pyruvic acid adducts was concurrent with the degradation of anthocyanidin monoglucosides.     

Prediction of wine color attributes from the phenolic profiles of red grapes (Vitis vinifera)

Knowledge about the relation between grape and wine phenolics is of key interest for the wine industry with respect to being able to predict wine quality from analyses of grapes. Prediction of the phenolic composition and color of experimentally produced red wines from the detailed phenolic composition of the corresponding grapes was investigated using a multivariate approach. Grape extracts and wines were produced from 55 different grape samples, covering 8 different Vitis vinifera cultivars: Alicante, Merlot, Syrah, Cinsault, Grenache, Carignan, Cabernet Sauvignon, and Mourvedre. The phenolic composition of the grapes and wines showed that the average ratios between wine and grape phenolics ranged from 0.25 to 7.9 for the different phenolic compounds. Most interestingly, the average ratios were low for anthocyanins (0.31) and tannins (0.32), intermediate for (+)-catechin (0.75) and polymeric pigments (0.98), and high for gallic acid (7.9). Individual wine phenolics in general correlated well with several grape phenolics, indicating that a multivariate approach might be advantageous for prediction of wine phenolics from grape phenolics analysis. However the use of multivariate prediction of individual wine phenolics from the complete grape phenolic composition only improved the prediction of wine polymeric pigments, whereas wine anthocyanins were predicted with the same precision as from the direct relation with grape anthocyanins. Prediction of color attributes of pH normalized experimental wines from the phenolic profiles of grapes was accomplished using a multivariate approach. The correlation between predicted and measured total wine color was high ( r = 0.958) but was very similar to the correlation coefficient obtained for the direct relation between grape anthocyanins and total wine color ( r = 0.961). Color due to copigmentation, color due to anthocyanins, and color intensity were also predicted well.     

Reaction between hydroxycinnamic acids and anthocyanin-pyruvic acid adducts yielding new portisins

Three new anthocyanin-derived pigments were found to occur in a 2-year-old Port red wine. Their structures were elucidated through LC/DAD-MS and NMR analysis and were found to correspond to a pyranoanthocyanin moiety linked to substituted cinnamyl substituents. The structures of these compounds are very similar to the one already reported for portisins, with a phenolic moiety replacing the catechin moiety. The newly formed anthocyanin-derived compounds display a bathochromic shift of the lambdamax ( approximately 540 nm) when compared with their anthocyanin-pyruvic acid adduct precursor (lambdamax = 511 nm), which may be due to the extended conjugation of the pi electrons in the structures of those pigments. Studies performed in model solutions helped to clarify the formation mechanism of these pigments that can result from the nucleophilic attack of the olefinic double bond of a hydroxycinnamic acid to the eletrophilic C-10 position of the anthocyanin-pyruvic acid adduct, followed by the loss of a formic acid molecule and decarboxylation. The chromatic characterization of these malvidin-3-glucoside-derived compounds revealed a higher resistance to discoloration against the nucleophilic attack by water and bisulfite when compared to malvidin-3-glucoside that is almost converted into its colorless hemiacetal form. However, the resistance to discoloration of these new pigments is not as high as the one reported for catechin-derived portisins. This could be explained by the presence of a smaller group (hydroxycinnamyl group), which does not protect so efficiently the chromophore against nucleophilic attack at the C-2 position. The occurrence of these pigments in red wine highlights new chemical pathways involving anthocyanin-pyruvic acid derivatives as precursors for the formation of new pigments in subsequent stages of wine aging that may contribute to its color evolution.     

Formation of hydroxyphenyl-pyranoanthocyanins in Grenache wines: precursor levels and evolution during aging

Grenache red wines were produced following three different winemaking techniques, that is, small-scale standard experimental wines and industrial-scale wines by both double-mash fermentation and fermentation in Ganimede vats. Wines were analyzed for their color properties, as well as the anthocyanin, flavonol, hydroxycinnamic acid, and pyranoanthocyanin profiles following alcoholic and malolactic fermentation. The evolution of pyranoanthocyanins and their corresponding precursors in the experimental wines was monitored at 6 and 10 months of aging. Wines produced by double-mash fermentation exhibited superior color properties compared to Ganimede wines and the experimental red wines, due to better extraction of flavonols and anthocyanins as well as a lower degree of polymerization. Pyranoanthocyanin formation varied within the different classes of pigments. Vitisins A and B were formed only during alcoholic fermentation. Pinotin A (i.e., the reaction product from malvidin 3-glucoside and caffeic acid) formation took place only during the aging process, whereas formation of hydroxyphenyl-pyranoanthocyanins derived from p-coumaric and ferulic acid followed two different pathways, that is, an enzymatically assisted production during fermentation and a pure chemical formation during aging.     

Effect of SO2 concentration on polyphenol development during red wine micro-oxygenation

A Merlot wine in 15 L research tanks was subjected to micro-oxygenation at 10 mL O2 per liter of wine per month over a 16 week period with additions of 0, 50, 100, and 200 mg/L SO2. A large decrease in monomeric anthocyanins and flavan-3-ols was seen in wines with a lower concentration of SO2, coupled with an increase in nonbleachable pigments; an increase in tannin, measured using precipitation with methyl cellulose; and a greater size and red coloration of a proanthocyanidin extract obtained using Sephadex LH-20. These changes were largely suppressed in wines initially treated with 200 mg/L SO2 and occurred more slowly in wines stored in bottles in the absence of O2. The concentration of SO2 is shown to regulate the polyphenol chemistry involved in the formation of polymeric pigments and changes in tannin structure affecting wine astringency.     

Role of vinylcatechin in the formation of pyranomalvidin-3-glucoside-+-catechin

Reactions between malvidin-3-glucoside (mv3glc) and 8-vinylcatechin were carried out to synthesize pyranomv3glc-(+)-catechin pigment and to study the formation of intermediates. A rapid decrease of mv3glc content concomitant with the formation of more complex structures such as mv3glc-vinylcatechin [precursor of pyranomv3glc-(+)-catechin pigment] and mv3glc-divinylcatechin was observed. On the other hand, 8-vinylcatechin undergoes acid-catalyzed dimerization in model wine solution, giving rise to 8-vinylcatechin dimers. These compounds were also found in the reaction between mv3glc and (+)-catechin mediated by acetaldehyde, which provides evidence for the formation of 8-vinylcatechin and its involvement in the formation of pyranoanthocyanins in aged red wines.     

Simulated digestion and antioxidant activity of red wine fractions separated by high speed countercurrent chromatography

Wine is an important source of dietary antioxidants because of its phenolic compound content. The antioxidant activity (AA) of pure monomer substances present in wines, such as phenolic acids, flavanols, and anthocyanins, has already been described, but the AA of polymeric phenols is still unknown. In this study, we have fractionated a red wine by countercurrent chromatography (CCC) into four fractions: fraction 1, made up of polymeric compounds; fraction 2, containing malvidin-3-glucoside; fraction 3, containing peonidin-3-glucoside; and fraction 4, containing vitisin A. The AA of these fractions was determined by oxygen radical absorbance capacity and ferric reducing ability assays. The weight of fraction 1 was the largest, so this was the largest contributor to the AA of the wine. However, the antioxidant powers (muM Trolox/g fraction) of fractions 2-4 were similar and higher than that of fraction 1. We also determined AA before and after in vitro gastric and intestinal digestions. After gastric digestion, the AA was 100-1000 times higher than the original fraction values. Gallic acid was determined in gastric and intestinal digested fractions. After intestinal digestion, the concentrations of simple phenols, such as caffeic acid, p-coumaric acid, and protocatechualdehyde, increased as they were released from the fractions under our conditions. Protocatechuic acid was determined in more intestinal digested fractions than in gastric digested fractions. These results partly explain the increase in AA after the digestion and indicate the relevance of polymeric polyphenolic compounds as precursors of smaller molecules with biological activity.     

Different sorption behaviors for wine polyphenols in contact with oak wood

The evolution of polyphenols of enological interest- monomeric anthocyanins, (+)-catechin, (-)-epicatechin, gallic acid, and trans-resveratrol-in the presence of oak wood was investigated in aging-model conditions. Disappearance kinetics showed that, except for gallic acid, all of the wine polyphenols tend to disappear from the model wine in presence of oak wood, to reach an equilibrium after 20 days of contact. At equilibrium, the higher disappearance rates were obtained for monomeric anthocyanins and trans-resveratrol with values of 20 and 50%, respectively. For monomeric anthocyanins, the rate of disappearance seemed to be independent of their nature. In order to evaluate the contribution of sorption to oak wood in the disappearance phenomena, sorption kinetics were determined for trans-resveratrol and malvidin-3-glucoside through the extraction and the quantification of the fraction sorbed to wood. These curves showed that the wood intake of trans-resveratrol and malvidin-3-glucoside followed a two-step behavior, with a higher rate during the first 2 days, likely due to a surface sorption mechanism, and then a slower rate to reach the equilibrium, which could be related to a diffusion mechanism. The comparison of disappeared and sorbed amounts at equilibrium showed that a minor part of the disappeared monomeric anthocyanins were sorbed by wood. In contrast, half of the concentration decrease of trans-resveratrol in wine finds its origin in a sorption mechanism by oak wood. Results in real wine show similar sorption kinetics.     

In vitro inhibition of human cGMP-specific phosphodiesterase-5 by polyphenols from red grapes

A moderate consumption of red wine may reduce the risk of cardiovascular diseases via wine-derived phenolic compounds. A variety of biological mechanisms have been proposed for wine-derived phenolic compounds including nitric oxide-mediated vasorelaxation. This study examined whether the vasodilating effect of wine-derived phenolic compounds was associated with the inhibition of phosphodiesterases (PDEs) and, in particular, PDE5. For this purpose, human recombinant PDE5A1 isoform was prepared by expression of the full-length cDNA of PDE5A1 into COS-7 cells. Red wine and the extracts from grape skin inhibited PDE5A1 activity, whereas the seed extracts had a negligible effect. The mixture of anthocyanins inhibited the enzyme activity (IC50 = 11.6 microM), with malvidin-3-O-beta-glucoside (IC50 = 35.4 microM) and malvidin (IC50 = 24.9 microM) the most potent among the monoglucosides and aglycons, respectively. trans-Resveratrol and trans-piceid exhibited negligible effects, whereas hydroxycinnamates were completely inactive. These results indicate that polyphenols-induced vasorelaxation may also be sustained by smooth muscle PDE inhibition by anthocyanins present in red wines and grapes.     

Identification of anthocyanin-flavanol pigments in red wines by NMR and mass spectrometry

Three newly formed Port wine pigments were isolated by Toyopearl HW-40(s) gel chromatography and semipreparative HPLC. Furthermore, the pigments were identified by mass spectrometry (LC/MS) and NMR techniques (1D and 2D). These anthocyanin-derived pigments showed UV-visible spectra different from those of the original grape anthocyanins. These pigments correspond to malvidin 3-glucoside linked through a vinyl bond to either (+)-catechin, (-)-epicatechin, or procyanidin dimer B3 [(+)-catechin-(+)-catechin]. NMR data of these pigments are reported for the first time.     

Influence of vine vigor on grape (Vitis vinifera L. Cv. Pinot Noir) anthocyanins. 2. Anthocyanins and pigmented polymers in wine

The relationships between grapevine (Vitis vinifera) vigor variation and resulting wine anthocyanin concentration and composition and pigmented polymer formation were investigated. The study was conducted in a commercial vineyard consisting of the same clone, rootstock, age, and vineyard management practices. Vine vigor parameters were used to designate vigor zones within two vineyard sites (A and B) to produce research wines (2003 and 2004) and conduct a model extraction experiment (2004 only) to investigate the vine-fruit-wine continuum. Wines and model extracts were analyzed by HPLC and UV-vis spectrophotometry. For the model extractions, there were no differences between sites for pomace weight, whereas juice volume was higher for site A. This was not related to a larger berry size. Site A had a higher anthocyanin concentration (milligrams per liter) in the model extracts than site B specifically for the medium- and low-vigor zones. For anthocyanin composition in the model extraction, site B had a greater proportion of malvidin-3-O-glucoside and less of the remaining anthocyanin glucosides (delphinidin, cyanidin, petunidin, and peonidin) compared to site A. In the wines, there was a vintage effect, with the 2003 wines having a higher anthocyanin concentration (milligrams per liter) than the 2004 wines. This appears to have been primarily due to a greater accumulation of anthocyanins in the fruit. In general, the medium-vigor zone wines had higher anthocyanin concentrations than either the high- or low-vigor zone wines. There was also vintage variation related to anthocyanin composition, with the 2003 wines having a higher proportion of delphinidin and petunidin glucosides and lower malvidin-3-O-glucoside compared to 2004. In both years, there were higher proportions of delphinidin and petunidin glucosides in wines made from low-vigor-zone fruit. Wines made from low-vigor zones showed a greater propensity to form vitisin A as well as pigmented polymers. Low-vigor-zone wines had a approximately 2-fold increase in pigmented polymer concentration (milligrams per liter) over high-vigor-zones wines. There was a strong positive relationship between pigmented polymer concentration, bisulfite bleaching resistant pigments, proanthocyanidin concentration, and color density in wines. Overall, differences found in the wines magnified variation in the fruit.