Relationship among antioxidant activity, vasodilation capacity, and phenolic content of red wines

The relationship among antioxidant activity, based on the electron-spin resonance determination of the reduction of Fremy's radical, vasodilation activity, and phenolic content was investigated in 16 red wines. The wines were selected to provide a range of origins, grape varieties, and vinification methods. Sensitive and selective HPLC methods were used for the analysis of the major phenolics in red wine: free and conjugated myricetin, quercetin, kaempferol, and isorhamnetin; (+)-catechin, (-)-epicatechin, gallic acid, p-coumaric acid, caffeic acid, caftaric acid, trans-resveratrol, cis-resveratrol, and trans-resveratrol glucoside. Total anthocyanins were measured using a colorimetric assay. The total phenolic content of the wines was determined according to the Folin-Ciocalteu colorimetric assay and also by the cumulative measurements obtained by HPLC. The 16 wines exhibited a wide range in the values of all parameters investigated. However, the total phenol contents, measured both by HPLC and colorimetrically, correlated very strongly with the antioxidant activity and vasodilation activity. In addition, the antioxidant activity was associated with gallic acid, total resveratrol, and total catechin. In contrast, only the total anthocyanins were correlated with vasodilation activity. The results demonstrate that the different phenolic profiles of wines can produce varying antioxidant and vasodilatant activities, which opens up the possibility that some red wines may provide enhanced health benefits for the consumer.     

Pulsed electric field-assisted vinification of aglianico and piedirosso grapes

Pulsed electric field (PEF) treatments were applied to increase the polyphenolic content of fresh red wines made from Aglianico and Piedirosso grapes. Prior to the fermentation/maceration step, the grape skins were treated at different PEF intensities (field strengths from 0.5 to 1.5 kV/cm and energy inputs from 1 to 50 kJ/kg), with their permeabilization being characterized by electrical impedance measurements. Furthermore, the release kinetics of the total polyphenols and anthocyanins were characterized during the maceration stage by spectroscopic and Folin-Ciocalteu colorimetric methods, respectively. Finally, the fresh wine, obtained after pressing, was characterized for total acidity, pH, reducing sugar, color intensity, total polyphenols, anthocyanins content, antioxidant activity, and volatile compound composition. PEF treatment on Aglianico grapes induced a significantly higher release of polyphenols (+20%) and anthocyanins (+75%), thus improving the color intensity (+20%) and the antioxidant activity of the wine (+20%) while preserving the other organoleptic characteristics. In contrast, there was only a minor impact on the polyphenolic release kinetics of Piedirosso grapes, despite the significant degree of cell membrane permeabilization.     

Levels of flavan-3-ols in French wines.

French wines are abundant sources of phenolic compounds. The content of several catechins, i.e., (+)-catechin, (-)-epicatechin, dimers B1, B2, B3, and B4, trimers C1, and trimer 2 (T2), of 160 French wines was determined by HPLC with UV detection. Red wines (n = 95) were found to have high levels of catechins, ranging from 32.8 to 209.8 mg/L (mean concentration 114.5 mg/L) for (+)-catechin, from 22.1 to 130.7 mg/L (mean concentration 75.7 mg/L) for (-)-epicatechin, from 7.8 to 39.1 mg/L (mean concentration 25.4 mg/L) for B1, from 18.3 to 93 mg/L (mean concentration 47.4 mg/L) for B2, from 21.4 to 215.6 mg/L (mean concentration 119.6 mg/L) for B3, from 20.2 to 107.2 mg/L (mean concentration 81.9 mg/l) for B4, from 8.6 to 36.9 mg/L (mean concentration 26.3 mg/L) for C1, and from 26.7 to 79.3 mg/L (mean concentration 67.1 mg/L) for T2. White and rosé wines (n = 57 and n = 8) were found to have low levels of (+)-catechin (mean concentrations 9.8 and 10.6 mg/L, respectively) and (-)-epicatechin (mean concentrations 5.3 and 6.5 mg/L, respectively). These data provide a basis for the epidemiological evaluation of catechin intake by the consumption of French wine.     

Identification of adducts between an odoriferous volatile thiol and oxidized grape phenolic compounds: kinetic study of adduct formation under chemical and enzymatic oxidation conditions

HPLC-MS and (1)H, (13)C, and 2D NMR analyses were used to identify new addition products between 3-sulfanylhexan-1-ol (3SH) and o-quinones derived from (+)-catechin, (-)-epicatechin, and caftaric acid. The kinetics of formation of these adducts were monitored in a wine model solution and in a must-like medium by HPLC-UV-MS with the aim of understanding the chemical mechanism involved in reactions between volatile thiols and o-quinones. One o-quinone-caftaric acid/3SH adduct, three o-quinone-(+)-catechin/3SH adducts, and three o-quinone-(-)-epicatechin/3SH adducts were characterized. Caftaric acid was oxidized faster than (-)-epicatechin and (+)-catechin when these phenolic compounds were incubated in a one-component mixture with polyphenoloxidase (PPO) in the presence of 3SH. Consequently, o-quinone-caftaric acid formed adducts with 3SH more rapidly than o-quinone-(+)-catechin and o-quinone-(-)-epicatechin in the absence of other nucleophilic species. Furthermore, o-quinone-(-)-epicatechin reacted faster than o-quinone-(+)-catechin with 3SH. Sulfur dioxide decreased the yield of adduct formation to a significant extent. Under chemical oxidation conditions, the rates and yields of adduct formation were lower than those observed in the presence of PPO, and o-quinone-caftaric acid was slightly less reactive with 3SH, compared to oxidized flavan-3-ols. The identification of o-quinone-caftaric acid/3SH and o-quinone-(+)-catechin/3SH adducts in a must matrix suggests that the proposed reaction mechanism is responsible for 3SH loss in dry wines during their vinification and aging process.     

Competition between (+)-catechin and (-)-epicatechin in acetaldehyde-induced polymerization of flavanols.

The reactions of (+)-catechin and (-)-epicatechin in the presence of acetaldehyde were studied in model solution systems. When incubated separately with acetaldehyde and at pH values varying from 2.2 to 4. 0, reactions were faster with (-)-epicatechin than with (+)-catechin. In mixtures containing both (+)-catechin and (-)-epicatechin with acetaldehyde, new compounds besides the homogeneous bridged derivatives were detected. These compounds were concluded to be hetero-oligomers consisting of (+)-catechin and (-)-epicatechin linked with an ethyl bridge. In this case, the reaction of (-)-epicatechin was faster than that of (+)-catechin. This was also observed in solutions containing the two flavanols and the (+)-catechin-ethanol intermediate. Under these conditions, the homogeneous (+)-catechin bridged dimers and heterogeneous dimers were obtained by action of the intermediate on (+)-catechin and (-)-epicatechin, respectively. In addition, the homogeneous (-)-epicatechin ethyl-bridged dimers were also detected, showing that ethyl linkages underwent depolymerization and recombination reactions.     

Antioxidant synergy and regeneration effect of quercetin, (-)-epicatechin,and (+)-catechin on alpha-tocopherol in homogeneous solutions of peroxidating methyl linoleate

Antioxidant interactions between flavonoids and alpha-tocopherol have been demonstrated by oximetry (oxygen concentration measured by ESR signal line width). In tert-butyl alcohol, a solvent in which flavonoids are weak retarders of peroxidation of methyl linoleate when initiated by alpha,alpha'-azoisobutyronitrile, quercetin and (-)-epicatechin were found to act synergistically with the chain-breaking antioxidant alpha-tocopherol. In chlorobenzene, a solvent in which flavonoids are chain-breaking antioxidants, quercetin and (+)-catechin each regenerated alpha-tocopherol, resulting in a co-antioxidant effect. The stoichiometric factor of the flavonoids as chain-breaking antioxidants in 1:1 mixtures with alpha-tocopherol was measured to be close to 1 for quercetin and slightly smaller for the catechins. The apparent inhibition rate constant, k(inh), for the mixture quercetin/alpha-tocopherol was measured to be 4.1 x 10(5) and 2.6 x 10(6) M(-1) s(-1) in tert-butyl alcohol and chlorobenzene, respectively, at 50 degrees C. A k(inh) of 4.4 x 10(5) M(-1) s(-1) was measured for (+)-catechin alone in chlorobenzene at 50 degrees C.     

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.     

Isomeric influence on the oxidative coloration of phenolic compounds in a model white wine: comparison of (+)-catechin and (-)-epicatechin

The reactions of (+)-catechin and (-)-epicatechin with glyoxylic acid were studied in a model white wine solution. When the reactions were performed in darkness at 45 degrees C, the (-)-epicatechin concentration decreased more rapidly than that of (+)-catechin, and the (-)-epicatechin sample had twice the 440 nm absorbance of the (+)-catechin sample after the 14 day incubation period. The main pigments generated were identified as xanthylium cation pigments regardless of the isomeric character of the phenolic compound. Using a combination of absorbance and ion current data, the xanthylium cation pigments generated from (-)-epicatechin were found to have combined molar absorptivity coefficients 1.8 times that of the xanthylium cation pigments generated from (+)-catechin. The implication of these results on the development of an index of white wine oxidation susceptibility is discussed.     

Catechin contents of foods commonly consumed in The Netherlands. 2. Tea, wine, fruit juices, and chocolate milk

Catechins, compounds that belong to the flavonoid class, are potentially beneficial to human health. To enable an epidemiological evaluation of catechins, data on their contents in foods are required. HPLC with UV and fluorescence detection was used to determine the levels of (+)-catechin, (-)-epicatechin, (+)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg) in 8 types of black tea, 18 types of red and white wines, apple juice, grape juice, iced tea, beer, chocolate milk, and coffee. Tea infusions contained high levels of catechins (102-418 mg of total catechins/L), and tea was the only beverage that contained GC, EGC, ECg, and EGCg in addition to (+)-catechin and (-)-epicatechin. Catechin concentrations were still substantial in red wine (27-96 mg/L), but low to negligible amounts were found in white wine, commercially available fruit juices, iced tea, and chocolate milk. Catechins were absent from beer and coffee. The data reported here provide a base for the epidemiological evaluation of the effect of catechins on the risk for chronic diseases.     

Yeast-induced inhibition of (+)-catechin and (-)-epicatechin degradation in model solutions

(+)-catechin and (-)-epicatechin degradation in water-alcohol solutions containing Fe2+ and tartaric acid was studied in the presence and absence of yeasts. On the basis of the results, yeast partially inhibited the degradation of both flavans, with much slower formation of browning products absorbing at 420 and 520 nm. In comparative terms, yeast was found to be more efficient toward the degradation products of (+)-catechin absorbing at the latter wavelength. Likewise, the presence of yeast decreased the yield of a group of colored compounds eluting at high retention times in HPLC and indicated these as important contributors to color darkening in white wines. This inhibitory effect may in part account for the resistance to browning observed over periods of several years in sherry wines subjected to biological aging under flor yeast.     

Effects of cultivar and processing method on the contents of catechins and procyanidins in grape juice

The aim of the presented work was to study the effect of pressing method, pasteurization, cultivar, and vintage on the content of (+)-catechin, (-)-epicatechin, and nine procyanidins in grape juice. The results showed that the concentration of these flavan-3-ols in the juice was influenced, in decreasing order of importance, by pressing method, cultivar, pasteurization, and vintage. Cold pressing without maceration was the least and hot pressing after maceration at 60 degrees C for 60 min the most effective method for extracting the flavan-3-ols. Pasteurization increased the concentration of catechins in cold-pressed juices, but it decreased concentrations in hot-pressed juices. The concentration of most procyanidins was increased by pasteurization. Among the white cultivars, Seyval and Niagara were highest in procyanidins and Elvira and Chardonnay were highest in catechins. Vincent, Foch, and Baco were the red cultivars highest in catechins, and Vincent also had the highest content of procyanidins.     

The extraction of anthocyanins and proanthocyanidins from grapes to wine during fermentative maceration is affected by the enological technique

The effect of three enological techniques (low temperature prefermentative maceration, must freezing with dry ice, and the use of a maceration enzyme) on the extraction of anthocyanins and proanthocyanidins from must to wine during fermentative maceration was studied to determine the extent to which these compounds are extracted and to assess the changes on their qualitative composition due to enological technique applied. The results showed that the dry ice treatment led to wines with high color intensity and high anthocyanin content, the maximum rate of extraction being observed the first 6 days of fermentative maceration. Regarding the effect of the different techniques on the quantitative and qualitative composition of proanthocyanidins, only the dry ice treatment seemed to favor the extraction of high molecular weight skin proanthocyanidins. The low temperature prefermentative maceration treatment led to the highest concentration of proanthocyanidins at the moment of pressing; however, this treatment, contrary to expectations, led to wines with the highest content of seed-derived proanthocyanidins. The use of the maceration enzyme also increased the concentration of proanthocyanidins during all of the fermentative process, as compared to a control wine, although the increase was not only due to skin proanthocyanidins but also seed proanthocyanidins. We have demonstrated in this study that maceration enzymes also facilitate seed phenolic extraction.     

Flavanol and flavonol contents of cocoa powder products: influence of the manufacturing process

Major brands of cocoa powder products present in the Spanish market were analyzed for monomeric flavanols [(+)-catechin and (-)-epicatechin] and flavonols [quercetin-3-glucuronide, quercetin-3-glucoside (isoquercitrin), quercetin-3-arabinoside, and quercetin]. In addition, the influence of the manufacturing process of cocoa powder products, in particular, the alkalinization treatment ( Dutching), on the original content of these flavonoids has been studied. (-)-Epicatechin was in the range of 116.02-730.26 microg/g, whereas (+)-catechin was in the range of 81.40-447.62 microg/g in the commercial cocoa products studied. Among flavonols, quercetin-3-arabinoside and isoquercitrin were the major flavonols in the cocoa powder products studied, ranging from 2.10 to 40.33 microg/g and from 3.97 to 42.74 microg/g, respectively, followed by quercetin-3-glucuronide (0.13-9.88 microg/g) and quercetin aglycone (0.28-3.25 microg/g). To our knowledge, these results are the first quantitative data in relation to the content of individualized flavonol derivatives in commercial cocoa powder products. The alkalinization treatment resulted in 60% loss of the mean total flavonoid content. Among flavanols, (-)-epicatechin presented a larger decline (67%, as a mean percentage difference) than (+)-catechin (38%), probably because of its epimerization into (-)-catechin, a less bioavailable form of catechin. A decline was also confirmed for di-, tri-, and tetrameric procyanidins. In the case of flavonols, quercetin presented the highest loss (86%), whereas quercetin-3-glucuronide, quercetin-3-arabinoside, and isoquercitrin showed a similar decrease (58, 62, and 61%, respectively). It is concluded that the large decrease found in the flavonoid content of natural cocoa powder, together with the observed change in the monomeric flavanol profile that results from the alkalinization treatment, could affect the antioxidant properties and the polyphenol biovailability of cocoa powder products.     

Inhibition of angiotensin converting enzyme activity by flavanol-rich foods

Angiotensin converting enzyme (ACE) activity was evaluated in the presence of flavanol-rich foods, i.e., wines, chocolates, and teas, and of purified flavonoids. All foods assayed inhibited ACE activity, red wines being more effective than white wine, and green tea more effective than black tea. The inhibition of ACE activity was associated with both phenolic and flavanol content in the foods. When isolated polyphenols were assayed, procyanidins (dimer and hexamer) and epigallocatechin significantly inhibited enzyme activity; similar concentrations of (+)-catechin, (-)-epicatechin, gallic acid, chlorogenic acid, caffeic acid, quercetin, kaempferol, and resveratrol were ineffective. When ACE activity was assayed in rat kidney membranes in the presence of chocolate extracts or purified procyanidins, it was observed that the inhibition depended on the chocolate content of flavanols and the number of flavanol units constituting the procyanidin. These experiments demonstrate that flavanols either isolated or present in foods could inhibit ACE activity. The occurrence of such inhibition in vivo needs to be determined, although is supported by the association between the consumption of flavanol-rich foods and reductions in blood pressure observed in several experimental models.     

Metabolism of grape seed polyphenol in the rat

The metabolism of grape seed polyphenol (GSP) has been investigated in rats by high-performance liquid chromatography analysis of the serum and urinary concentrations of the GSP metabolites (+)-catechin (CT), (-)-epicatechin (EC), 3'-O-methyl-(+)-catechin, and 3'-O-methyl-(-)-epicatechin. The serum concentration of these four metabolites reached a maximum 3 h after the oral administration of GSP. The urinary excretion of these GSP metabolites accounted for 0.254% (w/w) of the administered dose of GSP (1.0 g/kg), and the majority of these metabolites were excreted within 25 h of oral administration. The serum concentration and urinary excretion of these metabolites were also compared after the oral administration of different GSP monomers (gallic acid, CT, and EC), normal GSP, and the high molecular weight components of GSP (GSPH). No metabolites were detected in the serum of rats given GSPH. The urinary percentage excretion of the GSP metabolites derived from the respective monomers (CT or EC) did not vary with the administration of different substances (CT or EC, GSP, or GSPH). Taken together, these results suggest that only the monomers of GSP are absorbed and metabolized.     

Direct HPLC analysis of quercetin and trans-resveratrol in red wine,grape, and winemaking byproducts

A simple and fast reversed-phase HPLC method using diode array detection was developed and validated for the simultaneous determination of trans-resveratrol and quercetin in Sicilian red wine from the Nero d'Avola red grape variety. Investigation was also extended to the quantitative determination of resveratrol and quercetin in grape skins and winemaking byproducts obtained from the same cultivar. Samples were eluted using a C18 narrow-bore column under isocratic conditions in less than 20 min. Quantification of trans-resveratrol and quercetin in red wine was performed without any sample pretreatment, whereas the determination of these phenolic compounds in grape skins and wine pomage required a solvent extraction procedure. Linearity was demonstrated over the 0.39-12.5 and 0.45-57.6 microg/mL range for trans-resveratrol and quercetin, respectively. Detection limits in real samples were in the low ppm level (0.07 mg/L for trans-resveratrol and 0.12 mg/L for quercetin). The HPLC-UV/DAD method was applied for the routine analyses of red wine and grape skin and winemaking byproduct extracts to evaluate their trans-resveratrol and quercetin content. In particular, a very high content of quercetin was found in wine pomace, suggesting the use of this wine byproduct as a potential source of this health-promoting phenolic compound.     

Determination of phenolic constituents of biological interest in red wine by capillary electrophoresis with electrochemical detection

A simultaneous determination of trans-resveratrol, (-)-epicatechin, and (+)-catechin in red wine by capillary electrophoresis with electrochemical detection (CE-ED) is reported. The effects of the potential of the working electrode, pH and concentration of running buffer, separation voltage, and injection time on CE-ED were investigated. Under the optimum conditions, the analytes could be separated in a 100 mmol/L borate buffer (pH 9.2) within 20 min. A 300 microm diameter carbon disk electrode has a good response at +0.85 V (vs SCE) for all analytes. The response was linear over 3 orders of magnitude with detection limit (S/N = 3) ranging from 2 x 10(-7) to 5 x 10(-7) g/mL for all analytes. This method has been used for the determination of these analytes in red wine without enrichment, and the assay result was satisfactory.     

Phenolic composition of grape stems

Grape stems contain significant amounts of polyphenolic compounds, especially phenolic acids, flavonols, and flavanonols such as astilbin. The tannin content was characterized after the depolymerization reaction thiolysis. Tannins consisted of polymeric proanthocyanidins (up to 27 units) mainly consisting of (-)-epicatechin units along with smaller amounts of (+)-catechin, (-)-epicatechin gallate, and (-)-epigallocatechin. Flavanonols (astilbin) have been identified for the first time in stem and characterized by LC/MS and NMR. All phenolic compounds in grape stems were quantified by HPLC: quercetin 3-glucuronide was the most important, followed by catechin, caffeoyltartaric acid, and dihydroquercetin 3-rhamnoside (astilbin). Comparison was made of proanthocyanidin characteristics in different white and red grape varieties and also among parts of the cluster (skin, seed, and stem). Stem-condensed tannins were qualitatively intermediate between seed and skin but could not be differentiated between red and white varieties.     

Glyceraldehyde bridging between flavanols and malvidin-3-glucoside in model solutions

Hydroxyl radicals (.OH) seem to have an important role in the oxidation of wine constituents and the production of important electrophilic aldehydes and ketones. In this experiment, glyceraldehyde, a .OH oxidation product of glycerol, recently described in wine, reacts with (+)-catechin, (-)-epicatechin, and malvidin-3-glucoside (Mv3gl), in model solutions, yielding new condensed phenolic compounds. The adduct compounds formed were separated by means of reversed phase liquid chromatography and detected and characterized using UV-vis and electrospray ionization mass spectrometry. Flavanol-flavanol and anthocyanin-flavanol adducts linked with glyceraldehyde yielded compounds with m/z ratios for their main ions, in positive ion mode, of 653.2 for the (+)-catechin dimer or the (-)-epicatechin dimer and 855.5 for Mv3gl/(+)-catechin or Mv3gl/(-)-epicatechin dimers. The possible occurrence of these compounds in wine is suggested, and the potential role of these and related reactions in wine aging is discussed.     

Low temperature metabolism of apple phenolics and quiescence of Phlyctaena vagabunda.

The content of chlorogenic acid, (+)-catechin, (-)-epicatechin, phloretin glycosides, and quercetin glycosides in fresh and stored Golden Delicious apples (Malus domestica Borkh) was determined. The relative amount of phenolics in the peel, with the exception of chlorogenic acid and (-)-epicatechin, was higher than that in the flesh. In addition, quercetin glycosides were detected only in the skin. These compounds were tested for fungicidal activity against Phlyctaena vagabunda Desm., the causal agent of a postharvest rot. Chlorogenic acid only inhibited P. vagabunda spore germination and mycelial growth in vitro. Changes of apple phenolics and polyphenol oxidase activity during cold storage and the biological activity of these phenolics have also been analyzed with reference to the development of quiescent infections during cold storage plus shelf life at room temperature. The results obtained suggested that phloridzin and chlorogenic acid in combination with polyphenol oxidase activity could function to arrest P. vagabunda in quiescent infections associated with immature and ripening apple fruit.