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.     

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.     

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.     

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.     

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.     

Antioxidant action of glutathione and the ascorbic acid/glutathione pair in a model white wine

Glutathione was assessed individually, and in combination with ascorbic acid, for its ability to act as an antioxidant with respect to color development in an oxidizing model white wine system. Glutathione was utilized at concentrations normally found in wine (30 mg/L), as well as at concentrations 20-fold higher (860 mg/L), the latter to afford ascorbic acid (500 mg/L) to glutathione ratios of 1:1. The model wine systems were stored at 45 °C without sulfur dioxide and at saturated oxygen levels, thereby in conditions highly conducive to oxidation. Under these conditions the results demonstrated the higher concentration of glutathione could initially provide protection against oxidative coloration, but eventually induced color formation. In the period during which glutathione offered a protective effect, the production of xanthylium cation pigment precursors and o-quinone-derived phenolic compounds was limited. When glutathione induced coloration, polymeric pigments were formed, but these were different from those found in model wine solutions without glutathione. In the presence of ascorbic acid, high concentrations of glutathione were able to delay the decay in ascorbic acid and inhibit the reaction of ascorbic acid degradation products with the wine flavanol compound (+)-catechin. However, on depletion, the glutathione again induced the production of a range of different polymeric pigments. These results highlight new mechanisms through which glutathione can offer both protection and spoilage during the oxidative coloration of a model wine.     

The role of copper(II) in the bridging reactions of (+)-catechin by glyoxylic acid in a model white wine

The influence of copper(II) on the bridging reactions between (+)-catechin and glyoxylic acid was studied in a white wine-like medium. When the reaction was performed in darkness at 45 degrees C, copper(II) increased the maximum levels of carboxymethine-linked (+)-catechin dimer and xanthylium cation pigment as monitored by high-performance liquid chromatography/photodiode array detection (HPLC/DAD) and liquid chromatography/mass spectrometry (LC/MS). At 10 degrees C, similar results were observed except that the xanthene intermediate was monitored and found to also increase in concentration at higher copper(II) concentrations. The kinetics for the formation of these species suggested that copper(II) accelerated the bridging of two (+)-catechin units by glyoxylic acid. The acid group of glyoxylic acid allowed copper(II) to influence this reaction, as no copper(II) enhancement was observed when acetaldehyde was used in place of glyoxylic acid.     

Spectrophotometric method for exploring 3-methyl-2-butene-1-thiol (MBT) formation in lager

The disappearance of riboflavin absorbance at 445 nm from beers or model beers on light exposure is directly linked to light-struck character formation. The addition of (+)-catechin, (-)-epicatechin, tryptophol, or ascorbic acid was able to reduce, but not stop, absorbance loss or light-struck character formation in either model beer or mainstream lager that was exposed to light. When isohumulone was present in model beer, the inhibitory effect of (+)-catechin, (-)-epicatechin, or tryptophol decreased with increasing isohumulone. The spectrophotometric method used in this study is a simple and effective method for determining light-struck susceptibility.     

New polyphenolic compounds with xanthylium skeletons formed through reaction between (+)-catechin and glyoxylic acid

The reaction between (+)-catechin and glyoxylic acid was studied in a model solution system. The major (+)-catechin carboxymethine-linked dimer was isolated and shown to proceed to new polyphenolic compounds exhibiting absorption maxima around 440 and 460 nm. Three yellow pigments were obtained by incubation of the 8-8 colorless isomer. One was the previously reported xanthylium compound NJ2 with a maximum at 440 nm. The other two, showing absorption maxima at 460 nm, were obtained separately by incubation of the colorless dimer in hydroethanolic or methanolic medium. Structural elucidation of these two new yellow pigments was achieved by means of MS and 1D and 2D NMR techniques and showed that they were, respectively, ethyl and methyl esters of NJ2. The fact that these compounds were not obtained when NJ2 was incubated in hydromethanolic or ethanolic medium showed that esterification took place before the formation of the xanthylium chromophores. The detection of the esterified colorless compounds and the corresponding xanthene intermediates confirmed the postulated mechanism. New pigments exhibiting a strong absorption at 560 nm were also observed.     

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.     

trans-Resveratrol, quercetin, (+)-catechin, and (-)-epicatechin content in south Italian monovarietal wines: relationship with maceration time and marc pressing during winemaking

The concentrations of trans-resveratrol, (+)-catechin, (-)-epicatechin, and quercetin were evaluated by means of high-performance liquid chromatography-diode array detection in red wines obtained from Aglianico, Piedirosso, and Nerello Mascalese grapes. The trans-resveratrol and epicatechin concentrations did not differ significantly between experimental wines. The concentration of quercetin in Nerello Mascalese wines was more than twice that observed in Aglianico and Piedirosso wines. Nerello Mascalese wines also significantly differed from other wines in the (+)-catechin content, which was significantly higher than those found in the other two wines. During maceration, the maximum extraction of trans-resveratrol was reached after 12 days for Aglianico and Piedirosso, after which a decline was observed. On the contrary, in the case of Nerello Mascalese, the concentration of trans-resveratrol increased steadily throughout the whole maceration process. After 2 days of maceration, the maximum concentration of quercetin was observed in Aglianico must, whereas the maximum quercetin extraction was reached after 12 days for Piedirosso and 17 days for Nerello Mascalese. The maximum levels of (+)-catechin and (-)-epicatechin were generally observed after 12 days of maceration for all wines, although a decline of (-)-epicatechin occurred after maximum extraction in Aglianico and Piedirosso wines. Following marc pressing, a significant increase in the concentration of trans-resveratrol for Aglianico, (+)-catechin and (-)-epicatechin for Piedirosso, and (-)-epicatechin for Nerello Mascalese was observed.     

Isolation and structural characterization of new acylated anthocyanin-vinyl-flavanol pigments occurring in aging red wines

Three newly formed pigments were detected and isolated from a 2-year-old Port wine through TSK Toyopearl HW-40(S) gel column chromatography and characterized by UV-visible spectrophotometry, NMR, and mass spectrometry (ESI/MS). (1)H NMR and (13)C NMR data for these pigments obtained using 1D and 2D NMR techniques (COSY, NOESY, gHSQC, and gHMBC) are reported for the first time. The structure of the pigments was found to correspond to the vinyl cycloadducts of malvidin 3-coumaroylglucoside bearing either a procyanidin dimer or a flavanol monomer ((+)-catechin or (-)-epicatechin). Additionally, conformational analysis was performed for one of these newly formed pigment using computer-assisted model building and molecular mechanics. A chemical nomenclature is proposed to unambiguously name this new family of anthocyanin-derived pigments.     

Ascorbic acid-induced browning of (+)-catechin in a model wine system

The ability of ascorbic acid to induce browning of (+)-catechin in a model wine system has been studied. A significant increase in absorbance at 440 nm was observed over 14 days when ascorbic acid was incubated at 45 degrees C with (+)-catechin in a model wine base. The onset of browning was delayed for about 2 days, although the length of the lag period was dependent on the amount of molecular oxygen in the headspace of the reaction system. The lag period was not observed when a preoxidized solution of ascorbic acid was used, suggesting that a product of ascorbic acid oxidation is responsible for the onset of browning. Hydrogen peroxide, when added directly to (+)-catechin in the model system, was not capable of producing the same degree of browning as that generated by ascorbic acid. Liquid chromotography evidence is presented to show that different reaction products are produced by ascorbic acid and hydrogen peroxide.     

Study of the reactions between (+)-catechin and furfural derivatives in the presence or absence of anthocyanins and their implication in food color change

(+)-catechin was separately incubated with furfural or with 5-(hydroxymethyl)furfural, and the formation of new oligomeric bridged compounds having flavanol units linked by furfuryl or 5-hydroxymethylfurfuryl groups was observed. LC/ESI-MS analyses detected four dimeric adducts along with intermediate adducts in each solution, and reaction was faster with furfural than with hydroxymethylfurfural. In addition, new compounds exhibiting the same UV--visible spectra as xanthylium salts with absorption maxima around 440 nm were also detected. When malvidin 3-O-glucoside or cyanidin 3-O-glucoside was added to the mixtures, new oligomeric colorless and colored pigments involving both (+)-catechin and anthocyanin moieties were detected, showing thus that the two polyphenols competed in the condensation process. Among the obtained colored pigment adducts, two dimeric compounds in which the flavanol was bridged to the anthocyanin were observed. Their UV-visible spectra were similar to the spectrum of malvidin 3-O-glucoside, but their maximum in the visible region was bathochromically shifted.     

Kinetics of thermal modifications in a grape seed extract

The thermal modification kinetics of a commercial grape seed extract (GSE) was investigated. A GSE was exposed to 60, 90, and 120 °C for 5, 10, 15, 30, 45, and 60 min. The antioxidant activity (AA) and the absorbance at 420 nm (A(420)) were measured. (+)-Catechin, (-)-epicatechin, procyanidins B1 and B2, and gallic acid were identified and measured. After the thermal treatments, the AA did not show a significant difference (p > 0.05) and both procyanidins and gallic acid increased as well as A(420). (+)-Catechin and (-)-epicatechin decreased. To obtain the activation energy (E(a)) of the changes, a modified Weibull and a combined zero- and first-order model were compared, both followed by the Arrhenius equation. The Weibull model was more accurate. The E(a) values for browning and (+)-catechin, (-)-epicatechin, gallic acid, and procyanidins B1 and B2 were 170, 286, 42, 102, 249, and 95 kJ/mol, respectively. The results were valid at a confident level of 95%.     

New phenolic compounds formed by evolution of (+)-catechin and glyoxylic acid in hydroalcoholic solution and their implication in color changes of grape-derived foods

The reaction between (+)-catechin and glyoxylic acid in model solution system was investigated by LC/DAD and LC/ESI-MS analyses. The formation of phenolic compounds exhibiting absorption maxima near 300 nm and presenting a shoulder around 350 nm was observed. Their structures consisted of a (+)-catechin unit with one or two aldehyde groups linked at positions 6, 8 or 6 and 8 of the A ring. In addition, new yellow pigments exhibiting UV-visible spectra similar to those of xanthylium salts with absorption maxima at 450 and 280 nm were also detected. The major yellow compound was isolated and identified by ESI-MS and 1D and 2D NMR spectroscopy. The implication of these compounds in color change and browning observed during aging of grape-derived beverages is also discussed.     

Influence of procyanidins on the color stability of oenin solutions

The aim of the present work was to specify the influence of the polymerization degree on the color stability of anthocyanins using model solutions under higher thermal conditions simulating rapid food aging. Results showed that an increase in polymeric degree improves the color stability of oenin. Solutions containing a catechin tetramer, purified from brown rice, displayed a remarkable stability. Flavanols as monomers, (+)-catechin and (-)-epicatechin, appeared to decrease stability with the formation of a xanthylium salt leading to yellowish solutions. For the dimers, procyanidin B2 and B3, different behaviors on red color stability have been observed corresponding to their different susceptibility to cleavage upon heating. In the presence of the trimeric procyanidin C2, the red color appeared more stable. However, the HPLC chromatograms showed a decrease in the amplitude of the peaks of oenin and procyanidin C2. Concomitantly, a new peak appeared with a maximal absorption in the red region. This newly formed pigment probably came from the condensation of oenin and procyanidin C2.     

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.     

Rapid reversed phase ultra-performance liquid chromatography analysis of the major cocoa polyphenols and inter-relationships of their concentrations in chocolate

Chocolate and other cocoa-containing products are a rich source of polyphenols. This paper describes an ultra-performance liquid chromatography (UPLC) method that can separate and quantify in 3 min six of the major chocolate polyphenols: catechin; epicatechin; B2 (epicatechin-4beta-8-epicatechin); B5 (epicatechin-4beta-6-epicatechin); C1 (epicatechin-4beta-8-epicatechin-4beta-8-epicatechin); and tetramer D (epicatechin-4beta-8-epicatechin-4beta-8-epicatechin-4beta-8-epicatechin). A survey of 68 chocolate samples indicated that there was a strongly predictive relationship between epicatechin and the other individual polyphenols, especially procyanidin B2 (R 2 = 0.989), even though the chocolates came from varied sources and manufacturers. The relationship was less strong with catechin, and so further work to explore the reasons for this difference was performed. Chiral analysis on a subset of 23 chocolates showed that (-)-epicatechin had a predictive relationship with (+)-catechin in line with the other polyphenols, but not with (-)-catechin (the predominant form). This indicates that (-)-catechin is the most affected by manufacturing conditions, possibly formed through epimerization from (-)-epicatechin during processing. The results show that epicatechin concentrations can be used to predict the content of other polyphenols, especially B2 and C1, and total polyphenols content. Finally, the (-)-catechin content is not predictable from the epicatechin content, and it is concluded that this is the main form of polyphenol that varies according to manufacturing conditions and cocoa origin.     

Isolation and structural elucidation of some procyanidins from apple by low-temperature nuclear magnetic resonance

Procyanidin fractions from apple were separated according to the degree of polymerization using normal phase chromatography. Evaluation of physiological functionalities of procyanidins requires individual structural determination. However, it is difficult to elucidate the structure of procyanidins, in particular those with (+)-epicatechin (1) or (-)-catechin (2) units, and determine whether the interflavanoid bonds are 4beta-->8 or 4beta-->6 without cleavage and acetylation. Structural determination used LC-MS and low-temperature NMR. Nine procyanidins were separated by preparative HPLC consisting of three well-known procyanidins [procyanidin B1 (3), procyanidin B2 (4), and procyanidin C1 (5)] and six new procyanidins [epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-catechin (6); epicatechin-(4beta-->6)-epicatechin-(4beta-->8)-catechin (7); epicatechin-(4beta-->6)-epicatechin-(4beta-->8)-epicatechin (8); epicatechin-(4beta-->8)-epicatechin-(4beta-->6)-catechin (9); epicatechin-(4beta-->8)-epicatechin-(4beta-->6)-epicatechin (10); and epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-epicatechin (11)]. Compounds 6-11 were detected for the first time as apple constituents.