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.     

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.     

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.     

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.     

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.     

Interactions between cyanidin 3-O-glucoside and furfural derivatives and their impact on food color changes

The reaction between (+)-catechin and cyanidin 3-O-glucoside was investigated in the presence of furfural and 5-(hydroxymethyl)furfural using LC/DAD and LC/MS analysis, and the obtained results were compared with those recorded with malvidin 3-O-glucoside. The appearance of colorless and red and yellow compounds was observed showing that the two polyphenols competed in the condensation process with a predominant formation of the reddish adducts. The colored compounds formed in the case of cyanidin 3-O-glucoside seemed to be more stable than those formed when the reaction was conducted with malvidin 3-O-glucoside. The detection of these reddish and yellowish compounds constitutes a new support for the contribution of this kind of reaction in the color evolution of fruit-derived beverages. In addition, other unidentified compounds were also detected, showing the occurrence of other interaction pathways in addition to the polymerization process yielding oligomeric bridged derivatives and opening perspectives of further investigations of these model solutions.     

Role of aldehydic derivatives in the condensation of phenolic compounds with emphasis on the sensorial properties of fruit-derived foods

The reactions between (epi)catechin, mavidin 3-O-glucoside, and some aldehydes were investigated by LC/DAD and LC/ESI-MS analysis. The obtained results showed that the acetaldehyde-mediated condensation occurred more generally and glyoxylic acid, furfural, and 5-(hydroxymethyl)furfural (HMF) react in the same way in the first stages of the reactions. In terms of reactivity, reactions were faster with acetaldehyde than with glyoxylic acid, furfural, or HMF, where the reactions were slower. In the case of acetaldehyde, the obtained purple derivatives were more predominant and stable than the colorless adducts and no xanthylium salt was detected. Interactions involving glyoxylic acid yield purple adducts, which were obtained in small amount compared to the colorless ones. The latter were shown to proceed to more polymerized and yellowish derivatives. Finally, in the case of furfural and HMF, purple compounds involving flavanol and anthocyanin units were detected, and colorless compounds were shown to be predominant and to yield yellowish xanthylium salts.     

(+)-Catechin-aldehyde condensations: competition between acetaldehyde and glyoxylic acid

(+)-Catechin reaction with two aldehydes (acetaldehyde and glyoxylic acid) was studied in winelike model solution. The two aldehydes were reacted either individually or together with (+)-catechin and in molar excess. The reactions were followed by HLPC-UV and HPLC-ESI/MS to monitor (+)-catechin disappearance as well as dimer and polymer appearance. In all reactions a reaction order of close to 1 for (+)-catechin disappearance was observed. (+)-Catechin disappearance was slower in the presence of acetaldehyde (t(1/2) = 6.7 +/- 0.2 h) compared to glyoxylic acid (t(1/2) = 2.3 +/- 0.2 h). When the two aldehydes were reacted together, (+)-catechin disappearance was faster (t(1/2) = 2.2 +/- 0.5 h). When aldehydes were reacted separately, the dimer appearance was independent of the type of aldehyde used but the ethyl-bridged dimer disappearance was slower with acetaldehyde. When aldehydes were reacted together, the dimer appearance changed. Ethyl-bridged dimers appeared before carboxymethine-bridged dimers, and their disappearance occurred earlier. Copolymers containing both ethyl and carboxymethine bridges were also observed.     

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.     

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.     

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.     

Novel oxidations of (+)-catechin by horseradish peroxidase and laccase

Horseradish peroxidase (HRP; EC 1.11.1.7) catalyzed the H(2)O(2)-dependent oxidative coupling of (+)-catechin 1 to form three different biphenyl C-C dimers 2-4, whereas Rhus vernicifera laccase catalyzed the formation of two new catechin-hydroquinone adducts 5 and 6. Spectroscopic evidence showed that HRP dimers were linked through position 8 of the A-ring of one catechin moiety to C-5' of ring B in 2 and 4 and to C-2 of ring C in 3. The unusual catechin dicarboxylic acid dimer 4 was obtained by ortho cleavage of the E-ring. Hydroquinone served as both a shuttle oxidant and a reactant by coupling at C-2' and C-5' of the catechin B-ring during laccase oxidations. HRP and laccase oxidation products were compared to D,L-alpha-tocopherol and (+)-catechin for their abilities to inhibit iron-induced lipid peroxidation in rat brain homogenates and Fe(3+)-ADP/NADPH in rat liver microsomes, as measured by the intensity of thiobarbituric acid reactive substance. All metabolites exhibited anti-lipid peroxidation with IC(50) values approximately 2-8 times higher than those of standard compounds. Characteristic reaction products may prove to be novel markers for (+)-catechin antioxidant reactions in living systems.     

Characterization of a colorless anthocyanin-flavan-3-ol dimer containing both carbon-carbon and ether interflavanoid linkages by NMR and mass spectrometry

Direct addition of anthocyanins and flavan-3-ols was investigated in a model system by incubating malvidin 3-glucoside and (-)-epicatechin in ethanol. Analysis of reaction products by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC/ESI-MS) before and after thiolysis showed the formation of colorless dimers detected at m/z 781 in the negative ion mode, with retention times and spectroscopic characteristics identical to those of compounds detected in wine, which contain one malvidin 3-glucoside unit and one flavanol unit. On the basis of their resistance to thiolysis, these compounds were postulated to be bicyclic dimers linked with both carbon-carbon and ether bonds as observed in the case of A type proanthocyanidins. The major dimer analyzed by NMR experiments was identified as malvidin 3-glucoside(C2-O-C7,C4-C8)epicatechin, confirming this hypothesis. A similar assay was performed with (+)-catechin instead of (-)-epicatechin, and the formation of bicyclic dimers was also observed.     

Stabilizing effect of ascorbic acid on flavan-3-ols and dimeric procyanidins from cocoa

Cocoa flavanols and procyanidins have numerous biological activities. It is known that (-)-epicatechin, (+)-catechin, epicatechin-(4beta-8)-epicatechin (dimer B2), and epicatechin-(4beta-6)-epicatechin (dimer B5) are unstable at physiologic pH, degrading almost completely within several hours, whereas they are relatively stable at pH 5.0. The present study investigated the effects of ascorbic and citric acid on the stability of monomers and dimers in simulated intestinal juice (pH 8.5) and in sodium phosphate buffer (pH 7.4). The addition of ascorbic acid to the incubation mixture significantly increased the stability of the monomers and dimers, whereas the addition of citric acid provided no protective effects. LC-MS showed that with the degradation of dimer B2 and dimer B5, doubly linked A-type dimers were formed. The present results, although not directly transferable to in vivo conditions, suggest that ascorbic acid may stabilize cocoa flavanols and procyanidins in the intestine where the pH is neutral, or alkaline, before absorption.     

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.     

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.     

Impact of glutathione on the formation of methylmethine- and carboxymethine-bridged (+)-catechin dimers in a model wine system

This study was performed to assess the impact of glutathione on the reaction between (+)-catechin and carbonyl compounds in wine-related conditions. (+)-Catechin (0.50 mM) and either glyoxylic acid (0.25 mM) or acetaldehyde (0.25 mM) were added to a model wine system with 0.0, 0.25, and 2.5 mM of glutathione added. UPLC-DAD and LC-MS analysis showed that the formation of carbonyl-bridged (+)-catechin dimers was inhibited in the samples with a glutathione to carbonyl ratio of 10:1 compared to the samples without glutathione. At a ratio of 1:1, glutathione inhibited the acetaldehyde-bridged dimers but only had a minor impact on the glyoxylic acid-bridged dimers. Further investigations showed that this trend of inhibition by glutathione on the glyoxylic acid-derived dimer was independent of temperatures, 20 °C vs 45 °C, or the presence of metal ions, 0.2 mg/L copper(II) and 5 mg/L iron(II). (1)H NMR analysis and LC-MS analysis provided evidence that glutathione inhibited dimer formation via different mechanisms depending on the carbonyl compound. For acetaldehyde-derived dimers, the main mode of inhibition was the ability of glutathione to form a (methyl-glutathionyl-methine)-(+)-catechin complex. Alternatively, the formation of a glutathione-glyoxylic acid addition product impeded the reaction between glyoxylic acid with (+)-catechin. These results demonstrate that glutathione, at sufficient concentration, can have a substantial impact on carbonyl-derived polymerization reactions in wine-like conditions.     

Defining the ascorbic acid crossover from anti-oxidant to pro-oxidant in a model wine matrix containing (+)-catechin

An examination of the ascorbic acid-induced oxidation of (+)-catechin was carried out. Using varying concentrations of ascorbic acid in a model white winebase, it was observed that there are at least two distinct steps in its oxidation process. The first step involves the formation of species that absorb in the visible region of the spectrum, while the second step generates species of less or no absorbance in the visible region. The first step reaches an absorbance maximum when ascorbic acid is completely oxidized. In winebase solutions containing both ascorbic acid and (+)-catechin, the lag period prior to the onset of (+)-catechin oxidation was dependent on the concentration of ascorbic acid. It was also observed that the end of the lag period corresponds to the complete oxidation of ascorbic acid. Xanthylium cations were identified as a species responsible for the increase in absorbance at 440 nm post lag period. The implication of the results, for establishing a chemical basis to the ascorbic acid crossover from antioxidant to pro-oxidant, is discussed.     

Analysis of tea shoot catechins: spectrophotometric quantitation and selective visualization on two-dimensional paper chromatograms using diazotized sulfanilamide.

A highly specific and sensitive diazotized sulfanilamide reagent is synthesized for determination of tea catechins. The reagent is employed both as spray reagent for selective visualization of tea catechins on two-dimensional paper chromatograms (sensitivity <1 microg of d-(+)-catechin) and for their spectrophotometric quantification in the crude extracts of tea polyphenols isolated from fresh or dried tea shoots. The formation of yellow color (lambda(max) = 425 nm) between catechins and diazotized sulfanilamide was investigated and made the basis of a simple and sensitive spectrophotometric method for estimation of the total and individual catechins in different tea cultivars. At 425 nm, the absorbance was linear (r = 0.999) over the (0.4-8.0 microg/mL) concentration range of d-(+)-catechin.     

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.