Studies on the acetaldehyde-induced condensation of (-)-epicatechin and malvidin 3-O-glucoside in a model solution system.

The reaction between (-)-epicatechin, malvidin 3-O-glucoside, and acetaldehyde was studied in a model solution system. Ethyl-linked flavanol oligomers and anthocyanin-flavanol derivatives were observed, showing that the two polyphenols competed in the condensation process. Among the anthocyanin-ethyl-flavanol adducts, dimeric compounds in which the flavanol was linked to the anthocyanin with CH(3)-CH bridges were observed. In addition, trimeric and tetrameric products containing one anthocyanin and one, two, or three flavanols units were detected. A tetrameric product containing two anthocyanin and two flavanol units was also found as a doubly charged ion. No compound containing more than two malvidin 3-O-glucosides was detected, suggesting that only one anthocyanin A ring summit can be included in the polymerization process, which thus stops when both ends are occupied by an anthocyanin moiety. Thioacidolysis of the two isolated anthocyanin-ethyl-flavanol dimeric derivatives showed that anthocyanin-ethyl linkage was not sensitive to such reactants, whereas the flavanol-ethyl one was. In addition, flavanol-ethyl linkages involved in anthocyanin-ethyl-flavanol adducts were found to be less sensitive to those involved in flavan-ethyl dimers.     

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.     

Investigations on anthocyanins in wines from Vitis vinifera cv. pinotage: factors influencing the formation of pinotin A and its correlation with wine age

Pinotage red wines were found to contain a reaction product of malvidin 3-glucoside and caffeic acid, the so-called pinotin A. A total of 50 Pinotage wines from the vintages 1996-2002 were analyzed for the content of pinotin A, malvidin 3-glucoside, caffeic acid, and caftaric acid. Statistical analyses were performed to reveal variations in the content of these compounds and to determine the factors that influence pinotin A formation during wine aging. An exponential increase of the concentration of this aging product was observed with prolonged storage time. The most rapid synthesis of pinotin A was observed in 2.5-4 year old wines, although at this age malvidin 3-glucoside is already degraded to a large extent. This phenomenon is explained by the increased ratio of caffeic acid/malvidin 3-glucoside, which strongly favors the formation of pinotin A and makes side reactions less likely. Pinotin A formation proceeds as long as a certain level of malvidin 3-glucoside is maintained in the wines. In wines >5-6 years old degradation or polymerization of pinotin A finally exceeds the rate of its de novo synthesis.     

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.     

Characterization and estimation of proanthocyanidins and other phenolics in coffee pulp (Coffea arabica) by thiolysis-high-performance liquid chromatography

Fresh and 3-day-old coffee pulp of the Arabica variety were analyzed for polyphenol composition followed by characterization by two different methods. The first method consisted in subjecting coffee pulp powder to direct thiolysis. For the second method, coffee pulp was subjected to successive solvent extractions, followed by thiolysis. Quantification of phenolic compounds was then achieved by high-performance liquid chromatography (HPLC) analysis of thiolysis products. Four major classes of polyphenols were identified: flavan-3-ols (monomers and procyanidins), hydroxycinnamic acids, flavonols, and anthocyanidins. Differences in concentration of procyanidins were observed between fresh and 3-day-old coffee pulp. Constitutive units were mainly epicatechin, representing more than 90% of the proanthocyanidin units, with average degrees of polymerization in the range of 3.8-9.1. Monomer to hexamer units of flavan-3-ols from fresh coffee pulp were separated by normal-phase HPLC. Molecular size of oligomeric proanthocyanidins was obtained by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Results obtained confirm the presence of oligomers of the flavan-3-ol (-)-epicatechin.     

PH-dependent forms of red wine anthocyanins as antioxidants

Anthocyanins are one of the main classes of flavonoids in red wines, and they appear to contribute significantly to the powerful antioxidant properties of the flavonoids. In grapes and wines the anthocyanins are in the flavylium form. However, during digestion they may reach higher pH values, forming the carbinol pseudo-base, quinoidal-base, or the chalcone, and these compounds appear to be absorbed from the gut into the blood system. The antioxidant activity of these compounds, in several metal-catalyzed lipid oxidation model systems, was evaluated in comparison with other antioxidants. The pseudo-base and quinoidal-base malvidin 3-glucoside significantly inhibited the peroxidation of linoleate by myoglobin. Both compounds were found to work better than catechin, a well-known antioxidant. In a membrane lipid peroxidation system, the effectiveness of the antioxidant was dependent on the catalyst: In the presence of H(2)O(2)-activated myoglobin, the inhibition efficiency of the antioxidant was malvidin 3-glucoside > catechin > malvidin > resveratrol. However, in the presence of an iron redox cycle catalyzer, the order of effectiveness was resveratrol > malvidin 3-glucoside = malvidin > catechin. The pH-transformed forms of the anthocyanins remained effective antioxidants in these systems, and their I(50) values were between 0.5 and 6.2 microM.     

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.     

Identification and quantification of free radical scavengers in Pu-erh tea by HPLC-DAD-MS coupled online with 2,2'-Azinobis(3-ethylbenzthiazolinesulfonic acid) diammonium salt assay

Pu-erh tea, a well-known traditional beverage in China, has attracted more attention because of its beneficial health effects and special flavor and taste. Generally, it is believed that Pu-erh tea with a longer preservation period has better quality and taste. Antioxidant activity is one of the major beneficial activities of tea. In this study, a HPLC-DAD-MS coupled with 2,2'-azinobis (3-ethylbenzthiazolinesulfonic acid) diammonium salt (ABTS) assay was employed for identification and quantification of free radical scavengers in different samples of Pu-erh tea. Among 12 main peaks detected in Pu-erh raw tea, 11 compounds were identified as gallic acid, (-)-gallocatechin, (-)-epigallocatechin, (+)-catechin, caffeine, (-)-epicatechin, (-)-epigallocatechingallate, rutin, (-)-epicatechingallate, quercetin-3-glucoside, and kaempferol-3-glucoside by comparison of their UV and MS data with standard compounds or literature data, respectively. The contents of 12 investigated compounds were also determined or estimated using caffeine, (-)-epicatechin, or rutin as standard. ABTS assay showed that 10 out of 12 compounds were free radical scavengers. Their total amount was used as the marker for evaluation of free radical scavenging activities of different Pu-erh teas, which indicated that the activity of different Pu-erh teas varied; Pu-erh raw tea was stronger than the ripe one, and the activity decreased with the increase of preservation period.     

Varietal differences among the polyphenol profiles of seven table grape cultivars studied by LC-DAD-MS-MS

Polyphenols present in red table grape varieties Red Globe, Flame Seedless, Crimson Seedless, and Napoleon, and the white varieties Superior Seedless, Dominga, and Moscatel Italica were analyzed by HPLC-DAD-MS. The anthocyanins peonidin 3-glucoside, cyanidin 3-glucoside (and their corresponding p-coumaroyl derivatives), malvidin 3-glucoside, petunidin 3-glucoside, and delphinidin 3-glucoside were found. In addition, caffeoyltartaric acid, p-coumaroyltartaric acid, and the flavonols quercetin 3-glucuronide, quercetin 3-rutinoside, quercetin 3-glucoside, kaempferol 3-galactoside, kaempferol 3-glucoside, and isorhamnetin 3-glucoside were detected. Flavan-3-ols were also detected, and were identified as gallocatechin, procyanidin B1, procyanidin B2, procyanidin B4, procyanidin C1, catechin, and epigallocatechin. These phenolics were present only in the skin, as the flesh of these grape cultivars was almost devoid of these compounds. Anthocyanins were the main phenolics in red grapes ranging from 69 (Crimson Seedless) to 151 (Flame Seedless) mg/kg fresh weight of grapes, whereas flavan-3-ols were the most abundant phenolics in the white varieties ranging from 52 (Dominga) to 81 (Moscatel Italica) mg/kg fresh weight of grapes. Total phenolics ranged from 115 (Dominga) to 361 (Flame Seedless) mg/kg fresh weight of grapes. This means that a serving of unpeeled table grapes (200 g) could provide up to 72 mg of total phenolics (Flame Seedless). These results indicate that the intake of unpeeled table grapes should be recommended in dietary habits as a potential source of antioxidant and anticarcinogenic phenolic compounds.     

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.     

Structural characterization of new malvidin 3-glucoside-catechin aryl/alkyl-linked pigments

The condensation reaction between malvidin 3-glucoside and catechin mediated by isobutyraldehyde, benzaldehyde, and isovaleraldehyde was conducted in model solutions at two pH values (1.5 and 3.2). The formation of new alkyl/aryl-linked adducts corresponding to the structures malvidin 3-glucoside-isobutylcatechin, malvidin 3-glucoside-benzylcatechin, and malvidin 3-glucoside-3-methylbutylcatechin was respectively observed from each aldehyde. The structural characterization of these new structures has been elucidated by 1D and 2D NMR, mass spectrometry, and UV-vis techniques. These new adducts showed the same lambda(max) in the visible region at 540 nm, which is bathochromically shifted 15 nm when compared with the original anthocyanin (lambda(max) = 525 nm).     

Structural features of procyanidin interactions with salivary proteins

Procyanidin dimers and trimer C1 were synthesized, whereas (-)-epicatechin O-gallate and B2-3"-O-gallate were isolated from grape seeds. Human saliva was separated into two fractions. One of these was mainly alpha-amylase and the other mainly proline-rich proteins (PRPs). The procyanidin compounds were combined with each of the saliva protein fractions and with bovine serum albumin. The protein-polyphenol interactions were observed using nephelometry. (+)-Catechin had a higher tannin specific activity (TSA) for PRPs than (-)-epicatechin (1.45 versus 0.65 nephelos turbidity units/mg of polyphenol). This indicated the effect of the stereochemistry of flavan-3-ols on their interaction with proteins. Procyanidin dimers linked through a C(4)-C(8) interflavanoid bond had consistently greater TSA than their counterparts with a C(4)-C(6) linkage. Esterification of a galloyl group to the C(3) hydroxyl function of (-)-epicatechin or to the epicatechin moiety of procyanidin dimer B2 increased TSA. This was not as strong an effect for the dimer, probably as a result of the expected "closed" structure of B2-3"-O-gallate.     

Effect of postharvest ultraviolet irradiation on resveratrol and other phenolics of cv. Napoleon table grapes

In the skin of cv. Napoleon table grapes, the anthocyanins malvidin 3-glucoside (and its acetyl and p-coumaroyl derivatives), cyanidin 3-glucoside, peonidin 3-glucoside, cyanidin 3-glucoside, petunidin 3-glucoside, and delphinidin 3-glucoside were identified by HPLC-DAD-MS. In addition, quercetin 3-glucoside and 3-glucuronide, caffeoyltartaric, piceid, and resveratrol were also detected. The content of most phenolics remained quite constant during postharvest refrigerated storage (10 days at 0 degrees C) while the resveratrol derivatives increased 2-fold. Postharvest treatments of grapes with UVC and UVB light induced a large increase in resveratrol derivatives (3- and 2-fold, respectively). This means that a serving of mature Napoleon grapes (200 g) provides approximately 1 mg of resveratrol, which is in the range of the amount supplied by a glass of red wine. This can be increased to 2 or 3 mg of resveratrol per serving in grapes that have been irradiated with UVB or UVC, respectively. These results show that refrigerated storage and UV irradiation of table grapes can be beneficial in terms of increasing the content of potentially health-promoting phenolics.     

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.     

Interactions between grape anthocyanins and pyruvic acid, with effect of pH and acid concentration on anthocyanin composition and color in model solutions.

The formation of vitisin A, an anthocyanin formed naturally in small quantities in maturing port wines, was studied in model wine solutions at a range of pH values (2.0-4.5) and pyruvate concentrations [molar ratios of pyruvic acid to total anthocyanins (PA/TA) ranging from 12.20 to 172.40]. Additionally, the effect of vitisin A formation on the color changes of these model wines was evaluated. Vitisin A was formed through the interaction between malvidin 3-glucoside and pyruvic acid, and vitisin A in acylated forms, having the 6-position of the sugar acylated with acetic acid (3-acetylvitisin A) and p-coumaric acid (3-p-coumarylvitisin A), formed through the interaction between pyruvic acid and malvidin 3-acetylglucoside and malvidin 3-p-coumarylglucoside, respectively; their identities were confirmed by spectral analysis and FABMS. The maximum formation of these new anthocyanin derivatives was at pH 2. 7-3.0, at the higher pyruvic acid concentration (PA/TA of 172.40 units). The vitisins A caused changes in the color of the solution and expressed about 11 times (pH 3) to 14 times (pH 2) more color than the normal anthocyanins. On aging, the model solutions changed from a bluish red, attributable to the main anthocyanins present, to a slightly more orange red, attributable to the vitisin compounds. The aged models containing vitisins A were all much redder than the more red-brown color of the models aged without pyruvic acid.     

Effect of storage temperature and pyruvate on kinetics of anthocyanin degradation, vitisin A derivative formation, and color characteristics of model solutions

The formation of vitisin A, an anthocyanin formed naturally in small quantities in maturing port wines, was studied in model wine solutions at several storage temperatures (10, 15, 20, and 32 degrees C). Vitisin A was formed through the interaction between malvidin 3-glucoside and pyruvic acid, Acylated forms of vitisin A, having the 6-position of the sugar acylated with acetic acid (3-acetylvitisin A) and p-coumaric acid (3-p-coumarylvitisin A), were also formed through the interaction between pyruvic acid and malvidin 3-acetylglucoside and malvidin 3-p-coumarylglucoside, respectively. A maximum degradation of the anthocyanins was obtained at higher temperatures, and it followed a first-order kinetics both with and without pyruvic acid in the solution. Whereas at low temperatures (10 and 15 degrees C) the presence of pyruvic acid accelerated the kinetic reaction, at higher temperatures (20 and 32 degrees C) it decreased it. The activation energy values for the degradation of the three anthocyanins in model solutions without and with pyruvic acid were not significantly different from each other. At low temperatures the highest concentrations of vitisin A compounds were obtained. All solutions showed a decrease in L value, indicating that all solutions became darker. This change increased with increasing temperature. All model solutions increased in the hue angle, indicating that the solutions changed from a bluish-red to an orange-red or even brownish-red color. Samples without pyruvic acid remained lighter and became browner than those with pyruvic acid. A good correlation between the amount of vitisin A in the solution and hue angle was found, indicating that vitisin A may contribute the orange-red of solutions, compared to the browner control.     

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.     

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.     

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.     

Ellagitannins,flavonoids, and other phenolics in red raspberries and their contribution to antioxidant capacity and vasorelaxation properties

Analysis of extracts of Glen Ample raspberries (Rubus idaeus L.) by gradient, reverse phase HPLC with diode array and tandem mass spectrometry identified eleven anthocyanins, including cyanidin-3-sophoroside, cyanidin-3-(2(G)-glucosylrutinoside), cyanidin-3-glucoside, cyanidin-3-rutinoside, pelargonidin-3-sophoroside, pelargonidin-3-(2(G)-glucosylrutinoside), and pelargonidin-3-glucoside. Significant quantities of an ellagitannin, sanguiin H-6, with an M(r) of 1870 were detected along with lower levels of a second ellagitannin, lambertianin C, which has an M(r) of 2804. Other phenolic compounds that were detected included trace levels of ellagic acid and its sugar conjugates along with one kaempferol- and four quercetin-based flavonol conjugates. Fractionation by preparative HPLC revealed that sanguiin H-6 was a major contributor to the antioxidant capacity of raspberries together with vitamin C and the anthocyanins. Vasodilation activity was restricted to fractions containing lambertianin C and sanguiin H-6.