Wine Anthocyanins Research Articles

Development and validation of a capillary zone electrophoresis method for the quantitative determination of anthocyanins in wine

A capillary zone electrophoresis (CZE) method is proposed for the quantitative determination of anthocyanins in wine as an alternative to high-performance liquid chromatography. The CZE separation was carried out using a 46 cm (effective length)×75 μm I.D. fused-silica capillary at 10 °C and a 50 m M sodium tetraborate buffer at pH 8.4 with 15% of methanol as modifier. A voltage of 25 kV and a hydrodynamic injection of 300 mbar s were used. The electropherograms were recorded at 599 nm. It was found that SO 2 (antibacterial and antioxidant agent added to wine during its production) increased the absorbance of anthocyanins at 599 nm in a basic medium. Therefore, a concentration of 250 mg/l of SO 2 was added to the samples and the calibration solution before the analysis in order to avoid errors by this matrix effect. The analytical response was linear ( R=0.998) between 10 and 700 μg/ml of malvidin-3- O-glucoside. The limit of detection and the reproducibility (as a relative standard deviation, n=11) were 1 μg/ml and 1.5%, respectively. Finally, the CZE method was validated by the analysis of synthetic wine samples (errors less than 8%) and by the comparison of the results obtained in the analysis of different monovarietal wines by CZE with those obtained by the standard HPLC method. In this comparison, a good correlation ( R=0.998) with a slope of 1.005±0.044 and an intercept of −0.752±6.690 was obtained for malvidin-3- O-glucoside.

Variations in the profile and content of anthocyanins in wines made from cabernet sauvignon and hybrid grapes.

To detect adulteration of wine, it has been proposed that the ratio of acetylated to p-coumaroylated conjugates of nine characteristic anthocyanins can be used to determine whether a wine is derived from Cabernet Sauvignon or hybrid grapes. If the ratio is >3, then a wine is classified as being derived from Cabernet Sauvignon grapes. This test has significant commercial implications as it is being used to decide whether Cabernet Sauvignon-labeled wines are genuine and can be imported into Germany. To assess whether this is a valid approach, 24 wines were analyzed, 4 of which were made from hybrids and 20 from Cabernet Sauvignon, with vintages ranging from 1993 to 2000. Only 13 of the Cabernet Sauvignon wines contained all nine of the "characteristic" anthocyanins, and the ratio of acetylated to p-coumaroylated derivatives varied from 1.2 to 6.5. It is evident that the use of the anthocyanin ratio method is flawed and that examination of the whole anthocyanin profile and/or investigation of the proportion of monoglucoside and acetylated anthocyanins is a better approach to distinguish between hybrid and Cabernet Sauvignon wines.

Screening for potential pigments derived from anthocyanins in red wine using nanoelectrospray tandem mass spectrometry.

Red wine extracts were screened for potential wine pigments derived from anthocyanins, using a combination of nanoelectrospray tandem mass spectrometry techniques. Fourteen aglycons were considered to be of anthocyanidin origin on the basis of their MS/MS spectra. The proposed structures of the aglycons were anthocyanidin C-4 substituted with vinyl linkage between C-4 and the hydroxy group at C-5. The anthocyanidin derivatives identified in the wine extracts were vinyl, vinylmethyl, vinylformic acid, 4-vinylphenol, 4-vinylguaiacol, and vinylcatechin adducts of malvidin as well as vinylformic acid and 4-vinylphenol adducts of peonidin and petunidin. The presence of vinyl alcohol, 4-vinylcatechol, and 4-vinylsyringol adducts of malvidin was also proposed.

Anthocyanin Analysis by HPLC/ESI-MS

A high-performance liquid chromatography (HPLC) method for the separation of anthocyanins in red wine has been developed. Sixty-six different anthocyanins were detected in a single red wine sample produced in 1997 by coupling HPLC and an electrospray interface with a mass spectrometer. These 66 anthocyanins were mostly well-known mono- and diglucosides of wines. In addition, the rare acetic acid ester and coumaric acid ester of the diglucosides could be identified. Some recently described and some new anthocyanin-derived pigments formed by wine maturation were also detected. Catechin dimers as well as pyruvic acid and acetaldehyde derivatives of delphinidin, cyanidin, petunidin, peonidin, and malvidin were present in the one-year-old wine, the latter also in their acetylated and coumarylated forms.

Rapid HPLC Analysis of Phenolic Compounds in Red Wines

A new direct and rapid (18 min) reversed-phase high-performance liquid chromatography method for the separation of phenolic compounds (benzoic acids, flavan-3-ols, cinnamic acids, flavonols, and anthocyanins) in red wines is described. A column that allows for low pH conditions and high flow was used, with a gradient of two solvents: water and acetonitrile, both with 0.2% trifluoroacetic acid. To improve selectivity, each compound was monitored at its absorbance maximum. Precision, linearity, and sensitivity (limit of detection and limit of quantitation) were established. While this rapid method cannot resolve all wine constituents, it is appropriate for measuring major components and quantifying total amounts of particular classes of phenolic compounds. The method was applied to a set of new and aged red wines.

Antioxidant effect of red wine anthocyanins in normal and catalase-inactive human erythrocytes

Previous studies reported that aged red wine, but not novel red wine or white wine protects human red blood cells from oxidative damage induced in vitro by H 2O 2. Here, we demonstrate that the beneficial properties of aged red wine are due, at least in part, to the presence of anthocyanins. We firstly measured the “antioxidant power” of an Italian red wine (Taurasi, Avellino) and that of its anthocyanin fractions by using Ferric Reducing Antioxidant Power Assay. Subsequently, we demonstrate that fractions containing anthocyanins lower ROS (reactive oxygen species) and methemoglobin production in human erythrocytes treated with H 2O 2. Finally, we reported that the protective effects of anthocyanins were also confirmed in an experimental model in which RBCs were deprived of catalase activity by treatment with 4 mM sodium azide. The results obtained clearly demonstrate that red wine anthocyanins protect human RBCs from oxidative stress.

Improvement of the HPLC analysis of anthocyanins in red wines by use of recently developed columns.

Comparison of the separation performance of five newly developed pH-stable HPLC columns is described for the analysis of anthocyanins in red wines. Separation of twenty anthocyanins in a single run is described using the most appropriate method.

Malvidin-3-glucoside bioavailability in humans after ingestion of red wine, dealcoholized red wine and red grape juice

Dietary polyphenols, including anthocyanins, are suggested to be involved in the protective effects of red wine against cardiovascular diseases. Very little data are available concerning the bioavailability of anthocyanins, major sources of red pigmentation in red wine. The aim of this study was to compare changes in plasma malvidin-3-glucoside (M-3-G), a red wine anthocyanin, and its urinary excretion after ingestion of red wine, dealcoholized red wine and red grape juice. Six healthy male subjects were studied in a randomized cross over setting in a human nutrition research unit under controlled conditions. All subject consumed 500 mL of each beverage on separate days providing the following M-3-G quantities: red wine 68 mg, dealcoholized red wine 58 mg, and red grape juice 117 mg. M-3-G was measured by HPLC and photodiode detection. M-3-G was found in plasma and urine after ingestion of all the beverages studied. The aglycon, sulfate or glucuronate conjugates of M-3-G were not detected in plasma and urine. Increases in plasma M-3-G concentrations were not significantly different after the consumption of either red wine or dealcoholized red wine and were about two times less than those measured after consumption of red grape juice. This difference may be caused by the about two times higher M-3-G concentration determined in red grape juice. Area under the plasma concentration curves were as follows: 288 +/- 127 nmol x h/L (red wine), 214 +/- 124nmol x h/L (dealcoholized red wine) and 662 +/- 210 nmol x h/L (red grape juice) and showed a linear relationship with the amount of anthocyanin consumed (mean +/- SD). M-3-G is poorly absorbed after a single ingestion of red wine, dealcoholized red wine, or red grape juice and seems to be differentially metabolized as compared to other red grape polyphenols. Our results suggest that not anthocyanins such as M-3-G themselves but rather not yet identified anthocyanin metabolites and/or other polyphenols in red wine might be responsible for the observed antioxidant and health effects in vivo in subjects consuming red wine.

Value of high-performance liquid chromatographic analysis of anthocyanins in the differentiation of red grape cultivars and red wines made from them

A HPLC method that allows the separation of several anthocyanins present in red grapes and red wines, using a linear gradient of acetonitrile in water at pH 1.3, using perchloric acid as an acid modifier, is described. Data clearly show that the anthocyanins profile of red grapes may be complex, but quite different for each cultivar studied. Thus, those molecules may be used as chemotaxonomic markers for classifying red grape cultivars. However, the anthocyanin profile of red wines clearly differs from that presented by grapes employed in making it, because red wine contains a higher relative amount of malvidin-3- O-glucoside than grapes, and the relative amount of other anthocyanins in wines is usually lower than in grapes. Therefore, the use of anthocyanins present in wines to determine the grape cultivar used for winemaking needs a careful evaluation of the influence of different technological procedures on the anthocyanins fingerprint.

Determination of anthocyanins in wine based on flow-injection, liquid–solid extraction, continuous evaporation and high-performance liquid chromatography–photometric detection

A continuous method, easy to automate, for the determination of anthocyanins in wine based on the coupling of continuous liquid–solid extraction, evaporation, HPLC individual separation and photometric detection is proposed. The target analytes are removed from the wine in a continuous fashion using a C 18 minicolumn and eluted with an aqueous solution (pH 2) with 16% acetonitrile. The eluted fraction is concentrated by solvent evaporation assisted by heat and dragging off the vapour using a flow of N 2. For in-line preconcentration, a continuous evaporation module was designed and located in the manifold between the solid-phase minicolumn and the injection valve of the chromatograph. In this way, injection of the sample into the dynamic system leads the plug through it for liquid–solid extraction of the anthocyanins, partial evaporation of the eluent (with a preconcentration factor as required) and transport to the high-pressure injection valve of the chromatograph, where individual separation and subsequent photometric detection take place. The method thus developed for the determination of malvidin-3-glucoside, cyanidin-3-glucoside and peonidin-3-glucoside anthocyanins in Spanish red wines is more sensitive than the batch manual method based on the same steps, has better linearity of the calibrations curves with lower detection limits and much wider determination range for the most abundant anthocyanins in wine. In addition, the method can be fully automated with low acquisition and maintenance costs.

Method development for the determination of anthocyanins in red wines by high-performance liquid chromatography and classification of German red wines by means of multivariate statistical methods

A simple and fast HPLC method without sample pretreatment is described for the separation of anthocyanins in red wines using a new pH-stable stationary phase. The linearity between peak area and concentration and ruggedness of the method were checked. Investigations were made about the safekeeping of red wine samples concerning anthocyanins. Classification of 52 different wine samples was performed by multivariate statistical methods.

Effect of acetaldehyde and several acids on the formation of vitisin A in model wine anthocyanin and colour evolution

Summary It was found that the reaction between malvidin 3‐glucoside and added pyruvic acid (PA), leading to the formation of vitisin A in model wine solutions, was not prevented by the addition of either acetaldehyde (A) or several organic acids. The acylated forms of vitisin A (3‐acetylvitisin A and 3‐p‐coumarylvitisin A) were formed through the interaction of malvidin 3‐acetylglucoside and malvidin 3‐p‐coumarylglucoside. Disappearance of the three main anthocyanins (malvidin 3‐glucoside, malvidin 3‐acetylglucoside and malvidin 3‐p‐coumarylglucoside) from the model wine with time followed first order kinetics. Acetaldehyde had the effect of increasing the total amount of these losses but producing smaller amounts of vitisin A. During ageing model solutions developed some browness. The brownest solution was obtained without A and a reduced rate of browning was found in the presence of A. This latter result can be explained by the assumption of a superimposition of a blueing effect upon reactions of A with anthocyanins. In the presence of PA the formation of vitisin A compounds gave an intermediate colour, contributing a reddish hue to the solution. A good correlation (r2= 0.96) between the percentage of vitisin A, of the total anthocyanins and the hue angle was observed. The addition of large amounts of organic acids that are normally found in wine into the model solutions did not lead to the formation of new anthocyanins. The linear loss of PA in all model systems indicates that a first order reaction occurs and 35.35 times more PA than total anthocyanin was lost to form the new compounds.

Excessive nitrogen supply and shoot trimming can impair colour development in Pinot Noir grapes and wine

We investigated the effects of nitrogen supply and shoot trimming on mature, field-grown Pinot Noir (Vitis vinifera L.) vines. Ammonium nitrate (0, 30, 60, 90 kg N/ha) was applied at the beginning of flowering. Shoots were topped either once (at fruit set), or twice, (at fruit set and during the lag-phase of berry growth). Neither treatment affected grape berry and skin weights, yield and grape sugar, but high rates of nitrogen increased malic acid and reduced skin phenols, flavonols and anthocyanins. Malvidin-3-glucoside was the most abundant anthocyanin in skins and wine. It accounted for 75% of the total anthocyanins at the beginning of fermentation and its relative proportion increased to 95% in the finished wine. Increases in anthocyanin concentration at the beginning of fermentation and before malolactic fermentation, were followed by declines during the later stages of alcoholic and malolactic fermentation. High nitrogen supply decreased anthocyanins in the juice and wine, increased pH and increased the percentage of malvidin-3-glucoside. Repeated shoot topping gave lower wine total phenols and anthocyanins and thus enhanced the nitrogen effect. Wine susceptibility to oxidation increased with higher pH and lower anthocyanin content. The best treatment combination for fruit and wine quality, in terms of colour and oxidative stability, was low nitrogen/single topping and the worst combination was high nitrogen/repeated topping. These results suggest that a combination of high rates of nitrogen fertiliser and repeated shoot trimming can decrease potential fruit and wine quality.

Free radical scavenging effect of anthocyanins in red wines

Free radicals are extremely harmful to living organisms in that, they attack different constituents of the cell, leading to acceleration of the ageing process and sometimes even its destruction, or if the DNA is affected, irreversible malfunctions. It is now widely recognised that the phenolic compounds of wine have very high free radical scavenging potential. The aim of this paper is to determine which of these phenolic compounds are responsible for the strong free radical scavenging potential of red wines. In order to do so, a red wine was fractionated into phenolic fractions. After extraction and purification of these compounds from the wine, we have measured their free radical scavenging activities using an enzymatic method. The anthocyanic fraction showed a high free radical scavenging power in relation to the other tannic fractions. In order to explain this phenomenon, some pure anthocyanins were studied and a relationship between their free radical scavenging activity and their molecular structure was suggested.

Analysis of anthocyanins in red wine and fruit juice using MALDI-MS.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful new technique that will have a great impact on food analysis. This study is the first to demonstrate the applicability of MALDI-MS to perform both qualitative and quantitative analyses of anthocyanins in food. 2,4, 6-Trihydroxyacetophenone (THAP) was found to be a good matrix for analysis of anthocyanins, with the best spot-to-spot repeatability. After a simple sample preparation, the presence of anthocyanins as cations with molecular masses was found in ratios expected from their fruit sources. Quantification of anthocyanins should be possible by choosing appropriate internal standards. MALDI-MS responses were linear, groups of chemically similar anthocyanins had similar responses, and addition of an internal standard had no effect on relative responses of the other anthocyanins.

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.

Bioavailability of Red Wine Anthocyanins As Detected in Human Urine

Anthocyanins, which are natural plant pigments from the flavonoid family, represent substantiated constituents of the human diet. Many foods but especially red grapes and wines contain large amounts of flavonoids, which are mostly anthocyanins. The aim of our study was to determine the potential bioavailability, in human, of several anthocyanins from red wine. Six healthy, fasting volunteers, having a polyphenols-free diet, drank 300 mL of water every hour for 12 h and collected urine. Several weeks later, the same volunteers repeated the same procedure but replaced the water of the fourth drinking dose with white wine. Two weeks later, they repeated the procedure with red instead of white wine. In the 300 mL dose of red wine, the subjects received 218 mg of anthocyanins, which were detected in their urine by HPLC analysis with a photodiode array detector. Two of the compounds found among the wine anthocyanins were found unchanged in the urine. Other anthocyanin compounds, which seemed to have undergone molecular modifications, were detected in the urine after incubation with HCl. The anthocyanin level in the urine reached a peak within 6 h of the wine drinking. Within 12 h of the wine drinking, we found 1.5−5.1% of the ingested anthocyanins, in the urine. Keywords: Red wine; anthocyanins; antioxidants; absorption; urine; human

Changes in Anthocyanins and Color Characteristics of Pinot Noir Wines during Different Vinification Processes

Simple and polymeric anthocyanins of Pinot noir wines produced by different vinification processes were monitored by high-performance liquid chromatography (HPLC) from the onset of fermentation to bottling. Color characteristics were determined by tristimulus measurement using a spectrophotometer with a color determination and match program. Up to 12 individual polymeric anthocyanins were found. Extraction of monomeric anthocyanins was increased with higher fermentation temperature, but anthocyanin concentration declined gradually after fermentation. In all wines, the dominant type of anthocyanin was malvidin 3-glucoside. In contrast, the content of polymeric anthocyanins increased during fermentation and thereafter, and reached the highest concentrations at bottling. Fermentation temperature was a critical factor for formation of polymeric anthocyanins. The presence of total or individual polymeric anthocyanins was directly related to wine color intensity, and all the polymeric anthocyanins were importan...

Reversed-Phase Ion-Pair Chromatography of Anthocyanins in Red Wines

A reversed-phase ion-pair chromatographic method has been developed for the separation of anthocyanins from red wines. Separation of anthocyanin monoglucosides can be achieved with a gradient mode elution using tetrabutylammonium hydrogen sulfate as a counter-ion, phosphoric acid, and methanol—acetonitrile—water in the mobile phase (pH = 2). This technique allows a relatively fast separation and identification of different anthocyanins in one run without prior treatment of the wine or derivatization of the compounds; it also avoids both the use of low pH (below 2) and, consequently, the degradation of the column. The order of elution of anthocyanin monoglucosides does not change in this mode of chromatography; they elute in the order of their polarity, as is typical in reversed-phase chromatography (delphinidin, cyanidin, petunidin, peonidin, and malvidin monoglucoside). Therefore, this characteristic profile is useful for the identification of anthocyanins of Vitis vinifera in red wines.

Influence of Vinification Treatments on Aroma Constituents and Sensory Descriptors of Pinot noir Wines

Three vinification methods were compared to assess their effects on chemical composition, sensory descrip- tors, and headspace volatile constituents of Pinot noir wines. Two methods involved standard vinification at fermentation temperatures of 20°C (VM1) and 30°C (VM2), respectively, while the third method (VM3) included a two-stage pre-fermentation treatment involving heat extraction, followed by fermentation at 15°C in contact with bentonite. VM2 produced wines with highest color intensity, currant aroma, and currant flavor. VM3 wines, however, contained twice the concentration of anthocyanins of VM1 and VM2 wines, and possessed the most intense fruity aroma and flavor. Total ester concentration was four-fold higher in VM3 compared to VM1 and VM2. Several esters were responsible for this difference but isoamyl acetate was particularly dominant.