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

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

Suppression of lipopolysaccharide and galactosamine-induced hepatic inflammation by red grape pomace

Grape pomace is generated in the production process of wine and grape juices and is an industrial waste. This study investigated whether an intake of grape pomace was able to suppress chronic inflammation induced by lipopolysaccharide (LPS) and galactosamine (GalN) in vivo. When Sprague-Dawley rats were orally given methanolic extracts from red and white grape pomace, the extracts inhibited the LPS/GalN-evoked activation of nuclear factor-κB (NF-κB) dose-dependently, and red grape pomace exerted a stronger effect than white grape one. Next, rats were fed an AIN93 M-based diet containing 5% red grape pomace for 7 days, followed by the intraperitoneal injection of LPS and GalN. The intake of the red grape pomace-supplemented diet was found to suppress the LPS/GalN-induced activation of NF-κB and expression of inducible nitric oxide synthase and cyclooxygenase-2 proteins. These results suggest that red grape pomace may contain an abundance of effective compound(s) for anti-inflammatory action.     

Chemical characterization of red wine grape (Vitis vinifera and Vitis interspecific hybrids) and pomace phenolic extracts and their biological activity against Streptococcus mutans

Grapes are rich sources of potentially bioactive polyphenols. However, the phenolic content is variable depending on grape variety, and may be modified during vinification. In this study, we examined the chemical composition and biological activity of phenolic extracts prepared from several red wine grape varieties and their fermented byproduct of winemaking (pomace) on some of the virulence properties of Streptococcus mutans a well-known dental pathogen. Grape phenolic extracts were obtained from Vitis vinifera varieties Cabernet Franc and Pinot Noir and Vitis interspecific hybrid varieties Baco Noir and Noiret. The anthocyanins and flavan-3-ols content were highly variable depending on grape variety and type of extract (whole fruit vs fermented pomace). Nevertheless, all grape phenolic extracts remarkably inhibited glucosyltransferases B and C (70-85% inhibition) at concentrations as low as 62.5 microg/mL (P < 0.01). Furthermore, the glycolytic pH-drop by S. mutans cells was inhibited by the grape extracts without affecting the bacterial viability; an effect that can be attributed to partial inhibition of F-ATPase activity (30-65% inhibition at 125 microg/mL; P < 0.01). The biological activity of fermented pomace was either as effective as or significantly better than whole fruit grape extracts. The results showed that grape phenolic extracts, especially from pomace, are highly effective against specific virulence traits of S. mutans despite major differences in their phenolic content.     

Polyphenol screening of pomace from red and white grape varieties (Vitis vinifera L.) by HPLC-DAD-MS/MS

Phenolic compounds of 14 pomace samples originating from red and white winemaking were characterized by HPLC-MS. Up to 13 anthocyanins, 11 hydroxybenzoic and hydroxycinnamic acids, and 13 catechins and flavonols as well as 2 stilbenes were identified and quantified in the skins and seeds by HPLC-DAD. Large variabilities comprising all individual phenolic compounds were observed, depending on cultivar and vintage. Grape skins proved to be rich sources of anthocyanins, hydroxycinnamic acids, flavanols, and flavonol glycosides, whereas flavanols were mainly present in the seeds. However, besides the lack of anthocyanins in white grape pomace, no principal differences between red and white grape varieties were observed. This is the first study presenting comprehensive data on the contents of individual phenolic compounds comprising all polyphenolic subclasses of grapes including a comparison of several red and white pomaces from nine cultivars. The results obtained in the present study confirm that both skins and seeds of most grape cultivars constitute a promising source of polyphenolics.     

An LC-MS method for analyzing total resveratrol in grape juice, cranberry juice, and in wine.

Resveratrol is an antioxidant found in grapes, grape products, and some other botanical sources with antiinflammatory and anticancer properties. In grapes and wine, it occurs both as free resveratrol and piceid, the 3beta-glucoside of resveratrol. Here we report a liquid chromatography-mass spectrometry method to analyze total resveratrol (including free resveratrol and resveratrol from piceid) in fruit products and wine. Samples were extracted using methanol, enzymatically hydrolyzed, and analyzed using reversed phase HPLC with positive ion atmospheric pressure chemical ionization (APCI) mass spectrometric detection. Following APCI, the abundance of protonated molecules was recorded using selected ion monitoring (SIM) of m/z 229. An external standard curve was used for quantitation, which showed a linear range of 0.52-2260 pmol of trans-resveratrol injected on-column with a correlation coefficient 0.9999. The coefficient of variance of the response factor over the same concentration range was determined to be 5.8%, and the intra-assay coefficient of variance was determined to be 4.2% (n = 7). The limit of quantitation, defined as signal-to-noise 10:1, was determined to be 0.31 pmol injected on-column. The extraction efficiency of the method was determined to be 92%. The stability of resveratrol under different conditions was also examined. For example, resveratrol was stable for up to 5 days at 4 degrees C in the dark but was not stable at room temperature without protection from light. Resveratrol was detected in grape, cranberry, and wine samples. Concentrations ranged from 1.56 to 1042 nmol/g in Concord grape products, and from 8.63 to 24.84 micromol/L in Italian red wine. The concentrations of resveratrol were silmilar in cranberry and grape juice at 1.07 and 1.56 nmol/g, respectively.     

Piceid, the major resveratrol derivative in grape juices

The levels of trans-piceid, cis-piceid, trans-resveratrol, and cis-resveratrol have been measured in 36 grape juices using an HPLC system with spectral analysis of eluting peaks. The piceid (glucosides) were the major component in the grape juices. In red grape juices the average concentrations were 3.38 mg/L for trans-piceid, 0.79 mg/L for cis-piceid, 0.50 mg/L for trans-resveratrol, and 0.06 mg/L for cis-resveratrol. In white grape juices the levels were, on average, 0.18 mg/L for trans-piceid, 0.26 mg/L for cis-piceid, and 0.05 mg/L for trans-resveratrol, and cis-resveratrol was not detected in any sample. Levels of total resveratrol (trans- and cis-resveratrol and -piceid) found in red and in white grape juices are similar to those described in Spanish red and white wines. Due to their resveratrol content, as well as other phenolics, grape juices may have a beneficial health effect of interest to those who cannot drink wine.     

Survey of the trans-resveratrol and trans-piceid content of cocoa-containing and chocolate products

Dietary resveratrol (3,4',5-trihydroxystilbene) has been implicated in the health benefits associated with grapes and red wine, more specifically with potential benefits for metabolic syndrome, energy use, and increased endurance. Levels of trans-resveratrol and its glucoside, trans-piceid, were determined in 19 top selling commercially available cocoa-containing and chocolate products from the U.S. market. Amounts of trans-resveratrol and trans-piceid were closely correlated with the amount of nonfat cocoa solids (NFCS) in the cocoa-containing products. Among these products, trans-resveratrol levels were highest in cocoa powders (1.85 +/- 0.43 microg/g), followed by unsweetened baking chocolates (1.24 +/- 0.22), semisweet chocolate baking chips (0.52 +/- 0.14), dark chocolates (0.35 +/- 0.08), milk chocolates (0.10 +/- 0.05), and chocolate syrups (0.09 +/- 0.02). These cocoa-containing and chocolate products have about 3-5 times more trans-piceid than trans-resveratrol. Levels of trans-piceid were highest in the cocoa powders (7.14 +/- 0.80 microg/g), followed by unsweetened baking chocolates (4.04 +/- 0.14), semisweet chocolate baking chips (2.01 +/- 0.18), dark chocolates (1.82 +/- 0.36), milk chocolates (0.44 +/- 0.06), and chocolate syrups (0.35 +/- 0.06). On an equal weight basis, cocoa powder had about half as much trans-resveratrol as the average California red wine. On a per serving basis, cocoa-containing and chocolate products had less trans-resveratrol than red wine and grape juice but more than roasted peanuts. Overall, these cocoa-containing and chocolate products rank second after red wines and grape juice in foods with the highest levels of total trans-resveratrol in the diet.     

Characterization of the mean degree of polymerization of proanthocyanidins in red wines using liquid chromatography-mass spectrometry (LC-MS)

An HPLC-MS method for the characterization of proanthocyanidins (PA) has been refined. Further application to red wines provided interesting conclusions about the composition of the flavanol fraction and PA extractability during winemaking. The yield in PA extraction increases with the length of the postfermentative maceration (PFM), as well as the mean degree of polymerization (mDP) of wine flavanols. In early winemaking events mostly monomers to trimers are extracted from grape solids, whereas PFM is required for the significant extraction of higher oligomers. Nevertheless, at the end of a regular process of elaboration the mDP is not very high and does not usually exceed a value of 2.3, dimers and trimers being the predominant flavanols in red wines. With regard to groups of compounds, gallocatechins and prodelphinidins (located in the skins) are extracted rapidly in the first stages of the winemaking. On the contrary, long postfermentative macerations are required for the extraction of galloyled derivatives from the seeds. PA extractability is also dependent on the grape variety used for winemaking. Thus, wines made with Graciano grapes were found to require a longer process of PFM than those made from Tempranillo grapes to obtain similar yields in the extraction of flavanols.     

Fate of iprovalicarb, indoxacarb, and boscalid residues in grapes and wine by GC-ITMS analysis

The behavior in field and the transfer from grapes to wine during winemaking of iprovalicarb, indoxacarb, and boscalid was studied. The residue levels found in grapes were far below the MRLs set for grapes in EU, accounting at harvest time 0.81, 0.43, and 4.23 mg/kg for iprovalicarb, indoxacarb, and boscalid, respectively. The residue levels in the samples treated with boscalid may have residual problems (due to an accumulation effect) if repeated field treatments will be performed. Winemaking experiments showed a complete transfer of all pesticide from grapes to the must, while in wine the residues were low or negligible due to the adsorbing effect of lees and pomace. The clarification experiments showed a good removal of pesticide residues from the wine media, for all pesticides. The GC-ITMS method showed good performance with adequate recoveries ranging from 75 to 115%, and good method limits of quantitation (LOQs) and of determination (LODs) far below MRLs.     

Associations between the sensory attributes and volatile composition of Cabernet Sauvignon wines and the volatile composition of the grapes used for their production

The sensory properties of wine are influenced by the chemical composition of the grapes used to produce them. Identification of grape and wine chemical markers associated with the attributes perceived by the consumer of the wine will enable better prediction of the potential of a parcel of grapes to produce wine of a certain flavor. This study explores the relationships between Cabernet Sauvignon grape volatile composition and wine volatile profiles with the sensory properties of wines. Twenty grape samples were obtained from nine vineyard sites across three vintages and wines vinified from these parcels using controlled winemaking methods. The volatile composition of the grapes were analyzed by SBSE-GCMS, the wines were analyzed by SPME-GCMS, and these data sets were compared to that obtained from the sensory analysis of the wines. Statistical treatment of the data to account for vintage and region effects allowed underlying relationships to be seen between wine sensory attributes and wine or grape volatile components. The observed associations between grape or wine volatile compounds and wine sensory attributes has revealed target compounds and pathways whose levels may reflect the biochemical effects on grape composition by differing growth conditions during berry development and ripening. The compounds identified in this study may be useful grape or wine markers for potential wine sensory characteristics.     

Design and optimization of a semicontinuous hot-cold extraction of polyphenols from grape pomace

Grape pomace contains appreciable amounts of polyphenolic compounds such as anthocyanins and procyanidins which can be recovered for use as food supplements. The extraction of these polyphenols from the pomace is usually accomplished at slightly elevated temperatures, frequently employing hydroethanolic solvents. Due to governmental regulations and the cost involved in using ethanol as a solvent, as well as the loss in polyphenolics due to thermal degradation, improved extraction techniques are required. In this study, a semicontinuous extraction apparatus employing only water was developed to maximize the recovery of anthocyanins and procyanidins from red grape pomace (Vitis vinifera). Water is preheated prior to its entry to the extraction cell containing the grape pomace sample, where it is allowed to then flow continuously through the unheated extraction vessel prior to its collection at ambient conditions. Extraction variables that impacted the polyphenolic recovery included pomace moisture content (crude or dried), sample mass, water flow rate, and extraction temperature. A response surface method was used to analyze the results from the extraction, and the optimal conditions were found to be 140 °C and 9 mL/min water flow rate. These conditions can produce an extract containing 130 mg/100 g DW of anthocyanins and 2077 mg/100 g DW of procyanidins. Higher yields of polyphenolics were observed using crude (wet) rather than dried pomace, hence avoiding the need to dry the pomace prior to extraction. The described semicontinuous extraction method using only water as the extraction solvent under subcritical conditions allowed the efficient extraction of polyphenols from red grape pomace without the attendant loss of polyphenolic content due to having to heat the extraction vessel prior to commencement of extraction.     

Relationship of changes in rotundone content during grape ripening and winemaking to manipulation of the 'peppery' character of wine

Biosynthesis of the sesquiterpene rotundone in Vespolina grapes during berry ripening was investigated over two consecutive seasons, revealing that the compound accumulates from veraison to harvest and reaches relatively high concentrations (up to 5.44 μg/kg). Rotundone levels up to 1.91 μg/kg were also found in clones of Gruener Veltliner, a white grape variety known to give 'peppery' wines. These concentrations are higher than those reported for Syrah grapes and are similar to the levels found in some plants. Rotundone was shown to accumulate almost exclusively in berry exocarp, suggesting that skin contact during winemaking could be used to modulate the peppery character of red wine. However, rotundone yield after the winemaking process was relatively low. Indeed, only 10% of the rotundone present in grapes was extracted during fermentation, and only 6% was recovered in bottled wine. The results presented in this work provide key knowledge for manipulation of the peppery character of wine in order to optimize the intensity of this characteristic wine aroma.     

Vineyard and fermentation studies to elucidate the origin of 1,8-cineole in Australian red wine

Preliminary investigations revealed that the proximity of Eucalyptus trees to grapevines can directly influence the concentration of the aroma compound 1,8-cineole present in the corresponding red wines. For two different vineyards, the closer the grapevines were to the trees, the greater was the amount of 1,8-cineole in the wines elaborated from those grapes. This led us to carry out further studies to quantify the levels of 1,8-cineole found in grape berries, leaves, and stems at set distances from Eucalyptus trees over multiple vintages. Generally, the highest concentration of 1,8-cineole was found in the grapevine leaves, followed by grape stems and then grapes. In each sample type, we observed greater concentrations of 1,8-cineole in samples closer to the trees. Various fermentation treatments carried out with Shiraz grapes showed that matter other than grapes (MOG, e.g., Eucalyptus or grape leaves) could contribute significant amounts of 1,8-cineole to the finished wines. These studies confirmed that vineyard position and winemaking conditions can determine the 1,8-cineole concentration in red wine. The fermentation study also showed for the first time that the concentration of rotundone in red wine can be strongly influenced by grapevine leaves and stems in the ferment.     

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

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

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.     

Separation and HPLC-MS identification of phenolic antioxidants from agricultural residues: almond hulls and grape pomace

Almond hulls and grape pomace are residues abundantly generated by agricultural industries, which could be processed to obtain bioactive products. To this purpose, crude ethanol extracts from both agricultural byproducts were attained and subsequently fractionated in order to obtain an organic/water fraction (FOW). Extracts and fractions were analyzed for antioxidant power and their phenolic components tentatively identified by HPLC-MS. Chromatographic peaks of almond hull extracts showed the occurrence of hydroxybenzoic and cinnamic acid derivatives, with minor presence of flavan-3-ols (ECG, EGCG), whereas the FOW fraction offered the additional presence of epicatechin (EC) and glycosylated flavonols. In the composition for extracts of white and red grape pomace several of these compounds were also detected but basically consisted of glycosylated flavonols (quercetin, kaempferol). As a difference between both grape pomaces, myricetin glycosyde was found in that from the red variety, whereas flavan-3-ols (EC, afzelechin) were only identified in white pomace. When their FOW fractions were analyzed, gallic acid and some hydroxybenzoic acids were additionally detected. Antioxidant activity was assessed by DPPH and TBARS assays. Almond hulls showed inhibition percentages lower than 50% in both assays, while the inhibition percentage ranged from 80% to 90% in pomace extracts. Red grape pomace extract was the most efficient antioxidant, with an EC50 value of 0.91 g/L for TBARS and 0.20 g/L for DPPH. Even appearing as two quite different vegetal matrixes, the composition of phenolics in grape pomace and almond hulls is quite similar, the main difference being the major occurrence of flavonols in grape pomace. This fact could presumably explain the lower antiradical activity of hull extracts.     

Rapid method for proline determination in grape juice and wine

Proline is typically the most abundant amino acid present in grape juice and wine. The amount present is influenced by viticultural and winemaking factors and can be of diagnostic importance. A method for rapid routine quantitation of proline would therefore be of benefit for wine researchers and the industry in general. Colorimetric determination utilizing isatin as a derivatizing agent has previously been applied to plant extracts, biological fluids, and protein hydrolysates. In the current study, this method has been successfully adapted to grape juice and wine and proved to be sensitive to milligram per liter amounts of proline. At sugar concentrations above 60 g/L, interference from the isatin-proline reaction was observed, such that proline concentrations were considerably underestimated in grape juice and dessert wine. However, the method was robust for the analysis of fermentation samples and table wines. Results were within ±10% agreement with data generated from typical HPLC-based analyses. The isatin method is therefore considered suitable for the routine analysis required to support research into the utilization or release of proline by yeast during fermentation.     

Multielement composition of wines and their precursors including provenance soil and their potentialities as fingerprints of wine origin

The influence of the provenance soil and vinification process on the wine multielemental composition was investigated. For this purpose, two different vineyards from the Douro wine district, Portugal, were selected. Monovarietal grapes from a 10 year old vineyard were used to produce a red table wine, in a very modern winery. Polyvarietal grapes from a 60-70 year old vineyard were used to produce a red fortified wine, similar to Port, through a traditional vinification process. The multielement compositions (Al, As, B, Ba, Be, Ca, Cd, Co, Cr, Cs, Cu, Fe, Ga, Hf, Li, Mn, Mo, Nb, Ni, Pb, Rb, Sb, Sc, Sr, Ti, Th, Tl, U, V, W, Y, Zn, Zr, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu) of soil, grape juices (prepared in the laboratory), and samples collected in the different steps of each winemaking process were measured. Inductively coupled plasma mass spectrometry was used, after suitable pretreatment of the samples (by UV irradiation for liquid samples and high-pressure microwave digestion for soil). Both vinification processes influenced the multielement composition of the wines. Most of the elements presented similar or even lower concentrations in the wine as compared to that observed in the respective grape juice, probably as a result of precipitation or coprecipitation with suspended particles during fermentation and/or wine aging. Evidence of effective contamination during grape pressing, fermentation, and/or fining of wines (depending on the element) was observed for Cd, Cr, Cu, Fe, Ni, Pb, V, and Zn in the fortified wine and Al, Cr, Fe, Ni, Pb, and V in the table wine. Nevertheless, significant correlations were obtained between the multielement composition of the wine and the respective grape juice (R = 0.997 and 0.979 for the fortified and table wines, respectively, n = 31, P < 0.01), as well as between that in the wine (median of the two studied wines) and the provenance soil (R = 0.994, n = 19, P < 0.01), for the set of elements determined in common in the different types of samples. These results are promising concerning the usefulness of the elemental patterns of both soil and wine as fingerprints of the origin of the studied wines. Nevertheless, more wines from the same and other wine districts must be studied in order to consolidate this conclusion. The multielement compositions of the studied wines were compared with those of wines of different characteristics and origins, as well as with the respective legal threshold limit values, when available. Relatively low metal levels, below their threshold limit values, were found in all cases.     

Stilbene levels in grape cane of different cultivars in southern Chile: determination by HPLC-DAD-MS/MS method

Health benefits of trans-resveratrol and other stilbenes in grapes, must, and wine have been pointed out by numerous authors. Less attention has been paid to the presence of stilbene derivatives in viticultural residues, such as grape canes. The present work reports the first results of a systematic study of stilbene levels in different grape varieties and cultivation areas in Chile, to evaluate their potential as an alternative source of bioactive stilbenes. In all cane samples, the predominant stilbene is trans-resveratrol, followed by ε-viniferin and piceatannol. In canes of Pinot noir up to 5590 ± 172 mg kg(-1) of trans-resveratrol and up to 6915 ± 175 mg kg(-1) of total stilbenes were detected. The observed concentrations of stilbenes in canes of Pinot noir from southern Chile until now are higher than those reported previously for this red variety. However, the highest concentration of total stilbenes observed in the analyzed samples was in the canes of white variety Gewürztraminer with 7857 ± 498 mg kg(-1). Preliminary results indicate that these levels can evolve if canes are left for some months on the vineyard after pruning, observing an increase during the first 2 months and a decrease after this period.