Postharvest stilbene-enrichment of red and white table grape varieties using UV-C irradiation pulses

The red table grape varieties Flame, Red Globe, Crimson, and Napoleon, as well as the white varieties Superior, Dominga, and Moscatel Italica, were irradiated with a previously optimized UV-C postharvest irradiation protocol (510 W, 40 cm, 60 s). The induction kinetics of the stilbenes trans-resveratrol, trans-piceid, trans-piceatannol, trans-astringin, and viniferins was followed by using HPLC-DAD/MS/MS. The most inducible stilbenes were trans-resveratrol, trans-piceatannol, and viniferins. Both quantitative and qualitative differences were observed in both the stilbene induction kinetics and stilbene content in the seven table grapes analyzed here. The total resveratrol content ranged from 0.69 mg/100 g fw (Dominga) to 2.3 mg/100 g fw (Red Globe). The net resveratrol induction ranged from 3.4-fold (Flame) to 2315-fold (Red Globe). The highest viniferins content was observed in the variety Flame (0.73 mg/100 g fw), although the variety Red Globe presented the highest viniferins induction (175-fold). The highest content and induction of piceatannol (0.17 mg/100 g fw and 173-fold, respectively) was observed in the variety Flame. It should be stressed that taking into account the health-beneficial effects claimed for stilbenes, the UV-C irradiated table grapes can be considered as new functional fruits that can supply (a serving of unpeeled 200 g table grapes) the resveratrol content (depending on the variety) equivalent to more than seven glasses of red wine ( approximately 1.5 L) with high resveratrol content.     

Postharvest induction modeling method using UV irradiation pulses for obtaining resveratrol-enriched table grapes: a new "functional" fruit?

A modeling method for the induction of resveratrol synthesis by UV irradiation pulses in Napoleon table grapes is proposed. The method is based on the combination of four main parameters: irradiation power (IW), irradiation time (IT), irradiation distance (ID), and number of elapsed days to achieve the highest resveratrol accumulation (D(m)). Maximum resveratrol content (11-fold higher than untreated grapes) was achieved using the combination: IW = 510 W, IT = 30 s, ID = 40 cm, and D(m) = 3 days. Sensory characteristics and main features of irradiated grapes (color, weight, firmness, flavor, size, ripening index and vitamin C content) remained unaltered after 1 week of storage. UV induction signal migrated to the hidden side of the grape skin with a delay of 3 days as compared to the directly irradiated side. Phenolic compounds were not detected in Napoleon grape flesh. Resveratrol content per standard serving (200 g) of irradiated grape was about 3 mg, an amount more than 10-fold higher than that of untreated Napoleon grapes. This means that a serving of irradiated grape (unpeeled) could supply the resveratrol content equivalent to 3 glasses of a red wine with high resveratrol content ( approximately 1 mg/glass). Therefore, controlled UV irradiation pulses are useful as a simple postharvest treatment (and alternative to genetic engineering) to obtain possible "functional" grapes (with enhanced health-promoting properties) as a dietary source of high resveratrol content.     

Production of stilbenoids from the callus of Arachis hypogaea: a novel source of the anticancer compound piceatannol

A new source to produce a significant quantity of a naturally occurring polyphenol, piceatannol, was investigated in this study. Both resveratrol and piceatannol are recognized as important ingredients in functional foods due to their beneficial health effects. However, unlike resveratrol, the piceatannol concentration in plants is very low. Thus, calluses of peanuts, an easily obtainable source, were chosen as the material to induce piceatannol production under controlled conditions. To induce resveratrol and piceatannol, calluses were exposed to the ultraviolet (UV) irradiation. Significant quantities of resveratrol and piceatannol were produced by calluses upon UV irradiation in both static and suspension culture conditions. The amounts of piceatannol and resveratrol produced in 1 g of calluses ranged from 2.17 to 5.31 microg and from 0.25 to 11.97 microg, respectively, in static culture. In suspension culture, the amounts of induced piceatannol and resveratrol were somewhat lower. The quantities of induced piceatannol and resveratrol reached a maximum at 18 h after UV irradiation treatment in static culture. In contrast, the levels of resveratrol and piceatannol remained almost constant throughout the experiments in suspension culture. The piceatannol produced by calluses in all studies was much higher than the values reported in the literature, whereas the resveratrol produced was comparable to reported values.     

Pterostilbene, a new agonist for the peroxisome proliferator-activated receptor alpha-isoform, lowers plasma lipoproteins and cholesterol in hypercholesterolemic hamsters

Resveratrol, a stilbenoid antioxidant found in grapes, wine, peanuts and other berries, has been reported to have hypolipidemic properties. We investigated whether resveratrol and its three analogues (pterostilbene, piceatannol, and resveratrol trimethyl ether) would activate the peroxisome proliferator-activated receptor alpha (PPARalpha) isoform. This nuclear receptor is proposed to mediate the activity of lipid-lowering drugs such as the fibrates. The four stilbenes were evaluated at 1, 10, 100, and 300 microM along with ciprofibrate (positive control), for the activation of endogenous PPARalpha in H4IIEC3 cells. Cells were transfected with a peroxisome proliferator response element-AB (rat fatty acyl CoA beta-oxidase response element)-luciferase gene reporter construct. Pterostilbene demonstrated the highest induction of PPARalpha showing 8- and 14-fold increases in luciferase activity at 100 and 300 microM, respectively, relative to the control. The maximal luciferase activity responses to pterostilbene were higher than those obtained with the hypolipidemic drug, ciprofibrate (33910 and 19460 relative luciferase units, respectively), at 100 microM. Hypercholesterolemic hamsters fed with pterostilbene at 25 ppm of the diet showed 29% lower plasma low density lipoprotein (LDL) cholesterol, 7% higher plasma high density lipoprotein (HDL) cholesterol, and 14% lower plasma glucose as compared to the control group. The LDL/HDL ratio was also statistically significantly lower for pterostilbene, as compared to results for the control animals, at this diet concentration. Results from in vitro studies showed that pterostilbene acts as a PPARalpha agonist and may be a more effective PPARalpha agonist and hypolipidemic agent than resveratrol. In vivo studies demonstrate that pterostilbene possesses lipid and glucose lowering effects.     

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.     

Plant foods and herbal sources of resveratrol

Stilbenes, in particular trans-resveratrol and its glucoside, are widely reported to be beneficial to health, having been shown to possess antioxidative, anticarcinogenic, and antitumor properties. Major dietary sources include grapes, wine, peanuts, and soy; however, they can also be introduced into the diet through Itadori tea, which has long been used in Japan and China as a traditional herbal remedy for heart disease and strokes. Analysis of grapes, peanuts, and Itadori tea shows that they contain mainly trans-resveratrol glucoside. In contrast, red wines are primarily a source of the aglycones cis- and trans-resveratrol. While peanuts and grapes contain low levels of the stilbenes, Itadori tea and red wine both supply relatively high concentrations of resveratrol. For people who do not consume alcohol, Itadori tea may be a suitable substitute for red wine. However, further study on the potential biological effects of other endogenous compounds in Itadori tea is required and there is also a need for more information on the absorption and in vivo biomedical actions of free and conjugated resveratrol.     

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.     

Comparison of ozone and UV-C treatments on the postharvest stilbenoid monomer, dimer, and trimer induction in var. 'Superior' white table grapes

Postharvest treatment of seedless white table grapes (var. 'Superior') with different gas ozone concentrations (3.88 and 1.67 g/h) for 1, 3, and 5 h induced an increase in stilbenoid biosynthesis [trans-resveratrol, piceatannol, and viniferinas (resveratrol dehydrodimers and dehydrotrimers)] during storage at 22 degrees C and 95% relative humidity. The maximal resveratrol concentration was reached after 2 days of storage, and this amount was similar to that induced by optimized UV-C treatments (1 min, 510 W, 40 cm). Although similar resveratrol concentrations accumulated in grapes after both UV-C and O3 treatments (maximum ozone production and time), the ozone treatment was more efficient in inducing viniferins accumulation in grape berries. A sequence in the biosynthesis of stilbenoids was observed, starting with the resveratrol monomer, continuing with the resveratrol dehydrodimers epsilon-viniferin and delta-viniferin, and ending with four different resveratrol dehydrotrimers. These trimers were different from alpha-viniferin, a trimer previously reported to be induced in grapes after biotic and abiotic stresses. Two alpha-viniferin isomers were also detected in the ozone-treated grapes, although at very low concentrations that prevented their quantification.     

Ultraviolet-C and induced stilbenes control ochratoxigenic Aspergillus in grapes

This study investigated the efficacy of ultraviolet-C (254 nm) and induced stilbenes to inhibit Aspergillus carbonarius and Aspergillus tubingensis and control ochratoxin A production in grapes. In addition, the stilbene synthesis as a response to UV-C treatment and to infection of ochratoxigenic Aspergillus was compared. The initial microbial inactivation by a previously optimized UV-C illumination protocol for increasing trans-resveratrol content in grapes (50 W/m (2), 40 cm, 60 s) was similar on undamaged and damaged grapes, achieving 1.2 and 1.3 log conidia/100 g reductions, respectively. After 5 days of storage at 22 degrees C, UV-C treatment and the stilbenes induced by UV-C inhibited ochratoxigenic Aspergillus growth in undamaged grapes. UV-C elicited the biosynthesis of trans-resveratrol, while microbial infection and tissue damage triggered the biosynthesis of trans-piceid. trans-Resveratrol was not synthesized as a consequence of ochratoxigenic Aspergillus contamination. However, when trans-resveratrol was synthesized by UV-C, it contributed to inhibiting the development of ochratoxin A producing aspergilli. Furthermore, UV-C treatment also contributed to decrease ochratoxin A production by ochratoxigenic aspergilli. Therefore, UV-C is a promising emerging technology either for reducing the potential ochratoxigenic risk in grapes, which is of particular interest to the wine industry, and also for increasing trans-resveratrol content of grapes, which would provide an added value to the wine.     

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.     

Positive correlation between high levels of ochratoxin A and resveratrol-related compounds in red wines

The natural occurrence of ochratoxin A in red wines has been widely reported by several authors, as well as a that of group of stilbenes including cis- and trans-resveratrols and related glucosylated forms. In the present study 112 samples of retail red wines were collected from northern (17), central (46), and southern (49) Italy and were analyzed for both ochratoxin A and resveratrol-related stilbenes. The mean levels of total resveratrols and total piceids were 3.14 and 5.80 mg/L, respectively, whereas the ochratoxin A mean level was 0.64 microg/L. The Merlot wines showed the highest mean value of total stilbenes, followed by Negroamaro and Negroamaro blend, Aglianico, and Syrah, all with mean levels of >10 mg/L. Ochratoxin A was detected in 70, 59, and 100% of wine samples from northern, central, and southern Italy, with mean levels of 0.12, 0.07, and 1.36 microg/L, respectively. The highest values of ochratoxin A were recorded in Negroamaro- and Primitivo-based wine samples from southern Italy, showing also the highest content of stilbenes. In wine samples from southern Italy, a positive correlation was obtained between levels of ochratoxin A and total stilbenes (r = 0.74) as well as between ochratoxin A and total resveratrols (r = 0.50) and between ochratoxin A and total piceids (r = 0.74). These results suggest that toxic levels of ochratoxin A in red wine may be, at some extent, counterbalanced by the beneficial effects of resveratrol derivatives. Further investigation should be warranted in this regard.     

Determination of stilbenes (trans-astringin, cis- and trans-piceid, and cis- and trans-resveratrol) in Portuguese wines.

Stilbenes have been shown to have cancer chemopreventive activity and to protect lipoproteins from oxidative damage. A method is described for their direct determination in different types of wine using high-performance liquid chromatography with ultraviolet detection. In a survey of 120 commercial wines from Portugal and France, the highest concentrations of stilbenes were found in red wines. The glucosides of resveratrol were present in higher concentrations than the free isomers. Isolation from wine and characterization of trans-astringin in a large quantity are described for the first time.     

Determination of piceid and resveratrol in Spanish wines deriving from Monastrell (Vitis vinifera L.) grape variety

The presence of stilbenes in wine is becoming an important issue due to their claimed relation to a low incidence in coronary diseases and their increasing implication as cancer chemopreventive and neuroprotective agents. Total resveratrol content, quantified as glucoside and aglycone forms of resveratrol, has been determined in a survey of 45 Monastrell monovarietal Spanish red wine types (around 135 wine samples), belonging to Alicante and Bullas appellations. The average between ratio glucoside/aglycone forms of resveratrol in these wines was considerably high, ranging from 82 to 91% of resveratrol in its glycosidic form. This characteristic was observed in a high percentage of the studied wines, which were made under different winemaking procedures, and from different vintages (1995-2002). In addition, wines made using macerative fermentations with double amount of solid parts ("doble pasta") reached the highest levels of total stilbene content expressed as resveratrol equivalent, i.e., 30 mg/L (average of 18.8 mg/L). It can be concluded that high resveratrol glucoside concentration and low free isomer content can be considered characteristics of the Monastrell variety, as it happens to red wines deriving from other varieties grown at warm climates. This fact, also observed for other French and Portuguese red varieties, might play an important role in food habits involving these types of wines.     

Resveratrol, pterostilbene, and piceatannol in vaccinium berries

A study was conducted to determine the presence of resveratrol, pterostilbene, and piceatannol in Vaccinium berries. Samples representing selections and cultivars of 10 species from Mississippi, North Carolina, Oregon, and Canada were analyzed by gas chromatography/mass spectrometry. Resveratrol was found in Vaccinium angustifolium (lowbush blueberry), Vaccinium arboretum (sparkleberry), Vaccinium ashei (rabbiteye blueberry), Vaccinium corymbosum (highbush blueberry), Vaccinium elliottii (Elliott's blueberry), Vaccinium macrocarpon (cranberry), Vaccinium myrtillus (bilberry), Vaccinium stamineum (deerberry), Vaccinium vitis-ideae var. vitis-ideae (lingonberry), and Vaccinium vitis-ideae var. minor (partridgeberry) at levels between 7 and 5884 ng/g dry sample. Lingonberry was found to have the highest content, 5884 ng/g dry sample, comparable to that found in grapes, 6471 ng/g dry sample. Pterostilbene was found in two cultivars of V. ashei and in V. stamineum at levels of 99-520 ng/g dry sample. Piceatannol was found in V. corymbosum and V. stamineum at levels of 138-422 ng/g dry sample. These naturally occurring stilbenes, known to be strong antioxidants and to have cancer chemopreventive activities, will add to the purported health benefits derived from the consumption of these small fruits.     

Polyphenols newly extracted in red wine from southwestern France by centrifugal partition chromatography.

Polyphenols from the ethyl acetate extracts of red wine were successfully fractionated using a four-step process (solvent extraction, ion-exchange column chromatography, centrifugal partition chromatography, and semipreparative HPLC), which resulted in the isolation of 22 compounds belonging to different classes of polyphenols (stilbenes, cinnamic acids, flavonoids). Five of them are red wine constituents reported for the first time. The newly isolated compounds include resveratrol dimers, dihydroflavonols, and a cinnamic derivative.     

Changes in the phytoalexin content of various Vitis spp. in response to ultraviolet C elicitation.

The phytoalexin production potential of three American Vitis species and that of three cultivars of Vitis vinifera were evaluated in response to UV-C irradiation. Time course changes in resveratrol, piceid, epsilon-viniferin, and pterostilbene contents were studied within 3 days after a short UV-C irradiation. Results show that the two major stilbenes accumulated as a response to UV-C elicitation are resveratrol and epsilon-viniferin, a resveratrol dehydrodimer, the concentration of both compounds usually reaching quantities >100 microgram/g of fresh weight. In contrast, piceid and pterostilbene were constantly produced in low quantities. Owing to the results obtained, the role of stilbene phytoalexins in the resistance of grapevines to diseases is discussed.     

Phenolic composition of the Brazilian seedless table grape varieties BRS Clara and BRS Morena

The detailed phenolic composition (anthocyanins, flavonols, hydroxycinnamic acid derivatives, stilbenes, and flavan-3-ols) in the skin and flesh of the new BRS Clara and BRS Morena seedless table grapes has been studied using HPLC-DAD-ESI-MS/MS. The two grapes, especially BRS Morena, contained high amounts of phenolic compounds, mainly located in their skins and qualitatively not different from those found in Vitis vinifera grapes. In addition, BRS Morena (a teinturier variety) showed qualitatively different phenolic compositions in its skin and flesh, mainly affecting the anthocyanin and flavonol profiles. Consistent with high phenolic contents, high antioxidant capacity values were registered for both grape varieties, especially for BRS Morena. Proanthocyanidins and hydroxycinnamoyl-tartaric acids were the major phenolic compounds found in BRS Clara and were also important in BRS Morena, although anthocyanins were the main phenolic compounds in the latter case. These results suggest that the entire grapes, including the skin, may potentially possess properties that are beneficial to human health. In this context, the BRS Morena grape can be considered as a high resveratrol producer.     

Determination of stilbenes (delta-viniferin, trans-astringin, trans-piceid, cis- and trans-resveratrol, epsilon-viniferin) in Brazilian wines

Phenolics from grapes and wines can play a role against oxidation and development of atherosclerosis. Stilbenes have been shown to protect lipoproteins from oxidative damage and to have cancer chemopreventive activity. We describe a method for the direct determination of stilbenes in several red wines using high-performance liquid chromatography with UV detection. In a survey of 12 commercial wines from the south of Brazil (Rio Grande del Sul), levels of delta-viniferin are reported for the first time in different varieties of red wines. Brazilian red wine contains trans-astringin, trans-piceid, trans-resveratrol, cis-resveratrol (in high quantity: 5 times more than the trans form), epsilon-viniferin, and a compound isolated for the first time in wine, trans-delta-viniferin. Isolation and identification of delta-viniferin was achieved by NMR after extraction and fractionation of red wine phenolics. delta-Viniferin contributes, as well as cis-resveratrol and trans-piceid, to a significant proportion of stilbenes in wine dietary intake, particularly with Merlot varieties containing an average level of 10 mg/L for delta-viniferin, 15 mg/L for cis-resveratrol, and 13 mg/L for trans-piceid. The total stilbene intake from wine origin was estimated for the Brazilian population as 5.3 mg/day per person (on the basis of a regular wine consumption of 160 mL/day). delta-Viniferin can contribute to around 20% of total stilbenes in wine (average of 6.4 mg/L in red Brazilian wines). It would be important in the future to investigate the origins of the differences in wine stilbene levels in relation to the vine varieties, and the bioavailability of the newly extracted stilbene delta-viniferin in plasma after consumption of different types of wines.     

Resveratrol and a novel tyrosinase in Carignan grape juice

Polyphenol levels in wines are affected by the wine-making process. Resveratrol is one polyphenol which has been the subject of a commendable amount of recent research. In this work, we found that resveratrol is immediately degraded by tyrosinase. A novel tyrosinase was purified from Carignan grapes. The purification process included salting out and separation on a cation-exchange column, followed by gel filtration. Tyrosinase was purified in a homogeneous form by SDS-PAGE and was characterized: its specific activity toward 3-(3,4-dihydroxyphenyl)-L-alanine (DOPA) increased by a factor of 24 with an overall recovery of 3% of initial activity. The apparent molecular mass of the purified tyrosinase was 40 kDa as determined by SDS-PAGE, and 42 kDa as determined by gel filtration. Its activity was optimal at pH 6 and at 25 degrees C. The enzyme exhibited high activity toward phenylenediamine, epicatechin, pyrogallol, DOPA, and resveratrol. Tyrosinase activity was inhibited by KCN, thiourea, and SO(2). Resveratrol levels were stable following the removal of proteins from the juice, suggesting that early spraying of grapes with SO(2) is an important factor affecting the final amount of resveratrol in wine.     

Detection and quantitation of resveratrol in tomato fruit (Lycopersicon esculentum Mill.)

Resveratrol is a stilbene phytoalexin well-known for its presence in grape, wine, and peanut. As a result of its antioxidant and chemopreventative properties, it has gained much attention as a functional food ingredient. A gas chromatography-mass spectrometry method for the detection of resveratrol, its 3-glucopyranoside piceid, and the cis isomers of both compounds has been developed and used to quantitate the levels of these compounds in the skin of commercially available tomato fruit (Lycopersicon esculentum Mill.). The resveratrol concentration remains relatively stable during fruit maturation, reaching a maximum concentration in the skin of 18.4 +/- 1.6 microg/g dry weight at 4 weeks postbreaker. No stilbenes were detected in the flesh of tomato fruit.