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.     

Effects of elicitors on the production of resveratrol and viniferins in cell cultures of Vitis vinifera L. cv Italia

Methyl jasmonate, jasmonic acid and chitosan were tested as elicitors on cell suspension cultures obtained from Vitis vinifera cv Italia to investigate their effect on stilbene production. Stilbene accumulation in the callus, grown under nonelicited conditions, was also investigated. Calli and cell suspensions were obtained in a B5 culture medium supplemented with 0.2 mg L(-1) NAA and 1 mg L(-1) KIN. Stilbene determination was achieved by HPLC/DAD/MS. Whereas callus biosynthesized only piceid, cell suspensions elicited with jasmonates produced several stilbenes, mainly viniferins. In suspended cells, methyl jasmonate and jasmonic acid were the most effective in stimulating stilbene biosynthesis, whereas chitosan was less effective; in fact, the amount of stilbenes obtained with this elicitor was not significantly different from that obtained for the control cells. The maximum production of total stilbenes was at day 20 of culture with 0.970 and 1.023 mg g(-1) DW for MeJA and JA, respectively.     

Identification and quantification of stilbenes in fruits of transgenic tomato plants (Lycopersicon esculentum Mill.) by reversed phase HPLC with photodiode array and mass spectrometry detection

Reversed-phase high-performance liquid chromatography (RP-HPLC) with photodiode array (PDA) and mass spectrometry (MS) detection was employed to study the accumulation of stilbenes and other naturally occurring polyphenol intermediates of flavonoid pathway in tomato fruits of plants genetically modified to synthesize resveratrol. The transgenic tomato fruits were obtained by overexpression of a grapevine gene encoding the enzyme stilbene synthase in tomato plants (Lycopersicon esculentum Mill.). Stilbenes and flavonoids, either glycosylated or free, were simultaneosly identified by electrospray interface (ESI)-MS in negative ionization mode and were quantified by PDA detection at the wavelength corresponding to their maximum absorbance. The two detectors were coupled online with an HPLC system utilizing a narrow-bore C18 reversed-phase column, which was eluted by a multistep gradient of increasing concentration of acetonitrile in water containing 0.5% (v/v) formic acid. The results of these analysis revealed that the genetic modification of the tomato plants originated different levels of accumulation of four stilbenes (i.e., trans- and cis-piceid and trans- and cis-resveratrol) in their fruit depending on the stages of ripening. Either at immature or at mature stages of ripening the stilbenes were preferentially accumulated in the fruit peel as the glycosylated form. The highest amount of trans-piceid and trans-resveratrol were found in the peel of fruits harvested at mature stage of ripening. The variations in the levels of rutin, naringenin, and chlorogenic acid found in the samples extracted from the fruits of transgenic tomato plants, in comparison to that determined in the control lines, seemed to be related to the genetic transformation, whose effect on the flavonoid biosynthetic pathway needs to be elucidated by additional studies.     

Postharvest UV-C-irradiated grapes as a potential source for producing stilbene-enriched red wines

The use of postharvest controlled irradiation by UV pulses is proposed as a potential method to produce stilbene-enriched red wine. "Monastrell" grapes were UV-C-irradiated to increase stilbene content. The main inducible stilbenes were resveratrol and piceatannol, which are molecules with reported health-beneficial activities. The evolution of both compounds was followed in the different steps of an "analytical" traditional maceration wine-making process. The final wine made from UV-C-irradiated grapes was enriched about 2- and 1.5-fold in resveratrol and piceatannol, respectively, when compared to the control wine. In addition, no difference was detected regarding the standard enological parameters (color, acidity, etc.). It is strongly suggested that, with the use of more susceptible wine grapes to induce bioactive stilbenes upon UV-C irradiation, the stilbene-enrichment of wine can be much higher.     

Ethephon elicits protection against Erysiphe necator in grapevine

The grapevine (Vitis vinifera) is susceptible to many pathogens such as Botrytis cinerea, Plasmopara viticola, Erysiphe necator, and Eutypa lata. Phytochemicals are used extensively in vineyards to reduce pathogen infections, but the appearance of pesticide-resistant pathogen strains and the need for environmental protection require the use of alternative strategies. The phytohormone ethylene is assumed to play a role in the development of disease resistance. In the present study, we have treated grapevine foliar cuttings (Cabernet Sauvignon) with ethylene-releasing ethephon. This resulted in an increase in the number of pathogenesis-related protein (CHIT4c, PIN, PGIP, and GLU) gene copies and in an enhancement of phytoalexin biosynthesis by inducing the PAL and STS genes that correlated with the accumulation of stilbenes (antimicrobial compounds). Moreover, ethephon treatment triggered the protection of grapevine detached leaves and grapevine foliar cuttings against Erysiphe necator, the causal agent of powdery mildew (64% and 70%, respectively). These studies emphasize the major role of ethylene in grapevine defense.     

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.     

Profiling of resveratrol oligomers, important stress metabolites, accumulating in the leaves of hybrid Vitis vinifera (Merzling × Teroldego) genotypes infected with Plasmopara viticola

In the Vitaceae, viniferins represent a relatively restricted group of trans-resveratrol oligomers with antifungal properties, thus enabling plants to cope with pathogen attack. The aim of this study was to perform isolation and structural characterization of the whole class of viniferins accumulating in the leaves of hybrid Vitis vinifera (Merzling × Teroldego) genotypes infected with Plasmopara viticola . Infected leaves of resistant plants were collected 6 days after infection, extracted with methanol, and prepurified by flash chromatography using ENV+ and Toyopearl HW 40S resins. Further fractionation using normal-phase preparative chromatography and then reversed-phase preparative chromatography allowed isolation of 14 peaks. The isolated compounds were identified using advanced mass spectrometry techniques and extensive one- and two-dimensional nuclear magnetic resonance measurements, UV, CD, optical properties, and molecular mechanic calculations. The results demonstrated the presence in infected leaves of seven dimers (six stilbenes and one stilbenoid), of which four were new in grapevine (ampelopsin D, quadrangularin A, E-ω-viniferin, and Z-ω-viniferin), four trimers (three stilbenes and one stilbenoid), of which two (Z-miyabenol C and E-cis-miyabenol C) were new in grapevine, three tetramer stilbenoids, all new in grapevine, isohopeaphenol, ampelopsin H, and a vaticanol C-like isomer. The isolation of a dimer deriving from the condensation of (+)-catechin with trans-caffeic acid also indicated that other preformed phenolics are structurally modified in tissues infected with P. viticola.     

Constitutive expression of a grapevine stilbene synthase gene in transgenic hop (Humulus lupulus L.) yields resveratrol and its derivatives in substantial quantities

Resveratrol, a well-known phytoalexin and antioxidant, is produced by the action of stilbene synthase (STS) in some plant species. Hop (Humulus lupulus L.) plants of the Tettnang variety were transformed with a gene encoding for STS from grapevine. Under the control of the constitutive 35S cauliflower mosaic virus promoter, expression of the transgene resulted in accumulation of resveratrol and high levels of its glycosylated derivatives in leaves and inflorescences. Piceid, the predominant derivative, reached a concentration of up to 560 microg/g of fresh weight (f.w.) in hop cones, whereas no stilbenes were detected in nontransformed controls (wild-type). In transgenic plants the amounts of alpha- and beta-acids, naringenin chalcone, and prenylated flavonoids did not change significantly when compared with nontransformed plants. Transgenic plants showed normal morphology and flower development as did the nontransformed controls. The results clearly show that in hop constitutive expression of sts interferes neither with plant development nor with the biosynthesis of secondary metabolites relevant for the brewing industry. Since resveratrol is a well-known phytoalexin and antioxidant, sts transgenic hop plants could display enhanced pathogen resistance against microbial pathogens, exhibit new beneficial properties for health, and open new venues for metabolic engineering.     

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.     

Phytoalexins from the Vitaceae: biosynthesis, phytoalexin gene expression in transgenic plants, antifungal activity, and metabolism

Resistance of plants to infection by phytopathogenic microorganisms is the result of multiple defense reactions comprising both constitutive and inducible barriers. In grapevine, the most frequently observed and best characterized defense mechanisms are the accumulation of phytoalexins and the synthesis of PR-proteins. Particular attention has been given here to stilbene phytoalexins produced by Vitaceae, specifically, their pathway of biosynthesis (including stilbene phytoalexin gene transfer experiments to other plants) and their biological activity together with fungal metabolism.     

Optimization, in-house validation, and application of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for the quantification of selected polyphenolic compounds in leaves of grapevine (Vitis vinifera L.)

Polyphenols in grapevine can be constitutive or induced, depending upon cultivar, plant organ, and environmental influences. The aim of the presented work was to develop and optimize a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to study the pattern and amount of selected polyphenols in leaves of Vitis vinifera L. The method is simple and does not require any sample cleanup. It covers representative metabolites of the structure classes cinnamic acids, flavonoids, and stilbenes and enables the simultaneous separation and quantification of 13 polyphenols within 9 min at concentration levels between 0.1 and 3 μg/g. We present the method performance characteristics and its application to the quantification of polyphenols in grapevine leaves of the cultivars Riesling and Pinot noir. A total of 7 of 13 target polyphenols were detected at concentrations above the limits of quantification. Interestingly, instead of the expected trans-resveratrol, the investigated leaf samples of both cultivars contained cis-resveratrol-3-O-glucoside. The measurements also showed that Riesling leaves tended to contain higher concentrations of the selected polyphenols than Pinot noir. In view of its intended future use, the developed method has been shown to be a powerful and fast tool to study polyphenols in grapevine leaves subjected to environmental stress conditions.     

Hopeaphenol: the first resveratrol tetramer in wines from North Africa

Grapes and wines are now known to constitute a rich source of phenolics such as stilbenes and flavonoids. These compounds have been shown to have cancer chemopreventive activity and potential beneficial effects on cardiovascular diseases thanks to their antioxidant and antiplatelet properties. However, because little is known about African wines and their phenolic compositions, we investigated wine samples from North Africa. A three-step method was used for the fractionation of the Merlot variety wine: column chromatography followed by centrifugal partition chromatography and reversed-phase semipreparative high-performance liquid chromatography (HPLC). Six polyphenolic compounds of the Merlot variety (from Algeria) were isolated and identified by NMR spectroscopy, five of which are known (trans-resveratrol, trans-piceid, trans-epsilon-viniferin, pallidol, and astilbin) and one that is reported for the first time in wine, (+)-hopeaphenol, a stilbene tetramer. Furthermore, these molecules were quantified in 10 commercial wines from North Africa by means of an analytical HPLC system coupled with diode array detection. Differences in concentrations were found ranging in mg/L from 4.6 to 45 (trans-piceid), 0.66 to 3.45 (trans-resveratrol), 0.2 to 1.2 (trans-epsilon-viniferin), 0.2 to 9.2 (pallidol), 0.3 to 3.8 (hopeaphenol), and 10.8 to 24.22 (astilbin). Such a high level of pallidol and astilbin has never been recorded in wine. North African wines may contribute to a significant proportion of dietary intake of stilbene and astilbin, which may have health benefits.     

A simple extraction and chromatographic system for the simultaneous analysis of anthocyanins and stilbenes of Vitis species

A separation system has been developed to permit the simultaneous analysis of major anthocyanins and stilbenes from berries of Vitis species in <1 h. The system makes use of separation by HPLC and detection by UV-visible absorption and fluorescence, for anthocyanins and stilbenes, respectively, with the two detection systems linked in series. Monitoring the absorption at 520 nm permits ready identification and quantification of major anthocyanins. Chromatograms derived from fluorescence (330 nm excitation/374 nm emission) produce less clear data for the piceids and resveratrols, which are present in much lower amounts; peaks for these compounds can be verified by means of external or internal standards.     

Piceid (resveratrol glucoside) synthesis in stilbene synthase transgenic apple fruit

A stilbene synthase gene along with the selectable marker gene bar for herbicide resistance was transferred via Agrobacterium tumefaciens mediated transformation into apple (Malus domesticaBorkh.) cvs. 'Elstar' and 'Holsteiner Cox'. The stilbene synthase catalyzes the conversion of 1 molecule of p-coumaroyl-CoA and 3 molecules of malonyl-CoA into 3,4',5-trihydroxystilbene, commonly known as resveratrol. This phytoalexin has implications in both phytopathology and human health. Greenhouse-grown transgenic and nontransformed control plants were grafted onto dwarfing rootstock M27. Flowering and fruiting occurred within the following years, offering the opportunity to analyze transgenic apple fruit and fertility of transgenic plants as well as inheritance of the transgenes into the seedling progeny. Molecular analysis revealed that the stilbene synthase is expressed in transgenic plants and in the skin and flesh of transgenic apple fruit. After formation, resveratrol is modified by the addition of a hexose sugar. The resulting component was characterized as piceid. With the aim of characterizing the influence of the novel biosynthetic pathway on the accumulation of other phenolic compounds naturally present in apple fruit, the amounts of flavanols, flavonols, phloretin derivatives and hydroxycinnamic acids in wild type and transgenic fruit were determined by HPLC. In all investigated transformed lines that accumulated piceid, no negative correlation between levels of piceid and the above-mentioned compounds was observed, except for the flavonol contents, which slightly decreased. Inheritance of the transgenes was confirmed in the seedling progeny, which were obtained after pollination of transgenic plants with nontransgenic pollen and vice versa after pollination of nontransgenic plants with pollen obtained from transgenic plants. The fertility of stilbene synthase transgenic plants was demonstrated. To the authors' knowledge this is the first time that data are available on piceid synthesis in transgenic apple fruit and the effects of its accumulation on levels of other phenolic compounds present in the fruit.     

Polyphenol glucosylating activity in cell suspensions of grape (Vitis vinifera)

Stilbenes are phenolic molecules that have antifungal effects in the plant and antioxidant and anti-cancer effects when consumed in the human diet. Glycosylation of stilbenes increases their solubility and may make them more easily absorbed by the intestine. We have found an activity in extracts of cultured cells of Vitis vinifera (cv. Gamay Freaux) that glucosylates the stilbene resveratrol to form piceid. The Km for UDP-Glucose was 1.2 mM, and the Km for resveratrol was 0.06 mM, values similar to those of other phenolic glucosyltransferases. We investigated the resveratrol glucosylating activity of the enzyme extracted from cells grown under different light treatments (dark, visible light, light + ultraviolet (UVC) radiation) and found the activity to be unaffected or slightly reduced. In contrast, UVC light strongly stimulated extractable quercetin glucosyltransferase activity. These results, combined with analysis of phenolic compounds extracted from the differently treated cells, suggest that the resveratrol glucosyltransferase is distinct from the glucosyltransferase(s) active on other phenolics.     

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.     

Isolation and characterization of novel stilbene derivatives from Riesling wine

Besides the already known stilbenes trans-resveratrol as well as isomeric piceids seven novel stilbene derivatives have been isolated from a commercial Riesling wine. The newly identified compounds included the monostilbene 2,4,6-trihydroxyphenanthrene-2-O-glucoside, as well as two isomeric resveratrol-2-C-glucosides. In addition, four dimeric stilbenes, i.e., cis- and trans-epsilon-viniferin diglucoside as well as pallidol glucoside and pallidol diglucoside, have also been obtained for the first time from Riesling wine.     

Benzothiadiazole enhances resveratrol and anthocyanin biosynthesis in grapevine, meanwhile improving resistance to Botrytis cinerea

Pre-harvest multiple treatments of grapevine (cv. Merlot) with the plant activator benzothiadiazole (BTH, 0.3 mM) enhanced trans-resveratrol content in berries by about 40%. An even more striking effect was observed on anthocyanin synthesis, particularly on malvidine 3-glucoside, malvidine 3-(6-O-acetyl)glucoside and malvidine 3-(6-O-p-coumaroyl)glucoside, whose amounts were more than doubled. These data were obtained with a novel and time-saving HPLC method, set up for the simultaneous detection of stilbenes and anthocyanins, using an RF-10Axl fluorimetric detector instrument, with excitation at 330 nm and emission at 374 nm, and a SPD-Avp UV detector with absorption at 520 nm. Furthermore, BTH treatments induced systemic acquired resistance in grapevine, as assessed by inoculating clusters from treated and untreated plants with Botrytis cinerea. Disease severity, estimated according to the percentage of infected berries per cluster, was significantly reduced in grapes from BTH-treated plants. These results indicate that BTH treatments, besides improving the content of two important classes of nutraceuticals, with their well-known antioxidant, antitumoral, and phytoestrogenic activities, could be exploited in vineyard to protect grape against gray mould infection, thereby limiting an excessive use of fungicides     

Verteilung von Eisen in Blättern von Weinreben mit HCO3− induzierter Fe-Chlorose

Distribution of iron in vine leaves with HCO₃^? induced chlorosis The distribution of Fe in chlorotic and healthy leaves of grapevine has been studied. Application of HCO₃^? had no significant influence on the long distance transport of labelled Fe, the translocation of Fe from the vascular tissue to the intercostal cells (cells between leaf veins), however, was much affected by HCO₃^?. Leaf samples collected from three different sites showed in most cases higher Fe contents in the chlorotic leaves as compared with healthy leaves. The solubility of leaf Fe in diluted HCl, however, was lower in chlorotic leaves than in green leaves. Enzymatic dissolution of leaves into vascular tissue, intercostal cells and chloroplasts revealed that the Fe-content in the intercostal cells of green leaves was significantly higher than in the intercostal cells of chlorotic leaves. In addition the intercostal cells of chlorotic leaves had extremly high Ca and P-contents. The P content of green and chlorotic leaves was not related to the level of available P in the soil. It is therefore concluded that the high P content in chlorotic leaves is the sequence and not the cause of Fe-chlorosis. On each of the three sites investigated, higher clay contents were found under chlorotic grapevine plants than under healthy ones. It is assumed that because of this higher clay content, soil compaction may occur, resulting in an accumulation of CO₂ and in an increase of the HCO₃^? concentration in the soil solution.