Stilbene content of mature Vitis vinifera berries in response to UV-C elicitation

A method using HPLC analysis has been used to compare the level of resveratrol and its derivatives, piceid, pterostilbene and epsilon-viniferin, in grapevine berries of three Vitis vinifera varieties. The concentration of these compounds has been evaluated in healthy and Botrytis cinerea infected grape clusters, both in natural vineyard conditions and in response to UV elicitation.     

Delta-viniferin,a resveratrol dehydrodimer: one of the major stilbenes synthesized by stressed grapevine leaves

Delta-viniferin is a resveratrol dehydrodimer, an isomer of epsilon-viniferin. This compound has been reported as a molecule produced in vitro by the oxidative dimerization of resveratrol by plant peroxidases or fungal laccases. It was also recently identified in wines and in grape cell cultures. We have now identified this dimer by NMR, high-performance liquid chromatography-diode array detection (HPLC-DAD), and HPLC-mass spectrometry (MS) in grapevine leaves infected by Plasmopara viticola (downy mildew) or UV-C irradiated. Its concentration was higher than that of epsilon-viniferin and constitutes one of the most important phytoalexins derived from resveratrol.     

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.     

Inhibitory effects of resveratrol and pterostilbene on human colon cancer cells: a side-by-side comparison

The effects of resveratrol and pterostilbene (two structurally related stilbene compounds) on three human colon cancer cells were systematically compared. Cell viability tests indicated that IC(50) values of pterostilbene were 2-5-fold lower than those of resveratrol in all three cancer cells. Pterostilbene was also more potent in inhibiting colony formation of all three cancer cells. Annexin V/propidium iodide costaining assay and Western blotting analysis showed pterostilbene had a stronger apoptosis-inducing effect, which was evidenced by the higher percentage of annexin V positive cells and higher levels of cleaved caspase-3 and poly(ADP-ribose) polymerase proteins in cancer cells treated with pterostilbene compared with resveratrol. High-performance liquid chromatography analysis demonstrated that intracellular levels of pterostilbene were 2-4-fold higher than those of resveratrol after treatments with individual compounds at the same concentration. Overall, the results demonstrated that pterostilbene had more potent inhibitory effects on colon cancer cells than resveratrol, which may be associated with the superior bioavailability of pterostilbene to resveratrol.     

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.     

Capillary electrophoresis determination,synthesis, and stability of resveratrol and related 3-O-beta-D-glucopyranosides

Because of the health implications of resveratrol and piceid, which are widely present in foods, we focused our attention on the development of a highly efficient methodology for their characterization and measurement. On the basis of our experience in capillary electrophoresis for separation of polyphenolic compounds, we developed a general analytical method for piceid identification which was also applicable to complex natural mixtures such as red wine. In view of its very limited availability from natural sources, we first directed our attention to the development of a synthetic route suitable to produce an adequate amount of polydatin (piceid or resveratrol 3-O-beta-D-glucopyranoside). The latter was synthesized by a new one-step procedure which afforded the expected product in a good yield. The studied compounds were also investigated for their stability to UV irradiation.     

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.     

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.     

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.     

Cancer chemopreventive and antioxidant activities of pterostilbene,a naturally occurring analogue of resveratrol

Pterostilbene, a natural methoxylated analogue of resveratrol, was evaluated for antioxidative potential. The peroxyl-radical scavenging activity of pterostilbene was the same as that of resveratrol, having total reactive antioxidant potentials of 237 +/- 58 and 253 +/- 53 microM, respectively. Both compounds were found to be more effective than Trolox as free radical scavengers. Using a plant system, pterostilbene also was shown to be as effective as resveratrol in inhibiting electrolyte leakage caused by herbicide-induced oxidative damage, and both compounds had the same activity as alpha-tocopherol. Pterostilbene showed moderate inhibition (IC50 = 19.8 microM) of cyclooxygenase (COX)-1, and was weakly active (IC50 = 83.9 microM) against COX-2, whereas resveratrol strongly inhibited both isoforms of the enzyme with IC50 values of approximately 1 microM. Using a mouse mammary organ culture model, carcinogen-induced preneoplastic lesions were, similarly to resveratrol, significantly inhibited by pterostilbene (ED50 = 4.8 microM), suggesting antioxidant activity plays an important role in this process.     

Differentiation of young red wines based on chemometrics of minor polyphenolic constituents

For reasons of examining any possible discrimination of young red wines based on selected, minor polyphenols, a survey was carried out including 35 samples originating from three Hellenic native and three international Vitis vinifera cultivars from various regions of Greece. All samples were experimental wines vinified and stored under identical conditions, in an effort to minimize the effect of different winemaking technologies as well as aging. The polyphenols analyzed belonged to two categories: benzoic acid derivatives, including gallic acid, protocatechuic acid, vanillic acid, and syringic acid, and stilbenes, including astringin, piceid, and resveratrol (all trans isomers). Data handling employing discriminant analysis (DA) yielded very satisfactory categorization of samples in terms of both cultivar and geographical region of origin. This outcome was discussed with regard to the value of certain minor polyphenols that could serve as characteristic indices for discrimination of varietal red wines, after appropriate implementation of chemometrics.     

Isolation and identification of stilbenes in two varieties of Polygonum cuspidatum

The roots of two varieties of Polygonum cuspidatum (Hu Zhang and Mexican Bamboo) were analyzed for resveratrol and analogues. The roots of each variety were dried and ground into a powder. The powdered roots were then extracted with methanol and ethyl acetate. The ethyl acetate fraction of the Mexican Bamboo was then subjected to fractionation and purification using silica gel column chromatography and semipreparative HPLC. In addition to resveratrol (3,5,4'-trihydroxystilbene), three stilbene glucosides were identified by (1)H NMR, (13)C NMR, and MS. The stilbene glucosides were shown to be a piceatannol glucoside (3,5,3', 4'-tetrahydroxystilbene 4'-O-beta-D-glucopyranoside), resveratroloside (3,5,4'-trihydroxystilbene 4'-O-beta-D-glucopyranoside), and piceid (3,5,4'-trihydroxystilbene 3-O-beta-D-glucopyranoside). The levels of the piceatannol glucoside and piceid were twice as high in the Mexican Bamboo as compared to the Hu Zhang.     

Enzymatic Synthesis of Piceid Glucosides Using Maltosyltransferase from Caldicellulosiruptor bescii DSM 6725

Piceid is widely used in food, cosmetics, and pharmaceuticals because of its therapeutic benefits. However, the use of piceid as a drug is limited because of its low solubility. To increase solubility, we synthesized piceid glucosides using maltosyltransferase from Caldicellulosiruptor bescii . The MTase gene was cloned and expressed in Escherichia coli . The enzyme had a unique transfer specificity to the transfer of maltosyl units. Four piceid transglycosylation products were present and identified by thin-layer chromatography and recycling preparative high-performance liquid chromatography. The major product was purified by C(18) and gel filtration chromatography, and its molecular structure was determined using nuclear magnetic resonance spectroscopy to be α-d-maltosyl-(1→4)-piceid. The solubility of maltosyl piceid was 8.54 × 10(3) and 1.86 × 10(3) times those of natural resveratrol and piceid, respectively, suggesting that the transglycosylation greatly increased the water solubility. This suggests that dietary intake of this compound can enhance the bioavailability of resveratrol in the human body.     

Method for the quantitative extraction of resveratrol and piceid isomers in grape berry skins. Effect of powdery mildew on the stilbene content

A simple method for the quantitative extraction of resveratrol and its glycosides from grape berry skins has been developed. Optimal conditions for the extraction were investigated. Type of solvent, time, and temperature assayed influenced resveratrol and piceid yield. Adequate extraction was attained with ethanol/water (80:20 v/v) maintained at 60 degrees C for 30 min. Recovery (>96%) and reproducibility (6.83-15.13%) were satisfactory. After extraction, resveratrol and piceid isomers were quantified by high-performance liquid chromatography coupled to a ultraviolet-visible diode array detector. The amounts detected in 13 samples of 7 different varieties analyzed were, on average, 92.33 microg/g of dry skin for cis-piceid, 42.19 microg/g of dry skin for trans-piceid, and 24.06 microg/g of dry skin for trans-resveratrol. cis-Resveratrol was not detected in any sample. In grape berries infected by powdery mildew the contentw of these compounds were considerably increased and the degree of infection was positively related to their stilbene content.     

Inhibiting effects of resveratrol and its glucoside piceid against Venturia inaequalis, the causal agent of apple scab

Resveratrol is a phytoalexin produced in different unrelated plant species such as grapevine and peanut. The effect of resveratrol and its glucoside trans-resveratrol-3-O-beta-D-glycopyranosid (piceid) against Venturia inaequalis, the causal agent of apple scab, was evaluated using a newly established test based on enzymatically isolated cuticular membranes (CMs) from apple (Malus domestica Borkh.) leaves. The test substances resveratrol and piceid were either sorbed to CMs before inoculation with spores or were applied simultaneously with the Venturia inaequalis spores to the CMs, and their effect on germination, appressoria formation, and penetration was examined. Resveratrol had no influence on spore germination but a significant inhibiting effect on penetration when applied simultaneously as well as before. A percentage inhibition of 89.7 +/- 11.5 and 61.8 +/- 35.1 was observed for simultaneous and preapplication, respectively. The resveratrol glucoside piceid had a significant inhibitory effect on germination and completely inhibited penetration of the fungus at concentrations between 200 and 400 microg mL(-1) when applied simultaneously with the spores to the CMs. On piceid-enriched CM (preapplication), spores germinated but penetration was inhibited nearly completely (96.1 +/- 5.1%). Thus, in vitro experiments showed that resveratrol and its glucoside in fact could contribute to improving the pathogen resistance of apple leaves.     

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.     

Cellular and behavioral effects of stilbene resveratrol analogues: implications for reducing the deleterious effects of aging

Research suggests that polyphenolic compounds contained in fruits and vegetables that are rich in color may have potent antioxidant and anti-inflammatory activities. The present studies determined if stilbene (e.g., resveratrol) compounds would be efficacious in reversing the deleterious effects of aging in 19 month old Fischer 344 rats. Experiment I utilized resveratrol and six resveratrol analogues and examined their efficacies in preventing dopamine-induced decrements in calcium clearance following oxotremorine-induced depolarization in COS-7 cells transfected with M1 muscarinic receptors (MAChR) that we have shown previously to be sensitive to oxidative stressors. Experiment II utilized the most efficacious analogue (pterostilbene) from experiment I and fed aged rats a diet with a low (0.004%) or a high (0.016%) concentration of pterostilbene. Results indicated that pterostilbene was effective in reversing cognitive behavioral deficits, as well as dopamine release, and working memory was correlated with pterostilbene levels in the hippocampus.     

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.     

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.