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Aim: Stilbenes are well-known phytoalexins present in vine and wine. Stilbene glucosides have been identified in wine; however, other than piceid, these compounds have never been reported to be present in the woody parts of grapevine. The aims of this study were to investigate the presence of stilbene glucosides in the woody parts of the vine and to evaluate their cytotoxic activity, in comparison with that of resveratrol, in human hepatoma HepG2 cells. Methods and results: Stilbene glucosides were isolated from a Vitis vinifera root extract. The extract was partitioned with ethyl acetate and fractionated by polyamide gel column chromatography. Pure compounds were obtained by semipreparative high-performance liquid chromatography. These were then identified by mass spectrometry and nuclear magnetic resonance (NMR) analyses, including analysis of two-dimensional NMR spectra. In addition to resveratrol, five stilbene glucosides were found: resveratroloside, resveratrol rutinoside, trans-ε-viniferin diglucoside, cis-ε-viniferin diglucoside and piceid. Of these, the first four showed cytotoxic effects against HepG2 cells when the crystal violet assay was used to determine cell viability. Conclusion: In addition to resveratrol and piceid, this is the first report of the presence of four glucosylated derivatives of resveratrol (resveratroloside, resveratrol rutinoside, and trans- and cis-ε-viniferin diglucosides) in the woody parts of vine. These compounds showed significant cytotoxicity against HepG2 cells. Significance and impact of the study: Stilbenes are well-known biological compounds. Grapevine is one of the main sources of this family of polyphenols. Other than piceid, stilbene glucosides have been identified in wine but never in the woody parts of vine. This is the first study in which five glucosylated derivatives of resveratrol were isolated from woody parts of vine. They were also shown to exert antiproliferative effects in human hepatoma HepG2 cells.

Seven Tunisian red wines mainly from the Mornag appellation were analyzed for resveratrol and analogues. The wines of each variety were evaporated, concentrated, and then subjected to fractionation and purification using XAD16 and DOWEX column chromatography. In addition to resveratrol, seven stilbenes were identified by UPLC-MS. The stilbenes derived were shown to be piceatannol, piceid, a-viniferin, e-viniferin, hopeaphenol and isohopeaphenol. From the point of view of the presence of resveratrol derivatives, one wine, Sidi Zahia, was the richest qualitatively.

Stilbenoids are of great interest on account of their many promising biological activities, especially in regards to prevention and potential treatment of many chronic diseases associated with aging. The simple stilbenoid monomer, E-resveratrol, has received the most attention due to early in vitro and in vivo biological activities in anti-aging assays. Since Vitis vinifera, primarily in the form of wine, is a major dietary source of these compounds, there is a tremendous amount of research on resveratrol in wine and grapes. Relatively few biological studies have been performed on other stilbenoids from Vitis, primarily due to the lack of commercial sources of many of these compounds. The diverse stilbenoids from this economically important genus are an untapped source of health promoting compounds and because of this, numerous efforts for isolation, identification and quantification of additional stilbenoids have been ongoing. Additionally, due to their role as phytoalexins, stilbenoids play an important role in the defense against pathogens. Therefore, the compounds produced by highly resistant strains are of great interest for the development of resistant crops, natural spray reagents, and as new dietary supplements or pharmaceuticals. Since closely related species are likely to have similar metabolic pathways, a more thorough understanding of the chemical diversity of stilbenoids within Vitis is useful in this endeavor. In this review, we focus on stilbenoids found in the Vitis genus with the aim of aiding future stilbenoid chemistry, particularly in V. vinifera and wine. Additionally, we discuss the efforts to quantify stilbenoids in Vitis, with a focus on non-resveratrol stilbenoid compounds.

Aims : The aim of the present investigation was to establish glucuronidation of trans-resveratrol derivates in the liver. Stilbenes are naturally occurring polyphenolic compounds which have been reported to have potential preventive activities in human diseases. Trans-stilbenes, mainly found in grapes and red wine, are important in terms of biological activities. However, little is known about the metabolism of these compounds in human. Methods and results : The glucuronoconjugation of stilbenes was investigated using human liver microsomes and the structure of new metabolites was characterized by LC-MS and proton NMR. For the first time, the structure of the metabolites of trans-piceid and trans-piceatannol was established. The reaction led to the formation of two glucuronides for trans-piceid and three for trans-piceatannol. Significance and impact of study: This study is of particular relevance since the phenolic substances of red wine (especially stilbenes) might be responsible for the potential beneficial effects of moderate and regular wine consumption.

Polynucleotidyl transferases are enzymes involved in several DNA mobility mechanisms in prokaryotes and eukaryotes. Some of them such as retroviral integrases are crucial for pathogenous processes and are therefore good candidates for therapeutic approaches. To identify new therapeutic compounds and new tools for investigating the common functional features of these proteins, we addressed the inhibition properties of natural stilbenoids deriving from resveratrol on two models: the HIV-1 integrase and the eukaryote MOS-1 transposase. Two resveratrol dimers, leachianol F and G, were isolated for the first time in Vitis along with fourteen known stilbenoids: E-resveratrol, E-piceid, E-pterostilbene, E-piceatannol, (+)-E-ε-viniferin, E-ε-viniferinglucoside, E-scirpusin A, quadragularin A, ampelopsin A, pallidol, E-miyabenol C, E-vitisin B, hopeaphenol, and isohopeaphenol and were purified from stalks of Vitis vinifera (Vitaceae), and moracin M from stem bark of Milliciaexelsa (Moraceae). These compounds were tested in in vitro and in vivo assays reproducing the activity of both enzymes. Several molecules presented significant inhibition on both systems. Some of the molecules were found to be active against both proteins while others were specific for one of the two models. Comparison of the differential effects of the molecules suggested that the compounds could target specific intermediate nucleocomplexes of the reactions. Additionally E-pterostilbene was found active on the early lentiviral replication steps in lentiviruses transduced cells. Consequently, in addition to representing new original lead compounds for further modelling of new active agents against HIV-1 integrase, these molecules could be good tools for identifying such reaction intermediates in DNA mobility processes.

Natural extracts, and particularly plant by-product extracts, can represent an eco-friendly alternative to synthetic pesticides to control crop diseases. We produced an extract from a mix of grapevine trunk and roots that contain a high amount of complex stilbenes (grapevine phytoalexins), especially ε- and r-viniferins. This extract conferred protection to grapevines against Plasmopara viticola (the causal agent of downy mildew) by displaying antimicrobial activities towards zoospore mobility and sporulation and by stimulating plant defences. From the perspective of its application in the field, a formulated preparation of the extract was proposed using polysorbate and sophorolipids. The formulated extract reduced the development of various oomycetes impacting grapevine, potato, tomato and melon in semi-controlled conditions. Thus, such grapevine extract constitutes a promising strategy to protect major cultivated plants against downy mildews.

The investigation of natural and safe antioxidants from natural origin is highly encouraged since it was revealed that synthetic antioxidants have restricted use in foods due to their toxicological effects and suspected carcinogenic potential. Purification of most active phenolics from the halophyte Inula crithmoїdes was the objective fixed for this work. The separation of the flower phenolics was carried using centrifugal partition chromatography (CPC) and yielded 24 fractions. A bio-guided selection of the most active fractions was done based on their antioxidant activities. Fractions 2, 7, 11 and 19 were the most active ones, even more potent than positive controls BHT, BHA and ascorbic acid. The semi-preparative High Performance Liquid Chromatography (HPLC) purification of the antioxidant molecules from these active fractions and their identification by Nuclear Magnetic Resonance spectroscopy (NMR) revealed that the most potent phenolics of I. crithmoїdes are the chlorogenic acid and its two derivatives 3-p-coumaroyl-5-caffeoyl quinic acid and 1,5-di-O-caffeoylquinic acid, in addition to the quercetin and its derivative quercimeritrin. All identified compounds are powerful antioxidants, as they have many biological properties allowing their use in agro-food, pharmaceutical or cosmetic industries. Keywords: Inula crithmoїdes, CPC, Antioxidant activities, Phenolics, HPLC, MNR

Anthocyanin biosynthesis is regulated by environmental factors (such as light, temperature, and water availability) and nutrient status (such as carbon, nitrogen, and phosphate nutrition). Previous reports show that low nitrogen availability strongly enhances anthocyanin accumulation in non carbon-limited plant organs or cell suspensions. It has been hypothesized that high carbon-to-nitrogen ratio would lead to an energy excess in plant cells, and that an increase in flavonoid pathway metabolic fluxes would act as an “energy escape valve,” helping plant cells to cope with energy and carbon excess. However, this hypothesis has never been tested directly. To this end, we used the grapevine Vitis vinifera L. cultivar Gamay Teinturier (syn. Gamay Freaux or Freaux Tintorier, VIVC #4382) cell suspension line as a model system to study the regulation of anthocyanin accumulation in response to nitrogen supply. The cells were sub-cultured in the presence of either control (25 mM) or low (5 mM) nitrate concentration. Targeted metabolomics and enzyme activity determinations were used to parametrize a constraint-based model describing both the central carbon and nitrogen metabolisms and the flavonoid (phenylpropanoid) pathway connected by the energy (ATP) and reducing power equivalents (NADPH and NADH) cofactors. The flux analysis (2 flux maps generated, for control and low nitrogen in culture medium) clearly showed that in low nitrogen-fed cells all the metabolic fluxes of central metabolism were decreased, whereas fluxes that consume energy and reducing power, were either increased (upper part of glycolysis, shikimate, and flavonoid pathway) or maintained (pentose phosphate pathway). Also, fluxes of flavanone 3β-hydroxylase, flavonol synthase, and anthocyanidin synthase were strongly increased, advocating for a regulation of the flavonoid pathway by alpha-ketoglutarate levels. These results strongly support the hypothesis of anthocyanin biosynthesis acting as an energy escape valve in plant cells, and they open new possibilities to manipulate flavonoid production in plant cells. They do not, however, support a role of anthocyanins as an effective mechanism for coping with carbon excess in high carbon to nitrogen ratio situations in grape cells. Instead, constraint-based modeling output and biomass analysis indicate that carbon excess is dealt with by vacuolar storage of soluble sugars.

Aim: To investigate the antifungal efficacy of grape cane extracts enriched in stilbenes against Plasmopara viticola by in vivo experiments on grape plants. Methods and results: Experiments on grape plants were carried out from 2007 to 2008 at commercial vineyards in « Le Tourne » (southwest region of France) on Vitis vinifera L. cv Merlot plants and in a greenhouse, where 45-day-old plants (Merlot) were used. Grapevine plants were treated with stilbene extract (STE) or « Bordeaux mixture » (BM) as positive control. STE, prepared from Vitis vinifera canes, contained 33% of total stilbenes ranging from monomers to tetramers and was used at 5 g L-1. The degree of protection against downy mildew was determined as the percentage of infected leaf surface area and pathogen attack frequency. On young plants in greenhouse, P. viticola inoculation and three treatments with STE were carried out. After the treatments, the disease reduction was from 59 to 69% for pathogen attack frequency and from 83 to 88% for infected leaf surface for the two modes of infestation (artificial and secondary). STE triggered a similar intense decrease in downy mildew infection as recorded for BM. In the vineyard assay, downy mildew infection was allowed to occur naturally. Field grown plants were treated every 7th day for eight weeks or every 14th day for eight weeks during June and July. We observed that STE was also able to prevent P. viticola infection in vineyard but to a lesser extent. Treatment with STE every 7th day provided better results in terms of infection frequency and infected leaf area than that every 14th day. STE treatment of plants every 7th day in vineyard reduced the frequency of attack by 16 to 39% and leaf surface infection by 57 to 61%, which was lower than that recorded for BM. STE also displayed in vitro inhibitory effects on the release of zoospores from sporangia and the germination of zoospores of P. viticola. Zoospore germination was completely inhibited at a concentration of 100 mg L-1 and zoospore release was inhibited at 330 mg L-1. Conclusions: Treatment of young grapevine plants in greenhouse with stilbene extract from grapevine canes offered considerable protection against P. viticola that was identical to that provided by Bordeaux mixture. STE was also able to prevent P. viticola infection in vineyard but to a lesser extent. Treatment with STE every 7th day in vineyard provided better results than that every 14th day. Significance and impact of the study: Stilbenes from grapevine canes are good candidates as natural fungicides against downy mildew. Given the very large quantity of grape canes available each year, this strategy could be scaled up to control this grapevine disease in a sustainable manner.

Aim: The aim of the present investigation was to determine whether methyl jasmonate and ethylene could synergistically induce grapevine foliar cuttings (Vitis vinifera) defense mechanisms and enhance resistance to Erysiphe necator. Methods and results: We cotreated grapevine foliar cuttings (Cabernet- Sauvignon) with ethylene-releasing ethephon in association with methyl jasmonate. However, this cotreatment did not improve resistance to powdery mildew induced by MeJA or ethephon, alone. Quantitative PCR analysis performed on grape cell suspensions showed that the association ethephon/MeJA triggered an enhancement of phytoalexin biosynthesis by synergistically inducing PAL and STS genes. This gene expression was correlated with accumulation of stilbenes (antimicrobial compounds), assessed by HPLC analysis. However, ethephon seemed to inhibit MeJA-dependent induction of PR protein gene expression mainly for the first eighteen hours. Significance and impact of study: Since methyl jasmonate and ethephon can separately enhance grapevine resistance to Erysiphe necator, it was interesting to study the effect of the association of the two molecules on it. Although we observed a synergistic effect on phytoalexin production, no improved resistance against the fungus was obtained. These results can be exploited for the development of new pest control strategies in vineyard.

Two new phenanthrene derivatives, a phenanthrenequinone named arundiquinone (1) and a 9,10-dihydrophenanthrene named arundigramin (2) together with a known lignin dimer (3) and seven known stilbenoids (4–10) were isolated from the aerial parts of the Asian orchid Arundina graminifolia. The structures of the isolated compounds were elucidated by spectroscopic methods, including extensive 1D, 2D NMR (heteronuclear single quantum coherence (HSQC), heteronuclear multiple-bond correlation spectroscopy (HMBC), and HR-ESI-MS techniques, as well as comparison with respective literature reports. The cytoprotective activity of the isolated compounds were evaluated for their ability to reduce beta amyloid induced toxicity on undifferentiated PC12 cells. Compound 8 showed moderate cytoprotective activity at 0.5 µmol/L (71% of cell viability) while the other compounds showed no significant activity at the highest concentration tested.

Aim: Stilbenes are a group of polyphenols that contribute to the defense mechanisms of grapevine under stress conditions. The main active compounds are oligomeric stilbenes. The sourcing of such compounds is limited by plant raw material availability. In order to overcome this limitation, we proceeded to hemisynthesis of oligomeric stilbenes from natural resveratrol by oxidative coupling using metals. The reaction mixture and the obtained pure compounds were tested for their antimicrobial activity in vitro against P. viticola and B. cinerea, and compared with that of resveratrol and grapevine cane extract. Methods and results: Hemisynthesis was achieved in methanol using silver acetate (AgOAc) as oxidative coupling reagent. Four main compounds were obtained in the reaction mixture: δ-viniferin, parthenostilbenin B, oxistilbenins A and B. Results demonstrate the ability of oxidative coupling reaction to produce potent inhibitors of P. viticola and B. cinerea. The reaction mixture is enriched in antimicrobial compounds with high potency as it is at least eight times more active than grapevine cane extract against P. viticola. In addition, this reaction mixture reveals a high fungitoxicity against B. cinerea in contrast to the grapevine extract. Conclusion: The present findings indicate that metals could be used for producing highly antimicrobial compounds from natural resveratrol. Significance and impact of the study: In this study, a method to produce active dimeric stilbenes to protect grapevine was developed based on botanical ingredients. The combination of natural extracts, rich in resveratrol, and metals such as copper may be used as alternatives to Bordeaux mixture and chemical fungicides to protect crops.