Joseph Vercauteren

STRUCTURAL DETERMINATION OF COLOURLESS AND YELLOW DIMERS RESULTING FROM (+)-CATECHIN COUPLING CATALYSED BY GRAPE POLYPHENOLOXIDASE

Abstract (+)-Catechin oxidation was carried out in aqueous systems using grape polyphenoloxidase as catalyst. Eight fractions corresponding to the major products formed at pH 3 and 6 were purified using HPLC at the preparative and semi-preparative scale. Structural characterization using UV-visible detection and mass spectrometry indicated that they corresponded to (+)-catechin dimers including two yellow pigments. Mono- and bi-dimensional 1H and 13C NMR analyses provided structural hypotheses for five oxidation products whereas the other three fractions were mixtures of several isomers. Colourless products, with C-C or C-O interflavan linkages, were dehydrodicatechins of the B-type. One of the two pigments corresponds to dehydrodicatechin A, already identified in other oxidation models, and the other pigment is a new structure of the quinone-methide type.

Direct liquid chromatographic analysis of resveratrol derivatives and flavanonols in wines with absorbance and fluorescence detection

Abstract Wine contains a large number of polyphenols including stilbenes, flavanols and anthocyanins. Of these, stilbenes have been reported to have potential chemopreventive activities. We describe the simultaneous determination of six resveratrol derivatives in wines by liquid chromatography (LC) with fluorescence detection. The levels of pallidol, the symmetrical dimer of resveratrol, are reported for the first time. Quantifications were carried out at optimal wavelengths for each compound during separation. A total of 19 red and 30 white commercial wines from South-Western France were analysed, and the highest stilbene concentrations were found in red wines. In addition, the levels of catechins and two flavanonols recently isolated in red wine are reported.

Lepadiformine, a new marine cytotoxic alkaloid from Clavelina lepadiformis Müller

Abstract A cytotoxic alkaloid, lepadiformine 1, with a new heterocyclic skeleton, has been isolated from the ascidian Clavelina lepadiformis. The structure of 1 was established on the basis of chemical properties and by spectroscopic means : it includes a unique zwitterionic-like moiety.

Preparative isolation of polyphenolic compounds from Vitis vinifera by centrifugal partition chromatography

This study deals with a centrifugal partition chromatography developed for the separation of phenolic compounds from Vitis vinifera. EtOAc grape seed extracts were separated using the solvent system hexane-ethyl acetate-ethanol-water (1:8:2:7; v/v) in two fractions: one containing about 75% of flavanol monomers (catechin and epicatechin) corresponding to 18% of crude extract and another fraction B-type dimers (22% of crude extract). From the stalk extracts, we could separate stilbenoid compounds (resveratrol and its oligomers; 12% of crude extract) which were eluted in less than 30 min from flavanols (which required a few hours of additional elution). Using the same solvent system but in different ratios (4:5:3:3; v/v), we isolated the trans-resveratrol (7@1000; 90% purity).

Bisulfite addition to anthocyanins: revisited structures of colourless adducts

Abstract Decolourization of anthocyanins by sulfur dioxide is investigated through the reaction between sodium bisulfite and malvidin 3- O -glucoside. The structural elucidation of the C-4 sulfonate adducts is established by 1 H, 13 C and 33 S NMR spectroscopies.

Vasorelaxant effects of grape polyphenols in rat isolated aorta. Possible involvement of a purinergic pathway

The purpose of this study was to investigate the mechanism of the vascular relaxation produced by polyphenolic substances from red wine, with a particular focus on the possible involvement of purinoceptors. With this aim, relaxing responses induced by procyanidin from grape seeds (GSP), anthocyanins, catechin and epicatechin were assessed in rat isolated aortic rings left intact (+E) or endothelium‐denuded (−E). In preparations precontracted with noradrenaline, incubation with NG‐nitro‐l‐arginine methyl ester (100 μm, 30 min) fully inhibited the GSP‐induced relaxations. Concentration–effect curves to these substances (from 10−7 to 10−1 g/L) were determined in depolarized (60 mm KCl) preparations in control condition, after incubation with reactive blue 2 (an antagonist of P2Y purinoceptors, 30 μm), with apyrase (an enzyme which hydrolyses ATP and ADP, 0.8 U/mL) or with α,β‐methylene ATP (an inhibitor of ecto ATPases, 10 μm). In (+E) rings, relaxations (expressed as percentage of initial contraction) were 41 ± 2 and 37 ± 3 for GSP and anthocyanins, respectively. Only modest relaxations (ca. 10%) were observed in (−E) rings, as it was the case for catechin and epicatechin in (±E) rings. Reactive blue 2 or apyrase inhibited the GSP‐ and anthocyanin‐induced relaxations in (+E) rings, while α,β‐methylene ATP shifted to the left the relaxation curves obtained with GSP. These data confirm that modest relaxations observed with catechin and epicatechin are not endothelium‐dependent but that GSP and anthocyanins induce a relaxing effect, which is related to the integrity of the endothelium and the synthesis and release of nitric oxide (NO). Furthermore, the inhibition by apyrase and the increase by ecto‐ATPase inhibition of the GSP‐ and anthocyanin‐induced relaxation suggest that these substances could act via an initial release of nucleotides, which in turn could activate P2Y1 and/or P2Y2 purinoceptors of endothelial cells, trigger the synthesis and release of NO and then lead to relaxation.

Trans-resveratrol-3-O-β-glucoside (piceid) in cell suspension cultures of vitis vinifera

Suspension cultures of Vitis vinifera were found to produce catechins and stilbenes. When the cells were grown in a polyphenol-inducing medium, a stilbene glucoside (piceid) was isolated. By using spectroscopic methods, the structure was determined as trans-resveratrol-3-O-β-glucoside.

Production of 13C-labelled anthocyanins by Vitis vinifera cell suspension cultures

The use of plant cell cultures for producing isotopically (13C) labelled phenolic substances is reported. Vitis vinifera cells synthesize high levels of anthocyanins when they are cultured in a polyphenol synthesis-inducing medium. Three major anthocyanin monoglucosides found in red wine were identified in grape cells: cyanidin-3-O-beta-glucoside, peonidin-3-O-beta-glucoside, and malvidin-3-O-beta-glucoside. Kinetic study of the intracellular level of phenylalanine and its metabolites showed that it is preferable to add this precursor to grape cell suspensions after the 5th day of culture, i.e. at the beginning of the exponential growth phase. After adding phenylalanine to the culture medium, its uptake was complete and the accumulation of anthocyanins in grape cells was stimulated. Incorporation of [1-13C]-phenylalanine into anthocyanins was measured by means of 13C satellites in the proton NMR spectrum. The maximal rate of 13C enrichment anthocyanins obtained with this technique reached 65%. The production of 13C labelled phenolic compounds was undertaken in order to investigate their absorption and metabolism in humans.

DNA triplex stabilization property of natural anthocyanins.

The DNA triplex stabilization property of seven natural anthocyanins (five monoglucosides and two diglucosides) has been measured by the mean of triplex thermal denaturation experiments. We have noticed a difference between the diglucosides that do not modify this melting temperature and the monoglucosides (namely 3-O-beta-D-glucopyranoside of malvidin, peonidin, delphinidin, petunidin and cyanidin) which present a weak but significant stabilizing effect. It appears clearly that the difference between the two series could be due to the supplementary sugar moiety at the 5 position for the diglucosylated compounds, that would make them too crowded to allow interaction with the triplex. Among the monoglucoside series, the most active compounds are the only ones to embody a catechol B-ring in their structure that could be important for such an interaction. The need to have pure and fully characterized compounds to run these measurements, made it possible for us to unambiguously assign the 1H and 13C NMR spectra with the help of 2D NMR experiments. Thus, missing data of compounds not totally described earlier, are provided herein.

Multivariate statistical analysis of two-dimensional NMR data to differentiate grapevine cultivars and clones

Multivariate statistical methods were applied on two-dimensional NMR data (‘H-r3C) of polyphenol extracts from grapevine clones harvested in the Bordeaux region. An analysis of variance detected the most discriminating NMR spectral correlation volumes which were then used to perform principal component, hierarchical clustering and discriminant analyses. The results showed that clones are divided into three groups according to the cultivar and they can be differentiated inside each cultivar. This method was applied to polyphenol extracts from grape seeds and leaves. Nowadays, two-dimensional NMR is the only analytical tool which can differentiate grapevine clones. It could thus be used to check the identity of grapevine species, cultivars or clones as well as any other plants which produce polyphenols. Copyright 0 1996 Elsevier Science Ltd