New flavanol O-glycosides in grape and wine.

The presence of monomeric and dimeric flavan-3-ol monohexosides was investigated in grapes and wines. Polyphenol extracts were prepared from grape seeds and skins (Syrah, Merlot, and Cabernet-Sauvignon) sampled at three different developmental stages. Different wines (Tannat, Alicante, Syrah, Merlot, and Grenache) were also studied. The different samples obtained were analyzed by UPLC-ESI-IT-MS. Specific molecular ions corresponding to flavan-3-ol hexosides were detected by using targeted molecular ions with specific m/z values: 451 for (epi)catechin hexosides, and 739 for procyanidin dimer hexosides. 4'-O-ß-glucosyl-(+)-catechin and 7-O-ß-glucosyl-(+)-catechin were obtained by using a glucosyl transferase from apple, UGT71A15, and their structures determined by NMR. These glucosylated monomers and the dimers were identified in all analyzed grape seed and several skin extracts at the different stages and in wines made from different varieties.

p-Hydroxyphenyl-pyranoanthocyanins: An Experimental and Theoretical Investigation of Their Acid-Base Properties and Molecular Interactions.

The physicochemical properties of the wine pigments catechyl-pyranomalvidin-3-O-glucoside (PA1) and guaiacyl-pyranomalvidin-3-O-glucoside (PA2) are extensively revisited using ultraviolet (UV)-visible spectroscopy, dynamic light scattering (DLS) and quantum chemistry density functional theory (DFT) calculations. In mildly acidic aqueous solution, each cationic pigment undergoes regioselective deprotonation to form a single neutral quinonoid base and water addition appears negligible. Above pH = 4, both PA1 and PA2 become prone to aggregation, which is manifested by the slow build-up of broad absorption bands at longer wavelengths (λ ≥ 600 nm), followed in the case of PA2 by precipitation. Some phenolic copigments are able to inhibit aggregation of pyranoanthocyanins (PAs), although at large copigment/PA molar ratios. Thus, chlorogenic acid can dissociate PA1 aggregates while catechin is inactive. With PA2, both chlorogenic acid and catechin are able to prevent precipitation but not self-association. Calculations confirmed that the noncovalent dimerization of PAs is stronger with the neutral base than with the cation and also stronger than π-π stacking of PAs to chlorogenic acid (copigmentation). For each type of complex, the most stable conformation could be obtained. Finally, PA1 can also bind hard metal ions such as Al3+ and Fe3+ and the corresponding chelates are less prone to self-association.

Synthesis, Identification, and Structure Elucidation of Adducts Formed by Reactions of Hydroxycinnamic Acids with Glutathione or Cysteinylglycine.

Grape polyphenols, especially hydroxycinnamic acids such as caftaric and caffeic acid, are prone to enzymatic oxidation reactions during the winemaking process, forming o-quinones and leading to color darkening. Glutathione is capable of trapping these o-quinones and thus limiting juice browning. In this study, the addition of glutathione or cysteinylglycine onto caftaric or caffeic acid o-quinones formed by polyphenoloxidase-catalyzed reactions was investigated by UPLC-DAD-ESIMS and NMR data analyses. Complete identification of adducts has been achieved via NMR data. The results confirmed that the favored reaction is the substitution of the sulfanyl group of cysteine at C-2 of the aromatic ring. Several minor isomers, namely, the cis-isomer of the 2-S adduct and trans-isomers of the 5-S and 6-S adducts, and the 2,5-di-S-glutathionyl adducts were also identified and quantified by qNMR. With the exception of 2-(S-glutathionyl)- and 2,5-di(S-glutathionyl)-trans-caftaric acid, these products had never been formally identified. In particular, the 5-S and 6-S derivatives are reported here for the first time. The first formal identification of 2-S cis-derivatives is also provided. Moreover, NMR and UPLC-DAD-ESIMS analysis showed that signature UV and MS spectra can serve as markers of the conformation and substitution position in the aromatic ring for each of the isomers.

How to gain insight into the polydispersity of tannins: a combined MS and LC study.

In the context of the potential health benefits of food polyphenols, the bioavailability of tannins (i.e. proanthocyanidins) is a major issue, which is strongly influenced by the polydispersity and the degree of polymerisation of tannins. The average degree of polymerisation (DP) of tannins is usually determined using depolymerisation methods, which do not provide any information about their polymer distribution. Moreover, it is still a challenge to characterise tannin fractions of high polydispersity and/or containing polymers of high molecular weights, due to the limit of detection of direct mass spectrometry (MS) analysis methods. In the present work, the polydispersity of several tannin fractions is investigated by two complementary methods: a MALDI-MS method and a semi-preparative sub-fractionation. Using a combination of these methods we are able to gain insight into the DP distributions of the fractions consisting of tannins of medium and high DP. Moreover combining analyses can be useful to assess and compare the DP distributions of most tannin fractions.

Qualitative and semi-quantitative analysis of phenolics in Eucalyptus globulus leaves by high-performance liquid chromatography coupled with diode array detection and electrospray ionisation mass spectrometry.

INTRODUCTION: Eucalyptus species are widely cultivated in Mediterranean regions. Moreover, plants of this family have been utilized for medicinal purposes. A number of studies have been devoted to the identification of eucalypt phenolics, all of them have focused on specific families of compounds, and no exhaustive profiling has been reported in leaves of this plant. OBJECTIVE: To develop methods that allows the identification and quantification of different classes of phenolics in Eucalyptus globulus leaf. METHODOLOGY: Acetonic extract was fractionated by chromatography on a Sephadex LH-20 column using consecutive elution with ethanol, methanol and aqueous acetone (60%). High-performance liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI/MS) were applied to determine the structure of different compounds. Quantities were evaluated from peak areas in the HPLC profile, using external calibration curves. RESULTS: Fractionation of acetonic extract yielded three fractions: F1, F2 and F3. In total 39 phenolic compounds are detected. Among them: 16 hydrolyzable tannins, 3 terpenyl derivatives, 12 ellagic acid derivatives, 5 flavonols, 2 hydroxybenzoic acids and 1 formylated phloroglucinol. 26 compounds described in this study have not previously detected in leaves of this plant and this is the first report of quercetin 3-O-ß-galactoside-6"-O-gallate and cypellogin A and B, in E. globulus plant. Quantitatively, ellagic acid derivatives and sideroxylonal A or B are largely predominant. CONCLUSION: Fractionation of crude extract by chromatography on Sephadex LH-20 was efficient to separate different molecular weight compounds. HPLC-DAD-ESI/MS enabled detection of gallotannin, ellagitannin and flavonol derivatives, in leaves of E. globulus.

Comprehensive study of condensed tannins by ESI mass spectrometry: average degree of polymerisation and polymer distribution determination from mass spectra.

The determination of the molecular mass distribution of tannins is still a challenge. To elucidate it, mass spectrometry is potentially interesting, but many previous studies have highlighted that the mass spectra of a tannin fraction do not always reflect the actual abundance of different chain lengths. To clarify the potentialities offered by the MS approach, a comprehensive study involving different tannin fractions analysed under different conditions was conducted with an electrospray ionization (ESI) source. This study allowed optimised ESI-MS conditions to be established for analysing tannins but also it outlines the limits of detection encountered. If the detection of high molecular weight tannins seems difficult or even impossible, the spectral distortions brought about by this limitation are not totally related to the sole average degree of polymerisation of the tannin fraction studied but greatly depend on its polymer distribution. However, ESI-MS used under optimised conditions is a suitable method to study tannin composition of vegetable extracts which contain degree of polymerisations below 26.

Analysis by high-performance liquid chromatography diode array detection mass spectrometry of phenolic compounds in fruit of Eucalyptus globulus cultivated in Algeria.

A method based on high-performance liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS) following fractionation by chromatography on a Sephadex LH-20 column has been developed to determine the phenolic composition of fruit of Eucalyptus globulus growing in Algeria. The presence of 18 gallotannins, 26 ellagitannins, and 2 flavonols was established. Tentative identification is provided for these compounds on the basis of UV-visible spectra and mass spectrometry data. Most compounds described in this study have not previously detected in fruit of E. globulus. Moreover, this is the first report of methyl digalloyl diglucose, 3,3'-O-dimethylellagic acid 4-O-ß-glucopyranoside, ellagic acid hexose, methyl ellagic acid pentose, methyltetragalloylglucose, and valoneic acid isomers (sanguisorbic, flavogallic acid dilactone) in the genus Eucalyptus. Quantitatively, ellagic acid and its derivatives, including ellagitannins, are largely predominant.

Grapevine MATE-type proteins act as vacuolar H+-dependent acylated anthocyanin transporters.

In grapevine (Vitis vinifera), anthocyanins are responsible for most of the red, blue, and purple pigmentation found in the skin of berries. In cells, anthocyanins are synthesized in the cytoplasm and accumulated into the vacuole. However, little is known about the transport of these compounds through the tonoplast. Recently, the sequencing of the grapevine genome allowed us to identify genes encoding proteins with high sequence similarity to the Multidrug And Toxic Extrusion (MATE) family. Among them, we selected two genes as anthocyanin transporter candidates and named them anthoMATE1 (AM1) and AM3. The expression of both genes was mainly fruit specific and concomitant with the accumulation of anthocyanin pigment. Subcellular localization assays in grapevine hairy roots stably transformed with AM1 or AM3green fluorescent protein fusion protein revealed that AM1 and AM3 are primarily localized to the tonoplast. Yeast vesicles expressing anthoMATEs transported acylated anthocyanins in the presence of MgATP. Inhibitor studies demonstrated that AM1 and AM3 proteins act in vitro as vacuolar H(+)-dependent acylated anthocyanin transporters. By contrast, under our experimental conditions, anthoMATEs could not transport malvidin 3-O-glucoside or cyanidin 3-O-glucoside, suggesting that the acyl conjugation was essential for the uptake. Taken together, these results provide evidence that in vitro the two grapevine AM1 and AM3 proteins mediate specifically acylated anthocyanin transport.

Direct mass spectrometry approaches to characterize polyphenol composition of complex samples.

Lower molecular weight polyphenols including proanthocyanidin oligomers can be analyzed after HPLC separation on either reversed-phase or normal phase columns. However, these techniques are time consuming and can have poor resolution as polymer chain length and structural diversity increase. The detection of higher molecular weight compounds, as well as the determination of molecular weight distributions, remain major challenges in polyphenol analysis. Approaches based on direct mass spectrometry (MS) analysis that are proposed to help overcome these problems are reviewed. Thus, direct flow injection electrospray ionization mass spectrometry analysis can be used to establish polyphenol fingerprints of complex extracts such as in wine. This technique enabled discrimination of samples on the basis of their phenolic (i.e. anthocyanin, phenolic acid and flavan-3-ol) compositions, but larger oligomers and polymers were poorly detectable. Detection of higher molecular weight proanthocyanidins was also restricted with matrix-assisted laser desorption ionization (MALDI) MS, suggesting that they are difficult to desorb as gas-phase ions. The mass distribution of polymeric fractions could, however, be determined by analyzing the mass distributions of bovine serum albumin/proanthocyanidin complexes using MALDI-TOF-MS.

Interactions between yeast lees and wine polyphenols during simulation of wine aging. II. Analysis of desorbed polyphenol compounds from yeast lees.

In the first part of this work, the analysis of the polyphenolic compounds remaining in the wine after different contact times with yeast lees during simulation of red wine aging was undertaken. To achieve a more precise view of the wine polyphenols adsorbed on lees during red wine aging and to establish a clear balance between adsorbed and remnant polyphenol compounds, the specific analysis of the chemical composition of the adsorbed polyphenolic compounds (condensed tannins and anthocyanins) after their partial desorbtion from yeast lees by denaturation treatments was realized in the second part of the study. The total recovery of polyphenol compounds from yeast lees was not complete, since a rather important part of the initial wine colored polyphenols, especially those with a dominant blue color component, remained strongly adsorbed on yeast lees, as monitored by color tristimulus and reflectance spectra measurements. All anthocyanins were recovered at a rather high percentage (about 62%), and it was demonstrated that they were not adsorbed in relation with their sole polarity. Very few monomeric phenolic compounds were extracted from yeast lees. With the use of drastic denaturing treatments, the total recovery of condensed tannins reached 83%. Such tannins extracted from yeast lees exhibited very high polymeric size and a rather high percentage of galloylated residues by comparison with initial wine tannins, indicating that nonpolar tannins were preferentially desorbed from yeast lees by the extraction treatments.

Interactions between yeast lees and wine polyphenols during simulation of wine aging: I. Analysis of remnant polyphenolic compounds in the resulting wines.

Wine aging on yeast lees is a traditional enological practice used during the manufacture of wines. This technique has increased in popularity in recent years for the aging of red wines. Although wine polyphenols interact with yeast lees to a limited extent, such interactions have a large effect on the reactivity toward oxygen of wine polyphenolic compounds and yeast lees. Various domains of the yeast cell wall are protected by wine polyphenols from the action of extracellular hydrolytic enzymatic activities. Polysaccharides released during autolysis are thought to exert a significant effect on the sensory qualities of wine. We studied the chemical composition of polyphenolic compounds remaining in solution or adsorbed on yeast lees after various contact times during the simulation of wine aging. The analysis of the remnant polyphenols in the wine indicated that wine polyphenols adsorption on yeast lees follows biphasic kinetics. An initial and rapid fixation is followed by a slow, constant, and saturating fixation that reaches its maximum after about 1 week. Only very few monomeric phenolic compounds remained adsorbed on yeast lees, and no preferential adsorption of low or high polymeric size tannins occurred. The remnant condensed tannins in the wine contained fewer epigallocatechin units than the initial tannins, indicating that polar condensed tannins were preferentially adsorbed on yeast lees. Conversely, the efficiency of anthocyanin adsorption on yeast lees was unrelated to its polarity.