Syrah Grape Polyphenol Extracts Protect Human Skeletal Muscle Cells from Oxidative and Metabolic Stress Induced by Excess of Palmitic Acid: Effect of Skin/Seed Ripening Stage.

Metabolic skeletal muscle (SM) dysfunction, triggered by increased oxidative stress and mitochondrial impairment, is a pivotal contributor to obesity-associated insulin resistance (IR). Addressing obesity and SM IR demands substantial lifestyle changes including regular exercise and dietary adjustments that are difficult to follow over time. This prompted exploration of alternative approaches. Grape polyphenols (GPPs) have demonstrated a positive impact on metabolism, although few studies have focused on SM. Since grape polyphenolic content and composition depend on tissue and ripening, we explored the antioxidant potential of GPPs from skin (Sk) and seeds (Sd) extracted before veraison (Bv) and at mature (M) stages, on palmitate-induced IR in primary human SM cells. Despite their important difference in polyphenol (PP) content: Sd-BvPP > Sd-MPP/Sk-BvPP > Sk-MPP, all extracts reduced lipid peroxidation by 44-60%, up-regulated the heme-oxygenase 1 protein level by 75-132% and mitochondrial activity by 47-68%. Contrary to the other extracts, which improved insulin response by 50%, Sd-BvPP did not. Our findings suggest that compounds other than stilbenoids or anthocyanin-type molecules, present only in grape Sk, could play an active role in regulating SM oxidative and metabolic stress and insulin sensitivity, paving the way for further exploration of novel bioactive compounds.

Analysis of targeted phenolic ageing markers in Syrah red wines during bottle ageing: Influence of cork oxygen transfer rate.

A Syrah red wine ageing experiment was set up during 24-months and the influence of four micro-agglomerated corks were investigated. Specific phenolic ageing markers were selected and hemi-synthesized: vitisin B, malvidin-ethyl-catechin, and epicatechin-sulfonate. A targeted quantification method of these markers was then developed and validated by using ultra-high performance liquid chromatography - triple quadrupole mass spectrometry (UHPLC-QqQ-MS) operating in MRM (Multiple Reaction Monitoring). Results showed a significant decline in native grape polyphenol levels (anthocyanins, flavanols) as ageing progresses while pyranoanthocyanins, ethyl-linked pigments, and flavanol-sulfonates content increased. The cork oxygen transfer rate emerged as a pivotal factor and had significant effects on polyphenolic concentration evolution but had no significant impact on flavanol-sulfonate formation. These results provide valuable insights into the chemical evolution ongoing during wine ageing, accentuating the pivotal role of cork stopper selection in preserving wine quality over time.

Impact of phenolic composition and antioxidant parameters on the ageing potential of Syrah red wines measured by accelerated ageing tests.

Fourteen Syrah red wines with different initial composition and antioxidant properties (polyphenols, antioxidant capacity, voltammetric behaviour, colour parameters and SO2) were selected. Three different accelerated ageing tests (AATs) were then performed on these wines: thermal test at 60 °C (60 °C-ATT), enzymatic test with laccase (Laccase-ATT) and chemical test with H2O2 (H2O2-ATT). The results showed high correlations between the initial phenolic composition and antioxidant properties of the samples. Partial least squares (PLS) regressions were used in order to establish some models that can predict the AATs test results based on their different initial composition and antioxidant properties. The PLS regression models had overall very good accuracy and involved different explaining variables for each test. The models taking into account all the measured parameters and the phenolic composition alone showed good predictive capacities with correlation coefficients (r2) > 0.89.

Grape Polyphenols in the Treatment of Human Skeletal Muscle Damage Due to Inflammation and Oxidative Stress during Obesity and Aging: Early Outcomes and Promises.

Today, inactivity and high-calorie diets contribute to the development of obesity and premature aging. In addition, the population of elderly people is growing due to improvements in healthcare management. Obesity and aging are together key risk factors for non-communicable diseases associated with several co-morbidities and increased mortality, with a major impact on skeletal muscle defect and/or poor muscle mass quality. Skeletal muscles contribute to multiple body functions and play a vital role throughout the day, in all our activities. In our society, limiting skeletal muscle deterioration, frailty and dependence is not only a major public health challenge but also a major socio-economic issue. Specific diet supplementation with natural chemical compounds such as grape polyphenols had shown to play a relevant and direct role in regulating metabolic and molecular pathways involved in the prevention and treatment of obesity and aging and their related muscle comorbidities in cell culture and animal studies. However, clinical studies aiming to restore skeletal muscle mass and function with nutritional grape polyphenols supplementation are still very scarce. There is an urgent need for clinical studies to validate the very encouraging results observed in animal models.

Improved Analysis of Isomeric Polyphenol Dimers Using the 4th Dimension of Trapped Ion Mobility Spectrometry-Mass Spectrometry.

Dehydrodicatechins resulting from (epi)catechin oxidation have been investigated in different foods and natural products, but they still offer some analytical challenges. The purpose of this research is to develop a method using ultra-high performance liquid chromatography coupled with trapped ion mobility spectrometry and tandem mass spectrometry (UHPLC-ESI-TIMS-QTOF-MS/MS) to improve the characterization of dehydrodicatechins from model solutions (oxidation dimers of (+)-catechin and/or (-)-epicatechin). Approximately 30 dehydrodicatechins were detected in the model solutions, including dehydrodicatechins B with ß and ε-interflavanic configurations and dehydrodicatechins A with γ-configuration. A total of 11 dehydrodicatechins B, based on (-)-epicatechin, (+)-catechin, or both, were tentatively identified in a grape seed extract. All of them were of ß-configuration, except for one compound that was of ε-configuration. TIMS allowed the mobility separation of chromatographically coeluted isomers including dehydrodicatechins and procyanidins with similar MS/MS fragmentation patterns that would hardly be distinguished by LC-MS/MS alone, which demonstrates the superiority of TIMS added to LC-MS/MS for these kinds of compounds. To the best of our knowledge, this is the first time that ion mobility spectrometry (IMS) was applied to the analysis of dehydrodicatechins. This method can be adapted for other natural products.

Impact of Acetaldehyde Addition on the Sensory Perception of Syrah Red Wines.

Two experimental Syrah red wines with different polyphenol contents were used to study the impact of acetaldehyde addition on olfactory perception. Free acetaldehyde levels were measured in red wine by Head Space-Gas Chromatography-Mass Spectrometry (HS-GC-MS) to determine the acetaldehyde combination levels for those wines. Significant differences were observed for both sensory threshold and acetaldehyde combination for the wines. A descriptive sensory analysis of the wines was then performed by using a trained panel and a Hierarchical-Check-All-That-Apply (HCATA) analysis of the wines with or without acetaldehyde addition. The results show that classical cited sensory descriptors for acetaldehyde (overripe apple and oxidized apple) varied significantly between the control wines and those with acetaldehyde addition. Non-acetaldehyde related descriptors (fresh vegetable, fresh flowers, cocoa, and meat juice) were also significantly impacted in the samples with increasing acetaldehyde additions. This suggests possible interactions between acetaldehyde and other volatile compounds that can create antagonistic or synergistic effects between the molecules or at the olfactory receptor level.

UHPLC-Q-Orbitrap /MS2 identification of (+)-Catechin oxidation reaction dimeric products in red wines and grape seed extracts.

B-type procyanidin dimers and (+)-catechin dimeric oxidation products were analyzed in grape seed extracts and red wines (UHPLC-Q-Orbitrap MS). The different dimers had different fragmentation patterns according to their interflavan linkage position. Oxidation dimeric compounds had a specific fragment ion at m/z 393, missing for B-Type dimers fragmentations. A fragment ion at m/z 291 occurred and was specific for oxidation dimeric compounds with a COC linkage. Higher level oxidation products had abundant specific fragments: m/z 425, 397 and 245. These fragmentations were useful to identify them in complex samples such as grape seed extracts and wines. Three grape varieties and three ripening stages were selected and the corresponding seed extracts were obtained. The analyses revealed an increasing trend for the oxidation markers during grape ripening. The analysis of Syrah wines (2018, 2014, 2010) showed a decreasing trend of these molecules during wine ageing which might be due to further oxidation.

Study of the oxidative evolution of tannins during Syrah red wines ageing by tandem mass spectrometry.

Red wine is a very complex medium in which condensed tannins undergo many modifications during winemaking and bottle ageing. These reactions have an impact on the organoleptic properties. This work aimed to highlight tannins evolution related to wine evolution by studying three vintages of Syrah wines. An accelerated oxidation was also undertaken in order to evaluate the ability of this oxidation to imitate natural evolution. After chemical depolymerization of the tannins, the monitoring of 6 types of markers at two oxidation levels was investigated. An evolution of the tannin oxidation state during ageing evidenced by the increase of the markers of the second oxidation level was observed. In the 2018 oxidized wine sample, the first oxidation level markers were similar to the 2014 vintage but the second oxidation level markers were higher than other vintages, indicating a more advanced state of tannin oxidation.

Red Wine Oxidation Characterization by Accelerated Ageing Tests and Cyclic Voltammetry.

In order to obtain information on the oxidative behavior of red wines, oxygen consumption rates and electrochemical changes (cyclic voltammetry) were measured for nine red wines subject to three different accelerated ageing tests: chemical (with hydrogen peroxide), enzymatic (with laccase from Trametes versicolor), and temperature (at 60 °C). Oxidative behavior depended both on the wine sample and accelerated ageing test type. A good correlation was observed between electrochemical parameters of charges for reference/non-oxidized wines, in accordance with their antioxidant capacity, and the variation of charges after enzymatic and temperature tests, meaning that cyclic voltammetry could be used in order to predict these two oxidation tests and reflect the wine sensitivity towards respective oxidation targets. However, it was not possible to predict wine chemical oxidation test based on hydrogen peroxide from the electrochemical measurements.

Unambiguous NMR Structural Determination of (+)-Catechin-Laccase Dimeric Reaction Products as Potential Markers of Grape and Wine Oxidation.

(+)-Catechin-laccase oxidation dimeric standards were hemi-synthesized using laccase from Trametes versicolor in a water-ethanol solution at pH 3.6. Eight fractions corresponding to eight potential oxidation dimeric products were detected. The fractions profiles were compared with profiles obtained with two other oxidoreductases: polyphenoloxidase extracted from grapes and laccase from Botrytis cinerea. The profiles were very similar, although some minor differences suggested possible dissimilarities in the reactivity of these enzymes. Five fractions were then isolated and analyzed by 1D and 2D NMR spectroscopy. The addition of traces of cadmium nitrate in the samples solubilized in acetone-d6 led to fully resolved NMR signals of phenolic protons, allowing the unambiguous structural determination of six reaction products, one of the fractions containing two enantiomers. These products can further be used as oxidation markers to investigate their presence and evolution in wine during winemaking and wine ageing.

Polyphenolic Characterization of Merlot, Tannat and Syrah Skin Extracts at Different Degrees of Maturity and Anti-Inflammatory Potential in RAW 264.7 Cells.

(1) Background: Both sensory quality and healthy attributes of Vitis vinifera grapes used for winemaking are closely related with the polyphenolic composition of their skins. (2) Methods: In this study, the polyphenolic characterization (flavan-3-ols, procyanidins, flavonols, stilbenes, anthocyanins) was investigated by ultra performance liquid chromatography coupled to a triple quadrupole mass spectrometer (UPLC-QqQ-MS). Skins from Vitis vinifera Merlot, Tannat, and Syrah red grape varieties cultivated in the south of France at different stages of ripening in 2018 were used. The anti-inflammatory and the antioxidant potential of the extracts were evaluated by the measure of nitric oxide (NO) and the intracellular reactive oxygen species production (ROS) in lipopolysaccharide (LPS)-stimulated macrophages. (3) Results: 41 polyphenols were quantified in all samples. Generally, the flavan-3-ol and procyanidin content decreased during ripening whereas the anthocyanins and stilbenes increased. In addition, as a novelty of this work, a wide identification and characterization of monomeric and oligomeric stilbenes was assessed by using authentic standards isolated in our laboratory, some of them (parthenocissin A and miyabenol C) reported for the first time in Merlot, Tannat and Syrah cultivars. The before-veraison skin extracts of all studied varieties, exhibited higher NO and ROS productions inhibition (>50%) proving both antioxidant and anti-inflammatory properties.

Voltammetric Behavior, Flavanol and Anthocyanin Contents, and Antioxidant Capacity of Grape Skins and Seeds during Ripening (Vitis vinifera var. Merlot, Tannat, and Syrah).

Skin and seed grape extracts of three red varieties (Merlot, Tannat, and Syrah) at different stages of ripening were studied for their total phenolic content (TPC) by using the Folin-Ciocalteu assay and for their total antioxidant capacity (TAC) by using spectrophotometric and electrochemical assays. Flavanol and anthocyanin compositions were also investigated using Ultra Performance Liquid Chromatography coupled with Mass Spectrometry (UPLC-MS). Results showed that seeds had the highest phenolic content and the highest antioxidant potential compared to skins at all stages of ripening. The highest TPC and TAC values were measured in seeds at close to veraison and veraison ripening stages. In skins, the highest values were found at the green stage, it was in accordance with the flavanols content. The voltammetric measurements were carried out using disposable single walled carbon nanotubes modified screen-printed carbon electrodes (SWCNT-SPCE). Three peaks on voltammograms were obtained at different oxidation potentials. The first anodic peak that oxidized at a low potential describes the oxidation of ortho-dihydroxy phenols and gallate groups, the second peak corresponds to the malvidin anthocyanins oxidation and the second oxidation of flavonoids. The third voltammetric peak could be due to phenolic acids such as p-coumaric acid and ferulic acid or the second oxidation of malvidin anthocyanins. The high linear correlation was observed between antioxidant tests and flavanols in skins (0.86 ≤ r ≤ 0.94), while in seeds, 'r' was higher between electrochemical parameters and flavanols (0.64 ≤ r ≤ 0.8).

α-Glucosidase Inhibitory Activity of Tannat Grape Phenolic Extracts in Relation to Their Ripening Stages.

The present study aimed to screen grape extracts as novel α-glucosidase inhibitors to prevent type-2 diabetes and hyperglycemia. The total polyphenol content (TPC) was measured by Folin-Ciocalteu assay and the stilbene, anthocyanin and flavan-3-ol compounds were measured by Ultra High-Performance Liquid Chromatography coupled to Mass Spectrometry (UHPLC-MS). The α-glucosidase inhibitory of seed and skin Tannat grape extracts at four ripening stages were investigated. The highest TPC values were measured in seeds at the "veraison stage" (65.29 ± 5.33 g of Gallic Acid Equivalent (GAE) per kilogram of Fresh Weight (FW)). This was in accordance with the high flavan-3-ol contents measured for these two extracts (43.22 ± 2.59 and 45.45 ± 6.48 g/kg of seeds FW, respectively). The skin and seed extracts at the first stage of ripening exerted strong α-glucosidase inhibition, exceeding 95% (p < 0.05). A high linear correlation (R = 0.723, p ≤ 0.05) was observed between flavan-3-ol contents and the α-glucosidase inhibitory activity. The stilbene contents and this activity were moderately to strongly anti-correlated (R = -0.828, p ≤ 0.05 for trans-resveratrol). The enzyme kinetic studies revealed a mixed type of inhibition. This study brings promising results for the therapeutic potential of seed and skin Tannat grape extracts as a functional food product with anti-diabetic activity.

Red Wine Oxidation: Accelerated Ageing Tests, Possible Reaction Mechanisms and Application to Syrah Red Wines.

Wine oxidation and ageing involve many complex chemical pathways and reaction mechanisms. The purpose of this study is to set up new and reproducible accelerated red wine ageing tests and identify chemical oxidation or ageing molecular markers. Three accelerated and reproducible ageing tests were developed: a heat test (60 °C); an enzymatic test (laccase test; a chemical test (hydrogen peroxide test). Depending on the test, oxygen consumption was significantly different. For a young wine (2018), the oxygen consumption rate moved from 2.40 ppm.h-1 for the heat test to 3.33 ppm.h-1 for the enzymatic test and 2.86 ppm.h-1 for the chemical test. Once applied to two other vintages (2010 and 2014) from the same winery, the tests revealed different comportments corresponding to wine natural evolution. High resolution UPLC-MS was performed on forced ageing samples and compared to naturally aged red wines. Specific oxidation or ageing ion markers were found with significant differences between tests, revealing the specificity of each test and different possible molecular pathways involved. The hydrogen peroxide test seems to be closer to natural oxidation with an important decrease in absorbance at 520 nm and similar molecular ion variations for [M+H]+ = 291, 331, 347, 493, 535, 581, 639 Da.

Discrimination of rosé wines using shotgun metabolomics with a genetic algorithm and MS ion intensity ratios.

A rapid Ultra Performance Liquid Chromatography coupled with Quadrupole/Time Of Flight Mass Spectrometry (UPLC-QTOF-MS) method was designed to quickly acquire high-resolution mass spectra metabolomics fingerprints for rosé wines. An original statistical analysis involving ion ratios, discriminant analysis, and genetic algorithm (GA) was then applied to study the discrimination of rosé wines according to their origins. After noise reduction and ion peak alignments on the mass spectra, about 14 000 different signals were detected. The use of an in-house mass spectrometry database allowed us to assign 72 molecules. Then, a genetic algorithm was applied on two series of samples (learning and validation sets), each composed of 30 commercial wines from three different wine producing regions of France. Excellent results were obtained with only four diagnostic peaks and two ion ratios. This new approach could be applied to other aspects of wine production but also to other metabolomics studies.

Flavanol Glycoside Content of Grape Seeds and Skins of Vitis vinifera Varieties Grown in Castilla-La Mancha, Spain.

Glycosylated flavanols (monoglycosides and diglycosides) in skin and seed extracts of Vitis vinifera grapes grown in Castilla-La Mancha (Spain) were investigated using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-ESI-QQQ-MS/MS). Six grape varieties (Airén, Tempranillo, the recently identified Albillo Dorado, Montonera del Casar, Moribel, and Tinto Fragoso) were studied over two consecutive years (2016 and 2017). A total of twenty monomeric flavanol monoglycosides, four diglycosylated monomers, and three dimeric flavanol monoglycosides were detected in all grape samples. The diversity observed in the composition of glycosylated flavanol in the grape berries suggests a strong influence of variety and grape tissue (skin or seed). Monomeric flavanol glycosides were more abundant in grape seed extracts, in contrast with monoglycosylated dimeric forms. In addition, the glycosylated flavanol content was related to berry color in grape skins, with higher concentrations measured in black grape varieties.

Effect of polyvinylpolypyrrolidone treatment on rosés wines during fermentation: Impact on color, polyphenols and thiol aromas.

Fining treatment with polyvinylpolypyrrolidone (PVPP) is often used during winemaking of rosé wines. It can modulate the intensity and hue of their pink color and prevent some organoleptic degradations. In this paper, the effect of PVPP treatments on rosé wine during fermentation was investigated by measuring color, polyphenol content and thiol aromas. As expected, colorimetry results showed a decrease in color, indicating some adsorption of anthocyanins and other pigments. This was confirmed by UPLC-ESI-MS/MS analyses. Specific adsorption of certain families of polyphenols was evidenced. Flavonols, flavanols and anthocyanins, especially coumaroylated anthocyanins were preferentially adsorbed by PVPP. The thiol content (3-sulfanylhexyl acetate (3SHA) and 3-sulfanylhexan-1-ol (3SH)) was usually higher after PVPP treatments, in a dose dependent manner. A possible explanation is that the partial adsorption of some polyphenols at an early stage of fermentation would later limit the amount of quinone compounds able to trap thiol aromas.

Evolution of Flavanol Glycosides during Red Grape Fermentation.

Monomeric and dimeric flavanol glycosides were quantified by UHPLC-MRM in Syrah (SYR) and Grenache (GRE) grapes and in their corresponding wines for the first time. Quantities were extremely variable depending on grape tissue (seeds or skins) and during fermentation. Overall, 22 monomeric and dimeric mono- and diglycosides were determined with concentrations ranging from 0.7 nanograms to 0.700 micrograms per gram of grape tissue, and 0 to 60 micrograms per liter for wines. The evolution of the glycosides' composition during winemaking suggests that almost all these compounds originate in the grapes themselves and display different extraction kinetics during winemaking. One isomer of the monomeric (epi) flavanol monoglycosides seemed to be biosynthesized by yeasts during wine fermentation. The sharp decrease in concentration of some isomers at the late stages of fermentation or after pressing suggests that some grape glycosidase activities convert these compounds into non-glycosylated flavanols.

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.

Identification and Quantification of Flavanol Glycosides in Vitis vinifera Grape Seeds and Skins during Ripening.

Monomeric and dimeric flavanol glycosides were analyzed in Vitis vinifera grapes and seeds during ripening. An analytical method using ultra-high performance liquid chromatography coupled with a triple quadrupole mass spectrometry (UHPLC-ESI-QQQ-MS/MS) in multiple reaction monitoring (MRM) mode was employed. Three grape varieties (Merlot, Syrah and Tannat) were chosen and grape berries were sampled at different stages of development. Ten monoglycosylated and six diglycosylated flavanol monomers were detected. Twelve monoglycosylated and three diglycosylated flavanol dimers were also detected for all three grape varieties. All diglycosides were detected for the first time in Vitis vinifera grapes, though some of these compounds were only detected in skins or seeds. Furthermore, the evolution of all these compounds was studied, and a decrease in monomeric (epi) catechin monoglycosides was observed during ripening for Tannat, Merlot and Syrah grape skins. The dimers would appear to accumulate in skin tissues up to mid-summer (after veraison) and decrease when grape berries reached maturity.