Characterization of Crocetin Isomers in Serum Samples via UHPLC-DAD-MS/MS and NMR after Saffron Extract (Safr'Inside™) Consumption.

The therapeutic effects of saffron have been reported and described in relation to its major derivatives. Among them, in terms of saffron's properties, crocin and crocetin absorption and bioavailability have been the most studied. Nevertheless, the metabolism of these major compounds of saffron has not yet been entirely elucidated. Current data indicate that the phase 2 metabolism of crocetins go through conjugation reactions. Crocetins could also be present in isomeric forms such as other carotenoids. Nonetheless, there are still shadow areas in regard to the measurements of the different circulating forms of crocetins after oral saffron extract administration (Safr'Inside™). In using various approaches, we propose the identification of a new cis isomeric form of crocetin, the 6-cis-crocetin. This compound was found in human serum samples after an oral administration of saffron extract. The 6-cis-crocetin represents 19% of the total crocetin measured after 45 min of consumption. These data mark, for the first time, the presence of a cis isomeric form of crocetin in human serum samples. Moreover, this study led to the development of an analytical method that is able to identify and quantify both isomeric forms (trans and cis).

Reduced Production of Pro-Inflammatory and Pro-Catabolic Factors by Human Serum Metabolites Derived from a Patented Saffron Extract Intake.

Safe and anti-inflammatory plant-based natural products present an increasing focus in the treatment of chronic inflammatory diseases such as osteoarthritis or inflammatory bowel diseases. Among them, saffron, a spice derived from the stigma of Crocus sativus, could have anti-inflammatory properties and would be therefore a promising therapeutic agent for the treatment of such conditions. However, the anti-inflammatory molecular mechanisms of saffron in humans are still understudied and unclear. In this study, combining human serum metabolites and cell cultures, we evaluated the effect of circulating metabolites from the consumption of a patented saffron extract (Safr'InsideTM) on the chondrocytes and colon epithelial cell responses to inflammatory stress. Parametric or non-parametric Analysis of Variance with post hoc tests was performed. We demonstrated that human serum containing metabolites from saffron intake attenuated IL-1ß-stimulated production of PGE2 and MMP-13 in chondrocyte cells and limited the increase in ICAM-1, MCP-1, iNOS, and MMP-3 in human epithelial cells following combined IL-1ß and TNF-α inflammatory stimulation. Altogether, these data provide new findings into the mechanisms underlying the beneficial effects of saffron on chondrocytes and enterocyte cells at the cellular level and in the context of chronic inflammatory disorders.

Targeted 1-H-NMR wine analyses revealed specific metabolomic signatures of yeast populations belonging to the Saccharomyces genus.

This study aimed to explore the non-volatile metabolomic variability of a large panel of strains (44) belonging to the Saccharomyces cerevisiae and Saccharomyces uvarum species in the context of the wine alcoholic fermentation. For the S. cerevisiae strains flor, fruit and wine strains isolated from different anthropic niches were compared. This phenotypic survey was achieved with a special focus on acidity management by using natural grape juices showing opposite level of acidity. A 1H NMR based metabolomics approach was developed for quantifying fifteen wine metabolites that showed important quantitative variability within the strains. Thanks to the robustness of the assay and the low amount of sample required, this tool is relevant for the analysis of the metabolomic profile of numerous wines. The S. cerevisiae and S. uvarum species displayed significant differences for malic, succinic, and pyruvic acids, as well as for glycerol and 2,3-butanediol production. As expected, S. uvarum showed weaker fermentation fitness but interesting acidifying properties. The three groups of S. cerevisiae strains showed different metabolic profiles mostly related to their production and consumption of organic acids. More specifically, flor yeast consumed more malic acid and produced more acetic acid than the other S. cerevisiae strains which was never reported before. These features might be linked to the ability of flor yeasts to shift their metabolism during wine oxidation.

Endo metabolomic profiling of flor and wine yeasts reveals a positive correlation between intracellular metabolite load and the specific glycolytic flux during wine fermentation.

This study explored the intracellular metabolic variations between 17 strains of Saccharomyces cerevisiae belonging to two different genetic populations: flor and wine yeasts, in the context of alcoholic fermentation. These two populations are closely related as they share the same ecological niche but display distinct genetic characteristics. A protocol was developed for intracellular metabolites extraction and 1H-NMR analysis. This methodology allowed us to identify and quantify 21 intracellular metabolites at two different fermentation steps: the exponential and stationary phases. This work provided evidence of significant differences in the abundance of intracellular metabolites, which are strain- and time-dependent, thus revealing complex interactions. Moreover, the differences in abundance appeared to be correlated with life-history traits such as average cell size and specific glycolytic flux, which revealed unsuspected phenotypic correlations between metabolite load and fermentation activity.

Resveratrol, ε-Viniferin, and Vitisin B from Vine: Comparison of Their In Vitro Antioxidant Activities and Study of Their Interactions.

The control of oxidative stress with natural active substances could limit the development of numerous pathologies. Our objective was to study the antiradical effects of resveratrol (RSV), ε-viniferin (VNF), and vitisin B (VB) alone or in combination, and those of a standardized stilbene-enriched vine extract (SSVE). In the DPPH-, FRAP-, and NO-scavenging assays, RSV presented the highest activity with an IC50 of 81.92 ± 9.17, 13.36 ± 0.91, and 200.68 ± 15.40 µM, respectively. All binary combinations resulted in additive interactions in the DPPH- and NO-scavenging assays. In the FRAP assay, a synergic interaction for RSV + VNF, an additive for VNF + VB, and an antagonistic for RSV + VB were observed. The ternary combination of RSV + VNF + VB elicited an additive interaction in the DPPH assay and a synergic interaction in the FRAP- and NO-scavenging assays. There was no significant difference between the antioxidant activity of the SSVE and that of the combination of RSV + VNF. In conclusion, RSV presented the highest effects, followed by VNF and VB. The interactions revealed additive or synergistic effects, depending on the combination of the stilbenes and assay.

A novel flavonol glycoside and six derivatives of quercetin and kaempferol from Clematis flammula with antioxidant and anticancer potentials.

Clematis flammula leaves are traditionally used in Algeria to treat rheumatoid arthritis. Our aim was to identify the main compounds in this plant in order to characterize its antioxidant and anticancer activities. A new flavonol compound, kaempferol 3-O-[(6-O- caffeoyl)- glucosyl(1 â†’ 2)]-(6-Ocaffeoyl) glucoside-7-O-rhamnoside (6) along with six known flavonol molecules were isolated from an ethanolic extract of Clematis flammula leaves. The chemical structures of these flavonols were elucidated using NMR and high resolution-MS spectroscopies. Antioxidant activities of the extract were revealed through its elimination of superoxide radical (O2.-) produced enzymatically (49.7 ± 1.52% at 50 µg/ml) and non-enzymatically (34 ± 1.2% at 100 µg/ml), probably related to its inhibition of the xanthine oxidase form of the xanthine oxidoreductase (XOR) enzyme (25.05 ± 2.33 µg/mL at 100 µg/mL), but mostly to that of the NADH oxidase form of the enzyme (69.16 ± 4.0%). Cytotoxicity tests of the extract on human hepatoma cell line HepG2 and ovarian cancer cell lines A2780 and OVCAR3 were promising especially regarding A2780 cell line (IC50: 77.0 µg/mL), which was comparable to taxol (IC50:76.9 µg/mL).

Red Wine Extract Prevents Oxidative Stress and Inflammation in ARPE-19 Retinal Cells.

Age-related macular degeneration (AMD) is one of the most commonly occurring ocular diseases worldwide. This degenerative condition affects the retina and leads to the loss of central vision. The current treatments are focused on the late stage of the disease, but recent studies have highlighted the importance and benefits of preventive treatments and how good dietary habits can reduce the risk of progression to an advanced form of the disease. In this context, we studied whether resveratrol (RSV) or a polyphenolic cocktail, red wine extract (RWE), are able to prevent the initiating events of AMD (i.e., oxidative stress and inflammation) in human ARPE-19 retinal pigment epithelial (RPE) cells and macrophages. This study highlights that RWE and RSV can prevent hydrogen peroxide (H2O2) or 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH)-induced oxidative stress and can subsequently prevent DNA damage via the inhibition of the ATM (ataxia telangiectasia-mutated)/Chk2 (checkpoint kinase 2) or Chk1 signaling pathways, respectively. Moreover, ELISA assays show that RWE and RSV can prevent the secretion of proinflammatory cytokines in RPE cells and in human macrophages. Interestingly, RWE exhibits a greater protective impact compared to RSV alone, even though RSV was more concentrated when used alone than in the red wine extract. Our results suggest that RWE and RSV may have potential interest as preventive nutritional supplementations against AMD.

NMR metabolite quantification of a synthetic urine sample: an inter-laboratory comparison of processing workflows.

INTRODUCTION: Absolute quantification of individual metabolites in complex biological samples is crucial in targeted metabolomic profiling. OBJECTIVES: An inter-laboratory test was performed to evaluate the impact of the NMR software, peak-area determination method (integration vs. deconvolution) and operator on quantification trueness and precision. METHODS: A synthetic urine containing 32 compounds was prepared. One site prepared the urine and calibration samples, and performed NMR acquisition. NMR spectra were acquired with two pulse sequences including water suppression used in routine analyses. The pre-processed spectra were sent to the other sites where each operator quantified the metabolites using internal referencing or external calibration, and his/her favourite in-house, open-access or commercial NMR tool. RESULTS: For 1D NMR measurements with solvent presaturation during the recovery delay (zgpr), 20 metabolites were successfully quantified by all processing strategies. Some metabolites could not be quantified by some methods. For internal referencing with TSP, only one half of the metabolites were quantified with a trueness below 5%. With peak integration and external calibration, about 90% of the metabolites were quantified with a trueness below 5%. The NMRProcFlow integration module allowed the quantification of several additional metabolites. The number of quantified metabolites and quantification trueness improved for some metabolites with deconvolution tools. Trueness and precision were not significantly different between zgpr- and NOESYpr-based spectra for about 70% of the variables. CONCLUSION: External calibration performed better than TSP internal referencing. Inter-laboratory tests are useful when choosing to better rationalize the choice of quantification tools for NMR-based metabolomic profiling and confirm the value of spectra deconvolution tools.

Antifungal Activities of a Grapevine Byproduct Extract Enriched in Complex Stilbenes and Stilbenes Metabolization by Botrytis cinerea.

Grapevine co-products, as canes, represent a source of compounds of interest to control vineyard diseases with a sustainable approach. We chose to study an extract that we produced from grapevine trunk and roots. This extract, enriched in complex stilbenes, strongly reduced mycelial growth and spore germination of Botrytis cinerea, the fungal agent causing gray mold. The most active stilbenes were resveratrol, r-viniferin, and ε-viniferin. This grapevine extract also inhibited the production of Botrytis laccases. Conversely, Botrytis secretome metabolized resveratrol into δ-viniferin and pallidol (2 dimers); and ε-viniferin, a dimer, into hopeaphenol, r-viniferin, and r2-viniferin (3 tetramers). r-Viniferin and hopeaphenol (2 tetramers) were not metabolized. The biotransformed extract maintained an effective antimycelial activity. This study provides evidence that a grapevine extract enriched in oligomerized stilbenes exerts different anti-Botrytis activities, notwithstanding the ability of the fungus to metabolize some stilbenes.

In the shadow of resveratrol: biological activities of epsilon-viniferin.

Stilbenes are secondary metabolites belonging to the polyphenol family. Those compounds are derived from the glycosylation, prenylation, methoxylation, hydroxylation, or also oligomerization of the well-known trans-resveratrol. One of them, trans-epsilon-viniferin (ε-viniferin), is a trans-resveratrol dimer that arouses the interest of researchers in the field of human health. The biosynthesis of this molecule in various plant species, particularly high in the Vitaceae family, explains its presence in some red wines, which represent the main source of ε-viniferin in the human diet. Although bioavailability studies have shown poor absorption and high metabolism of this stilbene, multiple studies demonstrated its biological properties. The ε-viniferin exhibits strong activities against inflammatory and oxidative stress. Moreover, various studies have reported great activity of this compound not only in a wide range of disorders and diseases, such as cancer, obesity, and its associated disorders, but also in vascular diseases and neurodegeneration, for which the pathophysiology is closely related to the state of oxidation and inflammation. This review provides a state of art of the main activities of ε-viniferin demonstrated in vitro and in vivo, highlighting that this resveratrol dimer could be a promising candidate for future functional foods or supplement foods used for the management of many chronic diseases of concern in terms of public health.

Oxyresveratrol and Gnetol Glucuronide Metabolites: Chemical Production, Structural Identification, Metabolism by Human and Rat Liver Fractions, and In Vitro Anti-inflammatory Properties.

Stilbene metabolites are attracting great interest because many of them exhibit similar or even stronger biological effects than their parent compounds. Furthermore, the metabolized forms are predominant in biological fluids; therefore, their study is highly relevant. After hemisynthesis production, isolation, and structural elucidation, three glucuronide metabolites for oxyresveratrol (ORV) were formed: trans-ORV-4'-O-glucuronide, trans-ORV-3-O-glucuronide, and trans-ORV-2'-O-glucuronide. In addition, two glucuronide metabolites were obtained for gnetol (GN): trans-GN-2'-O-glucuronide and trans-GN-3-O-glucuronide. When the metabolism of ORV and GN is studied in vitro by human and rat hepatic enzymes, four of the five hemisynthesized compounds were identified and quantified. Human enzymes glucuronidated preferably at the C-2' position, whereas rat enzymes do so at the C-3 position. In view of these kinetic findings, rat enzymes have a stronger metabolic capacity than human enzymes. Finally, ORV, GN, and their glucuronide metabolites (mainly at the C-3 position) decreased nitric oxide, reactive oxygen species, interleukin 1ß, and tumor necrosis factor α production in lipopolysaccharide-stimulated macrophages.

Identifying early metabolite markers of successful graft union formation in grapevine.

Grafting is an important horticultural technique used for many crop species. However, some scion/rootstock combinations are considered as incompatible due to poor graft union formation and subsequently high plant mortality. The early identification of graft incompatibility could allow the selection of non-viable plants before planting and would have a beneficial impact on research and development in the nursery sector. In general, visible phenotypes of grafted plants (size, root number, etc.) are poorly correlated with grafting success, but some studies have suggested that some polyphenols could be used as markers of graft incompatibility several months or years after grafting. However, much of the previous studies into metabolite markers of grafting success have not included all the controls necessary to unequivocally validate the markers proposed. In this study, we quantified 73 primary and secondary metabolites in nine hetero-grafts and six homo-grafted controls 33 days after grafting at the graft interface and in both the scion and rootstock woody tissues. Certain biomarker metabolites typical of a high stress status (such as proline, GABA and pallidol) were particularly accumulated at the graft interface of the incompatible scion/rootstock combination. We then used correlation analysis and generalized linear models to identify potential metabolite markers of grafting success measured one year after grafting. Here we present the first attempt to quantitatively predict graft compatibility and identify marker metabolites (especially asparagine, trans-resveratrol, trans-piceatannol and α-viniferin) 33 days after grafting, which was found to be particularly informative for homo-graft combinations.

Trans-ε-Viniferin Encapsulation in Multi-Lamellar Liposomes: Consequences on Pharmacokinetic Parameters, Biodistribution and Glucuronide Formation in Rats.

Trans-ε-viniferin (εVin) is a resveratrol dimer exhibiting promising biological activities for human health. Its bioavailability being low, the development of encapsulation methods would be used to overcome this issue. The aim of this study was to measure the consequences of the encapsulation of εVin in multilamellar liposomes on its pharmacokinetic parameters, metabolism and tissue distribution in rats. After oral administration of εVin (20 mg/kg body weight), either as free or encapsulated forms, plasmas were sequentially collected (from 0 to 4 h) as well as liver, kidneys and adipose tissues (4 h after administration) and analyzed by LC-HRMS. The glucuronide metabolites (εVG) were also produced by hemisynthesis for their quantification in plasma and tissues. The encapsulation process did not significantly modify the pharmacokinetic parameters of εVin itself. However, a significant increase of the T1/2 was noticed for εVG after administration of the encapsulated form as compared to the free form. An accumulation of εVin and εVG in adipose tissues was noticed, and interestingly a significant increase of the latter in the mesenteric one after administration of the encapsulated form was highlighted. Since adipose tissues could represent storage depots, and encapsulation allows for prolonging the exposure time of glucuronide metabolites in the organism, this could be of interest to promote their potential biological activities.

1H-NMR Metabolomics as a Tool for Winemaking Monitoring.

The chemical composition of wine is known to be influenced by multiple factors including some viticulture practices and winemaking processes. 1H-NMR metabolomics has been successfully applied to the study of wine authenticity. In the present study, 1H-NMR metabolomics in combination with multivariate analysis was applied to investigate the effects of grape maturity and enzyme and fining treatments on Cabernet Sauvignon wines. A total of forty wine metabolites were quantified. Three different stages of maturity were studied (under-maturity, maturity and over-maturity). Enzyme treatments were carried out using two pectolytic enzymes (E1 and E2). Finally, two proteinaceous fining treatments were compared (vegetable protein, fining F1; pea protein and PVPP, fining F2). The results show a clear difference between the three stages of maturity, with an impact on different classes of metabolites including amino acids, organic acids, sugars, phenolic compounds, alcohols and esters. A clear separation between enzymes E1 and E2 was observed. Both fining agents had a significant effect on metabolite concentrations. The results demonstrate that 1H-NMR metabolomics provides a fast and robust approach to study the effect of winemaking processes on wine metabolites. These results support the interest to pursue the development of 1H-NMR metabolomics to investigate the effects of winemaking on wine quality.

Anthocyanins Promote Learning through Modulation of Synaptic Plasticity Related Proteins in an Animal Model of Ageing.

Anthocyanin-rich foods, such as berries, reportedly ameliorate age-related cognitive deficits in both animals and humans. Despite this, investigation into the mechanisms which underpin anthocyanin-mediated learning and memory benefits remains relatively limited. The present study investigates the effects of anthocyanin intake on a spatial working memory paradigm, assessed via the cross-maze apparatus, and relates behavioural test performance to underlying molecular mechanisms. Six-week supplementation with pure anthocyanins (2% w/w), administered throughout the learning phase of the task, improved both spatial and psychomotor performances in aged rats. Behavioural outputs were accompanied by changes in the expression profile of key proteins integral to synaptic function/maintenance, with upregulation of dystrophin, protein kinase B (PKB/Akt) and tyrosine hydroxylase, and downregulation of apoptotic proteins B-cell lymphoma-extra-large (Bcl-xL) and the phosphorylated rapidly accelerated fibrosarcoma (p-Raf). Separate immunoblot analysis supported these observations, indicating increased activation of extracellular signal-related kinase (ERK1), Akt Ser473, mammalian target of rapamycin (mTOR) Ser2448, activity-regulated cytoskeleton-associated protein (Arc/Arg 3.1) and brain-derived neurotrophic factor (BDNF) in response to anthocyanin treatment, whilst α-E-catenin, c-Jun N-terminal kinase (JNK1) and p38 protein levels decreased. Together, these findings suggest that purified anthocyanin consumption enhances spatial learning and motor coordination in aged animals and can be attributed to the modulation of key synaptic proteins, which support integrity and maintenance of synaptic function.

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.

Encapsulation of ε-Viniferin into Multi-Lamellar Liposomes: Development of a Rapid, Easy and Cost-Efficient Separation Method to Determine the Encapsulation Efficiency.

Onion-type multi-lamellar liposomes (MLLs), composed of a mixture of phosphatidylcholine and Tween 80, were analyzed for their ability to encapsulate ε-Viniferin (εVin), a resveratrol dimer. Their encapsulation efficiency (EE) was measured by UV-VIS spectroscopy using three different separation methods-ultracentrifugation, size exclusion chromatography, and a more original and advantageous one, based on adsorption filtration. The adsorption filtration method consists indeed of using syringe filters to retain the molecule of interest, and not the liposomes as usually performed. The process is rapid (less than 10 min), easy to handle, and inexpensive in terms of sample amount (around 2 mg of liposomes) and equipment (one syringe filter is required). Whatever the separation method, a similar EE value was determined, validating the proposed method. A total of 80% ± 4% of εVin was found to be encapsulated leading to a 6.1% payload, roughly twice those reported for resveratrol-loaded liposomes. Finally, the release kinetics of εVin from MLLs was followed for a 77 day period, demonstrating a slow release of the polyphenol.

Three Types of Elicitors Induce Grapevine Resistance against Downy Mildew via Common and Specific Immune Responses.

Three recognized plant defense stimulators (PDS), methyl jasmonate (MeJA), benzothiadiazole (BTH) and phosphonates (PHOS), were sprayed on grapevine Vitis vinifera cuttings and conferred resistance to the biotrophic pathogen Plasmopara viticola. The effects on molecular defense-related genes and polyphenol content (stilbenes and flavanols) were revealed at 6 and 8 days post-elicitation. The transcript accumulation was consistent with the signaling pathway specific to the elicitor, salicylic acid for BTH, and jasmonic acid for MeJA, with some cross-talks. PHOS tended to modulate the defense responses like BTH. Moreover, in response to a downy mildew inoculation, the leaves pre-treated with PHOS and BTH overproduced pterostilbene, and after MeJA treatment, piceids and ε-viniferin, compared to uninoculated elicitor-treated leaves. These results provide evidence of the different modes of action of PDS and their role in sustainable viticulture.

Red Wine Extract Inhibits VEGF Secretion and Its Signaling Pathway in Retinal ARPE-19 Cells to Potentially Disrupt AMD.

Age-related macular degeneration (AMD) is a degenerative disease of the retina where the molecular mechanism involves the production of vascular endothelial growth factor (VEGF), a factor of poor prognosis of the progression of the disease. Previous studies have shown that resveratrol, a polyphenol of grapevines, can prevent VEGF secretion induced by stress from retinal cells. Considering the fundamental role played by VEGF in development and progression of AMD, we investigate the potential effect of red wine extract (RWE) on VEGF secretion and its signaling pathway in human retinal cells ARPE-19. To examine the effect of RWE in ARPE-19, a quantitative and qualitative analysis of the RWE was performed by HPLC MS/MS. We show for the first time that RWE decreased VEGF-A secretion from ARPE-19 cells and its protein expression in concentration-dependent manner. RWE-induced alteration in VEGF-A production is associated with a down of VEGF-receptor 2 (VEGF-R2) protein expression and its phosphorylated intracytoplasmic domain. Subsequently, the activation of kinase pathway is disturbing and RWE prevents the phosphorylation of MEK and ERK 1/2 in human retinal cells ARPE-19. Finally, this study sheds light on the interest that the use of polyphenolic cocktails could represent in a prevention strategy.