The Moringin/α-CD Pretreatment Induces Neuroprotection in an In Vitro Model of Alzheimer's Disease: A Transcriptomic Study.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and represents the most common form of senile dementia. Autophagy and mitophagy are cellular processes that play a key role in the aggregation of ß-amyloid (Aß) and tau phosphorylation. As a consequence, impairment of these processes leads to the progression of AD. Thus, interest is growing in the search for new natural compounds, such as Moringin (MOR), with neuroprotective, anti-amyloidogenic, antioxidative, and anti-inflammatory properties that could be used for AD prevention. However, MOR appears to be poorly soluble and stable in water. To increase its solubility MOR was conjugated with α-cyclodextrin (MOR/α-CD). In this work, it was evaluated if MOR/α-CD pretreatment was able to exert neuroprotective effects in an AD in vitro model through the evaluation of the transcriptional profile by next-generation sequencing (NGS). To induce the AD model, retinoic acid-differentiated SH-SY5Y cells were exposed to Aß1-42. The MOR/α-CD pretreatment reduced the expression of the genes which encode proteins involved in senescence, autophagy, and mitophagy processes. Additionally, MOR/α-CD was able to induce neuronal remodeling modulating the axon guidance, principally downregulating the Slit/Robo signaling pathway. Noteworthy, MOR/α-CD, modulating these important pathways, may induce neuronal protection against Aß1-42 toxicity as demonstrated also by the reduction of cleaved caspase 3. These data indicated that MOR/α-CD could attenuate the progression of the disease and promote neuronal repair.

Improving the Phloroglucinolysis Protocol and Characterization of Sagrantino Wines Proanthocyanidins.

Proanthocyanidins are key metabolites that explain wine sensorial character (bitterness and astringency) and red wine color changes during aging. Therefore, a fast and accurate method to evaluate the degree of polymerization and the structural composition of the polymeric proanthocyanidins is a crucial analytical tool. Phloroglucinolysis is the most used method for this analysis but, unfortunately, the phloroglucinol adducts of the monomeric flavan-3-ols are not commercially available, making the results less accurate. The aim of this work was the isolation by semi-preparative high performance liquid chromatography (HPLC) of these non-commercial compounds and their use for the development of an accurate UHPLC-MS/MS protocol. The purity of each adduct was established via quantitative 1H-nuclear magnetic resonance (NMR) measurements with 3-trimethylsilyl-propionic-d4 acid sodium salt as the calibration standard. The developed method was applied to evaluate the proanthocyanidins profile of Sagrantino di Montefalco wines in comparison to other well-known tannic wines. Commercial, 6-8 years old Sagrantino wines were demonstrated to be very rich in epicatechin type B procyanidins, to have low galloylation %, and to have a high mean degree of polymerization of the proanthocyanidins with respect to the other analyzed wines.

Moringin, an isothiocyanate modulates multiple cellular signalling molecules in breast cancer cells.

Prohibitin (PHB) is a pleiotropic molecule with a variety of known functions and subcellular locations. PHB's function in breast cancer is poorly understood. Herein, we report that PHB is expressed in cancer types of diverse origin including breast cancer. The cancer patients with changes in PHB were reported to have significantly reduced 'overall survival' in comparison to the cases without alterations in PHB. The expression of PHB was increased by H2O2 and also by Moringin (MG), which is an isothiocyanate derived from the seeds of Moringa oleifera. MG interacted with PHB, DRP1, and SLP2 and inhibited the growth of MCF-7 and MDAMB-231 cells. The isothiocyanate triggered apoptosis in breast cancer cells as revealed by AO/PI assay, phosphatidylserine externalization, cell cycle analysis and DAPI staining. MG induced proapoptotic proteins expression such as cytochrome c, p53, and cleaved caspase-7. Further, cell survival proteins such as survivin, Bcl-2, and Bcl-xL were suppressed. A depolarization of membrane potential suggested that the apoptosis was triggered through mitochondria. The isothiocyanate suppressed the cancer cell migration and interacted with NF-κB subunits. MG suppressed p65 nuclear translocation induced by TNF-α. The reactive oxygen species generation was also induced by the isothiocyanate in breast cancer cells. MG also modulated the expression of lncRNAs. Collectively, the functions of PHB in breast cancer growth is evident from this study. The activities of MG against breast cancer might result from its ability to modulate multiple cancer-related targets.

α-Cyclodextrin/Moringin Induces an Antioxidant Transcriptional Response Activating Nrf2 in Differentiated NSC-34 Motor Neurons.

Oxidative stress is a common feature of neurodegenerative diseases. Different natural compounds mediate neuroprotective effects by activating the Nrf2 antioxidant response. Some isothiocyanates are Nrf2 activators, including Moringin (MOR). In this study, the transcriptional profile of differentiated NSC-34 motor neurons was evaluated after treatment for 48 h and 96 h with concentrations of 0.5, 5, and 10 µM of a new MOR formulation obtained with α-cyclodextrin (α-CD). All the concentrations increased gene expression and cytoplasmic protein levels of Nrf2 at 96 h. However, the highest dose also increased nuclear Nrf2 levels at 96 h. Then, Nrf2 interactors were selected using STRING, and common biological process (BP) terms between the groups were evaluated. α-CD/MOR was able to modulate BP related to responses to oxidative stress, proteostasis, and autophagy. Specifically, the treatment with 10 µM of α-CD/MOR for 96 h induced genes involved in glutathione synthesis and proteasome subunits and reduced the expression of genes related to endoplasmic reticulum stress. Moreover, this group showed the lowest levels of the apoptotic markers Bax, cleaved caspase 9, and cleaved caspase 3. These results indicate the beneficial effects of prolonged α-CD/MOR supplementation that are mediated, at least in part, by Nrf2 activation. Then, α-CD/MOR could be a valuable treatment against neurodegenerative diseases, in particular motor neuron degeneration.

Multivariate prediction of Saliva Precipitation Index for relating selected chemical parameters of red wines to the sensory perception of astringency.

Astringency is an essential sensory attribute of red wine closely related to the saliva precipitation upon contact with the wine. In this study a data matrix of 52 physico-chemical parameters was used to predict the Saliva Precipitation Index (SPI) in 110 Italian mono-varietal red wines using partial least squares regression (PLSr) with variable selection by Variable Importance for Projection (VIP) and the significance of regression coefficients. The final PLSr model, evaluated using a test data set, had 3 components and yielded an R2test of 0.630 and an RMSEtest of 0.994, with 19 independent variables whose regression coefficients were all significant at p < 0.05. Variables selected in the final model according to the decreasing magnitude of their absolute regression coefficient include the following: Procyanidin B1, Epicatechin terminal unit, Total aldehydes, Protein content, Vanillin assay, 520 nm, Polysaccharide content, Epigallocatechin PHL, Tartaric acid, Volatile acidity, Titratable acidity, Catechin terminal unit, Proanthocyanidin assay, pH, Tannin-Fe/Anthocyanin, Buffer capacity, Epigallocatechin PHL gallate, Catechin + epicatechin PHL, and Tannin-Fe. These results can be used to better understand the physico-chemical relationship underlying astringency in red wine.

Metabolomic Characterization of Pigmented and Non-Pigmented Potato Cultivars Using a Joint and Individual Variation Explained (JIVE).

Potatoes (Solanum tuberosum L.) are one of the most valuable agricultural crops, and the flesh of these tubers provides various classes of healthy compounds important for human nutrition. This work presents the results of a joint analysis of different chemical classes of compounds which provided insights on the metabolic characterization of pigmented and non-pigmented potato varieties collected from Italy. The identification of common or individual metabolic characteristics across the omic datasets (antioxidants, total polyphenolic content, polyphenols, and sugars) is conducted by Joint and Individual Variation Explained (JIVE), a data fusion multivariate approach. The common part of the multivariate model allowed the separation between non-pigmented and pigmented samples. Polyphenolic compounds were mainly responsible for the separation between purple-fleshed and red-skinned potatoes. An additional detailed analysis of the anthocyanin composition, including the acylated anthocyanins, allowed to pinpoint the diversities between the pigmented potato groups. Furthermore, the presence of an appreciable amount of hydroxycinnamic acids and anthocyanins in the purple-fleshed varieties, which are also characterized by a lower content of sugars, is found. Our results provide scientific evidence for the promotion of promising potato cultivars, which are characterized by a remarkable amount of various health benefit compounds.

Modeling grape taste and mouthfeel from chemical composition.

This research aims at predicting sensory properties generated by the phenolic fraction (PF) of grapes from chemical composition. Thirty-one grape extracts of different grape lots were obtained by maceration of grapes in hydroalcoholic solution; afterward they were submitted to solid phase extraction. The recovered PFs were reconstituted in a wine model. Subsequently the wine models, containing the PFs, were sensory (taste, mouthfeel) and chemically characterized. Significant sensory differences among the 31 PFs were identified. Sensory variables were predicted from chemical parameters by PLS-regression. Tannin activity and concentration along with mean degree of polymerization were found to be good predictors of dryness, while the concentration of large polymeric pigments seems to be involved in the "sticky" percept and flavonols in the "bitter" taste. Four fully validated PLS-models predicting sensory properties from chemical variables were obtained. Two out of the three sensory dimensions could be satisfactorily modeled. These results increase knowledge about grape properties and proposes the measurement of chemical variables to infer grape quality.

The contribution of varietal thiols in the diverse aroma of Italian monovarietal white wines.

Thanks to their low odor detection thresholds, free varietal thiols (VTs) play a key role in the primary aroma of wines, to which they confer an intense scent reminiscent of box tree, grapefruit, citrus fruits, passionfruit and cat urine odor. Excluding wines from a few VT-rich grapevine cultivars, VTs appear to be present in most cultivars at trace levels, although a comprehensive dataset is still missing. The low concentration of VTs combined with their high reactivity and matrix complexity make their determination in wines a challenging task. In this research an optimized liquid chromatography - tandem mass spectrometry (LC-MS/MS) method was validated and used for the quantification of 4-methyl-4-sulfanylpentan-2-one (4-MSP), 3-sulfanylhexan-1-ol (3-SH), 3-sulfanylhexyl acetate (3-SHA) and ethyl 3-sulfanylpropionate (E3SP) in 246 samples (vintage 2019) representative of 18 monovarietal Italian white wines. VTs were detected in all cultivars even though higher values of 3-SH were found in Lugana, Müller-Thurgau and Verdicchio cultivars. Müller-Thurgau wines showed the highest level of 4-MSP, that was mainly correlated to the odor descriptors of passionfruit and box tree/cat urine. The VTs composition of Müller-Thurgau was confirmed on a second set of 50 wines from different vintages. From a sensory perspective, the samples of Müller-Thurgau showed the best positive correlations between chemical variables and the odor descriptors thiol note, passion fruit and box tree/cat urine. These notes are significantly related to 4-MSP, suggesting that it could play a relevant olfactory role for the aroma of Müller-Thurgau wines. Sorting analysis allowed to group these wines according to their thiolic characteristics. The chemical variables and the odor descriptors attributable to the thiol notes are important for Müller-Thurgau and Lugana wines, while the contribution of thiol notes was sensorially negligible for the other wines.

Potent Antifungal Properties of Dimeric Acylphloroglucinols from Hypericum mexicanum and Mechanism of Action of a Highly Active 3'Prenyl Uliginosin B.

The success of antifungal therapies is often hindered by the limited number of available drugs. To close the gap in the antifungal pipeline, the search of novel leads is of primary importance, and here the exploration of neglected plants has great promise for the discovery of new principles. Through bioassay-guided isolation, uliginosin B and five new dimeric acylphloroglucinols (uliginosins C-D, and 3'prenyl uliginosins B-D), besides cembrenoids, have been isolated from the lipophilic extract of Hypericum mexicanum. Their structures were elucidated by a combination of Liquid Chromatography - Mass Spectrometry LC-MS and Nuclear Magnetic Resonance (NMR) measurements. The compounds showed strong anti-Candida activity, also against fluconazole-resistant strains, with fungal growth inhibition properties at concentrations ranging from 3 to 32 µM, and reduced or absent cytotoxicity against human cell lines. A chemogenomic screen of 3'prenyl uliginosin B revealed target genes that are important for cell cycle regulation and cytoskeleton assembly in fungi. Taken together, our study suggests dimeric acylphloroglucinols as potential candidates for the development of alternative antifungal therapies.

Two apples a day lower serum cholesterol and improve cardiometabolic biomarkers in mildly hypercholesterolemic adults: a randomized, controlled, crossover trial.

BACKGROUND: Apples are rich in bioactive polyphenols and fiber. Evidence suggests that consumption of apples or their bioactive components is associated with beneficial effects on lipid metabolism and other markers of cardiovascular disease (CVD). However, adequately powered randomized controlled trials are necessary to confirm these data and explore the mechanisms. OBJECTIVE: We aimed to determine the effects of apple consumption on circulating lipids, vascular function, and other CVD risk markers. METHODS: The trial was a randomized, controlled, crossover, intervention study. Healthy mildly hypercholesterolemic volunteers (23 women, 17 men), with a mean ± SD BMI 25.3 ± 3.7 kg/m2 and age 51 ± 11 y, consumed 2 apples/d [Renetta Canada, rich in proanthocyanidins (PAs)] or a sugar- and energy-matched apple control beverage (CB) for 8 wk each, separated by a 4-wk washout period. Fasted blood was collected before and after each treatment. Serum lipids, glucose, insulin, bile acids, and endothelial and inflammation biomarkers were measured, in addition to microvascular reactivity, using laser Doppler imaging with iontophoresis, and arterial stiffness, using pulse wave analysis. RESULTS: Whole apple (WA) consumption decreased serum total (WA: 5.89 mmol/L; CB: 6.11 mmol/L; P = 0.006) and LDL cholesterol (WA: 3.72 mmol/L; CB: 3.86 mmol/L; P = 0.031), triacylglycerol (WA: 1.17 mmol/L; CB: 1.30 mmol/L; P = 0.021), and intercellular cell adhesion molecule-1 (WA: 153.9 ng/mL; CB: 159.4 ng/mL; P = 0.028), and increased serum uric acid (WA: 341.4 µmol/L; CB: 330 µmol/L; P = 0.020) compared with the CB. The response to endothelium-dependent microvascular vasodilation was greater after the apples [WA: 853 perfusion units (PU), CB: 760 PU; P = 0.037] than after the CB. Apples had no effect on blood pressure or other CVD markers. CONCLUSIONS: These data support beneficial hypocholesterolemic and vascular effects of the daily consumption of PA-rich apples by mildly hypercholesterolemic individuals.This trial was registered at clinicaltrials.gov as NCT01988389.

Myrtle Seeds (Myrtus communis L.) as a Rich Source of the Bioactive Ellagitannins Oenothein B and Eugeniflorin D2.

The increasing popularity of "Mirto" liqueur, produced from Myrtus communis berries, has led to the planting of domesticated cultivars, expanding myrtle berry production. To promote the use of cultivated berries, the content in the nutraceutical compounds ellagitannins has been investigated both in spontaneous and cultivated fruits. Oenothein B and eugeniflorin D2, characterized by 1H and 13C NMR, were isolated and quantified using ultrahigh-performance liquid chromatography-diode array detector-tandem mass spectrometry (UPLC-DAD-MS/MS). The antifungal and anti-inflammatory activities of oenothein B were assayed in vitro. Large amounts of oenothein B and eugeniflorin D2 were detected in seeds (12 ± 2.4 and 5.8 ± 1.2 mg/g). The oenothein B concentration in liqueurs was 194 ± 22 mg/L. This macrocyclic ellagitannin dimer showed anti-Candida (minimal inhibitory concentration <8-64 µg/mL) and anti-inflammatory properties. Cultivated myrtle berries are a source of nutraceutical compounds. The high concentration of oenothein B in liqueur suggests a possible contribution to the organoleptic and biological properties of the beverage.

Extracts From Hypericum hircinum subsp. majus Exert Antifungal Activity Against a Panel of Sensitive and Drug-Resistant Clinical Strains.

During the last two decades incidences of fungal infections dramatically increased and the often accompanying failure of available antifungal therapies represents a substantial clinical problem. The urgent need for novel antimycotics called particular attention to the study of natural products. The genus Hypericum includes many species that are used in the traditional medicine to treat pathological states like inflammations and infections caused by fungi. However, despite the diffused use of Hypericum-based products the antifungal potential of the genus is still poorly investigated. In this study five Hypericum species autochthonous of Central and Eastern Europe were evaluated regarding their polyphenolic content, their toxicological safety and their antifungal potential against a broad panel of clinical fungal isolates. LC-MS analysis led to the identification and quantification of 52 compounds, revealing that Hypericum extracts are rich sources of flavonols, benzoates and cinnamates, and of flavan-3-ols. An in-depth screen of the biological activity of crude extracts clearly unveiled H. hircinum subsp. majus as a promising candidate species for the search of novel antifungals. H. hircinum is diffused in the Mediterranean basin from Spain to Turkey where it is traditionally used to prepare a herbal tea indicated for the treatment of respiratory tract disorders, several of which are caused by fungi. Noteworthy, the infusion of H. hircinum subsp. majus excreted broad antifungal activity against Penicillium, Aspergillus and non-albicans Candida isolates comprising strains both sensitive and resistant to fluconazole. Additionally, it showed no cytotoxicity on human cells and the chemical characterization of the H. hircinum subsp. majus infusion revealed high amounts of the metabolite hyperoside. These results scientifically support the traditional use of H. hircinum extracts for the treatment of respiratory tract infections and suggest the presence of exploitable antifungal principles for further investigations aimed at developing novel antifungal therapies.

Metabolomics assisted fingerprint of Hypericum perforatum chemotypes and assessment of their cytotoxic activity.

Hypericum perforatum is known as an important medicinal plant, used for the treatment of several diseases, while its pharmacological properties are attributed to the presence of a wide range of secondary metabolites. Due to the great chemotypic variability of Hypericum species in the nature, and the demand for standardized herbal products, a detailed phytochemical investigation was carried out on different parts (herba, leaf, flowers) from wild collected and cultivated populations, using advanced chromatographic tools. Liquid Chromatographic analysis (LC-MS/MS MRM) revealed significant variability in the secondary metabolites content of the examined methanolic extracts. The most common derivatives belong to 9 groups i.e. benzoic acids, phenylpropanoids, coumarins, flavones, flavonols, flavan-3-ols, anthocyanins, phloroglucinols and naphtodianthrones. The main polyphenolic compounds were catechin, epicatechin, quercetin, quercetin 3-O-rhamnoside, quercetin 3-O-glucoside, neochlorogenic acid, proanthocyanidins (A and B series) and cyanidin-3-O-glucoside. In addition, the content of the characteristic compounds hypericin and hyperforin in herba crude extracts ranged between 0.5 and 1.7 mg/g and 0.6-3.3 mg/g respectively. The cytotoxic activity of the crude extracts was assessed at concentrations ranged between 0.01 and 100 µg/mL, on Caco-2 intestinal cancer cell cultures, and a cytotoxic behavior was shown only at the highest concentration of 100 µg/mL.

The metabolomic profile of red non-V. vinifera genotypes.

Wild American genotypes represent an important part of the Vitis germplasm in relation to grape improvement. Today, these genotypes are currently involved in breeding programmes in order to introgress traits resistant to pests and diseases in V. vinifera cultivars. Nevertheless, the metabolic composition of their grapes has not been widely investigated. This study aimed to explore in detail the metabolomic profile in terms of simple phenolic, proanthocyanidin, anthocyanin and lipid compounds in two hybrids and five American genotypes. The results were compared with those of two V. vinifera cultivars. A multi-targeted metabolomics approach using a combination of LC-MS and LC-DAD methods was used to identify and quantify 124 selected metabolites. The genotypes studied showed considerable variability in the metabolomic profile according to the grape composition of V. vinifera and other Vitis genotypes. As regards the composition of anthocyanins, not all wild genotypes contained both mono- and di-glucoside derivatives. Wild genotype 41B and V. vinifera cultivars contained only monoglucoside anthocyanins. The proanthocyanidins of non-V. vinifera genotypes were mainly rich in oligomers and short-chain polymers. The analysis of lipids in wild Vitis genotypes, here reported for the first time, showed the existence of a certain diversity in their composition suggesting a strong influence of the environmental conditions on the general lipid pattern.

Wine metabolomics reveals new sulfonated products in bottled white wines, promoted by small amounts of oxygen.

The impact of minute amounts of oxygen in the headspace on the post-bottling development of wine is generally considered to be very important, since oxygen can either damage or improve the quality of wine. This project aimed to gain new experimental evidence about the chemistry of the interaction between wine and oxygen. The experimental design included 216 bottles of 12 different white wines produced from 6 different cultivars (Inzolia, Muller Thurgau, Chardonnay, Grillo, Traminer and Pinot gris). Half of them were bottled using the standard industrial process with inert headspace and the other half without inert gas and with extra headspace. After 60 days of storage at room temperature, the wines were analysed using an untargeted LC-MS method. The use of a detailed holistic analysis workflow, with several levels of quality control and marker selection, gave 35 metabolites putatively induced by the different amounts of oxygen. These metabolite markers included ascorbic acid, tartaric acid and various sulfonated compounds observed in wine for the first time (e.g. S-sulfonated cysteine, glutathione and pantetheine; and sulfonated indole-3-lactic acid hexoside and tryptophol). The consumption of SO2 mediated by these sulfonation reactions was promoted by the presence of higher levels of oxygen on bottling.

Use of instrumental acoustic parameters of winegrape seeds as possible predictors of extractable phenolic compounds.

The use of instrumental acoustic parameters produced during a compression test as reliable predictors of the extractable phenolic composition in intact winegrape seeds, determined by reference chemical methods, was evaluated by means of the analytical performance of calibration models. These models were developed only for those phenolic compounds most significantly and strongly correlated with the acoustic parameters. The analytical performance of the models was expressed in terms of standard error of cross-validation (SECV) and residual predictive interquartile amplitude (RPIQ), among other statistics. Several acoustic parameters showed satisfactory predictive accuracy for the percentage of galloylation in the terminal units, the content of (-)-epicatechin, and the mean degree of polymerization. Most of the reliable models developed are fairly recommended not for quantitative purposes but for fast screening (SECV% < 19, 1.6 < RPIQ < 2.1).

Study of Sangiovese Wines Pigment Profile by UHPLC-MS/MS.

The metabolic pigment composition of Sangiovese wines produced from grapes harvested at 20 different vineyards in Montalcino over three consecutive years (2008-2010) on a semi-industrial scale and of 55 commercial Brunello di Montalcino wines (2004-2007) was studied, using a targeted method capable of analyzing 90 pigments in an 11 min UHPLC-MS/MS chromatographic run. Interesting correlations were shown between various pigments formed during wine aging and those present in Sangiovese grapes. Vitisin B-like pigment and vitisin A-like pigment concentrations would seem to have a good correlation with ethyl-linked and direct-linked flavanol-anthocyanin concentrations, respectively. Moreover, the anthocyanic pattern recognition, genetically controlled by the plant variety, was shown to be inherited by the pigments formed during wine aging.

Identification of intermediates involved in the biosynthetic pathway of 3-mercaptohexan-1-ol conjugates in yellow passion fruit (Passiflora edulis f. flavicarpa).

Yellow passion fruit is one of the most well-known tropical fruits and much of its success comes from its typical aroma. Key compounds in explaining yellow passion fruit scent are volatile thiols. These molecules are reported to be present in several fruits and originate from non-volatile precursors. Such free thiols are particularly appreciated in white wines and considerable efforts have been made to try to maximise their production and understand their biosynthesis. Two main precursors have been identified so far: S-glutathionylated and S-cysteinylated precursors, the latter originating in the breaking down of the glycyl and glutamyl moieties of the former. Improving knowledge about this pathway is currently one of the main challenges in the field of aroma chemistry. Only S-cysteinylated precursors have been reported in the literature for yellow passion fruit, thus much of the biochemical pathway remains unknown. In this paper a combination of organic synthesis, MS and NMR experiments was developed in order to investigate this pathway in yellow passion fruit. The three missing stages leading to the S-cysteinylated precursor were clearly identified. Both intermediate species between S-glutathionyl and S-cysteinyl 3-mercaptohexan-1-ol were found, suggesting that the plant is capable of activating both metabolic routes. The information gained would appear to be crucial for study of this important pathway and for potentially extending this knowledge to other plants, in particular the grapevine.

A metabolomic approach to the study of wine micro-oxygenation.

Wine micro-oxygenation is a globally used treatment and its effects were studied here by analysing by untargeted LC-MS the wine metabolomic fingerprint. Eight different procedural variations, marked by the addition of oxygen (four levels) and iron (two levels) were applied to Sangiovese wine, before and after malolactic fermentation. Data analysis using supervised and unsupervised multivariate methods highlighted some known candidate biomarkers, together with a number of metabolites which had never previously been considered as possible biomarkers for wine micro-oxygenation. Various pigments and tannins were identified among the known candidate biomarkers. Additional new information was obtained suggesting a correlation between oxygen doses and metal contents and changes in the concentration of primary metabolites such as arginine, proline, tryptophan and raffinose, and secondary metabolites such as succinic acid and xanthine. Based on these findings, new hypotheses regarding the formation and reactivity of wine pigment during micro-oxygenation have been proposed. This experiment highlights the feasibility of using unbiased, untargeted metabolomic fingerprinting to improve our understanding of wine chemistry.

Development of reliable analytical tools for evaluating the influence of reductive winemaking on the quality of Lugana wines.

This paper presents methods for the definition of important analytical tools, such as the development of sensitive and rapid methods for analysing reduced and oxidised glutathione (GSH and GSSG), hydroxycinnamic acids (HCA), bound thiols (GSH-3MH and Cys-3MH) and free thiols (3MH and 3MHA), and their first application to evaluate the effect of reductive winemaking on the composition of Lugana juices and wines. Lugana is a traditional white wine from the Lake Garda region (Italy), produced using a local grape variety, Trebbiano di Lugana. An innovative winemaking procedure based on preliminary cooling of grape berries followed by crushing in an inert environment was implemented and explored on a winery scale. The effects of these procedures on hydroxycinnamic acids, GSH, GSSG, free and bound thiols and flavanols content were investigated. The juices and wines produced using different protocols were examined. Moreover, wines aged in tanks for 1, 2 and 3 months were analysed. The high level of GSH found in Lugana grapes, which can act as a natural antioxidant and be preserved in must and young wines, thus reducing the need of exogenous antioxidants, was particularly interesting. Moreover, it was clear that polyphenol concentrations (hydroxycinnamic acids and catechins) were strongly influenced by winemaking and pressing conditions, which required fine tuning of pressing. Above-threshold levels of 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA) were found in the wines and changed according to the winemaking procedure applied. Interestingly, the evolution during the first three months also varied depending on the procedure adopted. Organic synthesis of cysteine and glutathione conjugates was carried out and juices and wines were subjected to LC-MS/MS analysis. These two molecules appeared to be strongly affected by the winemaking procedure, but did not show any significant change during the first 3 months of post-bottling ageing. This supports the theory, already proposed in the literature, that there are other synthetic pathways for free thiol formation.