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One of the organ-specific functions of the liver is the excretion of bilirubin into the bile. Membrane transport of bilirubin from the blood to the liver is not only an orphan function, because there is no link to the protein/gene units that perform this function, but also a poorly characterised function. The aim of this study was to investigate the pharmacology of bilirubin uptake in the liver of the female Wistar rat to improve basic knowledge in this neglected area of liver physiology. We treated isolated perfused livers of female rats with repeated single-pass, albumin-free bilirubin boli. We monitored both bilirubin and bilirubin glucuronide in perfusion effluent with a bio-fluorometric assay. We tested the ability of nine molecules known as substrates or inhibitors of sinusoidal membrane transporters to inhibit hepatic uptake of bilirubin. We found that cyanidin 3-glucoside and malvidin 3-glucoside were the only molecules that inhibited bilirubin uptake. These dietary anthocyanins resemble bro-mosulfophthalein (BSP), a substrate of several sinusoidal membrane transporters. The SLCO-specific substrates estradiol-17 beta-glucuronide, pravastatin, and taurocholate inhibited only bilirubin glucuronide uptake. Cya-nidin 3-glucoside and taurocholate acted at physiological concentrations. The SLC22-specific substrates indo-methacin and ketoprofen were inactive. We demonstrated the existence of a bilirubin-glucuronide transporter inhibited by bilirubin, a fact reported only once in the literature. The data suggest that bilirubin and bilirubin glucuronide are transported to the liver via pharmacologically distinct membrane transport pathways. Some dietary anthocyanins may physiologically modulate the uptake of bilirubin into the liver.

The low temperature normally applied to prevent fruit decay during the storage of apples, can also triggers the onset of a chilling injury disorder known as superficial scald. In this work, the etiology of this disorder and the mechanism of action of two preventing strategies, such as the application of 1-MCP (1-methylcyclopropene) and storage at low oxygen concentration in ‘Granny Smith’ and ‘Ladina’ apple cultivars were investigated. The metabolite assessment highlighted a reorganization of specific metabolites, in particular flavan-3-ols and unsaturated fatty acids, while the genome-wide transcriptomic analysis grouped the DEGs into four functional clusters. The KEGG pathway and GO enrichment analysis, together with the gene-metabolite interactome, showed that the treatment with 1-MCP prevented the development of superficial scald by actively promoting the production of unsaturated fatty acids, especially in ‘Granny Smith’. ‘Ladina’, more susceptible to superficial scald and less responsive to the preventing strategies, was instead characterized by a higher accumulation of very long chain fatty acids. Storage at low oxygen concentration stimulated a higher accumulation of ethanol and acetaldehyde together with the expression of genes involved in anaerobic respiration, such as malate, alcohol dehydrogenase and pyruvate decarboxylase in both cultivars. Low oxygen concentration, likewise 1-MCP, through a direct control on ethylene prevented the onset of superficial scald repressing the expression of PPO, a gene encoding for the polyphenol oxidase enzyme responsible of the oxidation of chlorogenic acid. Moreover, in ‘Granny Smith’ apple, the expression of three members of the VII subgroups of ERF genes, encoding for elements coordinating the acclimation process to hypoxia in plants was observed. The global RNA-Seq pattern also elucidated a specific transcriptomic signature between the two cultivars, disclosing the effect of the different genetic background in the control of this disorder.

Anecdotal observations have shown that there is often incoherence between sugar accumulation and evolution of grape flavour during later ripening stages. For this reason, an experiment with Malvazija istarska (Vitis vinifera L.) was conducted to investigate the relationship between harvest date and changes in the abundance of wine volatile aroma compounds. Malvazija grapes were harvested and subsequently vinified at three harvest dates (H1 – H3) separated from each other by approximately 10 days, with grape quality parameters at each date suitable for production of standard still white wines. Wine volatile aroma compounds were analysed by comprehensive two- dimensional gas chromatography with time-of-flight mass spectrometry combined with conventional one- dimensional GC/MS and were statistically elaborated. Various terpenes and alcohols were found in the highest concentration in wines from the grapes harvested at H3. Most of fermentation aroma compounds showed no significant differences. The exceptions were ethyl-2-methylbutyrate, ethyl propanoate and isobutyl decanoate found in the highest concentrations in wines from H3 and isopropyl acetate and isoamyl decanoate in wines from H1. The obtained results confirmed that by choosing the harvest date it is possible to significantly influence the composition of volatile aroma compounds in white wine. This study was funded by Croatian Science Foundation under the projects IP-2020-02-4551 and DOK2021-02-5500.

Spontaneous fermentation in winemaking is a complex microbial reaction that involves influences from many factors, including the microbiota, the grape variety, climatic and soil conditions, and vinification technology. The perceived aroma of the produced wine is the result of interactions between all the volatile and nonvolatile metabolites. The study aimed to investigate the volatile metabolite profile of „Maraština“ wines produced by spontaneous fermentation, thus supporting the concept of terroir impact on the volatile aromas of the wine. The „Maraština“ grapes were harvested in 10 vineyards from two Croatian vine-growing regions: Northern Dalmatia and Central and Southern Dalmatia. A total of 10 experimental fermentation trails in three repetitions were set up on the laboratory scale. Volatile compounds from the wine samples were isolated by solid-phase extraction (SPE) and analyzed by an untargeted approach using two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GCxGC-TOF-MS) and a targeted approach by gas chromatography-tandem mass spectrometry (GC- MS/MS). Comprehensive two-dimensional GCxGC analysis provided a detailed characterization of total volatile metabolites in the wines due to its excellent separation ability. More than 900 compounds were detected after untargeted profiling and 222 of them were identified or tentatively identified by comparison of experimental retention indices and mass spectra with literature data from the NIST database. A total of 56 volatile compounds were identified and quantified using GC-MS/MS analysis, including terpenes (14), C13- norisoprenoids (3), esters (18), alcohols (4), phenols (4), aldehydes (2), ketones (2), acids (4), lactones (4) and indole (1). The Fischer ratio was useful for establishing the compounds responsible for the main differences between grapes harvested from different subregions. „Maraština“ wines from the Northern Dalmatia subregion had a significantly higher concentration of total volatile compounds than the wines from the Central and Southern Dalmatia subregion, especially the following compounds: cis-Rose oxide, trans-Rose oxide, β-Damascenone, TDN, ethyl leucate, diethyl succinate, phenylacetaldehyde, benzaldehyde and octanoic acid.

Information on the content and composition of phenols in monovarietal wines is of fundamental importance for adequate wine quality management and control, it is useful for predicting wine sensory properties and oxidative stability, and may indicate wine age and production technology. It may also be used for authentication of varietal origin, as well as a strong point in marketing. To investigate the possibility of wine varietal differentiation based on phenol composition despite the existing heterogeneity, almost 200 samples produced from grapes of various red and white varieties grown in various terroirs in three Croatian regions were subjected to targeted profiling of phenols by ultra-performance liquid chromatography-triple quadrupole mass spectrometry. Descriptive sensory analysis was performed, including evaluation of varietal typicity, to investigate if there is a relation between variety-typical sensory profiles and certain phenols. Many of identified phenols were attributed as discriminators between wines. Plavac mali was characterized by the most distinguished anthocyanin profile, with generally the lowest concentrations among the investigated red wines. On the other hand, the wines from this variety were generally the most abundant in non- anthocyanin phenols. Cabernet Sauvignon wine stood out with the highest values of acetylated to p-coumaroylated anthocyanins ratio and the percentage of the sum of the acylated anthocyanins. Teran wine was characterised by the highest concentration of anthocyanins in general, while Merlot exhibited the least distinguished profile. As for white wines, those from the region of Dalmatia made from Pošip and Maraština varieties were generally more abundant in phenols than others. Malvazija istarska wines contained the lowest concentration of the majority of flavonols and hydroxycinammates, while Graševina was distinguished by the lowest amounts of flavan-3-ols. The highest concentrations of hydroxycinammates were found in Muscat blanc wines. Linear discriminant analysis resulted in successful differentiation, with peonidin 3-(6”- acetyl)-glucoside and taxifolin as the most potent differentiators among red, and cis-piceid among white wines. Particular varietal typicity concepts were partially confirmed by tentatively relating the concentrations of certain phenols and their possible sensory effects. This work has been supported by the UIP-2014-09- 1194 (Croatian Science Foundation) and ADP 2017 (Trento, Italy) projects.

Grape quality and yield can be impaired by bunch rot, caused by the necrotrophic fungus Botrytis cinerea. Infection often occurs at flowering, and the pathogen stays quiescent until fruit maturity. Here, we report a molecular analysis of the early interaction between B. cinerea and Vitis vinifera flowers, using a controlled infection system, confocal microscopy and integrated transcriptomic and metabolic analysis of the host and the pathogen. Flowers from fruiting cuttings of the cultivar Pinot Noir were infected with green fluorescent protein (GFP)-labelled B. cinerea and studied at 24 and 96 hours post-inoculation (h.p.i.). We observed that penetration of the epidermis by B. cinerea coincided with increased expression of genes encoding cell-wall-degrading enzymes, phytotoxins and proteases. Grapevine responded with a rapid defence reaction involving 1193 genes associated with the accumulation of antimicrobial proteins, polyphenols, reactive oxygen species and cell wall reinforcement. At 96 h.p.i., the reaction appears largely diminished both in the host and in the pathogen. Our data indicate that the defence responses of the grapevine flower collectively are able to restrict invasive fungal growth into the underlying tissues, thereby forcing the fungus to enter quiescence until the conditions become more favourable to resume pathogenic development.

In order to investigate the effect of the addition of tannins to Malvazija istarska (Vitis vinifera L.) must during fermentation on the protein stability and selected aspects of wine quality, several vinification treatments were performed: addition of tannins during fermentation (TA), addition of bentonite at the end of fermentation (BE), combination of the addition of tannins and bentonite (TA+BE), and a control treatment (CO) without tannin and bentonite added during fermentation. Protein stability of wines was evaluated, additional doses required were determined and applied. Volatile aroma compounds were extracted from wine samples by SPE for the measurements by GC-MS. Hydroxycinnamoyltartaric acids and the corresponding free derivatives were determined by HPLC-DAD. Wines were evaluated by 100-point OIV method and quantitative descriptive sensory analysis. The most effective treatment in terms of achieving total protein stability was TA. The reduction of the total bentonite dose required in relation to CO was 19%. Treatment TA resulted in higher concentrations of the majority of important acetate and ethyl esters. Concentration of most acids detected were higher in wines obtained without tannin addition. TA+BE treatment preserved the highest concentration of hydroxycinnamoyltartaric acids, followed by BE, TA, and CO. Free hydroxycinnamic acid concentrations responded diametrically opposite. Significant differences in the intensity of wine sensory attributes were mainly detected among wines that were not stabilized after fermentation. Wines fermented with tannin addition gained higher scores, but the additional bentonite fining step after fermentation mostly decreased their sensory quality. This work has been supported in part by Croatian Science Foundation under the project UIP2014-09-1194.

The aim of this work was to investigate whether it is possible to differentiate red monovarietal wines produced in Croatia (EU) according to variety using compositional data on phenols, despite the existent geographical and pedoclimatic heterogeneity and variations in winemaking techniques. The focus was put on Croatian domestic grape varieties, such as Teran and Plavac mali, in order to provide evidence to support anecdotal knowledge on the existence of particular sensory varietal typicity constructs, which reached a reasonable consensus among professionals and experts familiar with these wines, but have not been scientifically confirmed. Selected typical samples of Croatian monovarietal wines produced from domestic Teran and Plavac mali, as well as introduced world- known Cabernet Sauvignon and Merlot varieties were collected from the producers in the Istria and Dalmatia regions of Croatia, and subjected to UPLC/MS/MS analysis of phenols. Wines were assessed sensorially by descriptive sensory analysis performed by a well-trained and experienced sensory panel. Successful differentiation according to variety was achieved by univariate and multivariate statistics. The typical intense color of Teran, especially with respect to Plavac mali wine, was confirmed to correlate with higher concentrations of anthocyanins. Plavac mali was characterized by very low concentration of acylated forms, while Cabernet Sauvignon contained less p-coumarate anthocyanins in relation to other monovarietal wines. The typical accentuated bitterness and astringency of Plavac mali wines coincided with higher concentrations of particular non-colored phenols, such as gallocatechin and taxifolin, with the latter emerging as the most potent differentiator of Plavac mali among the investigated phenols. Teran and Merlot contained higher amounts of piceids. This work has been supported in part by Croatian Science Foundation under the project UIP- 2014-09-1194.

Although many more or less successful virgin olive oil (VOO) varietal characterization and differentiation studies have been conducted based on the composition of sensory-active compounds in conjuction with sensory analysis data, the concept of VOO varietal typicity has received almost no attention up to date. The aim of this study was to investigate if the existent anecdotal varietal typicity concepts for two among the most important Istrian (Croatia) monovarietal VOOs can be scientifically explained and supported. VOO samples were sorted according to typicity grade by an internationally recognized sensory analysis panel, and put in relation with descriptive sensory and compositional analysis data for volatiles and phenols using univariate and multivariate statistics. Clear inter- and intra-varietal differences with respect to typicity were established. The complex odor typical for Buža VOO coincided with the abundance in lipoxygenase- derived (LOX) volatiles, especially (Z)-3- hexenal, combined with particular hydrocarbons, terpenes, and benzenoids. The typical Buža VOO gradually increasing pungency was tentatively related to the elevated level of pinoresinol. The simpler green odor typical for Istarska bjelica VOO coincided with the lower amounts of the majority of LOX volatiles, while the predominant role of p-HPEA-EDA in the formation of its intense bitterness and burning pungency was assumed. Intra-varietal typicity grades correlated strongly with hedonic liking, as well as with general VOO positive sensory attributes related to key C5 and C6 volatiles and phenols.

Comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GC×GC- TOF-MS) was combined with conventional mono-dimensional GC–MS to differentiate Croatian virgin olive oils (VOO) according to variety and geographical origin, based on the profile of volatile aroma compounds isolated by HS-SPME. More than 1000 compounds were detected after untargeted profiling and 256 were identified or tentatively identified, providing one of the most detailed profiles of volatile aroma compounds in VOO up to date. Among them, 131 volatile compounds were significantly different across monovarietal VOOs, while 60 were found useful for the discrimination according to geographical origin. Many major lipoxygenase and minor nonlipoxygenase-generated compounds were shown to have discriminating ability with respect to both factors. Multivariate statistical analysis extracted twenty-one volatile markers with the highest discriminant power for varietal differentiation. The approach reported may have practical application in better understanding, defining, managing, and communicating the varietal or geographical typicity of monovarietal VOOs.

Comprehensive two-dimensional gas chromatography (GCxGC) has emerged as a powerful analytical technique for unraveling the volatile composition of complex matrices. This work will present three applications of GCxGC Tof-MS in the fields of wine and olive oil, aimed to identify biomarkers which could be used in the quality control. In the first study this analytical technique was used to monitor, during shelf life in realistic condition, the headspace profile of aromatic compounds in white wines, through an analysis of untargeted metabolomics. The aim of this study was to assess the impact of the exposure to the light on large number of white wines bottled in bottles of different colors on their aromatic profiles. In the second study an investigation of Shiraz wine volatile composition from four vineyards located in the Riverina region of Australia was performed by accessing wines made from sequentially harvested grapes. Shiraz wines were vinified from 60 kg grape triplicates. Following a berry ripening model the first harvest (H1) was 12 days from the plateau of berry sugar accumulation and the second harvest (H2), 24 days after the plateau. These results indicate significant modulation of wine volatiles as a consequence of harvest dates, by altering lipoxygenase derived compounds and yeast metabolism, irrespective of vineyard cultural practices, within the same warm to hot climatic region. In the third study the volatile composition of EVOO from five olive cultivars was investigated with GCxGC Tof-MS in association with multivariate analyses. A possible varietal differentiation according to the profiles of volatile compounds was affirmed by PCA and potential cultivar-specific biomarkers were identified.

Autotoxic compounds are likely to be among the causes of apple replant disease, but their secretion is low during plant life. Using targeted metabolomics, the changes in soil phenolic profile were analyzed after the addition of apple roots, and their potential autotoxicity was assessed on apple seedlings. The addition of apple roots severely damaged the plants, attributed to autotoxic action of the phenolic compound phlorizin. Prolonged residence time of the roots in the soil before planting reduced their negative action, probably due to the degradation of phlorizin.

The ability of anthocyanins to permeate the gastric mucosa can be suggested as a possible explanation of the fast kinetics of plasma appearance of anthocyanins in rats and humans. This paper presents an in vivo experiment aimed to prove the involvement of the stomach in the absorption of grape anthocyanins in rats. The required analytical selectivity and sensitivity was achieved by high-performance liquid chromatography, diode array detection and mass spectrometry. Malvidin 3-glucoside appeared in both portal and systemic plasma after only 6 min. The average concentrations measured in portal and systemic plasma were 0.650±0.162 μM and 0.234±0.083 μM (mean±S.E.M.), respectively.

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.

In physiological conditions, the response to oxidative stress induced by strenuous physical training, is not always efficient. Furthermore, a weak muscle fiber antioxidant system can contribute to muscle wasting during ageing and pathological conditions. Natural antioxidant supplements could counteract the effects of oxidative stress. An in vitro model of exercise allowed us to mimic intense or moderate exercise by electrically stimulating cultured mouse myotubes with high- and/or low-frequency pulses; the resulting ROS production was then monitored. The herbal supplement Rhodiola rosea aqueous extract was characterized in terms of its polyphenol content and used to test for antioxidant activity against ROS, produced during muscle contraction. Our results showed that Rhodiola rosea extract reduced the oxidative stress produced by muscle contraction and brought values of ROS production back to physiological levels. We suggest that Rhodiola rosea root extract could exert a protective antioxidant role during intense physical exercise.

Strawberry (Fragaria × ananassa) fruit contains high concentrations of health-promoting phenolic compounds, playing important roles for the fruit ontogenic tolerance to fungi. In the highly susceptible cultivar Alba, the two major strawberry fungal pathogens, Colletotrichum acutatum and Botrytis cinerea, displayed disease symptoms only at red ripe stages because immature fruits are tolerant to diseases. We analyzed and compared the variation of 47 polyphenols in the surface of unripe and ripe Alba fruits upon 24 and 48 h of C. acutatum and B. cinerea infection or mock inoculation. Significant alteration in phenolic content was detected only in white infected fruit, with differences specific for each pathogen. The expression analysis of phenylpropanoid, flavonoid, and shikimate pathway genes showed in only a few cases correlation with the relative metabolite abundance. The alteration in phenolic content and the lack of consistency with gene expression data are discussed in light of previously reported metabolome data of different susceptible and resistant strawberry genotypes.

Different polyphenol compounds are ingested when consuming a serving of fruits rich in polyphenols, spanning from one-phenol hydroxybenzoic acid to more complex polymeric compounds. Only a minor quantity of the polyphenols (5 - 10%) is absorbed. The remainder reaches the colon and is extensively metabolized by gut microbiota to low-molecular weight metabolites. Their subsequent tissue distribution is still undefined, although these microbial metabolites are currently believed to play a role in human health and disease states. To fill this knowledge gap, we performed a pharmacokinetics experiment in which a single bolus of 23 polyphenol microbial metabolites (total 2.7 mu mol) was administered intravenously to rats to reliably reproduce a physiological postabsorption situation. Tissues and urine were collected shortly thereafter (15 s to 15 min) and were analyzed by UHPLC-MS/MS to quantitatively track these compounds. Remarkably, the brain was found to be a specific target organ for 10 of the 23 polyphenol metabolites injected, which significantly increased in the treated animals. In most cases, their appearance in the brain was biphasic, with an early wave at 2 min (4 compounds) and a second wave starting at 5 min; at 15 min, 9 compounds were still detectable. Most compounds were excreted into the urine. The concentrations in the brain of the treated animals were compared against those of the control group by Student's t test, with p-values < 0.1 considered to be statistically significant. These findings provide new perspectives for understanding the role of diet on brain chemistry. Our experimental approach has enabled us to obtain rich metabolomics information from a single experiment involving a limited number of animals.

A range of different polyphenols can be ingested in a bowl of polyphenol-rich fruit, going from one-phenol hydroxybenzoic acid to more complex polymeric compounds. Epidemiologically, polyphenol consumption has been associated with a reduced risk of cancer and cardiovascular disease and neurological protection against brain ageing. However, only a small proportion of native polyphenols (5-10%) are absorbed and the remainder reach the colon, where they are extensively metabolised by the gut microbiota. The colonic microbiota produces a relatively small number of polyphenol microbial metabolites from a large number of different dietary polyphenols. During subsequent tissue distribution, the target organs and the effective concentration circulating remain largely unreported. This Ph.D. thesis is divided into two parts: chemical analysis of food composition and in vivo bioavailability of polyphenol metabolites. Metabolomics offers an innovative approach that has recently been shown to be effective in both food chemistry and nutritional bioavailability studies. Polyphenol composition in strawberries is studied in the first part of this Ph.D. thesis, with the aim of evaluating nutritionally significant amounts of polyphenols before ingestion. In this context, a targeted method for quantitative analysis of multiple classes of phenols was developed. A high sensitivity MRM-based method for 135 phenolics with a wide dynamic range was obtained, providing valuable insight and assisting with the analysis of complex matrices such as fruit, and more in general food. Application of the method was tested in Fragaria spp., and along with another rapid method for the analysis of anthocyanins and ellagitannins, provided a general overview of polyphenol composition in strawberries. A total of 56 individual compounds were accurately identified and quantified, some of them for the first time, their concentration ranging from 1 ug/100 g for low abundant polyphenols to 40 mg/100 g of fresh fruit. Moreover the isolation of some ellagitannins and definition of their profile in Fragaria spp. was carried out during fruit ripening. Clarification of the main ellagitannin, agrimoniin, was obtained by isolation and it was ambiguously assigned as the main ellagitannin present in the diet. In the second part of this Ph.D. thesis the in vivo bioavailability of a dose of polyphenol microbial metabolites reflecting dietary consumption of fruit was studied. The focus was on the metabolites of polyphenols which can be found in the bloodstream after gut microflora metabolism. Their distribution was explored in rats in different organs, in particular in the brain, considering their possible neuropreventive properties. Development of a specific quantitative method for the quantification of selected polyphenol microbial metabolites made it possible to analyse complex biological samples resulting from in vivo trials with rats treated with a nutritionally significant dose of polyphenol microbial metabolites, intravenously injected. A high-throughput, sensitive and reproducible method for 23 polyphenol metabolites in six different biological matrices was developed. A purification protocol made it possible to obtain cleaner and more concentrated samples, with low limits of quantification. Specific organ-tropism was observed, mainly hepatotropism. Remarkably, in this study the brain was reported to be one of the target organs for these molecules, already being present at basal level or increasing their concentration after treatment. Furthermore, the amount of 10 out of 23 compounds significantly increased with a nutritionally significant dose.