Oxidative stress inhibition by resveratrol in alcohol-dependent mice.

OBJECTIVES: Uncontrolled ingestion of alcohol has dramatic consequences on the entire organism that are also associated with the oxidation process induced by alcohol and elevate radical oxygen species. Resveratrol, a nonflavonoid phenol, shows well-documented antioxidant properties. We investigated the potential antioxidant ability of this natural compound in a mouse model of alcohol addiction. METHODS: We administered (per os) for 60 d 10 mg · kg-1 · d-1 of resveratrol in alcoholic adult male mice. Oxidative stress was evaluated by measuring serum-free oxygen radicals defense and free oxygen radical levels. Resveratrol metabolites were measured in the serum of mice that were administered with resveratrol. Finally, the effect of resveratrol on the alcohol-induced alteration of brain-derived neurotrophic factors (BDNF) in the liver was investigated. RESULTS: Prolonged consumption of resveratrol strongly counteracts serum radical oxygen species formation caused by chronic alcohol intake without effects on natural, free oxygen radical defense. The presence of resveratrol metabolites in the serum only of animals supplemented with resveratrol potentiates the evidence that the antioxidant effect observed is due to the ingestion of the natural compound. Moreover, resveratrol supplementation can counteract alcohol-induced BDNF elevation in the liver, which is the main target of organ alcohol-induced damage. CONCLUSIONS: The consumption of resveratrol through metabolite formation may play a protective role by decreasing free radical formation and modulating the BDNF involved in hepatic disruption induced by chronic alcohol consumption. Further investigation into the mechanism underlying the protective effect could reinforce the potential use of resveratrol as a dietary supplement to prevent damage associated with chronic alcohol abuse.

Supplementation with dairy matrices impacts on homocysteine levels and gut microbiota composition of hyperhomocysteinemic mice.

PURPOSE: Several studies highlighted a correlation between folic acid deficiency and high plasma homocysteine concentration, considered a risk factor for multifactorial diseases. Natural folates represent an emerging alternative strategy to supplementation with synthetic folic acid, whose effects are controversial. The present work was, therefore, performed in hyperhomocysteinemic mice to study the impact of supplementation with dairy matrices containing natural folates on plasma homocysteine levels and faecal microbiota composition. METHODS: Forty mice were divided into six groups, two of which fed control or folic acid deficient (FD) diets for 10 weeks. The remaining four groups were fed FD diet for the first 5 weeks and then shifted to a standard control diet containing synthetic folic acid (R) or a FD diet supplemented with folate-enriched fermented milk (FFM) produced by selected lactic acid bacteria, fermented milk (FM), or milk (M), for additional 5 weeks. RESULTS: Supplementation with dairy matrices restored homocysteine levels in FD mice, although impacting differently on hepatic S-adenosyl-methionine levels. In particular, FFM restored both homocysteine and S-adenosyl-methionine levels to the control conditions, in comparison with FM and M. Next generation sequencing analysis revealed that faecal microbiota of mice supplemented with FFM, FM and M were characterised by a higher richness of bacterial species in comparison with C, FD and R groups. Analysis of beta diversity highlighted that the three dairy matrices determined specific, significant variations of faecal microbiota composition, while hyperhomocysteinemia was not associated with significant changes. CONCLUSIONS: Overall, the results represent a promising starting point for the applicability of food matrices enriched in natural folates to manage hyperhomocysteinemia.

Buckwheat Hull Extracts Inhibit Aspergillus flavus Growth and AFB1 Biosynthesis.

Fungal contamination poses at risk the whole food production chain - from farm to fork - with potential negative impact on human health. So far, the insurgence of pathogens has been restrained by the use of chemical compounds, whose residues have gradually accumulated determining toxic effects in the environment. Modern innovative techniques imply the use of natural and eco-sustainable bioactive plant molecules as pathogens and pests-control agents. These may be profitably recovered in large amounts at the end of industrial milling processes. This is the case of the non-digestible hull of common buckwheat (Fagopyrum esculentum Moench), a natural source of polyphenols, tocopherols, phytosterols and fatty acids. We extract these compounds from the hull of buckwheat; apply them to Aspergillus flavus - aflatoxin producer - under in vitro conditions, checking their ability to inhibit fungal growth and aflatoxin biosynthesis. Moreover, a solvent free method implying the adoption of supercritical CO2 as solvent was set up to extract lipophilic molecules from the buckwheat' hulls. Positive results in controlling fungal growth and aflatoxin biosynthesis let infer that the extracts could be further tested also under in vivo conditions.

Glucoraphanin and sulforaphane evolution during juice preparation from broccoli sprouts.

Broccoli sprouts are considered functional food as they are naturally enriched in glucoraphanin (GR) that is the biological precursor of the anticancer compound sulforaphane (SFN). Due to its health promoting value, also broccoli sprout juice is becoming very popular. The present study aimed to quantitatively assess the conversion of GR to its hydrolysis products, SFN and SFN-nitrile, during the juice preparation process. We demonstrated that SFN plus SFN-nitrile yield from glucoraphanin is quite low (≈25%) and that some SFN is lost during the juice preparation partially due to the spontaneous conversion to sulforaphane-amine or conjugation to GSH and proteins naturally present in the juice. Our results demonstrate that the detection of the sole SFN free form does not provide reliable information about the real concentration of this functional compound in the juice.

Olive polyphenol effects in a mouse model of chronic ethanol addiction.

OBJECTIVES: Alcohol addiction elicits oxidative imbalance and it is well known that polyphenols possess antioxidant properties. We investigated whether or not polyphenols could confer a protective potential against alcohol-induced oxidative stress. METHODS: We administered (per os) for two months 20 mg/kg of olive polyphenols containing mostly hydroxytyrosol in alcoholic adult male mice. Hydroxytyrosol metabolites as hydroxytyrosol sulfate 1 and hydroxytyrosol sulfate 2 were found in the serum of mice administered with polyphenols with the highest amount in animals treated with both polyphenols and alcohol. Oxidative stress was evaluated by FORT (free oxygen radical test) and FORD (free oxygen radical defense) tests. RESULTS: Alcoholic mice showed a worse oxidative status than nonalcoholic mice (higher FORT and lower FORD) but polyphenol supplementation partially counteracted the alcohol pro-oxidant effects, as evidenced by FORT. CONCLUSIONS: A better understanding of the antioxidant protection provided by polyphenols might be of primary interest for drug discovery and dietary-based prevention of the damage associated with chronic alcohol abuse.

Functional roles of the fatty acid desaturases encoded by KlOLE1, FAD2 and FAD3 in the yeast Kluyveromyces lactis.

Functional properties of cell membranes depend on their composition, particularly on the relative amount of saturated, unsaturated and polyunsaturated fatty acids present in the phospholipids. The aim of this study was to investigate the effect of cell membrane composition on cell fitness, adaptation and stress response in Kluyveromyces lactis. To this purpose, we have deleted the genes FAD2 and FAD3 encoding Δ12 and ω3 desaturases in Kluyveromyces lactis, thus generating mutant strains with altered fatty acid composition of membranes. These strains were viable and able to grow in stressing conditions like hypoxia and low temperature. Deletion of the Δ9 desaturase-encoding gene KlOLE1 resulted in lethality, suggesting that this enzyme has an essential role in this yeast. Transcription of the desaturase genes KlOLE1, FAD2 and FAD3 and cellular localization of the corresponding enzymes, have been studied under hypoxia and cold stress. Our findings indicate that expression of these desaturase genes and membrane composition were modulated by hypoxia and temperature stress, although the changes induced by these and other assayed conditions did not dramatically affect the general cellular fitness.

Unsaturated fatty acids-dependent linkage between respiration and fermentation revealed by deletion of hypoxic regulatory KlMGA2 gene in the facultative anaerobe-respiratory yeast Kluyveromyces lactis.

In the yeast Kluyveromyces lactis, the inactivation of structural or regulatory glycolytic and fermentative genes generates obligate respiratory mutants which can be characterized by sensitivity to the mitochondrial drug antimycin A on glucose medium (Rag(-) phenotype). Rag(-) mutations can occasionally be generated by the inactivation of genes not evidently related to glycolysis or fermentation. One such gene is the hypoxic regulatory gene KlMGA2. In this work, we report a study of the many defects, in addition to the Rag(-) phenotype, generated by KlMGA2 deletion. We analyzed the fermentative and respiratory metabolism, mitochondrial functioning and morphology in the Klmga2Δ strain. We also examined alterations in the regulation of the expression of lipid biosynthetic genes, in particular fatty acids, ergosterol and cardiolipin, under hypoxic and cold stress and the phenotypic suppression by unsaturated fatty acids of the deleted strain. Results indicate that, despite the fact that the deleted mutant strain had a typical glycolytic/fermentative phenotype and KlMGA2 is a hypoxic regulatory gene, the deletion of this gene generated defects linked to mitochondrial functions suggesting new roles of this protein in the general regulation and cellular fitness of K. lactis. Supplementation of unsaturated fatty acids suppressed or modified these defects suggesting that KlMga2 modulates membrane functioning or membrane-associated functions, both cytoplasmic and mitochondrial.

Aflatoxin control in maize by Trametes versicolor.

Aspergillus flavus is a well-known ubiquitous fungus able to contaminate both in pre- and postharvest period different feed and food commodities. During their growth, these fungi can synthesise aflatoxins, secondary metabolites highly hazardous for animal and human health. The requirement of products with low impact on the environment and on human health, able to control aflatoxin production, has increased. In this work the effect of the basidiomycete Trametes versicolor on the aflatoxin production by A. flavus both in vitro and in maize, was investigated. The goal was to propose an environmental loyal tool for a significant control of aflatoxin production, in order to obtain feedstuffs and feed with a high standard of quality and safety to enhance the wellbeing of dairy cows. The presence of T. versicolor, grown on sugar beet pulp, inhibited the production of aflatoxin B1 in maize by A. flavus. Furthermore, treatment of contaminated maize with culture filtrates of T. versicolor containing ligninolytic enzymes, showed a significant reduction of the content of aflatoxin B1.

Identification and quantification of phenolic compounds in grapes by HPLC-PDA-ESI-MS on a semimicro separation scale.

Reversed phase high performance liquid chromatography (RP-HPLC) on a semimicro separation scale was employed to develop a straightforward method for the simultaneous separation, identification, and quantification of phenolic compounds occurring in whole berries of Vitis vinifera, which comprise phenolic acids, flavonols, catechins, stilbenes, and anthocyanins. A C-18 narrow bore column of 150 x 2.0 mm I.D. and a semimicro photodiode array detector (PDA) cell of 2.5 microL, in conjunction with a mass spectrometry detector equipped with an electrospray ionization source (ESI-MS) to confirm peak identification, were employed. The C-18 narrow bore column was eluted by a multisegment gradient of increasing concentration of acetonitrile in water-formic acid solution that was optimized on the basis of the results of a study carried out to evaluate the influence of mobile phase composition and gradient shape on separation performance and detection sensitivity by ESI-MS. The identification of individual phenolic compounds was performed on the basis of their retention times and both UV-visible and mass spectra, acquired by a mass spectrometer (MS) equipped with an electrospray ionization (ESI) source, employed in conjunction with the PDA detector. Libraries comprising retention times, UV-visible, and mass spectra for major phenolic compounds expected in grape berries were made by subjecting solutions of each phenolic standard to the optimized RP-HPLC method. Quantification of individual compounds was performed by the external standard method using a six point regression graph of the UV-visible absorption data collected at the wavelength of maximum absorbance of each analyte determined by the PDA spectra. The RP-HPLC method was validated in terms of linearity of calibration graphs, limits of detection, limits of quantification, repeatability, and accuracy, which was evaluated by a recovery study. The developed method was successfully applied to identify the phenolic compounds occurring in the whole berries of nine red and one white grape of different varieties of Vitis vinifera, comprising some autochthonous varieties of south Italy such as Aglianico, Malvasia Nera, Uva di Troia, Negroamaro, Primitivo, and Susumaniello. Large differences in the content of phenolic compounds was found in the investigated grape varieties. As expected, only glycosilated flavonols were quantified, and the total amount of these compounds was higher in the whole berries of red grapes than in the white Moscato, where the most abundant phenolic compound was quercetin 3-O-glucoside. In almost all samples, the most and least abundant anthocyanins were malvidin 3-O-glucoside and cyanidin 3-O-glucoside, respectively, with the exception of Uva di Troia where the least abundant anthocyanin was delphinidin 3-O-glucoside.

Role of the spacer stereochemistry on the aggregation properties of cationic gemini surfactants.

The preparation and characterization of three stereoisomeric cationic gemini surfactants, 2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonio)butane dibromide, are described. The aggregation properties have been studied by fluorescence, electrical conductivity, and quasi-elastic light scattering. A conformational study of the surfactant headgroups has been performed by molecular mechanics calculations to investigate the effect of the stereogenic centers on the surfactant molecular shape and therefore on the different organizations of the monomers in the aggregates. Results show that the stereochemistry of the spacer strongly influences the aggregation behavior of the diasteromeric gemini in water.

Use of liposomes as a dispersed pseudo-stationary phase in capillary electrophoresis of basic proteins.

The suitability of liposome capillary electrophoresis for separating basic proteins at different pH values in the acidic domain was evaluated preparing liposomes consisting of large unilamellar vesicles of the phospholipid 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC), whose hydrodynamic diameter and size distribution were evaluated by dynamic light scattering measurements. The study was conducted evaluating different approaches of performing liposome capillary electrophoresis of proteins, using liposomes dispersed in the electrolyte solution of different composition and pH values ranging from pH 6.2 to 4.0. The liposomes were employed as a pseudo-stationary phase dispersed in the electrolyte solution, which was introduced into the capillary as a plug of volume equivalent to that of the capillary tube, whereas liposome-free electrolyte solutions were contained into the electrolyte vessels during electrophoresis. The study was performed with either bare fused-silica capillaries or capillaries previously treated with POPC that was employed as a dynamic coating agent. Such treatment was performed rinsing the capillary tube with a proper volume of electrolyte solution containing POPC and resulted in a significant reduction of the electroosmotic flow, indicative of the adsorptive behavior of liposomes onto the internal surface of bare fused-silica capillaries. The usefulness of using liposome dynamically coated capillaries for separating basic proteins in absence of the dispersed liposome pseudo-stationary phase filled into the capillary prior to injecting the protein sample was evaluated too. The results show that the presence of liposomes reduced drastically the untoward interactions between basic proteins and the capillary wall, also in capillary not subjected to the dynamic coating process with POPC. In addition, the use of liposomes as a pseudo-stationary phase dispersed in the electrolyte solution enhanced the separation of basic proteins also in POPC dynamically coated capillaries.