Faecal metabolomic fingerprint after moderate consumption of red wine by healthy subjects.

Faecal metabolome contains information on the metabolites found in the intestine, from which knowledge about the metabolic function of the gut microbiota can be obtained. Changes in the metabolomic profile of faeces reflect, among others, changes in the composition and activity of the intestinal microorganisms. In an effort to improve our understanding of the biological effects that phenolic compounds (including red wine polyphenols) exert at the gut level, in this foodomic study we have undertaken a metabolome characterization of human faeces after moderate consumption of red wine by healthy subjects for 4 weeks. Namely, a nontargeted metabolomic approach based on the use of UHPLC-TOF MS was developed to achieve the maximum metabolite information on 82 human faecal samples. After data processing and statistical analysis, 37 metabolites were related to wine intake, from which 20 could be tentatively or completely identified, including the following: (A) wine compounds, (B) microbial-derived metabolites of wine polyphenols, and (C) endogenous metabolites and/or others derived from other nutrient pathways. After wine consumption, faecal metabolome was fortified in flavan-3-ols metabolites. Also, of relevance was the down regulation of xanthine and bilirubin-derived metabolites such as urobilinogen and stercobilin after moderate wine consumption. As far as we know, this is the first study of the faecal metabolome after wine intake.

Phenolic Assesment of Uncaria tomentosa L. (Cat's Claw): Leaves, Stem, Bark and Wood Extracts.

The phenolic composition of extracts from Uncaria tomentosa L. from different regions of Costa Rica was studied using advanced analytical techniques such as UPLC/TQ-ESI-MS and (13)C-NMR. Samples from leaves, stems, bark and wood (n = 22) were subjected to extraction to obtain phenolic and alkaloid extracts, separately. Comparatively, higher values of total phenolic content were observed for leaves, stems and bark (225-494 gallic acid equivalents/g) than for wood extracts (40-167 gallic acid equivalents/g). A total of 32 non-flavonoid and flavonoid compounds were identified in the phenolic extracts: hydroxybenzoic acids (benzoic, salicylic, 4-hydroxybenzoic, prochatechuic, gallic, syringic and vanillic acids), hydroxycinnamic acids (p-coumaric, caffeic, ferulic and isoferulic acids), flavan-3-ols monomers [(+)-catechin and (-)-epicatechin)], procyanidin dimers (B1, B2, B3, B4, B5, B7 and two other of unknown structure) and trimers (C1, T2 and one of unknown structure), flavalignans (four unknown structures pertaining to the cinchonain family) and propelargonidin dimers (four unknown structures, reported for the first time in U. tomentosa). Additionally, alkaloid extracts obtained from the plant residue after phenolic extraction exhibited a content of tetracyclic and pentacyclic alkaloids ranging between 95 and 275 mg/100 g of dry material for bark extracts, and between 30 and 704 mg/100 g for leaves extracts. In addition, a minor alkaloid was isolated and characterized, namely 18,19-dehydrocorynoxinoic acid. Our results confirmed the feasibility of U. tomentosa as a suitable raw material for obtaining phenolic- and alkaloid-rich extracts of potential interest.

Assessment of the impact of the addition of antimicrobial plant extracts to wine: volatile and phenolic composition.

BACKGROUND: Antimicrobial plant phenolic-rich extracts have been proposed as alternative to sulfites in the control of the malolactic fermentation (MLF) during winemaking. This addition may affect wine organoleptic properties. In this paper, we have investigated the changes in wine volatile and phenolic composition, after MLF, of a red wine treated with antimicrobial extracts from eucalyptus leaves and almond skins. RESULTS: Although addition of both extracts led to statistically significant changes (P < 0.05) in the concentration of several esters, alcohols, C13 nor-isoprenoids and volatile phenols, only few of these volatile compounds showed values of odour activity > 1 aroma unit; that is to say, whose concentrations were higher than their corresponding odour thresholds. With regard to phenolic compounds, the addition of both extracts did not significantly modify the content of anthocyanins, which predicts minor changes in wine colour. However, the content of non-anthocyanin phenolics was significantly higher in the wines treated with antimicrobial extracts, especially for flavonols, being the dose-over-taste factor for these wines significantly higher. Finally, principal component analysis showed that wines were mainly differentiated on the basis of whether MLF was conducted or not, and its method of performance (inoculated/spontaneous). CONCLUSION: Addition of antimicrobial extracts leads to some compositional changes in the wine, whose relevance needs to be addressed in future experiments, including wine sensorial analysis.

Toward a predictive model of Alzheimer's disease progression using capillary electrophoresis-mass spectrometry metabolomics.

Alzheimer's disease (AD) is the most prevalent form of dementia with an estimated worldwide prevalence of over 30 million people, and its incidence is expected to increase dramatically with an increasing elderly population. Up until now, cerebrospinal fluid (CSF) has been the preferred sample to investigate central nervous system (CNS) disorders since its composition is directly related to metabolite production in the brain. In this work, a nontargeted metabolomic approach based on capillary electrophoresis-mass spectrometry (CE-MS) is developed to examine metabolic differences in CSF samples from subjects with different cognitive status related to AD progression. To do this, CSF samples from 85 subjects were obtained from patients with (i) subjective cognitive impairment (SCI, i.e. control group), (ii) mild cognitive impairment (MCI) which remained stable after a follow-up period of 2 years, (iii) MCI which progressed to AD within a 2-year time after the initial MCI diagnostic and, (iv) diagnosed AD. A prediction model for AD progression using multivariate statistical analysis based on CE-MS metabolomics of CSF samples was obtained using 73 CSF samples. Using our model, we were able to correctly classify 97-100% of the samples in the diagnostic groups. The prediction power was confirmed in a blind small test set of 12 CSF samples, reaching a 83% of diagnostic accuracy. The obtained predictive values were higher than those reported with classical CSF AD biomarkers (Aß42 and tau) but need to be confirmed in larger samples cohorts. Choline, dimethylarginine, arginine, valine, proline, serine, histidine, creatine, carnitine, and suberylglycine were identified as possible disease progression biomarkers. Our results suggest that CE-MS metabolomics of CSF samples can be a useful tool to predict AD progression.

Statistical evaluation of CZE-UV and CZE-ESI-MS data of intact α-1-acid glycoprotein isoforms for their use as potential biomarkers in bladder cancer.

α-1-acid glycoprotein (AGP) is a highly heterogeneous protein that presents a vast number of isoforms (molecules of the protein differing in its peptidic and/or glycosidic moieties). In the last years, several authors have studied the potential use of AGP as a cancer biomarker. These studies focus on the correlation of different features of AGP structure (i.e. fucosylation, antennarity) with cancer or on the total protein blood concentration. In this study, the potential of CZE-UV and CZE-ESI-MS analysis of intact AGP isoforms to study the correlation of this protein with bladder cancer is shown. Samples from 16 individuals (eight healthy, eight bladder cancer) were analyzed and characterized in great detail including data on intact protein isoforms and on released glycans. The analytical data were evaluated employing different statistical techniques (ANOVA; principal component analysis, PCA; linear discriminant analysis; and partial least squares-discriminant analysis). Statistical differences between the two groups of study were observed. The best results were obtained by linear discriminant analysis of the CZE-ESI-MS data for intact AGP isoforms (93.75% of correct classification). Due to MS characterization, it can be observed that differences between the samples are mainly due to higher abundance of AGP isoforms containing tri- and tetra-antennary fucosylated oligosaccharides in cancer patients. The results show the great potential of CE-MS in combination with advanced data processing for the use of intact protein isoforms as disease biomarkers.

Almond (Prunus dulcis (Mill.) D.A. Webb) polyphenols: from chemical characterization to targeted analysis of phenolic metabolites in humans.

In this paper, a survey of our studies on almond polyphenols including their chemical characterization and further bioavailability in humans is reported. Combination of analytical techniques (LC-DAD/fluorescence, LC/ESI-MS and MALDI-TOF-MS) allowed us, for the first time, the identification of A- and B-type procyanidin, propelargonidin and prodelphinidin polymers in almond skins. Glucuronide, O-methyl glucuronide, sulfate and O-methyl sulfate derivatives of (epi)catechin, as well as the glucuronide conjugates of naringenin and isorhamnetin, and sulfate conjugates of isorhamnetin, together with conjugates of hydroxyphenylvalerolactones were detected in plasma and urine samples after the intake of almond skin polyphenols. In addition, numerous microbial-derived metabolites, including hydroxyphenylpropionic, hydroxyphenylacetic, hydroxycinnamic, hydroxybenzoic and hydroxyhippuric acids were also identified. Depending of the type of metabolite, maximum urinary excretion was attained at different time in comparison to the control group in the course of the 24-h period of urine excretion, allowing us to establish the onset of microbial metabolism.

Targeted analysis of conjugated and microbial-derived phenolic metabolites in human urine after consumption of an almond skin phenolic extract.

A single-blind, placebo-controlled, and randomized trial study was carried out with 16 healthy volunteers (7 men and 5 women). The test group ingested an encapsulated almond skin phenolic extract (884 mg of total polyphenols/dose) containing flavan-3-ols, flavonols, and flavanones, whereas the placebo group ingested microcrystalline cellulose. Our aim in this study was to determine changes in the urinary excretion of conjugated and microbial-derived phenolic metabolites before (-2 to 0 h) and after (0-2, 2-6, 6-10, and 10-24 h) intake of the almond polyphenols compared with the placebo group. For the test group, maximum urinary excretion of (epi)catechin and naringenin conjugates derived from phase II metabolism was attained at 2-6 h after consumption of the almond skin extract and excretions differed from the placebo group during this time period (P ≤ 0.0001). However, excretion of conjugated metabolites of isorhamnetin was highest at 10-24 h and did not differ from the placebo group during this time (P > 0.05). Hydroxyphenylvalerolactones reached maximum urinary levels at 6-10 h after consumption of almond polyphenols, and excretion differed from the placebo group during this time period (P = 0.0004). For the test group, excretions of phenolic acids (hydroxyphenylpropionic, hydroxyphenylacetic, hydroxybenzoic, and hydroxycinnamic acids) did not differ from the placebo group at any time period of urine collection (P > 0.05). The findings presented in this work provide evidence concerning the bioavailability of almond skin polyphenols considering the effects of both phase II and microbial metabolism.

Angiotensin-converting enzyme activity in plasma and tissues of spontaneously hypertensive rats after the short- and long-term intake of hydrolysed egg white.

This paper evaluates the effects of the short- (1 g/kg) and long-term (0.5 and 1 g/kg/day) oral intake of egg white hydrolysed with pepsin (hEW) and the long-term oral intake (1 g/kg/day) of egg white (EW) on local angiotensin-converting enzyme (ACE) activities in plasma and other tissues of spontaneously hypertensive rats (SHR), as compared to the effect of the ACE inhibitor prototype captopril. The rats treated with hEW were classed in a different group than the control rats and the rats treated with EW by cluster analysis, taking into account their tissue ACE activities and their systolic blood pressure (SBP). Principal component analysis (PCA) showed that SBP in SHR was negatively related with ACE activity in plasma and positively related with ACE activity in aorta and kidney. ACE activity in plasma significantly increased after the long-term treatment with hEW (0.5 g/kg/day). ACE activity in aorta and kidney was significantly inhibited 4 h after the short-term administration of hEW. The long-term treatment with hEW caused local effects on ACE activity in aorta, kidney and lungs that followed a pattern similar, but less pronounced, than that caused by captopril.

Beta-carotene isomer composition of sub- and supercritical carbon dioxide extracts. Antioxidant activity measurement.

In the present work sub- and supercritical extraction conditions using carbon dioxide were studied in order to obtain extracts with different compositions from the green microalgae Dunaliella salina. Different compositions of beta-carotene isomers were identified in the extracts by using HPLC-DAD. Also, antioxidant activity of the extracts was measured using a TEAC assay. An experimental design was applied considering two factors, extraction pressure and temperature, in a wide range of values, trying to maximize the extraction yield. Higher yields were obtained at high pressures and low temperatures, that is, at higher CO2 densities. Attempts were made to correlate the antioxidant activity of the extracts with their chemical composition by means of principal component analysis. A certain relationship was found between their antioxidant activity and the isomeric composition of beta-carotenes. As a result, an original equation is proposed to predict the antioxidant activity of extracts from D. salina in terms of the ratio 9-cis-beta-carotene/all-trans-beta-carotene, the concentration of alpha-carotene, and, especially, the concentration of 9-cis-beta-carotene.

Optimization of the extraction of antioxidants from Dunaliella salina microalga by pressurized liquids.

In this work, extraction of antioxidant compounds from Dunaliella salina microalga is optimized by combining pressurized liquid extraction (PLE) and experimental design (three-level factorial design) with three different solvents (hexane, ethanol, and water). Two main factors were considered, the extraction temperature (40, 100, and 160 degrees C) and the extraction time (5, 17.5, and 30 min). As response variables, the extraction yield (percent dry weight/initial weight) and the antioxidant activity of the extracts (determined using the TEAC method) were used. The parameters of the model were estimated by multiple linear regression. Results showed that the extraction temperature was the factor having the strongest influence (positive) on the two response variables. The best yields were obtained with ethanol at the higher extraction temperature and time tested. Besides, although hexane extracts provided the best antioxidant activity, ethanol extracts were also very active. The chemical characterization of ethanol extracts was carried out using HPLC-DAD, and attempts have been made to correlate their chemical composition with the antioxidant activity measured. Results pointed out that the extracts contained, besides all-trans-beta-carotene and isomers, several different minor carotenoids that seemed to make a contribution to the antioxidant activity of the extracts.

Changes in the free amino acid contents of honeys during storage at ambient temperature.

This study was carried out to establish the changes in the free amino acid contents of floral honeys, honeydew honeys, and blend honeys during storage at room temperature and to test the capacity of the amino acids to distinguish the origin of the honeys after storage. For this purpose, 54 artisanal honeys (39 floral, 5 honeydew, and 10 blend) were studied. Samples were taken from recently collected honeys and at 3, 6, 9, 12, 16, 20, and 24 months after harvesting. The contents of most of the free amino acids were found to decrease with storage time, with the greatest reduction observed in the first 9 months. The contents of the amino acids aspartic acid, beta-alanine, and proline increased in the first few months after storage, reaching maximum values at 6 months, suggesting the possible existence of enzymatic activities. The application of stepwise discriminant analysis to the free amino acid content data demonstrated that the contents of the amino acids valine, beta-alanine, gamma-aminobutyric acid, serine, isoleucine, alpha-alanine, ornithine, and glutamine correctly assigned 87% of honeys to their group of origin: floral, honeydew, or blend.

Protein analysis of honeys by fast protein liquid chromatography: application to differentiate floral and honeydew honeys.

Fast protein liquid chromatography on a Superdex 75 HR column has been applied to analyze the proteins of 29 honeys, 12 of floral origin and 17 from honeydew. The molecular masses were comprised between 13100 and 94000 Da. Seven peaks have been separated; four of them were present in all of the honeys, and three were only present in some honeys. Direct observation of the chromatograms of the floral and honeydew honeys did not reveal any information about their botanical origins. However, both types of honeys can be distinguished with the percentages of the areas of four of the seven chromatographic peaks obtained.

Selective recovery of tagatose from mixtures with galactose by direct extraction with supercritical CO2 and different cosolvents.

A selective fractionation method of carbohydrate mixtures of galactose/tagatose, using supercritical CO(2) and isopropanol as cosolvent, has been evaluated. Optimization was carried out using a central composite face design and considering as factors the extraction pressure (from 100 to 300 bar), the extraction temperature (from 60 to 100 degrees C), and the modifier flow rate (from 0.2 to 0.4 mL/min, which corresponded to a total cosolvent percentage ranging from 4 to 18% vol). The responses evaluated were the amount (milligrams) of tagatose and galactose extracted and their recoveries (percent). The statistical analysis of the results provided mathematical models for each response variable. The corresponding parameters were estimated by multiple linear regression, and high determination coefficients (>0.96) were obtained. The optimum conditions of the extraction process to get the maximum recovery of tagatose (37%) were 300 bar, 60 degrees C, and 0.4 mL/min of cosolvent. The predicted value was 24.37 mg of tagatose, whereas the experimental value was 26.34 mg, which is a 7% error from the predicted value. Cosolvent polarity effects on tagatose extraction from mixtures of galactose/tagatose were also studied using different alcohols and their mixtures with water. Although a remarkable increase of the amount of total carbohydrate extracted with polarity was found, selective extraction of tagatose decreased with increase of polarity of assayed cosolvents. To improve the recovery of extracted tagatose, additional experiments outside the experimental domain were carried out (300 bar, 80 degrees C, and 0.6 mL/min of isopropanol); recoveries >75% of tagatose with purity >90% were obtained.

Profiling of Phenolic Metabolites in Feces from Menopausal Women after Long-Term Isoflavone Supplementation.

Phenolic compounds were screened by UPLC-ESI-MS/MS in the feces of 15 menopausal women before and after long-term isoflavone treatment. In total, 44 compounds were detected. Large intertreatment, interindividual, and intersample variations were observed in terms of the number of compounds and their concentration. Four compounds, the aglycones daidzein and genistein and the daidzein derivatives dihydrodaidzein and O-desmethylangolensin, were associated with isoflavone metabolism; these were identified only after the isoflavone treatment. In addition, 4-ethylcatechol, 3-hydroxyphenylacetic acid, and 3-phenylpropionic acid differed significantly in pre- and postintervention samples, whereas the concentration of 4-hydroxy-5-phenylvaleric acid showed a trend toward increasing over the treatment. The phenolic profiles of equol-producing and -non-producing groups were similar, with the exceptions of 3-hydroxyphenylacetic acid and 3-phenylpropionic acid, which showed higher concentrations in equol-non-producing women. These findings may help to trace isoflavone-derived metabolites in feces during isoflavone interventions and to design new studies to address their biological effects.

Comparative in vitro fermentations of cranberry and grape seed polyphenols with colonic microbiota.

In this study, we have assessed the phenolic metabolism of a cranberry extract by microbiota obtained from the ascending colon and descending colon compartments of a dynamic gastrointestinal simulator (SHIME). For comparison, parallel fermentations with a grape seed extract were carried out. Extracts were used directly without previous intestinal digestion. Among the 60 phenolic compounds targeted, our results confirmed the formation of phenylacetic, phenylpropionic and benzoic acids as well as phenols such as catechol and its derivatives from the action of colonic microbiota on cranberry polyphenols. Benzoic acid (38.4µg/ml), 4-hydroxy-5-(3'-hydroxyphenyl)-valeric acid (26.2µg/ml) and phenylacetic acid (19.5µg/ml) reached the highest concentrations. Under the same conditions, microbial degradation of grape seed polyphenols took place to a lesser extent compared to cranberry polyphenols, which was consistent with the more pronounced antimicrobial effect observed for the grape seed polyphenols, particularly against Bacteroides, Prevotella and Blautia coccoides-Eubacterium rectale.

Assessment of the spectrophotometric method for determination of angiotensin-converting-enzyme activity: influence of the inhibition type.

A set of in vitro assay conditions were selected for the determination of ACE-inhibitory activity, and the need was demonstrated to standardize this assay so that the results obtained by different authors may be comparable. The conditions selected were as follows: 10 mM HHL concentration in 0.2 M potassium phosphate buffer and 0.3 M NaCl and 26 mU of ACE/mL as reaction medium; incubation time, 80 min at 37 degrees C. The method was applied to the study of ACE-inhibitory activity of dairy product and wine samples. Of the samples assayed, it was infant formulae whey that produces the greatest ACE inhibition. Red wine also presents a high inhibition percentage. This latter sample has an important matrix effect that must be corrected in the calculation. ACE-inhibition type was also studied, using a yogurt whey and a Captropil solution as substrates. The whey produced noncompetitive inhibition and the Captropil competitive inhibition.

Study of low molecular weight phenolic compounds during the aging of sparkling wines manufactured with red and white grape varieties.

Thirty-two phenolic compounds of low molecular weight were identified in 36 white, blanc de noir, and rosé sparkling wines by using HPLC with photodiode array and mass spectrometry detection. Some of the identified compounds, such as cis- and trans-ethylcaftaric, cis- and trans-ethylcaffeic, and cis- or trans-ethyl-p-coumaric acids, 2R,3R-dihydroquercetin, 2R,3R-dihydrokaempferol 3-O-beta-d-glucoside, and a lignan derivative are described for the first time in sparkling wines manufactured with grapes of red varieties. This is also the first time that cis- or trans-diethylfertaric acids have been identified in wines. When cluster analysis was applied to the data of 19 of the 32 identified compounds, the greatest differences found in the low molecular weight phenolic compounds in sparkling wines were due to the grape variety from which they were manufactured, whereas aging time did not significantly influence phenolic composition. Nine phenolic compounds, that is, trans-p-coumaric and trans-caftaric acids, trans-resveratrol glucoside, cis-coutaric, trans-coutaric, cis-p-coumaric, and cis-caftaric acids, tryptophol, and syringic acid, permit the wines to be classified correctly in accordance with the grape variety from which they were manufactured.

Impact of the nonvolatile wine matrix composition on the in vivo aroma release from wines.

The impact of the nonvolatile wine matrix composition on the retronasal aroma release of four volatile compounds added to different types of wines has been evaluated. For this purpose, a tailor-made retronasal aroma trapping device (RATD) was used to entrap the exhaled breath of six panelists previously trained in a specific consumption procedure. Five wines of different composition (white wine, sparkling white wine, young red wine, aged red wine, and a sweet wine) were evaluated. Prior to the evaluation, with the exception of the sweet wine, the wines were adjusted to the same ethanol content and aromatized with a mixture of four target volatile compounds. Aroma release data were submitted to multivariate statistical analysis in order to relate wine chemical composition and aroma release during wine drinking. Results showed interindividual differences and a clustering of panelists among lower and higher aroma releasers, which was in agreement to the differences in their breathing capacity. A significant influence of the matrix composition in the low aroma releasers group during wine consumption was observed. The consumption of red wines provoked a significantly higher aroma release than the consumption of white and sweet wines. From the chemical composition determined in the wine samples (pH, total acidity, total polyphenols, neutral polysaccharides, residual sugar, and nitrogenous compounds), the amount of total polyphenols was better correlated with the observed effect.

Moderate consumption of red wine can modulate human intestinal inflammatory response.

In this study, 24 immune markers were analyzed in feces from healthy volunteers (n = 34) before and after consumption of a red wine (12% ethanol, 1758 mg/L total polyphenols) for 4 weeks. Analysis of the data permitted the differentiation of a six-volunteer subgroup showing unusually high basal values of cytokines. For this subgroup, consumption of wine significantly (P < 0.05) reduced the content of 16 markers to usual values, especially noticeable for those cytokines that promote initial inflammation (TNF-α, IL-6, and IFN-γ). On the contrary, no significant differences in the concentration of any immune marker were observed after wine consumption for the rest of the volunteers. Additionally, significant and negative correlations among cytokines IFN-γ, IL-8, and IL-6 and the total fecal content of phenolic metabolites were found for the high-cytokines-values subgroup, before wine intake. This study shows, for the first time, that moderate consumption of red wine could modulate inflammatory intestinal response in vivo.

Understanding the role of saliva in aroma release from wine by using static and dynamic headspace conditions.

The aim of this work was to determine the role of saliva in wine aroma release by using static and dynamic headspace conditions. In the latter conditions, two different sampling points (t = 0 and t = 10 min) corresponding with oral (25.5 °C) and postoral phases (36 °C) were monitored. Both methodologies were applied to reconstituted dearomatized white and red wines with different nonvolatile wine matrix compositions and a synthetic wine (without matrix effect). All of the wines had the same ethanol concentration and were spiked with a mixture of 45 aroma compounds covering a wide range of physicochemical characteristics at typical wine concentrations. Two types of saliva (human and artificial) or control samples (water) were added to the wines. The adequacy of the two headspace methodologies for the purposes of the study (repeatability, linear ranges, determination coefficients, etc.) was previously determined. After application of different chemometric analysis (ANOVA, LSD, PCA), results showed a significant effect of saliva on aroma release dependent on saliva type (differences between artificial and human) and on wine matrix using static headspace conditions. Red wines were more affected than white and synthetic wines by saliva, specifically human saliva, which provoked a reduction in aroma release for most of the assayed aroma compounds independent of their chemical structure. The application of dynamic headspace conditions using a saliva bioreactor at the two different sampling points (t = 0 and t = 10 min) showed a lesser but significant effect of saliva than matrix composition and a high influence of temperature (oral and postoral phases) on aroma release.