Gut microbial catabolism of grape seed flavan-3-ols by human faecal microbiota. Targetted analysis of precursor compounds, intermediate metabolites and end-products.

In vitro batch culture fermentations were conducted with grape seed polyphenols and human faecal microbiota, in order to monitor both changes in precursor flavan-3-ols and the formation of microbial-derived metabolites. By the application of UPLC-DAD-ESI-TQ MS, monomers, and dimeric and trimeric procyanidins were shown to be degraded during the first 10 h of fermentation, with notable inter-individual differences being observed between fermentations. This period (10 h) also coincided with the maximum formation of intermediate metabolites, such as 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone and 4-hydroxy-5-(3′,4′-dihydroxyphenyl)-valeric acid, and of several phenolic acids, including 3-(3,4-dihydroxyphenyl)-propionic acid, 3,4-dihydroxyphenylacetic acid, 4-hydroxymandelic acid, and gallic acid (5–10 h maximum formation). Later phases of the incubations (10–48 h) were characterised by the appearance of mono- and non-hydroxylated forms of previous metabolites by dehydroxylation reactions. Of particular interest was the detection of γ-valerolactone, which was seen for the first time as a metabolite from the microbial catabolism of flavan-3-ols. Changes registered during fermentation were finally summarised by a principal component analysis (PCA). Results revealed that 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone was a key metabolite in explaining inter-individual differences and delineating the rate and extent of the microbial catabolism of flavan-3-ols, which could finally affect absorption and bioactivity of these compounds.

Assessment of the dietary intake of total flavan-3-ols, monomeric flavan-3-ols, proanthocyanidins and theaflavins in the European Union

Dietary interventions with flavan-3-ols have shown beneficial effects on vascular function. The translation of these findings into the context of the health of the general public requires detailed information on habitual dietary intake. However, only limited data are currently available for European populations. Therefore, in the present study, we assessed the habitual intake of flavan-3-ol monomers, proanthocyanidins (PA) and theaflavins in the European Union (EU) and determined their main food sources using the EFSA (European Food Safety Authority) Comprehensive European Food Consumption Database. Data for adults aged 18–64 years were available from fourteen European countries, and intake was determined using the FLAVIOLA Flavanol Food Composition Database, developed for the present study and based on the latest US Department of Agriculture and Phenol-Explorer databases. The mean habitual intake of flavan-3-ol monomers, theaflavins and PA ranged from 181 mg/d (Czech Republic) to 793 mg/d (Ireland). The highest intakes of flavan-3-ol monomers and theaflavins were observed in Ireland (191/505 mg/d) and the lowest intakes in Spain (24/9 mg/d). In contrast, the daily intake of PA was highest in Spain (175 mg/d) and lowest in The Netherlands (96 mg/d). Main sources were tea (62 %), pome fruits (11 %), berries (3 %) and cocoa products (3 %). Tea was the major single contributor to monomer intake (75 %), followed by pome fruits (6 %). Pome fruits were also the main source of PA (28 %). The present study provides important data on the population-based intake of flavanols in the EU and demonstrates that dietary intake amounts for flavan-3-ol monomers, PA and theaflavins vary significantly across European countries. The average habitual intake of flavan-3-ols is considerably below the amounts used in most dietary intervention studies.

In vitro fermentation of grape seed flavan-3-ol fractions by human faecal microbiota: changes in microbial groups and phenolic metabolites

With the aim of investigating the potential of flavan-3-ols to influence the growth of intestinal bacterial groups, we have carried out the in vitro fermentation, with human faecal microbiota, of two purified fractions from grape seed extract (GSE): GSE-M (70% monomers and 28% procyanidins) and GSE-O (21% monomers and 78 % procyanidins). Samples were collected at 0, 5, 10, 24, 30 and 48 h of fermentation for bacterial enumeration by fluorescent in situ hybridization and for analysis of phenolic metabolites. Both GSE-M and GSE-O fractions promoted growth of Lactobacillus/Enterococcus and decrease in the Clostridium histolyticum group during fermentation, although the effects were only statistically significant with GSE-M for Lactobacillus/Enterococcus (at 5 and 10 h of fermentation) and GSE-O for C. histolyticum (at 10 h of fermentation). Main changes in polyphenol catabolism also occurred during the first 10 h of fermentation, however no significant correlation coefficients (P>0.05) were found between changes in microbial populations and precursor flavan-3-ols or microbial metabolites. Together these data suggest that the flavan-3-ol profile of a particular food source could affect the microbiota composition and its catabolic activity, inducing changes that could in turn affect the bioavailability and potential bioactivity of these compounds.

Associations between flavan-3-ol intake and CVD risk in the Norfolk cohort of the European Prospective Investigation into Cancer (EPIC-Norfolk)

Background: Dietary intervention studies suggest that flavan-3-ol intake can improve vascular function and reduce the risk of cardiovascular diseases (CVD). However, results from prospective studies failed to show a consistent beneficial effect. Objective: To investigate associations between flavan-3-ol intake and CVD risk in the Norfolk arm of the European Prospective Investigation into Cancer and Nutrition (EPIC-Norfolk). Design: Data was available from 24,885 (11,252 men; 13,633 women) participants, recruited between 1993 and 1997 into the EPIC-Norfolk study. Flavan-3-ol intake was assessed using 7-day food diaries and the FLAVIOLA Flavanol Food Composition database. Missing data for plasma cholesterol and vitamin C were imputed using multiple imputation. Associations between flavan-3-ol intake and blood pressure at baseline were determined using linear regression models. Associations with CVD risk were estimated using Cox regression analyses. Results: Median intake of total flavan-3-ols was 1034 mg/d (range: 0 – 8531 mg/d) for men and 970 mg/d (0 – 6695 mg/d) for women, median intake of flavan-3-ol monomers was 233 mg/d (0 – 3248 mg/d) for men and 217 (0 – 2712 mg/d) for women. There were no consistent associations between flavan-3-ol monomer intake and baseline systolic and diastolic blood pressure (BP). After 286,147 person-years of follow up, there were 8463 cardio-vascular events and 1987 CVD related deaths; no consistent association between flavan-3-ol intake and CVD risk (HR 0.93, 95% CI:0.87; 1.00; Q1 vs Q5) or mortality was observed (HR 0.93, 95% CI: 0.84; 1.04). Conclusions: Flavan-3-ol intake in EPIC-Norfolk is not sufficient to achieve a statistically significant reduction in CVD risk.

1H-13C HSQC NMR spectroscopy for estimating procyanidin/prodelphinidin and cis/trans-flavan-3-ol ratios of condensed tannin samples: correlation with thiolysis

Studies with a diverse array of 22 purified condensed tannin (CT) samples from nine plant species demonstrated that procyanidin/prodelphinidin (PC/PD) and cis/trans-flavan-3-ol ratios can be appraised by 1H-13C HSQC NMR spectroscopy. The method was developed from samples containing 44 to ~100% CT, PC/PD ratios ranging from 0/100 to 99/1, and cis/trans ratios from 58/42 to 95/5 as determined by thiolysis with benzyl mercaptan. Integration of cross-peak contours of H/C-6' signals from PC and of H/C-2',6' signals from PD yielded nuclei adjusted estimates that were highly correlated with PC/PD ratios obtained by thiolysis (R2 = 0.99). Cis/trans-flavan-3-ol ratios, obtained by integration of the respective H/C-4 cross-peak contours, were also related to determinations made by thiolysis (R2 = 0.89). Overall, 1H-13C HSQC NMR spectroscopy appears to be a viable alternative to thiolysis for estimating PC/PD and cis/trans ratios of CT, if precautions are taken to avoid integration of cross-peak contours of contaminants.

Metabolism of tea flavonoids in the gastrointestinal tract

There is considerable interest in the bioavailability of flavan-3-ols such as tea catechins and their bioactivity in vivo. Although flavanols such as catechin and epicatechin have long been characterized as powerful antioxidants in vitro, evidence suggests that these compounds undergo significant metabolism and conjugation during absorption in the small intestine and in the colon. In the small intestine these modifications lead primarily to the formation of glucuronide conjugates that are more polar than the parent flavanol and are marked for renal excretion. Other phase II processes lead to the production of O-methylated forms that have reduced antioxidant potential via the methylation of the B-ring catechol. Significant modification of flavanols also occurs in the colon where the resident microflora degrade them to smaller phenolic acids, some of which may be absorbed. Cell, animal and human studies have confirmed such metabolism by the detection of flavanol metabolites in the circulation and tissues. This review will highlight the major sites of flavanol metabolism in the gastrointestinal tract and the processes that give rise to potential bioactive forms of flavan-3-ols in vivo.

Regulation of NF-κB activity in astrocytes: effects of flavonoids at dietary-relevant concentrations

Neuroinflammation plays an important role in the progression of neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease. Sustained activation of nuclear transcription factor κB (NF-κB) is thought to play an important role in the pathogenesis of neurodegenerative disorders. Flavonoids have been shown to possess antioxidant and anti-inflammatory properties and we investigated whether flavonoids, at submicromolar concentrations relevant to their bioavailability from the diet, were able to modulate NF-κB signalling in astrocytes. Using luciferase reporter assays, we found that tumour necrosis factor (TNFα, 150 ng/ml) increased NF-κB-mediated transcription in primary cultures of mouse cortical astrocytes, which was abolished on co-transfection of a dominant-negative IκBα construct. In addition, TNFα increased nuclear localisation of p65 as shown by immunocytochemistry. To investigate potential flavonoid modulation of NF-κB activity, astrocytes were treated with flavonoids from different classes; flavan-3-ols ((−)-epicatechin and (+)-catechin), flavones (luteolin and chrysin), a flavonol (kaempferol) or the flavanones (naringenin and hesperetin) at dietary-relevant concentrations (0.1–1 μM) for 18 h. None of the flavonoids modulated constitutive or TNFα-induced NF-κB activity. Therefore, we conclude that NF-κB signalling in astrocytes is not a major target for flavonoids.

In vitro fermentation of a red wine extract by human gut microbiota: changes in microbial groups and formation of phenolic metabolites

An in vitro batch culture fermentation experiment was conducted with fecal inocula from three healthy volunteers in the presence and absence of a red wine extract. Changes in main bacterial groups were determined by FISH during a 48 h fermentation period. The catabolism of main flavonoids (i.e., flavan-3-ols and anthocyanins) and the formation of a wide a range of phenolic microbial metabolites were determined by a targeted UPLC-PAD-ESI-TQ MS method. Statistical analysis revealed that catechol/pyrocatechol, as well as 4-hydroxy-5-(phenyl)-valeric, 3- and 4-hydroxyphenylacetic, phenylacetic, phenylpropionic, and benzoic acids, showed the greatest increases in concentration during fermentation, whereas 5-(3′-hydroxyphenyl)-γ-valerolactone, its open form 4-hydroxy-5-(3′-hydroxyphenyl)-valeric acid, and 3,4-dihydroxyphenylacetic acid represented the largest interindividual variations in the catabolism of red wine polyphenols. Despite these changes, microbial catabolism did not produce significant changes in the main bacterial groups detected, although a slight inhibition of the Clostridium histolyticum group was observed.

Flavonoid intake in European adults (18 to 64 years)

Background Flavonoids are a group of phenolic secondary plant metabolites that are ubiquitous in plant-based diets. Data from anthropological, observational and intervention studies have shown that many flavonoids are bioactive. For this reason, there is an increasing interest in investigating the potential health effects of these compounds. The translation of these findings into the context of the health of the general public requires detailed information on habitual dietary intake. However, only limited data are currently available for European populations. Objective The objective of this study is to determine the habitual intake and main sources of anthocyanidins, flavanols, flavanones, flavones, flavonols, proanthocyanidins, theaflavins and thearubigins in the European Union. Design We use food consumption data from the European Food Safety Authority (EFSA) and the FLAVIOLA Food Composition Database to estimate intake of flavonoids. Results Mean (±SEM) intake of total flavonoids in Europe was 428±49 mg/d, of which 136±14 mg/d were monomeric compounds. Gallated flavan-3-ols (53±12 mg/d) were the main contributor. The lowest flavonoid intake was observed in Mediterranean countries (monomeric compounds: 95±11 mg/d). The distribution of intake was skewed in many countries, especially in Germany (monomeric flavonoids; mean intake: 181 mg/d; median intake: 3 mg/d). Conclusions The habitual intake of flavonoids in Europe is below the amounts found to have a significant health effect.

Impact of chemical structure of flavanol monomers and condensed tannins on in vitro anthelmintic activity against bovine nematodes

Plants containing condensed tannins (CT) may have potential to control gastrointestinal nematodes (GIN) of cattle. The aim was to investigate the anthelmintic activities of four flavan-3-ols, two galloyl derivatives and 14 purified CT fractions, and to define which structural features of CT determine the anti-parasitic effects against the main cattle nematodes. We used in vitro tests targeting L1 larvae (feeding inhibition assay) and adults (motility assay) of Ostertagia ostertagi and Cooperia oncophora. In the larval feeding inhibition assay, O. ostertagi L1 were significantly more susceptible to all CT fractions than C. oncophora L1. The mean degree of polymerization of CT (i.e. average size) was the most important structural parameter: large CT reduced larval feeding more than small CT. The flavan-3-ols of prodelphinidin (PD)-type tannins had a stronger negative influence on parasite activity than the stereochemistry, i.e. cis- vs trans-configurations, or the presence of a gallate group. In contrast, for C. oncophora high reductions in the motility of larvae and adult worms were strongly related with a higher percentage of PDs within the CT fractions while there was no effect of size. Overall, the size and the percentage of PDs within CT seemed to be the most important parameters that influence anti-parasitic activity.

Structural features of condensed tannins affect in vitro ruminal methane production and fermentation characteristics

An in vitro study was conducted to investigate the effects of condensed tannins (CT) structural properties, i.e. average polymer size (or mean degree of polymerization); percentage of cis flavan-3-ols and percentage of prodelphinidins in CT extracts on methane production (CH4) and fermentation characteristics. CT were extracted from eight plants in order to obtain different CT types: black currant leaves, goat willow leaves, goat willow twigs, pine bark, red currant leaves, sainfoin plants, weeping willow catkins and white clover flowers. They were analysed for CT content and CT composition by thiolytic degradation, followed by HPLC analysis. Grass silage was used as a control substrate. Condensed tannins were added to the substrate at a concentration of 40 g/kg, with or without polyethylene glycol (+ or −PEG 6000 treatment) to inactivate tannins, and then incubated for 72 h in mixed buffered rumen fluid from three different lactating dairy cows per run. Total cumulative gas production (GP) was measured by an automated gas production system. During the incubation, 12 gas samples (10 μl) were collected from each bottle headspace at 0, 2, 4, 6, 8, 12, 24, 30, 36, 48, 56 and 72 h of incubation and analyzed for CH4. A modified Michaelis–Menten model was fitted to the CH4 concentration patterns and model estimates were used to calculate total cumulative CH4 production (GPCH4). Total cumulative gas production and GPCH4 curves were fitted using biphasic and monophasic modified Michaelis-Menten models, respectively. Addition of PEG increased GP, GPCH4, and CH4 concentration compared to the −PEG treatment. All CT types reduced GPCH4 and CH4 concentration. All CT increased the half time of GP and GPCH4. Moreover, all CT decreased the maximum rate of fermentation for GPCH4 and rate of substrate degradation. The correlation between CT structure and GPCH4 and fermentation characteristics showed that the proportion of prodelphinidins within CT had the largest effect on fermentation characteristics, followed by average 27 polymer size and percentage of cis-flavan-3-ols.

Dietary flavonoid (-)epicatechin stimulates phosphatidylinositol 3-kinase-dependent anti-oxidant response element activity and up-regulates glutathione in cortical astrocytes

Flavonoids are plant-derived polyphenolic compounds with neuroprotective properties. Recent work suggests that, in addition to acting as hydrogen donors, they activate protective signalling pathways. The anti-oxidant response element (ARE) promotes the expression of protective proteins including those required for glutathione synthesis (xCT cystine antiporter, gamma-glutamylcysteine synthetase and glutathione synthase). The use of a luciferase reporter (ARE-luc) assay showed that the dietary flavan-3-ol (-)epicatechin activates this pathway in primary cortical astrocytes but not neurones. We also examined the distribution of NF-E2-related factor-2 (Nrf2), a key transcription factor in ARE-mediated gene expression. We found, using immunocytochemistry, that Nrf2 accumulated in the nuclei of astrocytes following exposure to tert-butylhydroquinone (100 mu M) and (-)epicatechin (100 nM). (-)Epicatechin signalling via Nrf2 was inhibited by wortmannin implicating a phosphatidylinositol 3-kinase-dependent pathway. Finally, (-)epicatechin increased glutathione levels in astrocytes consistent with an up-regulation of ARE-mediated gene expression. Together, this suggests that flavonoids may be cytoprotective by increasing anti-oxidant gene expression.

(-)Epicatechin stimulates ERK-dependent cyclic AMP response element activity and up-regulates GluR2 in cortical neurons

Emerging evidence suggests that the cellular actions of flavonoids relate not simply to their antioxidant potential but also to the modulation of protein kinase signalling pathways. We investigated in primary cortical neurons, the ability of the flavan-3-ol, (-)epicatechin, and its human metabolites at physiologically relevant concentrations, to stimulate phosphorylation of the transcription factor cAMP-response element binding protein (CREB), a regulator of neuronal viability and synaptic plasticity. (-)Epicatechin at 100-300 nmol/L stimulated a rapid, extracellular signal-regulated kinase (ERK)- and PI3K-dependent, increase in CREB phosphorylation. At micromolar concentrations, stimulation was no longer apparent and at the highest concentration tested (30 mu mol/L) (-)epicatechin was inhibitory. (-)Epicatechin also stimulated ERK and Akt phosphorylation with similar bell-shaped concentration-response characteristics. The human metabolite 3 '-O-methyl-(-)epicatechin was as effective as (-)epicatechin at stimulating ERK phosphorylation, but (-)epicatechin glucuronide was inactive. (-)Epicatechin and 3 '-O-methyl-(-)epicatechin treatments (100 nmol/L) increased CRE-luciferase activity in cortical neurons in a partially ERK-dependent manner, suggesting the potential to increase CREB-mediated gene expression. mRNA levels of the glutamate receptor subunit GluR2 increased by 60%, measured 18 h after a 15 min exposure to (-)epicatechin and this translated into an increase in GluR2 protein. Thus, (-)epicatechin has the potential to increase CREB-regulated gene expression and increase GluR2 levels and thus modulate neurotransmission, plasticity and synaptogenesis.

In situ analysis and structural elucidation of sainfoin (Onobrychis viciifolia) tannins for high-throughput germplasm screening

A rapid thiolytic degradation and cleanup procedure was developed for analyzing tannins directly in chlorophyll-containing sainfoin (Onobrychis viciifolia) plants. The technique proved suitable for complex tannin mixtures containing catechin, epicatechin, gallocatechin, and epigallocatechin flavan-3-ol units. The reaction time was standardized at 60 min to minimize the loss of structural information as a result of epimerization and degradation of terminal flavan-3-ol units. The results were evaluated by separate analysis of extractable and unextractable tannins, which accounted for 63.6−113.7% of the in situ plant tannins. It is of note that 70% aqueous acetone extracted tannins with a lower mean degree of polymerization (mDP) than was found for tannins analyzed in situ. Extractable tannins had between 4 and 29 lower mDP values. The method was validated by comparing results from individual and mixed sample sets. The tannin composition of different sainfoin accessions covered a range of mDP values from 16 to 83, procyanidin/prodelphinidin (PC/PD) ratios from 19.2/80.8 to 45.6/54.4, and cis/trans ratios from 74.1/25.9 to 88.0/12.0. This is the first high-throughput screening method that is suitable for analyzing condensed tannin contents and structural composition directly in green plant tissue.

Estimated dietary intakes and sources of flavanols in the German population (German National Nutrition Survey II)

Background/Objectives Data from intervention studies suggest a beneficial effect of flavanols on vascular health. However, insufficient data on their intake have delayed the assessment of their health benefits. The aim of this study was to estimate intake of flavanols and their main sources among people living in Germany. Subjects/Methods Data from diet history interviews of the German National Nutrition Survey II for 15,371 people across Germany aged 14–80 years were analyzed. The FLAVIOLA Flavanol Food Composition Database was compiled using the latest US Department of Agriculture and Phenol-Explorer Databases and expanded to include recipes and retention factors. Results Mean intake of total flavanols, flavan-3-ol monomers, proanthocyanidins (PA), and theaflavins in Germany was 386, 120, 196, and 70 mg/day, respectively. Women had higher intakes of total flavanols (399 mg/day) than men (372 mg/day) in all age groups, with the exception of the elderly. Similar results were observed for monomers (108 mg/day for men, 131 mg/day for women) and PA (190 mg/day; 203 mg/day), although intake of theaflavins was higher in men (74 mg/day; 66 mg/day). There was an age gradient with an increase in total flavanols, monomers, and theaflavins across the age groups. The major contributor of total flavanols in all subjects was pome fruits (27 %) followed by black tea (25 %). Conclusions This study demonstrated age- and sex-related variations in the intake and sources of dietary flavanols in Germany. The current analysis will provide a valuable tool in clarifying and confirming the potential health benefits of flavanols.