Epicatechin Metabolites Research Articles

Gene expression changes by high-polyphenols cocoa powder intake: a randomized crossover clinical study

PurposeTo assess the effect of the intake of a single dose of high-polyphenols cocoa on gene expression in peripheral mononuclear cells (PBMCs), and analyze conjugated (−)-epicatechin metabolites in plasma, which may be related with an antioxidant response in healthy human.MethodsA randomized, controlled, double-blind, cross-over, clinical trial in healthy young adults who consumed a single dose of high-polyphenols cocoa powder and maltodextrins as control, with a one-week washout period. Analysis of circulating metabolites, plasma antioxidant capacity and gene expression changes in PBMCs were performed under fasting conditions and 2-h after treatment using microarray in a subsample. Pathway analysis was conducted using Ingenuity Pathway Analysis (IPA).ResultsTwenty healthy participants (9 F) were included in the study. A significant increase in circulating (−)-epicatechin metabolites was found after cocoa intake in all participants without related changes in antioxidant capacity of plasma. The metabolites profile slightly varied across subjects. Treatments triggered different transcriptional changes in PBMC. A group of 98 genes showed changes in expression after cocoa treatment, while only 18 were modified by control. Differentially expressed genes included inflammatory cytokines and other molecules involved in redox balance. Gene and network analysis after cocoa intake converged in functions annotated as decreased production of reactive oxygen species (p = 9.58E−04), decreased leukocyte activation (p = 4E−03) and calcium mobilization (p = 2.51E–05).ConclusionsNo association was found between conjugated metabolites in plasma and antioxidant capacity. Changes in PBMCs gene expression suggest anti-inflammatory effects.

Use of LC-MS for the quantitative analysis of (poly)phenol metabolites does not necessarily yield accurate results: Implications for assessing existing data and conducting future research

Plant-derived, dietary (poly)phenols have potential effects on disease-risk reduction and primary disease prevention. The characterization of (poly)phenol absorption, distribution, metabolism and excretion (ADME) is recognized as crucial step to further advance nutritional and biomedical research of these compounds; and given that (poly)phenols are extensively metabolized after ingestion, accurate assessments of their in vivo metabolites is required. It has become common practice to use unmetabolized parent compounds as reference standards when quantifying (poly)phenol metabolites by LC-MS, although little is known about the accuracy of this approach. To investigate this situation with routinely used LC-MS conditions, the signal yielded by the flavan-3-ol (−)-epicatechin was compared to those of authentic standards of its phase II and microbiota-derived metabolites. The results obtained revealed underestimations up to 94% and overestimations up to 113% of individual epicatechin metabolites. Inaccurate quantitative estimates were also obtained when phase II metabolites of other (poly)phenols were quantified by reference to their unmetabolized parent compounds. This demonstrates the importance of using structurally-identical authentic metabolites as reference compounds when quantifying (poly)phenol metabolites by LC-MS. This is of importance, not just to the accuracy of ADME studies, but for the identification and validation of (poly)phenol metabolites as biomarkers of intake in epidemiological studies.

OP-39 - NADPH oxidase is a target for (-)-epicatechin in the prevention of fatty acid-induced insulin resistance

While diets rich in fruit and vegetables can decrease the risk for type 2 diabetes (T2D), diets rich in fat and carbohydrates can increase it. The flavanol-3-ol (-)-epicatechin (EC) can improve insulin sensitivity both in humans and animal models of T2D. NADPH oxidases and oxidative stress can contribute to the development of insulin resistance. This study investigated the capacity of EC and EC metabolites (ECM) to downregulate NADPH oxidases and oxidative stress, and its association to an improvement of insulin sensitivity. This was studied in in vivo (high fat-fed mice) and in vitro (HepG2 cells) conditions of hepatic lipid overload. EC decreased NOX3/NOX4 liver expression and mitigated oxidative stress in high fat-fed mice. In HepG2 cells, incubation with palmitate increased: i) lipid deposition, ii) NOX3/NOX4 expression, iii) NADPH oxidase activity, and iv) oxidative stress; promoting v) the activation of redox-sensitive kinases (JNK and IKK), and vi) impaired insulin responses. Physiological concentrations of EC and ECM, and NADPH oxidase inhibitors (apocynin, VAS2870) prevented all those deleterious effects of palmitate. The obtained results points to NADPH oxidases as an important target in the capacity of EC to prevent Western style diet-induced insulin resistance.

(-)-Epicatechin and its metabolites prevent palmitate-induced NADPH oxidase upregulation, oxidative stress and insulin resistance in HepG2 cells

While diets rich in fruit and vegetables can decrease the risk for type 2 diabetes (T2D), diets rich in fat and carbohydrates can increase it. The flavanol-3-ol (-)-epicatechin (EC) can improve insulin sensitivity both in humans and animal models of T2D. NADPH oxidases and oxidative stress can contribute to the development of insulin resistance. This study investigated the capacity of EC and EC metabolites (ECM) to downregulate NADPH oxidases and oxidative stress, and its association to an improvement of insulin sensitivity. This was studied in in vivo (high fat-fed mice) and in vitro (HepG2 cells) conditions of hepatic lipid overload. EC decreased NOX3/NOX4 liver expression and mitigated oxidative stress in high fat-fed mice. In HepG2 cells, incubation with palmitate increased: i) lipid deposition, ii) NOX3/NOX4 expression, iii) NADPH oxidase activity, and iv) oxidative stress; promoting v) the activation of redox-sensitive kinases (JNK and IKK), and vi) impaired insulin responses. Physiological concentrations of EC and ECM, and NADPH oxidase inhibitors (apocynin, VAS2870) prevented all those deleterious effects of palmitate. The obtained results points to NADPH oxidases as an important target in the capacity of EC to improve insulin sensitivity in conditions of liver lipid overload, as those associated with Western-style diets.

Profiling and identification of (-)-epicatechin metabolites in rats using ultra-high performance liquid chromatography coupled with linear trap-Orbitrap mass spectrometer.

(-)-Epicatechin (EC), an optical antipode of (+)-catechin (C), possesses many potential significant health benefits. However, the in vivo metabolic pathway of EC has not been clarified yet. In this study, an efficient strategy based on ultra-high performance liquid chromatography coupled with a linear ion trap-Orbitrap mass spectrometer was developed to profile and characterize EC metabolites in rat urine, faeces, plasma, and various tissues. Meanwhile, post-acquisition data-mining methods including high-resolution extracted ion chromatogram (HREIC), multiple mass defect filters (MMDFs), and diagnostic product ions (DPIs) were utilized to screen and identify EC metabolites from HR-ESI-MS1 to ESI-MSn stage. Finally, a total of 67 metabolites (including parent drug) were tentatively identified based on standard substances, chromatographic retention times, accurate mass measurement, and relevant drug biotransformation knowledge. The results demonstrated that EC underwent multiple in vivo metabolic reactions including methylation, dehydration, hydrogenation, glucosylation, sulfonation, glucuronidation, ring-cleavage, and their composite reactions. Among them, methylation, dehydration, glucosylation, and their composite reactions were observed only occurring on EC when compared with C. Meanwhile, the distribution of these detected metabolites in various tissues including heart, liver, spleen, lung, kidney, and brain were respectively studied. The results demonstrated that liver and kidney were the most important organs for EC and its metabolites elimination. In conclusion, the newly discovered EC metabolites significantly expanded the understanding on its pharmacological effects and built the foundation for further toxicity and safety studies. Copyright © 2017 John Wiley & Sons, Ltd.

Methylxanthines enhance the effects of cocoa flavanols on cardiovascular function: randomized, double-masked controlled studies

Cocoa flavanol intake, especially that of (-)-epicatechin, has been linked to beneficial effects on human cardiovascular function. However, cocoa also contains the methylxanthines theobromine and caffeine, which may also affect vascular function. We sought to determine whether an interaction between cocoa flavanols and methylxanthines exists that influences cocoa flavanol-dependent vascular effects. Test drinks that contained various amounts of cocoa flavanols (0-820 mg) and methylxanthines (0-220 mg), either together or individually, were consumed by healthy volunteers (n = 47) in 4 different clinical studies-3 with a randomized, double-masked crossover design and 1 with 4 parallel crossover studies. Vascular status was assessed by measuring flow-mediated vasodilation (FMD), brachial pulse wave velocity (bPWV), circulating angiogenic cells (CACs), and blood pressure before and 2 h after the ingestion of test drinks. Although cocoa flavanol intake increased FMD 2 h after intake, the consumption of cocoa flavanols with methylxanthines resulted in a greater enhancement of FMD. Methylxanthine intake alone did not result in statistically significant changes in FMD. Cocoa flavanol ingestion alone decreased bPWV and diastolic blood pressure and increased CACs. Each of these changes was more pronounced when cocoa flavanols and methylxanthines were ingested together. It is important to note that the area under the curve of the plasma concentration of (-)-epicatechin metabolites over time was higher after the co-ingestion of cocoa flavanols and methylxanthines than after the intake of cocoa flavanols alone. Similar results were obtained when pure (-)-epicatechin and the methylxanthines theobromine and caffeine were consumed together. A substantial interaction between cocoa flavanols and methylxanthines exists at the level of absorption, in which the methylxanthines mediate an increased plasma concentration of (-)-epicatechin metabolites that coincides with enhanced vascular effects commonly ascribed to cocoa flavanol intake. This trial was registered at clinicaltrials.gov as NCT02149238.

Inhibitory Activity of Catechin Metabolites Produced by Intestinal Microbiota on Proliferation of HeLa Cells.

Eleven kinds of catechin metabolites produced from (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCg) by intestinal microbiota were evaluated for inhibitory activity on the proliferation of HeLa cells, which are human cervical cancer cells. Among the catechin metabolites, 1-(3,4,5-trihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (EGC-M2), 4-hydroxy-5-(3,4,5-trihydroxyphenyl)valeric acid (EGC-M7), and 5-(3,4,5-trihydroxyphenyl)valeric acid (EGC-M9) were found to show inhibitory activity on HeLa cell proliferation as compared with control. The results suggested that three adjacent hydroxyl groups in the phenyl moiety may play an important role in the inhibitory activity. In addition, the inhibitory activity was also examined with four (-)-epicatechin (EC) metabolites possessing two adjacent hydroxyl groups in the phenyl moiety. Only 5-(3,4-dihydroxyphenyl)valeric acid (EC-M9) showed inhibitory activity and therefore valeric acid moiety likely contributes to the inhibitory activity. EGC-M9 showed the strongest inhibitory activity with IC50 of 5.58 µM. Thus, in this study it was found for the first time that several catechin metabolites derived from EGC, EGCg, and EC inhibit the proliferation of cervical cancer cells.

Does epicatechin contribute to the acute vascular function effects of dark chocolate? A randomized, crossover study.

Cocoa, rich in flavan-3-ols, improves vascular function, but the contribution of specific flavan-3-ols is unknown. We compared the effects of pure epicatechin, a major cocoa flavan-3-ol, and chocolate. In a randomized crossover study, twenty healthy men (40-80 years) were supplemented with: (1) 70g dark chocolate (150 mg epicatechin) with placebo capsules; (2) pure epicatechin capsules (2 × 50 mg epicatechin) with 75g white chocolate; and (3) placebo capsules with 75 g white chocolate (0 mg epicatechin). Vascular function (flow-mediated dilation (FMD) and augmentation index (AIx)) were measured before and 2 hours after interventions. Epicatechin metabolites time-profiles were measured in blood to calculate the bioavailability. Pure epicatechin did not significantly improve FMD (+0.75%; p = 0.10) or AIx (-2.2%; p = 0.23) compared to placebo. Dark chocolate significantly improved FMD (+0.96%; p = 0.04) and AIx (-4.6%; p = 0.02). Differences in improvements in FMD (+ 0.21%; p = 0.65) or Aix (-2.4%; p = 0.20) between pure epicatechin and dark chocolate were not significant. The bioavailability of epicatechin did not differ between pure epicatechin and dark chocolate (p = 0.14). Despite differences in epicatechin dose, improvements in vascular function after pure epicatechin and chocolate were similar and the bioavailability did not differ, suggesting a role for epicatechin.

Dietary Epicatechin Is Available to Breastfed Infants through Human Breast Milk in the Form of Host and Microbial Metabolites

Polyphenols play an important role in human health. To address their accessibility to a breastfed infant, we planned to evaluate whether breast milk (BM) (colostrum, transitional, and mature) epicatechin metabolites could be related to the dietary habits of mothers. The polyphenol consumption of breastfeeding mothers was estimated using a food frequency questionnaire and 24 h recalls. Solid-phase extraction-ultra performance liquid chromatography-tandem mass spectrometry (SPE-UPLC-MS/MS) was applied for direct epicatechin metabolite analysis. Their bioavailability in BM as a result of dietary ingestion was confirmed in a preliminary experiment with a single dose of dark chocolate. Several host and microbial phase II metabolites of epicatechin were detected in BM among free-living lactating mothers. Interestingly, a modest correlation between dihydroxyvalerolactone sulfate and the intake of cocoa products was observed. Although a very low percentage of dietary polyphenols is excreted in BM, they are definitely in the diet of breastfed infants. Therefore, evaluation of their role in infant health could be further promoted.

Synthesis and quantitative analysis of plasma-targeted metabolites of catechin and epicatechin.

Grape seed polyphenolic extract (GSPE) rich in the flavan-3-ols (+)-catechin and (-)-epicatechin beneficially modulates Alzheimer's Disease phenotypes in animal models. The parent molecules in the extract are converted to a series of methylated and glucuronidated derivatives. To fully characterize these metabolites and establish a robust quantitative assay of their levels in biological fluids, we have implemented a partial synthetic approach utilizing chemical methylation followed by enzymatic glucuronidation. Liquid chromatography/time-of-flight mass spectrometry (LC-TOF-MS) and nuclear magnetic resonance (NMR) spectroscopy were used to assign unequivocal structures to the compounds. An analytical method using solid-phase extraction and LC-MS/MS in selective reaction monitoring mode (SRM) was validated for their quantitation in plasma. These studies provide a basis for improvements in future work on the bioavailability, metabolism, and mechanism of action of metabolites derived from dietary flavan-3-ols in a range of interventions.

Microbial catabolism of procyanidins by human gut microbiota.

A major portion of ingested procyanidins is degraded by human microbiota in the colon into various phenolic compounds. These microbial metabolites are thought to contribute to the health benefits of procyanidins in vivo. The objective of this study was to identify and quantify the microbial metabolites of procyanidins after anaerobic fermentation with human microbiota. (-)-Epicatechin, (+)-catechin, procyanidin B2, procyanidin A2, partially purified apple and cranberry procyanidins were incubated with human microbiota at a concentration equivalent to 0.5 mM epicatechin. GC-MS analysis showed that common metabolites of all six substrates were benzoic acid, 2-phenylacetic acid, 3-phenylpropionic acid, 2-(3'-hydroxyphenyl)acetic acid, 2-(4'-hydroxyphenyl)acetic acid, 3-(3'-hydroxyphenyl)propionic acid, and hydroxyphenylvaleric acid. 5-(3',4'-Dihydroxyphenyl)-γ-valerolactones and 5-(3'-hydroxyphenyl)-γ-valerolactones were identified as the microbial metabolites of epicatechin, catechin, procyanidin B2, and apple procyanidins but not from the procyanidin A2 or cranberry procyanidin ferments. 2-(3',4'-Dihydroxyphenyl)acetic acid was only found in the fermented broth of procyanidin B2, A2, apple, and cranberry procyanidins. The mass recoveries of microbial metabolites range from 20.0 to 56.9% for the six substrates after 24 h of fermentation. Procyanidins, both B-type and A-type can be degraded by human gut microbiota. The microbial metabolites may contribute to the bioactivities of procyanidins.

Identification of epicatechin as one of the key bioactive constituents of polyphenol-enriched extracts that demonstrate an anti-allergic effect in a murine model of food allergy.

Polyphenols are naturally derived bioactive compounds with numerous reported health benefits. We have previously reported on the beneficial effect of a polyphenol-enriched apple extract in a murine model of food allergy. The objectives of the present study were to elucidate the class of bioactive polyphenols that exhibit a beneficial anti-allergic effect and to assess whether the protective effect matches the in vivo bioavailable metabolite concentrations. Female BALB/c mice were sensitised to ovalbumin (OVA) following the protocol of a well-established murine model of food allergy. They were fed diets containing polyphenol-enriched extracts or purified epicatechin for 8 d after the last sensitisation. The sensitised mice were orally challenged with OVA after the intervention. The allergy symptoms, in addition to allergen-specific serum Ig concentrations and gene expression profiles in the intestine, of the control and treated mice were compared. Plasma samples were collected to compare the concentrations of bioavailable epicatechin metabolites in the treatment groups. Polyphenol-enriched fruit extracts containing epicatechin exhibited a significant anti-allergic effect in vivo. This effect was unambiguously attributed to epicatechin, as oral administration of this purified polyphenol to sensitised mice by inclusion in their diet modulated allergy symptoms in a dose-dependent manner. Immune parameters were also affected by the administration of epicatechin. Bioavailability measurements in plasma indicated that the attenuation of allergy symptoms could be due to the higher concentrations of bioavailable epicatechin metabolites. In conclusion, epicatechin is a key bioactive polyphenol that has the ability to modulate allergy outcomes in sensitised mice.

Catabolism of (+)-Catechin and (−)-Epicatechin by Rat Intestinal Microbiota

Catabolism of (+)-catechin (+C) and (-)-epicatechin (EC) by rat intestinal microbiota was examined in vitro. +C and EC metabolites isolated were identified by LC-MS and NMR analyses. As a result, 4-hydroxy-5-(3-hydroxyphenyl)valeric acid (C-5 and EC-5), 4-oxo-5-(3,4-dihydorxyphenyl)valeric acid (EC-7), 4-oxo-5-(3-hydorxyphenyl)valeric acid (EC-8), and 1-(4-hydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (EC-13) were identified as new metabolites of +C or EC. From the measurement of optical rotation, each of the +C and EC metabolites possessing the same chemical structure and chiral carbon was inferred to have an enantiomeric relationship to each other and to maintain the configurations at the 3-position of the original catechins. On the basis of these findings together with previous information, the proposed metabolic pathway of +C and EC by rat intestinal microbiota was updated.

P69: Analysis of acute and chronic effects of (−)-epicatechin on NO bioavailability and organ redox state in the cardiovascular system in vivo

Background and Hypothesis Consumption of cocoa flavanols may have beneficial effects on cardiovascular health. (−)-Epicatechin, the major flavanol monomer present in cocoa, has been shown to positively affect endothelial function and a (−)-epicatechin-rich cocoa drink increased the circulating nitric oxide (NO) metabolites in humans. There is also evidence that flavanols and flavonoids may activate the expression and the activity of Nrf2-dependent phase II genes in cultured non-endothelial cells. We hypothesized that (−)-epicatechin may induce a systemic Nrf2-dependent antioxidant response, thereby contributing to maintain the redox state, NO bioavailability, and the functionality of the vasculature. Methods In vivo effects of acute and chronic oral administration of (−)-epicatechin were analyzed in C57/Bl6 mice. (−)-Epicatechin was administrated at 1–10 mg/kg body weight (BW) intragastrically and the effects analyzed after 1 h. Chronic effects were assessed after 5 days (−)-epicatechin administration of 2 and 10 mg/kg BW in drinking water. Vascular function was assessed by measuring reactive hyperemic response of the hindlimb by laser Doppler perfusion imaging (LDPI). Chronic inhibition of nitric oxide synthase (NOS) was achieved by administration of the specific inhibitor l -NNA in drinking water for 6 days. Circulating levels of (−)-epicatechin metabolites were determined with HPLC. In vitro effects of (−)-epicatechin and its main plasma metabolites present in humans were analyzed in human umbilical vein endothelial cells (HUVECs). The H 2 O 2 concentration in media was measured by a peroxide assay. The activation of Nrf2 by epicatechin was measured by immunohistochemistry and confocal microscopy of cells cultured on cover slips, Western blot analysis of nuclear and cytoplasmic extracts, as well as by ARE (antioxidant responsive element)-binding assay (TransAM). The expression of eNOS and of phase II genes was assessed in cells and organs by real time RT-PCR and Western blot analysis. Phosphorylation of eNOS was assessed by Western blot analysis. The concentration of reduced glutathione in mouse organs were assessed by the glutathione reductase recycling assay. Results Acute administration of (−)-epicatechin dose-dependently increased the circulating levels of epicatechin metabolites along with improved vascular response to reactive hyperemia of the hindlimb. This was paralleled by an increase in eNOS phosphorylation in the aorta of the mice, indicating that (−)-epicatechin application induces NOS activation. Chronic treatment with (−)-epicatechin improved vascular response in a NOS-dependent fashion, and increased cGMP levels in aorta and plasma of the mice, demonstrating enhanced NO bioavailability. Interestingly, we observed a parallel increase in the expression of Nrf2-dependent phase II genes, including glutamate cysteine ligase (Gclc), and GSH levels in the heart and lung of the mice. In vitro we found that (−)-epicatechin induced the translocation of Nrf2 into the nucleus of the cells and the binding to the ARE. Moreover,(−)-epicatechin and its main plasma metabolites increased expression of GCL (mRNA and protein), as well as eNOS expression and phosphorylation. Conclusions Taken together, our findings demonstrate that administration of (−)-epicatechin leads to an increase of systemic NO bioavailability and vascular response in vivo and activates eNOS and Nrf2-dependent pathways. Disclosure Nothing to disclose.

Chocolate may attenuate cognitive deterioration in Alzheimer's disease through prevention of gene expression related to depressive disorder

Chocolate has been shown to improve cognitive behavior. In particular, there is evidence that it may reduce dementia and attenuate cognitive deterioration in experimental models of Alzheimer's disease (AD) as well as in elderly patients with mild cognitive impairment. One condition associated with onset of experimental AD mouse models is characterized by depression and anxiety. A recent study suggests that the brain of a subject with major depressive disorder may be characterized by lower brain volume and major synaptic loss in humans. In addition, recent evidence suggests that experimental models of depression are characterized by decreased expression of downstream genes associated with transcriptional factor GATA1, which are genes associated with synaptic plasticity. Ongoing studies in our laboratory suggest that certain preparations of natural cocoa with enriched epicatechin levels may promote GATA1‐mediated transcription of genes associated with brain plasticity coincidental with promotion of long‐term potentiation. Our studies will clarify the role of chocolate epicatechin metabolites in the modulation of depression‐related genes in a mouse model of AD.

Chemical Synthesis and Characterization of Epicatechin Glucuronides and Sulfates: Bioanalytical Standards for Epicatechin Metabolite Identification

The monoglucuronides and sulfates of epicatechin, 3'-O-methylepicatechin, and 4'-O-methylepicatechin, respectively, were synthesized as authentic bioanalytical standards. Reversed-phase HPLC methods capable of baseline separation of the glucuronides and sulfates have been developed. Both the epicatechin glucuronides and sulfates were stable in the solid state when stored under ambient conditions and in aqueous solution when stored refrigerated. These results should prove invaluable to the research community as analytical standards as well as in future studies of the biological and pharmacological effects of epicatechin in humans.

Quantitative analysis of epicatechin metabolites in human serum using UHPLC-MS-MS

Flavanols such as epicatechin occurring in the seeds of Theobroma cacao (cocoa beans) are associated with beneficial health effects. When cocoa seed-based products are ingested, epicatechin is absorbed and converted into epicatechin glucuronide, epicatechin sulfates, and epicatechin methylsulfates, which can be found in human serum. A UHPLC-MS-MS method was developed for rapid quantitative analysis of epicatechin glucuronide, epicatechin sulfates, and epicatechin methylsulfates in serum. The concentrations of the epicatechin metabolites in blood serum were measured using a Shimadzu Nexera UHPLC with an LCMS-8030 triple quadrupole mass spectrometer with collision-induced dissociation and selected reaction monitoring.

Elucidation of (−)-epicatechin metabolites after ingestion of chocolate by healthy humans

After absorption in the gastrointestinal tract, (−)-epicatechin is extensively transformed into various conjugated metabolites. These metabolites, chemically different from the aglycone forms found in foods, are the compounds that reach the circulatory system and the target organs. Therefore, it is imperative to identify and quantify these circulating metabolites to investigate their roles in the biological effects associated with (−)-epicatechin intake. Using authentic synthetic standards of (−)-epicatechin sulfates, glucuronides, and O-methyl sulfates, a novel LC–MS/MS–MRM analytical methodology to quantify (−)-epicatechin metabolites in biological matrices was developed and validated. The optimized method was subsequently applied to the analysis of plasma and urine metabolites after consumption of dark chocolate, an (−)-epicatechin-rich food, by humans. (−)-Epicatechin-3′-β-d-glucuronide (Cmax 290±49nM), (−)-epicatechin 3′-sulfate (Cmax 233±60nM), and 3′-O-methyl epicatechin sulfates substituted in the 4′, 5, and 7 positions were the most relevant (−)-epicatechin metabolites in plasma. When plasmatic metabolites were divided into their substituent groups, it was revealed that (−)-epicatechin glucuronides, sulfates, and O-methyl sulfates represented 33±4, 28±5, and 33±4% of total metabolites (AUC0–24h), respectively, after dark chocolate consumption. Similar metabolites were found in urine samples collected over 24h. The total urine excretion of (−)-epicatechin was 20±2% of the amount ingested. In conclusion, we describe the entire metabolite profile and its degree of elimination after administration of (−)-epicatechin-containing food. These results will help us understand more precisely the mechanisms and the main metabolites involved in the beneficial physiological effects of flavanols.

Identification of O-methyl-(−)-epicatechin-O-sulphate metabolites by mass-spectrometry after O-methylation with trimethylsilyldiazomethane

(−)-Epicatechin, an abundant dietary polyphenol found mainly in cocoa and tea, is known to extensively undergo metabolism after ingestion giving rise to a complex series of conjugated metabolites including numerous isomers. In the present study, the combination of fractionation, chemical derivatization and various mass spectrometric approaches is described to determine the exact position of sulphate group in methylated epicatechin metabolites. Four O-methyl-(−)-epicatechin-O-sulphate metabolites isolated from human urine samples were derivatized under mild condition using trimethylsilyldiazomethane (TMSD) in the presence of methanol. The resulting methylated reaction products were then analyzed by high resolution and multistage mass spectrometry for the subsequent identification of the sulphate positional isomers. Results show that O-methylation affects the charge delocalization in negatively charged ions and hereby the fragmentation pattern of the sulphate isomers allowing the identification of diagnostic ions. In addition, this study demonstrates that methoxy derivatives of polyphenol metabolites can be prepared using TMSD. Subsequently, the localization of the sulphate group in the polyphenol metabolites can be achieved by analyzing the methoxy derivatives by multistage mass spectrometry. Using an enzymatic reaction for identification of the O-methyl position, and a chemical O-methylation with TMSD follow by high resolution and multistage tandem MS for the identification of the sulphate group position, we were able to identify the previously unknown O-methyl-(−)-epicatechin-O-sulphate. Accordingly, we identified 3′-O-methyl-(−)-epicatechin-5-O-sulphate and 3′-O-methyl-(−)-epicatechin-7-O-sulphate as the main O-methyl-(−)-epicatechin-sulfates(−)-epicatechin metabolites in humans.

Brain-Targeted Proanthocyanidin Metabolites for Alzheimer's Disease Treatment

While polyphenolic compounds have many health benefits, the potential development of polyphenols for the prevention/treatment of neurological disorders is largely hindered by their complexity as well as by limited knowledge regarding their bioavailability, metabolism, and bioactivity, especially in the brain. We recently demonstrated that dietary supplementation with a specific grape-derived polyphenolic preparation (GP) significantly improves cognitive function in a mouse model of Alzheimer's disease (AD). GP is comprised of the proanthocyanidin (PAC) catechin and epicatechin in monomeric (Mo), oligomeric, and polymeric forms. In this study, we report that following oral administration of the independent GP forms, only Mo is able to improve cognitive function and only Mo metabolites can selectively reach and accumulate in the brain at a concentration of ∼400 nM. Most importantly, we report for the first time that a biosynthetic epicatechin metabolite, 3'-O-methyl-epicatechin-5-O-β-glucuronide (3'-O-Me-EC-Gluc), one of the PAC metabolites identified in the brain following Mo treatment, promotes basal synaptic transmission and long-term potentiation at physiologically relevant concentrations in hippocampus slices through mechanisms associated with cAMP response element binding protein (CREB) signaling. Our studies suggest that select brain-targeted PAC metabolites benefit cognition by improving synaptic plasticity in the brain, and provide impetus to develop 3'-O-Me-EC-Gluc and other brain-targeted PAC metabolites to promote learning and memory in AD and other forms of dementia.