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Scope: The physiological relevance of contemporary cell culture studies is often perplexing, given the use of unmetabolized phytochemicals at supraphysiological concentrations. We investigated the activity of physiologically relevant anthocyanin metabolite signatures, derived from a previous pharmacokinetics study of 500 mg 13 C 5 -cyanidin-3-glucoside in eight healthy participants, on soluble vascular adhesion molecule-1 (VCAM-1) and interleukin-6 (IL-6) in human endothelial cells., Methods and Results: Signatures of peak metabolites (previously identified at 1, 6, and 24 h post-bolus) were reproduced using pure standards and effects were investigated across concentrations ten-fold lower and higher than observed mean (<5 μM) serum levels. Tumor necrosis factor-α (TNF-α)-stimulated VCAM-1 was reduced in response to all treatments, with maximal effects observed for the 6 and 24 h profiles. Profiles tested at ten-fold below mean serum concentrations (0.19-0.44 μM) remained active. IL-6 was reduced in response to 1, 6, and 24 h profiles, with maximal effects observed for 6 h and 24 h profiles at concentrations above 2 μM. Protein responses were reflected by reductions in VCAM-1 and IL-6 mRNA, however there was no effect on phosphorylated NFκB-p65 expression., Conclusion: Signatures of anthocyanin metabolites following dietary consumption reduce VCAM-1 and IL-6 production, providing evidence of physiologically relevant biological activity., (© 2017 The Authors. Molecular Nutrition & Food Research published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Scope: In vitro and in vivo studies suggest that dietary anthocyanins modulate cardiovascular disease risk; however, given anthocyanins extensive metabolism, it is likely that their degradation products and conjugated metabolites are responsible for this reported bioactivity., Methods and Results: Human vascular endothelial cells were stimulated with either oxidized LDL (oxLDL) or cluster of differentiation 40 ligand (CD40L) and cotreated with cyanidin-3-glucoside and 11 of its recently identified metabolites, at 0.1, 1, and 10 μM concentrations. Protein and gene expression of IL-6 and VCAM-1 was quantified by ELISA and RT-qPCR. In oxLDL-stimulated cells the parent anthocyanin had no effect on IL-6 production, whereas numerous anthocyanin metabolites significantly reduced IL-6 protein levels; phase II conjugates of protocatechuic acid produced the greatest effects (>75% reduction, p ≤ 0.05). In CD40L-stimulated cells the anthocyanin and its phase II metabolites reduced IL-6 protein production, where protocatechuic acid-4-sulfate induced the greatest reduction (>96% reduction, p ≤ 0.03). Similarly, the anthocyanin and its metabolites reduced VCAM-1 protein production, with ferulic acid producing the greatest effect (>65% reduction, p ≤ 0.04)., Conclusion: These novel data provide evidence to suggest that anthocyanin metabolites are bioactive at physiologically relevant concentrations and have the potential to modulate cardiovascular disease progression by altering the expression of inflammatory mediators., (© 2015 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background and Purpose: Anthocyanins are phytochemicals with reported vasoactive bioactivity. However, given their instability at neutral pH, they are presumed to undergo significant degradation and subsequent biotransformation. The aim of the present study was to establish the pharmacokinetics of the metabolites of cyanidin-3-glucoside (C3G), a widely consumed dietary phytochemical with potential cardioprotective properties., Experimental Approach: A 500 mg oral bolus dose of 6,8,10,3',5'-(13)C5-C3G was fed to eight healthy male participants, followed by a 48 h collection (0, 0.5, 1, 2, 4, 6, 24, 48 h) of blood, urine and faecal samples. Samples were analysed by HPLC-ESI-MS/MS with elimination kinetics established using non-compartmental pharmacokinetic modelling., Key Results: Seventeen (13)C-labelled compounds were identified in the serum, including (13)C5-C3G, its degradation products, protocatechuic acid (PCA) and phloroglucinaldehyde (PGA), 13 metabolites of PCA and 1 metabolite derived from PGA. The maximal concentrations of the phenolic metabolites (Cmax ) ranged from 10 to 2000 nM, between 2 and 30 h (tmax) post-consumption, with half-lives of elimination observed between 0.5 and 96 h. The major phenolic metabolites identified were hippuric acid and ferulic acid, which peaked in the serum at approximately 16 and 8 h respectively., Conclusions and Implications: Anthocyanins are metabolized to a structurally diverse range of metabolites that exhibit dynamic kinetic profiles. Understanding the elimination kinetics of these metabolites is key to the design of future studies examining their utility in dietary interventions or as therapeutics for disease risk reduction., (© 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.)

Background: Evidence suggests that the consumption of anthocyanin-rich foods beneficially affects cardiovascular health; however, the absorption, distribution, metabolism, and elimination (ADME) of anthocyanin-rich foods are relatively unknown., Objective: We investigated the ADME of a (13)C5-labeled anthocyanin in humans., Design: Eight male participants consumed 500 mg isotopically labeled cyanidin-3-glucoside (6,8,10,3',5'-(13)C5-C3G). Biological samples were collected over 48 h, and (13)C and (13)C-labeled metabolite concentrations were measured by using isotope-ratio mass spectrometry and liquid chromatography-tandem mass spectrometry., Results: The mean ± SE percentage of (13)C recovered in urine, breath, and feces was 43.9 ± 25.9% (range: 15.1-99.3% across participants). The relative bioavailability was 12.38 ± 1.38% (5.37 ± 0.67% excreted in urine and 6.91 ± 1.59% in breath). Maximum rates of (13)C elimination were achieved 30 min after ingestion (32.53 ± 14.24 μg(13)C/h), whereas (13)C-labeled metabolites peaked (maximum serum concentration: 5.97 ± 2.14 μmol/L) at 10.25 ± 4.14 h. The half-life for (13)C-labeled metabolites ranged between 12.44 ± 4.22 and 51.62 ± 22.55 h. (13)C elimination was greatest between 0 and 1 h for urine (90.30 ± 15.28 μg/h), at 6 h for breath (132.87 ± 32.23 μg/h), and between 6 and 24 h for feces (557.28 ± 247.88 μg/h), whereas the highest concentrations of (13)C-labeled metabolites were identified in urine (10.77 ± 4.52 μmol/L) and fecal samples (43.16 ± 18.00 μmol/L) collected between 6 and 24 h. Metabolites were identified as degradation products, phenolic, hippuric, phenylacetic, and phenylpropenoic acids., Conclusion: Anthocyanins are more bioavailable than previously perceived, and their metabolites are present in the circulation for ≤48 h after ingestion. This trial was registered at clinicaltrials.gov as NCT01106729.

Scope: Current research indicates that anthocyanins are primarily degraded to form phenolic acid products. However, no studies have yet demonstrated the metabolic conjugation of these anthocyanin-derived phenolic acids in humans., Methods and Results: Within the present study, a simulated gastrointestinal digestion model was used to evaluate the potential degradation of anthocyanins post-consumption. Subsequently, cyanidin (Cy) and pelargonidin and their degradation products, protocatechuic acid and 4-hydroxybenzoic acid, were incubated in the presence of human liver microsomes to assess their potential to form hepatic glucuronide conjugates. For structural conformation, phenolic glucuronides were chemically synthesised and compared to the microsomal metabolites. During the simulated gastric digestion, anthocyanin glycosides (200 μM) remained stable however their aglycone derivatives were significantly degraded (20% loss), while during subsequent pancreatic/intestinal digestion only pelargonidin-3-glucoside remained stable while cyanidin-3-glucoside (30% loss) and Cy and pelagonidin aglycones were significantly degraded (100% loss, respectively). Following microsomal metabolism, pelargonidin formed 4-hydroxybenzoic acid, which was further metabolised (65%) to form two additional glucuronide conjugates, while Cy formed protocatechuic acid, which was further metabolised (43%) to form three glucuronide conjugates., Conclusions: We propose that following ingestion, anthocyanins may be found in the systemic circulation as free or conjugated phenolic acids, which should be a focus of future dietary interventions., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the present study we investigated the metabolic conversion of cyanidin glycosides in human subjects using solid-phase extraction,HPLC-diode array detector, MS, GC, and enzymic techniques. Volunteers consumed approximately 20 g chokeberry extract containing 1.3 g cyanidin 3-glycosides (899 mg cyanidin 3-galactoside, 321 mg cyanidin 3-arabinoside, 51 mg cyanidin 3-xyloside and 50 mg cyanidin 3-glucoside). Blood samples were drawn at 0, 0.5, 1, and 2 h post-consumption of the extract. Urine samples were also collected at 0, 4-5,and 22-24h. We have confirmed that human subjects have the capacity to metabolise cyanidin 3-glycosides, as we observed at least ten individual anthocyanin metabolites in the urine and serum. Average concentrations of anthocyanins and anthocyanin metabolites in the urine reached levels of 17.9 (range 14.9-20.9) l.mol/l within 5 h post-consumption and persisted in 24h urine samples at levels of 12.1 (range 11.1-13.0) nmol/l. In addition, average total levels of anthocyanins and anthocyanin metabolites detected in the serum were observed at 5917 (range 197.3-986.1) nmol/ within 2h post-consumption. Cyanidin 3-galactoside accounted for 55.4% (9.9(range 7-2-12-6) l.mol/) and 66.0% (390.6 (range 119.4-661-9) nmol V) of the detected anthocyanins in the urine and serum samples,respectively. The metabolites were identified as glucuronide conjugates, as well as methylated and oxidised derivatives of cyanidin 3-galactoside and cyanidin glucuronide. Conjugation probably affects the biological activity of anthocyanins and these metabolic products are likely in part responsible for the reported health benefits associated with the consumption of anthocyanins.