Phenolic metabolites of anthocyanins following a dietary intervention study in post-menopausal women

Scope Numerous studies feeding anthocyanin-rich foods report limited bioavailability of the parent anthocyanins. The present study explores the identity and concentration of the phenolic metabolites of anthocyanins in humans. Methods and results Anthocyanin metabolites were quantified in samples collected from a previously conducted 12-wk elderberry intervention study in healthy post-menopausal women. Individual 1-, 2- and 3-h post-bolus urine samples and pooled plasma samples following acute (single bolus) and chronic (12-wk supplementation) anthocyanin consumption (500 mg/day) were analysed using HPLC-ESI-MS/MS. Twenty-eight anthocyanin metabolites were identified in urine and 21 in plasma (including sulfates of vanillic, protocatechuic and benzoic acid). Phenolic metabolites reached peak concentrations of 1237 nM in plasma, while anthocyanin conjugates only reached concentrations of 34 nM. Similarly, in urine, phenolic metabolites were detected at concentrations of 33 185 ± 2549 nM/mM creatinine, while anthocyanin conjugates reached concentrations of 548 ± 219 nM/mM creatinine. There was no evidence that chronic exposure had any impact on either the profile or quantity of metabolites recovered relative to acute exposure. Conclusion An extensive range of phenolic metabolites of anthocyanin was identified following elderberry consumption in humans, including 11 novel metabolites, which were identified at much higher concentrations than their parent compounds

The pharmacokinetics of anthocyanins and their metabolites in humans

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′-13C5-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 13C-labelled compounds were identified in the serum, including 13C5-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

Methods for isolating, identifying and quantifying anthocyanin metabolites in clinical samples

The metabolic fate of anthocyanins until recently was relatively unknown, primarily as a result of their instability at physiological pH and a lack of published methods for isolating and identifying their metabolites from biological samples. The aim of the present work was to establish methods for the extraction and quantification of anthocyanin metabolites present in urine, serum and fecal samples. 35 commercial and 10 synthetic analytes, including both known and predicted human and microbial metabolites of anthocyanins were obtained as reference standards. HPLC and MS/MS conditions were optimized for organic modifier, ionic modifier, mobile phase gradient, flow rate, column type and MS source and compound dependent parameters. The impact of sorbent, solvent, acid, preservative, elution and evaporation on SPE extraction efficiency was also explored. The HPLC-MS/MS method validation demonstrated acceptable linearity (r2, 0.997 ± 0.002) and sensitivity (LODs: urine, 100 ± 375 nM; serum, 104 ± 358 nM and feces 138 ± 344 nM) and the final SPE methods provided recoveries of 88.3 ± 17.8% for urine, 86.5 ± 11.1% for serum and 80.6 ± 20.9% for feces. Final methods were applied to clinical samples derived from an anthocyanin intervention study, where 36 of the 45 modeled metabolites were detected within urine, plasma or faecal samples. The described methods provide suitable versatility for the identification and quantification of an extensive series of anthocyanin metabolites for use in future clinical studies exploring absorption, distribution, metabolism and elimination

Identification of plasma and urinary metabolites and catabolites derived from orange juice (poly)phenols: analysis by high-performance liquid chromatography–high-resolution mass spectrometry

Orange juice is a rich source of (poly)phenols, in particular, the flavanones hesperetin-7-O-rutinoside and naringenin-7-O-rutinoside. Following the acute consumption of 500 mL of orange juice containing 398 μmol of (poly)phenols by 12 volunteers, 0–24 h plasma and urine samples were analyzed by targeted high-performance liquid chromatography–high-resolution mass spectrometry in order to identify flavanone metabolites and phenolic acid and aromatic catabolites. A total of 19 flavanone metabolites—comprising di-O-glucuronide, O-glucuronide, O-glucuronyl-sulfate, and sulfate derivatives of hesperetin, naringenin, and eriodictyol—and 65 microbial-derived phenolic catabolites, such as phenylpropanoid, phenylpropionic, phenylacetic, benzoic, and hydroxycarboxylic acids and benzenetriol and benzoylglycine derivatives, including free phenolics and phase II sulfate, glucuronide, and methyl metabolites, were identified or partially identified in plasma and/or urine samples. The data obtained provide a detailed evaluation of the fate of orange juice (poly)phenols as they pass through the gastrointestinal tract and are absorbed into the circulatory system prior to renal excretion. Potential pathways for these conversions are proposed

Human metabolism and elimination of the anthocyanin, cyanidin-3-glucoside: a 13C-tracer study

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 mug(13)C/h), whereas (13)C-labeled metabolites peaked (maximum serum concentration: 5.97 +/- 2.14 mumol/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 mug/h), at 6 h for breath (132.87 +/- 32.23 mug/h), and between 6 and 24 h for feces (557.28 +/- 247.88 mug/h), whereas the highest concentrations of (13)C-labeled metabolites were identified in urine (10.77 +/- 4.52 mumol/L) and fecal samples (43.16 +/- 18.00 mumol/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 fo

Exploring the Phytochemical Landscape of the Early-Diverging Flowering Plant Amborella trichopoda Baill.

Although the evolutionary significance of the early-diverging flowering plant Amborella (Amborella trichopoda Baill.) is widely recognized, its metabolic landscape, particularly specialized metabolites, is currently underexplored. In this work, we analyzed the metabolomes of Amborella tissues using liquid chromatography high-resolution electrospray ionization mass spectrometry (LC-HR-ESI-MS). By matching the mass spectra of Amborella metabolites with those of authentic phytochemical standards in the publicly accessible libraries, 63, 39, and 21 compounds were tentatively identified in leaves, stems, and roots, respectively. Free amino acids, organic acids, simple sugars, cofactors, as well as abundant glycosylated and/or methylated phenolic specialized metabolites were observed in Amborella leaves. Diverse metabolites were also detected in stems and roots, including those that were not identified in leaves. To understand the biosynthesis of specialized metabolites with glycosyl and methyl modifications, families of small molecule UDP-dependent glycosyltransferases (UGTs) and O-methyltransferases (OMTs) were identified in the Amborella genome and the InterPro database based on conserved functional domains. Of the 17 phylogenetic groups of plant UGTs (A-Q) defined to date, Amborella UGTs are absent from groups B, N, and P, but they are highly abundant in group L. Among the 25 Amborella OMTs, 7 cluster with caffeoyl-coenzyme A (CCoA) OMTs involved in lignin and phenolic metabolism, whereas 18 form a clade with plant OMTs that methylate hydroxycinnamic acids, flavonoids, or alkaloids. Overall, this first report of metabolomes and candidate metabolic genes in Amborella provides a starting point to a better understanding of specialized metabolites and biosynthetic enzymes in this basal lineage of flowering plants

Bioavailability of orange juice (poly)phenols: the impact of short-term cessation of training by male endurance athletes

Background: Physical exercise has been reported to increase the bioavailability of citrus flavanones. Objective: To investigate the bioavailability of orange juice (OJ) (poly)phenols in endurance-trained men before and after cessation of training for 7 days. Design: Ten fit endurance-trained males, with a maximal oxygen consumption of 58.2 ± 5.3 mL/kg/min, followed a low (poly)phenol diet for 2 d before drinking 500 mL of OJ, containing 398 µmol of (poly)phenols of which 330 µmol were flavanones. After the volunteers stopped training for 7 days the feeding study was repeated. Urine samples were collected 12 h pre- and 24 h post-OJ orange consumption. Bioavailability was assessed by the quantitative analysis of urinary flavanone metabolites and (poly)phenol catabolites using HPLC-HR-MS. Results: While training, 0-24 h urinary excretion of flavanone metabolites, mainly hesperetin-3-O-glucuronide, hesperetin-3´-sulfate, naringenin-4´-O-glucuronide, naringenin-7-O-glucuronide, was equivalent to 4.2% of OJ flavanone intake. This increased significantly to 5.2% when OJ was consumed after the volunteers stopped training for 7 days. Overall, this trend, although not significant, was also observed with OJ-derived colonic catabolites which after supplementation in the trained state were excreted in amounts equivalent to 51% of intake compared to 59% after cessation of training. However, urinary excretion of three colonic catabolites of bacterial origin, most notably, 3-(3´-hydroxy-4´-methoxyphenyl)hydracrylic acid, did increase significantly when OJ was consumed post- compared to pre-cessation of training. Data were also obtained on inter-individual variations in flavanone bioavailability. Conclusion: A 7-day cessation of endurance training enhanced, rather than reduced, the bioavailability of OJ flavanones. The biological significance of these differences and, whether or not they extend to the bioavailability of other dietary (poly)phenols, remains to be determined. Hesperetin-3´-O-glucuronide and the colonic microbiota-derived catabolite 3-(3´-hydroxy-4´-methoxyphenyl)hydracrylic acid are key biomarkers of the consumption of hesperetin-O-glycoside-containing OJ and other citrus products

The Metabolic Fate and Bioactivity of Anthocyanins in Humans

Anthocyanins, the class of flavonoid responsible for giving a red hue to many berries, have been associated with a decreased risk of cardiovascular disease. However, numerous intervention studies feeding anthocyanin-rich foods report limited (<1%) bioavailability of the parent anthocyanins in vivo. Due to the instability of anthocyanins at neutral pH, it is postulated that degradation products and metabolites of anthocyanins may be responsible for the perceived bioactive effects. The aims of the present thesis were: (1) To model and establish analytical methods for the extraction and quantification of putative anthocyanin metabolites in urine, serum and faecal samples. (2) To identify and explore the pharmacokinetics of anthocyanin metabolites via the analysis of urine, serum and faecal samples from two human interventions, feeding either (a) 500 mg of isotopically (13C5) labelled anthocyanin or (b) 500 mg elderberry anthocyanins for 12 wks. (3) To explore the impact of acute (500 mg) versus chronic (500 mg/day for 12 wks) anthocyanin consumption on their metabolism and (4) To investigate the anti-inflammatory activity of six anthocyanin metabolites at physiologically relevant concentrations (0.01 μM to 10 μM) using human umbilical vein endothelial cells (HUVECs). Following the consumption of 500 mg elderberry anthocyanins, 28 anthocyanin metabolites were identified in urine and 21 in plasma, with the phenolic metabolites within plasma identified at 45 fold higher levels than their parent compounds. Similar results were observed within the 13C-labelled anthocyanin intervention, where 17 13C-labelled compounds were identified in serum and 31 in urine. However, chronic consumption of anthocyanins had no impact on the formation of the metabolites. The cardiovascular bioactivity of anthocyanins may be linked to the antiinflammatory activity of their metabolites. IL-6 and VCAM-1 are cytokines and adhesion molecules integral to the initiation and progression of inflammation. In vitro, anthocyanin metabolites reduced CD40L and TNF-α stimulated expression of the inflammatory markers, sVCAM-1 and IL-6, indicating that the anti-inflammatory effects of anthocyanins are likely attributed to their metabolites. In conclusion, the present thesis provides a new understanding into the metabolism and bioactivity of anthocyanins, which should provide an informative insight into how the consumption of higher intakes of anthocyanins may contribute to optimising human health

Cardiovascular Mechanisms of Action of Anthocyanins May Be Associated with the Impact of Microbial Metabolites on Heme Oxygenase-1 in Vascular Smooth Muscle Cells

Anthocyanins are reported to have cardio-protective effects, although their mechanisms of action remain elusive. We aimed to explore the effects of microbial metabolites common to anthocyanins and other flavonoids on vascular smooth muscle heme oxygenase-1 (HO-1) expression. Thirteen phenolic metabolites identified by previous anthocyanin human feeding studies, as well as 28 unique mixtures of metabolites and their known precursor structures were explored for their activity on HO-1 protein expression in rat aortic smooth muscle cells (RASMCs). No phenolic metabolites were active when treated in isolation; however, five mixtures of phenolic metabolites significantly increased HO-1 protein expression (127.4-116.6%, p ≤ 0.03). The present study demonstrates that phenolic metabolites of anthocyanins differentially affect HO-1 activity, often having additive, synergistic or nullifying effects