Colon-available mango (poly)phenols exhibit mitigating effects on the intestinal barrier function in human intestinal cell monolayers under inflammatory conditions.

This study investigated the impact of in vivo available colon-mango (poly)phenols on stress-induced impairment of intestinal barrier function. Caco-2/HT29-MTX cells were incubated with six extracts of ileal fluid collected pre- and 4-8 h post-mango consumption before being subjected to inflammatory stress. (Poly)phenols in ileal fluids were analysed by UHPLC-HR-MS. Epithelial barrier function was monitored by measurement of trans-epithelial electrical resistance (TEER) and the production of selected inflammatory markers (interleukin-8 (IL-8) and nitric oxide (NO)) and the major mucin of the mucosal layer (MUC2). Post-mango intake ileal fluids contained principally benzoic acids, hydroxybenzenes and galloyl derivatives. There was a high interindividual variability in the levels of these compounds, which was reflected by the degree of variability in the protective effects of individual ileal extracts on inflammatory changes in the treated cell cultures. The 24 h treatment with non-cytotoxic doses of extracts of 4-8 h post-mango intake ileal fluid significantly reduced the TEER decrease in monolayers treated with the inflammatory cytomix. This effect was not associated with changes in IL-8 expression and secretion or claudine-7 expression. The mango derived-ileal fluid extract (IFE) also mitigated cytomix-dependent nitrite secretion, as a proxy of NO production, and the MUC2 reduction observed upon the inflammatory challenge. These insights shed light on the potential protective effect of mango (poly)phenols on the intestinal barrier exposed to inflammatory conditions.

Using Targeted Metabolomics to Unravel Phenolic Metabolites of Plant Origin in Animal Milk.

Milk holds a high nutritional value and is associated with diverse health benefits. The understanding of its composition of (poly)phenolic metabolites is limited, which necessitates a comprehensive evaluation of the subject. This study aimed at analyzing the (poly)phenolic profile of commercial milk samples from cows and goats and investigating their sterilization treatments, fat content, and lactose content. Fingerprinting of phenolic metabolites was achieved by using ultra-high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry (UHPLC-QqQ-MS/MS). Two hundred and three potential microbial and phase II metabolites of the main dietary (poly)phenols were targeted. Twenty-five metabolites were identified, revealing a diverse array of phenolic metabolites in milk, including isoflavones and their microbial catabolites equol and O-desmethylangolensin, phenyl-γ-valerolactones (flavan-3-ol microbial catabolites), enterolignans, urolithins (ellagitannin microbial catabolites), benzene diols, and hippuric acid derivates. Goat's milk contained higher concentrations of these metabolites than cow's milk, while the sterilization process and milk composition (fat and lactose content) had minimal impact on the metabolite profiles. Thus, the consumption of goat's milk might serve as a potential means to supplement bioactive phenolic metabolites, especially in individuals with limited production capacity. However, further research is needed to elucidate the potential health effects of milk-derived phenolics.

Exploring and disentangling the production of potentially bioactive phenolic catabolites from dietary (poly)phenols, phenylalanine, tyrosine and catecholamines.

Following ingestion of fruits, vegetables and derived products, (poly)phenols that are not absorbed in the upper gastrointestinal tract pass to the colon, where they undergo microbiota-mediated ring fission resulting in the production of a diversity of low molecular weight phenolic catabolites, which appear in the circulatory system and are excreted in urine along with their phase II metabolites. There is increasing interest in these catabolites because of their potential bioactivity and their use as biomarkers of (poly)phenol intake. Investigating the fate of dietary (poly)phenolics in the colon has become confounded as a result of the recent realisation that many of the phenolics appearing in biofluids can also be derived from the aromatic amino acids, l-phenylalanine and l-tyrosine, and to a lesser extent catecholamines, in reactions that can be catalysed by both colonic microbiota and endogenous mammalian enzymes. The available evidence, albeit currently rather limited, indicates that substantial amounts of phenolic catabolites originate from phenylalanine and tyrosine, while somewhat smaller quantities are produced from dietary (poly)phenols. This review outlines information on this topic and assesses procedures that can be used to help distinguish between phenolics originating from dietary (poly)phenols, the two aromatic amino acids and catecholamines.

A Systematic Review and Comprehensive Evaluation of Human Intervention Studies to Unravel the Bioavailability of Hydroxycinnamic Acids.

Significance: Hydroxycinnamic acids (HCAs) are the main phenolic acids in the western diet. Harmonizing the available information on the absorption, distribution, metabolism, and excretion (ADME) of HCAs is fundamental to unraveling the compounds responsible for their health effects. This work systematically assessed pharmacokinetics, including urinary recovery, and bioavailability of HCAs and their metabolites, based on literature reports. Recent Advances: Forty-seven intervention studies with coffee, berries, herbs, cereals, tomato, orange, grape products, and pure compounds, as well as other sources yielding HCA metabolites, were included. Up to 105 HCA metabolites were collected, mainly acyl-quinic and C6-C3 cinnamic acids. C6-C3 cinnamic acids, such as caffeic and ferulic acid, reached the highest blood concentrations (maximum plasma concentration [Cmax] = 423 nM), with time to reach Cmax (Tmax) values ranging from 2.7 to 4.2 h. These compounds were excreted in urine in higher amounts than their phenylpropanoic acid derivatives (4% and 1% of intake, respectively), but both in a lower percentage than hydroxybenzene catabolites (11%). Data accounted for 16 and 18 main urinary and blood HCA metabolites, which were moderately bioavailable in humans (collectively 25%). Critical Issues: A relevant variability emerged. It was not possible to unequivocally assess the bioavailability of HCAs from each ingested source, and data from some plant based-foods were absent or inconsistent. Future Directions: A comprehensive study investigating the ADME of HCAs derived from their most important dietary sources is urgently required. Eight key metabolites were identified and reached interesting plasma Cmax concentrations and urinary recoveries, opening up new perspectives to evaluate their bioactivity at physiological concentrations. Antioxid. Redox Signal. 40, 510-541.

Impact of polyphenol oxidase on the bioavailability of flavan-3-ols in fruit smoothies: a controlled, single blinded, cross-over study.

Flavan-3-ols are bioactive compounds found in a variety of fruits and vegetables (F&V) that have been linked to positive health benefits. Increasing habitual flavan-3-ol intake is challenged by the generally low consumption of F&V. While smoothies are a commonly endorsed, consumer-accepted means to increase the daily intake of these important foods, fruits used for smoothie preparation can have a high polyphenol oxidase (PPO) activity and thus potentially affect the content and bioavailability of flavan-3-ols. To assess whether or not consuming freshly prepared smoothies made with different PPO-containing fruit impacts the bioavailability of the flavan-3-ols, a controlled, single blinded and cross-over study was conducted in healthy men (n = 8) who consumed a flavan-3-ol-containing banana-based smoothie (high-PPO drink), a flavan-3-ol-containing mixed berry smoothie (low-PPO drink) and flavan-3-ols in a capsule format (control). The peak plasma concentration (Cmax) of flavan-3-ol metabolites after capsule intake was 680 ± 78 nmol L-1, which was similar to the levels detected after the intake of the low PPO drink. In contrast, the intake of the high PPO drink resulted in a Cmax of 96 ± 47 nmol L-1, 84% lower than that obtained after capsule intake. In a subsequent study (n = 11), flavan-3-ols were co-ingested with a high-PPO banana drink but contact prior to intake was prevented. In this context, plasma flavan-3-ol levels were still reduced, suggesting an effect possibly related to post-ingestion PPO activity degrading flavan-3-ols in the stomach. There was a substantial range in the PPO activity detected in 18 different fruits, vegetables and plant-derived dietary products. In conclusion, bioavailability of flavan-3-ols, and most likely other dietary polyphenol bioactives, can be reduced substantially by the co-ingestion of high PPO-containing products, the implications of which are of importance for dietary advice and food preparation both at home and in industrial settings.

5-(3',4'-Dihydroxyphenyl)-γ-Valerolactone Is a Substrate for Human Paraoxonase: A Novel Pathway in Flavan-3-ol Metabolism.

SCOPE: Dietary flavan-3-ols are known to mediate cardiovascular benefits. Currently, it is assumed that the levels of flavan-3-ol catabolites detected in humans, 5-(3',4'-dihydroxyphenyl)-γ-valerolactone (γVL) and 5-(3',4'-dihydroxyphenyl)-γ-valeric acid (γVA), and their corresponding phase II metabolites, are determined exclusively by the action of the gut microbiome. However, a family of human proteins, paraoxonase (PON), can theoretically hydrolyze γVL metabolites into the corresponding γVAs. This study aims to determine if PON is involved in γVL and γVA metabolism in humans. METHODS AND RESULTS: A rapid conversion of γVL into γVA is detected in serum ex vivo (half-life = 9.8 ± 0.3 min) that is catalyzed by PON1 and PON3 isoforms. Phase II metabolites of γVL are also reacted with PON in serum. Following an intake of flavan-3-ol in healthy males (n = 13), the profile of γVA metabolites detected is consistent with that predicted from the reactivity of γVL metabolites with PON in serum. Furthermore, common PON polymorphisms are evaluated to assess the use of γVL metabolites as biomarkers of flavan-3-ol intake. CONCLUSION: PONs are involved in flavan-3-ol metabolic pathway in humans. PON polymorphisms have a minor contribution to inter-individual differences in the levels of γVL metabolites, without affecting their use as a nutritional biomarker.

UHPLC-HRMS Spectrometric Analysis: Method Validation and Plasma and Urinary Metabolite Identification after Mango Pulp Intake.

After an acute intake of 300 g of mango purée by 10 subjects, 0 and 24 h urine and plasma samples were analyzed by high-performance liquid chromatography-high-resolution mass spectrometry. The method was first validated for 44 reference polyphenols in terms of linearity, specificity, limits of detection and quantification, intra-day and inter-day precision, recovery, and matrix effects in two biological matrices. After method validation, a total of 94 microbial-derived phenolic catabolites, including 15 cinnamic acids, 3 phenylhydracrylic acids, 14 phenylpropanoic acids, 12 phenylacetic acids, 28 benzoic acids, 2 mandelic acids, 15 hydroxybenzenes, and 5 hippuric acid derivatives, were identified or tentatively identified in urine and/or plasma. These results establish the value of the UHPLC-HRMS protocol and the use of authentic standards to obtain a detailed and accurate picture of mango polyphenol metabolites, together with their phase II conjugated metabolites, in human bioavailability studies.

Excretion by subjects on a low (poly)phenol diet of phenolic gut microbiota catabolites sequestered in tissues or associated with catecholamines and surplus amino acids.

Phenolic catabolites excreted by fasting subjects with a functioning colon and ileostomists on a low (poly)phenol diet have been investigated. Urine was collected over a 12 h fasting period after adherence to a low (poly)phenol diet for 36 h. UHPLC-HR-MS quantified 77 phenolics. Some were present in the urine of both groups in similar trace amounts and others were excreted in higher amounts by participants with a colon indicating the involvement of the microbiota. Most were present in sub- or low-µmol amounts, but hippuric acid dominated accounting on average for 60% of the total for both volunteer categories indicating significant production from sources other than non-nutrient dietary (poly)phenols. The potential origins of the phenolics associated with the low (poly)phenol diet, include endogenous catecholamines, surplus tyrosine and phenylalanine, and washout of catabolites derived from pre-study intakes of non-nutrient dietary (poly)phenols.

Bioavailability of orange juice (poly)phenols: ß-glucan-rich oat bran decreases urinary excretion of flavanone phase II metabolites and enhances excretion of microbiota-derived phenolic catabolites.

The impact of ß-glucan-rich oat bran on the bioavailability of orange juice (OJ) flavanones was investigated. Volunteers consumed 500 mL of OJ with and without 22 g of oat bran containing 6 g of ß-glucan (OB-6). Urine collected 12 h prior to and over a 0-24 h period post-supplementation was analysed by UHPLC-HRMS. Sixteen flavanone metabolites and thirty-nine colon-derived phenolic catabolites were identified and quantified. The major compounds were hesperetin-3'-glucuronide, along with hippuric acids and the C6-C3 phenolic acids 3-(3'-hydroxy-4'-methoxyphenyl)hydracrylic acid and 3-(4'-hydroxy-3'-methoxyphenyl)propanoic acid. A marked reduction in the 0-24 h excretion of flavanone metabolites from 29.7 µmol (9.3% recovery) to 9.3 µmol (2.9% recovery), occurred following consumption of OB-6 compared to OJ. This appeared not to be an effect of fiber on the rate of transport in the upper gut. After consumption of OJ there was a 163 ± 15 µmol excretion of colon-derived phenolic catabolites, equivalent to 43% of (poly)phenol intake and following OB-6 intake there was a further significant 30% increase. The ß-oat bran in OB-6 contained 5.8 µmol of free and 52 µmol of bound phenolic derivatives compared to 371 µmol of OJ (poly)phenols. The elevated excretion of phenolics after OB-6 consumption appears not to be due to bound phenolics in the bran, rather it is consequence, principally, of a bran-mediated increase in the quantities of flavanones passing from the upper to the lower bowel where they were subjected to microbiota-mediated catabolism. CLINICAL TRIAL REGISTRATION NUMBER: This trial was registered at clinicaltrials.gov as NCT04867655.

Flavan-3-ol-methylxanthine interactions: Modulation of flavan-3-ol bioavailability in volunteers with a functional colon and an ileostomy.

Flavan-3-ols, including the flavan-3-ol monomer (-)-epicatechin, are dietary bioactives known to mediate beneficial cardiovascular effects in humans. Recent studies showed that flavan-3-ols could interact with methylxanthines, evidenced by an increase in flavan-3-ol bioavailability with a concomitant increase in flavan-3-ol intake-mediated vascular effects. This study aimed at elucidating flavan-3-ol-methylxanthine interactions in humans in vivo by evaluating the specific contributions of theobromine and caffeine on flavan-3-ol bioavailability. In ileostomists, the effect of methylxanthines on the efflux of flavan-3-ol metabolites in the small intestine was assessed, a parameter important to an understanding of the pharmacokinetics of flavan-3-ols in humans. In a randomized, controlled, triple cross-over study in volunteers with a functional colon (n = 10), co-ingestion of flavan-3-ols and cocoa methylxanthines, mainly represented by theobromine, increased peak circulatory levels (Cmax) of flavan-3-ols metabolites (+21 ± 8%; p < 0.05). Conversely, caffeine did not mediate a statistically significant effect on flavan-3-ol bioavailability (Cmax = +10 ± 8%, p = n.s.). In a subsequent randomized, controlled, double cross-over study in ileostomists (n = 10), cocoa methylxanthines did not affect circulatory levels of flavan-3-ol metabolites, suggesting potential differences in flavan-3-ol bioavailability compared to volunteers with a functional colon. The main metabolite in ileal fluid was (-)-epicatechin-3'-sulfate, however, no differences in flavan-3-ol metabolites in ileal fluid were observed after flavan-3-ol intake with and without cocoa methylxanthines. Taken together, these results demonstrate a differential effect of caffeine and theobromine in modulating flavan-3-ol bioavailability when these bioactives are co-ingested. These findings should be considered when comparing the effects mediated by the intake of flavan-3-ol-containing foods and beverages and the amount and type of methylxanthines present in the ingested matrixes. Ultimately, these insights will be of value to further optimize current dietary recommendations for flavan-3-ol intake. CLINICAL TRIAL REGISTRATION NUMBER: This work was registered at clinicaltrials.gov as NCT03526107 (study part 1, volunteers with functional colon) and NCT03765606 (study part 2, volunteers with an ileostomy).

Human colonic catabolism of dietary flavan-3-ol bioactives.

Understanding the fate of ingested polyphenols is crucial in elucidating the molecular mechanisms underlying the beneficial effects of a fruit and vegetable-based diet. This review focuses on the colon microbiota-mediated transformation of the flavan-3-ols and the structurally related procyanidins found in dietary plant foods and beverages, plus the flavan-3-ol-derived theaflavins of black tea, and the post-absorption phase II metabolism of the gut microbiota catabolites. Despite significant advances in the last decade major analytical challenges remain. Strategies to address them are presented.

Revisiting the bioavailability of flavan-3-ols in humans: A systematic review and comprehensive data analysis.

This systematic review summarizes findings from human studies investigating the different routes of absorption, metabolism, distribution and excretion (ADME) of dietary flavan-3-ols and their circulating metabolites in healthy subjects. Literature searches were performed in PubMed, Scopus and the Web of Science. Human intervention studies using single and/or multiple intake of flavan-3-ols from food, extracts, and pure compounds were included. Forty-nine human intervention studies met inclusion criteria. Up to 180 metabolites were quantified from blood and urine samples following intake of flavan-3-ols, mainly as phase 2 conjugates of microbial catabolites (n = 97), with phenyl-γ-valerolactones being the most representative ones (n = 34). Phase 2 conjugates of monomers and phenyl-γ-valerolactones, the main compounds in both plasma and urine, reached two peak plasma concentrations (Cmax) of 260 and 88 nmol/L at 1.8 and 5.3 h (Tmax) after flavan-3-ol intake. They contributed to the bioavailability of flavan-3-ols for over 20%. Mean bioavailability for flavan-3-ols was moderate (31 ± 23%, n bioavailability values = 20), and it seems to be scarcely affected by the amount of ingested compounds. While intra- and inter-source differences in flavan-3-ol bioavailability emerged, mean flavan-3-ol bioavailability was 82% (n = 1) and 63% (n = 2) after (-)-epicatechin and nut (hazelnuts, almonds) intake, respectively, followed by 25% after consumption of tea (n = 7), cocoa (n = 5), apples (n = 3) and grape (n = 2). This highlights the need to better clarify the metabolic yield with which monomer flavan-3-ols and proanthocyanidins are metabolized in humans. This work clarified in a comprehensive way for the first time the ADME of a (poly)phenol family, highlighting the pool of circulating compounds that might be determinants of the putative beneficial effects linked to flavan-3-ol intake. Lastly, methodological inputs for implementing well-designed human and experimental model studies were provided.

Absorption, distribution, metabolism and excretion of apigenin and its glycosides in healthy male adults.

The bioavailability of apigenin and its O-glycosides in humans was investigated with apigenin-4'-glucuronide (Ap-4'-GlcUA), apigenin-7-glucuronide and apigenin-7-sulfate being identified as in vivo metabolites. Apigenin per se was poorly absorbed with metabolites equivalent to 0.5% of intake excreted in urine 0-24 h post-intake. Consumption of a parsley drink containing apigenin-7-O-(2″-O-apiosyl)glucoside resulted in the peak plasma concentration (Cmax) of Ap-4'-GlcUA occurring after 4 h, indicative of absorption in the lower gastrointestinal tract (GIT). Urinary excretion of the three metabolites corresponded to 11.2% of intake. Ingestion of dried powdered parsley leaves with yogurt extended the Cmax of Ap-4'-GlcUA to 6 h. Consumption of chamomile tea containing apigenin-7'-O-glucoside resulted in a 2 h Cmax of Ap-4'-GlcUA, in keeping with absorption in the upper GIT. Urinary excretion was equivalent to 34% of intake. Intake of the parsley drink provided information on intra- and inter-individual variations in the level of excretion of the apigenin metabolites. CLINICAL TRAIL REGISTRATION NUMBER: This trail was registered at clinicaltrials.gov as NCT03526081.

In Vitro Faecal Fermentation of Monomeric and Oligomeric Flavan-3-ols: Catabolic Pathways and Stoichiometry.

SCOPE: The study evaluates the influence of flavan-3-ol structure on the production of phenolic catabolites, principally phenyl-γ-valerolactones (PVLs), and phenylvaleric acids (PVAs). METHODS AND RESULTS: A set of 12 monomeric flavan-3-ols and proanthocyanidins (degree of polymerization (DP) of 2-5), are fermented in vitro for 24 h using human faecal microbiota, and catabolism is analyzed by UHPLC-ESI-MS/MS. Up to 32 catabolites strictly related to microbial catabolism of parent compounds are detected. (+)-Catechin and (-)-epicatechin have the highest molar mass recoveries, expressed as a percentage with respect to the incubated concentration (75 µmol L-1 ) of the parent compound, for total PVLs and PVAs, both at 5 h (about 20%) and 24 h (about 40%) of faecal incubation. Only A-type dimer and B-type procyanidins underwent the ring fission step, and no differences are found in total PVL and PVA production (≃1.5% and 6.0% at 5 and 24 h faecal incubation, respectively) despite the different DPs. CONCLUSION: The flavan-3-ol structure strongly affects the colonic catabolism of the native compounds, influencing the profile of PVLs and PVAs produced in vitro. This study opens new perspectives to further elucidate the colonic fate of oligomeric flavan-3-ols and their availability in producing bioactive catabolites.

(Poly)phenolic compounds and gut microbiome: new opportunities for personalized nutrition.

For decades, (poly)phenols have been linked to cardiometabolic health, but population heterogeneity limits their apparent efficacy and the development of tailored, practical protocols in dietary interventions. This heterogeneity is likely determined by the existence of different metabotypes, sub-populations of individuals metabolizing some classes of (poly)phenols differently. The gut microbiota plays a major role in this process. The impact of microbiota-related phenolic metabotypes on cardiometabolic health is becoming evident, although the picture is still incomplete, and data are absent for some classes of (poly)phenols. The lack of a complete understanding of the main microbial actors involved in the process complicates the picture. Elucidation of the mechanisms behind phenolic metabotypes requires novel experimental designs that can dissect the inter-individual variability. This paper, in addition to providing an overview on the current state-of-the-art, proposes wider metabotyping approaches as a means of paving the way towards effective personalized nutrition with dietary (poly)phenols.

Acute effect of oat ß-glucan on the bioavailability of orange juice flavanones.

The impact of ß-glucan on the bioavailability of orange juice (OJ) flavanones was investigated in a randomised controlled trial. Volunteers consumed 500 mL of OJ without or with either 3 g (OB-3) or 6 g (OB-6) of ß-glucan. Urine samples, collected 12 h before and over a 0-24 h period post-supplementation, were analysed by high-performance liquid chromatography-high resolution mass spectrometry. The overall 0-24 h urinary excretion of the 17 flavanone metabolites identified and quantified in urine after OJ ingestion corresponded to 29.7 µmol, and 25.0 and 9.3 µmol, respectively, after OB-3 and OB-6 intake. This corresponds to 9.3, 7.9, and 2.9% recoveries of the 318 µmol of the ingested flavanones. The acute ingestion of OJ with 6 g, but not 3 g of ß-glucan led to a significant reduction (p < 0.05) in the excretion of flavanone metabolites compared with consumption of OJ alone.

Validation of a high-throughput method for the quantification of flavanol and procyanidin biomarkers and methylxanthines in plasma by UPLC-MS.

Nutritional biomarkers are critical tools to objectively assess intake of nutrients and other compounds from the diet. In this context, it is essential that suitable analytical methods are available for the accurate quantification of biomarkers in large scale studies. Recently, structurally-related (-)-epicatechin metabolites (SREMs) and 5-(3',4'-dihydroxyphenyl)-γ-valerolactone metabolites (gVLMs) were identified as biomarkers of intake of flavanols and procyanidins, a group of polyphenol bioactives. This study aimed at validating a high throughput method for the quantification of SREMs and gVLMs in plasma along with methylxanthines (MXs), dietary compounds known to interact with flavanol and procyanidin effects. To accomplish this, a full set of authentic analytical standards were used to optimize a micro solid phase extraction method for sample preparation coupled to HPLC-MS detection. Isotopically-labelled standards for all analytes were included to correct potential matrix effects on quantification. Average accuracies of 101%, 93% and 103% were obtained, respectively, for SREMs, gVLMs and MXs. Intra- and inter-day repeatability values were <15%. The method showed linear responses for all analytes (>0.993). Most SREMs and gVLMs had limits of quantifications <5 nM while limits of quantification of MXs were 0.2 µM. All analytes were stable under different tested processing conditions. Finally, the method proved to be suitable to assess SREMs, gVLMs and MXs in plasma collected after single acute and daily intake of cocoa-derived test materials. Overall, this method proved to be a valid analytical tool for high throughput quantification of flavanol and procyanidin biomarkers and methylxanthines in plasma.

Development and validation of HPLC-MS2 methodology for the accurate determination of C4-C8 B-type flavanols and procyanidins.

Cocoa flavanols and procyanidins (CFs), natural dietary bioactives, have been studied extensively over the past two decades for their potential health benefits. Research on their safety and efficacy is critically dependent upon on the ability to reliably characeterize the research materials that are utilized, and with growing consumer availability of CF-based products, reliable methods for the detection of potential adulteration are of increasing importance. This research focused on the development of a high performance liquid chromatography-tandem mass spectrometry method (HPLC-MS2) using primary standards and 13C-labelled procyanidins as internal standards. The ability of MS2 detection to discriminate A- and B-type procyanidins was demonstrated. Method performances were validated for degrees of polymerization up to four in seven model food matrices. Accuracy ranged from 90.9 to 125.4% and precision was < 10% at lower concentrations. Finally, the method was applied to cocoa-based samples and compared to the AOAC 2020.05 analytical protocol, supporting the use of NIST 8403 as reference material for HPLC-MS2 analysis.

Ex vivo fecal fermentation of human ileal fluid collected after raspberry consumption modifies (poly)phenolics and modulates genoprotective effects in colonic epithelial cells.

Diets rich in fruit and vegetables are associated with a decreased incidence of colorectal cancer (CRC) due, in part, to the bioactive (poly)phenolic components and their microbiota-mediated metabolites. This study investigated how such compounds, derived from ingested raspberries in the gastrointestinal tract, may exert protective effects by reducing DNA damage. Ileal fluids collected pre- and post-consumption of 300 g of raspberries by ileostomists (n = 11) were subjected to 24 h ex vivo fermentation with fecal inoculum to simulate interaction with colonic microbiota. The impact of fermentation on (poly)phenolics in ileal fluid was determined and the bioactivity of ileal fluids pre- and post fermentation investigated. (Poly)phenolic compounds including sanguiin H-6, sanguiin H-10 and cyanidin-3-O-sophoroside decreased significantly during fermentation while, in contrast, microbial catabolites, including 3-(3'-hydroxyphenyl)propanoic acid, 3-hydroxybenzoic acid and benzoic acid increased significantly. The post-raspberry ileal fermentate from 9 of the 11 ileostomates significantly decreased DNA damage (~30%) in the CCD 841 CoN normal cell line using an oxidative challenge COMET assay. The raspberry ileal fermentates also modulated gene expression of the nuclear factor 2-antioxidant responsive element (Nrf2-ARE) pathway involved in oxidative stress cytoprotection, namely Nrf2, NAD(P)H dehydrogenase, quinone-1 and heme oxygenase-1. Four of the phenolic catabolites were assessed individually, each significantly reducing DNA damage from an oxidative challenge over a physiologically relevant 10-100 µM range. They also induced a differential pattern of expression of key genes in the Nrf2-ARE pathway in CCD 841 CoN cells. The study indicates that the colon-available raspberry (poly)phenols and their microbial-derived catabolites may play a role in protection against CRC in vivo.