Bioavailability and bioactivity of polyphenols and their microbial metabolites following simulated dynamic gastrointestinal digestion

This doctoral dissertation describes the first combined use of a human simulated gut digestion model together with intestinal and hepatic cell cultures to examine the profiles, bioactivities and bioavailabilities of microbial metabolites generated from polyphenols commonly present in the human diet (chlorogenic acid, caffeic acid, ferulic acid and rutin). Study 1 involved digestion of the above polyphenols using a continuous multi-reactor Computer Controlled Dynamic Human Gastrointestinal (GI) Model representing digestion in the stomach, the small intestine and the three colonic segments. The profiles of the microbial phenolic metabolites and short chain fatty acids generated by microbiota varied greatly according to the colonic compartment. Prolonged microbial metabolism over a 16 h period generated antioxidant capacity that matched that produced by the parent polyphenols, which differs from previous short-term batch fermentation studies showing low antioxidant potential of the metabolites. Study 2 utilized the human colorectal adenocarcinoma Caco-2 cell line to explore the anti-colon cancer effects of chlorogenic acid and its major microbial metabolites (caffeic, 3-phenylpropionic and benzoic acids) at physiologically relevant concentrations of each compound and an equimolar mixture of the compounds. The combination of chlorogenic acid and its metabolites enhanced the anti-cancer efficacy as anti-proliferative, apoptotic and cell cycle arrest effects were seen at several-fold lower concentrations of those compounds within the equimolar mixture than when they were provided singly. Alterations in mitochondrial DNA were not associated with the apoptotic events for chlorogenic acid and caffeic acid but a reduction of mitochondrial DNA content was involved in 3-phenylpropionic-mediated apoptosis. In Study 3, the digests from the human simulated gut digestion system were coupled with a co-culture of human intestinal Caco-2 and hepatic HepG2 cells to investigate the digestion and first pass metabolism of a polyphenol-rich potato extract (PRPE) containing chlorogenic acid, caffeic acid, ferulic acid and rutin as the major polyphenolic constituents. Digestion of PRPE in the GI model led to generation of the microbial-derived metabolites of the polyphenols (dihydrocaffeic, dihydroferulic, 3-hydroxybenzoic, 3-hydroxyphenylpropionic, coumaric, 3-hydroxyphenylacetic, phenylpropanoic and cinnamic acids). Following a 2 h incubation of the colonic digesta with Caco-2 cells, ferulic acid and dihydrocaffeic, dihydroferulic, 3-hydroxyphenylpropionic, 3-hydroxybenzoic and coumaric acids were noted to be poorly transported (3-15%). A two- to three-fold increase in the concentrations of ferulic, dihydrocaffeic, 3-hydroxyphenylpropionic and coumaric acids after 3 h incubation with HepG2 cells demonstrated a major contribution of hepatic metabolism in the generation of those compounds despite their poor Caco-2 cellular transport. Overall, the combined approach using simulated gut digestion and cell culture systems applied in the current work provides a unique platform for the detailed study of mechanisms involved in biotransformation, bioavailability and bioactivity of polyphenols and their metabolites, which is otherwise difficult to perform in vivo.Cette thèse de doctorat décrit le premier emploi combiné d'un modèle de digestion intestinale humaine simulée en association avec des cultures cellulaires intestinales et hépatiques afin d'examiner les profils, les bioactivités et les biodisponibilités des métabolites microbiens générés à partir de polyphénols communément présents dans l'alimentation humaine (l'acide chlorogénique [CGA], l'acide caféique, l'acide férulique et la rutine). L'étude 1 a porté sur la digestion des polyphénols ci-dessus à l'aide d'un modèle continu et dynamique gastro-intestinal (GI) humain contrôlé par ordinateur et à plusieurs réacteurs représentants la digestion dans l'estomac, l'intestin grêle et les trois segments du colon. Les profils des métabolites phénoliques et des acides gras à chaine courte générés par le microbiote ont varié considérablement en fonction du compartiment du colon. Le métabolisme microbien prolongé sur une période de 16 heures a généré une capacité antioxydante similaire à celle produite par les polyphénols parents; ceci diffère des études précédentes de fermentation discontinue à court terme montrant un faible potentiel antioxydant des métabolites. L'étude 2 a utilisé la lignée cellulaire d'adénocarcinome colorectal humain Caco-2 pour étudier les effets de l'acide chlorogénique et de ses principaux métabolites microbiens (les acides caféique, 3-phenylpropionique et benzoïque) sur le cancer du côlon à des concentrations physiologiquement pertinentes d'un mélange équimolaire des composés. La combinaison de CGA et de ses métabolites a amélioré l'efficacité antiproliférative de ces composés, puisque des effets antiprolifératifs, apoptotiques et d'arrêt du cycle cellulaire ont été observés à des concentrations plusieurs fois plus faibles dans le mélange équimolaire que lorsqu'ils étaient administrés séparément. Des altérations de l'ADN mitochondrial n'étaient pas impliquées dans les mécanismes d'action des évènements apoptotiques du CGA et de l'acide caféique, mais une réduction du contenu en ADN mitochondrial était impliquée dans l'apoptose médiée par l'acide 3-phenylpropionique. Dans l'étude 3, les digestats obtenus du système de digestion intestinale humain simulé ont été soumis à une coculture de cellules intestinales humaines Caco-2 et hépatiques HepG2 pour examiner la digestion et l'effet de premier passage d'un extrait de pomme de terre (PRPE) contenant les acides chlorogénique, caféique et férulique et la rutine comme principaux constituants polyphénoliques. La digestion du PRPE par le modèle GI a mené à l'apparition de métabolites générés par les microbes (les acides dihydrocaféique, dihydroférulique, 3-hydroxybenzoïque, 3-hydroxyphénylpropanoïque, coumarique, 3-hydroxyphénylacétique, phénylpropanoïque et cinnamique). Après 2 heures d'incubation du digestat colique avec les cellules Caco-2, l'acide férulique et les acides dihydrocaféique, dihydroférulique, 3-hydroxyphénylpropionique, 3-hydroxybenzoïque et coumarique ont été mal transportés à travers la monocouche de cellules Caco-2 (3 – 15%). Une augmentation de deux à trois fois supérieure des concentrations d'acides férulique, dihydrocaféique, 3-hydroxyphénylpropionique et coumarique après 3 heures d'incubation avec les cellules HepG2 a démontré une contribution majeure du métabolisme hépatique à la génération de ces composés malgré leur faible transport à travers les cellules Caco-2.Globalement, l'approche combinée utilisant les systèmes de digestion intestinale simulée et de culture cellulaire, développée dans le cadre des travaux actuels, offre une plate-forme unique pour l'étude détaillée des mécanismes impliqués dans la biotransformation, la biodisponibilité et la bioactivité des polyphénols et de leurs métabolites, ce qui est par ailleurs difficile à réaliser in vivo

Chlorogenic Acid and Its Microbial Metabolites Exert Anti-Proliferative Effects, S-Phase Cell-Cycle Arrest and Apoptosis in Human Colon Cancer Caco-2 Cells

Chlorogenic acid (CGA) decreases colon cancer-cell proliferation but the combined anti-cancer effects of CGA with its major colonic microbial metabolites, caffeic acid (CA), 3-phenylpropionic acid (3-PPA) and benzoic acid (BA), needs elucidation as they occur together in colonic digesta. Caco-2 cancer cells were treated for 24 h with the four compounds individually (50–1000 µM) and as an equimolar ratio (1:1:1:1; MIX). The effective concentration to decrease cell proliferation by 50% (EC50) was lower for MIX (431 ± 51.84 µM) and CA (460 ± 21.88) versus CGA (758 ± 19.09 µM). The EC50 for cytotoxicity measured by lactate dehydrogenase release in MIX (527 ± 75.34 µM) showed more potency than CA (740 ± 38.68 µM). Cell proliferation was decreased by 3-PPA and BA at 1000 µM with no cytotoxicity. Cell-cycle arrest was induced at the S-phase by CA (100 µM), MIX (100 µM), CGA (250 µM) and 3-PPA (500 µM) with activation of caspase-3 by CGA, CA, MIX (500 and 1000 µM). Mitochondrial DNA content was reduced by 3-PPA (1000 µM). The anti-cancer effects occurred at markedly lower concentrations of each compound within MIX than when provided singly, indicating that they function together to enhance anti-colon cancer activities

Absorption and Metabolism of Phenolics from Digests of Polyphenol-Rich Potato Extracts Using the Caco-2/HepG2 Co-Culture System

The bioactivity of dietary polyphenols depends upon gastrointestinal and hepatic metabolism of secondary microbial phenolic metabolites generated via colonic microbiota-mediated biotransformation. A polyphenol-rich potato extract (PRPE) containing chlorogenic, caffeic, and ferulic acids and rutin was digested in a dynamic multi-reactor gastrointestinal simulator of the human intestinal microbial ecosystem (GI model). Simulated digestion showed extensive degradation of the parent compounds and the generation of microbial phenolic metabolites. To characterize the transport and metabolism of microbial phenolic metabolites following digestion, a co-culture of intestinal Caco-2 and hepatic HepG2 cells was exposed to the PRPE-derived digests obtained from the colonic vessels. Following a 2 h incubation of the digesta with the Caco-2/HepG2 co-cultures, approximately 10–15% of ferulic, dihydrocaffeic, and dihydroferulic acids and 3–5% of 3-hydroxybenzoic, 3-hydroxyphenylpropionic, and coumaric acids were observed in the basolateral side, whereas 3-hydroxyphenylacetic acid, phenylpropanoic acid, and cinnamic acid were not detected. Subsequent HepG2 cellular metabolism led to major increases in ferulic, dihydrocaffeic, 3-hydroxyphenylpropionic, and coumaric acids ranging from 160–370%. These findings highlight the importance of hepatic metabolism towards the generation of secondary metabolites of polyphenols despite low selective Caco-2 cellular uptake of microbial phenolic metabolites

Microbial Biotransformation of a Polyphenol-Rich Potato Extract Affects Antioxidant Capacity in a Simulated Gastrointestinal Model

A multistage human gastrointestinal model was used to digest a polyphenol-rich potato extract containing chlorogenic acid, caffeic acid, ferulic acid, and rutin as the primary polyphenols, to assess for their microbial biotransformation and to measure changes in antioxidant capacity in up to 24 h of digestion. The biotransformation of polyphenols was assessed by liquid chromatography–mass spectrometry. Antioxidant capacity was measured by the ferric reducing antioxidant power (FRAP) assay. Among the colonic reactors, parent (poly)phenols were detected in the ascending (AC), but not the transverse (TC) or descending (DC) colons. The most abundant microbial phenolic metabolites in all colonic reactors included derivatives of propionic acid, acetic acid, and benzoic acid. As compared to the baseline, an earlier increase in antioxidant capacity (T = 8 h) was seen in the stomach and small intestine vessels as compared to the AC (T = 16 h) and TC and DC (T = 24 h). The increase in antioxidant capacity observed in the DC and TC can be linked to the accumulation of microbial smaller-molecular-weight phenolic catabolites, as the parent polyphenolics had completely degraded in those vessels. The colonic microbial digestion of potato-based polyphenols could lead to improved colonic health, as this generates phenolic metabolites with significant antioxidant potential