Intra- and Inter-individual Differences in the Human Intestinal Microbial Conversion of (-)-Epicatechin and Bioactivity of Its Major Colonic Metabolite 5-(3′,4′-Dihydroxy-Phenyl)-γ-Valerolactone in Regulating Nrf2-Mediated Gene Expression

Abstract

(-)-Epicatechin (EC) is one of the most popular polyphenols present in various food products in daily life. Upon intake, it is intensively metabolized by microbiota in the large intestine. In the present study, intra- and inter-individual variations in this gut microbial conversion of EC and the concomitant formation of its major metabolites, including 5-(3′,4′-dihydroxy phenyl)-γ-valerolactone (3,4-diHPV), were identified and quantified via liquid chromatography triple quadrupole mass spectrometry (LC-TQ-MS) in anaerobic fecal incubations. In addition, the bioactivity of EC and 3,4-diHPV in activating Nrf2-mediated gene expression was tested quantifying their effects in the U2OS Nrf2 CALUX assay (a reporter gene assay that is used to test the potency of chemicals in activation of Nrf2 signaling), and on the expression levels of Nrf2-related proteins in Hepa1c1c7 and Caco-2 cells via nanoLC-MSMS. A quantitative real-time polymerase chain reaction (RT-qPCR) was carried out to confirm selected Nrf2-regulated gene expressions at the mRNA level. Results obtained show that both intra- and inter-individual differences exist in human gut microbial EC degradation and 3,4-diHPV formation, with inter-individual differences being more distinct than intra-individual differences. The metabolite, 3,4-diHPV, showed higher potency in the U2OS Nrf2 CALUX assay than EC itself. Among the obviously altered Nrf2-related proteins, 14 and 10 Nrf2-associated proteins were upregulated to a higher extent upon 3,4-diHPV treatment than in the EC treated group for Hepa1c1c7 and Caco-2 cells, respectively. While only three and four of these Nrf2-associated proteins were induced at a higher level upon EC than upon 3,4-diHPV treatment for Hepa1c1c7 and Caco-2 cells, respectively. RT-qPCR results showed that indeed Nrf2-mediated genes (e.g., Nqo1 and Ugt1a) were only induced significantly in 3,4-diHPV treated and not in EC treated Hepa1c1c7 cells. Taken together, the results suggest that the major colonic EC metabolite, 3,4-diHPV, was more capable of inducing Nrf2-mediated gene expression than its parent compound EC. This implies that the evident inter- and intra-individual differences in the microbial conversion of EC to this major metabolite 3,4-diHPV may affect the overall health-promoting effects of EC consumption related to the Nrf2 pathway activation.

The Potential of Flavonoids and Flavonoid Metabolites in the Treatment of Neurodegenerative Pathology in Disorders of Cognitive Decline

“…EC and 3,4-diHPV both activate Nrf2-mediated gene expression, however, 3,4-diHPV shows higher potency in the upregulation of Nrf2 gene expression than EC. Conversion of EC to 3,4-diHPV by the gut microbiota improves the overall health-promoting effects of EC consumption due to this ability to selectively promote Nrf2 pathway activation [ 230 ].…”

Factors driving the inter-individual variability in the metabolism and bioavailability of (poly)phenolic metabolites: A systematic review of human studies

“…Indeed, many works where the source of IIV was classified as “undefined” are likely related to differences in the gut microbiota composition, but evidence of this still has to be provided. Faecal fermentation studies may be of help in classifying individuals according to the microbial metabolism of specific phenolics, as recently done for some flavan-3-ol monomers and avenanthramides [ [163] , [164] , [165] ].…”

Use of Physiologically Based Kinetic Modeling-Based Reverse Dosimetry to Predict In Vivo Nrf2 Activation by EGCG and Its Colonic Metabolites in Humans

“…These two microflora-derived metabolites were included since they were reported to be more potent in triggering Nrf2-mediated gene expression than the parent compound EGCG in our previous study . However, given that other gut microbiota metabolites of EGCG such as, for example, phenylvalerolactones and phenylvaleric acids might also have biological activities, it may be of interest to include also these metabolites in future PBK model development. Previous studies showed a proof of the principle for the inclusion of gut microbial metabolism in PBK modeling using kinetic parameters derived from in vitro anaerobic fecal incubations. , The model predicted the C max of GA and PG, after consumption of two capsules of the EGCG supplement (the recommended daily intake), to be less than 1.5% of the blood C max of EGCG and less than 14.1% when expressed in unbound EGCG equivalents in plasma.…”