Characterization of Metabolites of Hydroxycinnamates in the in Vitro Model of Human Small Intestinal Epithelium Caco-2 Cells

Abstract

Hydroxycinnamic acids are antioxidant phenolic compounds which are widespread in plant foods, contribute significantly to total polyphenol intakes, and are absorbed by humans. The extent of their putative health benefit in vivo depends largely on their bioavailability. However, the mechanisms of absorption and metabolism of these phenolic compounds have not been described. In this study, we used the in vitro Caco-2 model of human small intestinal epithelium to investigate the metabolism of the major dietary hydroxycinnamates (ferulate, sinapate, p-coumarate, and caffeate) and of diferulates. The appearance of metabolites in the medium versus time was monitored, and the various conjugates and derivatives produced were identified by HPLC-DAD, LC/MS, and enzyme treatment with beta-glucuronidase or sulfatase. Enterocyte-like differentiated Caco-2 cells have extra- and intracellular esterases able to de-esterify hydroxycinnamate and diferulate esters. In addition, intracellular UDP-glucuronosyltransferases and sulfotransferases existing in Caco-2 cells are able to form the sulfate and the glucuronide conjugates of methyl ferulate, methyl sinapate, methyl caffeate, and methyl p-coumarate. However, only the sulfate conjugates of the free acids, ferulic acid, sinapic acid, and p-coumaric acid, were detected after 24 h. The O-methylated derivatives, ferulic and isoferulic acid, were the only metabolites detected following incubation of Caco-2 cells with caffeic acid. These results show that the in vitro model system differentiated Caco-2 cells have the capacity to metabolize dietary hydroxycinnamates, including various phase I (de-esterification) and phase II (glucuronidation, sulfation, and O-methylation) reactions, and suggests that the human small intestinal epithelium plays a role in the metabolism and bioavailability of these phenolic compounds.

Transport and Metabolism of Ferulic Acid through the Colonic Epithelium

“…Caco-2 cells have already been used for studying ferulic acid transport and metabolism. Grown as tridimensional cultures, they were unable to conjugate ferulic acid (Konishi and Shimizu, 2003), whereas grown as monolayers, they exported ferulic acid as sulfate but not glucuronide conjugates (Kern et al, 2003). The results obtained, showing free ferulic acid as the major form transported followed by its glucuronide, and also the presence of sulfate and dihydroferulic acid in lower amounts, were also seen with everted sacs of rat colon.…”

Absorption and Metabolism of Chlorogenic Acids in Cultured Gastric Epithelial Monolayers

“…The hydrolysis of the relatively hydrophilic 5-O-CQAs, which are likely to be transported by the paracellular route, would require that gastric esterase(s) be located on the apical membrane facing into the lumen. However, we cannot rule out the possibility that esterase(s) may be secreted into the apical (lumen) medium as observed with Caco-2 esterase activity (Kern et al, 2003) or that some caffeic acid was released via intraepithelial hydrolysis. Serine hydrolases may be responsible for the cinnamoyl esterase activity of the gastrointestinal mucosa; these carboxylesterases share similar structural homology and mechanism of hydrolysis with cinnamoyl esterases such as FAEA from Aspergillus niger.…”

Ferulic acid and derivatives: molecules with potential application in the pharmaceutical field

“…Conjugation was required for its distribution in the organism. The conjugation reactions of FA occur mainly in the liver but may also occur in the intestinal mucosa or kidney, catalyzed by sulfotransferases (EC 2.8.2.1) and UDP glucuronosyl transferases (EC 2.4.1.17) (Piskula, Terao, 1998;Kern et al, 2003;Zhao et al, 2004). Studies in humans and rats showed that after oral intake of FA, the most abundant metabolites generated in plasma were FA-glucuronide (FA conjugate with glucuronic acid) and FA-sulfoglucuronide (FA conjugate with sulfate and glucuronide) in addition to unmodified FA.…”