Krenek, Kimberly Ann (2013-08). Chemical Characterization and Absorption of Phytochemicals From Mangifera indica L.. Doctoral Dissertation.
It was hypothesized that ester-linked gallic acid glycosides could be absorbed and metabolized in vivo due to the instability of an ester-linked glycosides at neutral pH. To evaluate in vivo absorption of Mangifera Indica, L. var. Keitt polyphenolics, it was first necessary to chemically characterize the compounds present using HPLC-MSn analysis. Mango pulp and extracts were also incubated with a pectinase, cellulase, pectinase with ss-glucosidase activity, and a pure ss-glucosidase to learn the extent of hydrolysis with potential application to enhancing bioavailability as a result of incubation to increase mango juice yield. After which the same extracts were assessed in differentiated Caco-2 cells to discern stability at physiological pH and to characterize metabolite formation in vitro. Finally, human urinary metabolites were characterized after 10 day consumption of 400 g in 11 individuals. Mass spectroscopic characterization and HPLC quantification of mango pulp revealed for the first time two monogalloyl glucosides (MGGs) with distinct differences in their glycoside linkages, with the ester form dominating, as well as the presence of five other phenolic acid glycosides; hydroxybenzoic acid glucoside, courmaric glucoside, ferulic acid glucoside, and sinapic acid mono and di-glucosides. Six oxygenated carotenoid derivatives were identified for the first time in a phytochemical extract, namely, a phytohormone, abscisic acid and its glycoside, two catabolism products of abscisic acid, dihydrophasic acids, and two hydroxy-dimethyl decadiene-dioic acid glucopiranosylesters. Gallotannins ranging from tetra-galloyl glucosides to nona-galloyl glucosides were also identified in the pulp, but not quantified. Clearzyme 200XL and Rapidase AR2000 were the most effective at increasing juice yield of mango pulp due to their pectinase action. Cz reduced the amount of ester-linked MGG by 70% after 4 hours of incubation while Rap hydrolyzed the ether linked MGG. The instability of ester-linked galloyl-glycosides at pH 7.4 was characterized by HPLC-MS and after only four hours of incubation a shift from HWM tannins (>8GG) to LMW (<8GG) occurred and resulted in 25 mg/L free gallic acid. Caco-2 cells metabolized gallic acid, MGG from a mango extract, and a gallotannin extract into O-methyl gallic acid indicating catechol-O-methyl transferase (COMT) as a major metabolizing enzyme for gallic acid. Urinary metabolites were identified by HPLC-MSn in dependant scans. O-methylgallic acid-O-sulfate was identified as the major metabolite 0-6 hours post consumption, followed by O-methylgallic acid at a lower concentration. The presence of gallic acid metabolites in the urine indicates absorption of ester-linked glycosides. Colonic metabolites were detected beginning 3-6 hours after consumption of mango, and were identified as pyrogalloyl derivatives. They are hypothesized to be the products of microbial breakdown of gallotannins. Daily consumption of mango for 10 days increased the concentration of O-methylgallic acid-O-sulfate, but was not significant.