Dongjie Wang (2011). IN VITRO AND IN VIVO INTESTINAL METABOLISM OF GRAPE SEED EXTRACT PHENOLIC COMPOUNDS PhD Thesis, School of Agriculture and Food Sciences, The University of Queensland.

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Author Dongjie Wang
School, Centre or Institute School of Agriculture and Food Sciences
Institution The University of Queensland
Publication date 2011-09
Thesis type PhD Thesis
Supervisor BRUCE D'ARCY
Total pages 195
Total colour pages 4
Total black and white pages 191
Language eng
Subjects 111103 Nutritional Physiology
100199 Agricultural Biotechnology not elsewhere classified
060199 Biochemistry and Cell Biology not elsewhere classified
039999 Chemical Sciences not elsewhere classified
070199 Agriculture, Land and Farm Management not elsewhere classified
Abstract/Summary Grape seed extract (GSE) is a rich source of biologically active phenolic compounds, including phenolic acids, and proanthocyanidins (PAC’s) in the form of monomers, and polymers of flavan-3-ols including (+)-catechin (C) and (-)-epicatechin (EC). GSE has been reported to have health benefits, including the potential of prevention and treatment of Alzheimer’s Disease and prevention of cardiovascular disorders. While promising preclinical data exist on the biological effects of GSE, information on the bioavailability (digestibility and metabolism, especially the metabolism by intestinal microbiota) and the in vivo distribution of GSE phenolic compounds and their metabolites remains limited. The overall aim of this research was to investigate the gastro-intestinal digestion and metabolism of GSE phenolics in vitro and in vivo, with a particular focus on the GSE metabolites of the intestinal microbiota. In the in vitro study, the gastrointestinal tract (GIT) digestion model was carried out using the gastric-pancreatic method, followed by Caco-2 permeability experiments to study the potential small intestinal absorption of intact GSE phenolic compounds (Chapter 4). Results showed that the type/number of GSE phenolic compounds did not change based on the recovery of all nine major phenolic compounds found in the original GSE samples by LC-DAD-MS analysis. However, there was a significant (P<0.05) change in the concentrations of phenolics due to gastric digestion, with a significant decrease in the concentration of PAC trimers and polymers, and a significant increase in the concentrations of monomers was observed (total monomer content was increased 40% to the original GSE sample). A decrease in simple phenolic compounds was observed following simulated small intestinal-pancreatic digestion, possibly due, in part to the alkaline intestinal conditions (pH = 7.4) that favour auto-oxidative processes. Intestinal permeability (assessed by Caco-2 monolayers) of the original GSE compounds was limited after 30 min, with most GSE phenolic compounds recovered in the apical compartment. This suggests the poor bioavailability of these polyphenol compounds observed in previous studies may be due to a combination of digestive sensitivity and limited trans-epithelial transport. The large amount of GSE phenolics remaining in the gut lumen is therefore available for further metabolism by intestinal microbiota. In order to further understand the metabolism of GSE along the GIT, the impacts of GSE phenolics on the fermentation of starch (Gelose 80) by ileal and fecal microbiota were investigated using six different GSE concentrations (0, 60, 125, 250, 500, 750 μg/mL). To achieve this, cumulative gas production, and short chain fatty acid (SCFA) and ammonium production from the fermentation were measured (Chapter 5). Results support the notion that the highest GSE concentration (250 μg/mL) produced no significant (P<0.05) effect on normal intestinal microbiota fermentation of starch in vitro. Next, an in vitro metabolism experiment, in the presence of GSE (250 μg/mL) and involving both ileal and fecal microbiota, was carried out, followed by a Caco-2 cell permeability study mimics absorption in the ileum and colon, to determine the amount of absorption of the small molecular weight metabolites that resulted from these fermentations (Chapter 6). Two metabolites with deprotonated molecular mass of 165/121 and 193/175 ([M-H]-) were detected and tentatively identified as 3-(3´-hydroxyphenyl)propionic acid (3-HPP) and 5-(4´-hydroxyphenyl)valeric acid (5-HPV) respectively, after both ileal and fecal fermentation. In addition, these two compounds passed through the Caco-2 cell monolayer after a 30 min permeability experiment, and were detected in the basolateral solution using LC-DAD-MS analysis, suggesting the systemic bioavailability of these microbial metabolites of GSE PACs. To confirm the in vivo relevance of GSE microbial metabolites, a rodent in vivo study was completed to examine the formation and bioavailability of GSE metabolites originating from metabolism by intestinal microbiota (Chapter 7). Sprage-Dawley rats were dosed daily for 10 days by intra-gastric gavage with a control (deionized water), low dose GSE solution (25 mg/kg BW) or a high dose GSE solution (250 mg/kg BW). Urine and blood were collected every 24 h for ten consecutive days before the rats were sacrificed. The accumulation of phenolic compounds in three target tissues, brain, large intestine, and cecum were assessed after the 11th dose. A significant (P<0.05) increase in urinary excretion and in the plasma levels of 3-(3´-hydroxyphenyl) propionic acid (3-HPP, 165/121 [M-H]-) and a possible compound, 5-(4´-hydroxyphenyl)valeric acid (5-HPV) with a deprotonated molecular weight of 193/175 ([M-H]-) was found for the GSE administration group using LC-TOF/MS. A significant (P<0.05) increase in the accumulation of 3-HPP in all three tissues was also detected by LC-MS/MS, indicating the potential physiological significance of this microbial metabolite in vivo. In conclusion, that the results of in vitro and in vivo studies converged nicely to enable the prediction of the metabolism of GSE phenolic compounds by intestinal microbiota, with 3-HPP and 5-HPV being identified as the two major metabolites, which may play important roles in the beneficial effects of GSE on human health.
Keyword Grape seed extract (GSE)
intestinal microbiota metabolism
gastric-pancreatic digestion
Caco-2 cell monolayer
urinary excretion
in vitro
in vivo
Additional Notes 34, 47, 49, 77

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Created: Tue, 12 Jun 2012, 15:07:54 EST by Ms Dongjie Wang on behalf of Library - Information Access Service