Study on the intestinal absorption mechanism of green tea catechins and hawthorn flavonoids using caco-2 cell monolayer model.

Zhang Li.Thesis (M.Phil.)--Chinese University of Hong Kong, 2003.Includes bibliographical references (leaves 148-159).Abstracts in English and Chinese.Acknowledgements --- p.IAbstract --- p.IIAbstract (in Chinese) --- p.IVPublications --- p.VList of Abbreviations --- p.VIList of Tables --- p.VIIList of Figures --- p.VIIITable of Contents --- p.XIIIChapter Chapter One. --- Introduction --- p.1Chapter 1.1 --- Flavonoids --- p.1Chapter 1.2 --- Tea --- p.4Chapter 1.2.1 --- Composition of green tea catechins (GTC) --- p.4Chapter 1.2.2 --- Pharmacological activity --- p.6Chapter 1.2.2.1 --- Anticarcinogenic activity --- p.6Chapter 1.2.2.2 --- Antioxidative activity --- p.7Chapter 1.2.2.3 --- Radical scavenge --- p.7Chapter 1.2.2.4 --- Cardiovascular activity --- p.8Chapter 1.2.3 --- Pharmacokinetics of GTC --- p.8Chapter 1.2.3.1 --- Absorption --- p.10Chapter 1.2.3.2 --- Distribution --- p.11Chapter 1.2.3.3 --- Elimination --- p.11Chapter 1.2.3.4 --- Metabolism --- p.12Chapter 1.2.3.4.1 --- Metabolism in the small intestine --- p.12Chapter 1.2.3.4.2 --- Metabolism in the liver --- p.13Chapter 1.2.3.5 --- Summary of the pharmacokinetics of GTC --- p.13Chapter 1.3 --- Hawthorn --- p.14Chapter 1.3.1 --- Composition of hawthorn --- p.14Chapter 1.3.2 --- Pharmacological activity --- p.16Chapter 1.3.2.1 --- Inotonic activity --- p.16Chapter 1.3.2.2 --- Antiarrhythmic activity --- p.17Chapter 1.3.2.3 --- Hypolipidemic activity --- p.17Chapter 1.3.2.4 --- Antihypertensive activity --- p.18Chapter 1.3.2.5 --- Antioxidative activity --- p.18Chapter 1.3.3 --- Pharmacokinetics of HF --- p.18Chapter 1.3.3.1 --- Absorption --- p.19Chapter 1.3.3.2 --- Distribution and elimination --- p.21Chapter 1.3.3.3 --- Summary of pharmacokinetic of HF --- p.22Chapter 1.4 --- Mechanisms of intestinal absorption --- p.22Chapter 1.4.1 --- Passive transcellular transport --- p.23Chapter 1.4.2 --- Paracellular transport --- p.23Chapter 1.4.3 --- Carrier-mediated transport --- p.23Chapter 1.5 --- ABC transporters --- p.24Chapter 1.5.1 --- Cellular location and tissue distribution --- p.25Chapter 1.5.2 --- Substrates and inhibitors of ABC transporters --- p.26Chapter 1.6 --- Oral absorption models --- p.31Chapter 1.6.1 --- Ussing chamber --- p.31Chapter 1.6.2 --- In situ intestinal perfusion model --- p.33Chapter 1.6.3 --- Cell culture model --- p.34Chapter 1.7 --- Aims of the study --- p.36Chapter Chapter Two. --- Transport mechanism of green tea catechins --- p.37Chapter 2.1 --- Introduction --- p.37Chapter 2.2 --- Materials --- p.38Chapter 2.2.1 --- Chemicals --- p.38Chapter 2.2.2 --- Materials for cell culture --- p.38Chapter 2.2.3 --- Instruments --- p.39Chapter 2.3 --- Methods --- p.39Chapter 2.3.1 --- Analytical methods --- p.39Chapter 2.3.1.1 --- Analytical methods for validation of Caco-2 model --- p.39Chapter 2.3.1.1.1 --- Fluorescence analysis of lucifer yellow --- p.39Chapter 2.3.1.1.2 --- HPLC analysis of propranolol --- p.39Chapter 2.3.1.1.3 --- HPLC analysis of verapamil --- p.40Chapter 2.3.1.1.4 --- HPLC analysis of quinidine --- p.40Chapter 2.3.1.2 --- Analytical methods for samples contained GTC --- p.41Chapter 2.3.1.2.1 --- HPLC analysis for each GTC --- p.41Chapter 2.3.1.2.2 --- Preparation of calibration curves for each GTC --- p.42Chapter 2.3.1.2.3 --- HPLC/MS analysis of samples containing mixtures of four GTC --- p.42Chapter 2.3.1.2.4 --- Preparation of calibration curves for samples containing GTC mixture --- p.43Chapter 2.3.1.2.5 --- Validation of the HPLC methods --- p.43Chapter 2.3.1.3 --- Identification of metabolites with HPLC/MS --- p.44Chapter 2.3.2 --- Determination of stability profile of GTC in phosphate buffer --- p.44Chapter 2.3.3 --- Cell culture --- p.45Chapter 2.3.4 --- Validation of Caco-2 cell monolayer model --- p.46Chapter 2.3.4.1 --- Integrity of Caco-2 cell monolayer at pH 6.0 --- p.46Chapter 2.3.4.2 --- Permeability of paracellular and transcellular markers at pH 6.0 --- p.46Chapter 2.3.4.3 --- Validation of the existence of P-glycoprotein (P-gp) transporterin Caco-2 monolayer model --- p.46Chapter 2.3.4.4 --- Cytotoxicity test --- p.47Chapter 2.3.5 --- Transport study of GTC using Caco-2 cell monolayer model --- p.48Chapter 2.3.5.1 --- Bi-directional transport experiment --- p.48Chapter 2.3.5.2 --- Preparation of different dosing formulations of GTC --- p.48Chapter 2.3.5.2.1 --- Preparation of individual pure GTC solutions --- p.48Chapter 2.3.5.2.2 --- Preparation of cocktail 1 solution --- p.49Chapter 2.3.5.2.3 --- Preparation of green tea extract solution --- p.49Chapter 2.3.5.2.4 --- Preparation of cocktail 2 solution --- p.50Chapter 2.3.5.3 --- Sample treatment --- p.50Chapter 2.3.5.3.1 --- Samples for direct analysis --- p.50Chapter 2.3.5.3.2 --- Samples for enzymatic hydrolysis treatment --- p.51Chapter 2.3.5.4 --- Further investigation of the transport mechanism of GTC --- p.51Chapter 2.3.5.4.1 --- Inhibition transport of EC and EGC --- p.51Chapter 2.3.5.4.2 --- Transport mechanism of metabolites of EC and EGC --- p.52Chapter 2.3.5.4.3 --- Metabolic competition between EGC and the other GTC --- p.52Chapter 2.3.6 --- Calculation --- p.53Chapter 2.3.7 --- Data analysis --- p.54Chapter 2.4 --- Results --- p.55Chapter 2.4.1 --- Validation of the HPLC methods --- p.55Chapter 2.4.2 --- Stability of the GTC --- p.55Chapter 2.4.3 --- Extract of green tea leaves --- p.55Chapter 2.4.4 --- Validation of Caco-2 model --- p.59Chapter 2.4.4.1 --- Integrity of Caco-2 cell monolayer --- p.59Chapter 2.4.4.2 --- Permeability of paracellular and transcellular markers at pH 6.0 --- p.59Chapter 2.4.4.3 --- Validation of P-glycoprotein --- p.60Chapter 2.4.4.4 --- Cytotoxicity test --- p.61Chapter 2.4.5 --- Transport study of GTC --- p.63Chapter 2.4.5.1 --- Bi-directional transport of individual pure GTC --- p.63Chapter 2.4.5.2 --- Bi-directional transport of GTC in different dosing formulations --- p.66Chapter 2.4.5.2.1 --- Absorption transport profile of GTC in different dosing formulations --- p.66Chapter 2.4.5.2.2 --- Secretion transport profile of GTC in different dosing formulations --- p.66Chapter 2.4.5.3 --- Identification of metabolites of each GTC formed during the transport in Caco-2 cell model --- p.71Chapter 2.4.6 --- Further investigation of the transport mechanism of GTC --- p.82Chapter 2.4.6.1 --- Inhibition transport of EC and EGC --- p.82Chapter 2.4.6.2 --- Transport mechanism of metabolites of EC and EGC --- p.82Chapter 2.4.6.3 --- Metabolic competition between EGC and the other GTC --- p.85Chapter 2.4.6.4 --- Contribution of GTC on the metabolism of EGC --- p.89Chapter 2.5 --- Discussion --- p.92Chapter 2.5.1 --- Stability of the four GTC --- p.92Chapter 2.5.2 --- Validation of Caco-2 cell model --- p.92Chapter 2.5.3 --- Bi-directional transport of GTC --- p.93Chapter 2.5.4 --- Structure related efflux --- p.97Chapter 2.5.5 --- Metabolism of GTC --- p.98Chapter 2.5.6 --- Relationship between metabolism and efflux transport of GTC --- p.99Chapter 2.5.7 --- Bi-directional transport of GTC in different dosing formulations …… --- p.100Chapter 2.5.7.1 --- Absorption transport profile of different dosing formulations --- p.100Chapter 2.5.7.2 --- Secretion transport profile of different dosing formulations --- p.101Chapter 2.6 --- Conclusion --- p.105Chapter Chapter Three. --- Transport mechanism of hawthorn flavonoids --- p.106Chapter 3.1 --- Introduction --- p.106Chapter 3.2 --- Materials --- p.107Chapter 3.2.1 --- Chemicals --- p.107Chapter 3.2.2 --- Materials for cell culture --- p.108Chapter 3.2.3 --- Instruments --- p.108Chapter 3.3 --- Methods --- p.109Chapter 3.3.1 --- Analytical methods for HF --- p.109Chapter 3.3.1.1 --- Analytical methods of individual pure compound of HF --- p.109Chapter 3.3.1.1.1 --- HPLC analysis of HP and IQ --- p.109Chapter 3.3.1.1.2 --- HPLC analysis of EC --- p.109Chapter 3.3.1.2 --- Preparation of calibration curves for individual pure HF --- p.109Chapter 3.3.1.3 --- HPLC/MS analysis of three HF in mixture --- p.110Chapter 3.3.1.4 --- Preparation of the calibration curves of three HF in mixture --- p.111Chapter 3.3.1.5 --- Validation of HPLC methods --- p.111Chapter 3.3.2 --- Analytical methods for identification of metabolites with HPLC/MS --- p.111Chapter 3.3.3 --- Cell culture --- p.112Chapter 3.3.4 --- Cytotoxicity test --- p.113Chapter 3.3.5 --- Transport studies of HF using Caco-2 monolayer model --- p.113Chapter 3.3.5.1 --- Bi-directional transport experiment --- p.113Chapter 3.3.5.2 --- Preparation of loading solutions in different dosing formulations of HF for Caco-2 cell model --- p.114Chapter 3.3.5.2.1 --- Preparation of individual pure HF solutions --- p.114Chapter 3.3.5.2.2 --- Preparation of cocktail 1 solution --- p.114Chapter 3.3.5.2.3 --- Preparation of hawthorn extract solution --- p.114Chapter 3.3.5.2.4 --- Preparation of cocktail 2 solution --- p.114Chapter 3.3.5.3 --- Sample treatment --- p.115Chapter 3.3.5.4 --- Further study of the transport mechanism of HF --- p.115Chapter 3.3.5.4.1 --- "Inhibition transport of EC, IQ, and HP" --- p.115Chapter 3.3.5.4.2 --- "Transport mechanisms of the metabolites of EC, HP, IQ" --- p.116Chapter 3.3.6 --- Calculation --- p.116Chapter 3.3.7 --- Data analysis --- p.117Chapter 3.4 --- Results --- p.118Chapter 3.4.1 --- Validation of the HPLC methods --- p.118Chapter 3.4.2 --- Cytotoxicity test --- p.118Chapter 3.4.3 --- Transport study of HF --- p.122Chapter 3.4.3.1 --- Bi-directional transport of individual pure HF --- p.122Chapter 3.4.3.2 --- Bi-directional transport of the HF in different formulations --- p.123Chapter 3.4.3.2.1 --- Absorption transport of different formulations of HF --- p.123Chapter 3.4.3.2.2 --- Secretion transport of different dosing forms --- p.123Chapter 3.4.3.3 --- Identification of metabolites of each HF formed during their transport in Caco-2 model --- p.126Chapter 3.4.4 --- Further study on the transport mechanism --- p.136Chapter 3.4.4.1 --- "Inhibition transport of EC, HP, IQ" --- p.136Chapter 3.4.4.2 --- Transport mechanism of metabolites of HF --- p.136Chapter 3.4.4.3 --- Transport profiles of HF metabolites upon the loading of different dosing formulations of HF --- p.138Chapter 3.5 --- Discussion --- p.140Chapter 3.5.1 --- Bi-directional transport of each HF --- p.140Chapter 3.5.2 --- Bi-directional transport of HF in different formulations --- p.141Chapter 3.6 --- Conclusion --- p.142Chapter Chapter Four. --- Limitations of the current study --- p.143Chapter Chapter Five. --- Overall conclusions --- p.146References --- p.148Appendices --- p.16