The Influence Of Gastrointestinal Physiology Factors On Main Ingredients Of Tea Polyphenols And The Study On Absorption Process Of EGCG

Objective:Green tea polyphenols, including (-)-epicatechin (EC), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin gallate (EGCG), are the major active components in green tea. Despite the well known beneficial effects of GTPs, the oral bioavailability of GTPs has been demonstrated to be very low. In the present study, to explore factors that affect the stability of tea polyphenols, and to Simulate the process of absorption in the gastrointestinal tract. To discuss the methods of improving oral bioavailability of Green tea polyphenols.To investigate whether tea polyphenols exerts influence on P-gp and discusses the mechanism. In order to avoid or use the influence of tea polyphenols on drug co-administration.Methods:1. Using liquid chromatography to examine the recovery of EGCG、ECG、EC which are Main components of tea polyphenols in the environment of vary pH, time, enzyme and Intestinal flora in gastrointestinal tract.2. To establish Caco-2 cell modle. Transepithelial electrical resistance (TEER) value was measured using an volt ohmmeter. Changing in the morphology of Caco-2 cell were observed by transmission electron microscopy. Using Automatic microplate reader to detect the apparent permeability coefficient of fluorescein.3. The cytotoxicity of tea polyphenols on Caco-2 cell was detected by CCK-8 test. And to evaluate dispensing solvents and samples on Caco-2 cells.4. Digestion model combining with Caco-2 cell model, to simulate the process of digestion and absorption in the gastrointestinal tract for tea polyphenols in vitro. This solution was analysed by HPLC. Apparent permeability coefficient (Papp) was taken as indexes to analyze the process of absorption for tea polyphenols in the gastrointestinal tract.5. Using efflux of Rhodamine 123 test to detect the activity of P-gp of Caco-2 cell. Using Real time PCR to detect the influence of concentration and incubation time of tea polyphenols for the expression level of P-gp MDR1 gene.Using WESTERN BLOT to detect the influence of concentration and incubation time of tea polyphenols for the expression level of P-gp and NF-κB.Results:1. In a certain pH range, pH had low effect to tea polyphenols.However, with the increase of pH and time, the stability of tea polyphenols were decreased when pH is 5.8～8.0. Pepsin, pancreatin, bile salt have no effect on tea polyphenols. gut bacteria have major effect on tea polyphenols, and tea polyphenols were almost degraded after 6 h. In the three components of tea polyphenols, ECG、EC were more stable than EGCG.2. After Caco-2 cell modle culture for 21 days,the transepithelial electrical resistance (TEER) value reached 400～600Ωcm2. The apparent permeability coefficient of fluorescein was 0.45249×10-6 cm/s which was Less than the permeability of experiment of 0.5×10-6 cm/s. Microvilli and cell junction of Caco-2 cell were can observed by transmission electron microscopy, indicating that the integrity of the monolayers might have occurred.3. With the increase of concentration of tea polyphenols,the survival rates of Caco-2 was smooth at first, and than dramatically decreased. When the concentration of tea polyphenols was 0.25 mg/ml, the cell survival rate was 77.0384%;When the concentration of tea polyphenols was 0.5 mg/ml,the cell survival rate was 17.9872%;. Using the solvent of dispensing drugs and samples to incubation Caco-2 cell 2 h, cell survival rate is above 88%.4. RPapp (PappBL-AP/PappAP-BL) of EGCG＞2. The apparent permeability coefficient of tea polyphenols is very low. And the permeability significantly decreased when tea polyphenols was perfused with P-gp inhibitor verapamil. The apparent permeability coefficient in intestines buffer is lower than that in Stomach buffer.5. The efflux of Rhodamine 123 increased significantly under verapamil which is the P-gp inhibitor. Expression level of MDR1 was decreased with the increase of concentration and incubation time of tea polyphenols.the expression level of P-gp and NF-κ B was decreased with the increase of concentration and incubation time of tea polyphenols.Conclusion:1. Tea polyphenols is Very Stable Under acid condition. However, with the increase of pH and time, the stability of tea polyphenols were decreased when pH is 5.8～8.0. Pepsin, pancreatin, bile salt have no effect on tea polyphenols. Intestinal flora can produce some enzyme, which can degrade tea polyphenols. EGCG have ester and more phenolic hydroxyl, which lead it to a more unstability condition than ECG and EC.2.After 20 days, the transepithelial electrical resistance (TEER) value and the apparent permeability coefficient of fluorescein reach to the standard. Microvilli and cell junction of Caco-2 cell can be observed by transmission electron microscopy. Which show that Caco-2 cell monolayer model was established.3. The concentration of tea polyphenols should be<0.25 mg/ml, to ensure the survival rates of Caco-2＞50%. The solvent of dispensing drugs and samples have no effect to Caco-2 cell.4. RPapp (PappBL-AP/PappAP-BL) of EGCG＞2, which indicate that the transportion of EGCG was influenced by transporter. The permeability significantly decreased when tea polyphenols was perfused with P-glycoprotein inhibitor verapamil Which indicate that EGCG may be the substrate of P-gp. The apparent permeability coefficient in intestines buffer is lower than that in Stomach buffer, Which show that EGCG is unstable in alkaline environment or alkaline environment can promote the degradation of tea polyphenols. The apparent permeability coefficient is very low. Perhaps the main reason is that it is difficult for tea polyphenols through the cell membrane for the low fat soluble of tea polyphenols;And the nature of tea polyphenols is unstable, easy degradation in neutral and alkaline conditions;Some free tea polyphenols can be combined with cell membranes or absorbed into the cell;P-glycoprotein and MRP are responsible for some tea polyphenols efflux.5. The efflux of Rhodamine 123 increased significantly under P-gp inhibitor verapamil, Which is indicated that the functions of P-gp was in a good condition. Expression level of MDR1、P-gp and NF-κB showed a concentration-dependent and time-dependent reduction in Caco-2 cells.Tea polyphenols may be down-regulating the expression levels of NF-κ B.And than NF-κ B down-regulating the expression levels of MDRl.and than then decrease expression level of P-gp.