Study On Intestinal Absorption Mechanism Of Major Components Of Cyperi Rhizoma With Jianchanbang By Rats Single Pass Intestinal Intestinal Perfusion In Situ And Caco-2 Cell Model

Objective:The present study was to investigate the intestinal absorption mechanism of major components?cyperotundone and?-cyperone?of Cyperi Rhizoma with Jianchanbang and to provide scientific basis for the clinical application and formulation design of Cyperi Rhizoma with Jianchanbang,using Caco-2 cell and in situ single pass intestinal perfusion as model.Methods:1.Establishment and evaluation of Caco-2 cell modelBy observing the morphology of cells,determine the activity of AKP,TEERand the permeability of fluorescein sodium markers,evaluate the success of the establishment of Caco-2 cell monolayer model in vitro.2.Cytotoxicity evaluation of extracts and major components of Rhizoma Cyperi with JianchanbangMTT cytotoxicity test was used to calculate the cell viability of petroleum ether part of Cyperi Rhizoma with Jianchanbang,cyperotunone,?-cyperone and its mixture?cyperotunone+?-cyperone?at different mass concentration.The safe concentration of the drug was determined by the corresponding concentration of the cell viability??90%?.3.Study on intestinal absorption mechanism of major components of Cyperi Rhizoma with Jianchanbang based on Caco-2 cell model.The contents of cyperotundone and?-cyperone in transport experiment samples were determined by HPLC-MS/MS.The effects of concentration,time,EDTA,P-gp inhibitor and MRP2 inhibitor on the transmembrane transport of cyperotundone and?-cyperone in Caco-2 monolayer cell model were investigated,and the forward transport characteristics before and after the compatibility of cyperotundone and?-cyperone in Caco-2 cell model were investigated.4.Study on intestinal absorption mechanism of major components of Cyperi Rhizoma with Jianchanbang in rats by in-situ single pass intestinal perfusion model.The contents of cyperotundone and?-cyperone in intestinal perfusion samples were determined by HPLC.The effects of different intestinal segments,concentration,EDTA and P-gp inhibitor on the intestinal absorption of cyperotundone and?-cyperone were investigated,and the effects of petroleum ether part of Rhizoma Cyperi with Jianchanbang simulation system and petroleum ether of Rhizoma Cyperi with Jianchanbang system on the intestinal absorption of cyperotundone and?-cyperone were investigated.Results:1.After 21 days of growth,there were no gap between the Caco-2 cells;The activity of AKP on AP side was about 8 times that of AKP on BL side,indicating that cell could polarize;TEER value of Caco-2 monolayer cell?600?·cm²;The P_apppp value of fluorescein sodium was?2.61±0.20?×10^-77 cm·s^-1,less than 10^-66 cm·s^-1,indicating that the cell integrity was good.2.MTT assay showed the concentration of methanol<2%;The safe concentration of petroleum ether part of Rhizoma Cyperi with Jianchanbang was 2.98⁴6.45?g·mL^-1;The safe concentration of cyperotundone was 3⁹0?g·mL^-1;The safe concentration of?-cyperone was 3¹20?g·mL^-1;The concentration of?-cyperone in each proportion remained unchanged at 30?g·mL^-1,the viability of cell was more than 90%after 4 hours of the combination of cyperotundone and?-cyperone at the ratio of 1:2 and 1:3.3.The transport amounts of cyperotundone and?-cyperone were concentration-and time-dependent,The Papp value of cyperotundone and?-cyperone>1×10^-66 cm·s^-1,and the efflux rate was 0.5¹.5.After EDTA,verapamil and MK-571were added,there were no significant difference in the P_apppp and ER values of cyperotundone and?-cyperone.After compatibility,the Papp values of AP-BL side of cyperotundone and?-cyperone increased significantly.4.The absorption of cyperotundone and?-cyperone was easy in the whole intestine,among which cyperotundone was the best in the ileum and?-cyperone was the best in the jejunum.Within a certain mass concentration range,there were no significant difference in the absorption parameters Ka and Peff of cyperotundone and?-cyperone,and the P_effff value>1.2×10^-3/cm·min^-1.The presence of verapamil and EDTA did not significantly improve the intestinal absorption of cyperotundone and?-cyperone;Compared with monomers,there were significant differences in absorption parameters K_a and P_effff values of cyperotundone and?-cyperone in the petroleum ether part of Rhizoma Cyperi with Jianchanbang simulation system and petroleum ether of Rhizoma Cyperi with Jianchanbang system.Conclusions:In this study,two intestinal absorption models were used to study the intestinal absorption mechanism of cyperotundone and?-cyperone.Passive diffusion was the main mechanism.Cell bypass and P-gp were not involved in their transport.Caco-2cell model study showed that the transmembrane transport of cyperotundone and?-cyperone were not affected by MRP2,and there might be a synergistic effect between cyperotundone and?-cyperone.In situ single pass intestinal perfusion model study found that there were a synergistic effect on intestinal absorption of cyperotundone and?-cyperone,and other components in the petroleum ether part of Rhizoma Cyperi with Jianchanbang might have a synergistic effect on intestinal absorption of cyperotundone and?-cyperone.