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 andα-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,α-cyperone and its mixture（cyperotunone+α-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 andα-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 andα-cyperone in Caco-2 monolayer cell model were investigated,and the forward transport characteristics before and after the compatibility of cyperotundone andα-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 andα-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 andα-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 andα-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﹥600Ω·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 2.98⁴6.45μg·mL^-1;The safe concentration of cyperotundone was 3⁹0μg·mL^-1;The safe concentration ofα-cyperone was 3¹20μg·mL^-1;The concentration ofα-cyperone in each proportion remained unchanged at 30μg·mL^-1,the viability of cell was more than 90%after 4 hours of the combination of cyperotundone andα-cyperone at the ratio of 1:2 and 1:3.3.The transport amounts of cyperotundone andα-cyperone were concentration-and time-dependent,The Papp value of cyperotundone andα-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 andα-cyperone.After compatibility,the Papp values of AP-BL side of cyperotundone andα-cyperone increased significantly.4.The absorption of cyperotundone andα-cyperone was easy in the whole intestine,among which cyperotundone was the best in the ileum andα-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 andα-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 andα-cyperone;Compared with monomers,there were significant differences in absorption parameters K_a and P_effff values of cyperotundone andα-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 andα-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 andα-cyperone were not affected by MRP2,and there might be a synergistic effect between cyperotundone andα-cyperone.In situ single pass intestinal perfusion model study found that there were a synergistic effect on intestinal absorption of cyperotundone andα-cyperone,and other components in the petroleum ether part of Rhizoma Cyperi with Jianchanbang might have a synergistic effect on intestinal absorption of cyperotundone andα-cyperone.