Study On Mechanism Of Absorption And Metabolism Of Ursolic Acid In Vitro

Objectives: To investigate the method of Caco-2 cell model and system of liver microsomes incubation for studying the mechanism of absorption and metabolism of UA in vitro respectively.and to refer the experimental and theories gist for a deep study of the pharmacokinetics and pharmacological mechanism .Methods: A reversed-phase high-performance liquid chromatography method with UV detection was developed for determination of UA on extracellular and intracellular of Caco-2 cell and in liver microsomes incubation. Establishing and valuating the characteristic of Caco-2 cell monolayer.Investigating the effect of uptake time,substrate concentration,system temperature and PH of culture media on the uptake of UA by Caco-2 cell. Checking the transport characteristic of whether P-gp inhibitor Verapamil exists or not, exploring excretion phenomenon in the experiment of uptake and transport on Caco-2 cell; Dexamethasone, phenobarbita andβ-naphthoflavone were selected to pretreat the SD rat in vivo. Rat liver microsomes calcium precipitation has been adopted to produce and establish the metabolic system of liver microsomes incubation. UA was taken as react substrate to investigate the metabolic behavior and characteristic in rat liver microsomes.Results:①C aco-2 cells cultured on transwell plate had formed a tight structure with a TEER value of Caco-2 monolayers being greater than 500Ω·cm2 and Dextran labeled rhodamine not being able to permeate.②In the Concentration range of 10~40μM,the uptake of UA by Caco-2 cell all linearly increased , which increased gradually within twenty minutes, and then tended to be stable. When its PH was 5.5, the uptake increased to its highest level and decreased with the increasing pH; The uptake of UA by Caco-2 cell showed no significantly statistical difference(P>0.05) when the temperature was 4℃,25℃and 37℃respectively.③the Apparent permeability coefficient of UA transported on Caco-2 cell monolayer model had taken significantly change when the sepecificity inhibitor of P-gp was added to model.and the PDR value decreased from 3.445 to 1.386.④The metabolism of UA in rat liver microsomes had showed the characteristics of enzyme kinetics with a significant correlation between peak area of a metabolite and concentration of UA in rat liver microsomes (r=0.975).⑤DEX is the classic inductor of CYP3A,which could promote the metabolism of UA in liver microsomes, which was significantly different from the control group (p<0.05). Whereas,the inhibitor of CPY1A and CYP2B,BNF and PB had no obvious effect on the metabolism of UA in liver microsomes(P>0.05).Conclusions: The study has established an integrated Caco-2 cell monolayer model in our laboratory, which meets the requirement of the drug transport experiment. The uptake of UA by Caco-2 cell has demonstrated that it greatly depends on concentration and time and may be greatly influenced by the value of PH despite the temperature. The transmembrance transport of UA had showed a significant direction. Verapamil could inhibit the excretion effect of transport of UA by P-gp significantly with an indication that P-gp might be the substrate of UA. The metabolism of UA in liver microsomes could be mediated by CYP3A. As for the issue about whether low oral bioavailability of UA may be mediated by excretion of P-gp and biotransformation of CYP3A .