| Rhynchophylline is a kind of hydroxyindole alkaloid extracted from Uncaria tomentosa?wild.?DC.It is the main alkaloid in the extract of Uncaria tomentosa?Wild.?DC.Previous studies showed that rhynchophylline had a significant inhibitory effect on inflammation.The bioavailability of rhynchophylline was not high.For oral drugs,the most important factor affecting the bioavailability of drugs is the transport absorption and drug transport principle through the digestive tract.Because of the low bioavailability of rhynchophylline,this paper takes rhynchophylline as the model drug to build the experimental method of rhynchophylline absorption and transport and explore its absorption mode in the body.Caco-2 cell model is widely used in the model of imitating human intestinal epithelial cells in vitro.Its expression of transporter,P-glycoprotein?P-gp?and hydrolase are similar to that of human intestinal epithelial cells,so it is often used in the experiment of absorption and transport principle of experimental drugs in human intestinal epithelial cells.Therefore,the purpose of this study is to observe the absorption and transport of rhynchophylline in Caco-2 cell model,and to provide reliable experimental data and theoretical basis for the research of rhynchophylline in the future.Objective:To construct and validat a Caco-2 cell model,and observe the law of absorption and transport of rhynchophylline in the Caco-2 cell model.Method:1.The changes of the morphology of Caco-2 cells were observed,the single layer TEER value of Caco-2 cells and the activity of alkaline phosphatase,a marker of intestinal brush border cells,were measured,and the Caco-2 cell model was established and verified.2.A high-performance liquid chromatography?HPLC?method was established to detect the content of rhynchophylline in Caco-2 cell model.It was used to determine the concentration of the tested drug in the basal?BL?and intestinal?AP?surfaces.The apparent permeability coefficient?Papp?and apparent permeability ratio?PDR?of these indexes were calculated.3.To observe the absorption of rhynchophylline in Caco-2 cell model at different concentrations?31.25,62.5 and 125mM?and at different times?0,30,60,90 and 120min?.4.The effect of verapamil,a P-glycoprotein inhibitor,on the absorption of rhynchophylline in Caco-2 cell model was investigated.Results:1.Under the inverted optical microscope,it was observed that the cells could form a good tight connection.The cell growth was uniform and the contour was clear.On the21st day,the TEER value increased to 984.45±55.15?*cm2.The resistance before and after the transport experiment was basically constant,indicating that it had enough integrity.On the 21st day,the ratio of AP/BL was 5.10±0.07,indicating that with the increase of culture time.The ratio of alkaline phosphatase activity on AP side to that on BL side was significantly increased.AP side was the main site of alkaline phosphatase accumulation,and polarization occurred at the same time.Caco-2 cell model was successfully established.2.Successfully established an accurate and reliable HPLC detection method for the detection of rhynchophylline in HBSS solution of Caco-2 cell model,the peak time reached 8.06 minutes,the time is more suitable,no significant Caco-2 model Solution interference.3.Rhynchophylline in the concentration range of 31.25,62.5 and 125?M,the Papp(10-6cm/s)B?A group and the Papp(10-6cm/s)A?B value of rhynchophylline are concentration-dependent.The ratio between Pap(10-6cm/s)B?A and Papp(10-6cm/s)A-B called PDR value>1.5.4.In the range of 0?120 min,the bidirectional transport volume increased with time,and the absorption was significantly enhanced at the 60 min time point,and Papp B-A was significantly higher than Papp A-B,indicating that the course of absorption is time-dependent and the efflux is greater than the absorption.5.P-gp protein inhibitor verapamil can increase the value of rhynchophylline Papp?AP?BL?about 1.63 times of the control group?without inhibitor??20.76±2.27?×10-6cm/s vs?12.74±0.54?×10-6cm/s,P<0.05).The P-gp protein inhibitor verapamil can significantly reduce the apparent permeability coefficient?Papp value?of rhynchophylline from the BL surface to the AP surface,and significantly increase the apparent permeability coefficient?Papp value?from the AP surface to the BL surface.After adding the P-gp inhibitor verapamil,the transport rate of rhynchophylline was increased from?51.56±1.36?×10-5nmol/s to?60.37±5.31?×10-5nmol/s in AP?BL The transfer rate in the direction of BL?AP decreased from?86.46±6.15?×10-5nmol/s to?72.74±4.08?×10-5nmol/s.Conclusion:1.The cultured Caco-2 cells grow uniformly,have clear outlines,and are tightly connected.The Caco-2 cell monolayer model is successfully established and can be used for drug transport experiments.2.Successfully established the high-performance liquid chromatography?HPLC?detection method for rhynchophylline,which was used for the determination of its content in Caco-2 cell model.3.Rhynchophylline induced the expression of P-glycoprotein in Caco-2 cells,and the amount of bilateral transport increased with the increase of time and drug concentration.The transport of rhynchophylline in Caco-2 cell model has obvious efflux,which reveals its equal permeability.4.Rhynchophylline is partially absorbed.The absorption method through the Caco-2cell model is mainly free diffusion with the participation of efflux protein P-gp,and there may be other efflux proteins involved.5.Rhynchophylline is P-gp substrate. |
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