Initial Study On Interaction Between The Extracts From Angelica Sinensis And P-glycoprotein In Vitro

To study the influence of extracts from Angelica Sinensis and its relative components on the function and expression of P-glycoprotein in Caco-2 cells,and the effect of these components on the rhodamine-123 bidirectional transport though Caco-2 cells monolayer.Caco-2 cells and ECV304 cells were cultured with MEM (Eagle's minimum essential medium) contains 10% FBS(Fetal Bovine Serum) in a humidified atmosphere of 95% air and 5% CO2 at 37℃. The expression of P-glycoprotein in the cells was evaluated by immunofluorescence assy.First,we preliminarily analyzed the water extract of Angelica Sinensis with the method of HPLC-UV. Then, MTT (diphenyltetrazolium bromide) assay was used to find the non-cytotoxicity dosage of different drugs in experiments in Caco-2 and ECV304 cells. The dosage at which the survival rate of cells is above 90% was considered as the highest non-cytotoxic dosage.The effect of the extracts from Angelica Sinensis and its active components on P-glycoprotein function was analyzed using Rh-123 assay, flowcytometry was used to determine the intracellular Rh-123 concentration.The verapamil and dexamethasone were the control groups.Dexamethasone was used as positive control of up regulation effect on the expression of P-glycoprotein,and Caco-2 cells without drug dealing were used as negative control. Flowcytometry was used to measure the expression of P-glycoprotein in Caco-2 cells.The Caco-2 cells monolayer model was builded,identified,and Cyclosporin A was used as positive control drug to posses the inhibitory effect on the function of p-glycoprotein. Then we investigated the effcts of four drugs from Angelica Sinensis on rhodamine-123 bidirectional transport though Caco-2 cells monolayer.Both ECV304 cells and Caco-2 cells grew well in the experimental conditions. The expression of P-glycoprotein is plentiful in Caco-2 cells but sparse in ECV304 cells. Caco-2 cells is suitable for studying four anti-tuberculosis drugs on the function and expression of P-glycoprotein, and ECV304 cells is fit to be the negative control.Through the preliminarily analyzed the water extract of Angelica Sinensis,the percentage of FA is 0.107%, meeting the need of pharmacopoeia of people's republic of china.For 72h's MTT assay,AW,AE and FA at 100μg/mL,BGT at 1μg/mL,Ver,Dex and CsA at 10μg/mL showed to be non-cytotoxic towards the Caco-2 cells, and the survival rates of cells were greater than 90%. Compared with the negative group,after incubated with cells for 1h,the AW showed the moderated induced effct on P-glycoprotein function at the concentrations of 10,100μg/mL,and BGT mildly decreased the rhodamine-123 efflux when the concentrations were no less than 0.5μg·ml-1.However, EA(10,100μg/mL) and FA(1(10,100μg/mL) showed slightly interaction with the rhodamine-123 efflux.At the protein level, Longer term (72h) co-incubation of the Caco-2 cells with different durgs,the fluorescent intensity of AE group was larger than the negative group.And the fluorescent intensity of BGT group and AE group were lower than the control groups.The FA group showed slight effect on the expression of P-glycoprotein. These results indicated that there were some synergistic reactions between the effects of the expression and funcntion of P-glycoprotein. The result of rhodamine-123 bidirectional transport was that the Papp of rhodamine-123 from BL to AP was increased by AW,compared with the negative group. Contrarily,the values of Papp of rhodamine-123 from BL to AP were decreaed after Caco-2 cells co-maintained with AE,BGT and FA. Moreover, the values of Papp of rhodamine-123 from BL to AP were raised after ineracted with these three drugs.This result demonstrated that P-glycoprotein could regulated by Angelica Sinensis.Though there were so many ingredients in Angelica Sinensis,the regulations to P-glycoprotein by AW,AE,BGT and FA were various.1 The P-glycoprotein could be instaneously induced by AW,and the expression of P-glycoprotein is up-regulated in 72h.What's more,the value of ER of rhodamine-123 was increased in rhodamine-123 bidirectional transport.This indicated that the absorbing,the plasma concentration and therapeutic effect might be reduced when the substrates of P-glycoprotein co-administrated with the Decoction of Angelica sinensis.2 The function,expression of P-glycopreotein and the,the ER of rhodamine-123 were inhibited by BGT contained in Angelica sinensis. 3 After incubated with AE and FA, the rhodamine-123 bidirectional transports in Caco-2 cells were inhibited in some extent,and the value of ER of rhodamine-123 concomitantly abated.However,both of them showed negligible effects on the function and expression on P-glycorotein,their role played in transport via P-glycoprotein need deepgoing trials in vivo.We still must pay attention to the interaction and adverse reaction while P-glycoprotein substrates co-cosumed with AE and FA.