Effects Of Meridine Guide Drug Vinegar-processed Radix Bupleuri On BRL

ObjectiveTo explore the mechanism of TCM meridine guide drug- vinegar processed Radix Bupleuri from the different segments of drug action by investigating the effect of vinegar processed Radix Bupleuri on the permeability, intracellular pH,membrane lipid composition,Morphological study,and P-gp reversal of BRL.MethodEffect of TCM meridine guide drug vinegar processing Radix Bupleuri on the proliferation of BRL in vitro was performed by reformed MTT assay. Intracellular fluorescence intensity was detected by flow cytometry,with the purpose of the effect of vinegar processed Radix Bupleuri on the permeability of BRL.The change of pHi was determined by fluorescent probe (BCECF/AM and FIT-Dextran).The fluorescence intensity was determinated by fluorescence spectrophotometer,and pHi of BRL were calculated according to the fluorescence intensity.The membrane protein and cholesterol content of BRL was determinated by automatic biochemical analyzer,and the membrane phospholipids by TLC-scanning application. Effect of TCM meridine guide drug vinegar processed Radix Bupleuri on membrane morphology of BRL was observated by electronic microscope lens. Effect of TCM meridine guide drug vinegar processing Radix Bupleuri on membrane p-glycoprotein expression was detected by flow cytometry.ResultIt suggested that the water extract of vinegar processed Radix Bupleuri had the strongest action on proliferation of BRL with the concentration range from 0.16mg/ml to 4mg/ml.we choose 10mg/mL,2 mg/mL,0.4mg/mL in the later experiment.The effect of drug on the permeability of BRL was different in different concentration and different time.Significant changes were found in BRL with 24h 5%drug plasm effect(p＜0.05).With the time going on,The permeability of BRL became increased.The result showed the difference between vinegar processed Radix Bupleuri group and control group were significant at level 0.01.The effect of drug on cytosolic(pHcyt) in BRL was different in different concentration or different time.Low concentration vinegar processing Radix Bupleuri has less effect on cytosolic(pHcyt),but middle and high concentration drug have different degree effect on pHcyt in different time.Significant changes were found in BRL with 24h drug effect (p＜0.01),the drug has the tendency of descent apparent vesicular pH (pHves) in BRL.Significant changes were found in BRL with 18h drug effect (p＜0.05),but no significant effect was found between different drug concentrations.The effect of drug on the contents of cholsterol, phospholipid and protein membrane of BRL was different in different concentration and different time.Significant changes were found in BRL with 12h drug effect and 24h high concentration drug effect(p＜0.01),the drug has the tendency of descent the contents of protein membrane in BRL. Significant changes were found in BRL with 24h low concentration drug effect and 48h low and middle concentration drug effect(p＜0.01),the drug has the tendency of ascent the contents of protein membrane in BRL. Significant changes were found in BRL with 3h low concentration drug effect(p＜0.01),the drug has the tendency of increase the contents of SM,PC and PE in membrane of BRL.Significant changes were found in BRL with 24h middle and high concentration drug effect(p＜0.01),the drug has the tendency of descent the contents of SM,PC and PE in membrane of BRL.there were no significant changes between vinegar processed Radix Bupleuri group and control group in other time.The result showed that The membrane morphological changes of BRL was little after different drug concentration effect;but the membrane of BRL were getting smooth in middle and high concentration drug effect.The expression of P-gp in BRL were descent,especially in low and middle concentration drug effect.ConclusionThe water extract of vinegar processed Radix Bupleuri had the strongest action on proliferation of BRL with the concentration range from 0.16mg/ml to 4mg/ml.It can increase the permeability and reduce p-glycoprotein expressionl of BRL.These factors may directly affect the drug in the cell or cell-to-cell distribution.The effect of drug on the contents of cholsterol,phospholipid and protein membrane of BRL was different in different concentration and different time.Significant changes of the contents cholsterol,phospholipid were found in BRL with 3h and significant changes of the contents protein were found in BRL with 12h,24h,48h.The basic cause needed further research.Vinegar-baked Radix Bupleuri had little effect on the the morphological shape and pH value of BRL.The results of this paper showed that vinegar-baked Radix Bupleuri. increased liver uptake of other drugs may through affected the permeability and the components of the BRL memberane,the detail mechanism needs further study.