Metabolism Of Paeoniflorin And Albiflorin

Red peony root and white peony root are two commonly used traditional Chinese medicines. The former is from the dried root of Paeonia lactiflora Pall. and Paeonia veitchii Lynch. The later is from the decorticated, boiled and dried roots of Paeonia lactiflora Pall..Paeoniflorin and albiflorin are the two main components in Red and white peony root. Our present paper described the preliminary research of paeoniflorin and albiflorin's metabolism from two dire-ctions in vitro and in vivo. The studies of metabolism in vitro were carried out by using microbial-biotransformation model,culture of intestinal bacteria from rat feces,liver microsome experiment. Using HPLC-MS/MS method, the tentative metabolites in vivo was also found by analyzing urine and bile samples in rats after oral administration of paeoniflorin and albiflorin. The mutual transformation between the two components and dependablity of the results in vivo and in vitro models were preliminary discussed. Further more, the binding rates of paeoniflorin and albiflorin with human and rat plasma proteins were determined by equilibrium dialysis method. This study is of great significance for clarify the behavior in vivo of paeoniflorin and albiflorin so as to red and white peony. It also made a valuable exploration in metabolism research of traditional Chinese medicine. Part one Microbiological Transformation of Paeoniflorin and AlbiflorinObjective:To investigate the microbiological transfor mation of paeoniflorin and albiflorin and isolate and identifed the metabolites.Methods : Eighteen species of bacteria strains were screened, the one with the strong convert ability was selected for transformation of paeoniflorin and albiflorin, respectively. The products were isolated with chromatography using silica gel, ODS as staionary phase. The purification was completed with preparation HPLC. The structures were elucidated with HPLC-MS and NMR.Results:Cunninghamella blakesleana (AS 3.970) and Syncephalastrum racemosum (AS 3.264) were found to convert paeoniflorin and albiflorin efficiently,respectively. Two pure products were obtained. Cunninghamella blakesleana could convert paeoniflorin to albiflorin,while Syncephalastrum racemosum could convert albiflorin to paeoniflorin.Conclusion:Paeoniflorin and albiflorin could be converted each other in definited condition.Part two Studies on the Metabolism of Paeoniflorin and Albiflorin by Intestinal BacteriaObjective : To study the in vitro metabolism of paeoniflorin and albiflorin by intestinal bacteria from rats.Methods : Paeoniflorin and albiflorin contents were measured by HPLC.The separations were performed on a C18 reverse phase column with the mobile phase consisting of CH₃OH-H₂O at the flow rate of 1.0 mL·min^-1.The detection wave length was at 230 nm. Paeoniflorin and albiflorin incubated with rat feces for different times. HPLC-MS and NMR were used to confirm the chemical structures of the metabolites.Results:Paeoniflorin was transformed into albiflorin by rat intestinal bacteria in vitro, and albiflorin was transformed into paeoniflorin. The contents of the metabolites could be changed with time.Conclusion:Paeoniflorin and albiflorin could be converted each other by intestinal bacteria from rats.Part three Studies on the Metabolism of Paeoniflorin and Albiflorin by liver microsomeObjective: To find the metabolites by culturing with iver microsome and prepare pure metabolites as far as possible.Methods:Paeoniflorin and albiflorin were added to the incubation liquid which was prepared according to the pertinent literature. The incubation solution was treated and analyzed by HPLC-DAD.Results:There was no metabolite found in the liver microsome for both rat and mouse afer trial and error.Conclusion : The metabolism of paeoniflorin and albiflorin by liver microsome are probably very weak.Part four Identification of metabolites of paeoniflorin and albiflorin in rat urine and bile by HPLC-MS/MS Objective:To identify the metabolites of paeoniflorin and albiflorin in rat urin and bile.Methods:After oral administration of paeoniflorin and albiflorin, the urine and bile of male rats were collected within 24h. The urine samples and bile were extracted and analyzed with HPLC-MS/MS.Results:Several new metabolites were found and identified in rat urine and bile.Conclusion:HPLC-MS/MS is proved to be a simple, rapid, sensitive and specific technique for identification of the metabolites of paeoniflorin and albiflorin in rat urine and bile.Part five Determination in vitro binding rate of paeoni-florin and albiflorin to rat and human plasma protein.Objective : To develop a high performance liquid chro-matography (HPLC) to determine the protein binding rates of paeoniflorin and albiflorin in human plasma and rat plasma,Methods:The equilibrium dialysis method was employed to determine the plasma protein binding rate of paeoniflorin and albiflorin. The plasma concentrations of paeoniflorin and albiflorin were measured by RP-HPLC.Results:The rat plasma protein binding rate of paeoniflorin at 1,5,10,15,20,30μg/ml were 60.45 %,67.60 %,78.66 %,74.07 %,76.29 %,74.27 % , respectively;the rat plasma protein binding rate of albiflorin at 1,5,10,15,20,30μg/ml were 58.94 %,78.54 %,80.82 %,82.43 %,80.36 %,79.63 %, respectively. The binding rate of paeoniflorin and albiflorin at 10μg/ml to human plasma protein were found to be 82.32% and 85.65%.Conclusion : The binding rates of paeoniflorin and albiflorin to human plasma protein and rat plasma protein were high relatively. The plasma protein binding rate of albiflorin is a little higher than paeoniflorin. Moreover the binding rate is identical proportional to plasma concentration of them. Paeoniflorin and albiflorin are dependant to concentration.While the concentration go to the plateau region, the binding rates of paeoniflorin and albiflorin to human plasma protein and rat plasma protein no more to increase.