Studies On The Separation Of The Monoterpene Glycoside Of Radix Paeoniae Rubra And Metabolism Of Paeoniflorin In Vitro

Radix Paeoniae Rubra(RPR),the dried root of Paeonia lactiflora Pall.or Paeonia veitchii Lunch.,has been widely used by Chinese medicine practitioners to treat cardiovascular,inflammation and female reproductive diseases.Based on the principle of Chinese medicine,historical literatures described RPR with the functions of tonifying blood,cooling blood,cleansing heat,and invigorating blood circulation.In recent years,plenty of studies showed that Radix Paeoniae Rubra had effects on coagulant,atherosclerosis,thrombo and protecting the heart and liver etc.,and it had broad application prospects.The main active components of Radix Paeoniae Rubra include paeoniflorin,albiflorin,hydroxy paeoniflorin,benzoyl paeoniflorin and other monoterpene glycosides,which are considered as total paeony glycoside(TPG)collectively.There are two aspects in this study,One is to extract,separate,isolate and identify the monoterpene glycoside from RPR.The other is the metabolism of paeoniflorin in vitro.To study the main influencing factor in TPG extraction process from the root of RPR and provide technology reference to Chinese traditional medicine scopes.First,we extracted TPG by way of heat reflux extraction,the extract solution was filtered and concentrated.The residue was dissolved into water and extracted by petroleum ether,ethyl acetate,water-saturated n-butanol sequentially.The fraction was concentrated and separated by TLC,silica gel column chromatography and the semi-preparing liquid chromatography.The target compound was further purified and crystallized.Six of them were identified on the basis of spectral analysis and physicochemical evidences.They are paeoniflorin,albiflorin,oxypaeoniflorin,lactiflorin and benzoic acid.The purity of them detected by HPLC was greater than 95%.The paper described the preliminary research of paeoniflorin’s metabolism in vitro.The studies of metabolism in vitro were carried out by using culture of intestinal bacteria from human feces and rat liver microsome experiment.Studies on the metabolism of paeoniflorin by intestinal bacteria.The culture of intestinal bacteria were produced from human feces,and then incubated with paeoniflorin by anaerobic culture to prepare metabolites.Paeoniflorin incubated with intestinal bacteria for 2,4,8,12,18,24,30,36 and 48h.HPLC and HPLC-MS/MS were used to determine the content of remaining paeoniflorin confirm the chemical structures of the metabolites.The result showed paeoniflorin had totally transformed at 12h.Paeoniflorin was transformed into 34 metabolites by human intestinal bacteria in vitro,and we have speculated the structure of 10 metabolites.Among them,7 metabolites found firstly in the system.In order to study the metabolism of paeoniflorin in rat liver microsomal incubation in vitro system,the paper established a rat liver microsomal incubation system in vitro and detection method of HPLC-DAD.In order to investigate the metabolites and their metablic pathways,we established the HPLC-MS conditions.The rat liver microsomes were prepared by ultracentrifugation method.The concentration of the protein was 25.88mg·ml-1 determined by Lowry’s method.Paeoniflorin was incubated in incubation system that contained rat liver microsomes and various coenzyme for 120 min at 37℃.Reaction was terminated by adding methanol into transformation samples.The transformation samples were vortexed and centrifuged.Then samples were injected into HPLC system for analysis.The results showed that the optimum extraction condition was 2mg/L liver microsomal,1mmol/L NADP,0.5mmol/L NADH,10mmol/mL G-6-P,1.0IU/mL G-6-P DH,4.0mmol/L Mg2+,paeoniflorin 0.1mg/mL.The result by HPLC-MS/MS was that Paeoniflorin was transformed into 23 metabolites by rat liver microsomes in vitro,and we have speculated the structure of 5 metabolites.Among them,5 metabolites found firstly in the system.This study is of great significance for clarify the behavior of paeoniflorin in vitro.It also made a valuable exploration in metabolism research of traditional Chinese medicine.The above research explored the possible metabolic pathways of paeoniflorin.It is very significance in indicating the clinical rational medication,as well as providing the evidence of theory and experiment for the further research of paeoniflorin and development and utilization.