Study On The Hypoglycemic Activity And Pharmacokinetics Of The Incremental Ingredients Of Zhimu

Objective:Anemarrhenae Rhizoma is the dry rhizome of Anemarrhena asphodeloides Bge.with the efficiency of clearing heat and purging fire,nourishing yin and moistening dryness.In the previous study,we have found that the salt Anemarrhena Rhizoma can decrease blood sugar significantly.Through the comparative study on the chemical composition before and after processing,we have identified 3incremental components: timosaponin A?,timosaponin B? and mangiferin.This thesis focus on the incremental components,timosaponin A?,timosaponin B?,mangiferin,to study the hypoglycemic activity in vitro and the pharmacokinetics,to confirm the relation between the incremental components and the efficiency,and explain the processing principle more,provide a theoretical basis for the rational use of salt Anemarrhenae Rhizoma in clinical practice.Materials and methods:Materials: Anemarrhenae Rhizoma herbs were purchased from Yixian County,Hebei Province.Anemarrhenae Rhizoma pieces were purchased from Sichuan Neautus Traditional Chinese Medicine Co.,Ltd.Professor Zhai Yanjun,in the medicine college of Liaoning University of Traditional Chinese Medicine?Dalian campus?,identified that as the dry rhizome of the Anemarrhena asphodeloides Bge.Methods:1.Study on the relationship between increasing Ingredients of salt Anemarrhena e Rhizoma and hypoglycemic activityUPLC-MS was used to establish a method for the determination of Timosaponin A?,Timosaponin B? and Mangiferin in salt Anemarrhenae Rhizoma,compare with t he contents of the three increasing Ingredients in salt Anemarrhenae Rhizoma of diffe rent extraction methods,analyze its transformation pathways and transformation produ cts,and explore the reasons for their composition transformation and the basic princip les of processing.UPLC-MS was used to determine the contents of three components of salt Anemarrhenae Rhizoma medusae after chloroform and ethyl acetate extraction.To explore the ultimate reason for the conversion of hypoglycemic active ingredientsafter Anemarrhenae Rhizoma salted,this article further extracted the ethanol extract of Anemarrhenae Rhizoma and used the chloroform and ethyl acetate equal volume extraction to extract Saponins?Timosaponin A?,Timosaponin B??and Dipyrazolone?Mangiferin?between different processed samples,so as to find the basis for studying the principle of salt Anemarrhenae Rhizoma processing.2.Study on the hypoglycemic effect of the incremental components from Salt Rhizoma AnemarrhenaeBy investigating the effects of timosaponin A?,timosaponin B? and mangiferin on the glucose consumption of HepG-2 cells and the protect and treatment function on NIT-1 insulin ? cell,to confirm the processing principle of enhancing the hypoglycemic effects of salt Anemarrhenae Rhizoma.HepG-2 cells were divided into blank control group,timosaponin A? group,timosaponin B? group,mangiferin group,metformin group,cultured in a 5% CO₂ incubator for 24 hours,starved for 12 hours,and then added the corresponding concentrations drug-containing or drug-free serum medium,then continue being cultured for 24 hours,a glucose kit was used to detect the glucose contents in the culture solution.After the glucose consumption test,CCK-8 reagent was added,and cultured for 2 hours,and the absorbance?OD?was measured with a microplate reade,and calculate the survival rate?IC?.Insulin was used to induce the insulin resistant HepG-2 cells,the effects of timosaponin A?,timosaponin B?,and mangiferin on glucose consumption of insulin resistant HepG-2 cells were compared with the above method.The NIT-1 islet ? cells were divided into blank control group,timosaponin A? group,timosaponin B? group,mangiferin group.Serum 1640 medium was added into blank group,STZ and corresponding concentrations of the drug serum medium were added into the rest of the groups,and cultured in a 5% CO₂ incubator for 48 hours,then cck-8 reagent was added,and culture was performed for 2 hours.The absorbance?OD?was measured by using a microplate reader;the NIT-1 islet ? cells were cultured as the above method,then the contents of nitric oxide,malondialdehyde,and superoxide dismutase were measured by using the kit respectively.2.Comparison of the pharmacokinetics of the incremental components from Salt Rhizoma AnemarrhenaeThe rats were respectively administered orally with the extracts of Anemarrhena Rhizoma and salt one,and timosaponin A?,timosaponin B? and mangiferin were used as indexes to be determined the concentration and absorption rate of the prototype drug in the plasma by Waters Xevo TQD liquid chromatography-mass spectrometry and DAS3.2.7 data analyze software.AUC_0-t,AUC_0-?of each index component were compared and analyzed,whether there is a significant difference.The rats were respectively administered orally with the extracts of Anemarrhena Rhizoma and salt one,whose feces and urine were collected at certain time,after gastric samples were processed,the contents of timosaponin A?,timosaponin B? and mangiferin prototype drug in that were detected by UPLC-TOF-MS,combined with the UNIFI database,analyze the best way of metabolism in rats of salt Anemarrhena.Result:1.Study on the hypoglycemic effect of the incremental components from Salt Rhizoma AnemarrhenaeTimosaponin A?,timosaponin B?,and mangiferin in each concentration group can increase the glucose consumption of normal HepG-2 cells,and there are significant differences compared with the blank control group?P<0.01 or P<0.05?.After STZ was used,compared with the blank control group,the glucose consumption of the model group was significantly reduced?P<0.05?,indicating that the HepG-2 cell insulin resistance model was established successfully.All concentrations of timosaponin A?,timosaponin B?,and mangiferin can increase the glucose consumption of insulin-resistant HepG-2 cells,and there are significant differences compared with the model control group?P<0.01 or P<0.05?.The result of cck-8 experiment suggest that timosaponin A??1.0?g/mL?,timosaponin B??1.0?g/mL?,and mangiferin?0.02?g/mL?and ensure the cell survival rate?IC value?was greater than 98%.Compared with the model group,timosaponin A?,timosaponin B? andmangiferin all can improve the NO level of NIT-1 islet ? cells,while the timosaponin B? group improved NIT best at 4 ?g/mL?P<0.05?;From the results of SOD activity,it was seen that the vitality of the model group was significantly lower than that of the control group?P<0.05?.Compared with the model group,the timosaponin A? group,the timosaponin B? group and the mangiferin group had a tendency to increase the SOD activity;the timosaponin B? group?20?g/ml+STZ?can significantly increased the vitality compared with the model group?P <0.05?;From the MDA activity results,we can see that the activity of the model group was significantly higher than that of the control group?P<0.05?.Compared with the model group,timosaponin A? group,timosaponin B? group and mangiferin group,there was a tendency to reduce MDA activity,and the mangiferin group played a particularly significant role?P<0.01?.It is presumed that the incremental components from salt Anemarrhenae Rhizoma could repair the damage of pancreatic islet ? cells by improving NO,MDA and SOD pathways.2.Study on pharmacokinetics of the incremental components from salt Rhizoma AnemarrhenaeThe rats were given intragastric administration of Anemarrhenae extracts.The peak concentration Cmax in plasma of timosaponin A? was 4.483±0.508?mg/L?,the half-life of t_1/2 was 1.818±0.467h;Cmax of salt one was 2.183±0.440?mg/L?,half-life t_1/2of salt one was 2.062±0.256 h,average residence time in plasma MRT_0-t,MRT_0-?were extended.The area under the plasma concentration-time curve of timosaponin A? in the blood-drug concentration-time curve was 16.463±4.203?mg/L*^h?,and the AUC_0-?was 16.48±4.216?mg/L*^h?.The AUC_0-tof salt Anemarrhenae was7.159±1.378?mg/L*^h?,and the AUC_0-?of salt Anemarrhenae was 7.163±1.382?mg/L*^h?.The peak concentration Cmax of timosaponin B? was 13.45±3.663?mg/L?,the half-life t_1/2 was 2.519±0.788 h,and the peak concentration of drug Cmax was12.717±2.257?mg/L?,the half-life t_1/2 salt Anemarrhenae was 2.665±0.516 h,and the mean residence time of plasma MRT_0-tand MRT_0-?were prolonged.The area under the plasma concentration-time curve of timosaponin A? in blood concentration-time curve was 75.596±29.438?mg/L*t1/^h?for AUC_0-tand 75.959±29.384?mg/L*^h?for AUC_0-?;AUC_0-tof salt Anemarrhenae was 101.817±25.414?mg/L*^h?,and AUC_0-?salt Anemarrhenae was 102.355±25.155?mg/L*^h?.The peak concentration Cmax of mangiferin was 33.917±2.559?mg/L?,the half-life of t_1/2 was 3.513±0.358 h,and the peak concentration of drug Cmax was 33.637±4.675?mg/L?.The half-life t_1/2 of salt Anemarrhenae was 5.134±1.045 h,and the mean residence time of plasma MRT_0-tand MRT_0-?were prolonged.The area under the concentration-time curve of mangiferin plasma concentration was 167.746±24.332?mg/L*^h?for the AUC_0-tand171.289±25.781?mg/L*^h?for the AUC_0-?;AUC_0-tof salt Anemarrhena was202.742 ± 53.526 mg/L *^h,AUC_0-?of salt Anemarrhena was 210.698 ± 55.206?mg/L * h?.The rats were given intragastric administration of Anemarrhenae extracts,and the excretion of urine and feces were collected at fix time.After a series of sample processing methods,UPLC-MS method was established to study the internal excretion pathway in rats.According to the contents of timosaponin A?,timosaponin B? and mangiferin in urine and feces at different time,calculate the cumulative excretion fraction in rat urine and feces of raw salt anemarrhenae and salt one?cumulative excretion/administration dose×100%?.It was also found that timosaponin A?,timosaponin B? and mangiferin in the urine and feces of rats were excreted in a prototype way.It is presumed that the timosaponin A?,timosaponin B? and mangiferin of Anemarrhena Anemarrhena may be eliminated mainly in the form of metabolites.Therefore,it is necessary to study the metabolites of Anemarrhena Anemarrhena and provide a theoretical basis for the final determination of its metabolic pathway and the basis of drug effect.Conclusion:components from salt anemarrhena,all have hypoglycemic effect,and the absorption can be enhanced because of salt processing,which is the material basis for the enhancement of the efficacy of salt Anemarrhena,is relative directly with the hypoglycemic effect that have been verified before.