| Traditional Chinese medicine (TCM) which has a long history in China,has beenfound and proven to have some unique advantages in some complex diseases in view ofnumbers of evidence from basic and clinical research.It’s advantage Relatively low toxicityand fewer adverse reactions are observed and gradually taken seriously by more and morecountry. In recent years, the usage of TCM is increased dramatically, But there are a lotof"obstacles"faced by natural medicines including TCM, such as low bioavailability,unsharp components, unclear metabolic process and so on,in their morden process of drugdevelopment.The main research objects in this topic are total anemarrhenae saponins andtimosaponin B–II,an active ingredient, overall extracted from rhizoma anemarrhenae,ancommonly used TCM in clinic.On basis of quality standards for semi-finished productsand finished products of the total anemarrhenae saponins, a kind of qualitative andquantitative analysis method was developed with HPLC and LC-MS/MS/MS technologyas a main monitoring way. The pharmacokinetical characteristics of timosaponin B-II andits metabolic mechanism in vitro and in vivo were researched, which includes:1. To establish the quality standards of the total saponins semi-finished products andfinished products for injection, a TLC was employed to identify Timosaponin B-II. TheTLC spots were clear,well-separated and specific yet without interference from negativecontrol. While the content of Timosaponin B-II, Timosaponin E1, Timosaponin B,in thetotal saponins semi-finished products and finished products was determined by HPLC,the methods can be used for the quality control simply, reliably.2. After a intravenous administration with5.4,1.8and0.9mg/kg three doses oftimosaponin B-II in SD rats, AUC0-tof timosaponin B-II was12560.62±2304.23,2363.64±184.44,843.70±34.28ng.h/mL and Cmaxof B-II was48996.55±9472.31ng/mL,12295.36±816.69ng/mL,3876.93±189.20ng/mL respectively. The area under theconcentration-time curve and peak concentration with dose performance positivecorrelation and show linear pharmacokinetic characteristics. Dose Cmaxand AUC0-twaspositively correlated, indicating that Timosaponin B-II intravenous linear pharmacokineticcharacteristics in rats. After oral administration of the timosaponin B-IIwith180mg/kg,90mg/kg,30mg/kg three dose groups, respectively, the AUC0-twas2291.38±1294.56ng.h/mL,331.01±218.25ng.h/mL,123.64±124.92ng.h/mL, Cmaxwas1654.56 ±1342.83ng/mL,442.69±353.38ng/mL,56.60±39.44ng/mL respectively. It indicated thatthe dependency of Cmaxand AUC0-ton dosage was not strong.Theconcentration-time curves for timosaponin B-II in Beagle dogs showed bimodalphenomenon or multimodal phenomenon,which indicated existence of enterohepaticcyclings potentially. Oral and intravenous administration, respectively, getting thebioavailability which is about0.75%, the timosaponin B-II in SD rats in vivo oralbioavailability is slightly higher than Beagle dogs, but are relatively low, and furthershowed that the timosaponin B-II weak absorption in the gastrointestinal tract as aprototype drug.The tissue distribution of Timosaponin B-II in SD rats: It was eliminated fast afterintravenous administration; there was no prototype drug accumulation after6hours, theconcentration of the drug in various organs and tissues in various organs were significantlylower than plasma concentrations, of which the drug concentrations in liver, heart and lungtissue were higher and lowest in brain. This may be related to absorption of slow metabolicprocesses. Drug absorption site is in the small intestine, with the highest concentrationbecause of the drug reside.Timosaponin B-II excretion study as a prototype drug: the drug from biliary excretionwas1.27%of total dose of in30hours, the urine excretion of drugs accounted for the totaldose of1.55%among0to96h, and accumulated excretion in feces was0.048%within96hours.3. The equilibrium dialysis method was used for the determination of timosaponin B-IIin human plasma protein binding, the results show that there was no significant difference(p>0.05) between drug concentration and plasma protein binding rate, but there are somedifferences between the duration of dialysis and plasma protein binding rate (p <0.05) after72hours. The results showed that the protein binding rate of timosaponin B-II of was morethan85%after72hours and there were a balance of the drug in the plasma and dialysisequilibrium, which indicated that the most of drugs were binded with plasma protein in thebodies. It may affect drug distribution to the organization Because of the free drugs waslow.4, LC-ESI-MS/MS was used to study the Timosaponin B-II metabolism in ratsmajor metabolites.Six metabolites including M1to M6were detected in the gastrointestinalcontents; M1, M2, M4were detected in the urine;2kinds of metabolites (M1、M4) detectedin the feces sample. The common metabolite samples were M1(timosaponinAIII) and M4. The parts of human livers were used as a source of microsomes in vitro incubationexperiments to verdict whether Timosaponin B-II has activity effects on CYP3A4theCYPlA2, CYP2C9and CYP2C19, CYP2E1and CYP2D6, thus affecting enzymessubstrate metabolism by drug-metabolizing enzymes. The results show that thetimosaponin B-II’s weak inhibition of drug metabolizing enzymes, which is unlikely tolead to clinical drug interactions because of its inhibition of drug-metabolizing enzymes. |
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