Quality Control And Pharmacokinetic Study Of Chuankezhi Injection

BackgroundThe pharmacokinetics of traditional Chinese medicines (TCMs), a branch of Chinese herbal pharmacology, has attracted more and more attention in recent years. And it mainly focuses on researching the in vivo status of natural bioactive components in organism, i.e. absorption, distribution, metabolism and excretion, which is pretty important for understanding clinical pharmacology and effective mechanism of TCM. Chuankezhi Injection is a second-class national new drug, composed of Epimedii folium and Morinda officinalis. This injection possesses many therapeutic effects such as tonifying kidney, relieving cough and asthma, allergy, enhancing the humeral and cellular immunity. However, there currently is no report about its pharmacokinetic study. In this study, epimedin A, B, C and icariin, which are dominant flavonoid glycosides in this drug, were determinated for quality evaluation of Chuankezhi Injection, and then the pharmacokinetics and bioavailability of the four bioactive components in rat were also investigated after intravenous and intramuscular administration. Developing a holistic and comprehensive quality evalutation method including quantitative analysis of these4flavonoid glycosides and chromatographic fingerprint can offer a more reliable and pratical approach for quality control of Chuankezhi Injection. Investigating the pharmacokinetic parameters of principal bioactive compounds and comparing the in vivo processes of analytes among different administration approaches as well as through various forms can facilitate the establishment of clinical application program of the drug.Objectives1To establish a liquid chromatography-mass spectrometry (LC-MS/MS) quantification method for simultaneous determination of epimedin A, B, C and icariin, and then to assess the quality of15batches of Chuankezhi Injection using the established quantitative method.2To develop an analytical method for simultaneously determining4flavonoid glycosides contained in biological matrix in order to investigate the pharmacokinetic characteristics of these active components and their bioavailability through comparing intramuscular and intravenous administration of Chuankezhi Injection.3To probe into the influence of morinda officinal is and other three compounds on the pharmacokinetics characteristics of epimedin A, B, C and icariin.Methods1A liquid chromatography-mass spectrometry (LC/MS/MS) method was established for simultaneous determination of epimedin A, B, C and icariin. Chromatographic separation was carried out on an Agilent Eclipse XDB-C18column (150×2.1mm,5μm), and the mobile phase was composed of acetonitrile and aqueous formic acid (35:65) with the flow rate at0.22mL·min-1and column temperature at40℃. Eluent was detected using a triple quadrupole tandem mass spectrometer (API3000) by multi-reaction monitoring (MRM) in the negative ion detection mode. Naringin was used as the internal standard. Fifteen batches of Chuankezhi Injection were analyzed using the established method after these samples were respectively diluted2000times with the mobile phase.2A quantitative analytical method of epimedin A, B, C and icariin in rat plasma was developed to investigate pharmacokinetic parameters of Chuankezhi Injection. First of all, a series of rat plasma samples were prepared for obtaining a calibration curve using solid-phase extraction cartridges (Agilent). And then rat whole blood samples were collected at a series of time points after rats were dosed via the intramuscular injection of Chuankezhi Injection at low, moderate and high levels. The same preparation procedures were used for obtaining a series of plasma samples after intravenous administration of Chuankezhi Injection at moderate level. All the samples were finally analyzed using LC-MS/MS. The acquired data were computed using the software of DAS2.0to get the pharmacokinetic parameters of4analytes according to non-compartmental model.3The influence of radix morindae officinalis and other compounds on pharmacokinetics characteristics of epimedin A, B, C and icariin was also investigated. Firstly, a reference solution was prepared by mixing the reference substances of epimedin A, B, C and icariin whose final concentration was the same as that in Chuankezhi Injection. And rats were randomly divided into3groups (6rats each group). Then one group was administrated by the intramuscular injection of Chuankezhi injection, the other group was dosed via the intramuscular injection of the abovementional stock solution, and the last the other group was dosed via the intramuscular injection of the epimedin A. And then whole blood samples of each rat were collected at a series of time points after administration. Rat plasma samples were obtained by centrifuging the whole blood samples at high speed prior to LC-MS/MS analysis. All data were processed with DAS2.0software to get pharmacokinetic parameters in order to exhibit the influence factor(s) by comparison of the pharmacokinetic parameters of the three groups.Results1The quantification of4flavonoid glycosides in Chuankezhi Injection: in this study, the lowest limit of quantification (LLOQ) of epimedin A, B, C and icariin was less than0.04ng·mL-1, the lowest limit of detection is less than0.05ng·mL-1. There was good linear relationship for all four flavonoid glycosides in the range of1.0-1000.0ng mL-1(r>0.9950). Their corresponding recoveries were between90.11%-100.3%, RSD<2.89%(n=6), and the repeatability, stability and precision were all satisfactory. The established quantitative method was used for quality evaluation of15batches of Chuankezhi Injection. As a result, the method was feasible for quantitative analysis of four analytes in the drug, and all the15samples were qualified according to related regulation of SFDA.2Determination of epimedin A, B, C and icariin in rat plasma and their pharmacokinetic parameters:for4analytes of epimedin A, B, C and icariin in concentration range of1-1000ng·mL-1, the peak area ratio of analyte to internal standard showed a good linearity with all correlation coefficients (r)>0.996; matrix effect was between90.71%-104.77%; intra-day precision (RSD<5.99%) and corresponding accuracy (96.9%-107.5%), and inter-day precision (RSD<10.16%) and corresponding accuracy (92.3%-105.0%) were all good. After divided into3groups, rats were injected via intramuscular administration of Chuankezhi Injection of three doses (0.17,0.50and1.51ml.kg), repectively. Pharmacokinetic parameters of4flavonoid glycosides were studied by means of the established method. As a result, Cmax and AUC of 4analytes disproportionately increased with dose. There was not a significant difference among the values of Tmax of the three groups. The concentration-time curve for each rat displayed a "two peak" profile; however, the phenomenon did not appear in each group after averaging of individual differences.3The influence to epimedin C after different administration modes:by comparison the pharmacokinetic parameters of the singal C group、flavonoids combination group and CKZI group, find that in the flavonoids combination group showed the "two peak" profiles, the AUC0-∞values of epimedin C in CKZI group were more than the singal group and the flavonoids combination group. The singal group had the shortest half-life value than the the flavonoids combination group and CKZI group.Conclusions1The quantitative method of four major flavonoid glycosides in Chuankezhi Injection was successfully established. The method proved to be sensitive, accurate, stable, rapid and suitable and can be used for quality control of Chuankezhi Injection. In the present study this method was satisfactory for quality evaluation of15batches of Chuankezhi Injection. And the result showed that the content of four flavonoid glycosides in15batches was relatively consistent, and the total amount of epimedin C and icariin contained in Chuankezhi Injection was within0.50-1.00mg, meeting the pharmaceutical standards of the State.2An analytical method was developed for quantitatively analyzing epimedin A, B, C and icariin in biological matrix. The method is accurate, specific, rapid, and suitable for the quantitative determination of the4flavonoids in rat plasma. After intramuscular administration of Chuankezhi Injection, the blood concentration of4flavones rapidly increases to Cmax and their concentration-time curves show two peaks. The half-life of epimedin A, B and icariin was half an hour approximately.3By investigating on the pharmacokinetic characteristics of the singal C group、flavonoids combination group and CKZI group, get a different mode of administration of epimendin C absorption, distribution, metabolism and other methods. In this study, the comparative method proved to be feasible and the result was convictive. The CKZI group and flavonoids combination can enhance the absorption of epimendin C and delay their elimination half-life.