| 1 ObjectivePharmacokinetic study of Traditional Chinese Medicine (TCM) is under the guide of TCM theory, using the principle and mathematical processing method of dynamics to research the regular pattern of the absorption, distribution, metabolism and excretion after TCM entered.The current TCM pharmacokinetic study still mainly on the monomer composition of single the TCM or TCM prescription, but because of the particularity of more targets, more composition, and more effect in TCM, using monomer composition pharmacokinetic behavior replace that of the TCM is not accurate and not comprehensive. So it is quite necessary to set up a method which could accurately, fully explain the pharmacokinetics of TCM prescription.In this study the injectable ShuangHuangLian (freeze-dried) for drug, establishing its HPLC fingerprint, exploring appropriate microdialysis sampling conditions, and researching the feasibility of the internal standard method to measure the probe recovery. Apply the above researches on the pharmacokinetic study in microdialysis and fingerprint technology coupled for TCM prescription.2 Method2.1 Establish fingerprint of injectable ShuangHuangLian (freeze-drying)The ShuangHuangLian chromatographic fingerprint by HPLC was established, which is suitable for detecting the ShuangHuangLian preparations and blood samples also sutiable for the internal standard. The chromatographic conditions were optimized and the methodology was investigated.2.2 Study on the recovery of microdialysis probeThe flow velocity of perfusion fluid,temperature, concentration, and the material quantity dialysis media contains were examined to study the influence of the probe recovery and the stability of recovery was also studied. Through the initial screening and reviewing the recovery of internal standard under different conditions, the internal standard was confirmed. The stability of the proportion between target composition and internal standard was confirmed.2.3 Study on pharmacokinetic of injectable ShuangHuangLian (freeze-drying)The microdialysis probe was implanted in rabbit ear vein,and the rabbit was carotid intubated. Use the microdialysis method and traditional blood drug concentration method for sampling at the same time, which sustaining for at least 3 hours. Dynamic monitor the recovery of the target composition by internal standard and calculate the free drug concentration in the plasma. Determinate the drug concentration after the samples were processed. Draw the c-t curver spectively, and calculate the pharmacokinetic parameters by compartment model.3 Result3.1 Established the HPLC chromatographic fingerprint of injection ShuangHuangLian (freeze-dried)Through optimizating the chromatographic condition and investigating the methodology, the HPLC chromatographic fingerprin of injectable ShuangHuangLian(freeze-drying) was established.Mobile phase:A:water (including volume fraction for 1% of ice acetic acid), B: acetonitrile;Velocity:1.0 ml/min;Detected wavelength:325 nm;Mobile phase gradient elution program:0 to 6 min, A:84%; 6 to 12 min, A:84%-72%; 12-25 min, A:72%.It was showed that under this chromatographic condition, the separation of each chromatographic peak was good, the symmetry was good, the linear range, the LOQ and the detection limit accord with experimental requirements.3.2 Research on the influence factors and dynamic monitoring by internal standard of probe recoveryThe the recovery reduced and exponentially decline with perfusion fluid velocity faster; In certain temperature range, recovery higher with higher temperatures of microdialysis media; In certain concentration range, recovery remained stable, not with concentration change; The greater the number of substances in microdialysis media, the lower of certain single component recovery; Probe recovery in one day stability for at least 7 hous, to meet the requirements in vivo study.Finally choosing puerarin as the internal standard in microdialysis. The result of in vitro simulation experiment showed that the internal standard recovery was stable with concentration change. But in the same concentration, the faster perfusion fluid velocity,the recovery lower; The proportion of target composition recovery and internal standard P value remained in a stable level with microdialysis media was ShuangHuangLian Linger's solution. 3.3 Pharmacokinetic study of injecable ShuangHuangLian(freeze-drying)Determined rabbit plasma protein combined rate by microdialysis in vitro. The result showed that microdialysis could be used in the pharmacokinetic study of ShuangHuangLian. Using 3 times methanol as protein precipitation to process the blood samples obtained by traditional blood drug concentration method achieved a better effect.The result of pharmacokinetic analysis showed that using microdialysis method and traditional drug concentration method has the fit result. The pharmacokinetic behavior of ShuangHuangLian in rabbit accord with two compartment model. Microdialysis method could be used in pharmacokinetic studies, but microdialysis method and traditional blood drug concentration method each has advantages and disadvantages, needing attention in choice.4 Conclusions4.1 the chromatographic fingerprint of injectable ShuangHuangLian (freeze-dried) using gradient elution could not only make the composition in preparations and plasma get better separation, also could satisfy the micro dialysis internal standard elution requirement.4.2 Perfusion fluid flow velocity, temperature and the number of substances contained in microdialysis media influenced the recovery of probe, the influence of microdialysis media concentration could be ignored.4.3 Choose puerarin as the internal standard to dynamic monitor the probe recovery. The proportion P of target composition recovery rate and internal standard keep stable with the change of drug concentration around the probe.4.4 Using microdialysis method to determine the plasma protein combine rate of ShuangHuangLian in vitro confirm that it could use the microdialysis to study the pharmacokinetic of ShuangHuangLian in rabbit. 4.5 Using the dialysis method and traditional blood drug concentration method at the same time in the pharmacokinetic study, it was showed that the pharmacokinetic behavior of ShuangHuangLian fit with two compartment model. It could be experiment many times in the same animal using microdialysis method, while the traditional blood drug concentration method only once. Acquired by microdialysis method of peak time has certain hysteresis, peak relatively blood drug concentration obtained numerical small. |
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