Performance Evaluation Of Home-made Microdialysis Probe And Its Application To Pharmacokinetics Research Of TCM

Microdialysis, a powerful sampling technique, has been widely applied to pharmacokinetic studies in various tissues, for it can offer tissue-specific, real-time information of free drug with less animals. Due to the semipermeability of the membrane on the tip of the microdialysis probe, large molecules such as protein are excluded, and relatively clean samples are collected which can be directly injected into an analysis system without pretreatment. So, microdialysis is an ideal tool for the in vivo sampling in pharmacokinetic studies of traditional Chinese medicine (TCM). However, the microdialysis probe is expendable and expensive, thus home-made probe is of great importance to facilitate the application of microdialysis technique.In this thesis, concentric and linear probe were assembled by ourselves. They were successfully used for the pharmacokinetic analysis of puerarin, which is the main bioactive and the most abundant component in the puerariae radix isoflavone mixture. The main contents and outcomes of this thesis are listed as follows:(1) With peoniflorin used as a model compound, in vitro microdialysis experiments were carried out to evaluate the influence of a series of experimental conditions (perfusate flow rate, concentration of peoniflorin, membrane and method used for determination of recovery) on the extraction efficiency of home-made concentric probes. The results revealed that extraction efficiency was different between probes. An exponential decrease in extraction efficiency parallel to the increase in perfusate flow rate was found. Extraction efficiency became concentration-dependent when the flow rate was 8.0μL/min. Statistically significant differences were found between extraction efficiency determined by direct dialysis and retrodialysis. After the home-made probe was applied to continuously sampling in vivo for 6 h, the permeability of the membrane on the probe did not vary significantly. These results showed that the home-made microdialysis probe was competent for in vivo microdialysis sampling.(2) Due to the native fluorescence of puerarin, a specific and sensitive post-column fluorescence detection was established to analysis the microdialysate samples from the jugular vein and liver of rats. The limit of detection in Ringer's solution was determined to be 0.0014μg/mL with an S/N ratio of 3. The probe recoveries both before and after in vivo sampling experiments, determined in vitro, showed that the home-made probes were robust and reusable. The mean recoveries determined in vivo by retrodialysis were (11.4±2.09) % and (12.7±3.15) % for concentric probes and linear probes, respectively. Detailed pharmacokinetic profiles of free puerarin in rat blood and liver after a single dosage of 30 mg/kg (i.v.) were revealed. The similar peak time and downtrend of puerarin in liver and blood suggested that it fast distributed into liver.(3) Oral administration is the most popular dosage mode of TCMs. Efflux transporters like P-glycoprotein might play a significant role in the absorption of drugs from the intestine. Control group was utilized to study the pharmacokinetics of free puerarin and its interaction in the presence of verapamil, which is a P-glycoprotein modulator. The results indicated that, following verapamil treatment, the area under the curve (AUC) for free puerarin in rat blood was 2.11 times as much as that for the group without verapamil. The peak concentration of free puerarin was also significantly increased. However, both of the the clearance and residence time did not show significant difference. It suggested that the interaction between puerarin and verapamil mainly took place during drug absorption and the the absorption of free puerarin from the intestine may be regulated by the P-glycoprotein.