| ObjectiveThis research is the subitem of the project (No.81373973) which was funded by National Natural Science Foundation of China. To study the preparation and determination of Buyang Huanwu decoction, and its formulation is made up Astragalus membranaceus, Angelica sinensis, Paeonia lactiflora, Ligusticum chuanxiong, Prunus persica, Carthamus tinctorius and Pheretima aspergillum. To establish an in vivo sampling method by microdialysis and investigate the pharmacokinetics of Buyang Huanwu decoction by intravenous administration, and analying the differences of parameters for group of different Astragalus. In this study, we also investigated the effects of various ischemic duration on post-ischemic and reperfusion rat cerebral blood flow by laser speckle imaging system. On this foundation, we examined the protective effects on cerebral isehemia-reperfusion injury in MCAO rats with different degree of injury after intravenous administration of Buyang Huanwu decoction. It can provide theoretical and experimental basis for the mechanism of Buyang Huanwu decoction in treatment of acute ischemic stroke.Methods1 Preparation of experimental Buyang Huanwu injection1.1 Preparation of experimental Buyang Huanwu Yiqi injectionTo establish the determination method of total saponins of Astragalus by UV-Vis. Through static and dynamic adsorption experiments, the adsorption characteristics of several macroporous adsorbing resin were studied. The purification process were optimized by orthogonal test and single factor experiment. The eluate was prepared into injection after series of treatments.1.2 Preparation of experimental Buyang Huanwu Huoxue injectionTo establish the determination method of FA and TMP by HPLC. Taking FA and TMP as index, orthogonal test was used to investigate effects of extraction time, extraction times and solvent dosage on the extraction process. The extract was purified by cationic resin 732. And then a stable experimental Buyang Huanwu Huoxue injection was prepared.2 Study on the in vitro and in vivo recovery of FA and TMP in cerebral and blood microdialysis probesTo establish a method for detecting FA and TMP in the dialysate by HPLC. The influence of the flow rates, medium concentration, temperature and Intra day reproducibility on the recovery of FA and TMP were investigated by using concentration difference method (incremental method and decrement method). In addition, the in vivo probe stability were investigated within 8 h after implantation.3 Study on pharmacokinetics of Buyang Huanwu decoction of blood and brain in rats after intravenous administration by UPLC-MSThe UPLC-MS method was established to measure the drug concentration of FA and TMP in rat’s brain and blood microdialysates. The blood and brain pharmacokinetic of Buyang Huanwu decoction were investigated by microdialysis when the drug was given by intravenous administration, and were analysis with statistical values and compared with groups. The pharmacokinetic parameters were calculated by software PK solver 2.0.4 Study on the pharmacodynamics of buyang huanwu decoction in MCAO rats after intravenous administration by LSCI4.1 To establish a method to observe the blood flow of MCAO rats by LSCILaser speckle imaging system was used to determine the effect of various ischemic duration on post-ischemic and reperfusion rat cerebral blood flow. At the same time, the dynamic changes of capillary flow and diameter, no reflow phenomenon and hypoperfusion were analyzed.4.2 Pharmacodynamic of Buyang Huanwu decoctionThe model of acute cerebral ischemia-reperfusion was made by improved method of Zea Longa’s thread occlusion. The protective effects on cerebral isehemia-reperfusion injury was investigated in MCAO rats with different degree of injury, which were given by Buyang Huanwu injection with different dosage of Astragalus. The local cerebral vessel diameter and blood flow of MCAO rats were in real-time monitored by LSCI. The neurology score, cerebral water content and cerebral infarct volume were tested.Results1 Preparation of experimental Buyang Huanwu injection1.1 Preparation of experimental Buyang Huanwu Yiqi injectionThe adsorption and desorption ability of D101 macroporous resin for total saponins of Astragalus are better than others. The results of orthogonal test showed that the crude herbal dose of the Astragalus extract was at 0.06 g/mL, the loading quantity of sample with its flow at 3 BV/h was 1 BV, and eluted with 10 BV 70% ethyl alcohol. Collecting the eluent, and then the total saponins of Astragulus extract was done. According to this scheme,3 batches of medicinal materials were verified, The average content of total saponins of Astragalus was 279.16 mg and RSD was 1.9%, indicating the purification process was feasible. The eluate was concentrated and extracted, solvent evaporation. The residue was dissolved with amount of injection water, sizing, filtering, sterilizing and refrigerating.1.2 Preparation of experimental Buyang Huanwu Huoxue injectionThe best extraction of Buyang huanwu decoction was to extract 2 times by using 12 times amount of 40% alcohol, and each time took 1.5 h. The extract was purified by cationic resin 732. The eluate was concentrated, with pH value adjusting, filtering, sterilizing and refrigerating.2 Study on the in vitro and in vivo recovery of FA and TMP in cerebral and blood microdialysis probesThe recovery obtained by incremental method was similar to by decrement method. The in vitro recovery rate of FA and TMP decreased with the increase of 1-2.5μL·min-1, and increased obviously with the temperature of 25-42℃ under the same conditions. The concentration of FA and TMP had no obvious effect on the probe recovery under the same flow rate, indicating the in vitro recovery of probe can be corrected by retrodialysis. In addition, the recovery of TMP and FA remained stable and showed similar trends under the condition of four concentration cycles, indicating that the intra day reproducibility of the concentration difference method were good. The recovery of brain and blood microdialysis probes had a good stability in vivo 8 h.3 Study on pharmacokinetics of Buyang Huanwu decoction of blood and brain in rats after intravenous administration by UPLC-MSThe results were as follows:TMP showed a good linear relationship at concentration of 7.71-771 ng/mL. The LLOQ of TMP was 7.71 ng/mL. The linear range was 13.8-1380 ng/mL for FA. The LLOQ of FA was 13.8 ng/mL. The specificity, precision, stability and matrix effect of methods met requirements of biological sample analysis.The results showed that FA and TMP could pass through the blood-brain barrier after intravenous administration. TMP was two compartment model while FA was one compartment model in brain and blood. Compared with Huoxue group, increased doses of Astragalus obviously reduced t1/2 of TMP in blood, butincreased Cmax and AUC in brain. Moreover, increased doses of Astragalus not only prolonged t1/2and MRT of FA, but improved Cmax and AUC in brain, indicating increased doses of Astragalus tended to maintain higher drug concentrations and longer acting time. Finally, brain targeting efficiency of FA was improved with doses of Astragalus increased. The results demonstrated that the composing prescriptions of Buyang Huanwu decoction emphasized high doses of Astragalus, which was reasonable in view of its effeet on the pharmacokinetics and brain target efficiency.4 Study on the pharmacodynamics of buyang huanwu decoction in MCAO rats after intravenous administration by LSCI4.1 To establish a method to observe the blood flow of MCAO rats by LSCIDuring 2 h middle cerebral artery occlusion (MCAO) ischemia, perfusion of cerebral blood flow decreased to 46.68±3.16% of the baseline, tending to the state of hypoperfusion. With the prolongation of ischemic time, no reflow phenomenon was aggravated. Hypoperfusion in 2 h MCAO group was approximately 10% less than that in 30 min MCAO group during the period of reperfusion.4.2 Pharmaeodynamic of Buyang Huanwu decoctionCompared with model group, high doze group had the function to protect celebral ischemia reperfusion in rats, which obviously reduced cerebral water content and cerebral infarct volume of 30 min MCAO group and improved the cerebral blood flow during the period of hypoperfusion. Its effect was similar to Nimodipine control group. However, the treatment effect of 2 h MCAO group weren’t obvious.GonclusionThe preparation technology of Buyang Huanwu decoction was feasible. The established method of determination were reliable and had a good reproducibility, which could meet the requirements of this study. Microdialysis sampling combined with UPLC-MS can be used for the local brain and blood pharmacokinetic study of Buyang Huanwu decoction after intravenous administration. Increased doses of Astragalus prolonged t1/2 and MRT of FA, improved Cmax and AUC for FA and TMP in brain, and increased brain target efficiency for FA, indicating the composing prescriptions of Buyang Huanwu decoction was reasonable with high doses of Astragalus. LSCI was introduced to evaluate the degree of cerebral ischemia-reperfusion injury. The results showed that Buyang Huanwu decoction had pharmacologic effects on resisting cerebral ischemical reperfusion injury. The pharmacological effect of Buyang Huanwu decoction were verified, demonstrating compatibility with high doze Astragalus had the function to protect celebral ischemia reperfusion in MCAO rats that were caused by short-term process of cerebral ischemia-reperfusion. The results confirmed that the composing prescriptions of Buyang Huanwu decoction emphasized high doses of Astragalus and assisted by Huoxue group, which was reasonable in view on mechanism of "benefiting qi and promoting circulation of blood" traditional Chinese medicine composition therapy. |
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