| ObjectiveThis research is the subitem of the project(No.81373973)which was funded by National Natural Science Foundation of China.To study the pharmacokinetics of Buyang Huanwu Decoction in cerebral ischemia reperfusion injury model of rats,compare the blood and brain pharmacokinetics process of active ingredients in Buyang Huanwu Decoction of different Astragalus membranaceus doses.With the activity of superoxide dismutase(SOD)and lactate dehydrogenase(LDH)as the indexes of antioxidant capacity,evaluate the pharmacodynamics process of Buyang Huanwu Decoction of different Astragalus membranaceus doses.It can provide reference for the use of Buyang Huanwu Decoction in clinical.Methods1.Preparation of Buyang Huanwu Decoction and determination of active ingredient contentThe Buyang Huanwu decoction was prepared by water extraction and ethanol precipitation.HPLC methods were established for the determination of formononetin in Yiqi group and paeoniflorin in Huoxue group.The contents of formononetin and paeoniflorin were determined.2.Establishment of the blood and brain microtialysis metnod of formononetin and paeoniflorinAn HPLC method was established for the determination of formononetin and paeoniflorin in microdialysate.The influence of the flow rates,temperature and medium concentration were investigated by using concentration difference method(incremental method and decrement method).In addition,the in vivo probe stability was investigated after implantation.3.Study on pharmacokinetics of Buyang Huanwu decoction of blood and brain in MCAO rats.An UPLC-MS/MS method was established for simultaneous determination of formononetin and paeoniflorin in microdialysate.The difference of blood and brain pharmacokinetics between low,medium and high Astragalus membranaceus dose of Buyang Huanwu decoction were investigated by microdialsis technique.4.Study on the anti-oxidant process of Buyang Huanwu Decoction in MCAO rats.The model of acute cerebral ischemia-reperfusion was made by improved method of thread occlusion.With the activity of superoxide dismutase(SOD)and lactate dehydrogenase(LDH)as the indexes of antioxidant capacity,evaluated the pharmacodynamics process of Buyang Huanwu Decoction within different Astragalus membranaceus doses.The correlation between the pharmacokinetic and anti-oxidant effect of Buyang Huanwu Decoction was analyzed.Results1.Preparation of Buyang Huanwu Decoction and determination of active ingredient contentBuyang Huanwu Yiqi decoction and Buyang Huanwu Huoxue decoction were prepared by water extraction and ethanol precipitation.The content of formononetin in Buyang Huanwu Yiqi decoction were 16.53～17.49μg/g in three batches.The content of paeoniflorin in Buyang Huanwu Huoxue decoction were 4.44～4.77 mg/g in three batches.2.Establishment of the blood and brain microtialysis metnod of formononetin and paeoniflorinThe Relative Loss rate of formononetin and paeoniflorin decreased with increasing flow rate and increased with increasing temperature,independent of drug concentration.When the flow rate was 1.5 μL/min,the Relative Recovery rate of blood and brain microdialysis probes of formononetin were(29.1±0.61)%,(29.8±1.1)%,the Relative Recovery rate of paeoniflorin(41.9±2.4)%,(42.1±0.85)%.The recovery of blood and brain microdialysis probes had a good stability in vivo 10 h,which met requirements of analysis.3.Study on pharmacokinetics of Buyang Huanwu decoction of blood and brain in MCAO rats.A rapid,sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)method has been developed for the simultaneous determination and pharmacokinetic investigation of formononetin and paeoniflorin in microdialysate.The method was validated over the concentration range of 3.13～626 ng/mL for formononetin and 17.88～1788 ng/mL for paeoniflorin,with a lowerlimit of quantitation(LLOQ)of 3.13 ng/mL and 17.88 ng/mL,respectively.The pharmacokinetic study indicated that formononetin and paeoniflorin was consistent with the one-compartment model in blood after intravenous injection of Buyang Huanwu decoction.The pharmacokinetic parameters of paeoniflorin:t1/2 blood,MRTblood,MRTbrain and Cmax in brain were improved after administration of high Astragalus membranaceus dose of Buyang Huanwu decoction,when compared with low and medium Astragalus membranaceus dose.The AUCbrain of paeoniflorin after intravenous injection of medium and high Astragalus membranaceus dose of Buyang Huanwu decoction were significantly increased than low Astragalus membranaceus dose.The brain targeting efficiency of paeoniflorin after intravenous injection of high Astragalus membranaceus dose of Buyang Huanwu decoction was higher than low Astragalus membranaceus dose(P<0.05).4.Study on the anti-oxidant process of Buyang Huanwu Decoction in MCAO rats.The activity of superoxide dismutase(SOD)and lactate dehydrogenase(LDH)were(259.55.±9.37)U/mL and(3636.26±146.09)U/L in nomal rats.The activity of superoxide dismutase(SOD)and lactate dehydrogenase(LDH)in cerebral ischemia reperfusion injury model of rats were significantly decreased to(175.58±6.19)U/mL and increased to(5810.54±114.40)U/mL,respectively.After intravenous injection of Buyang Huanwu decoction,the activity of superoxide dismutase(SOD)was improved and the activity of lactate dehydrogenase(LDH)was decreased.The correlation coefficient RSOD was smaller,futher more RLDH>RSOD.The Buyang Huanwu decoction containted high Astragalus membranaceus had a better anti-oxidant effect.There was a positive correlation between the anti-oxidation effect and the concentration of formononetin and paeoniflorin in plasma.ConclusionThe effect of Buyang Huanwu decoction on cerebral ischemic injury may be related to its antioxidant effect in vivo.In addition to formononetin and paeoniflorin,other components may also play a role in the anti-oxidant effect.Increased the dose of Astragalus membranaceus can improved the antioxidant ability of Buyang Huanwu decoction,and then enhanced the protective effect on cerebral ischemia reperfusion injury.This may be associated with high doses of astragalus,which can prolonged retentiont of active ingredients in vivo and brain tissue. |
PDF Full Text Request