辛芍组方效应成分在正常和脑缺血再灌注损伤大鼠肝微粒体中的酶促反应动力学分析
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摘要
目的:研究辛芍组方中效应成分灯盏乙素、灯盏甲素和芍药苷在正常大鼠和大脑中动脉闭塞(MCAO)局灶性脑缺血再灌注损伤大鼠肝微粒体中的酶促反应动力学,比较上述成分在不同状态大鼠肝微粒体中的体外酶代谢动力学差异。方法:制备不同状态大鼠的肝微粒体,将辛芍组方与大鼠肝微粒体进行孵育,选择UPLC-MS为分析手段,采用底物消除法,计算各成分在正常大鼠和MCAO大鼠肝微粒体中的酶反应动力学米氏常数(Km),最大反应速率(Vmax)及体外肝微粒体对药物的固有清除率(CLint),将各参数进行组间统计学分析。结果:灯盏乙素、灯盏甲素和芍药苷在正常大鼠体外肝微粒体中的Km[(0. 597±0. 065),(0. 166±0. 012),(0. 640±0. 046)μmol·L~(-1)]与MCAO大鼠中的Km[(0. 798±0. 031),(0. 213±0. 017),(0. 499±0. 029)μmol·L-1]均明显不同;与正常组相比,MCAO大鼠体内的灯盏乙素和芍药苷的Vmax和CLint均明显减少(P <0. 05,P <0. 01),灯盏甲素的Vmax也显著降低(P <0. 05)。结论:辛芍组方的效应成分在脑缺血再灌注损伤大鼠肝微粒体中的代谢速率降低、消除减慢。
Objective: To investigate and compare enzymatic kinetics of scutellarin,apigenin-7-Oglucronide and paeoniflorin from Xinshao fomula in liver microsomes of sham-operated rats and middle cerebral artery occlusion(MCAO) rats with focal cerebral ischemia-reperfusion injury. Method: Xinshao fomula were incubated respectively with liver microsomes of sham-operated rats and MCAO rats, UPLC-MS and substrate elimination method was employed,Michaelis constant(Km),maximum velocity of enzymatic reaction(Vmax) and intrinsic clearance(CLint) of these three components from Xinshao fomula in liver microsomes of sham-operated rats and MCAO rats were calculated, these parameters between different groups were evaluated by statistical analysis. Result: The Kmvalues of scutellarin,apigenin-7-O-glucronide and paeoniflorin in liver microsomes of MCAO rats were(0. 798 ± 0. 031),(0. 213 ± 0. 017),(0. 499 ± 0. 029) μmol·L~(-1),which were quite different to these in liver microsomes of sham-operated rats. Compared with the sham-operated group,Vmaxand CLintvalues of scutellarin and paeoniflorin in liver microsomes of MCAO rats were significantly reduced(P < 0. 05, P <0. 01),and Vmaxof apigenin-7-O-glucronide in liver microsomes of MCAO rat was also significantly reduced(P <0. 05). Conclusion: Metabolic rates of these three active components from Xinshao fomula in liver microsomes of MCAO rats with focal cerebral ischemia-reperfusion injury decrease with low elimination rate.
Objective: To investigate and compare enzymatic kinetics of scutellarin,apigenin-7-Oglucronide and paeoniflorin from Xinshao fomula in liver microsomes of sham-operated rats and middle cerebral artery occlusion(MCAO) rats with focal cerebral ischemia-reperfusion injury. Method: Xinshao fomula were incubated respectively with liver microsomes of sham-operated rats and MCAO rats, UPLC-MS and substrate elimination method was employed,Michaelis constant(Km),maximum velocity of enzymatic reaction(Vmax) and intrinsic clearance(CLint) of these three components from Xinshao fomula in liver microsomes of sham-operated rats and MCAO rats were calculated, these parameters between different groups were evaluated by statistical analysis. Result: The Kmvalues of scutellarin,apigenin-7-O-glucronide and paeoniflorin in liver microsomes of MCAO rats were(0. 798 ± 0. 031),(0. 213 ± 0. 017),(0. 499 ± 0. 029) μmol·L~(-1),which were quite different to these in liver microsomes of sham-operated rats. Compared with the sham-operated group,Vmaxand CLintvalues of scutellarin and paeoniflorin in liver microsomes of MCAO rats were significantly reduced(P < 0. 05, P <0. 01),and Vmaxof apigenin-7-O-glucronide in liver microsomes of MCAO rat was also significantly reduced(P <0. 05). Conclusion: Metabolic rates of these three active components from Xinshao fomula in liver microsomes of MCAO rats with focal cerebral ischemia-reperfusion injury decrease with low elimination rate.
引文
[1]王永林,黄勇,郑林,等.注射用辛芍冻干粉针药味配伍作用研究[J].中国实验方剂学杂志,2007,13(7):38-40.
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[12]贾佩佩,张晓旭,张智勇,等.染料木苷在大鼠肝微粒体中的代谢及酶促反应动力学研究[J].中国药学杂志,2015,50(9):797-801.
[14]向云亚.6-姜酚在大鼠肝微粒体中的代谢研究[D].广州:广州中医药大学,2013.
[15]XIA Z L,YING J Y,SHENG R,et al.In vitro metabolism of BYZX in human liver microsomes and the structural elucidation of metabolite by liquid chromatography-mass spectrometry method[J].J Chromatogr B Analyt Technol Biomed Life Sci,2007,857(2):266-274.
[16]Sinz M,Wallace G,Sahi J.Current industrial practices in assessing CYP450 enzyme induction:preclinical and clinical[J].AAPS J,2008,10(2):391-400.
[17]YANG X F,HE W,LU W H.Effects of scutellarin on liver function after brain ischemia/reperfusion in rats[J].Acta Pharmcaol Sin,2003,24(11):1118-1124.
[18]BING Y T,ZHU S Y,JIANG K.Reduction of thyroid hormones triggers down-regulation of hepatic CYP2Bthrough nuclear receptors CAR and TR in a rat model of acute stroke[J].Biochem Pharmcaol,2014,87(4):636-649.
[19]Feere D A,Velenosi T J,Urquhart B L.Effect of erythropoietin on hepatic cytochrome P450expression and function in an adenine-fed rat model of chronic kidney disease[J].Brit J Pharmcaol,2015,172(1):201-213.
[20]Wójcikowski J,Daniel W A.The brain dopaminergic system as an important center regulating liver cytochrome P450 in the rat[J].Expert Opin Drug Metab Toxicol,2009,5(6):631-645.
[21]YANG J,HAO C,YANG D,et al.Pregnane X receptor is required forinterleukin-6-mediated down-regulation of cytochrome P450 3A4 in human hepatocytes[J].Toxicol Lett,2010,197(3):219-226.
[2]郑林,牟景丽,黄勇,等.UPLC-MS/MS法同时测定注射用辛芍中7种指标成分的含量[J].中国新药杂志,2014,23(1):105-109.
[3]贵州省药品监督管理局.贵州省中药材、民族药材质量标准[M].贵阳:贵州科技出版社,2003:172.
[4]陆苑,张洁,兰燕宇,等.灯盏细辛-赤芍组方配比及给药途径的脑保护作用[J].中国实验方剂学杂志,2013,19(21):175-179.
[5]黄勇,王永林,兰燕宇,等.注射用辛芍对大鼠脑缺血再灌注损伤的保护作用和对脑微循环血流量的影响[J].中国新药杂志,2008,17(2):119-123.
[6]刘宗炎,董莉,董永喜,等.灯盏细辛与赤芍配伍组方对H2O2致PC12细胞损伤的保护作用[J].中国实验方剂学杂志,2014,20(5):180-183.
[7]郑林,牟景丽,唐丽,等.UPLC-MS法研究辛芍提取物的药动学及其绝对生物利用度[J].中国新药杂志,2014,23(7):819-823.
[8]巩仔鹏,胡建春,李梅,等.基于大鼠脑缺血再灌注损伤模型建立辛芍组方中灯盏乙素和芍药苷的PK-PD结合模型[J].中国实验方剂学杂志,2018,24(1):74-78.
[9]刘亭,刘香香,陈亭亭,等.灯盏细辛和赤芍配伍组方对大鼠脑缺血再灌注损伤的保护作用及对NF-κB通路的影响[J].中国实验方剂学杂志,2018,24(9):111-115.
[10]LI Y T,LU Y,HU J C,et al.Pharmcaokinetic comparison of scutellarin and paeoniflorin in shamoperated and middle cerebral artery occlusion ischemia and reperfusion injury rats after intravenous administration of Xin-Shao formula[J].Molecules,2016,21(9):E1191.
[11]曾苏.药物代谢学[M].杭州:浙江大学出版社,2008:140-152.
[12]贾佩佩,张晓旭,张智勇,等.染料木苷在大鼠肝微粒体中的代谢及酶促反应动力学研究[J].中国药学杂志,2015,50(9):797-801.
[14]向云亚.6-姜酚在大鼠肝微粒体中的代谢研究[D].广州:广州中医药大学,2013.
[15]XIA Z L,YING J Y,SHENG R,et al.In vitro metabolism of BYZX in human liver microsomes and the structural elucidation of metabolite by liquid chromatography-mass spectrometry method[J].J Chromatogr B Analyt Technol Biomed Life Sci,2007,857(2):266-274.
[16]Sinz M,Wallace G,Sahi J.Current industrial practices in assessing CYP450 enzyme induction:preclinical and clinical[J].AAPS J,2008,10(2):391-400.
[17]YANG X F,HE W,LU W H.Effects of scutellarin on liver function after brain ischemia/reperfusion in rats[J].Acta Pharmcaol Sin,2003,24(11):1118-1124.
[18]BING Y T,ZHU S Y,JIANG K.Reduction of thyroid hormones triggers down-regulation of hepatic CYP2Bthrough nuclear receptors CAR and TR in a rat model of acute stroke[J].Biochem Pharmcaol,2014,87(4):636-649.
[19]Feere D A,Velenosi T J,Urquhart B L.Effect of erythropoietin on hepatic cytochrome P450expression and function in an adenine-fed rat model of chronic kidney disease[J].Brit J Pharmcaol,2015,172(1):201-213.
[20]Wójcikowski J,Daniel W A.The brain dopaminergic system as an important center regulating liver cytochrome P450 in the rat[J].Expert Opin Drug Metab Toxicol,2009,5(6):631-645.
[21]YANG J,HAO C,YANG D,et al.Pregnane X receptor is required forinterleukin-6-mediated down-regulation of cytochrome P450 3A4 in human hepatocytes[J].Toxicol Lett,2010,197(3):219-226.