蒙药“诃子解草乌毒”的配伍比例研究

Study on Compatibility Proportion of Mongolian Medicine“Terminalia chebula Decomposing Poison of Aconitum kusnezoffii”

目的:探讨蒙药“诃子解草乌毒”的最佳配伍比例。方法:将40只大鼠随机分为空白对照组(0.05%羧甲基纤维素钠溶液)、生草乌组(以生药量计0.12g/kg)和生草乌-诃子(1∶3、1∶1、3∶1,质量比)组(生草乌给药量以生药计均为0.12g/kg),每组8只;每天灌胃给药1次,连续给药28d;末次给药0.5h后,测定大鼠血清中肌钙蛋白(cTn-I)、肌红蛋白(MB)含量,并观察其心脏病理结构变化,以考察诃子对草乌致心脏毒性的解毒作用。采用平衡透析法并联合液质联用技术测定草乌中3种乌头碱(乌头碱、新乌头碱和次乌头碱,质量浓度均为5、10、20、40、80、160、400ng/mL)与正常人血浆的血浆蛋白结合率;然后固定3种乌头碱的质量浓度均为100ng/mL,考察加入不同比例诃子中主要解毒成分单宁酸后(3种乌头碱与诃子的质量比均分别为1∶0.025、1∶0.075、1∶0.1、1∶0.5),对3种乌头碱血浆蛋白结合率的影响,以考察诃子解草乌致心脏毒性的可能机制。结果:在解毒实验中,与空白对照组比较,生草乌组大鼠血清中cTn-I、MB含量显著升高(P<0.05),心肌组织出现细胞排列紊乱、细胞固缩、胞浆炎染等明显病理结构改变。与生草乌组比较,生草乌-诃子(1∶3、1∶1、3∶1)组大鼠血清中cTn-I、MB含量均显著降低(P<0.05),并且生草乌-诃子(3∶1、1∶1)组含量与空白对照组差异无统计学意义(P>0.05);心肌组织病变都得到不同程度改善,其中生草乌-诃子(3∶1)组大鼠心肌组织结构与空白对照组相似。在机制考察试验中,在不同质量浓度下,草乌中3种乌头碱与正常人血浆的血浆蛋白结合率均约为30%,差异均无统计学意义(P>0.05),且无明显的浓度依赖性;加入单宁酸后草乌中3种乌头碱的血浆蛋白结合率均不同程度降低,当3种乌头碱与单宁酸按1∶0.1、1∶0.075、1∶0.5比例配伍时,血浆蛋白结合率显著降低(P<0.05),且有配伍比例的依赖性。结论:生草乌-诃子(3∶1)配伍使用对草乌致心脏毒性的解毒效果最好;在此配伍比例下,草乌中3种乌头碱与正常人血浆的血浆蛋白结合率相对较高,游离药物浓度相对较低。

OBJECTIVE: To investigate the best compatibility proportion of Mongolian medicine “Terminalia chebula decomposing the poison of Aconitum kusnezoffii”. METHODS:Totally 40 rats were randomly divided into blank control group (0.05% CMC-Na),raw A. kusnezoffii group(0.12 g/kg by crude drug)and raw A. kusnezoffii-T. chebula (1 ∶ 3,1 ∶ 1,3 ∶ 1,mass ratio) group (0.12 g/kg raw A. kusnezoffii by raw material), 8 rats in each group. They were given relevant medicine intragastrically once a day,for consecutive 28 d. 0.5 h after last medication,serum contents of cTn-I and MB were determined, and the changes of cardiological structure were observed;the detoxification effects of T. chebula on cardiotoxicity induced by A. kusnezoffii were investigated. Binding rates of 3 kinds of aconitine (concentrations of aconitine,mesaconitine and hypaconitine were 5,10,20,40,80,160,400 ng/mL) to binding rate of plasma protein with normal human plasma were determined by equilibrium dialysis combined with liquid chromtography-mass spectrometry. The concentrations of 3 kinds of aconitine were fixed as 100 ng/mL. After adding main detoxification component tannic acid in different proportions of T. chebula(1 ∶0.025,1 ∶0.075,1 ∶ 0.1,1 ∶0.5),the effects of them on plasma protein binding rate of 3 kinds of aconitine were investigated;the possible mechanism of T. chebula decomposing the poison of A. kusnezoffii inducing cardiotoxicity were investigated. RESULTS:In detoxification experiment,compared with blank control group,serum contents of cTn-I and MB were increased significantly in raw A. kusnezoffii group (P<0.05). There were obvious pathological changes in myocardial tissue,such as disorder of cell arrangement,cell shrinkage and cytoplasm staining. Compared with raw A. kusnezoffii group,serum contents of cTn-I and MB in raw A. kusnezoffii-T. chebula(1 ∶3,1 ∶1,3 ∶1)groups were decreased significantly (P<0.05), and there was no significant difference between the raw A. kusnezoffii-T. chebula(3 ∶ 1,1 ∶ 1) groups and blank control group (P>0.05);myocardial pathological changes were improved to varying degrees. The structure of myocardial tissue in raw A. kusnezoffii-T. chebula(3 ∶ 1) groups were similar to blank control group. In the mechanism investigation experiment under the condition of different concentrations,plasma protein binding rates of 3 kinds of aconitine with normal human plasma were about 30%,without statistical significance(P>0.05)and significant concentration-dependent manner. After adding tannic acid,plasma protein binding rate of 3 kinds of aconitine in A. kusnezoffii was decreased to different extent;when 3 kinds of aconitine were combined with tannic acid in the ratio of 1 ∶0.1,1 ∶0.075 and 1 ∶0.5,the plasma protein binding rate decreased significantly(P<0.05),in proportion-dependent manner. CONCLUSIONS:Compatible use of raw A. kusnezoffii-T. chebula (3 ∶ 1) show that best detoxification effect on A. kusnezoffii-induced cardiotoxicity. Under this compatibility ratio,the plasma protein binding rate of 3 kinds of aconitine in A. kusnezoffii with normal human plasma is relatively high and the free drug concentration is relatively low.