葛根素注射液溶血反应的改良溶血检查法

Improved hemolysis test method for detecting hemolytic reaction induced by puerarin injection

目的:对检测葛根素注射液溶血反应的改良溶血检查法进行评价。方法:取Beagle犬24只(体重6～8kg,雌雄各半),制备50%和2%新鲜Beagle犬红细胞悬液。应用中国药典2005年版记载的标准溶血检查法和本实验室建立的改良溶血检查法(其特点为增加葛根素浓度和延长孵育时间),观察Beagle犬红细胞在体外经不同浓度葛根素注射液和(或)丙二醇作用后的溶血反应,分别以生理盐水和蒸馏水为阴性和阳性对照计算溶血发生率并对结果进行统计学分析。结果:用药典标准方法,丙二醇浓度为3.3～16.7ml/L、葛根素浓度为0.33～1.67mg/ml,观察3h,Beagle犬红细胞未出现溶血;而采用改良方法,丙二醇浓度为100ml/L时发现4只犬红细胞溶血,溶血发生率为16.6%,与阴性对照组比较差异有统计学意义(P<0.05);丙二醇浓度为100ml/L、葛根素浓度为10mg/ml时,16只犬红细胞出现溶血,溶血发生率为66.7%,与阴性对照管比较差异有统计学意义(P<0.01)。相关性分析显示,溶血发生率与丙二醇浓度不存在相关性(r=0.571,P>0.05);与葛根素浓度存在显著相关性(r=0.952,P<0.01)。结论:改良溶血检查法似比常规溶血检查法能更有效地检测葛根素注射液所致的溶血反应。

Objective： To evaluate the improved hemolysis test method for detecting a hemolytic reaction induced by puerarin injection. Methods： Twenty-four Beagle dogs （ body weight 6-8 kg,the ratio of male to female was 1 ： 1 ） were obtained and 50% and 2% fresh erythrocyte suspensions were prepared. The standard hemolysis test method in the 2005 edition of the Chinese Pharmacopoeia and improved hemolysis test method establisbed by our laboratory were used in this study. The features of the improved method were increase in puerarin concentration and incubation time. Beagle dog erythrocytes were incubated with different concentrations of puerarin injection and/or propylene glycol, and the hemolytic reaction was observed. In addition, the normal saline and the distilled water served as the negative and positive control, respectively. The incidence of hemolysis was calculated and all results were analyzed statistically. Results： When using the standard hemolysis test method in the pharmacopoeia, no hemolysis occurred in Beagle dog erythrocytes within 3 hours following application of propylene glycol 3.3-16.7 ml/L and puerarin 0.33-1.67 mg/ml. When using improved hemolysis test method, hemolytic phenomenon occurred in 4 Beagle dogs＇ erythrocytes following application of propylene glycol 100 ml/L, and the incidence of hetrolysis was 16.6% ; compared with the negative control, the difference was statistically significant （P 〈 0.05） ; in addition, hemolytic phenomenon occurred in 16 Beagle dogs＇ erythrocytes following application of propylene glycol 100 ml/L and puerarin 10 mg/ml, the incidence of hemolysis was 66.7% ; the difference was statistically significant, compared with the negative control （ P 〈 0. 01 ）. Ccrrelation analysis showed that the incidence of hemolysis was not correlated with the concentration of propylene glycol （ r = 0.571, P 〉 0.05 ）, and significantly correlated with the concentration of puerarin （ r = 0. 952, P 〈0.01 ）. Conclusion： The improved hemolysis test method appeared to be more effective than the standard hemolysis test method in detection of a hemolytic reaction induced by puerarin injection.