夜仁合有效部位含量测定和改善PCPA睡眠剥夺大鼠作用机制研究

Content Determination of Effective Parts of Yerenhe and Its Mechanism in Improving PCPA-Induced Sleep Deprivation in Rats

目的:基于高效液相色谱法(HPLC)测定夜仁合有效部位中8种有效成分的含量,研究夜仁合有效部位对氯苯丙氨酸(P-chlorophenylalanine,PCPA)致睡眠剥夺大鼠睡眠时间和作用机制的影响。方法:首先采用HPLC对夜仁合有效部位成分进行含量测定,其次将48只SD大鼠分为空白组、模型组、阳性药组、夜仁合有效部位低剂量组、中剂量组和高剂量组,共6组,采用实验动物监测系统(CLAMS)和酶联免疫分析(ELISA)法检测PCPA睡眠剥夺大鼠睡眠时间、睡眠发生次数、睡眠发生持续时间和下丘脑及海马中5-羟色胺(5-HT)、5-羟吲哚乙酸(5-HIAA)的含量。结果:斯皮诺素、阿魏酸、槲皮苷、五味子醇甲、五味子甲素、五味子乙素、酸枣仁皂苷A和酸枣仁皂苷B有效成分的线性关系良好(r>0.9993),平均加样回收率96.82%~100.49%,RSD值0.78%~1.38%。与空白组相比,模型组大鼠的全天、日间和夜间的睡眠时间均降低(P<0.05,P<0.01)、全天和日间的睡眠发生次数均减少(P<0.05)、夜间睡眠持续时间减少(P<0.01)、下丘脑和海马中5-HT、5-HIAA含量降低(P<0.05,P<0.01)。与模型组相比,阳性药组、夜仁合有效部位中剂量组和高剂量组大鼠全天、日间和夜间的睡眠时间均增加(P<0.05,P<0.01);阳性药组、夜仁合有效部位低剂量组、中剂量组和高剂量组全天和夜间的睡眠发生次数均增加(P<0.05,P<0.01);阳性药组、夜仁合有效部位中剂量组和高剂量组大鼠下丘脑和海马中5-HT、5-HIAA含量增加(P<0.05,P<0.01)。结论:该含量测定方法简单、重复性、稳定性良好,可为夜仁合质量标准提供一定依据;夜仁合有效部位可以改善大鼠睡眠时间,作用机制可能与调节下丘脑和海马内5-HT、5-HIAA含量有关。

Objective:To study the effects of the effective parts of Yeheren on sleep time in P-chlorophenylalanine(PCPA)-induced sleep deprivation and to explore its mechanism based on the determination of 8 effective components in it.Methods:HPLC was used to determine the contents of components in the effective parts of Yeheren.48 SD rats were divided into blank group,model group,positive group,the Yeheren effective parts groups of low-dose,medium-dose and high-dose.CLAMS and ELISA were used to detect the sleep time,sleep frequency,duration of sleep and the contents of 5-hydroxytryptamine(5-HT)and 5-hydroxyindoeacetic acid(5-HIAA)in hypothalamus and hippocampus of PCPA deprived rats.Results:The linear relationship of the effective components of spinotin,ferulic acid,quercetin,schisandrol A,schizandrin A and schizandrin B was good(r>0.9993).The average recovery rate was 96.82%-100.49%with RSD values of 0.78%-1.38%.Compared to those in the control group,the sleep time of all day,daytime and night were decreased in the model group(P<0.05,P<0.01),the sleep frequency of all day and daytime were decreased(P<0.05),the sleep duration of night was decreased(P<0.01),and the contents of 5-HT and 5-HIAA in hypothalamus and hippocampus were decreased in the model group(P<0.05,P<0.01).Compared with those in the model group,the sleep time of all day,daytime and night were increased in the positive group and the Yeheren effective parts groups of medium-dose and high-dose(P<0.05,P<0.01);the sleep frequency of all day and daytime were increased in the positive group and the Yeheren effective parts groups of low-dose,medium-dose and high-dose(P<0.05,P<0.01);the contents of 5-HT and 5-HIAA in hypothalamus and hippocampus were increased in the positive group and the Yeheren effective parts groups of medium-dose and high-dose(P<0.05,P<0.01).Conclusion:The determination method is simple,reproducible and stable,which can provide a basis for the quality standard of Yerenhe.The effective components of Yerenhe can improve sleep,and the mechanism may be related to the content regulations of 5-HT and 5-HIAA in hypothalamus and hippocampus.