基于甲型H1N1流感病毒致MDCK细胞损伤保护作用的双黄连组方HPLC特征图谱谱效关系研究(1)
[摘要] 构建双黄连组方HPLC特征图谱与甲型H1N1流感病毒致MDCK(Madin-Darby canine kidney)细胞损伤保护作用的相关性。在双黄连原方的基础上按正交法L9(34)组成9个不同配比的组方，分别进行HPLC分析和MDCK细胞损伤的保护作用试验，采用细胞病变效应法(cytopathic effect， CPE)观察病毒感染力，MTT染色结果作为浓度筛选和细胞损伤保护作用的判定指标。除组方1外，其他各组方对甲型H1N1流感病毒致MDCK细胞损伤均具有显著的保护作用(P<0.01，P<0.001)，对所获得的HPLC数据和药效数据应用SPSS软件进行逐步回归分析，结果双黄连各组方17个特征共有色谱峰中与甲型H1N1流感病毒致MDCK细胞损伤的保护作用相关性较大的组分为2，3，6，8和12号峰。将组方数据与药效数据进行逐步回归分析，发现金银花、连翘与细胞损伤的保护作用呈正相关。通过HPLC图比对，2，3和12号峰来自金银花，而6和8号峰来自连翘。通过对照品保留时间进一步比较，确定了其中4个色谱峰分别为绿原酸、隐绿原酸、连翘酯苷B和3，4-二咖啡酰奎宁酸。推测双黄连组方中与甲型H1N1流感病毒致MDCK细胞损伤保护作用相关性较大的的主要成分为金银花和连翘中咖啡酰类衍生物。
[关键词] 双黄连口服液；抗病毒，谱效相关；绿原酸
[Abstract] To establish HPLC specific chromatogram and its correlation with the protection effect of Shuanghuanglian on MDCK (Madin-Darby canine kidney) cell injury induced by influenza A virus(H1N1). Nine recipes of Shuanghuanglian based on the official prescription were prepared according to orthogonal test for HPLC analysis and MDCK cells protection experiment separately (cytopathic effect (CPE) method was used for observing the virus infectivity and MTT staining results were used as the determining indexes for drug concentration selection and analyzing cell viability). The results suggested that all the other Shuang-Huang-Lian recipes except recipe1 demonstrate protecting effect on MDCK cell injury induced by influenza A virus (P<0.01， P<0.001). Stepwise regression analysis was used for analyzing the relationships between HPLC fingerprint and the protecting effect of Shuanghuanglian on influenza A virus induced MDCK cell injury. Peak 2， 3， 6， 8 and 12 were found to be strongly related with anti-influenza A virus efficacy. Stepwise regression analysis of recipes data and efficacy data showed that Lonicerae Japonicae Flos and Forsythiae Fructus were positively associated with the protecting effect of cells injury. From HPLC fingerprints， we found that peak 2， 3， 12 were from Lonicerae Japonicae Flos and peak 6， 8 were from Forsythiae Fructus. Four peaks were identified through comparing the retention time between the standard and Shuanghuanglian recipes， and they were chlorogenicacid， cryptochlorogenic acid， forsythoside B and 3，4-dicaffeoylquinic acid respectively. Caffeic acid derivatives in Lonicerae Japonicae Flos and Forsythiae Fructus were found to be greatly correlated with anti-influenza A virus efficacy and maybe the substance basis of Shuanghuanglian.
[Key words] Shuanghuanglian oral liquid； anti-influenza A virus； relationships between fingerprint and efficacy； chlorogenic acid (刘廷 王海丹 狄留庆 康安 赵晓莉 朱萱萱 李俊松)
[关键词] 双黄连口服液；抗病毒，谱效相关；绿原酸
[Abstract] To establish HPLC specific chromatogram and its correlation with the protection effect of Shuanghuanglian on MDCK (Madin-Darby canine kidney) cell injury induced by influenza A virus(H1N1). Nine recipes of Shuanghuanglian based on the official prescription were prepared according to orthogonal test for HPLC analysis and MDCK cells protection experiment separately (cytopathic effect (CPE) method was used for observing the virus infectivity and MTT staining results were used as the determining indexes for drug concentration selection and analyzing cell viability). The results suggested that all the other Shuang-Huang-Lian recipes except recipe1 demonstrate protecting effect on MDCK cell injury induced by influenza A virus (P<0.01， P<0.001). Stepwise regression analysis was used for analyzing the relationships between HPLC fingerprint and the protecting effect of Shuanghuanglian on influenza A virus induced MDCK cell injury. Peak 2， 3， 6， 8 and 12 were found to be strongly related with anti-influenza A virus efficacy. Stepwise regression analysis of recipes data and efficacy data showed that Lonicerae Japonicae Flos and Forsythiae Fructus were positively associated with the protecting effect of cells injury. From HPLC fingerprints， we found that peak 2， 3， 12 were from Lonicerae Japonicae Flos and peak 6， 8 were from Forsythiae Fructus. Four peaks were identified through comparing the retention time between the standard and Shuanghuanglian recipes， and they were chlorogenicacid， cryptochlorogenic acid， forsythoside B and 3，4-dicaffeoylquinic acid respectively. Caffeic acid derivatives in Lonicerae Japonicae Flos and Forsythiae Fructus were found to be greatly correlated with anti-influenza A virus efficacy and maybe the substance basis of Shuanghuanglian.
[Key words] Shuanghuanglian oral liquid； anti-influenza A virus； relationships between fingerprint and efficacy； chlorogenic acid (刘廷 王海丹 狄留庆 康安 赵晓莉 朱萱萱 李俊松)