银翘散精简方提取物的鉴定及体外抗流感病毒作用研究

The Identification of Fructus Forsythiae(Yinqiaosan)Smaller Parties Extracts and Their in Vitro Inhibitory Effects on Influenza Virus

目的：建立以高效液相色谱法（HPLC）同时测定银翘散精简方中绿原酸、连翘苷、橙皮苷、牛蒡苷含量的方法,观察银翘散精简方提取物体外抑制甲型流感病毒FM1株的作用。方法：采用Phenomsil ODS C18（250 mm×4.6 mm,5μm）色谱柱分离;甲醇-0.4%磷酸水溶液,梯度洗脱;流速：1.0 mL·min^-1;检测波长275 nm。采用血凝试验方法,测定银翘散精简方提取物体外和鸡胚内抑制流感病毒效价,分析银翘散精简方提取物对病毒增殖的影响。结果：绿原酸在1.89～189 mg·L^-1呈良好的线性关系（r=0.9986）,连翘苷在0.93～22.32 mg·L^-1呈良好的线性关系（r=0.9984）,橙皮苷在3.04～30.4 mg·L^-1呈良好的线性关系（r=0.9992）,牛蒡苷分别在5.05～151.5 mg·L^-1呈良好的线性关系（r=0.9983）。绿原酸的平均加样回收率为92.05%（n=6）,RSD 0.81%,连翘苷的平均加样回收率为102.56%（n=6）,RSD 1.15%,橙皮苷的平均加样回收率为96.97%（n=6）,RSD 1.72%,牛蒡苷的平均加样回收率为94.88%（n=6）,RSD 1.0%。银翘散精简方提取物可直接抑制甲型流感病毒FM1株的活性,作用时间从1 h可持续到24 h。银翘散精简方提取物对流感病毒感染鸡胚有一定的预防和治疗作用,且治疗作用优于阳性对照利巴韦林组（P〈0.05）。结论：该方法简便、准确,重复性好,可用于银翘散精简方的质量控制。银翘散精简方提取物可有效抑制甲型流感病毒FM1株。

Objective：To establish a method for determination content of caffeoylquinic acid,forsythin,arctium lappa glycoside and aurantiamarin in fructus forsythiae smaller parties by high performance liquid chromatography（HPLC）,observ the anti influenza A virus results of fructus forsythiae in vitro. Methods：Using Phenomsil ODS C18（250 mm×4.6 mm,5μm）chromatographic column separation. Methanol-0.4% phosphoric acid solution,gradient elution;Flow rate：1.0 mL/min;The detection wavelength was 275 nm. Using test method of coagulation,determination of fructus forsythiae powder compact extract in vitro and chicken embryos in inhibition of influenza virus titer,analysis of fructus forsythiae powder compact extract effect on virus proliferation. Results：Chlorogenic acid had good linear relationship between 1.89～189 mg·L^-1（r=0.9986）,forsythin had good linear relationship between 0.93 ～ 22.32 mg·L^-1（r=0.9984）,arctium lappa glycoside had good linear relationship between 3.04 ～ 30.4 mg·L^-1（r=0.9992）,aurantiamarin had good linear relationship between 5.05 ～ 151.5 mg·L^-1（r=0.9983）.Chlorogenic acid＇s average sample recovery rate was 92.05%（n=6）,RSD 0.81%,forsythin＇s average sample recovery rate was 102.56%（n=6）,RSD 1.15%,arctium lappa glycoside＇s average sample recovery rate was 96.97%（n=6）,RSD 1.72%,aurantiamarin＇s average sample recovery rate was 94.88%（n=6）,RSD 1.0%. Extracts from fructus forsythiae smaller parties can directly inhibit the activity of the influenza A virus FM1 strain,action time sustain from 1 h to 24 h. Extracts from fructus forsythiae smaller parties can prevent and treat the chicken embryos from influenza virus to a certain extent,and the therapy effect is better than the ribavirin control（P〈0.05）. Conclusion：Method is simple and accurate,and reproducible,can be used for quality control of fructus forsythiae. The extracts of fructus forsythiae smaller parties can effectively inhibit influenza A virus.