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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 m L·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 simultaneous determination of chlorogenic acid, forsythin, hesperidin and arctiin in Yinqiao Sanjie Decoction by high performance liquid chromatography (HPLC), and to observe the effect of Yinqiao Sanjing extract Effect of influenza virus FM1 strain. METHODS: The chromatographic column was separated on a Phenomsil ODS C18 (250 mm × 4.6 mm, 5 μm) column and eluted with methanol-0.4% phosphoric acid solution. The flow rate was 1.0 m L · min-1. The detection wavelength was 275 nm. The method of hemagglutination test was used to determine the inhibitory effect of extract of Yinqiaosi Sanjie extract on influenza virus in vitro and in chicken embryo. The effect of Yinqiao powder extract on virus proliferation was analyzed. RESULTS: Chlorogenic acid had a good linearity (r = 0.9986) at 1.89-189 mg · L-1 and a good linear relationship between forsythin at 0.93-22.32 mg · L-1 (r = 0.9984) Glycosides showed good linearity (r = 0.9992) at 3.04-30.4 mg · L-1 and good linearity at 5.05-151.5 mg · L-1 (r = 0.9983). The average recoveries of chlorogenic acids were 92.05% (n = 6), RSD 0.81%. The average recoveries of forsythin were 102.56% (n = 6), RSD 1.15% The average recoveries were 96.97% (n = 6) and RSD 1.72%. The average recoveries of arctiin were 94.88% (n = 6) and RSD 1.0%. Yinqiaosi concanavalin extract can directly inhibit the activity of FM1 strain of influenza A virus and the duration of action can be prolonged from 1 h to 24 h. Yinqiaosi concanavalin extract has some preventive and therapeutic effects on influenza virus infection of chicken embryos, and the therapeutic effect is better than the positive control ribavirin group (P <0.05). Conclusion: The method is simple, accurate and reproducible. It can be used for the quality control of Yinqiao Powder concise prescription. Yinqiao Sanjie extract can effectively inhibit the influenza A virus FM1 strain.