银翘散不同煎煮时间指纹图谱及对金黄色葡萄球菌抑菌活性的影响

Fingerprint and Antibacterial Activity Against Staphylococcus aureus of Yinqiao Powder Decoction in the Decocting Process

目的比较不同煎煮时间银翘散水煎液的体外抑菌活性,建立不同煎煮时间水煎液的指纹图谱,谱效关系筛选银翘散抑菌活性的物质基础。方法以金黄色葡萄球菌为受试菌,以万古霉素微生物药敏试纸为阳性对照,纸片扩散(K-B)法研究不同煎煮时间银翘散水煎液的抑菌活性,筛选抑菌作用最强的水煎液。采用高效液相色谱(HPLC)法,以Kromasil C_(18)色谱柱(4.6 mm×250 mm,5μm),乙腈-0.1%磷酸水溶液为流动相梯度洗脱,流速1.0 mL·min^(-1),检测波长230 nm,柱温30℃,进样量5.0μL,建立不同煎煮时间银翘散水煎液指纹图谱。采用主成分分析、Pearson双变量相关分析及正交偏最小二乘法(OPLS)法研究其谱效关系。结果除浸泡30 min的银翘散水煎液无抑菌活性外,其余水煎液均有一定抑菌活性,以煎煮30 min水煎液抑菌作用最强;不同煎煮时间银翘散水煎液指纹图谱共标定了25个共有峰,相似度在0.938以上,采用对照品比对法指认了其中17个色谱峰;与抑菌作用关系密切的14个共有峰分别为1、23、3、6、22、24、15、13、18、14、2、8、21、10号,其中23、3、6、22、24、15、21号分别为牛蒡苷、五福花苷酸、马钱苷酸、连翘苷、甘草素、连翘酯苷I、橙皮苷。结论本实验为以"谱-效"结合的思路研究银翘散的抑菌活性物质基础及质量控制方法提供了科学依据。

OBJECTIVE To compare the in vitro antibacterial activity and establish the fingerprint of Yinqiao Powder decoctions with different decocting time,and select the material basis of Yinqiao Powder antibacterial activity by spectrum-effect relationship.METHODS With Staphylococcus aureus as the test bacteria and vancomycin microbial drug sensitivity test paper as a positive control,paper diffusion(K-B)method was used to study the antibacterial activity of Yinqiao Powder decoctions with different decocting time,and screen the strongest antibacterial effect decoction.High performance liquid chromatography(HPLC)was performed on a Kromasil C_(18)(4.6 mm×250 mm,5μm)column using 0.1%phosphoric acid water and acetonitrile as mobile phase.Gradient elution was conducted at the flow rate of 1 mL·min^(-1),the detection wavelength was set at 230 nm,the column temperature was maintained at 30℃,and the injection volume was 5μL.HPLC fingerprint of Yinqiao Powder decoctions with different decocting time was established.The spectrum-effect relationship was studied by main component analysis,Pearson double variable correlation analysis,and orthogonal partial minimum double multiplication(OPLS).RESULTS The Yinqiao Powder with different decocting time had obvious antibacterial activity except the decoction immersing 30 min,and the antibacterial effect of decocting 30 min was strongest.The Yinqiao Powder decoction fingerprints marked 25 common peaks,with similarities above 0.938.Seventeen chromatographic peaks were identified by using the control comparison method.Fourteen common peaks(Nos.1,23,3,6,22,24,15,13,18,14,2,8,21,10)closely related to antibacterial effect,Among them,23,3,6,22,24,15,21 were arctiin,adoxosidic acid,loganic acid,phillyrin,liquiritigenin,forsythoside I,hesperidin.CONCLUSION It provides a scientific basis for studying the basis and quality control methods of Yinqiao Powder antibacterial active substances with the idea of"spectrum-effect"combination.