经方桂枝芍药知母汤物质基准的HPLC指纹图谱及清除DPPH谱效关系的研究

Spectrum-effect relationship between HPLC fingerprint and free radicals scavenging in Guizhi Shaoyao Zhimu Decoction

建立经典名方桂枝芍药知母汤物质基准的HPLC指纹图谱及清除自由基活性的谱效关系,为其质量评价与经方现代化研究提供依据。采用Shimadsu GL-science C18色谱柱(4. 6 mm×250 mm,5μm)进行检测,以0. 1%甲酸乙腈-0. 1%甲酸水为流动相,进行梯度洗脱,流速1. 0 m L·min-1,检测波长254 nm,柱温32℃,进样量20μL。测定10批桂枝芍药知母汤物质基准样品,利用中药色谱指纹图谱相似度评价系统建立经典名方桂枝芍药知母汤物质基准的HPLC指纹图谱;1,1-二苯基-2-三硝基苯肼(DPPH)法研究其清除自由基的活性,并通过Pearson双变量相关分析研究谱效关系。建立了桂枝芍药知母汤物质基准指纹图谱共有模式,标定了26个共有峰,相似度在0. 929~0. 998。采用对照品比对法指认了其中8个色谱峰:没食子酸、芒果苷、芍药苷、甘草苷、升麻素苷、5-O-甲基维斯阿米醇苷、肉桂酸、甘草酸铵。其中14(芍药苷),20,12(芒果苷),13,23(肉桂酸)号共有峰含量变化与自由基清除活性呈负相关。该指纹图谱具有良好的精密度、重复性、稳定性,各共有峰分离度良好,可用于桂枝芍药知母汤的质量评价;该指纹图谱与清除自由基活性的谱效关系研究对于桂枝芍药知母汤物质基础的研究具有一定参考价值。

To establish the spectrum-effect relationship between HPLC fingerprint and free radicals activity scavenging in Guizhi Shaoyao Zhimu Decoction(GSZD),and provide a basis for the quality evaluation and modernization of classical prescriptions.Shimadsu GL-science C18 column(4.6 mm×250 mm,5μm)was used with acetonitrile-0.1%formic acid solution as the mobile phase for gradient elution.The detective wave length was 254 nm;the column temperature was set at 32℃;the injection volume was 20μL;and the flow rate was 1.0 m L·min-1.10 batches of primary standard samples of GSZD were detected,and their HPLC fingerprint was established by using the similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine(TCM).The activity of scavenging free radicals was studied by 1,1-diphenyl-2-trinitrophenylhydrazine(DPPH)method,and the spectrum-effect relationship was studied by Pearson bivariate correlation analysis.The common mode of GSZD fingerprints was established,and 26 common peaks were marked,with similarities ranging from 0.929 to 0.998.Eight of the chromatographic peaks were identified by using the control comparison method:gallic acid,mangiferin,paeoniflorin,glycyrrhizin,asparagus,5-O-methylvisamicin,cinnamic acid,and ammonium glycyrrhetate.Among them,the content changes of No.14(paeoniside),20,12(mangiferin),13 and 23(cinnamic acid)common peaks were negatively correlated with free radical scavenging activity.The fingerprint showed high precision,repeatability and stability,and the common peaks were well separated,so it can be used for the quality evaluation of GSZD,and could provide reference for further studies on the material basis of GSZD.