白花蛇舌草中车叶草苷的提取分离及含量测定的研究

Study on extraction and isolation of asperuloside from Hedyotis diffusa Willd. and assay by HPLC

目的:建立白花蛇舌草中车叶草苷的提取分离工艺及车叶草苷的高效液相色谱含量测定方法。方法:用硅胶柱层析分离白花蛇舌草醇提取液中的车叶草苷,以甲醇-乙酸乙脂-水(3∶3∶2;4∶3∶2;5∶3∶2;6∶3∶2;7∶3∶2;8∶3∶2;9∶3∶2;10∶3∶2)进行洗脱。将馏分(甲醇-乙酸乙脂-水比例4∶3∶2;5∶3∶2;6∶3∶2)合并,经几次硅胶柱层析后再次合并相似馏分,回收洗脱剂,得无色结晶,即车叶草苷;将分离得到的车叶草苷作为对照品,使用HPLC法对白花蛇舌草中车叶草苷的含量进行测定。色谱柱为Kromasil C18(4.6mm×250mm,5μm)柱,流动相为乙腈-0.2%磷酸(5∶95),流速0.8mL.min-1,检测波长240nm,柱温30.0℃。结果:所得车叶草苷纯度在99%以上;车叶草苷进样量在0.4～2.0μg范围内线性关系良好(r=0.9998),平均回收率(n=6)为97.7%。结论:该方法结果准确、简便,为车叶草苷的提取分离及白花蛇舌草含量测定提供了新方法。

Objective：To establish a method of extraction and isolation of asperuloside from Hedyotis diffusa Willd. and assay by HPLC.Methods：Gel silica column chromatography was selected to isolate asperuloside from the extracts of Hedyotis diffusa;The eluant was methanol-ethyl acetate-water（3：3：2;4：3：2;5：3：2;6：3：2;7：3：2;8：3：2;9：3：2;10：3：2）;The fraction of distillate with methanol-ethyl acetate-water（4：3：2;5：3：2;6：3：2） were incorporate.The work of above-mentioned was repeated.The eluant was reclaimed,and the colourless crystal that was asperuloside was obtained.The asperuloside above was used as reference substance,and HPLC method was used for assaying the asperuloside content from Hedyotis diffusa.The separation was performed on a Kromasil C18（250 mm×4.6 mm,5 μm） column with acetonitrile-0.2% aqueous phosphoric acid（5：95） as mobile phase at a flow rate of 0.8 mL·min-1.The detection wavelength was set at 240 nm and the column temperature was maintained at 30 ℃.Results：The colourless crystal was asperuloside by assessment,and the purity was more than 99%.The linear range of asperuloside was 0.4-2.0 μg（r=0.9998）,while the average recovery（n=6） was 97.7%.Conclusion：The method is accurate and convenient.It is a new method on the extraction and assay of asperuloside.