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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 for the extraction and separation of glycosides from Hedyotis diffusa Willd. And the determination of glycosides in Hedyotis diffusa by high performance liquid chromatography. METHODS: The carotenoid in Hedyotis diffusa extract was separated by silica gel column chromatography and eluted with 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 fractions (methanol-ethyl acetate-water ratio 4: 3: 2; 5: 3: 2; 6: 3: 2) were combined and subjected to several silica gel column chromatographies to recover similar fractions again, Colorless crystal, namely carotenoid glycosides; the carotenoid glycosides isolated as a reference substance, the HPLC method Hedyotis diffusa cabbage glycosides content was determined. The column was Kromasil C18 (4.6 mm × 250 mm, 5 μm). The mobile phase was acetonitrile-0.2% phosphoric acid (5:95) with a flow rate of 0.8 mL.min-1. The detection wavelength was 240 nm and the column temperature was 30.0 ℃. Results: The purity of carotenoid obtained was over 99%. The injection of carprost glycoside showed a good linearity (r = 0.9998) within the range of 0.4 ~ 2.0μg, and the average recovery was 97.7% (n = 6). Conclusion: The method is accurate and simple, and provides a new method for the extraction and separation of carverine glycosides and the determination of Hedyotis diffusa.