HPLC法同时测定重楼属药用植物中10种核苷和碱基

Simultaneous determination of ten nucleosides and bases contents in plants of Paris L. by HPLC

目的对不同品种、不同产地的重楼属药用植物中核苷（胞苷、尿苷、肌苷、鸟苷、胸苷、腺苷、2′-脱氧腺苷）和碱基（尿嘧啶、鸟嘌呤、腺嘌呤）量进行对比研究,为合理开发利用重楼属药用植物提供理论依据。方法采用RP-HPLC法,色谱柱为Venusil MP C18（2）柱（250 mm×4.6 mm,5μm）,流动相为甲醇-水溶液,梯度洗脱,体积流量1.0 m L/min,柱温35℃,检测波长260 nm。运用上述方法对16批重楼药材进行测定,并对测定结果进行聚类分析。结果测定了重楼药材中10种核苷和碱基的量,在线性范围内线性关系良好（r2〉0.999 1）,平均回收率为96.04%-104.34%,RSD≤2.94%。采用聚类分析能将不同产地的16批重楼药材分为3类。16批重楼样品中10种核苷和碱基均可检测到,不同品种重楼药材中10种核苷和碱基的质量分数及组成结构比存在着显著性差异,各核苷和碱基总量平均值为735.390 1-1 612.807 3μg/g,质量分数高低排序为巴山重楼〉长药隔重楼〉卵叶重楼〉五指莲重楼〉狭叶重楼〉华重楼〉滇重楼〉花叶重楼〉小重楼〉毛重楼。同一品种不同产地重楼药材中10种核苷和碱基的质量分数及组成结构比亦存在着较大差异。结论该方法简单,准确可靠,重复性好,适用于重楼药材中10种核苷和碱基的测定。同时亦为全面客观地认识重楼药材功效物质基础,丰富和发展重楼药材多指标评价体系研究提供科学依据。

Objective In order to provide a theory basis for rational development and utilization of medicinal plants from Paris L., this study compared nucleosides （cytidine, uridine, inosine, guanosine, thymidine, adenosine and 2＇-desoxyadenossine） and bases （including uracil, guanine, and adenine） contents in the different plants of Paris L.which collected from different places. Methods RP-HPLC was applied in this study, Venusil MP C18（2） chromatographic column （250 mm × 4.6 mm, 5 μn） was used, the mobile phase was a methanol and water gradient, the flow rate was1.0 mL/min, the temperature of column was 35 ℃ and the UV detector was set at 260 nm. Sixteen batches of samples in Paris L. were measured by above method, and cluster analysis was used to analyze the results. Results Ten nueleosides and bases from the plants of Paris L. were measured, a good linear relationship was showed （r2 〉 0.999 1）, and the average recovery was 96.04%-104.34%, RSD ≤ 2.94%. The samples in Paris L. from different places were divided into three categories by cluster analysis. All of the ten nueleosides and bases were detected in the 16 batches of samples; However, the mass fraction and component ratio of the ten substances were significantly different from each variety. The average of total nucleosides and bases content was ranged from 735.390 1 to 1 612.807 3 μg/g, and the order from high to low was： P. bashanensis 〉 P.polyphylla var.pseudothibetica 〉 P. delavayi var. petiolata 〉 P. axialis 〉 P. polyphylla vat. stenophylla 〉 P. polyphylla var. chinensis 〉 P. polyphylla var. yunnanensis 〉 P. marmorata 〉 P. polyphylla var. minor 〉 P. mairei. The mass fraction and component ratio of the ten substances also had a significant difference between the samples collected from different regions which were the same variety. Conclusion This method is simple, accurate and reliable and has good reproducibility. It is appropriate for measuring the contents of the ten nucleosides and bases. Simultaneously, this study provids a scientific basis for an objective and complete knowledge of the material basis for the efficacy, and for an enrichment and development of evaluation indicators system of in the plants Paris L.