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目的检测附子离体培养愈伤组织和离体苗的生物碱量,建立附子乌头类总生物碱和同时测定新乌头碱、乌头碱、次乌头碱的分析方法。方法用酸性染料比色法测定乌头类总生物碱的量;反相高效液相色谱法测定新乌头碱、乌头碱、次乌头碱的量,色谱柱为Zorbaxextend-C18(150mm×4.6mm,5μm),流动相为乙腈-0.1%乙二胺,梯度洗脱,体积流量为1mL/min,检测波长为240nm,柱温30℃。结果愈伤组织和离体苗中总生物碱的量分别为3.972、2.904mg/g;新乌头碱、乌头碱、次乌头碱的量分别为:0.678、0.448、0.887mg/g,0.464、0.234、0.339mg/g。3种生物碱得到很好分离,线性关系良好。结论愈伤组织中生物碱的量高于离体苗,该方法快速简便、灵敏准确、回收率高,可以为附子质量控制提供科学依据。
Objective To detect the amount of alkaloids in the cultured callus and in vitro seedlings of Radix Aconiti Lateralis Preparata, and to establish the method for the simultaneous determination of aconitine, aconitine and hypaconitine in the aconite genus Aconite. Methods The amount of total alkaloid of aconitine was determined by acid dye colorimetric method. The amount of mesaconitine, aconitine and hypaconitine was determined by reversed-phase high performance liquid chromatography. The column was Zorbaxextend-C18 (150mm × 4.6mm, 5μm). The mobile phase was acetonitrile-0.1% ethylenediamine. The gradient elution was carried out. The volume flow rate was 1mL / min, the detection wavelength was 240nm and the column temperature was 30 ℃. Results The amount of total alkaloids in callus and in vitro seedlings were 3.972 and 2.904 mg / g, respectively. The amounts of mesaconitine, aconitine and hypaconitine were 0.678, 0.448 and 0.877 mg / g, 0.464, 0.234, 0.339 mg / g. The three alkaloids are well separated and have a good linear relationship. Conclusion The amount of alkaloids in callus is higher than that of in vitro seedlings. The method is rapid, simple, sensitive and accurate, and has high recovery rate. It can provide a scientific basis for the quality control of aconite.