博落回根中生物碱的组织化学定位研究

Histochemical localization study of alkaloids in root of Macleaya cordata

目的建立激光显微切割结合HPLC-MS/MS技术测定博落回根不同组织中血根碱、原阿片碱、别隐品碱、白屈菜红碱、二氢白屈菜红碱和二氢血根碱的定量分析方法,开展博落回根的组织化学研究。方法利用激光显微切割技术将两年生不同生长期博落回根的木栓层、皮层、韧皮部、木质部维管束和木射线进行切割,HPLC-MS/MS对切割的组织进行目标生物碱的检测。结果 6种生物碱均有较宽的线性范围和良好的线性关系（r2〉0.996 6）;日内、日间精密度RSD均小于2.4%;检出限和定量限最高分别为0.29、0.57 ng/m L;重复性试验RSD均小于13.2%;加样回收率为85.1%-133.9%,RSD值为9.4%~20.7%。苗期根的上述5个组织中6种生物碱的总量分别为0.14、0.10、0.07、0.16、0.11μg/mm2;花期分别为0.38、0.22、0.15、0.41、0.26μg/mm2;果期分别为0.46、0.29、0.27、0.55、0.22μg/mm2;果后期分别为0.52、0.29、0.24、0.41、0.23μg/mm2。结论基于激光显微切割和LC-MS/MS的组织中生物碱的定量分析方法分辨率高、专属性强、灵敏度好、准确可靠;博落回根中6种生物碱主要分布在木栓层和木质部维管束。

Objective To develop the quantitative analysis method combined with laser microdissection and LC-MS/MS technique for determinating sanguinarine, protopin, allocryptopine, chelerythrine, dihydrochelerythrine, and dihydrosanguinarine in the roots of Macleaya cordata, and performe the histochemical study on M. cordata roots. Methods The technology of laser-microdissection was used to dissect the cork, cortex, phloem, xylem vascular bundles, and xylem rays from 2-year-old M. cordata roots at different growth stages, the technique of LC-MS/MS was applied to detecting the contents of targeted alkaloids in the microdissected tissues. Results The six kinds of alkaloids possesed a wide linear ranges and a good linear relationship（r2 〉0.996 6）; The RSD of intra- and inter-day precision was all less than 2.4%; The highest limits of detection and quantification were respectively 0.29 and 0.57 ng/m L; The repeatability RSD was below 13.2%. The recovery varied from 85.1% to 133.9%, and its RSD ranged from 9.4% to 20.7%. The total amounts of six kinds of alkaloids in the above five tissues were respectively 0.14, 0.10, 0.07, 0.16 and 0.11 μg/mm2 for seedling stage root; 0.38, 0.22, 0.15, 0.41 and 0.26 μg/mm2 for flowering stage root; 0.46, 0.29, 0.27, 0.55 and 0.22 μg/mm2 for fruit stage root, and 0.52, 0.29, 0.24, 0.41 and 0.23 μg/mm2 for latering fruit stage root. Conclusion The integrated method is high-resolution, specific, sensitive, and reliable for the quantitative analysis of alkaloid in tissues; The six kinds of alkaloids are mainly located in corks and xylem vascular bundles from the roots of M. cordata.