基于全电性离子色谱的6味中药药性的蛋白质分子标记研究

Study of Protein Molecular Markers of Six TCM drugs based on the Full Electrical Property Ion Chromatography

目的:提出分子标记法检测中药药性物质基础研究的新思路,探讨全电性离子色谱技术用于筛选中药药性蛋白质分子标记的可行性。方法:联合应用阴、阳离子交换色谱技术,将特定pH条件下中药的全部水溶性蛋白的色谱峰呈现出来,形成全电性离子色谱图,平行比较不同药性中药的全电性离子色谱图,筛选出相同药性中药间共有的药性标识峰,对药性标识峰组分作SDS-PAGE,比较分析药性相关的共有蛋白条带,作为中药药性的蛋白质分子标记。结果:干姜、肉桂、仙茅3味热性中药在阴离子交换色谱的收集部分编号为30附近,存在多个共有特征峰;黄连、知母、金银花3味寒性中药在阴离子交换色谱的收集编号为35附近会存在多个共有特征峰;3味热性中药的共有特征峰组分在分子质量为50ku(1u=1.66054×10-27)附近存在一条共有条带,在分子质量约为31ku处,存在2条共有条带;3味寒性中药共有特征峰组分在分子质量为31ku附近,存在2条共有条带。结论:收集编号为35和30附近的共有色谱峰在3次重复中稳定呈现,可以作为6味测试中药的寒、热药性标识峰。标识峰的共有条带分别是3味寒性中药与3味热性中药所特有的,在3次重复中稳定呈现,可以作为6味测试中药药性的蛋白质分子标记。全电性离子色谱法是一种可行的中药药性分子标记研究的新方法。

In this paper, authors present a new method to study the material base of Chinese traditional medicine using molecular markers, and discuss the feasibility of selecting the right protein molecular markers for judging the medici- nal nature of TCM drugs using the full electrical property ion chromatography. In the study, the chromatographic peaks of the whole water-soluble protein in a TCM drug with a special pH value were picked up by combining both anion and cation exchange chromatography, or the full electrical property ion chromatogram. The mutual peaks marking TCM drugs were sorted out through the parallel comparison of the full electrical property ion chromatograms of different TCM drugs. SDS-PAGE was performed on the components of mutual peaks, and on the mutual protein bands that were the molecular markers. Results show that near the fraction number of 30 in the negative ion exchange chromatography, there are several mutual peaks for all the three TCM drugs tested with hot nature of dried ginger, cassia bark, and curculigo rhizome, and near the fraction number of 35, there are several mutual peaks for all the three TCM drugs with cold nature of coptis, anemarrhena, and honeysuckle flower. All the three TCM drugs tested with hot nature enjoy a mutual protein of a molecular weight of 51 kU, with two mutual bands at 31 kU on the SDS-PAGE gel. Meanwhile, the three TCM drugs with cold nature reveal only two mutual protein bands near the molecular weight of 31 kU. It is concluded that the mutual peaks at the fraction number of 30 and 35 show three repeats, indicating that these peaks can be taken as the medicinal peaks of six TCM drugs tested. The mutual protein bands of the marking peak components on SDS-PAGE gel are special for both the three TCM drugs with hot nature and the three TCM drugs with cold nature, with steady three repeats. It is believed that the protein bands can serve as the protein molecular markers judging the medicinal nature of the six TCM drugs tested. Full electrical property ion chromatography is a new approach desirable for screening the molecular medicinal markers for TCM drugs.