[关键词]
[摘要]
目的 建立并完善从中成药及保健品中提取DNA的有效方法,并在人参类制剂中进行验证。方法 使用中成药和保健品中常用的辅料(如淀粉、蔗糖、葡萄糖等)模拟CTAB法提取DNA过程,用以筛选影响提取DNA效率的因素;使用甲醇作为沉淀试剂优化传统的CTAB法,并使用MAS-PCR方法对市场上人参类中成药及保健品进行分子鉴定,以验证优化的技术。结果 中成药及保健品中的淀粉(包括药源性淀粉和辅料添加的淀粉)通过CTAB反应后生成极性较大的复合物,其与70%乙醇不能互溶,而且在碱性条件下对DNA有强烈的溶解作用,从而影响了CTAB法提取DNA的提取效率;利用甲醇作为沉淀试剂能够有效去除CTAB裂解过程中产生的高极性复合物的影响,70%甲醇-醋酸钠溶液能够有效提取中成药及保健品的DNA;利用优化的DNA提取方法和MAS-PCR法实现了11种市售人参类制剂的快速分子鉴定,结果显示8种与商品描述一致、1种有掺伪情况、2种为伪品。结论 使用甲醇作为沉淀试剂优化传统的CTAB法,可以较好地克服中药制剂中淀粉对DNA提取的不利影响,可以有效地实现基于DNA的中药制剂分子鉴定。
[Key word]
[Abstract]
Objective To establish an optimized method for extracting DNA from Chinese patent medicine (CPM) and health-care products and assuring the quality of DNA for successful molecular identification in ginseng preparations. Methods Commonly-used pharmaceutical excipients, such as starch, sucrose, and glucose in CPM and health-care products were employed to discover the potential cause of low quality of DNA extraction in classic CTAB method. Methanol was used to precipitate and purify the DNA extracted with the classic CTAB method in order to improve the quality and quantity of DNA thus extracted. Finally, thus extracted DNA was employed to conduct the molecular identification of CPM and health-care products with ginseng herbs and validate the optimized method. Results Starch in CPM and health-care products may react with CTAB and thus form a high polar complex that is insoluble in 70% ethanol but can strongly dissolve DNA, resulting a very low DNA extraction rate in classic CTAB method. Methanol is able to dissolve the high polar complex and thus the application of 70% methanol and NaAc solution guaranteed the successful extraction of DNA with high quality and quantity from CPM and health-care products. With the optimized CTAB method, the DNA samples of 11 kinds of CPM and health-care products containing ginseng or American ginseng were extracted and applied for molecular identification with MAS-PCR method. The results showed that the identified herb complied with the label description in eight samples, but did not in three samples, in which one would be adulteration and two would be counterfeits. Conclusion Application of 70% methanol as a purification and precipitation reagent in classic CTAB method effectively overcome the negative influence of starch contained in Chinese herbal preparations to DNA extraction and can make the DNA molecular identification of Chinese herbal preparations successful.
[中图分类号]
[基金项目]
澳门科学技术发展基金(071/2011/A3)