中草药中遗传相关物质的分析研究
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摘要
中草药中高质量DNA的提取和DNA片段的分离分析研究是利用DNA分子标记技术来鉴别中药材的两个基础研究。本文主要探索了中草药中高质量DNA的快速提取方法和毛细管电泳在DNA片段分离中的应用,为以后的毛细管电泳应用于中草药中遗传相关物质的研究提供实验依据。另外,本文还建立了新的胶束电动毛细管色谱法,为快速、高效地检测药物中的生物碱基含量提供了一种新方法。
本文主要做了以下工作:
1.首先探讨出了一种简便可行的何首乌高质量DNA的提取方法,再将该方法分别用于车前草、红车轴草、黄芩、博落回和紫锥菊DNA的提取,然后用大孔吸附树脂对不纯DNA进行了纯化分离,目的是探索出一种较广泛适用的中药材DNA分离纯化方法。结果表明所探讨的方法可用于何首乌高质量DNA的提取,大孔吸附树脂对不纯DNA有一定的纯化作用。
2.通过优化DNA提取各步骤,获得了一种快速的赤芍干叶片完整、高质量DNA的提取方法,并成功应用于四个产地赤芍DNA的PCR分析比较。
3.以羟丙基甲基纤维素和聚乙烯吡咯烷酮作为筛分介质,采用无胶筛分毛细管电泳法分离了λDNA HindⅢ中的7个片段和λDNA/EcoRⅠ+HindⅢ中的12个片段,结果较好,并成功应用于药用植物何首乌DNA样品的分析。
4.以35 mmol·L~(-1)SDS-10 mmol·L~(-1)硼砂(pH9.0)作缓冲溶液,采用胶束电动毛细管色谱法,8 min内分离和测定了腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶、茶碱和咖啡因6种生物碱基,并且成功应用于去痛片、注射用更昔洛韦、心肝宝胶囊3种药物和茶叶中生物碱基含量的测定。
Extraction of high-quality DNA from Chinese herbal medicines and analysis and separation of DNA fragment are two basic aspects in Chinese herbal medicines identification by DNA molecular marker technology. In this paper, the methods of rapid extracting high-quality DNA from medicinal plants and the applications of capillary electrophoresis in the separation of DNA fragment were studied, which supplied some reliable experimental references for the subsequent analysis of genetic related substances in Chinese herbal medicine. In addition, a new method of micellar electrokinetic capillary chromatography for the rapid and efficient detection of biological bases in drugs was also established.
This paper is mainly composed of the following parts:
1. A simple and feasible method of extracting high-quality DNA from Radix Polygni Multiflori was developed, which was used in extraction of DNA from Plantago asiatica L., Trifolium pratense L., Scutellaria baicalensis, Macleaya cordata, and Echinacea. Afterwards, the extracted crude DNA was purified by macroporous resin so as to explore a more extensive and applicable method for isolation and purification of DNA from Chinese herbal medicines. The results showed that the method is suitable for extracting high-quality DNA from Radix Polygni Multiflori, and macroporous resin is capable of purifying the impure DNA.
2. A fast method of extracting integrity and high-quality DNA from dry leaves of Radix Paeoniae Rubra was obtained by optimizing the process of DNA extraction, which was applied successfully in PCR analyzing comparison of four Radix Paeoniae Rubra with various origin.
3. Seven DNA fragments ofλDNA HindIII and twelve DNA fragments ofλDNA/EcoR I + HindIII were separated by none-gel capillary electrophoresis with the use of hydroxypropyl methyl cellulose and polyvinyl pyrrolidone as the sieving matrix. The same method was applied in analyzing DNA samples of Radix Polygni Multiflori, and the results were satisfactory.
4. Six biological bases, including adenine, guanine, cytosine, thymine, theophylline, and caffeine, were separated in 8 minutes by using micellar electrokinetic capillary chromatography in a buffer solution of 35 mmol·L~(-1) SDS-10 mmol·L~(-1) sodium tetraborate (adjusted to pH9.0). The same method was also used successfully in determination of biological bases in Qutong tablets, ganciclovir for injection, Xinganbao capsules, and tea.
本文主要做了以下工作:
1.首先探讨出了一种简便可行的何首乌高质量DNA的提取方法,再将该方法分别用于车前草、红车轴草、黄芩、博落回和紫锥菊DNA的提取,然后用大孔吸附树脂对不纯DNA进行了纯化分离,目的是探索出一种较广泛适用的中药材DNA分离纯化方法。结果表明所探讨的方法可用于何首乌高质量DNA的提取,大孔吸附树脂对不纯DNA有一定的纯化作用。
2.通过优化DNA提取各步骤,获得了一种快速的赤芍干叶片完整、高质量DNA的提取方法,并成功应用于四个产地赤芍DNA的PCR分析比较。
3.以羟丙基甲基纤维素和聚乙烯吡咯烷酮作为筛分介质,采用无胶筛分毛细管电泳法分离了λDNA HindⅢ中的7个片段和λDNA/EcoRⅠ+HindⅢ中的12个片段,结果较好,并成功应用于药用植物何首乌DNA样品的分析。
4.以35 mmol·L~(-1)SDS-10 mmol·L~(-1)硼砂(pH9.0)作缓冲溶液,采用胶束电动毛细管色谱法,8 min内分离和测定了腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶、茶碱和咖啡因6种生物碱基,并且成功应用于去痛片、注射用更昔洛韦、心肝宝胶囊3种药物和茶叶中生物碱基含量的测定。
Extraction of high-quality DNA from Chinese herbal medicines and analysis and separation of DNA fragment are two basic aspects in Chinese herbal medicines identification by DNA molecular marker technology. In this paper, the methods of rapid extracting high-quality DNA from medicinal plants and the applications of capillary electrophoresis in the separation of DNA fragment were studied, which supplied some reliable experimental references for the subsequent analysis of genetic related substances in Chinese herbal medicine. In addition, a new method of micellar electrokinetic capillary chromatography for the rapid and efficient detection of biological bases in drugs was also established.
This paper is mainly composed of the following parts:
1. A simple and feasible method of extracting high-quality DNA from Radix Polygni Multiflori was developed, which was used in extraction of DNA from Plantago asiatica L., Trifolium pratense L., Scutellaria baicalensis, Macleaya cordata, and Echinacea. Afterwards, the extracted crude DNA was purified by macroporous resin so as to explore a more extensive and applicable method for isolation and purification of DNA from Chinese herbal medicines. The results showed that the method is suitable for extracting high-quality DNA from Radix Polygni Multiflori, and macroporous resin is capable of purifying the impure DNA.
2. A fast method of extracting integrity and high-quality DNA from dry leaves of Radix Paeoniae Rubra was obtained by optimizing the process of DNA extraction, which was applied successfully in PCR analyzing comparison of four Radix Paeoniae Rubra with various origin.
3. Seven DNA fragments ofλDNA HindIII and twelve DNA fragments ofλDNA/EcoR I + HindIII were separated by none-gel capillary electrophoresis with the use of hydroxypropyl methyl cellulose and polyvinyl pyrrolidone as the sieving matrix. The same method was applied in analyzing DNA samples of Radix Polygni Multiflori, and the results were satisfactory.
4. Six biological bases, including adenine, guanine, cytosine, thymine, theophylline, and caffeine, were separated in 8 minutes by using micellar electrokinetic capillary chromatography in a buffer solution of 35 mmol·L~(-1) SDS-10 mmol·L~(-1) sodium tetraborate (adjusted to pH9.0). The same method was also used successfully in determination of biological bases in Qutong tablets, ganciclovir for injection, Xinganbao capsules, and tea.
引文
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