喜树发根培养及培养基中次生代谢产物的研究

喜树发根培养及培养基中次生代谢产物的研究

文档编号:SP033 本文档字数:14476,页数:29

摘要
喜树毛状根培养不仅能生产喜树碱，而且可把二次代谢物如喜树碱等分泌至培养基中，利用现代生物技术与现代分离技术综合开发喜树和喜树碱，这是喜树资源的保护开发的新突破。本文档先对喜树发根进行培养，然后使用氯仿提取喜树培养基中喜树碱等有效成分，并用HPLC检测法检测其有效成分，检测发现喜树分泌在培养基中的次生代谢产物除了喜树碱、羟基喜树碱、白桦脂酸外，还含有其他有效成分。另外的氯仿提取法与树脂吸附法对加了标液的空白培养基进行提取的对比实验，证明了树脂富集培养基中微量的喜树碱的效果比氯仿提取的效果好，而且富集到的种类要比氯仿提取的多。这个实验为以后用树脂作为吸附材料，从喜树发根体系的培养基中富集喜树碱做了很好的铺垫。
关键词：喜树碱喜树发根培养基氯仿提取 HPLC检测

Research on Cultivating of the Camptotheca Acuminata Hairy Roots and the Secondary Metabolite Camptothecme in the Culture Medium
Abstract: The camptotheca acuminata hairy root raise not can only produce the camptotheca acuminata alkali, moreover may secretion and so on two metabolite like camptotheca acuminata alkali to the culture medium, the use modern biological technology and the modern isolation technique comprehensive development camptotheca acuminata and the camptotheca acuminata alkali, this is the camptotheca acuminata resources protection development new breakthrough. The present paper carries on the raise first to the camptotheca acuminata roots of the hair, then in the use chloroform extraction camptotheca acuminata culture medium the camptotheca acuminata alkali and so on effective component, and examines the law with HPLC to examine its effective component, the examination discovers the camptotheca acuminata secretion in the culture medium secondary metabolite besides the camptotheca acuminata alkali, the hydroxyl camptotheca acuminata alkali, the white birch fatty acid, but also includes other effective components. Other chloroform extraction process and the resin adsorption law to added the sign fluid blank culture medium to carry on the extraction the opposite experiment, had proven the resin concentrated in the culture medium the micro camptotheca acuminata alkali effect to be better than the chloroform extraction effect, moreover concentrated the type must compared to chloroform extraction many. This experiment will take the adsorption material for later with the resin, concentrated the camptotheca acuminata alkali from the camptotheca acuminata roots of the hair system's culture medium to make the very good upholstery.

Key word: camptotheca acuminata alkali Camptotheca acuminata roots of the hair culture medium Chloroform extraction HPLC examination

目录
1 绪论 1
1.1 前言 1
1.2 喜树生物学特性与药用价值 2
1.2.1 生物学特征 2
1.2.2 药用价值 2
1.3 扩大喜树碱药源途径的研究 3
1.3.1 组织培养及喜树细胞悬浮培养 3
1.3.2 利用毛状根培养技术生产喜树碱 4
1.3.2.1 发根农杆菌的特性 4
1.3.2.2 毛状根的转化方法、培养和鉴定 5
1.4 光照对喜树培养的影响 5
1.4.1 光照对毛状根生长及其次生代谢产物合成因素的影响 5
1.4.2 光照对喜树愈合组织生理及喜树碱合成的影响 6
1.4.3 光强对喜树幼苗叶片次生代谢产物喜树碱的影响 6
1.5 大孔吸附树脂吸附原理 6
1.6 喜树碱的富集与检测 7
1.7 本课题研究的内容 7
2. 喜树发根培养 7
2.1 喜树枝条不同组织及基本培养基的筛选 7
2.2 不同种类、浓度的植物生长调节剂对喜树茎段腋芽、不定根的诱导 9
2.3 发根农杆菌感染喜树茎的培养 10
3 喜树发根培养基中次生代谢产物喜树碱提取 11
3.1 试验材料与仪器 11
3.1.1 试验材料 11
3.1.2 试验仪器 11
3.1.3 色谱条件 11
3.2 试验方法 11
3.2.1 标液的配置 11
3.2.2 喜树培养基中喜树碱等有效成分富集试验 12
4 HPLC法检测喜树碱等喜树有效成分 12
4.1 标准曲线的测定 12
4.2 空白养基中喜树碱的抽滤氯仿萃取与树脂吸附的对比 15
4.3 培养基中富集喜树碱等有效成分的HPLC检测结果 15
4.3.1 培养基中培养产物喜树碱等有效成分的检测 15
4.3.2 喜树培养基经抽滤处理后滤渣与滤液中喜树碱含量的对比 16
4.3.3 喜树培养基中喜树碱等有效成分的检测结果 17
5 总结与讨论 20
5.1 结论 20
5.2 存在问题与展望 20
5.2.1 存在的问题 20
5.2.2 展望 21
参考文献 22
英文摘要 24
致谢 25

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