【作者】 李春斌；

【导师】 王关林；

【作者基本信息】 大连理工大学 ， 生物化学工程， 2002， 博士

【摘要】 银杏（Ginkgo biloba L．）又称白果树，是地球上生长着的最古老的高等植物之一。银杏叶中主要含有黄酮类和萜内酯类两大类有效成分，它们具有扩张血管、增加血流量、改善血液循环动力障碍、降低脑血管阻力、抑制血栓形成及清除人体自由基等多种作用。尤其银杏内酯B是目前发现的最有效的特异的血小板活化因子（PAF）拮抗剂，已成为世界多国医药专家的研究热点。 目前人们主要从银杏叶中提取银杏黄酮和萜内酯，但由于银杏黄酮和银杏内酯的含量很低，尤其银杏叶中最主要的有效成分银杏内酯B主要存在于根中，在银杏叶中含量极低（＜0.1％），且是在生长状态的叶中含量最高，采叶影响树的生长；又因银杏叶中主要的有效成分受多种因素的影响又有很大差别，银杏叶革质化、蜡质化程度高，使得国内银杏叶提取物的质量很难达到国际标准。同时，银杏的大面积栽植占据了大量珍贵的耕地面积。为此，本研究利用植物细胞工程技术建立了银杏幼叶愈伤组织无性系和细胞悬浮培养系，并对银杏细胞悬浮培养系进行了多代筛选、驯化，对其培养条件的生理、生化调控进行了大量研究，建立起了生长旺盛且具有高黄酮和内酯含量的优良的银杏细胞悬浮培养系。 实验中发现基因型、外植体类型、树龄和季节等诸多因素对银杏愈伤组织培养物中的黄酮和萜内酯的含量都有较大影响。应选择树体自身黄酮和内酯含量高的树种，在4月中下旬银杏叶刚萌发时取3～5年生的幼树的嫩叶为外植体进行愈伤组织的诱导。通过对培养基种类、多种不同浓度的激素组合、紫外线照射、培养过程的温度和光照等条件的研究，采用分光光度法和HPLC法相结合的方法筛选出了银杏黄酮和萜内酯含量都较高的银杏幼叶愈伤组织无性系。 由于在固体培养基上进行培养的愈伤组织生长速率较慢，不适于培养物中目标次生代谢产物的工业化生产，实验中又进一步以固体培养基上的愈伤组织无性系为材料建立了银杏细胞悬浮培养系。对银杏细胞悬浮培养系的生理、生化调控研究结果表明：以B5培养基为基本培养基，附加NAA0.5～1.0mg／L、BA0.2～0.5mg／L、30g／L蔗糖和15g／L葡萄糖，培养液pH值为5.8～6.2，培养基中Cu²⁺提高十倍，Fe²⁺浓度增加0.5倍，氨态氮加倍，硝态氮减半，以30～40FWg／L的接种量接种于装有50ml培养液的150ml三角瓶中，置于25±1℃、3000Lux连续光照条件下进行悬浮培养， 大连理工大学博士学位论文在培养的第15天加人0．ito．2叭的苯丙氨酸，在培养的第20天收获，最有利于银杏细胞悬浮培养系中黄酮和格内酯的生产。细胞悬浮培养物中黄酮含量可达3．35％，前内酯含量可达0．0758％。 本实验还对银杏叶及银杏组织细胞培养物中有效成分的提取、纯化和检测方法进行了研究，发现将银杏叶粉碎成 40～60目，在料液比为 1：15的情况下以 70％乙醇为提取溶剂，先湖预处理205，然后用超声波强牌取stnin，重复操作2次，银杏叶中有效成分提取率可达98．5％。GBE粗提液经液一液法去杂后，将提取液中黄酮浓度稀释为1．ul．Zmtw左右，再以10倍柱体积的提取液控制吸附流速为 4倍树脂柱体规用 HPD 00大孔吸附树脂吸附，吸附完全后依次用去高于水、20％乙测涤，再用流速为2倍树脂柱体肌的90％乙醇解吸的方法获得的GBE精提物经分光光度法和 HPLC法检测黄酮着为 26．l％，蔬内酯为 6．2％，达到国际标准。 对银杏黄酮提取液采用微波强化水解、HP＊C检测：对银杏内酯采用10％甲醇回流提取、单柱层析纯化、y－pLC分析和二极管阵列检测器检测，以上方法减少了样品的预纯化时间，且操作简便，易于掌握，可在短时间内检测很多样品。这将为以银杏叶提取物为主要原料的厂家对其产品中有效成分含量的测定提供极大的方便。更多还原

【Abstract】 Ginkgo biloba L. is also called white fruitage tree, and it is one of the higher plants, which have the longest life. The leaves from Ginkgo biloba L. mainly included some effective constituents, such as flavonoids and ginkgolides, which can aggrandize the blood vessel, increase the outflow of blood, improve the kinetic of blood-circulating, decrease the resisting force of cerebrovascular, suppress thrombisis and clean free radical of human body. Ginkgolide B especially is the most effective agonist of plaque activation factor (PAF) and now become a hot research issue in the medicine field of the world.Now, Flavonoids and ginkgolides were mainly extracted from the leaves of Ginkgo biloba., but the leaves often contain high content of corious and cereous, and the effective constituent is very different under the various conditions. In addition, the constituent of ginkgolides is very low in the leaves, and thus the quality of extraction is difficult to reach the international standards. The large area plantation of Ginkgo biloba occupied the rare cropland. In order to solve the problem above, in this study, the callus and cell suspension culture clone were constructed using plant cell engineering technology. After several generation selection and domestication, the cell suspension clone, which can vigorous grow and contained higher content of flavonoides and ginkgolides, were constructed.The results showed the contents of flavone and ginkgolides in the callus culture substances were affected by the factors, such as genotype, expalnt category, season and so on. Thus, the trees with higher content of flavone and ginkgolides should be selected at first time.After a ten days in April, the germination leaves, which grow on 3-5 years old tree, was selected as explant inducting callus. After research on the kinds of culture media .various content of hermone, ultraviolet irridation, temperature and intensity of light in the culture, the callus clone with higher content of flavonoids and ginkgolides of Ginkgo biloba spiraster screened using photometer and HPLC combined.As the growth rate of callus is slow in the solid medium in the process of culture, callus is not fit for industrialization of secondary metabolite. The cell suspension culture line was established using the callus in the solid medium. The results of physiology and biochemical modulation on cell suspension culture line show that the primary medium was B5, pH value of culture was 5.8-6.2, and then NAA 0.5-1 .Omg/L. 6-BA 0.2~0.5mg/L, sucrose 30g/L and 15g/L glucose were added. In the experiment, thecontent of Cu + was increased ten times; the content of Fe + was increased half of one time; the content of ammoniacal nitrogen was increased one time and the content of nitrate nitrogen was decreased half of one time. The inoculation weight was 30-40 FW/L, and inoculate the 150ml flask which contained 50ml culture liquid. At 25? 癈, SOOOlux continuous light treatment, conducted suspension cell culture, at Hie 15th day, add 0.1~0.2g/L phenylalanine, after 20 days, the product will be harvest, which is the optimium condition of producing flavone and ginkgolides in the suspension cell culture solution. In the production, the content of flavonoides can reach 3.35%, the content of ginkgolides can reach 0.0758%.In this experiment, active constituents in the leaves, tissue and cell culture substration of Ginkgo biloba were extracted, purified and detected. The leaves of Ginkgo biloba were pulverized in 40-60 orders, and were extracted in 70% ethanol solution at the rate of material and solution 1:15, pretreated 20s by microwave, and extracted 5min by ultrasonic, and repeated 2 times above. The extraction rate of active constituent can reach 98.5%. After the extraction by liquid-liquid method diluted the concentration of flavone to 1.0~1.2mg/ml and using 10-times of volume colume at the flow velocity of 4 times of colume volume per hour absorbed by HPD 100, and then washed by distilled water and 20% ethanol, and using 3-times of volume colume at the f更多还原