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题名	青蒿器官培养生产青蒿素过程调控及青蒿素提取研究
作者	赵兵
学位类别	博士
答辩日期	2000-10
授予单位	中国科学院研究生院
导师	欧阳藩
关键词	青蒿毛状根 青蒿芽 生长 青蒿素合成 稀土元素 活性氧 乙稀 玻璃化 超声提取
其他题名	The process regulation of artemisinin production by artemisia annua L. organ culture and extraction of artemisinin enhanced by ultrasonic wave
学位专业	化学工艺
中文摘要	应用稀土元素显著促进了青蒿器官（青蒿芽和毛状根）生长和青蒿素合成，大幅度提高了青蒿素产量，并对稀土元素的生物学作用机制，如对细胞膜渗透性的影响、植物激素作用、对活性氧的清除作用以及对青蒿芽玻璃化减轻作用等，进行了较系统的研究。摇瓶悬浮培养中活性氧、乙烯对青蒿器官生长和青蒿素合成的影响的研究结果，为反应器培养时通过活性氧、乙烯的调控进一步提高单位体积青蒿素产量奠定了基础。对青蒿芽玻璃化现象进行了系统研究，阐明了青蒿芽发生玻璃化的主要因素，找到了一种能有效抑制玻璃化的方法。此外，还进行了超声强化提取青蒿素研究，青蒿素提取时间大幅度缩短，提取率显著增加，据此开发的青蒿素循环超声破碎浸提工艺及装置成功地解决了超声提取的工程放大问题。在青蒿器官培养生产青蒿素和青蒿素提取中，以上研究结果均为首次获得，对大规模青蒿器官培养生产青蒿素及提高青蒿素提取技术水平均具有重要的参考价值。1.稀土元素对青蒿丛生芽生长及青蒿素合成的促进作用在青蒿芽固体培养时，应用0.1mmol/L左右浓度的镧、铈、钕及混合稀土Re显著促进了青蒿芽的生长和青蒿素合成，但是固体培养基中高浓度的稀土却导致青蒿芽分化生长停止，甚至死亡。在青蒿芽摇瓶液体悬浮培养时，应用适宜浓度的镧、铈、钕及混合稀土Re同样显著地促进青蒿芽的生长和青蒿素合成，但其适宜的浓度范围却降为0.002-0.04 mmol/L。在摇瓶液体悬浮培养中同样发现高浓度的稀土能导致青蒿芽分化生长停止，甚至枯黄死亡。在青蒿从生芽生长过程中，活性氧的产生速度在不同的生长时期不同，以前指数期、静止期及凋亡期较高，而以指数期较低。稀土元素对青蒿芽体内活性氧的清除作用是稀土元素促进青蒿芽生长和青蒿素合成的重要原因之一。稀土元素可以完全取代6-BA和部分取代NAA，它们具有的激素作用在促进青蒿芽分化的同时亦促进了青蒿素的合成，因为青蒿素的合成以芽的分化为先决条件。这是稀土元素促进青蒿芽生长和青蒿素合成的另一个重要原因。青蒿芽摇瓶悬浮培养时，稀土元素的加入在青蒿芽生长过程中改变了细胞膜的通透性，一方面增加了营养物的利用，另一方面减轻了青蒿芽的玻璃化程度。这也是稀土元素促进青蒿芽生长和青蒿素合成的重要原因1.8升反应器培养中加入0.04mmol/L La~(3+)使生物量提高15%，青蒿素平均含量增加10.8%,单位体积青蒿素产量提高24.4%;加入0.005mmol/L Ce~(3+)使生物量提高了14.3%,青蒿素总产量提高了17.5%;加入0.01 mmol/L钕使生物量提高了15.9%，单位体积生物量达到19.96g Wd/L左右，月单位体积生物量达到27.22 g Wd/L.M,但对青蒿素含量无明显影响，单位体积青蒿素产量达到199.40mg/L或271.9mg/L.M,比对照提高了16.03%;加入0.04mmol/L Re使生物量提高了16.8%,青蒿素平均含量提高3.61%;单位体积青蒿素产量比对照提高了17.78%。2.稀土元素对青蒿毛状根生长及青蒿素合成的促进作用青蒿毛状根摇瓶悬浮培养中，适宜浓度的镧、铈、钕及混合稀土Re均能显著促进青蒿毛状根的生长和青蒿素的合成。稀土元素的促进作用与其对毛状根细胞膜渗透性的影响有关，这种影响有利于营养物的吸收和促进毛状根的分化。1.8升反应器培养中加入0.1mmol/L La~(3+)能使生物量增加15.8%，青蒿素平均含量提高22.8%，单位体积青蒿素产量增加42.2%。加入0.05 mmol/L Ce~(3+)能显著促进生长，使生物量提高18.8%，青蒿素平均含量提高24.81%，单位体积青蒿素产量提高48.32%。加入0.03mmol/L钕生物量提高了12.5%,平均含量提高29.6%，单位体积青蒿素产量比对照提高了45.77%。加入0.02mmol/L Re使生物量提高了6.8%，青蒿素平均含量提高了19.93%,单位体积青蒿素产量比对照提高了28.1%。3.活性氧、乙烯对青蒿丛生芽生长及青蒿素合成的影响青蒿芽悬浮培养过程中，活性氧清除剂SOD和CAT加入有助于芽的生长和青蒿素合成，而且SOD较CAT效果更为显著。高浓度的外源乙烯显著抑制青蒿芽的生长，而适宜浓度的外源乙烯却显著促进芽的生长和青蒿素合成。青蒿芽体内的乙烯水平与其生长及青蒿素合成密切相关，这从乙烯作用抑制剂Ag~+和乙烯合成酶抑制剂Co~(2+)的实验得到了证实。4.活性氧、乙烯对青蒿毛状根生长及青蒿素合成的影响青蒿毛状根悬浮培养过程中，外源活性氧Na_2S_2O_4和H_2O_2对毛状根的生长有明显的抑制作用，高浓度的外源活性氧甚至导致毛状根死亡。活性氧清除剂SOD和CAT加入有助于毛状根的生长和青蒿素合成，不同于青蒿芽的SOD和CAT效果相似。高浓度的外源乙烯显著抑制青蒿毛状根的生长，而适宜的较低浓度的外源乙烯却显著促进芽的生长和青蒿素合成。青蒿毛状根体内的乙烯水平与其生长及青蒿素合成密切相关，这从乙烯作用抑制剂Ag~+和乙烯合成酶抑制剂Co~(2+)的实验得到了证实。5.减轻青蒿丛生芽生长过程中玻璃化程度研究青蒿芽悬浮培养过程中芽含水量及细胞膜渗透性的变化研究表明芽细胞膜渗透性的降低是造成青蒿芽玻璃化的重要原因。利用山梨醇及甘露醇作为渗透胁迫剂，对降低芽的含水量及减轻芽的玻璃化程度无明显作用。多胺（精胺、亚精胺及腐胺）虽然显著降低了芽的含水量，但仅能部分减轻玻璃化程度。镧、铈、钕及混合稀土元素Re取代植物激素NAA和6-BA时，对芽的含水量影响较小；仅取代NAA时，对芽的含水量影响很小；仅取代6-BA时，对芽含水量的影响显著。培养基中6-BA是造成芽含水量过高，产生玻璃化的重要因素。稀土元素可以取代6-BA，降低青蒿芽的含水量，从而大大减轻芽的玻璃化程度，这是稀土元素能促进青蒿芽生长和青蒿素合成的又一个重要原因。首次发现了海带多糖能有效抑制青蒿芽玻璃化现象。海带多糖的加入虽然使芽含水量的降低没有稀土元素等明显，但使芽的形态能恢复到基本与固体培养相同。对芽含水量动力学研究表明加入海带多糖在培养20天以后，含水量急剧下降，而对照样品却急剧上升，这就是海带多糖能有效抑制玻璃化的原因之一，但海带多糖在青蒿芽体内的作用机制有待进一步研究。6.超声强化提取青蒿素筛选出石油醚为青蒿素提取较适宜的溶剂，对石油醚搅拌提取青蒿素的工艺条件进行了较系统研究，找到了较适宜的提取条件。超声波用于强化石油醚提取青蒿素不仅可以将提取时间缩短到30分钟，减少提取产物中杂质量，而且还可以提高青蒿素提取率、降低溶剂消耗。本实验为开发大规模超声提取工艺及设备，改进现有青蒿提取技术水平，提高回收率，增加青蒿素生产经济效益奠定了基础。
英文摘要	Artemisinin is a sesquiterpene lactone with an endoperoxide moiety and the formula as C_(15)H_(22)O_5. As an anti-malaria drug, artemisinin is more effective in treating cerebral malaria and chloroquine-resistant malaria than traditional drugs, such as sulfadoxine. At present, only commercial source of this drug is extracted from field-grown leaves and flowering tops of Artemisia annua L., which are subject to seasonal and somatic variation. Although the complete organic synthesis has been developed, it is not yet economically feasible because of its complexity, high toxicity and low yield. For above reasons, artemisinin production by plant organ culture has been considered an attractive alternative. To improve the artemisinin production yield in culture of Artemisia annua L. organ (hairy root and shoot), it is necessary to solve the problems, such as low biomass and artemisinin content. The growth and artemisinin accumulation in Artemisia annua L. shot culture in solid and liquid was enhanced by addition of proper concentration of rare earth elements (La~(3+), Ce~(3+), Nd~(3+) and Re). For the first time, however, high concentration rare earth element in culture media restrained shoot growth and artimisinin biosynthesis. The experimental results indicated that rare earth elements affected shoot growth by four plant physiological functions: they were the action of plant hormone, the scavenger of active oxygen in shoot, the effect on cell membrane osmosis and the inhibitor of shoot vitrification. The experimental results in 1.8 liter bioreactor also indicated that the addition of rare earth elements increased biomass from 14.3% to 16.8%, content of artemisinin from 3.61% to 12%, and total production of artemisinin from 16.03% to 24.4%. In the suspension cultures of hair roots, the addition of proper concentration of rare earth elements also improved hairy root growth and artemisinin production. Rare earth elements changed cell membrane osmosis which was beneficial to differentiation of hair root and led to the increase of biomass and artemisinin content. The results obtained in 1.8 liter bioreactor culture showed that the increment of 6.8%-18.8% biomass, 19.93%-29.57% average content of artemisinin and 28.1%-48.32% total production of artemisinin obtained due to the addition of rare earth elements. The regulation of active oxygen and ethylene in bioreactor culture process played important role increasing the artemisinin production yield. The experimental results showed that the formation rate of active oxygen was related to the growth of Artemisia annua L. shoot. Exogenous active oxygen, such as Na_2S_2O_4 and H_2O_2, dismutase (SOD) and catalase (CAT), can improve shoot growth and enhance the artemisinin production. Ethylene also affected on the growth and artemisinin biosynthesis of Artemisia annua L. shoot. The high concentration of exogenous ethylene in culture media can inhibit the shoot growth and artemisinin biosynthesis. However, the proper concentration can increase both the biomass and accumulation of artemisinin. The formation rate of active oxygen was also related to the growth of Artemisia annua L. hair root. As scavengers of activated oxygen, superoxide dismutase (SOD) and catalase (CAT) can improve hair root growth and enhance the artemisinin production. To improve the artemisinin production in culture of Artemisia annua L. hair root, a proper concentration of exogenous ethylene was used in liquid suspension culture process. Anti-vitrification in shoot culture of Artemisia annua L. was investigated. Polyamines (spermine, spermidine and putreacine) and rare earth elements can control the shoot vitrification at a certain extent by means of decreasing the water content of shoot. Nevertheless, the shoot morphology can not be returned to the original shape in solid culture process. The addition of kelp polysaccharide in liquid culture media can control shoot vitrification effectively, although the water content of shoot decreased slightly. A new ultrasonic extraction process of artemisinin from Artemisia annua L. was developed based on stirring extraction process. The major parameters, such as ultrasonic processing time, the strength of ultrasonic wave, and so on, were investigated. Comparing with traditional extraction processes, the ultrasonic process had shorter extraction time, less impurity in the product and high recovery of artemisinin.
语种	中文
公开日期	2013-09-26
页码	155
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/1925]
专题	过程工程研究所_研究所（批量导入）
推荐引用方式 GB/T 7714	赵兵. 青蒿器官培养生产青蒿素过程调控及青蒿素提取研究[D]. 中国科学院研究生院. 2000.

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