【作者】 张荣沭；

【导师】 赵敏；

【作者基本信息】 东北林业大学 ， 森林生物工程， 2008， 博士

【摘要】 本文改进并建立了青蒿素提取、分析方法;以此为基础评价了黑龙江省青蒿野生资源的利用价值;并分别于2006、2007和2008年从北纬23-30°间的云南、广西、四川、湖南等地,引进了若干优质种源,在北纬46°以上的黑龙江省不同地区栽培,筛选出适合本地生长、含量高的青蒿种源,建立了青蒿引种栽培技术;研究了影响青蒿素含量、药用生物量的主要因子,选择对青蒿素产量影响大的因子进行正交设计试验,确定了高产青蒿的施肥技术。为黑龙江引种青蒿,开展大规模栽培生产提供了理论依据。主要研究结果如下:1、改进建立了恒温振荡浸提法作为青蒿素的提取方法:精确称取青蒿粗粉1.000g,40℃干燥3 h,以20 mL石油醚（60～90℃）为溶剂,30℃恒温振荡提取24 h,频率120 r/min,连续提取4次,每次将前一次的提取液倾出加入10 mL提取溶剂,共提取4d,合并提取液,总量为50 mL。该方法应用设备简单、可操作性强;提取溶剂经济、毒性小、回收方便、可重复利用;提取物杂质量小、适合工业生产操作。2、改进建立了新的青蒿素柱前衍生HPLC-UV测定方法。按文献方法进行青蒿素测定,发现结果不稳定、操作繁琐。研究表明影响柱前衍生HPLC-UV法测定青蒿素含量结果的主要因素是溶解青蒿素标准品（或青蒿提取浸膏）的95%乙醇用量和碱、酸化过程的碱、酸用量。以此为基础改进建立了新的青蒿素柱前衍生HPLC-UV测定方法。3、评价了黑龙江省野生青蒿资源利用价值。对黑龙江省内不同地区野生青蒿中青蒿素含量进行测定表明,青蒿素含量均低于0.1%,远远低于青蒿素含量≥0.5%的药用标准,即黑龙江省野生青蒿资源无利用价值。4、建立了青蒿引种栽培技术。对比室内和野外育苗结果及育苗方法表明:黑龙江省引种青蒿,采用温室育苗,可延长青蒿的生长期45 d左右,集成板式纸筒育苗移栽方便、用土量小,成本低。不同地区引种青蒿相同时间收获,育苗和移栽时间越早青蒿素产量最高。引种的青蒿,营养生长可持续到9月初,生长期长近170 d,可获得巨大药用部位生物量;8月20日后陆续转入生殖生长,本地气候高温、光照强,此时可获得高含量的青蒿素。即提早育苗移栽,可延长其生长时间,在气温较高的月份积累最高的青蒿素含量并达到大的生物量,从而提高青蒿素产量。5、通过大田栽培和露天盆栽方法,研究了植物生长必需的多数营养因子、逆境胁迫、辐射青蒿种子、日光、昼夜温差、栽培密度、育苗和移栽时间对青蒿药用生物量和青蒿素含量的影响,结果如下:营养元素单因子对青蒿素产量的影响大小顺序为复合肥（NPK）＞K＞Ca＞Fe＞Mg＞B＞P＞Mn＞Cu＞N＞Mo＞Zn＞ck（对照）。最大可使青蒿素含量提高对照的49.3%、青蒿药用生物量提高对照的55.4%、青蒿素产量提高对照的111.8%。植物生长调节剂单因素对青蒿素产量影响大小顺序为:GA₃＞NAA＞氨基酸微肥＞B9＞KT。最大可使青蒿素含量提高对照的36.9%、青蒿药用生物量提高对照的93.3%、青蒿素产量提高对照的125.5%。栽培密度大小对青蒿素含量虽有影响但幅度不大,但种植密度大可提高青蒿素产量（源于生物量产量的贡献）。大田栽培施肥技术:氮肥720 kg/ha、磷肥1080 kg/ha、钾肥1080 kg/ha及钙肥360kg/ha,赤霉素、萘乙酸及氨基酸微肥喷施浓度分别为40 mg/L、5 mg/L及225倍液。青蒿素含量最高可达到:1.0484%,最低为:0.5598%。6、引种青蒿中青蒿素含量累积趋势与本地野生青蒿一致,但含量高低差异极大,说明青蒿素含量主要受遗传背景控制,其次受环境条件影响。青蒿素含量最高达到:1.0484%,最低为:0.5598%,远大于0.5%的药用标准,引种青蒿中青蒿素含量可达到药用标准,且药用部位生物量超过原产地。引种栽培的青蒿对本地的各种立地条件具有很强的适应性,说明引种青蒿栽培获得成功。因此黑龙江省可充分利用青蒿适应性强的特点,在荒地、废弃地、闲置地、石砾地,开展大规模栽培生产,是个很好的惠农惠林项目。更多还原

【Abstract】 In this dissertation,the modified extraction and analytical method of artemisinin is proposed,evaluating the value of wild Artemisia annua resource in Heilongjiang Province on the basis.Some high quality provenances of A.annua from Yunnan,Guangxi,Sichuan and Hunanmain in northern latitude 23～30⁰ were introduced in the different areas over northern latitude 46⁰ of Heilongjiang Province in 2006,2007and 2008,for selecting the one which adapted to the environment and climate of the cultivated area and has high artemisinin content. The technique of introduced A.annua cultivation is established.The main factors which affect A.annua’s artemisinin content and medicinal biomass yield are studied in detail and major of them are researched to establish fertilization technique of high A.annua yield by orthogonal design test.It provides theoretical basis for introducing A.annua and promoting a large-scale planting production in Heilongjiang Province,of which the concrete results are as below:1.The modified extraction method of constant-temperature oscillating digestion is proposed,of which the appropriate technical conditions are established as follows:drying the powder sample in a baker（40℃） to constant weight,weighing 1.000 g and puting it in a reactor,adding petroleum ether（60～90℃） 20 mL,oscillating digestion（120 r/min） at constant temperature of 30℃for 24 h.Do the same thing but using petroleum ether（60～90℃） 10mL to the residue,repeatedly 3 times.Put the extract of the four times together（50mL in total） as the artemisinin extract.This extraction,which performed with simple apparatus under easy operation,uses economical,low-toxicity,recyclable and easy-recovering solvent.It also provides extraction of a relatively high pureness,with which all the above makes it fit for mass production.2.The artemisinin content detecting method of the newly modified HPLC-UV with Postcolumn Derivation is proposed.Doing the test according to the references showed unstable results and very complex operation.According to our researches and experiments,the main factors that affect the results of HPLC-UV with postcolumn derivation are the amount of 95% ethanol,which is used as solvent for artemisinin standard sample（or epurated artemisinin）,and that of NaOH,HAc used during alkali-treating and acid-treating process,by which the newly modified method was proposed.3.Evaluated the developing value of wild A.annua resource in Heilongjiang Province. After detecting the artemisinin content of A.annua in different areas of Heilongjiang Province, we found that the artemisinin contents are lower than 0.1%,which is far lower than the medicinal criterion of artemisinin content≥0.5%.It means that the wild A.annua in Heilongjiang is useless. 4.The technique of introducing A.annua cultivation is established.Comparing with the methods and results of sowing seedling in fields and greenhouse,the later,can prolong the growth period about 45 days.What’s more,the integrated plate-type paper containers,which require very little soil and enable easy transplanting,is quite economical.If we harvest A. annua in different areas at the same time,the earlier we grow,the higher yield of artemisinin we have.The nutritious growth period can prolong to the beginning of September for introduced A.annua,which is nearly 170 days.Above all,great medicinal biomass can be obtained.After 20^th of August,the nutritious growth period of A.annua transfers to breeding growth period in succession,and in this period,high artemisinin content can be obtained because the local climate is at high temperature and strong illumination.It is that the growth period can be lengthened if growing seedlings and transplanting are done earlier.Therefore, not only high artemisinin content can be accumulated,but also great medicinal biomass can be obtained at the month of high temperature.Thus,artemisinin production is increased.5.Through field planting and outdoor pot experiments,we studied most of the essential nutritious factors for plants growth,adversity stress,irradiating the seeds of A.annua,sunshine, temperature difference between day and night,planting density,sowing seedling and transplanting time,which affect A.annua’s artemisinin content and medicinal biomass.The results are as below:Each factor of mineral nutrition has its own influence on the artemisinin production, generalizing the results,the sequence ranked:NPK > K > Ca > Fe > Mg > B > P > Mn > Cu > N > Mo > Zn > ck（control）.With proper fertilization,artemisinin content can be increased by 49.3%,medicinal biomass by 55.4%,artemisinin production by 111.8%as a maximum, compared with the control.Each factor of hormone analogue can influence artemisinin production differently.As for the effect of each factor,the sequence ranked:gibberellins（GA₃）> naphthlcetic acid （NAA）>animo acid micro fertilizer>B₉>6-furifuryl-aminopurine（KT）.When used properly, artemisinin content can be increased by 36.9%,medicinal biomass by 93.3%,artemisinin production by 125.5%as a maximum,compared with the control.Planting density also can influence artemisinin content but not greatly.However, relatively high planting density leads to high artimisinin production（mainly due to high medicinal biomass）.The fertilization technique of planting A.annua in field is as follow:N is 720 kg/ha,P is 1080 kg/ha,K is 1080 kg/ha,Ca is 360 kg/ha.Besides,animo acid micro fertilizer is diluted to 225 times,and GA₃,NAA is 40 mg/L and 5 mg/L,respectively.The highest artemisinin content is 1.0484%,and the lowest one is 0.5598%.6.The drifts of artemisinin content of introduced A.annua and local wild ones are the same,but their levels differ greatly.It means artemisinin content is mainly controlled by their genetic backgrounds and secondly controlled by the natural circumstance.The highest artemisinin content is 1.0484%,and the lowest one is 0.5598%of introduced A.annua,which are far higher than the medicinal criterion of artemisinin content≥0.5%.While,with artemisinin content reaching industrial standard and medicinal biomass exceeding that of their origin,introduced A.annua has showed great adaptation to the local natural conditions.It means introduced A.annua get great succeed.So considering A.annua’s advantage of great adaptation,starting mass planting production in wasteland,abandoned land,uncultivated land and wasteland with some debris will be a nice forestry and agriculture-benifiting project.更多还原