【作者】 张永强；

【导师】 赵志模； 丁伟；

【作者基本信息】 西南大学 ， 农药学， 2008， 博士

【摘要】 植物在生长发育过程中,其光合作用和呼吸作用都是极为复杂的生理生化反应,会产生大量的代谢产物。这些产物具有的某种生理活性或用途,一直在被人们加以利用。植物成分及其代谢产物是天然药物的重要组成部分。人类在长期与疾病作斗争的过程中,以身试药,日积月累,对天然药物主要是植物药的应用积累了丰富的经验。中草药在人们控制疾病的过程中发挥了十分重要的作用,而且这些中草药大多数对于控制害虫也有一定的效果。黄花蒿（Artemisia annua L.）是一年生草本植物,为菊科蒿属植物,是中药青蒿的主要药源植物。株高约40-150cm,全生育期210d左右。黄花蒿在我国各地都有分布,生态适应性广,常生于山坡、林地、荒地。药农常于秋季花盛开时割取地上部分,除去老茎,晾干即可药用。黄花蒿药用价值很高,其主要活性成分是青蒿素,主治疟疾、结核病潮热,还能治中暑、皮肤瘙痒、荨麻疹、脂溢性皮炎等病症。青蒿素的衍生物可生产很多系列药品。由于重庆独特的自然地理因素适合黄花蒿的生长,重庆是全球黄花蒿种植的最大基地,有“黄花蒿故乡”的美誉,开发黄花蒿的优势得天独厚。螨类,特别是植食性害螨,是严重为害我国农业生产的一类重要生物。它具有个体小、繁殖快、种群密度高、为害重等特点,可为害150余种作物。据不完全统计,我国每年用于柑橘、苹果、梨、桃、棉花、蔬菜、小麦、茶8类主要作物的杀螨剂防治费用就达90多亿元,而且随着螨类对化学药剂抗药性的逐年增加,防治费用也逐年加大。面对化学农药所产生的种种问题和杀虫杀螨剂常规研发途径的日益艰难,从生物活性天然产物中开发新的药剂已经成为国内外农药研究开发的热点问题之一。本研究得到了国家自然科学基金（30671392）和重庆市科技攻关项目（2001-6599）的资助。本文主要研究了生长期在4月、5月、6月、7月和9月的黄花蒿根、茎、叶等不同部位的多种溶剂提取物的提取率;各种提取物对朱砂叶螨,柑橘全爪螨和酢浆草岩螨的生物活性;7月份黄花蒿提取物对豇豆种子胚根和胚轴的生长抑制作用;追踪分离到黄花蒿杀螨活性物质东莨菪内酯;研究了东莨菪内酯对朱砂叶螨SOD,CAT,POD,GST,CarE,AChE,MAO,Ca²⁺-ATP等酶系比活力的影响。通过近3年的研究,获得了如下结果:1黄花蒿有机物质的提取率利用一系列溶剂对4月份采集的黄花蒿植株的根、茎、叶分别进行常规室温浸提、恒温浸提、室温搅拌提取和微波辅助萃取。4种提取方法对提取率的影响差异不大,从经济角度综合比较,采用室温搅拌提取较为适宜。对4、5、6、7、9月份采集的黄花蒿根、茎、叶分别用不同溶剂进行平行提取和顺序提取,从提取率来看,平行提取的提取率普遍高于顺序提取;无论是平行或是顺序提取,水的提取率都是最高的（根茎叶提取率均高于其他溶剂的提取率）,其次是丙酮,再次是乙醇。从不同的植株部位看,叶的提取率相对较高,其次是根,而茎最低。2植物源杀螨剂的生物活性测定方法的选择与评价生物测定方法对农药的研究与开发具有重要意义。基于此本研究首先从剂量大小、测定效果、测定效率和成本等方面对玻片毛细管法、玻片浸渍法、叶片浸渍法等三种方法进行了比较。结果表明玻片毛细管法测定的结果与FAO推荐使用的玻片浸渍法无明显差异,且其用药量大幅度下降,效率更高,适合于植物源杀螨剂的生物测定。3黄花蒿提取物的杀螨活性3.1对朱砂叶螨的生物活性为了明确黄花蒿杀螨活性物质的最佳提取工艺,选择合适的溶剂和最佳的植株部位;明确黄花蒿杀螨活性物质的动态分布状态,从而为综合开发利用黄花蒿提供科学依据。将分别采集于4、5、6、7和9月5个月份的黄花蒿植株,分成根、茎、叶3个部分,采用石油醚（30～60℃）、石油醚（60～90℃）、乙醇、丙酮和水溶剂的平行和顺序提取方法,共计获得135种提取物,用玻片毛细管法测定其对朱砂叶螨的生物活性。在杀螨活性方面,黄花蒿的杀螨活性随植株的生长呈增加的趋势,总体表现为7月份＞6月份＞5月份＞4月份,但9月份的杀螨活性与7月份相比有所下降。5个月份的黄花蒿叶丙酮平行提取物的活性都比相同月份的其他提取物强,药剂浓度为5mg·mL^-1时,处理朱砂叶螨48h的校正死亡率介于74%～100%之间。黄花蒿不同月份叶的丙酮平行提取物对朱砂叶螨的LC₅₀随植物材料的采集时间不同呈规律性变化,4月份、5月份、6月份、7月份和9月份黄花蒿叶的丙酮平行提取物对朱砂叶螨处理48h,LC₅₀分别为1.3817,0.9443,0.8376,0.4341和0.5986mg·mL^-1。3.2对柑橘全爪螨的生物活性对采自于6月份的黄花蒿根茎叶不同溶剂提取物对柑橘全爪螨的生物活性测定结果表明:在同等条件下,叶提取物中除石油醚Ⅱ顺序提取物和水顺序提取物外,其他提取物的生物活性均比根和茎的提取物活性有明显的提高。其中叶丙酮平行提取物对柑橘全爪螨在5mg·mL^-1处理48h后生物活性最高,校正死亡率为88.84%,而石油醚Ⅱ平行提取物在相同条件下对柑橘全爪螨的校正死亡率亦达84.27%。7月份黄花蒿叶的不同溶剂提取物对柑橘全爪螨的生物活性亦明显高于根和茎的活性。其中叶的丙酮平行提取物活性最高,5mg·mL^-1处理48h后,对柑橘全爪螨的校正死亡率达100%。黄花蒿7月份叶丙酮平行提取物对柑橘全爪螨的生物活性明显高于6月份,对柑橘全爪螨处理48h后,7月份的LC₅₀(0.4222mg·mL^-1)仅为6月份(0.9489mg·mL^-1)的44%。3.3对酢浆草岩螨的生物活性黄花蒿6月份根、茎、叶不同溶剂的提取物对酢浆草岩螨的活性普遍不高。采集于7月份的黄花蒿植株的不同部位,采用极性不同的几种溶剂,用平行和顺序提取的方法获得的提取物对酢浆草岩螨表现出优越于6月份的生物活性。其中7月份叶的丙酮平行提取物生物活性最高,处理48h,对酢浆草岩螨的校正死亡率为95.00%。7月份黄花蒿叶丙酮平行提取物对酢浆草岩螨处理48h,其LC₅₀是0.4715mg·mL^-1,而6月份的为0.9083mg·mL^-1。4 7月份黄花蒿提取物对豇豆种子的植物毒性前面已经证实了黄花蒿7月份植株总体上对朱砂叶螨、柑橘全爪螨和酢浆草岩螨都具有较强的杀螨活性,为了考察提取物的植物毒性作用,研究了7月份黄花蒿植株根、茎、叶不同溶剂的平行和顺序提取物对叶螨类主要寄主植物之—豇豆的种子发芽率,种子胚根、胚轴的生长抑制情况。结果表明:7月份黄花蒿根、茎、叶不同溶剂的平行和顺序提取物对豇豆种子发芽的影响存在一定的差异。其中茎的乙醇顺序提取物对豇豆种子发芽率的影响最大,表现出明显抑制作用,发芽率仅为23.81%,此外叶的丙酮顺序提取物对豇豆种子发芽率的影响也较大,发芽率为40.48%,而叶的丙酮平行提取物对豇豆种子发芽率影响甚微。7月份黄花蒿根、茎、叶的不同溶剂提取物对豇豆种子胚根、胚轴的生长抑制作用较弱;有些还表现出刺激生长作用。5黄花蒿杀螨活性物质追踪分离采用生物活性追踪法,测定了黄花蒿7月份叶的丙酮平行提取物柱层析所得不同组分对朱砂叶螨的生物活性。在最终分离出的20种组分中,组分17的杀螨活性最高,组分18和8次之。组分17,18和8的LC₅₀（48h）分别为0.1675,0.4368和0.3753mg·mL^-1,其中组分17的活性相对于7月叶丙酮提取物而言提高大约2.6倍。也测定了黄花蒿7月份叶的丙酮平行提取物柱层析所得的不同组分对柑橘全爪螨的杀螨活性,其中组分、17的杀螨活性最高,处理48h后,校正死亡率达到96.65%,与其他组分的杀螨活性存在显著差异。同样测定了柱层析最终分离出的20种组分对酢浆草岩螨的生物活性,其中组分19的杀螨活性最高,2.5mg·mL^-1浓度下,处理48h后的校正死亡率达到69.28%。而综合有效含量和生物活性两个因素考虑,组分17最具研究价值。因此对组分17进行了进一步的分离纯化,选择朱砂叶螨做为进一步活性追踪的供试螨类。组分17中含有三个主要的成分,其中17-3占的比例最高为77.64%,17-1次之,为16.21%,17-2所占比例最小,仅为5.31%。三种成分进行TLC分析,经碘显色,可见的斑点数均为一个,且无拖尾。三种成分对朱砂叶螨的生物活性测定结果表明,17-3具有较强的杀螨活性,处理48h,对朱砂叶螨的LC₅₀为0.1014mg·mL^-1,与整个第17组分对朱砂叶螨的LC₅₀为0.1675mg·mL^-1相比,毒力有所提高。对17-3进行重结晶实验,在一定的温度下分别用无水甲醇、丙酮、三氯甲烷、乙酸乙酯、正丁烷溶解,重结晶,放冰箱冷却静置得到晶体,从中选出最佳的温度和溶剂。结果表明,根据结晶晶形以及溶剂用量判断,以乙酸乙酯为溶剂在55℃下结晶的效果最好。随后用HPLC-MS、¹H-NMR、IR等手段,确定纯化后的17-3为东莨菪内酯scopoletin,分子量:192.17,分子式:C₁₀H₈O₄。生物测定结果表明,对17-3进行重结晶纯化所得的东莨菪内酯对朱砂叶螨的生物活性较17-3有所增强。6东莨菪内酯对朱砂叶螨的致死机理朱砂叶螨受东莨菪内酯处理后,其超氧化物歧化酶（SOD）活性随着处理时间的延长表现出先升高再下降的趋势,但整体上表现为受药剂处理的酶活性低于对照。所有处理测得的过氧化氢酶（CAT）活性均有所提高,表明朱砂叶螨在受到东莨菪内酯处理后,CAT在一定程度上被激活。POD活性均较对照有明显下降,且随药剂浓度的变化而略有差异。总体表现为朱砂叶螨受到东莨菪内酯处理后,其体内的POD活性受到了明显的抑制。朱砂叶螨在受到东莨菪内酯处理后谷胱甘肽-S-转移酶（GST）活力有所提高。随着处理时间的延长,处理组的酶活力都高于对照组,但整体上表现出下降的趋势,尤其是在处理48h后,酶活力均降至最低水平。东莨菪内酯激活了朱砂叶螨羧酸酯酶（CarE）的活性,且随着药剂浓度的增加酶活性被激活的程度也随之增大。而乙酰胆碱酯酶（AChE）活性表现出先上升再下降的趋势。Ca²⁺-ATP酶的活力有不同程度的下降,表明东莨菪内酯对Ca²⁺-ATP酶有一定的抑制作用。就单胺氧化酶（MAO）的活性而言,处理组与对照组的MAO的活力变化趋势接近,但均低于对照组,因此,MAO受到了一定程度的抑制。更多还原

【Abstract】 In the course of plant development and growth,a great number of metabolites were produced in the process of photosynthesis and respiration,and some of these products have physical activity or other use,have been exploited since ancient times.Plant components are important parts of natural drugs.Since ancient times,and during the fighting against the disease human body to test drugs,the natural medicinal plants mainly plant drugs application has accumulated a wealth of experience.In China,botanicals also known as the Chinese herbal medicines,and together with the traditional Chinese medicine constitute the cultural treasures of the Chinese nation,but also a valuable heritage of mankind.Artemisia annua L.is an annual herbaceous plant,belongs to Compositae Artemisia,is the main source of qinghao traditional Chinese medicine.The whole height was 40-150cm,specially over 200cm,and the growth period about 210d.Artemisia annua are distributed across in China, widely ecological adaptability,and often grew at the slopes,the woodlands and the wasteland.The medicine farmers often harvested the aerial parts when flowers in full bloom in the autumn,removed the old stems,dried,can be medicinal.Arternisia annua with high medicinal value and artemisinin derivatives can produce a lot of series medicines.Artemisinin indicated malaria,tuberculosis hot flashes,heat stroke,pruritus,urticaria,and seborrheic dermatitis,and other anti-mosquito.AS natural and geographical reasons,Chongqing is the world’s largest basement of Artemisia annua,with "Artemisia annua hometown" reputation.The development of Artemisia annua industry has unique advantages in Chongqing.Mites,particularly phytophagous pest mites,are one of the important organisms which seriously harmful to our agricultural production.It has the characters of small individual size,fast breeding,the high population density,great harmness,and it can harm more than 150 crops. According to incomplete statistics,China’s annual used for citrus,apples,pears,peaches,cotton, vegetables,wheat,tea etc 8 major crops,the acaricide control costs amounted to more than 9 billion yuan RMB.And as the elevation of mites resistance to chemicals,the control costs increase year after year.Faced with chemical pesticides generated by the various problems and insecticide or acaricide conventional means of research and development has become increasingly difficult, bioactive natural products coming into the mainstream of plant protection time has come.This study was funded by the National Natural Science Foundation（30671392）and the Chongqing Municipality scientific and technological project（2001-6599）,the main research concerning grown in April,May,June,July and September of Artemisia annua root,stem,leaves, solvent extraction rate,and the bioactivity of various extracts against Tetranychus cinnabarinus, Panonychus citri and Petrobia harti,extract of Artemisia annua in July inhibition action of cowpea seeds radicle and hypocotyl growth,tracking acaricidal substances scopoletin isolated from Artemisia annua,and the effect on SOD,CAT,POD,GST,CarE,ACHE,MAO,Ca²⁺-ATPase of T. cinnabarinus treated by scopoletin.Through nearly three years of research,the main findings were followed:1 The extraction rate of Artemisia annuaThe materials of Artemisia annua plant roots,stems,leaves were extracted by conventional room temperature extraction,extraction at room temperature,stirring extraction at room temperature and microwave-assisted extraction etc using a series of solvents.Four kinds of extraction methods on the extraction rate do not differ greatly from the impact,and from an economic point of comparison,stirring extract at room temperature more appropriate.The roots,stems and leaves of Artemisia annua in April,May,June,July and September were extracted with different solvent,respectively,from the extraction rate,the parallel extract approach extraction rate generally higher than the extract of the sequenced.Water extraction rate is the highest （root stems extraction rate is higher than other solvent extraction rate）,followed by acetone,and then ethanol.From different parts of the plant,the leaves the relatively high rate of extraction,followed by the roots,and stems the lowest.2 Suitable methods to botanical acaricide bioassayBioassay methods play a key role in the research and development of pesticide,new bioassay method is tantamount to the discovery of new pesticides.Based on this we compared a glass capillary method,slide impregnation method,leaf impregnation method.The results showed that glass capillary method was no significant difference with the FAO recommended slide impregnation method.And its pesticide usage was a significant decline and suitable for the botanical acaricide bioassay.3 Acaricidal activity of Artemisia annua extracts3.1 Bioactivities against Tetranychus cinnabarinusThe aim of this study was to determine the best extraction technique,the suitable solvent and the optimal plant parts and the acaricidal activities of Artemisia annua L.The acaricidal bioactivities against Tetranychus cinnabarinus of total 135 extracts of petroleum ether（30-60℃）,petroleum ether （60-90℃）,ethanol,acetone and water parallel and sequenced extracts from the leaves,stems and roots of Artemisia annua L.in different periods of April,May,June,July and September were determined by slide-capillary method in laboratory.The results showed that the acaricidal bioactivities elevated with the development of A.annua plant at the concentration of 5 mg·mL^-1.The general tendency exhibited the sequence of July＞June＞May＞April,but September decreased comparing with July.However,the most effective extracts in five months were all acetone parallel extract from A.annua leaf,and the corrected mortalities after treatment 48h were ranged from 74% to 100%.The median lethal concentrations(LC₅₀)against T.cinnabarinus of acetone parallel extracts from A.annua leaves in April,May,June,July and September were 1.3817,0.9443,0.8376, 0.4341,and 0.5986 mg·mL^-1,respectively after treatment48h.The Artemisia annua in July acetone parallel extract showed the highest activity,the growth cycle of Artemisia annua,the vegetative growth period is a crucial period of the defensive ability of the outside world is the strongest,with the experimental findings anastomosis.3.2 Bioaetivities against Panonychus citriLeaves of Artemisia annua in June except petroleum etherⅡsequenced extract and water sequenced extract,other than the activity of the extracts of the root extract significantly improved. One acetone parallel extract against Panonychus citri at the concentration of 5 mg·mL^-1treatment for 48 h,the corrected mortality was 88.84%,and petroleum etherⅡunder the same conditions against Panonychus citri also reached 84.27%.The different solvent extracts of leaves of Artemisia annua in July against Panonychus citri and their biological activities were significantly higher than the roots and stems.Leaf acetone parallel extract with the highest bio-activity,at the concentration of 5 mg·mL^-1for 48 h,the Panonychus citri corrected mortality was 100%.Leaves of Artemisia annua in July of the acetone parallel extract of Panonychus citri,biological activity was significantly higher than that in June,the Panonychus citri 48h after treatment,the LC₅₀(0.4222mg·mL^-1)in July,only 44%of June(0.9489mg·mL^-1).3.3 Bioactivities against Petrobia hartiThe acaricidal bioactivity against Petrobia harti of all solvents extracts of Artemisia annua in June was generally not too strong.Different parts of Artemisia annua in July by using several different polar solvent extracts,the biological activity against Petrobia harti shown superior to in June.Of which leaves in July parallel acetone extract was the highest biological activity,treatment for 48h,the corrected mortality was 95.00%.The acetone parallel extract of Artemisia annua in July against Petrobia harti treatment after 48h,the LC50 was 0.4715 mg·mL^-1,and in June was 0.9083 mg·mL^-1.4 Phytotoxicity of Artemisia annua extracts in July to cowpea seedsIt has been confirmed that the plant Artemisia annua in July showed a relative strong acaricidal activity against Tetranychus cinnabarinus,Panonychus citri and Petrobia harti.However,in order to clarify the toxic effects of plant extracts on the seed germination rate of cowpea,which was principal host plant of the spider mite,and also the radicle and hypocotyl growth inhibition.These extracts were the parallel and sequenced extracts of different solvents from the roots,stems and leaves of Artemisia annua in July.The results showed that,the impact of cowpea seed germination of Artemisia annua in July roots,stems and leaves different solvents extract varied with the solvent species and different extraction methods.Of which the impact of stem ethanol sequenced extract against cowpea seed germination rate was greatest,and that showed significant inhibition effect,and the germination rate was only 23.81%.Otherwise,the leaf acetone sequenced extract also showed strong inhibition effect on cowpea seed germination rate,of which the germination rate was 40.48%. The different solvent extracts of root stems and leaves Artemisia annua in July were tested their phytotoxicity against cowpea seeds,and the results showed that weak inhibition effect on radicle and hypocotyl growth,and maybe also exist some material which promoting radicle,hypocotyl growth.5 Bio-guided isolation of acaricidal active material from Artemisia annuaWith the bio-guided isolation method,the acaricidal activity of the different components isolated from Artemisia annua July parallel leaf acetone extracts by column chromatography were determined.Separated at the end of the 20 kinds of components,the acaricidal activity of component 17 was strongest,followed by component 18 and 8.LC₅₀（48h）of components 17,18 and 8 were 0.1675,0.4368 and 0.3753 mg·mL^-1,the active component 17 arising about 2.6 times compared with July of the acetone extract.The acaricidal activity against Panonychus citri of different components isolated by column chromatography from the acetone parallel extract of Artemisia annua leaf in July were determined in laboratory,in which the acaricidal activity of 17^thcomponent was highest treatment for 48 h, corrected mortality 96.65%,and there was significant differences with other components.Use bio-active guided isolation from the acetone extract of Artemisia annua by column chromatography,the acaricidal activity of the total 20 kinds components were also determined in laboratory,acaricidal activity of the 19^thcomponent was highest at 48 h after the treatment and the corrected mortality was 69.28%. Considered from the two factors of effective content and biological activity,component 17 is the most valuable component,17^thgroup contains three main components,of which the content of 17-3 was maximum 77.64%,17-1 second 16.21%,17-2 only 5.31%.TLC on the three components of the color of iodine, visible pot only one,and no smearing.The acaricidal activities against Tetranychus cinnbarinus of three components were determined in laboratory,the results showed that 17-3 has the strongest acaricidal activity treatment for 48 h,the LC₅₀for T.cinnabarinus was 0.1014 mg·mL^-1,elevated compared to the LC₅₀of 17^th0.1657 mg·mL^-1.According to the selected 17-3 re-crystallization experiments,select a suitable temperature with methanol,acetone,chloroform,ethyl acetate,n-butane dissolved,recrystallization,then put in iceberg cooling and crystal,and to choose the best temperature and solvent.According to crystal shape and the amount of solvents used,selected the best condition was ethyl acetate in 55℃. Followed by HPLC-MS,¹H-NMR,IR,and other means to determine 17-3 for the scopoletin, molecular weight:192.17,molecular formula:C₁₀H₈O₄.Bioassay results showed that the acaricidal activity against T.cinnabarinus of scopoletin was better than 17-3.6 Acaricidal mechanism of scopoletin against Tetranychus cinnabarinusTreatment after by scopoletin,the SOD activity of Tetranychus cinnabarinus was decreased compared with the control,and varied with the scopoletin concentration changed.Along with the activity of the extension of treatment time increased at first and then a downward trend,but the overall performance of the activity by the pharmaceutical treatment lower than that of control.All treatment measured by the activity of the catalase increase,which could be activated after treatment by scopoletin in T.cinnabarinus.POD activity decreased significantly compared with the control, and varied with the changes of scopoletin concentration.Overall,T.cinnabarinus treatment by scopoletin,the POD activity in vivo was inhibited obviously.With the extension of the treatment time,the overall vitality of GST on a declining trend, especially treatment after 48h,the activity to a lower level.But virtually all of the treatment groups were higher activity than control,in other words,GST activity of T.cinnabarinus treatment by scopoletin has improved.Scopoletin activated T.cinnabarinus carboxylesterase activity,and with the increasing concentration of scopoletin activity of the extent of activation also increased. Acetylcholinesterase activity increased first and then showed downward trend.Treatment by scopoletin the Ca²⁺-ATPase activity of T.cinnabarinus,compared with the control,had declined in certain degree,that is to say scopoletin could inhibit Ca²⁺-ATP enzyme activity in a certain extent. On monoamine oxidase activity,the treatment group and control group of MAO close to the vitality of the trend changes,but were all lower than the control group,therefore,the MAO activity of T. cinnabarinus was inhibited in a certain extent.更多还原