【作者】 崔俊；

【导师】 李孟楼；

【作者基本信息】 西北农林科技大学 ， 森林保护学， 2013， 博士

【摘要】 马桑内酯是马桑植物中的倍半萜内酯类活性物质，具有独特的苦毒素骨架，具有显著的神经毒性，被认为是昆虫行为的干扰剂、拒食剂和胃毒剂。羟基马桑毒素是马桑内酯类化合物的主要代表和活性最强的化合物，分析其构效关系、致毒机理，并对其进行结构改造是马桑内酯研究的热点。粘虫具有很强的迁飞性，是一种世界性分布的重要农业害虫。本研究优化了羟基马桑毒素的提取方法，并对羟基马桑毒素及其结构类似物进行了化学结构改造，通过活性测定筛选出了最优结构化合物。并以粘虫幼虫为试虫，初步分析了羟基马桑毒素引起粘虫麻痹和生长阻断的毒性机理。最后还分析了天敌昆虫花绒寄甲围心细胞的扫描电镜超微结构以及羟基马桑毒素对围心细胞形态的影响。主要研究结果如下：1、优化了羟基马桑毒素的提取分离技术。本研究首次以乙醇为溶剂采用超声波辅助提取技术从12.45Kg干燥马桑种籽中提取到6.60g羟基马桑毒素，提取率达到0.53‰。正交分析结果表明，其最佳提取工艺条件为料液比1︰15，提取时间120min，提取温度50°C，其中提取温度是用超声波萃取法提取羟基马桑毒素工艺的关键影响因素。2、对羟基马桑毒素进行了结构改造研究。采用酰化加成反应以羟基马桑毒素羟基为母体合成了7个2位羟基酰化衍生物，经过SicFinder Scholar查新可知所得7个化合物均为新物质。采用非限制性叶碟法测定了所得酰化产物及羟基马桑毒素对3龄粘虫的拒食和杀虫活性。结果表明2,4-二氯苯甲酰化羟基马桑毒素（2-(2,4-dichlorobenzoyloxy)-tutin，T7）的杀虫活性最强，是本研究中的最优化合物。构效分析表明，不同基团对羟基马桑毒素杀虫活性的影响不同，其中二氯苯甲酰基>氯烟酰基>甲氧苯甲酰基>氯苯甲酰基>对硝基苯甲酰基>磺酰基>二硝基苯甲酰基。3、对羟基马桑毒素结构类似物进行了结构改造。采用脱氧、加成和酰化等反应制备了23个具有羟基马桑毒素类似内酯环结构的衍生物。其中有9个穿心莲内酯衍生物、7个7-羟基香豆素衍生物和7个高丝氨酸内酯衍生物，经过SicFinder Scholar查新可知所得化合物中有9个新物质，另有5个化合物仅有CAS登录号，未见文献报道。活性分析结果表明15-氟代苯亚甲基-14-脱氧-11,12-烯穿心莲内酯衍生物（15-fluorobenzylidene-14-Deoxy-11,12-didehydroandrographolide，A4d）和15-溴代苯亚甲基-14-脱氧-11,12-烯穿心莲内酯衍生物（15-Bromoacylbenzylidene-14-deoxy-11,12-didehydroandrographolide，A4e）是穿心莲内酯衍生物中的最优化合物；7-甲烷磺酰基-香豆素和4-三甲基乙酰-香豆素（7-methane-sulfonyloxy-coumarine，C4）是香豆素衍生物中的最优化合物； N-4-硝基甲苯-高丝氨酸内酯（N-(4-nitrobenzoyl)-homoserinelactone，H1）是高丝氨酸内酯衍生物中的最优化合物。4、对羟基马桑毒素对粘虫的生长阻断活性、麻痹活性和拒食、毒杀活性进行了分析和致毒机理初探。结果表明，羟基马桑毒素对粘虫具有显著的拒食、麻痹和毒杀活性，并能降低试虫生长量，其生长阻断活性与毒素浓度呈正相关。羟基马桑毒素在体外培养过程中也能够显著影响浆细胞的伸展活性，诱导试虫产生细胞免疫响应。试虫GBP蛋白表达量增高可能是试虫产生麻痹活性和细胞免疫响应的原因之一。5、采用场发射扫描电镜观察了马桑内酯化合物对粘虫围食膜结构的影响。结果表明，在亚致死浓度下羟基马桑毒素及其结构类似物对粘虫围食膜均有不同程度的破坏作用。不同化合物对围食膜的破坏具有显著差异。SDS-PAGE法分析试虫围食膜的蛋白组成结果表明，羟基马桑毒素进入试虫肠道约12h就能导致44.3kD以上的围食膜蛋白被完全降解，而T7和A4d进入试虫肠道后均不同程度引起了一些特异性蛋白条带的增加，与几丁质竞争结合释放围食膜蛋白。通过ABI4800串联质谱（MALDI-TOF-TOF-MS）对所得差异蛋白进行质谱鉴定分析，结果成功鉴定出6个蛋白条带，分别为1个羧肽酶前体，1个肌钙蛋白，2个原肌球蛋白和2个肌动蛋白。6、采用场发射扫描电镜分析了不同虫态花绒寄甲围心细胞的形态。结果显示，花绒寄甲的背血管属于直管型，没有明显的膨大部分和分枝，并在胚胎期就完成了所有的分化和发育，具备了完整的功能。更多还原

【Abstract】 Coriaria lactone is a kind of sesquiterpene lactones extracted from plant Coriaria sinica.It has a unique picrotoxin scaffold and significant neurotoxicity. It is known as insectbehavior disruptor, antifeedant and stomach poison. Tutin is the main representative and mostactivated compound in coriaria lactone. The structure-activity relationships analyze of tutin,its toxic mechanism and structural modification have been areas of intense investigation ofcoriaria lactone. Mythimna separata is a world-wide agricultural pests with highly migratory.In this study, tutin extraction method was optimized, the derivatives of tutin and its analogueswere synthesized, and the optimal compounds were screened through the determination oftheir bioactivity. The mechanism of paralysis and growth blocking of tutin against Mythimnaseparata larva were preliminary analyzed. The SEM ultrastructure and the effect of tutinagainst Dastarcus helophoroides pericardial cells morphological analysis were carried out.The main results are as follows:1. The extraction method of tutin was optimized. This is the first time to use ultrasonicassisted extraction technology to extract tutin with ethanol as solvent from12.45Kg dryCoriaria seeds, the extraction ratio is0.53‰. The results of quadrature analysis showed that,the the optimum extraction condition is solid-liquid ratio as1︰15, in120min and50°C.The extraction temperature is the key factors to influencing the tutin ultrasonic extractionmethod.2. Structural modificationof tutin. Seven novel tutin acylation derivatives weresynthesized through addition reaction on the hydroxy at C-2of tutin. The result checkedthrought SicFinder Scholar showed that the seven derivatives are all new substances. Theantifeedant and insecticidal activity of tutin and their derivatives against M. separata weredetermined through non-chice leafdisc assay. T7,2-(2,4-dichlorobenzoyloxy)-tutin showedthe best activity among all the new compounds. The structure-activity relationship analysisshowed that, different moiety add in parent compound have different influence to theirbioactivity, and in which dichlorobenzoyl> chloronicotinoyl> methoxybenzoyl>chlorobenzoyl> nitrobenzoyl> sulfonyloxy> dinitrobenzoyl.3. Structural modification of tutin analogues. Twenty three derivatives with the lactonemoiety similar as tutin were synthesized through dehydration, addition and acylation reaction, in which contain9andrographis lactone derivatives, seven7-hydroxyl coumarin derivativesand7homoserine lactone derivatives. Novelty search through SicFinder Scholar showed thatthere are9new substances in them, while five compounds only have a CAS registry number,but have yet to be published. The activity analysis showed that15-fluorobenzylidene-14-Deoxy-11,12-didehydroandrographolide (A4d) and15-Bromoacylbenzylidene-14-deoxy-11,12-didehydroandrographolide (A4e) are the optimalcompounds of andrographolide derivatives;7-methane-sulfonyloxy-coumarine (C4) is theoptimal compound of7-hydroxycoumarin derivatives; N-(4-nitrobenzoyl)-homoserinelactone(H1) is the optimal compound of homoserine lactone derivatives.4. The growth blocking, paralysis and antifeedant activity of tutin against M. separatawere studied. The results showed that, tutin exhibited conspicuous antifeedant, paralysis andpoison activity to M. separate, and reduced the growth of larva. The growth blocking activitywas positively correlated with the toxin concentration. In vitro test, tutin significantlyaffected stretching activity of the plasma cell, and induced the immune response to testinsects. The increase of GBP gene expression may be respond for the tutin’s paralysis activityand immune response to test insects.5. The effect of tutin on the peritrophic membrane morphological features of M.separata was invealed by field emission scanning electron microscope. The results show that,tutin and its structural analogues have different levels of damaging on peritrophic membraneof M. separate in sublethal concentrations. Different compounds exhibited significantdifferences damage on peritrophic membrane. SDS-PAGE analysis showed that, tutin totallydegraded the peritrophic matrix proteins upper44.3kD in12h, while T7and A4d abundancethe proteins bands in different level. The ALDI-TOF-MS study identified6proteins thatexpressed differentially among tutin effect in12h. they are1Carboxypeptidase precursor,1troponin,2tropomyosins and2actins.6. Morphological features of pericardial cells of the different instars Dastarcushelophoroides revealed by field emission scanning electron microscopy analysis. The resultsshowed that, the dorsal vessel belongs to the straight tube, no obvious enlargement of parts andbranches, and embryonic differentiation and development, with the full functionality.更多还原