【作者】 郭新荣；

【导师】 张雅林； 李孟楼；

【作者基本信息】 西北农林科技大学 ， 农业昆虫与害虫防治， 2008， 博士

【摘要】 从天然产物中寻找具有杀虫活性的先导化合物，为人工合成和修饰合成开发活性更高的化合物奠定基础，是近年来新农药创制的热点。国内外关于马桑倍半萜内酯对昆虫的毒杀作用的研究报道甚少。本文对马桑倍半萜内酯的杀虫活性、试虫生理指标的影响、试虫解毒系统生理指标的影响及其致毒机理等方面作了研究，并进行了马桑倍半萜内酯对粘虫生理作用指示因子的筛选与评价的研究，主要结论如下。1、用提取分离鉴定的马桑倍半萜内酯成分中的羟基马桑毒素(tutin)、马桑亭(coriatin)和马桑宁(corianin)，采用载毒叶碟饲喂法研究试虫的中毒症状及试虫的拒食活性；用药剂点滴法处理粘虫以研究马桑倍半萜内酯对粘虫的毒杀作用。结果表明：羟基马桑毒素处理粘虫后中毒症状为：爬行缓慢，食量减少，进入昏迷；复苏，体变软，体表失水等，中毒症状明显；马桑亭和马桑宁处理后粘虫中毒症状不明显。不同比例(1：3、1：1、3：1)的羟基马桑毒素与马桑亭处理粘虫幼虫后，3、4、5龄期有不同程度进入昏迷期，且4龄期进入昏迷期数量最大，到6龄期全部解除昏迷；不同比例的羟基马桑毒素与马桑宁处理试虫，与相同配比的羟基马桑毒素和马桑亭处理比较，试虫在3、4龄期进入昏迷期数量有所减少，5、6龄期进入昏迷数量增加。马桑倍半萜内酯对粘虫拒食作用的影响差异很大。羟基马桑毒素处理后，对各龄期粘虫幼虫有明显拒食作用，随着龄期增长拒食作用更为明显；马桑亭和马桑宁对粘虫拒食作用不及羟基马桑毒素显著。点滴马桑倍半萜内酯对粘虫的触杀作用试验表明，以1．0mg／mL的羟基马桑毒素毒杀效果最强。不同比例的羟基马桑毒素和马桑亭、羟基马桑毒素和马桑宁饲喂处理对粘虫毒杀效果有明显差异。表现在试虫取食量及累积死亡率随着马桑亭或马桑宁在羟基马桑毒素配比中比例的提高，取食量下降速度和累积死亡率升高速度均加快，马桑宁的协同效应更为显著。2、采用试虫注射药剂和载毒叶碟饲喂处理粘虫，测定粘虫不同组织中的生理指标的变化情况。结果表明，羟基马桑毒素处理粘虫后，其体内蛋白质含量相对稳定，对蛋白酶的活性影响不明显；而马桑亭、马桑宁处理后，表现为刺激粘虫组织细胞分泌蛋白酶，干扰蛋白酶正常代谢，引起蛋白质积累，导致其含量增高。羧酸酯酶、乙酰胆碱酯酶、S-谷胱甘酞转移酶及多元酚氧化酶活性受马桑倍半萜内酯的影响有明显差别。羟基马桑毒素抑制粘虫羧酸酯酶使其活性降低；马桑亭和马桑宁对该酶抑制作用比羟基马桑毒素显著。羟基马桑毒素和马桑宁使粘虫乙酰胆碱酯酶活性出现曲线波动，马桑亭对其影响不明显。S-谷胱甘酞转移酶也是昆虫体内重要的代谢分解酶，羟基马桑毒素、马桑亭和马桑宁对S-谷胱甘酞转移酶和多元酚氧化酶活性的影响很小。3、参照人体的血液、肝功能及尿指标测定方法(全自动生化分析仪)测定了马桑倍半萜内酯处理粘虫后“血液”生理生化指标，分析总结了指标的变化规律。结果表明：与蛋白质代谢相关指标中的谷丙转氨酶、γ-谷氨酰转肽酶、总蛋白、白蛋白、尿素受羟基马桑毒素的影响不明显，谷草转氨酶、球蛋白均受羟基马桑毒素、马桑亭和马桑宁影响作用显著；与脂肪和能量代谢相关及其他代谢指标中的碱性磷酸酶、淀粉酶、肌酸激酶、肌酸激酶同工酶、a-乳酸脱氢酶、乳酸脱氢酶、肌酐、甘油三酯和钙含量等均升高；羟基马桑毒素对葡萄糖、总胆固醇、总胆红素、总胆汁酸影响不明显，而马桑亭和马桑宁使其降低。4、采用综合指标评价粘虫对马桑倍半萜内酯拒食敏感度、生理作用敏感度、血液生理生化解毒作用敏感度、马桑倍半萜内酯处理后粘虫生理生化活性变化过程响应因子的筛选与评价和粘虫生理生化属性的演变与模拟。5、通过马桑倍半萜内酯成分对粘虫作用后中毒症状、拒食和毒杀活性、生理作用，及粘虫对其成分响应能力的研究，分析比较3种马桑倍半萜内酯成分作用粘虫后各个生理指标影响差异。为确定马桑倍半萜内酯中有效杀虫成分及杀虫机理，寻找合成或结构修饰途径、开发植物源农药提供科学依据。本文首次将人体的血液、肝功能及尿指标测定方法应用于马桑对粘虫的毒理学研究中；关于马桑亭和马桑宁的杀虫活性研究在国内首次报道；探索性应用主成分分析和判别分析法筛选出适宜指示和评价马桑倍半萜内酯成分对粘虫生理生化作用的敏感指示因子；采用模糊数学提出和建立了昆虫生理生化指数评价模型，构建了不同内酯组分和处理方式下的这两种指数变化过程，证实了该指标评价模型能定量化的反应不同马桑倍半萜内酯对昆虫生理生化作用的变化过程。更多还原

【Abstract】 Searching leading compound with insecticidal activity from natural products for artificialand modified synthetic compound of higher activity is the focus of new pesticide productionin recent years.There has been few reports on the toxic effect of Coriaria lactone(CL)inChina.In this paper,pesticidal activity of CL,test insect physiological effect,detoxificationsystem and toxic mechanism were deeply studied.The test insect sensitivity degree was alsocomprehensively analyzed.1.In the activity tracing process,we fund that the sesquiterpene lactones in Coriariasinica had antifeeding and killing activity to armyworm(Mythimna separata Walker).Systematic research of toxic symptom,antifeeding activity and toxic effect were conducted toprove the active components and the difference of action mode,and provide accurate theorybasis for grasping the mechanism.The results showed that the toxic symptom of M.separatatreated by Tutin was significant,characterized by insects’ slowly crawling,decrease of foodintake,coma,body soften and losing of surface water after recovery.But the effect was notsignificant treated by Coriatin and Coriania.Treated by Tutin and Coriatin in differentproportion(1:3,1:1,3:1),most larvae in the 4th instar went into coma and recovered at 6thinstar.Treated by Tutin and Coriania,insects going into coma at 3rd and 4th instars decreasedand increased at 5th and 6th instars.The effect of sesquiterpene lactones on M.separata’s antifeeding is significantlydifferent.For instance,treated by Tutin larvae of all instars had obvious antifeeding activityand it increased with the growing instars.Treated by Coriatin and Coriania,it also hadantifeeding activity but not as obvious as Tutin.Consulting a great number of data,we know that there was few report of the antifeedingactivity of Coriatin and Coriania in China,just what we did in this paper.The contact toxicity of CL manifested the toxic effect of Tutin at 1.0mg/mL was the best.Different proportion of Tutin and Coriatin,Tutin and Coriania had great effect on the foodintaking and cumulative mortality.With the increasing of Coriatin,the effect on food intakingand cumulative mortality became significant.This paper only set cooperative toxicexperiment of 3 different proportion and preliminarily assert that Coriatin and Corianin had synergism effect on the antifeeding and killing activities of Tutin to M.separata.2.Reagent injection and Leaf-sandwiches method were used to test differentphysiological index changes of M.separata.The results showed the protease activity of M.separata was not obviously affected by treatment of Tutin,which made the protein contentcomparatively steady.Coriatin and Coriania influenced the protease secretion of M.separatatissue,interfered the normal metabolism of protease.and caused the accumulation of protein.Carboxylesterase,acetylcholinesterase,S-GST and PPO are important enzyme in insectcatabolism.The enzyme activity affected the role of reagent directly or indirectly.Tutin hadinhibitory effect on Carboxylesterase,which was less significant than those of Coriatin andCoriania.Acetylcholinesterase,as the target enzyme of some reagents,its activity showedvibration curve affected by Tutin and Cariania and was not sensitive to Coriatin.However itneeds further study to prove whether acetylcholinesterase is the target enzyme of Tutin andCariania.S-GST is also an important catabolism enzyme in insect but was not affected by CLat all.3.Due to its special physiological structure,insect has special decomposition,metabolism and detoxification mechanism of the agent that enters the tissue.So this papertried to study the insect detoxification system-blood physiological index.Reference to the testmethods of human blood,liver function and urine index,automatic biochemistry analyzerwere used to test the blood physiological index of M.separata treated by sesquiterpenelactones.The variation rules of index were systematically summarized.The results showed,the alanine transaminase(ALT),γ-glutamyltranspeptidase,total protein,albumin and urea ofthe protein metabolism index were affected by Tutin insignificantly.The aspartateaminotransferase(AST)and globulin were affected by Tutin,Coriatin and Coriania withsignificant effect.The content of alkaline phosphatase,direct bilirubin(DBIL),amylase,creatine kinase,creatine kinase isoenzyme,a-lactate dehydrogenase,lactate dehydrogenase,creatinine,triglyceride and calcium increased.Glucose,total cholesterol,total bilirubin,totalbile acid were not sensitive to Tutin,but Coriatin and Coriania made them decrease.4.The above results showed the effect of CL on physiological function and the responseof M.separata.To prove the difference of response ability,this paper used comprehensiveindexes to evaluate the antifeeding sensitivity,physiological sensitivity and bloodphysiological and biochemical sensitivity of M.separata treated by CL.The response factorsin the physiological and biological process were selected and evaluated.Insect attributeevolution and simulation were studied with different CL treatment.5.We studied the toxic symptom,antifeedant and toxic activity,physiological functionand responsive ability of M.separata treated by 3 CLs,compared the effect of 3 CLs to different physiological index to determine the toxic component and its mechanism,which canhelp to synthesize or modify structure of CL and offer scientific basis for exploring newbotanical pesticide.Test methods of human blood,liver function and urine index,automaticbiochemistry analyzer were firstly used to detect the toxic mechanism of CL on M.separatain our study.The toxicity of tutin and coriation were report in China for the first time.Weexpolred on the principal component analysis and discriminant analysis to select the avalibaleindex to indicate and evaluate the physiological influnce on M.separata treated by CL.Fuzzymathematics was used to propose and build 2 insect physiological index evaluation models.Different lactone components and treated methods were designed to measure the reseanabilityof the 2 models,which quantitatively reflected the different change process of insectphysiological fuction of diferent CLs.更多还原