【作者】 罗都强；

【导师】 张兴；

【作者基本信息】 西北农林科技大学 ， 农药学， 2002， 博士

【摘要】 从自然界存在的天然化合物中寻找农药活性化合物，直接利用其原料创制新农药，或从天然产物中寻找先导化合物，对其进行人工合成和修饰合成开发活性更高的化合物，是近年来新农药创制的主要途径之一，也是农药研究的热点。基于这一目的，对卫矛科植物雷公藤（Tripterygium，wilfordii）杀虫活性、有效成分、致毒机理及制剂加工等方面进行了研究。 1、采用生物活性追踪和化学分离相结合的方法，对雷公藤根皮中的杀虫活性成分进行了系统的研究。从雷公藤中分离出4种生物碱化合物，其中1种为首次报道的具有杀虫活性的生物碱化合物；5种二萜化合物和1种三萜化合物，其中一种为新的二萜化合物，5种为已知化合物；并首次明确了具有触杀活性的有效成分。通过现代波谱技术(HRMS、IR、¹HNMR¹³、CNMR、¹H-¹HCOSY、¹³C-¹HCOSY、NOESY、HMBC、HMQC等)确定了它们的结构。二萜化合物LX-1为雷酚内酯甲醚（Triptophenolide methyl ether）、LX-2为雷酚内酯（Triptophenolide）、LX-3为雷公藤内酯酮（Triptonlide）、LX-4为雷公藤甲素（Triptolide）和LX-11为雷酚δ-内酯甲醚（δ-Triptophenolide methyl ether）。LX-11为首次分离得到的新化合物。三萜化合物为LX-5为雷公藤内酯甲（Wilforlide A）。生物碱化合物LX-6为雷公藤定碱（Wilfordine）、LX-7为雷公藤新碱（Euonine）、LX-8为雷公藤吉碱（Wilforgine）、LX-9为雷公藤次碱（Wilforine）。LX-7生物碱的杀虫活性为首次发现。首次证实了雷公藤中除生物碱有杀虫活性外，二萜化合物也具有杀虫活性。对4龄粘虫（Mythimna separata）生物活性测定结果表明，二萜化合物LX-3、LX-4有拒食、胃毒和触杀作用。拒食中浓度AFC⁵⁰分别为124.06、88.63mg/L，胃毒麻痹中量ND₅₀分别为66.72、38.70μg/g，触杀中量分别为2.86、1.63μg/头；生物碱化合物LX-9、LX-6、LX-7、LX-8有拒食和胃毒麻痹作用，无触杀活性，拒食中浓度AFC₅₀分别为30.85、40.17、53.22、77.15mg/L，胃毒麻痹中量ND₅₀分别为20.67、82.70、128.93、262.80μ/g。 2、在杀虫活性追踪的过程中发现雷公藤生物碱和非生物碱均有杀虫活性，为探明其杀虫活性和作用方式的差异，为产业化提供理论依据，对其中毒症状和作用方式进行了系统的研究，结果表明，雷公藤总生物碱和非生物碱对害虫的中毒症状基本相似，因持续剂量、持续时间及进食速度差异而表现出拒食、麻痹、毒杀等症状；雷公藤总生物碱对5龄菜青虫（Plerls ge L．）、3龄粘虫（枷th。。n。sop。ta．．）＄113龄小菜蛾（PI；It。11a Xylostella卜．）的拒食中浓度仆Q；，分别为70．9卜功．50、1146．肥n；。八，毒杀r！。浓度＊。；，分别为36．52、13．69、102．99二g几：雷公藤非生物碱对这三种试虫的拒食r！。浓度八K。；；分别为99．17、47．46、1415．90mg／L，毒杀中浓度LC。；，分别为72．79、53．87和 1017．84mg／L；二者均在高浓度下拒食，低浓度毒杀；雷公藤总生物碱和非生物碱均有胃毒麻痹作用，总生物胃毒麻痹作用要大于非生物碱，总生物碱和非生物碱对5龄菜青虫和粘虫的胃毒麻痹中量ND。；；分别为29。02。74．45和 42．26、134．48 u g＊；雷公藤总生物碱无触杀活性，而＊生物碱有触杀活性，其对 5龄粘虫和菜青虫的触杀致死中量 LD。；；分别为 3．70、3．24 u g／头；雷公藤总生物碱杀卵活性要大于总生物碱，非生物碱对初孵幼虫的杀虫活性要小于总生物碱；同时，二者均无熏蒸作用。 3、在对杀虫活性成分、杀虫活性等研究的基础上，采用电镜技术和生化方法，以5龄粘虫为试虫，对雷公藤甲素触杀作用机理进行了初步探讨。结果表明：（1）雷公藤甲素可能对粘虫中肠消化酶活性无显著影l响，消化酶的降低不是造成昆虫死亡的原因。（2）雷公藤甲素对试虫的中肠肠壁细胞的内膜系统有一定的影，叼。雷公藤甲素作用于粘虫后，中肠肠壁细胞中最早发生形态变化的是线粒休，随着试虫中毒症状的加重，线离体峭模糊，出现空白亮区，并逐步消解；粗面内质网扩张，囊泡化；脂滴完全消失；o＼现了自噬泡，它是线粒体等各种细胞器残片山初级溶酶体包绕而成，外有单位膜包绕：杜状细胞顶端微绒毛及杯状细胞也无可见变化。这说明中肠肠壁细胞形态病变是代谢破坏的次生效应，中肠肠壁的细胞膜不是雷公藤作用的初始位点。（3）处理试虫比对照试虫的乙酚胆碱含量明显减少，这可能表明雷公藤甲素与神经突触递质的释放有关；雷公藤甲素不象有机磷、氨基甲酸酯等合成有机杀虫剂那样将乙酚胆碱酯酶活性显著抑制，因此，雷公藤甲素的作用机理不可能与有机磷、氨基甲酸酯的相似，乙酚胆碱酯酶可能不是雷公膝甲素作用的靶标酶；通过对酯酶活性测下和酯酶同工酶观察，二者变化的总趋势基本一致，对酯酶有显著影响。由此推测酯酶参与了雷公藤甲素的代谢过程；雷公藤甲素处理试虫后，粘虫Na”，k“－ATP酶活性与对照相比，明显降低。在兴奋期抑制率为19．3％：痉挛期和麻痹期活性几乎无差异，抑制率分别为50．狐和54．6％。这说明咐“，卜－川”P酶可能是雷公藤甲素的靶标之一更多还原

【Abstract】 The root bark of Tripterygium wilfordii Hook had been used for centuries by Chinese market gardeners to protect their crops against insect pest , it is one of widely used insecticidal plant in China . For the purpose of searching new environmental acceptable insecticides, active components , insecticidal activities , poisonous mechanism and formulation of Tripterygium wilfordii owere studied in this paper.1.Ten insecticidal constituents of T. wilfordii were isolated by means of phytochemistry combined with bioassay method. The structures of the compounds were determined by UV, IR, ’HNMR, "CNMR , ’H-’H COSY, ’H-’3C COSY , HMBC, HMQC and mass spectral analysis as triptophenolide methyl ether (LX-1), triptophenolide(LX-2), triptonide(LX-3), triptolide(LX-4), wilforlide A(LX-5), wilfordine(LX-6), euonine(LX-7), wilforgine(LX-8) and wilforine(LX-9) and 8-triptophenolide methyl ether(LX-11) .LX-11 is a new dipterpenoid compound. Laboratory bioassay showed the dipterenoids LX-3 and LX-4 had contact, stomach poisonous and antifeendant activities. The values of medium antifeeding concentration (AFC50) against the 4th larvae of Mythimna separata were 124. 06, 88. 63mg/L. Median stmoatch narcosis dose (ND50) were 66. 72, 38. 70ug/g. Median contact dose (LDJ were 2. 86,1.63ug/head, respectively. The alkaloids compounds LX-9, LX-6, LX-7, LX-8 had antifeedant, stmoatch narcosis activity, but no contact activity. The values of AFCa, were 30.85,40.17,53.22 and 77.15mg/L The NDb values were 20.67,82.70,1218.93,262.80 u g/g. The alkaloid compound LX-7 and dipternoid compounds LX-3, LX-4 were the first reported that had insecticidal activity , of which LX-3 and LX-4 were contact compoments of T. Wilfordii.2. The alkaloids and non-alkaloids of 71 wilfordii were extracted from the root bark of T. wilfordii and identified. The results of bioassay showed that the alkaloids had different insecticide activities with the non-alkaloids . The alkaloids and non-alkaloids from 71 wilfordii had antifeedant, toxicity and stomach narcosis activity .The values of AFC50 of the alkaloids and non-alkaloids against the 5th instars larvae of Pierisrapae , 3rd instars larvae of M. separata and Plutella xylostella were 70. 91, 39. 50, 1146. 82 and 99. 17, 47. 46, 1415. 90mg/L The LCSO values of poison were 36.52,13.69,102.99 and 72.79,53.87 , 1017.84mg/L. The LDSO values stomach narcosis to 5th instars larvae of Pier is rapae and Mythimna separata were 29.02,77.45 and 42. 26, 134. 48 n g/g. The alkaloids had no contact activity, but the non-alkaloids had. The medium contact dose (LD5<>) against 5th instars larval of P. rapae and M. separata were 3. 70, 3. 24 u g/head. When the concentration of them were 0. 5-20g/L, the unhatching of eggsratio was 34-60%.3. Triptolide is first found that had contact activity from T. wilfordii. The poisoned larvae of Mythimna separata became exciting, spasming , paralysising and finally dead.For the futher understanding the mechanism of action of triptolide on the molecular level and exploting the new environmental acceptable insectiides by means of biorational design, the mechanism of action of triptolide had also been studied in this paper. The activities of digestive enzymes of midgut of M. separata was examined with conventional biochemical methods, the results showed that the activities of protease, amylase and lipase of the poisoned larval midgut had no significant changes with the normal larval midgut ;The pathological changes were observed by electron microscope , the observation showed that the initial infected organellas were mitochondria. As the pathological process went on , the mitochondria had irregular enlarged, the bilayer membrane was not intact, the mitochondria critase was fuzziness;The cisterane of rough endoplasmic reticulum were enlarged, finally, vesiculation;LD compotely dissapper at the spasm .paralysis periods;The microvilli of the column cell and cotyloid cells had no changes.This suggested that the midgut cells were no the initalis target of triptoi更多还原