紫穗槐果实杀虫活性物质及其作用机理研究

发布时间：2018-08-19 15:22

【摘要】：紫穗槐为豆科紫穗槐属植物,多年生落叶小灌木。原产北美,后引入我国用于防风固沙,全国各地均有栽培,植物资源丰富。已有的研究表明紫穗槐果实具有触杀、胃毒、拒食等农用活性。本文运用气相色谱-质谱联用技术、柱层析、高压制备液相等现代分离技术和红外、紫外、质谱、核磁共振氢谱等波谱技术,对紫穗槐果实精油及黄酮类杀虫活性成分进行了鉴定；测定了紫穗槐果实精油和黄酮类活性成分对不同有害生物的生物活性；采用响应面法优化了紫穗槐果实总黄酮成分提取工艺,并研制了8%紫穗槐总黄酮微乳剂；建立了昆虫腺苷酸含量的高效液相测定方法,系统研究了紫穗槐总黄酮及8'-羟基鱼藤酮对有害生物腺苷酸含量及能荷的影响；探讨了8'-羟基鱼藤酮和鱼藤酮对昆虫线粒体复合酶Ⅰ的影响及作用机理。主要研究结果如下：采用GC-MS对水蒸气、石油醚、二氯甲烷和正己烷提取得到的紫穗槐果实精油进行分析鉴定,分别获得84种、112种、75种、93种化学成分。对比4种不同溶剂提取紫穗槐精油的成分发现,石油醚提取的精油数量和种类较多,3种有机溶剂提取精油含量较高的成分都是γ-依兰油烯、p-荜澄茄油烯、γ-荜澄茄烯。紫穗槐果实精油杀虫活性测试发现,4种精油对苹果黄蚜和淡色库蚊幼虫均具有毒杀活性,其中紫穗槐石油醚提取获得的精油的杀虫活性更为显著。紫穗槐果实提取溶剂选择试验结果表明,乙醇提取物,石油醚提取物,丙酮提取物,氯仿提取物和乙酸乙酯提取物均具有一定的杀虫活性。生物活性测定及毒力相对中位数强度分析显示乙醇提取物具有最高的杀虫活性,且与其他溶剂提取物相比具有显著差异。紫穗槐果实乙醇提取物,石油醚提取物,丙酮提取物,氯仿提取物和乙酸乙酯提取物对淡色库蚊4龄幼虫的毒力相对效力分别为1.00,1.46,1.51,1.61和3.80倍。采用活性追踪法,从紫穗槐果实乙醇提取物中分离纯化一乳白色针状高活性杀虫化合物,经紫外(UV)、红外光谱(IR)、核磁共振氢谱(1H-NMR)以及质谱(MS)分析鉴定为8'-羟基鱼藤酮。生测结果显示8’-羟基鱼藤酮对苹果黄蚜、小菜蛾、菜青虫、淡色库蚊幼虫以及溪流摇蚊幼虫均具有毒杀作用,且对苹果黄蚜、菜青虫以及淡色库蚊幼虫的杀虫效果优于对照鱼藤酮。8'-羟基鱼藤酮对苹果黄蚜、菜青虫幼虫以及淡色库蚊幼虫的LCso分别为0.213、131.531、4.290μg/mL,鱼藤酮对于3种幼虫的LC50分别为0.346、142.117、4.692μg/mL。采用单因素回归试验及响应面优化紫穗槐总黄酮提取工艺,结果表明乙醇浓度、提取时间对总黄酮提取所得率影响显著。回归模型预测总黄酮的最优工艺条件为：乙醇浓度为77.43%、提取时间为31.29min、料液比为1：21.22、超声功率为357.55W。通过筛选溶剂,乳化剂,获得了8%紫穗槐总黄酮微乳剂的制剂配方。并以8'-羟基鱼藤酮为主要成分,建立HPLC分析方法,制定了产品质量标准。测定8%紫穗槐黄酮微乳剂和8%紫穗槐黄酮乳油对苹果黄蚜和淡色库蚊4龄幼虫的生物活性,结果表明,将紫穗槐总黄酮加工成微乳剂后对苹果黄蚜和淡色库蚊4龄幼虫的毒杀活性明显高于乳油制剂。建立了高效液相色谱测定昆虫体内ATP, ADP和AMP含量的方法。研究发现紫穗槐总黄酮和8’-羟基鱼藤酮对淡色库蚊ATP含量、能荷及总腺苷酸库有影响,两者均能降低淡色库蚊ATP含量和能荷,且含量与药剂浓度呈反比例关系,即浓度越大ATP含量和能荷越小。探讨了8'-羟基鱼藤酮和鱼藤酮对昆虫线粒体复合酶Ⅰ的影响,结果显示8'-羟基鱼藤酮和鱼藤酮均能显著抑制淡色库蚊线粒体复合酶Ⅰ的酶活性；其中鱼藤酮的IC50为3.1375μM,而8'-羟基鱼藤酮的IC50为2.8592μM。抑制曲线表明,两者同为可逆性混合Ⅰ型性抑制。
[Abstract]:Sophora purpurea is a small perennial deciduous shrub of the genus Sophora in Leguminosae.It was originated in North America and introduced into China for windbreak and sand fixation.It is cultivated in various parts of the country and has abundant plant resources.Previous studies have shown that the fruit of Sophora purpurea has agricultural activities such as contact killing,gastrotoxicity and antifeeding.The fruit of Sophora Purea has been prepared by GC-MS with column chromatography and high pressure. The insecticidal components of essential oils and flavonoids from fruits of Amorpha fruits were identified by liquid chromatography and IR, UV, MS and 1H NMR spectroscopy, and the bioactivities of essential oils and flavonoids from fruits of Amorpha fruits to different pests were determined. A microemulsion of 8% total flavonoids from Amorpha fruticosa was prepared and a high performance liquid chromatographic method was established to determine the adenylate content of insects. The main results are as follows: 84, 112, 75 and 93 kinds of essential oils from fruits of Amorpha fruticosa extracted by water vapor, petroleum ether, dichloromethane and n-hexane were identified by GC-MS. The essential oils extracted by petroleum ether were compared with those extracted by four different solvents. The results showed that the essential oils from the fruits of Amorpha fruticosa had high insecticidal activity against the larvae of Culex pipiens pallens and Aphis flavescens, and the insecticidal activities of the essential oils from Amorpha fruticosa fruits were tested. The results of solvent selection showed that ethanol extract, petroleum ether extract, acetone extract, chloroform extract and ethyl acetate extract all had certain insecticidal activity. The relative toxicity of ethanol extract, petroleum ether extract, acetone extract, chloroform extract and ethyl acetate extract from fruits of Amorpha fruticosa to the 4th instar larvae of Culex pipiens pallens was 1.00, 1.46, 1.51, 1.61 and 3.80 times respectively. A milky white acicular insecticidal compound was isolated and purified and identified as 8'-hydroxy rotenone by UV, IR, 1H-NMR and MS. The bioassay results showed that 8'-hydroxy rotenone had toxic effects on apple yellow aphid, diamondback moth, cabbage caterpillar, Culex pipiens pallens larvae and chironomid mosquito larvae. The insecticidal efficacy of rotenone to Apple aphid, cabbage caterpillar and Culex pipiens pallens larvae was better than that of rotenone. The LCso of rotenone to Apple aphid, cabbage caterpillar larvae and Culex pipiens pallens larvae were 0.213, 131.531, 4.290 UG ug/mL, respectively. The LC50 of rotenone to three larvae were 0.346, 142.117 and 4.692 ug/mL, respectively. The results showed that the concentration of ethanol and the extraction time had a significant effect on the yield of total flavonoids. The formulation of 8% total flavonoids microemulsion of Amorpha fruticosa L. was obtained. The quality standard was established by HPLC with 8'-hydroxy rotenone as the main component. The bioactivity of 8% flavonoids microemulsion of Amorpha fruticosa L. and 8% flavonoids emulsifiable oil of Amorpha fruticosa L. against the 4th instar larvae of Aphis flavus and Culex pipiens pallens was determined. The results showed that the total flavonoids of Amorpha fruticosa L. The toxicity of the microemulsion to the 4th instar larvae of Culex pipiens pallens and Aphis flavus was significantly higher than that of the emulsifier. A high performance liquid chromatographic method was established for the determination of ATP, ADP and AMP in insects. The ATP content and energy charge of Culex pipiens pallens were decreased in inverse proportion to the concentration of the insecticide, i.e. the higher the concentration of ATP and the lower the energy charge. The effects of 8'-hydroxy rotenone and rotenone on mitochondrial complex enzyme I were studied. The results showed that both 8'-hydroxy rotenone and rotenone could significantly inhibit the enzyme of mitochondrial complex enzyme I of Culex pipiens pallens. The IC50 of rotenone was 3.1375 mu M, while that of 8'-hydroxy rotenone was 2.8592 mu M.
【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S482.39

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