紫穗槐果實殺蟲活性物質及其作用機理研究
發(fā)布時間：2018-08-19 15:22
【摘要】：紫穗槐為豆科紫穗槐屬植物,多年生落葉小灌木。原產北美,后引入我國用于防風固沙,全國各地均有栽培,植物資源豐富。已有的研究表明紫穗槐果實具有觸殺、胃毒、拒食等農用活性。本文運用氣相色譜-質譜聯(lián)用技術、柱層析、高壓制備液相等現(xiàn)代分離技術和紅外、紫外、質譜、核磁共振氫譜等波譜技術,對紫穗槐果實精油及黃酮類殺蟲活性成分進行了鑒定；測定了紫穗槐果實精油和黃酮類活性成分對不同有害生物的生物活性；采用響應面法優(yōu)化了紫穗槐果實總黃酮成分提取工藝,并研制了8%紫穗槐總黃酮微乳劑；建立了昆蟲腺苷酸含量的高效液相測定方法,系統(tǒng)研究了紫穗槐總黃酮及8'-羥基魚藤酮對有害生物腺苷酸含量及能荷的影響；探討了8'-羥基魚藤酮和魚藤酮對昆蟲線粒體復合酶Ⅰ的影響及作用機理。主要研究結果如下：采用GC-MS對水蒸氣、石油醚、二氯甲烷和正己烷提取得到的紫穗槐果實精油進行分析鑒定,分別獲得84種、112種、75種、93種化學成分。對比4種不同溶劑提取紫穗槐精油的成分發(fā)現(xiàn),石油醚提取的精油數量和種類較多,3種有機溶劑提取精油含量較高的成分都是γ-依蘭油烯、p-蓽澄茄油烯、γ-蓽澄茄烯。紫穗槐果實精油殺蟲活性測試發(fā)現(xiàn),4種精油對蘋果黃蚜和淡色庫蚊幼蟲均具有毒殺活性,其中紫穗槐石油醚提取獲得的精油的殺蟲活性更為顯著。紫穗槐果實提取溶劑選擇試驗結果表明,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殺蟲活性。生物活性測定及毒力相對中位數強度分析顯示乙醇提取物具有最高的殺蟲活性,且與其他溶劑提取物相比具有顯著差異。紫穗槐果實乙醇提取物,石油醚提取物,丙酮提取物,氯仿提取物和乙酸乙酯提取物對淡色庫蚊4齡幼蟲的毒力相對效力分別為1.00,1.46,1.51,1.61和3.80倍。采用活性追蹤法,從紫穗槐果實乙醇提取物中分離純化一乳白色針狀高活性殺蟲化合物,經紫外(UV)、紅外光譜(IR)、核磁共振氫譜(1H-NMR)以及質譜(MS)分析鑒定為8'-羥基魚藤酮。生測結果顯示8’-羥基魚藤酮對蘋果黃蚜、小菜蛾、菜青蟲、淡色庫蚊幼蟲以及溪流搖蚊幼蟲均具有毒殺作用,且對蘋果黃蚜、菜青蟲以及淡色庫蚊幼蟲的殺蟲效果優(yōu)于對照魚藤酮。8'-羥基魚藤酮對蘋果黃蚜、菜青蟲幼蟲以及淡色庫蚊幼蟲的LCso分別為0.213、131.531、4.290μg/mL,魚藤酮對于3種幼蟲的LC50分別為0.346、142.117、4.692μg/mL。采用單因素回歸試驗及響應面優(yōu)化紫穗槐總黃酮提取工藝,結果表明乙醇濃度、提取時間對總黃酮提取所得率影響顯著�；貧w模型預測總黃酮的最優(yōu)工藝條件為：乙醇濃度為77.43%、提取時間為31.29min、料液比為1：21.22、超聲功率為357.55W。通過篩選溶劑,乳化劑,獲得了8%紫穗槐總黃酮微乳劑的制劑配方。并以8'-羥基魚藤酮為主要成分,建立HPLC分析方法,制定了產品質量標準。測定8%紫穗槐黃酮微乳劑和8%紫穗槐黃酮乳油對蘋果黃蚜和淡色庫蚊4齡幼蟲的生物活性,結果表明,將紫穗槐總黃酮加工成微乳劑后對蘋果黃蚜和淡色庫蚊4齡幼蟲的毒殺活性明顯高于乳油制劑。建立了高效液相色譜測定昆蟲體內ATP, ADP和AMP含量的方法。研究發(fā)現(xiàn)紫穗槐總黃酮和8’-羥基魚藤酮對淡色庫蚊ATP含量、能荷及總腺苷酸庫有影響,兩者均能降低淡色庫蚊ATP含量和能荷,且含量與藥劑濃度呈反比例關系,即濃度越大ATP含量和能荷越小。探討了8'-羥基魚藤酮和魚藤酮對昆蟲線粒體復合酶Ⅰ的影響,結果顯示8'-羥基魚藤酮和魚藤酮均能顯著抑制淡色庫蚊線粒體復合酶Ⅰ的酶活性；其中魚藤酮的IC50為3.1375μM,而8'-羥基魚藤酮的IC50為2.8592μM。抑制曲線表明,兩者同為可逆性混合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.
【學位授予單位】：沈陽農業(yè)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：S482.39
本文編號：2192063
[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.
【學位授予單位】：沈陽農業(yè)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：S482.39
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