【作者】 韩俊艳；

【导师】 纪明山；

【作者基本信息】 沈阳农业大学 ， 农药学， 2011， 博士

【摘要】 本文采用HPLC-MS、GC-MS、1H-NMR、电镜显微技术等研究方法对药用植物细辛杀螨活性物质的提取、分离、结构鉴定、作用机理和剂型加工等进行了系统研究,明确了细辛的杀螨活性并阐明了其杀螨作用机理,纯化出了杀螨活性单体,鉴定了其化学结构,并研制出了一种细辛杀螨物质的实用剂型。本研究成果为药用植物细辛杀螨物质进一步开发应用奠定了坚实基础。研究了20种低毒药用植物甲醇提取物的杀螨活性。研究结果表明,槟榔、细辛、黄连、苦参和苦楝子等的提取物的生物活性较强,以10mg/mL浓度处理72h后雌成螨的校正死亡率达到100%,处理5d后卵的孵化率低于40%,对螨的LCso在0.47-2.14mg/mL之间,对卵的LC5o在0.88-2.20mg/mL之间。对杀螨活性最强的药用植物细辛进行了深入研究,结果表明：细辛甲醇提取物对二斑叶螨雌成螨和卵的触杀活性较强,成螨处理24 h和卵处理5d后,1mg/mL细辛甲醇提取物对雌成螨和卵的校正死亡率分别为66.5%和45.7%,对雌成螨与卵的LCso值分别为0.57 mg/mL与0.88 mg/mL。细辛甲醇提取物对二斑叶螨具有较强的产卵抑制作用,以1 mg/mL浓度处理,24与48 h的产卵抑制率分别为60.25%和61.45%,以4mg/mL浓度处理,24与48 h的产卵抑制率分别为92.89%与94.7%。细辛甲醇提取物对二斑叶螨还表现出驱避作用,以0.5 mg/mL处理,12 h后对螨的驱避率为57.14%,在大于2 mg/ml浓度时,从12至72h的驱避率均接近100%。细辛甲醇提取物还具有一定的胃毒毒性,但弱于触杀毒性。以1mg/mL浓度处理,48h的成螨校正死亡率和产卵抑制率分别为28.2%和53.21%。浓度增大,胃毒作用增强,在8mg/mL浓度下,48h的成螨校正死亡率和产卵抑制率分别为82.05%与92.66%。明确了细辛杀螨活性物质的作用机理。二斑叶螨进行触杀处理后,螨的体壁外观超微结构发生改变：体表失水、螨体皱缩、体壁肤纹间隙变大、肤纹突变小、表皮层和细胞内结构有不同程度的破坏。细辛提取物抑制二斑叶螨体内乙酰胆碱酯酶、Na+, K+-ATP酶、多功能氧化酶的酶活性,增强超氧化物歧化酶与过氧化氢酶活性,对谷胱甘肽S-转移酶表现出先升后降的趋势,对酯酶和单胺氧化酶没有显著影响,表明乙酰胆碱酯酶、Na+, K+-ATP酶、多功能氧化酶可能是细辛杀螨活性物质的作用靶标,同时氧化应激反应也与其杀螨作用机制有关。对细辛提取物进行了杀螨活性跟踪、分离和纯化,分离鉴定了杀螨活性单体。采用硅胶柱层析结合薄层层析、重结晶技术等方法得到杀螨活性单体,经HPLC-MS和GC-MS及1H-NMR等技术鉴定分析,确定杀螨活性物质为细辛脂素。生物活性测定结果表明细辛脂素的杀螨活性较强,LC5o为0.1237 mg/mL,毒力较细辛提取物增强。研制了细辛提取物乳油剂。配方组成：10%细辛提取物+80%溶剂(二甲苯)+10%复合乳化剂(3%农乳500+2%农乳600+5%吐温-80)。细辛乳油的乳化性、分散性、乳液的稳定性、低温稳定性以及热贮稳定性、挥发性等质量指标均符合要求。室内生测结果表明10%细辛提取物乳油400倍稀释液与15%哒螨灵3000倍稀释液对二斑叶螨的室内防效相当。对细辛挥发油的化学组成、杀螨活性进行了研究。经GC-MS分析,细辛挥发油中的9种主要成分占了86.15%,其中甲基丁香酚含量最高,为22.9%,其次是黄樟醚,含量为19.8%。生物测定结果表明细辛挥发油对二斑叶螨具有较强的熏蒸活性和驱避活性,在8μg/mL浓度下,24 h的螨死亡率为72.6%,在48 h后,达到100%,驱避率为86.1%。细辛挥发油对螨体内的谷胱甘肽S-转移酶和Na+,K+-ATP酶产生影响,细辛挥发油处理后,二斑叶螨体内谷胱甘肽S-转移酶活性随着时间的推移有先升高后降低的趋势,而Na+,K+-ATP酶活性降低,表明这两种酶酶活性的改变,可能与细辛挥发油对螨的毒性作用机制有关。更多还原

【Abstract】 In this paper, systematic studies were done for the extraction, separation, chemical identification, mechanism and dosage formulation of medial plant asarum acaridal substances using techniques of HPLC-MS, GC-MS,1H-NMR, Electron microscopy et al. The acaricidal activity to Tetranychus urticae was confirmed, and the acaricidal mechanisms were primarily elucidated, also, an acaricidal monomer was isolated, of which chemical structure was identified, meanwhile, a practical dosage formulation was prepared. The research results laid a solid foundation for further development of asarum acaricidal substances.The toxicity of methanol extracts of 20 medicinal plants to female adults and eggs of T. urticae were evaluated. Bioassay showed that Areca catechu L. Asarum heterotropoides var. mandshuricum, Coptis chinensis Franch, Melia toosendan Sieb. et Zucc, and Sophora flavescens Ait possessed better biological activity toward the female adults and eggs of T. urticae, with 100% modified mortality for female adults at concentration of 10 mg/mL after 72 h treatment, and≤40% egg hatching rate at concentration of 10 mg/mL after 5 days treatment.Asarum demonstrated excellent and was chosen for further acaricidal assay. After 24h treatment of 1mg/mL asarum extract for female adults (24 h) and eggs (5 d), the modified mortality was 66.5% for female adults and 45.7% for eggs, and the LD50S were 0.57 mg/mL and 0.88 mg/mL for female adults and eggs respectively. The asarum extract showed oviposition inhibition, at concentration 1 mg/mL, the inhibition rate was 60.25% and 61.45% at 24 h and 48 h respectively, while at 4 mg/mL, the inhibition rate was 92.89% and 94.7% at 24 h and 48 h respectively. The asarum extract also showed deterrent activity, at 0.5 mg/ml, the deterrent rate was 57.14% after 24 h, it declined to 50% after 72 h; at concentrations more than 2 mg/ml, the deterrent rates from 24 to 72h were nearly to 100%. The asarum extract also showed stomach toxicity, which was indicated by the mortality of female adults and oviposition inhibition, but this toxicity was weaker than the direct touch toxicity. At concentration of 1 mg/mL, the mortality of female adults was 28.2%, and oviposition inhibition was 53.1% after 48 h treatment. At 8mg/mL, the modified mortality was 82.05% for female adults, and oviposition inhibition rate reached to 92.66%.The acaricidal mechanisms of asarum extract were elucidated. The mite ultrastructure after asarum treatment showed various changes of the epidermis and cell structure, such as water loss in surface, mite body shrinkage, the integumentary lobes gap becoming bigger, and the granules becoming small. The asarum extract was found to inhibit acetylcholinesterase, Na+, K+-ATPase and mixed function oxidases; increase activities of superoxide dismutase and catalase; increase early and decrease latter the activity of glutathiones-transferase, there were no significant changes found for monoamine oxidase and esterase. The results hint acetylcholinesterase, Na+, K+-ATPase and mixed function oxidases are the targets of asarum extract, and oxidative stress is involved in the mechanism of toxicity to T. urticae.The main acaricidal monomer of asarum was isolated and identified. Tracking acaricidal activity, separation and purification of acaridal substances of asarum were done. Applying column chromatography and crystallizatin/recrytallization techniques, a monomeric compound was isolated. HPLC-MS and GC-MS and 1H-NMR analysis revealed the monomer was (-) asarinin. Bioassay showed (-) asarinin possessed good activity against T. urticae with LC50 of 0.1237 mg/mL, indicating the toxicity of (-) asarinin was more than that of asarum extract.Emulsifiable concentrate of asarum extract was prepared. The final resulted formulation was 10% asarum extract+80% solvent (xylene)+10% emulsifier mixture (3% pesticide emulsifier 500+2% pesticide emulsifier 600+5% tween-80). Emulsification, dispersion, the stability, low temperature stability, heat storage stability and volatility of the prepared EC met the requirements of EC quality. Bioassay results for determining indoor toxicity against T. urticae showed that 400 X diluted asarum extracts EC possessed comparable toxicity with 3000 X diluted 15% pyridaben EC.The chemical components and effect on T. urticae of asarum essential oils were primarily investigated. Chemical analysis by GC-MS showed that 9 major constituents composed 86.15% of the total peak areas, methyleugenol (22.9%) was the most abundant, followed by safrole (19.8%). Bioassay showed that the asarum essential oils possessed fumigant toxicity and deterrent activity, at 8μg/mL, the mortality was 72.6%,100% after 24 and 48 respectively, the deterrent rate 86.1%. Asarum essential oils affected some enzymes of T. urticae, they decreased the mite activity of Na+, K+-ATPase, and increased at first and decreased latter the activity of glutathione S-transferase, suggesting the two enzymes are involved in the toxicity mechanism of asarum essential oils.更多还原