精油主要活性成分单萜的杀螨功效及作用机制研究

The diseases caused by mites are seriously harmful to the healthy development of livestock and public health. There is an urgent need to develop new acaricides, especially the acaricides with fumigant activity to control mites in the environment. Previous studies on the acaricidal activity of ten essential oils showed that clove, palmarosa, geranium and tea tree had effective acaricidal effects. In order to further clarify the activity basis of the four essential oils, an animal model will be established by infecting rabbits with Sarcoptes mites. In vitro exposure and fumigation experiments will be conducted with the main components of the four monoterpenes from the oils, namely eugenol, geraniol, citronellol and terpinen-4-ol. Besides, the ovicidal effects of the monoterpenes are also to discover. The toxicity will be evaluated by the observation of ultrastructural lesions of the mites after treatment by transmission electron microscope. In order to revealing the specific pathways and targets of essential oils, iTRAQ combined with two-dimensional electrophoresis and mass spectrometry will be used to identify and analyze differentially expressed proteins before and after the treatment with the strongest acaricidal effect monoterpene. The study will lay a scientific foundation for the development of new acaricides, which has important scientific significance and application value.

螨虫病给畜牧业带来巨大的经济损失，同时威胁着人类健康和公共卫生安全，当前迫切需要开发新型杀螨剂，尤其具有熏蒸活性的杀螨剂以控制环境中的螨虫。前期对十种精油的杀螨活性研究发现，丁香油、玫瑰草油、天竺葵油和茶树精油有高效杀螨活性。为了进一步阐明四种精油杀螨的活性基础，本项目采用疥螨感染家兔建立动物模型，应用体外接触和熏蒸实验对四种精油主要成分单萜，即丁香酚、香叶醇、香草醇以及4-萜烯醇进行杀螨功效评价，同时研究其虫卵杀灭活性；利用透射显微镜观察经药物作用后螨虫的亚细胞结构变化，并采用iTRAQ技术联合二维电泳质谱鉴定和分析具最强杀螨功效单萜作用疥螨前后的差异表达蛋白，揭示精油抗螨虫的具体途径及作用靶点，为开发新型杀螨药物奠定科学基础，具有重要的科学意义和应用价值。

疥疮是一种由疥螨引起的传染性皮肤病，可导致人和动物严重的瘙痒和继发性感染，甚至死亡。由于化学杀螨剂的滥用，耐药性及环境污染等问题逐渐显现，且现有药物半衰期短且缺乏杀卵活性。因此，当前亟待开发新型杀螨剂解决以上问题。精油及其成分拥有低毒性、易降解和不易产生耐药等优点，有很好的应用前景。前期研究发现丁香、玫瑰草、香茅、茶树和薰衣草精油对疥螨有很强杀螨活性。因此，本项目对以上精油的6种精油单萜（香芹酚、丁香酚、香叶醇、柠檬醛、松油烯-4-醇和芳樟醇）进行杀螨和杀卵效力评价，再利用TMT标记定量蛋白质组学技术对其杀螨机制进行探究。. 体外触杀实验结果显示：0.5%的香芹酚、丁香酚、香叶醇、柠檬醛、松油烯-4-醇和芳樟醇的LT50值分别为6.7 min、33.8 min、48.1 min、6.1 h、22.3 h和39.9 h。表明6种精油单萜都有杀螨活性，其中香芹酚、丁香酚和香叶醇的杀螨效力最强。通过改进的滤纸浸药虫卵实验，测试了5%、2.5%和1%的6种精油单萜的杀卵活性。结果显示：香芹酚、丁香酚、香叶醇、柠檬醛、松油烯-4-醇和芳樟醇的EC50分别为0.5%、0.9%、2%、4.8%、5.1%和9.8%。表明6种精油单萜都有杀卵活性，其中香芹酚和丁香酚的杀卵效力最强。利用显微镜对6种精油单萜处理过的虫卵进行间隔12 h 拍照，观察虫卵内部发育情况。结果显示：香芹酚和丁香酚可以使虫卵在0 ~ 24 h内停止发育。表明这两个精油单萜对早期阶段的虫卵具有很好杀卵效果。利用TMT标记对经香芹酚和丁香酚作用后的疥螨进行蛋白质组学研究发现，两种单萜的杀螨机制可能与氧化磷酸化、胆碱能突触、药物代谢和核糖体等通路中蛋白相关。本项目发现香芹酚和丁香酚兼具杀螨虫和杀虫卵效力，为新型植物性杀螨剂的开发利用提供实验依据和理论支撑。