两种铁线莲属植物新颖结构杀虫活性皂苷的发现及杀虫机理研究

As the safety of agricultural products and environment was caused by residues and resistance of pesticide, the process of discovering novel botanical insecticide with environmental compatibility was more and more urgent. The applicant previously found the crude extracts of Clematis lasiandra and Clematis aethusifolia appeared significant insecticidal activity, and identified four new cytotoxic saponins from the above species. The primary evaluation of those saponins for their insecticidal activity, demonstrated that the saponins with a free carboxyl group at C-28 displayed significant stomach toxicity and antifeedant activity against 3 instar larvae Plutella xylostella. Based on the above research, we will use saponins’ characteristic NMR spectroscopy to quickly isolate insecticidal active novel saponins from the above two Clematis, and identify chemical structure for monomeric saponins using various spectroscopic methods. Apply P. xylostella as target insect, and use insect cytotoxicity, stomach toxicity, antifeedant activity, and the growth and development inhibition as the comprehend evaluation index, to analysis their insecticidal activity and structure activity relationship for the single saponin, determine the main pharmacophore for insecticidal active saponins, and finally provide 2 or 3 lead compounds for environment-friendly botanical pesticide. Through the methods of observation of toxic symptom, test the activity of midgut digestive enzymes, and histological observation of midgut tissue for the target insect after managed insecticidal active saponins, to analyze the impact of saponins on the target pest digestive system, clarify the insecticidal action site and target of saponins, and finally provide theoretical guidance for saponins to be developed as insecticides to control lepidopterous pests.

随着农药残留及抗药性增强带来的农产品质量安全及环境安全等问题，研发环境兼容性较好的新型植物源杀虫剂已迫在眉睫。申请者前期发现毛蕊铁线莲和芹叶铁线莲粗提物具有杀虫活性，并从中发现4个具细胞毒活性的新皂苷，杀虫活性显示具有C-28位自由羧基的皂苷对3龄小菜蛾表现出显著的胃毒和拒食活性。基于此，本课题拟以皂苷NMR特征谱学为导向，快速追踪分离两种铁线莲中具新颖结构的杀虫活性皂苷，运用各种波谱学手段鉴定单体皂苷的化学结构；以小菜蛾为指示害虫，采用昆虫细胞毒测试、胃毒、拒食和生长发育抑制等方法综合评价皂苷的杀虫活性，分析单体皂苷的构效关系，明确其主要药效团，为开发新型无公害植物源农药提供2-3个先导化合物；通过杀虫活性皂苷对靶标害虫的致毒症状观察、中肠消化酶活性测定及中肠组织病理学观察，分析皂苷对靶标害虫消化系统的影响，阐明皂苷杀虫作用部位与靶标，为大量有待开发的皂苷用于防治鳞翅目害虫提供理论指导。

随着农药残留及抗药性带来的诸多问题使得研发环境兼容性较好的新型植物源杀虫剂已迫在眉睫，从植物资源中挖掘和发现具有新颖结构的天然产物是新型农药创制的源头。项目主要研究内容涉及铁线莲属植物皂苷类化学成分的分离纯化、结构鉴定与构效关系、杀虫活性评价以及杀虫作用机理研究。重要结果为：①从芹叶铁线莲、毛蕊铁线莲和硬毛棘豆中分离鉴定了59个化合物，包括三萜皂苷24个，具有显著杀虫活性的皂苷14个；其中，新化合物13个，包括新三萜皂苷6个。②从铁线莲属植物中首次发现了具有达玛烷型结构的新原人参二醇型四环三萜皂苷：毛蕊铁线莲新苷A和B，其可作为西洋参等名贵中药材同类有效成分获取的资源替补。③明确了C-28位自由羧基是铁线莲皂苷杀虫活性的主要官能团，C-23位的羟基，以及C-3和C-28位糖基数目引起的分子结构对称性能够降低皂苷的胃毒活性；C-3位葡萄糖醛酸与C-30位的自由羧基是Azukisapogenol型三萜皂苷杀虫活性的主要官能团。④单糖链铁线莲皂苷对小菜蛾与蚜虫的作用方式主要包括胃毒、拒食/忌避、生长发育抑制三个方面，但无明显的触杀作用；皂苷对小菜蛾的致毒症状主要为虫体大小与颜色变化、尾部萎缩和蜕皮失败等，蚜虫主要为虫体颜色的变化；杀虫作用机理主要是通过抑制消化酶系的胃蛋白酶和α-淀粉酶，但皂苷类型与昆虫种类对害虫体内的酶活性影响显著；杀虫作用靶点为中肠线粒体、细胞核与微绒毛等细胞器。⑤从秦巴山区毛蕊铁线莲中发现了具有显著抗烟草花叶病毒活性且具有芳基糖取代的新黄酮苷，首次发现了具有抗植物病原细菌的8-O-4′结构骨架的新木脂素。研究结果充分体现了秦巴山区铁线莲属植物皂苷类次生代谢产物的结构独特性与农用活性开发价值，也为其他铁线莲属植物新颖糖苷的挖掘与农用活性研究提供了思路。