鹅绒藤属植物中菲并吲哚里西啶生物碱分离、结构修饰及抑菌机理研究

Blue and green mold caused by the phytopathogens penicillium italicum and P. digitatum, respectively, are the two most common postharvest disease of citrus fruits. The study will focus on the extraction and isolation of the phenanthroindolizidine alkaloids from three plants of the genus Cynanchum L. targeted by the antifungal effects on the two fungal phytopathogens. Synthesis and structural modification, structure—activity relationships and antifungal mechanism of those phenanthroindolizidine alkaloids are also study herein. Various kinds of chromatography columns and spectroscopy methods will be used to the isolation and identification of the antifungal phenanthroindolizidine alkaloids from the three plant extracts. Synthesis and structural modification of the main alkaloid components are based on the [2+4] cycloaddition of indole ring. Structure—activity relationships of the natural derivative phenanthroindolizidine alkaloids and their synthetic derivatives are discussed to determine the active group. As for antifungal mechanism, the influences of the main active constituents on the hyphae and ultrastructure morphology, macromolecular content, the activities of key metabolic enzyme and metabolomics analysis by 1H-NMR will help us to explore the antifungal mechanism of phenanthroindolizidine alkaloids. The project will provide reliable methods and theoretical basis for the application of natural plant constituents in the control of postharvest disease of fruits and vegetables.

由意大利青霉和指状青霉引起的青霉病和绿霉病是柑橘采后贮藏过程中最易发生的两种病害。本研究以抑制这两种病原菌活性为导向，研究三种鹅绒藤属植物中抑菌活性成分菲并吲哚里西啶生物碱类化合物的提取分离、结构鉴定、合成及结构修饰、构效关系及抑菌机理。采用多种柱色谱分离技术，从这三种鹅绒藤属植物中分离并鉴定菲并吲哚里西啶生物碱活性成分；通过[2+4]环加成合成吲哚环的新策略研究主要活性成分的全合成路线及进行结构修饰；比较天然植物中菲并吲哚里西啶生物碱与合成的系列产物的抑菌活性，明确其构效关系；从主要抑菌成分对菌丝形态和超微结构、生物大分子含量、代谢关键酶活性和菌丝体1H-NMR代谢组产物的影响，阐明其抑菌机理。为阐明天然植物活性成分防治果蔬采后病害研究提供可靠的实验方法和理论依据。

由意大利青霉(Penicillium italicum)和指状青霉(P. digitatum)侵染引起的柑橘青、绿霉病，占柑橘采后真菌腐烂的90%，给江西省柑橘生产造成巨大的经济损失。本项目前期发现三种鹅绒藤属植物(白薇、合掌消、徐长卿)乙醇提取物对柑橘采后青、绿霉病具有显著的抑菌效果。本项目主要研究白薇、合掌消及其抑菌活性成分抑制柑橘采后意大利青霉和指状青霉的作用机制。结果表明：1）从白薇抑菌提取物中分离鉴定出6个菲并吲哚里西啶生物碱成分：10 β-N-氧化-7-脱甲氧基娃儿藤碱(1)、9-脱氢-7-脱甲氧基娃儿藤碱(2)、9,14-脱氢7-脱甲氧基娃儿藤碱(3)、14羟基-N-氧化-7-脱甲氧基娃儿藤碱(4)、10α-N-氧化-7-脱甲氧基娃儿藤碱(5)和7-脱甲氧基娃儿藤碱(6)，其中7-脱甲氧基娃儿藤碱(7-demethoxytylophorine, DEM)为白薇中最主要抑菌生物碱；2）TMT定量蛋白组学揭示DEM对意大利青霉菌的抑制作用可能通过破坏TCA循环和氨基酸、脂肪酸、嘌呤等代谢途径；3）阐明了DEM通过膜质过氧化作用，致使菌丝体内ROS大量积累，通过扫描电镜和荧光显微镜观察发现，DEM处理的菌丝形态异常，坍塌破损，细胞内部结构杂乱分布，分区模糊或消失，导致菌丝体内能量代谢失衡，从而抑制菌丝生长及孢子萌发，达到减少柑橘采后绿霉病发生；4）研制出植物源抑菌复合涂膜应用于脐橙、新余蜜桔和金沙柚采后贮藏保鲜，显著减少柑橘采后真菌腐烂的发生，抑制果实的失重率、呼吸强度和丙二醛(MDA)含量，延缓柑橘果实采后营养品质的降解速率，维持果实贮藏中后期较高的抗氧化酶活性，提高果实的保鲜效果；5）基于转录组分析DEM诱导椪柑果实抗性提高的作用机制：DEM提高椪柑果实采后抗病性的诱导机制可能通过平衡AsA-GSH循环和提高ROS清除能力，减少果实青霉病害发生。相关成果发表在Food Chemistry、Postharvest Biology and Technology、Pesticide Biochemistry and Physiology、LWT - Food Science and Technology、果树学报等国内外Top期刊上，登记2项江西省科技成果，培养博士研究生3名，硕士研究生5名。