天然黄酮类化合物的抑藻活性、构效关系及其机制研究

Harmful algae blooms (HABs) induced by the increasing water eutrophication problems frequently occur in recent years. The chemical ecological method using plant allelochemicals as antialgal agents has attracted worldwide research attentions. Natural flavonoids, second metabolites which widely exist in plants and easy to be isolated, are believed to be novel antialgal allelochemicals. However, the relationship between antialgal abilities of flavonoids and their chemical structures is still unclear, meanwhile the mechanism of algal control by flavonoids has not yet to be fully understood. In this project, Microcystis sp. and Planktothrix sp. are chosen as experimental microorganisms to explore the law of algal inhibition by natural plant flavonoids, and the "chemical structures - antialgal ability" relationships of congeneric flavonoids will be systematically studied using different theoretical tools including 2D-QSAR, HQSAR and 3D-QSAR. By utilizing advanced biological analysis instruments such as FCM, PAM and BIA et al., together with modern molecular biology technologies including multiple fluorescent staining, RT-PCR and TUNEL et al., mechanisms of algal control by plant origin natural flavonoids will also be clarified from the following aspects: (1) physiological and morphological changes of target algal cells; (2) the observation of interactions between algal cells and chemicals; and (3) the programmed cell death (PCD) in phytoplankton. The implementation of this research project would provide a theoretical basis to the development of novel algaecides using natural flavonoids as leading compounds and be benefit to the water pollution control in aquatic environments with eutrophication problems.

近年来，水体富营养化日益加剧、有害藻华频发，利用植物化感物质控藻的化学生态学方法已引起广泛关注。天然黄酮类化合物资源丰富且易于提取，有望成为藻类防治领域的明星物质，但目前其抑藻活性构效关系及微观机制还不清楚。本项目拟选取微囊藻和浮丝藻为研究对象，揭示黄酮、黄酮醇、二氢黄酮和二氢黄酮醇等系列天然黄酮类化合物抑制有害藻类生长的规律，借助2D-QSAR、HQSAR、3D-QSAR等定量构效关系理论工具，系统研究黄酮天然分子抑藻活性与其分子结构之间的关系，利用流式细胞仪、浮游植物荧光仪、蛋白质互作仪等先进生物分析仪器，结合多重荧光染色、RT-PCR、TUNEL检测等现代分子生物学技术，从黄酮胁迫下藻细胞生理活性变化、抗逆形态、相互作用过程和黄酮诱导藻细胞产生类凋亡等方面，阐明植物天然产物黄酮类化合物抑藻的微观作用机制，为以天然黄酮为先导的高效新型抑藻剂开发和水体富营养化污染控制提供理论依据。

本项目以水华优势藻种铜绿微囊藻（Microcystis aeruginosa）为研究对象，揭示了系列天然黄酮类化合物抑制有害藻类生长的规律，借助 2D-QSAR和3D-QSAR 定量构效关系理论工具，系统研究了黄酮天然分子抑藻活性与其分子结构之间的关系；利用流式细胞仪、浮游植物荧光仪等先进生物分析仪器，从黄酮胁迫下藻细胞光合作用和细胞膜系统的动态变化情况，阐明植物天然产物黄酮类化合物抑藻的微观作用机制；通过微球化缓释技术开发了黄酮仿生化感抑藻剂，为以天然产物为先导的高效新型黄酮类抑藻开发提供技术指导。取得的主要研究成果包括：（1）系统研究了植物天然产物来源的系列黄酮类化合物抑制有害藻类生长的规律，发现5,4' -二羟基黄酮、山奈酚和木犀草素具有较强的抑藻活性；初步揭示了羟基数目对抑藻效果的影响规律。（2）在化感物质抑藻活性研究中创新性地引入定量构效关系手段，构建了噻唑烷二酮类化合物抑藻活性2D-QSAR模型并总结了其建模策略；进而对上述系列植物天然黄酮的抑藻活性进行QSAR分析，发现黄酮分子的极性越大、通透性越高以及疏水性越佳，其抑藻活性越高。（3）对天然黄酮化合物的抑藻活性进行3D-QSAR模型分析（CoMFA模型和CoMSIA模型），发现黄酮分子3位亲水基团、A环5位带负电氢键供体基团、B环4'位亲水性氢键供体基团以及3'、4'位带负电的基团取代有利于其抑藻活性的增加，反之，在A环6、7位引入带负电且为氢键供体基团或在B环2'、3'、5'和6'位引入氢键供体基团不利于黄酮化合物的抑藻活性。（4）研究了三种典型黄酮化合物5,4'-二羟基黄酮、芹菜素、木犀草素对铜绿微囊藻的细胞膜系统和光合作用的影响，发现木犀草素主要是通过中断光合系统II反应中心的电子传递，但5,4'-二羟基黄酮和芹菜素主要是影响藻细胞的形态和细胞膜系统，而三者均对铜绿微囊藻造成一定的氧化胁迫。（5）用医药微球化技术制备得到黄酮仿生抑藻剂，模拟了植物天然化感抑藻作用能持续缓慢地向水体中释放黄酮物质，较直接投加黄酮物质方式更为合理、可控。