蛇足石杉内生真菌中新生物碱类AChEI的发现及生物活性分析

Alzheimer's disease (AD), a neurodegenerative disease, was regard as the fourth leading cause of death after coronary disease, cancer and stroke. Acetylcholinesterase inhibitors (AChEI) are effective medications for AD in clinical setting at present. However, the existing anti-AD drugs of AChEI have serious adverse effects and so it is imperative to find new AChEI drugs for AD remedy. Endophyte was a large microbial population with abundance in biodiversity and became a great natural resources pool to find novel active natural compounds. Therefore, it was promising that new AChEI or lead compounds from endophytes. Three endophytic fungi were isolated from Huperzia serrata in our previous works and found they can produced others alkaloidal AChEI except for Huperzine A. In this study, the methods of modern chromatography and spectroscopy was used to separate and identify the alkaloidal AChEI from the three endophytes fungi of Huperzia serrata based on bioactivity analysis. Ultimately, we aim to find new alkaloidal AChEI from the endophytes and to lay the foundation for screening the efficient and safe drugs for AD remedy.

阿尔茨海默氏病（简称AD）是一种神经退行性疾病，被认为是继冠心病、癌症和中风之后人类的第四大死因。迄今，使用乙酰胆碱酯酶抑制剂（AChEI）类药物是目前临床上治疗AD有效手段之一，但现有的 AChEI药物毒副作用明显或疗效有限，因此寻找新型AChEI药物一直为该领域研究热点。植物内生菌是多样性十分丰富的生物类群，已成为寻找和发现新型天然活性化合物资源宝库，从植物内生菌中筛选新型AChEI药物或先导化合物将是一条行之有效的途径。本课题组前期从蛇足石杉中分离到3株内生真菌，产物分析表明其含有不同于石杉碱甲(HupA)的其他类型生物碱类AchEI。本项目以此3株内生真菌为对象，利用现代色谱和光谱技术结合生物活性检测追踪，系统开展生物碱类AChEI活性物质的追踪分离和结构解析，并对其生物活性进行研究，以期发现新型生物碱类AChEI，最终为筛选高效低毒的新型抗AD药物或先导化合物奠定物质基础。

本项目以此3株内生真菌为对象，利用现代色谱和光谱技术结合生物活性检测追踪，系统开展生物碱类AChEI活性物质的追踪分离和结构解析，并对其生物活性进行研究，以期发现新型生物碱类AChEI，最终为筛选高效低毒的新型抗AD药物或先导化合物奠定物质基础。项目从内生真菌中共获得29个单体化合物，其中17个为活性化合物。采用生物自显影、肉汤稀释法和DPPH•自由基清除率对分离到的化合物进行抗菌及抗氧化活性的检测。抗菌实验表明，9,12-十八二烯酸-2,3-二羟基丙酯、十八烷酸-2,3-二羟基丙酯、十六烷酸-2,3-二羟基丙酯、竹红菌甲素和痂囊腔菌素D等5个化合物具抑菌作用；抗氧化实验表明，齿孔醇、曲地酸乙酯（Ethyl asterrate）和D-甘露糖醇（D-mannitol）具有一定程度的抗氧化能力；体外AChEI活性实验表明，曲地酸、曲地酸甲酯和曲地酸乙酯的对AChE具较强的抑制能力。本课题还对蛇足石杉内生真菌Shiraia sp. Slf14的次级代谢产物HupA稳定性进行了初步探讨。结果表明，内生菌Shiraia sp. Slf14在传代过程中极不稳定，通过在斜面培养基上添加微量HupA的办法来可以保持其稳定性；同时，在液体发酵培养基中分别添加添加1.0 g/L的HSE和0.2 g/L的L-赖氨酸均能有效提高HupA的发酵产量。