LC-MS/MS导向红树林内生拟盘多毛孢菌抗肿瘤活性产物快速发现

Mangrove endophytic fungi, reportedly living in a mutualistic symbiosis with their hosts against the environmental high salinity stress, used to be recognized as rich sources of biologically active natural products that were indispensable for pharmaceutical or agrochemical applications. Due to its special ecological status, numerous metabolites with unforeseeable structures and promising bioactivities had been discovered. One fungal genus which is especially productive with regard to the accumulation of a diverse array of mostly bioactive compounds is Pestalotiopsis., which chemical investigation initiated from 1989. Since the discovery of the world's first billion dollar anticancer agent taxol from one strain of this fungal genus, interest to find bioactive compounds from the genus has increased considerably. Over the past few years, substantial advances in biotechnologically assisted chemistry have surmounted the barriers of limited methodology and sparked a resurgence in the discovery of interesting compounds with novel structures. Our previous chemical investigations of Pestalotiopsis spp. led to the discovery of over 50 different compounds with 40 of them being new natural products including polyketides and alkaloids. Application of LC-MS/MS-guided rapid dereplication and subsequent MTT bioassay-guided fractionation based protocol was developed for our further studies of antitumor metabolites from mangrove-associated endophytic fungi Pestalotiopsis. The isolation and purification of natural products was performed by using some chromatography techniques. The structures of all these will be unequivocally determined by one- and two-dimensional NMR spectroscopy, UV spectroscopy, IR spectroscopy, as well as by LC-MS/MS spectrometry. Their antitumor activies will be evaluated using MTT method against cells L5178Y, MCF-7, HL-60, A549, HepG-2, A375 and Hela. The research project is intended to lead the rapid discovery of antitumor drug candidates from mangrove-associated endophytic fungi Pestalotiopsis, which have established a strengthened function on broad-spectrum caner cells and lessened side effects. It is hoped that the more efficient and effective application of these natural products will promote the rapid microbial drug discovery process and construct a new perspective access to ecological sustainable development.

红树林内共生真菌与宿主植物在盐胁迫等特殊生境条件下互惠共生，产生较多结构新颖、活性显著多样的次生代谢物，为新药筛选、研发提供了丰富的模式结构化合物。拟盘多毛孢菌Pestalotiopsis中高效抗癌药物紫杉醇taxol的发现，使这属菌的药用开发潜力得到了证实，而从中寻找新型活性物质也成微生物学和天然药物化学的一个研究新热点。代谢产物快速排重分离与筛选出新的活性化合物是微生物药物研发的关键技术之一，本研究拟在前期工作从本属真菌中分离鉴定40种新化合物的基础上，以海南红树林内共生菌Pestalotiopsis为研究对象，LC-MS/MS导向代谢物快速排重与抗肿瘤活性导向追踪技术为依托、化学筛选为辅，采用先进色谱分离技术，以快速成功获得化学结构新颖、抗肿瘤活性显著的天然产物，运用现代波谱学进行结构鉴定，为高效低毒抗肿瘤药物的研发提供多个候选成分，也为微生物新药快速发现和生态可持续发展提供新的思路

本课题以海南红树林内共生拟盘多毛孢菌Pestalotiopsis为研究对象，代谢物快速排重与抗肿瘤活性导向追踪技术为依托、化学筛选为辅，采用先进的色谱分离技术，运用现代波谱学进行结构鉴定，以快速成功获得化学结构新颖活性显著的天然产物。.1、完成红树林微生物和拟盘多毛孢属真菌天然产物的文献调研工作，对本领域内相关工作进行系统综述；.2、完成微生物实验平台的搭建，实现海南红树林内生菌根真菌优势菌株的分离、纯化、和鉴定工作，获得拟盘多毛孢属真菌纯菌株，综合用形态学方法及分子生物学鉴定手段对获得的部分真菌进行种属鉴定与系统发育多样性分析，共获得本属真菌纯培养11株；.3、完成红茄苳内生拟盘多毛孢属真菌的初步分离鉴定，尝试在在细胞组分水平上，通过分析微生物细胞膜上磷脂脂肪酸的组分来鉴定微生物的种属，绘制海南红树林植物红茄冬共生拟盘多毛孢特征的脂肪酸甲酯图谱，鉴定挥发性成分18个；.4、完成高效锁定内生菌拟盘多毛孢代谢活性产物，运用各种现代分离技术追踪制备与分离，有针对性的分离纯化次生代谢产物30个，运用现代波谱学进行结构表征鉴定成分16个，其中2个新骨架化合物，1个新化合物；.5、与红树林内生菌一样，盐生药用植物狗牙根内生真菌也是一类特殊的耐盐微生物，我们于是增添了这方面的研究内容，并取得了阶段性成果，获得次级代谢产物15个，生物碱类成分10个，1个是新骨架；.6、完成初步抗菌活性筛选，以致病菌和厌氧菌为测试菌，采用滤纸片法和微量稀释法对分离获得的Pestalotiopsis菌真菌纯培养和狗牙根内生真菌代谢产物进行活性筛选，发现抗病原菌活性拟盘多毛孢菌株4株，弱活性代谢产物4个。.7、完成《红树林微生物天然产物化学研究》专著的撰写与出版；.8、课题计划立项3年内完成发表学术论文4-5篇，现已发表论文11篇，总影响因子≥20.0，均为SCI和EI收录，在国内外学术大会上作大会或分会报告6次，有关成果已获海南省科技进步奖三等奖、第四届海南省高等学校优秀科研成果二等奖、吴多泰博士科研成果三等奖，结题人成功入选教育部“新世纪优秀人才支持计划”人选，已按计划超额高质量完成。.以上研究将为微生物新药的快速发现和生态可持续发展提供新的思路，也将对海南省的医药事业和海南区域创新体系建设与经济、社会发展提供技术保障和新的思路。