功能化纳米Fe3O4材料靶向分离分析天然产物中酶抑制剂

Natural products have historically been invaluable as a source of therapeutic agents, however, the major bottleneck that continues to affect drug discovery is the target purification and analysis of active compounds from the complex extract, since natural products have many active compounds with different polarities and concentrations and sometimes the structures of the compounds would change because of the decomposition. As part of our continuous efforts on rapid screening and isolation of active compounds from natural products, the present work described a novel-immobilized enzyme strategy on Fe3O4 nanospheres for target purification and analysis of active compounds from natural products due to its high sensitivity and specifity, excellent suspension stability and convenient solid-liquid separation. PTP 1B, DPP IV and α-glucosidase (three drug targets for type 2 diabetes) were stably immobilized onto Fe3O4 magnetic nanospheres, respectively, and then the functional magnetic nanospheres were used to screen and isolate active compounds from the corresponding natural extracts. HPLC-MS/MS was used for compounds analysis and identification, while HSCCC was applied for the preparative purification of target compounds. Moreover, the structure-activity relationships and synergies of all active compounds were interpreted. The present work demonstrated a facile way with high efficiency and specificity to purify and identify active compounds from complex natural products.

天然产物是创新药物的主要源泉，其活性成分快速靶向分离分析是热点和难点问题。本项目针对天然产物成分复杂多样、难于快速分离分析、易变等特点，结合课题组在分离分析新方法研究中形成的基础和优势，开展基于功能化纳米Fe3O4材料的天然产物中酶抑制剂快速靶向分离分析新方法，即制备稳定、特异性强、磁响应强的酶(三种II型糖尿病药靶：蛋白酪氨酸磷酸酯酶-1B，二肽基肽酶IV和α-葡萄糖苷酶)负载的纳米Fe3O4材料，基于亲和吸附原理，在分子水平上快速靶向分离天然产物中酶抑制剂；配以HPLC-MS/MS分析鉴定；并采用HSCCC导向制备活性成分；建立集靶向分离-高通量分析-高效制备于一体的天然产物中酶抑制剂快速分离分析新方法，以期获得大量结构新颖的活性成分。同时系统研究各单体的构效关系以及化合物之间的协同作用机理。研究成果为天然产物活性成分的快速分离分析提供一个简便、高效、灵敏、特异性强的研究思路和方法。

天然产物活性成分结构多样，含量和极性差异明显，因此活性成分的快速靶向分离分析是难点问题。本项目基于课题组前期在分离分析新方法研究中形成的基础和优势，首先合成了靶标酶(α-葡萄糖苷酶、PTP1B、DPP IV、环氧化酶、黄嘌呤氧化酶、络氨酸酶等)和血清白蛋白负载的磁性亲和纳米材料，优化了合成和垂钓工艺，得到的磁性亲和材料稳定性强、灵敏度高、特异性强。接着基于亲和吸附原理，结合HPLC‒DAD‒QTOF MS/MS分析技术在分子水平上快速识别和鉴定黄芪等多种天然产物中的活性成分。考虑到离线筛选时存在试剂使用量大、操作步骤多等问题，我们扩展了固定化酶的应用，将其与SPR和亲和固相萃取结合，建立了快速联用筛选技术。接着建立了在线和离线二维HSCCC技术结合梯度洗脱、循环洗脱等模式靶向分离制备目标活性组分，对分离制备得到的化合物进行活性验证和构效关系分析，得到了一批活性较好的黄酮类化合物、萜类化合物和绿原酸类化合物。为了提升结构类似化合物的快速分离制备能力，我们还基于活性成分的结构特点设计合成了高吸附容量、高吸附速率的分子印迹材料，实现了同系物的快速分离提纯。本项目建立了集靶向识别‒高通量分析‒高效制备于一体的天然复杂体系中活性成分分离分析新方法，为天然产物活性成分的研究提供了简便、灵敏、特异性的研究思路。在该项目经费的资助下，发表SCI论文18篇，申请发明专利1项。培养3名博士研究生和2名硕士研究生，为天然产物活性成分识别及分离分析新技术研究积累人才。已圆满打完成项目计划书的研究任务，达到预期成果。