基于亲和阵列-超滤质谱技术靶向发现中药抗肿瘤ECSG的研究

Cancer, as one of the major disease, is used to be treated by the single-target drugs and multi-drug which exert unsatisfying therapeutic effects. The research of multi-targets anticancer drugs from traditional Chinese medicine resources is becoming a hot spot. It is discovered that there are many bio-active components with anti-tumor activity in herbs Chloranthus henryi Hemsl and sophora alopecuroides L. base on our previous research. But the anti-tumor activity of the single active compounds separated had less effect compared with the extract of these herbs. The effective compounds group (ECSG) can reveal the essence of traditional Chinese medicine. Thus, the method for finding ECSG targeted is a critical focus in the fields of Chinese medicine. Many conventional methods about screening anti-tomor ECSG are very waste of money, time-consuming and easy to lose bio-active component. This paper mainly study on the technique of ECSG screening by affinity array materials coupling with ultrafiltration - LC/MS technology and providing key technologies for the development of anti-tumor drugs. At first, the proper bio-targets such as microtubulin, type Ⅱ topoisomerase ect. four target proteins would be used to prepare affinity array materials by nanosized immobilization technology to capture target compounds. Then, the target compounds would be isolated and identified by ultrafiltration - LC/MS technology. The anti-tumor ECSG from the herbs Chloranthus henryi Hemsl and Sophora alopecuroides L would be quickly and efficiently targeted screen by the integrated method. Consequently, we intended to establish the most suitable method for anti-tumor drugs which are bio-active，multi-target and integrated structural identification with activity evaluation. Although ECSG is complex and low contents, it showed favorable application of targeted screening of anti-tumor ECSG in traditional Chinese medicine by affinity arrays coupling with ultrafiltration LC/MS technique.

癌症属重大疾病，临床单靶点抗肿瘤药物和多药联用存在不足。前期在国家自然科学基金资助下，多年研究确证特色中药宽叶金粟兰、苦豆子抗肿瘤活性强，有效成分复杂、活性单体不能体现中药抗肿瘤优势，有效成分群（ECSG）更能体现中药的优势和特色，从中药快速靶向获得ECSG是具有普适性的关键科学问题，高效筛选抗肿瘤ECSG有重要意义。传统ECSG筛选存在成本高、效率低、活性成分易丢失等不足。为此，本研究以微管蛋白、拓扑异构酶Ⅱ等商品化的成熟靶蛋白为靶标，利用纳米固定化技术制备组合靶标亲和阵列，以两种特色药材为例，利用超滤质谱技术分离鉴定ECSG，建立符合中医药特点抗肿瘤ECSG的靶向筛选方法，实现多靶点活性成分筛选和鉴定的一体化，探索复杂体系中结构复杂、含量低的ECSG分离筛选新方法，在大量前期工作基础上预期获得抗肿瘤ECSG和新结构活性成分，为发现创新抗肿瘤药物提供关键技术和先导化合物，具有示范意义。

.前期在国家自然科学基金资助下，多年研究确证特色中药宽叶金粟兰和苦豆子抗肿瘤活性强，但分离得到的单体化合物活性有限，有效成分群（ECSG）更能体现中药的优势和特色。为了快速筛选其中抗肿瘤ECSG，我们将β微管蛋白、DNA拓扑异构酶Ⅱ、基质金属蛋白酶2和端粒酶共4种关键靶蛋白作为组合靶标，以良好生物相容性的壳核PMMA/CS纳米粒为载体，利用纳米固定化技术成功制备了亲和靶向捕获材料，对宽叶金粟兰和苦豆子药材中抗肿瘤活性成分进行靶向识别，利用超滤技术实现分离，并结合追踪分离及HPLC/MS分析等手段进行结构鉴定。靶向筛选获得的单体化合物经活性初筛、复筛后从中药多穗金粟兰提取液中筛选得到由6个有效成分组成的ECSG，它们是3个倍半萜内脂，1个二萜，2个倍半萜二聚体。首次发现钓樟烷类倍半萜化合物具有较强的微管蛋白抑制作用，药理作用类似于紫杉醇。从中药苦豆子提取液中筛选得到3个生物碱，它们具有较好抗肿瘤活性，首次发现这些化合物能显著抑制TOPO酶。本项目所建立地从复杂体系中靶向筛选活性成分的新方法，能有效地保持了靶蛋白的原有活性，依据锁钥理论，特异性铺获目标化合物，结合HPLC/MS的分离和鉴定优势，简化了提取分离和活性验证步骤，大大提高复杂体系中靶蛋白抑制剂识别和分离的效率，同时兼具了耐用性，制备简单的特点。这种靶向捕获技术为实现复杂体系中活性化合物的快速筛选分离提供了新思路和新手段，在活性成分筛选分离方面拥有很大的应用潜能。