基于配体垂钓技术筛选三七叶中环氧合酶-2抑制剂  

Screening of cyclooxygenase-2 inhibitors in Panax notoginsengleaves based on ligand fishing technique

作　　者：张帆 王薇 曹颖 张祎[1] 吴丽杰 ZHANG Fan;WANG Wei;CAO Ying;ZHANG Yi;WU Lijie(College of Chinese Materia Medica,Tianjin University of Traditional Chinese Medicine,Tianjin 301617,China)

机构地区：[1]天津中医药大学中药学院,天津301617

出　　处：《色谱》2024年第12期1126-1135,共10页Chinese Journal of Chromatography

基　　金：国家自然科学基金(82104369).

摘　　要：市售环氧合酶-2(COX-2)抑制剂如美洛昔康、塞来昔布等与传统的非甾体抗炎药相比虽能够有效治疗炎症,但是也存在着引起肝毒性、心血管疾病的风险。中药三七、人参等被证实具有良好的抗炎作用且毒副作用小,因此从中药中筛选潜在的COX-2抑制剂对于开发安全有效的新型抗炎药具有重要意义。本研究以磁性纳米材料作为高效载体,采用配体垂钓法替代传统低效筛选手段,结合固定化金属亲和技术,解决了传统酶固定方法带来的酶活性降低或酶分子堆积等问题。首先合成了聚多巴胺(PDA)包覆的Fe_(3)O_(4),在PDA表面螯合Ni^(2+),利用金属离子亲和作用固定COX-2,制备了一种绿色环保、特异性好的垂钓工具。采用扫描电子显微镜、透射电子显微镜、X射线光电子能谱仪、热重分析仪、振动样品磁强计等方法对其结构进行了表征,结果显示磁性纳米材料为核壳结构,粒径为250~300 nm,具有较大的比表面积,且磁性强、稳定性好。以COX-2抑制剂塞来昔布为垂钓目标物对COX-2的固定条件及垂钓条件进行了优化。该法成功应用于三七叶中潜在COX-2抑制剂的筛选,最终得到了13种能与COX-2相互作用的成分,经液相色谱-质谱联用技术鉴定均为20(S)-原人参二醇型皂苷。与传统的筛选方法相比,本方法简便快速,酶活性高,能够实现对特定目标物的捕获与富集。该研究对新型抗炎药的合成与开发具有指导意义,为高效地从中药复杂体系中发现抗炎药物或先导化合物提供了借鉴,同时也为三七叶的资源化合理应用提供了新的思路。Human health is increasingly affected by chronic inflammation,which can subsequently develop into chronic diseases such as cancer,arthritis,diabetes,and diseases of the cardiovascular and nervous systems.Traditional non-steroidal anti-inflammatory drugs(NSAIDs)that inhibit cyclooxygenase-1(COX-1)produce side effects,such as irritation of the gastrointestinal tract and renal toxicity.While commercially available inhibitors of the cyclooxygenase-2(COX-2)enzyme,such as meloxicam and celecoxib,treat inflammation more-effectively than traditional NSAIDs,they associate with liver-toxicity and cardiovascular-disease risks.Modern pharmacological studies have shown that Panax ginseng,Panax notoginseng,and other traditional Chinese medicines have anti-inflammatory properties;these medicines mainly contain flavonoids,alkaloids,saponins,polysaccharides,and other anti-inflammatory components,are highly efficient,and exhibit few side effects,which is advantageous.Therefore,screening potential COX-2 inhibitors from traditional Chinese medicine is greatly significant for the development of safe and effective novel anti-inflammatory drugs.However,screening these traditional medicines in an easy,quick,accurate,and efficient manner presents problem that require urgent solutions because traditional screening methods are cumbersome and inefficient.In this study,we used the ligand fishing method instead of poorly efficient traditional screening methods,with magnetic nanoparticles used as the carrier owing to their advantageous fast separation speeds.In addition,we also maintained the spatial structure and activity of the fixed enzyme by combining immobilized metal affinity technology,and selected polydopamine(PDA)as a low-toxicity metal-chelating agent owing to its strongly adhesive nature and good chelating ability for various metal ions.The application of PDA solves the problems of metal-ion leakage and limited protein load associated with traditional chelating agents.Firstly,magnetic Fe_(3)O_(4) nanoparticles were hydrothermally sy

关 键 词：配体垂钓 环氧化酶-2抑制剂 三七叶 皂苷 抗炎 

分 类 号：O658[理学—分析化学]