Methods for isolation, purification and structural elucidation of bioactive secondary metabolites from marine invertebrates
Methods for isolation, purification and structural elucidation of bioactive secondary metabolites from marine invertebrates
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DOI：
10.1038/nprot.2008.182
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Proksch, Peter
中科院分区：
文献类型：
--
作者：
Ebada, Sherif S.;Edrada, Ru Angelie;Proksch, Peter
在过去的几十年里，海洋天然产物生物勘探已经产生了相当数量的候选药物。两种海洋天然产物最近被承认为新药：Prialt（也称为齐考诺肽）作为一种有效的止痛剂用于严重的慢性疼痛，Yondelis（也称为trabectedin或E-743）作为抗肿瘤剂用于治疗晚期软组织肉瘤。在该协议中，生物活性导向的分离，纯化和鉴定的海洋无脊椎动物，如海绵，被囊动物，软珊瑚和海百合的次级代谢产物的方法进行了讨论。为了实现这一目标，对通常冷冻干燥的海洋生物样品进行溶剂提取。接下来，通过液-液分配，然后通过各种色谱分离技术，包括薄层色谱法、真空液相色谱法、柱色谱法（CC）和制备型高效反相液相色谱法，对所获得的提取物进行分级。生物活性次级代谢产物的分离通常通过生物活性测定来监测，例如。例如，在一个实施例中，抗氧化剂（2，2-二苯基-1-苦基肼基）和细胞毒性（微量培养四唑）活性，最终产生活性成分。在进行适合于所分离化合物的物理和化学特性（特别是其脂或亲水特性）的分离程序时，应特别小心。从各种海洋无脊椎动物提取物中分离不同极性化合物的实例将在本协议中介绍。使用最近的光谱技术，特别是2D NMR和质谱分析实现结构解析。
In the past few decades, marine natural products bioprospecting has yielded a considerable number of drug candidates. Two marine natural products have recently been admitted as new drugs: Prialt (also known as ziconotide) as a potent analgesic for severe chronic pain and Yondelis (known also as trabectedin or E-743) as antitumor agent for the treatment of advanced soft tissue sarcoma. In this protocol, methods for bioactivity-guided isolation, purification and identification of secondary metabolites from marine invertebrates such as sponges, tunicates, soft corals and crinoids are discussed. To achieve this goal, solvent extraction of usually freeze-dried sample of marine organisms is performed. Next, the extract obtained is fractionated by liquid-liquid partitioning followed by various chromatographic separation techniques including thin layer chromatography, vacuum liquid chromatography, column chromatography (CC) and preparative high-performance reversed-phase liquid chromatography. Isolation of bioactive secondary metabolites is usually monitored by bioactivity assays, e. g., antioxidant (2,2-diphenyl-1-picryl hydrazyl) and cytotoxicity (microculture tetrazolium) activities that ultimately yield the active principles. Special care should be taken when performing isolation procedures adapted to the physical and chemical characteristics of the compounds isolated, particularly their lipo-or hydrophilic characters. Examples of isolation of compounds of different polarities from extracts of various marine invertebrates will be presented in this protocol. Structure elucidation is achieved using recent spectroscopic techniques, especially 2D NMR and mass spectrometry analysis.