Abstract
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.
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Acknowledgements
We are indebted to Professor Dr. W.E.G. Müller (University of Mainz) and Dr. W. Waetjen (Heinrich-Heine-University, Duesseldorf) for performing cytotoxicity (MTT) assays. We also acknowledge Dr. P. Tommes (Heinrich-Heine University, Duesseldorf) for his help in measuring mass spectra and Dr. W. Peters (Heinrich-Heine University, Duesseldorf), as well as Dr. V. Wray (Helmholtz Centre for Infection Research, Braunschweig), for performing NMR measurements. We also acknowledge Professor Dr. Gerhard Bringmann, Institute for Organic Chemistry, University of Wuerzburg, for performing CD spectral analysis. Continued financial support of our studies on bioactive marine natural products by the BMBF (Germany) as well as by numerous other grant agencies (e.g., DFG, DAAD, MOST, Egyptian Government Scholarship and others) is gratefully acknowledged.
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Ebada, S., Edrada, R., Lin, W. et al. Methods for isolation, purification and structural elucidation of bioactive secondary metabolites from marine invertebrates. Nat Protoc 3, 1820–1831 (2008). https://doi.org/10.1038/nprot.2008.182
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DOI: https://doi.org/10.1038/nprot.2008.182
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