Chemical Compound Isolation
For the isolation of biologically active compounds from organisms, several different steps need to be completed. The different steps required to obtain a biologically active compound are: Extraction, chromatographic purification, dereplication, structure elucidation and bioassay testing. The steps do not have to follow that particular order and many steps may be completed simultaneously. In the first step, the sample may be triturated and extracted with a suitable solvent or macerated. Some solvents used are methanol:chloroform, ethanol, acetonitrile, or others. The purpose is to remove organic compounds that have a medium polarity which are considered more "drug-like". Ideally,polar compounds like salts, peptides, sugars as well as very non-polar compounds like lipids are left behind to simplify chromatography since they are not generally considered "drug-like". Drying of the sample could be completed before extraction by lyophilisation to remove any excess water and therefore limit the amount of highly polar compounds extracted.
The next step depends on the methodology of individual laboratories. Bioassay-guided fractionation is a common method to find biologically active compounds. This involves testing the crude extract or preliminary fractions from chromatography in an assay or multiple assays, determining what fractions or crude extracts show activity in the specific assays, and further fractionating the active fractions or extracts. This step is than repeated where the new fractions are tested and the active fractions are further fractionated. This continues until the fraction only contains one compound. Dereplication is ideally performed as early as possible to determine if the active compound has been previously reported in order to prevent "rediscovering" a compound. This can be performed using Liquid Chromatography- Mass Spectrometry (LC-MS) data or Nuclear Magnetic Resonance (NMR) data obtained in the biological assay-guided process and than comparing the information to that found in databases of previously reported compounds.
Structure elucidation is performed by using NMR data obtained of the compound and High Resolution Mass Spectrometry (HR-MS) Data. Tandem Mass Spectrometry can also be useful, especially for large molecules like glycolipids, proteins, polysaccharides or peptides. Completed characterization for publication purposes may require Infrared (IR), Ultraviolet-visible (UV-vis), specific rotation and melting point data. Obtaining a crystal structure via X-ray crystallography can greatly accelerate and simplify structure elucidation however, obtaining crystals can be quite difficult.
There are many different bioassays available for testing. There are anticancer, antimicrobial, antiviral, anti-inflammatory, antiparasitic, anticholesterolemic, and many other differ assays. For MTT assay and cytosolic Lactate dehydrogenase (LDH) release are common cytotoxicity or cell viability assays.
Read more about this topic: Marine Pharmacognosy
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