Methods for isolation, purification and structural elucidation of bioactive secondary metabolites from marine invertebrates

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.

Cytotoxic Activity and Secondary Metabolite Characteristics of Sea Cucumber Actinopyga sp. Methanolic Extract

“…MTT-cytotoxic assessment is based on the formation of blue formazan crystal when MTT solution reacts with a cell line. The amount of crystal is directly proportional to the viable cell line (Ebada et al, 2008). The amount of formazan crystal in control cell (untreated cell) was more abundant than that of treated cell (Figure 1).…”

“…Cytotoxic test was performed using MTT [3-{4,5-dimetilthiazol-2yl)-2,5-difenil tetrazolium bromide] method according Ebada et al (2008) with some modifications. Breast cancer (T47D) and colon cancer (WiDr) cancer cell lines were used.…”

Sitotoksisitas Ekstrak Aseton Dan Kandungan Fukosantin Rumput Laut Sargassum

“…Uji sitotoksik dilakukan dengan metode MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) menggunakan 2 jenis sel yaitu sel HeLa (sel lestari kanker dinding rahim) dan sel T47D (sel lestari kanker payudara) menurut Ebada et al (2008) Hasil determinasi rumput laut yang dilakukan di Puslit Oseanologi Lembaga Ilmu Pengetahuan Indonesia, Jakarta menyatakan bahwa biota laut yang digunakan dalam penelitian ini adalah S. cinerum dan S. polycystum. Rendemen ekstrak kasar aseton yang dihasilkan dari 100 gram rumput laut S. cinerum dan S. polycystum yang telah dikeringbekukan masingmasing sebesar 0,135 g (0,134 %) dan 0,308 g (0,305 %).…”

Anti-Inflammatory and Analgesic Effects of the Marine-Derived Compound Comaparvin Isolated from the Crinoid Comanthus bennetti

“…Recently, several new medications based on substances from these marine organisms have undergone clinical and preclinical trials [1]. Natural ingredients obtained from the ocean, especially from marine invertebrates, have become important sources of new compounds [2][3][4]. Moreover, many studies have screened the anti-inflammatory properties and activities of marine compounds for the treatment of chronic illnesses [1,4,5].…”