Isolation and purification of marine bioactive compounds CAS-Croucher Funding Scheme for Joint Laboratories CAS-CF03/04.SC01 Isolation and purification of marine bioactive compounds from marine organisms from Sanya Bay Hong Kong University of Science and Technology & South China Sea Institute of Oceanography Introduction Marine organisms and their associated microbes are promising sources of antibiotics, antifoulants and marine drugs. In this joint project, our objectives are: To screen for marine bioactive compounds from sponges, gorgonian corals, mangrove plants and their associated microbes from Sanya Bay; To isolate and purify marine bioactive compounds using bioassay-guided separation assay system; and To identify chemical structures of bioactive compounds Sponge Acanthella cavernosa Fungus Fusarium sp. Bacterium P. luteoviolacea Materials & Methods Research Outcome (I) Screen for bioactive compounds (I) Bioactive compound screening Screened >30 sponges, 20 gorgonian corals, 10 mangrove trees, 3 bacteria & 3 fungi Obtained > 80 compounds, 20 were novel Crude extract Marine microbes Extract by solvents Culture Marine organism Test bioactivity Diterpenoids Polyoxygenated steroids Macrolactin Sesquiterpene (II) Bioactivity of pure compounds Antibacterial 96 well plate Extract Bacteria Inhibition zone Petri dish Barnacle Tubeworm Bryozoan Anti-larval settlement 24 well plate Swimming larvae Extract Cytotoxicity Cancer cell lines MDA-MB-231 MCF HeLa 96 well plate Extract Identified > 20 antifouling, 10 antibacterial & 10 antitumor compounds Antilarval sesquiterpene Antilarval diterpenoids EC50 = 0.004 – 21.06 μg/ml EC50 < 3.2 μg/ml Antilarval steriods EC50 = 6.25 – 15.6 μg/ml (II) Isolation of pure bioactive compounds Cytotoxic sesquiterpene Fractionate by HPLC Assay bioactivity of each fraction Antibacterial lactones IC50 = 8.87 μg/mL MIC against E. coli and B. subtilis = 0.1 µg/ml Further fractionate 1 2 3 4 5 6 Pure active compound Active extract (III) Bacterial culture collection (III) Identification of chemical structures Isolated > 600 microbes (130 bacteria, 123 actinomycetes, 27 fungi & others) Identified 150 bioactive strains Obtained HPLC profiles for microbial extracts Liquid N2 storage -80oC storage Multiple copies of stocks 1HNMR 13CNMR (IV) Others NMR LC-MS GC-MS Published 24 research articles Qiu et al. (2006) New polyoxygenated steroids from the South China Sea Gorgonian Echinogorgia aurantiaca. Pharmazie 61:645-647 Qi et al. (2006) Ten new antifouling briarane diterpenoids from the South China Sea gorgonian Junceella juncea. Tetrahedron 62:9123-9130 Lau et al. (2006) Marinomonas ostreistagni sp. nov., isolated from a pearl-oyster culture pond in Sanya, Hainan Province, China. Int J Syst Evol Microbiol 56:2271-2275 Yang et al. (2007) Effect of agitation on violacein production in Pseudoalteromonas luteoviolacea isolated from a marine sponge. Letters in Applied Microbiology,44 (6): 625-630. Qi et al. (2008) Antifouling and antibacterial compounds of the South China Sea gorgonians Subergorgia suberosa and Scripearia gracillis. Nat Prod Res 22(2): 154-166 Filed 3 US patents, 1 Chinese patent Trained 2 PhD students & 2 post-docs Developed further collaboration between the two research teams Conclusion Corals and sponges from Sanya Bay are good sources of bioactive compounds; their associated microbes enable better compound supply. Both physical and chemical culture condition greatly affect compound production and microbial growth. Optimal condition for microbial growth mismatches optimal condition for compound production. Large culture collection of microbes serves as goldmine of exploration of marine bioactive compounds Acknowledgements Financial support by CAS-Croucher Funding Scheme for Joint Laboratories (CAS-CF03/04.SC01)