1. 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 = – 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:
Qi et al. (2006) Ten new antifouling briarane diterpenoids from the South China Sea gorgonian Junceella juncea. Tetrahedron 62:
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:
Yang et al. (2007) Effect of agitation on violacein production in Pseudoalteromonas luteoviolacea isolated from a marine sponge. Letters in Applied Microbiology,44 (6):
Qi et al. (2008) Antifouling and antibacterial compounds of the South China Sea gorgonians Subergorgia suberosa and Scripearia gracillis. Nat Prod Res 22(2):
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)