Introduction CAS-Croucher Funding Scheme for Joint Laboratories CAS-CF03/04.SC01 CAS-CF03/04.SC01...
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Introduction Introduction CAS-Croucher Funding Scheme for Joint Laboratories CAS-Croucher Funding Scheme for Joint Laboratories CAS-CF03/04.SC01 CAS-CF03/04.SC01 Isolation and purification of marine bioactive Isolation and purification of marine bioactive compounds compounds from marine organisms from Sanya Bay from marine organisms from Sanya Bay Hong Kong University of Science and Technology & South China Sea Institute of Hong Kong University of Science and Technology & South China Sea Institute of Oceanography Oceanography Marine organisms and their associated microbes are promising sources Marine organisms and their associated microbes are promising sources of antibiotics, antifoulants and marine drugs. In this joint project, of antibiotics, antifoulants and marine drugs. In this joint project, our objectives are: our objectives are: 1) 1) To screen for marine bioactive compounds from sponges, gorgonian To screen for marine bioactive compounds from sponges, gorgonian corals, mangrove plants and their associated microbes from Sanya Bay; corals, mangrove plants and their associated microbes from Sanya Bay; 2) 2) To isolate and purify marine bioactive compounds using bioassay- To isolate and purify marine bioactive compounds using bioassay- guided separation assay system; and guided separation assay system; and 3) 3) To identify chemical structures of bioactive compounds To identify chemical structures of bioactive compounds Sponge Sponge Acanthella cavernosa Acanthella cavernosa Bacterium Bacterium P. luteoviolacea P. luteoviolacea Fungus Fungus Fusarium Fusarium sp. sp. Materials & Methods Materials & Methods Extract by Extract by solvents solvents (I) Screen for bioactive compounds (I) Screen for bioactive compounds Marine organism Marine organism Marine microbes Marine microbes Crude Crude extract extract Test Test bioactivity bioactivity Antibacterial Antibacterial 96 well plate 96 well plate Extrac Extrac t t Bacteria Bacteria Inhibit Inhibit ion ion zone zone Bacteria Bacteria Extrac Extrac t t Petri dish Petri dish Barnacle Barnacle Tubeworm Tubeworm Bryozoan Bryozoan Anti-larval settlement Anti-larval settlement 24 well plate 24 well plate Swimming larvae Swimming larvae Extrac Extrac t t Cytotoxicity Cytotoxicity Cancer cell Cancer cell lines lines - MDA-MB-231 MDA-MB-231 - MCF MCF - HeLa HeLa 96 well plate 96 well plate Extrac Extrac t t (II) Isolation of pure bioactive compounds (II) Isolation of pure bioactive compounds Active extrac t Fractiona te by HPLC 1 2 345 6 (III) Identification of chemical structures (III) Identification of chemical structures Assay bioactivity of each fraction NMR NMR Further fractionate Pure active compound GC-MS GC-MS LC-MS LC-MS 1 HNMR HNMR 13 13 CNMR CNMR Cultur Cultur e e Research Outcome Research Outcome (I) Bioactive compound screening (I) Bioactive compound screening (III) Bacterial culture collection (III) Bacterial culture collection (IV) Others Screened >30 sponges, 20 gorgonian corals, 10 Screened >30 sponges, 20 gorgonian corals, 10 mangrove trees, 3 bacteria & 3 fungi mangrove trees, 3 bacteria & 3 fungi Obtained > 80 compounds, 20 were novel Obtained > 80 compounds, 20 were novel (II) Bioactivity of pure compounds Sesquiterpene Sesquiterpene Diterpenoids Diterpenoids Macrolactin Macrolactin Polyoxygenated Polyoxygenated steroids steroids Identified > 20 antifouling, 10 antibacterial Identified > 20 antifouling, 10 antibacterial & 10 antitumor compounds & 10 antitumor compounds Isolated > 600 microbes Isolated > 600 microbes (130 bacteria, 123 (130 bacteria, 123 actinomycetes, 27 fungi actinomycetes, 27 fungi & others) & others) Identified 150 bioactive Identified 150 bioactive strains strains Obtained HPLC profiles Obtained HPLC profiles for microbial extracts for microbial extracts Liquid N Liquid N 2 storage storage -80 -80 o C C storage storage Multiple copies Multiple copies of stocks of stocks Antibacterial lactones Antibacterial lactones O O HO HO 1 3 7 9 11 13 15 19 23 24 HO OH 1 O O HO HO HO OH 2 O O HO HO OH 3 new MIC against E. coli and B. subtilis = 0.1 µg/ml Antilarval diterpenoids Antilarval diterpenoids R 5 O OR 1 R 2 O O OH A cO O R 4 O H R 3 O 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Antilarval Antilarval sesquiterpe sesquiterpe ne ne Cytotoxic Cytotoxic sesquiterpe sesquiterpe ne ne IC 50 = 8.87 μg/mL EC 50 < 3.2 μg/ml EC 50 = 0.004 – 21.06 μg/ml Antilarval steriods Antilarval steriods EC EC 50 50 = 6.25 = 6.25 – 15.6 – 15.6 μg/ml μg/ml HO HO O OH OH ss-24 HO HO O OH H ss-28 O HO H ss-8 O O H ss-9 Published 24 research articles Published 24 research articles Q Q iu et al. (2006) New polyoxygenated steroids from the South China Sea iu et al. (2006) New polyoxygenated steroids from the South China Sea Gorgonian Gorgonian Echinogorgia aurantiaca. Echinogorgia aurantiaca. Pharmazie 61:645-647 Pharmazie 61:645-647 Qi et al. (2006) Ten new antifouling briarane diterpenoids from the South Qi et al. (2006) Ten new antifouling briarane diterpenoids from the South China Sea gorgonian China Sea gorgonian Junceella juncea Junceella juncea . Tetrahedron . Tetrahedron 62:9123-9130 62:9123-9130 Lau et al. (2006) Lau et al. (2006) Marinomonas ostreistagni Marinomonas ostreistagni sp. nov., isolated from a pearl-oyster sp. nov., isolated from a pearl-oyster culture pond in Sanya, Hainan Province, China. Int J Syst Evol Microbiol culture pond in Sanya, Hainan Province, China. Int J Syst Evol Microbiol 56:2271-2275 56:2271-2275 Yang et al. (2007) Effect of agitation on violacein production in Yang et al. (2007) Effect of agitation on violacein production in Pseudoalteromonas luteoviolacea Pseudoalteromonas luteoviolacea isolated from a marine sponge. Letters in Applied isolated from a marine sponge. Letters in Applied Microbiology,44 (6): 625-630. Microbiology,44 (6): 625-630. Qi et al. (2008) Antifouling and antibacterial compounds of the South China Qi et al. (2008) Antifouling and antibacterial compounds of the South China Sea gorgonians Sea gorgonians Subergorgia suberosa Subergorgia suberosa and and Scripearia gracillis Scripearia gracillis . Nat Prod Res 22(2): 154- . Nat Prod Res 22(2): 154- 166 166 Filed 3 US patents, 1 Chinese patent Filed 3 US patents, 1 Chinese patent Trained 2 PhD students & 2 post-docs Trained 2 PhD students & 2 post-docs Developed further collaboration between the two Developed further collaboration between the two Corals and sponges from Sanya Bay are good Corals and sponges from Sanya Bay are good sources of bioactive compounds; their sources of bioactive compounds; their associated microbes enable better compound associated microbes enable better compound supply. supply. Both physical and chemical culture Both physical and chemical culture condition greatly affect compound condition greatly affect compound production and microbial growth. Optimal production and microbial growth. Optimal condition for microbial growth mismatches condition for microbial growth mismatches optimal condition for compound production. optimal condition for compound production. Large culture collection of microbes serves Large culture collection of microbes serves as goldmine of exploration of marine as goldmine of exploration of marine bioactive compounds bioactive compounds Conclusion Conclusion Acknowledgements Acknowledgements Financial support by CAS-Croucher Funding Financial support by CAS-Croucher Funding Scheme for Joint Laboratories (CAS- Scheme for Joint Laboratories (CAS- CF03/04.SC01) CF03/04.SC01)
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Transcript of Introduction CAS-Croucher Funding Scheme for Joint Laboratories CAS-CF03/04.SC01 CAS-CF03/04.SC01...
IntroductionIntroduction
CAS-Croucher Funding Scheme for Joint LaboratoriesCAS-Croucher Funding Scheme for Joint LaboratoriesCAS-CF03/04.SC01CAS-CF03/04.SC01
CAS-Croucher Funding Scheme for Joint LaboratoriesCAS-Croucher Funding Scheme for Joint LaboratoriesCAS-CF03/04.SC01CAS-CF03/04.SC01
Isolation and purification of marine bioactive compounds Isolation and purification of marine bioactive compounds from marine organisms from Sanya Bayfrom marine organisms from Sanya Bay
Hong Kong University of Science and Technology & South China Sea Institute of OceanographyHong Kong University of Science and Technology & South China Sea Institute of Oceanography
Marine organisms and their associated microbes are promising sources of antibiotics, Marine organisms and their associated microbes are promising sources of antibiotics, antifoulants and marine drugs. In this joint project, our objectives are:antifoulants and marine drugs. In this joint project, our objectives are:1)1)To screen for marine bioactive compounds from sponges, gorgonian corals, To screen for marine bioactive compounds from sponges, gorgonian corals, mangrove plants and their associated microbes from Sanya Bay; mangrove plants and their associated microbes from Sanya Bay; 2)2)To isolate and purify marine bioactive compounds using bioassay-guided separation To isolate and purify marine bioactive compounds using bioassay-guided separation assay system; and assay system; and 3)3)To identify chemical structures of bioactive compoundsTo identify chemical structures of bioactive compounds
Sponge Sponge Acanthella cavernosaAcanthella cavernosa
BacteriumBacteriumP. luteoviolacea P. luteoviolacea
Fungus Fungus FusariumFusarium sp. sp.
Materials & MethodsMaterials & Methods
Extract by Extract by solventssolvents
(I) Screen for bioactive compounds(I) Screen for bioactive compounds
Marine organismMarine organism
Marine microbesMarine microbes Crude Crude extractextract
Test bioactivityTest bioactivity
AntibacterialAntibacterial
96 well plate96 well plate
ExtractExtract
BacteriaBacteria
Inhibition Inhibition zonezone
BacteriaBacteria
ExtractExtract
Petri dishPetri dish
BarnacleBarnacleTubewormTubewormBryozoanBryozoan
Anti-larval settlementAnti-larval settlement
24 well plate24 well plate
Swimming larvaeSwimming larvae
ExtractExtract
CytotoxicityCytotoxicityCancer cell linesCancer cell lines- MDA-MB-231MDA-MB-231- MCFMCF- HeLaHeLa
96 well plate96 well plate
ExtractExtract
(II) Isolation of pure bioactive compounds(II) Isolation of pure bioactive compounds
Active extract
Fractionate by HPLC
1 2 3 4 5 6
(III) Identification of chemical structures(III) Identification of chemical structures
Assay bioactivity of each fraction
NMRNMR
Further fractionate
Pure active compound
GC-MSGC-MSLC-MSLC-MS
11HNMRHNMR
1313CNMRCNMR
CultureCulture
Research OutcomeResearch Outcome(I) Bioactive compound screening(I) Bioactive compound screening
(III) Bacterial culture collection(III) Bacterial culture collection
(IV) Others
Screened >30 sponges, 20 gorgonian corals, 10 mangrove Screened >30 sponges, 20 gorgonian corals, 10 mangrove trees, 3 bacteria & 3 fungitrees, 3 bacteria & 3 fungi
Obtained > 80 compounds, 20 were novel Obtained > 80 compounds, 20 were novel
(II) Bioactivity of pure compoundsSesquiterpeneSesquiterpene
DiterpenoidsDiterpenoids MacrolactinMacrolactinPolyoxygenated Polyoxygenated steroidssteroids
Identified > 20 antifouling, 10 antibacterial & 10 antitumor Identified > 20 antifouling, 10 antibacterial & 10 antitumor compoundscompounds
Isolated > 600 microbes (130 Isolated > 600 microbes (130 bacteria, 123 actinomycetes, bacteria, 123 actinomycetes, 27 fungi & others)27 fungi & others)
Identified 150 bioactive strainsIdentified 150 bioactive strains Obtained HPLC profiles for Obtained HPLC profiles for
microbial extractsmicrobial extracts德国CHRIS冻干机隔氮型液氮冻存系统 超低温冰箱(-86C) 大型冷库（菌种保藏库） 多种保藏形式德国CHRIS冻干机隔氮型液氮冻存系统 超低温冰箱(-86C) 大型冷库（菌种保藏库） 多种保藏形式
德国CHRIS冻干机隔氮型液氮冻存系统 超低温冰箱(-86C) 大型冷库（菌种保藏库） 多种保藏形式德国CHRIS冻干机隔氮型液氮冻存系统 超低温冰箱(-86C) 大型冷库（菌种保藏库） 多种保藏形式
德国CHRIS冻干机隔氮型液氮冻存系统 超低温冰箱(-86C) 大型冷库（菌种保藏库） 多种保藏形式德国CHRIS冻干机隔氮型液氮冻存系统 超低温冰箱(-86C) 大型冷库（菌种保藏库） 多种保藏形式
Liquid NLiquid N22
storagestorage -80-80ooC storageC storage Multiple copies of Multiple copies of stocksstocks
Antibacterial lactonesAntibacterial lactones
OO
HO
HO
1
379
1113
15 19
23 24HO
OH
1
OOHO
HO
HO
OH
2
OO
HO
HO
OH
3
macrolactin V (1) showed strong antibacterial activity against Escherichia coli, Bacillus subtilis, and Staphyloccocus aureus with MIC value of 0.1 μ g/ml
macrolactin S (2) showed strong antibacterial activity against E. coli and S. aureus with MIC values of 0.3 and 0.1 μ g/ml, respectively, while weak activity against B. subtilis (MIC 100 μ g/ml).
newMIC against E. coli and B. subtilis = 0.1 µg/ml
Antilarval diterpenoidsAntilarval diterpenoids
R5O OR1
R2
O
O
OH
AcOO
R4O
H
R3O1
2 3 456
789101112
1314
15 16
17
18
1920
R2 = CH2OHR2 = CH2OCH3R2 = CH2ClR2= CH2OAcR2 = COOMe
Antilarval Antilarval sesquiterpenesesquiterpene
Cytotoxic Cytotoxic sesquiterpenesesquiterpene
IC50 = 8.87 μg/mL
EC50 < 3.2 μg/mlEC50 = 0.004 – 21.06 μg/ml
Antilarval steriodsAntilarval steriodsECEC5050 = 6.25 – = 6.25 –
15.6 μg/ml15.6 μg/ml
HO
HO
O
OHOH
ss-24
HO
HO
O
OHH
ss-28
O
HOH
ss-8
O
OH ss-9
Published 24 research articlesPublished 24 research articles QQiu et al. (2006) New polyoxygenated steroids from the South China Sea Gorgonian iu et al. (2006) New polyoxygenated steroids from the South China Sea Gorgonian Echinogorgia Echinogorgia
aurantiaca.aurantiaca. Pharmazie 61:645-647 Pharmazie 61:645-647 Qi et al. (2006) Ten new antifouling briarane diterpenoids from the South China Sea gorgonian Qi et al. (2006) Ten new antifouling briarane diterpenoids from the South China Sea gorgonian Junceella Junceella
junceajuncea. Tetrahedron. Tetrahedron 62:9123-913062:9123-9130 Lau et al. (2006) Lau et al. (2006) Marinomonas ostreistagniMarinomonas ostreistagni sp. nov., isolated from a pearl-oyster culture pond in Sanya, sp. nov., isolated from a pearl-oyster culture pond in Sanya,
Hainan Province, China. Int J Syst Evol Microbiol Hainan Province, China. Int J Syst Evol Microbiol 56:2271-227556:2271-2275 Yang et al. (2007) Effect of agitation on violacein production in Yang et al. (2007) Effect of agitation on violacein production in Pseudoalteromonas luteoviolacea Pseudoalteromonas luteoviolacea isolated isolated
from a marine sponge. Letters in Applied Microbiology,44 (6): 625-630. 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 Qi et al. (2008) Antifouling and antibacterial compounds of the South China Sea gorgonians Subergorgia Subergorgia
suberosa suberosa and and Scripearia gracillisScripearia gracillis. Nat Prod Res 22(2): 154-166. Nat Prod Res 22(2): 154-166
Filed 3 US patents, 1 Chinese patentFiled 3 US patents, 1 Chinese patent Trained 2 PhD students & 2 post-docs Trained 2 PhD students & 2 post-docs Developed further collaboration between the two research Developed further collaboration between the two research
teamsteams
Corals and sponges from Sanya Bay are good Corals and sponges from Sanya Bay are good sources of bioactive compounds; their associated sources of bioactive compounds; their associated microbes enable better compound supply.microbes enable better compound supply.
Both physical and chemical culture condition greatly Both physical and chemical culture condition greatly affect compound production and microbial growth. affect compound production and microbial growth. Optimal condition for microbial growth mismatches Optimal condition for microbial growth mismatches optimal condition for compound production.optimal condition for compound production.
Large culture collection of microbes serves as Large culture collection of microbes serves as goldmine of exploration of marine bioactive goldmine of exploration of marine bioactive compoundscompounds
ConclusionConclusion
AcknowledgementsAcknowledgements Financial support by CAS-Croucher Funding Scheme for Financial support by CAS-Croucher Funding Scheme for
Joint Laboratories (CAS-CF03/04.SC01) Joint Laboratories (CAS-CF03/04.SC01)
