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)