Marine sponges are chemical factories that are sources of potential pharmaceuticals1' The isolation

1
Sponge squeezeHow sanitary can a stinky sponge
be?An analysis of antimicrobial activity in
Gulf of Maine specimen of the Halichondria sp.
marine spongeFrançoise MorisonDepartment of
Chemistry, Inco 590, University of New Hampshire,
Manchester
Abstract
Conclusion
Materials and Methods (contd)
Some of the results supported the hypothesis that
Halichondria sp. may produce toxic substances
Further study is needed to try and resolve the
uncertainties mentioned in the results, i.e.
concentration of the extracts and solvent
involvement in toxicity. Sufficient amount of
compound needs to be extracted to evaluate its
concentration. Solvent control volume and time of
evaporation must systematically be measured. NMR
analysis may be pursued in an attempt to
determine the structure of the active compound.
Structure elucidation may however require further
purification. Extraction and culture of bacteria
or separation of microalgae possibly present in
the sponge may provide additional information as
to the source of the active substance(s).
Many of the chemical substances found in marine
sponges are bioactive and research on secondary
metabolites is being pursued in the fields of
drug discovery and chemical ecology. Specimen of
Halichondria sp., a marine sponge commonly found
in the waters of the Gulf of Maine, were
investigated for their production and bioactivity
of such compounds. Three of the purified
fractions produced in this study showed to
inhibit growth of the gram (-) bacterium
Pseudomonas aeruginosa.
Extraction and purification The fresh sponge
samples (760 g, wet wt) were extracted with
methanol (1 L x 3). After concentration, the
methanol crude extract (300 ml) was submitted to
solvent-solvent partitioning into hexane, ethyl
acetate, and methylene chloride phases
successively. The ethyl acetate and methylene
chloride extracts were chromatographed on a
silica gel column using a mixture of 30 hexane
70 ethyl acetate as eluent.
Bioassay Four ethyl acetate fractions were
tested. Trypticase soy agar plates were swabbed
with Pseudomonas aeruginosa , Proteus mirabilis,
and Staphylococcus aureus. The extracts were
tested using the sterile filter paper disk
method. A blank disk and two standard antibiotic
disks (tetracycline 30µg and chloramphenicol
30µg) were used as control.
Introduction
Marine sponges are chemical factories that are
sources of potential pharmaceuticals(1). The
isolation by Werner Bergman of two marine sponge
nucleosides in the early 1950s led to the
synthesis of the anti-HIV drug AZT and the
anti-herpes drug Acyclovir (2). Since then,
thousands of new compounds have been reported
(1,3). Many have bioactivity against cancer,
pathogenic viruses, bacteria, and fungi (1). Some
are currently undergoing clinical trials (4). The
ecological function of these metabolites is
poorly understood. Their role in feeding
deterrence, allelopathy, and control of bacterial
attachment have been described (5,6). It is
speculated that some sponge metabolites may in
fact be synthesized by symbiotic microorganisms
that can represent up to 40 of the sponge
biomass (7). Little is known of the nature of
these associations. Two species of marine sponges
of the genus Halichondria are commonly found in
the Gulf of Maine. Both are described as having a
sulfur odor, which suggests potential
biosynthesis of toxic chemicals. A search of the
Napralert database determined that among marine
sponges occurring locally, species of
Halichondria were the most studied, but all
studies were done abroad. (8). A variety of
steroids were isolated from H. bowerbanki by an
Italian group (8). Extracts from both H. panicea
and H. bowerbanki that were tested by Andersson
et al. (Sweden) for antimicrobial activity were
reported inactive (8).
Literature cited
Results
Three of the fractions obtained from the ethyl
acetate extract showed some inhibition of
Pseudomonas aeruginosa. Inhibition was observed
on that bacterial species only. Some zones had an
irregular shape. These fractions were eluted from
the column successively and may therefore contain
the same compound. While results of this study
do not support what had been reported by
Andersson et al., the group had tested different
extracts (H2O, Pet. Ether, CHCl3, and methanol)
(8). Because the extract concentrations could not
be calculated, due to minimal immeasurable amount
of product extracted, it is not possible to
assess the significance of the zone of inhibition
observed. Furthermore, whether inconclusive zones
of inhibition observed on some plates are related
or not to possible toxicity of lingering solvent
remains unclear. No inhibition was observed when
solvent testing was repeated.
1. Bhakuni D.S., Rawat D.S. Bioactive Marine
Natural Products. New York Springer 2005.
382 p. 2. Harbor Branch Oceanographic Institution
Media Lab website. The pipeline and the finish
line the first wave of marine-derived drugs.
lthttp//www.marinebiotech.org/pipeline.htmlw1gt 3.
Kornprobst Jean-Michel.. Les medicaments de la
mer. Faculté de Pharmacie et Institut Substances
et Organismes de la mer (ISOmer) Université de
Nantes. 2001. 4. Simmons T. L., Andrianasolo E. ,
McPhail K., Flatt P. , Gerwick W. H. Marine
natural products as anticancer drugs. Molecular
cancer therapeutics online 2005 4(2) 333-342.
Avail. From http//mct.aacrjournals.org/cgi/repri
nt/4/2/333. 5. Kelly S.R., Jensen P.R., Henkel
T.P., Fenical W., Pawlik J.R. Effects of
Caribbean sponge extracts on bacterial
attachment. Aquatic microbial ecology 2003
31175-182. 6. Assmann M., Lichte E., Pawlik
J.R., Kock M. Chemical defenses of the Caribbean
sponge Agelas wiedenmayeri and Agelas conifera.
Marine ecology progress series 2000
207255-262. 7. Guyot Michele. Intricate aspects
of sponge chemistry. Zoosystema 2000 22(2). 8.
Napralert. Program of collaborative research in
the pharmaceutical sciences. College of Pharmacy.
University of Illinois at Chicago.lthttp//www.napr
alert.org/gt
Table 1. Three fractions inhibited
the growth of P. aeruginosa.
P. aeruginosa 1 P. mirabilis
S. aureus ________________________________
_______________________________ FM2-I-(2-3)
Inhibited Inconclusive2 Negative
FM2-II-(4) Inhibited
Negative Negative FM2-III-(5)
Inhibited Inconclusive2 Negative
FM2-IV-(6) Negative
Negative Negative Tetracycline
Resistant Resistant3
Sensitive Chloramphenicol Resistant4
Sensitive Sensitive 1-Notorious for
its resistance to antibiotics
4- expected 2-Inhibition
ring present around solvent control 3-Reported
occurrence of R-plasmid in strains of P.
mirabilis Reported antimicrobial sensitivity
are based on the guidelines of the Kirby-Bauer
method (1995).
Fraction FM2-I-(2-3) with ring of inhibition
Fraction FM2-II-(4) showing inhibition. Note
irregularity around one disk.
Materials and Methods
Collection of animal material A total of 6
organisms of the sponge genus Halichondria were
collected on March 20, 2007, within a 50 yard
radius at a depth of 3 feet off Laighton Point,
Pembroke, Maine (44o55N66o55W). Water
temperature was 36oF the water salinity was 32
ppt.
Acknowledgements
Blank disk with solvent
I want to thank Dr. Lorraine Doucet, Dr. Sarah
Kenick, Dr. Stephen Pugh, Professor Allan Ray,
and Keith Legro, for their help, advice and
support. I also want to acknowledge Dr. William
Sponholtz from Cushing Academy, Ma., for sharing
methods used in his own research. Thank you to
Dr. Richard Johnson from the Durham Chemistry
department for lending the equipment. Thanks
Jeremy Neal for sharing the dishwashing.
Halichondria specimen
Area of collection
Chloramphenicol (30µg) (no ring of inhibition)
For further information
Tetracycline (30µg) with small ring of inhibition
(lt14 mm)
Please dont hesitate to contact the author at
frhop_at_comcast.net