Biotechnology
Bioassays
Crude extract, chromatographic fractions and pure compounds were
assayed in vitro for antimicrobial activity using the agar disk diffusion
technique, against the following microorganisms: Staphylococcus aureus
(209P), Escherkhia coli (RL 65 and 57 b) and the ichthyopathogenic Vibrio
angnilldrnm. Extracts and fractions (500 mg to 10 mg, dissolved in
10 ml CH 2 C 12 ) were applied on paper disks (6 mm). The pure compound
was tested within the range 10 -1 -10 - 4 miüigrammes per millilitre.
The mimmun inhibitory concentration was determined by the agar
incorporation test. Cytotoxicity was assayed on KB cells. Screening for
antimalarial activity was performed at the laboratoire de biologie parasitaire, Muséum national d’histoire naturelle, Paris, France. Tests tor
anti-HlV activity were carried on at ULP/INSERM U74, Institut de virologie, Strasbourg, France.
Collection and cultivation
The sponge Xestospongia sp. was collected by scuba diving oh Nouméa,
Homophymia sp. was collected by scuba diving off Touho (New Caledonia).
Sponges were rinsed with sterile sea water and brushed. The resultant
suspension was then diluted in ten-fold series, the dilutions were spread
on marine agar medium (DIFCO 2216). The best plate was chosen for
isolation and further screening on Staphylococcus aureus, Vibrio angnillarum and Candida albicans.
The bacterial strains îsolated were cultivated at 27°C, in 4 x 3 1 batch
cultures (sea water DIFCO-peptone 18 g/l enriched medium) for 4 days
with rotatory shaking. Cells were harvested by centrifugation at
11 000 rpm for 15 minutes. The pellet was sonicated and extracted
successively with methanol and dichloromethane-methanol (8:2, v/v).
The organic phase was separated by centrifugation and the solvents
removed under reduced pressure.
The bacteria were identified upon the basis of 16S sequence. The Gram( + )
strain R 1588-10 isolated from the surface of the sponge Xestospongia
sp. was identified as Micrococcus luteus. The Gram (-) bacterial strain
15 31 E7 collected at the surface of the sponge Homopbymia sp. was identified as a new Pseudomonas sp. Voucher specimens are conserved at IRD,
Nouméa, New Caledonia.
Extraction
The dichloromethane-methanol (8:2, v/v) extract (0.9 g) of Micrococcus
luteus was fractionated on a silica gel column using a dichloromethanemethanol gradient. The dichloromethane fraction retained the antimicrobial activity; another silica gel column chromatography using
hexane-dichloromethane (15:85, v/v), furnished compound 1 (0.55 mg,
0.005% dry weight). The fraction eluted with methanol (8:2, v/v to
7:3, v/v) contained, as the major product, compound 2, which was
further purified on another silica gel column eluted with chloroformmethanol (85:15, v/v).
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Bioassays
Crude extract, chromatographic fractions and pure compounds were
assayed in vitro for antimicrobial activity using the agar disk diffusion
technique, against the following microorganisms: Staphylococcus aureus
(209P), Escherkhia coli (RL 65 and 57 b) and the ichthyopathogenic Vibrio
angnilldrnm. Extracts and fractions (500 mg to 10 mg, dissolved in
10 ml CH 2 C 12 ) were applied on paper disks (6 mm). The pure compound
was tested within the range 10 -1 -10 - 4 miüigrammes per millilitre.
The mimmun inhibitory concentration was determined by the agar
incorporation test. Cytotoxicity was assayed on KB cells. Screening for
antimalarial activity was performed at the laboratoire de biologie parasitaire, Muséum national d’histoire naturelle, Paris, France. Tests tor
anti-HlV activity were carried on at ULP/INSERM U74, Institut de virologie, Strasbourg, France.
Collection and cultivation
The sponge Xestospongia sp. was collected by scuba diving oh Nouméa,
Homophymia sp. was collected by scuba diving off Touho (New Caledonia).
Sponges were rinsed with sterile sea water and brushed. The resultant
suspension was then diluted in ten-fold series, the dilutions were spread
on marine agar medium (DIFCO 2216). The best plate was chosen for
isolation and further screening on Staphylococcus aureus, Vibrio angnillarum and Candida albicans.
The bacterial strains îsolated were cultivated at 27°C, in 4 x 3 1 batch
cultures (sea water DIFCO-peptone 18 g/l enriched medium) for 4 days
with rotatory shaking. Cells were harvested by centrifugation at
11 000 rpm for 15 minutes. The pellet was sonicated and extracted
successively with methanol and dichloromethane-methanol (8:2, v/v).
The organic phase was separated by centrifugation and the solvents
removed under reduced pressure.
The bacteria were identified upon the basis of 16S sequence. The Gram( + )
strain R 1588-10 isolated from the surface of the sponge Xestospongia
sp. was identified as Micrococcus luteus. The Gram (-) bacterial strain
15 31 E7 collected at the surface of the sponge Homopbymia sp. was identified as a new Pseudomonas sp. Voucher specimens are conserved at IRD,
Nouméa, New Caledonia.
Extraction
The dichloromethane-methanol (8:2, v/v) extract (0.9 g) of Micrococcus
luteus was fractionated on a silica gel column using a dichloromethanemethanol gradient. The dichloromethane fraction retained the antimicrobial activity; another silica gel column chromatography using
hexane-dichloromethane (15:85, v/v), furnished compound 1 (0.55 mg,
0.005% dry weight). The fraction eluted with methanol (8:2, v/v to
7:3, v/v) contained, as the major product, compound 2, which was
further purified on another silica gel column eluted with chloroformmethanol (85:15, v/v).
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