Identification of three light sensitive chemicals in a sponge, Phakellia flabellata, from the Central Section of the Great Barrier Reef

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Samples of the sponge Phakellia flabellata were collected from the central section of the Great Barrier Reef, Australia during both winter and summer months and transported to AIMS frozen (-20°C).Three extraction and isolation procedures were adopted:Procedure 1:The finely-cut wet sponge (1 kg) was homogenised with water (1 litre) in a blender to give a fine slurry. After stirring for 48 h at 25°C, the slurry was centrifuged 5000 g, 30 min) and the residue re-extracted with water (2 x 500 mL). Combined water was removed under reduced pressure and purified on a column of Sephadex G -10. The column was eluted with deionised water and the fractions having the absorption maximum at lambda max 348 nm were freeze-dried and repeated chromatography of residue on Sephadex G-10 with water as eluent afforded the compound, Debromohymenialdisine hydrochloride, as a pale yellow amorphous solid (10 mg).Procedure 2:The wet sponge (1 kg) was extracted with refluxing acetone (3 x 1L) and after evaporation of extracting solvent, the remaining aqueous slurry was extracted with diethyl ether (2 x 250 mL) and then with n-butanol (2 x 150 mL) in a separatory funnel. The butanolic extract was suspended in methanol (150 mL) and the methanol insoluble fraction was filtered and the residue purified by repeated chromatography on Sephadex LH-20 (chloroform and mixtures of chloroform and methanol with increment of methanol) afforded the compound, Hymenialdisine, as a pale yellow amorphous solid (5 mg). Procedure 3:The finely-cut wet sponge (1 kg) was homogenised as before with 10% aqueous methanol and extracted with refluxing 10% aqueous methanol (3 x 1L). Removal of the solvent under reduced pressure gave an orange-yellow residue (80 g). The residue was further extracted with refluxing methanol. The residue (10 g) obtained after evaporation of hot methanol soluble fraction was a complex mixture (TLC) and was not investigated further. The residue (80 g) from 10% aqueous methanol was treated with 90% aqueous methanol (3 x 500 mL) at room temperature (48 h) and removal of the solvent from the soluble portion furnished a yellow residue (28 g). Purification of this residue (10 g) by repeated chromatography on Sephadex LH-20 with chloroform and mixtures of chloroform and methanol furnished two major fractions. The first fraction was an inseparable mixture of two components Debromohymenialdisine hydrochloride and Hymenialdisine. The second fraction was pre-absorbed onto Kieselgel and gradient elution with dichloromethane and mixtures of dichloromethane and methanol provided compound, Debromohymenialdisine, as a pale yellow amorphous solid (3 mg).
The sponge, Phakellia flabellata, from the Great Barrier Reef was investigated for the presence of UV-absorbing metabolites. Interest in this animal focused on the fact that sponge extracts exhibit strong absorption in the UV A region. This research was part of a program searching for biologically active compounds and novel UV-absorbing compounds of lambda max 290-360 nm, for use as UV blockers in skin care preparations.

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Statement: Statement: Marine sponge taxonomic identification was performed at the Australian Institute of Marine Science (AIMS) Australia.All solvents used were of analytical grade. Size exclusion chromatography was accomplished with Sephadex LH-20 and G-10 (fine, particle size 20-100 µ; Pharmacia, Sweden) and column chromatography on Kieselgel-100 (230-400 mesh, Merck). Thin layer chromatography (TLC) was carried out with silica gel 60 PF254 (pre-coated plates, Merck) (n-BuOH:AcOH:H2O; 60:15:25) and (n-BuOH: AcOH; 95:5) (detection by spraying with 1% Ce(SO4)2 in 10% aqueous H2SO4 followed by heating. Fractions from column chromatography were monitored by High Pressure Liquid Chromatography (HPLC) on reverse phase µ bondpak C18 column (MeOH:H20); 20:80 or 55:45) with detection at 340 nm.Melting points are uncorrected and were determined on an Electrotherma melting point apparatus. Ultraviolet spectra were recorded employing a GBC model UV/VIS spectrophotometer and IR spectra with a Perkin Elmer FT-IR model instrument. The NMR spectra were measured in DMSO-d6 using a Brucker Aspect 3000 MHz instrument. Electron Impact Low-Resolution Mass Spectrometry (LREIMS) were recorded in a VG 7070 F mass spectrometer interfaced to an INCOS data system. High-Resolution Electron Impact Mass Spectrometry (HREIMS) measurements were carried out on the same instrument by using perfluorokerosene as the reference compound.

Notes

Credit
Bandaranayake, Wickramasinghe M, Dr (Principal Investigator)