HPTLC Coupled with Bioluminescence and Mass Spectrometry for Bioactivity-Based Analysis of Secondary Metabolites in Marine Sponges

摘要

Rapid analysis by coupling HPTLC with bioluminescence and mass spectrometry enables very fast response to bioactive substances in unknown samples. In this study marine sponges were screened for new bioactive compounds. After chromatographic separation of twelve methanolic marine sponge extracts the HPTLC plates were coated with bioluminescent bacteria (Vibrio fischeri) by a simple dipping procedure. If separated compounds were bioactive they inhibited or enhanced the bacterial luminescence and could be identified as dark zones on the luminescent background. This micro-biological detection revealed new compounds compared with physical (absorbance or fluorescence measurement) or chemical (micro-chemical derivatization) detection techniques. Effect-directed analysis turned out to be superior to target analysis in the search for natural products with a distinct effect. For identification of unknown bioactive zones the HPTLC system was coupled to a high-resolution mass spectrometer to obtain the exact masses of the unknowns. Thus, a Vibrio diaxhwei-bioactive zone was identified as avarone, a bioactive metabolite so far only known to be synthesized by the sponge Dysidea avara. This methodology proved very effective not only for detection but also for identification of unknown bioactive metabolites in sponges.