|Two species of bryozoa, Pterocella vesiculosa and Plumatella repens, were investigated in an endeavour to isolate and characterise novel secondary metabolites.
Analysis of fractions derived from chromatography of a dichloromethane extract of Pterocella vesiculosa, indicated the probable presence of β-carboline and pterocellin alkaloids. Continued separation led to further purification of some pterocellin alkaloids, however the characteristic ultraviolet chromatogram that is produced by pterocellins could not be found in any of the fractions analysed. Despite this, further investigations by thin layer chromatography, tandem mass spectrometry and high resolution mass spectrometry suggested structural similarities between metabolites from polar fractions and known pterocellin alkaloids. In the process of isolating these polar, pterocellin like alkaloids, a new thin layer chromatography solvent system was developed, which better differentiates the more polar metabolites in extracts compared to previously utilised solvent schemes.
LCMS analysis of chromatographic fractions resulted in the detection of eight β carboline alkaloids, as well as the identification of the previously published 5 bromo-8-methoxy-1-methyl-β-carboline. Further purification of the alkaloid containing fractions by size exclusion chromatography was achieved but individual alkaloids were unable to be isolated due to time constraints associated with the project. Molecular formulae for the eight alkaloids were obtained by the use of high resolution mass spectrometry. Tandem mass spectrometry helped to confirm these formulae and also suggested the types of functional groups attached to the β-carboline skeleton.
A biologically active extract of the freshwater species, Plumatella repens was investigated chemically as a continuation of a pilot study. A series of fractions from the pilot study were separated by size exclusion chromatography, with monitoring of activity against the murine P388 lymphocytic leukaemia cell line. Results of the assay indicated the presence of a highly active metabolite/s, however isolation of the metabolite/s responsible for the activity was not achieved. This was a result of delays in finding facilities to undertake the biological assays and varying degrees of P388 activity, which may suggest metabolite instability.
As part of this project, tissue culture was undertaken at AgResearch, at the Ruakura Research Centre, Hamilton. A method for assay of Plumatella repens samples was developed for bioassay guided fractionation.
The sterol composition of Plumatella repens was also investigated. Three sterols were identified and an additional two sterols were present, the structures of which could not be confirmed. The relative concentration of each of the sterols was also determined.