Natural Product Analysis of Coastal Marine Species of New Zealand

Abstract

A chemical survey of 58 marine samples obtained from three sites around Tauranga New Zealand was undertaken in an effort to identify new metabolites. MTT assays using HeLa and P388 cell lines were carried out to determine if any of the samples contained bioactive compounds.

For each sample, a crude extract was obtained and analysed by Liquid Chromatography Mass Spectrometry (LCMS). The chromatogram traces were examined and fives samples containing potential metabolites of interest were chosen for further investigation. Of the five samples two were identified from preliminary taxonomic analysis as Lyngbya sp. (cyanobacterium) and Cliona celata (a sponge). The compound targeted in Lyngbya sp. was thought to potentially be a new peptide and the Cliona celata compounds contained bromine atoms. These two samples were extracted in bulk and fractionated by reversed phase and size exclusion chromatography. From the mass spectral data and the taxonomy there were no identified samples on the MarinLit database. The separation yielded several fractions and these were analysed by LCMS to identify which ones contained the targeted compounds. Nuclear magnetic resonance (NMR) spectroscopy and tandem mass spectrometry was then used on the most concentrated (targeted compound) fractions to get more structural information. NMR spectroscopy of Cliona celata was found contain many impurities and the target compounds concentrations were not sufficient enough to give any structural information. The compound from Lyngbya sp. appeared to be a fatty acid. The sterol composition of Cliona celata was also analysed by gas chromatography mass spectrometry (GCMS). The three other samples analysed in more detail were Aplidium sp. (RI 2-13), Alcyonaria sp. (RI 2-14) and Ircinia sp. (RI 2-17). These samples were fractionated by small scale chromatography and then subjected to tandem mass spectrometry with some structural features being identified.

All samples from the chemical survey were tested against the HeLa and P388 cell lines using the MTT assay. After inconclusive results from initial tests, the method was modified to give more consistent results. Even with the modifications to the method, there were still inconsistencies in the results. Only one sample, the brown alga Xiphophora sp. gave an expected trend of decreasing cell metabolism with increasing sample concentration which would indicate cytotoxic activity. The experiment would need to be repeated in independent tests to confirm the result.