A NEW ASSAY FOR QUANTIFYING BROWN ALGAL PHLOROTANNINS AND COMPARISONSTO PREVIOUS METHODS

Quantitative measurement of phlorotannins (polyphenolics) in brown alg ae (Phaeophyta) by colorimetric assays can be confounded because: (1) most such assays also react to nonphlorotannin substances (interferenc es) and (2) the appropriate reference compound for such assays is not always clear, although phloroglucinol is typically used. We developed a new assay in which 2,4-dimethoxybenzaldehyde (DMBA) reacts specifica lly with 1,3- and 1,3,5-substituted phenols (e.g., phlorotannins) to f orm a colored product. This new assay, as well as eliminating the prob lem of measuring interferences, is inexpensive, rapid, and can be used with small sample volumes. We recommend it for all assays of phlorota nnins from one or a set of closely related species where the structura l types of phlorotannins present are likely to be similar among sample s. It is also appropriate for broader surveys of phlorotannin levels a cross many species, but in this case a reference must be chosen with c are. We also compared the DMBA assay to existing assays, including the Folin-Denis [both before and after the samples were mixed with polyvi nylpolypyrrolidone (PVPP)] and the Prussian blue assays. PVPP was not 100% efficient (and often much less) at removing phlorotannins from so lution, and its effectiveness varied among different phlorotannins. Th us, in contrast to previous studies, measuring phenolic levels in extr acts before and after treatment with PVPP will not necessarily result in an interference-free measure of phlorotannins. Based on an analysis of reactive substances in red and green algae (which do not contain p hlorotannins) in the Folin-Denis and Prussian blue assays, we estimate that the average level of interferences (nonphlorotannins) in brown a lgae measured in these two assays is on the order of 0.5% by dry weigh t.