A new assay for quantifying brown algal phlorotannins and comparisons to previous methods

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Abstract
Quantitative measurement of phlorotannins (polyphenolics) in brown algae (Phaeophyta) by colorimetric assays can be confounded because: (1) most such assays also react to nonphlorotannin substances (interferences) 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 specifically with 1,3-and 1,3,5-substituted phenols (e.g., phlorotannins) to form a colored product. This new assay, as well as eliminating the problem of measuring interferences, is inexpensive, rapid, and can be used with small sample volumes. We recommend it for all assays of phlorotannins from one or a set of closely related species where the structural types of phlorotannins present are likely to be similar among samples. It is also appropriate for broader surveys of phlorotannin levels across many species, but in this case a reference must be chosen with care. We also compared the DMBA assay to existing assays, including the Folin-Denis [both before and after the samples were mixed with polyvinylpolypyrrolidone (PVPP)] and the Prussian blue assays. PVPP was not 100% efficient (and often much less) at removing phlorotannins from solution, and its effectiveness varied among different phlorotannins. Thus, in contrast to previous studies, measuring phenolic levels in extracts 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 phlorotannins) in the Folin-Denis and Prussian blue assays, we estimate that the average level of interferences (nonphlorotannins) in brown algae measured in these two assays is on the order of 0.5% by dry weight.