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.
Similar content being viewed by others
References
Amico, V., Currenti, R., Oriente, G., Piatelli, M., andTringali, C. 1981. A phloroglucinol derivative from the brown algaZonaria tournefortii.Phytochemistry 20:1451–1453.
Arnold, T., Tanner, C., andHatch, W. 1995. Phenotypic variation in polyphenol content of the tropical brown algaLobophra variegata as a function of nitrogen.Mar. Ecol. Prog. Ser. 123:177–183.
Beress, A., Wassermann, O., Bruhn, T., Beress, L., Kraiselburd, E. N., Gonzalez, L. V., Demotta, G. E., andChavez, P. I. 1993. A new procedure for the isolation of antiHIV compounds (polysaccharides and polyphenols) from the marine algaFucus vesiculosus.J. Nat. Prod. Lloydia 56:478–488.
Bernays, E. A., Cooper Driver, G., andBilgener, M. 1989. Herbivores and plant tannins.Adv. Ecol. Res. 19:263–302.
Blackman, A. J., Rogers, G. I., andVolkman, J. K. 1988. Phloroglucinol derivatives from three Australian marine algae of the genusZonaria.J. Nat. Prod. 51:158–160.
Boettcher, A. A., andTargett, N. M. 1992. Role of polyphenolic molecular size in reduction of assimilation efficiency inXiphister mucosa.Ecology 74:891–903.
Butler, L. G. 1982. Relative degree of polymerization of sorghum tannin during seed development and maturation.J. Agric. Food Chem. 30:1090–1094.
Butler, L. G., Price, M. L., andBrotherton, J. E. 1982. Vanillin assay for proanthocyanidins (condensed tannins): Modification of the solvent for estimation of the degree of polymerization.J. Agric. Food Chem. 30:1087–1089.
Carlson, D. J., andMayer, L. M. 1983. Relative influences of riverine and macroalgal phenolic materials on UV absorbance in temperate coastal waters.Can. J. Fish. Aquat. 40:1258–1263.
Coley, P. D., Bryant, J. P., andChapin, F. S. III. 1985. Resource availability and plant antiherbivore defense.Science 230:895–899.
Feeny, P. P. 1976. Plant apparency and chemical defense, pp. 1–40,in J. W. Wallace and R. L. Mansell (eds.). Biochemical Interactions between Plants and Insects. Plenum Press, New York.
Gerwick, W., andFenical, W. 1982. Phenolic lipids from related marine algae of the order Dictyotales.Phytochemistry 21:633–637.
Goldstein, J. L., andSwain, T. 1963. Methods for determining the degree of polymerization of flavans.Nature 198:587–588.
Harborne, J. B. 1991. Plant defenses against mammalian herbivory, pp. 45–60,in P. T. Palo and C. T. Robbins (eds.). The Chemical Basis of Plant Defense. CRC Press, Boca Raton, Florida.
Haslam, E. 1989. Plant Polyphenols. Vegetable tannins Revisited. Cambridge University Press, Cambridge.
Hay, M. E., andFenical, W. 1988. Marine plant-herbivore interactions: the ecology of chemical defense.Annu. Rev. Ecol. Syst. 19:111–145.
Kellem, S. J., Tisch, M. H., andWalker, J. R. L. 1992. Screening of New Zealand native plants for enzyme inhibitory activities.N.Z. J. Bot. 30:199–203.
Loomis, W. D., andBataille, J. 1966. Plant polyphenolic compounds and the isolation of plant enzymes.Phytochemistry 5:423–438.
McMillan, C. 1984. The condensed tannins (proanthocyanidins) in sea grasses.Aquat. Bot. 8:351–357.
Mole, S., andWaterman, P. G. 1987a. A critical analysis of techniques measuring tannins in ecological studies I. Techniques for chemically defining tannins.Oecologia 72:137–147.
Mole, S., andWaterman, P. G. 1987b. A critical analysis of techniques for measuring tannins in ecological studies II. Techniques for biochemically defining tannins.Oecologia 72:148–156.
Phillips, D. W. andTowers, G. H. N. 1982. Chemical ecology of red algal bromophenols: I. Temporal, interpopulational and within-thallus measurements of lanosol levels inRhodomela larix (Turner) C. Agardh.J. Exp. Mar. Biol. Ecol. 58:285–293.
Price, M. P., andButler, L. G. 1977. Rapid visual estimation and spectrophotometric determination of tannin content ofSorghum grain.J. Agric. Food Chem. 25:1268–1273.
Putnam, L. J., andButler, L. G. 1985. Fractionation of condensed tannins by counter-current chromatography.J. Chromatogr. 318:85–93.
Ragan, M. A. 1985. The high molecular weight polyphloroglucinols of the marine brown algaFucus vesiculosus L: Degradative analysis.Can. J. Chem. 63:294–303.
Ragan, M. A., andGlombitza, K.-W. 1986. Phlorotannins, brown algal polyphenols.Prog. Phycol. Res. 4:129–241.
Ragan, M. A., andJensen, A. 1977. Quantitative studies on brown algal phenolics. I. Estimation of absolute polyphenol content inAscophyllum nodosum andFucus vesiculosus.J. Exp. Mar. Biol. Ecol. 34:245–258.
Ribéreau-Gayon, P. 1972. Plant Phenolics. Oliver & Boyd, Edinburgh. pp. 23–53.
Schlesinger, W. H. 1991. Biogeochemistry: An Analysis of Global Change. Academic Press, San Diego.
Steinberg, P. D. 1989. Biogeographical variation in brown algal polyphenolics and other secondary metabolites: comparison between temperate Australasia and North America.Oecologia 78:374–383.
Steinberg, P. D. 1992. Geographical variation in the interaction between marine herbivores and brown algal secondary metabolites, pp. 51–72,in V. J. Paul (ed.). Ecological Roles for Marine Secondary Metabolites. Cornell University Press, New York.
Steinberg, P. D., andvan Altena, I. 1992. Tolerance of marine invertebrate herbivores to brown algal phlorotannins in temperate Australasia.Ecol. Monogr. 62:189–222.
Stern, J. L., Hagerman, A. E., Steinberg, P. D., andMason, P. K. 1995. Phlorotannin-protein interactions. In press.
Swain, T. 1979. Tannins, pp. 657–682,in G. A. Rosenthal, and D. H. Janzen, (eds.). Herbivores: Their Interaction with Secondary metabolites, Academic Press, New York.
Swain, T., andHillis, W. E. 1959. The phenolic constitutents ofPrunus domestica. 1. The quantitative analysis of phenolic constituents.J. Sci. Food. Agric. 10:63–68.
Targett, N. M., Boettcher, A. A., Targett, T. E., andVrolijk, N. H. 1995. Tropical marine herbivore assimilation of phenolic-rich plants.Oecologia 103:170–179.
Targett, N. M., Coen, L. D., Boettcher, A. A., andTanner, C. E. 1992. Biogeographic comparisons of marine algal polyphenolics: evidence against a latitudinal trend.Oecologia 89:464–470.
Van Alstyne, K. L. 1995. Comparison of three methods for quantifying brown algal polyphenolic compounds.J. Chem. Ecol. 21:45–58.
van Altena, I. A., andSteinberg, P. D. 1992. Are differences in the responses between North American and Australasian marine herbivores to phlorotannins due to differences in phlorotannin structure?Biochem. Syst. Ecol. 20:493–499.
Yates, J. L., andPeckol, P. 1993. Effect of nutrient availability and herbivory on polyphenolics in the seaweedFucus vesiculosis.Ecology 74:1757–1766.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Stern, J.L., Hagerman, A.E., Steinberg, P.D. et al. A new assay for quantifying brown algal phlorotannins and comparisons to previous methods. J Chem Ecol 22, 1273–1293 (1996). https://doi.org/10.1007/BF02266965
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02266965