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Choosing appropriate methods and standards for assaying tannin

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Abstract

Tannins are chemically diverse polyphenolics that have multiple biological activities. Attempts to establish the ecological significance of tannins have been hindered by the complexities of tannin analysis. A multitude of analytical procedures for tannins has been described, but it is difficult for the nonspecialist to select appropriate methods. We have classified the most common procedures for determining tannin as either chemical assays, appropriate for determining the amount and the chemical nature of the tannin in a sample, or as protein-binding assays, suitable for determining the potential biological activity of the tannin in a sample. We have recommended procedures that are particularly reliable and straightforward for general use. We have also considered the problems encountered in selecting appropriate standards for tannin analysis and have recommended standards that are readily available.

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References

  • Armory, A.M., andSchubert, C.L. 1987. A method to determine tannin concentration by the measurement and quantification of protein-tannin interaction.Oecologia (Berlin) 73:420–424.

    Google Scholar 

  • Artz, W.E., Bishop, P.D., Dunker, A.K., Schanus, E.G., andSwanson, B.G. 1987. Interaction of synthetic proanthocyanidin dimer and trimer with bovine serum albumin and purified bean globulin fraction G-1.J. Agric. Food Chem. 35:417–421.

    Google Scholar 

  • Asquith, T.N., andButler, L.G. 1985. Use of dye-labeled protein as spectrophotometric assay for protein precipitants such as tannin.J. Chem. Ecol. 11:1535–1544.

    Google Scholar 

  • Austin, P.J., Suchar, L.A., Robbins, C.T., andHagerman, A.E. 1989. Tannin binding proteins in the saliva of deer.J. Chem. Ecol. 15:1335–1347.

    Google Scholar 

  • Baldwin, I.T., Schultz, J.C., andWard, D. 1987. Patterns and sources of leaf tannin variation in yellow birch (Betula allegheniensis) and sugar maple (Acer saccharum).J. Chem. Ecol. 13:1069–1078.

    Google Scholar 

  • Barbeau, W.E., andKinsella, J.E. 1983. Factors affecting the binding of chlorogenic acid to fraction I leaf protein.J. Agric. Food Chem. 31:993–998.

    Google Scholar 

  • Bate-Smith, E.C. 1972. Detection and determination of ellagitannins.Phytochemistry 11:1153–1156.

    Google Scholar 

  • Bate-Smith, E.C. 1973. Haemanalysis of tannins: The concept of relative astringency.Phytochemistry 12:970–912.

    Google Scholar 

  • Bate-Smith, E.C. 1977. Astringent tannins ofAcer species.Phytochemistry 16:1421–1426.

    Google Scholar 

  • Bate-Smith, E.C., andSwain, T. 1962. Flavonoid compounds, pp. 755–809,in H.S. Mason and A.M. Florkin (eds.). Comparative Biochemistry, Vol. 3. Academic Press, New York.

    Google Scholar 

  • Batzli, G.O. 1983. Responses of arctic rodent populations to nutritional factors.Oikos 40:396–406.

    Google Scholar 

  • Beart, J.E., Lilley, T.H., andHaslam, E. 1985. Plant polyphenols—secondary metabolism and chemical defence: Some observations.Phytochemistry 24:33–38.

    Google Scholar 

  • Bryant, J.P. 1981. Phytochemical deterrence of snowshoe hare browsing by adventitious shoots of four Alaskan trees.Science 213:889–890.

    Google Scholar 

  • Butler, L.G. 1982. Relative degree of polymerization of sorghum tannin during seed development and maturation.J. Agric. Food Chem. 30:1090–1094.

    Google Scholar 

  • Butler, L.G., Price, M.P., 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.

    Google Scholar 

  • Calderon, P., Van Buren, J., andRobinson, W.B. 1968. Factors influencing the formation of precipitates and hazes by gelatin and condensed and hydrolyzable tannins.J. Agric. Food Chem. 16:479–482.

    Google Scholar 

  • Eggum, B.O., andChristensen, K.D. 1975. Influence of tannin on protein utilization in feedstuffs with special reference to barley, pp. 135–143,in Breeding for Seed Protein Improvement Using Nuclear Techniques. Atomic Energy Commission, Vienna.

    Google Scholar 

  • Feeny, P.P. 1968. Effect of oak leaf tannins on larval growth of the winter moth Operophtera brumata.J. Insect Physiol. 14:805–817.

    Google Scholar 

  • Figliuolo, R., Naylor, S., Wang, J., andLangheim, J.H. 1987. Unusual nonprotein imino acid and its relationship to phenolic and nitrogenous compounds inCopaifera.Phytochemistry 26:3255–3260.

    Google Scholar 

  • Folin, O., andDenis, W. 1915. A colorimetric method for the determination of phenols (and phenol derivatives) in urine.J. Biol. Chem. 22:305–308.

    Google Scholar 

  • Foo, L., Jones, W.T., Porter, L.J., andWilliams, V.M. 1982. Proanthocyanidin polymers of fodder legumes.Phytochemistry 21:933–935.

    Google Scholar 

  • Glick, Z., andJoslyn, M.A. 1970a. Effect of tannic acid and related compounds on the absorbtion and utilization of proteins in the rat.J. Nutr. 100:516–520.

    Google Scholar 

  • Glick, Z., andJoslyn, M.A. 1970b. Food intake depression and other metabolic effects of tannic acid in the rat.J. Nutr. 100:509–515.

    Google Scholar 

  • Goldstein, J.L., andSwain, T. 1965. The inhibition of enzymes by tannins.Phytochemistry 4:185–192.

    Google Scholar 

  • Grove, J.F., andPople, M. 1979. The identification and characterization ofo-dihydric phenols.Phytochemistry 18:1071–1072.

    Google Scholar 

  • Haddock, E.A., Gupta, R.K., Al-Shafi, S.M.K., Layden, K., Haslam, E., andMagnolato, D. 1982. The metabolism of gallic acid and hexahydroxydiphenic acid in plants: Biogenetic and molecular taxonomic considerations.Phytochemistry 21:1049–1062.

    Google Scholar 

  • Hagerman, A.E. 1987. Radial diffusion method for determining tannin in plant extracts.J. Chem. Ecol. 13:437–449.

    Google Scholar 

  • Hagerman, A.E. 1988. Extraction of tannin from fresh and preserved leaves.J. Chem. Ecol. 14:453–462.

    Google Scholar 

  • Hagerman, A.E., andButler, L.G. 1978. Protein precipitation method for the quantitative determination of tannins.J. Agric. Food Chem. 26:809–812.

    Google Scholar 

  • Hagerman, A.E., andButler, L.G. 1980a. Determination of protein in tannin-protein precipitates.J. Agric. Food Chem. 28:944–947.

    Google Scholar 

  • Hagerman, A.E., andButler, L.G. 1980b. Condensed tannin purification and characterization of tannin-associated proteins.J. Agric. Food Chem. 28:947–952.

    Google Scholar 

  • Hagerman, A.E., andButler, L.G. 1981. Specificity of proanthocyanidin-protein interactions.J. Biol. Chem. 256:4494–4497.

    Google Scholar 

  • Hagerman, A.E., andKlucher, K.M. 1986. Tannin-protein interactions, pp. 67–76,in V. Cody, E. Middleton, and J. Harborne (eds.). Plant Flavonoids in Biology and Medicine: Biochemical, Pharmacological and Structure Activity Relationships. Alan R. Liss, New York.

    Google Scholar 

  • Hagerman, A.E., andRobbins, C.T. 1987. Implications of soluble tannin-protein complexes for tannin analysis and plant defense mechanisms.J. Chem. Ecol. 13:1243–1259.

    Google Scholar 

  • Harris, H.B., andBurns, R.E. 1970. Influence of tannin content on preharvest seed germination inSorghum.Agron. J. 62:835–836.

    Google Scholar 

  • Haslam, E. 1979. Vegetable tannins, pp. 475–523,in T. Swain, J.B. Harborne, and C.F. VanSumere (eds.). Biochemistry of Plant Phenolics (Recent Advances in Phytochemistry). Plenum Press, New York.

    Google Scholar 

  • Hatano, T., Kira, R., Yoshizaki, M., andOkuda, T. 1986. Seasonal changes in the tannins ofLiquidambar formosana reflecting their biogenesis.Phytochemistry 25:2787–2789.

    Google Scholar 

  • Howard, J.J. 1987. Leafcutting ant diet selection: The role of nutrients, water and secondary chemistry.Ecology 68:503–515.

    Google Scholar 

  • Ikawa, M., Dollard, C.A., andSchaper, T.D. 1988. Reaction of Folin-Ciocalteau phenol reagent with purines, pyrimidines, and pteridines and its relationship to structure.J. Agric. Food Chem. 36:309–311.

    Google Scholar 

  • Inoue, K.H., andHagerman, A.E. 1988. Determination of gallotannin with rhodanine.Anal. Biochem. 169:363–369.

    Google Scholar 

  • Jambunathan, R., andMertz, E.T. 1973. Relationship between tannin levels, rat growth, and distribution of proteins in sorghum.J. Agric. Food Chem. 21:692–696.

    Google Scholar 

  • Jung, H.-J.G., Batzli, G.O., andSeigler, D.S. 1979. Patterns in the phytochemistry of arctic plants.Biochem. Syst. Ecol. 7:203–209.

    Google Scholar 

  • Kumar, R., andSingh, M. 1984. Tannins: Their adverse role in ruminant nutrition.J. Agric. Food Chem. 32:447–453.

    Google Scholar 

  • Leatham, G.F., King, V., andStahmann, M.A. 1980. In vitro protein polymerization by quinones or free radicals generated by plant or fungal oxidative enzymes.Phytopathology 70:1134–1140.

    Google Scholar 

  • Makkar, H.P.S., Dawra, R.K., andSingh, B. 1987. Protein precipitation assay for quantitation of tannins: Determination of protein in tannin-protein complex.Anal. Biochem. 166:435–439.

    Google Scholar 

  • Martin, J.S., andMartin, M.M. 1983. Tannin assays in ecological studies. Precipitation of ribulose-1,5-bisphosphate carboxylase/oxygenase by tannic acid, quebracho, and oak leaf foliage extracts.J. Chem. Ecol. 9:285–294.

    Google Scholar 

  • McKey, D., Waterman, P.G., Mbi, C.N., Gartlan, J.S., andStruhsakar, T.T. 1978. Phenolic content of vegetation in two African rain forests: Ecological implications.Science 202:61–64.

    Google Scholar 

  • McManus, J.P., Davis, K.G., Lilley, T.H., andHaslam, E. 1981. The association of proteins with polyphenols.J. Chem. Soc. Chem. Commun. 1981:309–311.

    Google Scholar 

  • Mishra, A., Siradhana, B.S., andShivpuri, A. 1980. Phenols in relation to resistance of sorghum to anthracnose.Phil. Agric. 1:63.

    Google Scholar 

  • Mitaru, B.N., Reichert, R.D., andBlair, R. 1984. The binding of dietary protein by sorghum tannins in the digestive tract of pigs.J. Nutr. 114:1787–1796.

    Google Scholar 

  • Mole, S., andWaterman, P.G. 1987a. A critical analysis of techniques for measuring tannins in ecological studies. I. Techniques for chemically defining tannins.Oecologia 72:137–147.

    Google Scholar 

  • 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.

    Google Scholar 

  • Mole, S., andWaterman, P.G. 1988. Light-induced variation in phenolic levels in foliage of rain-forest plants. II. Potential significance to herbivores.J. Chem. Ecol. 14:23–34.

    Google Scholar 

  • Mole, S., Butler, L.G., Hagerman, A.E., andWaterman, P.G. 1989. Ecological tannin assays: a critique.Oecologia 78:93–96.

    Google Scholar 

  • Nishizawa, M., Yamagishi, T., Nonaka, G., Nishioka, I., andRagan, M.A. 1985. Gallotannins of the freshwater green algaSpirogyra sp.Phytochemistry 24:2411–2413.

    Google Scholar 

  • Oh, H.I., Hoff, J.E., Armstrong, G.S., andHaff, L.A. 1980. Hydrophobic interaction in tannin-protein complexes.J. Agric. Food Chem. 28:394–398.

    Google Scholar 

  • Owen-Smith, N., andCooper, S.M. 1987. Palatability of woody plants to browsing ruminants in a South African savanna.Ecology 68:319–331.

    Google Scholar 

  • Pederson, G.L. 1979. Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall.Anal. Biochem. 100:201–220.

    Google Scholar 

  • Pierpoint, W.S. 1969.O-Quinones formed in plant extracts. Their reactions with bovine serumalbumin.Biochem. J. 112:619–629.

    Google Scholar 

  • Porter, L.J., Hrstich, L.N., andChan, B.C. 1986. The conversion of procyanidins and prodelphinidins to cyanidin and delphinidin.Phytochemistry 25:223–230.

    Google Scholar 

  • 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.

    Google Scholar 

  • Price, M.L., Van Scoyoc, S., andButler, L.G. 1978. A critical evaluation of the vanillin reaction as an assay for tannin in sorghum grain.J. Agric. Food Chem. 26:1214–1218.

    Google Scholar 

  • Provenza, F.D., andMalechek, J.C. 1984. Diet selection by domestic goats in relation to black-brush twig chemistry.J. Appl. Ecol. 21:831–841.

    Google Scholar 

  • Putnam, L.J., andButler, L.G. 1985. Fractionation of condensed tannins by counter-current chromatography.J. Chromatogr. 318:85–93.

    Google Scholar 

  • Reed, J.D., Horvath, P.J., Allen, M.S., andVansoest, P.J. 1985. Gravimetric determination of soluble phenolics including tannins from leaves by precipitation with trivalent ytterbirum.J. Sci. Food Agric. 36:255–261.

    Google Scholar 

  • Robbins, C.T., Hanley, T.A., Hagerman, A.E., Hjeljord, O., Baker, D.L., Schwartz, C.C., andMautz, W.W. 1987. Role of tannins in defending plants against ruminants: Reduction in protein availability.Ecology 68:98–107.

    Google Scholar 

  • Robinson, T. 1980. The Organic Constituents of Higher Plants. Cordus Press, Amherst, Massachusetts.

    Google Scholar 

  • Sakar, S.K., andHowarth, R.E., 1976. Specificity of the vanillin test for flavanols.J. Agric. Food Chem. 24:317–320.

    Google Scholar 

  • Schaffert, R.E., Lechtenberg, V.L., Oswalt, D.L., Axtell, J.D., Pickett, R.C., andRhykerd, L.C. 1974. Effect of tannin on in vitro dry matter and protein disappearance in sorghum grain.Crop Sci. 14:640–643.

    Google Scholar 

  • Schultz, J.C., andBaldwin, I.T. 1982. Oak leaf quality declines in response to defoliation by gypsy moth larvae.Science 217:149–151.

    Google Scholar 

  • Schultz, J.C., Baldwin, I.T. andNothnagle, P.J. 1981. Hemoglobin as a binding substrate in the quantitative analysis of plant tannins.J. Agric. Food Chem. 29:823–826.

    Google Scholar 

  • Strumeyer, D.H., andMalin, M.J. 1975. Condensed tannins in grain sorghum: Isolation, fractionation, and characterization.J. Agric. Food Chem. 23:909–914.

    Google Scholar 

  • Swain, T., andHillis, W.E. 1959. The phenolic constituents ofPrunus domestica. I. The quantitative analysis of phenolic compounds.J. Sci. Food Agric. 10:63–68.

    Google Scholar 

  • Swain, T. 1965. Nature and properties of flavonoids, pp. 211–245,in T.W. Goodwin (ed.). Chemistry and Biochemistry of Plant Pigments. Academic Press, New York.

    Google Scholar 

  • Swain, T. 1979. Tannins and lignins, pp. 657–682,in G.A. Rosenthal and D.H. Janzen (eds.). Herbivores: Their Interaction with Secondary Plant Metabolites. Academic Press, New York.

    Google Scholar 

  • Tempel, A.S. 1982. Tannin-measuring techniques: A review.J. Chem. Ecol. 8:1289–1298.

    Google Scholar 

  • Tipton, K.W., Floyd, E.H., Marshall, J.G., andMcDevitt, J.B. 1970. Resistance of certain grain sorghum hybrids to bird damage in Louisiana.Agron. J. 62:211–213.

    Google Scholar 

  • Wall, M.E., Taylor, H., Ambrosio, L., andDavis, K. 1969. Plant antitumor agents. III. A convenient separation of tannins from other plant constituents.J. Pharm. Sci. 58:839–841.

    Google Scholar 

  • Waterman, P.G., Ross, J.A.M., andMcKey, D.B. 1984. Factors affecting levels of some phenolic compounds, digestibility and nitrogen content of the mature leaves ofBarteria fistulosa (Passifloraceae).J. Chem. Ecol. 10:387–401.

    Google Scholar 

  • Watterson, J.J., andButler, L.G. 1983. Occurrence of an unusual leucoanthocyanidin and absence of proanthocyandins in sorghum leaves.J. Agric. Food Chem. 31:41–45.

    Google Scholar 

  • Wesp, E.F., andBrode, W.R. 1934. The absorbtion spectra of ferric compounds. I. The ferric chloride-phenol reaction.J. Am. Chem. Soc. 56:1037–1042.

    Google Scholar 

  • Wisdom, C.S., Gonzalez-Coloma, A., andRundel, P.W. 1987. Ecological tannin assays. Evaluation of proanthocyanidins, protein binding assays and protein precipitating potential.Oecologia 72:395–401.

    Google Scholar 

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  1. Department of Chemistry, Miami University, 45056, Oxford, Ohio

    Ann E. Hagerman

  2. Department of Biochemistry, Purdue University, 47907, West Lafayette, Indiana

    Larry G. Butler

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Hagerman, A.E., Butler, L.G. Choosing appropriate methods and standards for assaying tannin. J Chem Ecol 15, 1795–1810 (1989). https://doi.org/10.1007/BF01012267

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