High Molecular Weight Plant Polyphenolics (Tannins) as Biological Antioxidants Ann E. Hagerman,* Ken M. Riedl, G. Alexander Jones, Kara N. Sovik, Nicole T. Ritchard, Paul W. Hartzfeld, and Thomas L. Riechel Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056 Representative condensed and hydrolyzable tannins and related simple phenolics were evaluated as biological antioxidants using cyclic voltammetry, the metmyoglobin assay, and the deoxyribose assay. The redox potentials of the tannins were similar to those of structurally related simple phenolics. However, the tannins were 15-30 times more effective at quenching peroxyl radicals than simple phenolics or Trolox. One of the tannins, polygalloyl glucose, reacted an order of magnitude more quickly with hydroxyl radical than mannitol. These results suggest that tannins, which are found in many plant-based foods and beverages, are potentially very important biological antioxidants. Keywords: Tannins; plant phenolics; antioxidant; total antioxidant activity; oxidative damage; dietary antioxidant INTRODUCTION Phenolic compounds are among the most widely distributed plant secondary products and are found in many plants used as foods and feeds. Recently, the ability of phenolic compounds to serve as antioxidants has been recognized (Decker, 1995), leading to specula- tion about the potential benefits of ingesting plant phenolics. The ability of small phenolics including flavonoids and phenolic acids to act as antioxidants has been extensively investigated (Rice-Evans et al., 1996), but the high molecular weight phenolics known as tannins have been largely neglected. Tannins are found in grains and legumes (Salunkhe et al., 1990), fruits (Foo and Porter, 1981), herbs (Haslam et al., 1989), and in beverages derived from plants. The average human consumption of tannins in U.S. is estimated to be at least 1 g/day (Pierpoint, 1990), so they could be a significant source of dietary antioxidants. Tannins are naturally occurring phenolic compounds which precipitate protein. In general, tannins are high molecular weight (M r > 500) and have many phenolic groups (Hagerman et al., 1997). There is significant chemical heterogeneity among the tannins, as illus- trated by the representative compounds shown in Figure 1. Compounds typical of the three principle groups of tannins, the condensed tannins (proanthocya- nidins), the hydrolyzable tannins, and the phlorotannins are depicted. Phlorotannins are found only in marine brown algae (Ragan and Glombitza, 1986) and are not widely consumed by humans. Our study was therefore limited to condensed tannins and hydrolyzable tannins. For monomeric phenolics, the ability to act as anti- oxidants is dependent on extended conjugation, number and arrangement of phenolic substitutents, and molec- ular weight. For example, the flavonoids with the most hydroxyl groups were most easily oxidized (Hodnick et al., 1988), and for simple flavonoid oligomers, the degree of polymerization was correlated with the ability to scavenge free radicals (Ariga and Hamano, 1990). We hypothesized that tannins, which are highly polymer- ized and have many phenolic hydroxyl groups, are likely to be very effective antioxidants. To test this hypoth- esis, we evaluated representative tannins using methods which provide insights into both efficacy and mechanism of potential antioxidants. Nonenzymatic antioxidants can either prevent free radical formation (e.g., by chelating redox active metal ions) or inhibit free radical chain-propagation reactions (e.g., by reacting with peroxyl radical to form stable free species) (Simic and Jovanovic, 1994). We evaluated tannins electrochemically to determine their ability to participate in biological redox reactions (Hodnick et al., 1988), with the deoxyribose assay system to determine their reactivity in Fenton (metal ion-catalyzed) systems (Aruoma, 1994), and with the metmyoglobin method to rank their abilities to react with active oxygen species represented by the stable radical cation ABTS •+ (Rice- Evans and Miller, 1994). MATERIALS AND METHODS Chemicals and Reagents. Methyl gallate, catechin, myo- globin, deoxyribose, 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid (Trolox), thiobarbituric acid, trichloroacetic acid, and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were obtained from Sigma Chemicals (St. Louis, MO). All other chemicals were reagent grade. Procyanidin (PC) was purified from Sorgum grain (Sorghum biocolor Moench variety IS8260, obtained from John Axtell, Purdue University, W. Lafayette, IN) according to Hagerman and Butler (1980). HPLC of phloroglucinol degradation products (Koupai-Abya- zani et al., 1992) and C-13 NMR (Porter, 1989) were used to establish that the material was a B-1 type procyanidin comprised of 16 epicatechin extender units and a catechin terminal unit with average M r of 4930 (Schofield et al., 1998). -1,2,3,4,6-Penta-O-galloyl-D-glucose (PGG) (Mr 940) was puri- fied from commercial tannic acid by solvent extraction (Hager- man et al., 1997) and was characterized by proton NMR and negative ion FAB-MS (Porter, 1989; Self et al., 1986). Al- * Address correspondence to this author [fax (513) 529-5715; e-mail hagermae@muohio.edu]. 1887 J. Agric. Food Chem. 1998, 46, 1887-1892 S0021-8561(97)00975-8 CCC: $15.00 © 1998 American Chemical Society Published on Web 04/15/1998