Improved Characterization of Sorghum Tannins Using Size-Exclusion Chromatography

Sorghum (Sorghum bicolor L. Moench) is a native grass species of the arid and semi-arid regions of Africa (Kimber 2000). Sorghum is a genetically diverse crop and contains some genotypes that have a pigmented testa and therefore contain tannins (Rooney et al 1980; Rooney and Miller 1982). Such lines have the dominant B1_B2_ genes and are the only sorghum types with tannins (Blakeley et al 1979), despite common misperceptions that all sorghums have tannins or that the presence of tannins is linked to seed color (Boren and Waniska 1992). Due to the high antioxidant activity, sorghum tannins have recently been examined for potential as a nutraceutical, especially cancer prevention (Awika and Rooney 2004). Generally, tannins which are polymers, are characterized on the basis of their monomeric composition, and their degree of polymerization, both of which can impact their functionality (Asquith et al 1983). Many analytical methods have been used to study their composition such as reversed phase (RP) HPLC (Putman and Butler 1989; Prior and Gu 2005), normal-phase (NP) HPLC (Gu et al 2002; Awika et al 2003), size-exclusion chromatography (SEC) (Williams et al 1983; Karchesy et al 1988; Kennedy and Taylor 2003), capillary electrophoresis (CE) (Bae et al 1994; Cifuentes et al 2001; Bonoli et al 2004), and mass spectrometry (Cheynier and Fulcrand 2003; Kruger et al 2003). It is generally agreed that sorghum tannins are composed of monomeric flavan-3-ols. Thus, recent research has focused on the degree of polymerization of these subunits, with one limitation being how well the largest polymers (i.e., a high degree of polymerization [DP]) can be resolved. Recently, NP-HPLC has resolved tannins to a DP of >10 and has significantly improved the separation and characterization of tannins from a number of sources, including sorghum (Hammerstone et al 1999; Gu et al 2002). While NP-HPLC was capable of excellent resolution, run times were lengthy (40 min) and NP-HPLC must be conducted in the total absence of water, which complicates switching instruments between types of HPLC such as RP-HPLC. Thus, the objectives of this study were to investigate the use of improved SEC procedures for analyzing sorghum tannins that required no special sample preparation (such as derivatization, tannin purification, etc.) and that would provide information on the Mw distribution of the tannins. MATERIALS AND METHODS