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Title:   Author  item
                                            Grabber, Johnitem
                                            Zeller, Wayneitem
                                            MUELLER HARVEY, IRENE - University Of Reading
  Submitted to: Journal of Agricultural and Food Chemistry
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 2/1/2013
                                    Publication  Date: 2/5/2013
                                    Citation: Grabber, J.H., Zeller, W.E., Mueller Harvey, I. 2013. Acetone enhances the direct analysis of Procyanidin- and Prodelphinidin-based condensed tannins in lotus species by the butanol-HCl-iron assay. Journal of Agricultural and Food Chemistry. 61:2669-2678.Interpretive Summary: Condensed tannins produced by many plant species are of considerable interest because of their multiple effects on biological systems. They can, for example, affect the availability of nutrients in foods, feeds, and decaying plant matter; influence the palatability and flavor of food and beverages; and serve as antioxidants beneficial to human health. The butanol-HCl assay is among the most specific and commonly used methods for quantifying condensed tannin concentrations in extracts prepared from foods, forages, and foliage of herbaceous and woody plants. The method, however, underestimates the total amount of tannin in plant materials. We therefore initiated work to improve the assay by testing the utility of adding various co-solvents to butanol-HCl. Having identified acetone as a suitable co-solvent, we then optimized conditions for a modified acetone-butanol-HCl assay and compared it to the standard assay for direct analysis of total condensed tannins in plant tissue. The modified acetone-butanol-HCl assay increased estimates of total condensed tannins in forage legumes by at least 2-fold over the standard assay run without acetone. Analysis of insoluble residues collected following butanol-HCl hydrolysis revealed that acetone addition improved quantification by completely solubilizing tannin from plant tissue. We anticipate the improved assay will be widely used by scientists to understand the effects of condensed tannins on biological systems and to enhance their use in foods and feeds.
                                Technical Abstract: The butanol-HCl spectrophotometric assay is widely used for quantifying extractable and insoluble condensed tannins (CT, syn. proanthocyanidins) in foods, feeds, and foliage of herbaceous and woody plants, but the method underestimates total CT content when applied directly to plant material. To improve CT quantitation, we tested various cosolvents with butanol-HCl and found that acetone increased anthocyanidin yields from two forage Lotus species having contrasting procyanidin and prodelphinidin compositions. A butanol-HCl-iron assay run with 50% (v/v) acetone gave linear responses with Lotus CT standards and increased estimates of total CT in Lotus herbage and leaves by up to 3.2-fold over the conventional method run without acetone. The use of thiolysis to determine the purity of CT standards further improved quantitation. Gel-state 13C and 1H–13C HSQC NMR spectra of insoluble residues collected after butanol-HCl assays revealed that acetone increased anthocyanidin yields by facilitating complete solubilization of CT from tissue.


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