Journal of Cereal Science 47 (2008) 29–40 Cross-linking of arabinoxylans via 8-8-coupled diferulates as demonstrated by isolation and identification of diarabinosyl 8-8(cyclic)-dehydrodiferulate from maize bran Mirko Bunzel a,à , Ella Allerdings a , John Ralph b,c , Hans Steinhart a a Institute of Biochemistry and Food Chemistry, Department of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany b US Dairy Forage Research Center, USDA-ARS, 1925 Linden Drive West, Madison, WI 53706-1108, USA c Biological Systems Engineering Department, University of Wisconsin, Madison, WI 53706, USA Received 14 October 2006; received in revised form 22 December 2006; accepted 28 December 2006 Abstract Dehydrodiferulates are likely the most important arabinoxylan cross-links in cereals and grasses in general. However, association of dehydrodiferulates and arabinoxylans has only been authenticated for 5-5- and 8-O-4-dehydrodiferulates to date. In the present study, a saccharide ester of 8-8(cyclic)-dehydrodiferulate was isolated from maize bran insoluble fibre following mild acidic hydrolysis by using Sephadex LH-20 chromatography, gel chromatography on Bio-Gel P-2, and RP-HPLC. Mass spectrometry, one- and two-dimensional NMR and analysis of the carbohydrate and phenolic constituents following further hydrolysis identified the isolated compound as the di- 5-O-L-arabinosyl ester of 8-8(cyclic)-dehydrodiferulic acid. From this finding it is apparent that 8-8(cyclic)-dehydrodiferulate exists as such in the plant cell wall and acts as an arabinoxylan cross-link. In addition, a fraction was isolated that contained two saccharide esters of 8-O-4-dehydrodiferulates. This fraction was comprised of two compounds, both built from 8-O-4-dehydrodiferulate, a 5-linked arabinofuranose and a 5-linked xylopyranosyl-(1-2)-arabinofuranose unit. These compounds show that, in addition to the 5-O-(trans- feruloyl)-L-arabinofuranosyl sidechain, the more complex b-D-xylopyranosyl-(1-2)-5-O-trans-feruloyl-L-arabinofuranosyl sidechains are involved in the formation of 8-O-4-dehydrodiferulates. r 2007 Elsevier Ltd. All rights reserved. Keywords: Diferulic acid; Ferulic acid; Cell-wall cross-linking; Arabinoxylans; Dietary fibre 1. Introduction Cereal grain arabinoxylans are important dietary fibre compounds and they also influence formation and proper- ties of dough and bread (Andersson and Aman, 2001). As cell wall constituents of grasses in general, arabinoxylans are also an important source of energy for ruminants (Hatfield et al., 1999). The physicochemical properties of arabinoxylans are dependent on the arabinose/xylose ratio, the distribution of the sidechains, the degree of polymer- isation, the extent of their cross-linking, and on their coupling to other polymeric cell wall components. Arabi- noxylan cross-linking and cross-coupling to other cell wall components such as lignin or proteins is achieved via hydroxycinnamates, especially via ferulate and its deriva- tives (Bunzel et al., 2004a; Geissmann and Neukom, 1971; Markwalder and Neukom, 1976; Piber and Koehler, 2005; Ralph et al., 1994, 2004). Ferulates acylate xylan arabino- syl units at their O-5-positions (Ishii, 1997). Radical coupling of feruloylated arabinoxylans results in the formation of dehydrodiferulates or higher ferulate dehydrooligomers such as dehydrotriferulates and dehy- drotetraferulates (Bunzel et al., 2003, 2005, 2006; Funk et al., 2005; Rouau et al., 2003). The ferulate moieties are coupled to produce new bonds that may occur at the 4-O-, 5- or 8-carbons, thus forming 5-5-, 8-8-, 8-5-, 8-O-4- and 4- O-5-linkages. Dehydrodiferulates are now routinely ana- lysed in a whole range of plant materials following alkaline ARTICLE IN PRESS www.elsevier.com/locate/jcs 0733-5210/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2006.12.005 Abbreviations: Ara, arabinose; DFA, dehydrodiferulic acid; TFA, trifluoroacetic acid; Xyl, xylose à Corresponding author. Tel.: +49 40 42838 4379; fax: +49 40 42838 4342. E-mail address: mirko.bunzel@uni-hamburg.de (M. Bunzel).