Eur Food Res Technol (2003) 217:128–133 DOI 10.1007/s00217-003-0709-0
ORIGINAL PAPER
Mirko Bunzel · John Ralph · Carola Funk · Hans Steinhart
Isolation and identification of a ferulic acid dehydrotrimer from saponified maize bran insoluble fiber Received: 4 March 2003 / Published online: 15 May 2003 Springer-Verlag 2003
Abstract Following saponification of maize bran insoluble fiber a ferulic acid dehydrotrimer was isolated using Sephadex LH-20 chromatography. Structural identification was carried out using UV-spectroscopy, mass spectrometry and 1D and 2D NMR experiments (1H, 13 C, DEPT, COSY, TOCSY, 13C-1H HSQC, HMBC). UV-spectroscopy indicated characteristics of ferulate structures, mass spectrometry showed a trimeric ferulate structure, and the NMR spectra provided diagnostic evidence for its being a 5-5/8-O-4-coupled dehydrotrimer. Ferulic acid dehydrodimers are mainly derived from diferulates which cross-link polysaccharides. Because of the involvement of a 5-5-dehydrodiferulic acid unit in the identified trimer, this novel dehydrotriferulic acid from cereal grain fiber need not imply the cross-linking of three polysaccharide chains; molecular modeling of the ferulate dehydrodimerization in earlier studies showed that the 55-diferulate, uniquely, can form intramolecularly. This first identified ferulic acid dehydrotrimer nevertheless reveals that polysaccharide chains can be more extensively cross-linked than previously recognized. Keywords Zea mays L. · Dietary fiber · Ferulic acid · Diferulic acid · Triferulic acid · Arabinoxylan · Cell wall cross-linking
M. Bunzel ()) · C. Funk · H. Steinhart Institute of Biochemistry and Food Chemistry, Department of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany e-mail:
[email protected] Tel.: +49-40-42838-4379, Fax: +49-40-42838-4342 J. Ralph Department of Forestry, University of Wisconsin, Madison, Wisconsin, 53706 USA Present address: J. Ralph, U.S. Dairy Forage Research Center, USDA-Agricultural Research Service, Madison, Wisconsin, 53706, USA
Introduction Cross-linking of plant cell walls via ferulate dehydrodimerization reactions is well established [1, 2, 3, 4]. Ferulates acylate various polysaccharides, notably arabinoxylans in grasses [1] and in cereal grain dietary fiber, particularly in the insoluble fiber fraction [5, 6]. Ferulate dehydrodimerization is therefore a mechanism for linking two polysaccharide chains, providing structural integrity to the cell wall, but inhibiting fiber degradability [7]. Being phenolic entities capable of radical coupling reactions, ferulates and their dehydrodimers may also be incorporated into lignin polymers [2, 8], furthering the cross-linking of the wall, and further limiting the degradability of the polysaccharides [9]. The dehydrodimerization reaction is via radical coupling [10]. As such, the reaction is somewhat combinatorial in nature, much like the radical coupling of monolignols to each other and to lignins [11]. A range of dehydrodiferulates can be found in a variety of materials [10, 12, 13, 14, 15, 16], as predicted and verified in 1994 [10]. The dehydrodiferulates are characterized by the new bond formed in the radical coupling reaction between the two ferulates (at their 4-O-, 5- or 8carbons) as 5-5-, 8-8-, 8-5-, 8-O-4-, and 4-O-5-coupled dehydrodimers. Following saponification, or during the coupling process in the plant itself, several forms of the 88- and 8-5-coupled dehydrodimers are possible. Characterization of the cross-linking of walls via ferulate has therefore become considerably more interesting, but more complex than it was before the 1994 study, when only the 5-5-coupled dehydrodimer 2 (Fig. 1) was known. We have recently provided the first detailed characterization of releasable diferulates from a range of cereal grains [16]. Cereal grains are one of the most important food groups. Their fiber properties and even some of their health benefits can likely be attributed to the nature of their cell wall polymers and their chemical architecture. We have also recently discovered disinapates and sinapate-ferulate cross-products in wild rice, rice, wheat and
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this paper, we report on the isolation and structural identification of a ferulate dehydrotrimer from maize bran insoluble fiber, and discuss its implications for cell wall cross-linking in cereal grains.
Materials and Methods
Fig. 1 Structures and numbering system used for the new dehydrotriferulic acid (1) along with dehydrodimers 2 (a 5–5-dehydrodiferulic acid) and 3 (an 8-O-4-dehydrodiferulic acid). The actual bonds formed in the radical coupling steps are in bold
other grains, implicating sinapates in similar polysaccharide cross-linking reactions in the wall [17]. The idea that ferulates might continue their radical polymerization reactions and form higher oligomers in the wall has long been discussed, but rarely in the literature. Certainly, dehydrogenation oligomers [18] and polymers can be made in vitro. However, ferulates encumbered by their direct attachment to an extensive polysaccharide chain were thought unlikely to be able to approach two other such ferulates. A possible exception is at the ends of chains, or if there is rather extreme clustering of ferulates on polysaccharide chains such that dimerization (crosslinking two polysaccharide chains) might create a dehydrodiferulate in sufficient proximity to another ferulate on one of the chains that a further coupling reaction could occur. This dehydrotrimer formation would still cross-link only two polysaccharide chains, not three. In view of the finding reported in this paper, such possibilities should be examined through the use of molecular modeling. Molecular modeling of the ferulate dehydrodimerization process led Hatfield et al. to conclude that one dimer was unique in that it could be formed intramolecularly [19]. If ferulates on arabinosyl residues were attached to xylose units in a chain with two intervening xylose units (i.e. located on one xylose unit and on the fourth xylose unit from it), and with no other arrangement, it appears to be possible to form intramolecular 5-5-dehydrodiferulate, and only that dehydrodimer. Thus the 5-5-diferulate is likely the only one that can form intramolecularly and this may explain the prevalence of this dimer in various plant cell walls when its formation from low molecular-weight ferulate ester models in vivo is rather minor; ferulates may simply be positioned on wall polymers such that this coupling is relatively favored. More recently, Fry et al. have provided evidence for higher ferulate oligomers in maize suspension cultures [20]. No actual trimers or higher oligomers were structurally characterized, but they were strongly implied. In
General. Sephadex LH-20 was from Amersham Pharmacia Biotech (Freiburg, Germany). Heat-stable a-amylase Termamyl 120 L (EC 3.2.1.1, from Bacillus licheniformis, 120 KNU/g), the protease Alcalase 2.4 L (EC 3.4.21.62, from Bacillus licheniformis, 2.4 AU/ g) and the amyloglucosidase AMG 300 L (EC 3.2.1.3, from Aspergillus niger, 300 AGU/g) were from Novo Nordisk, Bagsvaerd, Denmark. Methanol (HPLC grade) was from Baker (Deventer, Holland), methanol-d4 from Deutero (Kastellaun, Germany) and acetone-d6 and D2O from Cambridge Isotope Laboratories (Andover, Mass., USA). All other chemicals were from Merck (Darmstadt, Germany). Plant material. Maize bran (Zea mays L.) was kindly provided by Hammermhle Maismhle GmbH, Kirrweiler, Germany. Preparation of insoluble maize fiber. Maize bran was defatted and extracted with acetone for 4 h using Soxhlet equipment. The following procedure was performed four times: defatted maize bran (20 g, milled to a particle size
