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1802 <strong>Research</strong> <strong>Article</strong> Received: 23 February 2010 Revised: 19 April 2010 Accepted: 23 April 2010 Published online in Wiley Interscience: 14 June 2010 (www.interscience.wiley.com) DOI 10.1002/jsfa.4017 Identification of ferulate oligomers from corn stover Diane Dobberstein a and Mirko Bunzel b∗ Abstract BACKGROUND: Cross-links between plant cell wall polymers negatively impact forage digestibility. Hydroxycinnamates and their oligomers act as cross-links between polysaccharides and/or polysaccharides and lignin. Higher ferulate oligomers such as dehydrotrimers were identified in cereal grains but not in vegetative organs of grasses. The aim of this study was to characterize ester-linked hydroxycinnamate oligomers from corn stover with special emphasis on ferulate dehydrotrimers. RESULTS: With the exception of the 4-O-5-dehydrodiferulic acid all known ferulate dehydrodimers, including the recently described 8-8(tetrahydrofuran) dimer, were identified in the alkaline hydrolyzate of corn stover after chromatographic fractionation. Next to dehydrodimers, 18 cyclobutane dimers made up of ferulic acid and/or p-coumaric acid were identified by GC-MS of the dimeric size exclusion chromatography fraction. Ferulate dehydrotrimers were isolated by using multiple chromatographic procedures and identified by UV spectroscopy, MS and NMR. Four trimers were unambiguously identified as 5-5/8-O-4-, 8-O-4/8-O-4-, 8-8(aryltetralin)/8-O-4-, and 8-O-4/8-5-dehydrotriferulic acids, a fifth tentatively as 8-5/5-5dehydrotriferulic acid. CONCLUSION: The formation of ferulate dehydrotrimers is not limited to reproductive organs of grasses but also contribute to network formation in the cell walls of vegetative organs. Although radically coupled hydroxycinnamate dimers and oligomers were in the focus of researchers over the last decade, the earlier described cyclobutane dimers significantly contribute to cell wall cross-linking. c○ 2010 Society of Chemical Industry Keywords: ferulic acid; p-coumaric acid; dehydrotrimer; dehydrotriferulic acid; cyclobutane dimers; forage digestibility INTRODUCTION Forage digestibility and hence quality is widely controlled by the cell walls in the plant organs. Structural and matrix polysaccharides, lignin, and proteins are the major constituents of the walls. Cell wall composition and interactions between cell wall polymers mediated by, for example, polysaccharide or polysaccharide–lignin cross-links are important factors limiting their digestibility. 1–6 In grasses, hydroxycinnamates, particularly ferulate and p-coumarate, are minor constituents of the cell wall. While p-coumarates are mostly bound to lignin 7 and only to a lesser degree to arabinoxylans, 8,9 ferulates are primarily acylating the O-5 position of arabinose side-chains in arabinoxylans. 10 Radical- and light-induced coupling reactions of esterified ferulates lead to the formation of dimeric ferulate cross-links between cell wall arabinoxylans. These dimers are known as dehydrodiferulates (radical coupling) or cyclobutane ferulate dimers (light-induced coupling). More recently, dehydrotriferulates and dehydrotetraferulates were isolated from corn bran. 11 – 15 Theoretically, these compounds can cross-link up to four polysaccharide chains forming a strong network in the cell wall. Ferulates can also cross-couple with monolignols. 16,17 Thus, they are co-polymerized into lignins 18,19 and cross-link arabinoxylans with lignins. Although radicals can easily be generated from p-coumarate, radically formed dimers of p-coumarates have not been identified from plant materials. Radical transfer reactions with other phenolics in the cell wall are discussed to explain these findings. 20 Cyclobutane dimers of p-coumarates and mixed cyclobutane dimers of ferulates and p-coumarates, however, were identified in different grasses. 21 – 23 As these compounds are formed by a photochemical mechanism the formation of the cyclobutane dimers requires sunlight during plant growth. The formation of these compounds is therefore supposed to vary strongly depending on the localization of the considered organ or tissue in the plant. While ferulate oligomers such as trimers were isolated form corn bran they were not yet identified in vegetative organs, e.g. stems or leaves, widely used as forages. The aim of this study was to demonstrate that ferulate oligomers, especially ferulate trimers, are not exclusively involved in cell wall cross-linking of reproductive organs but also occur in vegetative organs, thus having a potential influence on forage digestibility. ∗ Correspondence to: Mirko Bunzel, Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Avenue, St Paul, MN 55108, USA. E-mail: mbunzel@umn.edu a Department of Biochemistry and Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany b DepartmentofFoodScienceandNutrition,UniversityofMinnesota,1334Eckles Avenue, St Paul, MN 55108, USA J Sci Food Agric 2010; 90: 1802–1810 www.soci.org c○ 2010 Society of Chemical Industry
Ferulate oligomers from corn stover www.soci.org mV 2500 2000 1500 1000 500 B1 B2 B3 B4 0 200 220 240 260 280 300 320 340 360 380 Figure 1. Separation of alkali-extracted hydroxycinnamate derivatives/oligomers from corn stover by size exclusion chromatography (detection at 325 nm). EXPERIMENTAL General Alcalase 2.4 L (EC 3.4.21.62, from Bacillus licheniformis, 2.4 AU g −1 ) was kindly donated by Novo Nordisk (Bagsvaerd, Denmark). Bio-Beads S-X3 was from Bio-Rad Laboratories (Munich, Germany), the solvent resistant BioBeads size exclusion chromatography (SEC) glass column ECOPLUS from Kronlab (Sinsheim, Germany). Sephadex LH-20 was from Amersham Pharmacia Biotech (Freiburg, Germany). SEC and Sephadex LH-20 chromatography instrumentation (L-6000 pump, L-7400 UV detector) was from Merck/Hitachi (Darmstadt, Germany). Phenyl-hexyl HPLC-columns were purchased from phenomenex (Aschaffenburg, Germany). Phenyl-hexyl-RP-HPLC was carried out using either of the following instrumentations: L-6200 intelligent pump, T-6300 column thermostat, L-7400 UV detector or L-7150 intelligent pump, L-7300 column oven, L-7455 photodiode array detector (Merck/Hitachi, Darmstadt, Germany). HPLC-MS instrumentation was from Hewlett-Packard (Waldbronn, Germany): HP Series 1100: autosampler G1313, pump G 1312A, mass spectrometer G 1946A, photodiode array detector 1314A. Nuclear magnetic resonance (NMR) experiments were performed on a Bruker DRX-500 (Rheinstetten, Germany) instrument. Gas chromatography–mass spectroscopy (GC-MS) was carried out on a Trace 2000 GC coupled to a PolarisQ ion trap mass spectrometer (ThermoQuest, Thermo Scientific, Dreieich, Germany) using a HP-5-MS fused-silica capillary column (Hewlett-Packard). Plant material and sample preparation Seeds (DK 233) were made available by Monsanto Agrar Deutschland GmbH (Düsseldorf, Germany). The plant material was provided by Professor F. Schwarz and Dr F. Zeller from the Technical University of Munich, Department of Animal Nutrition. The plant material was seeded on 28 April 2004 in a field trial in Freising, Germany, and harvested on 27 September 2004. The dry matter content of the grains at the time of harvest was 640 g kg −1 , representing a typical maturity stage used for the production of silage. The duration of sunshine from sowing to harvest was calculated as 1226 h. Immediately after harvesting, the cob was removed, and the corn stover was coarsely chopped, freeze-dried, and stepwise ground to pass a 0.5 mm screen. Dried material (280 g) was washed five times with water (3 L each, stirring for 10 min) followed by centrifugation. The residue was consecutively extracted in a Soxhlet apparatus for 8 h with ethanol and 6 h with acetone followed by a drying step. Residual material (200 g) was suspended in a phosphate buffer pH 7.5, and proteins min were partially degraded by using the protease alcalase (30 µL enzyme g −1 dry material, 30 min at 60 ◦ C, continuous agitation). The residue was centrifuged, washed with hot water, 95% (v/v) ethanol, and acetone, and dried. Alkaline hydrolysis and extraction Alkaline hydrolysis and extraction was performed according to a previously described procedure. 12 In brief, extracted and dried stover (165 g) was saponified (NaOH (2 mol L −1 ); 20 mL NaOH g −1 stover) under nitrogen, protected from light and under continuous stirring for 18 h. Following acidification of the mixture (pH < 2), liberated phenolic acids were extracted into diethyl ether. Ether extracts were extracted with NaHCO3 solution (50 g L −1 ). After acidification (pH < 2) of the combined aqueous layers the phenolic acids were re-extracted into diethyl ether. Ether extracts were dried over Na2SO4, evaporated to dryness and re-dissolved in tetrahydrofuran (10 mL). Fractionation of phenolic acids SEC was carried out by using Bio-Beads S-X3 (gel bed: 1.5 cm × 95 cm) swollen in tetrahydrofuran which was also used as mobile phase. Hydrolyzate dissolved in tetrahydrofuran (about 200 mg in 500 µL) was applied to the column. The flow rate was maintained at 0.25 mL min −1 for 360 min, increased to 0.5 mL min −1 between 360 and 395 min and further increased to 0.75 mL min −1 until all material was eluted. Fractions were collected according to the chromatogram monitored at 325 nm (Fig. 1). Fractions B2 and B3 from 20 runs were pooled, dried under a stream of nitrogen, redissolved in methanol (MeOH)/water 50/50 (v/v) (ultrasonic bath, addition of a few drops acetone to improve solubility), and used for Sephadex LH-20 chromatography. Sephadex LH-20 chromatography was performed as described previously 11 with some minor modifications. Fraction B2 (386 mg) was separated in two Sephadex runs whereas only a portion of fraction B3 (875 mg) was separated in a single run. In brief, the sample was applied to the column (gel bed: 2.5 cm × 85 cm) pre-conditioned with aqueous trifluoroacetic acid (TFA) (0.5 mmol L −1 )/MeOH 95/5 (v/v). Elution was carried out as follows: (1) elution with TFA (0.5 mmol L −1 )/MeOH 95/5 (v/v) for 72 h, flow rate: 1.5 mL min −1 ; (2) elution with TFA (0.5 mmol L −1 ))/MeOH 50/50 (v/v) for 72 h, flow rate: 1.0 mL min −1 ;(3)elutionwithTFA (0.5 mmol L −1 )/MeOH 40/60 (v/v) for 65 h, flow rate: 1.0 mL min −1 ; (4) rinsing step with TFA (0.5 mmol L −1 )/MeOH 10/90 (v/v). Detection was carried out at 280 and 325 nm. Fractions were collected over 12-min periods, combined according to the J Sci Food Agric 2010; 90: 1802–1810 c○ 2010 Society of Chemical Industry www.interscience.wiley.com/jsfa 1803
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