Gordon J Provan

Hydroxycinnamic acids in the digestive tract of livestock and humans

Hydroxycinnamic acids are consumed as water-soluble conjugates and in larger amounts bound to plant cell walls. Bound acids are primarily released by microbial action in the modified forestomach of ruminants and the hindgut of non-ruminant species, including humans. In the rumen, rapid hydrogenation of p-coumaric, ferulic and caffeic acids, followed by dehydroxylation at C4 and more slowly at C3 yields 3-phenylpropionic acid. Phenylpropionate is absorbed and undergoes β-oxidation in the liver to benzoic acid which is then excreted predominately (75-95%) as its glycine conjugate (hippuric acid), but also as the free acid or glucuronide. In non-ruminants, hydroxycinnamates may be absorbed unchanged in the upper digestive tract via a Na + -dependent saturable transport system or escape to the hindgut where they are subject to microbial transformations with further absorption of metabolites. Metabolites of p-coumaric acid found in rat urine are the unchanged compound and its glycine conjugate, the reduced derivative and the β-oxidation product, 4-hydroxybenzoic acid. Caffeic acid and its methyl ethers (ferulic and iso-ferulic acids) are interconvertable and share metabolites. As in the rumen, reduction of the C 3 side-chain, demethylation of ferulate and dehydroxylation at C4 are products of microbial action. Dehydroxylation at C3 is more rarely encountered. The resulting 3-hydroxyphenylpropionic acid is commonly found in the urine of all species and is the major metabolite in rats where relatively little chain-shortening occurs. A larger range of metabolites including C 6 -C 1 compounds have been detected in human urine. Metabolism of hydroxycinnamate dimers found as cross-links between polysaccharide chains has been little studied although evident differences in the ability to metabolise such compounds exist between the human and rumen microflora.

Characterisation of lignin from CAD and OMT deficient Bm mutants of maize

Internodes of the maize cell line W401 and bm 1 and bm 3 mutants expressed in W401 were harvested 5 days after anthesis (A5) and at silage (S) stage. The normal maize had a higher total phenolic (TP) content (80.5-90.5 g kg -1 cell wall DM) than both bm 1 and bm 3 mutants (74.4-86.4 and 66.0-84.2 g kg -1 cell wall DM, respectively). TP were inversely related to cellulase digestibility with values of 85.4-91.5, 89.3-92. and 91.3-94.1% for normal, bm 1 and bm 3 . Marked differences in p-coumaric acid concentrations were found ranging from 20.9 to 26.3 g kg -1 cell wall DM for normal, 14.9 to 15.3 g kg -1 for bm 1 to 10.1 to 14.4 g kg -1 for bm 3 . The ferulate pattern was entirely different with the bm 1 genotype providing the lowest tota1 (9.1-10.7 g kg -1 ) and etherified (1.9-2.3 g kg -1 ) values. Although the bm 3 contained more total ferulate (11.5-13.1 vs 10.9-11.7 g kg -1 ), the normal variety had a significantly greater amount of etherified ferulate (2.8-3.4 vs 3.2-4.1 g kg -1 ) implying a greater extent of cross-linking between wall polymers. Recovery of guaiacyl and syringyl residues was greatest in the normal maize with the bm 1 occupying the middle position between the two extremes. Calculated S: G ratios from 4 M NaOH digestion and NMR were in good agreement with the normal line giving the highest ratio, bm 1 intermediate and bm 3 the lowest. Colorimetric analysis revealed a large increase in the aldehyde content of the in situ bm 1 lignin compared to normal and bm 3 genotypes although NMR failed to reveal significant numbers of aldehydic resonances.

Characterisation of Lignin from Parenchyma and Sclerenchyma Cell Walls of the Maize Internode

Internodes of maize (Zea mays L, Co125), harvested 5 days after anthesis, were sectioned into five equal parts and samples of sclerenchyma and parenchyma cells mechanically isolated from each section. Phenolic acids and syringyl and guaiacyl degradation products of lignin were released from the walls of the two cell types by microwave digestion with 4 M NaOH. Aryl ether bonded units were selectively released by thioacidolysis. Total phenolic content of cell walls from the youngest (basal) sections were approximately two-thirds of those of the oldest, topmost sections (parenchyma 70·8–99·0 and sclerenchyma 72·5–114·1 mg g-1) indicating that the process of lignification was already well advanced amongst most of the cell walls of the youngest section. The total phenolic content was marginally, but significantly, greater (P<0·05) in sclerenchyma walls than in parenchyma walls at all stages of maturity. There was no significant difference in phenolic acid concentrations between cell types from the same section but p-coumaric acid concentration increased with maturity (P<0·001) in walls from both cell types. The increase in p-coumarate with age was matched by an increased recovery of syringyl units resulting in a constant coumaroyl: syringyl molar ratio. Recovery of acetosyringone was significantly greater (P<0·001) from sclerenchyma than parenchyma walls and, in sclerenchyma, acetosyringone as a proportion of total syringyl recovery, increased significantly with age (P=0·015). Digestion with NaOH and thioacidolysis released comparable amounts of guaiacyl residues but NaOH digestion released approximately twice the amount of syringyl residues. This difference may be explained by the retention of the ester-bond between p-coumaric acid and syringyl units during thioacidolysis but not during digestion with 4 M alkali. The similarity in phenolic composition suggested that both cell types, despite their considerable anatomical differences, were exposed to a common flux of lignin precursors during the later stages of lignification as illustrated by the internode sections. Differences between cell walls arose because of differences in the regiochemistry of precursor incorporation.

Structure‐specific functionality of plant cell wall hydroxycinnamates

Within the plant cell wall, 4-hydroxy-3-methoxycinnamic acid (ferulic acid) has a clear role in polymer cross-linking. However, why this function appears largely restricted to the monomethoxylated compound and not to other hydroxycinnamates appearing in the wall is less evident. Since radical coupling is the main mechanism by which hydroxycinnamate cross-linking occurs, the ease of parent radical formation and distribution of the unpaired electron were investigated. Ease of oxidation increased with increased substrate methoxylation, as did the amount of positive spin-density residing on the phenolic oxygen. The properties of the dimethoxylated hydroxycinnamate indicated that when esterified to the wall, coupling would result in C-O bond formation. This form of bonding is weaker and more flexible than the C-C bonding which would result from coupling of 4-hydroxy-3-methoxycinnamate and would not be desirable as a cross-link. Although the non-methoxylated compound could also couple via C-C bonds, ESR measurements of phenoxyl radical formation suggested that this compound would not easily participate in coupling reactions.

Extent of incorporation of hydroxycinnamaldehydes into lignin in cinnamyl alcohol dehydrogenase-downregulated plants

Down-regulation of cinnamyl alcohol dehydrogenase leads to an accumulation of cinnamaldehydes available for incorporation into the developing lignin polymer. Using electron spin resonance spectroscopy we have demonstrated that the parent radical of 4-hydroxy-3-methoxycinnamaldehyde is generated by peroxidase catalysed oxidation. The extent of radical generation is similar to that of 4-hydroxy-3-methoxycinnamyl alcohol and is increased by further aromatic methoxylation. From the distribution of the electron-spin density, it was predicted that the regiochemistry of 4-hydroxy-3-methoxycinnamaldehyde coupling would be similar to that of the corresponding alcohol, with the possibility of a higher degree of 8-O-4 linkages occurring. These predictions were confirmed by polymerisation studies, which also showed that after radical coupling the alpha,beta-enone structure was regenerated. This suggests that, although the cross-linking and physical properties of cinnamaldeyde rich lignins differ from that of normal lignins, cinnamaldehydes are incorporated into the lignin polymer under the same controlling factors as the cinnamyl alcohols.