Wendy R. Russell

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.

Anti-Inflammatory Implications of the Microbial Transformation of Dietary Phenolic Compounds

Due to the success of therapeutic anti-inflammatory compounds to inhibit, retard, and reverse the development of colon cancer, the identification of dietary compounds as chemopreventives is being vigorously pursued. However, an important factor often overlooked is the metabolic transformation of the food-derived compounds in the gut that may affect their bioactivity. Commonly consumed dietary phenolics (esterified ferulic acid and its 5-5′-linked dimer), which have the potential to undergo predominant microbial transformations (de-esterification, hydrogenation, demethylation, dehydroxylation, and dimer cleavage), were incubated with human microbiota. The metabolites were identified (high-performance liquid chromatography and nuclear magnetic resonance) and confirmed to be present in fresh fecal samples from 4 human volunteers. The potential anti-inflammatory properties were compared by measuring the ability of the parent compounds and their metabolites to modulate prostanoid production in a cell line in which the inflammatory pathways were stimulated following a cytokine-induced insult. The compounds were readily de-esterified and hydrogenated, but no dimer cleavage occurred. Only the monomer underwent demethylation and selective de-hydroxylation. The resultant metabolites had differing effects on prostanoid production ranging from a slight increase to a significant reduction in magnitude. This suggests that the microbial transformation of dietary compounds will have important inflammatory implications in the chemoprevention of colon cancer.

Extraction of phenolic-carbohydrate complexes from graminaceous cell walls

Abstract Barley straw and a preparation of perennial ryegrass were sequentially extracted with oxalic acid, dimethyl sulphoxide (DMSO) and a “cellulolytic” enzyme preparation Driselase and the fractions studied by methylation analysis, 13 C NMR and other methods. Oxalic acid, as expected, solubilised the bulk of the arabinose and ferulic acid in both samples, although appreciable amounts of xylose were also solubilised. DMSO yielded polymeric lignin-carbohydrate complexes (LCC), both of which consisted predominantly of a β (1 → 4) xylan. From the results of methylation analysis, sensitivity to oxalic acid hydrolysis and size-exclusion chromatography after alkaline hydrolysis, it was evident that lignin polymers were attached to arabinosyl and xylosyl residues by both ester and aryl-ether linkages. After cellulolytic hydrolysis of the residues, thioacidolysis analysis of the lignin component in the DMSO-soluble and Driselase-insoluble fractions from ryegrass revealed differences in every measurable aspect. The DMSO-soluble lignin was found to be more highly condensed, have a higher S/G ratio and have a higher terminal G/internal G ratio.

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.

Copper–homocysteine complexes and potential physiological actions

During the last 2 decades it was proposed that atherogenesis was closely related to the homeostasis of homocysteine (hCys) and/or copper. We hypothesized that the physiological action of hCys may be connected with its ability to form complexes with Cu. Our results showed the presence of two different Cu-hCys complexes. At a molar ratio Cu:hCys 1:1, a blue complex most probably consistent with a tentative dimeric Cu(II)(2)(hCys)(2)(H(2)O)(2) formula was formed, with tetrahedral Cu coordination and anti-ferromagnetic properties. The redox processes between Cu(II) and hCys, in a molar ratio > or =1:3 led to formation of a second yellow Cu(I)hCys complex. Both Cu-hCys complexes affected the metabolism of extracellular thiols more than hCys alone and inhibited glutathione peroxidase-1 activity and mRNA abundance. The biological action of hCys and Cu-hCys complexes involved remodeling and phosphorylation of focal adhesion complexes and paxillin. The adhesive interactions of monocytes with an endothelial monolayer led to the redistribution of both paxillin and F-actin after all treatments, but the diapedesis of monocytes through endothelial cell monolayer was both greater and faster in the presence of the tentative Cu(II)(2)(hCys)(2)(H(2)O)(2) complex. Together, these observations suggest that Cu-hCys complexes actively participate in the biochemical responses of endothelial cells that are involved in the aethiopathogenesis of atherosclerosis.

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.

Radical formation and coupling of hydroxycinnamic acids containing 1,2-dihydroxy substituents

Hydroxycinnamic acids involved in the deposition and cross-linking of plant cell-wall polymers do not usually contain 1,2-dihydroxy substituents, despite the presence of both 3,4-dihydroxycinnamic acid and 4,5-dihydroxy-3-methoxycinnamic acid as intermediates in the biogenesis of lignin. Since the O-methyl transferases, enzymes catalysing methylation, are targets for the genetic manipulation of lignin biosynthesis, the potential incorporation of these 1,2-dihydroxated substrates becomes increasingly significant. Using EPR spectroscopy, it was observed that 1,2-dihydroxy substituents did not have an inhibitory effect on radical formation. Increasing the extent of hydroxylation and methoxylation, resulted in an increased ease of substrate oxidation. Despite formation of the parent radicals, coupling did not proceed, under conditions that generally result in phenylpropanoid polymerisation. It is postulated that intermolecular radical-coupling reactions are inhibited due to rapid conversion to the o-quinone. In contrast, when methoxylated at C3, as in 4,5-dihydroxy-3-methoxycinnamic acid, radical coupling proceeds with the major product resulting from 8-O-3 radical coupling and formation of a substituted 2,3-dihydro-1,4-dioxin ring.

Inhibition of 15-lipoxygenase-catalysed oxygenation of arachidonic acid by substituted benzoic acids

Elevated levels of phospholipases, prostaglandin synthases and lipoxygenases in colonic cells at various stages of malignancy indicate a strong link between dietary lipids and colon cancer. Lipoxygenase-catalysed arachidonic acid metabolism plays a key role in colorectal carcinogenesis and has the potential to be modulated by phenolic compounds. Plant-based foods are rich sources of phenolic compounds and in the human colon they are predominantly available as simple phenolics such as the benzoic acids. Benzoic acids were determined in faecal waters from four volunteers consuming a western-style diet. Structure-activity relationships were established for the lipoxygenase-catalysed oxygenation of arachidonic acid using an oxygen electrode. All compounds studied inhibited this reaction (21-73%; p<0.001) and many of the structural features could be rationalised by computational modelling. No correlation was observed with the ability to act as reductants, supporting the hypothesis that their mode of inhibition may not be by a direct redox effect on the non-haem iron.

Predicting the macromolecular structure and properties of lignin and comparison with synthetically produced polymers.

Summary Determining the structure of the plant polymer lignin is not feasible because of the heterogeneous structure of this polymer and the difficulties encountered in its extraction. Therefore, computational chemistry can provide information, which may otherwise be unavailable. Based on experimental results, computational chemistry was used to mimic the processes involved in lignin formation. Synthetic polymers also were prepared from the three major lignin precursors and their physical properties were shown to give good correlation with computational models constructed to represent these polymers. Both computational and experimental results, demonstrated that the higher the degree of methoxylation, the more flexible the polymer and this was shown to be dependent on the type of inter-unit linkage. Using the same experimentally derived parameters, a model was constructed to represent a generalised lignin. This model was shown to be a flexible, closely packed structure and this was attributed to the predominance of 8-O-4 linkages, which allowed closer stacking of the aromatic rings.