Lorraine Scobbie

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.

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.

Pectic polysaccharides of mesophyll cell walls of perennial ryegrass leaves

Abstract Extraction of mesophyll cell walls from leaves of perennial ryegrass with CDTA, a chelating agent, removed 25 mg uronic acid g − , largely in the form of a polymer which spontaneously precipitated on removal of the CDTA or during subsequent purification. Methylation analysis, before and after reduction, showed that the precipitated polymer was a 1,4-linked homogalacturonan essentially free from neutral sugar residues, with a low degree of acetylation (3.6%) and methyl esterification (3.3%). Hot water (HW) extracted further acidic material (5 mg uronide g −1 cell wall) which could be resolved by ion-exchange chromatography into neutral mixed-linked glucan and bound rhamnogalacturonan fractions. The latter co-chromatographed with sugar residues typical of 3-, 4- and 6-linked galactan and arabinoxylan. Pectin esterase promoted the release from cell walls of HW-soluble rhamnogalacturonan by polygalacturonase, but had no effect on the release of CDTA-soluble uronide. The presence of both homogalacturonan and rhamnogalcturonan, typical of dicotyledons, suggested that the pectic polysaccharides of the Gramineae differed from those of other plants in amount only, rather than nature.

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.