Isoenzymes of p-coumarate: CoA ligase from cell suspension cultures of Glycine max.

Two isoenzymes of p-coumarate: CoA ligase were isolated from cell suspension cultures of soybean (Glycine max L., var. Mandarin). Separation and partial purification of the enzymes were achieved by precipitation with MnCl2 and (NH4)2SO4, and column chromatography on DEAE-cellulose, Sephadex G-100 and hydroxyapatite. The isoenzymes had approximately the same molecular weight, but differed significantly with respect to their substrate specificity, their inhibition constants for AMP, their dependence on pH and ionic strength for optimum activity, and their fractionation pattern during the purification procedure or upon analytical disc-gel electrophoresis. Both coumarate: CoA ligases were specific for the activation of various substituted cinnamic acids. Of the cinnamic acids tested, ferulic, sinapic, 5-hydroxyferulic, p-coumaric, and caffeic acids were the substrates with the lowest apparent Km values (on all the order of 1 to 4 x 10(-5) M) for isoenzyme 1. The lowest apparent Km values (from about 1 to 9 x 10(-5) M) for isoenzyme 2 were obtained for caffeic, p-coumaric, m-coumaric, and o-coumaric acids. Sinapic acid and several methoxycinnamic acids were efficient substrates of isoenzyme 1 but were not activated at all by isoenzyme 2. The possible roles of the two p-coumarate: CoA ligase isoenzymes in the phenylpropanoid metabolism of the cell cultures are discussed.     

Modeling suberization with peroxidase-catalyzed polymerization of hydroxycinnamic acids: cross-coupling and dimerization reactions

An anionic potato peroxidase (EC 1.11.1.7, APP) thought to be involved in suberization after wounding was isolated from slices of Solanum tuberosum in order to elucidate the first steps of dehydrogenative polymerization between pairs of different hydroxycinnamic acids (FA, CafA, CA and SA) present in wound-healing plant tissues. Use of a commercial horseradish peroxidase (HRP)-H2O2 catalytic system gave the identical major products in these coupling reactions, providing sufficient quantities for purification and structural elucidation. Using an equimolar mixture of pairs of hydroxycinnamic acid suberin precursors, only caffeic acid is coupled to ferulic acid and sinapic acid in separate cross-coupling reactions. For the other systems, HRP and APP reacted as follows: (1) preferentially with ferulic acid in a reaction mixture that contained p-coumaric and ferulic acids; (2) with sinapic acid in a mixture of p-coumaric and sinapic acids; (3) with sinapic acid in a mixture of ferulic and sinapic acids; (4) with caffeic acid in a reaction mixture of p-coumaric and caffeic acids. The resulting products, isolated and identified by NMR and MS analysis, had predominantly beta-beta-gamma-lactone and beta-5 benzofuran molecular frameworks. Five cross-coupling products are described for the first time, whereas the beta-O-4 dehydrodimers identified from the caffeic acid and sinapic acid cross-coupling reaction are known materials that are highly abundant in plants. These reactivity trends lead to testable hypotheses regarding the molecular architecture of intractable suberin protective plant materials, complementing prior analysis of monomeric constituents by GC-MS and polymer functional group identification from solid-state NMR, respectively.     

Multiform biosynthetic pathway of syringyl lignin in angiosperms

To clarify the pathway for biosynthesis of sinapyl alcohol in angiosperms, tracer experiments using stable isotopes were performed on robinia ( Robinia pseudoacacia L.), oleander ( Nerium indicum Mill.), magnolia ( Magnolia kobus DC.) and Arabidopsis thaliana (L.) Heynh. Precursors used in the experiment were (13)C- and (2)H ( D)-labeled [8-(13)C, 3-OCD(3)]ferulic acid and [8-(13)C, 3,5-OCD(3)]sinapic acid. The incorporation of labeled precursor into lignin was confirmed by gas chromatography-mass spectrometry of the products of derivatization followed by reductive cleavage. Crude extracts of differentiating xylem or stems from these plants were also assayed for 4-coumarate-CoA ligase (4CL; EC 6.2.1.12) activity using sinapic acid and ferulic acid as substrates. In robinia and oleander, 4CL activity toward sinapic acid was detected, and labeled sinapic acids were incorporated into syringyl lignin. These results indicate that robinia and oleander have a pathway that produces sinapyl alcohol from sinapic acid via sinapoyl-CoA. By contrast, in magnolia and Arabidopsis, 4CL activity toward sinapic acid could not be detected, and labeled sinapic acid was not incorporated into lignin. These results suggest that syringyl lignin biosynthesis in angiosperms operates via multiple pathways that depend on the species.     

Substrate preferences of O-methyltransferases in alfalfa suggest new pathways for 3-O-methylation of monolignols

Measurement of relative O-methyltransferase activities against all potential substrates in the monolignol pathway in developing alfalfa stem extracts revealed activities in the order: caffeoyl CoA > caffeoyl alcohol > 5-hydroxyferulic acid > caffeoyl aldehyde > 5-hydroxyconiferyl alcohol > 5-hydroxyferuloyl CoA > 5-hydroxyconiferaldehyde > caffeic acid. Maxima for all activities occurred in the seventh internode. In stem extracts from transgenic alfalfa with antisense downregulated caffeoyl CoA O-methyltransferase (CCoAOMT), activities with all substrates except for the two coenzyme A esters were unaffected. In contrast, downregulation of caffeic acid O-methyltransferase (COMT) reduced activities against the non-esterifed substrates in the order: 5-hydroxyconiferyl alcohol > 5-hydroxyferulic acid and caffeoyl alcohol > caffeoyl aldehyde > caffeic acid > 5-hydroxyconiferaldehyde. Recombinant COMT expressed in Escherichia coli exhibited the highest V(max)/K(m) values with 5-hydroxyconiferaldehyde and caffeoyl aldehyde, and the lowest with caffeic acid. These results indicate that COMT is unlikely to methylate caffeic acid during lignin biosynthesis in vivo, and provide enzymatic evidence for an alternative pathway to monolignols involving methylation of caffeoyl aldehyde and/or caffeoyl alcohol by COMT. The concept of independent pathways to guaiacyl and syringyl monolignols is discussed.     

Enhanced Lignin Monomer Production Caused by Cinnamic Acid and Its Hydroxylated Derivatives Inhibits Soybean Root Growth

Cinnamic acid and its hydroxylated derivatives (p-coumaric, caffeic, ferulic and sinapic acids) are known allelochemicals that affect the seed germination and root growth of many plant species. Recent studies have indicated that the reduction of root growth by these allelochemicals is associated with premature cell wall lignification. We hypothesized that an influx of these compounds into the phenylpropanoid pathway increases the lignin monomer content and reduces the root growth. To confirm this hypothesis, we evaluated the effects of cinnamic, p-coumaric, caffeic, ferulic and sinapic acids on soybean root growth, lignin and the composition of p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) monomers. To this end, three-day-old seedlings were cultivated in nutrient solution with or without allelochemical (or selective enzymatic inhibitors of the phenylpropanoid pathway) in a growth chamber for 24 h. In general, the results showed that 1) cinnamic, p-coumaric, caffeic and ferulic acids reduced root growth and increased lignin content; 2) cinnamic and p-coumaric acids increased p-hydroxyphenyl (H) monomer content, whereas p-coumaric, caffeic and ferulic acids increased guaiacyl (G) content, and sinapic acid increased sinapyl (S) content; 3) when applied in conjunction with piperonylic acid (PIP, an inhibitor of the cinnamate 4-hydroxylase, C4H), cinnamic acid reduced H, G and S contents; and 4) when applied in conjunction with 3,4-(methylenedioxy)cinnamic acid (MDCA, an inhibitor of the 4-coumarate:CoA ligase, 4CL), p-coumaric acid reduced H, G and S contents, whereas caffeic, ferulic and sinapic acids reduced G and S contents. These results confirm our hypothesis that exogenously applied allelochemicals are channeled into the phenylpropanoid pathway causing excessive production of lignin and its main monomers. By consequence, an enhanced stiffening of the cell wall restricts soybean root growth.     

The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 gene encodes an aldehyde dehydrogenase involved in ferulic acid and sinapic acid biosynthesis

Recent research has significantly advanced our understanding of the phenylpropanoid pathway but has left in doubt the pathway by which sinapic acid is synthesized in plants. The reduced epidermal fluorescence1 (ref1) mutant of Arabidopsis thaliana accumulates only 10 to 30% of the sinapate esters found in wild-type plants. Positional cloning of the REF1 gene revealed that it encodes an aldehyde dehydrogenase, a member of a large class of NADP(+)-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. Consistent with this finding, extracts of ref1 leaves exhibit low sinapaldehyde dehydrogenase activity. These data indicate that REF1 encodes a sinapaldehyde dehydrogenase required for sinapic acid and sinapate ester biosynthesis. When expressed in Escherichia coli, REF1 was found to exhibit both sinapaldehyde and coniferaldehyde dehydrogenase activity, and further phenotypic analysis of ref1 mutant plants showed that they contain less cell wall-esterified ferulic acid. These findings suggest that both ferulic acid and sinapic acid are derived, at least in part, through oxidation of coniferaldehyde and sinapaldehyde. This route is directly opposite to the traditional representation of phenylpropanoid metabolism in which hydroxycinnamic acids are instead precursors of their corresponding aldehydes.     

The changes in contents and composition of phenolic acids during cell xylem growth in scots pine

The contents and composition of alcohol soluble phenolic acids were studied during cell xylem growth in the course of wood annual increment formation in the stems of Scots pine. The cells of cambium zone, of two stages of expansion growth and the outset of secondary thickening zone (before lignification) were successively gathered from the stem segments of 25-old pine trees in the period of earlywood xylem formation with constant anatomical and histochemical control. The contents of free and bound forms of phenolic acids, isolated by 80% ethanol from tissues, as well as of their ethers and esters were calculated both per dry weight and per cell. The content and relation of the fractions and the composition of phenolic acid have been found to change significantly from cambium zone to the outset of tracheid secondary thickening. The character of the variations depends on a calculation method. According to the calculation per cell the amount of free and bound phenolic acids and in their composition of esters and especially ethers increased at the first step of expansion growth zone, decreased at the second one and rose again in the outset of secondary wall deposition. In dependence on the stage of cell development the pool of bound phenolic acids exceeded of free acid pool in 2-5 times. Sinapic and ferulic acids dominated in the composition of free hydroxycinnamic acids. The content and composition of hydroxycinnamic acids in ethers and esters depended on cell development phase. In cambium p-coumaric and sinapic acids were principal aglycons in ethers, at other stages these were sinapic and caffeic acids. The esters in cambium zone included essentially p-coumaric acid and at the other stages - sinapic and ferulic acids. At the first phase of growth benzoic acid was connected principally by ester bonds. The pool of these esters decreased from the first phase of growth to the outset of cell wall thickening and in proportion to this the level of free benzoic acid rose.     

Identification of glucosyltransferase genes involved in sinapate metabolism and lignin synthesis in Arabidopsis

Sinapic acid is a major phenylpropanoid in Brassicaceae providing intermediates in two distinct metabolic pathways leading to sinapoyl esters and lignin synthesis. Glucosyltransferases play key roles in the formation of these intermediates, either through the production of the high energy compound 1-O-sinapoylglucose leading to sinapoylmalate and sinapoylcholine or through the production of sinapyl alcohol-4-O-glucoside, potentially leading to the syringyl units found in lignins. While the importance of these glucosyltransferases has been recognized for more than 20 years, their corresponding genes have not been identified. Combining sequence information in the Arabidopsis genomic data base with biochemical data from screening the activity of recombinant proteins in vitro, we have now identified five gene sequences encoding enzymes that can glucosylate sinapic acid, sinapyl alcohol, and their related phenylpropanoids. The data provide a foundation for future understanding and manipulation of sinapate metabolism and lignin biology in Arabidopsis.     

Analysis of five rice 4-coumarate:coenzyme A ligase enzyme activity and stress response for potential roles in lignin and flavonoid biosynthesis in rice

4-Coumarate:coenzyme A ligase (4CL) catalyzes the conversion of hydroxycinnamates into corresponding CoA esters for biosynthesis of flavonoids and lignin. In this study, five members of the 4CL gene family from rice were cloned and analyzed. Recombinant 4CL data revealed that 4-coumaric acid and ferulic acid were the two main substrates of 4CL (Os4CL1/3/4/5) for monolignol biosynthesis in rice. Os4CL2 was specifically expressed in the anther and was strongly activated by UV irradiation, suggesting its potential involvement in flavonoid formation. Moreover, bioinformatics analysis showed that the existence of valine residue at the substrate-binding pocket may mainly affect rice 4CL activities toward sinapic acid.     

Changes in content and composition of phenolic acids during growth of xylem cells of Scots pine

The content and composition of alcohol soluble phenolic acids (PhAs) were studied during cell xylem growth in course of wood annual increment formation in the trunks of Scots pine. Cells of the cambium zone, two stages of expansion growth, and outset of secondary thickening zone (before lignification) within the period of formation of early wood xylem were subsequently isolated from trunk segments of 25-year-old trees with constant anatomical and histochemical control. The amount of free and bound forms of phenolic acids extracted from tissues by 80% ethanol, as well as their ethers and esters, were calculated both per dry weight and per cells. The substantial alteration in content, proportion of fractions and composition of acids has been found between the cambium zone and the outset of secondary thickening of tracheids, and the character of variation depended on the calculation method. The amount of free and bound PhAs and esters and especially ethers calculated per cell had increased at the first stage of extension growth, reduced at the second, and increased in the outset of secondary wall deposition. The pool of bound acids was more than acids by 2–5 times depending on the stage of development of the cells. Sinapic and ferulic acids dominate among free hydroxycinnamic acids. The composition and the content of hydroxycinnamic acids in esters and ethers also depended on the stage of development of the cells. p-Coumaric and sinapic acids were the main aglycons in ethers in the cambium and sinapic and caffeic acids were in the other stages. The esters from cambium included mostly p-coumaric acid and those at other stages of development were sinapic and ferulic acids. The esters included benzoic acid at the first stages of growth. The pool of these esters decreased from the first phase of growth until the outset of cell wall thickening. The level of free benzoic acid increased respectively.     

Soluble and wall-bound phenolics and phenolic polymers in Musa acuminata roots exposed to elicitors from Fusarium oxysporum f.sp. cubense

The accumulation of soluble and wall-bound phenolics and phenolic polymers in Musa acuminata roots exposed to cell wall-derived elicitor from the pathogen, Fusarium oxysporum, f.sp. cubense, race four, was investigated. The root tissue from the banana cultivar "Goldfinger" was found to respond strongly and rapidly towards the elicitor through the increased synthesis of phenolic compounds. Following elicitation, the conjugated and non-conjugated phenolic metabolites in the induced root tissue were extracted and quantified. Induced phenolic synthesis was rapid and reached near maximum values after 16 h. High-performance liquid chromatography revealed both compositional and quantitative differences between induced phenolics (p-coumaric, ferulic, and sinapic acids) and those constitutively present (p-coumaric- and ferulic acid). In addition, vanillic acid was found in the ester-bound fraction and protocatechuic acid in the cell-wall bound fraction of elicited tissue. The deposition and accumulation kinetics of polymerized phenolic monomers as lignin and lignin-like polymers was quantified over a time period of 0-36 h and found to reach maximum values after 24 h. Ionization difference UV spectra of lignin indicated compositional differences in the newly synthesized lignin fraction and correlated with increased concentrations of ferulic acid and sinapic acids esters. The results show that the increased flux through the phenylpropanoid pathway resulted in the synthesis of cinnamic acid and benzoic acid derivatives that were esterified and incorporated into the cell wall fraction as part of the anti-microbial defenses activated in the root tissue.     

Feruloyl esterases as a tool for the release of phenolic compounds from agro-industrial by-products

Agro-industrial by-products are a potential source of added-value phenolic acids with promising applications in the food and pharmaceutical industries. Here two purified feruloyl esterases from Aspergillus niger, FAEA and FAEB were tested for their ability to release phenolic acids such as caffeic acid, p-coumaric acid and ferulic acid from coffee pulp, apple marc and wheat straw. Their hydrolysis activity was evaluated and compared with their action on maize bran and sugar beet pulp. The specificity of both enzymes against natural and synthetic substrates was evaluated; particular attention was paid to quinic esters and lignin monomers. The efficiency of both enzymes on model substrates was studied. We show the ability of these enzymes to hydrolyze quinic esters and ester linkages between phenolic acids and lignin monomer.     

Alternations in phenolic acids content in developing rye grains in normal environment and during enforced dehydration

A series of high pressure liquid chroamtography analyses revealed the presence of five phenolic acids in rye caryopses (vanillic, caffeic, p-coumaric, ferulic and sinapic), three of which (p-coumaric, ferulic and sinapic) were found in the free phenolic fraction. Ferulic acid was predominant, both among free acids and total phenolic acids (i.e. free, liberated from soluble esters and glycosides). The highest content of the free phenolic acids in rye caryopses was observed at the beginning of development, when on 22 DAF it was estimated at 11.55 μg·g⁻¹ DW. During dehydratation the total level of free phenolic acids in rye caryopses decreased in all investigated samples. Although total phenolic acids contents in all samples of unripe rye caryopses always decreased after dehydration, in rye sample collected in full ripeness (57 DAF), the amount of these compounds increased after the enforced dehydration. It should be added that in ester-bound-soluble phenolic acids fraction (the largest part in the total phenolic acids fraction), irrespective of the total amount decrease, much increase of sinapic acid content in this fraction was observed after dehydratation treatment in all investigated samples of caryopses of various ripeness. During the development and ripening of rye caryopses, a gradual increase in the precocious germination ability of the grain was observed. The enforced dehydration stimulated the process of precocious germination of developing and ripening rye caryopses. A possible role of phenolics in preventing precocious germination and acclimation to dehydration of developing and ripening rye grains is discussed.     

A SURVEY FOR BENZOIC AND CINNAMIC ACIDS OF THE CYCADACEAE

A survey for cinnamic and benzoic acids in 22 species representing the 10 extant genera of the Cycadaceae revealed the presence of p-coumaric, caffeic, ferulic, p-hydroxybenzoic, protocatechuic, and vanillic acids in all of the species. Two genera had detectable quantities of 2,4-dihydroxy-benzoic acid. In addition sinapic acid was detected in two genera, indicating that it has a wider distribution in vascular plants than was previously indicated.     

Enzymatic synthesis of butyl hydroxycinnamates and their inhibitory effects on LDL-oxidation

The potential of the Aspergillus niger type A feruloyl esterase (AnFaeA) for the synthesis of various phenolic acid esters was examined using a ternary-organic reaction system consisting of a mixture of n-hexane, 1- or 2-butanol and water. Reaction parameters including the type of methyl hydroxycinnamate, the composition of the reaction media, the temperature, and the substrate concentration were investigated to evaluate their effect on initial rate and conversion to butyl esters of sinapic acids. Optimisation of the reaction parameters lead to 78% and 9% yield for the synthesis of 1-butyl and 2-butyl sinapate, respectively. For the first time, a feruloyl esterase was introduced in the reaction system as cross-linked enzyme aggregates (CLEAs), after optimisation of the immobilisation procedure, allowing the recycling and reuse of the biocatalyst. The inhibition of copper-induced LDL oxidation by hydroxycinnamic acids and their corresponding butyl esters was investigated in vitro. Kinetic analysis of the antioxidation process demonstrates that sinapate derivatives are effective antioxidants indicating that esterification increases the free acid's antioxidant activity especially on dimethoxylated compounds such as sinapic acid compared to methoxy-hydroxy-compounds such as ferulic acid.     

A new type of peroxisomal acyl-coenzyme A synthetase from Arabidopsis thaliana has the catalytic capacity to activate biosynthetic precursors of jasmonic acid

Arabidopsis thaliana contains a large number of genes that encode carboxylic acid-activating enzymes, including nine long-chain fatty acyl-CoA synthetases, four 4-coumarate:CoA ligases (4CL), and 25 4CL-like proteins of unknown biochemical function. Because of their high structural and sequence similarity with bona fide 4CLs and their highly hydrophobic putative substrate-binding pockets, the 4CL-like proteins At4g05160 and At5g63380 were selected for detailed analysis. Following heterologous expression, the purified proteins were subjected to a large scale screen to identify their preferred in vitro substrates. This study uncovered a significant activity of At4g05160 with medium-chain fatty acids, medium-chain fatty acids carrying a phenyl substitution, long-chain fatty acids, as well as the jasmonic acid precursors 12-oxo-phytodienoic acid and 3-oxo-2-(2'-pentenyl)-cyclopentane-1-hexanoic acid. The closest homolog of At4g05160, namely At5g63380, showed high activity with long-chain fatty acids and 12-oxo-phytodienoic acid, the latter representing the most efficiently converted substrate. By using fluorescent-tagged variants, we demonstrated that both 4CL-like proteins are targeted to leaf peroxisomes. Collectively, these data demonstrate that At4g05160 and At5g63380 have the capacity to contribute to jasmonic acid biosynthesis by initiating the beta-oxidative chain shortening of its precursors.     

Metabolic differences between gymnosperms and angiosperms in the formation of syringyl lignin

Sliced xylem tissue from shoots of both poplar and cherry reduces ferulic and sinapic acids to the corresponding aldehydes and alcohols, while tissue from gymnosperms such as Japanese red pine and ginkgo can reduce only ferulic acid. In young, less differentiated, xylem tissue and callus tissue of angiosperms the ability to reduce sinapic acid is markedly lower than that of the fully differentiated xylem.Both gymnosperm and angiosperm tissues reduced coniferyl and sinapyl aldehydes to the corresponding alcohols and, further, the peroxidases from both classes gave similar dehydrogenation polymers from a mixture of coniferyl and sinapyl alcohols. In agreement with these findings, sinapyl aldehyde and sinapyl alcohol, when fed to living plants and tissue cultures of gymnosperms, were shown to be readily converted to syringyl lignin which was not originally present.     

Competitive kinetics in free radical reactions of cinnamic acid derivatives. Influence of phenoxyl radicals reactions

Relative rates of consumption of caffeic, ferulic and sinapic acids by 2,2'-azobis(2-amidine propane) derived peroxyl radicals has been measured in parallel experiments employing a single substrate and in competitive experiments. Rates of consumption measured in independent experiments at low substrate concentrations (first order limit) follow the order: sinapic > ferulic > caffeic. In agreement with this, in competitive experiments employing simultaneously sinapic and caffeic acid the former compound is consumed considerably faster. On the other hand, in competitive experiments employing ferulic and caffeic acids over a wide range of experimental conditions, caffeic acid is consumed considerably faster than ferulic acid, a result that contrasts with that obtained when both compounds are reacted independently. These apparently anomalous results are interpreted in terms of secondary reactions of the phenol-derived radicals. In particular, hydrogen transfer among phenoxyl radicals and the phenols and fast reactions (disproportionation) of caffeic acid derived radicals could explain these discrepancies.     

4-Coumarate:coenzyme A ligase in black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis>) catalyses the conversion of sinapate to sinapoyl-CoA

4-Coumarate:coenzyme A (CoA) ligase (4CL, EC 6.2.1.12) in crude enzyme preparation from the developing xylem of black locust (Robinia pseudoacacia) converted sinapate to sinapoyl CoA. The sinapate-converting activity was not inhibited by other cinnamate derivatives, such as p-coumarate, caffeate or ferulate, in the mixed-substrate assay. The crude extract prepared from the developing xylem was separated by anion-exchange chromatography into three different 4CL isoforms. The isoform 4CL1 had a strong substrate preference for p-coumarate, but lacked the activity for ferulate and sinapate. On the other hand, 4CL2 and 4CL3 displayed activity toward sinapate and also possessed high activity toward caffeate as well as p-coumarate. The crude extract from the shoots exhibited a very similar substrate preference to that of the developing xylem; therefore, 4CL2 may be a major isoform in both crude enzyme preparations. These results support the hypothesis that sinapate-converting 4CL isoform is constitutively expressed in lignin-forming cells.     

Competitive kinetics in free radical reactions of cinnamic acid derivatives. Influence of phenoxyl radicals reactions

Relative rates of consumption of caffeic, ferulic and sinapic acids by 2,2′-azobis(2-amidine propane) derived peroxyl radicals has been measured in parallel experiments employing a single substrate and in competitive experiments. Rates of consumption measured in independent experiments at low substrate concentrations (first order limit) follow the order: sinapic > ferulic > caffeic. In agreement with this, in competitive experiments employing simultaneously sinapic and caffeic acid the former compound is consumed considerably faster. On the other hand, in competitive experiments employing ferulic and caffeic acids over a wide range of experimental conditions, caffeic acid is consumed considerably faster than ferulic acid, a result that contrasts with that obtained when both compounds are reacted independently. These apparently anomalous results are interpreted in terms of secondary reactions of the phenol-derived radicals. In particular, hydrogen transfer among phenoxyl radicals and the phenols and fast reactions (disproportionation) of caffeic acid derived radicals could explain these discrepancies.