Chemical interaction between polyphenols and a cysteinyl thiol under radical oxidation conditions

Chemical interaction between polyphenols and thiols was investigated under radical oxidation conditions using a model cysteinyl thiol derivative, N-benzoylcysteine methyl ester. The radical oxidation was carried out with a stoichiometric amount of 2,2-diphenyl-1-picrylhydrazyl (DPPH), and the decreases in the amounts of polyphenols and the thiol were measured by HPLC analysis. Cross-coupling products between various polyphenols and the thiol were examined by LC-MS in reactions that showed decreases in both the polyphenols and the thiol. The LC-MS results indicated that three phenolic acid esters (methyl caffeate, methyl dihydrocaffeate, and methyl protocatechuate) and six flavonoids (kaempferol, myricetin, luteolin, morin, taxifolin, and catechin) gave corresponding thiol adducts, whereas three polyphenols (methyl ferulate, methyl sinapate, and quercetin) gave only dimers or simple oxidation products without thiol substituents. Thiol adducts of the structurally related compounds methyl caffeate and methyl dihydrocaffeate were isolated, and their chemical structures were determined by NMR analysis. The mechanism for the thiol addition was discussed on the basis of the structures of the products.     

Highly efficient preparation of lipophilic hydroxycinnamates by solvent-free lipase-catalyzed transesterification

Various medium- or long-chain alkyl cinnamates and hydroxycinnamates, including oleyl p-coumarate as well as palmityl and oleyl ferulates, were prepared in high yield by lipase-catalyzed transesterification of an equimolar mixture of a short-chain alkyl cinnamate and a fatty alcohol such as lauryl, palmityl, and oleyl alcohol under partial vacuum at moderate temperature in the absence of solvents and drying agents in direct contact with the reaction mixture. Immobilized lipase B from Candida antarctica was the most effective biocatalyst for the various transesterification reactions. Transesterification activity of this enzyme was up to 56-fold higher than esterification activity for the preparation of medium- and long-chain alkyl ferulates. The relative transesterification activities found for C. antarctica lipase were of the following order: hydrocinnamate > cinnamate > 4-hydroxyhydrocinnamate > 3-methoxycinnamate > 2-methoxycinnamate approximately 4-methoxycinnamate approximately 3-hydroxycinnamate > hydrocaffeate approximately 4-hydroxycinnamate > ferulate > 2-hydroxycinnamate > caffeate approximately sinapate. With respect to the position of the hydroxy substituents at the phenyl moiety, the transesterification activity of C. antarctica lipase B increased in the order meta > para > ortho. The immobilized lipases from Rhizomucor miehei and Thermomyces lanuginosus demonstrated moderate and low transesterification activity, respectively. Compounds with inverse chemical structure, that is, 3-phenylpropyl alkanoates such as 3-(4-hydroxyphenyl)propyl oleate and 3-(3,4-dimethoxyphenyl)propyl oleate, were obtained by C. antarctica lipase-catalyzed transesterification of fatty acid methyl esters with the corresponding 3-phenylpropan-1-ols in high yield, as well.     

Antioxidant mechanism studies on ferulic acid: identification of oxidative coupling products from methyl ferulate and linoleate

In our studies of the chain-breaking antioxidant mechanism of natural phenols in food components, ferulic acid, a phenolic acid widely distributed in edible plants, especially grain, was investigated. The radical oxidation reaction of a large amount of ethyl linoleate in the presence of the methyl ester of ferulic acid produced four types of peroxides as radical termination products. The isolation and structure determination of the peroxides revealed that they had tricyclic structures which consisted of ethyl linoleate, methyl ferulate, and molecular oxygen. Based on the formation pathway of the products, a radical scavenging reaction occurred at the 3'-position of the ferulate radical with the four types of peroxyl radicals of ethyl linoleate. The produced peroxides subsequently underwent intramolecular Diels-Alder reaction to afford stable tricyclic peroxides.     

Antioxidant effects of phenolic rye (Secale cereale L.) extracts, monomeric hydroxycinnamates, and ferulic acid dehydrodimers on human low-density lipoproteins

Dietary antioxidants that protect low-density lipoprotein (LDL) from oxidation may help to prevent atherosclerosis and coronary heart disease. The antioxidant activities of purified monomeric and dimeric hydroxycinnamates and of phenolic extracts from rye (whole grain, bran, and flour) were investigated using an in vitro copper-catalyzed human LDL oxidation assay. The most abundant ferulic acid dehydrodimer (diFA) found in rye, 8-O-4-diFA, was a slightly better antioxidant than ferulic acid and p-coumaric acid. The antioxidant activity of the 8-5-diFA was comparable to that of ferulic acid, but neither 5-5-diFA nor 8-5-benzofuran-diFA inhibited LDL oxidation when added at 10-40 microM. The antioxidant activity of the monomeric hydroxycinnamates decreased in the following order: caffeic acid > sinapic acid > ferulic acid > p-coumaric acid. The antioxidant activity of rye extracts was significantly correlated with their total content of monomeric and dimeric hydroxycinnamates, and the rye bran extract was the most potent. The data suggest that especially rye bran provides a source of dietary phenolic antioxidants that may have potential health effects.     

Uptake and metabolism of hydroxycinnamic acids (chlorogenic, caffeic, and ferulic acids) by HepG2 cells as a model of the human liver

Hydroxycinnamic acids are antioxidant polyphenols common in the human diet, although their potential health benefits depend on their bioavailability. To study the hepatic uptake and metabolism, human hepatoma HepG2 cells were incubated for 2 and 18 h with caffeic, ferulic, and chlorogenic acids. Moderate uptake of caffeic and ferulic acids was observed versus a low absorption of chlorogenic acid, where esterification of the caffeic acid moiety markedly reduced its absorption. Methylation was the preferential pathway for caffeic acid metabolism, along with glucuronidation and sulfation, while ferulic acid generated glucuronides as the only metabolites. Ferulic acid appeared to be more slowly taken up and metabolized by HepG2 cells than caffeic acid, with 73% and 64% of the free, nonmetabolized molecules detected in the culture medium after 18 h, respectively. In conclusion, hydroxycinnamic acids can be metabolized by the liver as suggested by the results obtained using HepG2 cells as a hepatic model system.     

Black cohosh (Cimicifuga racemosa L.) protects against menadione-induced DNA damage through scavenging of reactive oxygen species: bioassay-directed isolation and characterization of active principles

The roots/rhizomes of Cimicifuga racemosa L. (Nutt.) (black cohosh) have traditionally been used to treat menopausal symptoms through an unknown mechanism of action. In an effort to determine if black cohosh had additional health benefits, methanol extracts were investigated for their potential to scavenge reactive oxygen species and to protect against menadione-induced DNA damage. These extracts effectively scavenged 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals. In addition, the extracts showed dose-dependent decreases in DNA single-strand breaks and oxidized bases induced by the quinone menadione using the comet (single-cell gel electrophoresis assay) and fragment length associated repair enzyme assays, respectively. Bioassay-directed fractionation of the methanolic extracts using the DPPH assay as a monitor led to the isolation of nine antioxidant active compounds: caffeic acid (1), methyl caffeate (2), ferulic acid (3), isoferulic acid (4), fukinolic acid (5), cimicifugic acid A (6), cimicifugic acid B (7), cimicifugic acid F (8), cimiracemate A (9), and cimiracemate B (10). Six of these antioxidants were found to reduce menadione-induced DNA damage in cultured S30 breast cancer cells with the following order of potency: methyl caffeate (2) > caffeic acid (1) > ferulic acid (3) > cimiracemate A (9) > cimiracemate B (10) > fukinolic acid (5). These data suggest that black cohosh can protect against cellular DNA damage caused by reactive oxygen species by acting as antioxidants.     

Microbial metabolites of ingested caffeic acid are absorbed by the monocarboxylic acid transporter (MCT) in intestinal Caco-2 cell monolayers

It was previously reported that m-coumaric acid, m-hydroxyphenylpropionic acid (mHPP), and 3,4-dihydroxyphenylpropionic acid (DHPP) are major metabolites of ingested caffeic acid formed by gut microflora and would be transported by the monocarboxylic acid transporter (MCT). We have directly measured their absorption characteristics in Caco-2 cells using a coulometric detection method involving HPLC-ECD. The proton-coupled directional transport of m-coumaric acid, mHPP, and DHPP was observed, and the transport was inhibited by an MCT substrate. The permeation of m-coumaric acid and mHPP was concentration-dependent and saturable: The Michaelis constant for m-coumaric acid and mHPP was 32.5 and 12.9 mM, respectively, and the maximum velocity for m-coumaric acid and mHPP was 204.3 and 91.2 nmol (min)(-1) (mg protein)(-1), respectively. By contrast, the permeation of DHPP was nonsaturable even at 30 mM and was inversely correlated with the paracellular permeability of Caco-2 cells. Our results demonstrate that these compounds are absorbed by the MCT, although DHPP is mainly permeated across Caco-2 cells via the paracellular pathway. MCT-mediated absorption of phenolic compounds per se and their colonic metabolites would exert significant impact on human health.     

LC/ES-MS detection of hydroxycinnamates in human plasma and urine

Hydroxycinnamates are components of many fruits and vegetables, being present in particularly high concentrations in prunes. An abundance of phenolic compounds in the diet has been associated with reduced heart disease mortality. However, little is known about the absorption and metabolism of these metabolites after normal foods are consumed. An LC--electrospray--MS method was developed to measure the concentration of caffeic acid in human plasma and urine, but it can also be applied to ferulic acid and chlorogenic acid. The limit of detection was found to be 10.0 nmol/L for caffeic acid and 12.5 nmol/L for ferulic and chlorogenic acids. The method was tested on samples of plasma and urine collected from volunteers who consumed a single dose of 100 g of prunes and increased levels were observed, demonstrating that the method is capable of detecting changes in hydroxycinnamate levels induced by dietary consumption.     

First chemical synthesis and in vitro characterization of the potential human metabolites 5-o-feruloylquinic acid 4'-sulfate and 4'-O-glucuronide

Feruloylquinic acids are a major class of biologically active phenolic antioxidants in coffee beans, but their metabolic fate is poorly understood. The present study investigated the phase II metabolism of feruloylquinic acids with selected human sulfotransferases (SULT1A1 and SULT1E1) and uridine 5'-diphosphoglucuronosyltransferases (UGT1A1 and UGT1A9). For unequivocal metabolite identification, the chemical synthesis of two potential human metabolites of 5-O-feruloylquinic acid, the 4'-sulfated and 4'-O-glucuronidated conjugates, has been performed for the first time. Following incubation with human SULT1A1 or SULT1E1, formation of 5-O-feruloylquinic acid 4'-O-sulfate was confirmed by matching its HPLC and MS data with those of the authentic standard. On the other hand, no glucuronide conjugates were detected after incubation with human uridine 5'-diphosphoglucuronosyltransferases. These results suggest that sulfation can take place on the ferulic acid moiety of feruloylquinic acids and may be a major metabolic pathway for feruloylquinic acids in humans.     

Curcumin bioavailability from enriched bread: the effect of microencapsulated ingredients

Human bioavailability of curcumin from breads enriched with 1 g/portion of free curcumin (FCB), encapsulated curcumin (ECB), or encapsulated curcumin plus other polyphenols (ECBB) was evaluated. Parental and metabolized curcuminoids and phenolic acids were quantified by HPLC/MS/MS in blood, urine, and feces collected over 24 h. The concentrations of serum curcuminoids were always below 4 nmol/L and those of glucuronides 10-fold less. Encapsulation delayed and increased curcuminoid absorption as compared to the free ingredient. Serum and urinary concentrations of ferulic and vanillic acid were between 2- and 1000-fold higher than those of curcuminoids, with ECBB eliciting the highest amounts. Fecal curcuminoids were 6-fold more abundant after ECB than FCB, while phenolic acids after ECBB quadruplicated those after ECB. Curcuminoid encapsulation increased their bioavailability from enriched bread, probably preventing their biotransformation, with combined compounds slightly reducing this effect. Phenolic acids are the major metabolites of curcuminoids and may contribute to their biological properties.     

Oxidative Cross-Linking of Pentosans by a Fungal Laccase and Horseradish Peroxidase: Mechanism of Linkage Between Feruloylated Arabinoxylans

The potential of a laccase from the fungus Pycnoporus cinnabarinus to cross-link feruloylated soluble wheat arabinoxylans was investigated using capillary viscometry, size-exclusion HPLC, and reverse-phase HPLC of phenolic compounds. The laccase results were compared with those for a hydrogen peroxide/horseradish peroxidase system. The oxidants provoked an increase in viscosity of a 0.2% (w/v) arabinoxylan solution. A gel was formed after 30 min with laccase. Hydrogen peroxide was consumed rapidly before a gel could be formed. Free ferulic acid, methyl ferulate, and vanillic acid inhibited the gelation, whereas fumaric acid had no effect. This suggests that the aromatic ring, and not the propenoic chain of ferulic acid, was the initiating site for arabinoxylan cross-linking. Ferulic acid and its 8-O-4′, 8-5′, and 5-5′ dehydrodimers were present in nonoxidized arabinoxylans. Upon oxidation, the 8-8′ and 8-5′ benzofuran dehydrodimers appeared and the 8-O-4′ and 8-5′ dimers increased. The production of dimers was proportional to the consumption of ester-bound ferulic acid. In cross-linked arabinoxylans, the major dimers were 8-5′ benzofuran, 8-8′, and 8-O-4′, whereas the 5-5′ dehydrodimer remained at the same level as in the nonoxidized solution.     

Antioxidant properties of ferulic acid and its related compounds

Antioxidant activity of 24 ferulic acid related compounds together with 6 gallic acid related compounds was evaluated using several different physical systems as well as their radical scavenging activity. The radical scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) decreased in the order caffeic acid > sinapic acid > ferulic acid > ferulic acid esters > p-coumaric acid. In bulk methyl linoleate, test hydroxycinnamic acids and ferulic acid esters showed antioxidant activity in parallel with their radical scavenging activity. In an ethanol-buffer solution of linoleic acid, the activity of test compounds was not always associated with their radical scavenging activity. Ferulic acid was most effective among the tested phenolic acids. Esterification of ferulic acid resulted in increasing activity. The activity of alkyl ferulates was somewhat influenced by the chain length of alcohol moiety. When the inhibitory effects of alkyl ferulates against oxidation of liposome induced by AAPH were tested, hexyl, octyl, and 2-ethyl-1-hexyl ferulates were more active than the other alkyl ferulates. Furthermore, lauryl gallate is most effective among the tested alkyl gallates. These results indicated that not only the radical scavenging activity of antioxidants, but also their affinity with lipid substrates, might be important factors in their activity.     

Influence of nitrogen fertilization on yield and phenolic compounds in wheat grains (Triticum aestivum L. ssp. aestivum)

Nitrogen (N) is an important plant nutrient and is crucial for the plant growth and grain yield formation of field crops such as wheat (Triticum aestivum L. ssp. aestivum). However, little is known about the influence of N on secondary metabolites in wheat grains which are supposed to be beneficial for human health due to their antioxidant potentials. Therefore, we investigated the influence of N fertilization on plant growth and yield performance of winter wheat, as well as on total phenolic concentration, antioxidant capacity, and the accumulation of (in)soluble phenolic acids in wheat grains during the grain‐filling phase. It was found that ferulic acid was the predominant phenolic acid in wheat grains. As expected, higher amounts of N fertilizer led to increasing grain yields, whereas the concentration of soluble ferulic acid decreased. In contrast, insoluble bound ferulic acid, total phenolic content, and antioxidant capacity were not affected by the N treatment. Insoluble phenolic compounds seemed to be less susceptible to variations in N supply.     

Metabolic profiling of root exudates of Arabidopsis thaliana

In addition to accumulating biologically active chemicals, plant roots continuously produce and secrete compounds into their immediate rhizosphere. However, the mechanisms that drive and regulate root secretion of secondary metabolites are not fully understood. To enlighten two neglected areas of root biology, root secretion and secondary metabolism, an in vitro system implementing root-specific elicitation over a 48-day time course was developed. After roots of Arabidopsis thaliana had been elicited with salicylic acid, jasmonic acid, chitosan, and two fungal cell wall elicitors, the secondary metabolites subsequently secreted were profiled. High-performance liquid chromatography was used to metabolically profile compounds in the root exudates, and 289 possible secondary metabolites were quantified. The chemical structures of 10 compounds were further characterized by (1)H and (13)C NMR: butanoic acid, trans-cinnamic acid, o-coumaric acid, p-coumaric acid, ferulic acid, p-hydroxybenzamide, methyl p-hydroxybenzoate, 3-indolepropanoic acid, syringic acid, and vanillic acid. Several of these compounds exhibited a wide range of antimicrobial activity against both soil-borne bacteria and fungi at the concentration detected in the root exudates.     

Preferential sorption of phenolic phytotoxins to soil: implications for altering the availability of allelochemicals

Allelopathy, secondary metabolite-mediated plant-to-plant interaction, is gaining application in current agricultural science as well as in invasion ecology. However, the role of sorption to soil in modifying the bioavailability of components in complex allelochemical mixtures is still obscure. Hence, the role of preferential sorption to soil in altering the chemical composition of plant exudates was studied in a silt loam soil using representative mixtures of plant phenolic acids, namely, hydroxybenzoic acid, vanillic acid, coumaric acid, and ferulic acid. The experiment was conducted using a batch equilibration technique, and data were fitted to a Freundlich isotherm. The concentration-dependent sorption coefficient (K(d)) at 10 microg mL(-)(1) was used to assess the sorption affinity of phenolic acids across different systems. Along with solid phase dissolution, all of the phenolic acids exhibited strong site-specific sorption, as evident from their nonlinear isotherms. Removal of organic matter substantially decreased the sorption affinity of all phenolic acids. Direct competition for sorption sites was observed even at low concentrations of phenolic acids. The K(d) of hydroxybenzoic acid was decreased more than 90% in the presence of coumaric acid. About 95% of sorbed vanillic acid was displaced into the soil solution in the presence of ferulic acid. Hydroxybenzoic acid did not affect the sorption affinity of other phenolic acids significantly, whereas ferulic acid showed low displacement by other phenolic acids. The displacement pattern indicated directional sorption of phenolic acids with -OH and -COOH groups. Soil organic matter was associated with preferential sorption. This is the first study to elucidate competitive sorption characteristics of plant secondary metabolites in soil matrix. The results demonstrate that preferential sorption to soil can alter the availability of plant exudates in mixtures and thus may mediate their phytotoxic effects.     

Metabolites in contact with the rat digestive tract after ingestion of a phenolic-rich dietary fiber matrix

Grape antioxidant dietary fiber (GADF) is a phenolic-rich dietary fiber matrix. The aim of this work was to determine which phenolic compounds come into contact with colonic epithelial tissue after the ingestion of GADF. By use of HPLC-ESI-MS/MS techniques phenolic metabolites were detected in feces, cecal content, and colonic tissue from rats. Free (epi)catechin (EC) was detected in all three sources, and more than 20 conjugated metabolites of EC were also detected in feces. Fourteen microbially derived phenolic metabolites were also identified in feces, cecal content, and/or colonic tissue. These results show that during transit along the digestive tract, proanthocyanidin oligomers and polymers are depolymerized into EC units. After ingestion of GADF, free EC and its conjugates, as well as free and conjugated microbially derived phenolic metabolites, come into contact with the intestine epithelium for more than 24 h and may be partly responsible for the positive influence of GADF on gut health.     

Nitrification Inhibitors from the root tissues of Brachiaria humidicola, a tropical grass

Nitrification inhibitory activity was found in root tissue extracts of Brachiaria humidicola, a tropical pasture grass. Two active inhibitory compounds were isolated by activity-guided fractionation, using recombinant Nitrosomonas europaea containing luxAB genes derived from the bioluminescent marine gram-negative bacterium Vibrio harveyi. The compounds were identified as methyl-p-coumarate and methyl ferulate, respectively. Their nitrification inhibitory properties were confirmed in chemically synthesized preparations of each. The IC50 values of chemically synthesized preparations were 19.5 and 4.4 microM, respectively. The ethyl, propyl, and butyl esters of p-coumaric and ferulic acids inhibited nitrification, whereas the free acid forms did not show inhibitory activity.     

Potential Antioxidant Activity of γ‐Oryzanol in Rice Bran as Determined Using an In Vitro Mouse Lymph Axillary Endothelial Cell Model

A mouse lymphatic endothelial cell (SVEC4‐10) in vitro model was developed and found to be effective in the study of antioxidant activity of γ‐oryzanol in rice bran. The critical and vital parameters in developing these cell models included the emulsion preparation of hydrophobic compounds, the consistent management of cell culture, and the selection of cell viability detection methods compatible with the cell lines and the test substances. The SVEC4‐10 cell line had a fast metabolism and consequently could be used to determine antioxidant activity of a test substance in a relatively rapid manner. Results showed that γ‐oryzanol, ferulic acid, and α‐tocopherol could interact with cells such that oxidative damage induced by tert‐butyl hydroperoxide on cellular mitochondrial activity lessened, and in some situations, γ‐oryzanol was a more effective antioxidant than α‐tocopherol. The results also suggested different antioxidant mechanisms among γ‐oryzanol, ferulic acid, and α‐tocopherol. The three major components of γ‐oryzanol generally had higher antioxidant activity than γ‐oryzanol, with 24‐methylene cycloartanyl ferulate relatively more effective. Synergistic antioxidant activity among γ‐oryzanol, ferulic acid, and α‐tocopherol was also found.     

Transepithelial transport of chlorogenic acid, caffeic acid, and their colonic metabolites in intestinal caco-2 cell monolayers

Both chlorogenic and caffeic acids exhibited nonsaturable transport in Caco-2 cells, whereas caffeic acid also showed proton-coupled polarized absorption. Thus, the absorption efficiency of caffeic acid was greater than that of chlorogenic acid. Polarized transport of caffeic acid was inhibited by substrates of MCT such as benzoic and acetic acids. Almost all of the apically loaded chlorogenic and caffeic acid was retained on the apical side, and the transepithelial flux was inversely correlated with the paracellular permeability of Caco-2 cells. These results indicate that transport was mainly via paracellular diffusion, although caffeic acid was absorbed to a lesser extent by the monocarboxylic acid transporter (MCT). Furthermore, m-coumaric acid and 3-(m-hydroxyphenyl)propionic acid, the main metabolites of chlorogenic and caffeic acid by colonic microflora, competitively inhibited the transport of fluorescein, a known substrate of MCT. This suggests that their absorption could also be mediated by MCT. These findings have exemplified the physiological importance of MCT-mediated absorption in both phenolic acids per se and their colonic metabolites.