Effect of cysteinyl caffeic acid, caffeic acid, and L-dopa on the oxidative cross-linking of feruloylated arabinoxylans by a fungal laccase

To study a way to covalently link arabinoxylans and proteins using a fungal laccase from the fungus Pycnoporus cinnabarinus, the effect of cysteinyl caffeic acid on the cross-linking of wheat arabinoxylans was investigated by means of capillary viscometry and RP-HPLC of alkali labile phenolic compounds. Cysteinyl caffeic acid provoked a delay in gelation and in the consumption of the esterified ferulic acid on arabinoxylans. When reacting free ferulic acid and cysteinyl caffeic acid with laccase, the ferulic acid consumption and the dehydrodimers production were also diminished. These results suggest that cysteinyl caffeic acid is oxidized while reducing the semiquinones of ferulic acid produced by laccase. Thus, ferulic acid could not be oxidized into dimers until all cysteinyl caffeic acid was consumed, preventing the cross-linking of feruloylated arabinoxylan chains. A similar mechanism is proposed in the case of caffeic acid and of L-Dopa.     

Absorption of phenolic acids in humans after coffee consumption

Despite extensive literature describing the biological effects of polyphenols, little is known about their absorption from diet, one major unresolved point consisting of the absorption of the bound forms of polyphenols. In this view, in the present work we studied the absorption in humans of phenolic acids from coffee, a common beverage particularly rich in bound phenolic acids, such as caffeic acid, ferulic acid, and p-coumaric acid. Coffee brew was analyzed for free and total (free + bound) phenolic acids. Chlorogenic acid (5'-caffeoylquinic acid), a bound form of caffeic acid, was present in coffee at high levels, while free phenolic acids were undetectable. After alkaline hydrolysis, which released bound phenolic acids, ferulic acid, p-coumaric acid, and high levels of caffeic acid were detected. Plasma samples were collected before and 1 and 2 h after coffee administration and analyzed for free and total phenolic acid content. Two different procedures were applied to release bound phenolic acids in plasma: beta-glucuronidase treatment and alkaline hydrolysis. Coffee administration resulted in increased total plasma caffeic acid concentration, with an absorption peak at 1 h. Caffeic acid was the only phenolic acid found in plasma samples after coffee administration, while chlorogenic acid was undetectable. Most of caffeic acid was present in plasma in bound form, mainly in the glucuronate/sulfate forms. Due to the absence of free caffeic acid in coffee, plasma caffeic acid is likely to be derived from hydrolysis of chlorogenic acid in the gastrointestinal tract.     

Absorption of 6-O-caffeoylsophorose and its metabolites in Sprague-Dawley rats detected by electrochemical detector-high-performance liquid chromatography and electrospray ionization-time-of-flight-mass spectrometry methods

Absorption and metabolism of a natural compound, 6-O-caffeoylsophorose (CS) from acylated anthocyanins in a red vinegar fermented with purple sweet potato, were clarified. The absorption of CS and conjugated CS in blood from orally administrated Sprague-Dawley rats at a dose of 400 mg/kg was investigated by electrochemical detection-high performance liquid chromatography. As a result, CS was successfully detected in rat plasma (AUC(0-6h), 108.6 ± 8.1 nmol h/mL) and was found to be an intact absorbable polyphenol. In addition, half of the absorbed CS was detected as its conjugates (AUC(0-6h), 50.7 ± 5.7 nmol h/mL) as well as caffeic and ferulic acids from CS. By a time-of-flight-mass spectrometric analysis of CS-administered plasma sample, glucuronide and methylated conjugates of CS were identified, in addition to glucuronide, methylated, or sulfate conjugates of caffeic and ferulic acids. Consequently, CS was absorbed in intact form into rat blood and partly degraded to caffeic and ferulic acids or metabolized by glucuronidation, methylation, or sulfatation.     

Antioxidant activities of phenolic acids on ultraviolet radiation-induced erythrocyte and low density lipoprotein oxidation

The exposure of mammalian cells to UV light induces various deleterious responses. Some of the major harmful effects are DNA damage, cell membrane peroxidation, systemic immune suppression, and aging acceleration. Reactive oxygen species and free radicals are believed to be largely responsible for some of the deleterious effects of UV upon cells. Typical administration of antioxidants has recently proved to represent a successful strategy for protecting the cells against UV-mediated oxidative damage. The objective of this study was to investigate the inhibitory effect of phenolic acids (caffeic acid, ferulic acid, gallic acid, and protocatechuic acid) on oxidative damage in human erythrocytes and low-density lipoprotein (LDL) induced by UVB radiation. The results revealed that the thiobarbituric acids reactive substances induced by UVB were decreased from 2.78 to 0.12-0.89 nmol MDA/mg protein in erythrocyte ghost and from 0.72 to 0.14-0.43 nmol MDA/mg protein in LDL by the addition of phenolic acids (100 muM). Caffeic acid, ferulic acid, and gallic acid exhibited over 85 and 60% inhibitory effect toward UVB-induced oxidation in erythrocytes and LDL, respectively. Phenolic acids, especially gallic acid, could maintain the normal glutathione levels and glutathione peroxidase activity in hemolysate from erythrocytes that were exposed to UVB radiation in comparison with untreated control. The results indicate that the antioxidant activities of caffeic acid and ferulic acid play a potential role in protection against UVB oxidative damage to human erythrocytes and LDL.     

Absorption, disposition, metabolism, and excretion of [3-(14)C]caffeic acid in rats

Male Sprague-Dawley rats ingested 140 × 10(6) dpm of [3-(14)C]trans-caffeic acid, and over the ensuing 72 h period, body tissues, plasma, urine, and feces were collected and the overall levels of radioactivity determined. Where sufficient radioactivity had accumulated, samples were analyzed by HPLC with online radioactivity and tandem mass spectrometric detection. Nine labeled compounds were identified, the substrate and its cis isomer, 3'-O- and 4'-O-sulfates and glucuronides of caffeic acid, 4'-O-sulfates and glucuronides of ferulic acid, and isoferulic acid-4'-O-sulfate. Four unidentified metabolites were also detected. After passing down the gastrointestinal tract, the majority of the radiolabeled metabolites were excreted in urine with minimal accumulation in plasma. Only relatively small amounts of an unidentified (14)C-labeled metabolite were expelled in feces. There was little or no accumulation of radioactivity in body tissues, including the brain. The overall recovery of radioactivity 72 h after ingestion of [3-(14)C]caffeic acid was ～80% of intake.     

Characterization of the p-coumaric acid decarboxylase from Lactobacillus plantarum CECT 748(T)

It was previously reported that cell cultures from Lactobacillus plantarum CECT 748 (T) were able to decarboxylate phenolic acids, such as p-coumaric, m-coumaric, caffeic, ferulic, gallic, and protocatechuic acid. The p-coumaric acid decarboxylase (PDC) from this strain has been overexpressed and purified. This PDC differs at its C-terminal end when compared to the previously reported PDC from L. plantarum LPCHL2. Because the C-terminal region of PDC is involved in enzymatic activity, especially in substrate activity, it was decided to biochemically characterize the PDC from L. plantarum CECT 748 (T). Contrarily to L. plantarum LPCHL2 PDC, the recombinant PDC from L. plantarum CECT 748 (T) is a heat-labile enzyme, showing optimal activity at 22 degrees C. This PDC is able to decarboxylate exclusively the hydroxycinnamic acids p-coumaric, caffeic, and ferulic acids. Kinetic analysis showed that the enzyme has a 14-fold higher K(M) value for p-coumaric and caffeic acids than for ferulic acid. PDC catalyzes the formation of the corresponding 4-vinyl derivatives (vinylphenol and vinylguaiacol) from p-coumaric and ferulic acids, respectively, which are valuable food additives that have been approved as flavoring agents. The biochemical characteristics showed by L. plantarum PDC should be taken into account for its potential use in the food-processing industry.     

Absorption of chlorogenic acid and caffeic acid in rats after oral administration

Absorption of orally administered chlorogenic acid (5-caffeoylquinic acid) and caffeic acid in rats was studied to obtain plasma pharmacokinetic profiles of their metabolites. Rats were administered 700 micromol/kg body weight of chlorogenic or caffeic acid, and blood was collected from the tail for 6 h after administration. Ingested caffeic acid was absorbed from the alimentary tract and was present in the rat blood circulation in the form of various metabolites. On the other hand, only traces of metabolites, supposedly caffeic and ferulic acids conjugates, were detected in rat plasma for 6 h after chlorogenic acid administration. Chlorogenic acid and small amounts of caffeic acid were found in the small intestine for 6 h after chlorogenic acid administration. These results suggest that chlorogenic acid is not well absorbed from the digestive tract, unlike caffeic acid, and subject to almost no structural changes to the easily absorbed forms.     

In vitro and in vivo stability of caffeic acid phenethyl ester, a bioactive compound of propolis

The in vitro biochemical stability of caffeic acid phenethyl ester in rat and human plasma was investigated and compared with the stability of other caffeic acid esters (chlorogenic acid and rosmarinic acid). The incubation of the compounds in rat plasma for up to 6 h showed that caffeic acid phenethyl ester, but not the other compounds, was hydrolyzed, whereas human plasma did not affect the stability of all the assayed compounds. The products in rat plasma were caffeic acid and an unknown compound, which was identified by mass spectrometry as caffeic acid ethyl ester, produced by transesterification in the presence of ethanol used as vehicle for standard compounds. Specific inhibitors of different plasma esterases allowed the identification of a carboxylesterase as the enzyme involved in the metabolism of caffeic acid phenethyl ester. The oral administration in rats of caffeic acid phenethyl ester in the presence of both ethanol and 2-(2-ethoxyethoxy)ethanol gave rise to a dramatic increase of caffeic acid, as well as low levels of caffeic acid phenethyl ester, caffeic acid ethyl ester, and caffeic acid 2-(2-ethoxyethoxy)ethyl ester, in urine collected within 24 h after treatment. These results suggest that caffeic acid phenethyl ester is hydrolyzed also in vivo to caffeic acid as the major metabolite and that its biological activities should be more properly assayed and compared with those of caffeic acid, its bioactive hydrolysis product. Moreover, alcohols should be carefully used in vivo as solvents for caffeic acid phenethyl ester, since they can give rise to new bioactive caffeic acid esters.     

Absorption of hydroxycinnamates in humans after high-bran cereal consumption

Hydroxycinnamic acids are a group of phenolic compounds that exhibit a wide range of in vitro chemoprotective and antioxidant properties. Cereals containing a high proportion of the bran layers are rich in ester-linked hydroxycinnamic acids, such as ferulic and diferulic acids. The present work investigated the absorption in humans of hydroxycinnamic acids from high-bran breakfast cereal (wheat). Plasma and urine samples from six volunteers were collected before and after cereal consumption and analyzed for total hydroxycinnamic acids content after beta-glucuronidase/sulfatase treatment both by HPLC-DAD and by LC-MS (SIM monitoring). High-bran cereal administration resulted in increased plasma ferulic and sinapic acid concentrations (maximum levels detected of approximately 200 and approximately 40 nM, respectively) with absorption peaks between 1 and 3 h. Increases of approximately 4-fold in ferulic acid and approximately 5-fold in feruloylglycine were detected in 24-h urine after consumption of the cereal. Most of the ferulic acid detected in urine and plasma was present as conjugates (feruloylglycine and/or glucuronides). Diferulic acids were undetectable. The data show that ferulic and sinapic acids are taken up in humans from dietary high bran wheat but that absorption is limited and may originate only from the free and soluble portions present in the cereal.     

A new LC/MS/MS rapid and sensitive method for the determination of green tea catechins and their metabolites in biological samples

A new rapid and sensitive method has been developed, using liquid chromatography in tandem mass spectrometry (LC-ESI-MS/MS) to identify green tea catechin metabolites in plasma and urine after oral intake of a green tea extract. (-)-Epigallocatechin-3-gallate (EGCG), (-)-epicatechin-3-gallate (ECG), (-)-epigallocatechin (EGC)-glucuronide, (-)-epicatechin (EC)-glucuronide, and EC-sulfate were identified in plasma, whereas in urine only the conjugated catechins were detected (EGC-glucuronide, EGC-sulfate, EC-glucuronide, and EC-sulfate). Standard calibration curves prepared in plasma were found to be linear in the range of 10.9-1379.3 nmol/L for EGCG, EGC, ECG, and EC. The accuracy and precision of this assay showed a coefficient of variation of <15%. The method allowed the detection and quantification limits (for 20 microL injection) from 1.1 to 2.6 nmol/L and 3.8-8.7 nmol/L, respectively, in plasma and 0.8-1.8 nmol/L and 2.6-6.0 nmol/L, respectively, in urine. This method can be applied for future clinical and epidemiological studies, allowing the identification of the active metabolites that will reach the target tissues.     

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.     

Plasma levels of caffeic acid and antioxidant status after red wine intake.

The aim of this study was to evaluate the bioavailability of caffeic acid and the modification of plasma antioxidant status following red wine intake. Five healthy male volunteers consumed 100, 200, and 300 mL of red wine corresponding to approximately 0.9, 1.8, and 2.7 mg of caffeic acid, respectively. Plasma samples collected at different times (0-300 min) were evaluated for their content of caffeic acid and their total antioxidant status. Both these parameters, i.e., plasma concentration of caffeic acid and antioxidant potential, were dose-dependent and the C(max) was reached at about 60 min after red wine intake. The results indicate that caffeic acid is bioavailable and it may be correlated with the antioxidant potential of plasma.     

Development of a stable isotope dilution analysis with liquid chromatography-tandem mass spectrometry detection for the quantitative analysis of di- and trihydroxybenzenes in foods and model systems

A straightforward stable isotope dilution analysis (SIDA) for the quantitative determination of the di- and trihydroxybenzenes catechol (1), pyrogallol (2), 3-methylcatechol (3), 4-methylcatechol (4), and 4-ethylcatechol (5) in foods by means of liquid chromatography-tandem mass spectrometry was developed. With or without sample preparation involving phenylboronyl solid phase extraction, the method allowed the quantification of the target compounds in complex matrices such as coffee beverages with quantification limits of 9 nmol/L for 4-ethylcatechol, 24 nmol/L for catechol, 3-methyl-, and 4-methylcatechol, and 31 nmol/L for pyrogallol. Recovery rates for the analytes ranged from 97 to 103%. Application of the developed SIDA to various commercial food samples showed that quantitative analysis of the target compounds is possible within 30 min and gave first quantitative data on the amounts of di- and trihydroxybenzenes in coffee beverage, coffee powder, coffee surrogate, beer, malt, roasted cocoa powder, bread crust, potato crisps, fruits, and cigarette smoke and human urine. Model precursor studies revealed the carbohydrate/amino acid systems as well as the plant polyphenols catechin and epicatechin as precursors of catechol and 5-O-caffeoylquinic acid, caffeic acid as a precursor of catechol and 4-ethylcatechol, and gallocatechin, epigallocatechin, and gallic acid as precursors of pyrogallol.     

Sulfated ferulic acid is the main in vivo metabolite found after short-term ingestion of free ferulic acid in rats

The bioavailability of ferulic acid (FA; 3-methoxy-4-hydroxycinnamic acid) and its metabolites was investigated in rat plasma and urine after an oral short-term ingestion of 5.15 mg/kg of FA. Free FA, glucuronoconjugates, and sulfoconjugates were quickly detected in plasma with a peak of concentration found 30 min after ingestion. Sulfoconjugates were the main derivates ( approximately 50%). In urine, the cumulative excretion of total metabolites reached a plateau 1.5 h after ingestion, and approximately 40% were excreted by this way. Free FA recovered in urine represented only 4.9 +/-1.5% of the native FA consumed by rats. Glucuronoconjugates and sulfoconjugates represented 0.5 +/- 0.3 and 32.7 +/- 7.3%, respectively. These results suggested that a part of FA incorporated in the diet was quickly absorbed and largely metabolized in sulfoconjugates before excretion in urine.     

苜蓿植株及根际土壤中主要酚酸和香豆素物质含量测定

通过高效液相色谱(HPLC)法测定不同试验地间18个苜蓿(Medicago sativa L.)品种植株及根际土壤中主要酚酸和香豆素物质的含量,分析其在不同品种苜蓿植株中的分布特征及不同试验地间差异。经过ASE 350型加速溶剂萃取仪萃取苜蓿植株及根际土壤中酚酸类和香豆素物质,萃取液存放于2oC冰箱,并用0.45μm有机滤膜过滤后通过HPLC测定自毒物质含量。结果显示,18个苜蓿品种中香豆素、阿魏酸、绿原酸、咖啡酸的含量存在差异,其中香豆素、绿原酸含量显著高于阿魏酸、咖啡酸含量。各苜蓿品种间单一自毒物质含量差异显著(P0.05),香豆素、阿魏酸、绿原酸和咖啡酸的总含量差异明显。武威试验地酚酸和香豆素物质平均含量比会宁试验地低4.01%。研究表明:不同苜蓿品种间香豆素、阿魏酸、绿原酸、咖啡酸物质总含量差异显著;苜蓿植株中香豆素、阿魏酸、绿原酸、咖啡酸物质总含量与根际土壤中含量差异极显著。     

HPLC analysis of rosmarinic acid in feed enriched with aerial parts of Prunella vulgaris and its metabolites in pig plasma using dual-channel coulometric detection

This paper describes a sensitive isocratic HPLC/ECD method developed for the determination of rosmarinic acid (RA) in plant material, animal feed, and pig plasma. The plasma sample preparation only includes protein precipitation and adjustment of the pH. The applicability of the method was tested on plasma samples of pigs that were exposed to a 91-day oral intake of RA via feed enriched by aerial parts of Prunella vulgaris. The plasma was directly analyzed using the method described as well as after enzymatic hydrolysis. When no hydrolysis step was included, RA and caffeic acid (CA) were quantified in the plasma. In hydrolyzed plasma samples, several other metabolites were determined, including dihydrocaffeic, ferulic, and dihydroferulic acid. The dual-channel coulometric detection employed, as an alternative to mass spectrometry, offers good selectivity and sensitivity owing to the electrochemical properties of the phenolic constituents.     

Determination of some phenolic acids in Majorana hortensis by capillary electrophoresis with online electrokinetic preconcentration

An online accumulation/mobilization preconcentration technique based on a dynamic pH junction technique and electrokinetic injection was employed for analysis of phenolic acids (sinapic, ferulic, coumarinic, caffeic, syringic, vanillic, and 4-hydroxybenzoic acid) in extracts from Majorana hortensis leaves. Samples were extracted by pressurized solvent extraction with acetone at 150 degrees C and 15 MPa. The capillary electrophoretic method employed 50 mmol.L (-1) sodium borate, pH 9.5, as the sample electrolyte, 50 mmol.L (-1) sodium phosphate, pH 2.5, as the background electrolyte, and 50 mmol.L (-1) sodium phosphate, pH 2.5, with 60 mmol.L (-1) sodium dodecyl sulfate as the mobilization electrolyte. The method allowed 720-fold to 5560-fold preconcentration of the phenolic acids during 30 min of electrokinetic accumulation with detection limits from 0.38 to 4.22 ng.mL (-1).     

Insulin-releasing properties of a series of cinnamic acid derivatives in vitro and in vivo

Cinnamic acid derivatives are naturally occurring substances found in fruits, vegetables, and flowers and are consumed as dietary phenolic compounds. In the present study, cinnamic acid and its derivatives were evaluated for insulin secreting activity in perfused rat pancreas and pancreatic beta-cells (INS-1) as well as an increase in [Ca(2+)]i in vitro. The presence of m-hydroxy or p-methoxy residues on cinnamic acid was a significantly important substituent as an effective insulin releasing agent. The introduction of p-hydroxy and m-methoxy-substituted groups in cinnamic acid structure (ferulic acid) displayed the most potent insulin secreting agent among those of cinnamic acid derivatives. In particular, the stimulatory insulin secreting activities of test compounds were associated with a rise of [Ca(2+)]i in INS-1. In perfused rat pancreas, m-hydroxycinnamic acid, p-methoxycinnamic acid, and ferulic acid (100 microM) significantly stimulated insulin secretion during 10 min of administration. The onset time of insulin secretion of those compounds was less than 1 min and reached its peak at 4 min that was about 2.8-, 3.3-, and 3.4-fold of the baseline level, respectively. Intravenous administration of p-methoxycinnamic acid and ferulic acid (5 mg/kg) significantly decreased plasma glucose and increased insulin concentration in normal rats and maintained its level for 15 min until the end of experiment. Meanwhile, m-hydroxycinnamic acid induced a significant lowering of plasma glucose after 6 min, but the effects were transient with plasma glucose concentration, rapidly returning to basal levels. Our findings suggested that p-methoxycinnamic acid and ferulic acid may be beneficial for the treatment of diabetes mellitus because they regulated blood glucose level by stimulating insulin secretion from pancreatic beta-cells.     

Determination of phenolic compounds in Perilla frutescens L. by capillary electrophoresis with electrochemical detection

A method based on capillary electrophoresis with electrochemical detection has been developed to analyze flavonoids and phenolic acids in Perilla frutescens L. for the first time. Catechin, ferulic acid, apigenin, luteolin, rosmarinic acid, and caffeic acid are major important active ingredients in the plant. Operated in a wall-jet configuration, a 300 microm diameter carbon-disk electrode was used as the working electrode, which exhibits a good response at 0.90 V (versus saturated calomel electrode) for the analytes. Under the optimum conditions, the analytes were baseline separated within 20 min in a 100 mmol/L borax buffer (pH 8.7). Notably, excellent linearity was obtained over 3 orders of magnitude with detection limits (S/N = 3) ranging from 2 x 10(-7) to 1 x 10(-6) g/mL for all analytes. This proposed method has been successfully applied to monitor the flavonoids and phenolic acids contents in the leaves and seeds of P. frutescens L. at different growth stages with relatively simple extraction procedures, and the assay results were satisfactory.     

Hydroxycinnamic acids and ferulic acid dehydrodimers in barley and processed barley

Hydroxycinnamic acid content and ferulic acid dehydrodimer content were determined in 11 barley varieties after alkaline hydrolysis. Ferulic acid (FA) was the most abundant hydroxycinnamate with concentrations ranging from 359 to 624 microg/g dry weight. p-Coumaric acid (PCA) levels ranged from 79 to 260 microg/g dry weight, and caffeic acid was present at concentrations of <19 microg/g dry weight. Among the ferulic acid dehydrodimers that were identified, 8-O-4'-diFA was the most abundant (73-118 microg/g dry weight), followed by 5,5'-diFA (26-47 microg/g dry weight), the 8,5'-diFA benzofuran form (22-45 microg/g dry weight), and the 8,5'-diFA open form (10-23 microg/g dry weight). Significant variations (p < 0.05) among the different barley varieties were observed for all the compounds that were quantified. Barley grains were mechanically fractionated into three fractions: F1, fraction consisting mainly of the husk and outer layers; F2, intermediate fraction; and F3, fraction consisting mainly of the endosperm. Fraction F1 contained the highest concentration for ferulic acid (from 77.7 to 82.3% of the total amount in barley grain), p-coumaric acid (from 78.0 to 86.3%), and ferulic acid dehydrodimers (from 79.2 to 86.8%). Lower contents were found in fraction F2, whereas fraction F3 exhibited the lowest percentages (from 1.2 to 1.9% for ferulic acid, from 0.9 to 1.7% for p-coumaric acid, and <0.02% for ferulic acid dehydrodimers). The solid barley residue from the brewing process (brewer's spent grain) was approximately 5-fold richer in ferulic acid, p-coumaric acid, and ferulic acid dehydrodimers than barley grains.