Urinary metabolites of flavonoids and hydroxycinnamic acids in humans after application of a crude extract from Equisetum arvense.

Flavonoids and hydroxycinnamic acids are polyphenolic compounds present in our daily diet in form of tea and vegetables as well as in herbal remedies used in phytomedicine. A wide range of in-vitro activities, in particular their antioxidant properties, have been studied intensively. However, in-vivo-data on absorption, bioavailability and metabolism after oral intake are scarce and contradictory. In order to examine the metabolism and renal excretion of these compounds a standardized extract from horsetail (Equisetum arvense) was administered to 11 volunteers following a flavonoid-free diet for 8 d. 24 h urine samples were collected and analyzed by HPLC-DAD. The putative quercetin metabolites, 3,4-dihydroxyphenylacetic acid or 3,4-dihydroxytoluene could not be detected in urine in any sample. The endogenous amount of homovanillic acid, generally regarded as one of the main quercetin metabolites, was 4 +/- 1 mg/d and did not increase significantly. However, hippuric acid, the glycine conjugate of benzoic acid, increased twofold after drug intake. Thus, the degradation to benzoic acid derivatives rather than phenylacetic acid derivatives seems to be a predominant route of metabolism. The results of this pilot study give rise to additional, substantial pharmacokinetic investigations in humans.     

Wine phenolic compounds influence the production of volatile phenols by wine-related lactic acid bacteria

Aims: To evaluate the effect of wine phenolic compounds on the production of volatile phenols (4‐vinylphenol [4VP] and 4‐ethylphenol [4EP]) from the metabolism of p‐coumaric acid by lactic acid bacteria (LAB). Methods and Results: Lactobacillus plantarum, Lactobacillus collinoides and Pediococcus pentosaceus were grown in MRS medium supplemented with p‐coumaric acid, in the presence of different phenolic compounds: nonflavonoids (hydroxycinnamic and benzoic acids) and flavonoids (flavonols and flavanols). The inducibility of the enzymes involved in the p‐coumaric acid metabolism was studied in resting cells. The hydroxycinnamic acids tested stimulated the capacity of LAB to synthesize volatile phenols. Growth in the presence of hydroxycinnamic acids, especially caffeic acid, induced the production of 4VP by resting cells. The hydroxybenzoic acids did not significantly affect the behaviour of the studied strains. Some of the flavonoids showed an effect on the production of volatile phenols, although strongly dependent on the bacterial species. Relatively high concentrations (1 g l^?1) of tannins inhibited the synthesis of 4VP by Lact. plantarum. Conclusions: Hydroxycinnamic acids were the main compounds stimulating the production of volatile phenols by LAB. The results suggest that caffeic and ferulic acids induce the synthesis of the cinnamate decarboxylase involved in the metabolism of p‐coumaric acid. On the other hand, tannins exert an inhibitory effect. Significance and Impact of the Study: This study highlights the capacity of LAB to produce volatile phenols and that this activity is markedly influenced by the phenolic composition of the medium.     

Phytochemical fingerprinting of vegetable Brassica oleracea and Brassica napus by simultaneous identification of glucosinolates and phenolics

Introduction – Brassica vegetables have been related to the prevention of cancer and degenerative diseases, owing to their glucosinolate and phenolic content. Objective – Identification of glucosinolates, flavonoids and hydroxycinnamic acids in representative varieties of kale, cabbage and leaf rape. Methodology – One local variety of each crop was evaluated in this study using a multi‐purpose chromatographic method that simultaneously separates glucosinolates and phenolics. Chromatograms were recorded at 330 nm for flavonoid glycosides and acylated derivatives and 227 nm for glucosinolates. Results – Eight glucosinolates were identified in kale and cabbage, which exhibited the same glucosinolate profile, and 11 glucosinolates were identified in leaf rape. Furthermore, 20 flavonoids and 10 hydroxycinnamic acids were detected in kale and cabbage, while 17 flavonoids and eight hydroxycinnamic acids were found in leaf rape. Conclusions – This study has provided a deeper and comprehensive identification of health‐promoting compounds in kale, cabbage and leaf rape, thus showing that they are a good source of glucosinolates and phenolic antioxidants. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

Phenolic acids act as signaling molecules in plant-microbe symbioses

Phenolic acids are the main polyphenols made by plants. These compounds have diverse functions and are immensely important in plant-microbe interactions/symbiosis. Phenolic compounds act as signaling molecules in the initiation of legumerhizobia symbioses, establishment of arbuscular mycorrhizal symbioses and can act as agents in plant defense. Flavonoids are a diverse class of polyphenolic compounds that have received considerable attention as signaling molecules involved in plant-microbe interactions compared to the more widely distributed, simple phenolic acids; hydroxybenzoic and hydroxycinnamic acids, which are both derived from the general phenylpropanoid pathway. This review describes the well-known roles attributed to phenolic compounds as nod gene inducers of legume-rhizobia symbioses, their roles in induction of the GmGin1 gene in fungus for establishment of arbuscular mycorrhizal symbiosis, their roles in inducing vir gene expression in Agrobacterium, and their roles as defense molecules operating against soil borne pathogens that could have great implications for rhizospheric microbial ecology. Amongst plant phenolics we have a lack of knowledge concerning the roles of phenolic acids as signaling molecules beyond the relatively well-defined roles of flavonoids. This may be addressed through the use of plant mutants defective in phenolic acids biosynthesis or knock down target genes in future investigations.Key words: Agrobacterium sp., flavonoids, legume-rhizobium symbioses, phenolic acids, plant defense, vesicular arbuscular mycorrhiza     

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.     

Human fecal water content of phenolics: the extent of colonic exposure to aromatic compounds

Phenolic compounds are not completely absorbed in the small intestine and so enter the colon, where they might exert physiological effects. To identify phenolics that are present in normal human colon, fecal water was prepared from 5 free-living volunteers with no dietary restrictions and analyzed by gas chromatography-mass spectrometry. Daily measurements were also performed on a single individual to examine the variation more closely. Levels of polyphenols were variable between individuals. Naringenin and quercetin had mean concentrations of 1.20 and 0.63 microM. All other flavonoids examined were present < or =0.17 microM. Simple phenolic and other aromatic acids were present at much higher concentrations. The major components were phenylacetic acid, 479 microM; 3-phenylpropionic acid, 166 microM; 3-(4-hydroxy)-phenylpropionic acid, 68 microM; 3,4-dihydroxycinnamic acid, 52 microM; benzoic acid, 51 microM; 3-hydroxyphenylacetic acid, 46 microM; and 4-hydroxyphenylacetic acid, 19 microM. Other phenolic acids ranged from 0.04 to 7 microM. Decreased dietary phenolic intake caused a decrease in polyphenol and monophenolic acid concentration in fecal water 24 h later. This study is the first to measure the range of aromatic compounds in human fecal water and demonstrates that phenolic acid concentrations are high. The biological effects of phenolics may play an important role in colon function.     

Phenolic compounds in medicinal plants

Compositions of phenolic substances were studied in leaves of 21 species of medicinal plants. Flavonoid levels varied from 1.94 to 5.42%, whereas total amounts of monomeric polyphenols and hydroxybenzoic acids were estimated as 0.27 to 0.57%, and hydroxycinnamic acids and their esters with quinic acid, 0.09 to 0.18%. Condensed and polymerized polyphenols were detected in amounts of 0.41 to 1.20%. Qualitative compositions of flavonoids in leaves of seven plants studied were presented. The developed analytical procedures may be useful for plant polyphenol studies and as the basis of hemotaxonomy.     

Phenolic Substances in Medicinal Plants

Compositions of phenolic substances were studied in the leaves of 21 species of medicinal plants. Flavonoid levels varied from 1.94 to 5.42%, whereas total amounts of monomeric polyphenols and hydroxybenzoic acids were estimated as 0.27 to 0.57%; hydroxycinnamic acids and their esters with quinic acid, 0.09 to 0.18%. Condensed and polymerized polyphenols were detected in amounts of 0.41 to 1.20%. Qualitative compositions of flavonoids in leaves of the seven plants studied were presented. The developed analytical procedures may be useful for plant polyphenol studies and as a basis for hemotaxonomy.     

Phenolic antioxidants attenuate neuronal cell death following uptake of oxidized low-density lipoprotein

Oxidative stress is implicated in neuronal loss associated with neurodegeneration such as in Parkinson’s disease, Alzheimer’s disease and age-related cognitive decline. Recent reports indicate that the consumption of flavonoid-rich fruits partly reverses the age-related neuronal and cognitive decline. In this study, cultured striatal neurons were exposed to oxidized lipids in the form of low-density lipoprotein (oxLDL) as a model for the induction of oxidative injury, and the abilities of phenolic antioxidants, flavonoids and hydroxycinnamic acid derivatives, to attenuate this neuronal damage were examined. OxLDL was demonstrated to enter neuronal cells and to be capable of eliciting neurotoxicity in a dose- and time-dependent manner, inducing DNA fragmentation and cell lysis. Flavonoids exert protective effects, which appear to be related to specific structural characteristics, particularly relevant being those defining their reduction potentials and partition coefficients. In summary, these data suggest a possible role for flavonoids in reducing neurodegeneration associated with chronic disorders in which oxidative stress is implicated.     

In vitro and in vivo conjugation of dietary hydroxycinnamic acids by UDP-glucuronosyltransferases and sulfotransferases in humans

Hydroxycinnamic acids are a class of phenolic antioxidants found widely in dietary plants. Their biotransformation in the human organism primarily involves Phase II conjugation reactions. In this study, activities of UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) towards major dietary hydroxycinnamic acids (caffeic, dihydrocaffeic, dihydroferulic, ferulic and isoferulic acids) were investigated. Conjugate formation was evaluated using human liver and intestinal S9 homogenates, and in vitro characterization was carried out using recombinant human UGTs and SULTs. Analysis of the kinetics of hydroxycinnamic acid conjugation in human S9 homogenates revealed that intrinsic clearance (V_max/K_m) is much greater for sulfation than for glucuronidation. Assessment of activity using a panel of recombinant human SULTs showed that SULT1A1 is most active in the sulfation of caffeic, dihydrocaffeic and isoferulic acids, while SULT1E1 is most active in the sulfation of ferulic and dihydroferulic acids. Only isoferulic acid was significantly glucuronidated by human liver S9 homogenates, explained by the high activity of liver-specific UGT1A9. Studies on the kinetics of active SULTs and UGTs demonstrated a markedly lower K_m for SULTs. To further corroborate our findings, we carried out an intervention study in healthy humans to determine the hydroxycinnamic acid conjugates in urine after consumption of hydroxycinnamate-rich coffee (200 ml). Analysis showed that sulfates are the main conjugates in urine, with the exception of isoferulic acid, which is mainly glucuronidated. These data suggest that sulfates are the predominant hydroxycinnamic acid conjugates in humans, and that SULT mediated sulfation is a major factor determining the bioavailability of hydroxycinnamic acids in vivo.     

Structure-function relationships of the antibacterial activity of phenolic acids and their metabolism by lactic acid bacteria

Aims: To determine structure–function relationships of antibacterial phenolic acids and their metabolites produced by lactic acid bacteria (LAB). Methods and Results: Minimum inhibitory concentrations (MICs) of 6 hydroxybenzoic and 6 hydroxycinnamic acids were determined with Lactobacillus plantarum, Lactobacillus hammesii, Escherichia coli and Bacillus subtilis as indicator strains. The antibacterial activity of phenolic acids increased at lower pH. A decreasing number of hydroxyl groups enhanced the activity of hydroxybenzoic acids, but had minor effects on hydroxycinnamic acids. Substitution of hydroxyl groups with methoxy groups increased the activity of hydroxybenzoic, but not of hydroxycinnamic, acid. Metabolism of chlorogenic, caffeic, p‐coumaric, ferulic, protocatechuic or p‐hydroxybenzoic acids by L. plantarum, L. hammesii, Lactobacillus fermentum and Lactobacillus reuteri was analysed by LC‐DAD‐MS. Furthermore, MICs of substrates and metabolites were compared. Decarboxylated and/or reduced metabolites of phenolic acids had a lower activity than the substrates. Strain‐specific metabolism of phenolic acids generally corresponded to resistance. Conclusions: The influence of lipophilicity on the antibacterial activity of hydroxybenzoic acids is stronger than that of hydroxycinnamic acids. Metabolism of phenolic acids by LAB detoxifies phenolic acids. Significance and Impact of the Study: Results allow the targeted selection of plant extracts for food preservation, and selection of starter cultures for fermented products.     

The effect of phenolic compounds on the activity of respiratory chain enzymes and on respiration and phosphorylation activities of potato tuber mitochondria

The effect of derivatives of benzoic and cinnamic acids, quereetin,p-benzoquinone, and 2,5-dimethylbenzoquinone on oxygen consumption mitoehondrial suspensions and on the activity of some respiratory chain enzymes was studied. Benzoquinone and 2,5-dimethylbenzoquinone highly significantly inhibited the respiration and phosphorylation rates and malate- and succinate dehydrogenase activities. Chlorogenic acid, similarly as the quinones, very significantly inhibited the activities of the studied dehydrogenases but did not affect cytochrome oxidase. Oxygen consumption by intact mitochondria was not inhibited, only the oxidativo phosphorylation was significantly uncoupled. Quereetin significantly enhanced dehydrogenase activities and completely inhibited cytochrome oxidase activity. The respiration and phosphorylation activities of the mitochondria were significantly inhibited by quereetin. The effect of the other phenolic compounds studied on respiration and phosphorylation activities was not significant. Succinate dehydrogenase activity was the most affected enzyme among the respiratory chain enzymes. It was significantly inhibited by all the above phenolic compounds at 1^-4M or 5 10^-5M concentrations with the exception of gallic acid.     

Some allelochemicals of Pteridium aquilinum and their involvement in resistance to Pieris brassicae

A number of polar and semi-polar compounds from bracken, Pteridium aquilinum L. Kühn, were screened for feeding deterrent activity to Pieris brassicae L. At natural concentrations, 2 out of 4 flavonoids tested were stimulatory and one was inhibitory; while 2 out of 4 benzoic, and 4 out of 5 cinnamic acid derivatives were inhibitory. Prunasin and its enzymatic hydrolysates had no effect on feeding. It was concluded that braken flavonoids and cyanogenic glycoside were unlikely to be involved in resistance of polar bracken extracts to P. brassicae, while benzoic and cinnamic acids and condensed tannins were probably involved. A number of other benzoic and cinnamic acid derivatives not occurring in bracken were screened, and distinct relationships between chemical structure and feeding deterrent activity to P. brassicae were demonstrated.     

Influence of Plant Phenolic Acids on Growth and Cellulolytic Activity of Rumen Bacteria

Isolated rumen bacteria were examined for growth and, where appropriate, for their ability to degrade cellulose in the presence of the hydroxycinnamic acids trans-p-coumaric acid and trans-ferulic acid and the hydroxybenzoic acids vanillic acid and 4-hydroxybenzoic acid. Ferulic and p-coumaric acids proved to be the most toxic of the acids examined and suppressed the growth of the cellulolytic strains Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes when included in a simple sugars medium at concentrations of >5 mM. The extent of cellulose digestion by R. flavefaciens and B. succinogenes but not R. albus was also substantially reduced. Examination of rumen fluid from sheep maintained on dried grass containing 0.51% phenolic acids showed the presence of phloretic acid (0.1 mM) and 3-methoxyphloretic acid (trace) produced by hydrogenation of the 2-propenoic side chain of p-coumaric and ferulic acids, respectively. The parent acids were found in trace amounts only, although they represented the major phenolic acids ingested. Phloretic and 3-methoxyphloretic acids proved to be considerably less toxic than their parent acids. All of the cellulolytic strains (and Streptococcus bovis) showed at least a limited ability to hydrogenate hydroxycinnamic acids, with Ruminococcus spp. proving the most effective. No further modification of hydroxycinnamic acids was produced by the single strains of bacteria examined. However, a considerable shortfall in the recovery of added phenolic acids was noted in media inoculated with rumen fluid. It is suggested that hydrogenation may serve to protect cellulolytic strains from hydroxycinnamic acids.     

Hydroxycinnamic acids do not prevent aortic atherosclerosis in hypercholesterolemic golden Syrian hamsters

The protective effect of hydroxycinnamic acids, i.e. caffeic acid (CA) and sinapic acid (SA) present in wine, and chlorogenic acid (CHA) present in apple, compared to a red wine phenolic extract (RWPE) was investigated in hamsters fed an atherogenic diet for 12 weeks. Five groups of 8 hamsters fed such a diet received by force-feeding RWPE, CA or SA in water, mimicking a moderate consumption of alcohol-free red wine. Controls received water and CHA force-feeding was extrapolated from apple consumption. Plasma cholesterol concentration was lower in group that received RWPE (-22%) and hydroxycinnamic acids had no effect. Plasma apolipoprotein Apo-A1 concentration was not affected; consumption of RWPE only decreased Apo-B concentration (-46%). Liver superoxide dismutase activity was 33% lower and glutathione peroxidase activity was 67% greater in the group receiving RWPE compared to controls; there was no effect when CA, SA or CHA were given. All the phenolic compounds significantly increased plasma antioxidant capacity (about 28% on average) compared with controls. Aortic fatty streak area was significantly reduced in the group receiving RWPE (-30%) in comparison with controls and hydroxycinnamic acids. Our findings demonstrate that chronic ingestion of the nonalcoholic components of red wine, mainly polyphenols, prevent the development of atherosclerosis in hamster and that wine hydroxycinnamic acids are not the phenolic compounds involved in such a beneficial effect.     

New insights into the antioxidant activity of hydroxycinnamic and hydroxybenzoic systems: Spectroscopic,electrochemistry, and cellular studies

Abstract

A series hydroxycinnamic and gallic acids and their derivatives were studied with the aim of evaluating their in vitro antioxidant properties both in homogeneous and in cellular systems. It was concluded from the oxygen radical absorbance capacity-fluorescein (ORAC-FL), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and cyclic voltammetry data that some compounds exhibit remarkable antioxidant properties. In general, in homogeneous media (DPPH assay), galloyl-based cinnamic and benzoic systems (compounds 7–11) were the most active, exhibiting the lowest oxidation potentials in both dimethyl sulfoxide (DMSO) and phosphate buffer. Yet, p-coumaric acid and its derivatives (compounds 1–3) disclosed the highest scavenging activity toward peroxyl radicals (ORAC-FL assay). Interesting structure–property– activity relationships between ORAC-FL, or DPPH radical, and redox potentials have been attained, showing that the latter parameter can be a valuable antioxidant measure. It was evidenced that redox potentials are related to the structural features of cinnamic and benzoic systems and that their activities are also dependent on the radical generated in the assay. Electron spin resonance data of the phenoxyl radicals generated both in DMSO and phosphate buffer support the assumption that radical stability is related to the type of phenolic system. Galloyl-based cinnamic and benzoic ester-type systems (compounds 9 and 11) were the most active and effective compounds in cell-based assays (51.13 ± 1.27% and 54.90 ± 3.65%, respectively). In cellular systems, hydroxycinnamic and hydroxybenzoic systems operate based on their intrinsic antioxidant outline and lipophilic properties, so the balance between these two properties is considered of the utmost importance to ensure their performance in the prevention or minimization of the effects due to free radical overproduction.     

Diverting phenylpropanoid pathway flux from sinapine to produce industrially useful 4-vinyl derivatives of hydroxycinnamic acids in Brassicaceous oilseeds

Sinapine (sinapoylcholine) is an antinutritive phenolic compound that can account for up to 2% of seed weight in brassicaceous oilseed crops and reduces the suitability of their protein-rich seed meal for use as animal feed. Sinapine biosynthesis draws on hydroxycinnamic acid precursors produced by the phenylpropanoid pathway. The 4-vinyl derivatives of several hydroxycinnamic acids have industrial applications. For example, 4-vinyl phenol (4-hydroxystyrene) is a building block for a range of synthetic polymers applied in resins, inks, elastomers, and coatings. Here we have expressed a modified bacterial phenolic acid decarboxylase (PAD) in developing seed of Camelina sativa to redirect phenylpropanoid pathway flux from sinapine biosynthesis to the production of 4-vinyl phenols. PAD expression led to a ∼95% reduction in sinapine content in seeds of both glasshouse and field grown C. sativa and to an accumulation of 4-vinyl derivatives of hydroxycinnamic acids, primarily as glycosides. The most prevalent aglycone was 4-vinyl phenol, but 4-vinyl guaiacol, 6-hydroxy-4-vinyl guaiacol and 4-vinylsyringol (Canolol) were also detected. The molar quantity of 4-vinyl phenol glycosides was more than twice that of sinapine in wild type seeds. PAD expression was not associated with an adverse effect on seed yield, harvest index, seed morphology, storage oil content or germination in either glasshouse or field experiments. Our data show that expression of PAD in brassicaceous oilseeds can supress sinapine accumulation, diverting phenylpropanoid pathway flux into 4-vinyl phenol derivatives, thereby also providing a non-petrochemical source of this class of industrial chemicals.