Black tea represents a major source of dietary phenolics among regular tea drinkers.

The phenolic composition and antioxidant activities [TEAC, ORAC, FRAP] of consumer brews (1 tea bag in 230 ml for 1 min) of seven different brands of black tea from the British market were investigated. The main phenolic compounds identified were epigallocatechin gallate, four theaflavins, as well as epicatechin gallate, theogallin (tentative assignment), quercetin-3-rutinoside and 4-caffeoyl quinic acid. Thearubigins represented an estimated 75-82% of the total phenolics. Further, polyphenol fractions were in decreasing order theaflavins, flavan-3-ols, flavonols, gallic acids and hydroxycinnamates. On average, a cup of a consumer brew of black tea is providing polyphenols at the level of 262mg GAE/serving, of which 65 mg were assigned to individual polyphenols. The antioxidant activity of black tea preparations is higher than that of most reported dietary agents on a daily basis. Correlations were observed between the antioxidant activities and the sum of all quantified polyphenols by HPLC analysis as well as with the total phenolics. Treatment of the black tea brew with simulated gastric juice resulted in a significant increase of the identified theaflavins implying a partial cleavage of thearubigins in the environment of the gastric lumen. Therefore, black tea can be considered to be a rich source of polyphenols and/or antioxidants.

Caffeic acid derivatives in artichoke extract are metabolised to phenolic acids in vivo.

The purpose of this study was to investigate the absorption and metabolism of hydroxycinnamates from artichoke extract by determining the urinary excretion of the conjugates. Ten healthy, non smoking volunteers (5 female, 5 male) were given three capsules containing artichoke extract every 4 h (0, 4, 8 h) following two days of a low-polyphenol diet. One capsule contained 320 mg of artichoke extract equivalent to 34.3 +/- 0.6 mg/g hydroxycinnamates (caffeic acid derivatives) and 5.6 +/- 0.1 mg/g flavonoids. Polyphenols and phenolic acids present in the artichoke extract were not detected in the urine either as conjugates or aglycones. However, ferulic, isoferulic, dihydroferulic and vanillic acid were identified as major metabolites after beta-glucuronidase treatment of urine. The amount excreted as well as the ratio to that of creatinine, a biomarker for the general excretion rate, increased significantly on the study day compared to the pre-supplementation day. Thus, the caffeic acid esters found in the artichoke extract capsule are absorbed, metabolised and excreted as methylated phenolic acids such as ferulic, isoferulic, dihydroferulic and vanillic acid.

Iron supplementation and oxidative damage to DNA in healthy individuals with high plasma ascorbate.

Previously, we have investigated the potential for a pro-oxidant interaction of iron and ascorbate in vivo in iron and ascorbate cosupplementation or ascorbate supplementation studies. In this study, for the first time, the effects of iron supplementation on oxidative damage to DNA in healthy individuals with plasma ascorbate levels at the upper end of the normal range were examined. Forty female and male volunteers (mean plasma ascorbate approximately equal to 70 micromol/L) were supplemented with a daily dose of syrup (ferrous glycine sulphate equivalent to 12.5 mg iron) for 6 weeks. Serum ferritin, transferrin bound iron, % saturation of transferrin and plasma ascorbate were assessed and the mean dietary intakes of all subjects were estimated through food frequency questionnaires. Oxidative damage to DNA bases from white blood cells was measured by gas chromatography/mass spectrometry with selected-ion monitoring (GC/MS-SIM), using isotope-labelled standards for quantification. Iron supplementation did not affect any of the iron status parameters. There were also no detrimental effects, over the period under investigation, in terms of oxidative damage to DNA. However, the effects of larger doses or of longer supplementation periods should also be investigated.

Tea flavonoids and cardiovascular health.

Tea is rich in antioxidant polyphenols (catechins, flavonols, theaflavins and thearubigins). Epidemiological evidence relating regular consumption of tea or related polyphenols to CHD is equivocal. Catechins are absorbed from tea, but low plasma concentrations are attained. The bioavailability of theaflavins and thearubigins is unknown. Tea does not reduce blood pressure or plasma lipids in well-controlled human trials. Tea polyphenols inhibit LDL lipid peroxidation in vitro, but the effect ex vivo is small. The plasma antioxidant potential increases after drinking green but not black tea. Tea consumption tended to reduce the development of aortic atherosclerosis in rabbits. Tea polyphenols exert marked effects on cells, and inhibit neutrophil migration and inflammatory responses, sometimes at low concentrations. These diverging results suggest potential beneficial effects, but emphasize the need for good human trials of tea using early markers of CHD before firm conclusions can be drawn.

Novel biomarkers of the metabolism of caffeic acid derivatives in vivo.

The purpose of this study was to investigate biomarkers of the bioavailability and metabolism of hydroxycinnamate derivatives through the determination of the pharmacokinetics of their urinary elimination and identification of the metabolites excreted. Coffee was used as a rich source of caffeic acid derivatives and human supplementation was undertaken. The results show a highly significant increase in the excretion of ferulic, isoferulic, dihydroferulic acid (3-(4-hydroxy-3-methoxyphenyl)-propionic acid), and vanillic acid postsupplementation relative to the levels presupplementation. Thus, ferulic, isoferulic, and dihydroferulic acids are specific biomarkers for the bioavailability and metabolism of dietary caffeic acid esters. Isoferulic acid is a unique biomarker as it is not a dietary component, however, dihydroferulic acid may well derive from other flavonoids with a structurally related B-ring. 3-Hydroxyhippuric acid has also been identified as an indicator for bioavailability and metabolism of phenolic compounds, and shows a highly significant excretion increase postsupplementation. The results reveal isoferulic acid (and possibly dihydroferulic acid) as novel markers of caffeoyl quinic acid metabolism.

Flavonoid B-ring chemistry and antioxidant activity: fast reaction kinetics.

Rapid scavenging of the model stable radical cation, ABTS(*+), has been applied to screen for the antioxidant activity of flavonoids. The reaction follows two distinct phases. For compounds with a monophenolic B-ring there is a rapid initial phase of reduction of ABTS(*+) within 0.1 s with no further change in the subsequent 2.9 s. In contrast, compounds with a catechol-containing B ring follow a fast initial scavenging phase with a slow secondary phase. Flavonoids with an unsubstituted B ring do not react within this time scale. The findings suggest that the structure of the B ring is the primary determinant of the antioxidant activity of flavonoids when studied through fast reaction kinetics.

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.

Potential problems of ascorbate and iron supplementation: pro-oxidant effect in vivo?

The comparison was undertaken between the effects of ascorbate versus ascorbate plus iron supplementation on DNA damage. Twenty healthy subjects with initial levels of plasma ascorbate of 67.2 +/- 23.3 micromol/l were randomly assigned to and cycled through one of three supplementation regimes: placebo, 260 mg/d ascorbate, 260 mg/d ascorbate plus 14 mg/d iron for 6 weeks separated by 8-week washout periods. Supplementation did not cause a rise in total oxidative DNA damage measured by GC-MS. However, a significant decrease occurred in levels of 8-oxo-7,8-dihydroguanine by ascorbate supplementation and 5-hydroxymethyl uracil by both ascorbate and ascorbate plus iron supplementation, relative to the pre-supplemental levels but not to the placebo group. In addition, levels of 5-hydroxymethyl hydantoin and 5-hydroxy cytosine increased significantly, only relative to pre-supplementation, by ascorbate plus iron treatment. No compelling evidence for a pro-oxidant effect of ascorbate supplementation, in the presence or absence of iron, on DNA base damage was observed.

Comparison of the phenolic composition of fruit juices by single step gradient HPLC analysis of multiple components versus multiple chromatographic runs optimised for individual families.

After minimal sample preparation, two different HPLC methodologies, one based on a single gradient reversed-phase HPLC step, the other on multiple HPLC runs each optimised for specific components, were used to investigate the composition of flavonoids and phenolic acids in apple and tomato juices. The principal components in apple juice were identified as chlorogenic acid, phloridzin, caffeic acid and p-coumaric acid. Tomato juice was found to contain chlorogenic acid, caffeic acid, p-coumaric acid, naringenin and rutin. The quantitative estimates of the levels of these compounds, obtained with the two HPLC procedures, were very similar, demonstrating that either method can be used to analyse accurately the phenolic components of apple and tomato juices. Chlorogenic acid in tomato juice was the only component not fully resolved in the single run study and the multiple run analysis prior to enzyme treatment. The single run system of analysis is recommended for the initial investigation of plant phenolics and the multiple run approach for analyses where chromatographic resolution requires improvement.

Isomerization of dietary lycopene during assimilation and transport in plasma.

Diets of individuals were supplemented with tomatoes, either cooked or as tomato pureé in order to compare uptake of lycopene from intact and homogenized fruit tissue matrices. Following a diet containing cooked tomatoes over three consecutive 7-day periods, little change in the carotenoid levels in plasma lipoproteins occurred. In contrast, a diet supplemented with concentrated tomato pureé, over a 2 week period, caused a significant (p < 0.05) increase in lycopene levels in plasma, showing that the lycopene within intact cells is less bioavailable than that from processed tissue. The isomeric composition of plasma lycopene was significantly different to that of the ingested pureé. A number of cis-isomers (predominantly 5-cis, 13-cis and 9-cis-) were detected in plasma, that are not present in the lycopene from pureé. The significance of the increase in lycopene following dietary supplementation with respect to bioavailability and the causes of isomerization are discussed.

Measurement of total antioxidant activity as a marker of antioxidant status in vivo: procedures and limitations.

The ability to apply a simple routine assay to report on the total antioxidant activity of plasma/serum and other body fluids and their responses to dietary intervention and nutritional supplementation is an attractive useful proposition. This article will attempt to address where we are, in the author's view, in this context, setting out the pros and cons of the available methodologies en route.

Interactions of the flavonoid naringenin in the gastrointestinal tract and the influence of glycosylation.

We have studied interactions in the gastrointestinal tract of flavonoids and the influence of glycosylation on their subsequent metabolism by examining the urinary recoveries of the flavonoid naringenin-7-glucoside and its aglycone, in the conscious rat model, after oral and intravenous administration. Absorption studies were also conducted using an in vitro isolated rat jejunum. The results show that ca. 10% of the administered dose of naringenin was recovered after oral dosing, the majority as naringenin glucuronide, whereas, after intravenous administration, the recovery of the glucuronide was ca. 20%. In contrast, after oral dosing of naringenin-7-glucoside, its hydrolysis product naringenin (0.5%) and naringenin glucuronide (12.7%) were detected. After intravenous dosing the majority of that identified in the urine was as the native glucoside. These findings suggest that, via the oral route, the glycoside group is cleaved by an intestinal enzyme prior to glucuronidation within the epithelium. This is substantiated by the urinary elimination of the native glucoside and the lack of detection of glucuronide after intravenous administration. Transport studies with isolated intestine showed that neither unchanged naringenin nor the 7-glucoside was absorbed in significant quantities across the gut wall. The major metabolite detected in both cases was naringenin glucuronide, thus supporting the notion that glucuronidation as well as hydrolysis can occur at the intestinal epithelium.

Urinary excretion of hydroxycinnamates and flavonoids after oral and intravenous administration.

The urinary recoveries of the hydroxycinnamates, ferulic acid (3-methoxy, 4-hydroxy cinnamic acid), and chlorogenic acid (the quinic acid ester of 3,4-dihydroxycinnamic acid), and three structurally related flavonoids were studied in the rat. For the latter, the aglycone quercetin was compared with its 3-glucoside (isoquercitrin) and 3-rhamnoglucoside (rutin). Doses of 50 mg/kg were administered via the oral and intravenous routes and urine collected over the subsequent 24-h period. Reverse phase HPLC with photo-diode array detection was used to analyze the unchanged compound and their metabolites excreted in the urine. Ferulic acid and isoquercitrin were orally absorbed (5.4 and 0.48% of administered dose, respectively) and are therefore bioavailable. In contrast, neither unchanged chlorogenic acid, rutin, quercetin, nor the conjugated metabolites in the form of glucuronide or sulphate were detected in the urine after oral dosing. All the flavonoids studied produced low total urinary recoveries after intravenous administration, 9.2% for quercetin-3-rhamnoglucoside, 6.7% for the 3-glucoside, and 2.4% for the aglycone, indicating that extensive metabolism to low molecular weight compounds or excretion via other routes may be occurring. Overall it can be stated that renal excretion is not a major pathway of elimination for intact flavonoids and hydroxycinnamates in the rat.

Tomato consumption modulates oxidative DNA damage in humans.

Consumption of a single serving of tomatoes by healthy human volunteers was sufficient to alter levels of oxidative DNA base damage in white cell DNA within 24 h. Levels of the mutagenic oxidized purine base 8-hydroxyguanine decreased, especially in those subjects whose initial levels of this base were higher than the mean. However, total DNA base damage remained unchanged since levels of 8-hydroxyadenine rose. The ability of tomato consumption to modulate oxidative DNA damage in the short term may indicate why daily consumption of fruits and vegetables is beneficial in decreasing cancer incidence.

Singlet oxygen quenching and the redox properties of hydroxycinnamic acids.

The singlet oxygen quenching rate constants (kq) for a range of hydroxycinnamic acids in acetonitrile and D2O solutions were measured using time resolved near infrared phosphorescence in order to establish their antioxidant activity. The magnitude of kq observed depends on both the nature of the substituent groups and solvent polarity. The variations in kq depend on the energy of the hydroxycinnamic acid/molecular oxygen charge transfer states, (O2delta- ...HCAdelta+). In D2O the values of kq range from 4x10(7) M(-1) s(-1) to 4x10(6) M(-1) s(-1) for caffeic acid and o-coumaric acid respectively. In acetonitrile, the charge transfer energy levels are raised and this is reflected in lower singlet oxygen quenching rate constants with a kq value of 5x10(6) M(-1) s(-1) for caffeic acid. The phenoxyl radical spectra derived from the hydroxycinnamic acids were determined using pulse radiolysis of aqueous solutions and the reduction potentials were found to range from 534 to 596 mV. A linear correlation is observed between reduction potential, and hence free energy for electron transfer, and log kq. These correlations suggest a charge transfer mechanism for the quenching of singlet oxygen by the hydroxycinnamic acids.

The polyphenolic content of fruit and vegetables and their antioxidant activities. What does a serving constitute?

Analysis of the major flavone, flavonol, anthocyanidin and hydroxycinnamic acid constituents (and their glycosides) of onion, tomato, egg plant and apple has been undertaken and the antioxidant activities of the phenolic extracts determined. The major phenolic antioxidant components of egg plant are chlorogenic acid in the flesh and a delphinidin conjugate in the skin. In the case of apple, the major phenolic antioxidants detected are chlorogenic acid, procyanidins/catechin compounds, rutin and phloridzin. Quercetin glycosides are well-known to be the major phenolic components of onion. Assessment of the antioxidant activities of a serving of 100g fresh weight fruit, vegetable and comparison with previously reported findings for 150 ml beverage (500 ml portion in the case of beer), expressed in micromol Trolox equivalents show that the antioxidant activities of 1 glass (150 ml) red wine equivalent to 12 glasses white wine equivalent to 2 cups of tea equivalent to 4 apples equivalent to 5 portions of onion equivalent to 5.5 portions egg plant equivalent to 3.5 glasses of blackcurrant juice equivalent to 3.5 (500 ml) glasses of beer equivalent to 7 glasses of orange juice equivalent to 20 glasses of apple juice (long life).

Luteolin-rich artichoke extract protects low density lipoprotein from oxidation in vitro.

Flavonoids represent a diverse group of phytochemicals which possess the capacity to act as antioxidants in vitro. This study examined the free radical scavenging properties of a luteolin-rich artichoke extract and some of its pure flavonoid constituents by assessing their ability to prevent Cu2+-mediated LDL oxidation. Artichoke extract retarded LDL oxidation in a dose-dependent manner as measured by a prolongation of the lag phase to conjugated diene formation, a decrease in the rate of propagation and a sparing of endogenous LDL alpha-tocopherol during oxidation. The pure aglycone, luteolin (1 microM), demonstrated an efficacy similar to that of 20 microg/ml artichoke extract in inhibiting lipid peroxidation. Luteolin-7-O-glucoside, one of the glycosylated forms in the diet, also demonstrated a dose-dependent reduction of LDL oxidation that was less effective than that of luteolin. Studies of the copper-chelating properties of luteolin-7-O-glucoside and luteolin suggest a potential role for chelation in the antioxidative effects of artichoke extract. Overall, the results demonstrate that the antioxidant activity of the artichoke extract relates in part to its constituent flavonoids which act as hydrogen donors and metal ion chelators, and the effectiveness is further influenced by their partitioning between aqueous and lipophilic phases.

Urinary detection of hydroxycinnamates and flavonoids in humans after high dietary intake of fruit.

The determination of the abilities of flavonoids, hydroxycinnamates and phenolics to scavenge free radicals in vitro suggests potent combined antioxidant activities of fruits, vegetables, beverages and grains. However, the key question of uptake in humans has only recently been approached consistently. The study described here demonstrates the uptake of hydroxycinnamates, for the first time, and other phenolic components, applying an HPLC method for their detection in the urine of subjects consuming levels of specific fruit equivalent to an approximate intake of 25 mg flavonol glycosides.

Structural dependence of flavonoid interactions with Cu2+ ions: implications for their antioxidant properties.

The flavonoids constitute a large group of polyphenolic phytochemicals with antioxidant properties in vitro. The interactions of four structurally related flavonoids (quercetin, kaempferol, rutin and luteolin) with Cu2+ ions were investigated in terms of the extent to which they undergo complex formation through chelation or modification through oxidation, as well as in their structural dependence. The ortho 3',4'-dihydroxy substitution in the B ring is shown to be important for Cu2+-chelate formation, thereby influencing the antioxidant activity. The presence of a 3-hydroxy group in the flavonoid structure enhances the oxidation of quercetin and kaempferol, whereas luteolin and rutin, each lacking the 3-hydroxy group, do not oxidize as readily in the presence of Cu2+ ions. The results also demonstrate that the reactivities of the flavonoids in protecting low-density lipoprotein (LDL) against Cu2+ ion-induced oxidation are dependent on their structural properties in terms of the response of the particular flavonoid to Cu2+ ions, whether chelation or oxidation, their partitioning abilities between the aqueous compartment and the lipophilic environment within the LDL particle, and their hydrogen-donating antioxidant properties.