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.     

Inhibition of advanced glycation end product formation on collagen by rutin and its metabolites

Several lines of evidence suggest that rutin, flavonoid in fruits and vegetables, or one of its metabolites may effectively modulate advanced glycation end product (AGE) formation. Following ingestion, rutin forms metabolites that include 3,4-dihydroxyphenylacetic acid (3,4-DHPAA), 3,4-dihydroxytoluene (3,4-DHT), m-hydroxyphenylacetic acid (m-HPAA), 3-methoxy-4-hydroxyphenylacetic acid (homovanillic acid, HVA) and 3,5,7,3',5'-pentahydroxyflavonol (quercetin). We studied the effects of rutin and its metabolites on the formation of AGE biomarkers such as pentosidine, collagen-linked fluorescence, N(epsilon)-carboxymethyllysine (CML) adducts, glucose autoxidation and collagen glycation, using an in vitro model where collagen I was incubated with glucose. Rutin metabolites containing vicinyl dihydroxyl groups, i.e., 3,4-DHT, 3,4-DHPAA and quercetin, inhibited the formation of pentosidine and fluorescent adducts, glucose autoxidation and glycation of collagen I in a dose-dependent manner, whereas non-vicinyl dihydroxyl group-containing metabolites, i.e., HVA and m-HPAA, were much less effective. All five metabolites of rutin effectively inhibited CML formation. In contrast, during the initial stages of glycation and fluorescent AGE product accumulation, only vicinyl hydroxyl group-containing rutin metabolites were effective. These studies demonstrate that rutin and circulating metabolites of rutin can inhibit early glycation product formation, including both fluorescent and nonfluorescent AGEs induced by glucose glycation of collagen I in vitro. These effects likely contribute to the beneficial health effects associated with rutin consumption.     

Neurosteroids: a new brain function?

The biosynthesis of neurosteroids proceeds through cholesterol side-chain cleavage, and gives rise to pregnenolone (P) and dehydroepiandrosterone (D). These steroids accumulate in the rat brain independently of the supply by peripheral endocrine glands. This led to the discovery of a steroid biosynthesis pathway in rat brain oligodendrocytes based on enzyme immunocytochemistry and conversion of radioactive precursors to C-21 steroids. Several biological functions have been proposed for P and D. They may serve as precursors of other steroids (such as progesterone and testosterone and their metabolites). They are implicated in the control of some behavioural activities. They have excitatory effects on neurons, and they modulate the function of GABAA-receptors. These observations may apply to all mammalian species including the human, and the physiological significance of neurosteroid synthesis needs further investigation. The relationship between steroids and cerebral function may be reconsidered in the light of a new fact: the existence of a biosynthetic pathway of these compounds from cholesterol, assured in the brain by the oligodendrocytes, glial cells which synthesize myelin.     

Protective effects of flavonoids and two metabolites against oxidative stress in neuronal PC12 cells

AimsRecent interest has focused on plant antioxidants as potentially useful neuroprotective agents. In most studies only the genuine forms of flavonoids were used, although they are rapidly metabolized. Therefore, we have compared protective activities of two flavonoids (luteolin, quercetin) and two of their bioavailable metabolites (3,4-DHPAA and 3,4-DHT) against oxidative stress, induced by peroxides (t-BHP, H₂O₂) and iron (FeSO₄), in neuronal PC12 cells.Main methodsWe have measured their effect on the prevention of cell death (MTT assay), glutathione depletion (GSH assay), lipid peroxidation (MDA assay) and production of ROS (DCF assay). Differentiated PC12 cells were used as a model system of neuronal cells. The compounds (concentration range 6–25 µmol/L) were tested in preincubation and coincubation experiments.Key findingsIn MTT and DCF assays all tested compounds showed excellent protection. When cells were exposed to peroxides, both metabolites increased GSH levels less efficiently than their parent flavonoids in both types of incubations. Following exposure to iron, only coincubation significantly prevented GSH depletion and the metabolites surprisingly mimicked the suppressive effect of flavonoids. MDA levels induced by all stressors were reduced more potently during coincubation than during preincubation with polyphenols. While the lipophilic metabolite 3,4-DHT exerted excellent antilipoperoxidant activity, the hydrophilic metabolite 3,4-DHPAA was less effective.SignificanceThese results demonstrate that most of the protective effects of flavonoids against oxidative stress in PC12 cells are continued despite biodegradation of the parent flavonoids. In general, the lipophilic metabolite 3,4-DHT was more active than the hydrophilic 3,4-DHPAA.     

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.     

Rapid, concurrent analysis of dopamine, 5-hydroxytryptamine, their precursors and metabolites utilizing high performance liquid chromatography with electrochemical detection: analysis of brain tissue and cerebrospinal fluid

Assays capable of measuring picomole quantities of dopamine (DA), 5-hydroxytryptamine (5-HT), several of their precursors and metabolites concurrently within 25 minutes were developed utilizing high performance liquid chromatography with electrochemical detection (LCEC). Several parameters of the LCEC were altered in order to separate the compounds while maintaining a short assay time. The final LCEC systems demonstrated biological utility in that the DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and the 5-HT metabolite 5-hydroxy-3-indoleacetic acid (5-HIAA) were detected in rat cerebrospinal fluid; in addition to these compounds, DA and 5-HT were measurable in the striatum, hypothalamus and median eminence of the rat brain. Pargyline decreased the concentrations of DOPAC, HVA and 5-HIAA and increased the 5-HT concentration in all three brain regions, and increased the DA concentration in the striatum. Probenecid increased all three acid metabolite concentrations in the hypothalamus and median eminence, while only the HVA and 5-HIAA concentrations were increased in the striatum. The DA and 5-HT concentrations were unaltered. The LCEC methods described in this paper should be useful in elucidating the mechanisms and roles of 5-HT and DA neurons in experimental paradigms of biological interest.     

Comparison of the bioavailability of quercetin and catechin in rats

Quercetin and catechin are present in noticeable amounts in human diet and these polyphenolic compounds are supposed to exert beneficial effects on human health. However, their metabolic fates in the organism have never been compared. In the present study, rats were fed a 0.25% quercetin or a 0.25% catechin diet. Quercetin and catechin metabolites were analyzed in plasma and liver samples by high-performance liquid chromatography coupled to an ultraviolet or a multielectrode coulometric detection. All plasma metabolites were present as conjugated forms, but catechin metabolites were mainly constituted by glucuronidated derivatives, whereas quercetin metabolites were sulfo- and glucurono-sulfo conjugates. Quercetin was more intensively methylated than catechin in plasma. The plasma quercetin metabolites are well maintained during the postabsorptive period (approximately 50 microM), whereas the concentration of catechin metabolites dropped dramatically between 12- and 24-h after an experimental meal (from 38.0 to 4.5 microM). In the liver, the concentrations of quercetin and catechin derivatives were lower than in plasma, and no accumulation was observed when the rats were adapted for 14 d to the supplemented diets. The hepatic metabolites were intensively methylated (90-95%), but in contrast to plasma, some free aglycones could be detected. Thus, it clearly appears that studies dealing with the biological impact of these polyphenols should take into account the feature of their bioavailability, particularly the fact that their circulating metabolites are conjugated derivatives.     

Molecular cytotoxic mechanisms of catecholic polychlorinated biphenyl metabolites in isolated rat hepatocytes

Polychlorinated biphenyl (PCB) and PCB metabolites are highly lipophilic and accumulate easily in the lipid bilayer and fat deposits of the body. The molecular cytotoxic mechanisms of these metabolites are still not understood. The aim of the present study was to compare the cytotoxicity and toxicological properties of six dihydroxylated metabolites using isolated rat hepatocytes. All of the metabolites were more cytotoxic than 4-chlorobiphenyl (4-ClBP) and less cytotoxic than phenyl hydroquinone (PHQ). The order of cytotoxic effectiveness of catecholic metabolites expressed as LC(50) (2h) was 3',4'-diCl-2,3-diOH-biphenyl>PHQ>4'-Cl-2,5-diOH-biphenyl, 4'-Cl-2,3-diOH-biphenyl>2',5'-diCl-3,4-diOH-biphenyl>2',3'-diCl-3,4-diOH-biphenyl>3',4'-diCl-3,4-diOH-biphenyl>4'Cl-3,4-diOH-biphenyl>4'-Cl-biphenyl; showing that the positions of hydroxyl and chlorine groups were important for their hepatotoxicity and that the two 2,3-diOH congeners were the most cytotoxic. Cytotoxicity for 3,4-diOH metabolites correlated with the number and position of chlorine atoms with the more chlorine atoms being more cytotoxic. The cytotoxic order of metabolites with two chlorine atoms being 2',5'>2',3'>3',4'. Borneol, an uridine diphosphate glucuronosyltransferases (UGT) inhibitor, increased the cytotoxicity of all tested metabolites; suggesting that glucuronidation was a major mechanism of elimination of these compounds. On the other hand entacapone, a catechol-O-methyl transferase (COMT) inhibitor, only increased the cytotoxicity of 3',4'-diCl-3,4-diOH-biphenyl, 3',4'-diCl-2,3-diOH-biphenyl and 4'-Cl-2,3-diOH-biphenyl. Hepatocyte GSH was depleted (oxidized and conjugated) by these metabolites before cytotoxicity ensued in a similar order of effectiveness to their cytotoxicity with PHQ being the most effective. Hepatocyte mitochondrial membrane potential also decreased before cytotoxicity ensued with a similar order of effectiveness as their cytotoxicity. These results suggest that catecholic cytotoxicity can be attributed to mitochondrial toxicity and oxidative stress. Semiquinone or benzoquinone species were also important in the cytotoxicity of catecholic metabolites.     

Lanosterol 14 alpha-demethylase (P45014DM): effects of P45014DM inhibitors on sterol biosynthesis downstream of lanosterol

Lanosterol 14 alpha-demethylase (P45014DM) is the cytochrome P450 enzyme complex responsible for an early step in cholesterol biosynthesis, namely the 14 alpha-demethylation of lanosterol. We have synthesized a novel series of steroidal substrate analogues, designed to be specific and potent inhibitors of P45014DM. We describe here the effects of these compounds on sterol biosynthesis downstream from lanosterol, focusing ultimately on their efficacy as inhibitors of cholesterol biosynthesis. Results using a radio-high performance liquid chromatography (HPLC) assay show that in rat liver microsomal preparations, with [24,25-3H]dihydrolanosterol as substrate, the compounds do indeed inhibit the biosynthesis of sterols downstream from lanosterol. A range of inhibitory potencies was observed, and the key enzyme being inhibited was believed to be P45014DM. Inhibitor efficacy was readily correlated with non-metabolized [24,25-3H]dihydrolanosterol, formation of 4,4-dimethyl-cholest-8-en-3 beta-ol, and formation of lathosterol, a sterol believed to be an excellent indicator of whole body cholesterol biosynthesis in humans.     

Inhibitory effect of quercetin metabolites and their related derivatives on copper ion-induced lipid peroxidation in human low-density lipoprotein

To determine the antioxidant activity of dietary quercetin (3,3',4', 5,7-pentahydroxyflavone) in the blood circulation, we measured the inhibitory effect of quercetin metabolites and their related derivatives on copper ion-induced lipid peroxidation of human low-density lipoprotein (LDL). Conjugated quercetin metabolites were prepared from the plasma of rat 1 h after oral administration of quercetin aglycone (40 micromol/rat). The rate of cholesteryl ester hydroperoxide (CE-OOH) accumulation and the rate of alpha-tocopherol consumption in mixtures of LDL solution (0.4 mg/ml) with equal volumes of this preparation were slower than the rates in mixtures of LDL with preparations from control rats. The concentrations of CE-OOH after 2 h oxidation in the mixtures of LDL with preparations of conjugated quercetin metabolites were significantly lower than those in the control preparation. It is therefore confirmed that conjugated quercetin metabolites have an inhibitory effect on copper ion-induced lipid peroxidation in human LDL. Quercetin 7-O-beta-glucopyranoside (Q7G) and rhamnetin (3,3',4', 5-tetrahydroxy-7-methoxyflavone) exerted strong inhibition and their effect continued even after complete consumption, similarly to quercetin aglycone. The effect of quercetin 3-O-beta-glucopyranoside (Q3G) did not continue after its complete consumption, indicating that the antioxidant mechanism of quercetin conjugates lacking a free hydroxyl group at the 3-position is different from that of the other quercetin conjugates. The result that 4'-O-beta-glucopyranoside (Q4'G) and isorhamnetin (3,4',5, 7-tetrahydroxy-3'-methoxyflavone) showed little inhibition implies that introduction of a conjugate group to the position of the dihydroxyl group in the B ring markedly decreases the inhibitory effect. The results of azo radical-induced lipid peroxidation of LDL and the measurement of free radical scavenging capacity using stable free radical, 1,1,-diphenyl-2-picrylhydrazyl, demonstrated that the o-dihydroxyl structure in the B ring is required to exert maximum free radical scavenging activity. It is therefore likely that conjugation occurs at least partly in positions other than the B ring during the process of metabolic conversion so that the inhibitory effect of dietary quercetin is retained in blood plasma after absorption.     

Role of quercetin and its in vivo metabolites in protecting H9c2 cells against oxidative stress

The aim of this study was to investigate the potential of quercetin and two of its "in vivo" metabolites, 3'-O-methyl quercetin and 4'-O-methyl quercetin, to protect H9c2 cardiomyoblasts against H(2)O(2)-induced oxidative stress. As limited data are available regarding the potential uptake and cellular effects of quercetin and its metabolites in cardiac cells, we have evaluated the cellular association/uptake of the three compounds and their involvement in the modulation of two pro-survival signalling pathways: ERK1/2 signalling cascade and PI3K/Akt pathway. The three flavonols associated with cells to differing extents. Quercetin and its two O-methylated metabolites were able to reduce intracellular ROS production but only quercetin was able to counteract H(2)O(2) cell damage, as measured by MTT reduction assay, caspase-3 activity and DNA fragmentation assays. Furthermore, only quercetin was observed to modulate pro-survival signalling through ERK1/2 and PI3K/Akt pathway. In conclusion we have demonstrated that quercetin, but not its O-methylated metabolites, exerts protective effects against H(2)O(2) cardiotoxicity and that the mechanism of its action involves the modulation of PI3K/Akt and ERK1/2 signalling pathways.     

Antioxidative properties of functional polyphenols and their metabolites assessed by an ORAC assay

We compared the antioxidative activities of polyphenol metabolites with those of intact functional polyphenols by an assay of the oxygen radical absorbance capacity (ORAC). The metabolites of ellagitannin geraniin, chlorogenic acid, and (-)-epigallocatechin gallate displayed more potent antioxidative activity than their respective original compounds. Our findings suggest that these metabolites may play important roles as biological antioxidants after their consumption.     

Attenuation of lipid peroxidation and hyperlipidemia by quercetin glucoside in the aorta of high cholesterol-fed rabbit

Antioxidative activity of dietary flavonoids is suggested to be, at least partly, responsible for a wide variety of their biological effects relating to anti-atherosclerosis. However, it is not known whether dietary flavonoids reach to the target site and act as antioxidants. In this study, we tried to evaluate the antioxidative effect of quercetin 3-O-beta-D-glucoside (Q3G), a typical flavonoid present in vegetables, in rabbit aorta. New Zealand White rabbits were fed a control diet (control group), 2.0% cholesterol diet (HC group) and 2.0% cholesterol plus 0.1% Q3G (HC + Q3G group) for one month. The amounts of total cholesterol, triacylglycerol and total fatty acids in both the plasma and aorta were significantly lower in the HC + Q3G group as compared with the HC group. Quercetin was detected in the aorta of the HC + Q3G group after enzymatic deconjugation, indicating that quercetin accumulated as conjugated metabolites in the aorta. The contents of TBA-reacting substances (TBARS) and cholesteryl ester hydroperoxides (CEOOH) in the aorta of the HC + Q3G group were significantly lower than those in the HC group. The aorta of HC + Q3G group was more resistant than that of HC group in copper ion-induced lipid peroxidation ex vivo. HC + Q3G group accumulated a higher amount of vitamin E per total cholesterol than HC group in the aorta. These results strongly suggest that quercetin glucosides accumulate in the aorta as their metabolites and attenuate lipid peroxidation occurring in the aorta, along with the attenuation of hyperlipidemia.     

Modulation of the mutagenic effects of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) in bacteria with rat-liver 9000 x g supernatant or monolayers of rat hepatocytes as an activation system

An in vitro protocol was designed to separate the process of metabolic activation from the mutational events. Cultured rat hepatocytes were first incubated with the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) or 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ). After the incubation period the medium was removed and further incubated with Salmonella typhimurium TA98. A high direct mutagenic activity of the culture medium was then measured. The half-lives of the mutagenic metabolites formed from IQ and MeIQ were in the order of 45 min. The presence of the cytochrome P450 inhibitors alpha-naphthoflavone and metyrapone during the pre-incubation period reduced the accumulation of mutagenic metabolites. No effects of ascorbate on the mutagenic effects of IQ and MeIQ were seen. (+)-Catechin, another antioxidant and free-radical scavenger, markedly enhanced the number of IQ/MeIQ-induced revertants when added to the hepatocytes. In contrast, (+)-catechin clearly decreased the number of revertants when 9000 X g supernatant from rat liver (S9) was used as an activation system. No marked effect of pentachlorophenol, an inhibitor of hepatocyte sulfation and bacterial O-acetylation, was seen using hepatocytes as an activation system, while the mutagenic activity of both IQ and MeIQ was reduced by 90% in the S9/Salmonella system. The addition of an inhibitor of glucuronidation, galactosamine, or the nucleophile glutathione caused no or only minor decreases in the genotoxic effects of the IQ compounds. With both S9 and hepatocytes as activation systems the relative mutagenic effects observed in the S. typhimurium strains TA98 and TA98 NR were in the same order of magnitude, while a large decrease was seen with TA98/1,8-DNP6. The results show that this in vitro test protocol may be useful as a tool to study mechanisms involved in the formation of mutagenic metabolites.     

Analysis of amphetamine-type stimulants and their metabolites in plasma, urine and bile by liquid chromatography with a strong cation-exchange column-tandem mass spectrometry

The aim of this work was to develop and validate a method for analysing amphetamine-type stimulants (ATSs) and their metabolites in plasma, urine and bile by liquid chromatography with a strong cation-exchange column-tandem mass spectrometry, and to apply it to the pharmacokinetic study of ATSs. 3,4-Methylenedioxymethamphetamine, methamphetamine, ketamine and their main metabolites, 4-hydroxy-3-methoxymethamphetamine, 3,4-methylenedioxyamphetamine, p-hydroxymethamphetamine, amphetamine and norketamine, were simultaneously quantified by the new method (50-5000 ng/ml). The coefficients of variation and the percent deviations for the eight compounds were in the range of 0.2 to 5.3% and -9.4 to +12.8%, respectively. The recoveries were over 90% in all biological samples tested. This method was effective for the separation and the identification of ATSs and their main metabolites having amine moieties in plasma, urine and bile, and was applicable to pharmacokinetic analysis of methamphetamine, ketamine and their main metabolites in biological samples. This analytical method should be useful for the pharmacokinetic analysis of ATSs.     

Regulation of sterol 27-hydroxylase and an alternative pathway of bile acid biosynthesis in primary cultures of rat hepatocytes

In man, hepatic mitochondrial sterol 27-hydroxylase and microsomal cholesterol 7-hydroxylase initiate distinct pathways of bile acid biosynthesis from cholesterol, the “acidic” and “neutral” pathways, respectively. A similar acidic pathway in the rat has been hypothesized, but its quantitative importance and ability to be regulated at the level of sterol 27-hydroxylase are uncertain. In this study, we explored the molecular regulation of sterol 27-hydroxylase and the acidic pathway of bile acid biosynthesis in primary cultures of adult rat hepatocytes. mRNA and protein turnover rates were approximately 10-fold slower for sterol 27-hydroxylase than for cholesterol 7-hydroxylase. Sterol 27-hydroxylase mRNA was not spontaneously expressed in culture. The sole requirement for preserving sterol 27-hydroxylase mRNA at the level of freshly isolated hepatocytes (0 h) after 72 h was the addition of dexamethasone (0.1 μM; > 7-fold induction). Sterol 27-hydroxylase mRNA, mass and specific activity were not affected by thyroxine (1.0 μM), dibutyryl-cAMP (50 μM), nor squalestatin 1 (150 nM-1.0 μM), an inhibitor of cholesterol biosynthesis. Taurocholate (50 μM), however, repressed sterol 27-hydroxylase mRNA levels by 55%. Sterol 27-hydroxylase specific activity in isolated mitochondria was increased > 10-fold by the addition of 2-hydroxypropyl-β-cyclodextrin. Under culture conditions designed to maximally repress cholesterol 7-hydroxylase and bile acid synthesis from the neutral pathway but maintain sterol 27-hydroxylase mRNA and activity near 0 h levels, bile acid synthesis from [¹⁴C]cholesterol remained relatively high and consisted of β-muricholate, the product of chenodeoxycholate in the rat. We conclude that rat liver harbors a quantitatively important alternative pathway of bile acid biosynthesis and that its initiating enzyme, sterol 27-hydroxylase, may be slowly regulated by glucocorticoids and bile acids.     

Metabolic conversion of dietary flavonoids alters their anti-inflammatory and antioxidant properties

The notion that dietary flavonoids exert beneficial health effects in humans is often based on in vitro studies using the glycoside or aglycone forms of these flavonoids. However, flavonoids are extensively metabolized in humans, resulting in the formation of glucuronide, methyl, and sulfate derivatives, which may have different properties than their parent compounds. The goal of this study was to investigate whether different chemical modifications of the same flavonoid molecule affect its biological and antioxidant activities. Hence, we studied the anti-inflammatory effects of several major human metabolites of quercetin and (-)-epigallocatechin-3-O-gallate (EGCG) by assessing their inhibitory effects on tumor necrosis factor α (TNFα)-induced protein expression of cellular adhesion molecules in human aortic endothelial cells (HAEC). HAEC were incubated with 1-30 μM quercetin, 3'- or 4'-O-methyl-quercetin, quercetin-3-O-glucuronide, and quercetin-3'-O-sulfate or 20-100 μM EGCG, 4'-O-methyl-EGCG, and 4',4'-di-O-methyl-EGCG, prior to coincubation with 100 U/ml of TNFα. 3'-O-Methyl-quercetin, 4'-O-methyl-quercetin, and their parent aglycone compound, quercetin, all effectively inhibited expression of intercellular adhesion molecule-1 (ICAM-1) with IC(50) values (concentration required for 50% inhibition) of 8.0, 5.0, and 4.4 μM, respectively; E-selectin expression was suppressed to a somewhat lesser but still significant degree by all three compounds, whereas vascular cell adhesion molecule-1 (VCAM-1) was not affected. In contrast, quercetin-3-O-glucuronide (20-100 μM), quercetin-3'-O-sulfate (10-30 μM), and phenolic acid metabolites of quercetin (20-100 μM) did not inhibit adhesion molecule expression. 4',4'-Di-O-methyl-EGCG selectively inhibited ICAM-1 expression with an IC(50) value of 94 μM, whereas EGCG (20-60 μM) and 4'-O-methyl-EGCG (20-100 μM) had no effect. The inhibitory effects of 3'-O-methyl-quercetin and 4',4'-di-O-methyl-EGCG on adhesion molecule expression were not related either to inhibition of NF-κB activation or to their antioxidant reducing capacity. Our data indicate that flavonoid metabolites have different biological and antioxidant properties than their parent compounds, and suggest that data from in vitro studies using nonmetabolites of flavonoids are of limited relevance in vivo.     

Assay systems for screening food and natural substances that have anti-hyperuricemic activity: uric acid production in cultured hepatocytes and purine bodies-induced hyperuricemic model mice

Hyperuricemia is characterized by the high uric acid (UA) level in serum (or plasma) and has been considered to be an important risk factor for gout. In the present study, we have attempted to construct an assay system for UA production in vitro employing cultured AML12 hepatocytes. UA levels in balanced salt solution (BSS) in the presence of UA precursor nucleosides, adenosine, inosine, guanosine and xanthine, at 12.5, 25, and 100 µM were significantly higher than BSS alone and their effects were dose-dependent, while all the UA precursors did not significantly increase intracellular UA levels. Hence, UA levels in BSS were thereafter adopted as an index of UA production. UA production from nucleosides was significantly higher than that from nucleotides (GMP, IMP and AMP). UA production from guanosine and inosine in combination (GI mixture) as well as nucleosides increased time-dependently and almost linearly up to 2 h. Selecting GI mixture, effects of allopurinol, a widely used anti-hyperuricemic agent, and quercetin, a well-known polyphenol in onion and strawberry, on UA production were examined. Both allopurinol and quercetin dose-dependently (0.1, 0.3 and 1 μM for allopurinol and 10, 30, and 100 μM for quercetin) and significantly reduced UA production in the hepatocytes. They also significantly reduced hyperuricemia induced by intraperitoneal injection of UA precursor purine bodies to mice at a single oral dose of 10 (allopurinol) or 200 (quercetin) mg/kg body weight. This assay system for UA production in cultured hepatocytes is considered to be useful to search for novel anti-hyperuricemic compounds in foods and natural resources with possibility to have human health benefits.     

On the Significance of Tyrosine for the Synthesis and Catabolism of Dopamine in Rat Brain: Evaluation by HPLC with Electrochemical Detection

Abstract: A chemical assay of tyrosine (Tyr) in nervous tissue is described. The method is based on a rapidly performed isolation of Tyr on small Sephadex G 10 columns, followed by reverse-phase HPLC in conjunction with amperometric detection. The method permitted the additional quantification of 3,4-dihydroxyphenylalanine, dopamine (DA), and its acidic metabolites. The method was applied to a study of the effects of γ-butyrolactone, haloperidol, haloperidol in combination with amfonelic acid, morphine, NSD 1015, and tyrosine methylester on the concentration of Tyr in the striatum, frontal cortex, hypothalamus, and cerebellum of rat brain. The effect of tyrosine methylester on DA and its acidic metabolites was investigated in the striatum and frontal cortex. Morphine and NSD 1015 were found to increase Tyr levels. γ-Butyrolactone, haloperidol, and haloperidol combined with amfonelic acid decreased the Tyr content in a manner related to their stimulatory effect on DA biosynthesis. These effects were restricted to DA-rich brain areas. It was concluded that during conditions of increased DA biosynthesis, the Tyr pool still possesses a considerable reserve capacity. The results bring into question the concept that brain Tyr is an important additional factor controlling catechol synthesis during increased tyrosine hydroxylase activity.     

Anti-oxidative compounds in barley tea

Five phenolic compounds, p-hydroxyacetophenone, 5,7-dihydroxychromone, naringenin, quercetin, and iso-americanol A, were found first time in the barley tea, together with the known compounds, p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, p-hydroxybenzoic acid, vanillic acid, and p-coumaric acid. The anti-oxidative properties were evaluated by measuring their peroxynitrite-scavenging activities. Among these compounds, 3,4-dihydroxybenzaldehyde, p-coumaric acid, quercetin, and isoamericanol A showed stronger activities than that of BHT (butylated hydroxytoluene) at 400 microM.