Studies on flavonoid metabolism. Biliary and urinary excretion of metabolites of (+)-[U-14C]catechin

1. The biliary and urinary excretion of (+)-[U-(14)C]catechin was studied in normal male rats after a single injection of the flavonoid. 2. In rats large amounts of radioactivity (33.6-44.3% of the dose in 24h) were excreted in the bile as two glucuronide conjugates [one of which was a (+)-catechin conjugate] and three other unconjugated metabolites. 3. Excretion of radioactivity in the urine when the bile duct was not cannulated amounted to 44.5% of the dose. 4. In both the urine and bile the new metabolites showed maximum excretion in the (1/2)-1(1/2)h after intravenous injection of [(14)C]catechin. 5. The metabolites m-hydroxyphenylpropionic acid, p-hydroxyphenylpropionic acid, delta-(3-hydroxyphenyl)-gamma-valerolactone and delta-(3,4-dihydroxyphenyl)-gamma-valerolactione originate from the action of the intestinal micro-organisms on the biliary-excreted metabolites of (+)-catechin. These phenolic acid and lactone metabolites are then reabsorped and excreted in the urine. 6. It is proposed that, depending on the route of administration of (+)-catechin, there exists an alternative pathway, involving biliary excretion, for the metabolism of (+)-catechin.     

Studies on flavonoid metabolism. Metabolism of (+)-catechin in the guinea pig

1. Administration of (+)-catechin to the guinea pig gives rise to a number of phenolic acids and lactones, which have been identified by chromatographic and spectrophotometric methods. The major phenolic acid metabolite is m-hydroxybenzoic acid and the major lactone metabolite is delta-(3-hydroxyphenyl)-gamma-valerolactone. 2. The phenolic acid and lactone metabolites are excreted in both free and conjugated forms, including their glucuronides and to a lesser degree their ethereal sulphates. 3. Administration of certain of the metabolites isolated has permitted certain sequential relationships of these intermediates to be established. 4. Degradation of (+)-catechin in the guinea pig is effected at least in part by the gut microflora and is suppressed by aureomycin plus phthaloyl-sulphathiazole.     

Protective mechanisms of pycnogenol in ethanol-insulted cerebellar granule cells

Pycnogenol (PYC), a patented combination of bioflavonoids extracted from the bark of French maritime pine (Pinus maritima), inhibits apoptosis and necrosis of developing neurons exposed acutely to ethanol (EtOH). The present study shows that the protective mechanisms of PYC in EtOH-exposed postnatal day 9 cerebellar granule cells (P9 CGCs) include (1) reduction of reactive oxygen species (ROS) production; (2) counteraction of suppressed copper/zinc superoxide dismutase (Cu/Zn SOD) and glutathione peroxidase/reductase (GSH-Px/GSSG-R) system activities; (3) upregulation of Cu/Zn SOD protein expression; (4) mitigation of the EtOH-mediated exacerbation of catalase (CAT) activity; and, (5) specific binding and inhibition of active caspase-3. These results indicate that the mechanisms by which PYC antagonizes EtOH-induced oxidative stress include oxidant scavenging and modulation of endogenous, cellular proteins. Using findings from the present and previous studies, a model delineating the mechanisms of EtOH effects on the system of antioxidant enzymes in developing CGCs is presented.     

Facilitated cellular uptake and suppression of inducible nitric oxide synthase by a metabolite of maritime pine bark extract (Pycnogenol)

Many natural products exhibit anti-inflammatory activity by suppressing excessive nitric oxide (NO) production by inducible NO synthase (iNOS). The maritime pine bark extract Pycnogenol has been formerly shown to decrease nitrite generation, taken as an index for NO, but so far it was not clear which constituent of the complex flavonoid mixture mediated this effect. The purpose of this study was to elucidate whether the in vivo generated Pycnogenol metabolite M1 (δ-(3,4-dihydroxyphenyl)-γ-valerolactone) displayed any activity in the context of induction of iNOS expression and excessive NO production. For the first time we show that M1 inhibited nitrite production (IC(50) 1.3 μg/ml, 95% CI 0.96-1.70) and iNOS expression (IC(50) 3.8 μg/ml, 95% CI 0.99-14.35) in a concentration-dependent fashion. This exemplifies bioactivation by metabolism because the M1 precursor molecule catechin is only weakly active. However, these effects required application of M1 in the low-micromolar range, which was not consistent with concentrations previously detected in human plasma samples after ingestion of maritime pine bark extract. Thus, we investigated a possible accumulation of M1 in cells and indeed observed high-capacity binding of this flavonoid metabolite to macrophages, monocytes, and endothelial cells. This binding was distinctly decreased in the presence of the influx inhibitor phloretin, suggesting the contribution of a facilitated M1 transport into cells. In fact, intracellular accumulation of M1 could explain why in vivo bioactivity can be observed with nanomolar plasma concentrations that typically fail to exhibit measurable activity in vitro.     

Activity of monomeric, dimeric, and trimeric flavonoids on NO production, TNF-alpha secretion, and NF-kappaB-dependent gene expression in RAW 264.7 macrophages

Flavonoids are potent antioxidants and have been associated with lowering the risk of cardiovascular diseases. In this study, the effect of flavonoids (monomers, dimers and a trimer) as well as French maritime pine bark extract, Pycnogenol, on NO production, tumor necrosis factor-alpha (TNF-alpha) secretion and nuclear factor (NF)-kappaB activity was compared. Monomers and dimers repressed NO production, TNF-alpha secretion and NF-kappaB-dependent gene expression induced by interferon gamma, whereas the trimeric procyanidin C2 and Pycnogenol enhanced these parameters. In addition, in unstimulated RAW 264.7 macrophages, both procyanidin C2 and Pycnogenol increased TNF-alpha secretion in a concentration- and time-dependent manner. These results demonstrate that procyanidins act as modulators of the immune response in macrophages.     

Single and multiple dose pharmacokinetics of maritime pine bark extract (Pycnogenol) after oral administration to healthy volunteers

Background

Since plant extracts are increasingly used as phytotherapeutics or dietary supplements information on bioavailability, bioefficacy and safety are warranted. We elucidated the plasma kinetics of genuine extract components and metabolites after single and multiple ingestion of the standardized maritime pine bark extract Pycnogenol (USP quality) by human volunteers.

Methods

Eleven volunteers received a single dose of 300 mg pine bark extract, five volunteers ingested 200 mg daily for five days to reach steady state concentrations. Plasma samples were obtained before and at defined time points after intake of the extract. Samples were analyzed by HPLC with ion-pair reagents and simultaneous UV and electrochemical detection.

Results

We quantified total plasma concentrations of catechin, caffeic acid, ferulic acid, taxifolin and the metabolite M1 (δ-(3,4-dihydroxy-phenyl)-γ-valerolactone). Additionally, we describe plasma time courses and steady state appearance of ten so far unknown compounds, U1 to U10. After single ingestion, compounds derived from the extract were rapidly absorbed and the majority of them were detectable over whole experimental period of 14 h. The analysis of steady state plasma samples revealed significant phase II metabolism.

Conclusion

We present the first systematic pharmacokinetic analysis of compounds derived from maritime pine bark extract. Beyond the known constituents and metabolites we uncovered the plasma time courses of ten unknown compounds. In concert with our previous detection of anti-inflammatory bioefficacy of these plasma samples ex vivo we suggest that constituents and metabolites of Pycnogenol bear potential for disclosure of novel active principles.     

PYCNOGENOL in chronic venous insufficiency.

The aim of out study was to investigate the efficacy of Pycnogenol - a French maritime pine bark extract - in the treatment of chronic venous insufficiency (CVI). The study consisted of a double-blind phase - in which 20 patients were recruited and randomly treated with placebo or Pycnogenol (100 mg 2 3/day for 2 months) - and an open phase - in which other 20 patients were treated with Pycnogenol at the same dose schedule. In total, 40 patients were enrolled; 30 of them were treated with Pycnogenol and 10 with placebo. Pycnogenol significantly improved the legs' heaviness and subcutaneous edema; the venous pressure was also significantly reduced by the Pycnogenol treatment, thus adding further clinical evidence to its therapeutic efficacy in patients with CVI. Pycnogenol was effective, probably by either stabilizing the collagenous subendothelial basal membrane or scavenging the free radicals, or by a combination of these activities. Clinically, capillary leakage, perivascular inflammation and subcutaneous edema were all reduced. The safety of use of Pycnogenol is demonstrated by the lack of side effects or changes in blood biochemistry and hematologic parameters. Pycnogenol can be therefore recommended both for prevention and treatment of CVI and related veno-capillary disturbances.     

Synthesis and radical-scavenging activity of a dimethyl catechin analogue

Catechin analogue 1 with methyl substituents ortho to the catechol hydroxyl groups was synthesized to improve the antioxidant ability of (+)-catechin. The synthetic scheme involved a solid acid catalyzed Friedel–Crafts coupling of a cinnamyl alcohol derivative to 3,5-dibenzyloxyphenol followed by hydroxylation and then cyclization through an intermediate orthoester. The antioxidative radical scavenging activity of 1 against galvinoxyl radical, an oxyl radical, was found to be 28-fold more potent than (+)-catechin.     

Synthesis and antioxidant activity of a procyanidin B3 analogue

Proanthocyanidin, an oligomer of catechin, is a natural antioxidant and a potent inhibitor of lectin-like oxidized LDL receptor-1, which is involved in the pathogenesis of arteriosclerosis. We synthesized proanthocyanidin analogue 1, in which the geometry of one catechin molecule in procyanidin B3, a dimer of (+)-catechin, is constrained to be planar. The antioxidant activities of the compounds were evaluated in terms of their capacities to scavenge galvinoxyl radicals, and results demonstrate that while procyanidin was 3.8 times more potent than (+)-catechin, the radical scavenging activity of proanthocyanidin analogue 1 was further increased to 1.9 times that of procyanidin B3. This newly designed proanthocyanidin analogue 1 may be a promising lead compound for the treatment of arteriosclerosis and related cerebrovascular diseases.     

Pycnogenol, French maritime pine bark extract, improves endothelial function of hypertensive patients

A placebo-controlled, double-blind, parallel group study was performed with 58 patients to investigate effects of French maritime pine bark extract, Pycnogenol, on patients with hypertension. Supplementation of the patients with 100 mg Pycnogenol over a period of 12 weeks helped to reduce the dose of the calcium antagonist nifedipine in a statistically significant manner. The intake of Pycnogenol decreased endothelin-1 concentrations significantly compared to placebo while concentrations of 6-keto prostaglandin F1a in plasma were significantly higher compared to placebo. Values for nitric oxide (NO) in plasma increased in both groups, but the differences were not significant. Angiotensin II concentrations in plasma were lowered in the placebo group to a larger extent than in the Pycnogenol group. Heart rate, electrolytes and blood urea nitrogen were not changed during treatment in both groups of patients. Unwanted effects observed in both groups were of mild and transient nature, such as gastrointestinal problems, vertigo, headache and nausea. Differences in rate of side effects were not statistically significant between the two groups. Study results support a supplementation with Pycnogenol for mildly hypertensive patients.     

Antidiabetic effect of Pycnogenol French maritime pine bark extract in patients with diabetes type II

A double-blind, placebo-controlled, randomized, multi-center study was performed with 77 diabetes type II patients to investigate anti-diabetic effects of the French maritime pine bark extract, Pynogenol. Supplementation with 100 mg Pycnogenol for 12 weeks, during which a standard anti-diabetic treatment was continued, significantly lowered plasma glucose levels as compared to placebo. HbA1(c) was also lowered; however, the difference as compared to placebo was statistically significant only for the first month. In the Pycnogenol-group endothelin-1 was significantly decreased, while 6-ketoprostaglandin F(1a) in plasma was elevated compared to placebo. Nitric oxide levels in plasma increased during treatment in both groups, but, differences did not reach statistical significance. Pycnogenol was well-tolerated with ECG, electrolytes, creatinine and blood urea nitrogen remaining unchanged in both groups. Mild and transient unwanted effects were reported for both groups without significant differences. Supplementation of Pycnogenol to conventional diabetes treatment lowers glucose levels and improves endothelial function.     

Matrix metalloproteinase inhibition by green tea catechins

We have investigated the effects of different biologically active components from natural products, including green tea polyphenols (GTP), resveratrol, genistein and organosulfur compounds from garlic, on matrix metalloproteinase (MMP)-2, MMP-9 and MMP-12 activities. GTP caused the strongest inhibition of the three enzymes, as measured by fluorescence assays using gelatin or elastin as substrates. The inhibition of MMP-2 and MMP-9 caused by GTP was confirmed by gelatin zymography and was observed for MMPs associated with both various rat tissues and human brain tumors (glioblastoma and pituitary tumors). The activities of MMPs were also measured in the presence of various catechins isolated from green tea including (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate(ECG), (-)-epigallocatechin (EGC), (-)-epicatechin (EC) and (+)-catechin (C). The most potent inhibitors of these activities, as measured by fluorescence and by gelatin or casein zymography, were EGCG and ECG. GTP and the different catechins had no effect on pancreatic elastase, suggesting that the effects of these molecules on MMP activities are specific. Furthermore, in vitro activation of proMMP-2 secreted from the glioblastomas cell line U-87 by the lectin concanavalin A was completely inhibited by GTP and specifically by EGCG. These results indicate that catechins from green tea inhibit MMP activities and proMMP-2 activation.     

Antioxidant activity and biologic properties of a procyanidin-rich extract from pine (Pinus maritima) bark, pycnogenol.

There is growing interest in the biologic activities of plant extracts such as that obtained from the bark of the French maritime pine Pinus maritima, Pycnogenol. Pycnogenol (PYC) is a standardized extract composed of a mixture of flavonoids, mainly procyandins and phenolic acids. Studies indicate that PYC components are highly bioavailable. Uniquely PYC displays greater biologic effects as a mixture than its purified components do individually indicating that the components interact synergistically. PYC has been reported to have cardiovascular benefits, such as a vasorelaxant activity, angiotensin-converting enzyme (ACE) inhibiting activity, and the ability to enhance the microcirculation by increasing capillary permeability. Investigations of the cellular mechanisms of these therapeutic effects have demonstrated that PYC has strong free radical-scavenging activity against reactive oxygen and nitrogen species. The oligomeric components of PYC contribute significantly to the ESR free radical signal. PYC also participates in the cellular antioxidant network as indicated by its ability to regenerate the ascorbyl radical and to protect endogenous vitamin E and glutathione from oxidative stress. PYC modulates NO metabolism in activated macrophages by quenching the NO radical and inhibiting both iNOS mRNA expression and iNOS activity. The spectrum of different effects of NO in the circulation and the nervous system suggest the potential applications of PYC in immune and circulatory disorders as well as in neurodegenerative disease. PYC can bind to proteins, altering their structure and thereby modulating the activity of key enzymes and proteins involved in metabolic pathways. PYC effects redox-sensitive signal transduction pathways and alters gene expression. Aspects of PYC's activity are presented and discussed together with possible future implications and directions in the field of flavonoid research.     

The combined effect of pycnogenol with ascorbic acid and trolox on the oxidation of lipids and proteins

Pycnogenol (PYC), a procyanidin-rich extract of French maritime pine bark (Pinus pinaster) has strong antioxidant potential and promotes cellular health. The aim of this study was to investigate a possible cooperation of natural antioxidant PYC with synthetic antioxidants ascorbic acid and trolox in the model system of lipid peroxidation determined as conjugated dienes formation in liposomes and on the oxidation of proteins (in BSA and plasma proteins) determined as protein carbonyls. The present study shows that PYC and trolox significantly increased inhibition of lipid peroxidation initiated by copper acetate and tert-butylhydroperoxide in concentration and time dependence compared with untreated unilamellar liposomes. PYC and trolox added simultaneously to the oxidized liposomes exerted an additive preventive effect. PYC s inhibitory effect on formation of carbonyl compounds in BSA and plasma proteins, oxidized by two oxidative systems--H2O2/FeSO4 and HOCl, were studied in co-operation with other synthetic antioxidants--ascorbic acid and trolox. We found the synergistic or additive effect of PYC with mentioned antioxidants.     

Pycnogenol and vitamin E inhibit ethanol-induced apoptosis in rat cerebellar granule cells

Pycnogenol (PYC), a patented combination of bioflavonoids extracted from the bark of French maritime pine (Pinus maritima), scavenges free radicals and promotes cellular health. The protective capacity of PYC against ethanol toxicity of neurons has not previously been explored. The present study demonstrates that in postnatal day 9 (P9) rat cerebellar granule cells the antioxidants vitamin E (VE) and PYC (1) dose dependently block cell death following 400, 800, and 1600 mg/dL ethanol exposure (2) inhibit the ethanol-induced activation of caspase-3 in the same model system; and (3) reduce neuronal membrane disruption as assayed by phosphatidylserine translocation to the cell surface. These results suggest that both PYC and VE have the potential to act as therapeutic agents, antagonizing the induction of neuronal cell death by ethanol exposure.     

Polyphenols and red wine as antioxidants against peroxynitrite and other oxidants

The antioxidant capacity of polyphenols (+)-catechin, (-)-epicatechin and myricetin, and of different types of red wines (Cabernet Sauvignon, Malbec and blended wine) was evaluated by three assays. (a) NADH oxidation by peroxynitrite (ONOO-): the ONOO- scavenging activity was higher for myricetin (IC50=35 microM) than for (+)-catechin (IC50=275 microM) and (-)-epicatechin (IC50=313 microM). (b) Peroxynitrite initiated chemiluminescence in rat liver homogenate: (-)-epicatechin (IC50=7.0 microM) and (+)-catechin (IC50=13 microM) were more potent than myricetin (IC50=20 microM) in inhibiting the chemiluminescence signal. (c) Lucigenin chemiluminescence in aortic rings: (-)-epicatechin (IC50=15 microM) and (+)-catechin (IC50=18 microM) showed higher antioxidant capacity than myricetin (IC50=32 microM). All the assayed red wines were able to scavenge the oxidants and free radical species that generate the signal in each assay. Cabernet Sauvignon was the red wine with the highest antioxidant capacity in comparison with Malbec and blended wine. It is concluded that the use of sensitive biological systems (as the aortic ring chemiluminescence) provides important information in addition to the results from chemical (NADH oxidation by peroxynitrite) and biochemical (homogenate chemiluminescence) assays and offers advances in the physiological role of polyphenols.     

Bioflavonoid effects on the mitochondrial respiratory electron transport chain and cytochrome c redox state

The polyphenolic structure common to flavonoids enables them to donate electrons and exert antioxidant activity. Since the mitochondrial electron transport chain consists of a series of redox intermediates, the effect of flavonoids in a complex mixture of polyphenols, as well as related pure flavonoids, was evaluated on the rat liver mitochondrial electron transport chain. A French maritime pine bark extract (PBE), a complex mixture of polyphenols and related pure flavonoids, was able to reduce cytochrome c reversibly, possibly by donation of electrons to the iron of the heme group; the donated electrons can be utilized by cytochrome c oxidase. Among single flavonoids tested, (-)-epicatechin gallate had the greatest ability to reduce cytochrome c. In addition, PBE competitively inhibited electron chain activity in both whole mitochondria and submitochondrial particles. A 3.5-fold increase in the apparent Km value for succinate was calculated from reciprocal plots. Among the flavonoids tested, taxifolin and (-)-epicatechin gallate showed minor inhibitory effects, while (+/-)-catechin and (+)-epicatechin were ineffective. Activities of NADH-ubiquinone, succinate-ubiquinone, and ubiquinol-cytochrome c reductases were inhibited by low concentrations of PBE to a similar extent. However, inhibition of cytochrome c oxidase activity required 4-fold higher PBE concentrations. These results suggest that flavonoids reduce cytochrome c and that PBE inhibits electron transport chain activity mainly through NADH-ubiquinone, succinate-ubiquinone, and ubiquinol-cytochrome c reductases.     

Activity of matrix metalloproteinases in normal and transformed mouse fibroblasts exposed to antioxidants

The effects of two antioxidants on the activity of matrix metalloproteinases (MMP) secreted by normal (3T3) and transformed (3T3-SV40) mouse fibroblasts were examined. We compared the action of N-acetylcysteine (NAC) and alpha-lipoic acid (ALA) on two gelatinases, MMP-2 and MMP-9. Gel zymography demonstrated that activity of MMP-2 was higher in normal 3T3 cells, whereas, in transformed 3T3-SV40 cells, the MMP-9 activity was higher. NAC treatment for 2–6 h completely suppressed MMP-2 and MMP-9 activity in both cell lines. The inhibitory effect did not depend on NAC concentration within the range of 1–10 mM. ALA (1.2 mM) did not affect the cells very dramatically; it decreased the MMP-2 activity in both types of cells. MMP-9 activity in the presence of ALA was decreased in 3T3 cells and slightly increased in 3T3-SV40 cells. The activity of the membrane bound and intracellular MMP was not changed under the same conditions. In conclusion, the altered activity of MMP in the presence of antioxidant may influence the intracellular signaling and cell functions.     

Protective mechanisms of Pycnogenol® in ethanol‐insulted cerebellar granule cells

Pycnogenol® (PYC), a patented combination of bioflavonoids extracted from the bark of French maritime pine (Pinus maritima), inhibits apoptosis and necrosis of developing neurons exposed acutely to ethanol (EtOH). The present study shows that the protective mechanisms of PYC in EtOH‐exposed postnatal day 9 cerebellar granule cells (P9 CGCs) include (1) reduction of reactive oxygen species (ROS) production; (2) counteraction of suppressed copper/zinc superoxide dismutase (Cu/Zn SOD) and glutathione peroxidase/reductase (GSH‐Px/GSSG‐R) system activities; (3) upregulation of Cu/Zn SOD protein expression; (4) mitigation of the EtOH‐mediated exacerbation of catalase (CAT) activity; and, (5) specific binding and inhibition of active caspase‐3. These results indicate that the mechanisms by which PYC antagonizes EtOH‐induced oxidative stress include oxidant scavenging and modulation of endogenous, cellular proteins. Using findings from the present and previous studies, a model delineating the mechanisms of EtOH effects on the system of antioxidant enzymes in developing CGCs is presented. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2004     

Antioxidants and herbal extracts protect HT-4 neuronal cells against glutamate-induced cytotoxicity

Antioxidant therapy has been shown to be beneficial in neurological disorders including Alzheimer's disease and cerebral ischemia. Glutamate-induced cytotoxicity in HT-4 neuronal cells has been previously demonstrated to be due to oxidative stress caused by depletion of cellular glutathione (GSH). The present study demonstrates that a wide variety of antioxidants inhibit glutamate-induced cytotoxicity in HT-4 neuronal cells. Low concentrations of alpha-tocopherol and its analogs were highly effective in protecting neuronal cells against cytotoxicity. Purified flavonoids and herbal extracts of Gingko biloba (EGb 761) and French maritime pine bark (Pycnogenol) were also effective. We have previously shown that pro-glutathione agents can spare GSH and protect cells from glutamate insult in a C6 glial cell model. The protective effects of nonthiol-based antioxidants tested in the HT-4 line were not mediated via GSH level modulation. In contrast, protective effects of thiol-based pro-glutathione agents alpha-lipoic acid (LA) and N-acetyl cysteine (NAC) corresponded with a sparing effect on GSH levels in glutamate-treated HT-4 cells. Glutamate-induced cytotoxicity in HT-4 cells is a useful model system for testing compounds or mixtures for antioxidant activity.