Search

The aim of our study was to evaluate the effect of a standardized formulation of a polyphenolic extract of grapes (Leucoselect-Phytosome [LP]) on low-density lipoprotein (LDL) susceptibility to oxidation in a group of heavy smokers. A randomized, double-blind, crossover study was undertaken in 24 healthy male heavy smokers, aged > or = 50 years. Enrolled subjects were given 2 capsules twice daily for 4 weeks (phase 1). Each capsule contained 75 mg of a grape procyanidin extracts and soy-phosphatidlcholine or placebo consisiting of 75 mg lactose and soy-phosphatidlcholine. A wash out period of 3 weeks was then followed by 4 weeks of the opposite treatment (phase 2). Blood samples were taken at baseline and at the end of each phase and assayed for plasma lipids and LDL susceptibility to oxidation. Compliance was good, and no adverse effects were recorded. Subjects did not show significant modification of total cholesterol (TC), triglycerides (TG), high-density lipoprotein-cholesterol (HDL-C) and LDL-C during LP treatment. Among oxidative indices, thiobarbituric acid reactive substances (TBARS) concentration was significantly reduced in subjects taking LP (-14.7% +/- 21.1% v +5.0% +/- 18.1%, P <.01), and the lag phase prolonged (+15.4% +/- 24.4% v -0.1% +/- 16.0%, P <.05) compared with placebo and basal values. The antioxidant potential of grape seed extract polyphenols may prove effective in a model of oxidative stress (smoking); however more investigational data are needed before use in wider clinical settings.

Acrolein (ACR), the carbonyl toxin produced by lipid peroxidation, is significantly increased in Alzheimer's disease brain. Since ACR is one of the most reactive and neurotoxic aldehydes, and human brain contains both carnosine (beta-alanine-L-histidine) and homocarnosine (gamma-aminobutyryl-L-histidine), the aim of this work was first to evaluate the quenching ability of the two peptides towards ACR and then to characterize their reaction products by electrospray ionization tandem mass spectrometry (ESI-MS/MS; infusion experiments; positive-ion mode). The reaction progress of ACR with carnosine or homocarnosine was studied in phosphate buffer, by monitoring ACR consumption (by reverse-phase LC) and formation of the reaction products by ESI-MS/MS at different incubation times. N-Acetylcarnosine was used as reference compound to identify the sites of reaction. Both the dipeptides were able to quench ACR by almost 60% at 1 h and by more than 85% after 3 h incubation. Different reaction products between ACR and carnosine/homocarnosine were detected after 3 and 24 h, to indicate a complex reaction pathway involving sequential addition of 1, 2 and 3 moles of ACR/mole of the dipeptide to both the beta-alanine and histidine residues. The ESI mass spectra of ACR/carnosine reaction mixtures indicate formation of several molecular species, among which the predominant are: (a) the 14-membered macrocyclic derivatives, deriving from the formation of the iminic bond between the terminal amino group followed by intramolecular Michael addition of the C(3) of the ACR moiety to histidine; (b) the N(beta)-(3-formyl-3,4-dehydropiperidino) derivatives arising from the Michael addition of two acrolein molecules to the amino group of beta-alanine, followed by an aldol condensation and dehydration.The reaction of homocarnosine with ACR follows the same pathway, giving rise to the formation of homologous adducts. The results of this study shed light on the mechanism, until now never demonstrated, through which carnosine and homocarnosine detoxify the highly reactive aldehyde acrolein in a buffer system, and represent the starting point for further studies aimed at elucidating the biological role of these dipeptides in brain., (Copyright 2003 John Wiley & Sons, Ltd.)

The oxidation of endogenous antioxidant nutrients in human plasma was determined to examine their activities against free radicals generated in the aqueous and lipid compartments of plasma. Free radicals were induced at a constant rate in the aqueous compartment by the hydrophilic radical generator, 2,2'-azobis-(2-amidinopropane)dihydrochloride (AAPH; 10-20 mmol/L) and in the lipid compartment by the lipophilic radical generator, 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN; 1-2 mmol/L). The depletion of endogenous plasma antioxidant nutrients (lutein, cryptoxanthin, beta-carotene, lycopene, alpha-tocopherol, ascorbic acid, uric acid) was determined after incubation with either AAPH or MeO-AMVN at 37 degrees C using HPLC. The oxidation of the aqueous and lipid compartments of plasma was selectively monitored by a fluorimetric method using either the hydrophilic probe, 2',7'-dichlorodihydrofluorescein (DCFH) or the lipophilic probe, 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a, 4a-diaza-s-indacene-3-undecanoic acid (BODIPY 581/591). When plasma was incubated with AAPH, the rates of consumption of the antioxidant nutrients were as follows: ascorbic acid > alpha-tocopherol > uric acid > lycopene > lutein > cryptoxanthin > beta-carotene. When plasma was incubated with MeO-AMVN, alpha-tocopherol and carotenoids were depleted at similar rates and ahead of the major water-soluble antioxidants. Our study indicates that the antioxidant nutrients present in both the lipid and aqueous compartments can remove free radicals generated in plasma, and their activity depends on the localization of the attacking radical species.

When human plasma was exposed to the hydrophilic radical initiator, AAPH, (-)-epigallocatechin-(3)-gallate (EGCG) dose-dependently inhibited the aqueous compartment oxidation (IC(50)=0.72 microM) (monitored by DCFH oxidation) and spared the lipophilic antioxidants, alpha-tocopherol, and carotenoids, but not ascorbic acid. When radicals were selectively induced in the lipid compartment by the lipophilic radical initiator, MeO-AMVN, EGCG spared alpha-tocopherol, but not carotenoids and inhibited the lipid compartment oxidation (monitored by BODIPY 581/591) with a potency lower than that found in the aqueous compartment (IC(50)=4.37 microM). Our results indicate that EGCG, mainly localized in the aqueous compartment, effectively quenches aqueous radical species, thus limiting their diffusion into the lipid compartment and preventing lipid-soluble antioxidant depletion. Further, ESR experiments confirmed that EGCG recycled alpha-tocopherol through a H-transfer mechanism at the aqueous/lipid interface affording an additional protective mechanism to the lipid compartment of plasma.

Oxidation of polyunsaturated fatty acids containing phospholipids in tissue generates lipid hydroperoxides, which are further degraded to several products, among which unsaturated aldehydes such as 4-hydroxy-trans-2-nonenal (HNE) play an important role in mediating the pathological effects of oxidative stress. While the reaction of HNE with glutathione (GSH) is a well recognized pathway of detoxification in biological systems, no data are available on HNE interactions with carnosine, a dipeptide (beta-alanyl-L-histidine) present in high concentration in skeletal muscle. The aim of this work was to study the quenching ability of carnosine towards HNE and to characterize the reaction products by electrospray ionization tandem mass spectrometry (ESI-MS/MS), using GSH as a model peptide. GSH incubation with HNE in 1 mM phosphate buffer (pH 7.4) results in the complete disappearance of HNE within 1 h owing to the formation of a Michael adduct, S-(4-hydroxynonanal-3-yl)glutathione. The reaction of HNE with carnosine was studied in different molar ratios and monitored up to 24 h by high-performance liquid chromatography (HPLC) (HNE consumption), MS/MS (infusion) and liquid chromatography mass spectrometry (LC/MS) experiments. Carnosine, although less reactive than GSH, significantly quenched HNE (48.2 +/- 0.9% HNE consumption after 1 h; carnosine:HNE molar ratio 10 : 1). Two reaction products were identified: the Michael adduct, N-(4-hydroxynonanal-3-yl)carnosine involving the imidazolic nitrogen of histidine, and the imine adduct, involving the amino group of the beta-alanine residue. Definitive structure assignment was achieved by chemical reduction with NaBH(4) and multinuclear magnetic resonance experiments. To understand whether carnosine acts as a quencher of unsaturated aldehydes in biological matrices, rat skeletal muscle homogenate was incubated with HNE and the formation of conjugated adducts was determined by LC/MS analysis. Three main products were detected and identified as Michael adducts of HNE with GSH, carnosine and anserine (the N-methylated derivative of carnosine, present in high concentrations in rat muscle). The results indicate that beside GSH, histidine-containing dipeptides could be involved in the detoxification pathway of reactive aldehydes from lipid peroxidation generated in skeletal muscle during physical endurance., (Copyright 2002 John Wiley & Sons, Ltd.)

The aim of this study was to understand the mechanism of action through which carnosine (beta-alanyl-L-histidine) acts as a quencher of cytotoxic alpha,beta-unsaturated aldehydes, using 4-hydroxy-trans-2,3-nonenal (HNE) as a model aldehyde. In phosphate buffer solution (pH 7.4), carnosine was 10 times more active as an HNE quencher than L-histidine and N-acetyl-carnosine while beta-alanine was totally inactive; this indicates that the two constitutive amino acids act synergistically when incorporated as a dipeptide and that the beta-alanyl residue catalyzes the addition reaction of the histidine moiety to HNE. Two reaction products of carnosine were identified, in a pH-dependent equilibrium: (a) the Michael adduct, stabilized as a 5-member cyclic hemi-acetal and (b) an imine macrocyclic derivative. The adduction chemistry of carnosine to HNE thus appears to start with the formation of a reversible alpha,beta-unsaturated imine, followed by ring closure through an intra-molecular Michael addition. The biological role of carnosine as a quencher of alpha,beta-unsaturated aldehydes was verified by detecting carnosine-HNE reaction adducts in oxidized rat skeletal muscle homogenate.

Male rats were given the nitrooxybutyl ester of flurbiprofen HCT1026 (15 mg/Kg) by oral administration and plasma and brain levels of the parent drug and its potential metabolites (HCT1027 and flurbiprofen) were determined at different times post-administration by a validated HPLC method (UV-DAD detection; LOQ: 0.13 nmoles/ml and 0.3 nmoles/g respectively in plasma and brain tissue). Structural confirmation of the analytes was achieved by MS monitoring of their de-protonated (negative ion mode) or cationized/protonated (positive ion mode) molecular ions and of the relative fragment ions obtained by collision-induced dissociation (CID) experiments. The results indicate that flurbiprofen is the only metabolite found at measurable levels in both plasma and brain, while HCT1026 or its de-nitrated metabolite HCT1027 were always below the limit of detection at all the observation times. The same was observed after administration of the higher dose of HCT1026 (100 mg/Kg, i.p.). In orally-treated animals the time-course of flurbiprofen formation strictly parallels that of NOx (nitrite/nitrate) in plasma but not in brain, where the levels were always in the range of the controls. These data indicate that the NO molecule is not released from the parent drug within the brain.

The metabolism of a nitroderivative of acetylsalicylic acid, benzoic acid, 2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester (NCX4016), the lead compound of a new class of NO-releasing non steroidal-antiinflammatory drugs has been studied in vitro in rat liver subcellular fractions (S 9000xg, microsomes, cytosol). Samples were extracted with CH3CN (2 vol.) containing 1% H3PO4 (2 M), vortexed for 3 min and then centrifuged for 5 min at 5000 rpm. Supernatants were diluted with 0.02 M phosphoric acid and analysed by reverse-phase LC. Linearity of calibration for NCX4016 and metabolites was observed over the range 0.25-50 microg/ml with coefficients of determination greater than 0.9996. Extraction efficiency from spiked liver samples ranged from 85 to 95% for all the analytes. In the S 9000xg fraction, NCX4016 undergoes rapid metabolization, with the formation of salicylic acid (SA) and [3-(nitrooxymethyl)phenol] (HBN). HBN is then rapidly metabolised to 3-hydroxybenzylalcohol (HBA), and mainly to a new metabolic species, whose formation takes place specifically in the liver cell cytosol. LC-MS analysis (electrospray ionisation) of the cytosol extract in negative and positive-ion modes furnished deprotonated [M-H]- and protonated [M+H]+ molecular ions at m/z 412 and 414, respectively, accompanied by the typical clusters with sodium. MS/MS analysis in negative-ion mode, by selection and collision of the ion at m/z 412, gave a fragmentation pattern characterized by the ions at m/z 272 and 254, which allowed to assign the structure of 1-(glutathion-S-yl)methylene-3-hydroxy-benzene, a conjugated product between GSH and the benzyl carbon atom of HBN. In rat liver cytosol HBN is completely metabolised to this thioether adduct within 30 min incubation; the process is enzymatically mediated by GSH transferase and strictly dependent on GSH availability. The relevance of this new metabolic pathway in NCX4016 detoxification by rat liver is discussed.

The antioxidant/photoprotective potential of a standardized Krameria triandra (KT) root extract (15% neolignans) has been evaluated in different cell models, rat erythrocytes and human keratinocytes cell lines, exposed to chemical (cumene hydroperoxide, CuOOH) and physical (UVB radiation) free radical inducers. The extract was significantly more active (IC50 0.28 +/- 0.04 microg/ml) than the typical chain-breaking antioxidant alpha-tocopherol (IC50 = 6.37 +/- 0.41 microg/ml) in inhibiting the CuOOH-induced hemolysis in rat blood cells. The KT constituent 2-(2,4-dihydroxyphenyl)-5-(E)-propenylbenzofuran, was the most active (IC50 = 0.03 +/- 0.005 microg/ml), followed by eupomatenoid 6 (IC50 = 0.29 +/- 0.06 microg/ml) and conocarpan (IC50 = 0.77 +/- 0.08 microg/ml). The same order of potency was observed in red blood cells exposed to UVB irradiation in continuo, with IC50 values 0.78 +/- 0.08 microg/ml for KT extract, 0.18 +/- 0.02 microg/ml for 2-(2,4-dihydroxyphenyl)-5-(E)-propenylbenzofuran, 0.95 +/- 0.11 microg/ml for eupomatenoid 6, and 3.8 +/- 0.39 microg/ml for conocarpan. In cultured human keratinocytes exposed to UVB radiation (50 mJ/cm2), KT extract (2.5-20 microg/ml) significantly and dose-dependently restrained the loss in cell viability and the intracellular oxidative damage: glutathione (GSH) depletion and the rise in dichlorofluorescein (DCF), marker of peroxide accumulation, were suppressed by 20 microg/ml KT and in parallel cell morphology maintained. The cytoprotective effect of the extract was confirmed in a more severe model of cell damage: exposure of keratinocytes to higher UVB doses (300 mJ/cm2), which induce a 50% cell death. In keratinocyte cultures supplemented with 10 microg/ml, cell viability was almost completely preserved and more efficiently than with (-)-epigallocatechin 3-gallate and green tea. The results of this study indicate the potential use of Rhatany extracts, standardized in neolignans, as topical antioxidants/radical scavengers against skin photodamage.

Restenosis after percutaneous transluminal coronary angioplasty is caused by neointimal hyperplasia, which involves impairment of nitric oxide (NO)-dependent pathways, and may be further exacerbated by a concomitant aging process. We compared the effects of NO-releasing-aspirin (NCX-4016) and aspirin (ASA) on experimental restenosis in both adult and elderly rats. Moreover, to ascertain the efficacy of NCX-4016 during vascular aging, we fully characterized the release of bioactive NO by the drug. Sprague-Dawley rats aged 6 and 24 months were treated with NO releasing-aspirin (55 mg/kg) or ASA (30 mg/kg) for 7 days before and 21 days after standard carotid balloon injury. Histological examination and immunohistochemical double-staining were used to evaluate restenosis. Plasma nitrite and nitrate and S-nitrosothiols were determined by a chemiluminescence-based assay. Electron spin resonance was used for determining nitrosylhemoglobin. Treatment of aged rats with NCX-4016 was associated with increased bioactive NO, compared with ASA. NO aspirin, but not ASA, reduced experimental restenosis in old rats, an effect associated with reduced vascular smooth muscle cell proliferation. NCX-4016, but not ASA, was well tolerated and virtually devoid of gastric damage in either adult or old rats. Thus, impairment of NO-dependent mechanisms may be involved in the development of restenosis in old rats. We suggest that an NCX-4016 derivative could be an effective drug in reducing restenosis, especially in the presence of aging and/or gastrointestinal damage.

The lipophilic radical initiator (MeO-AMVN) and the fluorescent probe C11BODIPY581/591 (BODIPY) were used to measure the lipid compartment oxidizability of human plasma. Aqueous plasma oxidizability was initiated by the aqueous peroxyl radical generator, AAPH, and 2',7'-dichlorodihydrofluorescein (DCFH) was employed as the marker of the oxidative reaction. The distribution in aqueous and lipid compartments of the two radical initiators was determined by measuring the rate of consumption of the plasma hydrophilic and lipophilic endogenous antioxidants. In the presence of AAPH (20 mM), the order of consumption was: ascorbic acid > alpha-tocopherol > uric acid > beta-carotene, indicating a gradient of peroxyl radicals from the aqueous to the lipid phase. When MeO-AMVN was used (2mM), beta-carotene was consumed earlier than uric acid and almost at the same time as alpha-tocopherol, reflecting the diffusion and activation of MeO-AMVN in the lipophilic phase. The rate of BODIPY oxidation (increase in green fluorescence) significantly increased after the depletion of endogenous alpha-tocopherol and beta-carotene, whereas it was delayed for 180 min when AAPH was used instead of MeO-AMVN. The measurement of lipid oxidation in plasma was validated by adding to plasma the two lipophilic antioxidants, alpha-tocopherol and beta-carotene, whose inhibitory effects on BODIPY oxidation were dependent on the duration of the preincubation period and hence to their lipid diffusion. DCFH oxidation induced by AAPH only began after uric acid, the main hydrophilic plasma antioxidant, was consumed. In contrast, when MeO-AMVN was used, DCFH oxidation was delayed for 120 min, indicating its localization in the aqueous domain. In summary, the selective fluorescence method reported here is capable of distinguishing the lipophilic and hydrophilic components of the total antioxidant capacity of plasma.

The inhibitory properties of procyanidins (a standardized oligomeric catechin fraction) from Vitis vinifera L. seeds on the respiratory burst and on the release of granule components myeloperoxidase, beta-glucuronidase and elastase were studied in activated human neutrophils. Procyanidins strongly inhibit superoxide generation with an IC(50) of 7.2 microM, through a direct scavenging of superoxide and prevent the release from calcium ionophore activated neutrophils of beta-glucuronidase (IC(50) = 13.9 microM), myeloperoxidase (IC(50) = 7.2 microM) and elastase (IC(50) = 5.4 microM). In addition they dose-dependently inhibit the activity of myeloperoxidase released from calcium ionophore-stimulated cells with an IC(50) value of 2 microM. The monomeric constitutive unit (+)-catechin was far less active than procyanidins in all the models tested. These results evidence that procyanidins efficiently restrain the inflammatory response of activated neutrophils in vitro and whenever absorbed in vivo can prevent their oxidative discharge at the site(s) of their adhesion.

Electron spin resonance (ESR) spectroscopy was applied for the unequivocal detection/quantitation of nitric oxide (NO) as nitrosylhemoglobin (HbFe(II)NO) released from nitroaspirin, benzoic acid,2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester (NCX-4016; NO-ASA), the lead of a new class of nonsteroidal anti-inflammatory drugs. In both in vitro and in vivo experiments, the paramagnetic complex was detected at 100 K in the venous blood of the rat (microwave power, 20 mW) and characterized by a three-line hyperfine structure with coupling constants (A(x) and A(z)) of 17 G at g(x)=2.066 and g(z)=2.009. The kinetics of NO release from the drug were first determined in vitro by incubating rat blood with 1 mM NO-ASA and confirmed by the two-line hyperfine structure obtained with the labeled compound ((15)N-NO-ASA). In in vivo studies, the hematic levels of HbFe(II)NO were determined after oral (p.o.) and intraperitoneal (i.p.) administration of the drug (100 and 200 mg kg(-1)). In p.o. treated animals, the complex was detectable at 1 h post-dosing and its formation was maximal at 4-6 h, where the antithrombotic activity peaks. In i.p. treated animals, HbFe(II)NO complex peaks at the second hour to decline thereafter: in these animals, the ESR technique was applied to also detect nitrosylmyoglobin as an index of NO diffusion/compartmentalization in myocardial tissue. The results of this study emphasize the great potentiality of ESR spectroscopy for the study of the release, the metabolic fate and distribution of NO from nitrovasodilators.

The aim of this work was to compare in the rat the cardioprotective efficacy and the total plasma antioxidant activity of a standardised Ginkgo biloba L. extract (GB) as such (300 mg/kg/day) or complexed with phosphatidylcholine (GB-PC; 1:2 w/w), after a 5 days oral administration. At the end of the treatment, the total plasma antioxidant defence was determined by the TRAP and FRAP assays, and the hearts from all groups of animals subjected to moderate ischemia (flow reduction to 1 ml/min for 20 min) and reperfusion (15 ml/min for 30 min). The recovery of left ventricular developed pressure (LVDP) at the end of reperfusion was 35-40% of the preischemic values in both control and vehicle rats, 50.2% in the GB group and 72.5% in the GB-PC pre-treated animals. Creatine kinase (CK) outflow in the perfusate from the hearts of GB and GB-PC treated animals were restrained to a different extent vs. controls (by 71% GB-PC; by 22% GB); the rate of prostacyclin (6-keto-PGF1 alpha) release was far greater in GB-PC than in GB hearts. In parallel, the GB extract significantly increased the total antioxidant plasma capacity (by 24.5% TRAP; 27.9% FRAP) only when complexed with phospholipids. This indicates an increased bioavailability of phenolic antioxidants when suitably embedded within a lipophilic carrier. The results of this study demonstrate that complexation of Ginkgo biloba with phospholipids induces in the rat, even after a short treatment a greater resistance of the heart to ischemia/reperfusion damage in respect to the native extract, due to an increased plasma antioxidant activity.

Liquid Chromatography-Ion Trap Mass Spectrometry with an atmospheric pressure chemical ionization (APCI) interface in the negative and positive-ion modes in parallel to UV-diode-array detection (DAD), was applied for the rapid detection/characterization in crude extracts of the water-soluble antioxidant phenolics from Helichrysum stoechas. APCI-MS provides unequivocal molecular weight data of these compounds and useful information about their structures (diagnostic fragments ions), which were confirmed by the UV-DAD fingerprints. This combined approach allows the identification of ten constituents, including the three naturally occurring isomers of caffeoylquinic acid (CGAs), namely neo-chlorogenic acid, chlorogenic acid and crypto-chlorogenic acid, 2 isomeric dicaffeoyl quinic acids, 2 isomeric naringenin glucosides, quercetin, kaempferol and apigenin glucosides and a tetrahydroxychalcone-glucoside. The water-soluble extract from H. stoechas, standardized in both total polyphenol and kaempferol-3-glucoside content, exhibits strong antioxidant activity in vitro when tested in both artificial membrane systems (phosphatidylcholine liposomes) and in a cell model (rat erythrocytes).

Besides erythema and sunburn reactions, UVB stress can promote erythrocyte extravasation from skin capillaries and hemolysis, and photosensitized hemoglobin can in turn lead to an overload of free radicals in dermis which exacerbates photodamage. The objective of this study was to investigate in rat erythrocytes (RBC) the pattern of events leading to membrane peroxidation and hemolysis following UVB insult (1.5-8.5 J/cm2), and the protective action of grape seed procyanidins. UVB causes a dramatic dose-dependent decrease of intracellular glutathione (paralleled by the formation of pro-oxidant ferryl-hemoglobin), of intramembrane vitamin E and of membrane fluidity, then a rise of conjugated dienes (CD), and thiobarbituric acid-reactive substances (TBARS) and finally a strong hemolytic effect. Procyanidins prevent membrane peroxidation (but not intracellular GSH depletion nor ferryl-hemoglobin formation), with a minimal effective concentration of 0.1 microM (IC50 for TBARS and CD after 120 min UVB exposure: 0.71 microM and 0.56 microM) and dose-dependently delay the onset of hemolysis, by 30 min at 0.1 mciroM, by 90 and 120 min at 0.5 and 1.0 microM. Epigallocatechin-3-O-gallate (EGCG) and catechin, typical constituents of the fraction, were significantly less potent. This since procyanidins (1 microM) inhibit the formation of phospholipid hydroperoxides of the inner (phosphatidylserine, phosphatidylethanolamine) and outer (phosphatidylcholine) layers of the RBC membrane (HPLC analysis), suppress the decrease in membrane fluidity due to lipid and protein thiol oxidation and spare vitamin E from consumption in a dose-dependent manner (0.1-1 microM). Hence procyanidins, preserving membrane phospholipids, since their strong antilipoperoxidant activity, may maintain in vivo the integrity of RBC in sub-epidermal capillaries and effectively counteract in dermis the onset/exacerbation of the UVB-induced skin photodamage.

In this work we describe the development of specific markers for determination of both the membrane and intracellular damage induced by free radicals generated by UVB radiation (5-150 mJ/cm2) in cultured keratinocytes. This using simple, specific and sensitive fluorescent probes: cis-parinaric acid (PNA) to monitor membrane lipid peroxidation and 2',7'-dichloro-dihydrofluorescein diacetate (DCFH-DA) to evaluate the intracellular redox status, in parallel to the fluorimetric determination of the main intracellular antioxidant glutathione. To validate the methodologies, the changes in the intracellular oxidative status following exposure to low doses UVB were measured in both control and N-acetylcysteine-protected cells, in parallel with morphological analyses. UVB induces an early reduction of GSH inside the cell correlated with an increase in the intracellular peroxide content. The effects were time- and dose-dependent. In addition, using a sensitive fluorescent method, we quantitated the release of proteases, a family of proteolytic enzymes greatly involved in the onset/perpetuation of the free radical-induced skin damage from keratinocytes exposed to suberythemal UVB doses (5-15 mJ/cm2). The use of these fluorescent probes provides a reliable tool to detect the early signs of damage in keratinocyte cultures (when the apoptotic phenomenon has not yet been triggered) useful for future screening of protective molecules.

The aim of this work was to investigate in the rat the protective effect of an oral administration (one week) of Panax ginseng (PG) extract (10 mg/ml in drinking water; 1.6 g/kg/day) on myocardial post-ischemic damage induced by hyperbaric oxygen (HBO) and on the loss in functionality of the endothelium in aorta ring preparations. The hearts from control rats (no-HBO and no-HBO-PG), and from rats exposed to HBO and to HBO after PG treatment were isolated and subjected to mild ischemia and then reperfused. HBO greatly worsens the post-ischemic damage in controls, as demonstrated by the rise of left ventricular end diastolic pressure (LVEDP) and coronary perfusion pressure (CPP). PG significantly restrained the increase of LVEDP and CPP in respect to HBO-untreated rats, as well as that of CPP induced by injection of angiotensin II during pre-ischemia. In HBO control rats the reduction of the vasorelaxant effect of acetylcholine on norepinephrine precontracted aortic rings, was markedly recovered by PG; a similar trend was observed in aortic rings challenged with the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (56% recovery). These results strongly indicate that PG prevents the myocardial ischemia/reperfusion damage and the impairment of endothelial functionality induced by reactive oxygen species arising from HBO exposure, through an antioxidant intervention. The in vitro radical scavenging activity of PG seems to be too weak (0.05-0.5 mg/ml) to explain by itself the cardiac and extra-cardiac protective effects, and this suggests a role also for an indirect antioxidant action of the drug (endothelial nitric oxide synthase stimulation).

Aim of this work was to study the efficacy of procyanidins from Vitis vinifera seeds, a standardized mixture of polyphenol antioxidants, on cardiac mechanics following ischemia/reperfusion stunning in the rat, after 3 weeks supplementation. Young and aged male rats were fed a diet enriched with procyanidins complexed (1:3 w/w) with soybean lecithin (2.4%); control animals (CTR-young and CTR-aged) received an equal amount of lecithin and 2 additional groups of animals the standard diet. At the end of the treatment, the total plasma antioxidant defense (TRAP), vitamin E, ascorbic acid and uric acid were determined in plasma and the hearts from all groups of animals subjected to moderate ischemia (flow reduction to 1 ml/min for 20 min) and reperfusion (15 ml/min for 30 min). In both young and aged rats supplemented with procyanidins the recovery of left ventricular developed pressure (LVDP) at the end of reperfusion was 93% (p < 0.01) and 74% (p < 0.01) of the preischemic values and the values of coronary perfusion pressure (CPP) were maintained close to those of the preischemic period. Also creatine kinase (CK) outflow was restrained to baseline levels, while a 2-fold increase in prostacyclin (6-keto-PGF1alpha) in the perfusate from hearts of young and aged rats was elicited during both ischemia and reperfusion. In parallel, procyanidins significantly increased the total antioxidant plasma capacity (by 40% in young and by 30% in aged rats) and the plasma levels of ascorbic acid, while tend to reduce vitamin E levels; no significant differences were observed in uric acid levels. The results of this study demonstrate that procyanidins supplementation in the rat (young and aged) makes the heart less susceptible to ischemia/reperfusion damage and that this is positively associated to an increase in plasma antioxidant activity.

A study was undertaken for the characterization and quantitative determination of the main urinary metabolites of the non-steroidal anti-inflammatory drug (NSAID) nimesulide (4-nitro-2-phenoxy-methanesulfonanilide) in man following single oral administration (200 mg). Urines were collected from six healthy volunteers at 12, 24, 48, 72 and 96 h post-administration and submitted to liquid liquid extraction before (free metabolites) and after enzymatic hydrolysis (conjugated metabolites). The structure of the metabolites, isolated by TLC separation, was elucidated by mass spectrometry (electron impact ionization) and confirmed by synthesis. Five metabolites were identified: they arise from hydroxylation to the phenoxy nucleus (M1 = hydroxynimesulide); reduction of the nitro group to an amino derivative (M2); concomitant hydroxylation and reduction (M3); N-acetylation of the M2 (M4) and of the M3 (M5) metabolites. Quantitation was by reverse phase high performance liquid chromatography (Supelcosil LC-18 DB column; mobile phase: sodium phosphate buffer (pH 3.0, 50 mM)-acetonitrile (gradient elution); flow rate: 1 ml min(-1); UV detection, 230 nm), procedure which allows in a single chromatographic run the simultaneous determination of the unchanged drug and of its metabolites. The urinary excretion of the drug and metabolites (free + conjugated) in the overall 96 h-interval accounts for approximately 40% of the administered dose: 17.55 +/- 3.6% M1; 0.72 +/- 0.43% M2; 2.45 +/- 1.22% M3; 19.07 +/- 4.3% M5. The bulk of the metabolites was in conjugated form. Percentages excretion of the unchanged drug and of M4 metabolite were below 0.5%. The described method is suited to specifically and quantitatively measure nimesulide and metabolites in human urine with acceptable precision and accuracy.

The protective effect of procyanidins (catechin oligomers) from Vitis vinifera seeds on the UVB-promoted photo-oxygenation of polyunsaturated fatty acids (PUFAs) (linoleic, linolenic and arachidonic acids, 35 mM) has been studied in 0.5 mM sodium dodecylsulfate micelles exposed to UVB radiation (1-8 J cm-2) and determined qualitatively by Fourier transform infrared spectroscopy (FT-IR) and quantitatively by UV spectroscopy (conjugated dienes and total lipid hydroperoxides). Procyanidins (1-10 muM) strongly and dose-dependently inhibit the formation of PUFA hydroperoxides (IC50 = 4-6 muM), with a potency comparable to that of vitamin E. In these micellar systems, a strong co-operative interaction (synergistic effect) between procyanidins and vitamin E (1:1 molar ratio) in PUFA protection was demonstrated; this, as shown by electron spin resonance (ESR) studies, is a result of the ability of procyanidins to regenerate alpha-tocopherol from the alpha-tocopheroxyl radical (generated by a UV Solar Simulator), through a hydrogen transfer mechanism. HPLC monitoring of vitamin E in PUFAs micelles oxidatively stressed by UVB, confirms that procyanidins save vitamin E from consumption. All these findings, if properly extrapolated to an in vivo situation, indicate that this class of radical scavengers can be used as adjuvants of physical/chemical sunscreens in skin protection from photodamage.

The sparing/recycling effect of a highly purified, high molecular weight fraction of catechin oligomers (procyanidins) from Vitis vinifera seeds on alpha-tocopherol was studied in both homogeneous solution and in heterogeneous phase (phosphatidylcholine liposomes and red blood cells). By HPLC and electron spin resonance (ESR) spectroscopy we evidenced that tocopheroxyl radical, induced by reaction of alpha-tocopherol with the stable radical DPPH (2,2-diphenyl-1-picrylhydrazyl) is recycled by procyanidins. In addition procyanidins significantly and dose-dependently spare vitamin E from consumption (HPLC monitoring) during the autooxidation phase of the HO-induced peroxidation of phosphatidylcholine, by 23% at the lowest concentration (0.5 microM) and by 65.5% at 3 microM. In this membrane model the combination of 0.5 microM procyanidins and 2 microM alpha-tocopherol results in a marked delay in the appearance of conjugated dienes in respect to the single antioxidants (synergistic interaction), while catechin showed to be active only at 5 microM. In red blood cells oxidatively stressed by UVB exposure, procyanidins at 0.1-1.0 microM prevent vitamin E loss, markedly decrease membrane lipid peroxidation, linearly related to the concentration of vitamin E in the membranes, and significantly delay the onset of hemolysis (catechin protects between 5 and 10 microM).

The protective effect of hydroxynimesulide, the main metabolite of the nonsteroidal antiinflammatory drug nimesulide, on red blood cells (RBCs, 0.2%; 3.5 x 10(7) cell/ml) hemolysis induced by cumene hydroperoxide (CuOOH; 50 microM) was evaluated by turbidimetric and morphological analyses. Hydroxynimesulide inhibits the CuOOH-induced hemolysis in a dose dependent fashion: the protective effect, calculated after 150 min incubation (100% hemolysis in the controls), starts at 1 micron (% hemolysis 85.2 +/- 3.4%) and increases at the higher concentrations (63.5 +/- 3.9% at 5 microM; 43.5 +/- 6.3% at 10 microM; and, 14.5 +/- 4.3% at 20 microM). In addition, in the samples protected with 10 microM and 20 microM, there is a significant delay (30 and 60 min) in the onset of the hemolytic response. Inhibition of hemolysis is the result of protection of RBC membrane integrity, both on lipid (cis-Parinaric acid fluorescence quenching was delayed by 53 +/- 10 sec vs. the controls at 1 micron, by 115 +/- 15 sec at 5 microM, with a lag phase of 240 +/C- 18 sec at 10 microM) and protein constituents, as determined by SDS-PAGE electrophoresis. In hemolysis experiments, the efficacy of hydroxynimesulide is comparable to that of alpha-tocopherol and a cooperative interaction between hydroxynimesulide and alpha-tocopherol (both at 10 microM) has been observed. These results indicate that hydroxynimesulide protects RBC membranes by directly quenching reactive oxygen species generated by hemoglobin/peroxide interaction. Evidence for a direct radical scavenging intervention of the metabolite comes from HPLC studies, which demonstrate a time-dependent consumption of hydroxynimesulide, with the concomitant formation of two main reaction (addition/oxidation) products.

An isolated rabbit heart Langendorff preparation paced electrically was used to evaluate the effects of a highly purified, high molecular weight fraction of oligomeric procyanidines isolated from Vitis vinifera seeds on myocardial reperfusion injury after 40 minutes of low flow (1 ml/min) ischemia. Infusion of the heart with 100 or 200 micrograms/ml procyanidines dose-dependently reduced ventricular contracture during ischemia (LVEDP values decreased by 28% and 51%), decreased coronary perfusion pressure (CPP), improved cardiac mechanical performance upon reperfusion, increased the release of 6-keto-PGF1 alpha into the perfusate in both the pre-ischemic and the reperfusion periods (by 68% at 200 micrograms/ml), and suppressed rhythm irregularity. This antiarrhythmogenic action was confirmed in a more severe model of ischemia (flow rate 0.2 ml/ min). The cardioprotective agent allopurinol infused at 20 micrograms/ml had effects on the contractility and on the release of 6-keto-PGF1 alpha comparable to those of 200 micrograms/ml procyanidines. The results of the second part of this study show that procyanidines are potent scavengers of several reactive oxygen species involved in the ischemia/reperfusion damage: the superoxide anion (IC50 = 5.64 microM: rate constant K = 7.55 x 10(5) M-1 s-1, determined by the phenazine methosulfate/NADH method); the hydroxyl radical (IC50 = 28 microM; rate constant K = 1.2 x 10(12) M-1 s-1, determined by the electron spin resonance spectroscopy); peroxyl radicals (IC50 = 0.025 microM and 0.35 microM, determined using two different lipid substrates, phosphatidylcholine liposomes and methyl linoleate micelles by UV spectroscopy at 233 nm). Finally, procyanidines interact with Fe2+ and Cu2+ ions (the catalysts of HO. radicals production) giving rise to strong complexes, with stability constants (log K) ranging from 9.35 to approximately 9.

The protective effect of caffeoyl derivatives (echinacoside, chlorogenic acid, chicoric acid, cynarine, and caffeic acid, typical constituents of Echinacea species) on the free radical-induced degradation of Type III collagen has been investigated. The macromolecule was exposed to a flux of oxygen radicals (superoxide anion and hydroxyl radical) generated by the xanthine/xanthine oxidase/Fe2+/EDTA system and its degradation assessed qualitatively by SDS-PAGE and quantitatively as the amount of soluble peptides (according to the 4-hydroxyproline method) released from native collagen after oxidative stress. The SDS-PAGE pattern of native collagen is markedly modified by free radical attack, with formation of a great number of peptide fragments with molecular masses below 97 kDa: in the presence of microM concentrations of echinacoside, there is a complete recovery of the native profile. Collagen degradation was, in fact, dose-dependently inhibited by all the compounds, with the following order of potency: echinacoside approximately chicoric acid > cynarine approximately caffeic acid > chlorogenic acid, with IC50 ranging from 15 to 90 microM. These results indicate that this representative class of polyphenols of Echinacea species protects collagen from free radical damage through a scavenging effect on reactive oxygen species and/or C-, N-, S-centered secondary radicals, and provide an indication for the topical use of extracts from Echinacea species for the prevention/treatment of photodamage of the skin by UVA/UVB radiation, in which oxidative stress plays a crucial role.

The superoxide and hydroxyl radical scavenging activities of nimesulide (CAS 51803-78-2) and its main metabolite 4-hydroxynimesulide (CAS 109032-22-6) were investigated by Electron Spin Resonance (ESR) spectroscopy, with the spin trapping technique. Hydroxynimesulide is a good scavenger of both O2 degrees- (IC50 = 40 mumol/l) and HO degree (IC50 = 54.8 mumol/l) radicals, and its high reactivity towards HO degree was confirmed by the rate constant for reaction with HO degree (K = 8.9 x 10(10) mol-1 l s-1) determined by competition kinetic studies with 5,5-dimethyl-l-pyrroline-N-oxide. Nimesulide, which has been shown by ESR to be inactive as a superoxide quencher, has a rate constant of reaction with HO degree slightly greater than that of its metabolite (3.3 x 10(11) mol-1 l s-1). In the HO degree-induced peroxidation of phosphatidylcholine (PC) liposomes, both compounds act as potent preventive antioxidants, but the HO degree entrapping capacity of the parent drug was again greater than that of hydroxynimesulide (IC50 2.12 vs 3.84 mumol/l). The metabolite is also a potent scavenger of the peroxyl radical (ROO degree) in the propagation phase of PC peroxidation (marker conjugated dienes), with an IC50 = 2.67 mumol/l; at 5 mumol/l it induces a lag time in the decomposition of PC hydroperoxides (PC-OOH) into aldehydic products of 40 h longer than in the controls (markers: conjugated dienes and total carbonyl functions). In PC liposomes, in the presence of preformed PC-OOH, the metabolite prevents PC peroxidation stimulated by 5 mumol/l Fe2+, via the Fenton reaction (marker: conjugated dienes), at the micromolar level (IC50 = 17 mumol/l) through an anti-free radical activity and a free iron chelating mechanism. Hydroxynimesulide in fact interacts with Fe2+ ions, giving rise to a strong complex, with a stability constant (log K) estimated to be around 8/9. In addition, hydroxynimesulide efficiently protects ex vivo synovial fluid lipids from the oxidative stress induced by Fe2+/ascorbate, already at 10 mumol/l (marker: thiobarbituric acid reactive substances). These results provide evidence for strong antioxidant and iron-chelating properties of 4-hydroxynimesulide, which can act synergistically with the specific HO degree scavenging activity of the parent drug in preventing and limiting in vivo the free radical-mediated tissue damage in both acute and chronic inflammatory situations.

Triterpene and steroid saponins and sapogenins of medicinal plants (Aesculus hippocastanum L., Hedera helix L., Ruscus aculeatus L.) are claimed to be effective for the treatment/prevention of venous insufficiency. In this work we evaluated the inhibitory effects of these plant constituents on the activity of elastase and hyaluronidase, the enzyme systems involved in the turnover of the main components of the perivascular amorphous substance. The results evidence that for Hedera helix L., the sapogenins only non-competitively inhibit hyaluronidase activity in a dose-dependent fashion, showing comparable IC50 values (hederagenin IC50 = 280.4 microM; oleanolic acid IC50 = 300.2 microM); both the saponins hederacoside C and alpha-hederin are very weak inhibitors. The same behaviour is observed for serine protease porcine pancreatic elastase: the glycosides are devoid of inhibitory action, while genins are potent competitive inhibitors (oleanolic acid IC50 = 5.1 microM; hederagenin IC50 = 40.6 microM). Constituents from Aesculus hippocastanum L. show inhibitory effects only on hyaluronidase, and this activity is mainly linked to the saponin escin (IC50 = 149.9 microM), less to its genin escinol (IC50 = 1.65 mM). By contrast, ruscogenins from Ruscus aculeatus L., ineffective on hyaluronidase activity, exhibit remarkable anti-elastase activity (IC50 = 119.9 microM; competitive inhibition). The mechanism of elastase inhibition by triterpene and steroid aglycones, with a nitroanilide derivative as substrate, is discussed.

The scavenging by procyanidines (polyphenol oligomers from Vitis vinifera seeds, CAS 85594-37-2) of reactive oxygen species (ROS) involved in the onset (HO degrees) and the maintenance of microvascular injury (lipid radicals R degrees, RO degrees, ROO degrees) has been studied in phosphatidylcholine liposomes (PCL), using two different models of free radical generation: a) iron-promoted and b) ultrasound-induced lipid peroxidation. In a) lipid peroxidation was assessed by determination of thiobarbituric acid-reactive substances (TBARS); in b) by determination of conjugated dienes, formation of breakdown carbonyl products (as 2,4-dinitrophenylhydrazones) and loss of native phosphatidylcholine. In the iron-promoted (Fenton-driven) model, procyanidines had a remarkable, dose-dependent antilipoperoxidant activity (IC50 = 2.5 mumol/l), more than one order of magnitude greater than that of the monomeric unit catechin (IC50 = 50 mumol/l), activity which is due, at least in part, to their metal-chelating properties. In the more specific model b), which discriminates between the initiator (hydroxyl radical from water sonolysis) and the propagator species of lipid peroxidation (the peroxyl radical, from autooxidation of C-centered radicals), procyanidines are highly effective in preventing conjugated diene formation in both the induction (IC50 = 0.1 mumol/l) and propagation (IC50 = 0.05 mumol/l) phases (the scavenging effect of alpha-tocopherol was weaker, with IC50 of 1.5 and 1.25 mumol/l). In addition, procyanidines at 0.5 mumol/l markedly delayed the onset of the breakdown phase (48 h), totally inhibiting during this time the formation of degradation products (the lag-time induced by alpha-tocopherol was only of 24 h at 10 mumol/l concentration). The HO degrees entrapping capacity of these compounds was further confirmed by UV studies and by electron spin resonance (ESR) spectroscopy, using DMPO as spin trapper: procyanidines markedly reduced, in a dose-dependent fashion, the signal intensity of the DMPO-OH radical spin adduct (100% inhibition at 40 mumol/l). The results of the second part of this study show that procyanidines, in addition to free radical scavenging action, strongly and non-competitively, inhibit xanthine oxidase activity, the enzyme which triggers the oxy radical cascade (IC50 = 2.4 mumol/l). In addition procyanidines non-competitively inhibit the activities of the proteolytic enzymes collagenase (IC50 = 38 mumol/l) and elastase (IC50 = 4.24 mumol/l) and of the glycosidases hyaluronidase and beta-glucuronidase (IC50 = 80 mumol/l and 1.1 mumol/l), involved in the turnover of the main structural components of the extravascular matrix collagen, elastin and hyaluronic acid.(ABSTRACT TRUNCATED AT 400 WORDS)

Fast atom bombardment (FAB-MS) and fast atom bombardment tandem mass spectrometry (FAB-MS/MS) techniques (negative ions) have been successfully applied for identification of the constituents responsible for the antihyaluronidase activity of Echinacea angustifolia roots, whose extracts are widely employed for the adjuvant therapy of chronic inflammatory diseases. Crude extracts from different solvents were tested for antihyaluronidase activity, and those with the greatest inhibitory action (the ethylacetate, butylacetate and chloroform fractions, IC50 0.44, 0.50 e 0.62 mg/ml) were directly analyzed by MS. Full scan mass spectra produced intense molecular anions: collisional activation of these resulted in tandem mass spectra rich in significant product ions. Four main caffeoyl conjugates were detected and identified by tandem mass spectrometry (daughter and parent ion mode): 2,3-O-dicaffeoyltartaric acid (chicoric acid) and 5-O-dicaffeoylquinic acid (cynarine) and 2-O-caffeoyltartaric acid (caffaric acid) in the ethylacetate fraction. Among these caffeoyl conjugates, chicoric and caftaric acids had the greatest antihyaluronidase activity: IC50 = 0.42 and 0.61 mM, while the IC50 of cynarine and chlorogenic acid were 1.85 and 2.25 mM.

Liver fluke infection (Fasciola hepatica) depresses the drug-metabolizing capacity of the hepatic mixed function oxidase (MFO) and glucuronosyltransferase (GT) enzyme systems, throughout a free radicals mediated lipid peroxidation process. Glutathione (GSH, CAS 70-18-8) administered chronically (100 mg/kg i.p. once daily for 40 days) to experimentally infested rats from the onset to the maximal development of the infection (40th day), greatly reduced the damage to membrane lipids of the liver tissue (primary event of the disease), as judged by malonic dialdehyde (MDA) content (decreased by 80%) and diene conjugation absorption (delta E 1% value falls from 1.94 to 0.67). As a consequence, serum glutamate-oxaloacetate (GOT) and glutamate-pyruvate (GPT) transaminases levels, liver GSH and phospholipid (PL) contents, cytochrome P-450, NADPH-cytochrome-P-450 reductase and some typical cytochrome P-450-dependent activities (p-nitroanisole O-demethylase, aniline hydroxylase, as well as UDP-glucuronosyltransferase (GT) activity, all markedly affected in the acute stage of the disease, tend to recover to the control values. The efficacy of GSH in preventing the impairment of the hepatic drug metabolizing capacity was also demonstrated by using as substrate the widely employed flukicidal agent nitroxinil (3-iodo-4-hydroxy-5-nitrobenzonitrile). The in vitro cytochrome P-450-dependent nitroxinil detoxification (reduction to 3-iodo-4-hydroxy-5-aminobenzonitrile), drastically impaired in infested animals (-80%), is markedly restored (3-fold increase) in GSH-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

An in vitro assay based on the oxidation of phosphatidylcholine liposomes (PCL) with which we can distinguish the anti-HO zero activity from the antilipoperoxidant (R zero, ROO zero, RO zero) has been developed for testing the free-radical-scavenging ability of antiinflammatory drugs. PCL were exposed to a flux of hydroxyl radicals generated by water sonolysis for different periods, and the spontaneous lipid peroxidative phenomenon (propagation and breakdown phases) was followed for the subsequent 24 hours. Lipid peroxidation was assayed by simultaneous measurements of a) conjugated dienes, by UV spectroscopy (absorbance and second derivative at 233 nm), b) loss of lipid substrate (PC), by HPLC, and c) breakdown products (total carbonyl functions as 2,4-dinitrophenylhydrazones). Diclofenac, nimesulide and indomethacin, added to PCL at the starting of the different stages of lipid peroxidation, were tested for their overall and specific anti-radical properties. All the drugs exhibited a remarkable scavenging activity against oxy and lipid radicals, determined as percent inhibition of the formation of conjugated dienes, at concentrations easily attainable in vivo (IC50:diclofenac 2.5 microM, nimesulide 4.92 microM, indomethacin 6.85 microM), diclofenac being the most effective in quenching the R zero and ROO degree species responsible for the propagation phase. By contrast, the antioxidant activity of nimesulide and indomethacin, less potent as alkyl and peroxyl radical scavengers, is due to their ability to restrain the induction phase of the radical chain reaction mediated by hydroxyl radicals (IC50:nimesulide 1.85 microM, indomethacin 3.57 microM).

The antioxidant activity of nimesulide and its main metabolites, 4'-hydroxynimesulide (M1) and 2-(4'-hydroxyphenoxy)-4-N-acetylamino-methansulfonanilide (M2), was investigated using 2 in vitro models: NADPH-supported lipid peroxidation in rat liver microsomes (marker MDA formation) and xanthine/xanthine oxidase, iron-promoted depolymerisation of hyaluronic acid, determined by gel permeation chromatographic analysis (marker molecular weight distribution). In the lipid peroxidation model, all the compounds inhibited MDA formation in a concentration-dependent manner, although with different potencies; the maximum scavenging effect was observed for M1 [50% inhibitory concentration (IC50) = 30 mumol/L; M2 IC50 = 0.5 mmol/L; nimesulide = 0.8 mmol/L]. Nimesulide was more active than its metabolites in preventing OH-induced depolymerisation of hyaluronic acid, with a 50% effective concentration of approximately 230 mumol/L, which was fairly comparable to that of tenoxicam. This protective effect was due to the OH.-entrapping capacity of the drug, which, in the Fenton-driven model, is easily converted, via OH. attack, to M1 and putatively to 2-hydroxy-4-nitro-methansulfonanilide.