Role of the catechol group in the antioxidant and neuroprotective effects of virgin olive oil components in rat brain.

The aim of the present study was to determine the role of the catechol group in the antioxidant and neuroprotective effects of minor components of virgin olive oil in rat brain tissue. Hydroxytyrosol ethyl ether (HT, 2 OH), tyrosol ethyl ether (Ty, 1 OH) and 3,4-di-ortho-methylidene-hydroxytyrosol ethyl ether (MET, no OH) were compared. Oxidative stress was induced with ferrous salts (lipid peroxidation induction), diethylmaleate (depletion of glutathione) and hypoxia-reoxygenation in brain slices. Lipid peroxidation was inhibited in direct proportion to the number of OH groups: HT>Ty>MET. Exposure to HT led to partial recovery of the glutathione system after chemical inhibition or hypoxia-reoxygenation. All three compounds inhibited cell death in hypoxia-reoxygenation experiments (HT≥Ty>MET). Peroxynitrite formation (3-nitrotyrosine) and inflammatory mediators (prostaglandin E2 and interleukin 1ß) were inhibited by all three compounds. In conclusion, the presence of OH groups in the molecule of these phenolic compounds from virgin olive oil is a determinant factor in their antioxidant effect in brain tissue, but this antioxidant effect is not the only explanation for their neuroprotective effect.

Antiplatelet effect of new lipophilic hydroxytyrosol alkyl ether derivatives in human blood.

PURPOSE: To investigate the in vitro antiplatelet and anti-inflammatory effects of five alkyl hydroxytyrosol (HT) ether derivatives in human whole blood and compare these effects with those of HT. METHODS: Blood samples from healthy volunteers were incubated with HT and HT alkyl ether derivatives (ethyl, butyl, hexyl, octyl and dodecyl). Maximum intensity of platelet aggregation was induced with collagen, arachidonic acid or ADP. Calcium-induced thromboxane B(2) and nitric oxide production, LPS-induced prostaglandin E(2) and nitric oxide production and LPS-induced interleukin 1ß production were measured. RESULTS: All compounds inhibited platelet aggregation, thromboxane B(2) and inflammatory mediators in a concentration-dependent manner. The concentrations of each compound that inhibited the corresponding variable by 50 % compared to control samples (IC(50)) were in the range of 10(-7)-10(-6) M for HT hexyl ether; for the other compounds, these values were in the range of 10(-5) M. The IC(50) for thromboxane B(2) production was in the range of 10(-4) M. The effects of HT alkyl ether derivatives were greater than those of HT. These compounds increased nitric oxide production. There was no direct relationship between the effects of these compounds and alkyl chain length. Maximum effects were observed in the C4-C6 range. CONCLUSIONS: Alkyl ether derivatives of HT exert antiplatelet and anti-inflammatory effects that are greater than those of HT.

Effects of terutroban, a thromboxane/prostaglandin endoperoxide receptor antagonist, on retinal vascularity in diabetic rats.

BACKGROUND: The aim of the present study is to investigate the effectiveness of terutroban, a selective antagonist of the thromboxane/prostaglandin endoperoxide receptor, in preventing retinal ischaemia in a model of diabetes in rats. METHODS: Experimental diabetes was induced with streptozotocin. Rats were distributed into five groups (n = 20): (1) non-diabetic rats, (2) rats with diabetes (DR) treated with vehicle, (3) DR treated with aspirin (2 mg/kg/day p.o.), (4) DR treated with terutroban (5 mg/kg/day p.o.), (5) DR treated with terutroban (30 mg/kg/day p.o.). The follow-up period was 3 months. The main assessment was the percentage of retinal surface covered with vessels permeable to peroxidase. Platelet aggregation, aortic prostacyclin and nitric oxide production, plasma levels of lipid peroxides (thiobarbituric-acid-reactive substances) and 3-nitrotyrosine and serum levels of IL-6 were evaluated. RESULTS: Diabetes induced a reduction in retinal vascularity (76.9%), aortic prostacyclin (37.8%) and nitric oxide production (35.0%), and increased platelet aggregation, lipid peroxides, 3-nitrotyrosine. When compared with vehicle-treated DR, terutroban increased the percentage of retinal surface covered by PVPP (38% for terutroban-5 and 61% for terutroban-30), aortic prostacyclin (188% for terutroban-5 and 146% for terutroban-30) and nitric oxide production (320% for terutroban-5 and 390% for terutroban-30). Moreover, terutroban reduced platelet reactivity (27.8­95.1%, according to the inducer), lipid peroxides (60.7% for terutroban-5 and 50.0% for terutroban-30), 3-nitrotyrosine (43.8% for terutroban-5 and 36.8% for terutroban-30) and IL-6 concentration (18.0% for terutroban-30). The effect of terutroban in retinal, nitrosative and aortic parameters was significantly higher than that of aspirin. CONCLUSIONS: Terutroban significantly protected retinal vascularity from ischaemia in experimental diabetes, and this result could be attributed not only to its antiplatelet/antithrombotic activities but also to its vascular properties.

Neuroprotective effect of alkyl hydroxytyrosyl ethers in rat brain slices subjected to a hypoxia-reoxygenation model.

The aim of the present study was to investigate the antioxidant and possible neuroprotective and antioxidant effects of five alkyl hydroxytyrosyl (HT) ethers (ethyl, butyl, hexyl, octyl and dodecyl) in rat brain slices. None of the compounds modified lipid peroxidation or glutathione concentrations (GSH) in oxygenated samples. The effects of oxidative stress were investigated with ferrous salts to induce lipid peroxidation and diethylmaleate (DEM) to reduce GSH. All compounds inhibited lipid peroxidation with an inhibitory concentration 50% (IC(50)) one tenth that of HT. These compounds, especially the butyl derivative, prevented GSH depletion after incubation with DEM. We also explored the neuroprotective effect of these compounds in an experimental model of hypoxia-reoxygenation in rat brain slices. All compounds showed neuroprotective and antioxidant effects. Our results established a relationship between these effects and the length of the carbon chain (maximum effect in the range of C4-C8).

Lack of enantiomeric influence on the brain cytoprotective effect of ibuprofen and flurbiprofen.

R(-) enantiomers of the 2-arylpropionic acid derivatives ibuprofen and flurbiprofen weakly inhibit cyclooxygenase (COX) activity. However, a possible cytoprotective effect has been proposed. The aim of the study is to investigate the possible mechanism of this effect. An in vitro hypoxia-reoxygenation model in rat brain slices was used (n=6 rats per group). After reoxygenation, we measured LDH efflux (neuronal death), brain prostaglandin E(2) (PGE(2)) concentration, interleukins (IL)-1ß and 10, oxidative and nitrosative stress (lipid peroxides, glutathione, 3-nitrotyrosine, and nitrites/nitrates). Anti-COX activity was measured in human whole blood. Racemic, R(-), and S(+) enantiomers of ibuprofen and flurbiprofen were tested. All compounds had a cytoprotective effect with IC(50) values in the range of 10(-5) M. R(-) enantiomers did not significantly inhibit brain PGE(2). The concentration of IL-1ß was reduced by 53.1% by the racemic form, 30.6% by the S(+) and 43.2% by the R(-) enantiomer of ibuprofen. The IL-10 concentration increased significantly only with S(+)-flurbiprofen (33.1%) and R(-)-flurbiprofen (26.1%). Lipid peroxidation was significantly reduced by all three forms of flurbiprofen. Nitrite + nitrate concentrations were reduced by racemic, S(+), and R(-)-flurbiprofen. Peroxynitrite formation (3-nitrotyrosine) was significantly reduced by racemic and S(+)-ibuprofen. COX inhibition is not the main mechanism of cytoprotection for these compounds. Their influence on inflammatory mediators and oxidative and nitrosative stress could account for the potential cytoprotective effect of R(-) enantiomers.

Differences in the in vitro antiplatelet effect of dexibuprofen, ibuprofen, and flurbiprofen in human blood.

BACKGROUND: In this study, we compared the in vitro pharmacodynamic profile of dexibuprofen, ibuprofen, and flurbiprofen to identify possible differences in antiplatelet activity. METHODS: In whole blood samples from healthy volunteers, we measured platelet aggregation induced by adenosine diphosphate, collagen and arachidonic acid, platelet thromboxane B(2) (TxB(2)), lipopolysaccharide-induced prostaglandin E(2), leukocyte 6-keto-prostaglandin F(1α) (PGF(1α)), and nitric oxide induced by both constitutive and inducible pathways before and after incubation with increasing concentrations of acetylsalicylic acid, dexibuprofen, ibuprofen, or flurbiprofen. The concentration that inhibited (IC(50)) or increased each variable by 50% was calculated. RESULTS: All 3 drugs inhibited platelet aggregation in a dose-dependent manner, TxB(2), prostaglandin E(2), and 6-keto-PGF(1α), and increased calcium-induced nitric oxide production. Dexibuprofen showed greater antiplatelet potency than ibuprofen and flurbiprofen, and its profile was similar to that of aspirin. For example, IC(50) values for arachidonic acid-induced platelet aggregation were 0.85 ± 0.06 µM for dexibuprofen, 14.76 ± 1.22 µM for ibuprofen, 6.39 ± 0.51 µM for flurbiprofen, and 0.38 ± 0.03 µM for aspirin. All drugs inhibited both thromboxane and prostacyclin synthesis, but the IC(50) anti-TxB(2)/IC(50) anti-6-keto-PGF(1α) ratio was 0.21 ± 0.03 for dexibuprofen, 1.05 ± 0.08 for ibuprofen, 0.79 ± 0.11 for flurbiprofen, and 0.46 ± 0.06 for aspirin. All drugs increased calcium-dependent nitric oxide production. CONCLUSIONS: The aryl propionic acid derivative dexibuprofen was the most potent antiplatelet drug, and its pharmacodynamic profile is similar to aspirin.

Effects of propofol on the leukocyte nitric oxide pathway: in vitro and ex vivo studies in surgical patients.

This study was designed to evaluate the mechanism by which propofol modifies leukocyte production of nitric oxide (NO) in humans. In vitro experiments used whole blood from healthy volunteers (n = 10 samples/experiment). Ex vivo experiments studied the effects of an intravenous dose of 2.5 mg propofol per kilogram body weight followed by intravenous infusion of 4 mg kg(-1) h(-1) in surgical patients in ASA class I or II (n = 20). In whole blood, neutrophils and plasma, we measured NO production and the activities of the enzymes nitric oxide synthase [inducible (iNOS) and constitutive (cNOS)] and cyclooxygenase [constitutive (COX-1) and inducible (COX-2)]. Concentrations of interleukins (IL-1beta, IL-6, and IL-10) and tumor necrosis factor-alpha (TNFalpha) were measured in plasma. In blood from healthy donors, propofol increased NO production and cNOS activity. The concentration of propofol that increased NO production by 50% (EC(50)) was 23.5 microM, and the EC(50) of propofol for cNOS was 18.6 microM. In blood from surgical patients, propofol increased NO production by 52% and cNOS activity by 57%. Propofol inhibited iNOS activity in vitro; the concentration that reduced activity by 50% (IC(50)) was 19.9 microM. In surgical patients propofol inhibited iNOS activity by 53%. COX-1 and COX-2 activities were inhibited in vitro (IC(50) 32.6 and 187 microM, respectively) and in surgical patients (53 and 81% inhibition, respectively). Plasma concentrations of IL-1beta, IL-6, and TNFalpha were significantly reduced in surgical patients (32, 23, and 21% inhibition, respectively). None of these parameters were modified in a group of patients (n = 10) anesthetized with sevoflurane. We conclude that propofol stimulated constitutive NO production and inhibited inducible NO production, possibly by curtailing the stimulation of iNOS by inflammatory mediators in surgical patients.

Dietary virgin olive oil reduces oxidative stress and cellular damage in rat brain slices subjected to hypoxia-reoxygenation.

We investigated how virgin olive oil (VOO) affected platelet and hypoxic brain damage in rats. Rats were given VOO orally for 30 days at 0.25 or 0.5 mL kg(-1) per day (doses A and B, respectively). Platelet aggregation, thromboxane B2, 6-keto-PGF(1alpha), and nitrites + nitrates were measured, and hypoxic damage was evaluated in a hypoxia-reoxygenation assay with fresh brain slices. Oxidative stress, prostaglandin E2, nitric oxide pathway activity and lactate dehydrogenase (LDH) activity were also measured. Dose A inhibited platelet aggregation by 36% and thromboxane B2 by 19%; inhibition by dose B was 47 and 23%, respectively. Virgin olive oil inhibited the reoxygenation-induced increase in lipid peroxidation (57% in control rats vs. 2.5% (P < 0.05) in treated rats), and reduced the decrease in glutathione concentration from 67 to 24% (dose A) and 41% (dose B). Brain prostaglandin E2 after reoxygenation was 306% higher in control animals, but the increases in treated rats were only 53% (dose A) and 45% (dose B). The increases in nitric oxide production (213% in controls) and activity of the inducible isoform of nitric oxide synthase (175% in controls) were both smaller in animals given VOO (dose A 84%; dose B 12%). Lactate dehydrogenase activity was reduced by 17% (dose A) and 42% (dose B). In conclusion, VOO modified processes related to thrombogenesis and brain ischemia. It reduced oxidative stress and modulated the inducible isoform of nitric oxide synthase, diminishing platelet aggregation and protecting the brain from the effects of hypoxia-reoxygenation.

Differences in the influence of the interaction between acetylsalicylic acid and salicylic acid on platelet function in whole blood and isolated platelets: influence of neutrophils.

The aim of this study was to characterize the influence of the interaction between acetylsalicylic acid (ASA) and salicylic acid (SA) on the inhibition by ASA of platelet aggregation in platelets isolated from whole blood, and to determine whether leukocytes influence this pharmacological interaction. This in vitro study was done in human blood from which we prepared samples of whole blood, platelet-rich plasma (PRP), PRP plus mononuclear leukocytes, and PRP plus neutrophils. The variables recorded were maximum platelet aggregation intensity, thromboxane B2 (TxB2) production, and nitric oxide (NO) production (N=10 different samples in each type of experiment). Different concentrations of ASA and SA were incubated with all samples. In PRP, the concentration of ASA that inhibited maximum aggregation by 50% (IC50) (281+/-16microM) increased with increasing SA concentration to a maximum of more than 2mM when 500microM SA was used. In whole blood, the IC50 for ASA (24.9+/-1.2microM) decreased with decreasing SA concentrations to 7.9+/-0.8microM with 50microM SA and 15.6+/-0.9microM with 125microM SA, and increased to 46.2+/-2.6microM with 250microM SA and 96.3+/-7.2microM with 500microM SA. In experiments with PRP+neutrophils the IC50 of ASA increased in the presence of all concentrations of SA. The antagonistic interactions were also reflected in the changes in TxB2 production in all samples. In samples of neutrophils incubated with ASA, the curve for NO production was shifted to the right, a finding that paralleled the changes in platelet aggregation. In conclusion, the influence of the interaction between ASA and its metabolite SA on platelet aggregation difference depending on the type of sample, and was antagonistic in PRP but partially agonistic in whole blood. Nitric oxide synthesis showed an additive effect of the two compounds.