J. Muñoz-Marín

Effects of Hydroxytyrosol and Hydroxytyrosol Acetate Administration to Rats on Platelet Function Compared to Acetylsalicylic Acid

Virgin olive oil (VOO) contains the polyphenols hydroxytyrosol (HT) and hydroxytyrosol acetate (HT-AC). This study investigated the antiplatelet effect of HT and HT-AC in healthy rats and compared their effects to acetylsalicylic acid (ASA). All compounds were administered orally for 7 days. HT and HT-AC inhibited platelet aggregation in whole blood, with a 50% inhibitory dose (ID50) of 48.25 mg/kg per day for HT, 16.05 mg/kg per day for HT-AC, and 2.42 mg/kg per day for ASA. Platelet synthesis of thromboxane B2 was inhibited by up to 30% by HT and 37% by HT-AC; the ID50 of this effect for ASA was 1.09 mg/kg per day. Vascular prostacyclin production was inhibited by up to 27.5% by HT and 32% by HT-AC; the ID50 of this effect for ASA was 6.75 mg/kg per day. Vascular nitric oxide production was increased by up to 34.2% by HT, 66% by HT-AC, and 64% by ASA. We conclude that HT and HT-AC administered orally inhibited platelet aggregation in rats and that a decrease in thromboxane synthesis along with an increase in nitric oxide production contributed to this effect.

Role of the inhibition of oxidative stress and inflammatory mediators in the neuroprotective effects of hydroxytyrosol in rat brain slices subjected to hypoxia reoxygenation

The aim of this study was to analyze the mechanism of the neuroprotective effect of hydroxytyrosol (HT) in an experimental model of hypoxia-reoxygenation in rat brain slices. After reoxygenation the increase in lactate dehydrogenase efflux was inhibited by HT in a concentration-dependent manner and dose-dependent inhibition after oral administration to rats for 7 days (1, 5 and 10 mg/kg per day). Maximum inhibition was 57.4% in vitro and 38.7% ex vivo. Hydroxytyrosol reduced oxidative stress parameters: it inhibited lipid peroxidation and increased enzymatic activities related with the glutathione system both in vitro and after oral administration to rats. The increase in prostaglandin E2 and interleukin 1β after reoxygenation were inhibited after incubation of brain slices with HT and after oral administration. The accumulation of nitric oxide in brain slices was reduced in a concentration-dependent manner. In conclusion, HT exerts a neuroprotective effect in a model of hypoxia-reoxygenation in rat brain slices, both in vitro and after 7 days of oral administration to rats. HT exerts an antioxidant activity and lowered some inflammatory markers in this model.

Antioxidant effects of a single dose of acetylsalicylic acid and salicylic acid in rat brain slices subjected to oxygen-glucose deprivation in relation with its antiplatelet effect.

The aim of the present study was to analyze the relative participation of the antiplatelet and the antioxidant effects of acetylsalicylic acid (ASA) and salicylic acid (SA) after a single dose (1 or 10 mg/kg i.p.) in an in vitro model of anoxia in slices of rat brain. After 20 min of drug administration, blood and brain were obtained (n=6 rats per group). We measured: lipid peroxidation, glutathione levels and lactate dehydrogenase efflux (LDH), ASA and SA concentrations and platelet aggregation in whole blood. An increase in lipid peroxidation (80%) and in LDH efflux (520%) and a decrease in glutathione levels (35%) were observed after 120 min anoxia in saline-treated rats. SA reduced this oxidative stress and LDH efflux, but it did not modify platelet aggregation. ASA strongly inhibited platelet aggregation but exerted a poor antioxidant effect. ASA was not detectable in brain tissue. We conclude that repeated doses of ASA are necessary to obtain a tissular antioxidant effect, probably when liver generates enough SA.

Effects of dexibuprofen on platelet function in humans: comparison with low-dose aspirin.

Background:The aim of the current study is to evaluate the antiplatelet effect of dexibuprofen in healthy volunteers in comparison with low-dose aspirin. Methods:Healthy volunteers (n = 12) were treated in a crossover manner with 100 mg daily aspirin or with 800 mg daily dexibuprofen. Blood samples were obtained within 24 h; 3, 7, and 14 days after repeated doses; and 24 h after the last dose. In each sample, the authors measured platelet aggregation, thromboxane B2, 6-keto-prostaglandin F1α, and nitric oxide. Results:The antiplatelet effect of dexibuprofen (maximal inhibition of aggregation was 48–55% for adenosine diphosphate and 90–95% for collagen and arachidonic acid) was equal to the effect of aspirin. The main difference between the two drugs was in the degree of recovery of platelet function. The effect of aspirin persisted for 24 h after the last dose (remaining inhibition 50%, respect to the pretreatment value), whereas platelet aggregation had returned to baseline pretreatment values within 24 h after dexibuprofen was stopped. Conclusions:Both aspirin and dexibuprofen inhibited platelet function with a similar intensity, but dexibuprofen exerted a reversible effect for 24 h after the last dose.

Influence of vitamin E on the antiplatelet effect of acetylsalicylic acid in human blood

We analysed the in vitro interaction between acetylsalicylic acid and vitamin E on the principal antiplatelet sites of action of acetylsalicylic acid, i.e., platelet aggregation, prostanoid production in platelets and leukocytes, and nitric oxide synthesis. Aggregation was measured in whole blood and in platelet-rich plasma (PRP) with ADP, collagen or arachidonic acid as platelet inducers, and we measured the production of thromboxane B2, prostacyclin and nitric oxide. Vitamin E potentiated the antiplatelet effect of acetylsalicylic acid in both whole blood and PRP. In PRP induced with collagen the IC50 for acetylsalicylic acid alone was 339 ± 11.26, and that of acetylsalicylic acid + vitamin E was 0.89 ± 0.09 (P < 0.05). Vitamin E did not enhance inhibition of platelet thromboxane production by acetylsalicylic acid. Vitamin E spared or even increased prostacyclin levels, and acetylsalicylic acid + vitamin E diminished the inhibition of prostacyclin synthesis by acetylsalicylic acid (IC50 acetylsalicylic acid alone = 1.81 ± 0.15 µM; IC50 acetylsalicylic acid + vitamin E = 12.92 ± 1.10 µM, P < 0.05). Vitamin E increased the effect of acetylsalicylic acid on neutrophil nitric oxide production 42-fold (P < 0.05). We conclude that vitamin E potentiates the antiplatelet effect of acetylsalicylic acid in vitro, and thus merits further research in ex vivo studies.

Cytoprotective effect of nonsteroidal antiinflammatory drugs in rat brain slices subjected to reoxygenation after oxygen-glucose deprivation.

The aim of this study was to assess the possible neuroprotective effect of the main nonsteroidal antiinflammatory drugs (NSAIDs) in an experimental model of hypoxia-reoxygenation in rat brain slices. After reoxygenation the increase in lactate dehydrogenase (LDH) efflux was inhibited by nimesulide, celecoxib and meloxicam with an IC(50) in the 10(-6)M range, by flurbiprofen, ibuprofen and diclofenac in the 10(-5)M range, and by salicylic acid, indomethacin, acetylsalicylic acid and mefenamic acid the 10(-4)M range. The effect of other NSAIDs was seen with an IC(50) greater than 10(-3)M. A statistically significant linear correlation between the values of LDH efflux and prostaglandin E(2) was found for NSAIDs whose IC(50) of cytoprotection (LDH efflux) was below 10(-4)M. The concentration of interleukin 10 was increased with nimesulide, celecoxib, meloxicam, flurbiprofen, ibuprofen and diclofenac. Flurbiprofen and diclofenac significantly inhibited the production of lipid peroxides. The increase in brain nitrite levels was significantly reduced with celecoxib, flurbiprofen, diclofenac and salicylic acid. Concentrations of 3-nitrotyrosine were significantly reduced with celecoxib, flurbiprofen, ibuprofen, salicylic acid and ketorolac. In conclusion, NSAIDs with the greatest cytoprotective effect (nimesulide, celecoxib and meloxicam) may exert their effect mainly through the blockade of cyclooxygenase-2 (COX-2) activity. Other compounds with neuroprotective activity may complement their lower anti-COX-2 effect with a slight increase in interleukin 10 and reduced oxidative and nitrosative stress in our model of hypoxia-reoxygenation in rat brain slices.

Effects of triflusal on oxidative stress, prostaglandin production and nitric oxide pathway in a model of anoxia-reoxygenation in rat brain slices.

Acetylsalicylic acid (ASA) is the most widely used drug in the prevention of ischemic vascular accidents, mainly because of its antithrombotic effect. Recently, evidence of a neuroprotective effect has appeared. The aim of this study was to evaluate the neuroprotective effect of triflusal, a fluorinated derivative of ASA, in a model of anoxia-reoxygenation in rat brain slices. Rats (n=10 per group) were treated for 7 days with 1, 10 or 50 mg/kg/day p.o. of triflusal or ASA or solvent (control group), then brain slices were obtained and subjected to a period of anoxia followed by 180 min of reoxygenation. We measured oxidative stress parameters (lipid peroxidation, glutathione system), prostaglandins (PGE(2)), nitric oxide pathway activity (NO) (nitrites+nitrates, constitutive and inducible NO synthase activity) and cell death (lactate dehydrogenase (LDH) efflux). Triflusal decreased cell death in rat brain slices subjected to reoxygenation after anoxia by 21%, 42% and 47% with 1, 10 and 50 mg/kg/day, respectively. This effect was proportionately greater than the effect of ASA (0%, 25% and 24%). The antioxidant effects of triflusal on the biochemical mechanisms of cell damage studied here were also greater than the effects of ASA: lipid peroxidation was reduced by 29%, 35% and 36% with triflusal, and 0%, 19% and 29% with ASA. Inducible NO synthase activity was reduced by 25%, 27% and 30% with triflusal, and 0%, 25% and 24% with ASA. Triflusal can be considered an alternative to ASA as a neuroprotective agent, at least in the experimental model of anoxia-reoxygenation used in the present study.

Cytoprotective effect of hydroxytyrosyl alkyl ether derivatives after oral administration to rats in a model of glucose-oxygen deprivation in brain slices.

This study was designed to determine whether the oral administration of hydroxytyrosol (HT) alkyl ether derivatives has a neuroprotective effect in rats. The animals were treated for 7 days with HT or ethyl, butyl, hexyl, octyl, and dodecyl HT ether. A method of in vitro hypoxia-reoxygenation in brain slices was used. Hexyl, octyl, and dodecyl HT derivatives reduced brain cell death (LDH efflux). Lipid peroxidation and nitrite concentrations were inhibited most by hexyl, octyl, and dodecyl derivatives. Concentrations of 3-nitrotyrosine were reduced by HT butyl, hexyl, octyl, and dodecyl ether derivatives. Interleukin-1β was significantly reduced in brain slices from rats treated with all HT ether derivatives. LDH efflux showed a linear correlation with brain concentrations of lipid peroxides, nitrites plus nitrates, and interleukin 1β. The reduction in oxidative and nitrosative stress and decreased production of pro-inflammatory interleukins may be the basis for the observed neuroprotective effects.

Hydroxytyrosyl alkyl ether derivatives inhibit platelet activation after oral administration to rats

The low lipophilicity of hydroxytyrosol (HT) has motivated efforts to synthesize homologous series with better lipid solubility, such as the ethers, which are more lipophilic than HT. Because HT inhibits platelet aggregation, the aim of the study was to assess the possible anti-platelet effect of five HT ether derivatives (ethyl, butyl, hexyl, octyl and dodecyl) after oral administration to rats. Whole blood collagen-induced platelet aggregation and calcium-induced thromboxane B2 (TxB2), aortic 6-keto-prostaglandin F1α (6-keto-PGF1α) and nitrites+nitrates, plasma concentration of lipid peroxides (TBARS) and red blood cell content of reduced glutathione (GSH) were measured. The administration of 20 mg/kg/day inhibited platelet aggregation, TxB2 and TBARS in a non-linear manner related to the length of the carbon chain, with a cut-off effect in the hexyl derivative. Aortic nitrite and red blood cell GSH production were also increased. The aortic production of 6-keto-PGF1α was unaltered except in the group treated with the dodecyl derivative. The administration of 50 mg/kg/day showed a similar pharmacodynamic profile but without the non-linear effect. In conclusion, HT ethers, especially the hexyl derivative, are a potential alternative to hydroxytyrosol, and their effect merits additional research to determine their role in the prophylaxis of vascular disease.

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).