Inhibition of Endothelial Inflammatory Response by HT-C6, a Hydroxytyrosol Alkyl Ether Derivative.

Hydroxytyrosol (HT) is a bioactive phenolic compound naturally present in olives and extra virgin olive oil (EVOO) which is described as an antioxidant, antitumoral and antiangiogenic molecule. Previous studies of semi-synthetic HT-derivatives presented the hydroxytyrosyl alkyl ether HT-C6 as one of the most potent derivatives studied in the context of antioxidant, anti-platelet and antiangiogenic assays, but its direct effect on inflammation was not reported. In this work, we use RT-qPCR measure of gene expression, protein analysis by Western-blot and immunofluorescence techniques, adhesion and migration functional assays and single-cell monitoring of reactive oxygen species (ROS) in order to explore in vitro the ability of HT-C6 to interfere in the inflammatory response of endothelial cells (ECs). Our results showed that HT-C6 strongly reduces the TNF-α-induced expression of vascular cell adhesion molecule 1 (VCAM1), intercellular cell adhesion molecule 1 (ICAM1), E-selectin (SELE), C-C motif chemokine ligand 2 and 5 (CCL2 and CCL5) in HUVECs, impairing the chemotactic and adhesion potential of these cells towards THP-1 monocytes in vitro. In this work, we define a mechanism of action underlying the anti-inflammatory effect of HT-C6, which involves the abrogation of nuclear factor kappa B (NF-κB) pathway activation in ECs. These results, together with the ability of HT-C6 to reduce ROS formation in ECs, point to this compound as a promising HT-derivative to be tested in the treatment of atherosclerosis.

Antioxidant Effect of Hydroxytyrosol, Hydroxytyrosol Acetate and Nitrohydroxytyrosol in a Rat MPP+ Model of Parkinson's Disease.

3,4-Dihydroxyphenyl ethanol, known as hydroxytyrosol (HTy), is a phenylpropanoid found in diverse vegetable species. Several studies have demonstrated that HTy is a potent antioxidant. Thus, our study is aimed to evaluate the antioxidant effect of HTy and its derivatives, hydroxytyrosol acetate (HTyA) and nitrohydroxytyrosol (HTyN), in a model of oxidative stress induced by 1-methyl-4-phenylpyridinium (MPP+) in rats. Rats were administered intravenously (i.v.) in the tail with 1 mL saline solution or polyphenol compound (1.5 mg/kg) 5 min before intrastriatal infusion of 10 µg MPP+/8 µL. We found that rats injured with MPP+, pretreatment with HTy, HTyA or HTyN significantly decreased ipsilateral turns. This result was consistent with a significant preservation of striatal dopamine levels and decreased lipid fluorescence products (LFP), a marker of oxidative stress. Brain GSH/GSSG ratio, from rats pretreated with HTy or HTyN showed a significant preservation of that marker, decreased as a consequence of MPP+-induced oxidative damage. These results show an antioxidant effect of HTy, HTyA and HTyN in the MPP+ model of Parkinson's disease in the rat.

Evaluation of the Bioavailability and Metabolism of Nitroderivatives of Hydroxytyrosol Using Caco-2 and HepG2 Human Cell Models.

Considering that nitrocatechols present putative effects against Parkinson's disease, the absorption and metabolism of nitroderivatives of hydroxytyrosol (HT) were assessed using human cell model systems. The test compounds nitrohydroxytyrosol (NO2HT), nitrohydroxytyrosyl acetate (NO2HT-A), and ethyl nitrohydroxytyrosyl ether (NO2HT-E) were efficiently transferred across human Caco-2 cell monolayers as an intestinal barrier model, NO2HT-A and NO2HT-E being better (p < 0.05) absorbed (absorption rate (AR) = 1.4 ± 0.1 and 1.5 ± 0.2, respectively) than their precursor, NO2HT (AR = 1.1 ± 0.1). A significant amount of the absorbed compounds remained unconjugated (81, 70, and 33% for NO2HT, NO2HT-A, and NO2HT-E, respectively) after incubation in Caco-2 cells, being available for hepatic metabolism. Nitrocatechols were extensively taken up and metabolized by human hepatoma HepG2 cells as a model of the human liver. Both studies revealed extensive hydrolysis of NO2HT-A into NO2HT, whereas NO2HT-E was not hydrolyzed. Glucuronide (75-55%), methylglucuronide (25-33%), and methyl derivatives (0-12%) were the main nitrocatechol metabolites detected after metabolism in Caco-2 and HepG2 cells. In conclusion, NO2HT, NO2HT-A, and NO2HT-E show high in vitro bioavailability and are extensively metabolized by hepatic cells.

Effect of intracerebral hydroxytyrosol and its nitroderivatives on striatal dopamine metabolism: A study by in vivo microdialysis.

AIMS: The natural phenolic oil compound hydroxytyrosol (HTy) is widely studied because of its antioxidant and neuroprotective properties. Nitroderivatives of HTy have been studied in order to evaluate their putative effects on catechol-O-methyltransferase (COMT) activity. MAIN METHODS: To study its effect on dopamine metabolism, nitrohydroxytyrosol and its lipophilic derivatives (nitrohydroxytyrosyl acetate and ethyl nitrohydroxytyrosyl ether), were administered into the rat corpus striatum through a microdialysis probe. Other catechols (HTy and the known COMT inhibitor Ro 41-0960) were also studied for comparison. KEY FINDINGS: The olive oil phenolic compounds (nitroderivatives and HTy) increased extracellular levels of 3,4-dihydroxyphenylacetic acid during the perfusion with similar maximum values to that of Ro 41-0960 when comparing to basal dialysate levels (approximately 140%). None of the compound series produced a decrease in the homovanillic acid extracellular levels below 75%. Among all novel compounds studied, both lipophilic nitrocatechols (nitrohydroxytyrosyl acetate and ethyl nitrohydroxytyrosyl ether) showed a long-acting effect over time once the perfusion through the microdialysis probe ended. SIGNIFICANCE: In accordance with the actual design of novel COMT inhibitors with a long profile, our results suggest a certain influence of the side chain substituent on the COMT activity that could provide new lipophilic COMT inhibitors.

Differences in the Neuroprotective Effect of Orally Administered Virgin Olive Oil (Olea europaea) Polyphenols Tyrosol and Hydroxytyrosol in Rats.

The neuroprotective effect of virgin olive oil (VOO) polyphenols has been related to their antioxidant effect. The main objective was to analyze how tyrosol and hydroxytyrosol contribute to the antioxidant and neuroprotective effects of VOO in a model of hypoxia-reoxygenation in rat brain slices. Rats were treated per os (po) (10 or 20 mg/kg/day) with hydroxytyrosol ethyl ether (HTEE), tyrosol ethyl ether (TEE), or 3,4-di-o-methylidene-hydroxytyrosol ethyl ether (MHTEE), used as a negative control for antioxidant effects. Lipid peroxidation was inhibited with HTEE, TEE, and MHTEE (from 5.0 ± 1.5 to 2.6 ± 1.5, 4.5 ± 1.5, and 4.8 ± 1.5 nmol/mg protein, respectively). However, all three compounds had similar neuroprotective effects: from 2.8 ± 0.07 to 1.8 ± 0.02 arbitrary units for HTEE, 1.4 ± 0.09 arbitrary units for TEE, and 1.3 ± 0.2 arbitrary units for MHTEE. All three compounds inhibited 3-nitrotyrosine production (from 3.7 ± 0.3 to 1.2 ± 0.03 nmol/0.1 g tissue for HTEE, 1.0 ± 0.2 nmol/0.1 g tissue for TEE, and 1.3 ± 0.1 nmol/0.1 g tissue for MHTEE), prostaglandin E2 production (from 55.7 ± 2.2 to 46.4 ± 1.9 pg/0.1 g tissue for HTEE, 24.7 ± 1.3 pg/0.1 g tissue for TEE, and 27.6 ± 2.6 pg/0.1 g tissue for MHTEE), whereas only HTEE inhibited IL1ß production (from 35.7 ± 1.5 to 21.6 ± 0.8 pg/0.1 g tissue). Pearson correlation coefficients related neuroprotective effect with an antioxidant effect for HTEE (R = 0.72, p < 0.001), and inhibition of nitrosative stress (R = 0.78, 0.67, and 0.66 for HTEE, TEE, and MHTEE, respectively, p < 0.001) and inflammatory mediators (R = 0.72, 0.79, and 0.64 for HTEE, TEE, and MHTEE, respectively, p < 0.001) with all three compounds.

Antioxidant activity of alkyl hydroxytyrosyl ethers in unsaturated lipids.

The antioxidant activity of ethyl and octyl hydroxytyrosyl ethers toward lipids was determined using the Rancimat and open cup methods at high temperatures and 50 °C, respectively. The effect of the unsaturation of the matrix was evaluated using sunflower, soya, and fish refined oils. The antioxidant activities of alkyl hydroxytyrosyl ethers (HTy ethers), hydroxytyrosyl esters, and free hydroxytyrosol are similar, and are much higher than that of α-tocopherol at the same millimolar concentration. The relationship between the induction period and the concentration of the HTy ethers is a sigmoidal curve; an accurate concentration of HTy ethers is necessary to achieve maximum activity, as it increases with the level of matrix unsaturation. The presence of tocopherols in commercial oils affects the antioxidant effect of HTy ethers. Thus, the addition of a low concentration of HTy ethers results in a positive effect, whereas the effect of the addition of high amounts of ethers is slightly less than that of the phenol alone. The addition of HTy ethers to commercial refined oils increases the stability of the oils and preserves tocopherols and polyunsaturated fatty acids from oxidation, enabling the oils to maintain their nutritional properties for longer periods of time.

Synthesis and antioxidant evaluation of isochroman-derivatives of hydroxytyrosol: structure-activity relationship.

Isochroman-derivatives of the natural olive oil phenol hydroxytyrosol (HT) have been synthesised via Oxa-Pictet-Spengler reaction in high yields. Lipophilicity and antioxidant activity were determined to establish the structure-activity relationship of isochromans compared to HT, BHT and α-tocopherol. Antioxidant capacity was tested in two different media: bulk oils, using the Rancimat test, and brain homogenates, by measuring malondialdehyde (MDA) levels as a lipoperoxidation biomarker. In addition, other antioxidant assays (FRAP, ABTS and ORAC) were carried out. Rancimat and MDA results show that antioxidant activity was related with lipophilicity, directly in brain homogenates and inversely in the oils, in agreement with the polar paradox. Free o-diphenolic groups positively determined the activity in the oils, whereas reducing and radical-scavenging activities were related to the number of free hydroxyl moieties. BHT and α-tocopherol showed lower antioxidant activity than isochromans and HT. We conclude that HT-isochromans present significant potential as bioactive compounds.

Synthesis and antioxidant activity of nitrohydroxytyrosol and its acyl derivatives.

A series of nitroderivatives has been synthesized from hydroxytyrosol, the natural olive oil phenol, to increase the assortment of compounds with putative effects against Parkinson's disease. Nitrohydroxytyrosyl esters were obtained from nitrohydroxytyrosol using a chemoselective one-step, high-yield, transesterification procedure. The antioxidant activity of these new series of nitrocatechols was evaluated using FRAP, ABTS, and ORAC assays and compared to that of free hydroxytyrosol. The nitro functional group induced a significant increase in the antioxidant activity of nitrohydroxytyrosol compared to hydroxytyrosol. Regarding nitroester derivatives, variable antioxidant activity was observed depending on the acyl side-chain length; shorter chains maintained or even enhanced the antioxidant activity compared to nitrohydroxytyrosol, decreasing the activity with longer side chains in keeping with their lipophilic nature. Therefore, it may be concluded that nitroester derivatives of hydroxytyrosol, which may be obtained by a simple, high-yield reaction, have elevated antioxidant activity and thus present potential bioactivity.

In vivo striatal measurement of hydroxytyrosol, and its metabolite (homovanillic alcohol), compared with its derivative nitrohydroxytyrosol.

Phenolic compounds were measured by in vivo brain microdialysis in rat striatum. Basal extracellular levels of hydroxytyrosol, homovanillic alcohol and nitro-hydroxytyrosol were not detectable by HPLC with electrochemical detection. However, systemic administration of hydroxytyrosol (20 and 40mg/kg, i.p.) showed a clear increase in the extracellular level of this compound. This increase was accompanied by an increase in the extracellular level of homovanillic alcohol, a metabolite of hydroxytyrosol formed by catechol-O-methyltransferase activity. Perfusion of hydroxytyrosol (20µM) through the microdialysis cannula also produced an increase in the extracellular level of homovanillic alcohol. Systemic administration of nitro-hydroxytyrosol (20 and 40mg/kg, i.p.) produced a small increase in the extracellular level of this compound. Our data show that hydroxytyrosol is a more brain penetrant phenolic compound than nitro-hydroxytyrosol. Accordingly, there is high cerebral metabolism of hydroxytyrosol to produce homovanillic alcohol by catechol-O-methyltransferase activity, that is saturated at the higher administered dose of hydroxytyrosol.

Comparative evaluation of the metabolic effects of hydroxytyrosol and its lipophilic derivatives (hydroxytyrosyl acetate and ethyl hydroxytyrosyl ether) in hypercholesterolemic rats.

Hydroxytyrosol (HT), a virgin olive oil phenolic phytochemical with proven health benefits, has been used to generate new lipophilic antioxidants to preserve fats and oils against autoxidation. The aim of this work is to comparatively evaluate the physiological effects of HT and its lipophilic derivatives, hydroxytyrosyl acetate (HT-Ac) and ethyl hydroxytyrosyl ether (HT-Et), in high-cholesterol fed animals. Male Wistar rats (n = 8) were fed a standard diet (C group), a cholesterol-rich diet (Chol group) or a cholesterol-rich diet supplemented with phenolic compounds (HT group, HT-Ac group and HT-Et group) for 8 weeks. Body and tissue weights, the lipid profile, redox status, and biochemical, hormonal, and inflammatory biomarkers were evaluated. Plasma levels of total cholesterol, LDL cholesterol, glucose, insulin and leptin, as well as malondialdehyde in serum increased in Chol compared to C (p < 0.05). Rats fed the test diets had improved glucose, insulin, leptin and MDA levels and antioxidant capacity status, with HT-Ac being the most effective compound. The studied phenolic compounds also modulated TNF-α and IL-1ß plasma levels compared to Chol. HT-Ac and HT-Et improved adipose tissue distribution and adipokine production, decreasing MCP-1 and IL-1ß levels. Our results confirm the metabolic effects of HT, which are maintained and even improved by hydrophobic derivatives, particularly HT-Ac.

Selective cytotoxic activity of new lipophilic hydroxytyrosol alkyl ether derivatives.

Recent data suggest that hydroxytyrosol, a phenolic compound of virgin olive oils, has anticancer activity. This communication reports the synthesis of decyl and hexadecyl hydroxytyrosyl ethers, as well as the cytotoxic activity of hydroxytyrosol and a series of seven hydroxytyrosol alkyl ether derivatives against A549 lung cancer cells and MRC5 non-malignant lung fibroblasts. Hydroxytyrosyl dodecyl ether (HTDE) showed the highest selective cytotoxicity, and possible mechanisms of action were investigated; results suggest that HTDE can moderately inhibit glycolysis, induce oxidative stress, and cause DNA damage in A549 cells. The combination of HTDE with the anticancer drug 5-fluorouracil induced a synergistic cytotoxicity in A549 cancer cells but not in non-malignant MRC5 cells. HTDE also displayed selective cytotoxicity against MCF7 breast cancer cells versus MCF10 normal breast epithelial cells in the 1-30 µM range. These results suggest that the cytotoxicity of HTDE is more potent and selective than that of parent compound hydroxytyrosol.

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.

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-1ß 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 1ß. The reduction in oxidative and nitrosative stress and decreased production of pro-inflammatory interleukins may be the basis for the observed neuroprotective effects.

Digestive stability of hydroxytyrosol, hydroxytyrosyl acetate and alkyl hydroxytyrosyl ethers.

The digestive stability of two natural antioxidant compounds present in virgin olive oil, hydroxytyrosol (HTy) and hydroxytyrosyl acetate (HTy-Ac) and a new series of hydroxytyrosyl ethers (methyl, ethyl and butyl hydroxytyrosyl ethers) was evaluated by a simulated digestion procedure. High recovery of all compounds after gastric digestion was obtained, although they showed a statistically significant lower stability after pancreatic-bile salts digestion. HTy-Ac was partially hydrolyzed into free HTy, whereas after intestinal digestion, HTy was converted into 3,4-dihydroxyphenyl acetic acid (DOPAC), and HTy-Ac was hydrolyzed to HTy and subsequently transformed into DOPAC. In contrast, no chemical modification of hydroxytyrosyl ethers during in vitro digestion was observed. In summary, HTy, HTy-Ac and hydroxytyrosyl ethers show high and interesting digestive stability and the new synthetic hydroxytyrosyl ethers showed enhanced chemical stability compared to HTy and HTy-Ac.

Alkyl hydroxytyrosyl ethers show protective effects against oxidative stress in HepG2 cells.

Alkyl hydroxytyrosyl ethers (methyl, ethyl, propyl, and butyl ethers) have been synthesized from hydroxytyrosol (HTy) in response to the increasing food industry demand of new lipophilic antioxidants. Having confirmed that these compounds reach portal blood partially unconjugated and thus are effectively absorbed, their potential antioxidant activity was evaluated in the human hepatocarcinoma cell line (HepG2). The effects of 0.5-10 µM alkyl hydroxytyrosyl ethers on HepG2 cell integrity and redox status were assessed as well as the protective effect against oxidative stress induced by tert-butylhydroperoxide (t-BOOH). Cell viability (Crystal violet) and cell proliferation (BrdU assay) were measured as markers of cell integrity, concentration of reduced glutathione (GSH), generation of reactive oxygen species (ROS), and activity of antioxidant enzymes glutathione peroxidase (GPx) and glutathione reductase (GR) as markers of redox status and determination of malondialdehyde (MDA) as a marker of lipid peroxidation. Direct treatment of HepG2 with alkyl hydroxytyrosyl ethers induced slight changes in cellular intrinsic antioxidants status, reducing ROS generation and inducing changes in GPx and GR activities. Pretreatment of HepG2 cells with alkyl hydroxytyrosyl ethers counteracted cell damage induced by t-BOOH, partially after 2 h and completely after 20 h, by increasing GSH and decreasing ROS generation, MDA levels, and antioxidant enzyme (GPx and GR) activity. According to these results the alkyl hydroxytyrosyl ethers show clear protective effects against oxidative stress, related to their lipophilic nature, that are similar to or even higher than those of their precursor, HTy.

Transepithelial transport and metabolism of new lipophilic ether derivatives of hydroxytyrosol by enterocyte-like Caco-2/TC7 cells.

Intestinal transport and metabolism of a series of ether derivatives of the natural antioxidant hydroxytyrosol with differing alkyl chain lengths (methyl, ethyl, propyl, and butyl) were evaluated at 1, 2, and 4 h using a two-compartment transwell system containing human enterocyte (differentiated Caco-2/TC7) monolayers, which simulates the small intestinal barrier. All four ether derivatives were transferred across the enterocyte monolayers with Papp(apical-basolateral) values between 32.6 and 43.5 cm/s × 10(-6). One hour after apical loading, the predominant forms of the compounds on the basolateral side were unmodified molecules. Glucuronides and methylated metabolites were also present in both the apical and basolateral compartments, with conjugated metabolites preferentially transported to the basolateral side. The rate of metabolism increased according to the lipohilicity of the ether derivative (butyl > propyl > ethyl > methyl). In conclusion, hydroxytyrosyl ethers are rapidly absorbed across, and partially metabolized by, Caco-2/TC7 cell monolayers, in keeping with their lipophilic nature.

Uptake and metabolism of new synthetic lipophilic derivatives, hydroxytyrosyl ethers, by human hepatoma HepG2 cells.

As a response to the increasing demand by the food industry for new synthetic lipophilic antioxidants, hydroxytyrosyl methyl, ethyl, propyl and butyl ethers have been synthesized from hydroxytyrosol, with similar or even higher antioxidant activity than free hydroxytyrosol. The uptake and metabolism of hydroxytyrosyl ethers with different alkyl side chain lengths (methyl, ethyl, propyl, and butyl) was studied after incubation for 2 and 18 h with HepG2 cells as a model of the human liver. LC-DAD and LC-MS were used for the identification of metabolites in culture media, cell lysates and samples hydrolyzed with beta-glucuronidase and sulfatase. In vitro conjugation reactions of pure phenols were also performed. The results show an extensive uptake and metabolism by HepG2 cells after 18 h of incubation. A direct relationship between the lipophilic nature of the compound and the biotransformation yield was observed. Similar ratio of methyl and glucuronide forms were detected after 2 h of incubation while at 18 h high amounts of methylglucuronides and glucuronide metabolites were identified together with low amounts of methyl conjugates. In conclusion, alkyl hydroxytyrosyl ethers could be metabolized by the liver, their metabolic rate being higher for the more lipophilic compounds.

Virgin olive oil polyphenol hydroxytyrosol acetate inhibits in vitro platelet aggregation in human whole blood: comparison with hydroxytyrosol and acetylsalicylic acid.

Hydroxytyrosol acetate (HT-AC) is a polyphenol present in virgin olive oil (VOO) at a proportion similar to hydroxytyrosol (HT) (160-479 micromol/kg oil). The present study was designed to measure the in vitro platelet antiaggregating activity of HT-AC in human whole blood, and compare this effect with that of HT and acetylsalicylic acid (ASA). The experiments were designed according to the standard procedure to investigate the activity of ASA. HT-AC and HT inhibited platelet aggregation induced by ADP, collagen or arachidonic acid in both whole blood and platelet-rich plasma (PRP). ASA and HT-AC had a greater effect in whole blood than in PRP when ADP or collagen was used as inducer. ASA and HT-AC had a greater effect in PRP+leucocytes than in PRP alone. All three compounds inhibited platelet thromboxane B2 and leucocyte 6-keto-prostaglandin F1alpha (6-keto-PF1 alpha) production. The thromboxane/6-keto-PGF1alpha inhibition ratio (as an indirect index of the prostanoid equilibrium) was 10.8 (SE 1) for HT-AC, 1.0 (SE 0.1) for HT and 3.3 (SE 0.2) for ASA. All three compounds stimulated nitric oxide production, although HT was a weaker effect. In our experiments only concentrations higher than 500 microm (HT) or 1 mm (HT-AC and ASA) inhibited 3-nitrotyrosine production. All three compounds inhibited the production of TNFalpha by leucocytes, with no significant differences between them. In quantitative terms HT-AC showed a greater antiplatelet aggregating activity than HT and a similar activity to that of ASA. This effect involved a decrease in platelet thromboxane synthesis and an increase in leucocyte nitric oxide production.

New lipophilic tyrosyl esters. Comparative antioxidant evaluation with hydroxytyrosyl esters.

New lipophilic esters of tyrosol, a naturally occurring phenol with interesting biological properties, have been synthesized in good yields by a chemoselective procedure, using lipase from Candida antarctica or p-toluenesulfonic acid as catalysts. Their antioxidant activities have been evaluated by the Rancimat test in lipophilic food matrices, as well as by FRAP and ABTS assays in methanolic solutions, and compared with those of previously synthesized hydroxytyrosyl esters. Free tyrosol, hydroxytyrosol, butylhydroxytoluene, and alpha-tocopherol were used as standards. All methods used for the antioxidant activity evaluation emphasized the high influence of the ortho-diphenolic structure on the antioxidant capacity, tyrosol and its derivatives being less active than hydroxytyrosol and its analogues and even less than BHT and alpha-tocopherol. In addition, the Rancimat test revealed a lower activity for ester derivatives than for their respective reference compounds (HTy or Ty), in agreement with the polar paradox. On the other hand, FRAP and ABTS methods reported an opposite behavior between the synthetic esters and their respective references. Thus, hydroxytyrosyl esters were more active than HTy, whereas tyrosyl esters were less active than Ty. The length and nature of the acyl side chain did not seem to play an important role in the antioxidant activity of either the hydroxytyrosyl or tyrosyl ester series, since no significant differences were observed among them.

Neuroprotective effect of hydroxytyrosol and hydroxytyrosol acetate in rat brain slices subjected to hypoxia-reoxygenation.

Hydroxytyrosol (HT) and hydroxytyrosol acetate (HT-AC) are two well-known phenolic compounds with antioxidant properties that are present in virgin olive oil (VOO). Because VOO has shown neuroprotective effects in rats, the purpose of the present study was to investigate the possible neuroprotective effect of HT and HT-AC in a model of hypoxia-reoxygenation in rat brain slices after in vitro incubation of these compounds or after 7 days of oral treatment with 5 or 10 mg/kg per day. Lactate dehydrogenase (LDH) efflux to the incubation medium was measured as a marker of brain cell death. HT and HT-AC inhibited LDH efflux in a concentration-dependent manner, with 50% inhibitory concentrations of 77.78 and 28.18 microM, respectively. Other well-known antioxidants such as vitamin E and N-acetyl-cysteine had no neuroprotective effect in this experimental model. After 1 week of treatment, HT (5 and 10 mg/kg per day p.o.) reduced LDH efflux by 37.8% and 52.7%, respectively, and HT-AC reduced LDH efflux by 45.4% and 67.8%. These data are additional evidence of the cytoprotective effect of VOO administration, and provide a preliminary basis for further study of these polyphenols as potential neuroprotective compounds.