José L. Espartero

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.

Structural analysis of the exopolysaccharides produced by Lactobacillus spp. G-77.

The exopolysaccharide produced by a ropy strain of Lactobacillus spp. G-77 in a semi-defined medium, was found to be a mixture of two homopolymers composed of D-Glc. The two poly-saccharides were separated and, on the basis of monosaccharide and methylation analyses, 1H, 13C, 1D and 2D NMR experiments, one of the polysaccharides was shown to be a 2-substituted-(1-3)-beta-D-glucan, identical to that described for the EPS from Pediococcus damnosus 2.6 (M.T. Dueñas-Chasco, M.A. Rodríguez-Carvajal, P. Tejero-Mateo, G. Franco-Rodríguez, J. L. Espartero, A. Irastorza-Iribar, and A.M. Gil-Serrano, Carbohydr. Res., 303 (1997) 453-458), and the other polysaccharide was shown to consist of repeating units with the following structure [formula: see text]

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.

Synthesis of Hydroxytyrosyl Alkyl Ethers from Olive Oil Waste Waters

The preparation of a new type of derivatives of the naturally occurring antioxidant hydroxytyrosol is reported. Hydroxytyrosyl alkyl ethers were obtained in high yield by a three-step procedure starting from hydroxytyrosol isolated from olive oil waste waters. Preliminary results obtained by the Rancimat method have shown that these derivatives retain the high protective capacity of free hydroxytyrosol.

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.

Anti-apoptotic activity of hydroxytyrosol and hydroxytyrosyl laurate.

Hydroxytyrosol (HyT) is a polyphenol primarily released in olive mill wastewater and in olive oil. In animal and cell model studies, HyT and its metabolites have strong antioxidant and antimicrobial activities, as well as beneficial effects on the cardiovascular system and in several human diseases. Differently, many researchers reported that HyT down-regulates tumor cell viability and cell cycle progression, and induces reactive oxygen species (ROS) production and apoptosis. In this study we have investigated the effects of HyT and the corresponding ester hydroxytyrosyl laurate in U937 cells, a human monocytoid cell line, and in C2C12 myoblasts, a murine proliferating muscle cell model, after apoptotic death induction. Inverted, light and transmission electron microscopy have been utilized to characterize cell death patterns. H2O2, at the concentrations known to induce apoptosis, was utilized as cell death trigger. The results obtained show that laur-HyT has a protective antioxidant effect against H2O2 treatment, greater than HyT, so having a role in the prevention of apoptotic death in normal and tumor cells. These data suggest these compounds as good candidate for novel therapeutic strategies.

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.

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.

A generalization of the base effect on the diastereoselective synthesis of sulfinic and phosphinic esters

Abstract Various chiral secondary alcohols have been used to study the dependence of the stereochemical outcome of sulfinate and phosphinate ester synthesis on the nature of the base used to catalyse the reaction. From this study it has been shown that the achiral stereodirecting base effect determined in the DAG methodology is a general behaviour in the asymmetric synthesis of sulfinate and phosphinate esters.

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.