Neuroprotective effect of hydroxytyrosyl alkyl ether derivatives

MI Lopez-Leiva1, JA Lopez-Villodres2, A Madrid3, L Romero1, A Guerrero2, S Cabrerizo4, JP De la Cruz2, JA Gonzalez-Correa2,1. 1Faculty of Health Sciences. University of Malaga, Research Group LIAIT. Department of Pharmacology, Spain, 2School of Medicine, Research Group LIAIT. Department of Pharmacology, Spain, 3University Hospital Carlos Haya, UGC Pediatric, Spain, 4Hospital Ronda, Health Primary Care, Spain

Virgin olive oil was shown to have a neuroprotective effect in healthy rats. Moreover, this effect has also been described for pure Hydroxytyrosol (HT) and HT acetate. Because of these effects, including neuroprotection, efforts have been made to synthesize HT derivatives with a better hydrophile/lipophile balance in order to improve its pharmacodynamic profile and increase its bioavailability.

The aims of the present study was to investigate the antioxidant and possible neuroprotective and antioxidant effects of five alkyl hydroxytyrosyl (HT) ethers (ethyl (E), butyl (B), hexyl (H), octyl (O) and dodecyl (D)) in rat brain slices and to assess whether a neuroprotective effect of hydroxytyrosol (HT) alkyl ether derivatives occurs after oral administration to rats.

Methods: Male Wistar rats weighing 200-250 g were used. All animals were housed at the under standard temperature and light/dark conditions, with access to food and water ad libitum. “In vitro” study planned in three parts. First we tested all compounds in rat brain slices without chemical stimulation. Secondly, oxidative stress was induced with chemicals: ferrous salts (50 µM) for lipid peroxidation and diethylmaleate (10 mM) to reduce the glutathione (GSH) system. Then we explored the neuroprotective effect of these compounds in an experimental model of hypoxia-reoxygenation in rat brain slices (De la Cruz et al., 2004). For the “ex vivo” study used 6 groups of animals (6 animals per group) were used: a control group (treated with saline p.o.), a group treated with HT (10 mg/kg/day p.o.) and four groups treated with HT alkyl ethers (ethyl, butyl, octyl and dodecyl HT ether) at dose of 10 mg/kg/day p.o. All compounds were given once per day for seven days by gavage. End of treatment the rats were anesthetized with 40 mg/kg of sodium pentobarbital and killed by decapitation. Analytical techniques: tissue damage was measured by examining LDH efflux to the incubation solution. Oxidative stress by TBARs and glutathione levels. Inflammatory markers by determining, PGE2, interleukin1ß and nitric oxide production in brain tissue. Statistical analysis was performed using the t student test and ANOVA.

Results: Hydroxytyrosol and alkyl HT ethers reduced brain cell death in a concentration-dependent manner (values of IC50 mcM respect reduction LDH efflux: 65,14±6,03 (HT), 36,90±5,51 (E), 7,25±0,45 (B), 24,8±4,13 (H), 34,01±5,85 (O) and 60,81±7,59 (D)). Butyl HT ether was the most potent derivative with IC50 values significantly higher than HT. Administration of hydroxytyrosol alkyl ethers to rats reduced brain cell death in all the experimental periods of hypoxia-reoxygenation. After the reoxygenation period only HT and HT octyl and dodecyl ether derivatives significantly reduced LDH efllux with respect to brain slices from non-treated rats (26.60%, 45.33% and 66.00%, respectively)

Conclusion: alkyl HT ether derivatives compounds showed a neuroprotective effect that was related with their antioxidant effect. Our results established a relationship between these effects and the length of the carbon chain (maximum effect in the range of C4-C8).