A simple analytical method for the quantitative determination of phenols, flavones, and lignans in virgin olive oils was developed. The polar fraction was isolated from small amounts of oil sample (2.5 g) by solid-phase extraction (SPE) using diol-phase cartridges, and the extract was analyzed by reversed-phase HPLC coupled with diode array UV detection. Chromatographic separation of pinoresinol, cinnamic acid, and 1-acetoxypinoresinol was achieved. Repeatability (RSD < 6.5%), recovery (> 90%), and response factors for each identified component were determined. SPE on amino-phase cartridges was used for isolating acidic phenols and as an aid for phenol identification. For the first time, 2-(4-hydroxyphenyl)ethyl acetate was detected in olive oils. The aldehydic structure of the ligstroside aglycon was confirmed by NMR spectroscopy. The colorimetric determination of total o-diphenolic compounds by reaction with molybdate was consistent with their HPLC determination. Differences between results obtained by liquid-liquid extraction and SPE were not statistically significant.

Antioxidant activities of lipophilic hydroxytyrosyl acetate, palmitate, oleate, and linoleate were compared with those of hydroxytyrosol, alpha-tocopherol, and butylhydroxytoluene (BHT) in both glyceridic matrix and biological systems. Aliquots of a glyceridic matrix spiked with various concentrations of antioxidant were subjected to accelerated oxidation in a Rancimat apparatus operated at 90 degrees C. The relationships between induction time (IT) and antioxidant concentration (mmol/kg) presented by hydroxytyrosol and hydroxytyrosyl acetate, palmitate, oleate, and linoleate were similar. Hydroxytyrosol and its esters showed greater antioxidant activity than alpha-tocopherol or BHT. We also evaluated the capacity of hydroxytyrosyl esters to protect proteins and lipids against oxidation caused by peroxyl radicals, using a brain homogenate as an ex vivo model. All tested compounds showed a protective effect in these systems, which was greater in preventing the generation of carbonyl groups in protein than of malondialdehyde in lipid. Inclusion of a lipophilic chain in the hydroxytyrosol molecule enhanced its antioxidant capacities in this biological model.

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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.

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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.

In olive oils, relationships between oxidative stability, glyceridic composition, and antioxidant content were investigated. Lipid matrices, obtained by purification of olive and high-oleic sunflower oils, were spiked with hydroxytyrosol, alpha-tocopherol, and mixtures of them and then subjected to oxidation in a Rancimat apparatus at 100 degrees C. At the same concentration of antioxidants, induction time (IT) decreased as the unsaturation rate of the matrix increased, but only fair correlations were found with fatty acid composition. Oxidative susceptibility (OS(TAG)) was calculated as a function of the relative oxidation rate of the triacylglycerols, and a linear relationship-IT (h) = (a + b)OS(TAG)-between induction time and this parameter showed a good correlation coefficient (r > 0.990, p < 0.001). In the case of matrices with a single antioxidant, origin ordinate (a) and slope (b) can be calculated as a function of the antioxidant concentration. In matrices spiked with mixtures of hydroxytyrosol and alpha-tocopherol, a simple relationship between the coefficients a and b and the concentration of antioxidants cannot be established because additive and subtractive effects occur depending on the relative concentrations of both antioxidants. However, approximate values for these coefficients can be obtained, allowing the estimation of the oil stability. In various olive oils, an acceptable agreement was found between the IT experimentally determined and that calculated from the oil composition. These results confirmed that the Rancimat stability of olive oils mainly depends on triacylglycerol composition and concentrations of o-diphenols and alpha-tocopherol.

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.