Distribution of hydroxytyrosol and hydroxytyrosol acetate in olive oil emulsions and their antioxidant efficiency

We employed a kinetic method to determine the distributions of the antioxidants hydroxytyrosol (HT) and hydroxytyrosol acetate (HTA) between the oil, aqueous, and interfacial regions of a model food emulsion composed of stripped olive oil, acidic water, and a blend of Tween 80 and Span 80 [hydrophilic–lipophilic balance (HLB) = 8.05] as an emulsifier. HT is oil-insoluble, but HTA is both oil- and water-soluble (partition constant P(O)(W) = 0.61). Results indicate that, at a given emulsifier volume fraction Φ(I), the fraction of HTA in the interfacial region is higher than that of HT. The percentage of both antioxidants increases with an increasing Φ(I), so that % HT > 40% at Φ(I) = 0.005 and % HT > 80% at Φ(I) = 0.04. HTA appears to be a better antioxidant than HT, as shown by an accelerated oxidative test (Schaal oven method). A correlation between their distribution in the emulsion and their efficiency was established.     

Antioxidant activity of hydroxytyrosol acetate compared with that of other olive oil polyphenols

Hydroxytyrosol acetate was synthesized, and the antioxidant activity of this olive oil component was assessed in comparison with that of other olive oil components, namely hydroxytyrosol, oleuropein, 3,4-DHPEA-EA, and alpha-tocopherol in bulk oil and oil-in-water emulsions. The activity of the compounds was also assessed by scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. Hydroxytyrosol acetate had a weaker DPPH radical scavenging activity than hydroxytyrosol, oleuropein, or 3,4-DHPEA-EA but it had a radical scavenging activity similar to that of alpha-tocopherol. In oil, the antioxidant activity of hydroxytyrosol acetate was much higher than that of alpha-tocopherol or oleuropein, but in an emulsion 3,4-DHPEA-EA and alpha-tocopherol were more effective as antioxidants than hydroxytyrosol acetate. The antioxidant activity of hydroxytyrosol acetate was rather similar to that of hydroxytyrosol in oil and emulsions despite the difference in DPPH radical scavenging activity.     

Nutritional benefit of olive oil: the biological effects of hydroxytyrosol and its arylating quinone adducts

Olive oil is the essential component of the Mediterranean diet, a nutritional regimen gaining ever-increasing renown for its beneficial effects on inflammation, cardiovascular disease, and cancer. A unique characteristic of olive oil is its enrichment in oleuropein, a member of the secoiridoid family, which hydrolyzes to the catechol hydroxytyrosol and functions as a hydrophilic phenolic antioxidant that is oxidized to its catechol quinone during redox cycling. Little effort has been spent on exploring the biological properties of the catechol hydroxytyrosol quinone, a strong arylating electrophile that forms Michael adducts with thiol nucleophiles in glutathione and proteins. This study compares the chemical and biological characteristics of hydroxytyrosol with those of the tocopherol family in which Michael adducts of arylating desmethyltocopherol quinones have been identified and correlated with biologic properties including cytotoxicity and induction of endoplasmic reticulum stress. It is noted that hydroxytyrosol and desmethyltocopherols share many similarities, suggesting that Michael adduct formation by an arylating quinone electrophile may contribute to the biological properties of both families, including the unique nutritional benefit of olive oil.     

Structural characterization of the metabolites of hydroxytyrosol, the principal phenolic component in olive oil, in rats

Hydroxytyrosol is quantitatively and qualitatively the principal phenolic antioxidant in olive oil. Recently it was shown that hydroxytyrosol and five metabolites were excreted in urine when hydroxytyrosol was dosed intravenously or orally in an olive oil solution to rats. The conclusive identification of three metabolites of hydroxytyrosol by MS/MS as a monosulfate conjugate, a 3-O-glucuronide conjugate, and 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid) has been established in this investigation. The structural configurations of the glucuronide conjugate and 4-hydroxy-3-methoxyphenylacetic acid were confirmed by (1)H NMR. The radical scavenging potencies of homovanillic acid, homovanillic alcohol, hydroxytyrosol, and the metabolites were examined with the radical 2,2-diphenyl-1-picrylhydrazyl. These studies showed them to be potent antioxidants with SC(50) values of 14.8 and 11.4 microM for homovanillic acid and homovanillic alcohol, respectively. The 3-O-glucuronide conjugate was more potent than hydroxytyrosol, with an SC(50) of 2.3 in comparison to 11.0 microM, and the monosulfate conjugate was almost devoid of radical scavenging activity.     

Lipid-lowering and antioxidant effects of hydroxytyrosol and its triacetylated derivative recovered from olive tree leaves in cholesterol-fed rats

This study was designed to test the lipid-lowering and antioxidative activities of triacetylated hydroxytyrosol compared with its native compound, hydroxytyrosol, purified from olive tree leaves. Wistar rats fed a standard laboratory diet or a cholesterol-rich diet for 16 weeks were used. The serum lipid levels, the thiobarbituric acid-reactive substances (TBARS) level, as an indicator of lipid peroxidation, and the activity of superoxide dismutase (SOD) as well as that of catalase (CAT) were examined. The cholesterol-rich diet induced hypercholesterolemia that was manifested in the elevation of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C). Administration of hydroxytyrosol and triacetylated hydroxytyrosol (3 mg/kg of body weight) decreased the serum levels of TC, TG, and LDL-C significantly and increased the serum level of high-density lipoprotein cholesterol (HDL-C). Furthermore, the content of TBARS in liver, heart, kidney, and aorta decreased significantly when hydroxytyrosol and its triacetylated derivatives were orally administered to rats compared with those fed a cholesterol-rich diet. In addition, triacetylated hydroxytyrosol and hydroxytyrosol increased CAT and SOD activities in the liver. These results suggested that the hypolipidemic effect of triacetylated hydroxytyrosol and hydroxytyrosol might be due to their abilities to lower serum TC, TG, and LDL-C levels as well as to their antioxidant activities preventing the lipid peroxidation process.     

Metabolism of the olive oil phenols hydroxytyrosol, tyrosol, and hydroxytyrosyl acetate by human hepatoma HepG2 cells

To study the potential hepatic metabolism of olive oil phenols, human hepatoma HepG2 cells were incubated for 2 and 18 h with hydroxytyrosol, tyrosol, and hydroxytyrosyl acetate, three phenolic constituents of olive oil. After incubation, culture media and cell lysates were hydrolyzed with beta-glucuronidase and sulfatase and analyzed by LC-MS. In vitro methylation, glucuronidation, and sulfation of pure phenols were also performed. Methylated and glucuronidated forms of hydroxytyrosol were detected at 18 h of incubation, together with methylglucuronidated metabolites. Hydroxytyrosyl acetate was largely converted into free hydroxytyrosol and subsequently metabolized, yet small amounts of glucuronidated hydroxytyrosyl acetate were detected. Tyrosol was poorly metabolized, with <10% of the phenol glucuronidated after 18 h. Minor amounts of free or conjugated phenols were detected in cell lysates. No sulfated metabolites were found. In conclusion, olive oil phenols can be metabolized by the liver as suggested by the results obtained using HepG2 cells as a hepatic model system.     

Synthesis of the antioxidant hydroxytyrosol using tyrosinase as biocatalyst

Hydroxytyrosol (HTyr), a natural ortho-diphenolic antioxidant with health-beneficial properties that mainly occurs in virgin olive oil and olive oil mill waste waters (also known as vegetative waters), has been enzymatically synthesized using mushroom tyrosinase. This o-diphenol (not commercially available) was obtained from its monophenolic precursor tyrosol (commercially available) in the presence of both tyrosinase and ascorbic acid. The reaction synthesis is continuous, easy to perform, and adaptable to a bioreactor for industrial purposes. The HTyr concentration is time-predicted, and the yield of reaction can be 100%. The synthesis method reported here is an alternative approach to obtain this compound in an environmentally friendly way.     

Evaluation of the antioxidant capacity of individual phenolic compounds in virgin olive oil

Virgin olive oil has a high resistance to oxidative deterioration due to its tryacylglycerol composition low in polyunsaturated fatty acids and due to the presence of a group of phenolic antioxidants composed mainly of polyphenols and tocopherols. We isolated several phenolic compounds of extra virgin olive oil (phenyl-ethyl alcohols, lignans, and secoiridoids) by semipreparative high-performance liquid chromatography (HPLC) and identified them using ultraviolet, atmospheric pressure chemical ionization, and electrospray ionization MS detection. The purity of these extracts was confirmed by analytical HPLC using two different gradients. Finally, the antioxidant capacity of the isolated compounds was evaluated by measuring the radical scavenging effect on 1,1-diphenyl-2-picrylhydrazyl radical, by accelerated oxidation in a lipid model system (OSI, oxidative stability instrument), and by an electrochemical method.     

Direct analysis of total antioxidant activity of olive oil and studies on the influence of heating

Aim of this study was to evaluate the total antioxidant activity (TAA) of extra virgin olive oil (EVOO) and the effect of heating on the alpha-tocopherol content and TAA in relation to the presence of polyphenols, heating time, and temperature. Experiments included the measurement by ABTS decolorization assay of antioxidant capacity of alpha-tocopherol and 14 simple phenolic compounds present in EVOO, either dissolved in ethanol or added to refined olive oil, and the evaluation of TAA, total phenols, and alpha-tocopherol of six commercial EVOO and three olive oils. Finally, four experimental oils were prepared from refined olive oil containing a fixed amount (300 ppm) of alpha-tocopherol and increasing amounts of polyphenols (25, 125, 225, and 326 ppm) extracted from EVOO. The thermal stability of experimental oils under domestic heating conditions (heating time from 30 to 120 min, heating temperature from 160 to 190 degrees C) was studied by evaluating the loss of alpha-tocopherol and TAA according to a Latin square design. Results indicate that TAA of commercial oils is mainly due to their phenol and alpha-tocopherol content. Heating experiments suggest that polyphenols from EVOO are effective stabilizers of alpha-tocopherol during olive oil heating, thus contributing to the nutritional value of cooked foods.     

Contribution of the phenolic fraction to the antioxidant activity and oxidative stability of olive oil

Antioxidant activity of the phenolic fraction of extra virgin olive oil (EVOO) was measured by means of a chemical and an electrochemical method. Both methods were tested in predicting the oxidative spoilage and stability to oxidation of 22 EVOO samples and resulted correlated with peroxide values and oxidative stability measured by Rancimat. The main phenolic compounds of EVOOs were detected by HRGC. To study the contribution of single polyphenols (PPs) to antioxidant activity of phenolic fraction and oxidative stability of EVOOs, multivariate statistical analyses were applied on HRGC data. An isomer of oleuropein aglycon was shown to affect significantly antioxidant activity of phenolic fraction but not oil stability to oxidation. No individual compounds was identified as the main cause of the overall antioxidant activity, and the total polyphenol determination by the Folin reagent was better correlated to antioxidant activity and oxidative stability than each tested PP or PPs groups such as o-diphenols.     

Static-dynamic superheated liquid extraction of hydroxytyrosol and other biophenols from alperujo (a semisolid residue of the olive oil industry)

Hydroxytyrosol and other olive biophenols (OBPs) such as tyrosol, verbascoside, apigenin-7-glucoside, and alpha-taxifolin have been extracted from alperujo by using static-dynamic superheated liquids. Multivariate methodology has been used to carry out a detailed optimization of the extraction. Under the optimal working conditions no further extraction of the target analytes was achieved after 27 min (up to 2800 and 1500 mg/kg of hydroxytyrosol and tyrosol, respectively), so complete removal of them within this interval was assumed. The extract was injected into a chromatograph-photodiode array detector assembly for individual separation-quantification. The efficacy of ethanol/water mixtures to extract OBPs from alperujo has been demonstrated and compared with that of a conventional stirring-based method. These less toxic extractant mixtures are of interest with a view to future human uses of OBPs.     

Unsaturated fatty alcohol derivatives of olive oil phenolic compounds with potential low-density lipoprotein (LDL) antioxidant and antiobesity properties

A new route for the synthesis of fatty alcohol derivatives of hydroxytyrosol and other olive oil phenolic compounds was developed to allow the preparation of unsaturated derivatives. The biological activity of synthesized compounds was evaluated. Most of the compounds presented a significant antioxidant activity on low-density lipoprotein (LDL) particles. The activity of the tested products was significantly influenced by the number and position of unsaturations as well as modifications on the polar head of the synthesized compounds. Some of them presented modulation of food intake in rats and, due to their molecular similarity with CB(1) endogenous ligands, the endocannabinoid system and PPAR-α were also evaluated as potential targets. The pharmacodynamics could not be totally explained by CB(1) and PPAR-α receptor interactions because only two of the four compounds with biological activity showed a CB(1) activity and all of them presented low PPAR-α affinity, not justifying its whole in vivo activity. The hydroxytyrosol linoleylether (7) increased LDL resistance to oxidation with a capacity similar to that of hydroxytyrosol and was the most active in vivo compound with a hypophagic effect comparable to that of oleoylethanolamine. We consider that this compound could be a good lead compound for future drug development in obesity treatments.     

Analysis of total contents of hydroxytyrosol and tyrosol in olive oils

The most abundant phenolic compounds in olive oils are the phenethyl alcohols hydroxytyrosol and tyrosol. An optimized method to quantify the total concentration of these substances in olive oils has been described. It consists of the acid hydrolysis of the aglycons and the extraction of phenethyl alcohols with a 2 M HCl solution. Recovery of the phenethyl alcohols from oils was very high (<1% remained in the extracted oils), and the limits of quantification (LOQ) were 0.8 and 1.4 mg/kg for hydroxytyrosol and tyrosol, respectively. Precision values, both intraday and interday, remained below 3% for both compounds. The final optimized method allowed for the analysis of several types of commercial olive oils to evaluate their hydroxytyrosol and tyrosol contents. The results show that this method is simple, robust, and reliable for a routine analysis of the total concentration of these substances in olive oils.     

Production in large quantities of highly purified hydroxytyrosol from liquid-solid waste of two-phase olive oil processing or "Alperujo"

The effect of hydrothermal treatment of two-phase olive waste (alperujo) on the solubilization of hydroxytyrosol was studied. Different conditions of saturated steam were assayed. A high amount of hydroxytyrosol was solubilized and increased with increasing steaming temperature and time, reaching 1.4-1.7 g/100 g of dry alperujo. The effect of acidic (H(2)SO(4)) and basic (NaOH) catalysts was also evaluated. Acid-catalyzed treatment was more effective at milder conditions, whereas the alkali-catalyzed conditions were not very suitable. In the present study, the extracted hydroxytyrosol was purified by means of a new, simple, and inexpensive chromatographic system, under international patent application (PCT/ES02/00058). From 1000 kg of alperujo, with 70% humidity, can be obtained approximately 4.5-5 kg of hydroxytyrosol. After a purification process, at least 3 kg of hydroxytyrosol, at 90-95% purity, would be obtained. The purified compound was identified by HPLC/UV and (1)H and (13)C NMR analyses, and its antioxidant activity was tested on refined olive oil without antioxidants by Rancimat method. The oxidative stability of refined olive oil was increased by a factor of 1.71 in the presence of 100 ppm of hydroxytyrosol.     

Mild photochemical synthesis of the antioxidant hydroxytyrosol via conversion of tyrosol

Hydroxytyrosol, a naturally occurred orthodiphenolic antioxidant molecule found in olive oil and olive mill wastewaters, was obtained from the wet hydrogen peroxide photocatalytic oxidation of its monophenolic precursor tyrosol. The liquid-phase oxidation of tyrosol to hydroxytyrosol was performed by use of an iron-containing heterogeneous catalyst (Al-Fe)PILC with the assistance of UV irradiation at 254 nm and at room temperature. The spectroscopic and HPLC data of the synthesized compound proved to coincide fully with those of a pure sample obtained by continuous countercurrent extraction. This reaction was found to be light-induced. The hydroxytyrosol synthesis reaction reached its maximum yield of 64.36% under the optimized operating conditions of 3.6 mM tyrosol, 0.5 g L(-1) catalyst, and 10(-2) M H2O2 with the assistance of UV light. Increasing the initial hydrogen peroxide concentration more than 10(-2) M has a diminishing return on the reaction efficiency. Catalyst can be recuperated by means of filtration and then reused in a next run after regeneration since its activity did not significantly decrease (<10%). The reaction synthesis is operationally simple and could find application for industrial purposes.     

Changes in phenolic composition and antioxidant activity of virgin olive oil during frying

The concentration of hydroxytyrosol (3,4-DHPEA) and its secoiridoid derivatives (3,4-DHPEA-EDA and 3,4-DHPEA-EA) in virgin olive oil decreased rapidly when the oil was repeatedly used for preparing french fries in deep-fat frying operations. At the end of the first frying process (10 min at 180 degrees C), the concentration of the dihydroxyphenol components was reduced to 50-60% of the original value, and after six frying operations only about 10% of the initial components remained. However, tyrosol (p-HPEA) and its derivatives (p-HPEA-EDA and p-HPEA-EA) in the oil were much more stable during 12 frying operations. The reduction in their original concentration was much smaller than that for hydroxytyrosol and its derivatives and showed a roughly linear relationship with the number of frying operations. The antioxidant activity of the phenolic extract measured using the DPPH test rapidly diminished during the first six frying processes, from a total antioxidant activity higher than 740 micromol of Trolox/kg down to less than 250 micromol/kg. On the other hand, the concentration of polar compounds, oxidized triacylglycerol monomers (oxTGs), dimeric TGs, and polymerized TGs rapidly increased from the sixth frying operation onward, when the antioxidant activity of the phenolic extract was very low, and as a consequence the oil was much more susceptible to oxidation. The loss of antioxidant activity in the phenolic fraction due to deep-fat frying was confirmed by the storage oil and oil-in-water emulsions containing added extracts from olive oil used for 12 frying operations.     

New filtration systems for extra-virgin olive oil: effect on antioxidant compounds, oxidative stability, and physicochemical and sensory properties

The purpose of this work was to evaluate some new filtration systems in relation to the quality of extra-virgin olive oil (EVOO). Filtration processes were undertaken using a polypropylene filter bag and two different inert gas flows as filter aids (argon and nitrogen). Qualitative and quantitative variations of the glyceride composition, antioxidant and pro-oxidant compounds, and water content were correlated with the oxidative stability to establish the effect on EVOO shelf life. The influence on physicochemical and sensorial properties was also evaluated. After filtration, the oxidative stability was reduced. The behavior of the polyphenols and water content on the filtration process could explain the lowest oxidative stability of filtered EVOO. Moreover, the results of the sensorial analysis confirmed that filtration using inert gases did not decrease the intensity of the main positive sensory attributes. The results could help olive-oil producers to improve EVOO quality and establish optimal storage conditions.     

Synthesis of tritium-labeled hydroxytyrosol, a phenolic compound found in olive Oil

(3,4-Dihydroxyphenyl)ethanol, commonly known as hydroxytyrosol (1), is the major phenolic antioxidant compound in olive oil, and it contributes to the beneficial properties of olive oil. Bioavailability and metabolism studies of this compound are extremely limited, in part, related to unavailability of radiolabeled compound. Studies with radiolabeled compounds enable use of sensitive radiometric analytical methods as well as aiding elucidation of metabolic and elimination pathways. In the present study a route for the formation of hydroxytyrosol (1), by reduction of the corresponding acid 2 with tetrabutylammonium boronate, was found. Methods for the incorporation of a tritium label in 1 were investigated and successfully accomplished. Tritiated hydroxytyrosol (1t) was synthesized with a specific activity of 66 Ci/mol. The stability of unlabeled and labeled hydroxytyrosol was also investigated.     

氮素形态, 光合作用, 光呼吸

Under high light conditions, ammonium nutrition has a negative effect on plant growth. This suggests that the adverse effects of ammonium nutrition on plant growth may be related to carbon gain, photosynthesis, and photorespiration. However, there is no consistent evidence of a specific mechanism that could explain the plant growth reduction under ammonium supply. It is generally accepted that during the light reaction, a surplus of nicotinamide adenine dinucleotide hydrogen phosphate （NADPH） is produced, which is not completely used during the assimilation of CO2, Nitrate reduc- tion in the leaf represents an additional sink for NADPH that is not available to ammonium-grown plants. Nitrate and ammonium nutrition may use different pathways for NADPH consumption, which leads to differences in photosynthesis and photorespiration. The morphological （i.e., cell size, mesophyll thickness, and chloroplast volume） and enzymic （i.e., ribulose-1,5-bisphosphate carboxylase/oxygenase （Rubisco）, phosphoenolpyruvate carboxylase （PEPCase）, and glutamine synthetase/glutamate synthetase （GS/GOGAT）） differences that develop when plants are treated with either nitrate or ammonium nitrogen forms are related to photosynthesis and photorespiration. The differences in photorespiration rate for plants treated with nitrate or ammonium are related to the conversion of citrate to 2-oxoglutarate （2-OG） and photorespiratory CO2 refixation.     

Effect of olive stoning on the volatile and phenolic composition of virgin olive oil

Olive stoning during the virgin olive oil (VOO) mechanical extraction process was studied to show the effect on the phenolic and volatile composition of the oil. To study the impact of the constitutive parts of the fruit in the composition of olive pastes during processing, the phenolic compounds and several enzymatic activities such as polyphenoloxidase (PPO), peroxidase (POD), and lipoxygenase (LPO) of the olive pulp, stone, and seed were also studied. The olive pulp showed large amounts of oleuropein, demethyloleuropein, and lignans, while the contribution of the stone and the seed in the overall phenolic composition of the fruit was very low. The occurrence of crushed stone in the pastes, during malaxation, increased the peroxidase activity in the pastes, reducing the phenolic concentration in VOO and, at the same time, modifying the composition of volatile compounds produced by the lipoxygenase pathway. The oil obtained from stoned olive pastes contained higher amounts of secoiridoid derivatives such as the dialdehydic forms of elenolic acid linked to (3,4-dihydroxyphenyl)ethanol and (p-hydroxyphenyl)ethanol (3,4-DHPEA-EDA and p-HPEA-EDA, respectively) and the isomer of the oleuropein aglycon (3,4-DHPEA-EA) and, at the same time, did not show significant variations of lignans. The stoning process modified the volatile profile of VOO by increasing the C6 unsaturated aldehydes that are strictly related to the cut-grass sensory notes of the oil.