Hydroxytyrosol 4-beta-D-glucoside,an important phenolic compound in olive fruits and derived products

There is increasing interest in olive polyphenols because of their biological properties as well as their contribution to the color, taste, and shelf life of olive products. However, some of these compounds remain unidentified. It has been shown that hydroxytyrosol 4-beta-D-glucoside (4-beta-D-glucosyl-3-hydroxyphenylethanol) coeluted with hydroxytyrosol [(3,4-dihydroxyphenyl)ethanol] under reversed phase conditions in the phenolic chromatograms of olive pulp, vegetation water, and pomace of olive oil processing. A method to separate this compound from hydroxytyrosol by HPLC has been developed. The concentration of this glucoside increased in olive pulp with maturation and could be the main phenolic compound in mature olives. In contrast, the presence of this compound was not detected in olive oil by using HPLC-MS. The compound must be considered both in table olives and olive oil processing because of its glucose and hydroxytyrosol contribution to these products.     

Phenolic compounds in Spanish olive oils.

Phenolic compounds in Spanish virgin olive oils were characterized by HPLC. Simple phenols such as hydroxytyrosol, tyrosol, vanillic acid, p-coumaric acid, ferulic acid, and vanillin were found in most of the oils. The flavonoids apigenin and luteolin were also found in most of the oils. The dialdehydic form of elenolic acid linked to tyrosol and hydroxytyrosol was also detected, as were oleuropein and ligstroside aglycons. The structure of a new compound was elucidated by MS and NMR as being that of 4-(acetoxyethyl)-1,2-dihydroxybenzene. Changes of phenolic compounds in virgin olive oils with maturation of fruits were also studied. Hydroxytyrosol, tyrosol, and luteolin increased their concentration in oils with maturation of fruits. On the contrary, glucoside aglycons diminished their concentration with maturation. No clear tendency was observed for the rest of the phenolic compounds identified.     

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.     

Antioxidant activity of olive pulp and olive oil phenolic compounds of the arbequina cultivar

The aim of this study was to characterize antioxidant activities of phenolic compounds that appear in olive pulp and olive oils using both radical scavenging and antioxidant activity tests. Antiradical and antioxidant activities of olive pulp and olive oil phenolic compounds were due mainly to the presence of a 3,4-dihydroxy moiety linked to an aromatic ring, and the effect depended on the polarity of the phenolic compound. Glucosides and more complex phenolics exhibited higher antioxidant activities toward oxidation of liposomes, whereas in bulk lipids aglycons were more potent antioxidants with the exception of oleuropein. Lignans acted as antioxidants only in liposomes, which could partly be due to their chelating activity, because liposome oxidation was initiated by cupric acetate. The antioxidant activity of virgin olive oil is principally due to the dialdehydic form of elenolic acid linked to hydroxytyrosol (3,4-DHPEA-EDA), a secoiridoid derivative (peak RT 36, structure unidentified), and luteolin.     

Changes in the phenolic composition of virgin olive oil from young trees (Olea europaea L. cv. Arbequina) grown under linear irrigation strategies.

This study reports the HPLC profiles of phenolic compounds of virgin olive oils obtained from young olive trees (Olea europaea L. cv. Arbequina) and how the application of a linear irrigation strategy affected these. Hydroxytyrosol, tyrosol, vanillic acid, vanillin, 4-(acetoxyethyl)-1,2-dihydroxybenzene, p-coumaric acid, the dialdehydic form of elenolic acid linked to hydroxytyrosol and to tyrosol, lignans, and the oleuropein aglycon were found in all the oils. Hydroxytyrosol, tyrosol, vanillic acid, and p-coumaric acid contents in the oils were unaffected by linear irrigation. The concentration of lignans was lower in the oils from the least irrigated treatment and the concentration of vanillin increased as the amount of irrigation water applied to olive trees increased. However, 4-(acetoxyethyl)-1,2-dihydroxybenzene, the dialdehydic form of elenolic acid linked to hydroxytyrosol and to tyrosol, and the oleuropein aglycon, all of them hydroxyphenyl derivatives, decreased as the level of irrigation water increased. The latter three compounds represented the most considerable part of the phenolic fraction of the oils and they were shown to be correlated to the oxidative stability, the bitter index (K(225)), and the bitter, pungent, and sweet sensory attributes. Linear irrigation strategy changed the profile of the oil phenolic compounds and, therefore, changed both the organoleptic properties and the antioxidant capacity of the product.     

Qualitative and semiquantitative analysis of phenolic compounds in extra virgin olive oils as a function of the ripening degree of olive fruits by different analytical techniques

Capillary electrophoresis (CE) can be effectively used as a fast screening tool to obtain qualitative and semiquantitative information about simple and complex phenolic compounds of extra virgin olive oil. Three simple phenols (tyrosol, hydroxytyrosol, and vanillic acid), a secoiridoid derivative (deacetoxy oleuropein aglycon), and two lignans (pinoresinol and acetoxypinoresinol) were detected as the main compounds in extra virgin olive oils by high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE). Spectrophotometric indices, radical scavenging activity, and oxidative stability of extra virgin olive oil samples obtained from olives hand-picked at different ripening degrees were statistically correlated with the CZE and HPLC quantification. The concentration of phenols in extra virgin olive oil decreased with ripeness of olive fruits. The high correlations found between CZE and the other analytical results indicate that CE can be applied as a rapid and reliable tool to routinely determine phenolic compounds in extra virgin olive oils.     

Comparison of the concentrations of phenolic compounds in olive oils and other plant oils: correlation with antimicrobial activity

The antimicrobial activity of different edible vegetable oils was studied. In vitro results revealed that the oils from olive fruits had a strong bactericidal action against a broad spectrum of microorganisms, this effect being higher in general against Gram-positive than Gram-negative bacteria. Thus, olive oils showed bactericidal activity not only against harmful bacteria of the intestinal microbiota (Clostridium perfringens and Escherichia coli) also against beneficial microorganisms such as Lactobacillus acidophilus and Bifidobacterium bifidum. Otherwise, most of the foodborne pathogens tested (Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica, Yersinia sp., and Shigella sonnei) did not survive after 1 h of contact with olive oils. The dialdehydic form of decarboxymethyl oleuropein and ligstroside aglycons, hydroxytyrosol and tyrosol, were the phenolic compounds that statistically correlated with bacterial survival. These findings were confirmed by testing each individual phenolic compound, isolated by HPLC, against L. monocytogenes. In particular, the dialdehydic form of decarboxymethyl ligstroside aglycon showed a potent antimicrobial activity. These results indicate that not all oils classified as "olive oil" had similar bactericidal effects and that this bioactivity depended on their content of certain phenolic compounds.     

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.     

Acid hydrolysis of secoiridoid aglycons during storage of virgin olive oil.

The main change found in the phenolic composition of virgin olive oils of Arbequina, Hojiblanca, and Picual varieties during storage in darkness at 30 degrees C was the hydrolysis of the secoiridoid aglycons. This reaction gave rise to an increase in the free phenolics hydroxytyrosol and tyrosol in the oil. Filtration of oil and acidity influenced the hydrolysis to a large extent. Thus, the addition of commercial oleic acid to Hojiblanca and Picual oils increased the hydrolysis rate of the secoiridoid aglycons. In contrast, the concentration of lignans 1-acetoxypinoresinol and pinoresinol remained constant during storage. It must also be stressed that the total molar concentration of the phenolic compounds analyzed in the oils changed slightly (<20% reduction) after one year of storage, which is important from a nutritional point of view. However, the transformation of the secoiridoid aglycons into free phenolics may have consequences on oil taste and antioxidant capacity.     

Analytical evaluation of virgin olive oil of first and second extraction

Virgin olive oils from percolation (first extraction) have been compared with the corresponding oils from centrifugation (second extraction). The former were characterized by (i) higher contents of total phenols, o-diphenols, hydroxytyrosol, tyrosol-aglycons, tocopherols, trans-2-hexenal, total volatiles, and waxes; (ii) higher values of resistance to autoxidation and of turbidity; (iii) higher sensory scores; (iv) higher ratios of campesterol/stigmasterol, trans-2-hexenal/hexanal, and trans-2-hexenal/total volatiles; (v) lower contents of chlorophylls, pheophytins, sterols, and aliphatic and triterpene alcohols; (vi) lower alcoholic index and color indices; (vii) similar values of acidity, peroxide index, and UV (ultraviolet) spectrophotometric indices; (viii) similar percentages of saturated and unsaturated fatty acids, triglycerides, and diglycerides; and (ix) similar values of glyceridic indices. Stigmastadienes, trans-oleic, trans-linoleic, and trans-linolenic acid isomers were not detected in the two genuine oil kinds. Hence, the qualitative level of the first extraction oil was superior to the second extraction one.     

Electron paramagnetic resonance investigations of free radicals in extra virgin olive oils

Free radicals in olive oils were identified and quantified by EPR, by means of the spin-trapping technique making use of alpha-phenylbutylnitrone (PBN) as spin trap. The radical species were identified as PBN-trapped hydroxyl radicals (PBN-*OH) in the water microdroplets inside the fat medium. The largest radical concentration was 12.5 microM identical with 100%. The following were the relative concentrations of the radicals under different conditions: (1) Two oils, produced by continuous centrifugation, aged for 1 year, showed a 25-30% increase in the radicals compared to nonaged oils; 1-year-old oil, produced by pressure, did not differ from the nonaged oil. (2) Radical production was markedly reduced by N(2) bubbling; it was increased by heating, whereas it showed a biphasic pattern by air bubbling over time. (3) Radical concentration as a function of the UV irradiation time increased up to a maximum, after which it decreased and finally remained constant. The phenolic and oxygen contents were related to the radical content. This study demonstrates that the EPR technique is suitably applied to the detection of free radicals in olive oil and that storage, handling, and stress conditions of the oils significantly influence the radical concentration.     

Determination of esters of fatty acids with low molecular weight alcohols in olive oils

A simple and precise analytical method was developed for the simultaneous determination of squalene and methyl, ethyl, propyl, and butyl esters of fatty acids present in olive and olive pomace oils. A fraction containing squalene and fatty acid alkyl esters was isolated from the oil by solid phase extraction on silica gel cartridges and quantitatively analyzed by gas chromatography. A modification of the procedure allowed the isolation of squalene and esters separately. Repeatability and recovery of the method were good. The method was applied to extra and lampant virgin olive oil categories and also to oils obtained from olive pomace by second centrifugation and solvent extraction. Extra virgin olive oils contained low amounts of fatty acid methyl and ethyl esters, while oils obtained from altered olive or olive pomace showed high concentrations of fatty acid alkyl esters, mainly ethyl esters. Correlation between oil acidity and ethyl esters concentration was poor.     

Rapid evaluation of phenolic component profile and analysis of oleuropein aglycon in olive oil by atmospheric pressure chemical ionization-mass spectrometry (APCI-MS)

Epidemiological studies have linked the Mediterranean diet with a low incidence of cardiovascular diseases. Olive oil, the major fat component of this diet, is characterized by antioxidant properties related to their content in catecholic components, particularly oleuropein aglycon. Therefore quantification of these components in edible oils may be important in determining the quality, and consequently its commercial value. The present method allows us to obtain the profile of the phenolic components of the oil from the methanolic extracts of the crude olive oil. In particular tyrosol, hydroxytyrosol, elenolic acid, deacetoxyligstroside and deacetoxyoleuropein aglycons, ligstroside and oleuropein aglycons, and 10-hydroxy-oleuropein are clearly identified by atmospheric pressure chemical ionization-mass spectrometry (APCI-MS). Moreover, oleuropein and its isomers present in the oil are quantified by APCI-MS/MS analysis of the extracts without preliminary separation from other phenolic compounds.     

Relationships between oxidative stability, triacylglycerol composition, and antioxidant content in olive oil matrices

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.     

Development of a phenol-enriched olive oil with both its own phenolic compounds and complementary phenols from thyme

Besides affecting the oil's sensorial characteristics, the presence of herbs and spices has an impact on the nutritional value of the flavored oils. The aim of the study was to develop a new product based on the phenol-enrichment of a virgin olive oil with both its own phenolic compounds (secoiridoid derivatives) plus additional complementary phenols from thyme (flavonoids). We studied the effect of the addition of phenolic extracts (olive cake and thyme) on phenolic composition, oxidative stability, antioxidant activity, and bitter sensory attribute of olive oils. Results showed that flavonoids from thyme appeared to have higher transference ratios (average 89.7%) from the phenolic extract to oil, whereas secoiridoids from olive presented lower transference ratios (average 35.3%). The bitter sensory attribute of the phenol-enriched oils diminished with an increase of the concentration of phenols from thyme, which might denote an improvement in the consumer acceptance.     

Evaluation of the influence of thermal oxidation on the phenolic composition and on the antioxidant activity of extra-virgin olive oils

A comparison between the results obtained by using HPLC-UV, HPLC-MS, and CE-UV for characterizing the deterioration of extra-virgin olive oil during heating (180 degrees C) was investigated, taking into account phenolic compounds. The concentration of several compounds belonging to four families of phenols (simple phenols, lignans, complex phenols, and phenolic acids) was determined in the samples after the thermal treatment by all three techniques. Hydroxytyrosol, elenolic acid, decarboxymethyl oleuropein aglycon, and oleuropein aglycon reduced their concentration with the thermal treatment more quickly than other phenolic compounds present in olive oil. HYTY-Ac and Lig Agl were demonstrated to be quite resistant to this kind of treatment, and the behavior of lignans could be outstanding, as they belong to the family most resistant to thermal treatment. Several "unknown" compounds were determined in the phenolic profiles of the oils after the thermal treatment, and their presence was confirmed in refined olive oils. The oxidative stability index (OSI time) was reduced from 25 to 5 h after 3 h of heating, whereas the peroxide value showed a minimum after 1 h of heating.     

Correlations of the phenolic compounds and the phenolic content in some Spanish and French olive oils

The total content of phenolic compounds (TAP) in 29 different monocultivar olive oil samples from France (Aglandau and Tanche) and Spain (Cornicabra, Picual, and Verdial) was assessed by the colorimetric Folin-Ciocalteu method. Also, individual phenolic compounds were determined and quantified by liquid chromatography coupled to mass spectrometry (LC-MS). The French olive oil samples had a lower TAP compared to Spanish samples. The quantity of individual phenolics was similar except for pinoresinol, which was lower in the French olive oil samples. TAP moderately correlated to the sum of quantified compounds (r = 0.64 and p < 0.01) Partial least-squares (PLS) regression analysis emphasized the importance of hydroxytyrosol and the total amount of quantified phenolic compounds by LC-MS in the prediction of the total amount of phenolic compounds as determined by the Folin-Ciocalteu method. The amount of alpha-tocopherol was generally different among the cultivars (Tanche > Picual > Verdial > Aglandau > Cornicabra). Of all quantified phenolic compounds in French olive oil samples, only luteolin correlated well to the altitude of the olive orchards (r = 0.76, p < 0.01).     

Supplementation of plasma with olive oil phenols and extracts: influence on LDL oxidation

Phenols present in olive oil may contribute to the health effects of the Mediterranean lifestyle. Olive oil antioxidants increase the resistance of low-density lipoproteins (LDL) against oxidation in vitro, but human intervention studies have failed to demonstrate similar consistent effects. To better mimic the in vivo situation, plasma was incubated with either individual olive oil phenols or olive oil extracts with different phenolic compositions, and LDL was subsequently isolated and challenged for its resistance to oxidation. The results show that the ortho-dihydroxy phenols (hydroxytyrosol and oleuropein-aglycone) are more efficient than their mono-hydroxy counterparts (tyrosol and ligstroside-aglycone) in increasing the resistance of LDL to oxidation. However, the concentration of antioxidants required to inhibit LDL oxidation when added to whole plasma was substantially higher as compared to previous data where antioxidants are directly added to isolated LDL. In conclusion, this study supports the hypothesis that extra virgin olive oil phenols protect LDL in plasma against oxidation. The explanation that in vitro studies show protective effects in contrast to the lack of effect in the majority of human studies may be that the dose of the phenols and thus their plasma concentration in humans was too low to influence ex vivo LDL oxidizability. Further studies are required to gain a better understanding of the potential health benefits that extra virgin olive oil may provide.     

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.     

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.