Isolation and characterization of the antioxidant component 3,4-dihydroxyphenylethyl 4-formyl-3-formylmethyl-4-hexenoate from olive (Olea europaea) leaves

Storage of olive (Olea europaea) leaves for 22 h at 37 degrees C in closed plastic bags caused the content of a nonglycosidic secoiridoid, 3,4-dihydroxyphenylethyl 4-formyl-3-formylmethyl-4-hexenoate (3,4-DHPEA-EDA) to rise from 15% to 50% of the phenolic extract with corresponding falls in the content of oleuropein and two oleuropeindials, which were identified as precursors of 3,4-DHPEA-EDA. Pure product was isolated from one set of stored olive leaves in a 0.16% yield. Storage of olive leaves under various conditions showed that the moisture present in closed plastic bags was important for the formation of 3,4-DHPEA-EDA. The time taken to reach the maximum concentration of the product varied widely for different samples of olive leaves, with a shorter time for the sample with lower initial oleuropein content. The oleuropeindial precursors of the product were readily hydrolyzed to carboxylic acid derivatives, which have been identified by NMR. The antiradical activity of 3,4-DHPEA-EDA, evaluated by scavenging of 2,2-diphenyl-1-picrylhydrazyl radicals, was comparable to that of alpha-tocopherol.     

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.     

Interactions of ferric ions with olive oil phenolic compounds

The ferric complexing capacity of four phenolic compounds, occurring in olives and virgin olive oil, namely, oleuropein, hydroxytyrosol, 3,4-dihydroxyphenylethanol-elenolic acid (3,4-DHPEA-EA), and 3,4-dihydroxyphenylethanol-elenolic acid dialdehyde (3,4-DHPEA-EDA), and their stability in the presence of ferric ions were studied. At pH 3.5, all compounds formed a reversible 1:1 complex with ferric ions, but hydroxytyrosol could also form complexes containing >1 ferric ion per phenol molecule. At pH 5.5, the complexes between ferric ions and 3,4-DHPEA-EA or 3,4-DHPEA-EDA were relatively stable, indicating that the antioxidant activity of 3,4-DHPEA-EA or 3,4-DHPEA-EDA at pH 5.5 is partly due to their metal-chelating activity. At pH 7.4, a complex containing >1 ferric ion per phenol molecule was formed with hydroxytyrosol. Oleuropein, 3,4-DHPEA-EA, and 3,4-DHPEA-EDA also formed insoluble complexes at this pH. There was no evidence for chelation of Fe(II) by hydroxytyrosol or its derivatives. At all pH values tested, hydroxytyrosol was the most stable compound in the absence of Fe(III) but the most sensitive to the presence of Fe(III).     

Compositional and tissue modifications induced by the natural fermentation process in table olives

Olive fruits contain high concentrations of phenols that include phenolic acids, phenolic alcohols, flavonoids, and secoiridoids. The final concentration of phenols is strongly affected by brine conditions. The factors involved in modification by brine are still partially unknown and can include hydrolysis of secoiridoid glucosides and the release of hydrolyzed products. In this study olives from various Italian cultivars were processed by natural fermentation (e.g., without a preliminary treatment of olives with NaOH) using a selected Lactobacillus strain. Processed olives are characterized by a low phenolic concentration of phenols, consisting mainly of phenyl alcohols, verbascoside, and the dialdehydic form of decarboxymethylelenolic acid linked to (3,4-dihydroxyphenyl)ethanol (3,4-DHPEA-EDA), whereas a high level of phenols occurs in olive brine from all the cultivars studied. Olives of the Coratina cultivar, control and with fermentation by Lactobacillus pentosus 1MO, were analyzed in a frozen hydrated state by cryo scanning electron microscopy and energy-dispersive X-ray microanalysis, on both surface and transversal freeze-fracture planes. Structural modifications, found in olives after fermentation, may explain the phenol release in brine.     

Phenolic and volatile compounds of extra virgin olive oil (Olea europaea L. Cv. Cornicabra) with regard to fruit ripening and irrigation management

This study investigated the effect of both the degree of ripening of the olive fruit and irrigation management-rain-fed, two different regulated deficit irrigations (RDI), the method proposed by the Food and Agriculture Organization of the United Nations (known as FAO), and 125 FAO (125% FAO)-on the phenolic and volatile composition of Cornicabra virgin olive oils obtained during two crop seasons. Secoiridoid phenolic derivatives greatly decreased upon increase of both irrigation and ripening, for example, the 3,4-DHPEA-EDA content decreased from 770 to 450 mg/kg through fruit ripening under rain-fed conditions and from 676 to 388 mg/kg from rain-fed conditions to FAO irrigation treatment (at a ripeness index of approximately 4). Moreover, secoiridoid derivatives of hydroxytyrosol decreased more than those of tyrosol. The levels of major volatile components decreased in the course of ripening but were higher in irrigated olive oils: for example, the E-2-hexenal content ranged between 4.2 and 2.6 mg/kg (expressed as 4-methyl-2-pentanol) over fruit maturation under rain-fed conditions and between 8.0 and 3.5 mg/kg under FAO scheduling. It is important to note that where water was applied only from the beginning of August (RDI-2), when oil begins to accumulate in the fruit, the resulting virgin olive oil presented a phenol and volatile profile similar to those of the FAO and 125 FAO methods, but with a considerable reduction in the amount of water supplied to the olive orchard.     

How heating affects extra virgin olive oil quality indexes and chemical composition

Two monovarietal extra virgin olive oils from Arbequina and Picual cultivars were subjected to heating at 180 degrees C for 36 h. Oxidation progress was monitored by measuring oil quality changes (peroxide value and conjugated dienes and trienes), fatty acid composition, and minor compound content. Tocopherols and polyphenols were the most affected by the thermal treatment and showed the highest degradation rate although their behavior was different for each cultivar. Alpha-tocopherol loss was more important in Arbequina oil whereas, total phenol content loss was greater in Picual oil. The later showed an important decrease in hydroxytyrosol (3,4-DHPEA) and its secoiridoid derivatives (3,4-DHPEA-EDA and 3,4-DHPEA-EA), while lignans decrease was lesser. For Arbequina oil these compounds remained stable, and a lowering tendency was observed for tyrosol (p-HPEA) and its derivatives (p-HPEA-EDA and p-HPEA-EA). In general, flavone content showed a decrease during heating, being higher for Arbequina oil. On the other hand, oleic acid, sterols, squalene, and triterpenic alcohols (erythrodiol and uvaol) and acids (oleanolic and maslinic) were quite constant, exhibiting a high stability against oxidation. From these results, we can conclude that despite the heating conditions, VOO maintained most of its minor compounds and, therefore, most of its nutritional properties.     

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.     

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.     

Evaluation of phenolic compounds in virgin olive oil by direct injection in high-performance liquid chromatography with fluorometric detection

Hydrophilic phenols are the most abundant natural antioxidants of virgin olive oil (VOO), in which tocopherols and carotenes are also present. The prevalent classes of hydrophilic phenols found in VOO are phenyl alcohols, phenolic acids, secoiridoids such as the dialdehydic form of decarboxymethyl elenolic acid linked to (3,4-dihydroxyphenyl)ethanol or (p-hydroxypheny1)ethanol (3,4-DHPEA-EDA or p-HPEA-EDA) and an isomer of the oleuropein aglycon (3,4-DHPEA-EA), lignans such as (+)-1-acetoxypinoresinol and (+)-pinoresinol, and flavonoids. A new method for the analysis of VOO hydrophilic phenols by direct injection in high-performance liquid chromatography (HPLC) with the use of a fluorescence detector (FLD) has been proposed and compared with the traditional liquid-liquid extraction technique followed by the HPLC analysis utilizing a diode array detector (DAD) and a FLD. Results show that the most important classes of phenolic compounds occurring in VOO can be evaluated using HPLC direct injection. The efficiency of the new method, as compared to the liquid-liquid extraction, was higher to quantify phenyl alcohols, lignans, and 3,4-DHPEA-EA and lower for the evaluation of 3,4-DHPEA-EDA and p-HPEA-EDA.     

Phenolic compounds profile of cornicabra virgin olive oil

This study presents the phenolic compounds profile of commercial Cornicabra virgin olive oils from five successive crop seasons (1995/1996 to 1999/2000; n = 97), determined by solid phase extraction reversed phase high-performance liquid chromatography (SPE RP-HPLC), and its relationship with oxidative stability, processing conditions, and a preliminary study on variety classification. The median of total phenols content was 38 ppm (as syringic acid), although a wide range was observed, from 11 to 76 ppm. The main phenols found were the dialdehydic form of elenolic acid linked to tyrosol (p-HPEA-EDA; 9 +/- 7 ppm, as median and interquartile range), oleuropein aglycon (8 +/- 6 ppm), and the dialdehydic form of elenolic acid linked to hydroxytyrosol (3,4-DHPEA-EDA; 5 +/- 8 ppm). In many cases the correlation with oxidative stability was higher when the sum of the dialdehydic form of elenolic acid linked to hydroxytyrosol (3,4-DHPEA-EDA) and oleuropein aglycon (r (2) = 0.91-0.96) or the sum of these two and hydroxytyrosol (r (2) = 0.90-0.97) was considered than was observed with HPLC total phenols (r (2)= 0.91-0.95) and especially with colorimetric determination of total polyphenols and o-diphenols (r (2) = 0.77-0.95 and 0.78-0.92, respectively). 3,4-DHPEA-EDA, p-HPEA-EDA, the aglycons of oleuropein and ligstroside, and HPLC total phenols content presented highly significant differences (p = 0.001-0.010) with respect to the dual- and triple-phase extraction systems used, whereas colorimetric total polyphenols content did not (p = 0.348) and o-diphenols showed a much lower significant difference (p = 0.031). The five variables that most satisfactorily classified the principal commercial Spanish virgin olive oil varieties were 1-acetoxypinoresinol, 4-(acetoxyethyl)-1,2-dihydroxybenzene (3,4-DHPEA-AC), ligstroside aglycon, p-HPEA-EDA, and RT 43.3 contents.     

Novel secoiridoids with antioxidant activity from Australian olive mill waste

A new biophenolic secoiridoid was identified in Australian Frantoio olive mill waste (OMW) extracts. Isolation, purification, and structure elucidation were performed. Hydroxytyrosyl acyclodihydroelenolate, the first nonaldehydic acyclic secoiridoid, is reported. A second compound was identified as p-coumaroyl-6'-secologanoside (comselogoside), and although it has been identified recently in OMW and leaves, this is the first time it has been identified in both OMW and olive fruits. UV, mass spectral, and NMR data are given for both compounds. The two compounds were quantified by HPLC-DAD, and their antioxidant potential was assessed against the classical olive biophenols, hydroxytyrosol and oleuropein, by the in vitro DPPH radical scavenging assay.     

Effect of the maturation process of the olive fruit on the phenolic fraction of drupes and oils from Arbequina, Farga, and Morrut cultivars

The purpose of the work was to investigate the effect of the maturation process of the olive fruit on the phenolic fraction of drupes and oils from Arbequina, Farga, and Morrut cultivars. The level in the phenolic content of olive drupes declines rapidly during the black maturation phase. A general decreasing trend was observed too in the phenolic content of olive oils during the ripening process in the three varieties studied. Important differences in the high-performance liquid chromatography profile between varieties were observed. These included the presence of very low amounts of lignans in olive oils proceeding from the Morrut cultivar, and the presence of three peaks after elution of 3,4-DHPEA-EDA in the Farga and Morrut cultivars, which could be used as differentiating parameters. Sensory profile differences were observed between olive cultivars and due to the ripening process.     

Influence of cultivar and concentration of selected phenolic constituents on the in vitro chemiopreventive potential of olive oil extracts

One of the main olive oil phenolic compounds, hydroxytyrosol (3,4-DHPEA), exerts in vitro chemopreventive activities (antiproliferative and pro-apoptotic) on tumor cells through the accumulation of H(2)O(2) in the culture medium. However, the phenol composition of virgin olive oil is complex, and 3,4-DHPEA is present at low concentrations when compared to other secoiridoids. In this study, the in vitro chemopreventive activities of complex virgin olive oil phenolic extracts (VOO-PE, derived from the four Italian cultivars Nocellara del Belice, Coratina, Ogliarola, and Taggiasca) were compared to each other and related to the amount of the single phenolic constituents. A great chemopreventive potential among the different VOO-PE was found following this order: Ogliarola > Coratina > Nocellara > Taggiasca. The antiproliferative and pro-apoptotic activities of VOO-PE were positively correlated to the secoiridoid content and negatively correlated to the concentration of both phenyl alcohols and lignans. All extracts induced H(2)O(2) accumulation in the culture medium, but this phenomenon was not responsible for their pro-apoptotic activity. When tested in a complex mixture, the olive oil phenols exerted a more potent chemopreventive effect compared to the isolated compounds, and this effect could be due either to a synergistic action of components or to any other unidentified extract constituent.     

Feasible application of a portable NIR-AOTF tool for on-field prediction of phenolic compounds during the ripening of olives for oil production

Olive fruits of three different cultivars (Moraiolo, Dolce di Andria, and Nocellara Etnea) were monitored during ripening up to harvest, and specific and total phenols were measured by HPLC (High Pressure Liquid Chromatography). On the same olive samples (n = 450), spectral detections were performed using a portable NIR (Near Infrared)-AOTF (Acousto Optically Tunable Filter) device in diffuse reflectance mode (1100-2300 nm). Prediction models were developed for the main phenolic compounds (e.g., oleuropein, verbascoside, and 3,4-DHPEA-EDA) and total phenols using Partial Least Squares (PLS). Internal cross-validation (leave-one-out method) was applied for calibration and prediction models developed on the data sets relative to each single cultivar. Validation of the models obtained as the sum of the three sample sets (total phenols, n = 162; verbascoside, n = 162; oleuropein, n = 148; 3,4-DHPEA-EDA, n = 162) were performed by external sets of data. Obtained results in term of R(2) (in calibration, prediction and cross-validation) ranged between 0.930 and 0.998, 0.874-0.942, and 0.837-0.992, respectively. Standard errors in calibration (RMSEC), cross-validation (RMSECV), and prediction (RMSEP) were calculated obtaining minimum error in prediction of 0.68 and maximum of 6.33 mg/g. RPD ratios (SD/SECV) were also calculated as references of the model effectiveness. This work shows how NIR-AOTF can be considered a feasible tool for the on-field and nondestructive measurement of specific and total phenols in olives for oil production.     

Effect of enzyme treatment during mechanical extraction of olive oil on phenolic compounds and polysaccharides

The effect of the use of cell-wall-degrading-enzyme preparations during the mechanical extraction process of virgin olive oil on the phenolic compounds and polysaccharides was investigated. The use of the enzyme preparations increased the concentration of phenolic compounds in the paste, oil, and byproducts. Especially, the contents of secoiridiod derivatives such as the dialdehydic form of elenolic acid linked to 3,4-dihydroxyphenylethanol (3,4-DHPEA-EDA) and an isomer of oleuropein aglycon (3,4-DHPEA-EA), which have high antioxidant activities, increased significantly in the olive oil. Furthermore, the use of an N(2) flush during processing strongly increased the phenolic concentration. Analyses of the pectic polymers present in the paste showed that the use of pectinolytic enzyme preparations increased the yield of the buffer soluble pectins and the proportion of molecules with a lower molecular mass. Also, the content of uronic acids in the buffer soluble extract increased considerably due to the use of the enzyme preparations. Analysis of the polymeric carbohydrates in the vegetation waters showed the presence of mainly pectic polymers. The addition of commercial enzyme preparations increased the uronic acid content of the polysaccharides in the vegetation water substantially compared to the blank. This study showed that the addition of cell-wall-degrading enzymes did improve the olive oil quality; however, mechanisms remained unclear.     

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.     

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.     

Chemical and cellular antioxidant activity of phytochemicals purified from olive mill waste waters

The isolation and identification of a phytocomplex from olive mill waste waters (OMWW) was achieved. The isolated phytocomplex is made up of the following three phenolic compounds: hydroxytyrosol (3,4-DHPEA), tyrosol (p-HPEA) and the dialdehydic form of decarboxymethyl elenolic acid, linked with (3,4-dihydroxyphenyl)ethanol (3,4-DHPEA-EDA). The purification of this phytocomplex was reached by partial dehydration of the OMWW, followed by liquid-liquid extraction with ethyl acetate and middle pressure liquid chromatography (MPLC) on a Sephadex LH-20 column. The phytocomplex accounted for 6% of the total phenolic content of the OMWW. The phytocomplex and individual compounds were tested for antioxidant capacity by the oxygen radical absorbance capacity (ORAC) method. The ORAC phytocomplex produced 10,000 ORAC units/g dry weight, whereas the cellular antioxidant activity, measured by the cellular antioxidant activity in red blood cell (CAA-RBC) method, demonstrated that the phytocomplex and all of the components are able to permeate the cell membrane thus exhibiting antioxidant activity inside the red blood cells. Our phytocomplex could be employed in the formulation of fortified foods and nutraceuticals, with the goal to obtain substantial health protective effects due to the suitable combination of the component molecules.     

Factors influencing phenolic compounds in table olives (Olea europaea)

The Mediterranean diet appears to be associated with a reduced risk of several chronic diseases including cancer and cardiovascular and Alzheimer's diseases. Olive products (mainly olive oil and table olives) are important components of the Mediterranean diet. Olives contain a range of phenolic compounds; these natural antioxidants may contribute to the prevention of these chronic conditions. Consequently, the consumption of table olives and olive oil continues to increase worldwide by health-conscious consumers. There are numerous factors that can affect the phenolics in table olives including the cultivar, degree of ripening, and, importantly, the methods used for curing and processing table olives. The predominant phenolic compound found in fresh olive is the bitter secoiridoid oleuropein. Table olive processing decreases levels of oleuropein with concomitant increases in the hydrolysis products hydroxytyrosol and tyrosol. Many of the health benefits reported for olives are thought to be associated with the levels of hydroxytyrosol. Herein the pre- and post-harvest factors influencing the phenolics in olives, debittering methods, and health benefits of phenolics in table olives are reviewed.     

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.