Article pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. 2018, 66, 7337−7346
Oleocanthalic Acid, a Chemical Marker of Olive Oil Aging and Exposure to a High Storage Temperature with Potential Neuroprotective Activity Annia Tsolakou,† Panagiotis Diamantakos,† Iliana Kalaboki,† Antonio Mena-Bravo,‡ Feliciano Priego-Capote,‡ Ihab M. Abdallah,§ Amal Kaddoumi,§ Eleni Melliou,† and Prokopios Magiatis*,† Downloaded via KAOHSIUNG MEDICAL UNIV on July 29, 2018 at 15:13:42 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
†
Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, 15771 Athens, Greece ‡ Department of Analytical Chemistry, University of Córdoba, 14071 Córdoba, Spain § Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, 720 South Donahue Drive, Auburn, Alabama 36849, United States S Supporting Information *
ABSTRACT: The investigation of olive oils stored for a period of 24 months under appropriate conditions (25 °C, dark place, and airtight container) led to the identification of a new major phenolic ingredient, which was named oleocanthalic acid. The structure of the new compound was elucidated using one- and two-dimensional nuclear magnetic resonance in combination with tandem mass spectrometry. The new compound is an oxidation product of oleocanthal and is found in fresh oils in very low concentrations. The concentration of oleocanthalic acid increased with storage time, while the oleocanthal concentration decreased. A similar increase of the oleocanthalic acid/oleocanthal ratio was achieved after exposure of olive oil to 60 °C for 14 days. Although the presence of an oxidized derivative of decarboxymethylated ligstroside aglycon had been reported, it is the first time that its structure is characterized. The isolated compound could induce the expression of amyloid-β major transport proteins as well as tight junctions expressed at the blood−brain barrier, suggesting that oleocanthalic acid could be beneficial against Alzheimer’s disease. KEYWORDS: olive oil, phenolics, stability, NMR, LC−MS/MS, oleocanthal, oleocanthalic acid, Alzheimer’s disease
■
INTRODUCTION Olive oil, since 2012 in the European Union, is officially recognized as a food that, under specific conditions, can bear a health claim related to protection of blood lipids from oxidative stress.1 The factor determining if an olive oil can have on its label the above health claim is the concentration of hydroxytyrosol and its derivatives, including all of the secoiridoid phenolics coming from oleuropein and ligstroside. A series of compounds, such as oleocanthal (1), oleacein, oleuropein, and ligstroside aglycons, dialdehydic, monoaldehydic, and enolic forms (known also as oleuropeindials, ligstrodials, oloekoronal, and oleomissional), have been recognized as belonging to the class of phenolics that are measured to support the health claim. We have developed2−5 an analytical method based on onedimensional (1D) quantitative nuclear magnetic resonance (qNMR) spectroscopy that is able to measure the concentration of all of the above ingredients in one experiment, and since 2012, we have analyzed more than 4000 samples with the above method.2−7 A critical question raised by most olive oil producers is what happens to the major phenolic ingredients during storage and which parameters play the most important role and, consequently, which are the levels of phenolic compounds in olive oil that could guarantee that an olive oil will qualify for the health claim during all of its shelf life. © 2018 American Chemical Society
There are a number of previous works that have tried to investigate the chemical composition and product stability (or shelf life) parameters.8−11 It is known for over a decade that storing extra virgin olive oil (EVOO) at room temperature leads to chemical transformations of the complex phenolic compounds, through hydrolytic or oxidation mechanisms.12 Previous stability studies of EVOO13,14 have indicated the presence of oxidized products coming from the known phenolic derivatives, such as oleocanthal and oleacein; however, their precise structure has not been elucidated as well as the factors that determine their formation. To provide more answers to the above questions, we randomly selected 29 samples of olive oil from different varieties, geographic origin, and harvest time and monitored the levels of the phenolic compounds related to the health claim for up to 24 months under three different storage temperatures in airtight containers. The complete results of this study are a subject of a separate publication, but interestingly during the monitoring period, we noticed the appearance or increase of a new compound. This fact directed our investigation toward its isolation Received: Revised: Accepted: Published: 7337
January 30, 2018 April 14, 2018 June 14, 2018 June 14, 2018 DOI: 10.1021/acs.jafc.8b00561 J. Agric. Food Chem. 2018, 66, 7337−7346
Article
Journal of Agricultural and Food Chemistry
The elution was made for the first 20 fractions with 95:5 cyclohexane/ EtOAc, the next 20 fractions with the same solvents at 90:10, the next with ratios of 80:20, 70:30, 60:40, and 50:50. The final 20 fractions were eluted with 100% EtOAc. The fractions 82−125 containing the oleocanthalic acid (80 mg) were identified with spotting on normalphase silica gel TLC plates, developed with 100% EtOAc (Rf values: oleocanthalic acid, 0.15; oleacein, 0.56; and oleocanthal, 0.67, in 40:60 cyclohexane/EtOAc). LC−QTOF MS/MS Confirmatory Analysis. Samples of pure oleocanthal, oleacein, and oleocanthalic acid were reconstituted in acetonitrile to obtain a concentration of around 500 μg mL−1, and then aliquots were diluted in acetonitrile up to 10 μg mL−1. These aliquots were injected (5 μL) without any additional pretreatment into the LC−MS/MS system. This system allows for the simultaneous spraying of a mass reference solution that enabled continuous calibration of detected m/z ratios. The analytical column was a reversed-phase C18 Mediterranea Sea (50 × 0.46 mm inner diameter and 3 μm particle size) from Teknokroma (Barcelona, Spain). A pre-column, 40 × 3.0 mm inner diameter, from Phenomenex (Torrance, CA, U.S.A.) was inserted prior to the analytical column for preservation. The mobile phases were mobile phase A, 0.1% formic acid in water, and mobile phase B, 0.1% formic acid in acetonitrile. The gradient program, at 0.8 mL min−1 constant flow rate, was as follows: initially, the mobile phase was 80:20 A/B, the first gradient was from 20 to 45% phase B in 3 min, followed by a ramp of 9 min from 45 to 70% phase B, and, finally, a new gradient was applied in 3 min to reach 100% phase B. The total analysis time was 15 min, and an additional 5 min was required to re-establish and equilibrate the initial conditions. The column temperature was constant at 20 °C during the analysis. The operating conditions were as follows: gas temperature, 300 °C; drying gas, nitrogen at 10 L min−1; nebulizer pressure, 50 psi; sheath gas temperature, 360 °C; sheath gas flow, nitrogen at 12 L min−1; capillary voltage, 3000 V in negative ionization mode; nozzle voltage, 1000 V; skimmer, 65 V; octopole radio-frequency voltage, 750 V; and fragmentor voltage, 130 V. Data were acquired in centroid mode in high resolution (2 GHz). Full scan was carried out at 1 spectrum s−1 within the m/z range of 50−1200 with subsequent activation of the three most intense precursor ions per scan (only single or double charged ions were allowed) by MS/MS using a collision energy of 15 eV. MS/MS scanning was carried out at 1 spectrum s−1 within the m/z range of 50−1200. MassHunter Workstation software (version 7.00 qualitative analysis, Agilent Technologies, Santa Clara, CA, U.S.A.) was used for processing the raw LC−QTOF data files. Targeted extraction of molecular features was carried out by searching the molecular formulas of oleocanthal, oleacein, and oleocanthalic acid. The isotopic distribution of valid molecular features should be defined by two or more ions with a peak spacing tolerance of m/z 0.0025 plus 10 ppm in terms of mass accuracy. Because mass acquisition was in negative ionization mode, precursor ions were mainly [M − H]− ions, but other adducts (HCOO− and Cl−) were also taken into account together with the neutral loss by dehydration. The three target phenols were tentatively confirmed by identifying the structure of representative product ions detected in MS/MS spectra. Mass accuracy errors in MS and MS/MS acquisition were set at 5 and 10 ppm, respectively. Accelerated EVOO Aging. Pure oleocanthal (50 mg) was dissolved in EVOO (100 mL) with zero phenolic concentration and placed in an oven at 60 °C, in an open vial exposed to atmospheric O2 for 14 days. The samples were analyzed using the above-described qNMR method every 2 days. Cell Culture. The immortalized mouse brain endothelial cell line, bEnd3, was obtained from the American Type Culture Collection (ATCC, Manassas, VA, U.S.A.). bEnd3 cells, passage 25−35, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin G (100 IU/mL), streptomycin (100 μg/mL), 1% (w/v) non-essential amino acids, and 2 mM glutamine. Cultures were maintained in a humidified atmosphere (5% CO2/95% air) at 37 °C, and media were changed every other day. Western Blot Analysis. bEnd3 cells were seeded in 10 mm dishes for western blot analysis and allowed to grow to 70% confluency before
and structure elucidation by 1D and two-dimensional (2D) nuclear magnetic resonance (NMR) and tandem mass spectrometry (MS/MS). In addition, we investigated the main factors related to its formation and especially the role of the temperature and time of olive oil storage as well as the presence or not of oxygen in the headspace, aiming to show that this compound can be potentially used as a new marker of aging and heat exposure of olive oil during storage. Moreover, on the basis of the previously described in vitro and in vivo protective activities of oleocanthal on Alzheimer’s disease (AD),15−18 we investigated the activity of the new compound on the expression of amyloid-β (Aβ) transport proteins, mainly, P-glycoprotein (P-gp) and low-density lipoprotein receptorrelated protein 1 (LRP1), as well as tight junctions.
■
MATERIALS AND METHODS
Chemicals and Standards. All solvents used for extraction and isolation were of analytical grade and purchased from Merck (Darmstad, Germany). Acetonitrile and formic acid used for analysis were of liquid chromatography−mass spectrometry (LC−MS) grade from Scharlab (Barcelona, Spain). Deionized water (18 MΩ cm) from a Millipore Milli-Q water purification system (Bedford, MA, U.S.A.) was used to prepare the aqueous mobile phase. Oleocanthal and oleacein were isolated as previously described using normal-phase column chromatography and preparative thin-layer chromatography (TLC) performed on an EVOO extract obtained after liquid−liquid extraction.2 Instruments. NMR spectra were recorded on Bruker DRX 400 and Bruker AC 200 spectrometers [1H (400 MHz) and 13C (50 MHz)]. LC−MS/MS analysis was performed on a system consisting of an Agilent (Palo Alto, CA, U.S.A.) 1200 series liquid chromatograph coupled to an Agilent 6540 ultra-high-definition (UHD) accurate-mass quadrupole time-of-flight (QTOF) hybrid mass spectrometer equipped with a dual electrospray ionization (ESI) source. Olive Oil Sample Origin and Storage Conditions. For this study, we used 29 EVOO samples of different total phenolic concentrations, which were monitored periodically, with regard to their phenolic content, for a period of up to 2 years. The samples were stored in dark glass bottles, with 5% headspace. The bottles were placed either inside a dark cabinet, with an average temperature of 25 °C, or in a refrigerator at 4 or −18 °C, and three replicates of each sample were analyzed every 6 months. Information about the origin and variety of the analyzed oils is provided in Supplementary Table 1 of the Supporting Information. NMR Quantitative Analysis of Phenolic Compounds in Olive Oil. Direct measurements of the phenolic compounds were made using the protocol of Karkoula et al.2 More specifically, olive oil (5.0 g) was mixed with cyclohexane (20 mL) and acetonitrile (25 mL). The mixture was homogenized using a vortex mixer for 30 s and centrifuged at 4000 rpm for 5 min. A part of the acetonitrile phase (25 mL) was collected, mixed with 1.0 mL of a syringaldehyde internal standard solution (0.5 mg/mL) in acetonitrile, and evaporated under reduced pressure using a rotary evaporator (Buchi, Flawil, Switzerland). The residue of the above procedure was dissolved in CDCl3 (750 μL), and an accurately measured volume of the solution (550 μL) was transferred to a 5 mm NMR tube. Typically, 32 scans were collected into 32 000 data points over a spectral width of 0−16 ppm with a relaxation delay of 1 s and an acquisition time of 1.7 s. Prior to Fourier transformation (FT), an exponential weighting factor corresponding to a line broadening of 0.3 Hz was applied. The spectra were phasecorrected and integrated automatically using Topspin. Contained oleocanthal was quantified by integrating the peak of the aldehydic proton at 9.23 ppm. Oleocanthalic Acid (2) Isolation. An EVOO sample (400 g), 2 years after its production, was extracted with 4:5 cyclohexane/ acetonitrile (4 L), and the acetonitrile phase was evaporated under reduced pressure using a rotary evaporator. The extract was then submitted to column chromatography using normal-phase silica gel. 7338
DOI: 10.1021/acs.jafc.8b00561 J. Agric. Food Chem. 2018, 66, 7337−7346
Article
Journal of Agricultural and Food Chemistry
Figure 1. 1H NMR spectrum of isolated oleocanthalic acid.
Figure 2. Comparison between the aldehydic and aromatic proton region of (top) oleocanthal and (bottom) oleocanthalic acid. and electrotransferred onto a 0.45 μm polyvinylidene difluoride (PVDF) membrane. Membranes were blocked with 2% BSA and incubated overnight with monoclonal antibodies for P-gp (C-219, BioLegend, San Diego, CA, U.S.A.), LRP1 (Abcam, Cambridge, MA, U.S.A.), claudin-5 (clone 4C3C2, Invitrogen, Carlsbad, CA, U.S.A.), and ZO1 (Invitrogen). Proteins were normalized to total protein. For detection, the membranes were washed free of primary antibodies and incubated with horseradish peroxidase (HRP)-labeled secondary immunoglobulin G (IgG) anti-mouse antibody for P-gp, claudin-5, and ZO1 and anti-rabbit antibody for LRP1 (all from Santa Cruz Biotechnology, Dallas, TX, U.S.A.). The bands were visualized using a
treatment with oleocanthalic acid in a humidified atmosphere (5% CO2/95% air) at 37 °C. On the day of treatment, cells were treated with 0, 1, 2.5, 5, and 10 μM oleocanthalic acid, dissolved in dimethyl sulfoxide (DMSO), for 72 h. At the end of the treatment period, cells were lysed with radioimmunoprecipitation assay (RIPA) buffer containing complete mammalian protease inhibitors, followed by centrifugation at 21000g for 1 h at 4 °C. The supernatant was collected as the protein extract and stored at −80 °C until the time of analysis. Protein concentrations were determined by the bicinchoninic acid (BCA) method. For western blot analysis, 25 μg of protein was resolved on TGX stain-free acrylamide 10% gels (Bio-Rad, Hercules, CA, U.S.A.) 7339
DOI: 10.1021/acs.jafc.8b00561 J. Agric. Food Chem. 2018, 66, 7337−7346
Article
Journal of Agricultural and Food Chemistry Table 1. 1H and 13C NMR Data of Oleocanthal and Oleocanthalic Acid oleocanthal 1H NMR (δ, ppm; J, Hz)a 1 3 4a 4b 5 6a 6b 7 8 9 10 1′ 2′ 3′ 4′ and 8′ 5′ and 7′ 6′
9.23, (1H, d, J = 2.0 Hz) 9.64, (1H, brs) 2.99 (1H, ddd, J = 18.4, 8.5, and 1.0 Hz) 2.73 (1H, ddd, J = 18.4, 5.5, and 0.8 Hz) 3.62 (1H, m) 2.61 (1H, dd, J = 16.0, 6.8 Hz) 2.68 (1H, dd, J = 16.0, 8.4 Hz)
oleocanthalic acid 1H NMR (δ, ppm; J, Hz)a 9.25 (1H, d, J = 1.9 Hz) 2.77 (1H, dd, J = 16.6, 8.0 Hz) 2.65 (1H, ddd, J = 18.4, 8.5, and 1.0 Hz) 3.50 (1H, m) 2.66 (1H, m) 2.71 (1H, m)
6.63 (1H, q, J = 7.1 Hz) 2.08 (3H, d, J = 7.1 Hz) 4.20 (2H, m) 2.83 (2H, t, J = 6.8 Hz)
6.63 (1H, q, J = 7.1 Hz) 2.03 (3H, d, J = 7.1 Hz) 4.20 (2H, m) 2.82 (1H, t, J = 6.9 Hz)
7.05 (2H, d, J = 8.5 Hz) 6.77 (2H, d, J = 8.5 Hz)
7.03 (2H, d, J = 8.6 Hz) 6.75 (2H, d, J = 8.6 Hz)
oleocanthalic acid 13C NMR (δ, ppm)a 195.8 177.0 36.4 29.3 36.4 172.3 143.1 154.5 15.3 64.9 34.2 129.9 130.1 115.8 154.4
a
Measured in CDCl3.
Figure 3. EICs for [M − H]− ions from purified fractions of (a) oleacein (m/z 319.1181, 1.1 ppm), (b) oleocanthalic acid (m/z 319.1181, 0.8 ppm), and (c) oleocanthal (m/z 303.1238, 1.0 ppm).
EVOO samples.12 This observation has led to the investigation of the reasons for the transformations taking place and the impact of the storage conditions. For example, storage container studies19,20 pointed out that tin can be followed by glass vessels as the best storage medium for olive oil. In other stabilities of EVOO studies,13,14 there are indications of the presence of oxidized products, coming from known phenolic derivatives, such as oleocanthal and oleacein. Investigation of olive oils stored for long periods up to 24 months under appropriate conditions (25 °C, dark place, and airtight
Pierce chemiluminescence detection kit (Thermo Scientific, Waltham, MA, U.S.A.). Quantitative analysis of the immunoreactive bands was performed using ChemiDoc V3 (Bio-Rad), and band intensity was measured by densitometric analysis.
■
RESULTS AND DISCUSSION It is known for over a decade that storing EVOO in room temperature leads to chemical decomposition of the complex phenolic compounds, through hydrolytic or oxidation mechanisms.12 The percentage of phenolic loss within 21 months of storage has been reported to be between 43 and 73% in different 7340
DOI: 10.1021/acs.jafc.8b00561 J. Agric. Food Chem. 2018, 66, 7337−7346
Article
Journal of Agricultural and Food Chemistry
Figure 4. MS/MS fragmentation spectra provided by LC−QTOF analysis of (a) oleocanthal, (b) oleacein, and (c) oleocanthalic acid.
199 ppm that was in the case of compound 1. The chemical shift of the new carbon signal was compatible with a carboxyl group that was confirmed by the increase of the molecular weight by 16 amu, corresponding to the addition of an oxygen atom. The stereochemistry of the double bond at positions 8 and 9 was found to be E as in the case of oleocanthal based on the nuclear overhauser effect spectroscopy (NOESY) correlation between H-9 and aldehyde H-1. The absolute configuration at C-5 was the same as in compound 1 because both compounds showed the same optical rotation. On the basis of the above observations, it can be confirmed that the previously reported14,21−24 but undescribed oxidized derivative of oleocanthal is the carboxylic derivative, for which we propose the name oleocanthalic acid. The complete description of the proton and carbon signals and the comparison to the corresponding chemical shifts of oleocanthal are reported in Table 1. Confirmatory Analysis of Purified Phenols by LC− QTOF MS/MS. To confirm the presence of oleocanthalic acid as well as oleocanthal and oleacein in olive oil samples included in the present study, the pure compounds were analyzed by LC−QTOF MS/MS using the method described in the Materials and Methods. Oleocanthal and oleacein are characterized by the dialdehydic structure, with the difference of esterification to tyrosol or hydroxytyrosol. The hypothesis here is that oleocanthal is partially oxidized to form oleocanthalic acid. Therefore, the molecular formula of oleocanthal, C17H20O5, is slightly altered by the addition of an oxygen atom to give C17H20O6 for oleocanthalic acid, which also fits the molecular formula of oleacein. Chromatographically, oleacein and
container) led to the discovery of a new major phenolic ingredient, which was named oleocanthalic acid (2). The structure of the new compound was elucidated using 1D and 2D NMR in combination with MS/MS. The new compound is an oxidation product of oleocanthal (1) and was detected in fresh oils in very low concentrations. The concentration of oleocanthalic acid, as demonstrated below, increased with storage time, while the oleocanthal concentration decreased, and in several cases, after 24 months, it was the major phenolic ingredient found in the olive oil. Structure Elucidation of Oleocanthalic Acid (2). The 1 H NMR spectrum of oleocanthalic acid (2) (Figure 1) showed significant similarities with the spectrum of oleocanthal (1) but also showed two major differences. The first difference was that compound 2 presented only one aldehydic signal at 9.25 ppm, slightly deshielded in comparison to compound 1 and corresponding to the conjugated aldehyde of position 1, while the second aldehyde group at position 3 was absent (Figure 2). In addition, the peaks corresponding to protons H-4 had a significant change in their chemical shift, indicating that the main point of differentiation was the functional group at position 3. Thorough investigation with correlation spectroscopy (COSY), heteronuclear multiple-quantum coherence (HMQC), and heteronuclear multiple-bond correlation (HMBC) experiments led to the assignment of each proton and carbon peak and mainly showed that the aldehyde group at position 3 of oleocanthal had been replaced by another functional group with carbonyl observed at 177 ppm. More specifically, H-5 showed two J3 correlations with two carbonyls: one at 172.3 ppm and one at 177 ppm, instead of 7341
DOI: 10.1021/acs.jafc.8b00561 J. Agric. Food Chem. 2018, 66, 7337−7346
Article
Journal of Agricultural and Food Chemistry oleocanthalic acid are properly separated, which avoids the possibility of coelution. Extracted ion chromatograms (EICs) for [M − H]− ions from purified fractions of oleacein with m/z 319.1181 (1.9 ppm), oleocanthal with m/z 303.1242 (1.3 ppm), and oleocanthalic acid with m/z 319.1181 (1.9 ppm) showed three peaks at 6.6, 7.8, and 8.3 min (Figure 3), respectively. The pH of the mobile phases (around pH 3.0 by use of formic acid as the ionization agent) ensures that oleocanthalic acid is not as carboxylate ion, which would explain its elution after oleocanthal as a result of a higher retention with the reverse phase. The presence of a unique peak in the three purified fractions proved the efficiency of the isolation process. MS/MS fragmentation with TOF detection was enabled to obtain high-resolution information to proceed with identification and confirmation of the candidate compounds. Fragmentation of the precursor ion m/z 303.1238 (oleocanthal) generated several representative product ions as a result of the activation of the molecule at the weakest bond. Thus, the ion detected at m/z 137.0597 (C8H9O2) corresponded to the tyrosol moiety by β-phenyl ester fragmentation via McLafferty rearrangement. Two other fragments were detected at m/z 139.0390 (C7H7O3) and m/z 123.0445 (C7H7O2), which were assigned to the dialdehydic moiety, released after separation of tyrosol and its main fragment, respectively, as shown in Figure 4a. The fifth ion at m/z 59.0141 (C2H3O2) fits the acetoxy fragment associated with the ester bond. Fragmentation of oleacein is quite similar to that presented for oleocanthal as Figure 4b illustrates. The fragmentation of oleacein led to two main ion products at m/z 139.0403 (C7H7O3) and m/z 59.0144 (C2H3O2), corresponding to the dialdehydic moiety and the acetoxy fragment released after separation of hydroxytyrosol by analogy to oleocanthal. Besides, one ion at m/z 123.0450 was clearly identified as the main fragment of hydroxytyrosol when this is activated by MS/MS, which allowed for confirmation of the identity of oleacein. Despite oleacein and oleocanthalic acid having the same molecular formula, the fragmentation mechanism was completely different, as elucidated in Figure 4c. The oxidation of one of the aldehydic groups alters the activation process, which is clearly simplified. In fact, the MS/MS spectrum of oleocanthalic acid is clearly dominated by two main product ions at m/z 199.0611 (C9H11O5) and m/z 111.0086 (C5H3O3), while other minor fragments are also detected. Partial oxidation of the dialdehydic structure completely alters the fragmentation mechanism by MS/MS. This profile is indicative of a simple fragmentation that leads to these two main product ions without any other chemical alteration. The product ion m/z 199.0611 was formed by fragmentation of oleocanthalic acid through the ester bond; particularly, this ion fit the deacetoxy monoaldehydic acid moiety, resulting after oxidation of the dialdehydic structure. The product ion m/z 111.0086 is a subfragment of the previous product ion that gives the most intense signal of the MS/MS spectrum. This ion is formed by release of the C4H6O fragment and loss of the hydroxyl group (Figure 5). Monitoring the Phenolic Concentration by qNMR. During the “cabinet” storage, our results using qNMR confirmed previous works, with a loss of 42.8 ± 16.1% of the oleocanthal content of the samples after 18 months (measured for 29 samples) or up to 70% after 24 months (measured for 5 samples) (Supplementary Table 2 of the Supporting Information). The studied samples contained oleocanthal with an initial concentration ranging from 0 up to 469 mg/kg. Supplementary Figures 1−4 of the Supporting Information show the evolution of selected
Figure 5. Fragmentation scheme of oleocanthalic acid to confirm the detected compound in aged oils.
samples, with the reduction of oleocanthal and the increase of oleocanthalic acid. Figure 6a shows the increase of the
Figure 6. Comparison of the oleocanthalic acid/oleocanthal ratio change with time with (a) normal aging at 25 °C and (b) “accelerated” aging in open vials at 60 °C. The results are expressed as the molar ratio measured by the integration in the 1H NMR spectrum of the aldehyde peaks in comparison to the internal standard. In the normal aging, the values are the mean values of the 29 studied samples.
oleocanthalic acid/oleocanthal ratio during storage for up to 24 months. The samples stored at 4 or −18 °C did not show any increase of the oleocanthalic acid/oleocanthal ratio, even at 24 months, showing that the formation of oleocanthalic acid in mainly dependent upon the temperature of storage. 7342
DOI: 10.1021/acs.jafc.8b00561 J. Agric. Food Chem. 2018, 66, 7337−7346
Article
Journal of Agricultural and Food Chemistry
Figure 7. 1H NMR spectra of the accelerated aging process, measured every 2 days, starting from (bottom) day 2 of the experiment to (top) day 14, showing the oxidation of oleocanthal to oleocanthalic acid, in olive oil with PV = 8 mequiv of O2/kg, heated at 60 °C in an open vial.
Accelerated Aging and Oleocanthal Oxidation. A similar increase of the oleocanthalic acid/oleocanthal ratio, mimicking the 2 year storage period, was achieved after exposure of olive oil to 60 °C for 14 days in open vials, showing that the oleocanthalic acid/oleocanthal ratio can be potentially used as a marker of exposure to high temperatures and aging of olive oil. More specifically, we dissolved pure oleocanthal to a sample of EVOO with naturally zero phenolic content and peroxide value (PV) = 8 mequiv of O2/kg, stored it for 14 days at 60 °C, and measured its oleocanthal and oleocanthalic acid concentrations every 2 days. The result of this heat treatment was the oxidation of oleocanthal and the formation of its oxidized product, oleocanthalic acid. A percentage of contained oleocanthal was oxidized to oleocanthalic acid, as shown in Figure 6b, while the oleocanthalic acid concentration was increased steadily. These results were obtained by quantitative 1H NMR spectra analysis and integration of the aldehydic proton peaks, as shown in Figure 7. As seen in Figure 7, the whole region of the aldehydic protons remains the same, except the appearance of the peak of oleocanthalic acid and the decrease of the peaks of oleocanthal. It is clear that, under these specific conditions, there is no observable formation of other related oxidized compounds. The same happens in the whole 1H NMR spectrum. The only new signals are those belonging to oleocanthalic acid. However, we cannot exclude the formation of other oxidation products that are not extracted with acetonitrile (or other polar solvents) from olive oil (e.g., highly lipophilic oxidized derivatives). It should be
noted that oxidation is only one of the decomposition procedures for oleocanthal. The other major procedure is the hydrolysis reaction, leading from oleocanthal to free tyrosol. This reaction was also observed in most cases of the 29 studied olive oil samples during the 24 months of storage. Oleocanthalic acid is also susceptible to hydrolysis of the ester bond, exactly in the same way as oleocanthal. For this reason, the oleocanthalic acid/ oleocanthal ratio is independent of the hydrolytic procedure but dependent only upon the oxidative pathway. Heat Exposure and Oxygen. To investigate the role of several factors leading to the formation of oleocanthalic acid, we set up a series of experiments, including heating in the presence or not of atmospheric oxygen. In addition, to examine if there is a link between olive oil PV and the formation of oleocanthalic acid, we applied steady heat without the presence of oxygen in samples with different peroxide levels. In a first experiment, pure oleocanthal diluted in zero phenolic olive oil with PV = 8 mequiv of O2/kg was exposed to 60 °C for 20 days in either an open vial or an airtight vial without headspace. As shown in Supplementary Figure 5 of the Supporting Information, the absence of oxygen led to a much smaller amount of oleocanthalic acid in comparison to the open vial, showing the crucial role of oxygen. In the airtight closed vial, only 25% of oleocanthal was oxidized to oleocanthalic acid, while in the open vial, 80% was oxidized. Interestingly, heat exposure of oleocanthal diluted in organic solvent failed to give oleocanthalic acid. More specifically, when 7343
DOI: 10.1021/acs.jafc.8b00561 J. Agric. Food Chem. 2018, 66, 7337−7346
Article
Journal of Agricultural and Food Chemistry
Figure 8. Changes in the oleocanthal/oleocanthalic acid ratio caused by different PVs of the olive oil sample that were used for the accelerated aging experiment for 14 days at 60 °C in closed airtight vials: (top) PV = 30 mequiv of O2/kg and (bottom) PV = 8 mequiv of O2/kg.
pure oleocanthal was diluted in dichloromethane and heated at 60 °C for 14 days in half-filled, closed vials, the formation of oleocanthalic acid could not be observed, even in traces. The above procedure showed that the oxidation of oleocanthal to oleocanthalic acid is not a direct reaction with atmospheric oxygen but requires the presence of other constituents of olive oil, which act as intermediate oxidizing agents. The headspace oxygen when combined with increased heat exposure increases the formation of lipid peroxides that, in turn, play a role in the oxidation of oleocanthal. It was confirmed by heating airtight closed bottles containing olive oil with a low peroxide count. Even in that case, we monitored the oxidation of a small proportion of oleocanthal to oleocanthalic acid. The role of peroxides was confirmed in a third experiment, where pure oleocanthal was added in two olive oil samples in airtight vials: one with low peroxides (PV = 8 mequiv of O2/kg) and one with high peroxides (PV = 30 mequiv of O2/kg) and heated at 60 °C for 14 days. The results as shown in Figure 8 showed that the oxidation of oleocanthal to oleocanthalic acid was accelerated by the presence of lipid peroxides. Interestingly, the PV was reduced in parallel to oleocanthalic acid formation. There seems to be a combination of available atmospheric oxygen and lipid peroxides present in olive oil samples, which accelerates the formation of oleocanthalic acid. It should be mentioned that the two oils used in this experiment were carefully selected from the same variety having zero phenolic content; however, the first oil was fresh with low PV, and the second oil was 1 year old and had naturally developed a high PV. In this way, we were able to study the behavior of oleocanthal when added to those oils, which presented high similarity but significant difference in the PVs. The fact that the oleocanthalic acid formation was accelerated in the high PV oil and that the PV was reduced in parallel with the oleocanthalic acid formation provides strong evidence that the lipid peroxides are implicated in the reaction mechanism. The aldehyde oxidation to carboxylic
acid with hydrogen peroxide or hydroperoxides is a well-studied reaction with a well-known mechanism.25,26 However, it has not been studied in the case of lipid or alkyl peroxides found in olive oil. Potential Activity against AD: In Vitro Studies. Recently, we have reported oleocanthal as a potential molecule against AD.15−18 In vitro and in vivo studies have demonstrated oleocanthal to increase Aβ clearance across the blood−brain barrier (BBB) caused by its effect on upregulating Aβ major transport proteins P-gp and LRP1 expressed in the endothelial cells of the BBB. Besides, oleocanthal induced the tight junction claudin-5, which could be responsible for BBB increased intactness in AD mice brains. The isolation of oleocanthalic acid in the current study prompted us to investigate its activity on the induction of P-gp, LRP1, and expressions of tight junctions. As shown in panels a−d of Figure 9 and consistent with our findings with compound 1,15−18 compound 2 significantly increased the expression of P-gp, LRP1, ZO1, and claudin-5, however to a variable degree, with the effect more pronounced at the lower range of examined concentrations. Oleocanthalic acid significantly induced P-gp by 48 and 15% at 1.0 and 2.5 μM, respectively, but has no effect at higher concentrations (Figure 9a). For LRP1, on the other hand, while the effect was only significant at 2.5 μM, oleocanthalic acid increased LRP1 expression by approximately 50% in the concentration range of 1−10 μM (Figure 9b). For its effect on tight junctions, oleocanthalic acid significantly increased ZO1 expression by 47 and 75% and claudin-5 expression by 11% at 1 and 2.5 μM, respectively (panels b and c of Figure 9). This finding suggests that, at low concentrations, oleocanthalic acid could be beneficial against AD and Aβ-related pathology. Concerning the structure−activity relationships for the effect on upregulation of Aβ major transport proteins in vitro, it seems that the aldehyde group of oleocanthal at position 3 is not critical and can be replaced by other groups, such as the carboxyl group. While further 7344
DOI: 10.1021/acs.jafc.8b00561 J. Agric. Food Chem. 2018, 66, 7337−7346
Article
Journal of Agricultural and Food Chemistry
Figure 9. (a−d) Representative western blot and densitometry analysis of (a) P-gp, (b) LRP1, (c) ZO1, and (d) claudin-5 expressions in bEnd3 cells treated for 72 h with increasing concentrations of oleocanathalic acid. Data are presented as the mean ± standard deviation (SD) of three independent experiments. (∗) p < 0.05 and (∗∗∗) p < 0.001 compared to the control group (0 concentration).
Funding
confirmatory in vitro and in vivo studies in AD models are necessary, this interesting bioactivity of oleocanthalic acid suggests that an older oil could maintain a part of its protective activity against AD, despite oleocanthal loss.
■
The authors thank the European Regional Development fund for financial support through the project “ARISTOIL”. Notes
The authors declare no competing financial interest.
ASSOCIATED CONTENT
■
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b00561. Evolution of sample 1 (Supplementary Figure 1), sample 2 (Supplementary Figure 2), sample 3 (Supplementary Figure 3), and sample 4 (Supplementary Figure 4) from the (bottom) beginning of the research to (top) end after 24 months, changes in the oleocanthal/oleocanthalic acid ratio in the same sample with PV = 8 mequiv of O2/kg heated in (top) open vial and (bottom) closed vial without headspace for 20 days (Supplementary Figure 5), information about the origin and variety of the analyzed oils (Supplementary Table 1), and oleocanthal content of the samples after 24 months (Supplementary Table 2) (PDF)
■
REFERENCES
(1) European Food Safety Authority (EFSA). Scientific opinion on the substantiation of health claims related to polyphenols in olive and protection of LDL particles from oxidative damage. EFSA J. 2011, 9, 2033. (2) Karkoula, E.; Skantzari, A.; Melliou, E.; Magiatis, P. Direct measurement of oleocanthal and oleacein levels in olive oil by quantitative 1H NMR. Establishment of a new index for the characterization of extra virgin olive oils. J. Agric. Food Chem. 2012, 60, 11696−11703. (3) Karkoula, E.; Skantzari, A.; Melliou, E.; Magiatis, P. Quantitative Measurement of Major Secoiridoid Derivatives in Olive Oil Using qNMR. Proof of the Artificial Formation of Aldehydic Oleuropein and Ligstroside Aglycon Isomers. J. Agric. Food Chem. 2014, 62, 600−607. (4) Diamantakos, P.; Velkou, A.; Killday, K. B.; Gimisis, Th.; Melliou, E.; Magiatis, P. Oleokoronal and oleomissional: New major phenolic ingredients of extra virgin olive oil. Olivae 2015, 122, 22−35. (5) Magiatis, P.; Melliou, E.; Killday, B. A new ultra rapid screening method for olive oil health claim evaluation using selective pulse NMR spectroscopy. In Magnetic Resonance in Food Science: Defining Food by Magnetic Resonance; Capozzi, F., Laghi, L., Belton, P. S., Eds.; Royal Society of Chemistry: London, U.K., 2015; pp 84−92, DOI: 10.1039/ 9781782622741-00084. (6) Ben Mansour, A.; Gargouri, B.; Melliou, E.; Magiatis, P.; Bouaziz, M. Oil quality parameters and quantitative measurement of major
AUTHOR INFORMATION
Corresponding Author
*Telephone: +30-210-7274052. E-mail:
[email protected]. ORCID
Feliciano Priego-Capote: 0000-0003-0697-719X Amal Kaddoumi: 0000-0001-9792-7766 Prokopios Magiatis: 0000-0002-0399-5344 7345
DOI: 10.1021/acs.jafc.8b00561 J. Agric. Food Chem. 2018, 66, 7337−7346
Article
Journal of Agricultural and Food Chemistry
(25) Sato, Κ.; Hyodo, M.; Takagi, J.; Aoki, M.; Noyori, R. Hydrogen peroxide oxidation of aldehydes to carboxylic acids:an organic solvent-, halide- and metal-free procedure. Tetrahedron Lett. 2000, 41, 1439− 1442. (26) Shaikh, T. M.; Hong, F. E. Efficient method for the oxidation of aldehydes and diols with tert-butylhydroperoxide under transition metal-free conditions. Tetrahedron 2013, 69, 8929−8935.
secoiridoid derivatives in Neb Jmel olive oil from various Tunisian origins using qNMR. J. Sci. Food Agric. 2016, 96, 4432−4439. (7) Kulišić Bilušić, T.; Melliou, E.; Giacometti, J.; Č aušević, A.; Č orbo, S.; Landeka, M.; Magiatis, P. Phenolics, fatty acids, and biological potential of selected Croatian EVOOs. Eur. J. Lipid Sci. Technol. 2017, 119, 1700108. (8) Krichene, D.; Salvador, M. D.; Fregapane, G. Stability of Virgin Olive Oil Phenolic Compounds during Long-Term Storage (18 Months) at Temperatures of 5−50 °C. J. Agric. Food Chem. 2015, 63, 6779−6786. (9) Brkić Bubola, K.; Koprivnjak, O.; Sladonja, B.; Belobrajić, I. Influence of storage temperature on quality parameters, phenols and volatile compounds of Croatian virgin olive oils. Grasas Aceites 2014, 65, e034. (10) Cinquanta, L.; Esti, M.; La Notte, E. Evolution of Phenolic Compounds in Virgin Olive Oil during Storage. J. Am. Oil Chem. Soc. 1997, 74, 1259−1264. (11) Krichene, D.; Allalout, A.; Mancebo-Campos, V.; Salvador, M. D.; Zarrouk, M.; Fregapane, G. Stability of virgin olive oil and behaviour of its natural antioxidants under medium temperature accelerated storage conditions. Food Chem. 2010, 121, 171−177. (12) Gómez-Alonso, S.; Mancebo-Campos, V.; Salvador, M. D.; Fregapane, G. Evolution of major and minor components and oxidation indices of virgin olive oil during 21 months storage at room temperature. Food Chem. 2007, 100, 36−42. (13) Kotsiou, K.; Tasioula-Margari, M. Monitoring the phenolic compounds of Greek extra-virgin olive oils during storage. Food Chem. 2016, 200, 255−262. (14) Rios, J. J.; Gil, M. J.; Gutierrez-Rosales, F. Solid-phase extraction gas chromatography−ion trap−mass spectrometry qualitative method for evaluation of phenolic compounds in virgin olive oil and structural confirmation of oleuropein and ligstroside aglycons and their oxidation products. J. Chromatogr. A 2005, 1093, 167−176. (15) Abuznait, A. H.; Qosa, H.; Busnena, B. A.; El Sayed, K. A.; Kaddoumi, A. Olive-oil-derived oleocanthal enhances β-amyloid clearance as a potential neuroprotective mechanism against Alzheimer’s disease: In vitro and in vivo studies. ACS Chem. Neurosci. 2013, 4, 973− 982. (16) Qosa, H.; Batarseh, Y. S.; Mohyeldin, M. M.; El Sayed, K. A.; Keller, J. N.; Kaddoumi, A. Oleocanthal enhances amyloid-β clearance from the brains of TgSwDI mice and in vitro across a human bloodbrain barrier model. ACS Chem. Neurosci. 2015, 6, 1849−1859. (17) Batarseh, Y. S.; Mohamed, L. A.; Al Rihani, S. B.; Mousa, Y. M.; Siddique, A. B.; El Sayed, K. A.; Kaddoumi, A. Oleocanthal ameliorates amyloid-β oligomers’ toxicity on astrocytes and neuronal cells: In vitro studies. Neuroscience 2017, 352, 204−215. (18) Batarseh, Y. S.; Kaddoumi, A. Oleocanthal-rich extra-virgin olive oil enhances donepezil effect by reducing amyloid-β load and related toxicity in a mouse model of Alzheimer’s disease. J. Nutr. Biochem. 2018, 55, 113−123. (19) Méndez, A. I.; Falqué, E. Effect of storage time and container type on the quality of extra-virgin olive oil. Food Control 2007, 18, 521−529. (20) Dabbou, S.; Gharbi, I.; Dabbou, S.; Brahmi, F.; Nakbi, A.; Hammami, M. Impact of packaging material and storage time on olive oil quality. Afr. J. Biotechnol. 2011, 10, 16929−16936. (21) Daskalaki, D.; Kefi, G.; Kotsiou, K.; Tasioula-Margari, M. Evaluation of phenolic compounds degradation in virgin olive oil during storage and heating. J. Food Nutr. Res. 2009, 48, 31−41. (22) Tasioula-Margari, Μ.; Tsabolatidou, Ε. Extraction, separation, and identification of phenolic compounds in virgin olive oil by HPLC− DAD and HPLC−MS. Antioxidants 2015, 4, 548−562. (23) Armaforte, E.; Mancebo-Campos, V.; Bendini, A.; Desamparados Salvador, M.; Fregapane, G.; Cerretani, L. Retention effects of oxidized polyphenols during analytical extraction of phenolic compounds of virgin olive oil. J. Sep. Sci. 2007, 30, 2401−2406. (24) Rovellini, P.; Cortesi, N. Liquid chromatography−mass spectrometry in the study of oleuropein and ligstroside aglycons in virgin olive oil: Aldehydic forms and their oxidized products. Riv. Ital. Sostanze Grasse 2002, 77, 1−14. 7346
DOI: 10.1021/acs.jafc.8b00561 J. Agric. Food Chem. 2018, 66, 7337−7346
