Investigation of Phenols Activity in Early Stage ... - ACS Publications

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Investigation of Phenols Activity in Early Stage Oxidation of Edible Oils by Electron Paramagnetic Resonance and 19F NMR Spectroscopies Using Novel Lipid Vanadium Complexes As Radical Initiators Chryssoula Drouza,*,† Anthi Dieronitou,†,‡ Ioanna Hadjiadamou,‡ and Marios Stylianou† †

Department of Agricultural Sciences, Biotechnology, and Food Science, Cyprus University of Technology, Limassol 3036, Cyprus Department of Chemistry, University of Cyprus, Nicosia 1678, Cyprus

‡

S Supporting Information *

ABSTRACT: A novel dynamic method for the investigation of the phenols activity in early stage oxidation of edible oils based on the formation of α-tocopheryl radicals initiated by oil-soluble vanadium complexes is developed. Two new vanadium complexes in oxidation states V and IV were synthesized by reacting 2,2′-((2-hydroxyoctadecyl)azanediyl)bis(ethan-1-ol) (C18DEA) with [VO(acac)2] and 1-(bis(pyridin-2-ylmethyl)amino)octadecan-2-ol (C18DPA) with VOCl2. Addition of a solution of either complex in edible oils resulted in the formation of α-tocopheryl radical, which was monitored by electron paramagnetic resonance (EPR) spectroscopy. The intensity of the α-tocopheryl signal in the EPR spectra was measured versus time. It was found that the profile of the intensity of the α-tocopheryl signal versus time depends on the type of oil, the phenolic content, and the storage time of the oil. The time interval until the occurrence of maximum peak intensity be reached (tm), the height of the maximum intensity, and the rate of the quenching of the α-tocopheryl radical were used for the investigation of the mechanism of the edible oils oxidation. 19F NMR of the 19F labeled phenolic compounds (through trifluoroacetate esters) and radical trap experiments showed that the vanadium complexes in edible oil activate the one electron reduction of dioxygen to superperoxide radical. Superperoxide reacts with the lipids to form alkoperoxyl and alkoxyl lipid radicals, and all these radicals react with the phenols contained in oils. KEYWORDS: EPR/NMR, α-tocopheryl radical, olive oil, vanadium complexes, prooxidant/antioxidant

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INTRODUCTION Minor antioxidant compounds in extra virgin olive oil (EVOO), including phenols, such as α-tocopherol, tyrosol, and hydroxytyrosol, exhibit beneficial effects on human health.1−5 The phenolic compounds protect oils from oxidation, attributing to extent storage life.6,7 To monitor the oil stability, several methods have been developed to evaluate the phenol content.8 These compounds inhibit oxidation mainly through radical scavenging, with hydroxytyrosol to be the best antioxidant of all phenols contained in the polar fraction of the olive oil.9,10 The unsaturated fatty acids in edible oils are oxidized by dioxygen with a self-catalyzed mechanism. This is initiated by the decomposition of peroxides by exposure to light or heat, or the presence of catalysts such as copper or iron metal ions.11 To predict the oil self-life in actual storage conditions, oil degradation is accelerated by applying extreme experimental conditions such as elevated temperatures. These extreme conditions are harsh for the oil samples leading to the decomposition of the oil components to products, which significantly interfere with the measurement. Steady-state techniques such as those used in Rancimat test are performed under accelerated conditions require high temperatures, which may induce loss of phenolic compounds due to thermal evaporation of volatile molecules. In addition, the reaction mechanism changes at high temperatures, and a clear © 2017 American Chemical Society

correlation between test at high temperatures and actual lifetime is lost.12 Although this technique can provide information about oxidative stability of oils, it does not provide any details for the mechanism of the phenols reaction in lipid auto-oxidation.13 Direct generation of radicals provides milder conditions than heating for accelerated self-life testing of oils.14 Metal ions have been used as radical initiators in the form of salts. Apparently, they are insoluble in oils requiring the addition of emulsifiers, thus limiting the repeatability of the method.15−19 Moreover, the indirect steady state measurements for the oxidative stability and antioxidant activity of the edible oils, based on the addition of either radical traps or radicals (for example DPPH•) and the recording of the electron paramagnetic resonance (EPR) or UV−vis or luminescence etc. spectra, provide limited information about the reactivity of the oil components and the mechanisms of the reactions of the edible oil antioxidants with the molecular oxygen.20−27 Thus, there is a need for a different approach using lipid soluble radical initiators for the study of the mechanism of phenols participation in lipid oxidation reactions, allowing for monitoring of the redox species of oil in real time. The Received: Revised: Accepted: Published: 4942

March 13, 2017 June 2, 2017 June 5, 2017 June 5, 2017 DOI: 10.1021/acs.jafc.7b01144 J. Agric. Food Chem. 2017, 65, 4942−4951

Article

Journal of Agricultural and Food Chemistry

Oil Samples. Nine oil samples of sunflower, pomace olive oils, and EVOO commercial or provided by Cypriot olive oil mills were used for the study as follows: four EVOOs, one pomace olive oil, and one sunflower oil. Syntheses. Synthesis of 2,2′-((2-Hydroxyoctadecyl)azanediyl)bis(ethan-1-ol) (C18DEA). Diethanolamine (2.65 g, 0.0252 mol) and 1,2-epoxyoctadecane (5.77 g, 0.0215 mol) were dissolved in 50 mL of isopropanol. The solution was refluxed for 4 days. Then it was cooled at −18 °C for 2 days. White crystals formed upon cooling, were filtered, and dried under vacuum. The yield was 5.98 g (74%). The product was recrystallized from alcohols or toluene. Synthesis of 1-(Bis(pyridin-2-ylmethyl)amino)octadecan-2-ol (C18DPA). Bis(pyridin-2-ylmethyl)amine (1.25 g, 6.27 mmol) and 1,2-epoxyoctadecane (1.04 g, 3.87 mmol) were dissolved in 30 mL of isopropanol. The solution was refluxed for 4 days. Then the solution was acidified with HCl (6M) and evaporated to dry. The resulting brown oily residue was washed with water and dissolved in hot isopropanol. Addition of acetone at −18 °C gave a brown oily residue. The solvent was decanted, and an aqueous solution of NaOH was added until pH was above 10. The product was extracted with CH2Cl2. The solvent was removed in a rotary evaporator, yielding 1.00 g of a brown oily residue (55%) that solidifies upon standing. Synthesis of [VOC18DEA], 1. C18DEA (0.63 g, 1.7 mmol) and [VO(acac)2] (0.45, 1.7 mmol) were dissolved in CH2Cl2 (2 mL) and CH3CN (30 mL) under heating. The solution was stirred in an open vessel for 3 days at room temperature. The solvent was evaporated giving a green solid. The solid was recrystallized twice with toluene yielding 0.30 g of a yellow crystalline material (40%). Synthesis of [VO(Cl)C18DPA], 2. C18DPA (1.00 g, 2.14 mmol) and VOCl2 (2.14 mmol, 1.00 M CH3CN solution) were dissolved in warm CH3OH (30 mL). Triethylamine (0.22 g, 2.1 mmol) was added to the warm solution and the solution was evaporated to dry. To the brown residue water (30 mL) was added and the compound was extracted with CH2Cl2 (2 × 30 mL). The organic solvent was evaporated to give a brown solid that redissolved in water and re-extracted with CH2Cl2 as above. The organic solvent was evaporated to give 0.70 g of brown solid (55%). EPR Spectroscopy. The cw X-band EPR spectra of the compounds were acquired on an ELEXSYS E500 Bruker spectrometer at resonance frequency ∼9.5 GHz and modulation frequency 100 MHz. The optimization of the spin Hamiltonian parameters and EPR data simulation were performed by using the software package easy spin 4.5.2.53 2D EPR spectra of the magnetic field versus time were recorded using ∼100 gauss field sweep width and 60−180 s time intervals. For each spectrum, 20 scans were acquired. Diagrams of double integral of the signal versus time were constructed for calculation of the time of maximum intensity (tm) and the magnitude of the intensity at tm time (vide infra). Double integral is analogous to the number of spins in assay. EVOO1 and sunflower and pomace olive oil samples were used for the study. Stock solutions of 1 (7.0 mM) and 2 (7.0 mM) were prepared by dissolving the appropriate amount of material in CHCl3. The assays for EPR measurements were prepared by adding 50−200 μL either of 1 or 2 solution in 0.500 g oil, with the mixing time consisting the initial time of the reaction, time = 0 min. Then the mixture was transferred to a quartz tube 5 mm O.D, and the intensity of the radical versus time was monitored by EPR spectroscopy at room temperature (RT). The best concentration of 1 or 2 used for this experiment corresponds to the complex concentration resulting in (i) the maximum EPR signal intensity of the α-tocopheryl radical formed in oil solution and (ii) full oxidation of oil phenols in less than 30 min as described by 19F NMR experiment. In addition, the lipidic fraction of oils, which resulted from the oil after the polar part had been removed, reacted with 1 or 2 and measured by EPR as above. Specifically, the oil (5.00 g) was dissolved in n-hexane (5 mL) and extracted with CH3CN (2 × 5 mL). Then the lipidic phase of the oil after it was reacted with 1 or 2 was measured. NMR Spectroscopy. Edible oil samples, EVOO1, pomace olive oil, and sunflower oils, were used to test the method and analyzed according to the following analytical methods (vide infra).

synthesis of new metalorganic radical initiators is of high significance because of their numerous applications. In particular, they are used in organic synthesis as C−C bond and C−H activation catalysts,28,29 have anticancer properties,30 and are used in analysis and characterization of biochemical and food materials through the formation of paramagnetic species.31−34 Among them, the oxidation stability of olive oil can be evaluated.35,36 Herein, a new dynamic method is introduced based on novel lipid metal radical initiators for the investigation of the mechanism of oils oxidation at early stage. This method involves the addition of oil-soluble vanadium complexes in oils, in catalytic quantities, to activate the dioxygen contained in oils and generate radicals. These radicals are trapped by αtocopherol generating α-tocopheryl radicals monitored by EPR spectroscopy versus time. The time interval until the occurrence of maximum peak intensity be reached (tm), the height of the maximum intensity, and the rate of the quenching of the α-tocopheryl radical are correlated with the total phenolic content and consequently with the oxidative stability of the oils. The direct measurement of the α-tocopheryl radical versus time in oils consists an advantage of this method and is more suitable for the exploration of the kinetics of the oil oxidation compare with the indirect steady state methods performed by the addition of radical traps. Furthermore, no heating or light is required; thus, the degradation of the phenolics and other thermosensitive biomolecules is avoided. The radicals are initiated by the addition of either the new synthesized vanadium complex in oil. Also, the novel initiators are highly soluble in oil assuring that the chemical environment around phenols is similar to the bulk pure oil solution in contrast to emulsified metal salts, which alter the homogeneous distribution of the polar phenols. Vanadium ion has been chosen because is a good radical producer37−39 and dioxygen activator40−45 and has been used in several applications including radical polymerization,46 C−H activation,47,48 and as pharmaceutical.49−51 The structures of vanadium complexes, containing a C18 aliphatic chain, have been designed to optimize their solubility in edible oils. The edible oils content in minor phenolic/hydroxyl compounds was measured as trifluoroacetate esters, and the composition of the solutions was monitored in real time by 19F NMR spectroscopy, a method that has recently been developed by us.52 The relationship of the EPR signal intensity with the phenolic content and the storage-time of oil is discussed. In addition, the mechanisms of the oxidation reactions are investigated.

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EXPERIMENTAL PROCEDURES

Materials. Chemicals. Diethanolamine 98%, bis(pyridin-2ylmethyl)amine (or di-(2-picolyl)amine) 97%, 1,2-epoxyoctadecane 85%, vanadyl acetyl acetonate [VO(acac)2] 98%, triethylamine 99%, trifluoroacetic anhydride 99% (Aldrich), pyridine, maleic acid standard f or quantitative NMR grade, pentan-2-ol 98%, chloroform-d 99%, DPPH• (2,2-diphenyl picrazyl radical), α-tocopherol 98%, and tyrosol (or 2-(4-hydroxyphenyl)ethanol)) 98% were bought from Aldrich and used as received. DMPO (5,5-dimethyl-1-pyrroline N-oxide) 97% was bought from Sigma and used as received. CH2Cl2, CHCl3, CH3CN, isopropropanol, ethanol, methanol, and toluene solvents were reagent grade and bought from Aldrich or MERK. Chloroform-d solvent was distilled over CaH2 and used for preparing the solutions for all NMR experiments. Toluene used for the DPPH• experiments had been previously distilled over CaH2. 4943

DOI: 10.1021/acs.jafc.7b01144 J. Agric. Food Chem. 2017, 65, 4942−4951

Article

Journal of Agricultural and Food Chemistry Preparation of the Reagents. Stock solutions of trifluoroacetic anhydride and pyridine, 1.35 M each, were prepared in deuterated chloroform. Solution of 1 (7.00 mM) was prepared by dissolving the appropriate amount of crystalline material in CDCl3. Stock solutions of pentan-2-ol (0.140 M) in CDCl3 used as internal standards had been previously quantified based on the integrals of 1H NMR spectra of pure maleic acid analytical standard. Oil Esterification Experiments. The oil sample (0.250 g) and pentan-2-ol (25.0 μL, 0.140 M) were allowed to react with excess of trifluoroacetic anhydride (120 μL, 1.35 M in CDCl3) and pyridine (40.0 μL, 1.35 M in CDCl3). The mixture was shaken for 1 min, and 300 μL of CDCl3 was added. The solution was transferred to a 5 mm NMR tube, and the 19F NMR spectrum was recorded. The experiments were performed in duplicate. The spectra were recorded using 16 μs pulse (90°), an acquisition time 2.24 s, delay time 5.0 s, a spectral width of 2737 Hz, and 128 scans (+ 4 dummy scans) at 470.4 MHz. The chemical shifts are expressed in δ scale (ppm). The region of the 19F NMR chemical shifts of the compounds under study was from −76.5 to −74.5 ppm. The calibration for 19F NMR signals was done using CFCl3 as reference. DPPH• Experiments. The spectra were recorded on a Photonics UV−vis spectrophotometer Model 400, equipped with a CCD array. Experiment of Vanadium Complexes with DPPH•. The rate of DPPH• disappearance was measured at 515 nm (dry methanol over Mg) and 519 nm (dry toluene over CaH2) for time intervals from 1 s to 30 min. Stock solutions of each compound (12.0 mM) were prepared in dry toluene or methanol and kept at room temperature. The final concentrations of the tested compounds were in the range of 80−300 μM, while that of DPPH• was 100 μM. The samples were incubated at 25 °C for 4 min, and the reaction was initiated by the addition of the DPPH• solution. The measurements were conducted in triplicate. Second-order rate constants were calculated for the radical scavenging capacity (RSC) of the compounds. In this study, the decay of DPPH• from the medium has been assumed to follow pseudo-firstorder kinetics under the conditions of the reaction of DPPH• with complex, wherein one of the reactants is in large excess compared to the other so that the concentration of the minor component decreases exponentially.54 The [DPPH•] concentration is calculated from eq 1:

[DPPH•] = [DPPH•]0 e−k obsdt

O.D. quartz tube, and the intensity of the radical was monitored by EPR spectroscopy versus time at RT. The same experiment was repeated in CH2Cl2 without the addition of oil, consisting the blank.

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RESULTS AND DISCUSSION Syntheses. Reaction of excess of a secondary amine, diethanolamine, or bis(pyridin-2-ylmethyl)amine with 1,2epoxyoctadecane results in the synthesis C18DEA and C18DPA (Scheme 1).

Scheme 1. Synthetic Routes of the Ligands and Numbering

Reaction of equimolar quantities of C18DEA with [VO(acac)2] or C18DPA with VOCl2 in CH3OH results in the formation of vanadium(V) 1 or the vanadium(IV) 2 complex, respectively (Scheme 2). Scheme 2. Synthetic Routes of the Vanadium Complexes

(1)

where [DPPH•] is the radical concentration at time t, [DPPH•]0 is the radical concentration at time zero, and kobsd is the pseudo-first-order rate constant. The pseudo-first-order rate constant kobsd is linearly dependent on the concentration of the complex, and from the slope of their plot, second-order rate constants (k2) were calculated to evaluate the RSC of each compound. Quantification of the RSC of Oils with DPPH• AssayKinetics. To determine the RSC of oils as the rate of the DPPH• radical decay, the experimental procedure described by Espin et al. was followed.54 Oils (0.450 g) and 1 (100 μL, 7.00 mM) were mixed, and after 30 min, dilutions of the mixture with dry toluene resulted in solutions with oil content ranging from 70.0−370 mg/mL. The reaction started by the addition of 400 μL of DPPH• 10−4 M toluene solution, and the absorption was recorded at 519 nm versus time. Similar experiments performed as above using dilutions of pure oils in dry toluene followed by the addition of DPPH• solution. Experiments were performed in triplicate. Absorption values were recorded for time increments of 2 s resulting in a total of 300 points per spectrophotometric recording of the disappearance of DPPH• in the presence of the oil antioxidants versus time. Second-order constants k2 were calculated as the slope of the correlation of the kobsd with the oil content as previously described. DMPO (5,5-Dimethyl-1-pyrroline N-oxide) Trap Experiments. Samples of 1 (7.0 mM) were prepared by dissolving the appropriate amount of material in CH2Cl2. The assays were prepared by adding 200 μL of the solution of 1 to a solution of 0.500 g of oil (EVOO1 or pomace) and 100 μL of DMPO (30.0 mM DMPO stock solution in CH3OH), with the mixing time consisting the initial time of the reaction, time = 0 min. Then the mixture was transferred to a 5 mm

Chemical characterization and the purity of the organic molecules was done by CHN elemental analysis and 1H NMR spectroscopy as below: C18DEA, Elem. Anal. Experimental C, 70.61; H, 12.63; N, 3.76. Predicted C, 70.72; H, 12.68; N, 3.75. 1 H (CDCl3) δ ppm: 5.38 (O2−H), 4.75 (O1−H), 3.75, 3.55 (m, C1−H, C4−H), 2.80, 2.45 (m, C1−H, C4−H), 1.25 (carbon chain protons), 0.90 (t, C20−H); C18DPA, Elem. Anal. Experimental C, 76.81; H, 10.54; N, 8.93. Predicted C, 77.04; H, 10.56; N, 8.98. 1H (CDCl3) δ ppm: 5.34 (O2−H), 8.50− 7.33 (pyridine protons), 3.96, 3.41 (C2−H, C4−H), 2.59, 2.34 (m, C3−H), 1.25 (carbon chain protons), 0.90 (t, C20−H). 4944

DOI: 10.1021/acs.jafc.7b01144 J. Agric. Food Chem. 2017, 65, 4942−4951

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Journal of Agricultural and Food Chemistry The complexes were recrystallized twice for high-purity solids to be obtained as confirmed by elemental analysis, (1, Elem. Anal. Experimental C, 60.42; H, 10.18; N, 3.22. Predicted C, 60.39; H, 10.14; N, 3.20; 2, Elem. Anal. Experimental C, 62.99; H, 8.49; N, 7.35. Predicted C, 63.31; H, 8.50; N, 7.38) and thin layer chromatography (TLC) (1, in CHCl3:n-Hexane (1:1) gave one peak, Rf = 0.2; 2, TLC in CHCl3:n-Hexane (1:1) gave one peak, Rf = 0.16). The vanadium complexes are stable for about 1 week at room temperature and for at least six months stored at −18 °C. Storage of the compounds for more than a week showed the formation of organic radicals in solid state as evident by EPR spectroscopy. To ensure high purity of metal complexes, solids stored for more than one month in the freezer were recrystallized before their use and examined by thin-layer chromatography. The complexes were also characterized by 51V NMR and EPR spectroscopies. The 51V NMR spectrum of a CDCl3 solution of 1 gave one peak at −370 ppm (Figure S1), which is characteristic for 5-coordinated trialkoxy-amine vanadium(V) complexes.55 The X-band EPR spectrum of a frozen CH2Cl2 solution of 2 gave an anisotropic vanadium hyperfine coupled signal. The spectrum was simulated considering a rhombic symmetry with the following parameters, gx = gy = 1.975, gz = 1.944, Ax = −54.7 × 10−4, Ay = −59.3 × 10−4, and Az = −161 × 10−4 cm−1 (Figure S2). The Az value agrees with the expected value (−160 × 10−4 cm−1) for an equatorial environment consisted of 1Cl−, 1RO−, and 2Npy donor atoms.56 Reactivity of 1 and 2 with DPPH•. A large number of vanadium complexes exhibit RSC.57,58 Among them, [VO(acac)2] exerts a moderate RSC, therefore, it has been included in the study for comparison. The absorbance at 519 nm of solutions of C18DEA, C18DPA, 1, 2, and [VO(acac)2] in toluene with DPPH• radical versus time are shown in Figure S3, and the second-order rate constants (k2) are shown in Table 1. Although [VO(acac)2] inhibits the DPPH• radical

Figure 1. X-band EPR spectrum of 2 at (A) 0.2 mM in EVOO1 at 270 K, (B) 0.2 mM in EVOO1 at 114 K, and (C) 0.6 mM in CH2Cl2 at 114 K.

centered radical. By increasing the temperature, the signal of the vanadium radical decreases and the signal of the αtocopheryl radical becomes the predominant signal above 270 K (Figure 1). On the other hand, solutions of 2 in oleic acid at RT gave the expected isotropic 8 lines (Figure S5) indicating that the absence of an isotropic spectrum in oil above 270 K is not caused by metal−metal interactions. The disappearance of the signal of V(IV) at temperatures >270 K and its reappearance at low temperatures are due to either the strong interactions between the vanadium complex with the radicals in solution or the reversible electron transfer between 2 and the αtocopheryl radical, a well-documented temperature dependent reaction for model vanadium−hydroquinone complexes.37 This behavior of the vanadium(IV) ion is indicative for the existence of a synergism between vanadium(IV) ions and the phenols of oil toward the activation of the dioxygen possibly through the mechanisms described in the literature.40,59 This mechanism might be related to the pro-oxidant activity of the phenols in oils.60−62 Variable temperature EPR experiment using complex 1 did not give any vanadium EPR signal but only the signal of the α-tocopheryl radical at 114 K. It is expected the vanadium complexes in different oxidation states V(IV) and V(V) to interact differently with phenols as evident by the literature.40,59 However, addition of either compound in oils leads to the same result, which is the radical generation inducing the oxidation of the phenols and the lipids. Thus, this valence variability of the vanadium complexes used as initiators does not influence the propagation of the free radicals. The intensity of the α-tocopheryl radical signal varies with the time, increases at the beginning to reach a maximum value and then decays to zero (Figure 2). The maximum intensity as well the stability of the radical depends on the type of the edible oil and the quantity of 1 or 2 added in the oil. However, it is independent of the type of the complex (1 or 2), and the results are the same despite of the different oxidation state of the vanadium atom in the compounds 1, V(V), or 2, V(IV). The 51V NMR spectrum of 1 at RT and the X-band EPR spectrum of 2 at 120 K show that both complexes are intact. It is possible that atmospheric O2 is the oxidant that is activated by the vanadium complexes to form reactive oxygen species (ROS). To test this hypothesis, we compare the spectra of the reaction product of 1 with the oil samples after bubbling either with N2 or with air and for the pomace oil are shown in Figure 3. The intensity of the signal increases with the increase of the

Table 1. Rate Constants (k2) for the RSC of the Molecules under Study k2 (M/s) compounds

CH3OH

[VO(Cl)C18DPA] (2) [VOC18DEA] (1) C18DPA C18DEA [VO(acac)2] α-tocopherol71

0.9 ± 0.3 −0.38 ± 0.11

toluene 0.26 0.018 0.13 0.47 6.6

± ± ± ± ±

0.09 0.01 0.10 0.15 0.3

560 ± 80

(k2= 6.6 M−1 s−1), complexes 1 and 2 either do not show any activity or show very little increase of the peak intensity at 519 nm. For methanolic solutions of 1, the absorbance of the DPPH• at 515 nm slightly increases due to the formation of radicals. Apparently, complexes 1 and 2 not only do not inhibit DPPH• radical, but also initiate the formation of the radicals in solution. EPR of the Mixtures of 1 or 2 with Edible Oils. Addition of small quantities (7.00 × 10−7 up to 28.0 × 10−7 moles) of 1 or 2 in 0.500 g of edible oil (EVOO1, sunflower, and pomace olive oil) results in a signal in X-band EPR spectra at RT assigned to the α-tocopheryl radical (Figure S4). The EPR signals of 2 in oil solutions at 114 K and at 270 K are shown in Figure 1. At 114 K, the only peaks existing in the spectrum are originated from the anisotropic vanadium 4945

DOI: 10.1021/acs.jafc.7b01144 J. Agric. Food Chem. 2017, 65, 4942−4951

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Journal of Agricultural and Food Chemistry

Figure 4. Double integral of the α-tocopheryl radical signal in X-band EPR spectra versus time for the edible oils (0.450 g) and 1 (100 μL, 7.00 mM) (filled circles) for the lipidic fraction of the oils (filled squares) at RT. EVOO1 (black), sunflower oil (red), and pomace olive oil (blue).

Figure 2. 2D X-band EPR spectra of field (mT) versus time (s) of EVOO1 (0.500 g) after addition of 1 (100 μL, 7.00 mM solution in CH2Cl2) at RT. The spectra show the formation and degradation of αtocopheryl radical.

Table 2. Determined Values for the Time at Maximum Height (tm) and the Ratio of the α-Tocopheryl Signal Intensities for Pure EVOO1, Sunflower and Pomace Olive Oils, and Their Lipidic Fractions

quantity of O2 present in the solution. Apparently, dioxygen is the oxidant in these reactions.

tm (s) oil samples

pure oil

lipidic fraction

EPR signal intensity ratio at tm (pure oil/lipidic fraction)

pomace olive oila EVOO1a sunflowera

3267 ± 67

1706 ± 81

1.2

708 ± 44 869 ± 43

60 ± 60 325 ± 43

1.8 0.99

a

Oils bought from the market.

respective pure oils. The ratio of the α-tocopheryl peak intensity for pure oil/lipidic fraction is 0.98 for the case of sunflower oil, which is smaller than either the ratio for the EVOO1 or the pomace olive oil to their lipidic fractions. The same features were observed in the respective diagrams of the α-tocopheryl signal intensity versus time for the fresh oils and the same oils left in storage for one or more years due to the oxidation of the phenols (Table 3, Figure S6 and S7). To better understand the mechanism of this reaction in complex systems such as the edible oils, the experiments were repeated in the simpler model system of α-tocopherol and tyrosol solutions in oleic acid (Figure 5). Addition of 1 to either a solution of α-tocopherol or an equimolar solution of α-

Figure 3. X-band EPR spectra of pomace olive oil (0.500 g) 10 min after the addition of 1 (100 μL, 7.00 mM) at RT. The oil had previously been bubbled for 5 min either with N2 (brown dot line) or with air (red dashed line). The spectra of the reaction mixture without any gas treatment (black continues line).

The intensity of the α-tocopheryl signal is not directly dependent on the α-tocopherol concentration in the solution. For example, the EPR α-tocopheryl signal of the pomace olive oil exhibits intensity twice as high as the signal of the sunflower oil, although it contains only 192.4 mg/kg compared to the 564.6 mg/kg α-tocopherol of the sunflower oil. The total phenolic content controls the height of the maximum intensity and the time interval (tm) at which α-tocopheryl radical reaches the maximum intensity. The intensities of the signals of the α-tocopheryl radicals versus time for the three different types of the edible oils as well the lipidic fraction of the same oils are shown in Figure 4 and Table 2. The removal of the polar fraction from the edible oils resulted in the decrease of the maximum intensities of the EPR signals, whereas the signals maximize earlier and the decay of the radical is faster than the signals in the spectra of the

Table 3. Determined Values for the Time at Maximum Height (tm) and the Ratio (Fresh/Old) of the α-Tocopheryl Signal Intensities for Pure EVOOs, Pomace Olive Oil, and the Same Oils After 18−24 Months of Storage tm (s) oil samples pomace olive oila EVOO1a EVOO2b EVOO3b EVOO4b a

4946

fresh oil ∼6 months old

oil storage time 18−24 months

EPR signal intensity ratio at tm (fresh/old)

3249 ± 43

658 ± 22

1.2

60 60 60 325 ± 45

1.9 6.6 2.5 2.6

902 2180 1120 2180

± ± ± ±

67 35 42 51

Oils bought from the market. bOils provided by local olive oil mills. DOI: 10.1021/acs.jafc.7b01144 J. Agric. Food Chem. 2017, 65, 4942−4951

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Journal of Agricultural and Food Chemistry

Figure 5. Double integral of the α-tocopheryl radical signal in X-band EPR spectra versus time of a solution of oleic acid (0.455 g) 1 (100 μL, 1.38 M) and α-tocopherol (100 μL, 1.42 mM) (black filled circles) and of 1 (100 μL, 1.38 M), α-tocopherol (100 μL, 1.42 mM) and tyrosol (100 μL, 1.50 mM) (red filled squares) at RT.

tocopherol and tyrosol gives a α-tocopheryl radical signal in the EPR spectra, which increases in intensity with time to reach a maximum value after 30 min. In both solutions, the signal remains stable for more than 1 h; however, the intensity of the signal generated by the mixture of α-tocopherol and tyrosol is the half of that generated by the solution of the pure αtocopherol. α-Tocopherol is a better radical inhibitor than any other phenol in oil and reacts directly with the radicals in solution. The decrease of the α-tocopheryl radical with addition of tyrosol probably indicates the reduction of the radical by the phenol and probably the regeneration of the α-tocopherol molecule, as has been shown in antioxidant studies for αtocopherol/phenolic extracts in model systems in the literature.63 These results are different from those found for the edible oils, where in the absence of phenols the intensity of the signal is decreased. This is attributed to the large number of different molecules in oil interacting with α-tocopheryl facilitating the decay of the radical. This is in agreement with the longer stability of α-tocopheryl radical in the model experiment than in the one with the oils. These results show that model experiments cannot really simulate the neighboring environment around radicals in edible oils; therefore, more experiments were undertaken to clarify the mechanism of the reaction of 1 or 2 with the oil redox components. 19 F NMR Spectroscopy. The content of the phenols in edible oils during the oxidation reaction was directly measured by 19F NMR spectroscopy. The phenols in the edible oils 30 min after the addition of 1 were esterified with (CF3CO)2O according to the experimental procedures and determined by 19 F NMR spectroscopy. The spectra (Figure 6, Figures S8−S9) show the disappearance of the signal at −74.73 ppm assigned to α-tocopherol. The peaks of the less reactive phenols (from −74.71 to −74.94 ppm) disappeared when twice the quantity of 1 was added in the oil. This is in agreement with the results from the EPR studies, where the α-tocopheryl signal has less intensity and shorter lifetime in the absence of the polar phenols than at their presence, indicating that the polar phenols do not recycle α-tocopherol in oil but directly react with peroxides. Furthermore, new peaks emerge between −75.20 and −75.25 ppm assigned to saturated alcohols originated from lipids peroxidation. The phenolic content of the oils was measured for the fresh oils (∼6 months old) and for the same oils from 19 months of storage by 19F NMR spectroscopy (Table 4). Data show that during storage the concentration of α-tocopherol is slightly reduced, while the concentration of polar phenols is

Figure 6. 19F NMR spectra of EVOO1 (0.250 g): without addition of 1 (black continues line), 30 min after addition of 1 (50.0 μL, 7.00 mM) (red dashed line), and 30 min after addition of 1 (100 μL, 7.00 mM) (green dot line) at RT.

Table 4. Quantification of α-Tocopherol and Polar Phenols for Fresh Oils Fresh ∼6 Months and Stored for 13 Months, for EVOO1, Pomace Olive Oil, and Sunflower, and the Same Oils after 19 Months of Storage α-tocopherol (mg/kg)

oil samples pomace olive oila EVOO1a sunflowera a

polar phenols TEb (mg/kg)

fresh oil ∼6 months old

oil storage time 19 months

fresh oil ∼6 months old

oil storage time 19 months

192 ± 3

162 ± 4

263 ± 2

61.1 ± 0.4

91 ± 1 565 ± 5

89 ± 3 517 ± 5

175 ± 2 147 ± 1

53.5 ± 0.9 82 ± 2

Oils bought from the market. bTyrosol equivalents in mg/kg.

significantly decreased (Figure S10). This is in agreement with the results from the EPR experiment for the lipidic fraction of oils, which showed that the reduction of the maximum EPR α-tocopheryl signal and the earlier tm times originated from the decrease of the polar phenols concentration as shown in Table 2. In contrast to the accelerated oxidation caused by the addition of 1, the slow oxidation of phenols occurring during storage does not destroy α-tocopherol. This suggests that under these conditions α-tocopherol might be regenerated by the polar phenols. Similarly, other researchers have found that results for oxidative stability obtained by accelerated oxidation methods are not totally consistent with those obtained by the actual shelf life auto-oxidation of oils.64,65 Radical Scavenging Capacity of Oils. Total phenols of each oil were evaluated for their RSC to scavenge the DPPH• radical (Figure S11). The RSC is expressed as the second order k2 value, which is analogous to the most reactive phenols in oil. Thus, sunflower oil containing the highest α-tocopherol content exhibits the highest k2 of all oils. Comparison of k2 value, and thus of α-tocopherol, with the maximum intensity of the EPR α-tocopheryl signal for the above oils did not give any correlation. This is in agreement with the results from the EPR experiments, which show that the EPR signal is not analogous to the concentration of the α-tocopherol. Futhermore, k2 is drastically reduced after the addition of 1 in the oils (Figure S11). This certifies that the oil phenols have 4947

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Journal of Agricultural and Food Chemistry been completely oxidized as is also evident by the results from the 19F NMR experiment. Radical Trap Experiments. DMPO has been used as radical trap to evaluate the intermediates of the mechanism of the oxidation reactions initiated by 1 or 2. The radical trap experiments are also used to explore if the oxidation mechanism driven by 1 or 2 is going through the formation of the alkoperoxyl and alkoxyl lipid radicals and thus is analogous with the one occurring by other radical initiators reported in the literature.24,66 Reaction of a CH2Cl2 solution either of 1 or 2 with DMPO methanolic solution gives a sextet peak (AN = 1.31 and AH = 0.82 mT) assigned to the adduct of DMPO with the methoxyl radical (Figure S12).67 This supports the strong oxidizing nature of the two vanadium complexes toward the formation of radicals and is in agreement with the results of the DPPH• experiment. The reaction is slow in CH2Cl2 taking more than 6 h for observation of a strong EPR signal. On the contrary, addition of 1 in a solution of EVOO1 and DMPO immediately gave a strong signal, which was monitored by EPR spectroscopy versus time (Figure 7).

Figure 8. X-band EPR spectra of a solution of 1 (200 μL, 7.00 mM, in CH2Cl2) 0.500 g of pomace olive oil and DMPO (100 μL, 30.0 mM DMPO in CH3OH). (A) Spectra at 5 min, (i) real spectra, (ii), (iii) Deconvolution of the simulated spectra of the two components of the experimental spectra [AN = 1.37 and AH = 1.06 mT (DMPO-OOR) and with AN = 1.31, AHβ = 0.65, and AHγ = 0.17 mT (DMPO-OR)]. (B) After 30 min (black continues line), and the simulation spectrum considering two species in 12:1 ratio, with parameters AN = 1.38 and AH = 1.11 mT for DMPO-OOR) and with AN = 1.47 and AH = 2.15 mT for DMPO-CRR′R″). The nonsimulated peaks are assigned to nitrones formed by the decomposition of the DMPO adducts.

Figure 7. X-band EPR spectra of a solution of 1 (200 μL, 7.00 mM, in CH2Cl2) 0.500 g of pomace olive oil and DMPO (100 μL, 30.0 mM DMPO in CH3OH) versus time.

complexes act synergistically with the oil phenols as indicated by the variable temperature EPR spectroscopy; thus, the thermodynamically unfavored one electron reduction of the oxygen is facilitated.69 The superoxide radical first reacts with the lipids forming alkoperoxyl and alkoxyl lipid radicals. These radicals react with the most reactive phenol in oil, the αtocopherol, resulting in the formation of α-tocopheryl radical and lipid alcohols.70 As evidenced by the 19F NMR spectra, double quantity of the radical initiator was needed for the polar phenols to be oxidized. After the α-tocopherol is consumed, the rest of the phenols eventually continue the work of αtocopherol, quenching the radicals. Concluding, two lipid-vanadium(IV) and vanadium(V) complexes, soluble in edible oils, have been synthesized and characterized. The α-tocopherol contained in the oils traps the radicals forming α-tocopheryl radical, and the kinetics of the reaction were monitored by EPR spectroscopy. The intensity of the signal and the lifetime of the radical depend on the type of oil, the storage-period, and the phenol content of the oil. The radical trap experiments show that the mechanism of the reaction goes through the oxidation of the aliphatic unsaturated fatty acids. 19F NMR experiments show that at the presence of metal ions the polar phenols of the oil rather directly react with the fatty acid radicals than recycling the α-tocopherol. Further investigation is under way for gaining further insight into the mechanism of phenols participation in lipid peroxidation in food, and the possible use of these complexes for mining more information for the fate of the redox components of oils during storage and food processing.

The X-band EPR spectrum at 5 min (Figure 8A(i)) gave an overlapping signal of three species, a minor unknown carbon adduct of DMPO, 33% of a alkoperoxyl lipid radical adduct of DMPO (DMPO-OOR) (AN = 1.37 and AH = 1.06 mT) and 77% of the alkoxyl lipid radical adduct of DMPO (DMPO-OR) (AN = 1.31, AHβ = 0.65, and AHγ = 0.17 mT). The parameters obtained from the simulated spectra [Figure 8A (ii), (iii)], are in agreement with the literature.66,67 At times longer than 30 min, the signal of DMPO-OR radical decays. The major signals are originated from DMPO-OOR, and the unknown carbon DMPO adduct radical (DMPOCRR′R″) in 12:1 ratio, respectively.66 The spectrum at 30 min was simulated (Figure 8B) with the following parameters AN = 1.38 and AH = 1.11 mT for the DMPO-OOR and AN = 1.47 and AH = 2.15 mT for the DMPO-CRR′R″. At the same time, a triplet assigned to nitrones and originated from the decomposition of DMPO emerged and became the major species at 180 min (Figure 7). After 3 h, the color of the solution at the top of the tube became reddish due to the excessive formation of nitroxides. Overall, the above experiments show that in edible oil, either 1 or 2 activate dioxygen to superoxide radical. Although the vanadium complexes also produce radicals in CH2Cl2−CH3OH solutions, their reactivity in edible oils is much higher attributed to the prooxidant activity of phenols.40,60−62,68 The metal 4948

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Furthermore, these compounds will be used as radical initiators for measurement of the antioxidant activity of phenols in food.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b01144. Additional figures (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Chryssoula Drouza: 0000-0002-2630-4323 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

Supported by Research Promotional Foundation of Cyprus and the European Structural Funds ANABAΘMIΣH/ΠAΓIO/ 0308/32. Notes

The authors declare no competing financial interest.

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REFERENCES

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