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Direct Analysis of Lipophilic Antioxidants of Olive Oils Using Bicontinuous Microemulsions Eisuke Kuraya,*,†,‡ Shota Nagatomo,‡ Kouhei Sakata,‡ Dai Kato,§ Osamu Niwa,§ Taisei Nishimi,∥ and Masashi Kunitake*,‡ †

Science and Technology Division, Okinawa National College of Technology, 905 Henoko, Nago, Okinawa 905-2192, Japan Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto, Kumamoto 860-8555, Japan § National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan ∥ Japan Technological Research Association of Artificial Photosynthetic Chemical Process (ARPChem), Itopia Hashimoto Building 7F, 2-11-9 Iwamoto-cho, Chiyoda-ku, Tokyo 101-0032, Japan ‡

S Supporting Information *

ABSTRACT: Quantitative analyses of olive oil for lipophilic antioxidants, such as α-tocopherol and phenolics, by simple electrochemical measurements were conducted in a bicontinuous microemulsion (BME), which was bicontinuously composed of saline and toluene microphases with a surfactant system. Lipophilic antioxidants in oils were directly monitored in BME solutions using a lipophilic, fluorinated nanocarbon-film electrode (F−ECR). The combination of a well-balanced BME and extremely biased electrodes, such as strongly hydrophilic indium/tin oxide and strongly lipophilic (hydrophobic) F−ECR, allowed individual monitoring of hydrophilic and lipophilic antioxidants in the same BME solution without any required extraction. Furthermore, values for the charge Q, integrated from observed currents, showed good linear relationships with the results of conventional assays for antioxidant activity, namely, total phenolics and oxygen radical absorbance capacity assays, even with practical food samples. This proposed methodology provided a very simple, rapid, easily serviceable, and highly reproducible analysis that possesses great potential for applications to a wide range of chemical mixtures, in terms of analyte and media, beyond food oils.

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attributed to biological compounds that possess antioxidant activity.8 Antioxidants in foods and biological samples have been investigated by conventional chemical assays9 as well as electrochemical analyses10−14 in aqueous media, organic solvents,15 or emulsions.16−18 Among them, olive oils have been analyzed electrochemically in organic solvents11,19 or emulsions.18 Analytical evaluations of antioxidant capacities of olive oils have also been performed by detecting the main antioxidant concentrations. Considering the attention focused on antioxidants for evaluating olive oil functionality, measuring the antioxidant capacity of EVOO is an important food industry task. Several methods for identification and quantification of antioxidant substances in EVOO have been reported.20 Different antioxidants can be separated by chromatographic methods, such as high performance liquid chromatography

live oils produced by mechanical pressing of fruit from Olea europaea L., especially extra virgin olive oil (EVOO), contain various antioxidants that can scavenge oxygen radicals in biological systems.1 Among vegetable oils, EVOO is very rich in phenolic compounds, and the most important group of dietary phenolics is the polyphenols, such as flavonoids and phenolic acids. Tocopherols and carotenoids constitute a lipophilic antioxidant group and are also important compounds that contribute to overall oxidative stability.2,3 The bioavailable natural products of olive oil exert beneficial effects on physiological processes related to health and disease.4 Various studies, both in vivo and in vitro, have demonstrated that olive oil phenolic compounds beneficially alter microbial activity, oxidative processes, and inflammation.5 For instance, olive oil phenolics have been found to decrease reactive oxygen species production and elicit significant free-radical scavenging effects.1 Additionally, human intake of phenolic-rich EVOO considerably decreases oxidative DNA damage in vivo.6,7 These beneficial effects on undesirable oxidation have been partly © XXXX American Chemical Society

Received: September 10, 2015 Accepted: December 5, 2015

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DOI: 10.1021/acs.analchem.5b03445 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry (HPLC), with subsequent quantitation by UV detection or mass spectrometry (MS).19−21 Because of its greater simplicity, this approach, using spectrometric or spectrofluorometric measurement, has garnered increased attention. Total phenolic content, determined by the Folin-Ciocalteu method,22 and the oxygen radical absorbance capacity (ORAC) assay23 are widely used to determine a food or oil’s antioxidant capacity, assessing a sample’s overall antioxidant properties. Generally, phenolics are amphiphilic molecules, making it feasible to achieve maximal partitioning into a polar organic solvent, such as methanol.18,24 However, it is very likely that some compounds, only slightly soluble in methanol, might not be partitioned completely into the methanol phase. As a significant antioxidant capacity might thus remain in the olive oil, the ORAC value of methanolic extracts would be underestimated. Moreover, turbidity of some solutions hampers spectrometric measurements and does not yield reproducible results.3 Bicontinuous microemulsions (BMEs), in which water and oil phases coexist bicontinuously at the microscopic scale, can dissolve hydrophilic, amphiphilic, and lipophilic compounds simultaneously.25−28 In our previous research, a simple electrochemical analytical technique using a BME solution was proposed for evaluating antioxidant activity. The method allowed evaluation of hydrophilic, amphiphilic, and lipophilic antioxidants individually without extraction.29 Further investigation was quite important with respect to the quantitative and qualitative performance of this method against real samples if we are to reveal the advantages of this method compared with other conventional assays. The present study reports the use of an electrochemical method involving a BME solution (BMEEC) to evaluate antioxidant activities of mainly lipophilic antioxidants in olive oils and EVOO. Their antioxidant activities were also evaluated through total phenolic content determinations and ORAC assays for comparison with this BME-EC method. The proposed method can be considered a direct analysis of total antioxidant activity that is based on the electrochemical behavior of compounds.

Figure 1. Structural formulas of gallic acid, Trolox, α-tocopherol, tyrosol, and glyceryl trioleate.

Preparation of BME Solutions with Oils and Antioxidants. α-Tocopherol/toluene standard solutions (4.8, 2.4, 1.2, 0.59, and 0 mg/mL) and GTO and refined olive oil (ROO)/toluene standard solutions (0.5 g/mL) were prepared prior to mixing with a BME standard solution. Each ROO and GTO sample solution, in the presence of a certain α-tocopherol concentration, was prepared by 10-fold dilution of the αtocopherol standard solution with the GTO or ROO/toluene standard solution. For oil-less samples, BME solutions with αtocopherol without GTO and ROO were also prepared for comparison with oil samples by simply adding α-tocopherol/ toluene stock solution. Moreover, each BME solution with an olive oil was prepared by 10-fold dilution of 0.5 g/mL olive oil/ toluene solution with BME stock solution. Therefore, the oil concentration in BME was 5% (w/v). Because of high oil viscosities, oil weight was considered more accurate than relative volumes. BME solutions in the presence of GA and α-tocopherol were prepared by adding a GA aqueous solution and a 50 mM αtocopherol in 2-butanol to a BME solution. BME solutions of olive or model oils were prepared by blending 10% (by vol) of oil/toluene solution with 90% of BME solution through vortexing at room temperature for 1 min. No obvious changes in BME solutions’ phase structure were observed by addition up to 5% (w/v) oil samples. Concentration of GA in BME solutions was defined as apparent concentration in homogeneous saline, and concentrations of tyrosol and α-tocopherol in BME solutions were defined as apparent concentrations in toluene solution. Extraction of Phenolic Compounds for Ordinary Analysis. Extraction was performed by a procedure essentially similar to that reported by Benedetti.18 A solution, typically 10 mL of methanol and 5 g of an olive oil, was vortexed for 2 min at room temperature and then centrifuged for 10 min at 5000 × g. The supernatant was drawn off, the extraction was repeated once more, and the two supernatants were combined. Prior to liquid chromatographic-MS (LC-MS) analysis, the supernatant

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MATERIALS AND METHODS Chemicals and Samples. Folin-Ciocalteu reagent, gallic acid (GA) as a hydrophilic standard, α-tocopherol as a lipophilic antioxidant, toluene, sodium dodecyl sulfate (SDS), and 2-butanol were obtained from Nacalai Tesque, Inc. (Kyoto, Japan) at the highest grade available. Fluorescein disodium, ammonia solution, and NaCl were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Heptane, 6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) as a standard for ORAC assays, 2,2′-azobis(2-methyl propionamidine) dihydrochloride (AAPH), methanol, formic acid, acetonitrile, tyrosol, and glyceryl trioleate (triolein, GTO) were of analytical grade from Sigma-Aldrich, Inc. (St. Louis, MO, USA). The structures of the antioxidants and the major components in olive oils are shown in Figure 1. Distilled and deionized water were used in preparation of all solutions. Olive oil samples were purchased at local stores. Preparation of BME Systems. In accordance with previous reports,29 a mother emulsion solution, comprising phosphate buffer (pH 7.0), saline, sodium dodecyl sulfate surfactant, 2-butanol (cosurfactant), and toluene, was prepared as a macroscopically three-phase solution, consisting of macrosaline, BME, and macro oil phases. After allowing the solution to stand at 25 °C for more than 2 h, the middle phase was gently extracted as a BME solution. B


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Analytical Chemistry

constant from 2.5 to 3.0 min, before returning to starting conditions. MS was performed using a Micromass Quattro (QM) micro API triple quadrupole mass spectrometer (Waters Corp.) in negative electrospray mode. The desolvation gas was nitrogen, and the QM parameters were 120 °C source temperature, 350 °C desolvation temperature, 50 L/h cone gas flow, and 600 L/h desolvation gas flow. The QM capillary voltage was 3500 V. The SIR transition for tyrosol was m/z 137.21, with a 0.1 s dwell time and 29 V cone. Data were analyzed using MassLynx and QuanLynx software (Waters Corp.). Determination of Total Phenolic Content. Total phenolic content of samples was determined using the FolinCiocalteu reagent, with GA as a standard.22 Briefly, 10 μL of the Folin-Ciocalteu reagent was added to 20 μL of the sample in a 96 well microplate. After mixing, 40 μL of 10% aqueous sodium bicarbonate was added, and the mixture was allowed to stand for 30 min with intermittent shaking. The 750 nm absorbance was measured using a Varioskan Flash Multimode Reader (Thermo Fisher Scientific Oy, Vantaa, Finland). Total phenolic content was expressed as GA equivalents (GAE) in mg GA/kg oil. ORAC Assay. Oil antioxidant activities were determined by ORAC assay using a fluorimetric detection method.24,37 Briefly, AAPH was used as a peroxyl radical generator, Trolox as a standard, and fluorescein as a fluorescent probe. First, 35 μL of diluted sample, blank, or Trolox calibration solutions (0−50 μM) was mixed with 115 μL of 110.7 nM fluorescein in a 96well microplate and incubated at 37 °C for 15 min before injection of 50 μL of 32 mM AAPH solution. Fluorescence decay was measured at 37 °C every 2 min for 2 h at 485 and 525 nm for excitation and emission, respectively, using a Varioskan Flash Multimode Reader. The final ORAC values were calculated using the net area under decay curves, and the results were expressed as Trolox equivalents/kg oil (TE/kg oil).

was diluted with methanol and passed through a 0.2-μm polytetrafluoroethylene filter (Agilent Technologies, Inc., Santa Clara, CA, USA). Electrochemical Measurement in a BME System (BMEEC). A fluorinated electron cyclotron resonance (F-ECR) electrode was prepared by ECR sputtering with a CF4 plasma treatment for a short period. The resulting nanocarbon film on an F-ECR electrode has a nanocrystalline sp2 and sp3 mixedbond structure with an atomically flat surface. The fluorinated surface is easily prepared without losing the high activity and surface flatness of the electrode, which exhibits excellent electrochemical performance when used to study various biomolecules.30−34 The surfaces of hydrophilic ITO and lipophilic F-ECR electrodes contact predominantly with hydrophilic and lipophilic antioxidants in the microsaline and oil phases, respectively. Using a previously described procedure,29 cyclic voltammograms (CVs) were recorded at room temperature without degassing, using an ordinary threeelectrode system with an ALS Electrochemical Analyzer potentiostat (ALS600E, ALS Co., Ltd., Tokyo, Japan), from 0 to 1.5 V at a scan rate of 0.1 V/s. The electrode potential was recorded against a saturated calomel electrode (+244 mV vs SHE at 25 °C, ALS Co., Ltd.), and the current was measured against the response from a BME solution as a blank. A platinum wire electrode was used in BME solution as a counter electrode, and the geometrical electrode areas of F-ECR electrodes were regulated using an O-ring (7.7 mm i.d.). α-Tocopherol Determination by Supercritical Fluid Chromatography. For α-tocopherol content analysis in oil samples, α-tocopherol separation and quantification were carried out using supercritical fluid chromatography (SFC).35,36 SFC was conducted on an Acquity Ultra Performance Convergence Chromatograph (UPC2 ; Waters Corp., Milford, MA, USA) consisting of a degasser, binary gradient pump, autosampler at 10 °C, and column oven at 50 °C. Separations were achieved on an Acquity UPC2 BEH column (3.0 mm i.d. × 100 mm and 1.7 μm particle size, Waters Corp.). The mobile phase was CO2 (solvent A) and methanol with 0.2% formic acid (by vol, solvent B) at a flow rate of 2.5 mL/min. Gradient elution was performed using 99% A for 0.3 min, 95% A for 1.5 min, and 99% A for 0.2 min, reaching run completion at a total run time of 2 min. Oil samples were dissolved in heptane prior to injection of a 2 μL volume into the UPC2. The α-tocopherol present in an oil sample was calculated from respective α-tocopherol standard curves constructed from chromatograms detected at 294 nm. The αtocopherol present in extracts was determined against a standard α-tocopherol curve. Instrument control and data handling was managed using Empower 3 (Waters Corp.) operating in the Microsoft Windows software environment. Determination of Concentration of Tyrosol by LC-MS. Tyrosol in olive oils was quantified using LC-MS with selective ion recording (SIR). Chromatographic separations were performed using an ultraperformance liquid chromatographic (UPLC) system with an Acquity UPLC BEH C18 column (2.1 mm i.d. × 50 mm, 1.7 μm part. size, Waters Corp.). The autosampler was set at 4 °C and column compartment at 35 °C. Flow rates of 0.2 mL/min were maintained throughout all experiments and injection volumes at 2 μL. Solvent A was water with 0.1% NH4OH (by vol) and solvent B acetonitrile. Chromatographic separation of tyrosol required initial conditions of 85/15 A/B (v/v), from time = 0 to 2 min. From 2 to 2.5 min solvent ratios were changed to 5/95 A/B and then held

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RESULTS AND DISCUSSION Evaluation of Fatty Acid Contents and Antioxidant Activities. The good performance of BME electrochemistry with an F-ECR electrode for the assay of antioxidant activity in olive oils was confirmed by selecting and assaying several oil samples, including ROO, mixed refined and virgin olive oils (RVOO), and EVOOs. These samples included some antioxidants and fatty acids with a wide variety of concentrations. Therefore, prior to electrochemical analysis, those olive oil samples were elaborated by ordinary content analysis to reveal the availability and quantitative ability of this BME-EC method, as above, by comparison. Olive oils consist of various glycerides, with triglycerides the major component and very minor amounts of mono- and diglycerides. Quantitative olive oil characterizations are generally evaluated by their triglyceride fatty acid compositions. Generally, the major fatty acid in olive oils is oleic acid, a monounsaturated fatty acid, at roughly 60%. Olive oils contain ∼25% saturated fatty acids, with palmitic acid the major component. Unsaturated fatty acids besides oleic acid, such as linoleic and linolenic acids, might be significant markers for distinguishing types and origins of olive oils. Here, αtocopherol and tyrosol concentrations were measured using chromatography because they are known as major olive oil antioxidant species. In particular, α-tocopherol is often used as an antioxidant marker. Among the samples used here, the lowest and highest α-tocopherol contents were 105 and 203 C


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Analytical Chemistry Table 1. Concentrations of α-Tocopherol and Tyrosol and Antioxidant Activities of Tested Olive Oils

samples source concentrations of antioxidant species α-tocopherol ± SD (mg/kg)a tyrosol ± SD (mg/kg)b antioxidant activities total phenolics ± SD (GAE mg/kg)c ORAC value ± SD (mmol TE/kg)d BME-EC Q0−1.2V ± SD (μC/cm2)e

refined olive oil (ROO)

refined olive oil and virgin olive oil (R-VOO)

1

2

extra virgin olive oils samples (EVOO) 3

4

5

Imagine, Inc. (Japan)

Spain

Spain

Italy

J-Oil Mills, Inc. (Japan)

203 ± 5 not detected ( 0.996). BME solutions in the presence of α-tocopherol produced a simple linear correlation between peak currents and α-tocopherol added, with the line passing through the origin. In the case of model GTO oil, peak currents from α-tocopherol increased linearly with increased αtocopherol in the oil, and the line passed through the origin as well. However, the slope of peak currents against α-tocopherol concentrations in BME with GTO was approximately half the value compared with the BME value in the presence of αtocopherol alone. In a BME solution, the redox reaction is generally ruled by a simple diffusion of electroactive species.29 Therefore, the decreased slope observed here indicated the reduction of the α-tocopherol diffusion coefficient in BME because of increased viscosity imparted by GTO. This effect was easily confirmed by the fact that peak current slopes E


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Analytical Chemistry

and concentration. The relation of peak current slopes from three BME systems were essentially the same as those for the related Q0−1.2V values (Figure 5A). However, the apparent native α-tocopherol concentration in ROO 1, estimated from the intercept, was 335 mg/kg and ca. 150% higher than that estimated from the peak current. This was interpreted to indicate the existence of unknown antioxidants, other than native α-tocopherol and tyrosol, in ROO 1 because tyrosol was not detected by SFC. Electrochemical Measurements of Olive Oils in BME Solutions. Quantitative analyses of olive oils 1−5 were conducted in BME solutions with the addition of individual oils. Figure 6A shows CVs of BME solutions in the presence of

Figure 5. Plots of peak currents of α-tocopherol at 0.69 V in CVs for BME solutions (red line with diamonds) with 5% (w/v) in oil samples of GTO (blue dashed line with circles) and ROO 1 (green line with squares, A) and Q0−1.2V, integrated from currents from 0 to 1.2 V (B) against α-tocopherol content in oil samples. CVs measured in BME solution using an F-ECR electrode at 0.1 V/s, corresponding to CVs in Figure 4. Each plot is measured three times, and average values with error bars are shown.

increased with decreased GTO content in BME solutions. (Data are not shown.) Interestingly, BME solutions with ROO 1 exhibited a linear correlation of peak currents from α-tocopherol with a very similar peak current slope related to the α-tocopherol concentration and a certain intercept. The good agreement of these slopes appeared to indicate very similar diffusion coefficients between BME solutions for GTO and ROO 1. The intercept for BME solutions with ROO indicated the existence of natural α-tocopherol in ROO 1. The α-tocopherol concentration in ROO 1 was estimated, from the intercept in Figure 5A, at 215 mg/kg, which was in good agreement with the value, 203 mg/kg, measured by SFC analysis. Figure 5B illustrates linear correlations of the charge integrated from oxidative peak currents (Q) used in CVs of BME solutions with α-tocopherol concentrations in oil samples of GTO and ROO 1. The Q0−1.2V values, integrated from the currents from 0 to 1.2 V in the CVs shown in Figure 4, were proportional to the α-tocopherol concentration, with a high correlation coefficient (R2 > 0.998). These results correlated as highly as the correlation observed between the peak current

Figure 6. CVs (A) of BME solutions in the presence of 5% (w/v) olive oils, showing ROO 1, R-VOO 2, and EVOOs 3−5, and CV (B) of BME solution in 0.1 mM tyrosol using an F-ECR electrode at 0.1 V/s.

5% (w/v) olive oils 1−5. The oxidative peaks at 0.69 V, normally attributed to α-tocopherol oxidation, were observed for all samples (1−5). Moreover, oxidative peaks were also observed at 0.96 V for samples 2−5 except for ROO 1. Especially, EVOOs 3−5 showed high current responses at 0.96 V. The chemical assignment of the peak at 0.96 V is unknown. In addition, increased oxidative currents beyond 1.0 V were observed for samples 2−5. The origin of currents beyond 1.0 V might be attributed to tyrosol, a common antioxidant in olive oils. Certainly, CVs of tyrosol in BME produced a broad oxidative current, which starts to appear above 0.5 V (Figure 6B). F


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Analytical Chemistry In fact, α-tocopherol concentrations in 2−5, estimated from peak currents in CVs, were several times higher than values obtained by SFC. Model GTO oil and ROO 1 allowed analysis of α-tocopherol concentration by BME−EC measurement. In the case of other olive oils that contained antioxidative species besides α-tocopherol, it appeared difficult to analyze for individual concentrations of α-tocopherol or other antioxidants because of overlapping currents from other antioxidants, such as tyrosol. As shown in Table 1, tyrosol was not found in ROO 1. Electrochemical Determination of Antioxidant Activities of Olive Oils in Terms of Electrical Charges. Although qualitative and quantitative analysis for each antioxidant species proved difficult, effective quantitative evaluation of total antioxidant activities in real olive oils remains significant for food analysis. As an estimate of an olive oil’s antioxidative power, a Q0−1.2V value was obtained by integration of oxidative currents in each CV, and the values were compared with total phenolics and ORAC values of the same oil. As a single quantitative value, Q might prove advantageous over peak current evaluations. Figure 7 shows correlations of total phenolics and ORAC values for olive oil samples 1−5 with Q0−1.2V values measured

with no need for extraction, and thus eliminated possible uncertainties regarding sufficient extraction. Ninfali et al. have mentioned the problem of low methanol solubility for antioxidant species in olive oils.3

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CONCLUSION The present results clearly demonstrated the high applicability and reliability of a BME−EC method for quantitative analysis of antioxidants in olive oils. It should be emphasized that highly reproducible results were obtained with these reusable F-ECR electrodes. Surprisingly, the F-ECR electrodes were able to be reused more than 100 times because of a chemically inert carbon surface and cleaning effects of a BME solution.29−34 The BME−EC technique proposed here for analysis of antioxidant chemicals in olive oils exhibited a reliable and good performance that was comparable with conventional techniques. The lack of an extraction step in this method is a major improvement compared with other antioxidant assays. As a very simple, rapid, easily serviceable, and highly reproducible analysis, this methodology has the potential for application to a wide range of mixtures, in terms of analyte and media, beyond food oils.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03445. Experimental flow of quantitative analyses of antioxidant activities, details of preparation of F-ECR and ITO electrodes, setup of the electrochemical cell and analytical method, and table of composition fatty acids of olive oils (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Phone: 81-980-55-4043. Fax: 81-980-55-4044. E-mail: [email protected]. *Phone: 81-96-342-3674. Fax: 81-96-342-3679. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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Figure 7. Correlations of total phenolics content (solid circle, dotted line) and ORAC values (open circle, solid line) against Q0−1.2V values for olive oil samples 1−5. Q0−1.2V values obtained from corresponding CVs in Figure 6A.

ACKNOWLEDGMENTS We gratefully acknowledge Megumi Kubota, Shina Nakada, and Akiko Touyama for sample preparation, ORAC assays, and LC−MS analyses. This work was partially supported by a Grant-in-Aid for Scientific Research on Innovative Areas: “New Polymeric Materials Based on Element-Blocks (No.2401)” (24102006) from the Ministry of Education, Culture, Sports, Science and Technology. The work was also conducted in part at the Nano-Processing Facility, AIST, Japan.

electrochemically in BME solutions. Total phenolics and ORAC values were expressed as GAE and TE, respectively. The Q0−1.2V values showed good linear relation to both the total phenolics and ORAC assays. The correlation coefficient R2 was 0.935 and 0.962, respectively, as indicated by the regression line. In agreement for the three methods, EVOO 3−5 revealed higher antioxidant activities than ROO 1 and R−VOO 2. It was demonstrated here that BME−EC techniques allowed evaluation of the oils’ overall antioxidant activities, which corresponded well with results by conventional methods. Furthermore, it should be emphasized that conventional assays, such as total phenolics and ORAC assays, require extraction into methanol prior to assay. In contrast, the present BME−EC method provided a straightforward approach for oil analysis,

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