Antioxidant Properties and Efficacies of Synthesized Alkyl Caffeates

Article pubs.acs.org/JAFC

Antioxidant Properties and Efficacies of Synthesized Alkyl Caffeates, Ferulates, and Coumarates Ann-Dorit Moltke Sørensen,*,† Erwann Durand,‡ Mickael̈ Laguerre,‡ Christelle Bayrasy,‡ Jérôme Lecomte,‡ Pierre Villeneuve,‡ and Charlotte Jacobsen† †

Division of Industrial Food Research, National Food Institute (DTU Food), Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark ‡ CIRAD, UMR IATE, Montpellier F-34398, France S Supporting Information *

ABSTRACT: Caffeic, ferulic, and coumaric acids were lipophilized with saturated fatty alcohols (C1−C20). The antioxidant properties of these hydroxycinnamic acids and their alkyl esters were evaluated in various assays. Furthermore, the antioxidant efficiency of the compounds was evaluated in a simple o/w microemulsion using the conjugated autoxidizable triene (CAT) assay. All evaluated phenolipids had radical scavenging, reducing power, and metal chelating properties. Only caffeic acid and caffeates were able to form a complex with iron via their catechol group in the phenolic ring. In the o/w emulsion, the medium chain phenolipids of the three homologues series were most efficient. The antioxidant properties and efficacies were dependent upon functional groups substituted to the ring structure and were in the following order: caffeic acid and caffeates > ferulic acid and ferulates > coumaric acid and coumarates. Moreover, the results demonstrated that the test system has an impact on the antioxidative properties measured. KEYWORDS: phenolipids, lipophilization, caffeic acid, ferulic acid, coumaric acid, conjugated autoxidizable triene (CAT) assay, cut-off effect

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INTRODUCTION Lipid oxidation in food systems is a critical factor that affects food quality, nutrition, safety, color, and consumers’ acceptance. Moreover, the industry is becoming increasingly interested in incorporating healthy lipids such as omega-3 polyunsaturated fatty acids into food systems. An adverse effect of this is that it increases the susceptibility of the product to oxidative deterioration. However, all food products containing unsaturated lipids can undergo oxidative deterioration. Hence, developing effective approaches to limit lipid oxidation in food systems constitutes both a challenge and a need. Most foods are complex matrices, e.g., in the form of emulsions into which lipids are incorporated. In emulsions, antioxidants may partition into at least three different phases: the aqueous phase, the oil phase, and the oil−water interface. Since lipid oxidation in emulsions is initiated at the interface, the location of antioxidants in a given emulsion system is crucial for their effectiveness.1 Extensive work has been carried out to evaluate the effectiveness of antioxidants in emulsions. The so-called polar paradox hypothesis was the first hypothesis introduced on antioxidant efficacies in emulsions. The polar paradox hypothesizes that apolar antioxidants are more efficient in emulsions than polar antioxidants.2 The hypothesis is explained by differences in the antioxidants’ affinity toward the different phases. Thus, apolar antioxidants are more efficient in emulsions due to their ability to orient themselves closer to the oil−water interface, whereas polar antioxidants are diluted in the aqueous phase.3 Several studies have supported the polar paradox hypothesis.4−6 However, later research on the effect of © 2014 American Chemical Society

a range of different antioxidants in different emulsion systems contradicts the polar paradox hypothesis.7−10 In 2009, the cut-off concept was introduced based on results obtained with a lipophilized phenolic compound (phenolipid), chlorogenic acid,7 which was lipophilized to different degrees with saturated unbranched fatty alcohols. Thereby, it was possible to evaluate the antioxidant effectiveness related to the hydrophobicity of the antioxidant. Later, this theory was corroborated with alkyl rosmarinates.11 Antioxidant efficiencies of alkyl chlorogenates and alkyl rosmarinates increased with the increment of alkyl chain length until it reached a threshold, critical chain length (CCL). Further extension of the alkyl chain length resulted in a decreased antioxidant efficacy. Thus, the cut-off hypothesis relies on a nonlinear relationship between antioxidant activity and hydrophobicity and is related to the saturated alkyl chain length esterified to the phenolic compound.7,11 The efficacy of antioxidant homologues was related to the partitioning of these antioxidants in an emulsion system. It was assumed that antioxidants of CCL were in the highest concentration at the oil−aqueous surface where lipid oxidation is initiated. In addition, antioxidant homologues with chain length below and above CCL were driven away from the oil−aqueous surface.12 In this study, simple phenolic compounds were lipophilized with saturated fatty alcohols of different alkyl chain lengths. Received: Revised: Accepted: Published: 12553

February 6, 2014 November 17, 2014 December 2, 2014 December 2, 2014 dx.doi.org/10.1021/jf500588s | J. Agric. Food Chem. 2014, 62, 12553−12562

Journal of Agricultural and Food Chemistry

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Figure 1. Molecular structure of caffeic, ferulic, and coumaric acids and synthesized alkyl caffeates, ferulates and coumarates. Methyl Caffeate, Methyl Feulate, and Methyl Coumarate Synthesized with Amberlite IR-120H. Phenolic acid (3 g, caffeic acid (16.7 mmol), ferulic acid (15.4 mmol), and coumaric acid (18.3 mmol)) were solubilized in methanol (150 mL, 3.7 mol) in a dark, sealed bottle in an orbital shaker. The reaction between phenolic acid and methanol was catalyzed by adding 5% sulfonic resin Amberlite IR120H (w/w of total weight of both substrates, dried 4 h at 103 °C). Phenolipids Synthesized with Sulfuric Acid. Phenolic acid (caffeic, ferulic, and coumaric acids) was solubilized in THF (tetrahydrofuran) in a dark, sealed bottle in an orbital shaker. THF was applied as a solubilization medium due to the poor solubility of phenolic acids in longer chain alcohols (lipophilic medium). Alcohol was added to the phenolic acid-THF solution in a mole ratio of 1 mol phenolic acid to 3−5 mol of alcohol. Excess of alcohol was reduced as much as possible due to difficulties in the purification of synthesized phenolipids. The purification difficulties were due to the similarity in polarity of synthesized phenolipids and unreacted alcohols. Pure sulfuric acid (5% v/v of total volume of alcohol and THF) was added to the reactants in THF. Purification of Synthesized Phenolipids. Methyl esters were purified one way, and all other phenolipids were purified in different ways depending on the phenolic acid from which they were synthesized. The different ways applied to purify the synthesized phenolipids are described below. The purity of the purified phenolipids was evaluated quantitatively by HPLC-MS13 and qualitatively by TLC. The HPLC-MS analysis of the synthesized compounds revealed a purity ≥95%. Purification of Methyl Caffeate, Ferulate, and Coumarate. When the reaction was completed (>90% conversion), the reaction was stopped by removal of Amberlite IR-120H and molecular sieves in a centrifugation and filtration step. Then THF and unreacted methanol were evaporated, the mixture was diluted in ethyl acetate, and unreacted phenolic acid was removed by washing the ethyl acetate solution with two different saturated salt solutions (sodium bicarbonate (3 times), sodium chloride (2 times)), and water (2 times). To remove traces of water, the ethyl acetate solution was dried over sodium sulfate. Finally, the purified methyl ester was obtained by evaporating off ethyl acetate. Purification of Butyl, Octyl, Dodecyl, Hexadecyl, Octadecyl, and Eicosyl Esters. When the reaction was completed (>90% conversion), molecular sieves were removed from the reaction mixture in a centrifugation and filtration step. Then, THF was evaporated, and the reaction mixture was diluted in ethyl acetate. To neutralize the reaction mixture in ethyl acetate, this solution was washed with water

The aim of the study was to evaluate the antioxidant properties of hydroxycinnamic acids with very similar structures and their alkyl esters (phenolipids) using different in vitro antioxidant assays. Moreover, the efficacy of the antioxidants was evaluated in a microemulsion using the conjugated autoxidizable triene (CAT) assay. The phenolics selected only differ in one chemical group on the phenolic ring (Figure 1). Caffeic acid has an o-dihydroxyl (catechol) group, ferulic acid has a hydroxyl and a methoxyl group, and coumaric acid has one hydroxyl group substituted to the benzene ring. It is hypothesized that the structure will impact antioxidant efficacy and critical chain length for the different phenolipids: caffeates, ferulates, and coumarates.

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MATERIALS AND METHODS

Materials. Phenolic acids (caffeic, ferulic, and coumaric), alcohols, Amberlite IR-120H, and molecular sieves were purchased from SigmaAldrich (Saint Quentin, France). All solvents used were of HPLC or analytical grade and were purchased from Sigma (Saint Quentin, France). Silica columns for flash chromatography were purchased from Acros organics (Geel, Belgium). Tung oil (872 g/mol), Brij 35 (a nonionic polyoxyethylene surfactant, estimated Mw 1198 g/mol), AAPH (2,2′-azobis-2-methyl-propanimidamide, dihydrochloride), phosphate buffer solution (PBS, pH 7.2), alumina, BHT (butylated hydroxytoluene), and trolox were purchased from Sigma-Aldrich (Steinheim, Germany). EDTA (Titriplex, ethylenedinitrilo-tetraacetic acid disodium salt dehydrate) was purchased from Merck (Darmstadt, Germany). Synthesis of Caffeates, Ferulates, and Coumarates (Phenolipids). Esterification of caffeic, ferulic, and coumaric acids was catalyzed by acid either added as the strongly acidic sulfonic resin Amberlite IR-120H or as pure sulfuric acid to the reaction medium. The composition of the reaction mixtures depending on catalyst applied is described below. Molecular sieves (3 Å, 4−8 mesh, 40 mg/ mL, Aldrich, St. Louis, MO, USA) were added to the reaction mixtures to remove water generated during reaction. The reaction mixtures were stirred in an orbital shaker (250 rpm, 55 °C) during the whole reaction time. Samples (20 μL) from the reaction mixtures were regularly withdrawn, mixed with methanol (980 μL), filtered (0.45 μm syringe filter Millex-FH, Millipore Corp., Bedford, MA, USA), and analyzed by reverse phase HPLC with UV detection at 328 nm. 12554

dx.doi.org/10.1021/jf500588s | J. Agric. Food Chem. 2014, 62, 12553−12562


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were also diluted in PBS (pH 7.2). The final concentration of antioxidant was 100 μM. Iron, as FeSO4, was solubilized in 0.50 M HCl and buffer (1:1, v/v) and added to pure buffer and to the antioxidant−buffer solution. The final concentration of iron was 100 μM. Each solution was measured using a spectrophotometer (UV1800, Shimadzu Scientific Instruments, Columbia, MD, USA), and a spectrum was recorded in the UV−vis region (200−800 nm). Conjugated Autoxidizable Triene (CAT) Assay. Stock solutions of the different hydroxycinnamic acids, their alkyl esters, and trolox were prepared in methanol. Various volumes of these antioxidant methanolic solutions were added to 1.8 mL of PBS (pH 7.2) and then filled up to 2 mL with pure methanol. These buffered solutions with antioxidant (50 μL) were transfered into a microtiterplate, (UV-Star 96-well microplate, Greiner, Frickenhausen, Germany). The microplate was then preheated (37 °C) and stirred in the microplate reader (Synergy 2, BioTek, Winooski, VT, USA) for 5 min at medium speed. Brij 35-PBS (34 μM, 25 mL) was added to 5 mg of stripped tung oil in a brown glass bottle. Brij 35−PBS−tung oil was premixed for at least 10 s using a vortex apparatus before the mixture was homogenized with a POLYTRON PT1200E (Kinematica AG, Lucerne, Switzerland) at high speed for 4 min. Each well was then filled with 100 μL of the tung oil-in-PBS microemulsion; the plate was again preheated and stirred for 1 min. The oxidation of tung oil was initiated by adding 50 μL of 4 mM AAPH in PBS. Finally, each microplate well contained 200 μL of the following mixture: 115 μM stripped tung oil, 17 μM Brij 35, 1 mM AAPH, and various concentrations of phenolics, phenolipids, or trolox (0.2−0.8 μM). The progress of lipid oxidation was immediately followed by measuring the decrease in absorbance at 273 nm. Measurements were performed each minute for 6 h at 37 °C with 5 s stirring before each measurement. Each antioxidant concentration was measured in triplicate on the plate and via independent measurements (three different microplates), n = 9. Results were expressed as CAT value (mean ± SD). This method was developed by Laguerre et al.16 For further details about the calculations, see refs 7 and 16 by Laguerre et al. Removal of Tocopherols from Tung Oil. Tung oil was stripped from tocopherols using an alumina packed glass column. A glass column was packed with 25 g of alumina oxide in hexane. The excess of hexane was removed. Tocopherols were removed from the tung oil by passing 25 mL (200 mg/mL) of tung oil in hexane through the packed column. Pure hexane (50 mL) was then loaded to the column to get the loaded tung oil through the column. After passing the tung oil through the column, hexane in the stripped tung oil was removed using a vacuum rotary evaporator. Finally, a trace of hexane in the stripped tung oil was removed under nitrogen. The stripped tung oil was bottled (5 mg/bottle) in brown glass tubes, flushed with nitrogen, and stored at −80 °C until use (CAT Assay). Furthermore, the absence of tocopherols in the stripped tung oil was checked by HPLC according to the AOCS method.17 Data Treatment. All measurements were performed in triplicate and reported as average ± SD. In the separate method descriptions, calculations are described, or there are references to data treatment. Statistics. The obtained results were analyzed by one-way ANOVA (GraphPad Prism, version 4.03, GraphPad Software Inc.). Bonferroni multiple comparison post-test was used to test differences between samples. For statistics, a significance level of p < 0.05 was used. When a significant difference was observed between samples, they are denoted with different lowercase letters in the text, table and figures.

several times and dried over sodium sulfate to remove traces of water. Then, the remaining alcohol and phenolic acid were eliminated by flash chromatography (CombiFlash Companion system, Teledyne Isco Inc., Lincoln, NE, USA). Separation was carried out on a silica column (pore size 60 Å, Acros organics, Geel, Belgium) using an elution gradient of two solvents with different polarities. Because of the different polarities of the phenolic acids and their alkyl esters, a different elution gradient was applied for caffeates (chloroform and methanol, 0−100%), ferulates, and coumarates (dichloromethane and ethyl acetate, 5−100%), respectively. For some phenolipids, alcohol was still present after flash chromatography. In these cases, preparative TLC with different migration solvents was applied (caffeates, 96% chloroform and 4% methanol; ferulates, 95% dichloromethane and 5% ethyl acetate; and coumarates, 90% dichloromethane and 10% ethyl acetate). Antioxidant Properties. The antioxidative properties of the phenolic acids and their synthesized alkyl esters were evaluated using three different in vitro antioxidant assays: radical scavenging (DPPH), iron chelating activity, and reducing power. All antioxidants were evaluated in 4 concentrations (final concentrations: 50, 100, 200, and 400 μM) and were measured in triplicate (n = 3). The concentrations were selected based on earlier antioxidant property analysis and storage experiments with similar compounds.14 Radical Scavenging. Antioxidants were solubilized in methanol in different concentrations, and BHT was included in this assay as a positive control in the same concentrations. The antioxidant solution (150 μL) was diluted 1:1 (v/v) with 0.1 mM DPPH in ethanol (96%, 150 μL). Then, 200 μL of this mixture was transferred to a well in the microplate.15 The absorbance at 517 nm was measured after 30 min (RT, darkness) in a microplate reader (Synergy 2 BioTek, Winooski, VT, USA). Results are expressed as inhibition percentages.

⎛ A − A0 ⎞ Inhibition [%] = ⎜1 − s ⎟ × 100 Ab ⎠ ⎝ where As is the measured absorbance after the antioxidant-DPPH reaction, A0 is the measured absorbance of antioxidant in ethanol without DPPH (sample control), and Ab is the absorbance measured of DPPH without the antioxidant added. Reducing Power. Antioxidants were solubilized in methanol in different concentrations, and ascorbic acid was included in this assay as a positive control in the same concentrations. Antioxidant methanolic solutions (200 μL) were transferred to test tubes, and 0.2 M phosphate buffer (200 μL) and 1% potassium ferricyanide (200 μL) were added. These mixtures were incubated at 50 °C for 20 min. After incubation, 10% TCA (trichloroacetic acid, 200 μL) was added and mixed. This reaction mixture (228 μL) was transferred to an Eppendorf tube and mixed with an equal amount of water (228 μL). Ferric chloride (0.1%, 46 μL) was added to the reaction mixture, and it was incubated for 10 min at RT. After incubation, the reaction mixtures (200 μL) were transferred to the wells in the microplate,15 and absorbance was measured at 700 nm in a microplate reader (Synergy 2 BioTek, Winooski, VT, USA). Results are shown relative to ascorbic acid (AbsSample/AbsAscorbic acid). Iron Chelating Ability. Antioxidants were solubilized in warm water in different concentrations. Because of solubility problems with long alkyl chain phenolipids, only caffeic acid, ferulic acid, coumaric acid, and their methyl esters were evaluated for their chelating ability of ferrous chloride. EDTA was included in this assay as a positive control in the same concentrations as the phenols and phenolipids. For further details, refer to Sørensen et al.14 The absorbance was measured at 562 nm (UV-1800 Shimadzu, Columbia, MD, USA), and results are expressed as chelating activities. Antioxidant (Hydroxycinnamic Acids and Their Alkyl Esters)−Iron Interactions. Interactions between iron and the antioxidant were evaluated using a spectrophotometer according to the method developed by Sørensen et al.9 Methanolic solutions of caffeic acid, ferulic acid, coumaric acid, and their methyl esters were diluted in a buffer solution: 10 mM sodium acetate−imidazole (pH 7). Moreover, methanolic solutions of caffeic acid and methyl caffeate

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RESULTS Antioxidant Properties. All the synthesized antioxidants were evaluated for different antioxidant properties as radical scavenger (DPPH), reducing power, and iron chelation in in vitro assays. Radical Scavenging Properties. The phenolics and synthesized phenolipids were all able to scavenge free radicals measured by the DPPH assay (Figure 2A−C). Caffeic acid and caffeates had similar radical scavenging activities in the 12555



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ferulic acid and alkyl ferulates was independent of their concentration in the evaluated range (50−400 μM, Supporting Information, Figure A). Coumaric acid and alkyl coumarates had low DPPH inhibition medium, short chain alkyl ferulates, and BHT (Figure 2B). BHT had significantly lower DPPH inhibition than ferulic acid (∼1.5−4 fold) and alkyl ferulates (∼1.4−3 fold) at the different concentrations, but significant differences in DPPH inhibition between ferulic acid and alkyl ferulates were more unclear. The ranking of 12556



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and iron−methyl caffeate in two different buffers are shown in Figure 4. The absorbance measured was different for the two different buffers used. When either caffeic acid and iron or methyl caffeate and iron were solubilized in phosphate buffer, no absorbance was observed in the visible area, 400−800 nm (Figure 4A and B). In contrast, when the other buffer, sodium acetate−imidazole (10 mM), was used, both the combination of caffeic acid and iron or methyl caffeate and iron resulted in low absorbances in the visible area of the spectrum (Figure 4C and D). Moreover, these solutions changed from no color to blue or black color when iron was added. Phosphate buffer with caffeic acid and with and without iron added had peaks at the same wavelengths; however, when iron was added the absorbance was slightly higher in the UV area than that without iron added (Figure 4A). When methyl caffeate was solubilized in phosphate buffer, the spectra changed when iron was added compared to that when no iron was present (Figure 4B). This may indicate other types of interactions with iron. Furthermore, when iron was added to phosphate buffer without antioxidant, the spectrum changed, whereas iron added to sodium acetate−imidazole buffer did not alter the spectrum (Figure 5). Thus, phosphate buffer may also participate in interactions with the iron added. The other compounds evaluated, ferulic acid, methyl ferulate, coumaric acid, or methyl coumarate solubilized in sodium-acetate imidazole buffer, were not influenced by the addition of iron since similar absorbance spectra were obtained with and without iron addition (data not shown). This indicates that a catechol structure is needed to form a complex with iron, while a monophenol (coumaric acidlike structure) or a monophenol combined to a methoxyl group in ortho position (ferulic acid-like structure) does not allow the formation of such an iron complex. The possible interactions between iron and caffeates will be discussed in more detail later. Antioxidant Activity Evaluated by the CAT Assay (o/w Emulsion). The antioxidant effect of the hydroxycinnamic acids and their alkyl esters was evaluated in the CAT assay. In the CAT assay, lipid oxidation and the effect of the tested antioxidants are evaluated from the decrease in conjugated trienes over time. The results for the three series of homologues are shown in Figure 6. Similar to the other assays evaluated, the highest antioxidant effect was obtained with alkyl caffeates followed by alkyl ferulates with CAT values of 1.3−5.0 and 0.2−1.5, respectively. Coumarates had close to zero antioxidant effect due to the low CAT values measured (0.05−0.36). For caffeic acid and alkyl caffeates, an increase in antioxidant capacity with increasing alkyl chain length up to octyl caffeate (C8) was observed, whereas further increase in the alkyl chain length resulted in a decreased antioxidant

Figure 3. Reducing power properties of hydroxycinnamic acids and their alkyl esters at 100 μM calculated relative to ascorbic acid (positive control in the assay). (A) Caffeic acid and caffeates, (B) ferulic acid and ferulates, and (C) coumaric acid and coumarates. C0 is phenolic acid, and C1 to C20 indicates the length of the alkyl chain grafted to the phenolic acids (phenolipids). Error bars indicate the standard deviation (n = 3). Significant differences between different compounds are denoted with different lowercase letters (significance level was p < 0.05).

Table 1. Iron Chelating Ability [%] of Caffeic Acid, Ferulic Acid, Coumaric Acid, and Their Methyl Esters (50−400 μM)a concentration [μM] compounds caffeic acid methyl caffeate ferulic acid methyl ferulate coumaric acid methyl coumarate EDTA

50 7.37 11.6 12.3 3.92 0.05 0.80 65.5

± ± ± ± ± ± ±

0.57 0.44 0.80 1.51 0.22 0.35 3.26

100 b,c b b c,d d d a

16.0 22.3 14.4 13.0 1.11 5.93 94.2

± ± ± ± ± ± ±

0.71 1.23 1.30 1.85 0.08 0.57 2.29

200 c b c c d d a

39.8 59.3 24.2 27.1 1.47 14.7 99.3

± ± ± ± ± ± ±

2.85 1.53 2.64 3.01 1.56 1.03 0.80

400 c b d d f e a

78.2 ± 0.73 b 75.1 ± 8.79 b 36.6 ± 2.95 d 46.0 ± 3.50 c 4.30 ± 0.60 f 26.9 ± 3.16 e 100 ± 0.04 a

a

EDTA was included as a positive control. The analyses were performed in triplicate (n = 3). When a significant difference was observed between samples (within each column, i.e., concentrations), it is denoted with different lowercase letters (significance level was p < 0.05). 12557



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Figure 4. UV−vis spectra (200−800 nm) recorded for caffeic acid and methyl caffeate solubilized in different buffers with and without iron addition. (A) Phosphate buffer (pH 7.2), caffeic acid (100 μM) and ± Fe (100 μM), (B) phosphate buffer (pH 7.2), methyl caffeate (100 μM) and ± Fe (100 μM), (C) sodium acetate−imidazole buffer (10 mM, pH 7.0), caffeic acid (100 μM) and ± Fe (100 μM), and (D) sodium acetate−imidazole buffer (10 mM, pH 7.0), methyl caffeate (100 μM), and ± Fe (100 μM).

compounds is primarily due to resonance stabilization of the phenoxyl radical and various substitutions, e.g., electronegative hydroxyl groups may increase radical stability. Thus, di- and trihydroxyl substitutions have greater antioxidant activity than monohydroxyl substitution due to greater resonance stabilization.18,19 Moreover, Dueñas et al.20 have evaluated antioxidant activities of O-methylation of the catechol group in quercetin and catechins using the FRAP assay (ferric reducing power) and ABTS•+ assay (radical scavenging). The O-methylation of one of the hydroxyls in the catechol group reduced the antioxidant activity compared to that of the parent compounds.20 Thus, destroying the catechol structure lowered the antioxidant activity of the compound. Therefore, the efficacy based on radical scavenging and reducing power assays of the evaluated antioxidants is expected to be highest for caffeic acid followed by ferulic acid and coumaric acid. The obtained results confirmed this ranking in activity of the evaluated hydroxycinnamic acids (Figures 2 and 3). Moreover, Kikuzaki et al.21 have also reported that caffeic acid had the highest DPPH scavenging activity followed by ferulic acid and coumaric acid. This ranging of the three types of alkyl ester is also observed in another study where short saturated alkyl esters of caffeic and ferulic acids were evaluated.22 In addition, ester of sinapic acid has shown antioxidant activities in between those of alkyl caffeates and ferulates.23 In the DPPH assay, caffeic acid and alkyl caffeates showed similar activities, whereas ferulic acid and coumaric acid showed higher activities than their alkyl esters (Figure 2). However, among the alkyl esters there was no clear pattern with respect to the effect of the chain length on the DPPH

capacity (Figure 6). Thus, the most efficient antioxidant among the alkyl caffeates was octyl caffeate followed by dodecyl caffeate (not significantly different), and a cut-off effect was observed with C8−C12 as critical chain length. Octadecyl caffeate and eicosyl caffeate were significantly less efficient antioxidants than caffeic acid. The same clear cut-off effect as that for alkyl caffeates, with octyl caffeate being the best of the homologues series, was not observed for alkyl ferulates and alkyl coumarates, even though a global nonlinear tendency was observed between the CAT values and the alkyl chain lengths. The ranking of the alkyl ferulates was as follows: C12a = C8a = C4a ≥ C1a,b ≥ C0b = C16b (Figure 6). Alkyl coumarates had limited antioxidant efficacy in this assay. The most efficient antioxidant among the alkyl coumarates was dodecyl coumarates, and the ranking of the alkyl coumarates according to their antioxidant efficiency was as follows: C12a ≥ C8a,b = C4a,b ≥ C0b = C16b = C1b (Figure 6). Even though the antioxidative effects of coumaric acid and alkyl coumarates were limited, an improvement in the efficacy was observed with esterification. Dodecyl coumarate had approximately 5.6 times higher CAT value than coumaric acid.

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DISCUSSION Antioxidant Properties and Molecular Structure. Antioxidant properties depend upon the molecular structure of the compounds.18 Apart from the different degrees of lipophilization (alkyl chain length), three different molecular structures were evaluated: caffeic acid, ferulic acid, and coumaric acid (Figure 1). The antioxidant activity of phenolic 12558



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caffeic and ferulic acids had higher activities than their alkyl esters, and the activities decreased with increasing alkyl chain lengths for alkyl caffeates and alkyl ferulates (Figure 3). However, this is contrary to findings reported by Garrido et al.22 who used the FRAP assay to measure reducing power, which was similar to the IC50 findings, i.e., compounds with low IC50 had high reducing power. Phenylethanoid glycosides are ester derivatives of phenolic acids and are widely distributed in nature. These phenylethanoid glycosides are more lipophilic compounds compared to phenolic acids. Interestingly, some phenylethanoid glycosides of caffeic and ferulic acids, namely, acyl derivatives, have shown lower (∼2-fold) IC50 values than those of their corresponding phenolic acid,24 whereas alkyl esters of caffeic acid showed similar IC50 values as those of caffeic acid.22 Thus, some of these alkyl and glycoside esters of phenolic compounds may serve as good antioxidants in emulsions or other complex systems due to increased lipophilicity, which is important in multiphases systems. Both antioxidant assays in this study are homogeneous systems; whereas the DPPH assay is measured in ethanol, the reducing power assay is measured in distilled water. Thus, solubility may influence the results obtained. The evaluated compounds are more soluble in solvent than water, i.e., less polar. Therefore, the reduced activity with increased alkyl chain length in the reducing power assay can at least partly be explained by the reduced solubility of the more lipophilic alkyl esters in water. In the DPPH assay, the ethanolic medium did not influence the solubility of the more lipophilic alkyl esters. Additionally, the results obtained in the DPPH assay are in accordance with results obtained earlier by Kikuzaki et al.21 In their study, ferulic acid also had higher scavenging activity than alkyl ferulates, and no dependency in lipophilicity and antioxidant activity of the different alkyl ferulates was observed.21 Thus, these data confirm the earlier assumption regarding solubility issues that the ethanolic solution does not have the same impact on the solubility of the more lipophilic alkyl esters as the water solution. In addition, the lower activity observed for alkyl ferulates and alkyl coumarates than their corresponding acids in the DPPH and reducing power assays has also been reported earlier for other phenolipids, namely, dihydrocaffeates25 and rutin esters.26 These findings may indicate that the esterification site for the alkyl chain play an active role for the antioxidant properties measured in these assays. Additionally, López-Giraldo et al.27 have evaluated the kinetics of the reaction of chlorogenic acid and its alkyl esters with the DDPH radical. For the different compounds, strong differences were observed. Hence, pathways of DPPH stabilization were suggested to be different for chlorogenic acid and its alkyl esters, which were confirmed by LC-MS characterization of the reaction products obtained. Another antioxidant property is chelation of transition metal irons, which are prooxidants. Therefore, if they are chelated their interactions with the lipid substrate or lipid hydroperoxides is supposed to be inactivated.28 The iron chelating assay can thus reveal the ability of the compounds to chelate iron. This assay is, like the reducing power assay, a homogeneous solution of water wherein the antioxidants are solubilized. Solubility was indeed an issue for this assay. It seemed like the methanolic stock solutions of the different antioxidants interfered with the assay since the results obtained were not reproducible. The assay was repeated with the antioxidant solubilized only in water. Only caffeic acid, ferulic

Figure 5. Spectra (200−800 nm) recorded for different buffers with and without iron addition. (A) Phosphate buffer (pH 7.2) ± Fe (100 μM) and (B) sodium acetate−imidazole buffer (10 mM, pH 7.0) ± Fe (100 μM).

Figure 6. Influence of the alkyl chain length of phenolic alkyl esters on their antioxidant activity in stripped tung oil-in-water microemulsions oxidized by AAPH at 37 °C (CAT assay). Symbols: ●, caffeic acid and caffeates; ■, ferulic acid and ferulates; and ▲, coumaric acid and coumarates. Error bars indicate the standard deviation (n = 3).

radical scavenging activity. These findings are similar to observations reported by Garrido and co-workers,22 who had measured the IC50 for different short chain saturated alkyl esters (methyl, ethyl, propyl, and butyl) of caffeic and ferulic acids using the DPPH assay. They reported similar IC50 values for caffeic acid (16.6 μM) and caffeates (13.5−14.5 μM), whereas ferulic acid (44.6 μM) was reported with lower IC50 than ferulates (56.3−74.7 μM). For the two evaluated alkyl esters, the efficiency was as follows for caffeates and ferulates: ethyl (lowest IC50) > methyl > butyl > propyl and butyl > propyl > ethyl > methyl.22 In contrast, in the reducing power assay, 12559



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in the simple antioxidant assays. Moreover, several components present in the emulsions may interfere with the antioxidants and thus alter their efficacy. The CAT assay developed by Laguerre et al.17 measures the antioxidant activity in a microemulsified system with AAPH used as a peroxyradical initiator. This assay gave different results than those obtained with the DPPH (radical) assay. In the CAT assay, there was an effect of the alkyl chain length grafted to caffeic acid, which was not observed in the DPPH assay. The difference between the results obtained with the CAT assay and the DPPH assay is suggested to be due to the influence of the antioxidant partitioning in the microemulsion. However, the influence of the phenolic structure was similar to the findings with the DPPH assay; caffeic acid was the most efficient antioxidant followed by ferulic acid and then with less activity coumaric acid. For the three homologues series of antioxidants, the trend was that the antioxidant efficiency was highest for the phenolipids with medium chain length. However, only alkyl caffeates showed significant differences between the different chain lengths. Other reported results from the CAT assay have also shown that the CCL was obtained for medium chain length phenolipids. For alkyl chlorogenates and rosmarinates, the antioxidant efficiency was highest with a chain length of 12 and 8 carbon atoms, respectively.7,11 Moreover, the antioxidant efficiency of alkyl rosmarinates is 3−5 times higher than that of alkyl chlorogenates and caffeates. This is suggested to be due to the substituted groups in the ring structure. Rosmarinic acid has two catechol groups, whereas chlorogenic acid and caffeic acid only have one. The most efficient alkyl caffeate in the CAT assay was octyl caffeate. Recent studies with alkyl caffeates in food emulsions have shown that the effect of the chain length depended upon the system it was added into. In milk emulsion, the most efficient alkyl ferulate was methyl ferulate (C1),35 and the most efficient alkyl caffeates were methyl caffeate (C1) and butyl caffeate (C4), whereas in mayonnaise the most efficient were butyl caffeate (C4), octyl caffeate (C8), and dodecyl caffeate (C12).36 Similarly, the CCL for alkyl rosmarinates has also been shown to be influenced by the different emulsion systems. Despite this, the CCL for alkyl chlorogenates in emulsion and fibroblasts was similar , dodecyl chlorogenate (C12).37 In addition, a study reported by Anselmi et al.38 shows that ferulic acid lipophilized with different saturated unbranched and branched alkyl chain lengths resulted in different threedimensional structures. Different anchorages of the phenolic with the different alkyl chains were observed, which resulted in a specific folding of some of the alkyl esters. This structure resulted in the highest antioxidative capacity of these molecules (octyl ferulate and dodecyl ferulate) in rat liver microsomes. It was assumed that it was due to the orientation of the phenoxy group outside the membrane surface, which may have favored the radical scavenging in the microsomes.38 In milk emulsion, octyl and dodecyl ferulate were reported to be intermediate to strong prooxidants.35 Unfortunately, the three-dimensional structure of the molecules was not analyzed. However, if the alkyl chain has folded toward the ethylenic protons as reported by Anselmi et al.,38 this is not a favorable structure in milk emulsions due to their prooxidative effects. Hence, CCL can be a variable parameter dependent upon the molecule and its three-dimensional structure, as well as the structure and composition of the emulsion system. Therefore, the effect of

acid, coumaric acid, and their methyl esters were evaluated. It is known that phenolic compounds containing a catechol group are able to chelate iron in the form of an iron-phenolic complex. Only caffeic acid and its esters contain a catechol group in their molecular structure. Nevertheless, all the evaluated hydroxycinnamic acids and their methyl esters had iron chelating activity (Table 1). UV−vis spectra (200−800 nm) of caffeic acid and methyl caffeate in sodium acetate− imidazole buffer revealed different absorbance patterns when iron was added (Figure 4). Furthermore, absorbance was detected in the visible area when iron was added. Change in the absorbance at 587 and 680 nm has been reported to correspond to iron−gallyl and iron−catechol complexes, respectively.29 Thus, the obtained spectra indicated the formation of an iron−catechol complex with caffeic acid and methyl caffeate. Moreover, the absorbance spectra for these compounds changed in the area of 250−400 nm upon iron addition. The absorbance spectra in the area of 250−400 nm are characterizing the ring substitutions. Similar results have been reported by Andjelković et al.30 who evaluated the formation of a complex between caffeic acid and iron in Tris buffer. When EDTA was added to the iron−caffeic acid complex, the absorbance spectrum changed again and was similar to that obtained for caffeic acid solubilized in Tris buffer without iron.30 On the basis of the results reported by Andjelković et al.30 with caffeic acid, iron, and EDTA and the fact that the catechol group was a part of the complex formed with iron, the changes in this area of the spectra were due to the formed complex. Ferulic acid, methyl ferulate, and methyl coumarate had chelating ability but no change in their UV−vis spectra when iron was added. None of these compounds has a catechol or gallyl group. Therefore, the similar spectra obtained with and without iron were expected. The chelating ability measured for those compounds is therefore suggested to be due to other types of interactions with iron. Earlier findings support this suggestion since both caffeic acid and coumaric acid were shown to form nanoparticles in the presence of iron.9 Moreover, the complex formed between iron and the catechol group in caffeic acid and methyl caffeate were not formed when sodium acetate−imidazole buffer was substituted with phosphate buffer. A change in the UV region of the spectrum with phosphate buffer upon iron addition was also observed. Thus, this indicated interaction between phosphate and iron that impeded the complex formation between iron and the catechol group. A similar observation for phosphate buffer and iron has also been reported by Andjelković et al.30 when they evaluated the interaction with iron and different phenolics. Therefore, the choice of buffer is crucial when evaluating the chelating ability of different compounds30 due to the changed reactivity with different buffers. Studies have shown that the most efficient antioxidants in limiting lipid oxidation in food emulsions during storage are not always comparable to antioxidant properties measured in in vitro antioxidant assays.31,32 Hence, these antioxidant assays are only indicators of the properties of the compound studied and do not provide information about the efficiency of the antioxidants in emulsions or complex food systems. Antioxidant Activity in Emulsions. In contrast to the above-discussed antioxidant assays, emulsions are heterogeneous systems. In general, the efficacy of antioxidants in food emulsion is dependent on their reactivity, properties, and partitioning in the emulsion.33,34 These factors are not included 12560



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CCL, critical chain length; EDTA, ethylenedinitrilo-tetraacetic acid; PBS, phosphate buffer solution; TCA, trichloroacetic acid; THF, tetrahydrofuran

antioxidants has to be studied in each emulsion system where antioxidant protection is needed. In conclusion, the evaluated hydroxycinnamic acids and their alkyl esters showed antioxidant properties and activity. The antioxidant properties were affected by the molecular structure. Caffeic acid was a stronger iron chelator than ferulic acid and coumaric acid due to its ability to form complexes with iron. Moreover, caffeic acid also had higher radical scavenging activity and reducing power than the other hydroxycinnamic acids. The CCL in the CAT assay was C8−C12 for alkyl caffeates. Additionally, esterification of caffeic acid with saturated short alkyl chains (methyl, ethyl, propyl, and butyl) was shown to improve the cell protective activity against oxidative stress, whereas caffeic acid, ferulic acid, and alkyl esters of ferulic acid were not able to protect the cells.22 Moreover, esters of caffeic and ferulic acids have also shown excellent inhibition of tumor cell proliferation.39 Thus, the lipophilicity of phenolic compounds is a crucial factor that can determine their efficiency as antioxidants in multiphase systems such as food emulsions and cellular systems. Hence, the synthesized phenolipids may serve as good antioxidants in food emulsions, especially alkyl caffeates, due to their free radical scavenging properties and antioxidant efficacy. However, their activity will also depend upon the partitioning of the antioxidant in the food emulsion as well as the interaction with other compounds present in the food emulsion. Our present and previous findings seem to suggest that the effect of alkyl esterification on the antioxidant activity may differ from one phenolic compound to another. Thus, antioxidant protection has to be further studied in each specific food emulsion with the different phenolic compounds.

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(1) Waraho, T.; McClements, D. J.; Decker, E. A. Mechanisms of lipid oxidation in food dispersions. Trends Food Sci. Technol. 2011, 22, 3−13. (2) Porter, W. L. Paradoxical behavior of antioxidants in food and biological systems. Toxicol. Ind. Health 1993, 9 (1−2), 93−122. (3) Frankel, E. N.; Huang, S.-W.; Kanner, J.; German, J. B. Interfacial phenomena in the evaluation of antioxidants: Bulk oils vs emulsions. J. Agric. Food Chem. 1994, 42, 1054−1059. (4) Huang, S.-W.; Frankel, E. N.; Schwarz, K.; German, J. B. Effect of pH on the antioxidant activity of α-tocopherol and trolox in oil-inwater emulsions. J. Agric. Food Chem. 1996, 44, 2496−2502. (5) Huang, S.-W.; Frankel, E. N.; Schwarz, K.; Aeschbach, R.; German, J. B. Antioxidant activity of carnosic acid and methyl carnosate in bulk oils and oil-in-water emulsions. J. Agric. Food Chem. 1996, 44, 2951−2956. (6) Frankel, E. N.; Huang, S. W.; Prior, E.; Aeschbach, R. Evaluation of antioxidant activity of rosemary extracts, carnosol and carnosic acid in bulk vegetable oils and fish oil and their emulsions. J. Sci. Food Agric. 1996, 72 (2), 201−208. (7) Laguerre, M.; Giraldo, L. J. L.; Lecomte, J.; Figueroa-Espinoza, M. C.; Barea, B.; Weiss, J.; Decker, E. A.; Villeneuve, P. Chain length affects antioxidant properties of chlorogenate esters in emulsion: the cutoff theory behind the polar paradox. J. Agric. Food Chem. 2009, 57 (23), 11335−11342. (8) Lue, B.-M. Enzymatic Lipophilization of Bioactive Compounds in Ionic Liquids. Ph.D. Thesis, Department of Molecular Biology, Aarhus University, Denmark, 2009. (9) Sørensen, A.-D. M.; Haahr, A.-M.; Becker, E. M.; Skibsted, L. H.; Bergenståhl, B.; Nilsson, L.; Jacobsen, C. Interactions between iron, phenolic compounds, emulsifiers, and ph in omega-3-enriched oil-inwater emulsions. J. Agric. Food Chem. 2008, 56, 1740−1750. (10) Yuji, H.; Weiss, J.; Villeneuve, P.; Giraldo, L. J. L.; FigueroaEspinoza, M.-C.; Decker, E. A. Ability of surface-active antioxidants to inhibit lipid oxidation in oil-in-water emulsion. J. Agric. Food Chem. 2007, 55, 11052−11056. (11) Laguerre, M.; Giraldo, L. J. L.; Lecomte, J.; Figueroa-Espinoza, M.-C.; Baréa, B.; Weiss, J.; Decker, E. A.; Villeneuve, P. Relationship between hydrophobocity and antioxidant ability of “phenolipids” in emulsion: a parabolic effect of the chain length of rosmarinate esters. J. Agric. Food Chem. 2010, 58, 2869−2876. (12) Laguerre, M.; Sørensen, A.-D. M.; Bayrasy, C.; Lecomte, J.; Jacobsen, C.; Decker, E. A.; Villeneuve, P. Role of Hydrophobicity on Antioxidant Activity in Lipid Dispersions from the Polar Paradox to the Cut-off Theory. In Lipid Oxidation: Challenges in Food Systems, 1st ed.; Logan, A., Nienaber, U., Pan, X., Eds.; AOCS Press: Urbana, IL, 2013; pp 261−296. (13) Giraldo, L. J. L.; Laguerre, M.; Lecomte, J.; Figueroa-Espinoza, M.-C.; Barouh, N.; Barea, B.; Villeneuve, P. Lipase-catalyzed synthesis of chlorogenate fatty acid esters in solvent-free medium. Enzyme Microb. Technol. 2007, 41, 721−726. (14) Sørensen, A.-D. M.; Nielsen, N. S.; Yang, Z.; Xu, X.; Jacobsen, C. The effect of lipohilization of dihydrocaffeic acid on its antioxidative properties in fish-oil-enriched emulsion. Eur. J. Lipid Sci. Technol. 2012, 114, 134−145. (15) Farvin, K. H. S.; Andersen, L. L.; Nielsen, H. H.; Jacobsen, C.; Jakobsen, G.; Johansson, I.; Jessen, F. Antioxidant activity of cod (Gadus morhua) protein hydrolysates: Part 1 - In vitro assays and evaluation in 5% fish oil-in-water emulsion. Food Chem. 2014, 149, 326−334. (16) Laguerre, M.; Lopez-Giraldo, L. J.; Lecomte, J.; Barea, B.; Cambon, E.; Tchobo, P. F.; Barouh, N.; Villeneuve, P. Conjugated autoxidizable triene (CAT) assay: A novel spectrophotometric method

ASSOCIATED CONTENT

S Supporting Information *

Results for the radical scavenging properties and reducing power with all the measured concentrations (50, 100, 200, and 400 μM). This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

The study is a part of the project entitled “Phenolipids as Antioxidants in Omega-3 Model and Real Food Systems − Effect of Alkyl Chain Length and Concentration” with project number 10-093655 financed by the Danish Research Council, Technology and Production. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Nathalie Barouh for her assistance with the purification of some of the synthesized phenolipids. ABBREVIATIONS USED AAPH, 2,2′-azobis-2-methyl-propanimidaminde dichloride; BHT, butylated hydroxytoluene; C0, phenolic acid; C1, methyl; C4, butyl; C8, octyl; C12, dodecyl; C16, hexadecyl; C18, octadecyl; C20, eicosyl; CAT, conjugated autoxidizable triene; 12561



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Antioxidant Properties and Efficacies of Synthesized Alkyl Caffeates

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