Surface-Active Properties of Lipophilic AntioxidantsTyrosol and Hydroxytyrosol Fatty Acid Esters: A PotentialExplanation for the Nonlinear Hypothesis of the AntioxidantActivity in Oil-in-Water Emulsions

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ "This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of Agricultural and Food Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/jf1009928 1 Surface-Active Properties of Lipophilic Antioxidants 1 Tyrosol and Hydroxytyrosol Fatty Acid Esters: a 2 Potential Explanation for the Nonlinear Hypothesis of 3 the Antioxidant Activity in Oil-in-Water Emulsions 4 RICARDO LUCAS,†FRANCISCO COMELLES,‡ DAVID ALCÁNTARA,† OLIVIA S. MALDONADO,† 5 MELANIE CURCUROZE,† JOSE L. PARRA‡ AND JUAN C. MORALES*,† 6 † Instituto de Investigaciones Químicas, CSIC – Universidad de Sevilla, 49 Avda Americo Vespucio, 7 41092 Sevilla, Spain and ‡ Institut de Química Avançada de Catalunya, CSIC, 18-26 Jordi Girona, 08034 8 Barcelona, Spain 9 10 Running Title. Surface-active properties of tyrosol and hydroxytyrosol esters 11 12 * Corresponding author: Dr. J. C. Morales (telephone 34-954-489561; fax 34-954-460565; e-mail: 13 [email protected]). 14 RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required 15 according to the journal that you are submitting your paper to) 16 2 1 ABSTRACT 2 Our group has recently observed a nonlinear tendency in antioxidant capacity of different 3 hydroxytyrosol fatty acid esters in fish oil-in-water emulsions, where a maximum of antioxidant efficiency 4 appeared for hydroxytyrosol octanoate. These results appear to disagree with the antioxidant polar 5 paradox. Since the physical location of the antioxidants in an oil-water interface has been postulated as an 6 important factor in explaining this behaviour, we have prepared a series of tyrosol and hydroxytyrosol 7 fatty acid esters with different chain length and studied their surface active properties in water, since 8 these physicochemical parameters could be directly related to the preferential placement at the interface. 9 We have found that tyrosol and hydroxytyrosol fatty acid esters are relevant surfactants when the right 10 hydrophilic-lipophilic balance (HLB) is attained, and in some cases, as efficient as emulsifiers commonly 11 used in industry, such as Brij 30® or Tween 20®. Moreover, a nonlinear dependency of surfactant 12 effectiveness is observed with the increase in chain length of the lipophilic antioxidants. This tendency 13 seems to fit quite well with the reported antioxidant activity in emulsions, and the best antioxidant of the 14 series (hydroxytyrosol octanoate) is also a very effective surfactant. This potential explanation of the 15 nonlinear hypothesis will help in the rational design of antioxidants used in oil-in-water emulsions. 16 17 18 KEYWORDS Tyrosol, hydroxytyrosol, antioxidants, lipophilic, emulsions, surface activity, surfactants. 19 20 3 INTRODUCTION 1 2 Lipid oxidation is still today an important problem for cosmetic and food industries. This is especially 3 relevant when the lipidic substrates are composed of unsaturated or poly-unsaturated fatty acids that are 4 very sensitive to oxidation processes (1, 2) such as in fish lipids. Lipid oxidation alters their organoleptic 5 properties (taste, odor, color and texture), depletes their nutritional properties and at the same time toxic 6 compounds are produced. This phenomenon is much more accentuated in oil-in-water emulsions since a 7 large interfacial area is produced during the emulsification process where the lipid oxidation has been 8 suggested to be initiated (3, 4). 9 Antioxidants have been used to control food oxidation for years. Their effectiveness depends on their 10 chemical reactivity (as radical scavengers or metal chelators), the interaction with other food 11 components, the environmental conditions (such as pH or concentration) and the physical location of the 12 antioxidant in different food systems (4-6). A general working hypothesis for antioxidant activity was 13 formulated two decades ago, the antioxidant polar paradox: hydrophilic antioxidants are more effective in 14 bulk oils whereas lipophilic antioxidants are more effective in systems of high surface-to-volume ratio, 15 such as emulsions, micelles or membranes (7, 8). This behaviour was explained by the concept of 16 interfacial oxidation (9). Accordingly, lipophilic antioxidants would have more affinity for the oil-water 17 interface in emulsions and therefore, would inhibit lipid oxidation more efficiently (10). In contrast, polar 18 antioxidants would concentrate into the air-oil interface in bulk oils and would be more efficient in this 19 type of matrices. An alternative explanation proposes that bulk lipids contain surface active minor 20 components (e.g., free fatty acids, monoacylglycerols) which form reverse micelles that stabilize water 21 droplets. In this scenario, the polar antioxidants are thought to aggregate at these microemulsion 22 droplets (11). Since many natural phenolic antioxidants are highly polar, their lipophilization could extend 23 their application in oil-based foods and cosmetics, and make them more efficient in emulsions. 24 4 Among the natural polyphenols, olive oil phenols, and particularly tyrosol and hydroxytyrosol, have 1 shown highly potent antioxidant activity in oils and oil-in-water emulsions, even higher than several 2 commonly used food antioxidants such as -tocopherol, BHT or ascorbyl palmitate (12-14). Moreover, 3 hydroxytyrosol displays interesting biological properties such as inhibition of human low-density 4 lipoprotein (LDL) oxidation (a critical step in atherosclerosis) (15) and anticancer properties (16). Our 5 group and others have prepared lipophilic derivatives of hydroxytyrosol (17-20) and tyrosol (21). These 6 new compounds also display remarkable antioxidant capacity when tested in cell lines (17, 19) and in 7 food matrices such as oils and oil-in-water emulsions (18, 19, 22). The antioxidant polar paradox 8 explains the antioxidant capacity of these phenolic antioxidants from a general perspective but some 9 discrepancies were found especially in emulsions. An increase in the chain length of hydroxytyrosol fatty 10 acid esters correlated with an increase in antioxidant activity in a fish oil-in-water emulsion system but 11 only up to a certain length. In fact, hydroxytyrosol octanoate exhibited the highest antioxidant capacity, 12 higher than the butyl and the lauroyl hydroxytyrosol esters, indicating a nonlinear tendency (22). Indeed, 13 a “nonlinear trend hypothesis” has been recently proposed Laguerre et al. (23). They observed a 14 nonlinear dependency on antioxidant capacity in emulsion systems for chlorogenic acid alkyl esters where 15 maximum antioxidant efficiency was detected for the corresponding phenolic dodecyl ester (23, 24), and 16 also for rosmarinic acid alkyl esters where a maximum was detected for the corresponding phenolic octyl 17 ester (25). All these series of lipophilic phenolic antioxidants display a parabolic shape when antioxidant 18 capacity was plotted against alkyl chain length. 19 Our working hypothesis is based on the suggestion by Heins et al. (26) that an antioxidant with notable 20 surface-active properties would possess better ability to inhibit lipid oxidation in emulsions since it would 21 concentrate at the oil-in-water interface. Therefore, the antioxidant would act as a shield for the oil 22 placed in the interior of the micelle. The amphiphilic character of these recently called “phenolipids” (25) 23 could envisage certain surface-active properties that would lead to a non-ionic surfactant. In fact, it is 24 important to mention that different polar head groups have been used in non-ionic surfactants such as 25 5 carbohydrates and amino acids, leading to n-alkyl polyglucosides (27), sorbitan esters (28), sugar fatty 1 acid esters (29) and amino acid-based surfactants (30). Nevertheless, phenols have barely been used as 2 such polar heads of surfactants. To the best of our knowledge, the only examples where the polar head is 3 a phenol group are the alkyl esters of p-hydroxyphenylacetic acid (31) and, more recently, the 4 chlorogenic fatty acid esters (24). In both cases, a decrease of the interfacial tension in a water5 hexadecane interface has been described. 6 In this work we have prepared a series of tyrosol and hydroxytyrosol fatty acid esters and studied their 7 surfactive properties in water (Figure 1). Different chain lengths from C2 to C18 have been utilized in 8 the synthesis of the new derivatives in order to obtain a diversity of hydrophilic-lipophilic balance values 9 (HLB) and examine the tendency of surfactant efficiency with chain length. At the same time, this study 10 has allowed us to evaluate phenols as the polar head groups of non-ionic surfactants, and compare phenol 11 and di-orthophenol structures. Critical micelle concentration (CMC) and surface tension in water have 12 been analyzed and discussed. 13 14 MATERIALS AND METHODS 15 16 Materials. All chemicals were obtained from chemical suppliers and used without further purification, 17 unless otherwise noted. Vinyl alkyl esters were provided by TCI Chemicals. Tyrosol was provided by 18 Sigma-Aldrich and hydroxytyrosol was prepared from their corresponding carboxylic acid by reduction 19 with lithium aluminum hydride (32). Immobilized lipase Novozym435® was a gift from Novozymes. All 20 reactions were monitored by TLC on precoated Silica-Gel 60 plates F254, and detected by heating with 21 Mostain (500 ml of 10% H2SO4, 25g of (NH4)6Mo7O24• 4H2O, 1g Ce(SO4)2•4H2O). Products were 22 purified by flash chromatography with silica gel60 (200-400 mesh). NMR spectra were recorded on 300, 23 400 or 500 MHz NMR equipment, at room temperature for solutions in CDCl3 or CD3OD. Chemical 24 shifts are referred to the solvent signal and are expressed in ppm. 25 6 Synthesis of tyrosol and hydroxytyrosol fatty acid esters. Tyrosol fatty acid esters 3, 4 and 8 have 1 been previously described (21). Hydroxytyrosol fatty acid esters 9, 10, 12, 13, 14, 16 and 17 have been 2 previously described (17, 19, 20, 22). General procedure for the synthesis of 3-17. Candida antarctica 3 lipase (Novozym 435) (180 mg) was added to a mixture of tyrosol or hydroxytyrosol (1 eq) and the 4 acylating agent (20 eq.) in 45 mL of t-butyl methyl ether using a dry round bottom flask, and the mixture 5 was stirred for 60 min at 40 ºC. The enzyme was decanted and separated. The solvent was evaporated 6 and the product was purified by flash column chromatography. Characterization data for the new 7 prepared compounds (5, 6, 7, 11 and 15) can be found in the supporting information. 8 Surface tension and CMC determination. Surface tension measurements were performed at 23 ºC 9 by means of the Wilhelmy plate method in a Krüss K12 tensiometer. Samples were prepared by 10 successive dilutions of an initial concentrated solution. Prior to each surface tension measurement 11 samples were left 30 min in repose to attain the equilibrium. The possible aggregation properties of 12 tyrosol and hydroxytyrosol derivatives were evidenced from the adsorption isotherms obtained when 13 surface tension is plotted graphically against logarithm of concentration. The typical surfactant profile 14 consists in a linear decrease of the surface tension when the compound concentration increases, followed 15 by a surface tension stabilization when the concentration corresponding to the saturation of the interface 16 is attained. The intersection of the two linear portions in the graph determines the critical micelle 17 concentration (CMC). 18 Determination of HLB values. The hydrophilic-lipophilic balance (HLB) values were calculated 19 following the equation described by Griffin (33) for non-ionic surfactants. HLB = 20 x (Hydrophilic 20 group molecular weight) / (surfactant molecular weight). 21 Calculation of aggregation parameters. The area occupied per molecule adsorbed at the water/air 22 interface (in Å2) can be obtained from the equation: A = 1016 / NA• , where NA is the Avogadro’s 23 number and  is the adsorption at the saturated interface expressed in mol/cm2, calculated according the 24 Gibbs equation:  = -(d/dlog C)/2.303 n RT, where n is the number of molecular species in solution (n = 25 7 1 for non-ionic compounds as in our case) and (d/dlog C) is the slope of the linear portion of the graph 1 before the CMC concentration. 2 3 4 8 RESULTS 1 2 Preparation of tyrosol and hydroxytyrosol fatty acid esters. Some of the compounds for both series 3 have been synthesized and characterized previously by our group and others (17, 19, 20), using an 4 organic acid catalyst, such as p-toluenesulfonic acid, or using an enzymatic catalyst such as a lipase. We 5 have prepared the full series by enzymatic acylation of tyrosol and hydroxytyrosol using immobilized 6 lipase B from Candida antarctica (Novozym435®). The reactions were carried out in tert-butylmethyl 7 ether and the acylating agents were the corresponding vinyl esters of the different alkyl chains. Yields 8 were very high in all cases after enzyme filtration and short column chromatography of the reaction 9 mixture. New compounds tyrosol hexanoate 5, tyrosol octanoate 6, tyrosol decanoate 7, hydroxytyrosol 10 hexanoate 11 and hydroxytyrosol myristate 15 have been fully characterized by NMR spectroscopy and 11 mass spectrometry. 12 Surface tension measurements for tyrosol fatty acid esters. The graphs of surface tension/log of 13 compound concentration for the tyrosol series are represented in Figure 2. It can be observed that for 14 tyrosol 1, tyrosol acetate 3 and tyrosol butyrate 4, surface tension decreases but does not show a plateau. 15 Higher concentrations could not be tested since these samples become insoluble. Whereas these 16 compounds show surface activity reducing the surface tension, they do not behave as surfactants since no 17 self-aggregation (micelle formation) occurs. In contrast, when the alkyl chain length of the acyl group 18 increases, an adequate hydrophilic/lipophilic balance is attained and consequently, a typical curve shape 19 of surfactant is observed for tyrosol hexanoate 5, octanoate 6 and decanoate 7. Tyrosol laurate 8 showed 20 very low solubility in water, not allowing the corresponding surface tension measurements. 21 Surface tension measurements for hydroxytyrosol fatty acid esters. The representation of surface 22 tension/log of product concentration for the series of hydroxytyrosol fatty acid esters can be observed in 23 Figure 3. For these series even hydroxytyrosol esters with short alkyl chains (hydroxytyrosol acetate 9 24 and hydroxytyrosol butyrate 10) show a certain surface-active behaviour although the decrease in the 25 15 Figure 1. Figure 2. 30 35 40 45 50 55 60 65 70 75 0.001 0.01 0.1 1 10 100 1000 Concentration (mM) Surface tension (mN/m) ) 3 4 5 6 1 7 16 Figure 3. Figure 4. 0.001 0.01 0.1 1 10 4 6 8 10 12 14 n (carbons in the acyl alkyl chain) CMC (mM) 20 30 40 50 60 70 80 1E-05 0,0001 0,001 0,01 0,1 1 10 100 1000 Concentration (mM) Surface tension (mN/m) 9 2 10 11 12 13 14 15 16 17 Figure 5. 20 30 40 50 60 70 0 2 4 6 8 10 12 14 16 18 alkyl chain length (carbon atom)  (CMC) (mN/m) Figure 6. a) Oil Oil Oil ROS● ROS● ROS● Oil Oil Oil ROS● ROS● ROS● ROS● ROS● ROS● ROS●ROS●ROS● b) c) Emulsifier Phenolipids ROS●Radical oxygen species a) Oil Oil Oil ROS● ROS● ROS● Oil Oil Oil ROS● ROS● ROS● ROS● ROS● ROS● ROS●ROS●ROS● b) c) Emulsifier Phenolipids ROS●Radical oxygen species 18 Table 1. MW, HLB, CMC, surface tension at the CMC, C20, pC20,  and area per molecule of prepared tyrosol and hydroxytyrosol fatty acid esters and several common non-ionic surfactants. Compound MW HLB CMC(mM) cmc (mN/m) C20 (mM) pC20  (mol/cm2) A (Å2) T hexanoate (5) 236.3 10.2 0.9 44.0 0.32 3.49 3.240 x 10-10 51.3 T octanoate (6) 264.4 9.2 0.073 41.1 0.029 4.54 4.712 x 10-10 35.3 T decanoate (7) 292.4 8.3 0.013 41.5 0.0055 5.26 5.175 x 10-10 32.0 HT acetate (9) 196.2 14.0 3.0 57.7 - - 2.187 x 10-10 76.0 HT butyrate (10) 224.3 12.2 1.5 45.8 0.75 3.12 3.50 x 10-10 47.5 HT hexanoate (11) 252.3 10.9 1.2 39.6 0.34 3.47 3.91 x 10-10 42.5 HT octanoate (12) 280.4 9.8 0.38 30.5 0.03 4.52 3.43 x 10-10 48.4 HT decanoate (13) 308.4 8.9 0.09 28.0 0.007 5.15 4.21 x 10-10 39.4 HT laurate (14) 336.5 8.1 0.0055 39.0 0.0015 5.82 3.97 x 10-10 41.9 HT myristate (15) 364.5 7.5 0.0035 59.5 - - 4.034 x 10-10 41.2 HT palmitate (16) 392.6 7.0 0.002 62.0 - - 4.286 x 10-10 38.8 Brij 30 ® (Polyoxyethylene (4) lauryl ether)a 362.5 9.4 0.0035 30.0 0.0024 5.62 3.80 x 10-10 44.0 Tween 20 ® (polyoxyethylene sorbitan monolaurate) b,d 1227.5 16.6 0.0169 35.0 0.0025 5.61 3.560 x 10-10 46.6 n-octyl glucoside c,d 292.4 11.1 25 ~30.0 2.792 2.55 4.0 x 10-10 41.0 a From ref. 33. b From ref. 34. c From ref. 35. d C20 and pC20 were calculated as noted in the experimental section. 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