Assessing Interactions between Lipophilic and Hydrophilic

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Assessing interactions between lipophilic and hydrophilic antioxidants in food emulsions Erwann Durand, Yu Zhao, John Coupland, and Ryan J. Elias J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04152 • Publication Date (Web): 19 Oct 2015 Downloaded from http://pubs.acs.org on October 30, 2015

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Journal of Agricultural and Food Chemistry

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ASSESSING INTERACTIONS BETWEEN LIPOPHILIC AND

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HYDROPHILIC ANTIOXIDANTS IN FOOD EMULSIONS

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Erwann Durand1, Yu Zhao, John N. Coupland, Ryan J. Elias*

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Department of Food Science, The Pennsylvania State University, University Park, PA 16802,

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USA.

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CORRESPONDING AUTHOR FOOTNOTE:

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*Ryan J. Elias

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Phone: +1 (814) 865-5371

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Fax:

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Email: [email protected]

+1 (814) 863-6132

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1

Present Address: CIRAD, UMR IATE, Montpellier F-34398, France

1 ACS Paragon Plus Environment


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ABSTRACT

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Dietary lipids containing high concentrations of polyunsaturated fatty acids are

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considered to be beneficial to human health, yet their incorporation within formulated foods is

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complicated by their susceptibility to oxidation. Lipid oxidation in foods is inhibited through

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the incorporation of antioxidants, yet the list of antioxidants approved for food use is small,

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and consumers frequently demand foods without synthetic additives. As a consequence, food

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processors are now tasked with improving the efficacy of approved, “natural” (i.e., non-

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synthetic) antioxidants; a rational strategy for doing so involves localizing the antioxidants at

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the interface where oxidation usually occurs and regenerating the consumed antioxidants after

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the oxidation event has occurred. The present study describes a procedure to evaluate

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antioxidant interactions in oil-in-water food emulsions, which is based on controlled oxidation

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reactions induced in the dispersed oil phase by the lipophilic radical generator, 2,2'-

29

azobis(2,4-dimethylvaleronitrile). The extent of lipid oxidation is measured spectroscopically

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by following the loss of an oxidatively labile, lipophilic probe (methyl eleostearate), the

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synthesis of which is described here. Using this procedure, the ability of various aqueous

32

phase solvated antioxidants (ascorbic acid, gallic acid, (-)-epicatechin, (-)-epigallocatechin-3-

33

gallate) to regenerate lipid phase solvated α-tocopherol was evaluated. In all cases, the test

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compounds were able to inhibit oxidation reactions; however, these effects were not

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profoundly synergistic, and the maximum synergistic interaction observed was only ~3%

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using ascorbic acid.

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KEYWORDS: natural antioxidants, α-tocopherol, O/W emulsion, methyl eleostearate

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INTRODUCTION

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Lipid oxidation is arguably the most important non-microbial spoilage mechanism in

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foods, resulting in enormous economic loss due to off flavors and aromas, decreases in

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polyunsaturated fatty acids (PUFA) content, and the generation of products that are known to

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adversely affect human health 1–3. Food processors face significant challenges as they attempt

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to deal with lipid oxidation. Many consumers demand that their foods simultaneously contain

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high levels of PUFA, yet increasingly reject foods that contain synthetic antioxidants.

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Unfortunately, the number of approved antioxidants that can be added to foods is currently

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small. Attempts to expand the number of natural (non-synthetic) antioxidants through

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screening of natural products is important work; however, a sensible alternative approach is to

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more intelligently use the “natural” antioxidants that are currently available. An example of

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such an approach is to design food systems that promote synergistic interactions between

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various antioxidants, thereby extending their effectiveness.

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The highly lipophilic α-tocopherol (TCP) is both an endogenous component of plant-

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based materials and a known chain-breaking antioxidant. Indeed, TCP is known to be an

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essential chain-breaking antioxidant in vivo and is widely recognized as the major antioxidant

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in biological membranes

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dynamics in these systems is still debated 6, it is clear that TCP is strategically situated in

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areas of high oxidative stress (i.e., the membrane interface). Moreover, it has been suggested

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that the antioxidant ability of TCP can be augmented by a secondary molecule involved in an

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hydrogen atom transfer to the oxidized TCP (TCP•)

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ascorbic acid (ASC), appear to be involved in the regeneration of TCP by reducing the

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tocopheroxyl radical (TCP•) under biological conditions

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regeneration is proposed as an important mechanism for the synergetic protection widely

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observed in membrane systems 11–14, LDLs 15, in vitro on methyl linoleate 16, in micelles 17,18,

4,5

. While the question of TCP’s precise location, orientation, and

7,8

. Indeed, aqueous reductants, such as

9,10


(Figure 1). Antioxidant


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as well as in vivo 19. Paradoxically, this synergetic interaction is dependent upon the proximity

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of TCP (an extremely lipophilic molecule) and ASC (an extremely hydrophilic molecule).

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However, in the systems previously described, the molecules may be put into mutual contact.

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For example, in micelles, it is assumed that the phytyl side chain of TCP is oriented toward

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the lipophilic core of the micelle and the chromanol, anchored to the interface by its hydroxyl

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group, is positioned near the aqueous phase. Antioxidant synergy is thus observed as water-

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soluble ASC is able to replenish the TCP reservoir via one-electron reduction of TCP•

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radicals. This interaction between TCP radicals and ASC has been demonstrated

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experimentally using pulse radiolysis studies in water-isopropyl alcohol-acetone solutions and

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liposomal systems 9,20, as well as in micellar 10,21,22, biphasic solution (dichloromethane-water)

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23

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directly with flavonoid compounds in SDS micelles 25.

and biological systems

24

. More recently, Bo Zhou et al. showed that TCP• could react

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To our knowledge, no study has demonstrated similar antioxidant interactions in oil-in

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water (O/W) emulsions, which consist of relatively large lipid droplets (d~100–1000 nm).

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Indeed, determining a given molecule’s distribution and reactivity in such optically opaque

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emulsions is experimentally difficult by conventional methods. Specifically, no direct

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interaction between TCP• and water-soluble reductants has been established in O/W

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emulsions, although this has been assumed based on electrochemical calculations and

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observations in simple micellar systems 22,26. In an O/W emulsion system, the mechanism for

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antioxidant synergy is proposed to begin with the initial TCP oxidation, with the resulting

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TCP• becoming oriented at the surface of lipid droplets and, subsequently, being reduced by a

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water-soluble reductant in the pseudo phase interface layer to regenerate TCP; such a

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mechanism is difficult to show directly. Herein, we provide a novel and facile procedure to

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promote and evaluate this interaction in O/W emulsions.


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Aliphatic azo compounds are commonly used as initiators to study the kinetics of lipid

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oxidation since they generate radicals at a reproducible and constant rate upon heating or

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irradiation 27,28. By using either water-soluble or lipid-soluble azo initiators, radical-mediated

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reactions can be initiated in specific microenvironments. For example, a mixture of TCP and

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ascorbic acid showed an additive protective effect with respect to lipid oxidation in a

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liposome that was oxidized in the presence of water-soluble azo-initiator, which generates

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free radicals in the aqueous phase. In contrast, the same mixture showed a synergistic

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protective effect in the presence of oil-soluble azo-initiator, which generates free radicals

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within the lipophilic domain

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azobis(2,4-dimethylvaleronitrile) (AMVN), a widely used lipophilic azo initiator 28. By doing

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so, the radical (R-C·) formed during AMVN thermo-decomposition will react quickly with

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triplet oxygen, which is predicted to concentrate within oil, leading to the formation of

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peroxyl radicals (R-COO·); this peroxyl radical will abstract a hydrogen atom from TCP to

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form a hydroperoxide and TCP•. The half-life of this phenoxyl radical (TCP•) is long due to

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resonance stabilization such that it can interact via one-electron reduction with an aqueous

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soluble reductant (e.g., ascorbic acid), thus replenishing the TCP reservoir. Methyl

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eleostearate was synthesized and incorporated into O/W emulsions as a model oxidizable lipid

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in order to assess lipid oxidation rates in this system while simultaneously determining

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antioxidant synergy. This fatty acid consists of a highly oxidizable conjugated triene with

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strong UV absorptivity, thus its oxidation can be simply observed by following its absorbance

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loss at 270 nm. These properties allow for an efficient, easy, rapid and direct assessment of

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lipid oxidation.

13,29

. Thus, in our proposed model, oxidation is induced by 2,2'-

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In this study, the capacity for four model water soluble reductants, ascorbic acid, gallic

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acid, (-)-epicatechin, (-)-epigallocatechin-3-gallate (Figure 2) to regenerate α-tocopherol in

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the lipid phase of O/W emulsions was evaluated using the above described method. 5 ACS Paragon Plus Environment


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MATERIALS AND METHODS

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Materials

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n-Tetradecane was obtained from Fisher Scientific (Pittsburgh, PA, USA). α-

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Tocopherol, sodium caseinate (from bovine milk), isopropanol, hexane, 2-methyl-t-butyl-

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ether, tung oil, (-)-epicatechin, gallic acid, (-)-epigallocatechin-3-gallate, ascorbic acid, and

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sodium phosphate dibasic heptahydrate were obtained from Sigma (St. Louis, MO, USA).

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Sodium phosphate monobasic monohydrate was obtained from EMD Chemicals (Merck

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KGaA, Darmstadt, Germany). 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) was obtained

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from Wako Pure Chemical Industries (Osaka, Japan). All materials were reagent grade and

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were used as received.

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Preparation and physical characterization of nanoemulsions containing α-tocopherol

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and AMVN

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Sodium caseinate solutions (1 wt% in pH 7, 100 mM phosphate buffer) were prepared

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and stirred overnight to ensure the protein was sufficiently hydrated. Then, α-tocopherol,

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methyl eleostearate and AMVN were dissolved directly within tetradecane by sonication

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(Branson B3510R-MTH, power density ~58 W/L, 40 kHz, 1 min at 25°C). The lipid phase

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(10 wt%) was then mixed with sodium caseinate solutions using a high-speed blender

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(Brinkmann Polytron, Brinkmann Instruments Inc., Westbury NY, USA) to produce a coarse

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emulsion (final α-tocopherol concentration: 75 µM, AMVN 3 mM). The coarse emulsion was

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processed through a microfluidizer (M-110Y Microfluidizer, Microfluidics, Newton, MA,

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USA) configured with 75 and 200 µm interaction chambers in series at 1200 bar (5 passes) to

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produce fine nanoemulsions. The particle size distribution of the emulsion was measured by

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static light scattering (Horiba LA-920, Horiba Instruments Inc., West Chicago, IL, USA). All


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nanoemulsions prepared had similar droplet sizes (d32 = 210 – 230 nm) and were physically

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stable over the course of the experiment, even in the presence of the oxidizing agent.

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Experimental procedure for synthesis of methyl eleostearate from tung oil

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Eleostearic acid contains conjugated trienes with a strong UV absorption at 270 nm

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and is oxidatively labile. Oxidative degradation of the conjugated trienes is observed upon

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loss of absorbance at 270 nm. Eleostearic acid was isolated from tung oil using a method

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adapted from

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absolute ethanol (250 mL) in a 500 mL round bottom flask under an argon atmosphere in the

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dark. The reaction mixture was stirred and refluxed for 3 hours to saponify the oil. The

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mixture was cooled to room temperature, to which 200 mL of distilled water was added, and

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the aqueous phase was washed three times with hexane (150 mL). The aqueous phase was

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then acidified (pH 2) with sulfuric acid solution (50 % diluted, 9M) and extracted three times

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with diethyl ether (200 mL). The combined diethyl ether solution was dried over anhydrous

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sodium sulfate and evaporated using a rotovap at 15 ºC in the dark. The resulting eleostearic

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acid was purified by crystallization. The crude powder obtained after evaporation was

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dissolved in acetone at room temperature and recrystallized twice at -20 ºC. After vacuum

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filtration, a white powder of eleostearic acid (8.4 g) was obtained and dried under vacuum for

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further esterification. The product was identified by 1D NMR (1H and

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MHz, CDCl3): δ 9.244 (s, br, 1H), 6.36-6.24 (dd, 1H), 6.12-5.97 (q, 2H), 5.96-5.85 (dd, 1H),

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5.68-5.56 (dt, 1H), 5.36-5.25 (dt, 1H), 2.30 (t, 2H), 2.16-2.01 (m, 4H), 1.60 (q, 2H), 1.41-1.20

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(m, 12H), 0.83 (t, 3H) ppm. 13C NMR (75 MHz, CHCl3): δ 179.66 (COOH), 135.1-125.42 (5

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CH), 33.8-22.1 (10 CH2), 13.9 (1 CH3) ppm.

30–32

. Tung oil (34 g) and potassium hydroxide (40.5 g) were dissolved in

161 162


13

C): 1H NMR (300


163

Methyl esterification of eleostearic acid

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The procedure was deeply inspired by the esterification reaction described by 33

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Kamarudin et al.

where a catalytic amount of SOCl2 was used. Pure eleostearic acid (1 g)

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was dissolved in methanol (25 mL) in a 100 mL round bottom flask and refluxed under argon

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in the dark. Next, SOCl2 (12.4 µL, 2 wt%) was added to initiate the esterification reaction at

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room temperature. The reaction was stopped after 4 hours by quenching with saturated

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sodium bicarbonate solution (20 mL). The reaction mixture was extracted by hexane 3 times

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and the extract solution was dried over anhydrous sodium sulfate and evaporated under

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vacuum using a rotovap at 15ºC. Finally, the product was identified by 1D NMR (1H): 1H

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NMR (300 MHz, CHCl3): δ 6.36-6.24 (dd, 1H), 6.12-5.97 (q, 2H), 5.96-5.85 (dd, 1H), 5.68-

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5.56 (dt, 1H), 5.36-5.25 (dt, 1H), 3.61 (s, 3H), 2.30 (t, 2H), 2.16-2.01 (m, 4H), 1.60 (q, 2H),

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1.20-1.41 (m, 12H), 0.83 (t, 3H) ppm.

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Oxidation of α-tocopherol and methyl eleostearate in emulsion

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Aliquots (20 mL) of emulsions containing α-tocopherol (75 µM) and methyl

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eleostearate (1 mM) were subjected to oxidation by the thermal decomposition of the lipid

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radical initiator (AMVN, 3 mM) at 40°C. For experiments with aqueous reductants, aliquots

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(200 µL) of aqueous soluble reductant in buffer was added (final reductant concentration: 75

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µM or 10 µM) before thermal oxidation was initiated. Samples aliquots (1 mL) from

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emulsions were withdrawn periodically for analysis. The emulsion samples were extracted

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with hexane (1 mL) and isopropanol (700 µL), vortexed (1 min) and then centrifuged

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(2500×g, 1 min) to separate the phases. To assess the remaining α-tocopherol, 500 µL from

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the upper organic phase was collected and immediately analyzed by HPLC (described below).

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To assess the remaining methyl eleostearate, 70 µL from the upper organic phase was


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collected, combined with 630 µL of hexane and immediately analyzed in HPLC (described

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below).

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TCP and methyl eleostearate analysis

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The HPLC analysis was performed in isocratic mode with hexane:tert-butylmethyl

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ether (90:10, v:v) at 1 mL/min (430 psi). The HPLC system consisted of a Waters 1525 binary

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pump, a Waters 717 Plus autosampler, and a Waters 2487 dual λ absorbance detector (Waters,

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Milford, MA, USA). Chromatographic separation occurred on an Agilent Zorbax RX-SIL

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column (4.6 mm ID, 250 mm, 5 µm) (Agilent, Santa Clara, CA, USA), which was held in a

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column heater at 25 °C. TCP and methyl eleostearate were detected at 295 and 270 nm,

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respectively. Standard curves were constructed by measuring the peak area at 295 nm for TCP

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and 270 nm for methyl eleostearate at different concentrations in hexane solutions. A linear

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correlation was obtained between peak area and concentration (R2 = 0.99).

200 201

RESULTS AND DISCUSSION

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Depletion of TCP in absence or in presence of hydrophilic antioxidants in O/W

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emulsions.

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The ability of ASC, GA, EGCG, and EC to prevent the loss of TCP was assessed in

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caseinate-stabilized tetradecane-in-water emulsions (Figure 3). These water-soluble

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reductants have already been shown to regenerate TCP• in non-emulsion (e.g., micellar)

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systems

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AMVN, a lipophilic azo compound, was used as the radical generator in O/W emulsions,

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which resulted in a constant rate of TCP depletion (zeroth-order reaction; kappox-toc=0.26

210

µmol/h; t1/2=171.3 ± 3.6 min). The oxidation of TCP was substantially slowed in the presence

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of all of the aqueous reductants. ASC was observed to stabilize TCP during the first two hours

25,34

, thus significantly extending of the effective life of TCP. In the present study,



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of the experiment, after which the apparent oxidation kinetics were the same as in the ASC-

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free control. The trend with GA, EGCG and EC was slightly different; these compounds

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decreased the rate TCP depletion without a distinct lag period, as was the case with ASC. GA

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increased the TCP half-life with relatively efficient protection within the first four hours.

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Beyond this time, the oxidation rate of TCP reached the same rate as the GA-free control, and

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TCP was fully oxidized after nearly 10 hours. Both EC and EGCG significantly delayed TCP

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depletion without any observable lag period, resulting in an increase in TCP half-life

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(t1/2=12.95 hours with EC and t1/2=19.68 hours with EGCG). This protective effect may be

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due either by the hydrophilic antioxidants reacting with the lipophilic TCP• (i.e., the basis of

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synergetic effect), or with the lipophilic radicals (R-C·, R-COO·, etc.) generated by the azo

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initiator (i.e., an additive effect), or a combination of both mechanisms.

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Evaluating antioxidant synergy in methyl eleostearate-containing emulsions.

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The introduction of an oxidizable lipid (i.e., methyl eleostearate) into the oil droplets

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provides a means by which to measure the extent of synergetic interaction between TCP and

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aqueous phase reductants. As can be seen in Figure 4, in the absence of a hydrophilic

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reductant, the presence of TCP resulted in a lag phase with respect to lipid oxidation that

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persisted until the molecule was fully consumed; at this point, the rate of methyl eleostearate

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oxidation rose sharply until it reached the same rate as the TCP-free control, and was

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completely oxidized after 342 min (5.7 hours).

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The kinetics of methyl eleostearate oxidation in the presence of the aqueous

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compounds ASC, GA, EGCG or EC, with or without TCP in the oil droplet, was measured

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(Figure 5). The addition of ASC alone did not affect the rate of methyl eleostearate oxidation,

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thereby demonstrating that this highly hydrophilic molecule is unable to interact with the

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oxidizing species generated in the lipid droplets; this is consistent with the accepted

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mechanism by which dispersed lipids are thought to oxidize (i.e., oxidation reactions occur 10 ACS Paragon Plus Environment

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within the droplet or at the droplet interface). However, the other aqueous-dispersed

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compounds (GA, EGCG and EC) all delayed lipid oxidation in the absence of TCP with

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varying degrees of efficacy. GA alone inhibited methyl eleostearate oxidation within the first

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six hours, and beyond this point, the rate of methyl eleostearate oxidation was the same as the

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GA-free control. EGCG and EC strongly inhibited methyl eleostearate oxidation for a

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relatively longer period than GA. In the presence of TCP, the rate of methyl eleostearate

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oxidation was slower compared to the individual aqueous reductants. To determine if the

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interaction between aqueous phase compounds (ASC, GA, EGCG, EC) and droplet-bound

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TCP was synergistic or merely additive, the degree of synergetic interaction (SI) was

247

calculated according to the following equation (Eq. 1):

248 249

= (1 −

( ( – ( ( – (

× 100

(Eq. 1)

250 251

Where A is the area under the residual eleostearate-time curve and the subscripts refer to the

252

antioxidant present (or “blank” in the absence of added antioxidant). A positive SI value

253

indicates synergism, while a negative value indicates antagonistic interaction. ASC, GA, and

254

EC showed no statistically significant synergistic interaction with TCP under the experimental

255

conditions described (Table 1). EGCG demonstrated a small but statistically significant

256

degree of antagonistic interaction with TCP. Thus, we can provide no evidence that these

257

aqueous antioxidants are capable of working synergistically with TCP to delay oxidation

258

reactions in oxidatively labile O/W emulsions.

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When ASC and TCP were present in the same system, their synergetic interaction was

260

calculated to be 3 ± 1.9%. ASC appears to contribute to the overall stability of TCP within

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the first two hours where, conceivably, it is able to regenerate TCP from TCP•. The reduction

262

of TCP• by ASC is thermodynamically favored due to the low reducing potential of ASC 11 ACS Paragon Plus Environment


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(E0 ∼ 0.28 V) relative to that of TCP (E0 ∼ 0.5 V)

35

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not appear to confer significant protection of the oil droplets against oxidation, and the overall

265

effect of the combined antioxidants could be reduced to the sum of two independent effects.

; however, this interaction period does

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We noted earlier that GA allows the TCP to persist in the lipid phase up to 10 hours.

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Yet, no synergetic effect between GA and TCP was observed (synergistic interaction

268

calculated to be -3.6 ± 2.4 %). In presence of GA and TCP, lipid oxidation was observed to

269

progress as two distinct kinetic periods. A first phase with an efficient protective effect (i.e.,

270

before 12 hours), followed by a second phase wherein methyl eleostearate oxidation is

271

identical to that observed without any antioxidant. The very low depletion (ca. 8%) in methyl

272

eleostearate during the first phase appears to be related to the presence of TCP in the oil phase

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that remains for ca. 10 hours in presence of GA. However, without direct observation of the

274

TCP•, it is impossible to state unequivocally whether GA may be involved or not in the

275

regeneration of TCP. In the other word, the regeneration of the TCP• will not be necessary

276

associated to a significant protection of the lipid oxidation. Indeed, if one molecule of GA is

277

used to regenerate one molecule of TCP•, that means this GA molecule would not be

278

available to react with the oxidizing species. In this association, it seems that GA can delay

279

the oxidation of the TCP in the same way it can protect the methyl eleostearate against

280

oxidation (i. e. reduce oxidizing species).

281

It has been previously observed that both EC and EGCG dramatically increase the

282

TCP half-life, but co-addition of EC or EGCG with TCP in methyl eleostearate-containing

283

emulsions did not confer a synergistic effect with respect to lipid oxidation inhibition

284

(synergetic interaction was calculated at -2 ± 2.3 % and -6 ± 2.1 % for EC and EGCG,

285

respectively). Thus, the presence of EC (or EGCG) delayed methyl eleostearate oxidation in

286

TCP containing emulsions, although this effect was merely additive, if not slightly

287

antagonistic. Here again, we can’t provide evidence of antioxidant interaction because one


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may argue that the expected positive effect of the possible TCP regeneration could be

289

counterbalanced by the loss of the EC (or EGCG) capacity to directly reduce the oxidizing

290

species.

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In summary, this work clearly showed a lack of synergistic interaction between the

292

lipophilic chain-breaking antioxidant TCP and various aqueous phase dispersed antioxidants

293

(ASC, GA, EC, EGCG) in conventional O/W emulsions. Using EPR, pulse radiolysis or laser

294

flash photolysis, many authors have directly observed TCP• reduction by such aqueous phase

295

antioxidants in simple organic solvent solubilized systems and micellar systems 9,20–22,25. TCP

296

regeneration has also been shown in membrane models and postmortem meat

297

consistent with the generally accepted mechanism in vivo (i.e., ASC reduction of TCP• in

298

biological membranes). However, to our knowledge, the direct observation of TCP• and

299

aqueous phase antioxidant interaction by EPR has not been reported for O/W emulsions,

300

perhaps due to methodological challenges. Some have argued that such interactions occur in

301

O/W emulsions based on conventional lipid oxidation markers but, in the absence of direct

302

evidence of TCP• and aqueous phase antioxidant interactions, it is difficult to conclude that

303

TCP regeneration is a relevant mechanism. Based on our findings using a novel method, in

304

which the loss of an oxidatively labile lipid is directly observed in a model food O/W

305

emulsion and radicals are generated in lipid droplets, we are unable to demonstrate efficient

306

interaction with respect to lipid oxidation. This could be due to the fact that the extremely

307

lipophilic molecule TCP (logP ~ 11) tends to partition deep within lipid droplet cores and, as

308

such, is physically isolated from emulsion interfaces36,37 where oxidation reactions are

309

thought to occur oxidation reactions 38. Such internalization of TCP would not be predicted in

310

the non-emulsion systems described above (TCP dissolved in organic solvent, TCP dispersed

311

within SDS micelles, TCP present within membranes, etc.). Thus, the remoteness of TCP

312

from oil-water interfaces in actual O/W emulsions, which have relatively large lipid droplet


10,24

, which is


313

diameters, may prevent antioxidant synergism between aqueous phase and lipid phase

314

antioxidants. Future work is needed to identify novel approaches to encourage TCP

315

regeneration in actual food O/W emulsions, possibly by encouraging the localization of TCP

316

at lipid droplet interfaces.

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ABBREVIATIONS USED

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PUFA, polyunsaturated fatty acids; EPR, electron paramagnetic resonance; NMR, nuclear

320

magnetic resonance; CDCL3, deuterated chloroform; UV, ultraviolet; HPLC, high-

321

performance liquid chromatography; SOCl2, thionyl chloride; AMVN, 2,2'-azobis(2,4-

322

dimethylvaleronitrile); TCP, α-tocopherol; TCP•, tocopheroxyl radical; ASC, ascorbic acid;

323

GA, gallic acid; EC, (-)-epicatechin; EGCG, (-)-epigallocatechin-3-gallate; SDS, sodium

324

dodecyl sulfate.

325 326

FUNDING SOURCE

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This material is based upon work that is supported by the National Institute of Food and

328

Agriculture, U.S. Department of Agriculture, under award number 2014-67017-21645.



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REFERENCES

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1.

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ed.). New York: Marcel Dekker Inc., 2002.

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2.

Frankel, E. N. Lipid oxidation; The Oil Press, Dundee, Scotland, 1998.

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432 433


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434

Table 1: Synergetic interaction between α-tocopherol (75 µM) and water-soluble reductant

435

(ASC: ascorbic acid, GA: gallic acid, EC: (-)-epicatechin, EGCG: (-)-epigallocatechin-3-

436

gallate, 75 µM) in caseinate-stabilized O/W emulsion.

437

Figure 1: Principle of α-tocopherol (TCP) regeneration by a reductant.

438

Figure 2: Structures of test water-soluble compounds (ASC: ascorbic acid, GA: gallic acid,

439

EC: (-)-epicatechin, EGCG: (-)-epigallocatechin-3-gallate).

440

Figure 3: Depletion of TCP (75 µM, 1.5 µmol) upon oxidation induced by the AMVN (3

441

mM) decomposition at 40ºC in absence ( ) or in presence ( ) of hydrophilic antioxidants in

442

caseinate-stabilized O/W emulsion.

443

Figure 4: Oxidation (induced by 3 mM AMVN at 40ºC) of methyl eleostearate (1 mM) in

444

caseinate-stabilized O/W emulsion, without antioxidant (dotted line ●) and in presence of 75

445

µM TCP (line ▲). Depletion of TCP during the experiment (▐, 75 µM, 1.5 µmol).

446

Figure 5: Oxidation (induced by 3 mM AMVN at 40ºC) of methyl eleostearate (1 mM) in

447

caseinate-stabilized O/W emulsion, without antioxidant (dotted line ●), in presence of 75 µM

448

α-tocopherol (line ▲), in presence of 75 µM water soluble reductants (line ●) or in presence

449

of 75 µM α-tocopherol and 75 µM water soluble reductants (line ▲).

450



451

Table 1 Compound

Synergetic interaction

ASC

+2.9 ± 1.9 %

GA

-3.6 ± 2.4 %

EC

-2.1 ± 2.3 %

EGCG

-5.6 ± 2.1 %

452


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453


Figure 1 TCP oxidation

α-tocopherol (TCP)

454

R

R

A

AH

TCP reduction


α-tocopheroxyl radical (TCP)


455

Figure 2 ASC

GA

EGCG

EC

456


Page 24 of 28

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457


Figure 3 100

100

90

ASC

80

Remaining tocopherol (%)


90 70 60 50 40 30 20 10

GA

80 70 60 50 40 30 20 10 0

0 0

1

2

3

4

5

6

0

7

1

2

Time (hour)

5

6

7

8

9

100

90

90

EGCG

80



4

Time (hour)

100

70 60 50 40 30 20 10

EC

80 70 60 50 40 30 20 10 0

0 0

458

3

5

10

15

20

25

0

5

10

Time (hour)

15 Time (hour)


20

10


459

Page 26 of 28

Figure 4

100 90


Remaining methyl eleostearate (%)

80 70 60 50 40 30 20 10 0 0

3

6

9

12

15

Time (hour)

460 461


18

21

24

Page 27 of 28

462


Figure 5

90 80 70 60 50 40 30 20

ASC

10 0 0

3

6

9

12 15 Time (hour)

18

21

24



100

80 70 60 50 40 30 20

GA

10 0

3

6

9

12 15 Time (hour)

18

21

24

6

9

12 15 Time (hour)

18

21

24

100 Remaining methyl eleostearate (%)


90

0

100 90 80 70 60 50 40 30 20

EGCG

10 0

90 80 70 60 50 40 30 20

EC

10 0

0

463

100

3

6

9

12 15 Time (hour)

18

21

24

0

3

464



465

TABLE OF CONTENTS GRAPHIC

466

467


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Assessing Interactions between Lipophilic and Hydrophilic

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