Comparison of Antioxidant Evaluation Assays for Investigating

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A Comparison of Antioxidant Evaluation Assays for Investigating Antioxidative Activity of Gallic Acid and Its Alkyl Esters in Different Food Matrices Natthaporn Phonsatta, Pawinee Deetae, Pairoj Luangpituksa, Claudia Grajeda Iglesias, Maria Cruz FigueroaEspinoza, Jerome Lecomte, Pierre Villeneuve, Eric A Decker, Wonnop Visessanguan, and Atikorn Panya J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02503 • Publication Date (Web): 27 Jul 2017 Downloaded from http://pubs.acs.org on July 30, 2017

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Journal of Agricultural and Food Chemistry

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A Comparison of Antioxidant Evaluation Assays for Investigating Antioxidative Activity of

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Gallic Acid and Its Alkyl Esters in Different Food Matrices

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Natthaporn Phonsatta1,3, Pawinee Deetae2, Pairoj Luangpituksa3, Claudia Grajeda-Iglesias4,

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Maria Cruz Figueroa-Espinoza4, Jérôme Le Comte5, Pierre Villeneuve5, Eric A. Decker6,7,

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Wonnop Visessanguan1 and Atikorn Panya1*

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1

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Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Rd., Klong Luang,

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Pathumthani 12120, Thailand; 2 Division of Food Science and Technology, Faculty of Agro-

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Industry, King Mongkut's Institute of Technology Ladkrabang, Bangkok 10520, Thailand; 3

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Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400,

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Thailand. 4 Montpellier SupAgro, UMR 1208 IATE, Montpellier F-34060, France. 5 CIRAD,

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UMR 1208 IATE, Montpellier F-34060, France. 6 Department of Food Science, University of

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Massachusetts, Chenoweth Laboratory, 100 Holdsworth Way, Amherst, MA 01003, USA,

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7

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King Abdulaziz University, P. O. Box 80203 Jeddah 21589 Saudi Arabia

Food Biotechnology Research Unit, National Center for Genetic Engineering and

Bioactive Natural Products Research Group, Department of Biochemistry, Faculty of Science,

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*

Corresponding author

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Running title: Relationship between antioxidant evaluation assays and food matrices

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ACS Paragon Plus Environment


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ABSTRACT The addition of antioxidants is one of the strategies to inhibit lipid oxidation, a major

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cause of lipid deterioration in foods leading to rancidity development and nutritional losses.

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However, several studies have been reported that conventional antioxidant assays e.g. TPC,

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ABTS, FRAP, and ORAC could not predict antioxidant performance in several foods. This study

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aimed to investigate the performance of two recently developed assays e.g. the conjugated

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autoxidizable triene (CAT) and the apolar radical-initiated conjugated autoxidizable triene

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(ApoCAT) assays to predict the antioxidant effectiveness of gallic acid and its esters in selected

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food models in comparison with the conventional antioxidant assays. The results indicated that

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the polarities of the antioxidants have a strong impact on antioxidant activities. In addition,

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different oxidant locations demonstrated by the CAT and ApoCAT assays influenced the overall

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antioxidant performances of the antioxidants with different polarities. To validate the

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predictability of the assays, the antioxidative performance of gallic acid and its alkyl esters was

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investigated in oil-in-water (O/W) emulsions, bulk soybean oils, and roasted peanuts as the lipid

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food models. The results showed that only the ApoCAT assay could be able to predict the

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antioxidative performances in O/W emulsions regardless of the antioxidant polarities. This study

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demonstrated that the relevance of antioxidant assays to food models was strongly dependent on

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physical similarities between the tested assays and the food structure matrices.

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Keywords: CAT, ApoCAT, Lipid oxidation, Antioxidant, Food matrix, Gallic acid, Gallates


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INTRODUCTION Lipid oxidation is an important reaction common to both biological and food systems. In

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food containing lipids, lipid oxidation has been considered as the deteriorative reaction1. To solve

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the lipid oxidation problem, numerous strategies have been incorporated into food products. One

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of the most effective strategies is the addition of antioxidant2. Antioxidants can prevent lipid

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oxidation by various mechanisms including scavenging free radical, chelating metal ions,

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decomposing lipid peroxides, quenching reactive oxygen species, preventing formation of

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peroxides, breaking the antioxidative chain reaction, and reducing localized O2 concentrations3.

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Recently, there is no standardized and/or single method to determine the antioxidant

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capacity of substances4. Nowadays, various methods have been used to determine antioxidant

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activities based on both non-competitive and competitive reaction schemes5. For non-competitive

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assays, the ABTS radical scavenging activity (ABTS) and ferric ion reducing antioxidant power

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(FRAP) assays measure the antioxidative ability of a substance to reduce an oxidant directly. On

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the contrary, the competitive assays measure the ability of an antioxidant to prevent formation of

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an oxidizable substrate from an oxidant. For example, the oxygen radical absorbance capacity

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(ORAC) assay, one of the competitive antioxidant evaluation assays, determines the ability of an

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antioxidant to inhibit the degradation of fluorescein in the presence of peroxyl radicals generated

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from the thermally decomposition of an azo compound such as AAPH6. In the past, researchers

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have been tried to establish a standardized antioxidant evaluation assay. In 2007, the USDA

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released the database for the ORAC values of 277 food items. In addition, the ORAC values of

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49 food items were added into the database in 2010 for a total of 326 food items7. However, the



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ORAC database was then removed from the USDA’s Nutrient Data Laboratory (NDL) database

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due to various evidences supporting that the ORAC assay had no relevance to the antioxidative

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abilities of bioactive compounds, including polyphenols on human health8. In addition to the

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ORAC assay, there were some evidences that other homogeneous assays such as the DPPH

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radical scavenging activity (DPPH) and FRAP assays had also poorly predicted the antioxidant

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effectiveness of compounds related to food systems2, 9.

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From these reasons, a heterogeneous antioxidant assay called the conjugated

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autoxidizable triene (CAT) assay was developed based on tung oil-in-water emulsion10.

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Heterogeneous assays would contain a dispersed lipid phase, such as an emulsion, vs a

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homogenous solvent system. In this assay, the concept of antioxidant distribution/location has

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been integrated into the test under the assumption that an antioxidant located near the oxidation

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site could effectively prevent lipid oxidation induced by peroxyl radicals formed from the

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thermo-degradation of 2,2'-Azobis (2-amidinopropane) dihydrochloride (AAPH). However, the use

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of a water-soluble radical initiator (AAPH) raised a question of distribution of oxidants in the

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system, which might not be a suitable way to mimic lipid oxidation dynamics of oil-in-water

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emulsions where most radicals would be in the lipid phase. Thus, the apolar radical-initiated

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conjugated autoxidizable triene (ApoCAT) assay was developed to eliminate the use of a water

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soluble free radical initiator by using a lipid soluble free radical initiator (Dimethyl 2,2'-azobis (2-

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methylpropionate so called AIBME)11.

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To date, there are no studies supporting the relationship between homogeneous and heterogeneous antioxidant evaluation assays, and their ability to predict the antioxidant activity


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in food systems. Therefore, the aims of this study were to investigate the antioxidant activities of

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gallic acid and its alkyl esters using various antioxidative evaluation assays (TPC, ABTS, FRAP,

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ORAC, CAT and ApoCAT) and investigate the relationship among these antioxidant evaluation

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assays and their ability to predict the antioxidant effectiveness in various food models including

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an oil-in-water emulsion, bulk soybean oil and roasted peanut.

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MATERIALS AND METHODS

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Chemicals Reagents

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Trichloroacetic acid (TCA), butylated hydroxyl toluene, thiobarbituric acid, cumene

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hydroperoxide and 1,1,3,3,- tetrahydroxypropane were purchased from Merck (USA).

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Ethylenediaminetetraacetic acid (EDTA), 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), silicic acid,

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Folin-Ciocalteau’s reagent, sodium carbonate, ferrous sulfate, ferric chloride, ferrozine, Tung oil,

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Brij 35, ascorbic acid, polyoxyethylene sorbitan monolaurate (TWEEN® 20), barium chloride,

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ammonium thiocyanate, 2-Thiobarbituric acid and butylated hydroxytoluene (BHT) were

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purchased from Sigma-Aldrich (St. Louis, USA). Absolute ethanol was purchased from RCI

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Labscan (Thailand). 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), dimethyl 2,2'-

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azobis(2-methylpropionate) or AIBME and activated charcoal were purchased from Wako

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Chemical (Japan). Medium chain triglyceride (MCT) mixture (Miglyol®812N) was purchased from

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Sasol Germany Gmbh (Witten, Germany). Gallic acid, methyl gallate, propyl gallate, butyl

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gallate, octyl gallate, and laury gallate were purchased from Sigma-Aldrich (St. Louis, USA). Cold



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pressed perilla oil was purchased from a local market in Thailand. Deionized water was used for

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the preparation of all solutions. All organic solvents used in this study were analytical grade.

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Total Phenolic Content (TPC) Assay Total phenolic content (TPC) of gallic acid and its alkyl esters was determined using

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Folin-Ciocalteu method with some modifications12. The reaction was performed in 96-well

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microplates. Samples (30 µl) were mixed with 150 µl of Folin-Ciocalteau’s reagent (10-time

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dilution) and 120 µl of sodium carbonate (7.5% w/v). The mixtures were mixed using a microplate

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mixer (Thermomixer comfort; eppendorf, Germany) at 600 rpm for 1 min, and kept in the dark

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for 30 min. The absorbance of mixture was read at 765 nm using a microplate reader (Spectra

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MAX 190, Molecular Devices, USA). Gallic acid was used as a standard phenolic compound.

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Results were expressed as gallic acid equivalent.

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ABTS Radical Scavenging (ABTS) Assay The free radical scavenging activity of plants extracts was performed using the ABTS

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assay12. ABTS cation radicals (ABTS•+) were generated by reacting 7 mM ABTS stock solution

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(10 ml) with 140 mM potassium persulfate (179 µl). Then, the mixture was incubated at room

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temperature for 16 h in the dark. The ABTS•+ solution was diluted to obtain OD of 0.700±0.050 at

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a wavelength of 734 nm before use. Twenty microliters of samples were mixed with 280 µl of the

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ABTS•+ solution and then incubated in the dark at room temperature for 6 min. The absorbance of


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mixture was then read at 734 nm using a microplate reader (Spectra MAX 190, Molecular

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Devices, USA). The amount of the absorbance decrease was expressed against the standard curve

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of various concentrations of ascorbic acid. Results were expressed as ascorbic acid equivalent.

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Ferric Reducing Antioxidant Power (FRAP) Assay The ferric reducing antioxidant power (FRAP) assay was performed using the modified

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method as described by Deetae et al. (2012)12. The FRAP reagent was freshly prepared by mixing

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300 mM acetate buffer (pH 3.6), 10 mM TPTZ in 40 mM HCl and 20 mM FeCl3.6H2O at a mole

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ratio of 10:1:1. The reactions were started by mixing 30 µl of samples with 270 µl of FRAP

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reagent. The reaction was performed in 96-well microplates. The total volume of reaction was 300

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µl. The microplates containing reaction mixtures were kept in the dark at 37 °C for 8 min. The

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increase in absorbance of mixtures was read at 595 nm using a microplate reader (Spectra MAX

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190, Molecular Devices, USA). Using ascorbic acid as a standard curve, results were expressed as

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ascorbic acid equivalent.

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The Oxygen Radical Absorbance Capacity (ORAC) Assay The oxygen radical absorbance capacity (ORAC) assay method was performed with some

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modifications13. Fluorescein was used as the fluorescent probe, and AAPH was used as the

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peroxyl radical generator. The final reaction mixture consisted of fluorescein (160 µl, 78 nM),

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sample (20 µl) and AAPH (20 µl, 12 mM). All reagents were prepared in 75 mM phosphate buffer



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solution, pH 7.4. The ORAC assay was done in black 96-well plates (Eppendorf Microplate 96/U-

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PP). Fluorescent intensity of the mixture was recorded each minute with 5 s agitation using a

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fluorescent microplate reader (SynergyMX, Biotek, USA) with an excitation wavelength of 485

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nm and an emission wavelength of 520 nm in kinetic mode for 3 h. Using trolox as a standard

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curve, results were expressed as trolox equivalent.

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The Conjugated Autoxidizable Triene (CAT) Assay

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The conjugated autoxidizable triene (CAT) assay method was performed with some

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modifications10. Conjugated triene triacylglycerols from Tung oil were used as the oxidizable UV

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probe for an oxidation reaction. All of the reagents and samples were dissolved and diluted in

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phosphate buffer (50 mM, pH7.2). The CAT assay protocol was composed of 3 steps described as

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follows.

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1. Oil Stripping

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Tung oil was striped prior to remove the minor polar components. Briefly, 100 g of silicic

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acid was washed with distilled water for three times with a total volume of 3 L, then the washed

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silicic acid was filtered with Whatman filter paper and dried at 110 °C for 20 h. The glass

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chromatographic column (3.0 cm internal diameter×35 cm height) was packed sequentially with

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22.5 g of washed silicic acid followed by 5.63 g of activated charcoal and 22.5 g of washed silicic

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acid. The washed silicic acid and activated charcoal were suspended in 100 ml and 70 ml of n-

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hexane, respectively, before packing into a glass column. Thirty grams of Tung oil were


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dissolved in 30 ml of n-hexane, and passed through the column by eluting with 270 ml of n-

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hexane. The collected triacylglycerols were kept in an ice bath and covered with an aluminum

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foil in order to minimize lipid oxidation during stripping process. To remove the n-hexane from

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the collected stripped Tung oil after the elution completed, the rotary evaporator (BÜCHI

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Rotavapor R-114, Switzerland) was performed at 35 °C under vacuum. The nitrogen flushing was

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adapted to remove traces of hexane remaining in the oil. After flushing for 10 min, 3 ml of

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stripped oil was transferred into vials and flushed with nitrogen again. The aliquots of stripped

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Tung oil were kept at -80 °C before use14.

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2. Emulsion Preparation

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Phosphate buffer (PB) solution (50 mM, pH 7.2) containing 34 µM Brij 35 was added to

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6±1 mg of stripped Tung oil. The mixture was then homogenized (Ultra-TurraxT25 basic, IKA®

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Werke, Germany) at 6,500 rpm for 90 s. The emulsion was centrifuged at 10,000 x g, 10 °C for 5

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min to remove large emulsion droplets.

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3. Reaction Mixture

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A 50-µl volume of 50 mM PB containing samples (desirable concentration) was

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transferred to a UV-Star 96-well plate followed by 125 µl of emulsion. The 96-well plate

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containing antioxidants and emulsions was mixed at 1,200 rpm, 37 °C for 5 min by a microplate

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thermomixer (Thermomixer comfort, Eppendorf, Germany). After mixing, 25 µl of 8 mM AAPH

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solution in PB solution was added into the mixtures. The final mixture volume was 200 µl with



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17 µM Brij 35, 1 mM AAPH and various concentrations of antioxidants. Changes in the

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absorbance at 273 nm in of mixtures were then recorded using a microplate reader (Spectra MAX

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190, Molecular Devices, USA) in kinetic mode for 3 h. The CAT values were expressed as trolox

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equivalent by calculating the relationship between net protection areas under the curve (AUC)

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and concentrations of antioxidants compared to that of trolox (6-hydroxy-2,5,7,8-

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tetramethylchroman-2-carboxylic acid).

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The Apolar Radical-Initiated Conjugated Autoxidizable Triene (ApoCAT) Assay The ApoCAT assay was performed with slight modifications from previously published

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methods11. Based on the original CAT assay, all procedures of the ApoCAT assay were

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performed in the same manner as the original CAT assay except for the free radical initiator.

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Instead of using AAPH, dimethyl 2,2'-azobis (2-methylpropionate) or AIBME, an lipid-soluble

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free radical initiator was used in this assay. To deliver the lipid radical initiator into emulsion

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mixtures, medium chain triglycerides (MCT) emulsions were prepared. Briefly, 10 mM PB

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solution (pH 7.2) containing 34 µM Brij 35 was added to 6 ± 1 mg of MCT oil. The mixture was

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then homogenized at 6,500 rpm for 90 s. The AIBME radical initiator was prepared by dissolving

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in the MCT emulsions to obtain a final concentration of 8 mM. To remove oil residues and other

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non-stable parts, the emulsion was centrifuged at 10,000 x g, 10 °C for 5 min. The MCT

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emulsion containing 8 mM V-601 was collected from the supernatant. To begin the oxidation of


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the ApoCAT assay, 25 µl of the MCT emulsion with AIBME was added into the prepared Tung

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oil emulsions with antioxidants as described in the CAT assay.

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Evaluation of Antioxidative Performance of Gallic Acid and Its Esters in Conventional

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Perilla Oil-in-water (O/W) Emulsions

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In this study, oil-in-water (O/W) emulsions were used to represent a heterogeneous food

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model for evaluating the antioxidative performance of gallic acid and its alkyl esters. Perilla oil

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was stripped prior to use in the same manner as Tung oil as described in the CAT assay. The O/W

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emulsion was prepared with slightly modifications. Briefly, 1% (w/w) of stripped perilla oil was

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mixed with TWEEN® 20 at the emulsifier/oil ratio of 1:10 (w/w) in PB (10 mM, pH 7.0) by

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homogenizing at 9,500 rpm for 2 min (Ultra-Turrax T25 basic, IKA® Werke, Germany). Then, the

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coarse emulsion mixture was homogenized with ultrasonic treatment using a 20 kHz 130 W

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ultrasonic processor (Sonic, Vibra-cell™ VCX 130, USA) equipped with a 13 mm diameter tip

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(with 100% amplitude). Total pulse duration was 2 min (30 s each pulse, with 10 s interval)14.

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The perilla O/W emulsions with gallic acid and its alkyl esters at the final concentration

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of 50 µM were prepared. One milliliter of the O/W emulsions containing tested antioxidants was

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then transferred into 96-deep well plates. Aluminum foil sealing tapes (Corning® USA) were used

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to cover the microplate in order to minimize the evaporation of emulsions. The plates were then

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incubated at 30 °C under the dark. The emulsion samples were taken periodically by pipetting

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though the pierce-able film to determine the oxidation products. Lipid hydroperoxide values (PV)



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and thiobarbituric acid reactive substances (TBARs) were evaluated as the indicators of primary

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and secondary lipid oxidation products, respectively.

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The oxidative stability of emulsions was expressed as the induction periods (IP) or

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oxidation lag time obtained from the formation of hydroperoxides and TBARs. The induction

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periods were defined as the first data point that statistically increased from the previous data

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points during the oxidation studies. Comparisons of the means were performed using Duncan’s

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multiple range tests.

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Measurement of Lipid Hydroperoxide Value (PV)

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Formation of lipid hydroperoxides was determined with some modifications15. Fifty

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microliter of emulsions were first mixed with 450 µl of isooctane: isopropanol (3:1, v/v) then

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centrifuged (3,200 x g) for 3 min to break emulsions. Then, 50 µl of the upper layer of organic

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solvent phase was collected and 200 µl of methanol: butanol (2:1, v/v) was added. The reaction

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begins with the addition of 20 µl of ammonium thiocyanate reagent. The ammonium thiocyanate

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reagent was prepared by mixing FeSO4 (0.144 M) with BaCl2 (0.132 M) at the ratio 1:1 (v/v). After

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that, the mixture was centrifuge at 3,200 x g for 2 min. The upper clear phase of centrifuged

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mixture was mixed with 3.94 M of ammonium thiocyanate solution at the ratio of 1:1(v/v). The

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reaction mixture was left at room temperature for 20 min, and the absorbance was recorded at

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510 nm using a microplate reader (Spectra MAX 190, Molecular Devices, USA). Cummene

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hydroperoxide was used as a standard lipid hydroperoxide.


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Thiobarbituric Acid Reactance (TBAR) Assay Thiobarbituric acid reactance (TBAR) assay was performed with slightly modifications2.

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Briefly, TBA reagent was prepared by mixing 20% of TCA, 0.5% (w/v) of TBA and 0.2% (w/v) of

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EDTA in 30 mM HCl. Immediately before analysis, 30 ml of TBA reagent was mixed with 450

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µl of 3%(w/v) BHT in absolute ethanol solution. Emulsion (150 µl) was reacted with the TBA

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reagent (300 µl) in 96-deep well plates. The plates containing mixture were covered and heated at

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90 °C for 20 min. Then, 96-deep well plates were transferred into an ice bath to stop the reaction

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for 10 min. Then, the plates were centrifuged at 3,200 x g for 5 min. The supernatant was

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collected to measure the absorbance at 532 nm. A calibration curve was prepared with 1,1,3,3-

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tetrahydroxypropane as a standard for quantification.

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Evaluation of Antioxidative Performance of Gallic Acid and Its Esters in Bulk Soybean Oil

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and Roasted Peanuts

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In addition to O/W emulsions, the antioxidative performances of gallic acid and its alkyl

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esters were investigated in other food matrices including bulk soybean oil and roasted ground

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peanuts using the Rancimat method. A commercial refined soybean oil (TVO public company

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limited, Thailand) and freshly prepared roasted ground peanuts were purchased locally. The

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soybean oil and roasted ground peanuts were mixed with the ethanolic solution of gallic acid and

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its alkyl esters to obtain the final concentration of 2 millimoles per kilogram. For soybean oil

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samples, ethanol was evaporated out using a rotary evaporator operating at 35 °C under vacuum

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for 10 min. For roasted grounded peanut, samples were hand shaken to obtain homogeneous



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mixture for 3 min after the addition of antioxidants. After that, the roasted grounded peanut

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samples were dried in a hot air oven 35 °C for 30 min to remove the remaining ethanol.

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Soybean oil and roasted grounded peanut with or without gallic acid and its alkyl esters

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were placed in a Rancimat machine (Rancimat 743, Metrohm CH-9101, Herisau, Switzerland) to

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determine the oxidative stability of the samples. The instrument was set at 110 °C and air flow

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rate of 20 L/h16. The sample size of bulk soybean oils and roasted ground peanuts were three

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grams and one gram, respectively. Samples were weighed into the reaction tube and placed in the

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Rancimat instrument. The increasing of conductivity by trapping lipid oxidation products in

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water was monitored kinetically. The induction times (IT) of oxidation were calculated using 743

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Rancimat software (Metrohm CH-9101, Herisau, Switzerland).

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Statistical Analysis All analyses were performed on triplicate samples. The relationship between antioxidant

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evaluation assays were analyzed by principle component analysis (PCA) using SPSS 17

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(http://www.spss.com; SPSS Inc., Chicago, IL).

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RESULTS AND DISCUSSION

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Antioxidant Activities of Gallic Acid and Its Alkyl Esters in Homogeneous Antioxidant

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Assays

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TPC, ABTS, FRAP, and ORAC assays represent examples of homogeneous antioxidant

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assays that have been commonly used to evaluate the ability of compounds to donate hydrogen

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atoms and/or electrons. Similar to ABTS and FRAP assay, the total phenolic content (TPC) assay

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also evaluates the electron donating ability of compounds by evaluating reduction of molybdate

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in Folin-Ciocalteu (F-C) reagent via oxidation-reduction reactions in comparison to gallic acid as a

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phenolic standard17. Dependent upon the nature of the reaction, these assays can be categorized

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into competitive (the ORAC assay) and non-competitive systems (the TPC, ABTS, and FRAP

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assays). In the ORAC assay, antioxidants have to compete with peroxyl radicals generated from

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AAPH in order to prevent the oxidation of the fluorescence compound fluorescein, while in the

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TPC, ABTS, and FRAP assays antioxidants are able to react directly with oxidants (e.g. ABTS•+).

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Thus, antioxidants performing well in the ORAC assay should have not only the ability or

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capacity to give electrons, but also high reactivity to radicals.

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The antioxidants performing in the non-competitive assays (TPC, ABTS, and FRAP

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assays) exhibited a similar behavioral pattern that increasing alkyl chain lengths of a free acid

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form of gallic acid (G0) to methyl gallate (G1) dramatically decreased the antioxidant activity

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(Table 1). Further decreases in the antioxidant activities were observed when alkyl chain lengths

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were increased. Gallic acid had the highest TPC, ABTS, and FRAP values followed by methyl



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gallate (G1), propyl gallate (G3), butyl gallate (G4), octyl gallate (G8), and lauryl gallate (G12),

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respectively.

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However, in the ORAC assay, increases in alkyl chain lengths from G0 to G3 improved

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the antioxidant activity as indicated by the ORAC value. Propyl gallate (G3) exhibited

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significantly higher ORAC value than gallic acid (G0). This result was consistent with Alamed

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and co-workers (2009) that propyl gallate exhibited higher ORAC value than gallic acid2. It should

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be noted that further increases in alkyl chain lengths beyond G3, antioxidant activities decreased

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as shown in Table 1.

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In terms of the kinetic points of view, decreasing patterns of the absorbance of

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fluorescein during the ORAC assay indicated that all antioxidants exhibited clear oxidation lag

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times. Results indicated that chain-breaking behaviors were observed regardless of alkyl chain

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lengths (data not shown). This result suggested that peroxyl radicals generated from AAPH could

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react with gallic acid in the same manner as other alkyl gallate esters. However, the higher

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concentrations of antioxidant, the longer lag times of fluorescein were observed.

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Since the ABTS and FRAP assays were performed in aqueous solutions the antioxidant

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values of gallate esters decreased according to their alkyl chain length. This phenomenon could

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be explained that the polarities of gallate alkyl esters were decreased resulting in their lower

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solubility in the aqueous system. Thus, the reduction of solubility of gallate alkyl ester could

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influence their ability to scavenge free radical as compare to gallic acid. Moreover, the gallate

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alkyl esters could be self-aggregated by the hydrophobic effect as explained by self-assembly of


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the nonpolar substances in aqueous or polar solvents18. The aggregation of gallate alkyl esters

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might occur as micelles, lamellar structures and other association colloids19.

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With the increasing of alkyl chain lengths, G1, G3, and G4 exhibited higher ORAC

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values than G0. This result might be explained by the esterification reaction increasing the radical

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scavenging activity of substances, which was consistent to the study of Lu and co-workers20.

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However, the ORAC value decreased at G8 and observed lowest at G12, it might be due to the

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aggregation of lipophilic molecules in water phase, which may decrease the solubility of

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antioxidants resulting in lower antioxidant activities.

330 331

Antioxidant Activity of Gallic Acid and Its Alkyl Esters in Heterogeneous Antioxidant

332

Assays

333

The conjugated autoxidizable triene (CAT) assay has been developed to determine the

334

antioxidant activity in oil-in-water emulsions10. Since the CAT assay is conducted in oil-in-water

335

emulsion, the locations of antioxidant in the system have to be accounted for their antioxidative

336

performances. According to the polar paradox hypothesis, the non-polar antioxidant is more

337

active in emulsified systems21. Results showed that butyl gallate (G4) exhibited the highest value

338

of the CAT assay followed by propyl gallate (G3), methyl gallate (G1), lauryl gallate (G12), octyl

339

gallate (G8) and gallic acid (G0), respectively (Table 2). According to this result, it was

340

inconsistent with the polar paradox hypothesis. The non-linear trend was observed after increased

341

the alkyl chain length to G4. Interestingly, the antioxidant activity of alkyl gallate esters was

342

sharply dropped at G8 before increasing again at G12. According to the AAPH (water soluble



343

radical) used in the CAT assay, it was hypothesized that an overestimation of the antioxidant

344

capacity of water soluble antioxidants, and on the contrary, an underestimation of the antioxidant

345

performance of a lipid soluble antioxidant may be observed. Therefore, the ApoCAT assay, a

346

modified version of the CAT assay, has been developed11. Instead of using AAPH, a lipid soluble

347

radical initiator (AIBME) was used to initiate oxidation reaction.

348

The results indicated that lauryl gallate (G12) exhibited the highest ApoCAT value followed by

349

octyl gallate (G8), butyl gallate (G4), propyl gallate (G3), methyl gallate (G1) and gallic acid (G0)

350

(Table 2). Unlike the CAT assay, the linear trend between antioxidant values and alkyl chain

351

lengths was observed in the ApoCAT assay.

352

There were some other experiments observing the nonlinear trend of antioxidant

353

behaviors in emulsion systems. For example, the antioxidant activity of chlorogenic acid and its

354

alkyl ester in the CAT assay dramatically decreased when the alkyl chain was greater than 12

355

carbons22. Furthermore, other studies also observed the nonlinear trend against the polar paradox

356

hypothesis using various antioxidants with different polarities in emulsion systems15, 23.

357

According to the nonlinear trend of antioxidant behaviors, the cut-off hypothesis was introduced

358

to explain the observation that an intermediate alkyl chain length exhibited the highest

359

antioxidative activity in emulsions. This chain length was termed the critical chain length or CCL

360

and typically ranges from 8 to12 carbons in oil-in-water emulsions. The cut-off effect of

361

antioxidants with >12 carbons in oil in water emulsion has been hypothesized to be due to three

362

possible mechanisms of action including reduced mobility, internalization in the emulsion

363

droplet and the self-aggregation. In the reduced mobility hypothesis, when the alkyl chain of


Page 18 of 41

Page 19 of 41


364

antioxidant was lengthened, the ability to move toward the oxidation sites was reduced due to

365

the strongly bounding to the molecular environment by hydrophobic interactions19. Another

366

mechanism of action explaining the cut-off hypothesis is internalization. The internalization

367

hypothesis explained that increasing the hydrocarbon chain from medium to long chain resulting

368

in the internalization of antioxidants into the lipid droplet core. Consequently, the poor

369

antioxidative performance is observed due to the antioxidant compounds located far away from

370

the oil/water interface where the oxidation occurs19. For the last hypothesis, self-aggregation,

371

antioxidant activity is decreased when the antioxidants aggregate with each other or other

372

surfactants in the emulsion to form micelles in the water phase of emulsions. When the self-

373

aggregation occurs, the concentration of antioxidants at the oxidation sites may be decreased,

374

resulting in the lower ability of antioxidant to prevent lipid oxidation in emulsified systems19.

375

In contrast, the cut off effect was not found in the ApoCAT assay. Due to the lipid soluble

376

free radical initiator used in the ApoCAT assay, free radical would locate at oil-water interphase.

377

Thus, the scavenging reaction between the antioxidant and free radical in water phase was not

378

occurred and the overestimation of water soluble antioxidant may not observe in this assay.

379

More insight was provided by calculating the ratio between ApoCAT and CAT values for

380

each antioxidant. The ApoCAT/CAT ratios of gallic acid (G0), methyl gallate (G1), propyl gallate

381

(G3), butyl gallate (G4), octyl gallate (G8) and lauryl gallate (G12) are illustrated in Figure 1. The

382

ratio of ApoCAT/CAT was able to predict which antioxidants interacted with free radicals in

383

water- and/or lipid-phases. As expected, the lipophilic antioxidants (G8 and G12) performed better

384

in ApoCAT system as their calculated values were above 1. In contrast, it might be summarized



385

that G0, G1, G3 and G4 exhibited the preferential activity toward water soluble free radical. This

386

result was consistent to the study on the effect of oxidant location on antioxidant capacities using

387

ApoCAT and CAT ratio from Panya and co-workers which demonstrated that the esters forms of

388

antioxidants had higher ratio than their acid forms11. It is worth mentioned that this

389

ApoCAT/CAT ratio could be applied for characterizing the unknown antioxidant or natural crude

390

extracts.

391

From the kinetic point of views of the CAT assay (Figure 2), lag phase was observed with

392

only methyl gallate (G1) and octyl gallate (G8) while no clearly lag phases were observed with

393

gallic acid (G0), propyl gallate (G3), butyl gallate (G4), nor lauryl gallate (G12). Results indicated

394

that only G1 and G8 showed the antioxidant behaviors as chain breaker while others showed

395

retarder behaviors in the CAT assay. The observed induction period or lag phase of conjugated

396

trienes degradation would indicate the chain breaker of antioxidants. From the kinetic point of

397

view (Figure 2 and 3), results from some antioxidants showed lag phase which could be defined

398

as chain breaker while others with no lag phase observed could be identified as retarder.

399

However, in the ApoCAT assay, no lag phases were observed for none of the tested compounds

400

(Figure 3). Results indicated that gallic acid and its alkyl esters exhibited the antioxidant

401

behaviors as retarders in the ApoCAT assay. According to the CAT assay, the result was

402

consistent to previous study of Laguerre and co-workers10 who also found the retarder antioxidant

403

behavior of gallic acid.

404


Page 20 of 41

Page 21 of 41


405

Antioxidative Performances of Gallic Acid and Its Alkyl Esters in Oil-in-Water (O/W)

406

Emulsions

407

The ability of gallic acid and its alkyl esters to prevent lipid oxidation in oil-in-water

408

(O/W) emulsions was evaluated using the induction periods obtained from the formation of

409

primary lipid oxidation products (peroxide value or PV value) and secondary lipid oxidation

410

products (thiobarbituric acid reactance or TBAR value). The induction periods calculated based

411

on the PV and TBAR values of O/W emulsion treated with 50 µM of gallic acid (G0), methyl

412

gallate (G1), propyl gallate (G3), butyl gallate (G4), octyl gallate (G8), and lauryl gallate (G12) are

413

shown in Figure 4. The linear trend of antioxidative performance in O/W emulsions was

414

observed according to the alkyl chain lengths. This observation could be explained by the polar

415

paradox hypothesis that the non-polar antioxidants are more active than their polar homologues

416

in O/W emulsion systems.

417 418 419

Antioxidative Performances of Gallic Acid and Its Alkyl Esters in Bulk Soybean Oils Bulk soybean oil was used as the bulk oil model to evaluate the antioxidant performances

420

of gallic acid and its alkyl esters. In bulk oil, lipid oxidation mechanisms are different from

421

emulsified system due to many factors1. It was hypothesized that polar antioxidants are more

422

effective in bulk oil than their non-polar homologues24.

423 424

The antioxidative performances in bulk soybean oil of gallic acid (G0), methyl gallate (G1), propyl gallate (G3), butyl gallate (G4), octyl gallate (G8), and lauryl gallate (G12) were



425

evaluated using Rancimat test. Result showed that G0 exhibited the highest induction time

426

followed by G4, G8, G12, G3, and G1, respectively (Figure 4).

427

The result was consistent to the polar paradox hypothesis that gallic acid (polar

428

homologue) was the most effective antioxidant in bulk soybean oil compared to its non-polar

429

homologues (gallate esters). However, increasing alkyl chain length to G3, the performances of

430

gallate esters in bulk soybean oil were decreased. As illustrated in Figure 4, the result indicated

431

that antioxidant performances of gallic acid and its alkyl esters in bulk soybean oil were

432

independent of polarities. This result was in agreement with the study on chlorogenic acid and its

433

alkyl esters which hydrophobicity did not exert a strong influence on antioxidant capacity25. It is

434

worth mentioned that there might be some other minor components and water remained in

435

soybean oil. Thus, the association colloids could form in bulk soybean oil resulting in the high

436

susceptibility of bulk soybean oil to oxidation reaction1. This result could give a good provide

437

that not only the intrinsic factors such as hydrophobicity of antioxidant influences on the

438

antioxidant capacity, but also the extrinsic factors of environmental system could exert the high

439

influencing of the antioxidant capacity.

440 441

Antioxidative Performances of Gallic Acid and Its Alkyl Esters in Roasted Peanuts

442

According to the studies of antioxidative performances of gallic acid and its alkyl esters

443

in oil-in-water emulsions and bulk soybean oil, the results showed that the antioxidant ability of

444

our substances to protect lipid oxidation was not dependent only on the antioxidant’s intrinsic

445

factors. However, the extrinsic factors such as the nature of food system also play the key role on


Page 22 of 41

Page 23 of 41


446

the antioxidative performances. To better understand the antioxidative performances of

447

substances in various food models, roasted peanut was chosen as a low moisture food containing

448

lipid model.

449

The antioxidative performances of gallic acid and its alkyl esters were determined in

450

roasted peanut using Rancimat test. Roasted peanuts treated with lauryl gallate (G12) exhibited

451

the highest induction time followed by octyl gallate (G8), butyl gallate (G4), methyl gallate (G1),

452

propyl gallate (G3) and gallic acid (G0), respectively (Figure 4).

453

The information on the antioxidant effectiveness in low moisture food is limited26. This

454

result suggested that the antioxidant effectiveness in roasted peanuts increased with increasing

455

the hydrophobicity of antioxidants. Results were in agreement with the study of rosmarinic acid

456

and its alkyl esters in cracker that similar to gallic acid, rosmarinic acid exhibited the lowest

457

antioxidant efficacy to inhibit lipid oxidation in a model cracker system. The reason for these

458

results might be due to the exclusion of polar antioxidants from the lipid phase resulting in the

459

separation of antioxidants from lipid oxidation reaction site27. On the contrary, increasing the

460

hydrophobicity of antioxidant would make antioxidants more capable to migrate to lipid

461

oxidation site thus the more effectiveness to prevent lipid oxidation were observed in low

462

moisture food.

463 464

Relationship between Antioxidant Activities in Various Antioxidant Assays and

465

Antioxidative Performances in Food Models of Gallic Acid and Its Alky Esters

466 467

The relationship between antioxidant activities in various antioxidant assays and antioxidant performances in different food matrices with gallic acid and its alkyl esters were



468

investigated using principle component analysis (PCA). As illustrated in Figure 5a, from ten

469

parameters, they could be reduced into two components (PC1 and PC2) explaining 95.40 % of

470

total variance. Each component was responsible for 52.38% and 43.02%, respectively. The PC1

471

represented the ApoCAT value, induction periods from PV and TBAR values of oil-in-water

472

emulsions and the oxidative stability of roasted peanuts (IT_RP), while the PC2 explained the

473

TPC, ABTS and FRAP values, and the oxidative stability of bulk soybean oils (IT_SBO). The

474

PCA score plot of gallic acid and it alkyl esters as shown in Figure 5b suggests that antioxidant

475

activities of lauryl gallate (G12) and octyl gallate (G8) were strongly explained by the assay in

476

PC1. In contrast, the antioxidant activity of gallic acid was highly correlated with the assays

477

categorizing in PC2. However, the antioxidant activities of methyl gallate (G1), propyl gallate

478

(G3), and butyl gallate (G4) were intermediate correlate with the assays in both PC1 and PC2.

479

The results were consistent with previous studies that the ORAC assay was not related to

480

the TPC, ABTS and FRAP assays28-30. However, a good relationship between the ORAC assay

481

and others were observed 31. This information suggested that the correlation between the ORAC

482

and other methods depended on the different kinetics and reaction mechanisms of the various

483

antioxidants present in the assays.

484

Furthermore, the results were in agreement with the study on relationship between radical

485

scavenging activity and antioxidant activity in foods which reported that the ORAC assay was

486

not able to predict the antioxidant activity of compounds in O/W emulsions and in cooked ground

487

beef2. The lack of relationship between antioxidant activities obtained from a homogeneous assay

488

such as TPC, ABTS, FRAP, and ORAC and antioxidant activities of compounds in food might


Page 24 of 41

Page 25 of 41


489

be due to the complexity of food. Thus, food matrices are the important part providing the

490

multitude factors influencing on the ability of compounds to inhibit lipid oxidation in food

491

system.

492

However, our results showed that the CAT assay was not able to predict antioxidant

493

performance in all three types of food systems (O/W emulsions, bulk soybean oils, and roasted

494

peanuts). The CAT assay could not predict the actual antioxidant activity in foods due to the

495

unrealistic oxidation reaction occurring in the tested systems. The CAT assay used AAPH which

496

is water soluble radical initiator to initiate lipid oxidation thus making the radical locating in

497

water phase of emulsion. Thus, the reaction scheme of the CAT assay might be different from

498

lipid oxidation in complex food systems in which lipid oxidation occurs at the oil/water interface

499

regions of emulsion droplets1. To solve this problem, the ApoCAT assay has been developed to

500

use the lipid soluble radical initiator instead of AAPH. Thus, in ApoCAT test system, the lipid

501

oxidation could occur at the interphase of water-oil droplets, and thus the phenomenon might

502

relate to lipid oxidation in actual food systems.

503

Interestingly, as illustrated in Figure 5, our results exhibited a close relationship between

504

the ApoCAT assay with the induction periods of PV and TBAR formation in O/W emulsions and

505

the oxidative stability of roasted peanuts (IT_RP). However, the ApoCAT had a poor correlation

506

with the oxidative stability of bulk soybean oils (IT_SBO). This result could explain that lipid

507

oxidation mechanisms in bulk oil systems might be different from emulsion systems.

508 509

From the PCA score plot (Figure 5b), results suggested that high polar antioxidant (G0) exhibited high antioxidant values in non-competitive homogeneous assays while non-polar



510

antioxidant (G8 and G12) exhibited high antioxidant value in heterogeneous assays. In addition,

511

the correlations among all tested methods showed that the ApoCAT assay was significantly

512

correlated with the oxidative induction periods of O/W emulsions (R2=0.99) and the oxidative

513

induction times of roasted peanuts (R2=0.89) (Figure 6).

514

In conclusion, to be able to accurately predict antioxidant performances in complex food

515

systems using the rapid determination of antioxidants, new knowledge on influences of food

516

matrices on oxidation and antioxidant mechanisms should be taken into consideration. In this

517

study, we demonstrated that the ApoCAT assay was able to predict the activities of antioxidant

518

in O/W emulsions and roasted peanut, suggesting that the heterogeneous antioxidant evaluation

519

assays could predict the antioxidant activities in food models having similar matrix to the tested

520

system not only in terms of food structures, but also in terms of oxidant locations.

521 522

ACKNOWLEDGMENTS

523

We would like to express our thanks to Junior Research Fellowship Program, The Franco-Thai

524

Scholarship Program, and Thailand Research Fund (Grant No. TRG5780061) for supporting this

525

research project.

526 527


Page 26 of 41

Page 27 of 41


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guava fruit extracts. J. Food Comp. Anal. 2006, 19 (6–7), 669-675.

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Perez, D. D.; Leighton, F.; Aspee, A.; Aliaga, C.; Lissi, E., A comparison of methods

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Page 32 of 41

616

Table list

617

Table 1 Antioxidant activities of gallic acid and its alkyl esters using homogeneous antioxidant

618

evaluation assays

619

Table 2 Antioxidant activities of gallic acid and its alkyl esters using heterogeneous antioxidant

620

evaluation assays

621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637


Page 33 of 41


638

Figure list

639

Figure 1 The ratio between the ApoCAT and CAT values of gallic acid (G0), methyl gallate (G1),

640

propyl gallate (G3), butyl gallate (G4), octyl gallate (G8) and lauryl gallate (G12)

641

Figure 2 Antioxidant performance of gallic acid and its esters in oil-in-water emulsions

642

(Emulsion) expressed by thiobarbituric acid reactance (TBAR) values, and in oxidative induction

643

times of bulk soybean oils (SBO) and roasted peanut treated (RP) measured by the Rancimat test at

644

110 °C with airflow rate of 20 L/h

645

Figure 3 Kinetic decay of conjugated triene in the CAT assay treated with gallic acid (a), methyl

646

gallate (b), propyl gallate (c), butyl gallate (d), octyl gallate (e), lauryl gallate (f)

647

Figure 4 Kinetic decay of conjugated triene in the ApoCAT assay treated with gallic acid (a),

648

methyl gallate (b), propyl gallate (c), butyl gallate (d), octyl gallate (e), lauryl gallate (f)

649

Figure 5 PCA loading (a) and score (b) plots obtained from total phenolic content (TPC assay),

650

antioxidant activities (ABTS, FRAP, ORAC, CAT and ApoCAT assays), the oxidative induction

651

periods of PV and TBAR formation in oil-in-water emulsions, and the oxidative induction time of

652

bulk soybean oils (IT_SBO) and roasted peanuts (IT_RP)

653

Figure 6Scatter plots between ApoCAT values with the oxidative induction times of peanuts and

654

the oxidative induction periods of oil-in-water emulsions from TBAR formation



655

656 657

Page 34 of 41

Antioxidants

TPC (Gallic acid equivalent)

ABTS (Ascorbic acid equivalent)

FRAP (Ascorbic acid equivalent)

ORAC (Trolox equivalent)

Gallic acid (G0)

1*d

2.91±0.14d

2.25±0.03d

1.53±0.17c

Methyl gallate (G1)

0.091±0.00c

1.08±0.04c

0.73±0.01c

1.76±0.20cd

Propyl gallate (G3)

0.087±0.01c

1.09±0.01c

0.77±0.04c

1.88±0.17d

Butyl gallate (G4)

0.088±0.00c

0.98±0.02c

0.73±0.03c

1.78±0.13cd

Octyl gallate (G8)

0.060±0.01b

0.65±0.93b

0.56±0.03b

1.23±0.11b

Dodecyl gallate (G12)

0.055±0.00a

0.09±0.00a

0.30±0.00a

0.32±0.04a

Table 1 a-d

Means within a column with different letters are significantly different (p

Comparison of Antioxidant Evaluation Assays for Investigating

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