Biological and Nonbiological Antioxidant Activity of Some Essential

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Biological and non-biological antioxidant activity of some essential oils. Renato Pérez-Rosés, Ester Risco, Roser Vila, Pedro Peñalver, and Salvador Canigueral J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00986 • Publication Date (Web): 23 May 2016 Downloaded from http://pubs.acs.org on May 27, 2016

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Journal of Agricultural and Food Chemistry

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Biological and non-biological antioxidant activity of some essential oils

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Renato Pérez-Rosés a, Ester Risco a,b, 1, Roser Vila a, Pedro Peñalver c

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and Salvador Cañigueral a,*

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a

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Unitat de Farmacologia, Farmacognòsia i Terapèutica, Facultat de Farmàcia, Universitat de Barcelona. Av. Joan XXIII, 27-31. E-08028 Barcelona, Spain.

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b

Phytonexus, S.L. Na Jordana, 11. E-46240 Carlet (València), Spain.

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c

Lidervet, S.L. Plaça García Lorca, 17, Baixos. E-43006 Tarragona, Spain.

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E-mail addresses of all contributing authors: Author

e-mail

Renato Pérez-Rosés

[email protected]

Ester Risco

[email protected]

Roser Vila

[email protected]

Pedro Peñalver

[email protected]

Salvador Cañigueral*

[email protected]

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* Corresponding author. Tel.: +34 934024531; Fax: +34 934035982. E-mail address:

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[email protected] (S. Cañigueral)

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1

Present address: Phytonexus, S.L.

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ACS Paragon Plus 1 Environment


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Abstract

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Fifteen essential oils, four essential oil fractions and three pure compounds (thymol,

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carvacrol and eugenol), characterized by gas chromatography (GC) and gas

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chromatography–mass spectrometry (GC-MS), were investigated for biological and

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non-biological antioxidant activity. Clove oil and eugenol showed strong DPPH (2,2-

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diphenyl-1-picrylhydrazyl) free-radical scavenging activity (IC50 = 13.2 µg/mL and

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11.7 µg/mL, respectively) and powerfully inhibited reactive oxygen species (ROS)

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production in neutrophils stimulated by PMA (phorbol 12-myristate 13-acetate) (IC50 =

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7.5 µg/mL and 1.6 µg/mL) or H2O2 (IC50 = 22.6 µg/mL and 27.1 µg/mL). Nutmeg,

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ginger and palmarosa oils were also highly active on this test.

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Essential oils from clove and ginger, as well as eugenol, carvacrol and bornyl acetate

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inhibited NO (nitric oxide) production (IC50 < 50.0 µg/mL). The oils of clove, red

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thyme and Spanish oregano, together with eugenol, thymol and carvacrol showed the

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highest myeloperoxidase (MPO) inhibitory activity. Isomers carvacrol and thymol

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displayed a disparate behaviour in some tests. All in all, clove oil and eugenol offered

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the best antioxidant profile.

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Keywords: essential oils, clove oil, eugenol, antioxidant, DPPH, ROS, flow cytometry,

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NO, myeloperoxidase.

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Introduction

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Aromatic plants have been used since ancient times for their preservative and medicinal

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properties, as well as aromatising and flavouring agents for food. These properties are,

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at least in part, attributed to the essential oils, which are complex mixtures of volatile

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compounds, mainly terpenes, in addition to some other non-terpene substances, such as


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phenylpropanoids. Modern society looks at food not only for the basic nutrition it

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provides but also for the health benefits it brings about. The latter is coupled with a

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clear trend in consumer preference for natural food ingredients and additives, which are

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perceived to be healthy, with names that are familiar to the consumer.1 Essential oils fit

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perfectly into this trend, they are reservoirs of bioactive compounds and they are aligned

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with current consumer preference for natural products.2

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Essential oils have shown good antibacterial and antifungal properties useful against

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infectious diseases in humans and animals. In addition, essential oils are also active

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against the more serious foodborne pathogens such as Salmonella spp., E. Coli and

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Listeria monocytogenes.3,4 Essential oils also act along the animal digestive tract to

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improve appetite and digestion, and are able to modulate the intestinal microbiota. One

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advantage of essential oils is that they occur in nature as complex mixtures, hence

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microorganism resistance is less likely to become a problem than with single synthetic

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compounds.5

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Due to human health and safety concerns, the European Union (EU) banned the use of

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all antibiotics as animal growth promoters in EU member states since the beginning of

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2006. Proposed alternatives include essential oils.6 Nevertheless, its mechanism of

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action, which goes beyond its antibacterial activity, is only partially known. In

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particular, immunomodulatory and antioxidant activities may also be relevant.7

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Essential oils can act as immune enhancers and, consequently, support gut health of

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farm animals raised in an antibiotic-free production environment. For example,

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supplementing certain essential oils to piglets improved their immune status after

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weaning, as indicated by the increase in lymphocyte proliferation rate, phagocytosis

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rate, as well as IgG, IgA, IgM, C3 and C4 serum levels.8



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Antioxidant activity has been described in several essential oils and it has been recently

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reviewed.9,10 This property can contribute to food and feed preservation. In addition, the

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use of antioxidant essential oils as additives in feedstuffs for farm animals may be

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relevant for product quality: essential oils may improve the dietary value and lead to a

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better oxidative stability and longer shelf-life of fat, meat and eggs.5

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Moreover, the antioxidant activity of essential oils may play a role in the prevention of

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some diseases, such as brain dysfunction, heart disease and immune system decline.

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Increasing evidence has suggested that these diseases may result from cellular damage

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caused by free radicals.10 In addition, reactive oxygen species (ROS) and reactive

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nitrogen species (RNS) play important roles in inflammation as messenger molecules.

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Thus, essential oils can also act as anti-inflammatory agents.10,11

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The in vitro evaluation of the antioxidant activity of the essential oils can be done using

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two main types of assays:12 those mainly related to the chemical properties of the

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constituents, usually called non-biological, and those known as biological tests,

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performed on a biological substratum, such as cells or enzymes. One of the best known

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in the former group is the assay on the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH).

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In the group of biological tests, the production of reactive oxygen species (ROS) or

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reactive nitrogen species (RNS) at cellular level, or the activity of enzymes, such as

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glutathione reductase, glutathione peroxidase, Nitric oxide (NO) synthase or

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myeloperoxidase (MPO), are usually assessed. The information obtained from the two

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types of tests is different, but complementary.

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In the literature on in vitro antioxidant activity of essential oils, the number of published

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studies varies greatly depending on the assay concerned. For example, there are a great

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number of papers using the DPPH assay. They show that the essential oil constituents

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having the highest activity are phenols, either terpenic or phenylpropanoid.9


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On the contrary, published literature that examines essential oil effects on ROS

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production by flow cytometry assays in living cells is scarce. Only the essential oils of

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Melaleuca alternifolia and threes species of Cymbopogon have been studied. Results

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showed antioxidant activity in stimulated human leukocytes for the former.

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Nevertheless, also prooxidant activity can be observed, either when using unstimulated

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cells, in the case of M. alternifolia, or at high concentrations in the oils of Cymbopogon

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winterianus and C. citratus (1000 µg/mL or higher, human lymphocytes).13,14

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The number essential oils investigated for their activity on leukocyte NO production is

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limited, and none of the oils studied here has been previously studied. However, the

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activity for some of their constituents has been described, such is the case of eugenol,

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carvacrol, thymol, linalool, sabinene, limonene, and bornyl acetate.15-19

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Links between essential oils or their constituents and MPO activity have been somehow

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explored in several in vivo experiments, especially in inflammation models. MPO

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activity was reduced by carvacrol in a periodontitis model in rats20 and the essential oils

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of Eucalyptus globulus and Melaleuca alternifolia also decreased the MPO activity in a

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dermal inflammation model in mice.21 It is noteworthy, however, that the in vitro

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activity of essential oils on MPO has been scarcely investigated. For example, the oils

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of Cymbopogon citratus (DC.) Stapf and C. winterianus Jowitt significantly reduced the

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MPO activity in PMA stimulated human leukocytes.22 It has been also reported that

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eugenol caused a significant reduction in MPO release by human neutrophils, but at

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high concentrations, between 0.625 mM (102.6 µg/mL) and 2.5 mM (410.5 µg/mL).23

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Finally, it should be noted that papers dealing with in vitro testing of essential oils or

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their constituents in the isolated MPO enzyme were not found in literature.

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From the above overview, it is concluded that the knowledge on the biological

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antioxidant activity of essential oils is limited and, in some cases, practically inexistent.



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This knowledge could help to a better understanding of the immunomodulatory activity

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of this group of substances, which can be relevant for their applications in medicine, as

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well as feed additives.7 Thus, the objective of the present study was to examine the

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antioxidant effect of a group of essential oils, fractions and pure constituents by

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different in vitro tests, mainly biological.

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2. Materials and methods

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2.1. Chemicals and reagents and instruments

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Water was freshly taken daily from a Milli-Q system (Millipore, Bedford, MA).

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Dimethyl sulfoxide (DMSO), 2,2-diphenyl-1-picrylhydrazyl (DPPH), o-dianisidine

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dihydrochloride, quercetin, absolute ethanol, Hanks’ balanced salt solution (HBSS),

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modified Hanks’ balanced salt solution without Ca2+ and Mg2+ (modified HBSS), 2′,7′-

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dichlorofluorescin diacetate (DCFH-DA), hydrogen peroxide solution (H2O2) 30%,

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phorbol 12-myristate 13-acetate (PMA), ethylenediaminetetraacetic acid tetrasodium

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salt dihydrate (EDTA-Na4 · 2H2O), sodium azide (NaN3), propidium iodide, ammonium

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chloride (NH4Cl), potassium bicarbonate (KHCO3), paraformaldehyde, Griess reagent

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(modified), L-arginine, lipopolysaccharides (LPS) from Escherichia coli 0127:B8, NG-

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methyl-L-arginine acetate salt (L-NMMA), and sodium nitrite (NaNO2) were obtained

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from Sigma Chemical Company (St. Louis, MO, USA).

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A microtiter plate spectrophotometer Benchmark Plus (Bio-Rad Laboratories, USA), a

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flow-cytometer Cytomics FC 500 MPL system (Beckman coulter, Inc., Bea, CA, USA)

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and the flow-cytometry software Summit v4.2 were employed for the analysis.

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2.2. Essential oils and compounds studied

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Essential oils from nutmeg (Myristica fragrans Houtt.), niaouli (Melaleuca

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quinquenervia (Cav.) S. T. Blake), clove (Syzygium aromaticum (L.) Merr. & L. M.

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Perry), tarragon (Artemisia dracunculus L.), coriander (Coriandrum sativum L.), juniper

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(Juniperus communis L.), tea tree (Melaleuca alternifolia (Maiden & Betche) Cheel),

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ginger (Zingiber officinale Roscoe), rosemary (Rosmarinus officinalis L.), bay laurel

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(Laurus nobilis L.), palmarosa (Cymbopogon martini (Roxb.) Will. Watson), cajuput

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(Melaleuca cajuputi Powell), lemon (Citrus limon (L.) Burm. f.), red thyme (Thymus

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zygis L.) and Spanish oregano (Coridothymus capitatus (L.) Rchb. f. = Thymbra

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capitata (L.) Cav.) were tested. The terpenic fractions from nutmeg, clove and lemon

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were also included in our research. Finally, the pure compounds eugenol, carvacrol, and

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thymol, as well as a mixture of bornyl (76.8%) and isobornyl (21.7%) acetates, were

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also investigated. All samples except eugenol (Sigma-Aldrich) were supplied by

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Lidervet S.L. (Tarragona, Spain) and obtained from commercial sources (Lluch Essence

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S.L., Barcelona, and Ernesto Ventós S.A., Barcelona). All samples were kept in sealed

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airtight glass vials, protected from the light, at 4 ºC.

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2.3. Essential oil characterization

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The essential oils and fractions were characterized by its composition and some physical

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constants. The analyses of the composition of the oils were carried out by gas

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chromatography (GC–FID) and gas chromatography coupled to mass spectrometry

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(GC–MS) as previously described.7 The main constituents of the oils and fractions are

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shown in Table 1. Additional data on the characterisation of the samples can be found in

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Pérez-Rosés et al. (2015).7

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2.4. Sample preparation for activity testing

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Recent findings on the incompatibility of Tweens for solubilisation of essential oils for

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antioxidant activity testing were taken in account.24

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For the DPPH antioxidant assay, samples were dissolved in absolute ethanol to give

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solutions that ranged from 5.0 x 10-2 % to 9.8 x 10-5 % (v/v).

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For assays on the inhibitory activity of ROS and NO production, samples were initially

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dissolved (1% v/v) in modified HBSS supplemented with 10% (v/v) DMSO and 1.5%

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(v/v) E-484 (glyceryl polyethyleneglycol ricinoleate). Thirteen dilutions, ranging from

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1.50 x 10-2 to 7.80 x 10-7 % (v/v), were tested on the ROS assay, while nine dilutions,

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ranging from 5.00 x 10-3 to 7.80 x 10-7 % (v/v); were investigated on the NO test.

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Dilutions were prepared with modified HBSS.

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For MPO inhibition tests, samples were initially dissolved at 1% (v/v) with DMSO.

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Nine dilutions with HBSS, which ranged from 1.00 x 10-2 to 7.81 x 10-6 % (v/v), were

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examined.

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2.3. Radical scavenging activity

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The radical scavenging activity of samples were evaluated using 2,2-diphenyl-1-

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picrylhydrazyl (DPPH•)25 in flat bottom microtiter plate.

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Quercetin was used as positive control of antioxidant activity while absolute ethanol

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was used as negative control. After incubation for 30 min at room temperature in the

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dark, absorbance at 515 nm was measured. Scavenging activity on DPPH• free stable

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radical by the samples was calculated as percentage.

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2.4. Isolation of human leukocytes

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Leukocytes were isolated through a controlled haemolytic shock with an ammonium

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chloride solution from buffy coats obtained from blood of healthy donors at the Blood

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and Tissue Bank of Catalonia.26 The pellet was suspended in modified HBSS.

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2.5. In vitro ROS production assessment by flow cytometry

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ROS production was quantified by oxidation of 2’,7’-dichlorofluorescin-diacetate

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(DCFH-DA) by flow cytometry in stimulated human leukocytes. The method described

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by Perez-Garcia et al., 1996,27 was followed with minor modifications to adapt it to

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microtiter plates.

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A human leukocyte suspension was incubated in the darkness with DCFH-DA (10 µM)

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and sodium azide (1 mM) for 10 min at 37 ºC. After centrifugation for 5 min at 300 x g,

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supernatant was removed and the cell pellet resuspended in 4 mL of HBSS. In a

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microtiter plate, 200 µL of this suspension of human leukocytes (approximately 106

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cells) were added to all wells. Twenty µL of each treatment dilution or quercetin (1

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µg/mL, positive control) were added. After incubation for 5 min at 37 ºC, 20 µL of the

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stimulant of ROS production, either hydrogen peroxide (H2O2, 100 µM in modified

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HBSS) or phorbol 12-myristate 13-acetate (PMA, 10 µM in DMSO) were placed in all

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wells, except those designated as base line controls, where 20 µL of modified HBSS

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were added. Microtiter plates were again incubated for 5 min at 37 ºC. Finally, 50 µL of

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paraformaldehyde (1% w/v) were added to all wells. Just before flow cytometry

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analysis, 2 µL of propidium iodide (10 µg/mL) were added in all wells with the purpose

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of discriminating viable cells. Plates were analysed by flow cytometry. Cellular viability

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was well over 95% in all experiments. The gate for neutrophils was established based

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on the size and granularity. The acquisition process was stopped when 20,000 viable



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neutrophils (negative to propidium iodide) were acquired, and the fluorescence

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histogram of dichlorofluorescein (515-540 nm) was obtained. Inhibition of ROS

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production in treated cells was calculated.

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2.6. Nitric oxide assay

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In a 96-well U bottomed microtiter plate, all experimental wells received an aliquot of

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200 µL of a suspension of human leukocytes (approximately 106 cells). Then, 20 µL of

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the different treatment dilutions or modified HBSS (negative controls) were added.

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Microtiter plates were incubated for 10 min at 37 ºC. Then, 20 µL of LPS (3 mg/mL)

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and 20 µL of L-Arg (2 mg/mL) were added to all wells, the plate was incubated for 1 h

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at 37 ºC and centrifuged (12 min, 2700 rpm). Aliquots of 100 µL of supernatant from

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each well were transferred to a 96-well flat bottomed microtiter plate and mixed with

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100 µL of Griess reagent.28,29 After 15 min at room temperature, absorbance was

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measured at 540 nm. The amount of nitrite was calculated from a NaNO2 standard

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curve. Supernatant from leucocytes not exposed to LPS was used as negative control

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while NG-methyl-L-arginine acetate salt (L-NMMA), a well-known NOS inhibitor was

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used as positive control. Results were expressed as inhibition of NO production.

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2.7. MPO inhibition assay

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Ten millilitres of a human leukocyte suspension (approximately 2x106 cells/mL) were

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treated with 0.5 mL of lipopolysaccharides from Escherichia coli 0127:B8 (50 µg/mL)

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and incubated for 30 min at 37 ºC. The supernatant (containing the enzyme) was

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centrifuged 7 min at 2500 x g and kept for later use. MPO enzymatic activity was

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determined by measuring the rate of oxidation of o-dianisidine dihydrochloride by

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H2O2.30 In a microtiter plate, 100 µL of the cell free supernatant plus 10 µL of the


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different treatments were added, including quercetin (positive control) and HBSS

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(negative control). After 6 min incubation at 37 ºC, 100 µL of o-dianisidine

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dihydrochloride (1.25 mg/mL final concentration, supplemented with 0.004% H2O2)

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were added and incubated at room temperature for 5 min. Aliquots of 50 µL from each

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well were transferred to a new flat bottomed microtiter plate, and were completed with

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150 µL HBSS and 30 µL NaN3 (2% w/v). Absorbance was determined at 460 nm.

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Inhibition of MPO activity was calculated.

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2.8. MPO release from human leukocytes

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About 200 µL of human leukocytes suspended in HBSS (approximately 106 cells) were

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added to a 96-well U bottomed microtiter plate, 20 µl of the different treatment dilutions

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or quercetin (1 mg/mL, positive control) were added and the plate was incubated for 10

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min at 37 ºC. Then, 10 µl of LPS (50 µg/mL, for stimulation of MPO release from

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leukocytes) or modified HBSS (negative controls)

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incubated at 37 ºC for 30 min and centrifuged for 10 min at 2500 x g at 4 ºC. MPO

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enzymatic activity was determined in the supernatant by measuring the rate of oxidation

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of o-dianisidine dihydrochloride by H2O2, as described for the MPO inhibition assay.

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Results were expressed as inhibition of MPO activity.

were added and the plate was

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2.9. Data processing and statistics

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The results were expressed as the mean ± standard deviation (SD) of at least four

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independent experiments. One-way analysis of variance (ANOVA) followed by a

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Dunnett´s test was used. A paired t test was employed for detecting statistical

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differences between the two MPO assays for each treatment. In all cases, a difference

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was considered significant when p

Biological and Nonbiological Antioxidant Activity of Some Essential

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