Biological and Nonbiological Antioxidant Activity of Some Essential Oils

May 23, 2016 - ABSTRACT: Fifteen essential oils, four essential oil fractions, and three pure compounds (thymol, carvacrol, and eugenol), characterize...
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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 is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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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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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