research article antioxidant, antibacterial, and cytotoxic...

Research ArticleAntioxidant Antibacterial and Cytotoxic Activities of theEthanolic Origanum vulgare Extract and Its Major Constituents

John Coccimiglio1 Misagh Alipour2 Zi-Hua Jiang3

Christine Gottardo3 and Zacharias Suntres134

1Department of Biology Lakehead University 955 Oliver Road Thunder Bay ON Canada P7B 5E12Faculty of Medicine and Dentistry Department of Pediatrics University of Alberta Edmonton AB Canada3Department of Chemistry Lakehead University 955 Oliver Road Thunder Bay ON Canada P7B 5E14Medical Sciences Division Northern Ontario School of Medicine Lakehead University 955 Oliver RoadThunder Bay ON Canada P7B 5E1

Correspondence should be addressed to Zacharias Suntres zsuntresnosmca

Received 5 October 2015 Accepted 22 February 2016

Academic Editor Hesham A El Enshasy

Copyright copy 2016 John Coccimiglio et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Oregano is a perennial shrub that grows in the mountains of the Mediterranean and EuroIrano-Siberian regions This study wasconducted to identify the major constituents of the ethanolic Origanum vulgare extract and examine the cytotoxic antioxidantand antibacterial properties of the extract but more importantly the contribution of its specific major constituent(s) or theircombination to the overall extract biological activity Gas chromatographymass spectroscopy analysis showed that the extractcontained monoterpene hydrocarbons and phenolic compounds the major ones being carvacrol and thymol and to a lesserextent p-cymene 1-octacosanol creosol and phytol A549 epithelial cells challenged with the extract showed a concentration-dependent increase in cytotoxicity A combination of thymol and carvacrol at equimolar concentrations to those present in theextract was less cytotoxic The A549 cells pretreated with nonlethal extract concentrations protected against hydrogen-peroxide-induced cytotoxicity an antioxidant effect more effective than the combination of equimolar concentrations of thymolcarvacrolInclusion of p-cymene andor 1-octacosanol did not alter the synergistic antioxidant effects of the carvacrolthymol mixture Theextract also exhibited antimicrobial properties againstGram-positive andGram-negative bacterial strains including clinical isolatesIn conclusion the oregano extract has cytotoxic antioxidant and antibacterial activities mostly attributed to carvacrol and thymol

1 Introduction

Herbs and spices contain a wide array of phytochemicalswith strong biological and pharmacological properties [1]One of them oregano is a perennial shrub native to thedry rocky calcareous soils in the mountainous area of theMediterranean and EuroIrano-Siberian regions but it is alsocultivated for its uses as a herb and therapeutic propertiesStudies examining the antioxidant activities of different formsof oregano (fresh dry and ready-to-use herb blend pastes)showed that oregano retains its strong antioxidant capacityin both fresh and dry form [2] The leaves and dried herb oforegano as well as its essential oil are traditionally used for

respiratory disorders indigestion and rheumatoid arthritis[3ndash6]

The antibacterial and antioxidant properties of oreganohave been attributed mainly to carvacrol and thymol whichare themajor components of its essential oil [7] Antibacterialeffects have been reported for oregano against Clostridiumperfringens Pseudomonas aeruginosa and Staphylococcusaureus [8ndash10] Studies comparing the antioxidant propertiesof Mediterranean food spices and common food additiveshave shown that extracts from oregano were more effectivethan butylated hydroxyanisole (BHA) and butylated hydrox-ytoluene (BHT) in inhibiting lipid peroxidation [11] Theuse of synthetic antioxidants to prevent free radical damage

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 1404505 8 pageshttpdxdoiorg10115520161404505

2 Oxidative Medicine and Cellular Longevity

can involve questionable nutritional value and toxic sideeffects while natural antioxidants present in many plantsreduce oxidative damage and help in preventingmutagenesiscarcinogenesis and aging due to their radical scavengingactivities [12] The role of free radicals has been implicatedin several pathological conditions including cancer car-diovascular diseases neurodegenerative disorders and drugtoxicity [13ndash15]

This study was carried out to identify the main com-ponents of Origanum vulgare from Mt Parnon SouthernGreece assess the cytotoxic antioxidant and antimicrobialproperties of the oregano extract and delineate the contribu-tion of the extractrsquos major components towards such effects

2 Materials and Methods

21 Herb Material and Chemicals The wild-grown herb Ori-ganum vulgarewas collected and authenticated byDr Z Sun-tres from the southern slope of Mt Parnon (37∘191015840N 22∘391015840E)in Kynouria Peloponnese during the early summer of 2009and voucher specimens are stored in the Department ofChemistry LakeheadUniversity Dr C Gottardorsquos laboratoryMTT (3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide) and all other chemicals (ie thymol carvacrolp-cymene and 1-octacosanol) were purchased from Sigma-Aldrich (Oakville ON Canada) The chemicals used were ofanalytical reagent grade

22 Preparation of Extracts Ground leaves from Origanumvulgare (55 g) were extracted in 100mL of ethanol at roomtemperature for three times each for 24 h The combinedethanol solution (300mL) was concentrated using a rotaryevaporator in vacuo (at 28∘C) resulting in an organic oil(104 g 19 from the dried sample)

23 Gas ChromatographyMass Spectrometry (GC-MS) Twohundred (200)120583g of dried extract was dissolved in a 11mLethanolThe sample and standards (1120583L)were loaded into theVarian 300 GC-MS The samples were analyzed by a Varianmodel-450 gas chromatograph coupled with a Varian model300-MS quadruple GC-MS mass spectrometer This wasattachedwith a factor four capillary column (VF-5ms 30mmtimes 025mm ID DF = 025120583m) Heliumwas used as the carriergas with a flow rate of 10mLmin Samples were introducedthrough a split mode method This involved a one in tensplit by a Varian 450 autosampler with a high temperatureinjection port (280∘C) The oven temperature was initially50∘C for 1min This was increased to a final temperature of280∘C at a rate of 10∘CminThe final 280∘C temperature washeld for 6min Electron ionization conditions were used withionization energy of 70 eV The scan range was from 70 to600 amu Lastly the GC-MS interface temperature was set to270∘C

The components were identified based on the comparisonof their RI (retention indices) and mass spectra (NIST08Mass Spectrum Library) of the GC-MS system Syntheticcomponents (Sigma-Aldrich Co Oakville ON Canada)were used as references for retention time calculations Major

peaks with respective Mass Spectrum Library matches areshown in Table 1 All determinations were performed intriplicate

24 Cell Culture Human alveolar type II-like epithelialA549 cells (American Type Culture Collection ManassasVA USA) were maintained in Corning Costar 02 120583m ventcap cell culture flasks (Corning NY USA) with standardDulbeccorsquos modified Eaglersquos medium nutrient mixture F-12Ham supplementedwith 10 iron-fortified bovine calf serum(SAFC Biosciences Lenexa KS USA) 2mM l-glutamineand antibioticantimycotic (100UmL penicillin 100 120583gmLstreptomycin and 025 120583gmL amphotericin B) from Gibco(Carlsbad CA USA) Cultures were incubated at 37∘C in ahumidified atmosphere of 5 CO

2in air and subcultured

when they were 80 confluent Prior to plating cell countsand viabilities were assessed using a Vi-Cell XR Cell ViabilityAnalyzer (Beckman Coulter Mississauga ON Canada)

25 Cytotoxic and Antioxidant Properties of Oregano Extractsand Major Extract Components The A549 cells were seededinto sterile flat-bottom 96-well plates at 10000 cellswelland grown to 80 confluence overnight before treatmentsbegan To determine the cytotoxicity of ethanolic extractsand their individual components cells were treated with thedifferent treatments (thymol carvacrol p-cymene andor1-octacosanol dissolved in 1 ethanol [the vehicle solutionhad no effect on the antioxidant and cytotoxic activities ofthe extract andor its constituents]) in media To determinethe antioxidant effects of the oregano extracts and theirindividual components (thymol carvacrol p-cymene andor1-octacosanol dissolved in 1 ethanol) A549 cells were firstpretreated for 24 h with the different treatments in mediafollowed by treatment with 500120583M hydrogen peroxide TheMTT assay a commonly used measure of cell viability wasused to assess cell survival as described by the manufacturer(Sigma-Aldrich Co Oakville ON Canada) Viabilities ofchallenged cells were assessed relatively to control cells

26 Bacterial Strains P aeruginosa (ATCC 25619 and clin-ical isolates) Bordetella bronchiseptica (ATCC 4617 ATCC10580) Escherichia coli (ATCC 25922 ATCC 700973) Bur-kholderia cenocepacia (ATCC 25608 and clinical isolates)Acinetobacter lwoffii (ATCC 17925)Acinetobacter baumannii(ATCC 19606) Moraxella catarrhalis (ATCC 8176) Bacillussubtilis (ATCC 6633) and S aureus (ATCC 29213 and clinicalisolates) were purchased from Cedarlane (Burlington ONCanada) or obtained from the Clinical Microbiology Lab-oratory of Memorial Hospital (Sudbury ON Canada) Forexperimentation the strains were inoculated onto MuellerHinton II agar plates and incubated for 24 h at 37∘C

27 The Minimum Inhibitory Concentrations (MIC) of Ore-gano Extract against Gram-Positive and Gram-NegativeStrains The inhibitory concentrations were determined bythe agar dilution method as previously described [16] Bac-terial inocula were prepared from an overnight culture inMuellerHinton II agar and adjusted to contain an equivalence

Oxidative Medicine and Cellular Longevity 3

Table 1 Composition of Origanum vulgare assessed by GC-MS analysis

Peak Retention time (min) area Compound

1 8959 6900 1-Methyl-4-(1-methylethyl) benzene-(p-cumene)

2 9664 1904 1-Methyl-4-(1-methylethyl)-14-cyclohexadiene(120574-terpinene)

3 13113 2110 1-Methoxy-4-methyl-2-(1-methylethyl) benzene(creosol)

4 14072 25008 2-(1-Methylethyl)-5-methylphenol(thymol)

5 14263 59468 2-Methyl-5-(1-methylethyl)-phenol(carvacrol)

6 19327 0560 371115-Tetramethyl-2-hexadecen-1-ol(phytol)

7 25600 4050 1-Octacosanol

of a 05 McFarland standard with phosphate-buffered salineOne (1)120583L of the adjusted inocula was then delivered ontoMueller Hinton II agar plates containing twofold serialdilutions of oregano from 25000 120583gmL to 3100 120583gmL usingthe Replianalyzer system (Oxoid Inc Nepean ON Canada)The lowest concentration of oregano that prevented theappearance of a visible growth within the inoculation areaafter 24 h at 36∘C was defined as the MIC

28 Carvacrol and Thymol Cellular Uptake The cellularuptake of carvacrol and thymol was examined followingexposure of A549 cells (3 times 106 cells) to 56 120583M carvacrolor 233 120583M thymol (ie equimolar concentrations to thosefound in the alcoholic extract) or a combination of the twocomponents Treated cells were removed at 1 4 8 12 and24 h washed twice with PBS and lysed with NP-40 lysisbuffer Cytosolic and membrane fractions were separated viacentrifugation at 10000 rpm for 12 minutes Membrane andcytosolic fractions were dissolved in diethyl ether evacuatedwith nitrogen gas capped and crimped The samples wereanalyzed by gas chromatographymass spectrometry (GC-MS) as previously described

29 Statistical Analysis All data were expressed as meansplusmn SD of triplicate measurements Data were evaluated byone way analysis of variance (ANOVA) If the 119865 values weresignificant Studentrsquos 119905-test was used to compare all groupsThe level of significance was accepted at 119901 lt 005

3 Results

31 GC-MS Analysis In the present study the GC-MSanalysis of the ethanol extract of wild-growing herb Origa-num vulgare obtained from Southern Greece revealed anabundance ofmonoterpene hydrocarbons and phenolic com-pounds with the main constituents being carvacrol (2-meth-yl-5-(1-methylethyl)phenol) (5946) followed by thymol (5-methyl-2-(1-methylethyl)phenol) (2500) p-cymene (690)and 1-octacosanol (405) (Table 1)

LC50 = 14 120583gmL

Oregano extract (120583gmL)

Unt

reat

ed

100

200

390

780

156

313

625

125

250

0

25

50

75

100

125

MTT

cell

surv

ival

()

Figure 1The cytotoxic properties of the ethanolicO vulgare extractin A549 cells All experiments were repeated three times in tworeplicates as outlined in Section 2

32 Chemotherapeutic Properties of the Oregano ComponentsIn order to examine the chemotherapeutic properties ofthe oregano extracts A549 human lung adenocarcinomaepithelial cells were treated with increasing concentrationsof oregano ethanolic extracts (0ndash250120583gmL final concen-tration) and cell viability was assessed 24 h after treatmentTreatment of A549 cells with oregano extract resulted ina concentration-dependent decrease in cell viability with acalculated LC

50= 14 120583gmL (Figure 1)

To assess the contribution of the major componentsisolated from the oregano extract on cytotoxicity the cellviability of A549 cells challenged with thymol carvacrolp-cymene 1-octacosanol or a mixture containing all fourmajor constituents was examined As shown in Figure 2(a)challenge of A549 cells with increasing concentrations ofthymol carvacrol p-cymene or 1-octacosanol alone resultedin a concentration-dependent decrease in cell viability with


ThymolCarvacrolp-Cymene1-Octacosanol

0

25

50

75

100

Viab

ility

()

10050 150 200 2500

Concentration (120583M)

(a)

A

BC BC BC

BCD

0

25

50

75

100

Viab

ility

()

Con

trol

20120583

Mth

ymol

(T)

56120583

M ca

rvac

rol (

C)

7nM

p-c

ymen

e (p-

C)

14

nM1

-oct

acos

anol

(O)

T +

C

T +

C +

p-C

T +

C +

p-C

+ O

Extr

act

(b)

Figure 2The cytotoxic effects of carvacrol thymol p-cymene and 1-octacosanol in A549 cells All experiments were repeated three times intwo replicates as outlined in Section 2 The thymol and carvacrol concentrations are equimolar to those present in the ethanolic extract (A)Significantly different from control 119901 lt 005 (B) significantly different from thymol-treated group 119901 lt 005 (C) significantly different fromcarvacrol-treated group 119901 lt 005 (D) significantly different from carvacrol + thymol + p-cymene + 1-octacosanol-treated group 119901 lt 005

CarvacrolCarvacrol + thymol

0

5

10

15

20

Carv

acro

l upt

ake (

)

1 4 80 2412

Time (h)

(a)

0

5

10

15

20

25

Thym

ol u

ptak

e (

)

1 4 80 2412

Time (h)

ThymolThymol + carvacrol

lowast

lowast

lowast

lowast

(b)

Figure 3 Uptake of carvacrol and thymol in A549 cells All experiments were repeated three times in two replicates as outlined in Section 2lowast denotes significant difference from thymol-treated group 119901 lt 005

thymol being the most cytotoxic Challenge of A549 cellswith a combination of thymol carvacrol p-cymene and1-octacosanol at equimolar concentrations to the extract(56120583M carvacrol 233 120583M thymol 719 nM p-cymene and138 nM 1-octacosanol) was less cytotoxic than the extractitself ( viability of mixture versus oregano extract 6265 plusmn197 versus 4959 plusmn 226) It should be noted that the contri-bution of p-cymene and 1-octacosanol to the cytotoxicity ofthe mixture was negligible (Figure 2(b))

33 Carvacrol and Thymol Cellular Uptake To investigatethe synergistic effect of carvacrol and thymol cytotoxicitythe cellular uptake of carvacrol and thymol was examined(note 56120583M carvacrol resulted in cell viability of 9420 plusmn256 while 233 120583M thymol resulted in 8260 plusmn 212and their combination resulted in 6265 plusmn 197 viability)Incubation of cells with carvacrol or thymol alone resultedin a time-dependent increase in their uptake (Figures 3(a)and 3(b)) Coincubation of cells with a mixture of carvacrol


0 018 037 073 146 29324h oreganoPretreatment (120583gmL)

24h H2O2

Posttreatment (500120583M)

+++++

++++++

minus

lowast

lowast

lowast

0

25

50

75

100

125

Viab

ility

()

Figure 4 The antioxidant effect of the ethanolic O vulgare extractagainst H

2

O2

-induced cytotoxicity in A549 cells All experimentswere repeated three times in two replicates as outlined in Section 2lowastdenotes significance from the control group treated with H

2

O2

only119901 lt 005

and thymol did not have any effect on the uptake of carvacrol(Figure 3(a)) a small but significant increase in the uptake ofthymol was observed (Figure 3(b))

34 Antioxidant Protective Effects of the Oregano ExtractTo examine the antioxidant effects of the oregano extractnoncytotoxic concentrations of extract that correspondedto greater than 95 cell viability were used As shown inFigure 4 pretreatment of A549 cells with the oregano extractat concentrations ranging from 0 to 293120583gmL resulted ina concentration-dependent protective effect against H

2O2

Preincubation of A549 cells with nontoxic concentrations ofcarvacrol or thymol showed that both phenolic isomers pro-tected against hydrogen-peroxide induced cytotoxicity withcarvacrol producing a better protective effect than thymol(Figure 5(a)) Preincubation of cells with a combination ofboth isomers at equimolar concentrations (12 120583M carvacroland 5 120583M thymol) to those present in the oregano extract (atthe 293 120583gmL concentration) resulted in a less protectiveeffect than that produced by the extract ( viability ofcarvacrol + thymol mixture versus oregano extract 3714 plusmn077 versus 6960 plusmn 500) (Figure 5) Inclusion of p-cymeneandor 1-octacosanol to the carvacrol and thymol mixturedid not contribute to the antioxidant effects of the mixture(data not shown) however the inclusion of the other oreganoextracts to the carvacrolthymol mixture was not examined

35 Antimicrobial Properties of the Oregano Extract Theantibacterial properties of the ethanolic oregano extract weremeasured using an agar dilutionmethodThe oregano extractinhibited the growth of reference ATCC Gram-negative andGram-positive bacterial strains with varying degree but notrends were realised (Table 2) The inhibitory concentrationswere 2- to 4-fold lower against nonmucoid than mucoidclinical isolates of P aeruginosa and exhibited greater effectsagainst clinical isolates of B cenocepacia compared to theATCC reference strain (Table 2)

4 Discussion

Results from the GC-MS analysis of the ethanol extractof wild-growing herb Origanum vulgare obtained fromSouthern Greece revealed an abundance of monoterpenehydrocarbons and phenolic compounds with the main con-stituents being carvacrol and thymol (Table 1) This findingis consistent with that reported by other investigators whofound that in spite of the high variability of individualcompounds in the essential oil ofO vulgare from 23 localitiesscattered all over Greece the sum of carvacrol thymol p-cymene and 120574-terpinene was consistent amounting to gt80[10 17 18] Plants collected from the northern region ofGreece were rich in thymol (303ndash428 of total oil) whereasthose from the southern part of the country were rich incarvacrol (574ndash696 of total oil) [17] p-Cymene (1-methyl-4-(1-methylethyl)-benzene) and 120574-terpinene (1-methyl-4-(1-methylethyl)-14-cyclohexadiene) are the precursors of car-vacrol and thymol in species of Origanum [19]

Studies examining the effect of oregano extract on cancerprevention and cytotoxicity are limited [20] In the presentstudy challenge of A549 human lung adenocarcinomaepithelial cells with oregano ethanolic extracts (0ndash250 120583gmLfinal concentration) resulted in a concentration-dependentdecrease in cell viability with a calculated LC

50= 14 120583gmL

(Figure 1) It is not knownwhether the cytotoxicity of oreganoextract is attributed to a specific component or combinationof components Results shown in Figure 2 demonstrated thatchallenge of A549 cells with thymol carvacrol p-cymeneor 1-octacosanol alone resulted in a concentration-dependentdecrease in cell viability with thymol being more cytotoxicthan the other three compounds Challenge of A549 cellswith a combination of the four compounds at equimolarconcentrations (56120583M carvacrol 233 120583M thymol) to thosepresent in the oregano extract was less cytotoxic than theextract itselfThe cytotoxicity of the oregano extract is mostlyattributed to presence of carvacrol and thymol p-cymene and1-octacosanol although cytotoxic at high concentrations didnot contribute to the cytotoxicity of the mixture most likelyattributed to their low potency and very low availability inthe mixture (nM range) It appears that other unmeasuredethanolic constituents at lower concentrations than thosedetermined in this study might possess higher potencies andplay a role in the overall effects of the oregano extracts

The mechanisms by which thymol and carvacrol causecell death in mammalian cell lines have not been thoroughlyinvestigated Results from an in vitro study showed thatcarvacrol is very potent inhibitor of cell growth in A549 cellline as evidenced by the concentration-dependent decreasesin cell number degeneration of cell morphology and adecrease in total protein amount [21] Thymol induces celldeath in humanosteosarcoma and astrocytes andmay involveapoptosis via mitochondrial pathways [22 23] Whetherthe synergistic effects of carvacrol and thymol regardingcell viability are related to a combination of the purportedmechanisms is under investigation It is evident from theresults of this study that coincubation of carvacrol andthymol increased the uptake of the more cytotoxic thymoland enhanced the cytotoxicity of the mixture Carvacrol is


Viab

ility

()

ThymolCarvacrol

Thymol or carvacrol conc (120583M)

AB

AB

0

20

40

60

0 1 5 10 50 100

(a)

Viab

ility

()

lowast

lowastlowast

0

20

40

60

80

+++++minus+minus+minus

minus++minusminus

+minusminusminusminus

500120583M H2O2

5120583M thymol

10120583M carvacrol

Extract(b)

Figure 5 The antioxidant effect of (a) carvacrol and thymol and (b) thymol andor carvacrol Equimolar concentration to carvacrol andthymol is present in the extract at the 293 120583gmL concentration as well as in ethanolic extract against H

2

O2

-induced cytotoxicity in A549cells All experiments were repeated three times in two replicates as outlined in Section 2 (A) denotes significance from the control grouptreated with H

2

O2

only 119901 lt 005 (B) denotes significance from the thymol pretreated group challenged with H2

O2

119901 lt 005 lowast denotessignificance from the control group treated with H

2

O2

only 119901 lt 005 denotes significance from the control group pretreated with thymoland carvacrol mixture and challenged with H

2

O2

119901 lt 005

slightlymore lipophilic than thymol with partition coefficientin octanolwater (119875ow) of 364 and 330 respectively [24ndash26] suggesting that carvacrol is partitioned deeper in thecytoplasmic membrane thereby causing an expansion of themembrane [24] altering its permeability [27]

Oxidative stress describes the outcome of an increasedreactive oxygen species (ROS) production andor a decreasein their elimination ROS (eg superoxide anion hydro-gen peroxide hydroxyl radical and lipid peroxides) canbe formed as normal products of aerobic metabolism butcan be produced at elevated rates under pathophysiologicalconditions ROS attack biological macromolecules such asmembrane lipids nucleic acids carbohydrates and proteinsresulting in damage [13ndash15 28] Pretreatment of A549 cellswith the oregano extract at nontoxic concentrations rangingfrom 0 to 293 120583gmL resulted in a concentration-dependentprotective effect against H

2O2 H2O2has been used as a

model of oxidative stress because it can be generated in vivoby the spontaneous andor enzymatic dismutation of thesuperoxide anion radical The antioxidant effectiveness oforegano extracts in vitro is perhaps due to their ability to act asreducing agents and free radical scavengers as quenchers ofsinglet O

2formation and to complex with prooxidant metal

ions [29 30] Phenols (thymol carvacrol) monocyclic hydro-carbons (terpinolene R-terpinene and 120574-terpinene) belongto the most active natural antioxidants found in the essentialoils [29 30] Indeed in our study nontoxic concentrations ofboth phenolic isomers protected against hydrogen-peroxideinduced cytotoxicity with carvacrol producing a better pro-tective effect than thymol However a combination of bothisomers at equimolar concentrations (12 120583M carvacrol and

5 120583M thymol) to those present in the oregano extract (at the293 120583gmL concentration) resulted in a less protective effectthan that produced by the extract (Figure 4) Although inclu-sion of p-cymene andor 1-octacosanol to the carvacrol andthymol mixture did not contribute to the antioxidant effectsof the mixture other components present in the extract atlower levels might possess higher antioxidant potencies andplay a role in the overall effects of the oregano extractsFor example 120573-caryophyllene a constituent in oregano atvery low levels has a higher inhibitory capacity on lipidperoxidation than probucol 120572-humulene and 120572-tocopherol[31 32]

Gram-negative pathogens like P aeruginosa and B ceno-cepacia are commonly found opportunistic bacilli in ourenvironment that establish infections in patients sufferingfrom pulmonary diseases like cystic fibrosis [33 34] Theresults of this study showed that the ethanolic extracts oforegano exhibited antibacterial properties as indicated bytheir ability to inhibit nonmucoid andmucoid clinical isolatesof P aeruginosa and clinical isolates of B cenocepacia It isimportant to note that the extracts were less effective againstthe mucoid clinical isolates of P aeruginosa possibly dueto the secretion of negatively charged alginate-rich matrixto the surroundings which inhibit antibiotic penetrationEarlier work by Lambert et al [8] credits the activity oforegano essential oil and its active constituents (carvacrol andthymol) to interference with the pH gradient and membranepermeability Burt et al [35] have previously described thatcarvacrol may also be involved in inhibiting E coli flagellinA study examining the separate and combined antibacterialactivities of the main chemical constituents of oregano and


Table 2 Minimum Inhibitory Concentrations (MIC) of bacterial strains

Bacterial strains Source Ethanolic oregano extract (120583gmL)Pseudomonas aeruginosa

25619 ATCC 25Mucoid clinical isolate 1 Cystic fibrosis isolate 25Mucoid clinical isolate 2 Cystic fibrosis isolate 25Mucoid clinical isolate 3 Cystic fibrosis isolate 25Mucoid clinical isolate 4 Cystic fibrosis isolate 25Mucoid clinical isolate 5 Cystic fibrosis isolate 125Nonmucoid clinical isolate 1 Cystic fibrosis isolate 63Nonmucoid clinical isolate 2 Cystic fibrosis isolate 63Nonmucoid clinical isolate 3 Cystic fibrosis isolate 25Nonmucoid clinical isolate 4 Cystic fibrosis isolate 125

Bordetella bronchiseptica10580 ATCC 1254617 ATCC 125

Escherichia coli25922 ATCC 25700973 ATCC 125

Burkholderia cenocepacia25608 ATCC 25Clinical isolate 1 Cystic fibrosis isolate 125Clinical isolate 2 Cystic fibrosis isolate 63Clinical isolate 3 Cystic fibrosis isolate 125Clinical isolate 4 Cystic fibrosis isolate 125

Acinetobacter lwoffii 17925 ATCC 125Acinetobacter baumannii 19606 ATCC 125Moraxella catarrhalis 8176 ATCC 125Bacillus subtilis 6633 ATCC 63Staphylococcus aureus

29213 ATCC 25Clinical isolate 1 Cystic fibrosis isolate 125Clinical isolate 2 Cystic fibrosis isolate 25

other spices (namely eugenol cinnamaldehyde thymol andcarvacrol) showed that each component possessed antibacte-rial properties and the components acted synergistically witheach other [8 36] The mechanism by which the oreganoextract and individual components produce the antimicrobialeffect on mucoid and nonmucoid bacteria is currently underinvestigation

In conclusion the results of this study showed thatthe ethanolic extract of Origanum vulgare possesses strongcytotoxic antioxidant and antibacterial activities which areattributed mostly to the presence of the isomeric phenolicconstituents carvacrol and thymol

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

Acknowledgments

This work was supported by research funds from the North-ern Ontario School of Medicine

References

[1] P Ninfali G Mea S Giorgini M Rocchi and M BacchioccaldquoAntioxidant capacity of vegetables spices and dressings rele-vant to nutritionrdquo British Journal of Nutrition vol 93 no 2 pp257ndash266 2005

[2] S M Henning Y Zhang N P Seeram et al ldquoAntioxidantcapacity and phytochemical content of herbs and spices in dryfresh and blended herb paste formrdquo International Journal ofFood Sciences and Nutrition vol 62 no 3 pp 219ndash225 2011

[3] D Ivanova D Gerova T Chervenkov and T YankovaldquoPolyphenols and antioxidant capacity of Bulgarian medicinal


plantsrdquo Journal of Ethnopharmacology vol 96 no 1-2 pp 145ndash150 2005

[4] Y T Lin Y I Kwon R G Labbe and K Shetty ldquoInhibitionof Helicobacter pylori and associated urease by oregano andcranberry phytochemical synergiesrdquo Applied and Environmen-tal Microbiology vol 71 no 12 pp 8558ndash8564 2005

[5] G BMahady S L PendlandA Stoia et al ldquoIn vitro susceptibil-ity ofHelicobacter pylori to botanical extracts used traditionallyfor the treatment of gastrointestinal disordersrdquo PhytotherapyResearch vol 19 no 11 pp 988ndash991 2005

[6] C C Rosenbaum D P OrsquoMathuna M Chavez and K ShieldsldquoAntioxidants and antiinflammatory dietary supplements forosteoarthritis and rheumatoid arthritisrdquo Alternative Therapiesin Health and Medicine vol 16 no 2 pp 32ndash40 2010

[7] K H C Baser ldquoBiological and pharmacological activities ofcarvacrol and carvacrol bearing essential oilsrdquoCurrent Pharma-ceutical Design vol 14 no 29 pp 3106ndash3119 2008

[8] R J W Lambert P N Skandamis P J Coote and G-J ENychas ldquoA study of the minimum inhibitory concentration andmode of action of oregano essential oil thymol and carvacrolrdquoJournal of AppliedMicrobiology vol 91 no 3 pp 453ndash462 2001

[9] V K Juneja and M Friedman ldquoCarvacrol cinnamaldehydeoregano oil and thymol inhibit Clostridium perfringens sporegermination and outgrowth in ground Turkey during chillingrdquoJournal of Food Protection vol 70 no 1 pp 218ndash222 2007

[10] A Alexopoulos A C Kimbaris S Plessas et al ldquoAntibacterialactivities of essential oils from eight Greek aromatic plantsagainst clinical isolates of Staphylococcus aureusrdquoAnaerobe vol17 no 6 pp 399ndash402 2011

[11] M Martınez-Tome A M Jimenez S Ruggieri N FregaR Strabbioli and M A Murcia ldquoAntioxidant properties ofMediterranean spices compared with common food additivesrdquoJournal of Food Protection vol 64 no 9 pp 1412ndash1419 2001

[12] A T H Mossa and G A M Nawwar ldquoFree radical scavengingand antiacetylcholinesterase activities ofOriganummajorana Lessential oilrdquo Human and Experimental Toxicology vol 30 no10 pp 1501ndash1513 2011

[13] K Jomova andM Valko ldquoAdvances in metal-induced oxidativestress and human diseaserdquo Toxicology vol 283 no 2-3 pp 65ndash87 2011

[14] S R Steinhubl ldquoWhy have antioxidants failed in clinical trialsrdquoAmerican Journal of Cardiology vol 101 no 10 pp S14ndashS192008

[15] Z E Suntres ldquoRole of antioxidants in paraquat toxicityrdquoToxicology vol 180 no 1 pp 65ndash77 2002

[16] M Alipour M Halwani A Omri and Z E Suntres ldquoAntimi-crobial effectiveness of liposomal polymyxin B against resistantGram-negative bacterial strainsrdquo International Journal of Phar-maceutics vol 355 no 1-2 pp 293ndash298 2008

[17] S Kokkini R Karousou A Dardioti N Krigas and T LanarasldquoAutumn essential oils of Greek oreganordquo Phytochemistry vol44 no 5 pp 883ndash886 1997

[18] D Vokou S Kokkini and J-M Bessiere ldquoGeographic variationof Greek oregano (Origanum vulgare ssp hirtum) essential oilsrdquoBiochemical Systematics and Ecology vol 21 no 2 pp 287ndash2951993

[19] S Burt ldquoEssential oils their antibacterial properties and poten-tial applications in foodsmdasha reviewrdquo International Journal ofFood Microbiology vol 94 no 3 pp 223ndash253 2004

[20] I Savini R Arnone M V Catani and L Avigliano ldquoOriganumvulgare Iinduces apoptosis in human colon cancer caco

2

cellsrdquoNutrition and Cancer vol 61 no 3 pp 381ndash389 2009

[21] A T Koparal and M Zeytinoglu ldquoEffects of carvacrol on ahuman non-small cell lung cancer (NSCLC) cell line A549rdquoCytotechnology vol 43 no 1ndash3 pp 149ndash154 2003

[22] H-T Chang S-S Hsu C-T Chou et al ldquoEffect of thymol onCa2+ homeostasis and viability in MG63 human osteosarcomacellsrdquo Pharmacology vol 88 no 3-4 pp 201ndash212 2011

[23] S-S Hsu K-L Lin C-T Chou et al ldquoEffect of thymol onCa2+ homeostasis and viability in human glioblastoma cellsrdquoEuropean Journal of Pharmacology vol 670 no 1 pp 85ndash912011

[24] A Ultee M H J Bennik and R Moezelaar ldquoThe phenolichydroxyl group of carvacrol is essential for action against thefood-borne pathogen Bacillus cereusrdquoApplied and Environmen-tal Microbiology vol 68 no 4 pp 1561ndash1568 2002

[25] S Griffin S G Wyllie and J Markham ldquoDetermination ofoctanol-water partition coefficient for terpenoids using rever-sed-phase high-performance liquid chromatographyrdquo Journalof Chromatography A vol 864 no 2 pp 221ndash228 1999

[26] F J Weber and J A M de Bont ldquoAdaptation mechanismsof microorganisms to the toxic effects of organic solvents onmembranesrdquo Biochimica et Biophysica Acta (BBA)mdashReviews onBiomembranes vol 1286 no 3 pp 225ndash245 1996

[27] J Sikkema J A M de Bont and B Poolman ldquoInteractionsof cyclic hydrocarbons with biological membranesrdquo Journal ofBiological Chemistry vol 269 no 11 pp 8022ndash8028 1994

[28] Z E Suntres ldquoLiposomal antioxidants for protection againstoxidant-induced damagerdquo Journal of Toxicology vol 2011 Arti-cle ID 152474 16 pages 2011

[29] N V Yanishlieva E M Marinova M H Gordon and VG Raneva ldquoAntioxidant activity and mechanism of action ofthymol and carvacrol in two lipid systemsrdquo Food Chemistry vol64 no 1 pp 59ndash66 1999

[30] G Ruberto and M T Baratta ldquoAntioxidant activity of selectedessential oil components in two lipid model systemsrdquo FoodChemistry vol 69 no 2 pp 167ndash174 2000

[31] L De Martino V De Feo C Formisano E Mignola and FSenatore ldquoChemical composition and antimicrobial activity ofthe essential oils from three chemotypes of origanum vulgarel ssp hirtum (Link) ietswaart growing wild in campania(Southern Italy)rdquoMolecules vol 14 no 8 pp 2735ndash2746 2009

[32] M A Calleja J M Vieites T Montero-Melendez et al ldquoTheantioxidant effect of 120573-caryophyllene protects rat liver fromcarbon tetrachloride-induced fibrosis by inhibiting hepaticstellate cell activationrdquo British Journal of Nutrition vol 109 no3 pp 394ndash401 2013

[33] P Drevinek and E Mahenthiralingam ldquoBurkholderia ceno-cepacia in cystic fibrosis epidemiology and molecular mecha-nisms of virulencerdquo Clinical Microbiology and Infection vol 16no 7 pp 821ndash830 2010

[34] NHoslashiby O Ciofu andT Bjarnsholt ldquoPseudomonas aeruginosabiofilms in cystic fibrosisrdquo FutureMicrobiology vol 5 no 11 pp1663ndash1674 2010

[35] S A Burt R van der Zee A P Koets et al ldquoCarvacrol inducesheat shock protein 60 and inhibits synthesis of flagellin inEscherichia coli O157H7rdquo Applied and Environmental Microbi-ology vol 73 no 14 pp 4484ndash4490 2007

[36] R-S Pei F Zhou B-P Ji and J Xu ldquoEvaluation of combinedantibacterial effects of eugenol cinnamaldehyde thymol andcarvacrol against E coli with an improved methodrdquo Journal ofFood Science vol 74 no 7 pp M379ndashM383 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


can involve questionable nutritional value and toxic sideeffects while natural antioxidants present in many plantsreduce oxidative damage and help in preventingmutagenesiscarcinogenesis and aging due to their radical scavengingactivities [12] The role of free radicals has been implicatedin several pathological conditions including cancer car-diovascular diseases neurodegenerative disorders and drugtoxicity [13ndash15]

This study was carried out to identify the main com-ponents of Origanum vulgare from Mt Parnon SouthernGreece assess the cytotoxic antioxidant and antimicrobialproperties of the oregano extract and delineate the contribu-tion of the extractrsquos major components towards such effects

2 Materials and Methods

21 Herb Material and Chemicals The wild-grown herb Ori-ganum vulgarewas collected and authenticated byDr Z Sun-tres from the southern slope of Mt Parnon (37∘191015840N 22∘391015840E)in Kynouria Peloponnese during the early summer of 2009and voucher specimens are stored in the Department ofChemistry LakeheadUniversity Dr C Gottardorsquos laboratoryMTT (3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide) and all other chemicals (ie thymol carvacrolp-cymene and 1-octacosanol) were purchased from Sigma-Aldrich (Oakville ON Canada) The chemicals used were ofanalytical reagent grade

22 Preparation of Extracts Ground leaves from Origanumvulgare (55 g) were extracted in 100mL of ethanol at roomtemperature for three times each for 24 h The combinedethanol solution (300mL) was concentrated using a rotaryevaporator in vacuo (at 28∘C) resulting in an organic oil(104 g 19 from the dried sample)

23 Gas ChromatographyMass Spectrometry (GC-MS) Twohundred (200)120583g of dried extract was dissolved in a 11mLethanolThe sample and standards (1120583L)were loaded into theVarian 300 GC-MS The samples were analyzed by a Varianmodel-450 gas chromatograph coupled with a Varian model300-MS quadruple GC-MS mass spectrometer This wasattachedwith a factor four capillary column (VF-5ms 30mmtimes 025mm ID DF = 025120583m) Heliumwas used as the carriergas with a flow rate of 10mLmin Samples were introducedthrough a split mode method This involved a one in tensplit by a Varian 450 autosampler with a high temperatureinjection port (280∘C) The oven temperature was initially50∘C for 1min This was increased to a final temperature of280∘C at a rate of 10∘CminThe final 280∘C temperature washeld for 6min Electron ionization conditions were used withionization energy of 70 eV The scan range was from 70 to600 amu Lastly the GC-MS interface temperature was set to270∘C

The components were identified based on the comparisonof their RI (retention indices) and mass spectra (NIST08Mass Spectrum Library) of the GC-MS system Syntheticcomponents (Sigma-Aldrich Co Oakville ON Canada)were used as references for retention time calculations Major

peaks with respective Mass Spectrum Library matches areshown in Table 1 All determinations were performed intriplicate

24 Cell Culture Human alveolar type II-like epithelialA549 cells (American Type Culture Collection ManassasVA USA) were maintained in Corning Costar 02 120583m ventcap cell culture flasks (Corning NY USA) with standardDulbeccorsquos modified Eaglersquos medium nutrient mixture F-12Ham supplementedwith 10 iron-fortified bovine calf serum(SAFC Biosciences Lenexa KS USA) 2mM l-glutamineand antibioticantimycotic (100UmL penicillin 100 120583gmLstreptomycin and 025 120583gmL amphotericin B) from Gibco(Carlsbad CA USA) Cultures were incubated at 37∘C in ahumidified atmosphere of 5 CO

2in air and subcultured

when they were 80 confluent Prior to plating cell countsand viabilities were assessed using a Vi-Cell XR Cell ViabilityAnalyzer (Beckman Coulter Mississauga ON Canada)

25 Cytotoxic and Antioxidant Properties of Oregano Extractsand Major Extract Components The A549 cells were seededinto sterile flat-bottom 96-well plates at 10000 cellswelland grown to 80 confluence overnight before treatmentsbegan To determine the cytotoxicity of ethanolic extractsand their individual components cells were treated with thedifferent treatments (thymol carvacrol p-cymene andor1-octacosanol dissolved in 1 ethanol [the vehicle solutionhad no effect on the antioxidant and cytotoxic activities ofthe extract andor its constituents]) in media To determinethe antioxidant effects of the oregano extracts and theirindividual components (thymol carvacrol p-cymene andor1-octacosanol dissolved in 1 ethanol) A549 cells were firstpretreated for 24 h with the different treatments in mediafollowed by treatment with 500120583M hydrogen peroxide TheMTT assay a commonly used measure of cell viability wasused to assess cell survival as described by the manufacturer(Sigma-Aldrich Co Oakville ON Canada) Viabilities ofchallenged cells were assessed relatively to control cells

26 Bacterial Strains P aeruginosa (ATCC 25619 and clin-ical isolates) Bordetella bronchiseptica (ATCC 4617 ATCC10580) Escherichia coli (ATCC 25922 ATCC 700973) Bur-kholderia cenocepacia (ATCC 25608 and clinical isolates)Acinetobacter lwoffii (ATCC 17925)Acinetobacter baumannii(ATCC 19606) Moraxella catarrhalis (ATCC 8176) Bacillussubtilis (ATCC 6633) and S aureus (ATCC 29213 and clinicalisolates) were purchased from Cedarlane (Burlington ONCanada) or obtained from the Clinical Microbiology Lab-oratory of Memorial Hospital (Sudbury ON Canada) Forexperimentation the strains were inoculated onto MuellerHinton II agar plates and incubated for 24 h at 37∘C

27 The Minimum Inhibitory Concentrations (MIC) of Ore-gano Extract against Gram-Positive and Gram-NegativeStrains The inhibitory concentrations were determined bythe agar dilution method as previously described [16] Bac-terial inocula were prepared from an overnight culture inMuellerHinton II agar and adjusted to contain an equivalence














3 Results


LC50 = 14 120583gmL


Unt

reat

ed

100

200

390

780

156

313

625

125

250

0

25

50

75

100

125

MTT

cell

surv

ival

()



50= 14 120583gmL (Figure 1)




0

25

50

75

100

Viab

ility

()

10050 150 200 2500


(a)

A

BC BC BC

BCD

0

25

50

75

100

Viab

ility

()

Con

trol

20120583

Mth

ymol

(T)

56120583

M ca

rvac

rol (

C)

7nM

p-c

ymen

e (p-

C)

14

nM1

-oct

acos

anol

(O)

T +

C

T +

C +

p-C

T +

C +

p-C

+ O

Extr

act

(b)



0

5

10

15

20

Carv

acro

l upt

ake (

)

1 4 80 2412

Time (h)

(a)

0

5

10

15

20

25

Thym

ol u

ptak

e (

)

1 4 80 2412

Time (h)


lowast

lowast

lowast

lowast

(b)






24h H2O2


+++++

++++++

minus

lowast

lowast

lowast

0

25

50

75

100

125

Viab

ility

()


2

O2


2

O2

only119901 lt 005



2O2



4 Discussion



50= 14 120583gmL




Viab

ility

()

ThymolCarvacrol


AB

AB

0

20

40

60

0 1 5 10 50 100

(a)

Viab

ility

()

lowast

lowastlowast

0

20

40

60

80


minus++minusminus


500120583M H2O2

5120583M thymol

10120583M carvacrol

Extract(b)


2

O2


2

O2


O2


2

O2


2

O2

119901 lt 005




















Competing Interests


Acknowledgments


References























2























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























3 Results


LC50 = 14 120583gmL


Unt

reat

ed

100

200

390

780

156

313

625

125

250

0

25

50

75

100

125

MTT

cell

surv

ival

()



50= 14 120583gmL (Figure 1)




0

25

50

75

100

Viab

ility

()

10050 150 200 2500


(a)

A

BC BC BC

BCD

0

25

50

75

100

Viab

ility

()

Con

trol

20120583

Mth

ymol

(T)

56120583

M ca

rvac

rol (

C)

7nM

p-c

ymen

e (p-

C)

14

nM1

-oct

acos

anol

(O)

T +

C

T +

C +

p-C

T +

C +

p-C

+ O

Extr

act

(b)



0

5

10

15

20

Carv

acro

l upt

ake (

)

1 4 80 2412

Time (h)

(a)

0

5

10

15

20

25

Thym

ol u

ptak

e (

)

1 4 80 2412

Time (h)


lowast

lowast

lowast

lowast

(b)






24h H2O2


+++++

++++++

minus

lowast

lowast

lowast

0

25

50

75

100

125

Viab

ility

()


2

O2


2

O2

only119901 lt 005



2O2



4 Discussion



50= 14 120583gmL




Viab

ility

()

ThymolCarvacrol


AB

AB

0

20

40

60

0 1 5 10 50 100

(a)

Viab

ility

()

lowast

lowastlowast

0

20

40

60

80


minus++minusminus


500120583M H2O2

5120583M thymol

10120583M carvacrol

Extract(b)


2

O2


2

O2


O2


2

O2


2

O2

119901 lt 005




















Competing Interests


Acknowledgments


References























2























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















0

25

50

75

100

Viab

ility

()

10050 150 200 2500


(a)

A

BC BC BC

BCD

0

25

50

75

100

Viab

ility

()

Con

trol

20120583

Mth

ymol

(T)

56120583

M ca

rvac

rol (

C)

7nM

p-c

ymen

e (p-

C)

14

nM1

-oct

acos

anol

(O)

T +

C

T +

C +

p-C

T +

C +

p-C

+ O

Extr

act

(b)



0

5

10

15

20

Carv

acro

l upt

ake (

)

1 4 80 2412

Time (h)

(a)

0

5

10

15

20

25

Thym

ol u

ptak

e (

)

1 4 80 2412

Time (h)


lowast

lowast

lowast

lowast

(b)






24h H2O2


+++++

++++++

minus

lowast

lowast

lowast

0

25

50

75

100

125

Viab

ility

()


2

O2


2

O2

only119901 lt 005



2O2



4 Discussion



50= 14 120583gmL




Viab

ility

()

ThymolCarvacrol


AB

AB

0

20

40

60

0 1 5 10 50 100

(a)

Viab

ility

()

lowast

lowastlowast

0

20

40

60

80


minus++minusminus


500120583M H2O2

5120583M thymol

10120583M carvacrol

Extract(b)


2

O2


2

O2


O2


2

O2


2

O2

119901 lt 005




















Competing Interests


Acknowledgments


References























2























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















24h H2O2


+++++

++++++

minus

lowast

lowast

lowast

0

25

50

75

100

125

Viab

ility

()


2

O2


2

O2

only119901 lt 005



2O2



4 Discussion



50= 14 120583gmL




Viab

ility

()

ThymolCarvacrol


AB

AB

0

20

40

60

0 1 5 10 50 100

(a)

Viab

ility

()

lowast

lowastlowast

0

20

40

60

80


minus++minusminus


500120583M H2O2

5120583M thymol

10120583M carvacrol

Extract(b)


2

O2


2

O2


O2


2

O2


2

O2

119901 lt 005




















Competing Interests


Acknowledgments


References























2























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Viab

ility

()

ThymolCarvacrol


AB

AB

0

20

40

60

0 1 5 10 50 100

(a)

Viab

ility

()

lowast

lowastlowast

0

20

40

60

80


minus++minusminus


500120583M H2O2

5120583M thymol

10120583M carvacrol

Extract(b)


2

O2


2

O2


O2


2

O2


2

O2

119901 lt 005




















Competing Interests


Acknowledgments


References























2























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























Competing Interests


Acknowledgments


References























2























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































2























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article antioxidant, antibacterial, and cytotoxic...

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