chemical composition antioxidant and antimicrobial activities of the essential oils of ...

This article was downloaded by: [Linköping University Library]On: 24 August 2014, At: 16:17Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK

Journal of Essential Oil Bearing PlantsPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/teop20

Chemical Composition Antioxidant and AntimicrobialActivities of the Essential Oils of Matricaria aureaLoefl. Growing in TunisiaFethi Bel Hadj Khedera, Mohamed Ali Mahjouba, Fatma Zaghrounib, Sadok Kwajac, AhmedNoureddine Helala & Zine Mighriaa Laboratoire de Genome, Diagnostique immunitaire et Valorisation, Faculté de pharmacie,5000 Monastir, Tunisiab Laboratory of parasitology and Mycology CHU Farhat Hached 4019 Sousse, Tunisiac Laboratory of Analysis and Control of chemical Pollutant and Microbiology, Faculty ofPharmacy, 5000 Monastir, TunisiaPublished online: 20 Aug 2014.

To cite this article: Fethi Bel Hadj Kheder, Mohamed Ali Mahjoub, Fatma Zaghrouni, Sadok Kwaja, Ahmed Noureddine Helal& Zine Mighri (2014) Chemical Composition Antioxidant and Antimicrobial Activities of the Essential Oils of Matricaria aureaLoefl. Growing in Tunisia, Journal of Essential Oil Bearing Plants, 17:3, 493-505, DOI: 10.1080/0972060X.2014.884777

To link to this article: http://dx.doi.org/10.1080/0972060X.2014.884777

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) containedin the publications on our platform. However, Taylor & Francis, our agents, and our licensors make norepresentations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of theContent. Any opinions and views expressed in this publication are the opinions and views of the authors, andare not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon andshould be independently verified with primary sources of information. Taylor and Francis shall not be liable forany losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoeveror howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use ofthe Content.

This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in anyform to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

http://www.tandfonline.com/loi/teop20

http://www.tandfonline.com/action/showCitFormats?doi=10.1080/0972060X.2014.884777

http://dx.doi.org/10.1080/0972060X.2014.884777

http://www.tandfonline.com/page/terms-and-conditions

http://www.tandfonline.com/page/terms-and-conditions

Chemical Composition Antioxidant and Antimicrobial Activities ofthe Essential Oils of Matricaria aurea Loefl. Growing in Tunisia

Fethi Bel Hadj Kheder 1*, Mohamed Ali Mahjoub 1, Fatma Zaghrouni 2,Sadok Kwaja 3, Ahmed Noureddine Helal 1 and Zine Mighri 1

1 Laboratoire de Genome, Diagnostique immunitaire et Valorisation,Faculté de pharmacie, 5000 Monastir, Tunisia

2 Laboratory of parasitology and Mycology CHU Farhat Hached 4019 Sousse, Tunisia3 Laboratory of Analysis and Control of chemical Pollutant

and Microbiology, Faculty of Pharmacy, 5000 Monastir, Tunisia

Abstract: The present study describes the chemical composition, antibacterial, antifungal and antioxidantactivities of essential oils isolated by steam distillation from the aerial parts and roots of the Tunisian Matricariaaurea Loefl. The chemical composition of essential oils was analyzed by gas chromatography/mass spectrometry(GC-MS). 105 components were identified, of which 1,5 bis (dicyclohexylphosphino)-pentane (44.7 %) wasthe main compound in leaves. These oils were subjected to screening for their possible antioxidant activities byusing the ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) and DPPH (2,2-diphenyl-1-picrylhydrazyl radical) methods and compared to the synthetic antioxidant Trolox. The inhibitory activity ofM. aurea Loefl. essential oils against Gram-positive bacteria was significantly higher than against Gram-negativeones, Both pathogenic yeasts Candida albicans ATCC 9008 and Candida parapsilosis CECT 13009 were allresistant to the tested oils. Our results demonstrate that the essential oils of M. aurea possessing antioxidantand antimicrobial activities might be a natural potential source of preservative used in food and other alliedindustries.

Key words: Matricaria aurea Loefl; Essential oils; antimicrobial activity; antioxidant activity.

IntroductionEssential oils of plants are of growing interest

both in the industry and scientific researchbecause of their antibacterial, antifungal, andantioxidant properties and make them useful asnatural additives in foods 8. Free radical oxidationof the lipid components in food due to the chainreaction of lipid peroxidation is a major strategicproblem for food manufacturers. Due toundesirable influences of oxidized lipids on thehuman organisms, it is essential to decrease lipidperoxidation products in food 21. Reactive oxygen

species are reported to be a causative agent ofvarious diseases such as arthritis, asthma,dementia, mongolism, carcinoma and Parkinson’sdisease 28. Plant essential oils and their extractshave had a great usage in food flavoring,fragrance, pharmaceutical industries 19 and in folkmedicine as herbal remedies for rheumatic andneuralgic diseases 11.

Flowers of Matricaria aurea Loefl. are usedfor respiratory canals, stomachaches, anti-spasmodic antipyretic, anticough, antinfluenza,antiasthma and antiflatulents proprieties 12,15.

ISSN Print: 0972-060XISSN Online: 0976-5026

*Corresponding author (Fethi Bel Hadj Kheder)E-mail: < [email protected] > © 2014, Har Krishan Bhalla & Sons

TEOP 17 (3) 2014 pp 493 - 505 493

Received 25 July 2012; accepted in revised form 18 January 2013

Dow

nloa

ded

by [

Lin

köpi

ng U

nive

rsity

Lib

rary

] at

16:

17 2

4 A

ugus

t 201

4

Leaves and flowers are used against fever,coughing and heart diseases: an infusion is madefrom 1 to 2 tsp leaves and flowers in one cupwater. 1-3 cups can be taken daily until improve-ment Occurs 16. Matricaria aurea Loefl. is usedin traditional medicine to treat diabetes 20.

The aim of this work is to establish the chemicalcomposition of M. aurea essential oils, isolatedby steam distillation from the aerial parts and rootsand to evaluate the antioxidant capacity of theseplant parts essential oils using DPPH and ABTS+

tests, as as well as to screen their antimicrobialactivities.

Material and methodsPlant material

Plant material was collected at the floweringstage in February 2008, in Sousse (Tunisia). Thefresh plant was separated in four parts: roots,leaves, stems and flowers. Each part was dividedin little peaces and weighed before extraction ofessential oils. A Voucher specimens ASTER-23344 was deposited in the Laboratory.

Essential oils extractionFresh quantity of each organ of the plant M.

aurea Loefl were divided in to little pieces andweighed before the extraction of volatilecompounds and submitted to a steam distillationfor 5-6 h. The oils were collected throughdichloromethane extraction and they were stored(prior to analysis). After drying the extract overanhydrous Na2 SO4, the solvent was removed, oilsamples were calculated.

Analysis of the essential oilsGas chromatography

HP 5890-series II equipped with: flameionization detector (FID), HP -5, 30 m x 0.25 mmID, 0.25 μm film thicknesses fused capillarycolumn and HP Innowax 30 m x 0.25 ID, 0.25μm film thickness fused capillary column. Thecarrier gas was nitrogen (1.2 mL/min). The oventemperature program was 1 min isothermally at50°C, then 50-280°C at rate of 5°C /min and heldisothermally for 1 min. The injection porttemperature was 250°C, detectors: 280°C. Volumeinjected: 0.1 μL of 1 % solution diluted in hexane.

Percentages of the constituents were calculatedby electronic integration of FID peak areaswithout the use of response factor correction.

Gas chromatography-Mass spectrometryThe volatile constituents analysis was done on

a Hawlet-Packard GC: 5890 series II; MSD 5972)and the fused-silica HP-5 MS capillary column(30 m x 0.25 μm) was directly coupled with theMass spectrometer. The carrier gas was heliumwith a flow rate of 1.2 mL/min. Oven temperaturewas programmed 50°C for 1 min, then 50-280°Cat rate of 5°C/min and subsequently, heldisothermally for 20 min. Injector port: 250°C,detector : 280°C, split ratio 1:50. Volume injected:0.1 μL of 1 % solution (diluted in hexane). HP5972 recorded at 70 eV; scan time 1.5 s; massrange 40-300 amu. Software adopted to handlemass spectra and chromatograms was aChemStation®.

The oils components were identified throughthe comparison of their mass spectra to those of acomputer library (Wiley 275 Library). Furtherconfirmation was done by referring to retentionindex data generated from a series of alkanes (C9-C28)

Antioxidant activityChemicals and reagents

2,2-azinobis (3-ethylbenzothiazoline-6-sulpho-nic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin–Ciocalteu,catechin and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) werepurchased from Sigma-Aldrich; potassiumpersulphate, sodium acetate, and sodiumcarbonate, ethanol and were for gradient purity.

Cation radical ABTS+• scavenging activityIn this study we adopted the standard method

described by Miller, et. al.,24. This method basedon the ability of a compound to scavenge thestable cation radical (ABTS+•). ABTS+• cationradical form is the result of the reaction betweenABTS (20 mg), potassium persulfate (K2S2O8)dissolved in demonized water. The solution wasincubated at room temperature in the dark forminimum 12 hours. A concentrated ABTS+• stock

Fethi Bel Hadj Kheder et al., / TEOP 17 (3) 2014 493 - 505 494

Dow

nloa

ded

by [

Lin

köpi

ng U

nive

rsity

Lib

rary

] at

16:

17 2

4 A

ugus

t 201

4

solution was diluted with phosphate bufferedsaline (PBS), pH 7.4 to give a final absorbanceof 0.70 ± 0.02 with a wavelength of 734 nm andat room temperature. Then, different concen-trations of samples were prepared by dilution withethanol for the volatile fractions.

Sample absorbance was compared to a maximalabsorbance of 990 μL of the ABTS+• solution and10 μL of Ethanol. Ten micro liters of studiedsolution were added to 990 μL of ABTS+• solutionat the absorbance of 734 nm was measured after5, 10, 15 and 20 min. Each measure was repeatedthree times and the inhibition percentage ofdifferent samples concentrations (mg/mL) wascalculated in times previously mentioned.

DPPH scavenging activityThe Hydrogen atom or electron donating ability

of the extracts was measured from the bleachingof purple colored ethanol solution of DPPH.These spectrophotometric assays were performedas described elsewhere 4,7,31. The DPPH solutionwas prepared in absolute ethanol to give anabsorbance of 0.70 ± 0.02. 0.5 mL with a wave-length of 520 nm in a spectrophotometerSchimadzu 160-UV.

The reaction for scavenging DPPH radicals wascarried out at room temperature in the dark for30 min. A mixture of 0.5 ml of DPPH solutionsand 0.5 mL of ethanol was taken as a reference.Inhibition of free-radical DPPH in percent (1%)was calculated thus:

where Ablanc is the absorbance of the controlreaction (containing all reagents except the testcompound) and Asample is the absorbance of thetest compound.

Decrease in absorption induced by the testedextracts was compared to that of the positivecontrol Trolox. All measurements were performedin triplicate. Results were expressed in inhibitionpercentage of different sample concentrations(mg/mL) at 30 min.

TEAC assayIn this study TEAC assay was performed with

minor modifications according to the original

method proposed by Van Den Berg et. al., 33 withminor modifications. Solutions were prepared asthe ABTS+• scavenging activity section, Fourconcentrations was used and each measure wasrepeated three times. The TEAC values of theantioxidant were calculated at 5, 10, 15 and 20minutes by relating this decrease in absorbanceto that of a Trolox solution on a molar basis. Theantioxidant activity of the samples was expressedas milligrams of samples equivalents per gram ofdry weight.

Determination of total phenolic contentThe concentration of total phenols compounds

in each sample was determined with Folin–Ciocalteu reagent following the colorimetricmethod adopted by Wojdylo, et. al.34. All sampleswere prepared at a concentration of 1 mg/Ml. 0.75mL of Folin-Ciocalteu reagent were added andmixed with 100 μL of extract. 0.75 mL ofsaturated sodium carbonate solution were addedto the mixture and then mixed gently. Afterstanding at 25°C for 90 min, the absorbance wasread at 725 nm using an UV-Vis spectrophoto-meter.

All Measurements were carried out in triplicateand calculations were based on a calibration curveobtained with catechin. calibration (0.01-0.05 mg/mL) curve was plotted using catechin. The levelsof total phenols were expressed as milligrams ofcatechin equivalents per 1 gram of dry weight.

Antimicrobial activityMicrobial strains

The antimicrobial and antifungal activities ofvolatile fractions were individually tested bypaper disc diffusion method and by determinationof minimum inhibitory concentration (MIC)against a panel of microorganisms (bacteria andyeast) : Vibrio Harveyi ATCC 14126, Vibrio vulni-ficus ATCC 27562, Pseudomonas aerugi-nosa,ATCC 27853; Vibrio alginolyticus ATCC33787, Vibrio fluvialis ATCC 33809, Micrococcusluteus NCIMB 8166, Escherichia coli ATCC35218, Staphylococcus aureus ATCC 25923,Salmonella wien ATCC 1408, Bacilus cocusATCC 11778, Vibrio parahaemolyticus ATCC17802, and against clinically isolated strains from

1% = (Ablanc- Asample)/ Ablanc*100


Dow

nloa

ded

by [

Lin

köpi

ng U

nive

rsity

Lib

rary

] at

16:

17 2

4 A

ugus

t 201

4

the parasitology and mycology laboratory ofFarhat Hached university hospital: Candidaalbicans ATCC 9008, Candida parapsiloisCECET 13009, Candida tropicalis, Candidakruseii and Candida glabrata. All the strainswere grown on Muller Hinton Agar (MHA) forthe bacteria and sabauraud Dextrose Agar withchloromphenicol for yaests.

Preparation of the inoculumsCulture media

Bacteria were assayed on Mueller Hinton Agar(MHA, Pronadisa Hispanalab, g/L): beef infusion2.0; acid casein peptone 17.5; starch 1.5;bacteriological agar 17.0. Sabouraud DextroseAgar (SDA, Merck, g/L): peptone, 10.0; glucose,20.0; agar-agar, 17.0 with chloramphenicol 0.5 gwas used for yeasts. The inoculum used for allthe assays reached the microbial density of theorder of 106–107 CFU/mL for the bacteria andyeasts: The strains preserved in the nutrient agarat 4°C, were revived in nutrient solution andincubated at 37°C during 18-24 h., 0.1 ml of eachculture was added to 10 mL BHIB (Brain HeartInfusion Broth, Pronadisa Hispanalab, S.A.).Yeasts: The strains preserved at 4°C in theSabouraud agar supplemented withchloramphenicol were revived in nutrient solutionand incubated at 27 ± 1°C for 24-48 h, 0.1 mL ofeach culture was added to 10 mL sterile physio-logical water.

Diffusion methodThe antimicrobial essay of Matricaria aurea

essential oil. was carried out by the disc diffusionmethod 1,5,23. Five hundred microliters of theinoculums were spread over plates containingsterile Mueller-Hinton agar (pH: 7.2) and thepaper filter discs (5 mm) impregnated with 0.04,0.5 and 3 mg of the oil were placed on the surfaceof the media. The plates were left 30 min at roomtemperature to allow the diffusion of the oil andincubated at 37°C for 18 h. Each measure wasreputed in three times. The inhibition zone aroundthe disc was measured. Two controls were alsoincluded in the test, the first was Ampecellin(15μg) for bacteria and the second wasAmphoterecin B (100 μg) for yeasts: Ampicillin

used to control the sensitivity of the testedbacteria, and the developing inhibition zones werecompared with those of reference discs.

Minimum inhibitory concentrationA broth microdilution method was used to

determine the minimum inhibitory concentration(MIC) (NCCLS, 2001). All tests were performedin BHIB (Brain Heart Infusion Broth, PronadisaHispanalab, S.A.). The essential oils weredissolved in 10 % dimethylsulphoxide (DMSO).A serial doubling dilution of each oil was preparedin a 96 well microtiter plate over the range of0.05-100 μg/mL. Overnight broth cultures of eachstrain were prepared, the final concentration ineach positive well was adjusted to 2-106 CFU/mL and the plates were incubated at 37°C for 24h. The MIC was defined as the lowest concen-tration of the essential oil at which the bacteriaor yeast does not show visible growth. TheAmpecellin and 10 % DMSO solution served aspositive and negative controls, respectively.

ResultsFresh flowers, stems, roots and leaves of M.

aurea were subjected to steam distillation and thecolorless and yellowish essential oils wereobtained with respective yields: 0.03 %; 0.05 %;0.1 % and 0.06 %, w/w. Results obtained by GC-MC and GC-FID analysis of the essential oilsfrom M. aurea are presented in table1.

Eighty seven compounds were identified in thewhole part of M. aurea, representing 87.77 %,77.23 %, 72.41 %, 89.84 % from flowers, stems,roots and leaves respectively. The essential oil ofeach part of the studied plant has a specificqualitative and quantitative composition. Indeed,the composition of flowers showed richness inhydrocarbons (67.32 %), alcohols (16.62 %),oxygenated sesquiterpenes (3.87 %), aromaticcompounds (5.34 %). While the essential oilcompounds obtained from stems of M. aureaLoefl were characterized by the prevalence ofhydrocarbons (28.56 %), oxygenated diterpenes(2.48 %), monoterpenes hydrocarbons (0.44 %),and aromatic compounds (3.64 %). On the otherhand, the majority of roots constituentsrepresented especially by fatty acid and fatty acid


Dow

nloa

ded

by [

Lin

köpi

ng U

nive

rsity

Lib

rary

] at

16:

17 2

4 A

ugus

t 201

4

esters (17.92 %). The leaves were rich in hydro-carbons (59.97 %).

The Diterpenes were present in less percentagein the different parts of the studied plant. The fattyacid and aromatic compounds however,represented an important proportion compared tothe rest.

The flower essential oil compounds showed asmain constituents 2-ethoxy-6-ethyl-4,4,5trimethyl-1,3 dioxa4sila2boracyclohex5ene(37.99 %), (2,5 bis1,1dimethyleth) thiophene(10.95 %), n-eicosane (6.59 %), 1-Oleylglycerol(5.59 %). 5,7-dimethyloctahydrocoumarin wasrather the major compound in the stems (19.16%), followed by dodecanoic acid (13.72 %), 2-ethoxy-6-ethyl-4,4,5-trimethyl-1-3-dioxa-4-sila-2-boracyclohex-5-ene (37.99 %).

Essentials oils of roots showed 2-ethoxy-6-ethyl-4,4,5-trimethyl-1,3-dioxa-4-sila-2-boracyclohex-5-ene (18.45 %), pentadecanoicacid (16.03 %), n-eicosanol (9.55 %), phytol (5.13

%) as main constituents. The most importantcompound detected in leaves was 1,5-bis(dicyclohexylphosphino)-pentane (44.7 %).

Three major products were common in thedifferent parts of the tested plant, such as the 5,7-dimethyloctahydrocoumarin which was concen-trated especially in its stems (19.16 %), 2-ethoxy-6-ethyl-4,4,5-trimethyl-1,3-dioxa-4-sila-2-boracyclohex-5-ene witch was concentrated in itsstems (37.99 %), 1,5-bis (dicyclohexyl-phosphino)-pentane which was concentratedespecially in its leaves (44.7 %). Some compo-nents were specific to one part only.

Indeed 1-oleylglycerol (5.59 %), 2 farnesol(2.04 %) existed only in the flowers, dodecanoicacid (13.72 %) and 7-methyl-3,4-octadiene (1.45%) were detected only in the stems. Whereas,acetic acid (13.42 %), lavandulol (0.07 %),jasmolin (0.05 %), 15-Hexadecanolide (0.43 %)and 9-Octadecenoic acid (0.65 %) were detectedonly in the leaves of this plant.

Table 1. Chemical composition of Matricaria aurea L. essential oils

No. Compounds RIa RIb Flowers Stems Roots Leaves

1 2-Cyclopentenol 1327 802 0.27 - - 0.172 1,7-Octadiene 2,3,3-trimethyl 778 609 0.34 0.05 - -3 1-Hepten-4-ol 1052 660 0.17 - - -4 Cyclohexane-octyl 732 677 0.14 - - -5 1,3-Pentadiene-4-methyl 796 717 0.1 - - -6 Octanal-7-dimethyl-3,7-hydroxy 1096 727 - 0.03 - -7 Nonanal 1102 796 - 0.2 0.04 -8 2-Hexyne 1038 800 0.12 - - -9 Phenylethyl alcohol 1925 804 - 0.07 - -

10 3,3,5-Trimethyl-Cyclohexene 1182 804 - 0.05 - -11 Acetic acid 1477 827 - - - 13.4212 4-Methyl-1-penten-3-ol 1390 838 0.08 0.11 - -13 Ether ethylic 1377 903 0.08 - - -14 Camphene 957 962 - 0.03 0.14 -15 1,14-Tetradecanediol 1826 965 - - 0.09 -16 β-Myrcene 1168 981 - 0.16 0.1 -17 Pinane 1075 994 1.25 0.03 - -18 Benzyl alcohol 1865 1024 - 0.02 0.18 0.2319 Phenyl acetaldehyde, 1646 1024 - 0.04 - -20 Nonyl alcohol 1167 1033 0.14 - - -21 Hex-4-ene-1-ol 888 1050 0.09 - - -22 3-Carene 1017 1052 - 0.14 - -


Dow

nloa

ded

by [

Lin

köpi

ng U

nive

rsity

Lib

rary

] at

16:

17 2

4 A

ugus

t 201

4

table 1. (continued).


23 Pentadecane 1504 1079 0.17 0.14 0.04 -24 Octahydrocoumarin 5,7-dimethyl 2465 1138 5.29 19.16 3.95 0.0525 3-Decyne 1078 1140 - 0.03 - 0.0826 Lavandulol 1169 1154 - - - 0.0727 Citronellal 1485 1160 - 0.09 - 0.0528 Cyclododecane 1242 1200 0.79 - - -29 Octanoic acid 2098 1234 - - 0.05 -30 Vanillin 2589 1298 - 0.04 0.17 -31 Tetradecanal 1940 1321 - 0.31 - -32 6-Methyl-5-hepten-2-one 1337 995 - 0.16 - -33 Ethyl chloroacetate 1344 806 - 0.34 - -34 1-Hexadecanol 2364 1380 - - - 0.135 2-(3'-Hydroxy butyl) cyclooctanone 1382 920 - 0.16 - -36 2-Methyl-1-naphthol 2248 1390 - 0.19 - -37 Farnesene 1725 1403 - 0.02 - -38 Epoxy cyclododecane 1242 1409 0.13 0.06 - -39 Isocitronellene 1965 1452 - 0.11 - 0.0940 β-Cadinene 1519 1464 0.14 - 0.32 0.0941 Dihydroactinidiolide 2348 1485 0.15 - 0.28 -42 trans-2-Hexenal 801 1486 0.18 - 0.1 -43 β-caryophyllene oxide 1999 1500 0.15 - 0.15 -44 Caryophyllene oxide 1962 1514 1.54 - 0.36 0.0645 Citronellol 1764 1228 0.27 - 0.03 -46 Spathulenol 2129 1545 0.1 - 0.13 0.1747 (Z)-13-Octadecenal, 2370 1558 - - 0.15 -48 β-Farnesol 2044 1569 2.04 - - 0.1749 β-Citronellol 1762 1575 - 0.11 0.13 -50 trans-Farnesol 1701 1603 - - 0.19 0.0551 Gossonorol 1632 1613 - 0.43 - -52 γ-Muurolen 1684 1614 0.62 - - 0,1953 β-Eudesmol 2246 1639 0.77 - - -54 α-Bisabolol oxide 2235 1649 1.16 3.13 4.64 2.6455 (2,5-Bis1,1-8 dimethyleth) 1161 1662 10.95 0.02 - 0.38

thiophene56 Heptadecane 1700 1689 0.47 - 0.06 -57 (Z,E)-Farnesol 1743 1697 - 0.24 - 0.4958 Tetradecanoic acid 2690 1737 - - 0.19 -59 1,5-Bis (dicyclohexylphosphino)- 500 1743 5.29 19.16 3.95 44.7

pentane60 Octadecane 1800 1768 - - 0.93 -61 cis-Tetrahydroionol 1433 1783 0.45 0.89 - 0.9462 Hexahydro pseudoionone 1550 1827 0.12 0.37 0.19 0.1763 14-Pentadecenoic acid 3181 1848 0.49 0.63 0.04 -64 6,7-Dimethoxycoumarin, 4-methyl 1699 1851 0.37 - - 0.32


Dow

nloa

ded

by [

Lin

köpi

ng U

nive

rsity

Lib

rary

] at

16:

17 2

4 A

ugus

t 201

4

table 1. (continued).


65 Pyridine 1193 1860 0.59 - 0.12 -66 2-Methyl-3-vinylbenzofuran 1599 1879 - - 5.18 1.7267 7-Methyl-3,4-octadiene 1176 1890 - 1.45 - -68 2-Acetyl- naphthoquinone, 1444 1905 - - 0.17 -69 2-Ethoxy-6-ethyl-4,4,5- trimethyl- 1727 1915 37.99 6.49 18.45 12.97

1,3-dioxa-4-sila-2-boracyclohex-5-ene70 15-Hexadecanolide 1842 1957 - - - 0.4371 9-Octadecenoic acid 2102 1967 - - - 0.6572 Hexadecanoic acid 2860 1968 - - 0.89 -73 n-Eicosane 2000 1978 6.59 0.6 - 0.4274 6-Methyl-3-phenyl-cinnoline 2000 3.69 3.61 - -75 Linoleic acid 2130 2058 2.92 - - 3.6276 Phytol 2571 2059 - - 5.13 -77 n-Eicosanol 2275 2078 - - 9.55 4.4678 Bergamotene 1431 2135 0.17 1.03 - 1.2879 Oleic acid 3184 2174 0.16 - 0.14 -80 Dotriacontane 3200 2181 - 0.36 0.05 0.0881 Lauric acid 2517 2320 - 13.72 - -82 Hexadecenoic acid 2860 2343 1.37 0.24 - -83 2-(Hexadecyloxy) ethanol, 2136 2392 0.31 - - -84 4-Dihydro-5,7-dimethoxy-4-methyl 1699 2407 0.4 - - 7.08

coumarin85 Pentadecanoic acid 1878 2691 - 2.79 16.03 -86 1-Oleoyl glycerol 2097 2888 5.59 - - -87 Methyl allyl sulfide 922 2990 0.16 - - 1.98

Total identified 87.77 77.23 72.41 89.84Total monoterpene 0.27 0.55 0.59 0.05Oxygenated containing monoterpene 0.27 0.11 0.35 0.05Monoterpene hydrocarbons - 0.44 0.24 -Total Sesuiterpene 6.49 1.85 5.15 4.83Oxygenated containing Sesquiterpene 3.87 0.37 4.64 3.3Sesquiterpene Hydrocarbons 2.62 1.48 0.51 1.53diterpene - - 5.13 -Total terpene 6.71 2.29 11.42 4.83Ketones 0.12 0.37 0.36 0.17Carboxylic acid 4.94 14.59 17.34 17.69Alcohols 16,62 1,17 5.88 6,75Aldehyde - 0.67 0.18 -Aromatic compounds 5.34 3.64 - 7.15Esters 5.59 0.43Hydrocarbon 67.32 28.56 23.48 59,97

a: retention index relative to n-alcanes on the polar columnb: retention index relative to n-alcanes on the DB-5 column-: Not detect


Dow

nloa

ded

by [

Lin

köpi

ng U

nive

rsity

Lib

rary

] at

16:

17 2

4 A

ugus

t 201

4

Antioxidant activityCation radical ABTS+• scavenging activity

Results of our investigation on ABTS+• cationradicals scavenging activity of M. aurea Loeflessential oil, were represented in table 2.Compared to Trolox, maximal inhibitionpercentage values calculated after 20 min ofreaction indicated difference in the antioxidantactivity for the four tested essential oils. Theessential oil of leaves showed a moderateinhibition, with value of 21.2 % at a concentrationof 1 mg/mL. Essential oil of leaves showed alsofeeble ABTS+• inhibition with a value of 13.4 %at a concentration of 1 mg/mL, after 20 minutesof reaction. The least ABTS+• inhibitor was theessential oil of stems with a value of 5.7 % at aconcentration of 1 mg/mL in comparison withTrolox ( 95.59 %) (table 2).

At low concentration, essential oils of leaves,stems, roots, flowers were inactive with feebleinhibition percentages of 1.5,-0.1, 0.6 and 5.1 %respectively at the concen-tration of 0.03 mg/mL,after 20 min of reaction. Although, at high concen-tration, some samples showed a pro-oxidantbehavior.

DPPH scavenging capacityThe results of the antioxidant activity of M.

aurea essential oils against DPPH radicals wererepresented in table 3. Compared to Trolox as astandard reference product, we note that the fouressential oils seemed to be moderately activeagainst DPPH radicals. It attended inhibitionpercentages of 19.55; 24.53; 18.7 and 1.64 % forstems, flowers, leaves and roots respectively, atthe concentration of 1 mg/mL.

Table 2. ABTS+ inhibition percentages in the presence of differentconcentrations of Matricaria aurea Loefl. essential oils

Concentration ABTS+• % inhibition(% μμμμμg/ml) Time (min)

5 10 15 20

Leaves 1.00 11.9±6.8 17.7±2.6 21.9±5.7 21.2±3.60.50 6.6±3.8 10.9±1.7 11.6±1.8 121±1.60.25 8.6±4.9 8±3.7 7.9±4.1 7.4±4.00.12 2.5±1.4 2.5±2.2 1.9±2.8 2.1±3.10.06 -1.1±0.6 13.2±7.90 -1.1±0.1 -1.9±0.30.03 1.4± 0.8 2.5±1.70 1.4±0.8 1.5±1.0

Stems 1.00 6.9±3.4 9.4±7.3 10.8±3.3 5.7±11.70.50 2.1±2.0 2.5±5.9 3.6±2.1 3.6±1.90.25 0.4±2.2 1.2±2.4 1.0±2.3 0.3±2.20.12 2.5±1.5 2.5±2.1 2.2±2.0 2.1±1.80.06 2.9±1.5 2.5±1.1 2.2±1.5 1.7±1.50.03 -2.8±3.5 0±2.7 0.0±2.6 -0.1±2.2

Flowers 1.00 6.2±0.8 4.2±0.4 4.2±0.4 10±0.50.50 0.3±2.2 5.7±0.4 6.9±0.3 8.8±0.30.25 3±1.8 3.4±0.3 3.3.±0.4 2.5±0.40.12 3.3±1.4 3±0.6 2.8±0.8 5.4±0.40.06 5.2±3.2 -1±0.5 -0.7±0.4 -1.8±0.60.03 3.5±0.5 5±0.2 5.1±0.3 5.1±0.3

Roots 1.00 6.1±6.9 10.5±5.7 11.7±6.0 13.4±5.60.50 2.3±6.7 7.9±8.8 8.2±10.2 8.2±10.80.25 -3.1±3.8 -0.8±2.9 1.0±3.2 0.1±4.50.12 2.7±2.8 3.4±4.7 3.5±2.0 2.5±1.80.06 2.7±0.7 1.8±1.8 2.7±1.5 1.4±2.2


Dow

nloa

ded

by [

Lin

köpi

ng U

nive

rsity

Lib

rary

] at

16:

17 2

4 A

ugus

t 201

4

Table 3. Inhibition percentage of DPPH scavenging of Matricaria aurea Loefl. essential oils

Concentrations Inhibition % of DPPH radicals(mg/mL) 0.03 0.06 0.125 0.25 0.5 1

Leaves 1.99±1.80 1.06±3.11 3.05±9.95 2.48±7.14 18.34±5.83 18.7±4.32Stems 4.97±2.81 12.73±3.41 3.41±1.81 8.17±1.30 9.24 ±3.82 19.55±0.50Flowers 2.34±2.71 4.69±2.61 8.81±5.63 8.17±1.30 18.42±7.14 24.53±0.90Roots 17.46±4.94 12.94±1.64 10.54±2.71 4.31±1.64 3.63±1.64 1.64±4.84Trolox 94.085±0.58 94.085±0.58 94.085±0.09 94.085±0.19 95.39±0.84 95.59±0.38

We note that the essential oils of flowers werethe most active with inhibition percentage of24.53 % at a concentration of 1 mg/mL.

The essential oils of roots seem to be the leastinhibitor of DPPH radicals in comparison to thoseof the other plant parts (Table 3).

TEAC assaysThe values of the TEAC analysis measured

using ABTS method at 20 minutes show thatleaves possessed the highest antioxidant capacityat a concentration of 0.5 mM, followed by roots(which showed average values of 0.32 mM thusshow pro-oxidant activity), then flowers (0.24 %),then stems (0.14 %) (Table 4).

Determination of total phenolic contentThe total phenolic content of the different

essential oils were evaluated, using the Folinciocalteux method. Among tested samples leaveshad the highest phenolic content with an amountof 94.63 mg of Catechin/g of extract. Stemspresented the lowest phenolic content with a valueof 41.48 mg of Catechin/g of extract. Flowers androots essential oils have an important phenoliccontent with respective values of 79.41 and 91.91mg of Catechin per g of sample (Table 4).

Antimicrobial activityThe antibacterial activities of the volatile

Table 4. Trolox equivalent antioxidant capacity (TEAC) andPhenolic content (TCP) in Matricaria aurea Loefl essential oils

Organ Leaves Roots Stems Flowers

TEAC (Trolox/g ) 0.516 0.329 0.14 0.24Phenolic contentmg catechin/g 94.63 91.91 41.48 79.41

fractions were tested in vitro by using discdiffusion and liquid dilution methods with themicroorganisms as seen in Table 5.

The results of the bioassays showed thatessential oils of the fourth organs (stems, leaves,flowers, and roots) exhibited a significantantibacterial activity against most of the bacteriatested (Vibrio harveii, Micrococcus luteusEcherichia coli, Staphylococcus aureus, andBacillus coccus). Visible growth of Staphylo-cocus aueus and Micrococcus luteus wasprevented at a concentration of 500 μg/mL whilefor E. coli it was at a concentration of 1 mg/mL.

DiscussionIn the case of flowers, 33 compounds were

identified representing 87.77 % of the volatilefraction includes a high hydrocarbon fraction(67.32) from which 21.89, 10.21, 9.52, 5.40 and5.16 % are attributed respectively to nonadecane,(Z)-8-Hexacosene, 3-methyldocosane, (Z)-2-Tetracosene and (Z)-2-pentacosene. Terpenespresent 6.71 % which are divided to 6.49 % ofsesquiterpenes and 0.27 % of monoterpenes.Carboxylic acids represent 4.94 %.

In the case of stems, 44 compounds of oil wereidentified. This oil is characterized by thepresence of terpenes (2.29 %) and especially bythe presence of sesquiterpenes (1.85 %).Carboxylic acids represent 4.94 % of the oil and


Dow

nloa

ded

by [

Lin

köpi

ng U

nive

rsity

Lib

rary

] at

16:

17 2

4 A

ugus

t 201

4

Tabl

e 5.

Ant

imic

robi

al a

ctiv

ity o

f the

ess

entia

l oils

of

Mat

rica

ria

aure

a L

oefl.

Mic

roor

gani

sms

Lea

ves

Flow

ers

Roo

tsSt

ems

Am

BR

efer

ence

str

ains

DD

aM

ICb

DD

aM

ICD

Da

MIC

DD

aM

ICD

DM

IC

Vibr

ioha

rvey

i ATC

C 1

4126

1210

015

>100

014

100

1620

0Vi

brio

vul

nific

us A

TCC

275

6212

>800

20>8

0016

250

15>8

00Ps

eudo

mon

as a

erug

inos

a AT

CC

278

53N

d>1

000

12>1

000

12>1

000

10>8

00Vi

brio

alg

inol

ytic

us A

TCC

337

8710

>800

13>8

0012

>800

14>8

00Vi

brio

fluv

ialis

ATC

C 3

3809

10>1

000

1350

012

>800

13>8

00M

icro

cocc

us lu

teus

NC

IMB

8166

1850

2010

021

5017

75Es

cher

ichi

a co

li AT

CC

352

1811

100

1175

1250

09

>500

Stap

hylo

cocc

us a

ureu

s ATC

C 2

5923

1775

1950

22nd

1940

Salm

onel

la w

ien

ATC

C 1

408

950

012

100

1280

011

ndBa

cilu

s coc

us A

TCC

117

7819

250

17>8

0020

1018

35Vi

brio

par

ahae

mol

ytic

us A

TCC

178

028

>800

10>8

0011

>500

9>1

000

Can

dida

alb

ican

s ATC

C 9

008

650

0na

>100

0na

950

(-)

>100

020

1C

andi

da p

arap

silo

sis C

ECT

1300

99

950

na>1

000

12>1

000

9>1

000

121

Clin

ical

ly is

olat

ed s

train

sC

andi

da tr

opic

alis

1095

011

8511

950

1190

013

1C

andi

da g

labr

ata

1380

014

>100

07

850

12>1

000

181

Can

dida

Kre

usei

i15

8010

209

40na

250

151

DD

: Dia

met

er o

f zon

e of

inhi

bitio

n (m

m) i

nclu

ding

dis

c di

amet

er o

f 6 m

m;

(-):

no a

ctiv

ity d

itect

ednd

: not

det

erm

ined

a : T

este

d at

a c

once

ntra

tion

of 1

000

μg/m

LM

ICb :

min

imum

inhi

bito

ry co

ncen

tratio

nVa

lues

giv

en a

s μg/

mL

Am

B: A

mph

oter

ecin

B (1

00 μ

g)AT

CC

: Am

eric

an T

ype

Cul

ture

Col

lect

ion

NC

IMB

: Nat

iona

l Col

lect

ions

of I

ndus

trial

Mar

ine

and

Food

Bac

teria

CEC

ET: S

pani

sh T

ype

Cul

ture

Col

lect

ion


Dow

nloa

ded

by [

Lin

köpi

ng U

nive

rsity

Lib

rary

] at

16:

17 2

4 A

ugus

t 201

4

hydrocarbons represent 28.56 % of the oil, 3-methyldocosane and tetracosane are the maincompounds (11.73 and 6.52 %).

In the case of roots , 37 compounds of oil wereidentified. It represent 72.41 % and the maincompound is 2-ethoxy-6-ethyl-4,4,5- trimethyl-1,3-dioxa-4-sila-2-boracyclohex-5-ene (28.56%).While In the case of leaves, 33 compounds ofoil were identified represent (89.84 %). The maincompounds is1,5-bis (dicyclohexylphosphino)-pentane (44.7 %).

The species Matricaria aurea Loefl growingin Tunisia was characterized by variety of chemi-cal compound; this can be attributed to themediterranien climate of the country.

Although of the important antioxidant capacity,we note that the majority of tested samples presenta moderate total phenolic content. This anti-oxidant activity may be caused by other meta-bolites. These metabolites may be vitamins asvitamin E and C, carotenoids metals ions as Se,Zn, Cu, Mn and Fe or enzymes implicated inantioxidant mechanisms.

A positive linear correlation was observedbetween the total antioxidant activity (TEAC) andphenolic content method.

Some authors have found positive correlationsbetween the total content of phenolic compoundsand antioxidant activity in essentials oils 22.

It revealed there is a relationship between totalphenol contents and the antioxidant activity.Polyphenolic antioxidants are products ofsecondary metabolism in plants, and the anti-oxidant activity is mainly due to their redoxproperties and chemical structure, which can playan important role in chelating transitional metals,inhibiting lipoxygenase and scavenging freeradicals 9. In addition, the synergism between theantioxidants in the mixture makes the antioxidant,not only dependent on the concentration, but alsoon the structure and interaction between anti-oxidants 29.

These results of antimicrobial activity are inaccordance with previously published data

reported by Bougatsos, et. al.3 Kokoska, et. al.18

and Yayli, et. al.36. The higher resistance amongGram negative bacteria could be due to thedifferences in the cell membrane of these bacterialgroups. Indeed, the external membrane of Gramnegative bacteria renders their surfaces highlyhydrophilic 30, whereas the lipophilic ends of thelipoteichoic acids of the cell membrane of Grampositive bacteria may facilitate penetration byhydrophobic compounds 6,32. According toDelaquis, et. al.10 fractions rich in long chainalcohols were active against bacteria. Theantimicrobial properties of alcohols were knownto increase with molecular weight 25,27. Thecompounds present in the greatest proportions arenot necessarily responsible for the higher shareof the total activity. Moreover, some study provedthat volatile fractions have a greater antibacterialactivity than the major component 14,26. Thissuggests that the minor components, such as β-eudesmol, β-cadinene and β-myrcene are criticalto the activity and may have a synergistic effector potentiating influence.

In conclusion, Essential oils of Matricariaaurea Loefl growing in Tunisia are very rich inchemical compounds especially phenolic contentcoumarin products which has contributed to amoderate antioxidant and antibacterial activity butfeeble antifungal activity was detected.

AcknowledgmentsThe authors are grateful to Ministry of

Scientific Research of Tunisia for supporting thiswork, Dr. Zohra Marzouk (Laboratory of Botanic,Faculty of Pharmacy, Monastir-Tunisia), Pr.Amina Bakhrouf (Laboratory of EnvironmentMicrobiology), Dr. Dhouha Saiidana, (Laboratoryof Diagnostic and Valorization- High School ofBiotechnologies Monastir- Tunisia), Dr SihemChkiwa and Hayet Belhadj Kheder) for their helpin the botanical identification, assistance in anti-bacterial assays and for their providing languagehelp.

References1. Bauer, A.W., Kirby, W.M.M., Sherries, J.C., Tuck, M. (1996). Antibiotic susceptibility testing

by a standardized disc method. American Journal of Clinical Pathology. 45: 493-6.


Dow

nloa

ded

by [

Lin

köpi

ng U

nive

rsity

Lib

rary

] at

16:

17 2

4 A

ugus

t 201

4

2. Burjai, T. (2008). Ethnopharmacological survey of medicinal plants in Jordan. Mujib NatureReserve and surrounding area. J. Ethnopharmacol. 120: 63-71.

3. Bougatsos, C., Meyer, J.J.M., Magiatis, P., Vagias, C., ChinouI.B. (2003). Composition andantimicrobial activity of the essential oils of Helichrysum kraussii Sch. Bip and Helichrysumrugulosum Less from South Africa. Flavour Frag. J. 18: 48-51.

4. Buritis, M. and Bukar, F. (2000). Antioxidant activity of Nigelle sativa essential. PhytotherRes. 14: 323-328.

5. Chabbert, Y.A. (1972). Les antibiotiques en bactériologie médicale, in: Daguet, G.L., Chabbert,Y.A. (Eds), Techniques en bactériologie, Tome 3. Flammarion Ed, Paris, p.158.

6. Cox, S.D., Mann, C.M., Markham, J.L., Bell H.C.,Gustafson, J.E., Warmington, J.R., Wyllie,S.G. (2000). The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (teatree oil). J. Appl Microbiol. 88: 170-175.

7. Cuendent, M., Hosettmann, K., Potterat, O. (1997). Iridiod glucoside with free radicalscavengeng properties from Fragraea blumei. Helv. Chim Acta. 80: 1144-1152.

8. Pattnaik, S., Subramanyam, V.R., Bapaji, M., Kole, C. R. (1997). Antibacterial and antifungalactivity of aromatic constituents of essential oils. Microbios. 89: 39-46.

9. Decker, E. A. (1997). Phenolics: Prooxidantsor antioxidants? Nutr Rev. 155: 396-398.10. Delaquis, P.J. Stanich, K., Girard B., Mazza, G. (2002). Antimicrobial activity of individual

and mixed fractions of dill, cilantro, coriander and eucalyptus essential oils. Int. J. Food Microbiol.74: 101-109.

11. Elbeyreth, M., ellya, A., Nnick, D.D., Dupont, F. (2008). Plants used as remedies antirheumaticand antineuralgic in the traditional medicine of Lebanon. J. Ethnopharmacol. 120: 315-334.

12. Elfraim, L. Amar, Z. (2002). Ethnopharmacological survey of traditional drugs sold in theKingdom of Jordan. J. Ethnopharmacol. 82: 131-145.

13. Ghannoum, M.A., Rex, J.H., Galgiani, J.N. (1996). Susceptibility testing of fungi: currentstatus of correlation of in vitro data with clinical outcome. J. Clin. Microbiol. 34: 489-495.

14. Gill, A.O., Delaquis, P., Russo, P., Holley, R.A. (2002). Evaluation of antilisterial action ofcilantro oil on vacuum packed ham. Int. J. Food Microbiol. 73: 83-92.

15. Hudaib, M., Ohammad, M., Bustanjib, Y., Ayyema, R., Yousef, M., Abuirjeied, M., Burjai,T. (2008). Ethnopharmacological survey of medicinal plants in Jordan. Mujib Nature Reserveand surrounding area. J. Ethnopharmacol. 120: 63-71.

16. Said, O., Khalil, K., Ulder, S., Azaizeh, H. (2002). Ethnopharmacological survey of medicinalherbs in Israel, the Golan Heights and the West Bank region. J. Ethnopharmacol. 83: 251-265.

17. Hatano, T., Kagawa, H., Yasuhara, T., kuda, T. (1988). Two new flavonoids and other consti-tuents in licorice 330 root. Their relative astringency and radical scavenging effects. Chem.Pharm Bull. 36: 2090-2097.

18. Kokoska, L., Polesny, Z., Rada, V., Povim, A., Vanek, T. (2002). Screening of some Siberianmedicinal plants for antimicrobial activity. J. Ethnopharmacol. 82: 51-53.

19. Kusmenoglu, S., Baser, K.H.C., Ozek, T. (1995). Constituents of the essential oils from thehulls of Pistacia vera L. J. Essent. Oil Reseach. 7: 441-442.

20. Williamson, E.M., Okpako, D.T. (1996). Pharmacological Methods in Phytotherapy Research.Evans, F.J 1, 1555. Wiley, New York,

21. Karpin´ska, M., Borowski, J., Danowska-Oziewicz, M., (2001). The use of natural antioxidantsin ready-to-serve food. Food Chem. 72: 5-9.

22. Li, C., Yong, S., Liu, Y , Sun, H. Jin, W., Li, Z., Zhao. X.H., Le Pan, R. (2009). Essential oilcomposition, antimicrobial and antioxidant properties of Mosla chinensis Maxim. Food chem.115: 801-805.

23. Marmonier, A.A. (1987). Techniques usuelles Antibiotiques, Technique de diffusion en gélose


Dow

nloa

ded

by [

Lin

köpi

ng U

nive

rsity

Lib

rary

] at

16:

17 2

4 A

ugus

t 201

4

méthode des disques, Bactériologie médicale. SIMEP SA-PARIS, France, Chap 4, pp. 238-244.24. Miller , N.J., Rice-Evans, C.A., Avies, M.J., Gopinathan, V., Milner, A. (1993). A novel method

for measuring antioxidant capacity and its application to monitoring the antioxidant status inpremature neonates. Clin. Sci. 84: 407-412.

25. Morton H.E., Alcohols, I. Block, S.S. (1983). Ed., Disinfection, Sterilization and Preservation.Lea and Febiger: Philadelphia, 225-239.

26. Mourrey, A., Canillac N. (2002). Anti-Listeria monocytogenes activity of essential oils compo-nents of conifers. Food Control 13, 289–92.oil.Phytother Res. 14: 323-328.

27. Pauli, A. (2001). Antimicrobial properties of essential oil constituents. Int. J. Arom. 11: 126-133.

28. Perry, G., Raina, A.K., Nunomura, A., Wataya, T., Sayre, L.M., Smith, M.A. (2000). Howimportant is oxidative damage? Lessons from Alzheimer’s disease. Free Radical Biol. Med. 28:831-834.

29. Rice-Evans, C.A., Sampson, J., Bramley, P.M., Holloway, D.E. (1997). Why do we expectcarotenoids to be antioxidants in vivo? Free Radical Res. 26 (15) 381-398.

30. Smith-Palmer, A., Stewart, J., Fyfe L. (1998). Antimicrobial properties of plant essential oilsand essences against five important food-borne pathogens. Lett. Appl. Microbiol. 26: 118-122.

31. Tsuchiya, H., Sato, M., Miyazaki, S. et al. (1996). comparative study on the antibacterial activityof phytochemical flavones against methicillin-resistant Staphyloccus aureus. J. Ethnopharmacol.50: 27-34.

32. Ultee, A., Kets E.P.W., Smid E.J.(1999). Mechanisms of actions of carvacrol on the food-borne pathogen Bacillus cereus. Appl. Environ Microbiol. 65: 4606-4610.

33. Van den Berg, R., Haenen Guido, R.M., Van den Berg, M., Bast, H, A. (1999). Applicabilityof an improved Trolox equivalent antioxidant capacity (TEAC) assay for evaluation of antioxidantcapacity measurements of mixtures. Food Chem. 66: 511-517.

34. Wojdyyo, A., Szmianski, J., Zemerys, R. (2007). Antioxidant activity and phenolic compoundsin 33 selected herbs. Food Chem. 105: 940-949.

35. Yayli, N., Yacar, A., Gulec, C., Usta, A., Kolayli, S., Coskuncelebi, K., Karaoglu, S. (2005).Composition and antimicrobial activity of essential oils from Centaurea sessilis and Centaureaarmena. Phytochemistry 66: 1741-174.


Dow

nloa

ded

by [

Lin

köpi

ng U

nive

rsity

Lib

rary

] at

16:

17 2

4 A

ugus

t 201

4

chemical composition antioxidant and antimicrobial activities of the essential oils of ...

Documents