Antimicrobial, Antioxidant Activity of the Essential Oil of Bay Laurel from Hatay, Turkey

Antimicrobial, Antioxidant Activity of theEssential Oil of Bay Laurel from Hatay, Turkey

Ebru Sebnem Yilmaz 1,, Mahir Timur 2*, and Belma Aslim 3

1 Mustafa Kemal University, Faculty of Art and Science, Department of Biology,Tayfur Sokmen Campus, 31024-Antakya, Hatay, Turkey

2 Mustafa Kemal University, Faculty of Art and Science, Department of Chemistry,Tayfur Sokmen Campus, 31024-Antakya, Hatay, Turkey

3 Gazi University, Molecular Biology Research Center, Golbasi, Ankara, Turkey

Abstract: The aim of this study was to evaluate the antimicrobial and antioxidant activity of theessential oil (EO) extracted from the leaves of Laurus nobilis L. The oil was analyzed by gas chromatographyand mass spectrometry (GC-MS). Twenty seven components were identified, representing 96.6 % of the EO.The main compounds identified were 1,8-cineole (51.8 %), α-terpinyl acetate (11.2 %), and sabinene (10.1%). The oil was screened for possible antioxidant activity using two complementary test systems: DPPH (2,2-diphenylpicrylhydrazyl) free radical-scavenging and the β-carotene/linoleic acid assay. Both of these in vitromethods showed that the EO was a less powerful reducing agent than the well-known synthetic antioxidants,butylated hydroxytoluene and ascorbic acid. Also, the antimicrobial activity of the EO was tested against apanel of food-spoiling bacteria and one yeast strain. The minimum inhibitory concentration values formicroorganisms that were sensitive to L. nobilis EO ranged from 125-2000 μg/mL.

Key words: Laurus nobilis; Essential oil; Anti-microbial activity; Antioxidant activity; GC-MS.

IntroductionFor many years, various medical plants have

been used to treat diseases all over the world.Turkey is an important international floristiccenter because of its geographic location, climate,and the presence of nearly 10,000 natural plantspecies. According to a study by the World HealthOrganization (WHO) based on pharmacopoeiasand medical plants in 91 countries, there arealmost 20,000 medical plant species 1. Thecharacteristics of plants that inhibit the growth ofmicroorganisms and are therefore important forhuman health have been studied since 1926 2,3 .Traditional medical treatments used in daily life

are now being used alongside empirical methods.Laurus nobilis L. (bay) is an evergreen tree orshrub that belongs to the Lauraceae family and iscultivated in many temperate and warm parts ofthe world, particularly the Mediterraneancountries of Turkey, Greece, Spain, Portugal andMorocco, and in Mexico. In Turkey, the plant isnatively cultivated in coastal areas to an altitudeof 600-800 m4. Both the leaves and berries arewidely used. Dried leaves, also called ‘sweet bay’,and the essential oils (EO) they contain have astrong spicy aroma and are widely used as flavorenhancers for foods such as meats, soups, saucesand confectionery 5. Chemical studies have

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

*Corresponding author (Mahir Timur)E-mail: < [email protected] > © 2013, Har Krishan Bhalla & Sons

TEOP 16 (1) 2013 pp 108 - 116 108

Received 24 April 2012; accepted in revised form 29 August 2012

identified 1,8-Cineole as the major component ofthe essential oil in laurel leaves 6.

Recent studies show that laurel leaves and theirEO possess functional activities. Hence, severalstudies have evaluated the potential role of laurelEO as an antimicrobial agent 7,8,9 and others haveexamined the antioxidant properties of some leafextracts 10,11.

The present study examined the antimicrobialand antioxidant activities of EO from laurelleaves. Antimicrobial activity was measured usingagar-well diffusion and broth microdilutionmethods. Antioxidant activity was determinedusing two complementary assays: DPPH (2,2-diphenylpicrylhydrazyl) free radical-scavengingand the β-carotene/linoleic acid assay. Thechemical composition of the EO was alsoevaluated using GC-MS.

ExperimentalChemicals

Nutrient Broth (NB) was obtained from OxoidLtd. (Basingstoke, UK). Tryptic Soy Broth (TSB),Yeast Extract Potato Dextrose Agar (YEPDA),Tween 40, chloroform, anhydrous sodiumcarbonate, Folin-Ciocalteu’s phenol reagent andethanol were purchased from Merck (Darmstadt,Germany). Erythromycin, cefoxitin andflucanazole were acquired from Bioanalyse Co.,Ltd. (Ankara, Turkey). β-carotene, dimethyl-sulphoxide (DMSO), 2,2-diphenyl-1-picrylhydrazyl (DPPH), gallic acid, 2,6-di-tert-butyl-4-methylphenol (BHT), ascorbic acid (AA)and linoleic acid were purchased from Sigma-Aldrich GmbH (Steinheim, Germany).

Plant materialsLaurus nobilis L. leaves were harvested in

Antakya-Hatay, Turkey, during July and October2007. They were separated from their stem partsand stored air-dried until use.

Isolation of EOAir-dried samples were subjected to steam

distillation for 4 h in a Clevenger-type apparatus(yield = 2.3–2.5 %, v/w). The EO was dried overanhydrous CaCl2 and stored in sealed vials at lowtemperature before analysis. The EO yield was

estimated according to the amount of dry leavesusing the following equation 12.

RHE (%) = (mHE/mS) x 100

where mHE = EO mass (g), mS = dry leaves (g),and RHE = EO yield (%).

Gas chromatography analysisThe chemical composition of the EO was

analyzed using GC-MS, performed using aHewlett Packard GCD (model 6890) and aHewlett Packard MS (model 5972) equipped witha mass selective detector (MSD). An HP-5 column(bonded and cross-linked 5% phenyl-methylpolysiloxane, 30 m × 250 μm, coatingthickness: 0.25 μm) and an HP 18593B automaticinjection system were also used. EO (30 μL) weretransferred into 1 ml of diethyl ether (Merck) andinjected into the GC-MS sampling port. Thechromatogram was produced by holding the oventemperature at 45°C for 5 min initially and thenincreasing the temperature to 130°C at a rate of2°C/min, followed by 3°C/min to 170°C. Theoven was then set at 220°C (reached at a rate of10°C/min) and the temperature was kept constantfor 5 min. The MSD conditions were as follows:capillary direct interface temperature = 250°C;ionization energy = 70 eV; mass range = 33-330amu; EM voltage (Atune +200); and scan rate =5 scans/s. Helium (99.9 %) was used as the carriergas with a flow rate of 1.5 mL/min.

Identification of the components was based ona comparison of their mass spectra with those inthe Wiley GC-MS and NBS spectra libraries.Relative percentage amounts of the separatedcompounds were calculated automatically fromthe peak areas of the total ion chromatograms.

Microbial strains and preparation of inoculatesThe EO was tested against individual micro-

organisms, including Escherichia coli 0157:H7,Staphylococcus aureus ATCC 25923, Salmonellaenteritidis ATCC 13076, Shigella sonnei RSKK8177, Listeria monocytogenes ATCC 7644,Pseudomonas aeruginosa ATCC 29212,Campylobacter jejuni ATCC 33291,Staphylococcus epidermidis ATCC 12228, and

Ebru Sebnem Yilmaz et al., / TEOP 16 (1) 2013 108 - 116 109

Candida albicans ATCC 16231. All strains wereprovided by Gazi University, Biology Depart-ment, Biotechnology Culture Collection (Ankara,Turkey). NB, TSB and YEPDA were used forculturing the test microorganisms.

Inoculums of the microbial strains wereprepared using 24- or 48-hour-old culturessuspended in sterile 0.9 % sodium chloride andadjusted by comparison with 5.0 McFarlandStandards.

Determination of minimum inhibitory concen-tration (MIC)

The microdilution broth susceptibility was usedto determine the MIC 13. In brief, a stock solutionof the EO was prepared in 10 % DMSO, and serialdilutions were prepared to yield concentrationsof 7.8-2000 μg/mL. Test tubes were prepared bydispensing 950 μL of NB, 1000 μl EO (dissolvedin 10 % DMSO) and 50 μL of inoculant into thetube. The final volume in each tube was 2 mL. Apositive control (containing inoculum but no EO)and a negative control (containing EO but noinoculum) were included in each tube. Thesolutions in the tubes were mixed and thenincubated at the optimal temperature for 24 h forbacteria and 48 h for yeast. After incubation, theMIC of each sample was determined by visualinspection of the tubes. The lowest concentrationof the active ingredient that inhibited growth, asdetected by a lack of visual turbidity, was takento be the MIC. The minimum bactericidal andfungicidal concentration was determined bypreparing subcultures from the tubes that did notshow any growth. Each test was performed intriplicate.

Inhibitory effects assayed using the agar welldiffusion method

The inhibitory effect of the EO on the testbacteria was determined using the agar diffusionmethod 14. Bacterial cultures were grown at 37°Cfor 24 h in NB. C. jejuni ATCC 33291 wascultured overnight at 42°C, L. monocytogenesATCC 7644 was cultured overnight at 37°C inTSB, and C. albicans ATCC 16231 was culturedfor two days at 30°C in YEPDA. The culturesuspensions were adjusted by comparison with

5.0 McFarland Standards. Petri dishes containing20 mL of Nutrient Agar, which had beenpreviously inoculated with 200 μL of culturesuspension, were prepared. Wells (7.0 mm) weremade in the agar and the EO (20 μL; diluted inethanol to a test concentration (1/5) was added.Ethanol alone (20 μL) was used as the control.Antimicrobial discs containing erythromycin (15μg/disc), cefoxitin (30 μg/ disc) and the antifungalflucanazole (20 μg/disc) were used as positivecontrols. The inoculated plates were thenincubated under optimal conditions. Afterincubation, the diameter of the inhibition zone wasmeasured using calipers. Measurements weremade from the edge of the zone to the edge of thewell.

DPPH free radical-scavenging assayThis spectrophotometric assay uses the stable

DPPH radical as the reagent 15. Radical-scavenging activity is measured using the methodof Blois, which is based on the reduction of DPPHin methanol 16. Briefly, 1 mL of EO (at variousconcentrations in methanol) was added to 1 mLof the DPPH at 0.004 % methanol solution. Themixture was then shaken vigorously and left tostand at room temperature for 30 min in the dark.The absorbance was then measured at 517 nmagainst a blank using a spectrophotometer(Hitachi, U-1800, Tokyo, Japan). The percentageinhibition of DPPH free radical formation (I %)was calculated using the formula:

I% = (Ablank – Asample)/Ablank × 100

where Ablank is the absorbance of the controlreaction (containing all reagents apart from thetest compound) and Asample is the absorbance ofthe test compound. The EO concentration causing50 % inhibition (IC50) was calculated by plottingpercentage inhibition against EO concentration.All tests were carried out in triplicate. BHT andAA were used as positive controls.

βββββ-Carotene-linoleic acid assayIn this assay, the antioxidant activity is

determined by measuring the inhibition of avolatile organic compound and the conjugated


diene hydroperoxide products of linoleic acidoxidation 17. The antioxidant activity of the EOwas evaluated using the spectrophotometric β-carotene bleaching test 18. A stock solution of β-carotene-linoleic acid was prepared as follows:β-carotene (0.5 mg) was dissolved in 1 mL ofchloroform (HPLC grade). Linoleic acid (25 μL)and Tween 40 (200 mg) were then added. Thechloroform was evaporated in a vacuumevaporator, and 100 mL of distilled water wasadded with oxygen (30 min at a flow rate of 100mL min-1) with vigorous shaking. Aliquots (2.5mL) of this reaction mixture were then dispensedinto test tubes and 350 μL of EO (2 g/L stock)was added. The emulsion system was incubatedfor up to 48 h at room temperature. The procedurewas repeated using the synthetic antioxidants,BHT and AA as positive controls, and a blankcontaining 350 μL of ethanol alone. Theabsorbance of the mixtures was then measured at490 nm and the anti-oxidative capacity of the EOcompared with that of the controls and the blank.

Total Phenolic Content (TPC)The TPC of the EO was analyzed using the

Folin-Ciocalteu reagent according to the methodof Singleton and Rossi, with some modifications,using gallic acid as the standard 19. In brief, theEO solution (0.1 mL) was mixed with 0.2 mL of50 % Folin-Ciocalteu reagent. The mixture wasthen allowed to react for 3 min, followed by theaddition of 1 mL aqueous 2 % Na2CO3. Themixture was then vortexed vigorously. After afurther 45 minute incubation at room temperature,the absorbance of each mixture was measured at760 nm. The same procedure was applied tostandard solutions of gallic acid. The total phenolcontent was expressed as μg gallic acidequivalents/mg of EO.

Statistical analysisAll experiments were performed in triplicate

and data were expressed as mean values.Statistical analysis was performed using SPSS11.0 Bivariate Correlation Analysis (SPSS Inc.,Chicago, IL, USA.) and statistical significancewas determined at P < 0.05. The Pearson rankcorrelation test was used for comparisons between

the broth microdilution and agar well diffusionmethods used to determine the antimicrobialactivity of the EO.

Results and discussionMany secondary metabolites of plants,

including a wide variety of phytochemicals, havebeen isolated and identified 20,21. Of these,aromatic EO show useful antibacterial, antifungal,antiviral and anti-inflammatory properties.Generally, these EO have a terpenoid structureand their effects are mediated via a combinationof constituents, which in some oils may number>100 different compounds 22 .

In the present study, the aerial portion of theBay plant was steam distilled for 3 h in aClevenger-type apparatus to yield 2.5 %, v/wyellow L. nobilis EO. All the test microorganismswere sensitive to the EO, but E. coli O157:H7, C.albicans ATCC 16231, S. enteritidis ATCC 13076and L. monocytogenes ATCC 7644 were the mostsensitive. Both the inhibition zones around theagar wells and the MIC values for the strainssensitive to L. nobilis EO ranged from 6.0-33.0mm and 125->2000 μg/ml, respectively (Table 1).

E. coli O157:H7, C. albicans ATCC 16231, S.enteritidis ATCC 13076 and L. monocytogenesATCC 7644 had MIC values of 125, 250 and 500μg/ml, respectively, and showed the largestgrowth inhibition halos in the agar well diffusionassays (33.0, 26.0, 24.0 and 22.0 mm,respectively). The inhibition zones in each assayshowed a significant correlation with the MICvalues (P < 0.05). The control (absolute alcohol)did not show an inhibitory effect on any of thebacteria tested. The sensitivity of the testedmicroorganisms to erythromycin, cefoxitin andflucanazole are shown in Table 1. The EO showedboth antimicrobial and antifungal activity againstnine different microbial species.

E. coli O157:H7, S. enteritidis, L. monocyto-genes and C. jejuni are some of the most importantfood-contaminating pathogenic bacteria. Manycompounds are used to prevent the growth of thesepathogens in foods, and the use of plant extractsand/or EO is gaining popularity 23,24 . Also, foodscientists found that the terpenoids present in plantEO are useful for the control of L. monocytogenes


25. Erdogrul found that extracts of L. nobilisinhibited the growth of some bacteria and fungi,particularly Mycobacterium smegmatus, Yersiniaenterocolitica, S. aureus, L. monocytogenes andBacillus megaterium 26. Dadalýoglu andEvrendilek indicated that EO from bay laurel hasvery strong antimicrobial activity against E. coliO157:H7, L. monocytogenes, S. typhimurium andS. aureus. L. nobilis (L.), which is a natural florafrom Turkey and is widely used as a traditionalmedicine and a flavor enhancer in foods, may alsobe used as a preservative against food-bornepathogens 7.

The composition of L. nobilis EO obtained byGC-MS analysis is shown in Table 2. Twenty-seven compounds were identified, representing96.6 % of the total oil. The oil yield of the plantwas 2.3-2.5 %, v/w. GC-MS analysis identified1.8-cineole (51.8 %), α-terpinyl acetate (11.2 %)and sabinene (10.1 %) as the major compounds.Other important compounds were α-terpineol (5.2%), terpinen-4-ol (3.1 %) and eugenol (0.4 %).Sabinene and 1,8-cineole are the majorcomponents of the EO obtained from Turkish L.nobilis in Hatay 7,27. Akgul et al., reported thatbay leaves contained mostly 1,8-cineole, eugenol,acetyl eugenol, methyl eugenol and terpineol 28.Previous studies report similar results 29,30. It isclaimed that the qualitative and quantitative

differences in EO composition depend on morethan one component and on different conditions31. The present study supports the finding that 1,8-cineole is a major component of L. nobilis EO ofTurkish origin. Plant EOs are active against a widerange of organisms. Studies of EO from variousaromatic plant species 32 show that the most activecomponents of the oils are 1,8-cineole andterpinene-4-ol, which is agreement with the resultsof the present study. In addition, 1,8-cineole showsactivity against Gram-positive and Gram-negativebacteria, and yeast, including L . monocytogenes.Oke et al.33 also published results similar to thosedescribed in the present study.

Bullerman et al., showed that cinnamaldehydeand eugenol completely inhibit fungal growth andaflotoxin production 34. Politeo et al., indicatedthat EO from laurel, eugenol and methyl eugenolmay be considered the main mediators ofantioxidant activity 10. The antioxidant activity ofeugenol has been reported in several studies 35-37.There are few reports regarding the radical-scavenging activity of the constituents of baylaurel leaves 38.

In the present study, the antioxidant activity ofL. nobilis EO was tested using the DPPH radical-scavenging and β-carotene/linoleic acid bleachingassays. The effect of antioxidants on DPPH radicalscavenging is thought to be due to their hydrogen-

Table 1. Antimicrobial activity of essential oil of L. nobilis

Inhibition zone diameter (mm)Test Bacteria MIC(μg/ml) Agar Well Antibiotics

Diffusion Erythromycin Cefoxitin Flucanosol

E. coli O157:H7 125 33.0±0.2 R 16.4±0.2 NS.C. jejuni ATCC 33291 500 20.0±0.3 13.9±0.6 6.4±0.2 NS.S. sonnei RSKK 8177 1800 6.0±0.1 R 11.2±0.2 NS.S. aureus ATCC 25923 >2000 10.0±0.2 2.1±0.7 10.9±0.2 NS.L. monocytogenes ATCC 7644 500 22.0±0.2 16.8±0.2 4.0±0.2 NS.P. aureginosa ATCC 29212 >2000 8.2±0.1 R R NS.S. enteritidis ATCC 13076 250 24.0±0.2 R R NS.S. epidermidis ATCC 12228 1200 13.0±0.4 1.4±0.2 10.8±0.4 NS.C. albicans ATCC 16231 250 26.0±0.2 NS. NS. 10.7±0.3

Values represent averages ± standard deviations for triplicate experimentsR = no inbition zone; NS = Not Studied.


donating ability 39. As the radical is scavenged byantioxidants through the donation of hydrogen toform stable DPPH-H molecules, the color changesfrom purple to yellow, which causes a decreasein the measured absorbance 39,40. The free radical-scavenging properties of EO are shown in Table3. Low IC50 values correspond to a higherantioxidant capacity 11 L. nobilis EO (IC50 = 59.2± 2.3 μg/mL) showed weak scavenging activityagainst DPPH radicals. The DPPH-scavengingability of the EO was also lower than that of thesynthetic antioxidants BHT and AA (21.3 ± 1.5μg/mL and 5.8 ± 0.10 μg/mL, respectively).

Politeo et al., 10 measured the DPPH radical-scavenging activity of laurel EO (89.6 %inhibition) and compared it with that of volatileaglycones (91.4 % inhibition). The results of thepresent study showed that the radical-scavengingactivity of the test samples was: AA > BHT > EO;similar results were obtained by Politeo et al.,.Conforti et al., and Santoyo et al., also studiedthe DPPH-radical scavenging activity of differentlaurel extract fractions 10,11,39.

The β-carotene bleaching method is based onthe loss of the yellow color of β-carotene due toits reaction with radicals formed by the oxidation

Table 2. Chemical compositions of L. nobilis essential oil

No Compounds RT Composition (%)

1 α-Thujene 9.4 0.22 α-Pinene 9.7 3.73 Sabinene 12 10.14 β-Pinene 12.1 2.85 Myrcene 13.1 0.96 α-Phellandrene 13.8 0.57 1,8-Cineole 15.7 51.88 γ-Terpinene 17.3 0.59 trans-Sabinene hydrate 17.8 0.6

10 cis-Sabinene hydrate 19.9 0.411 Linalool 20.2 1.912 Pinacarvone 24.1 0.113 Terpinen-4-ol 25.3 3.114 α-Terpinenol 26.2 5.215 Bornyl acetate 30.7 0.116 Pseudolimonene 31.9 0.417 α-Terpinyl acetate 33.1 11.218 Eugenol 33.3 0.419 Neryl acetate 33.6 0.320 β-Elemene 34.4 0.421 Methyl eugenol 34.8 0.822 Germacrene 37 0.123 Bicyclogermacrene 37.4 0.224 β-Eudesmol 41.3 0.325 Elemol 41.3 0.126 Eremanthin 48.7 0.127 1,2 Benzenedicarboxylic acid 70.0 0.4

Total 96.6

RT: Retention time


Table 3. Antioxidant activity of L. nobilis essential oila and positive controls

Material DPPH IC50 βββββ-carotene bleaching Total phenol contents(μμμμμg/ml) (RAA) (%) (μμμμμg/mg)

Essential oil 59.2±2.3 76.8±0.5 112.3±0.3BHT 21.3±1.5 100±0.0 NSb

AA 5.8±0.1 95.6±2.1 NSb

a Values represent averages ± standart deviations for triplicate experiments.b Not studied

of linoleic acid in an emulsion. The rate of β-carotene bleaching slows down in the presenceof antioxidants 41. The relative antioxidant activity(RAAs) of the EO was calculated using thefollowing equation:

RAA = Asample/ABHT,

where ABHT is the absorbance of the control(BHT) and Asample is the absorbance of the EO.The calculated RAAs for the EO and standardantioxidant compounds (BHT and AA) are givenin Table 3.

The inhibition values for linoleic acid oxidationwere estimated to be 76.8 ± 0.5 %, 100 %, and95.6 ± 2.1 % in the presence of EO, BHT andAA, respectively. There was a relationshipbetween the DPPH-scavenging ability and β-carotene bleaching.

EO showed weaker antioxidant activity in bothassays. Santoyo et al., found that supercriticalfluid extracts from laurel leaves showed varyingdegrees of antioxidant activity, whereas Confortiet al., reported that β-carotene bleaching by thewild-type L. nobilis extract was greater than thatby the cultivated-type extract.

The TPC of the EO was determined spectro-photometrically according to the method of Folin-Ciocalteu and the results were expressed as gallicacid equivalents. Gallic acid is a water-solublepolyhydroxyphenolic compound found in various

natural plants, such as grapes, strawberries,bananas, and many other fruits 30,42. The standardcurve equation used was: y (absorbance) = 0.0067× (μg gallic acid) + 0.3005, R2 = 0.9999. Theabsorbance value was inserted in the aboveequation and the total amount of phenoliccompound was calculated. As seen in Table 3, theTPC of the EO was 112.3 ± 0.3 μg/mg. Accordingto these results, there is a relationship betweenTPC and antioxidant activity. Phenolic consti-tuents are very important to plants because of theirscavenging activity (due to the presence ofhydroxyl groups) and may contribute directly toantioxidant activity 43. In the present study, 1,8-cineole (51.8 %), which is a phenolic component,was identified as the major compound; therefore,the EO from L. nobilis showed high antioxidantand antimicrobial activity. The results of thepresent study show that EO from L. nobilis is avaluable antioxidant.

ConclusionsThe results of this in vitro study suggest very

important implications for the antibacterial,antifungal and antioxidant potential of L. nobilisEO. Laurel EO can be used as an easily accessibleand rich source of natural antioxidants, as a foodsupplement, against selected food-borne pathogenbacteria, or in the pharmaceutical industry. Furtherwork is necessary to determine the effects ofdifferent laurel extracts.

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