Food and Nutrition Sciences, 2014, 5, ***-***

Published Online January 2014 (http://www.scirp.org/journal/fns)

Antioxidant and Antimicrobial Activities of Essential Oils

Extracted from Laurus nobilis L. Leaves by Using
Solvent-Free Microwave and Hydrodistillation
Sedef Nehir El1*, Nural Karagozlu2, Sibel Karakaya1, Serpil Sahın3
1
Department of Food Engineering, Faculty of Engineering, Ege University, Izmir, Turkey; 2Department of Food Engineering, Faculty
of Engineering, Celal Bayar University, Manisa, Turkey; 3Department of Food Engineering, Faculty of Engineering, Middle East
Technical University, Ankara, Turkey.
Email: *sedef.el@ege.edu.tr

Received *************** 2014

Copyright © 2014 Sedef Nehir El et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accor-
dance of the Creative Commons Attribution License all Copyrights © 2014 are reserved for SCIRP and the owner of the intellectual
property Sedef Nehir El et al. All Copyright © 2014 are guarded by law and by SCIRP as a guardian.

ABSTRACT
In this study, laurel essential oils were obtained by using solvent-free microwave extraction (SFME) and hydro-
distillation (HD) methods from Laurus nobilis leaves and determined their antioxidant and antimicrobial activity.
Extraction time was reduced by about 43% in SFME at 622 W and 67% in SFME at 249 W compared to hydro-
distillation. Essential oil of laurel was extracted by SFME at 622 W (100%) and 249 W (40%) power levels and
HD inhibited oxidation generated by ABTS radical by 93.88%, 94.13% and 92.06%, respectively. Trolox equiv-
alent antioxidant capacities (TEAC) of essential oils were 0.18 mM/mL oil for SFME at 622 W, 1.36 mM/mL oil
for SFME at 249 W and 2.40 mM/mL oil for HD (p < 0.05). Essential oils of L. nobilis were extracted by SFME at
100% and 40% power levels and HD inhibited linoleic acid peroxidation by 70.57%, 63.53% and 89.18% respec-
tively. Inhibition effects of laurel essential oils obtained by SFME at different power levels and HD on DPPH
radical cation oxidation were not significantly different. The strongest antioxidant activity against DPPH radical
was found in the essential oil obtained by SFME at 100% power level. Essential oils displayed antimicrobial ac-
tivity against Staphylococcus aureus 6538P, Escherichia coli O157:H7 and Salmonella typhimurium NRRL E 4463
except Listeria monocytogenes. The inhibitory effect on Staphylococcus aureus 6538P survival of laurel oil ob-
tained from SFME by using lower power level was found to be lower than that obtained from SFME at 100%
power level and HD (p < 0.05).

KEYWORDS
ABTS; Antimicrobial Activity; Antioxidant Activity; DPPH; Laurel; Laurus nobilis; Solvent-Free Microwave
Extraction; Hydrodistillation

1. Introduction compounds as an alternative to synthetic substances, has

been taken much attention in recent years. As a result,
Current research indicates that many chronic diseases,
interest has focused on different extracts of plants as
including cardiovascular diseases and at least some types
sources of natural antioxidants since they are rich bioac-
of cancer, are initiated by free radical oxidation of lipids,
tive organic chemicals and biodegradable to nontoxic
nucleic acids, or proteins [1]. Therefore, investigating the
products. Herbs and spices are important natural antioxi-
beneficial effects of dietary antioxidants has been the dants. Their antioxidant activity has been attributed to the
main goal of many studies. In addition, the need, which presence of polar phenolic compounds and essential oils.
is supported by the consumer’s concern about natural Particularly, many herbs and spices have been evaluated
products, for natural antioxidants and/or antimicrobial as sources of natural antioxidants and antimicrobial
*
Corresponding author. compounds [2-5].

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2 Antioxidant and Antimicrobial Activities of Essential Oils Extracted from Laurus nobilis L.
Leaves by Using Solvent-Free Microwave and Hydrodistillation

In ancient times, the plant named as “Daphne” was de- provided as dried leaves form by KÜTAŞ Agricultural
fined as Laurus nobilis by Goodyer in 1655. Leaves of L. Products Inc. (Izmir, Turkey). The specimens were col-
nobilis are also widely used as a spice, antiseptic, insec- lected in November 2006 and September 2007 from lo-
ticide and stomachic, and used in the past to treat rheu- cation namely Kemalpaşa (Izmir, Aegean region). DPPH
matism in European folk medicine. L. nobilis, commonly (2,2-Diphenyl-1-picrylhydrazyl, D9132), linoleic acid
known as bay, sweet bay and laurel, is an evergreen tree (L1376), phosphate buffer (P4417), hemoglobin (H2500),
native to the Mediterranean region especially in Turkey, FeCl2 (372870), methanol were purchased from Sigma-
Greece, Spain, Italy and France. Laurel leaves have been Aldrich (Germany), ABTS [2-2’-Azino-bis (3-ethyl-
used as flavoring ingredients in Mediterranean cuisine. benzothiazoline-6-sulfonic acid] Diammonium salt
They have been traditionally added to meat, fish and (11557) was obtained from Fluka. Ammonium thiocya-
poultry meals and also used in food industry as flavor nate was purchased from Merck.
enhancing ingredients in pickle, sauce and canned foods
in world and also in Turkey [6,7]. In addition to enhanc- 2.2. Extraction Methods
ing flavor, certain spices and their essential oils can pro-
long the storage life of foods by their antioxidant and/or 2.2.1. Solvent-Free Microwave Extraction (SFME)
antimicrobial activities [7-9]. Conforti et al. [10] indi- A domestic microwave oven with an interior cavity size
cated that beneficial effect of laurel leaves and their es- of 29 × 37 × 40 cm and operating at 2450 MHz was used
sential oils against digestive disorders such as flatulent for SFME (White-Westinghouse, KM90WP Model,
colic and increasing effect on gastric fluid secretion have Pittsburg, USA). The maximum power of the oven was
been shown in recent studies. Anticonvulsive and anti- 622 W (100%) that was determined by IMPI-2L test [16].
epileptic activities of the leaf extract have been con- Before SFME, 150 g of laurel leaves were soaked in 700
firmed. Although, L. nobilis does not have important uses ml distilled water at room temperature (25˚C) for 1 h to
in traditional medicine, recent studies have revealed that hydrate the external layers of the herb. At the end of the
L. nobilis leaves and their essential oil could possess soaking period excess water was drained off. The mois-
some functional activities. Hence, several studies have tened herb was placed in a flat-bottom flask connected to
screened the potential capacity of laurel essential oil as a Clevenger apparatus and SFME procedure was started.
an antimicrobial agent [11,12] and also the antioxidant Extractions were performed at 622 W, 85 min (100%)
property of some leaves extracts [6,3,13]. Improvements and 249 W, 130 min (40%) conditions. The essential oils
in the production technology of essential oils are quite were collected in amber colored vials, dehydrated with
important to obtain the highest yield and product quality. anhydrous sodium sulfate, capped under nitrogen and
It is well known that extraction procedure has a great kept at 4˚C until being analyzed.
impact on the quality of final product such as extraction
efficiency, retention of volatile compounds and bioactive 2.2.2. Hydrodistillation (HD)
components [14]. New technologies have been developed In conventional hydrodistillation, Clevenger apparatus
for obtaining essential oils to minimize the loss of vola- was used. A hemispherical heater (Thermal Laboratory
tile compounds, to improve extraction efficiency and to Equipments, Istanbul, Turkey) with a maximum power of
reduce the process time. Solvent-free microwave extrac- 200 W was used in the experiments. Distillation was
tion (SFME) is one of these new techniques that combine performed using dry leaves to water ratio of 1:10 for 195
microwave heating with dry distillation at atmospheric min. The essential oil was collected in amber colored
pressure for the isolation of essential oils [15]. vials, dehydrated with anhydrous sodium sulfate, capped
In this study, antioxidant activity (radical scavenging under nitrogen and kept at 4˚C until being analyzed.
activity against ABTS and DPPH radicals and inhibition
of linoleic acid peroxidation) and antimicrobial activity 2.3. Essential Oil Composition Analysis
of essential oils obtained from leaves of L. nobilis by
using solvent-free microwave (SFME) at different power Identification and quantitative analysis of essential oil
levels (622 W and 40 W) and hydrodistillation (HD) components were performed using gas chromatography
were investigated. Variation in the composition of the (Agilent Technologies 6890 N Network GC System, Palo
essential oils during SFME and hydrodistilation (HD) Alto, CA, US) and gas chromatography coupled to mass
was also examined. spectrometry (Agilent Technologies 6890 N Network GC
System coupled to Agilent Technologies 5973 Network
2. Materials and Methods Mass Selective detector, Palo Alto, CA,US). In order to
perform quantative analysis with Flame Ionization De-
2.1. Samples and Chemicals tector (FID) with the Mass Selective Detector (MSD)
Laurel (Laurus nobilis) cultivated in Turkey was kindly were placed.

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 Antioxidant and Antimicrobial Activities of Essential Oils Extracted from Laurus nobilis L. 3
Leaves by Using Solvent-Free Microwave and Hydrodistillation

2.4. Antioxidant Activity lank: Absorbance of the DPPH.

2.4.1. ABTS Free Radical-Scavenging Assay
2.4.3. Inhibition of the Linoleic Acid Peroxidation
ABTS radical scavenging activities of the samples were
Inhibition effect of the samples on the peroxidation of
determined by the method of Re et al. [17]. Briefly,
linoleic acid was determined by the method of Kuo et al.
ABTS was dissolved in water to obtain 7 mM concentra-
[20]. 10 µL of sample, 0.37 mL of 0.05 M phosphate
tion. ABTS radical cation was produced by reacting
buffer (pH 7.0) containing 0.05% Tween 20 and 4 mM of
ABTS stock solution with 2.45 mM potassium persulfate
linoleic acid were mixed in a test tube. This mixture
(final concentration) and allowing the mixture to stand in
equilibrated at 37˚C for 3 min. The peroxidation of li-
the dark at room temperature for 12 - 16 hours before use.
For the determination of antioxidant activity, the ABTS noleic acid in the mixture was initiated by adding 20 µl
radical solution was diluted with ethanol to an absor- of 0.035% hemoglobin prepared in water. Then, mixture
bance of 0.70 (±0.02) at 734 nm. After the addition of 1.0 incubated at 37˚C in a shaking water bath under 100 rpm
ml diluted ABTS radical solution to 10 µl of the sample, for 10 min. Reaction was stopped by the addition of 5
absorbance reading was recorded 5 min after initial mix- mL of 0.6% HCl prepared in ethanol. The hydroperoxide
ing using spectrophotometer (Cary 50 Scan UV-Visible formed was determined according to ferric thiocyanate
Spectrophotometer, Victoria, Australia). Percent inhibi- method. Absorbance readings were taken at 480 nm with
tion was calculated by using the following equation: a spectrophotometer (Cary 50 Scan UV-Visible Spectro-
photometer, Victoria, Australia). Antioxidant activity of
 (  )
1 − Asample AABTS solution  × 100
Inhibition % = (1) the sample was calculated according to the equation be-
low;
Equation (1) is Asample: Absorbance reading obtained
for the sample, AABTS solution: Absorbance reading obtained Inhibition ( % ) =1 − ( As − A0 A100 − A0 )  × 100 (3)
for ABTS solution.
Trolox equivalent antioxidant capacity (TEAC) Equation (3) is A0 = Absorbance obtained for reaction
The Trolox equivalent antioxidant capacity assay mixture that does not contain hemoglobin, A100 = Absor-
based on the reaction of ABTS radical with Trolox was bance obtained for reaction mixture that does not contain
performed in order to compare radical scavenging activ- sample, As = Absorbance obtained for reaction mixture.
ity of a sample to that of Trolox. The antioxidant activi-
ties of the samples were estimated within the range of the 2.5. Antimicrobial Activity
dose-response curve of Trolox and expressed as the Tro- Gram-positive and gram-negative bacterial species used
lox equivalent antioxidant capacity. The latter is defined in this study were kindly obtained from the culture col-
as the concentration of Trolox having the antioxidant lection of Microbiology Laboratory in Food Engineering
capacity equivalent to 1.0 mmol/l solution of the sub- Department of Ege University. The bacteria species in-
stance under investigation. In this study, the TEAC val- clude: Listeria monocytogenes Scott A, Staphyloccous
ues were expressed as µmol TEAC µl−1 sample [18]. aureus 6538P, Esherichia coli O157:H7 and Salmonella
typhimurium NRRLE 4463. Trypton Soya Broth (TSB,
2.4.2. DPPH Free Radical-Scavenging Assay Oxoid CM 129) was used as media for the development
The free radical scavenging activity was determined us- of the strains of pathogen cultures, whereas Plate Count
ing 1,1-Diphenyl-2-picryl-hydrazyl radical (DPPH) ac- Agar (Oxoid CM 325) was used for the enumeration.
cording to Braca et al. [19]. DPPH is a stable free radical, Inocula used in the antimicrobial assay were obtained
which has an unpaired valence electron at one atom of a from cultures grown on TSB at 35˚C for 24 h. Essential
nitrogen bridge. In this experiment, 50 µL of each extract oils were sterilized using filtration 0.45 µm millipore
was added to 950 µL of 0.030 mg/mL methanol solution filters. Antimicrobial tests were then carried out by the
of DPPH. Then, the mixture was shaken vigorously and disc diffusion method using 100 µL of suspension con-
left in darkness for 5 min. Finally; the absorbance of the taining 108 CFU/ml of pathogen bacteria spread on Nu-
mixture was measured against methanol (blank) at 515 trient Agar (NA Oxoid CM0003). The sterilized paper-
nm by using spectrophotometer (Cary 50 Scan UV-Visi- discs (5 mM in diameter) were impregnated aseptically
ble Spectrophotometer, Victoria, Australia). The DPPH with 10 µL of essential oil placed on the inoculated agar.
scavenging activity was expressed as the inhibition of the Three discs were placed on each petri plate. Sterilized
free radical DPPH; water was used as a control. Plates were kept at ambient
1 − ( Asample Ablank ) × 100
Inhibition ( % ) = (2) temperature for 1 h and then incubated at 37˚C for 24 h.
Antimicrobial activity was evaluated by measuring the
Equation (2) is Asample: Absorbance of the sample, Ab- zone of inhibition against the test organisms [21].

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4 Antioxidant and Antimicrobial Activities of Essential Oils Extracted from Laurus nobilis L.
Leaves by Using Solvent-Free Microwave and Hydrodistillation

2.6. Statistical Analysis one of the spectrophotometric methods that have been
applied to the measurement of the total antioxidant activ-
All experiments were conducted in triplicate and parallel.
ity [17]. Inhibition effects and TEAC values determined
Six values for each sample were expressed as mean ±
for laurel essential oils obtained by SFME methods at
standard deviation. Statistical analysis was performed
different power levels and hydrodistillation on ABTS
using SPSS for Windows (Version 10.0). Analysis of
radical cation oxidation were shown in Table 2. Al-
variance (one way ANOVA) was performed, and Tukey
though ABTS radical scavenging effect of laurel essen-
HSD multiple range test were used to determine signifi-
tial oils obtained by two extraction methods was found to
cant differences at p < 0.05 [22].
be high and similar, TEAC values of these essential oils
were significantly different (p < 0.05). Antioxidant activ-
3. Results and Discussion
ity of essential oil obtained by HD showed the greatest
This study is the part of our previous investigation TEAC value which indicated the strongest antioxidant
project dealing with extraction of essential oils from activity. The reason of the similarity between the inhibi-
some spices using novel technologies and determination tion effects of SFME and hydrodistillation extracts can
of composition, yield and physical properties. Part of our be explained by the fact that diluted essential oils are not
study related composition, yield and physical properties being used in the inhibition analysis (ABTS decoloriza-
of the essential oil of L. nobilis obtained by SFME and tion assay). In the ABTS decolorization assay, instead of
HD is presented Table 1. Maximum yields obtained us- the dose-response relationship, the inhibition capacity of
ing SFME at 622 W, 249 W power levels and hydrodis- the intact material against ABTS radical cation oxidation
tillation method were found to be 0.0235, 0.022 and is measured. Therefore, it is not possible to distinguish
0.022 mL oil/g laurel, respectively. No significant dif- differences among inhibition values that are above 90%.
ference was obtained between essential oil yields ob- However, in the TEAC method essential oils are diluted
tained by SFME and hydrodistillation methods. It was to three different concentrations to obtain three different
found that the time needed for the complete extraction of percentage inhibition values within the range of the dose-
essential oil of laurel at 622 W power was 85 min, while response curve of Trolox and expressed as the TEAC
it was 130 min in 249 W power. In the case of hydrodis- [24]. As far as our literature survey could ascertain,
tillation, the process time was 195 min. In addition, ex- ABTS radical scavenging activity of essential oils ob-
traction time was reduced by about 43% in SFME at 622 tained from L. nobilis have not been reported. There are
W and 67% in SFME at 249 W compared to hydrodistil- few reports regarding with antioxidant activities of the
lation. Both of the methods yielded essential oils with the extracts obtained from L. nobilis leaves. Kang et al. [2]
main component 1,8-cineole (630 - 731 mg/mL oil). The studied alkyl peroxy radical scavenging activity of the
main components of the essential oil of laurel were fol- leaves of L. nobilis cultivated in Turkey. They reported
lowed by α-terpinyl acetate (90 - 115 mg/mL oil), sabi- that only the ethylacetate soluble subfraction obtained
nene (45 - 55 mg/mL oil), α-pinene (30 - 40 mg/mL oil), from ethanol soluble fraction of L. nobilis leaves showed
4-terpineol (35 - 40 mg/mL oil) and β-pinene (~30 the most potent alkyl peroxy radical scavenging activity.
mg/mL oil). It was also found that the essential oil was The compound with the highest antioxidant activity iso-
composed mainly of oxygenated compounds (≥75%) lated from the ethylacetate soluble subfraction was iden-
while monoterpene hydrocarbons constituted about ≥15% tified as isoquercetrin. Ünver et al. [25] reported that
of the essential oil. The oxygenated compounds detected antioxidant activity of L. nobilis extract calculated as
in laurel essential oil were found to be mainly composed TEAC value against ABTS radical was 1.001 mmol TE/g
of ethers (70% - 75%), especially of 1,8-cineole (~70%). extract. In the study of Shan et al. [26] ABTS radical
The rest of the oxygenated constituents of the oil were scavenging activity of methanolic extract of L. nobilis
determined as alcohols, aldehydes, ketones, esters, lac-
was found to be 34.3 mmol Trolox/100g DW. Compari-
tones, and phenols (Table 1).
son of our result with the data reported by Ünver et al.
[25] and Shan et al. [26] could not be possible since the
3.1. Antioxidant Activity extraction methods and sample characteristics used in
Three different methods were used to determine antioxi- these studies were different.
dant activity. These methods are based on the generation
of a different radical and inhibition extent of the sca- 3.1.2. Antioxidant Activity against DPPH Radical
venging by antioxidant compounds which are hydrogen DPPH is a stable nitrogen-centered free radical, the color
or electron donors [17,23]. of which changes from violet to yellow upon reduction
by either the process of hydrogen- or electron-donation.
3.1.1. Antioxidant Activity against ABTS Radical Substances, which are able to perform this reaction, can
Generation of the ABTS radical cation forms the basis of be considered as antioxidants and therefore radical sca-

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 Antioxidant and Antimicrobial Activities of Essential Oils Extracted from Laurus nobilis L. 5
Leaves by Using Solvent-Free Microwave and Hydrodistillation

Table 1. Composition of L. nobilis essential oils obtained from SFME and HD.

Concentration (mg/ml)

Hydrodistillation SFME-622 W SFME-249 W

Compounds 195 min 85 min 130 min

1 α-thujene 3.45 3.29 2.67
2 α-pinene 38.98 38.89 32.62
3 camphene 2.87 2.92 2.43
4 sabinene 47.03 53.45 46.69
5 β-pinene 32.35 33.28 30.21
6 α-phellandrene 1.58 -- 0.90
7 α-terpinene 4.92 4.46 4.25
8 p-cymene 12.79 13.41 12.70
9 limonene -- -- 8.17
10 1,8-cineola 630.24 731.75 653.05
11 γ-terpinene 8.89 8.18 8.74
12 terpinolene 2.92 2.30 2.60
13 linalool 2.40 2.17 2.48
14 pinocarveol/trans-pinocarveol 9.83 11.30 12.07
15 camphor 0.89 -- 1.03
16 sabina ketone 3.67 4.40 5.72
17 pinocarvone 6.30 8.03 8.03
18 borneol 8.81 11.46 12.25
19 4-terpineol 36.78 40.47 42.56
20 α-terpineol 2.22 2.90 3.06
21 myrtenal 10.09 12.50 12.49
22 cuminal 3.06 3.43 3.50
23 carvone 2.75 3.01 3.33
24 bornyl acetate 4.54 4.83 4.37
25 cumic alcohol 1.62 2.64 2.47
26 pseudolimonene 7.14 8.02 8.64
27 α-terpynil acetate 90.28 107.30 115.32
28 eugenol 12.15 16.36 17.32
29 β-elemene -- 1.31 1.93
30 methyl eugenol 12.08 14.76 14.93
31 β-caryophyllene trans/cis-methyl -- 2.28 3.10
32 isoeugenol -- 1.63 1.00
33 β-eudesmol dehydrocostuslacto 4.04 4.84 3.89
34 ne (?) 0.97 5.15 1.83
35 eremanthin -- 3.05 --
% total 91.26 88.69 88.75

SFME = Solvent-free microwave extraction; HD = Hyrodistillation.

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6 Antioxidant and Antimicrobial Activities of Essential Oils Extracted from Laurus nobilis L.
Leaves by Using Solvent-Free Microwave and Hydrodistillation

Table 2. Scavenging effect of L. nobilis essential oils obtained from SFME and HD on ABTS and DPPH radicals and linoleic
acid peroxidation1,2.

ABTS DPPH Linoleic acid peroxidation

Extraction methods
Inhibition % TEAC mM/ml oil Inhibition % Inhibition %
a a a
SFME (at 100% power level) 93.9 ± 1.12 0.18 ± 0.012 91.1 ± 1.62 70.6 ± 5.89a
SFME (at 40% power level) 94.1 ± 3.15a 1.4 ± 0.03 b 76.3 ± 14.82a 63.5 ± 17.8a
a c a
HD 92.1 ± 2.31 2.4 ± 0.09 83.3 ± 6.63 89.2 ± 1.02a
1
Results are given as mean ± standard deviation (n = 6); 2Different superscript denotes statistically significant differences (p < 0.05) among data in the same
column; TEAC = Trolox equivalent antioxidant capacity, SFME = Solvent-free microwave extraction.

vengers [27]. Inhibition effects of laurel essential oils compounds in the volatile aglycone fraction and the es-
obtained by SFME at different power levels and HD on sential oil of laurel, eugenol (0.3% and 2.5% respectively)
DPPH radical cation oxidation were shown in Table 2. and methyl eugenol (10% in essential oil) considered as
No significant difference was found for the essential oils the main contributors to the antioxidant activity. In our
obtained by SFME (at 100% and 40% power level) and previous investigation project eugenol content of the es-
HD. Hinneburg et al. [28] studied antioxidant activity of sential oils obtained by SFME at 100% and 40% power
hydrodistilled extracts of nine herbs and spices. They levels and HD were 16.36 mg/mL oil (1.41 of the identi-
reported that extracts obtained from basil and laurel dis- fied compounds), 17.32 mg/mL oil (1.59% of the identi-
played the highest antioxidant activity in the ABTS and fied compounds) and 12.153 mg/mL oil (1.21% of the
DPPH radical scavenging assays than the iron-chelating identified compounds) respectively. Methyl eugenol
assay. Antioxidant activities of herbs and spices have content of the essential oils were 14.757 mg/ml for
been mainly attributed to phenolic compounds and es- SFME at 100% power level, 14.933 mg/ml for SFME at
sential oils they contain [2]. It was reported that essential 40% power level and 12.075 mg/ml for HD which
oil of laurel has more than 40 components. The major representing 1.27%, 1.37% and 1.20% of the identified
components of laurel essential oil that determine the spe- compounds in the oil, respectively [31].
cific odor of the plant and its essential oil are reported to
be as 4-terpineol, alfa-terpenyl acetate, and methyleuge- 3.1.3. Inhibition of the Linoleic Acid Peroxidation
nol [3]. Misharina and Polshkov [29] reported that, Lipid peroxidation is an early event in the deterioration
probably, terpinenes and other compounds (having vari- of food quality during processing and/or storage and in
ous functional groups and differing in redox properties) the development of degenerative diseases (e.g., cardi-
stabilize the system of laurel essential oil and inhibit the ovascular diseases and age-related disorders).
oxidation of reactive substances (e.g., monoterpene hy- The most important reaction in lipid peroxidation is
drocarbons and trans-anethol). They also reported that the autoxidation of unsaturated fatty acids, which is
the composition of volatile compounds in laurel leaves known to proceed by a radical chain reaction [3]. Essen-
largely depends on the age of the plants, their origin, and tial oils of L. nobilis obtained from SFME at two power
the time of collection of the leaves, and the duration and levels and HD were analyzed in order to determine their
conditions of storage. In our project, compositions of inhibitory effects on the linoleic acid peroxidation. Es-
laurel essential oil obtained from both SFME and HD sential oils of L. nobilis extracted by SFME at 100%
extraction methods were found to be similar. Main com- power level and 40% power level inhibited linoleic acid
ponents of laurel essential oil are 1,8-cineole (630 - 730 peroxidation by the percentages of 70.57 ± 5.89 and
mg/mL oil), α-terpinyl acetate (90 - 115 mg/mL oil), 63.53 ± 17.8 respectively (p > 0.05) (Table 2). Although
sabinene (45 - 55 mg/mL oil), α-pinene (30 - 40 mg/mL essential oil obtained by HD showed greater inhibitory
oil), 4-terpineol (35 - 40 mg/mL oil) and β-pinene (~30 effect on the linoleic acid peroxidation (89.18% ± 1.02%)
mg/mL oil) [15]. Politeo et al. [30] evaluated the anti- than those obtained by SFME, this difference was not
oxidant activity of laurel volatile aglycones and com- found to be statistically significant. Hinneburg et al. [28]
pared its antioxidant activity with the antioxidant activity reported that 1 g of laurel extract obtained by hydrodis-
of laurel essential oil and well known synthetic antioxi- tillation was as effective as about 212 mg of Trolox in
dant butylated hydroxytoluene (BHT) by using DPPH the prevention of lipid peroxidation. To determine anti-
radical scavenging method and ferric reducing/antioxi- oxidant activity of methanolic extracts of laurel leaves,
dant power assay (FRAP). They found that the decrease Simic et al. [12] used the lipid peroxidation inhibition in
in the concentration of volatile aglycones caused a reduc- liposomes induced by Fe+2/ascorbate system. Maximum
tion in the antioxidant activity. Among the identified antioxidant activity obtained for defatted and crude ex-

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 Antioxidant and Antimicrobial Activities of Essential Oils Extracted from Laurus nobilis L. 7
Leaves by Using Solvent-Free Microwave and Hydrodistillation

tracts were reported as 68.4% and 64.6%, respectively. togenes. Muñoz et al. [35] evaluated antimicrobial prop-
erties of oregano, rosemary and laurel extracts obtained
3.2. Antimicrobial Activity by supercritical fluid extraction on the growth and viabil-
ity of Listeria monocytogenes CECT 4032 in laboratory
Two gram-positive (Staphylococcus aureus 6538P and
medium and broccoli juice at 30˚C and 8˚C. They found
Listeria monocytogenes Scott-A) and two gram-negative
that rosemary and oregano extracts reduced the counts of
bacteria (Escherichia coli O157:H7, Salmonella typhi-
L. monocytogenes CECT 4032 below the detection limits
murium NRRL E 4463) were used the possible antimi-
(ca 1.0 lo cfu/mL) after 4 h at 30˚C in laboratory medium.
crobial activity of HD and SFME extracts of laurel leaves.
Laurel essential oils obtained from HD and SFME at However, significant reductions in growth rate and in-
100% and 40% power levels inhibited the survival of all crease in lag phase of L. monocytogenes were observed
microorganisms studied except Listeria monocytogenes in the presence of some of the laurel and oregano extracts
Scott-A. Laurel essential oils obtained by SFME at 100% at both temperatures. Effect of extraction methods on the
power level and HD against S. aureus 6538P inhibition survival of Salmonella typhimurium NRRL E 4463 was
zones (4.5 mM) (Table 3). However, inhibition effect of the same (2.5 mM). Similarly, antimicrobial activity of
laurel oil obtained by SFME at 40% W (3.5 mM) was essential oils obtained from SFME at two power levels
weaker than other oils obtained from SFME at 100% and HD against Escherichia coli O157:H7 were not
power level and HD (p < 0.05) (Table 2). Simic et al. [32] found to be significantly different. Antimicrobial activi-
found a relationship between the high presence of 1,8 ties of spices, their active components and their essential
cineole and sabinene in L. nobilis oil and moderate-high oils on the growth of pathogenic bacteria and other mi-
antifungal activity of this oil. Derwich et al. [33] reported croorganisms leading to spoilage of foods, have been
that Staphlococcus aureus was the most sensitive strain studied intensively in recent years. It is well known that,
among the bacteria tested (Staphylococcus intermedius sage, anise, daphne, mustard, black pepper, thyme, la-
and Klebsiella Pneumoniae) to the oil of Lauris nobilis vender, laurel, oregano, fennel, cumin, mint, garlic and
the strongest inhibition zone of 13 mm. Although inhibi- cinnamon have inhibitory effects on the survival of bac-
tion zone that we determined for S. aureus 6538P was teria such as Bacillus cereus, B. subtilis, Clostridium
smaller (4.5 mm) than that of reported by Derwich et al. botulinum, Escherichia coli, Staphylococcus aureus, Lis-
[33]. S. aureus was the most sensitive strain to the essen- teria monocytogenes and several fungi [36-38]. Sağdıç et
tial oils obtained from SFME at 100% power level and al. [36] and Özcan and Erkmen [39] studied antimicrobi-
HD. It was reported that essential oils have greater anti- al activity of spices including cumin, Helichrysum com-
microbial activity on gram-positive bacteria than gram- pactum Boiss. (HC), myrtle, oregano, sage, laurel and
negative. This result explained the higher resistance thyme against E. coli O157:H7. Both studies data showed
among gram-negative bacteria than gram-positive bacte- that inhibitory activity of laurel on the growth of E. coli
ria could be due to the differences in the cell membranes O157:H7 were not determined. Sağdıç and Özcan [40]
of these bacterial groups. Indeed, the external membrane reported that hydrosols of laurel showed no antimicrobial
of gram-negative bacteria renders their surfaces highly activity against Bacillus amyloliquefaciens ATCC 23842,
hydrophobic, whereas the lipophilic ends of the lipotei- Bacillus brevis FMC 3, Bacillus cereus FMC 19, Bacil-
choic acids of the cell membrane of gram-positive bacte- lus subtilis var. niger ATCC 10, Enterobacter aerogenes
ria may facilitate penetration by hydrophobic compounds CCM 2531, Escherichia coli ATCC 25922, Escherichia
[34]. However, all extracts we studied did not display coli O157:H7 ATCC 33150, Klebsiella pneumoniae
any inhibitory effect on the survival of Listeria monocy- FMC 5, Proteus vulgaris FMC 1, Salmonella enteritidis,

Table 3. Antimicrobial activity of L. nobilis essential oils obtained from SFME and HD1.

Diameter of the zones of inhibition in mm (5 mm disc)

SFME
HD
Control 100% (622 W) 40% (249 W)
Listeria monocytogenes Scott-A 0a 0a 0a 0a
S. aureus 6538P 0a 4.5b 3.5c 4.5b
S. typhimurium NRRL E4463 0a 2.5b 2.5b 2.5b
E. coli O157:H7 0a 3.5b 3.5b 3.5b
1
Different superscript denotes statistically significant differences (p < 0.05) among data in the same row.

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8 Antioxidant and Antimicrobial Activities of Essential Oils Extracted from Laurus nobilis L.
Leaves by Using Solvent-Free Microwave and Hydrodistillation

Salmonella gallinarum, Salmonella typhimurium, Staphlo- of Alkyl Peroxy Radical Scavenging Compound from
coccus aureus ATCC 2392, Staphylococcus aureus Leaves of Laurus nobilis,” Chemical and Pharmaceutical
Bulletin, Vol. 25, No. 1, 2002, pp. 102-108.
ATCC 28213 and Yersinia enterocolitica ATCC 1501.
Dadalıoglu and Evrendilek [38] reported that oregano, [3] S. Santoyo, R. Lloría, L. Jaime, E. Ibañez, F. J. Señoráns
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oxidant and Antimicrobial Compounds from Laurus no-
Turkey exhibited inhibitory activity on Escherichia coli bilis L. Chemical and Functional Characterization,” Eu-
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[4] M. Elmastas, İ. Gülçin, Ö. Işıldak, Ö. İ. Küfrevioğlu, K.
4. Conclusion İbaoğlu and H. Y. Aboul-Enein, “Antioxidant Capacity of
Bay (Laurus nobilis L.) Leave Extracts,” Journal of the
In conclusion, no significant differences were obtained in Iranian Chemical Society, Vol. 3, No. 3, 2006, pp. 258-
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hydrodistillation, but SFME can be preferred to HD since [5] P. Pillai and K. Ramaswamy, “Effect of Naturally Occur-
extraction time was reduced considerably. In the future, ing Antimicrobials and Chemical Preservatives on the
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DPPH radical cation oxidation were not significantly http://dx.doi.org/10.1016/j.foodchem.2004.02.025
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SFME and HD showed similar acted as antimicrobial
matography/Mass Spectrometry Analysis of Laurus nobi-
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