Chemical Composition and Antimicrobial Activity of Essential Oils of Lavender (Lavandula angustifolia) and Lavandin (Lavandula x intermedia) Grown in Western Romania

Abstract

The purpose of this study was to determine the chemical composition and antimicrobial properties of essential oils (EOs) isolated from lavender (L. angustifolia Miller) and lavandin (Lavandula x intermedia) harvested in 2011 in western Romania. The essential oils, isolated by steam distillation from inflorescences arrived at full flowering stage, were analyzed by gas chromatography coupled with mass spectrometry (GC-MS). The essential oil of L. angustifolia Miller analyzed contained as main components caryophyllene (24.1%), beta-phellandrene (16%) and eucalyptol (15.6%), while the essential oil of Lavandula x intermedia contains camphor (32.7%) and eucalyptol (26.9%). The antimicrobial activity was evaluated by the Kirby-Bauer method. Antimicrobial tests showed antimicrobial activity against Shigella flexneri, Staphylococcus aureus, E. coli and Salmonella typhimurium, while Streptococcus pyogenes is not sensitive to the action of the two essential oils. The study revealed that essential oils isolated and analyzed from lavender (L. angustifolia Miller) and lavandin (Lavandula x intermedia) display significant bactericidal effects against microorganisms such as Shigella flexneri, Staphylococcus aureus and E. coli even in the absence of active principles like linalool and linalyl acetate, considered responsible for the antibacterial and antifungal properties of essential oils obtained from different species of Lavandula. The results suggest once again that the antimicrobial activity of EOs is a resultant of the antibacterial properties of the major and minor components in their chemical composition.

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INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY
ISSN Print: 1560–8530; ISSN Online: 1814–9596
12–1270/2013/15–4–772–776
http://www.fspublishers.org
Full Length Article
To cite this paper: Jianu, C., G. Pop, A.T. Gruia and F.G. Horhat, 2013. Chemical composition and antimicrobial activity of essential oils of lavender
(Lavandula angustifolia) and lavandin (Lavandula x intermedia) grown in Western Romania. Int. J. Agric. Biol., 15: 772‒776
Chemical Composition and Antimicrobial Activity of Essential Oils of
Lavender (Lavandula angustifolia) and Lavandin (Lavandula x
intermedia) Grown in Western Romania
Călin Jianu1*, Georgeta Pop2, Alexandra T. Gruia3 and Florin George Horhat4
1Department of Food Science, Faculty of Food Processing Technology, Banat's University of Agricultural Sciences and
Veterinary Medicine, Calea Aradului 119, RO-300645 Timisoara, Romania
2Faculty of Agriculture, Banat's University of Agricultural Sciences and Veterinary Medicine, Calea Aradului 119, RO-
300645 Timisoara, Romania
3Center for Transplant Immunology, County Hospital Timisoara, Blvd. Iosif Bulbuca 10, RO-300736 Timisoara, Romania
4Department of Microbiology, Victor Babes University of Medicine and Pharmacy, Timisoara, Romania, 2nd Eftimie
Murgu Square, RO-300041 Timisoara, Romania
*For correspondence: calin.jianu@gmail.com
Abstract
The purpose of this study was to determine the chemical composition and antimicrobial properties of essential oils (EOs)
isolated from lavender (L. angustifolia Miller) and lavandin (Lavandula x intermedia) harvested in 2011 in western Romania.
The essential oils, isolated by steam distillation from inflorescences arrived at full flowering stage, were analyzed by gas
chromatography coupled with mass spectrometry (GC-MS). The essential oil of L. angustifolia Miller analyzed contained as
main components caryophyllene (24.1%), beta-phellandrene (16%) and eucalyptol (15.6%), while the essential oil of
Lavandula x intermedia contains camphor (32.7%) and eucalyptol (26.9%). The antimicrobial activity was evaluated by the
Kirby-Bauer method. Antimicrobial tests showed antimicrobial activity against Shigella flexneri, Staphylococcus aureus, E.
coli and Salmonella typhimurium, while Streptococcus pyogenes is not sensitive to the action of the two essential oils. The
study revealed that essential oils isolated and analyzed from lavender (L. angustifolia Miller) and lavandin (Lavandula x
intermedia) display significant bactericidal effects against microorganisms such as Shigella flexneri, Staphylococcus aureus
and E. coli even in the absence of active principles like linalool and linalyl acetate, considered responsible for the antibacterial
and antifungal properties of essential oils obtained from different species of Lavandula. The results suggest once again that the
antimicrobial activity of EOs is a resultant of the antibacterial properties of the major and minor components in their chemical
composition. © 2013 Friends Science Publishers
Keywords: Lavender; Lavandin; Essential oil; Steam distillation; GC-MS analysis; Antimicrobial activity
Introduction
L. angustifolia Miller or true lavender is a perennial shrub of
the family Lamiaceae (Lis-Balchin, 2002). The main
growing countries are Bulgaria and France and on smaller
areas in Morocco, the former republics of Yugoslavia,
Hungary, Italia, Russia, Spain, Romania, Ukraine, Turkey,
and others (Zheljazkov, 2012). Their main use is the
extraction of the essential oil (EO) isolated from flower
heads harvested in July-August and processed fresh. The
yield is between 0.6-1% (Burdock, 1998).
Lavandula × intermedia (lavandin) is a sterile hybrid
obtained from L. angustifolia Miller and L. latifolia (L.)
(spike lavender) (Raghavan, 2007). Lavandin was produced
to serve as raw material to obtain EO. Its yield of EO can be
five times higher than that of L. angustifolia (Lis-Balchin,
2002).
The EO obtained from L. angustifolia flowers is
composed primarily of linalyl acetate, linalool, lavandulol,
1,8-cineol, lavandulyl acetate and camphor (Lis-Balchin and
Hart, 1999), while the EO from L. x intermedia contains
linalool, linalyl acetate, camphor, 1,8-cineol and borneol
(Lis-Balchin, 2002).
The EO obtained from L. angustifolia has various
medical applications due to its sedative, carminative, anti-
depressive and anti-inflammatory properties (Cavanagh,
2005), while the oil isolated from L. x intermedia due to its
high content of camphor is used mainly in the production of
perfumes and soap (Lis-Balchin, 2002). In addition to these
applications, both EOs are used in the food industry as
natural flavorings in baked goods, alcoholic and
nonalcoholic beverages, puddings etc. (Burdock, 1998).
Besides these properties numerous studies have
reported that both EOs possess antimicrobial and cytotoxic

Composition and Antimicrobial Activity of Lavender and Lavandin Essential Oils / Int. J. Agric. Biol., Vol. 15, No. 4, 2013
773
activities against several species of bacteria (Lis-Balchin
and Deans, 1997; Rota et al., 2004; Soković et al., 2007;
Hussain et al., 2010; Soković et al., 2010; Blazeković et al.,
2011; Stanojević et al., 2011; Zheljazkov et al., 2012). Also
both EOs showed fungistatic effects against Candida
albicans, Microsporum canis, Aspergillus fumigates,
Fusarium oxysporum etc. (Blazekovic et al., 2011;
Stanojević et al., 2011; Şerban et al., 2011).
The purpose of this study is to determine the chemical
composition and antimicrobial properties of EOs isolated
from lavender (L. angustifolia Miller) and lavandin
(Lavandula × intermedia) harvested in western Romania.
Knowledge and understanding of the chemical composition
and antibacterial properties of the EOs could contribute to
accessing new natural antiseptic with applications in the
food, pharmaceutical and cosmetics industries.
Materials and Methods
Raw Materials
The plant material used in the study was obtained from the
experimental lots of Banat’s University of Agricultural
Sciences and Veterinary Medicine of Timişoara in July
2011. The lavender (L. angustifolia Miller) and lavandin
(Lavandula × intermedia) inflorescences were harvested
manually, at the maximum flowering stage, when the EO
content and quality are considered the best (Guitton et al.,
2010; Zheljazkov et al., 2012). Voucher specimens were
collected from each plant that were identified and deposited
in the herbarium of the Department of Agricultural
Technologies, Faculty of Agronomy, Banat’s University of
Agricultural Sciences and Veterinary Medicine of
Timişoara, Romania (Number VSNH.BUASTM-81 and
VSNH.BUASTM-82).
Isolation of Essential Oils
Fresh plant material was used for the extraction of EOs. EOs
was extracting by steam distillation according to the method
previously described by (Craveiro et al., 1976), separated by
decantation then dried on anhydrous sodium sulfate and
stored for the GC-MS and antimicrobial activity analyses in
hermetically sealed vials at 4°C.
Gas Chromatography-Mass Spectrometry
Oil samples were analyzed by gas chromatography with a
HP6890 gas chromatograph, coupled with a HP 5973 mass
spectrometer. The gas chromatograph has a split-splitless
injector and a capillary column Factor Four TM VF-35 ms,
35% phenylmethyl phase, 30 m

0.25 mm, 0.25 μm film
thickness. The gas chromatography conditions include a
temperature range of 50 to 250°C with 4°C/min, with a
solvent delay of 5 min. The temperature of the injector was
maintained at 250°C. The inert gas was helium at a flow of
1.0 mL/min, and the volume of injected sample in the
splitless mode was 2 μL. The MS conditions were the
following: ionization energy, 70 eV; quadrupole
temperature, 100°C; scanning velocity, 1.6 scan/s; weight
range, 40-500 amu.
The percent composition of the essential oils was
calculated. The qualitative analysis was based on the percent
area of each peak of the sample compounds. The mass
spectrum of each compound was compared with the mass
spectrum from the spectra library NIST 98 (USA National
Institute of Science and Technology software).
Determination of Antimicrobial Activity
The essential oils were tested on the following strains
Staphylococcus aureus (ATCC 25923), Shigella flexneri
(ATCC 12022), Salmonella typhimurium (ATCC 14028),
Escherichia coli (ATCC 25922), Streptococcus pyogenes
(ATCC 19615).
The antibacterial activity of the essential oils was
determined by using the Kirby-Bauer method (Bauer and
Kirby, 1966). Briefly, the test was performed in sterile Petri
dishes (100 mm diameter) containing an appropriate solid
sterile media. The Gram positive and negative bacteria were
cultivated on Mueller-Hinton agar (Sanimed: 20779,
Romania). The surface of the plates was inoculated with 200
μL of bacterial suspension. Sterile filter paper (Whatman
No. 1) discs (6 mm in diameter) containing 5, 10, 15, 20 μL
of the tested essential oils were placed in the centre of the
agar surface. A disc containing 10 μL of sterile broth media
was used as the negative control. Two different reference
antibiotics, rifampicin and tetracycline, (Oxoid, UK)
amended discs, at 100 μg mL-1 concentrations, were used as
the positive control for comparison. Each individual Petri
dish was immediately covered to prevent eventual
evaporation. After allowing the essential oils to diffuse
across the surface for 1 h at room temperatures, the plates
were sealed with sterile parafilm and incubated at 37°C for
24-48 h. The antibacterial activities of the oils and
antibiotics were demonstrated by a clear zone of inhibition
around the disc. The zone of inhibition was measured using
electronic digital Vernier calipers. Each test was performed
in triplicate on at least three separate experiments.
Statistical Analysis
Data distributions were expressed as mean values and
standard deviations (SD). The Student’s t-test was used to
compare the differences between the sample mean sizes of
the inhibitory zones, at the same volume, in lavender and
lavandin groups. The one-way ANOVA test was used to
assess mean differences between sample mean sizes of the
inhibitory zones at different volumes of the same oil. All
tests of significance were two-tailed. StataIC 11 statistical
software (StataCorp LP, Texas, USA, version 2009) was
used for data analysis. A p-value <0.05 was considered
statistically significant.

Jianu et al. / Int. J. Agric. Biol., Vol. 15, No. 4, 2013
774
Results and Discussion
The yield of EO (% v/w) was 2.75% for Lavandula ×
intermedia and 1.13% for L. angustifolia Miller; the
chemical components identified are reported in Table 1.
22 components were identified in the EO obtained
from L. angustifolia Miller, representing 99.9% of the
total, the major components being caryophyllene
24.12%, beta-phellandrene 16% and eucalyptol (1,8-
cineol) 15.69%. The EO of Lavandula × intermedia has
as major component camphor 32.7% and eucalyptol
26.9%, 24 components being identified in this case
representing 98.26% of the total.
EOs obtained from various species of Lavandula have
Table 1: Chemical composition of L. angustifolia Miller and Lavandula × intermedia oils from western Romania
No.
Compound
% of total
L. angustifolia Miller
Lavandula × intermedia
1.
alpha-thujene
0.4
0.38
2.
alpha-pinene
0.78
2.31
3.
camphene
1.37
1.34
4.
sabinene
0.31
0.77
5.
beta-pinene
0.94
1.84
6.
beta-myrcene
2.03
1.43
7.
carene
0.76
1.78
8.
D-limonene
2.1
3.07
9.
beta-phellandrene
16.00
3.87
10.
eucalyptol (1,8-cineole)
15.69
26.9
11.
gamma-terpinene
0.48
0.38
12.
terpineol
-
0.92
13.
terpinolene
-
1.05
14.
linalool
tr
tr
15.
terpinen-4-ol
9.57
-
16.
camphor
-
32.70
17.
borneol
5.07
7.11
18.
alpha-terpineol
6.00
1.48
19.
1,6-octadien-3-ol, 3,7-dimethyl-, 2-aminobenzoate
tr
tr
20.
alpha-bergamotene
-
0.26
21.
santalene
4.5
0.94
22.
caryophyllene
24.12
4.88
23.
beta-sesquiphellandrene
0.39
-
24.
1,6-cyclodecadiene, 1-methyl-5-methylene-8-(1-methylethyl)-
4.7
0.47
25.
bicyclo[4.4.0]dec-1-ene, 2-isopropyl-5-methyl-9-methylene
-
0.22
26.
naphthalene, 1,2,3,4,4a,5,6,8a-octahydro-7-methyl-4-methylene-1-(1-methylethyl)-
4.16
-
27.
naphthalene, 1,2,3,4,4a,7-hexahydro-1,6-dimethyl-4-(1-methylethyl)-
0.53
-
28.
alpha-bisabolol
-
4.16
Identified from total area
99.9
98.26
tr - trace (<0.05%)
Table 2: Effects of L. angustifolia Miller and Lavandula × intermedia oils against bacteria expressed by the mean sizes of
the inhibitory zones
Test microorganism
Amount of essential oil [μL]
5
10
15
20
Salmonella typhimurium
(ATCC 14028)
Lavandin (Lavandula × intermedia)
na
na
10 ± 0.18
13.99 ± 0.16
Lavender (L. angustifolia Miller)
na
na
na
na
Shigella flexneri
(ATCC 12022)
Lavandin (Lavandula × intermedia)
11.41 ± 0.12
18.23 ± 0.12
22.05 ± 0.16
26.09 ± 0.19
Lavender (L. angustifolia Miller)
na
na
16.1 ± 0.16)
20.35 ± 0.20
Staphylococcus aureus
(ATCC 25923)
Lavandin (Lavandula × intermedia)
8.28 ± 0.14
12.12 ± 0.18
16.12 ± 0.24
19.92 ± 0.19
Lavender (L. angustifolia Miller)
7.04 ± 0.14
11.01 ± 0.19
14.95 ± 0.2
19.96 ± 0.33
Escherichia coli
(ATCC 25922)
Lavandin (Lavandula × intermedia)
8.98 ± 0.19
10.84 ± 0.12
12.74 ± 0.11
21.03 ± 0.16
Lavender (L. angustifolia Miller)
7.03 ± 0.16
10.06 ± 0.16
18.59 ± 0.15
20.2 ± 0.23
Streptococcus pyogenes
(ATCC 19615)
Lavandin (Lavandula × intermedia)
na
na
na
na
Lavender (L. angustifolia Miller)
na
na
na
na
Inhibitions are expressed in mm and include the diameter of the paper disc (6 mm). Data distributions were expressed as mean values and standard
deviations (SD) (n = 9). Rifampicin and tetracycline was used as positive control; na: no activity

Composition and Antimicrobial Activity of Lavender and Lavandin Essential Oils / Int. J. Agric. Biol., Vol. 15, No. 4, 2013
775
a very different chemical composition, the major
components reported in the literature being linalool,
linalyl acetate, fenchone, eucalyptol and borneol
(Bouzouita et al., 2005; Imeloune et al., 2009; Soković et
al., 2010; Stanojević et al., 2011; Zheljazkov et al., 2012).
In contrast in the composition of the EOs analyzed in this
study linalool and linalyl acetate are not found or their
presence is observed only in traces. Similarly the absence of
these compounds has also been reported in other studies
(Hui et al., 2010; Abroomand Azar et al., 2011).
These changes in the essential oil composition might
arise from several environmental (climatic, seasonal,
geographical) and genetic differences (Stanojević et al.,
2011).
Experimental data obtained from the evaluation of
antimicrobial activity are reported in Table 2. Comparing
the effects of the two EOs, for the same volumes against
selected bacteria, the mean sizes of the inhibitory zones
were statistically significantly higher for lavandin (p< 0.001,
t test), except for Staphylococcus aureus (20 μL) (p > 0.05, t
test) and E. coli (15 μL), where the results were significantly
higher for lavender (p < 0.001, t test). When comparing the
effects of the two EOs (at different volumes) there was a
statistical significant difference for lavandin between the
mean sizes of the inhibitory zones when testing against
Salmonella typhimurium, Shigella flexneri, Staphylococcus
aureus and E. coli (p < 0.001, oneway ANOVA test). No
effect was observed against Streptococcus pyogenes. In the
case of lavender statistically significant differences were
recorded between the mean sizes of the inhibitory zones
when testing against Shigella flexneri, Staphylococcus
aureus and E. coli (p < 0.001, oneway ANOVA test). No
effects were observed against Salmonella typhimurium and
Streptococcus pyogenes.
The antimicrobial activity of the two EOs analyzed by
us is comparable with data reported in previous studies
(Hammer et al., 1999; Soković et al., 2007;
Hanamanthagouda et al., 2010; Blazekovic et al., 2011;
Şerban et al., 2011) although they had in their composition
only trace amounts (<0.05%) of linalool while linalyl
acetate was not identified, these two active compounds
being considered by a number of studies as possessing a
strong antimicrobial effect (Dorman and Deans, 2000;
Aridogan et al., 2002; Soković et al., 2007; De Martino et
al., 2009; Soković et al., 2010; Blazekovic et al., 2011). In
the composition of the lavender EO studied we find
however other components recognized for their antibacterial
efficacy: caryophyllene (Oztürk et al., 2009), terpinen-4-ol
(Dorman and Deans, 2000; Kotan et al., 2007), borneol, α-
pinene, terpineol (Dorman and Deans, 2000). Camphor the
major component of the Lavandin EO analyzed has
antibacterial properties itself (Magiatis et al., 2002; Soković
et al., 2007; Mahboubi and Kazempour, 2009). Moreover, it
has been demonstrated that eucalyptol, one of the primary
components of both EOs studied, presents antimicrobial
activity against bacteria such as Staphylococcus aureus,
methicillin-resistant S. aureus, E. coli and Candida albicans
(Hendry et al., 2009). The same study also suggests the
synergistic effect of minor components in the chemical
composition of the EOs in relation to its antimicrobial
activity, similar results having been reported in other
studies, along with additive and antagonistic effects (Gill et
al., 2002; Mourey et al., 2002). EOs represent complex
mixtures of chemical compounds with different
antimicrobial properties, and for these reasons it is very
difficult to reduce their antimicrobial effect to one or several
active principles (Bouzouita et al., 2005).
Conclusion
The study demonstrates that lavender and lavandin EOs
presents significant bactericidal effects against
microorganisms such as Shigella flexneri, Staphylococcus
aureus and E. coli, even in the absence of active principles
like linalool and linalyl acetate, credited with strong
antimicrobial and antifungal effects. The study suggests
once again that the antimicrobial activity of the EOs is a
resultant of the antibacterial properties of the major and
minor components in their chemical composition. In these
conditions further research is required for the elucidation of
the mechanisms determining the increase of the bioactivity
of Eos, but also of the synergistic relationship between their
components. Explanation of these mechanisms will allow
the easy accessing of new sources of natural antiseptics with
applications in the food, cosmetics and pharmaceutical
industries.
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(Received 24 November 2012; Accepted 25 march 2013)