Antioxidant, analgesic and anti-inflammatory effects of lavender essential oil

Abstract

Several studies have investigated the antinociceptive, immunomodulatory and anti-inflammatory properties of compounds found in the lavender essential oil (LEO), however to date, there is still lack of substantial data. The objective of this study was to assess the antioxidant, anti-inflammatory and antinociceptive effects of lavender essential oil. The 1,1-diphenyl-2-picrylhydrazyl radical decolorization assay was used for antioxidant activity evaluation. The anti-inflammatory activity was tested using two models of acute inflammation: carrageenan-induced pleurisy and croton oil-induced ear edema. The antinociceptive activity was tested using the pain model induced by formalin. LEO has antioxidant activity, which is dose-dependent response. The inflammatory response evoked by carrageenan and by croton oil was reduced through the pre-treatment of animals with LEO. In the pleurisy model, the drug used as positive control, dexamethasone, was more efficacious. However, in the ear swelling, the antiedematogenic effect of the oil was similar to that observed for dexamethasone. In the formalin test, LEO consistently inhibited spontaneous nociception and presented a similar effect to that of tramadol. The results of this study reveal (in vivo) the analgesic and anti-inflammatory activities of LEO and demonstrates its important therapeutic potential.

Full text

An Acad Bras Cienc (2015) 87 (2 Suppl.)
Anais da Academia Brasileira de Ciências (2015) 87(2 Suppl.): 1397-1408
(Annals of the Brazilian Academy of Sciences)
Printed version ISSN 0001-3765 / Online version ISSN 1678-2690
http://dx.doi.org/10.1590/0001-3765201520150056
www.scielo.br/aabc
Antioxidant, analgesic and anti-inammatory effects of lavender essential oil
GABRIELA L. DA SILVA1, CAROLINA LUFT1, ADROALDO LUNARDELLI1, ROBSON H. AMARAL2,
DENIZAR A. DA SILVA MELO1, MÁRCIO V.F. DONADIO1, FERNANDA B. NUNES1, MARCOS S. DE
AZAMBUJA1
, JOÃO C. SANTANA1, CRISTINA M.B. MORAES1, RICARDO O. MELLO1, EDUARDO
CASSEL3, MARCOS AURÉLIO DE ALMEIDA PEREIRA3 and JARBAS R. DE OLIVEIRA1
1Laboratório de Pesquisa em Biofísica Celular e Inamação, Pontifícia Universidade Católica do
Rio Grande do Sul, Avenida Ipiranga, 6681, Partenon, 90619-900 Porto Alegre, RS, Brasil
2Laboratório de Análises Clínicas, Instituto de Cardiologia do Rio Grande do Sul, Avenida
Princesa Isabel, 395, Santana, 90620-001 Porto Alegre, RS, Brasil
3Laboratório de Operações Unitárias, Pontifícia Universidade Católica do Rio Grande do Sul,
Faculdade de Engenharia, Departamento de Engenharia Química,
Avenida Ipiranga, 6681, Prédio 30, Sala 208, Partenon, 90619-900 Porto Alegre, RS, Brasil
Manuscript received on January 26, 2015; accepted for publication on March 18, 2015
ABSTRACT
Several studies have investigated the antinociceptive, immunomodulatory and anti-inammatory properties
of compounds found in the lavender essential oil (LEO), however to date, there is still lack of substantial
data. The objective of this study was to assess the antioxidant, anti-inammatory and antinociceptive
effects of lavender essential oil. The 1,1-diphenyl-2-picrylhydrazyl radical decolorization assay was
used for antioxidant activity evaluation. The anti-inammatory activity was tested using two models of
acute inammation: carrageenan-induced pleurisy and croton oil-induced ear edema. The antinociceptive
activity was tested using the pain model induced by formalin. LEO has antioxidant activity, which is
dose-dependent response. The inammatory response evoked by carrageenan and by croton oil was
reduced through the pre-treatment of animals with LEO. In the pleurisy model, the drug used as positive
control, dexamethasone, was more efcacious. However, in the ear swelling, the antiedematogenic effect
of the oil was similar to that observed for dexamethasone. In the formalin test, LEO consistently inhibited
spontaneous nociception and presented a similar effect to that of tramadol. The results of this study reveal
(in vivo) the analgesic and anti-inammatory activities of LEO and demonstrates its important therapeutic
potential.
Key words: antioxidants, inflammation, lavender, nociception.
Correspondence to: Jarbas Rodrigues de Oliveira
E-mail: jarbas@pucrs.br
INTRODUCTION
Lavender genus is an important member of the
Lamiaceae family. Lavandula species are widely
distributed in the Mediterranean region and
cultivated in France, Italy and Spain. The Lavandula
augustifolia Mill. specie is well known among
people as a powerful aromatic and medicinal herb.
The plant is used in traditional and folk medicines
of different parts of the world for the treatment of
several gastrointestinal, nervous and rheumatic

An Acad Bras Cienc (2015) 87 (2 Suppl.)
1398 GABRIELA L. DA SILVA et al.
disorders (Hajhashemi et al. 2003, Gören et
al. 2002). Lavandula augustifolia essential oil
(LEO) and its major components, (-)-linalool and
linalyl acetate, also presented anti-inammatory
properties in rats (Peana et al. 2002). In an in vitro
study, (-)-linalool decreased the production and the
release of nitric oxide (NO) without interference
in the prostaglandins pathway (Peana et al. 2006).
Several studies have investigated the antinoci-
ceptive, immunomodulatory and anti-inammatory
properties of compounds found in the lavender es-
sential oil (Peana et al. 2002, Kim and Cho 1999).
These studies investigated the effects of different
constituents of essential oils, such as α–pipine,
α-terpinene, terpin-4-ol, α-terpineol, linalyl acetate
and linalool. Taken together, these studies conclud-
ed that the compounds present in the lavender es-
sential oil may have direct or indirect anti-inam-
matory or antinociceptive activities.
Although a growing number of investigations
have been conducted in these last years, there is
a lack of more substantial data on the effects
and mechanisms of action of lavender essential
oil. In this work, the in vitro antioxidant activity
of lavender essential oil was investigated.
Furthermore, the anti-inammatory effects were
evaluated by using different models of acute
inammation and the antinociceptive activity was
tested by using the formalin-induced pain model.
In all of them, the lavender essential oil effect
was compared to well-known anti-inammatory
and analgesic drugs. Attempts have also been
made in order to investigate some mechanisms of
action. Considering the potential pharmacological
effects of the lavender essential oil, we also
investigated the possible toxic effects in rats using
histopathological, biochemical and hematological
parameters.
MATERIALS AND METHODS
Plant Material and GC/MS analySeS
Lavender oil was purchased from the
Bioessencia (Brazil). The oil was analyzed by
gas chromatography-mass spectrometry (GC/
MS). The equipment used was the Brand Agilent
Technologies, model 7890A GC system, equipped
with a data processor. Capillary column HP - 5 MS
(30 m x 0.250 mm; lm thickness 0.25 μm), 60 to
325/350°C temperature. Helium was the carrier gas
used. The mass spectrometry (MS) analysis was
performed in equipment from Agilent Technologies
Brand, model 5975C VL MSD, operating at 70 eV,
and temperature of the ion source was maintained
at 250ºC. The injected volume was 5 μL of diluted
sample (1:1) in n-hexane for each analysis.
Identication of components in the oil was based
on GC retention indexes relative by comparison
of the fragmentation patterns of mass spectra with
those reported in the literature (Adams 2001).
aniMalS
The animals were cared for and used in accordance
with the Guiding Principles in the Care and
Use of Animals approved by the Council of the
American Physiologic Society. Male Swiss mice
(30 ± 5 g), female Wistar rats (200 ± 20 g) and
male Wistar rats (200 ± 20 g) were obtained from
the Fundação Estadual de Pesquisa e Produção
em Saúde (Porto Alegre, Brazil). Animals were
housed under conditions of optimum light (12
hours light-dark cycle), temperature (22 ± 1°C) and
humidity, with food and water provided ad libitum.
All experiments were carried out between 8:00
a.m. and 7:00 p.m. The Ethics Committee of the
Pontifícia Universidade Católica do Rio Grande
do Sul approved all experimental procedures. All
efforts were made in order to minimize animal
suffering.
BleaChinG of the free-radiCal 1,1-diPhenyl-
2-PiCrylhydrazyl (dPPh teSt)
The antioxidant activity of the lavender essential
oil was determined using the stable 1,1-diphenyl-
2-picrylhydrazyl radical (DPPH), according to
procedures previously described (Aquino et al.
2001). DPPH has an absorption band at 515

An Acad Bras Cienc (2015) 87 (2 Suppl.)
LAVENDER ESSENTIAL OIL PROPERTIES 1399
nm, which disappears upon reduction of an anti-
radical compound. An aliquot containing different
amounts of the oil was added to freshly prepared
DPPH solution (150 μM in methanol); the
concentrations of lavender essential oil employed
were 0.1 mg/mL; 1.0 mg/mL; 10 mg/mL; 20 mg/
mL; 40 mg/mL; 60 mg/mL; 80 mg/mL; 100 mg/
mL; 120 mg/mL and 150 mg/mL. An equal volume
of methanol was added to control tubes. After
starting the reaction, and a 60 minutes incubation
period at room temperature, the absorbance was
read against a blank at 515 nm. All experiments
were carried out in triplicate. We expressed the
radical scavenger activity in terms of the amount of
antioxidants necessary to decrease the initial DPPH
absorbance by 50% (IC50). The IC50 value was
determined graphically by plotting the percentage
disappearance of DPPH as a function of the sample
concentration. The antioxidant activity in percent (I
%) was calculated as follows:
I (%) = (Ablank −Asample)/Ablank × 100
Where Ablank is the absorbance of the control reaction
(containing all reagents except the test compound),
and Asample is the absorbance of the test compound.
oral aCute toxiCity
Female Wistar rats were used. The animals were
randomly divided into ve groups (n=6). The rst
group (control group) received saline solution per
oral route (p.o.). Groups 2 to 5 were treated with
lavender essential oil at the doses of 0.6, 1.5, 3.0 and
5.0 g/kg, respectively. Animals were observed for
14 days after treatment. During these 14 days, the
appearance of general toxic signs were observed.
In this period of observation, the following
parameters were also measured: weight changes,
food consumption and mortality recording. The
surviving animals were euthanized by decapitation
and then blood collection for hematological and
biochemical analysis was carried out. The animals
that died during the 14 days had their lungs, liver
and kidneys collected for histological analysis.
The lethal dose 50 (LD50) was calculated by linear
regression analysis.
heMatoloGiCal and BioCheMiCal analySiS
Leukocyte counting was performed using an
automatic hematological analyzer (Cell Dyn
1700, Abbott). The differential leukocyte counting
was performed with optical microscope after
staining and, in each case, 100 cells were counted.
Biochemical analyses were made in an automatic
biochemical analyzer. The blood was centrifuged
at 1000 x g for 10 minutes to obtain the serum.
The following parameters were analyzed: alanine
aminotransferase (ALT), aspartate aminotransferase
(AST), urea, creatinine, sodium, potassium and
total protein.
hiStoloGiCal exaMination of the tiSSueS
Liver, kidneys and lungs were carefully dissected.
Small slices of these freshly harvested tissues were
fixed in buffered formaldehyde solution (10%),
dehydrated by serial ethanol solution, diaphanized
with ethanol–benzene and enclosed with parafn.
Micrometer sections, cut by a microtome (Leica
Leitz 1512), were stained with hematoxylin–eosin
and examined under a light microscope.
PleuriSy
Female Wistar rats were used. Animals were
treated with lavender essential oil 0.6 mg/kg p.o.
(LEO) (n=13), with saline (carrageenan control
– CC) (n=15) or dexamethasone 0.5 mg/kg by
subcutaneous route (s.c.) (DEX) (n=10) 1 hour
prior to the pleurisy induction. Dexamethasone, a
steroidal anti-inammatory, was used as a reference
drug. Pleurisy was induced by the intrapleural route
(i.pl.) injection of 200 μL of a 1% (w/v) carrageenan
solution, according to the method described
previously (Pinheiro and Calixto 2002). A control
group (saline control – SC) (n=10) was treated with
saline p.o. and received an i.pl. injection of saline
solution (200 μL). Four hours after i.pl. injection

An Acad Bras Cienc (2015) 87 (2 Suppl.)
1400 GABRIELA L. DA SILVA et al.
of carrageenan/saline, the animals were euthanized
in a CO2 chamber and the pleural exudate was
collected by pleural cavity lavage with 2 mL of
saline solution (NaCl 0.9%) containing EDTA 1%.
The samples of the pleural lavage were collected
to determine exudation, total proteins, total and
differential leukocyte counts. Exudate volumes
were measured and the results were calculated by
subtracting the volume injected into the pleural
cavity (2 mL) from the total volume recovered
(Lunardelli et al. 2006). The total cell count in
each sample was estimated after dilution with Türk
solution (1:20), using a Neubauer cell counting
chamber (Boeco, Germany). Cytospin preparations
of the samples were stained with May–Grunwald–
Giemsa for the differential leukocyte count, which
was performed under the immersion objective of a
light microscope. After the cell count, the samples
of the pleural lavage obtained from control and
treated animals were collected, separated and stored
at −20°C. Protein concentration was measured by
the biuret technique.
ear edeMa
To estimate the topical and oral anti-inammatory
activity of the LEO, the mouse ear edema model
was used (n=6). Briey, 65 μL of acetone solution
containing 2% croton oil was applied topically to
the right ear of male mice. The left ear received
an equal volume of acetone. Six hours after the
application of croton oil, the mice were euthanized
and a plug (6 mm diameter) was removed from
both, treated (right) and untreated (left) ears. The
edematous response was measured by the weight
difference between the two plugs. LEO was applied
topically to the right ear (50 μL/ear) 60 minutes
before the croton oil treatment (LEO topic). To
evaluate the oral anti-inammatory activity, the
other group was treated orally with the lavender
essential oil 0.6 g/kg p.o. (LEO p.o.) diluted
in DMSO. Another group was treated only with
diluent (DMSO) and was used as vehicle control
(VC, DMSO p.o.). Dexamethasone (DEX) was
used as a reference drug (0.5 mg/kg s.c. - 60 min
before). The control group (C) had no pre-treated.
forMalin teSt
To assess the antinociceptive activity of the LEO,
the formalin-induced nociception model was
employed. In this test, a diluted formalin solution
(in which formaldehyde is the active ingredient)
was injected into the paw of a rodent, and pain-
related behaviors were observed. Male Wistar rats
were used in these experiments. The animals were
divided into three groups (n=6) and pre-treated p.o.
with lavender, indomethacin or tramadol, 1 hour
before formalin injection, and placed in individual
cages (22.0 x 15.0 x 12.5 cm) which served as the
observation chamber after the injection of formalin.
In order to reduce variability, animals were placed
in open cages observation chamber for 30 minutes
to allow them to get used to the environment
and then were removed in order to receive to the
formalin administration. Nociceptive behavior was
induced by injecting 50 μL 2% formalin (s.c.) into
the surface of the right hind paw (a control group
received an injection of 50 μL of saline). Animals
were then returned to the observation chamber. A
mirror was placed behind the chamber to enable
observation of the formalin-injected paw. Rats were
observed for nociceptive behavior immediately
after formalin injection every 5 minutes until 60
minutes after injection. Nociceptive flinching
behavior was quantied as the number of inches
of the injected paw during the observation period.
Phase I was dened as the rst 10 minutes and the
second phase was dened as the remaining time. At
the end of the experiments, the rats were euthanized
in a CO2 chamber.
The anti-nociceptive effects produced by
lavender essential oil (0.6 g/kg p.o. 1 hour prior)
were compared to the opioid-like analgesic μ
agonist, tramadol (30 mg/kg p.o. 1 hour prior),
and with a non-steroidal anti-inammatory non-
selective COX inhibitor, indomethacin (5 mg p.o. 1
hour prior). The dose of reference drugs used was

An Acad Bras Cienc (2015) 87 (2 Suppl.)
LAVENDER ESSENTIAL OIL PROPERTIES 1401
aCute toxiCity Study
The acute treatment with LEO by oral administration
at doses up to 1.5 g/kg did not produce any sign of
toxicity or death in rats the 14 days of observation.
There were no changes in the behavior of animals
chosen based on data reported in literature (García-
Hernández et al. 2007, Mohajer et al. 2005).
StatiStiCal analySiS
The results were evaluated statistically using
SPSS (Statistical Package for Social Sciences)
18.0 software. The Shapiro-Wilk test was used for
analysis of the data distribution. After conrmation
of normal data, differences among control and
experimental groups were compared by analysis
of variance (ANOVA) followed by Bonferroni
post hoc test. In the formalin test, the differences
were evaluated by repeated measures ANOVA.
The results were expressed as the mean ± standard
error of the mean (SEM). The level of statistical
signicance was dened as p < 0.05.
RESULTS
GC and GC/MS analySeS
The LEO showed a diverse composition with
28 constituents, comprising approximately 82%
of the total oil composition, reported in Table I.
The components found are in agreement with the
British Pharmacopoeia and, as reported in the
literature, the oil is predominantly constituted by
linalol (32.52%) and linalyl acetate (21.57%),
demonstrating the authenticity sample.
dPPh-radiCal-SCavenGinG aCtivity
Figure 1 shows the absorbance reduction when
DPPH solution was tested against various
concentrations of the LEO. The greatest inhibitory
activity was observed at the concentrations of 150,
120 and 100 mg/mL. No signicant differences
were found between these doses (p > 0.05). The
lavender essential oil concentration resulting in
a 50% inhibition of the free radical (IC50) was
51.05 mg/mL. This value was calculated by linear
regression using geometric means.
TABLE I
The percentage composition of the total oil from
Lavandula angustifolia.
RT (min) Compound Percentage (%)
7.012 α-phellandrene 0.05
7.370 α-pipene 2.45
8.029 camphene 0.381
9.523 β-pinene 0.720
11.075 α-phellandrene 0.116
11.397 α-pinene 0.046
11.800 (+)-4-carene 0.725
12.693 D-limolene 6.477
12.767 Eucalyptol 1.816
13.191 4-carene 0.655
14.253 3-carene 0.367
14.722 β-cis-terpineol 0.036
17.423 linalyl acetate 21.57
17.702 octen-1-ol acetate 0.170
19.225 camphora 3.934
20.323 borneol 1.763
21.817 α-terpinol 4.301
22.153 cyclohexanol 1.195
24.204 camphene 0.277
25.822 linalol 32.52
31.240 α-bourbonene 0.069
32.316 α-bisabolene 0.112
32.704 α-cedreno 0.041
32.880 caryophyllene 1.593
34.366 α-caryophyllene 0.101
36.358 naphthalene 0.071
36.885 cis-α-bisabolene 0.171
44.280 α-bisabolol 0.346
RT = retention time. Compounds with more than 1% are
highlighted.

An Acad Bras Cienc (2015) 87 (2 Suppl.)
1402 GABRIELA L. DA SILVA et al.
TABLE II
Effects of the Lavandula augustifolia essential oil (LEO) administered orally on serum biochemical and hematological
parameters in female Wistar rats.
Control LEO
0.6 mg/kg
LEO
1.5 mg/kg
LEO
3.0 mg/kg
LEO
5.0 mg/kg
Urea (mg/dL) 56.75 ± 3.60 47.75 ± 5.56 49.8 ± 4.27 51.5 ± 9.19 48.00 ± 7.07
Creatinine (mg/dL) 0.94 ± 0.15 0.79 ± 0.07 0.74 ± 0.11 0.94 ± 0.37 0.82 ± 0.06
Total protein (g/dL) 7.05 ± 0.24 6.3 ± 0,42 7.28 ± 0.76 8.15 ± 0.64 6.65 ± 0.21
AST (U/L) 114.50 ± 17.54 117.00 ± 15.93 225.40 ± 62.28* 195.50 ± 68.29* 223.50 ± 34.65*
ALT (U/L) 60.50 ± 9.88 68.40 ± 12.50 134.20 ± 33.15* 117.00 ± 32.53* 81.50 ± 4.95*
Na+ (mEq/L) 146.75 ± 8.10 146.50 ± 12.8 142.40 ± 3.65 141.00 ± 2.83 142.00 ± 1.41
K+ (mEq/L) 6.28 ± 1.28 5.53 ± 0.52 6.34 ± 0.76 6.00 ± 0.14 5.75 ± 0.07
Leukocytes (x103/µL) 6400 ± 916.52 6133 ± 736.36 3000 ± 400.00* 2200 ± 282.84* 2900 ± 141.42*
ALT = alanine aminotransferase, AST = aspartate aminotransferase, Na+ = sodium, K+ = potassium. The results are expressed as
the mean ± SDM. * p < 0.05.
Figure 1 - Linear regression of radical scavenging effect of lavender essential oil (LEO)
concentration on 2,2-diphenyl-1-picrylhydrazyl radical test.
treated with doses of 0.6 g/kg and 1.5 g/kg. In
higher doses (3.0 g/kg and 5.0 g/kg) the main signs
of toxicity observed were: atypical locomotion,
anorexia, ataxia, piloerection, hypo activity and
respiratory depression. The lethal 50 dose (LD50)
found was 3.55 g/kg. The hematological and
biochemical prole of control and treated groups
are presented in Table II. The lavender essential
oil administration did not induce changes in urea,
creatinine, total protein, sodium and potassium.
However, an increased transaminases (ALT and
AST) serum level and a decreased total leukocyte
were observed in all treated animals, with the
exception of the 0.6 g/kg dose.

An Acad Bras Cienc (2015) 87 (2 Suppl.)
LAVENDER ESSENTIAL OIL PROPERTIES 1403
hiStoloGiCal analySiS
Histological analyses of control rats showed normal
structures. Leo did not cause any histological
changes to the kidneys or the liver. However, the
animals treated with the higher doses (3.0 g/kg
and 5.0 g/kg) showed inammatory inltration in
the lungs. The essential oil caused enlargement of
lobules due to inammatory process and lesion of
bronchiolar mucosa.
PleuriSy
The results shown in Figure 2 demonstrate that
edematogenic response evoked by i.pl. injection
of carrageenan in rats was signicantly reduced
through the pre-treatment of animals with the
Lavandula angustifolia oil (0.6 g/kg, p.o.),
administered 1 hour prior to the pleurisy induction.
The essential oil caused a marked reduction in the
volume (Fig. 2a) and total protein concentration
(Fig. 2b) in the collected exudates. There was
also a reduction in the total leukocytes counting
(Fig. 2c) and in the number of polymorphonuclear
leukocytes (Fig. 2d), that migrated into the
pleural cavity. Dexamethasone, the drug used as
reference, produced similar effects, but it has been
signicantly more effective than the essential oil.
ear edeMa
The oral administration of the LEO (0.6 g/kg) or
the topical application (50 μL/ear), 60 minutes
before croton oil, inhibited the development of ear
Figure 2 - Effect of pre-treatment with the lavender essential oil (LEO, 0.6 g/kg, p.o.) and dexamethasone (DEX,
0.5 mg/kg s.c.) on pleurisy induced by carrageenan (2 mg/cavity) on exudation (a), total protein concentration on
exudates (b), number of total leukocytes (c) and PMN leukocytes (d). Values represent the mean ± SEM. * p < 0.05
vs. saline control group (SC); # p < 0.05 vs. carrageenan control group (CC); $ p < 0.05 vs LEO group.

An Acad Bras Cienc (2015) 87 (2 Suppl.)
1404 GABRIELA L. DA SILVA et al.
edema. The inhibitory effect of the oil was similar
to the inhibition caused by dexamethasone (Fig. 3).
forMalin teSt
Formalin injection produced a typical pattern of
flinching behavior. Two phases of spontaneous
licking behavior were observed after the formalin
injection. The rst phase started immediately after
administration of formalin and then diminished
gradually after about 10 minutes. The second phase
started about 15 minutes and lasted for a period of
1 hour.
We demonstrated that, when administered prior to
the 2% formalin injection, the lavender essential oil
consistently inhibited the spontaneous nociception
and presented a similar effect to tramadol. The
administration of lavender essential oil (Fig. 4a)
or tramadol (Fig. 4b) significantly reduced the
number of inches in the rst phase. In contrast,
the administration of indomethacin was not able to
reduce the inching behavior in this stage (Fig. 4c).
In the second phase, all treatments inhibited the
nociceptive behavior, but only indomethacin was
effective throughout the entire observation period.
Figure 3 - Comparison of topical and oral anti-inammatory
effects of lavender essential oil in croton oil-induced ear
edema. C = control group (mice not treated); VC = vehicle
control group (mice treated with DMSO p.o.); LEO p.o. =
mice treated with lavender essential oil 0.6 g/kg p.o.; LEO
topic = mice treated with lavender essential oil topically 50 μL/
ear; DEXA = mice treated with dexamethasone 0.5 mg/kg s.c.
Values represent the mean ± SEM. * p < 0.05 vs. control group
(C) and vehicle control group (VC).
Figure 4 - Dose- and time-response curves of flinches
induced by injection of formalin 2% s.c. into the surface of
the right hind paw. Effect of treatments on the nociceptive
behavior. (a) Lavender essential oil 0.6 g/kg, p.o., 1
hour prior. (b) Tramadol 30 mg/kg, p.o., 1 hour prior. (c)
Indomethacin 5 mg/kg, p.o., 1 hour prior. Values represent
the mean ± SEM. * p < 0.05 compared to the formalin
group.

An Acad Bras Cienc (2015) 87 (2 Suppl.)
LAVENDER ESSENTIAL OIL PROPERTIES 1405
DISCUSSION
Many compounds of plant origin have potent
antioxidant activities, thus in the present study a
screening of antioxidant activity was carried out
prior to performing in vivo tests. The free radical
scavenging capacity of the lavender essential
oil was determined using the stable radical,
DPPH. As expected, the lavender essential oil
presented antioxidant activity and this ability was
concentration-dependent.
Despite the potential pharmacological
activities attributed to LEO, there is insufcient
data about its toxicity. We performed a study of
acute toxicity that showed that lavender essential
oil has some toxic effects in doses up to 1.5 g/kg,
although the dose of 0.6 g/kg appears to be well
tolerated orally. Histopathological examination
revealed some alterations in lungs of animals
treated with high doses (3.0 g/kg and 5.0 g/kg).
A lesion of bronchiolar mucosa was observed,
associated with inammatory inltration. These
data are consistent with clinical signs of toxicity
observed before death (hyperventilation followed
by respiratory depression). However, considering
that such changes were found only in animals
treated with high doses, this data can be taken as
a result of an overload of the evaluated product.
Furthermore, the treatment with lavender essential
oil caused a decrease in the total leukocytes and an
increase in transaminases (AST and ALT) at doses
higher than 0.6 g/kg. For this reason, a dose of 0.6
g/kg was considered safe and can be used in further
studies.
Carrageenan-induced pleurisy is a well
characterized experimental model of inammation
that permits the quantication and correlation of
both exudate and cellular migration with changes of
other inammatory parameters (Santos et al. 2010).
This model has been widely used to investigate the
pathophysiology of acute inammation and also to
evaluate the efcacy of drugs in inammation (Paul
et al. 2009). Our results revealed that treatment with
lavender essential oil was effective in reducing the
responses induced by carrageenan. However, the
drug used as reference, dexamethasone, was more
effective in preventing these effects. It has been
recently shown that carrageenan activates the toll-
like receptor 4 (TLR4) (Bhattacharyya et al. 2008).
TLR4 is an important receptor in the initiation of
the inflammatory response in human cells (Lin
and Yeh 2005). The identication of TLR4 as the
receptor membrane that interacts with carrageenan
strengthens the hypothesis that the effects of
carrageenan are attributable to the activation of
NF-kB.
The anti-inammatory activity of lavender oil
was further evaluated by the inhibition of croton oil-
induced ear edema. The fact that carrageenan and
croton oil have different inammatory mechanisms,
led us to suggest a possible mechanism of action for
LEO. The croton oil is a highly irritating substance
that stimulated an inammatory response in the
epidermis. Tetradecanoyl-phorbol acetate is a
phorbol ester derivative of croton oil (Pitot 1979).
TPA-induced inflammation is associated with
alterations in cytokine production and increased
production of prostaglandins and leukotrienes.
These effects are thought to be mediated by
protein kinase C (Passos et al. 2013). Many protein
kinases C are activated by diacylglycerol and by
high levels of intracellular calcium, both produced
by activation of receptors coupled to G protein.
Protein kinase C mediates a number of intracellular
signal transduction pathways implicated in
the pathogenesis of inflammation, including
phospholipase A2-dependent arachidonic acid
release and eicosanoid production.
The anti-inammatory action of LEO in croton
oil-induced ear edema model, compared to the
reference drug, dexamethasone, was greater than
the effect observed in the pleurisy model. This
nding suggests that the mechanism involved in
the anti-inammatory effect of lavender may be
related to a G protein-coupled receptor and/or
interference in the system of intracellular second
messenger phospholipase C/inositol phosphate.

An Acad Bras Cienc (2015) 87 (2 Suppl.)
1406 GABRIELA L. DA SILVA et al.
The receptors involved in pain are also
activated in inammation. Pain is not a single entity,
but differs in its underlying causes, symptoms and
neurobiological mechanisms. On a coarse scale,
three major forms of pain can be distinguished.
Nociceptive or acute pain originates from the acute
activation of primary nociceptive nerve fibers,
and inammatory pain originates from all forms
of inammation. The neuropathic pain originates
from the damage of peripheral or central nerves
and neurons. Inammatory and neuropathic pain
can turn into chronic pain syndromes, in which
plastic changes in nociceptive processing have
occurred that are no longer readily reversible by
pharmacological treatment (Zeilhofer 2007).
Acute and chronic pain can be relieved
effectively by non-steroidal anti-inflammatory
drugs that inhibit cyclooxygenases (COX-1
and COX-2) and, consequently, are widely used
(Hoogstraate et al. 2003). The opioid analgesic
drugs remain the most effective therapy available
for the treatment of moderate to severe pain;
however, the problems arising from unwanted side-
effects persist. A range of new therapies has been
developed in recent decades to treat inammation
and chronic inammatory diseases. Nonetheless,
the number of such drugs remains small and in
addition to the fact that they all display side effects
that limit their use. Thus, the development of new
pain management strategies can allow clinicians to
have additional options for the management pain.
The antinociceptive properties of lavender
species have been demonstrated by many authors
(Hajhashemi et al. 2003, Barocelli et al. 2004).
In this study, we evaluated the antinociceptive
effect of Lavandula angustifolia Mill. essential oil
using the formalin-induced pain model. There is a
correlation between nociceptive and inammatory
effects in this model. This relationship is dependent
on the concentration of formalin injected into the
rat’s hind paw. Thus, only high concentrations
of formalin, which produce significant plasma
extravasation, are suitable for demonstrating the
antinociceptive effects of anti-inammatory agents
(Yashpal and Coderre 1998). In a pilot test in
our laboratory, we found that this concentration
would be 2%, since the antinociceptive effects
of indomethacin parallel their anti-inammatory
action were observed throughout the second phase.
Subcutaneous injection of formalin, a
chemical irritant, into the rat’s paw results in a
reproducible biphasic response (Rocha-González
et al. 2005). The reaction in the rst phase (phase
1, 0–5 minutes) is caused by the initial tissue injury
and a direct activation of peripheral nociceptors
by formalin. The second phase (phase 2, 15–60
minutes) is mediated by a low level of peripheral
nerve activity, whose effects are then enhanced in
the spinal level by central sensitization (Yashpal
and Coderre 1998). It has been well documented
that inammatory mediators such as neuropeptides,
kinins, protons, prostanoids, serotonin and other
substances are released during the inammation
produced by tissue damage. These mediators cause
a painful inammatory response.
In this work, we demonstrated that, in addition
to anti-inammatory activity, the oral treatment with
lavender oil produces signicant antinociception.
In the rst phase, lavender essential oil, as well as
tramadol, presented antinociceptive effects. Indeed,
indomethacin, a non-selective COX inhibitor, was
able to inhibit the antinociceptive behavior only
in the second phase. This data supports a relevant
role for cyclooxygenases in this model. With this
rationale one would think that, if lavender treatment
caused a similar behavior to tramadol and different
from indomethacin, then possibly the mechanism
of action of lavender essential oil is not involved
with inactivation of COX. However, it could be
related to opioidergic neurotransmission. These
findings are in agreement with previous study
(Barocelli et al. 2004) which showed that treatment
with naloxone, an opioid antagonist, prevents the
analgesic action of lavender essential oil.
The opioid receptors belong to the family of
receptors coupled to G protein and inhibit adenylate
cyclase, reducing the content of intracellular AMPc.
These receptors also exert effects on ion channels

An Acad Bras Cienc (2015) 87 (2 Suppl.)
LAVENDER ESSENTIAL OIL PROPERTIES 1407
via G protein. Thus, the opioids or its agonists
promotes the opening of potassium channels and
inhibit the opening of calcium channels. The
fact that lavender oil presented similar effect to
tramadol and have been able to inhibit the action of
croton oil (a protein kinase C activator), suggesting
that the mechanism of action can be related to the
activation of channels via protein G.
In conclusion, the results of the present study
show the analgesic and anti-inammatory activities
of the lavender oil. Furthermore, the effectiveness of
the oil without evidence of signicant toxic effects
supports the interest for application of the lavender
essential oil as a therapeutic agent. Further studies
should be conducted to evaluate and characterize
the receptors involved in the antinociceptive and
anti-inammatory effects.
RESUMO
Diversos estudos têm investigado as propriedades
antinociceptivas, imunomoduladoras e anti-inamatórias
dos compostos encontrados no óleo essencial de
lavanda (OEL), contudo, até o momento, ainda faltam
dados mais substanciais. O objetivo deste estudo foi
avaliar os efeitos antioxidantes, anti-inamatórios e
antinociceptivos do óleo essencial de lavanda. Para
avaliação da atividade antioxidante foi realizado o ensaio
de descoloração do radical 1,1-difenil-2-picrilhidrazil.
A atividade anti-inamatória foi testada utilizando dois
modelos de inamação aguda: indução de pleurisia por
carragenina e edema de orelha induzido pela aplicação
de óleo de cróton. A atividade antinociceptiva foi
avaliada utilizando o modelo de dor provocada por
formalina. OEL apresenta atividade antioxidante, sendo
esta resposta dose-dependente. A resposta inamatória
provocada pela carragenina e pelo óleo de cróton foi
reduzida com o pré-tratamento dos animais com OEL.
No modelo de pleurisia, a droga usada como controle
positivo, dexametasona, foi mais ecaz. Entretanto, no
edema de orelha, o efeito antiedematogênico do óleo
foi similar ao observado na dexametasona. No teste da
formalina, o OEL inibiu consistentemente a nocicepção
espontânea e apresentou efeito similar ao do tramadol.
Os resultados deste estudo revelam a atividade (in vivo)
analgésica e anti-inamatória do OEL, demonstrando
seu importante potencial terapêutico.
Palavras-chave: antioxidante, inamação, lavanda,
nocicepção.
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