PHYTOTHERAPY RESEARCH

Phytother. Res. (2010)

Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.3325

Acute Effects of Bergamot Oil on

Anxiety-related Behaviour and Corticosterone
Level in Rats ptr_3325 1..5

Somrudee Saiyudthong1,2* and Charles A. Marsden3

1
Department of Physiology, Faculty of Medicine, Srinakharinwirot University, Bangkok, Thailand
2
Neuroscience Research Center, Srinakharinwirot University, Bangkok, Thailand
3
School of Biomedical Sciences, University of Nottingham, Nottingham, UK

Bergamot essential oil (BEO), Citrus aurantium subsp. bergamia (Risso) Wright & Arn. (Rutaceae), is used
widely in aromatherapy to reduce stress and anxiety despite limited scientific evidence. A previous study showed
that BEO significantly increased gamma-aminobutyric acid levels in rat hippocampus, suggesting potential
anxiolytic properties. The aim of this study was to investigate the effect of BEO (1.0%, 2.5% and 5.0% w/w)
administered to rats on both anxiety-related behaviours (the elevated plus-maze (EPM) and hole-board tests)
and stress-induced levels of plasma corticosterone in comparison with the effects of diazepam. Inhalation of
BEO (1% and 2.5%) and injection of diazepam (1 mg/kg, i.p.) significantly increased the percentage of open
arm entries on the EPM. The percentage time spent in the open arms was also significantly enhanced following
administration of either BEO (2.5% and 5%) or diazepam. Total arm entries were significantly increased with
the highest dose (5%), suggesting an increase in locomotor activity. In the hole-board test, 2.5% BEO and
diazepam significantly increased the number of head dips. 2.5% BEO and diazepam attenuated the corticos-
terone response to acute stress caused by exposure to the EPM. In conclusion, both BEO and diazepam
exhibited anxiolytic-like behaviours and attenuated HPA axis activity by reducing the corticosterone response
to stress. Copyright © 2010 John Wiley & Sons, Ltd.

Keywords: essential oil; elevated plus-maze; anxiety; HPA axis; stress.

Bergamot, Citrus aurantium subsp. bergamia (Risso)

INTRODUCTION
Anxiety disorders are among the most common type of
psychiatric disorders and include generalized anxiety
(GAD), panic disorder (PD), social anxiety disorder
(SAD) and post-traumatic stress disorder (PTSD)
(Kessler et al., 2005). Various classes of anxiolytic drugs
have been specifically prescribed for each disorder
(Hoffman and Mathew, 2008). In addition, aroma-
therapy, one of the most popular complementary thera-
pies, has been used extensively to relieve stress and
anxiety. In human studies, aromatherapy has been shown
to reduce anxiety in both healthy subjects (Höferl et al.,
2006; Rho et al., 2006; Takeda et al., 2008) and patients
(Imanishi et al., 2007). In animal studies, many essential
oils, used in aromatherapy, show anxiolytic effects. For
example, lavender oil has anxiolytic effects in rats placed
in the open field (Shaw et al., 2007), rose oil demonstrates
anxiolytic-like properties using the elevated plus-maze
(de Almeida et al., 2004) and essential oil from Citrus
aurantium L. induces anxiolytic activity in mice again
detected using the elevated plus-maze (Carvalho-Freitas
and Costa, 2002). Although the effects of various essen-
tial oils on anxiety have been examined, bergamot essen-
tial oil (BEO), also suggested to reduce anxiety, has been
subjected to very limited investigation.
* Correspondence to: S. Saiyudthong, Department of Physiology, Faculty
of Medicine, Srinakharinwirot University, Bangkok, Thailand.
E-mail: somrudee@swu.ac.th
Copyright © 2010 John Wiley & Sons, Ltd.
Wright & Arn. (Rutaceae), has been used in many situ-
ations including the food, drug and cosmetic industries.
In aromatherapy, BEO, normally extracted from berga-
mot peel, has been suggested to reduce stress and
anxiety (Price and Price, 1999). Despite the growing
application of this extract, there are only a few system-
atic investigations on these biological properties. A
previous study showed that intraperitoneal administra-
tion of BEO significantly increased the extracellular
levels of the inhibitory amino acid neurotransmitter
gamma-aminobutyric acid (GABA) in rat hippocampus
(Morrone et al., 2007). This effect of BEO on the
GABAergic system could indicate anxiolytic related
behavioural effects as the existing benzodiazepine
anxiolytic drugs act by increasing GABA function in
the brain. There are, however, no studies investigat-
ing anxiety-related behaviour following BEO
administration.
The aims of the present study were to investigate in
the rat the effects of BEO on two anxiety-related behav-
ioural models, the elevated plus-maze and the hole-
board, and to determine whether BEO altered the
plasma corticosterone response to stress induced by
exposure to the elevated plus-maze.
MATERIALS AND METHODS
Animals. Male Wistar rats weighing 200–250 g
(National Laboratory Animal Centre, Mahidol Univer-
Received 30 January 2009
Revised 22 August 2010
Accepted 16 September 2010
 S. SAIYUDTHONG AND C. A. MARSDEN
sity, Bangkok, Thailand) were housed in groups of four
in a room with a 12 : 12 h light/dark cycle (light on at
0700 h) and maintained at a constant temperature of
22 ⫾ 0.5°C. Laboratory pellets (National Laboratory
Animal Center, Thailand) and water were available ad
libitum.Animals were used after an adaptation period of
1 week. The rats were inexperienced to the elevated
plus-maze and hole-board apparatus as well as naïve to
essential oil and drugs. For the behavioural test and
corticosterone measurement, the rats were divided into
six groups (n = 10), saline (i.p), saline (inhalation, INH),
diazepam 1 mg/kg (i.p.) and BEO (1.0%, 2.5%, and 5.0%
w/w, INH).All experiments in this study were carried out
according to the Animal Ethics Committee of Srina-
kharinwirot University, which is in compliance with the
International Guiding Principles for Biomedical
Research Involving Animals provided by the National
Research Council of Thailand.
Chemicals. Diazepam (10 mg/2 mL ampoule), pur-
chased from Roche (Welwyn Garden City, UK), was
diluted with 0.9% saline solution. BEO, extracted from
Kaffir Lime peels and without bergapten, was supplied
by Make Scent Limited (Bangkok, Thailand). The BEO
samples were analysed using GC and confirmed using
GC-MS to identify the major compounds (limonene,
linalool and linalyl acetate). The GC-MS analysis was
carried out using an Agilent Technologies mass spec-
trometer (Model 6890 N), with a thermo conductivity
detector with a HP-INNOWAX (30 m ¥ 0.25 mm ¥
25 mm) column. The column temperature was kept at
50°C and programmed to 230°C (4°C/min). The pulse
split ratio was adjusted at 10:1, under an injector tem-
perature of 230°C. Electron impact ionization was at
70 eV. Emulsions (water/oil) of BEO 1.0%, 2.5% and
5.0% (w/w) were freshly prepared just before the start
of the experiments. Control inhalations were performed
using 0.9% saline.
Elevated plus-maze test. The procedures used for the
elevated plus-maze were as described previously
(Handley and Mithani, 1984; Pellow et al., 1985). The
elevated plus-maze comprised two open arms (45 ¥
15 cm) and two enclosed arms (45 ¥ 15 ¥ 10 cm) that
extended from a common central platform (10 ¥ 10 cm).
The apparatus was made from black Perspex and
elevated to a height of 70 cm above floor level. For the
test, the rats were placed individually in the centre of
the cross of the elevated plus-maze and then allowed to
explore for 5 min. The behavioural parameters mea-
sured were the percentage entries into the open arms,
the percentage time spent in the open arms together
with total arm entries. The behaviour was recorded on a
video for later analysis.
Hole-board test. The procedures used for the hole-
board were modified from that described by File and
Wardill (1975). The hole-board apparatus, raised to a
height of 7 cm above floor level, was made of wood
covered with dark Formica (62 ¥ 62 ¥ 36 cm) with 16
holes, each 4 cm in diameter, equally spaced in the floor.
For the hole-board experiments, each rat was placed in
the centre of the hole-board and allowed to freely
explore the apparatus for 5 min. The number of head
dips and the time spent head dipping were recorded by
Copyright © 2010 John Wiley & Sons, Ltd.
a video camera for later analysis. A head dip was scored
when the head was introduced into the holes at least to
the level of the eyes.
Inhalation. The inhalation apparatus used in this study
was modified from that described by the previous study
(de Almeida et al., 2004). The floor was made of a stain-
less steel grid. The apparatus was made of acrylic fibre
and consisted of front and back walls with four holes
(2 cm in diameter each) into which cotton soaked with
0.9% saline or BEO was placed.The upper side, contain-
ing 30 small holes for ventilation, could be opened to
allow the animals to be placed into the box.After a 7 min
exposure for each rat, the cotton was removed and the
inside cleaned before placing another rat into the box.
Determination of plasma corticosterone. Enzyme
immunoassay (EIA) for corticosterone was carried out
using a corticosterone kit (Immunodiagnostic Systems
Ltd, Tyne & Wear, UK). Briefly, plasma samples were
defrosted, centrifuged at 5000 ¥ g for 5 min at 4°C.
Thirty microlitres of samples was diluted using 270 mL
of diluents and then vortexed. One hundred microlitres
of standard, control or diluted samples were transferred
to antibody-coated plates and 100 mL of enzyme conju-
gate added. The plate was covered and incubated at 4°C
for 24 h, then washed three times with wash buffer. Two
hundred microlitres of TMB (tetramethylbenzidine)
substrate were added and incubated at 25°C for 30 min.
Absorbance at 450 nm was measured after the addition
of 100 mL of HCl to stop the reaction.
Experimental procedures. The behavioural experi-
ments were performed in the early part of the light
phase (0900–1200 h) (Büttner and Wollnik, 1984). Rats
were transferred to the behavioural observation room
about 30 min prior to the experiments. The behavioural
assessment was later scored by two observers from the
video tapes. Either diazepam 1 mg/kg or 0.9% saline
was administered intraperitoneally 30 min before expo-
sure to the behavioural experiments. For the inhalation
procedure, BEO in the form of an aqueous emulsion
(1%, 2.5% or 5%) was applied to the cotton and
inserted into the inhalation apparatus. The animals were
exposed to the vapour for 7 min. For the control group,
0.9% saline was applied to the cotton. At the end of the
elevated plus-maze experiment, the animals were
immediately decapitated. Trunk blood samples were
taken and put into heparinized tubes and then centri-
fuged (1500 ¥ g, 5 min, room temperature). Plasma was
collected and subsequently stored at -20°C until
required for the corticosterone EIA.
Statistical analysis. All data are expressed as mean ⫾
SEM. The data were analysed by one-way ANOVA with
a post hoc Dunnett’s test when appropriate. Differences
were considered statistically significant when p < 0.05.
RESULTS AND DISCUSSION
The results in the present study demonstrate for the
first time that BEO has an anxiolytic effect, using two
behavioural measurements of anxiety, the elevated plus-
Phytother. Res. (2010)
 ACUTE EFFECTS OF BERGAMOT OIL ON ANXIETY-RELATED BEHAVIOUR
Figure 1. Effects of bergamot essential oil (BEO inhalation, INH;
1%, 2.5% and 5% w/w) and diazepam (DZP; 1 mg/kg, i.p.) com-
pared with controls (0.9% saline (CONT) either i.p. or INH) on
percent time spent in open arm (A), percent open arm entries (B)
and total arm entries (C). Data are expressed as mean ⫾ SEM
(n = 10). * p < 0.05 and ** p < 0.01 compared with CONT (i.p.)
and # p < 0.05 and ## p < 0.01 compared with CONT (INH).
maze test and the hole-board test. The results with BEO
were similar to those observed following diazepam
treatment. The BEO also reduced the corticosterone
response to stress exposure.
The elevated plus-maze is a well-established animal
model used extensively to identify potential new anxi-
olytic drugs (Handley and Mithani, 1984; Pellow et al.,
1985; Dawson and Tricklebank, 1995). In this test, it is
assumed that animals feel safe in the closed arms but
fear and anxiety during exploration of the open arms.
The percentage entries into the open arms and time
spent in the open arms are generally used as indices of
anxiety and drugs increasing these measures show anxi-
olytic properties. The inhalation of the intermediate
dose (2.5% BEO), significantly increased both the per-
centage of entries in the open arms (p < 0.01) and time
spent on the open arms (p < 0.01), indicating the anxi-
olytic effect (Fig. 1A, B). The lowest dose (1% BEO)
Copyright © 2010 John Wiley & Sons, Ltd.
80
A
Time head dipping
60
40
20
B 50
*
Number head dipping
#
40
30
20
10
0
CONT i.p. CONT INH DZP 1 mg/kg 1% BEO 2.5% BEO 5% BEO
Figure 2. Effects of bergamot essential oil (BEO inhalation, INH;
1%, 2.5% and 5% w/w) and diazepam (DZP; 1 mg/kg, i.p.) com-
pared with controls (0.9% saline (CONT) either i.p. or INH) on
time head dipping (A) and number head dipping (B). Data are
expressed as mean ⫾ SEM (n = 10). * p < 0.05 compared with
CONT (i.p.) and # p < 0.05 compared with CONT (INH).
increased the percentage of entries in the open arms (p
< 0.05) but did not change the percentage of time spent
in the open arms. The highest dose (5% BEO) increased
total arm entries (p < 0.05), suggesting increased loco-
motor activity in the elevated plus-maze.A saturation of
the total arm entries with (5% BEO) was not observed
(Fig. 1C). Further studies with higher doses may clarify
this issue. Diazepam produced a similar profile to 2.5%
BEO, increasing the percentage of entries in the open
arms (p < 0.05) and the percentage of time spent in the
open arms (p < 0.01). These findings replicate previous
studies showing that acute diazepam treatment
increases the percentage open: total entries and time
and time spent in the end of the open arms on the
elevated plus-maze (Wright et al., 1992; Nishino et al.,
2008). Moreover, the results also showed that total arm
entries were significantly increased after the inhalation
of the highest dose of BEO, suggesting an increase in
locomotor activity (p < 0.05) (Fig. 1).
The hole-board test has been used widely to assess
anxiety, emotionality or responses to stress in animals
(Takeda et al., 1998; Adamec et al., 2006; Harada et al.,
2006). An increase in head-dipping behaviour in the
hole-board test reflects an anxiolytic-like state (Takeda
et al., 1998). In the present study, 2.5% BEO signifi-
cantly increased the number of head dips (p < 0.05).
Diazepam (1 mg/kg) also produced a significant
increase in head dipping, in agreement with a previous
study (Wei et al., 2007) (p < 0.05) (Fig. 2). The data from
the present study indicate that 2.5% BEO has a signifi-
cant anxiolytic-like effect in this paradigm.
The mechanism underlying the anxiolytic properties
of BEO is unknown but may be due to alterations in
Phytother. Res. (2010)
 S. SAIYUDTHONG AND C. A. MARSDEN
Figure 3. Effects of bergamot essential oil (BEO inhalation, INH;
1%, 2.5% and 5% w/w) and diazepam (DZP; 1 mg/kg, i.p.) com-
pared with controls (0.9% saline (CONT) either i.p. or INH) on
plasma corticosterone following a 5 min exposure to the
elevated plus-maze test. Data are expressed as mean ⫾ SEM
(n = 10). * p < 0.05 compared with CONT (i.p.) and # p < 0.05
and compared with CONT (INH).
GABA function, the major inhibitory neurotransmitter
in the brain. Our behavioural findings are supported by
a previous study (Morrone et al., 2007) showing that
intraperitoneal administration of BEO significantly
increased the extracellular levels of GABA in the hip-
pocampus of freely moving rats. It has been suggested
that anxiety disorders are associated with dysfunction
of GABAergic systems possibly due to reduced
receptor sensitivity and/or decreased levels of the neu-
rotransmitter (Nutt, 2001). Benzodiazepines bind to a
benzodiazepine-binding site on the GABAA receptor
complex, resulting in the facilitation of the action of
GABA and anxiolytic effects. A previous study showed
that lavender oil, mainly containing linalyl acetate and
linalool, potentiated the GABAA receptor response
caused by a low concentration of GABA, suggesting the
anxiolytic, anticonvulsant and sedative activity
(Aoshima and Hamamoto, 1999).These two compounds
are also present in BEO. Therefore, future studies need
to investigate whether the anxiolytic properties of BEO
are mediated by GABAA receptors.
The stress hormone corticosterone was measured to
investigate the response of the HPA axis to BEO. The
present data show that inhalation of 2.5% BEO, a dose
that produced anxiolytic activity in the behavioural
experiments, attenuated the activity of HPA axis by
reducing the corticosterone response to the stress of
exposure to the elevated plus-maze (Fig. 3). Similarly,
plasma corticosterone levels of rats treated with diaz-
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studies need to focus on the mechanism of action of
BEO involved in these anxiolytic properties.
Acknowledgements
This research was supported by a grant from Faculty of Medicine,
Srinakharinwirot University. Our thanks go to Dr Reinaldo Nóbrega
de Almeida, Department of Physiology and Pathology/LTF, Univer-
sidade Federal da Paraiba, for suggesting the inhalation apparatus. We
also wish to thank colleagues from Faculty of Medicine, Srinakharin-
wirot University, Associate Professor Dr Surangrat Srisurapanon for
advice concerning the corticosterone measurements and Dr Alfredo
Villarroel for invaluable comments.
Conflict of Interest
The authors have declared that there is no conflict of interest.
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