Molecules 2015, 20, 18620-18660; doi:10.

3390/molecules201018620
OPEN ACCESS

molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Review

A Systematic Review of the Anxiolytic-Like Effects of Essential

Oils in Animal Models
Damio Pergentino de Sousa 1, Palloma de Almeida Soares Hocayen 2, Luciana Nalone Andrade 3
and Roberto Andreatini 2,*

1
Departamento de Cincias Farmacuticas, Universidade Federal da Paraba, CP 5009, Joo Pessoa-PB,
CEP 58051-970, Brazil; E-Mail: damiao_desousa@yahoo.com.br
2
Departamento de Farmacologia, Universidade Federal do Paran, CP 19031, Curitiba-PR,
CEP 81540-970, Brazil; E-Mail: pallomaas@yahoo.com.br
3
Departamento de Fisiologia, Universidade Federal de Sergipe, CEP 49100-000, So Cristvo-SE,
Brazil; E-Mail: lulisynalone@hotmail.com

* Author to whom correspondence should be addressed; E-Mail: randreatini@ufpr.br;

Fax: +55-41-3266-2042.

Academic Editor: Derek J. McPhee

Received: 19 August 2015 / Accepted: 2 October 2015 / Published: 14 October 2015

Abstract: The clinical efficacy of standardized essential oils (such as Lavender officinalis),
in treating anxiety disorders strongly suggests that these natural products are an important
candidate source for new anxiolytic drugs. A systematic review of essential oils, their
bioactive constituents, and anxiolytic-like activity is conducted. The essential oil with the
best profile is Lavendula angustifolia, which has already been tested in controlled clinical
trials with positive results. Citrus aurantium using different routes of administration
also showed significant effects in several animal models, and was corroborated by different
research groups. Other promising essential oils are Citrus sinensis and bergamot oil, which
showed certain clinical anxiolytic actions; along with Achillea wilhemsii, Alpinia zerumbet,
Citrus aurantium, and Spiranthera odoratissima, which, like Lavendula angustifolia, appear
to exert anxiolytic-like effects without GABA/benzodiazepine activity, thus differing in their
mechanisms of action from the benzodiazepines. The anxiolytic activity of 25 compounds
commonly found in essential oils is also discussed.
Molecules 2015, 20 18621

Keywords: essential oil; terpene; natural products; anxiolytic; anxiety; tranquillizer; sedative;
relaxant; elevated plus maze; lavender

1. Introduction

Generalized anxiety disorder (GAD) is characterized by a high and free-floating anxiety, revealing
both psychic (e.g., tension, insomnia, etc.), and somatic symptoms (e.g., muscle tension, dry mouth, etc.).
GAD maintains a significant prevalence and causes important personal, family, and social impairments,
which makes adequate treatment of GAD patients essential.
Pharmacological treatment of GAD usually employs benzodiazepines (e.g., diazepam and clonazepam),
azaspirone (buspirone), and antidepressants (e.g., paroxetine). More recently, the anticonvulsant pregabalin
has been introduced to treat GAD. However, all of these drug treatments have important drawbacks, such
as abuse/dependence liability, retrograde amnesia (benzodiazepines), clinical effect delay (buspirone and
antidepressants), sexual dysfunction (antidepressants), sedation (benzodiazepines and pregabalin), and
dizziness (pregabalin), all of which affect clinical adherence [1]. Though it is not surprising that nearly
43% of patients with significant anxiety symptoms already use some form of alternative treatment [2],
the above facts highlight the need for new anxiolytic drugs.
Medicinal plants are cited frequently as a potential source for new drugs, including for GAD. However,
despite numerous pre-clinical studies, in well controlled studies very few medicinal plants have showed
consistent clinical efficacy for GAD [3]. One interesting exception is a standardized essential oil from
Lavender officinalis, called Silexan, which has been found effective for GAD patients [4], and reinforces
the attention given to essential oils to source new GAD treatments. When we recognize that extensive
pre-clinical research for discovery of new anxiolytic drugs has resulted disappointingly in very few
molecules, their importance increases [5]. Certain essential oils have shown anxiolytic-like effects in
animal models [68]. Essential oils are obtained from natural raw plant material thru steam distillation,
from the epicarp of citrus fruits through mechanical processes, or by dry distillation after separation of
the aqueous phaseif anyby physical processes (ISO 9235:2013). The pharmacological activity of
essential oils on the central nervous system has been reported in previous reviews [911]. An earlier
systematic review [6] focuses more on methodological issues. In the interim however, several studies have
been publicized (Table 1). The aim of this review was to conduct a systematic investigation of pre-clinical
essential oil studies in animal anxiety models.

2. Results and Discussion

The essential plant oils found with our search strategy where anxiolytic-like effect was found were:
Acantholippia deserticola, Achillea umbelata, Achillea wilhemsii, Alpinia zerumbet, Angelica sinensis,
Chamaecypais obtuse, Casimiroa pringlei, Citrus aurantium, Citrus aurantium subsp. bergomia (bergamot),
Citrus junos, Citrus latifolia, Citrus limon, Citrus reticulate, Citrus sinensis, Coriandrum sativum var.
microcarpum, Cymbopogon citratus, Ducrosia anethifolia, Celastarus paniculatus, Chamaecyparis obtuse,
Copaifera reticulata Ducke, Dennettia tripetala, Ducrosia anethifolia, Foeniculum vulgare, Lavendula
angustifolia, Lippia alba, Ocimum sanctum L., Ocimum basilicum L. essential oil, Piper guineense, propolis,
Molecules 2015, 20 18622

rose, Spiranthera odoratissima A. St. Hil, Santalum album L., Stachys tibetica, and Thujopsis dolabrata.
Table 1 shows the studies included, describing their main characteristics (essential oil, administration
route, species and genus, animal model used, group control used, control for motor activity), and results.
In addition, we present certain studies which evaluate the anxiolytic-like effects of constituents isolated
from their essential oils (Table 2).

2.1. Abies sachalinensis

The effect of Abies sachalinensis essential oil (dark green needles) was evaluated in the elevated
plus-maze [12]. Male mice treated acutely with inhaled Abies sachalinensis essential oil exhibited an
increase in open arm exploration (% time spent and entries) in a 10-min session. However, when the
essential oil was administered intraperitoneally it did not induce any change in the open arm explorations.
These results indicate that the route of administration influences the anxiolytic-like effect of the essential
oil, and the authors proposed that this difference may be related to the brain concentration of its constituents
(e.g., -pinene), and/or the olfactory sense [12].

2.2. Acantholippia deserticola

Female rats treated (i.p., acute, only during diestrous phase) with Acantholippia deserticola essential
oil revealed increases in open arm exploration (% time and entries). However, these effects were seen at
dose that also decreased square crossings in the open-field test, indicating motor impairment or
sedative/depressant effect. Furthermore, the essential oil has a narrow effective range, with toxic effect
(tonic-clonic convulsion) at a dose slightly above the effective dose in the elevated plus-maze [13].

2.3. Achillea umbelata

Achillea umbelata essential oil (aerial parts) was administered orally (acute) to male mice tested in
the light/dark test [14]. An increase in time spent in the light side, and a decrease in the number of
transitions was observed. The experimenters also described a certain paralyzing effect of the treatment.
Moreover, the essential oil decreased motor activity in the open-field test. The authors suggested that
the effect was more likely related to toxic effect than anxiolytic-like effect [14].

2.4. Achillea wilhemsii

Male rats treated acutely with A. williemsii essential oil (i.p.) displayed increased open arm exploration
(number of entries, and % time spent) in the elevated plus-maze at a dose that did not change the total
number of entries but decreased the closed arm entries [15]. The latter result indicates a motor impairment
that may have influenced the anxiety indexes. Although flumazenil blocked the positive effect of diazepam
(positive control), it did not change the effect of A. williemsii, indicating a non GABA/benzodiazepine
mediation. Also, naloxone did not block the effect of A. williemsii.
Molecules 2015, 20 18623

Table 1. Summary of studies with essential oils in animal models of anxiety (only essential oils with at least one positive result are included).
Essential Oils Administration Specie Anxiety Model Observed Effect Motor Activity Mechanism of Action Observation Reference
Abies Inhalation (acute) Anxiolytic-like 10-min session
Mouse Elevated plus-maze Not evaluated [12]
sachalinensis i.p. (acute) No effect DR+
Female only
Acantholippia
i.p. Rat Elevated plus-maze Anxiolytic-like? Decrease Toxicity? [13]
deserticola
DR+
Achillea Toxicity?
Oral (acute) Mice Light/Dark Anxiolytic-like? Decrease [14]
umbelata DR+
Achillea Not mediated by BDZ
i.p. (acute) Rat Elevated plus-maze Anxiolytic-like Decrease? DR+ [15]
wilhemsii Not mediated by opioid receptors
i.p. (acute) Mouse Elevated plus-maze No effect Decrease DR+ [16]
Decrease 5-HTP and fluoxetine Motor activity evaluated trough rearing
Inhalation (acute) Mouse Elevated plus-maze Anxiolytic-like No change [17]
induced jumping (5-HT action) DR+
Inhalation (3 days) Elevated plus-maze Anxiolytic-like No change
Alpinia zerumbet Mouse DR [18]
Inhalation (1 day) Light/Dark No effect No change
Anxiolytic-like effect is dependent of
Inhalation
Mouse Elevated plus-maze Anxiolytic-like No change duration of inhalation (30120 min) [12]
(5150 min)
DR+
Elevated plus-maze Anxiolytic-like
Inverted U curve
Oral (acute) Mouse Light/Dark Anxiolytic-like No change [19]
DR+
Angelica sinensis Stress-induced hyperthermia Anxiolytic-like
Inverted U curve
Oral (acute) Rat Social Interaction Putative Anxiolytic-like Increase [19]
DR+
Casimiroa Elevated plus-maze No effect Positive effect when compared to caffeine only
Oral (acute) Rat No change [20]
pringlei Holeboard (compared to control) DR+
Celastarus Elevated plus-maze Anxiolytic-like
Oral (repeated) Rat No change [21]
paniculatus Vogel Anxiolytic-like
Molecules 2015, 20 18624

Table 1. Cont.
Essential Oils Administration Specie Anxiety Model Observed Effect Motor Activity Mechanism of Action Observation Reference
10-min session
Inhaled (acute) Mouse Elevated plus-maze Anxiolytic-like Not evaluated [22]
DR+
Chamaecyparis
Reversed
obtusa Associated with IL-6 and Ccl2
Inhaled (repeated) Rat Elevated plus-maze anxiogenic-like effect Not evaluated DR [23]
cytokines reductions
of mother separation
Elevated plus-maze
Oral Mouse Anxiolytic-like No change DR+ [24]
Open Field
Marble-burying
Oral Mouse Anxiolytic-like No change DR+ [25]
Light/Dark
Social interaction
Citrus aurantium
Inhalation rats Open Field Anxiolytic-like No change DR+ [26]
L.
Elevated plus-maze
Oral Mouse Light/Dark Anxiolytic-like No change 5-HT1A-receptors DR+ [27]
i.p. (acute) Mouse Elevated plus-maze Anxiolytic-like Not evaluated DR+ [28]
Reduced diazepam anxiolytic-effect
i.p. (acute) Mouse Elevated plus-maze Anxiolytic-like Not evaluated GABA partial agonist? [29]
DR+
inverted U-shaped curve
Citrus aurantium DR+
Rat Elevated plus-maze Anxiolytic-like? Increase? [30]
subsp. bergamia decrease increase corticosterone induced by
behavioral test
Elevated plus-maze Anxiolytic-like
Citrus junos Inhalation Mouse No change DR+ [31]
Light/Dark Anxiolytic-like
Coriandrum Decrease anxiogenic-
Not tested in naive rats
sativum var. Inhalation (repeated) Rats Elevated plus-maze like effect of icv Not evaluated [32]
DR
microcarpum beta-amyloid (142)
Molecules 2015, 20 18625

Table 1. Cont.
Essential Oils Administration Specie Anxiety Model Observed Effect Motor Activity Mechanism of Action Observation Reference
Inhalation Rat
Elevated plus-maze Anxiogenic-like No change DR [33]
(continuous for 1 week) (male/female)
Lemon 5-HTergic
Inhalation Mouse Elevated plus-maze Anxiolytic-like? Decrease (5-HT1A) DR [34]
GABA-A/BZP and DAminergic
inverted U-shaped curve
Citrus limon Oral (acute) Mouse Elevated plus-maze Anxiolytic-like? Decrease [35]
DR+
Marble-burying Anxiolytic-like inverted U-shaped curve
Citrus latifolia Oral Mouse No change [36]
Light/Dark Anxiolytic-like DR+
Marble-burying Anxiolytic-like
Citrus reticulata Oral Mouse No change DR+ [36]
Light/Dark No effect
Melaleuca alternifolia essential oil used as
Light/Dark Anxiolytic-like
Citrus sinensis Inhalation Rat No change neutral odor control [37]
Elevated plus-maze Anxiolytic-like
DR+
Copaifera
Oral (acute) Rat Elevated plus-maze Anxiolytic-like No change DR+ [38]
reticulata
Open Field
Oral Mouse Elevated plus-maze Anxiolytic-like No change DR+ [39]
Cymbopogon
Light/Dark
citratus
Marble-burying
Oral Mouse Anxiolytic-like No change GABA-A /BDZ DR+ [40]
Light/Dark
Inconsistency in data showed in figure and text
Dennettia Mice Anxiolytic-like *
i.p. (acute) Elevated plus-maze Not evaluated Gender not considered [41]
tripetala (male/Female) (see observation)
DR+
Ducrosia
Oral (acute) Mouse Elevated plus-maze Anxiolytic-like No change DR+ [42]
anethfolia
Molecules 2015, 20 18626

Table 1. Cont.
Essential Oils Administration Specie Anxiety Model Observed Effect Motor Activity Mechanism of Action Observation Reference
Elevated plus-maze Anxiolytic-like
Foeniculum inverted U-shaped curve
Oral Mouse Staircase test Anxiolytic-like No change [43]
vulgare DR+
Open-field Anxiolytic-like
Lavandula Geller conflict Anxiolytic-like
i.p. Mouse No change DR+ [44]
officinalis Vogel conflict Anxiolytic-like
Inhalation Mouse Elevated plus-maze No effect DR [34]
Anxiolytic in male and female
Lavandula
Inhalation (24 h) Gerbil Anxiolytic-like Inclusion of ethological measures
angustifolia Elevated plus-maze [45]
Inhalation (7 days) (male/female) Anxiolytic-like One-tailed test
DR+
Increase immobility (sedation)
Not evaluated
Inhalation rat Open Field Anxiolytic-like Inclusion one group also exposed during the open-field [46]
(total locomotion)
DR+
Reduction in c-fos increases with open field exposition
Inhalation rat Open Field Anxiolytic-like No change [47]
DR
Nervous sheep: anxiogenic; calm sheep:
Reaction to stress
Inhalation Sheep Mixed results change anxiolytic-like effect [48]
(isolation)
DR
Anxiolytic-like effect correlated with
Lavandula
Inhalation Mouse Elevated plus-maze Anxiolytic-like No change linalool/linalyl acetate content [49]
angustifolia
DR
Similar effect in stressed and non-stressed mice
Inhalation Mouse Elevated plus-maze Anxiolytic-like [50]
DR
Serotonergic system
Elevated plus-maze Anxiolytic-like (5-HT1A) Neutral odor control
Inhalation Mouse No change [51]
Marble-burying Anxiolytic-like Not mediated by DR+
GABA-A/BDZ
Similar effects in anosmic and normal mice
Inhalation Mouse Marble-burying Anxiolytic-like No change [52]
DR+
Molecules 2015, 20 18627

Table 1. Cont.
Essential Oils Administration Specie Anxiety Model Observed Effect Motor Activity Mechanism of Action Observation Reference
Lavandula Increase hippocampal 5-HT Similar effects in anosmic and normal mice
Inhalation Mouse Elevated plus-maze Anxiolytic-like [53]
angustifolia turn-over DR
Elevated plus-maze
Anxiolytic-like
Open Field
Anxiolytic-like Some results could be influenced by motor
Elevated zero-maze
Lavandula i.p. (repeated) rat Anxiolytic-like Mixed results activity changes [54]
Social interaction test
angustifolia Anxiolytic-like DR+
Novelty-induced suppressed
(silexan) Anxiolytic-like
feeding latency test
Non selective inhibition of
Oral (repeated) Mouse Elevated plus-maze Anxiolytic-like No change DR+ [55]
voltage operated calcium channels
i.p. Mouse Elevated-plus-maze Anxiolytic-like No change DR+ [56]
Lippia alba
i.p. (acute) Rat Elevated T-maze Anxiolytic-like No change DR+ [57]
Litsea cubeba Oral (7 days) Mouse Elevated plus-maze Anxiolytic-like? Decrease DR+ [58]
Ocimum inverted U-shaped curve
Inhalation (acute) Mouse Light/Dark Anxiolytic-like? Decrease [59]
gratissimum L. DR+
Decrease anxiogenic-like
Ocimun Not tested in naive rats
Inhalation (repeated) Rat elevated plus-maze effect of icv beta-amyloid No change [60]
sanctum L. DR+
(142)
Decrease anxiogenic-like
Ocimum Not tested in naive rats
Inhalation (repeated) Rat elevated plus-maze effect of icv beta-amyloid No change [60]
basilicum L. DR+
(142)
Piper guineense Inhalation (acute) Mouse Light/Dark Anxiolytic-like? Mixed results DR+ [61]
Effect in stressed mice
Mouse Elevated plus-maze Anxiolytic-like No change [62]
DR+
Propolis Oral (14 days)
Elevated plus-maze Female (estrous phase not specified)
Rat Anxiolytic-like? Increase [63]
Open-field DR+
Molecules 2015, 20 18628

Table 1. Cont.
Essential Oils Administration Specie Anxiety Model Observed Effect Motor Activity Mechanism of Action Observation Reference
Geller conflict Anxiolytic-like
Rose centifolia i.p. Mouse No change DR+ [64]
Vogel Anxiolytic-like
Inhaled Rat Elevated plus-maze Anxiolytic-like Not evaluated DR+ [65]
Rose Ethanol as control
Inhalation (acute) Mouse Elevated plus-maze No effect No change [34]
DR
Prolonged Inhalation (24 h) Elevated plus-maze No effect
Rose damascena Gerbil Mixed DR [66]
Repeated Inhalation (14 days) Light/Dark Anxiolytic-like?
Santalum Anxiolytic-like in No effect in non-stressed mice
Inhalation (acute) Mouse Elevated plus-maze Not evaluated [67]
album L. stressed mice DR
Spiranthera Elevated plus-maze Anxiolytic-like 5-HTergic
odoratissima Oral (acute) Mouse Light/Dark Anxiolytic-like No change (5-HT1A) DR+ [68]
A. St. Hil. Open-field Anxiolytic-like Not mediated by GABA-A/BDZ
Elevated plus-maze Anxiolytic-like
Social Interaction Anxiolytic-like
Oral (3 days)
Stachys tibetica Rat Light/Dark Anxiolytic-like No change DR+ [69]
Oral (7 days)
Holeboard Anxiolytic-like
Elevated plus-maze Anxiolytic-like
Thujopsis 10 min session
Elevated plus-maze Anxiolytic-like Decrease [70]
dolabrata DR+
Anxiolytic-like?sedation/motor activity impairment could be a confounding variable; Toxicity?Putative toxic effect; DR (dose/concentration-response design)Plus
(+) where at least two doses/concentrations tested; Minus () where only one dose/concentration tested; * Statistical significant difference cited in the text but not showed
in the graphic.
Molecules 2015, 20 18629

Table 2. Constituents isolated from essential oils with anxiolytic-like effect.

Compound Experimental Protocol Anxiolytic-Like Effect and/or Mechanism Animal Tested Reference
1,4-Cineole

Elevated plus-maze test Increased exploration of the open arms

O Mice [71]
Holeboard test Increased head dipping

-Asarone
O
H3C Elevated plus-maze test Decreases in open-arm exploration
Holeboard test Increased time spent for head dips Rats [72]
O O
Open field test Increase in the total number of line crossings
CH 3 CH 3

-Pinene

Increased open arm exploration in the elevated

Elevated plus-maze test Mice [73]
plus maze

-Caryophyllene
Increased open arm exploration
Elevated plus-maze test
Increased time spent in light side, and number
Light/dark test
of transitions Mice [68,74]
Marble-burying test
Decreased number of marbles buried
Open-field test
Increased time spent in the center

Citral

Myrcene Elevated plus-maze test No effect was observed Mice [75]

Carvacrol

OH
Increased % time spent and % entries in the
Elevated plus-maze test Mice [76]
open arms
Molecules 2015, 20 18630

Table 2. Cont.
Compound Experimental Protocol Anxiolytic-Like Effect and/or Mechanism Animal Tested Reference
Carvacryl acetate

O Elevated plus-maze test

Increased mouse motor activity
Light-dark box test Mice [77]
O Anxiolytic-like effect
Marble-burying test

(R)-(-)-Carvone

O
Reduced avoidance latency without any effect in
Elevated-T maze test Rats [57]
escape time

Citronellol

Increased punished behavior at dose that did not

Geller conflict test
change unpunished behavior
OH
Mice [64]

2-Phenethyl alcohol
OH
Vogel conflict tests Increased punished licking

Increased time spent and % entries in the open

(E)-Methyl isoeugenol
H3CO Elevated plus-maze test arms
Light/dark test Increased number of transitions and time spent in Mice [78]

H3CO Open field test light side

Increased crossing of the center
Nerol Increased number of entries and time of
Elevated plus-maze test permanence in the open arms
Open field test Decrease in motor activity
Mice [79]
Light/dark test Increased time of permanence in the room clear
OH
Rota rod test No modification in time spent or number of falls
in the revolving bar
Molecules 2015, 20 18631

Table 2. Cont.
Compound Experimental Protocol Anxiolytic-Like Effect and/or Mechanism Animal Tested Reference
Isopropyl N-methylanthranilate

O O

NH

Light/dark test Increased time spent in the light side without

Mice [80]
Open field test effect on the number of crossing
Methyl N-methylanthranilate
O O

NH

Isopulegol

Increased number of entries and time spent in the

Elevated plus-maze test
open arms
Holeboard test Mice [81]
Increase number of head dips
OH Open field test
Did not change the number of crossing

(R)-(+)-Limonene

Increase in time spent and in the number of

Elevated plus-maze test entries in the open arms
Mice [31,82]
Light/dark test Increased time spent in the light side of the
light/dark apparatus

(+)-Limonene epoxide
O

Marble burying test Reduction in number of buried marbles Mice [83]

Linalool
OH

Increased time spent in the light side and

Light/dark test
increased social interaction Mice [84]
Elevated plus-maze test
Increased number of visits to the open arms

Linalool oxide
Increased number of crossings and time spent in
Light/dark test Mice [85]
O the light side
OH
Molecules 2015, 20 18632

Table 2. Cont.
Compound Experimental Protocol Anxiolytic-Like Effect and/or Mechanism Animal Tested Reference
Myrtenol
Increase in open arm exploration
H3C Elevated plus-maze test
OH Elevated time spent in the light side of Rats [86]
H3C Light/dark test
light/dark apparatus

Phytol Increased social interaction and decreased

Elevated plus-maze test Mice [87]
OH number of marbles buried

Increased mouse motor activity

Pulegone Elevated plus-maze test Increased ambulatory activity of mice in a
Open field test dose-dependent and bell-shaped manner
Rota rod test Decreased performance on the
Mice [88]
Grasping test Rota rod apparatus
O
Conditioning place Decreased grasping strength
preference (CPP) test Decreased percentage of time spent in the least
preferred compartment
Thymol
Reduced struggle latency and increased
Brief mechanical restraint struggle bouts, which would be indicative of
Quail [89]
Open-field test fear reduction
OH
No effect on motor activity

Vanillin
H O Increase in the percentile ratio of open arm to
total arm entries and reduction in the time spent
Elevated plus-maze test
in the closed arms Rats [90]
Bright and dark arena
Increased number of bright chamber entries,
OCH3
time spent, and rears in bright arena
OH

2.5. Alpinia zerumbet

In the first study with Alpinia zerumbet essential oil (leaves), De Arajo and collaborators [16]
observed no effect in mice (gender not cited) tested in the elevated plus-maze (50100 mg/kg, i.p.),
although they did find a sedative/depressant effect in the open-field test,. On the other hand, Murakami
and collaborators [17] found that acute inhalation using Alpinia zerumbet essential oil (leaves) increased
the time spent by male mice in the open arms of the elevated plus-maze at a dose which did not affect
motor activity. The same group published two additional studies showing similar results. Satou and
collaborators [18] observed that inhalation of this essential oil by mice increased the time spent in the
open arms of the elevated plus-maze (three days of administration), this at a dose which did not change
motor activity (calculated through rearing frequency in an observation box). Apparently, no significant
effect was seen in the light/dark test (one day of administration). Satou and collaborators [91] also found
that male mice treated with Alpinia zerumbet essential oil (leaves) at a single inhalation exhibit an increase
in open arm exploration (% entries and time spent) in the elevated plus-maze, without changes in the
total distance travelled. They also showed that this anxiolytic-like effect is dependent on inhalation time
(90 to 120-min). Together, these results suggest an anxiolytic-like effect for A. zerumbet essential oil.
Molecules 2015, 20 18633

2.6. Angelica sinensis

Chen and collaborators [19] studied the effect of A. sinensis essential oil in mice tested in two
animal models of anxiety (elevated plus-maze and light/dark transition), and a single test for detection
of anxiolytic-like drugs (stress-induced hyperthermia). Mice treated orally with A. sinensis essential oil
exhibited an increase in open arm exploration (% time spent in), and a decrease in protected head dips
on the elevated plus-maze. An inverted U shaped response curve was observed. In the light/dark test, a higher
dose of A. sinensis essential oil increased the number of transitions, while an intermediate dose increased
the time spent in the lighted side. A. sinensis essential oil also reduced stress-induced hyperthermia, with a
similar pattern being seen in the elevated-plus maze (U shaped curve). These effects were seen at doses that
did not modify motor activity in the models (e.g., number of closed arm entries in the elevated plus-maze).
Min and collaborators [92] also evaluated the potential anxiolytic-like effect of A. sinensis essential
oil in rats submitted to social interaction tests. A. sinensis (oral, acute) increased social interaction in
unfamiliar and familiar test conditions, while diazepam was effective in unfamiliar test conditions only.
Moreover, similar to what was observed by Chen and collaborators [19] in the elevated plus-maze, A. sinensis
essential oil exhibited an inverted U shaped response in the social interaction test, since the high dose
(42 mg/kg) only reduced aggressive behaviors, while the intermediate dose (21 mg/kg) increased social
interaction. However, in the social interaction test, the increased motor activity induced by the intermediate
and higher doses may be a confounding factor. In the holeboard test, only the lower dose increased
head-dipping; an indication of anxiolytic-like effect.

2.7. Chamaecyparis obtusa

The putative anxiolytic-like effect of Chamaecyparis obtusa essential oil was tested in mother-separated
male rats submitted to the elevated plus-maze test [23]. At 14 to 28 days (postnatal) the rats were separated
from their mothers, and treated with essential oil (inhalation for 1 or 2 h, once a day). Fluoxetine was
used as the control. The maternal separation induced an anxiogenic-like behavior in the elevated
plus-maze (decreased percentage of time and entries in the open arms). Both Chamaecypais obtuse
essential oil (1 h or 2 h), and fluoxetine reversed this anxiogenic-like effect. This anxiolytic-like effect
was associated with reduction in interleukin 6 and Ccl2 cytokines. However, no control for motor activity
change was used. Furthermore, the authors used a modified criteria for arm entry in the elevated
plus-maze (2 paws instead the common 4 paws criteria).
Kasuya and collaborators [22] evaluated the effect of C. obtusa essential oil (inhalation for 90 min) in
isolated male mice submitted to elevated plus-maze. They observed an increase in open arm exploration
(% time and entries) in a 10-min session, associated with an increase in fast nerve growth factor receptor
(NGFR). This effect in the elevated plus-maze was dose dependent with an inverted U shaped curve.
Although these results could suggest an anxiolytic-like effect, no measurement of motor activity
was described.

2.8. Casimiroa pringlei

Oral acute administration of Casimiroa pringlei essential oil in male rats induced an increase in %
time spent in the open arms (elevated plus-maze), and increased holes exploration in the holeboard test,
Molecules 2015, 20 18634

the dosage did not impair motor activity in the open-field [20]. However, these effects were significantly
different from rats treated with caffeine alone, which did not permit conclusions with regard to its effect
on anxiety.

2.9. Celastarus paniculatus

Rajkumar and collaborators [21] observed that rats (male and female) treated for 14 days (p.o.) with oil
from Celastarus paniculatus seeds showed an increase in punished licking (Vogels test) and open arms
exploration in the elevated plus-maze, without effect on motor activity in the open-field test. These results
indicated an anxiolytic-like effect for this essential oil.

2.10. Citrus Genus

2.10.1. Citrus sp.

Umezu [93] studied the effect of acute administration (i.p.) of several essential oils in mice submitted
to conflict procedures (Geller and Vogel tests), revealing no effect for either orange oil (Citrus sp.),
chamomile (Matricaria chamomilla), or Ylang-ylang (Cananga odorata). The positive effect observed
with diazepam and rose oil (see below) indicated the procedures sensitivity.

2.10.2. Citrus aurantium (Sour/Bitter Orange)

Carvalho-Freitas and Costa [24] observed that C. aurantium essential oil administration (acute oral) in
mice increased exploration of the open arms (time spent) in the elevated plus-maze at a dose that did not
impair motor activity (open-field and rota rod test), which is indicative of anxiolytic-like effect.
Pultrini and collaborators [25] studied the effect of C. aurantium in acute and repeated (15 days) oral
administration in mice. The acute administrations induced an anxiolytic-like effect in the light/dark
transition tests (increased time spent in the light side, and in the number of transitions) and in marble
burying (decreased number of marbles buried), while repeated administration showed effects in the
marble-burying test only. Repeated diazepam administrations did not increase light/dark transitions. For
the rota rod tests, no C. aurantium effect was seen, suggesting no motor impairment. Thus, the results
suggest an anxiolytic-like effect for acute and repeated C. aurantium essential oil treatment.
Costa and collaborators [27] also observed that both acute and repeated (14 day, p.o.) of C. aurantium
essential oil administration induced anxiolytic-like effect in the light/dark transition model. Acute and
chronic administrations increased both time spent in the light side, and the number of transitions, at a
dosage that did not impair the motor activity of the mice [27]. Both in acute and chronic treatments, the
lower doses were effective while higher doses were not. This anxiolytic-like effect of C. aurantium
essential oil was reversed by 5-HT1A antagonist WAY100635 but not by flumazenil, a benzodiazepine
antagonist, suggesting serotonergic mediation. Moreover, no effect on dopamine and/or serotonin levels
was seen in the cortex, hypothalamus, pons, or striatum.
In this line, inhaled C. aurantium essential oil increased social interactions for rats (time spent in
active social interaction), and increased exploration time in the open arms of the elevated plus-maze,
suggesting an anxiolytic-like effect at a dose that did not impair motor activity in the open-field test [26].
Molecules 2015, 20 18635

Saketi and collaborators [28] observed that acute administration (i.p.) of C. aurantium L. essential oil
in male mice increased open arm explorations (percentage of time spent and percentage of entries) in the
elevated plus-maze. Fluoxetine exhibited the same anxiolytic effects. The authors claim that the effect
of C. aurantium L. is related to serotonergic transmission based on a fluoxetine + C. aurantium L.
interaction. However, C. aurantium L. did not change the anxiolytic effect of fluoxetine in the elevated
plus-maze, suggesting no drug interaction. Moreover, no measurement of motor activity was presented
in the results.
The same group evaluated the co-administration effects of C. aurantium L. with diazepam [29]. They
found that C. aurantium L. essential oil (i.p.) increased the open arms exploration (increase of percentage
time spent) of male mice submitted to the elevated plus-maze. Although diazepam increased open arms
exploration (percentage of time spent and percentage of entries), co-administration with C. aurantium L.
reduced the anxiolytic effect of diazepam. These results indicate that C. aurantium L. may exert an
anxiolytic-like effect acting as partial agonist at the GABA-A receptor/benzodiazepine site. However,
flumazenil did not alter the effect of C. aurantium [27]. One observation concerning this study is that the
authors cited the total number of entries (closed + open) as being used for motor activity measurement, but
there is no further mention of it in the text.

2.10.3. Citrus aurantium subsp. bergamia (Bergamot)

Saiyudthong and Marsdem [30] evaluated the effect of bergamot oil in the elevated plus-maze and
holeboard tests. Male rats treated with bergamot oil (inhalation for 7 min) showed increase in open arm
exploration (% time and % entries) in the elevated plus-maze, and increased head dipping in the holeboard
test. The dose-response curve exhibited an inverted U shape in both models, with the intermediate dose
showing better results. Further, the higher dose increased the total number of arm entries, which might
indicate an excitatory effect, although this parameter may be influenced by anxiety changes. No other
measurement of motor activity was taken. It is interesting that bergamot oil and diazepam also decreased
corticosterone levels after the elevated plus-maze test. Thus, bergamot essential oil exhibits an anxiolytic-
like profile, although future studies must evaluate motor activity properly. This putative anxiolytic-like
effect was corroborated in a clinical trial showing that bergamot oil inhalation reduces anxiety in patients
awaiting minor surgery [94]. Interestingly, an in vivo study using micro-dialysis, observed that systemic
bergamot oil administration (i.p.) increased aspartate, taurine, and glycine release in the hippocampus.
However, local bergamot administration also increased glutamate and hippocampal GABA release [95].

2.10.4. Citrus latifolia and C. reticulata

Gargano and collaborators [36] studied the effect of systemic (oral) administrations of C. latifolia
(containing 58% limonene and 13% beta-pinene), and C. reticulata (containing 90% limonene) essential
oils in the marble-burying test and the light/dark test. It was observed that mice treated with C. latifolia
decreased the number of marbles buried (marble burying test) and increased the time spent in the light side
(light/dark test) with an inverted U-shaped curve. The dosage did not impair performance in the rota-rod
test. C. reticulata was effective only in the marble burying test (reduced number of marbles buried).
Imipramine (marble burying) and diazepam (light/dark test) were used as positive controls. The data
Molecules 2015, 20 18636

suggest an anxiolytic-like effect for both essential oils, but with different profiles (perhaps reflecting
differing mechanisms of action).

2.10.5. Citrus junos

Satou and collaborators [31] evaluated the effect of C. junos essential oil from pericarps in isolated
male mice tested in the light/dark test and the elevated plus-maze. Apparently the same mice were tested
in the light/dark test (first day), open-field (second day) and elevated plus-maze (third day). Inhaled
C. junos essential oil increased the time spent and the number of entries in the light side of the light/dark
apparatus. In the elevated plus-maze, the essential oil induced an increase in open arms exploration (time
spent and % entries). These effects were seen at doses that did not impair open-field activity, indicating
an anxiolytic-like effect for C. junos essential oil.

2.10.6. Citrus limon

Inhaled lemon oil increased the time spent and the entries on the open arms of mice tested on the
elevated plus-maze, without effects on motor activity (number of entries in closed arms), suggesting an
anxiolytic-like effect [34]. However, in the same study, lemon oil inhalation reduced locomotion and
exploration in the open field, which would be suggestive of a sedative/depressant effect. Considering
that flumazenil (an antagonist of the benzodiazepine site at the GABA-A receptor), WAY 100635
(a 5-HT 1A receptor antagonist), and apomorphine (a non-selective dopamine agonist) all blocked lemon
oils effect in the elevated plus-maze, the effect may be meditated by either GABA-A/benzodiazepine,
serotonergic, and/or dopaminergic neurotransmissions.
On the other hand, Ceccarelli and collaborators [33] evaluated the effects of prolonged lemon essential
oil exposition in male and female rats. The rats were exposed throughout one week (continuous home-cage
exposure), and then tested in the elevated plus-maze. Both male and female rats exhibited a reduction in
open arms exploration (% time spent in the open arms), indicating an anxiogenic-like effect. It has been
previously shown that repeated stressful stimuli have anxiogenic-like effects [96], and thus, continuous
lemon essential oil exposition may be a stressful stimulus inducing anxiogenic-like effect.
Using Citrus limon essential oil (from the leaves), containing limonene (52.7%) and linalool (1.7%),
Lopes Campelo and collaborators [35] observed mixed results in the elevated plus-maze. At a low dose
(50 mg/kg, p.o.) mice increased % time spent in the open arm, but decreased the percentage of open arm
entries, while an intermediate dose (100 mg/kg) increased both parameters, and the highest dose (150 mg/kg)
decreased both parameters. However, all doses decreased motor activity in the open field, suggesting a
sedative/depressant effect that could interfere with the elevated plus-maze behavior.

2.10.7. Citrus sinensis (Sweet Orange)

Inhalation of Citrus sinensis essential oil (sweet orange, containing 97% limonene) by male rats induced
an increase in open arm exploration (% time spent and % number of entries) in the elevated plus-maze,
and the % time spent in the lit chamber of the light/dark test [37]. There was no effect on motor activity,
as measured by the total distance travelled in the elevated plus-maze. Melaleuca alternifolia essential oil
Molecules 2015, 20 18637

was used as a neutral odor control. These results suggest an anxiolytic-like effect, which was corroborated
in a clinical study with normal volunteers submitted to an anxiety-provoking experimental situation [97].

2.11. Copaifera reticulata Ducke

It was found that acute administration (p.o.) of Copaifera reticulata Ducke essential oil in rats increased
conventional parameters of open arm exploration (% time spent and % entries), and ethological measures
(rearing, peeping out, and end open arm activity) in the elevated plus-maze. No change was seen in the
number of closed arm entries, indicating no impairment of motor activity [38]. These results suggest an
anxiolytic-like effect for this oil.

2.12. Coriandrum sativum Var. microcarpum

Volatile oil extracted from Coriandrum sativum var. microcarpum was tested for anxiolytic-like action
in an animal model of Alzheimer disease [32]. This oil contains linalool (69%) and -pinene (6.5%). Male
rats treated with -amyloid (142) were submitted to an elevated plus-maze 60 min after the last volatile
coriander oil inhalation (21 days, 60 min each day). It was observed that -amyloid (142) decreased open
arms exploration (percentage of time spent and number of open arm entries), and that volatile coriander
oil reversed these changes. Although these results may suggest an anxiolytic-like effect, the influence of
a putative motor impairment must be evaluated. Moreover, there was no group treated exclusively with
volatile coriander oil.

2.13. Cymbopogon citratus

Male mice treated orally with C. citratus essential oil (leaves) (mainly composed of citral and
-myrcene) displayed increased open arm exploration (% entries and time spent in the open arms) in the
elevated plus-maze, and time spent in the light side of the light/dark test [39]. No motor effect was seen in
the open-field or rota-rod test. The pattern was similar to the positive control diazepam. Thus, the results
suggested an anxiolytic-like effect for this essential oil. Costa and collaborators [40] also observed an
anxiolytic-like effect in the light/dark test (increased time spent in the lighted side, and number of
transitions at a dosage which did not impair rota rod performance), after acute administration. This effect
was not seen after repeated (21 days) treatment, suggesting tolerance development. Furthermore, the
anxiolytic-like effect appeared to be mediated by GABA-A/benzodiazepine transmission, since it was
blocked by flumazenil but not by WAY100635 pretreatment. However, it is interesting to note that Vale
and collaborators [75] did not observe anxiolytic-like effect in the elevated plus-maze with isolated citral,
limonene, or myrcene, which suggests that other constituents contribute to the anxiolytic-like effect of
C. citratus. In this study, diazepam, used as a positive control, increased open arms exploration, which
corroborate the sensitivity of the procedure used.

2.14. Dennettia tripetala

Oyemitan and collaborators [41] studied the effect of D. tripetala essential oil and its main compound
1-nitro-2-phenylethane (BPNE) in the elevated plus-maze. Male and female mice treated with D. tripetala
essential oil and BPNE (both i.p.) apparently showed an increase in open arm exploration (% arm entries
Molecules 2015, 20 18638

and % time spent). These results suggest that the anxiolytic-like effect of D. tripetala essential oil is related
to BPNE. However, no measure of motor activity was taken and some problems in the manuscript (e.g.,
significant effects of the essential oil described in the text were not shown in the figure concerning the
elevated plus-maze test) making it difficult to draw an overall conclusion.

2.15. Ducrosia anethifolia

Evaluating the potential anxiolytic-like effect of Ducrosis anethifolia, essential oil oral administration
in male mice, Hajhashemi and collaborators [42] found an increase in open arm exploration (time spent
and entries) in the elevated plus-maze, at a dosage that did not impair motor activity (number of beam
breaks in the automated chamber). This profile suggests an anxiolytic-like effect for Ducrosis anethifolia
essential oil.

2.16. Foeniculum vulgare

Mesfin and collaborators [43] studied the putative anxiolytic-like effect of essential oil from aerial
parts of Foeniculum vulgare in oral administrations to male mice. Acute Foeniculum vulgare essential
oil administrations increased open arms exploration (% time spent and entries) in the elevated plus-maze,
and decreased rearing in the staircase test. Furthermore, the essential oil increased center exploration
(number of square crossings and time spent) in the open-field test. These effects showed an inverted U
shape dose-response curve, and they were seen at doses that did not change motor activity in the elevated
plus-maze (number of closed arm entries), staircase test (number of steps climbed), or open-field test
(total number of square crossings). Thus, the results indicated an anxiolytic-like effect for Foeniculum
vulgare essential oil [43]. This essential oil presents among its constituents certain substances that have
also displayed anxiolytic-like effect such as: limonene, -pinene, 1,8-cineole, and, at a lower concentration,
linalool (see below).

2.17. Lavendula angustifolia

Umezu and collaborators [44] evaluated the effect of systemic lavender oil administration (i.p.) to mice
in two conflict tests: the Geller and Vogel conflict tests. They observed that lavender had an anti-conflict
effect: increased punished response at a dose that did not change unpunished response in the Geller test,
and increased punished licking in the Vogel test. Further, the effects of the main constituents of lavender
essential oil (linalool, linalyl acetate, borneol, camphene, cineol, terpinen-4-ol, -pinene and -myrcene)
were also evaluated, and the only compound that showed a clearly anti-conflict effect was linalool. These
results suggest that linalool may be the main active component of lavender.
Bradley and collaborators [45] studied the effect of lavender in Mongolian gerbils submitted to an
adapted version of the elevated plus-maze. The use of gerbils is based on findings that this specie has a
greater homology to humans in respect to the distribution of neurokinin-1 (NK-1) receptors than rats and
mice. Male and female Mongolian gerbils were bred in an enriched environment with food supplemented
by fruit and sunflower seeds; the gerbils were exposed to inhaled lavender continuously for 24 h, or for
7 days. Although some slight differences were seen between male and female gerbils, on the whole,
acute lavender treatment exerted anxiolytic-like effects in ethological measures (e.g., decrease risk
Molecules 2015, 20 18639

assessment behavior), however not in spatiotemporal measures (% entries or % time spent in open arms).
On the other hand, chronic continuous lavender exposure increased the % entries in the open arms, and
decreased the stretch-attend posture (another ethological measure). These results suggest that lavenders
anxiolytic-like effect did not decrease with repeated exposure, but appears to increase. It must be noted
that the data were analyzed statistically using a one-tailed test, which may have influenced the conclusions.
Shaw and collaborators [46] suggested an anxiolytic-like effect for lavender essential oil using the
open-field test. Male rats were exposed to various schedules of lavender essential oil inhalation and at
30 min or 1 h, before or during the test; all rats received an injection previous to the inhalation procedure.
The higher doses of lavender reduced peripheral movement and defecation, effects that were also seen
in the diazepam treated group. However, no data was given for central (considered the measure related
to anxiety), or total motor activity (indicative of sedative/depressant effect and/or motor impairment).
Moreover, certain lavender groups displayed decreased mobility, indicating for the oil a sedative or
depressant, rather than an anxiolytic-like effect. This interpretation is consistent with reductions in
rearing and grooming as seen in some of the experiments in this study.
Another study from Shaw and collaborators [47] evaluated the effect of lavender essential oil and
chlordiazepoxide in c-fos expression after open field exposition testing. Again, lavender essential oil
(inhaled for 1 h), and chlordiazepoxide reduced peripheral movement and defecation of the rats. However,
lavender in this study did not affect mobility. Further, lavender and chlordiazepoxide decreased open-field
induced increased c-fos expression in the paraventricular nucleus of hypothalamus, and in the dorsomedial
hypothalamic nucleus. In addition, chlordiazepoxide also decreased open-field induced increased c-fos
expression in the ventromedial hypothalamus, lateral hypothalamic area, accumbens shell and core, and
the caudate-putamen striatum. The authors concluded that lavender exert anxiolytic-like effects which may
be mediated by a different neural substrate than benzodiazepines.
Chioca and collaborators [51] observed that male mice acutely exposed to inhaled lavender essential
oil displayed reduced marble-burying behavior and increased open arm exploration (% number of entries
and time spent in the open arms), an effect that was not seen with amyl acetate (banana odor), that is used
as a negative odor control (odor not inducing behavior changes). Diazepam, used as positive control,
exerted anxiolytic effect in both models. The effect of lavender on marble-burying was not blocked by
picrotoxin (GABA antagonist) pre-treatment, nor changed [3H] flunitrazepam binding. On the other hand,
WAY 100635 (a 5-HT1A antagonist) blocked the anxiolytic-like effect of lavender in marble-burying
test. Lavender essential oil also reduced 8-OH-DPAT (5-HT1A agonist) induced serotonergic syndrome.
These results indicate that the anxiolytic-like effect of lavender essential oil may be mediated by
serotonergic transmission.
A subsequent study by the same group replicated the anxiolytic-like effect of lavender essential oil in
the marble-burying test, and it was observed that this effect was similar in mice with normal olfactory
function, as compared to mice with anosmia, which suggests that odor perception was not crucial. The
normal (non-anosmic), and anosmic mice (both treated with vehicle) results did not differ [52]. Using a
similar approach, Takahashi and collaborators [53] observed that anosmic and non-anosmic male mice
showed similar behavior in the elevated plus-maze after lavender inhalation. However, anosmic mice
showed increased open arms exploration (time spent) in the elevated plus-maze; beyond that of non-anosmic
mice. Inhaled lavender essential oil induced an anxiolytic-like effect in normal mice compared to vehicle
Molecules 2015, 20 18640

treated mice. They also found an increase in hippocampal serotonin turn-over after lavender inhalation,
reinforcing the proposal of a serotonergic mediation of the anxiolytic-like effects of lavender [51].
In a very interesting study, Takahsahi and collaborators [49] evaluated the anxiolytic-like effect of
essential oils from different species of lavender (e.g., L. officinallis and L. latifolia), which differ in linalool
and linalyl acetate contents. They observed that male mice treated with inhaled lavender essential oils
that contain linalool and linalyl acetate spent more time in the open arms of the elevated plus-maze than
the controls. These effects were seen without changes in the distance travelled in the maze, indicating
an anxiolytic-like effect. However, there is no relationship between linalool content and anxiolytic-like
effect, yet a positive correlation (r = +82) was seen between linalool and linalyl acetate content and
increased open arm exploration. These results suggest that linalyl acetate, which showed no anxiolytic-like
action per se, increased the anxiolytic-like effect of linalool.
Another study from Takahashi and co-workers [50] found that lavender essential oil inhalation exerted
anxiolytic-like effect in the elevated plus-maze (10-min session), both in non-stressed and stressed male
mice. However, stress (water immersion for 24 h, 12 cm deep) had no effect on elevated plus-maze
behaviors. The divergent effects of lavender in stressed and non-stressed mice are observed in the expression
of mRNA of some proteins (fast nerve growth factor receptor, NGFR, and the activity regulated
cytoskeletal-associated protein, Arc).
Silexan is a standardized essential oil produced from L. angustifolia flowers containing 36% linalool
and 34% linalyl acetate [55]. Repeated administration (7 days, i.p.) of Silexan showed anxiolytic-like
effects in male rats in the elevated plus-maze (increase open arms exploration), zero-maze (increased
exploration of open areas), social interaction (increased social interaction in a familiar environment),
novelty-induced suppression feeding (decreased latency for eating starts in a novel environment), and
the open-field square (increased central area exploration). However, some contradictory effects on motor
activity were seen: increased square crossing in the open-field test (excitatory effect), decreased number of
closed arm entries in the elevated plus-maze (motor impairment), and no effect on the square closed-field
arena with light beam sensors [54]. Overall, these results suggest an anxiolytic-like effect, although it
might be influenced by motor activity changes.
An anxiolytic-like effect for Silexan was also found by Schuwald and collaborators [55]. The authors
observed that male mice treated with Silexan (orally for 3 days) showed an increase in open arms exploration
(time spent and entries in open arms) in the elevated plus-maze. Diazepam and pregabalin were used as
positive controls and exhibited the same effects. They also observed that the anxiolytic-like effect of
Silexan is related to non-specific inhibition of voltage operated calcium channels showing similarities
with pregabalin (although Silexan did not bind to the 2- subunits of the P/Q-type voltage operated
calcium channels, the target of pregabalin). Finally, the authors stated that Silexan did not bind to SERT,
DAT, NET, MAO-A, or GABA-A receptors. Thus, action on calcium homeostasis may contribute to the
anxiolytic-like effect of Silexan (and thus, lavender essential oil).
It is interesting to note that rats trained to discriminate between saline and diazepam (two-lever operant
behavior) did not generalize to the diazepam cue when treated with Silexan (330 mg/kg, i.p.), suggesting a
that Silexan induces a different interoceptive stimuli [98]. These results reinforce the hypothesis that Silexan
(thus, lavender essential oil) did not share the same mechanism of action as benzodiazepines [47,51,55].
Certain negative results were also found. Inhalation of lavender oil did not increase open arm explorations
of mice tested on the elevated plus-maze [34]. Hawken and collaborators [48] observed that lavender
Molecules 2015, 20 18641

essential oil exerted opposite effects in sheep for their stress response: lavender increases agitation,
vocalization, and escape attempts in isolated stressed nervous sheep (sheep that exhibit increase motor
activity and vocalization frequency when isolated or in human presence). However, lavender essential oil
decreases these behaviors in calm sheep. Nervous sheep that inhaled lavender also showed increased
plasma cortisol at 30 min after isolation stress. These unexpected findings in nervous sheep could be
related to systemic delivery of the essential oil (individual mask), or to odor novelty, although plasma
cortisol levels (before isolation testing, and after lavender administration) did not differ from the control.
The authors proposed that the divergent response to stress (isolation and human proximity) is genetically
determined; therefore genetic background may also influence the response to lavender essential oil.
Moreover, considering that only a single concentration of lavender essential oil was administered, it may
have been too low to reduce the stress reaction in nervous sheep. Future replications of this interesting
bi-directional effect of lavender essential oil in a standardized rodent animal model for anxiety, while
using a neutral odor and dose/concentration-response design would be very important.
Silexan has shown clinical anxiolytic effect in controlled studies with GAD patients [99,100], and
those with sub-syndrome anxiety disorder [4]. Further, Silexan administration shows reduced 5-HT1A
receptor binding in certain brain areas (e.g., hippocampus) in healthy volunteers [101], reinforcing the
hypothesis that serotonin mediates the anxiolytic effect of lavender essential oil.

2.18. Lippia alba

Hatano and collaborators [57] administered Lippia alba essential oil (i.p.) to male rats which were then
submitted to the elevated-T maze. L. alba reduced avoidance latency without any effect on escape time, a
profile similar to the reference drug diazepam. Moreover, L. alba essential oil did not impair motor activity
in the open-field test. Vale and collaborators [56] observed that acute administration of three chemotypes
of Lippia alba essential oil (i.p.) increased male mice exploration of open arms (% entries and % time
spent) in the elevated plus-maze. An anesthetic effect study of Lippia alba essential oil on silver catfish
(Rhamdia quelen), suggested a GABAergic effect [102], explaining its anxiolytic-like effect. Taken
together, the results suggest an anxiolytic-like effect for L. alba essential oil.

2.19. Litsea cubeba

Male mice treated orally for seven days with Litsea cubeba essential oil showed increased open arms
exploration (time spent and number of entries), and a decrease in total distance traveled in the open arms
of the elevated plus-maze test [58]. This result suggests a sedative effect influencing the behavior of mice
in the elevated plus-maze.

2.20. Ocimum basilicum L. and Ocimun sanctum L. Essential Oil

Gradinariu and collaborators [60] evaluated the effects of O. sanctum L and O. basilicum L essential
oils in male rats treated (i.c.v.) with beta-amyloid (142). Similar to that observed by Cioanca and
collaborators [32], beta-amyloid (142) induced an anxiogenic-like behavior in the elevated plus-maze
(decreasing the number of entries in the open arms). O. sanctum L. and O. basilicum L. (essential oil
inhalations, 60 min per day, for 21 days) reversed this effect at doses that did not change motor activity
Molecules 2015, 20 18642

(number of crossing in the elevated plus-maze), while increasing the percentage of open arms time
spent. These essential oils contain linalool, and 1,8-cineole, which may well contribute to the behavioral
effects [60]. However, there was no group treated only with essential oils.

2.21. Ocimum gratissimum L.

Tankam and Ito [59] administered the essential oil from O. gratissimun L to male mice tested in the
light/dark and open-field tests. The essential oil was administered by inhalation during the behavioral
test (embedded filter paper disk adhered to apparatus wall). It was observed that treated mice showed an
increase in the number of transitions and time spent in the light side of the light/dark apparatus. The
dose-response curve showed an inverted U shape. However, O. gratissimun essential oil administration
also decreased motor activity in the open-field test, indicating a sedative/depressant effect that could
contribute to increased time in the light side of the light/dark apparatus, but would impair the number of
transitions (the opposite of the result observed). It is interesting to note that thymol, the main component
of O. gratissimun essential oil, also showed anxiolytic-like effects (see below).

2.22. Piper guineense

Inhaled essential oil from dried fruits of P. guineense (containing linalool and 3,5-dimethoxytoluene)
increased the time spent by mice in the light side of the light/dark apparatus and the number of
transitions [61]. However, mixed results were seen for motor activity, since the essential oil reduced the
number of crossing in the open-field test (indicating impairment) although not changing latency to the
first transition in the light/dark test (indicating absence of impairment).

2.23. Propolis

Li and collaborators [62] studied the effect of propolis essential oil on stressed male mice (restraint
stress) submitted to the elevated plus-maze. Propolis (administered orally for 14 days) reversed the
anxiogenic-like effects of restraint stress in the elevated plus-maze test (decreased % time and entries
spent in the open arms, and any increases in protective head dipping and rearing). These effects were
seen at doses that did not change motor activity in the open-field testing (number of transitions), or in
the elevated plus-maze test (number of closed arms entries). Propolis also reversed increases in plasma
corticosterone and ACTH when induced by restraint stress.
Another study also showed an anxiolytic-like effect for propolis essential oil. Reis and collaborators [63]
observed that female rats (estrous phase not specified) treated (intraperitoneally) with propolis increased
center exploration in the open-field, and open arm exploration (% time and entries) in the elevated plus-maze
tests. However, sometimes propolis also increased motor activity (distance travelled in the open-field
test and number of closed arm entries in the elevated plus-maze) which may have influenced the results.
Thus, propolis may exert anxiolytic-like effects, although motor activity changes must be observed to
draw a clearer conclusion.
Molecules 2015, 20 18643

2.24. Rose

Umezu and collaborators [64] evaluated the effect of rose oil (Rose centifolia) and diazepam (both
acute, i.p.) in the Geller conflict and Vogel conflict tests. Both treatments increased the punished behavior
of mice in both tests, and were without effect for unpunished behavior in the Geller test, suggesting an
anxiolytic-like effect for rose oil. Inhaled rose oil, (acute, 7 min), caused an increase in exploration of
the open arms (time spent and number of entries) in the elevated plus-maze test although no measurement
of motor activity was taken [65].
Bradley and collaborators [66] evaluated the behavioral effects on gerbils (male and female) of prolonged
single (24 h), and repeated (2 weeks) rose oil (Rose damascena) inhalation. They did not observe any
significant effect on % time and entries into the open arms of the elevated plus-maze, but there were effects
on ethological measures (increases in head dips and rearing). On the other hand, they observed an increase
in % time spent in the light side of the light/dark test (no male/female difference was found). However,
rose oil also increased latency to first entrance in the dark compartment, which has been related to a
sedative/depressant effect. Diazepam was effective in both models, corroborating the sensitivity of the
procedure and suggesting that different mechanisms of action mediate the effects of benzodiazepine, vs.
the effects of rose oil.
Contrary to studies showing anxiolytic-like effects for rose oil, Komiya and collaborators [34] observed
that inhaled rose oil did not increase the time spent or the number of entries of mice in the open arm of the
elevated plus-maze test. Yet, C. limon increased open-arm exploration, which confirmed the sensitivity of
the procedure.
Thus, the results with rose oil are mixed and they may be influenced by a sedative/depressant
effect. However, some of its main constituents (citronellol and 2-phenethyl alcohol) have shown
anxiolytic-like effect.

2.25. Santalum album L.

Sandalwood oil (Santalum album L.) was studied in stressed male mice submitted to the elevated
plus-maze [67]. Stressed mice were isolated for 1 week and immersed in 1-cm deep water for 24h. Stressed
mice treated with Santalum album L oil (90-min inhalation) showed an increase in open arms exploration
(percentage of entries and time spent) during a 10-min session. This effect was observed when the oil
was administered before (24 h before elevated plus-maze), or after the water stress. However, this effect
was not observed in non-stressed mice. The data suggest an anxiolytic-like effect that is sustained for
24 h. However, no control for motor activity was employed.

2.26. Spiranthera odoratissima A. St. Hil.

Spiranthera odoratissima A. St. Hil. essential oil acutely administered to male mice (p.o.) showed an
anxiolytic-like profile: in the elevated plus-maze (increase % entries and time spent in the open arms),
holeboard (increase head dipping), light-dark (increased transitions between compartments and time spent in
the light side), and the open-field tests (increased center exploration), without effects on motor activity [68].
It was observed that the anxiolytic-like effect of S. odoratissima is partially blocked by NAN-190 (5-HT1A
Molecules 2015, 20 18644

antagonist) but not by flumazenil, suggesting that it is related to 5-HT1A receptors but not to the
benzodiazepine binding site.

2.27. Stachys tibetica

Kumar and collaborators [69] studied the putative anxiolytic-like effect of oral administration of
S. tibetica essential oil in rats (male and female). Acute and repeated (3 and 7 days) S. tibetica essential
oil administration induced an increase in open arm exploration (% time and entries), and a decrease in
protected head dipping in the elevated plus-maze test. Acute treatment increased social interaction in
familiar and unfamiliar conditions, and decreased aggressive behavior. Acute S. tibetica essential oil also
exerted an anxiolytic like effect in the holeboard (decreased latency to first head dip, and increases in
number and duration of dips), and in the light/dark tests (increased time spent in the light side and in the
number of transitions). These effects were seen at doses that did not decrease activity in these models
(e.g., no decrease of square crossing in the holeboard test) except a decrease in the number of closed arm
entries in the elevated plus-maze (with 7-days of treatment). On the whole, these results suggest an
anxiolytic-like effect.

2.28. Thujopsis dolabrata

Matsuura and collaborators [70] used anxiety induced by restraint stress to evaluate the effect of
Thujopsis dolabrata (hiba) essential oil inhalation. Immobilization of the rats induced a decrease in the
number of entries in the open arms of the elevated plus-maze (10 min sessions). Hiba essential oil reversed
the anxiogenic effect of stress at a dose that did not decrease the total distance moved. These results suggest
an anxiolytic-like effect for Thujopsis dolabrata essential oil (although only a 10-min session of elevated
plus-maze was employed).

2.29. Constituents from Essential Oils with Anxiolytic-Like Activity

2.29.1. 1,4-Cineole

1,4-Cineole is a minor component of the essential oils of certain aromatic plants (e.g., Salvia spp.).
Considering that other monoterpenes exhibit anxiolytic-like effect, Gomes and collaborators [71]
evaluated the effect of 1,4-cineole in animal models of anxiety. They observed that 1,4-cineole (oral),
and diazepam increased male mice exploration of the open arms (% entries and % time spent) in the
elevated plus-maze, and increased head dipping in the holeboard test. These effects of 1,4-cineole were
seen at a dose that did not affect motor activity in the open field or time of permanency in rota rod tests.
Flumazenil pre-treatment blocked the effect of diazepam but not the anxiolytic-like effect of 1,4-cineole,
indicating that its anxiolytic-like effect is independent from the benzodiazepine site.

2.29.2. -Asarone

Lee and collaborators [72] evaluated the effect of -asarone, (a major component of Acorus gramineus),
in corticosterone-induced anxiety in the elevated plus-maze and holeboard tests. Male rats treated with
repeated corticosterone administration (21 days, subcutaneously) displayed a decrease in the percentage
Molecules 2015, 20 18645

of time spent and the number of entries in the open arms of the elevated plus-maze test. Moreover,
corticosterone administration decreased the head dipping time in the holeboard test. Previous treatment
with -asarone dose-dependently reversed the anxiogenic-like effect of corticosterone. These effects were
seen at a dose that did not change motor activity in the elevated plus-maze test (the number of closed arm
entries). However, the motor effects in the open field test were mixed, since no effect was observed in
the total distance travelled (in cm), but a decrease in line crossing was found after corticosterone
administration (which is reversed by -asarone). The same pattern was seen with diazepam, the positive
control used. This anxiolytic-like effect of -asarone is associated with reversion of CRH levels, and
decreases in BDNF (and its receptor TrkB) mRNA induced by corticosterone treatment.

2.29.3. -Pinene

-Pinene is an important constituent of some essential oils with anxiolytic-like profiles (e.g.,
Ducrosis anethifolia, Chamaecyparis obtusa), and thus, it is evaluated alone in animal models of anxiety.
Satou and collaborators [73] observed that male mice treated with -pinene inhaled for 90 min/day
(for 1, 3, or 5 days) displayed increased open arm exploration (time spent and % entries) in the elevated
plus maze. There was no change in this effect of -pinene over 5 days of treatment. Although these results
suggest an anxiolytic-like effect, no measurement of motor activity was taken. The effect of -pinene
may be related to potentiation of GABA inhibition [103].

2.29.4. -Caryophyllene

-Caryophyllene is a major component of S. odoratissima essential oil [68] and it is also present in
propolis essential oil [62]. -Caryophyllene has presented anxiolytic-like effects, increasing open arm
exploration (increase % entries and % time spent) in the elevated plus maze, and exploration of the light
side of the Light/Dark test (increase time spent in light side, and number of transitions). The dosage
reported, did not change motor activity in the elevated plus-maze (number of closed arms entries), or in
the holeboard test (number of line crossed). However, differently from the essential oil, the anxiolytic-like
effect of -caryophyllene was not blocked by 5-HT1A antagonist, which indicates that another component
may also contribute to the anxiolytic-like effect of Spiranthera odoratissima A. St. Hil. [68]. Considering
that -caryophyllene is an agonist of type 2 cannabinoid receptors (CB2), Bahi and collaborators [74]
evaluated the role of CB2 receptors in the anxiolytic-like effect of -caryophyllene. They observed an
anxiolytic-like effect for caryophyllene in male mice in the elevated plus-maze test (increase % open arm
time and entries), in the marble-burying test (decreased number of marbles buried), and in the open-field
test (increase time spent in the center), yet without any effect on motor activity (number of closed arm
entries in elevated plus-maze or the number of line crossings in the open-field test). These effects were
reversed by treatment with AM630, an antagonist of CB2 receptors, indicating that CB2 receptor activation
plays an important role in the anxiolytic-like effect of -caryophyllene [74].

2.29.5. Citral and Myrcene

As cited above, citral and myrcene, two constituents of Lippia alba, were studied in the elevated plus
maze and no effect was observed [75].
Molecules 2015, 20 18646

2.29.6. Carvacrol

Carvacrol is a monoterpene phenol found in the essential oils of oregano and thyme [76]. Acute oral
administration of carvacrol in male mice exerted an anxiolytic-like effect in the elevated plus maze
(increased % time spent and % entries in the open arms) without impairment on motor activity in the
open-field test, or motor coordination in the rota rod test [76]. The anxiolytic-like effect of carvacrol is
prevented by flumazenil pre-treatment, suggesting benzodiazepine site mediation.

2.29.7. Carvacryl Acetate

Pires and collaborators [77] evaluated the effect of carvacryl acetate, which is derived from carvacrol
(a component of Origanum vulgare). Carvacrol has shown anxiolytic-like effect in animal models [76].
Male mice treated with carvacryl acetate (i.p.) showed an anxiolytic-like effect in the elevated plus maze
(increase time spent and number of entries in the open arms), the light/dark test (increased time spent and
number of entries in the light side), and the marble burying test (decrease in the number of marbles buried).
The doses of carvacryl acetate given did not reduce motor activity in the open field test. Buspirone and
diazepam were used as positive controls. The anxiolytic-like effects of cavacryl acetate were blocked by
pre-treatment with flumazenil (benzodiazepine site antagonist), but not with WAY100635 (5-HT1A
antagonist), indicating a GABA-A/benzodiazepine mediation.

2.29.8. (R)-()-Carvone

Male rats were treated with (R)-()-carvone for 14 days (i.p.), and then submitted to the elevated-T
maze [57] test. Carvone reduced avoidance latency without any effect on escape time, a profile similar
to the reference drug diazepam, and to Lippia alba essential oil. No treatment effects were seen in motor
activity in the open-field test. The results suggest an anxiolytic-like effect for carvone.

2.29.9. Citronellol and 2-Phenethyl Alcohol

Citronellol and 2-phenethyl alcohol are important constituents of rose oil and thus their activity in
animal models were evaluated [64]. Both compounds exhibit an anxiolytic-like effect in male mice as
tested in the Geller conflict test (increasing punished behavior at a dose that did not change unpunished
behavior), and in the Vogel test (increased punished licking). Citronellol exerted an inverted U curve in
the Vogel test. These results suggest that citronellol and 2-phenethyl alcohol are the active constituents
of rose oil [63]. Citronellol may act by increasing GABAs inhibitory effect [103].

2.29.10. (E)-Methyl Isoeugenol

(E)-Methyl isoeugenol is an important constituent of Pimenta pseudocaryophyllus essential oil. Male

mice treated orally with (E)-methyl isoeugenol showed anxiolytic-like effect in the elevated plus maze
(increase time spent and % entries in the open arms), in the light/dark test (increased number of transitions
and time spent in light side), and in the open-field test (increased center crossings) without effects on
motor activity in the open-field or wire tests [78]. Yet, (E)-methyl isoeugenol did not protect mice against
convulsions induced by pentylenetetrazole (a GABA functional antagonist); and its anxiolytic-like effect
Molecules 2015, 20 18647

was blocked by WAY100635 (a 5-HT1A antagonist). Thus, 5-HT neurotransmission has an important role
for the anxiolytic-like effect of (E)-methyl isoeugenol.

2.29.11. Nerol

Nerol is a monoterpene found in Lippia spp. and Melissa officinalis L. Marques and collaborators [79]
evaluated the acute effect of nerol (i.p.) in male mice in the elevated plus-maze, the light-dark test, the
rota-rod and the open field tests. Nerol exerted an anxiolytic-like effect in the elevated plus-maze
(number of entries and time spent in the open arms), and the light-dark test (time spent in the light side).
Although Nerol did not impair rota-rod performance, it did reduce motor activity, grooming and rearing in
the open-field test. This later result suggests a motor impairment or sedative effect which could influence
the behavior of mice in the anxiety models.

2.29.12. Isopropyl N-Methylanthranilate and Methyl N-Methylanthranilate

These substances were isolated from essential oil of Choisya ternate and were tested in male mice
submitted to the light/dark test [80]. These volatile alkaloids (administered intraperitoneally) increased
the time spent in the lighter side without affecting the number of crossings. Also, these alkaloids did not
affect the number of square crossings in the open field test. The results suggest anxiolytic-like effect for
both isopropyl N-methylanthranilate and methyl N-methylanthranilate.

2.29.13. Isopulegol

Systemic administration of isopulegol (i.p.) in male mice increased the number of entries and the time
spent in the open arms of the elevated plus maze, although it also decreased the number of closed arm
entries [81], which suggests motor impairment. Isopulegol also increased the number of head dips in the
holeboard test. In the open field test, isopulegol did not change the number of crossings. An anxiolytic-like
effect is suggested, although the putative motor effect might have influenced the results.

2.29.14. (R)-(+)-Limonene and (+)-Limonene Epoxide

Limonene is an important constituent of some essential oils (e.g., Citrus aurantium, Citrus junos,
Citrus sinensis, and Lippia alba) which has shown anxiolytic-like effect in animal models [2427,56,57],
and clinical studies [97,104]. Lima and collaborators [82] observed that (R)-(+)-limonene inhalation exerts
an anxiolytic-like effect in male mice tested in the elevated plus-maze (increases in time spent, and the
number of open arm entries), at a dose which did not interfere with motor activity (number of entries in
the closed arms). Flumazenil pre-treatment did not block the anxiolytic-like effect of limonene, indicating
that the benzodiazepine receptor site did not mediate this effect. Similar anxiolytic-like effect of (+)-limonene
was also found by Satou and collaborators [31] who observed an increase in open arm exploration (increase
in time spent and % entries) in the elevated plus-maze, and an increase in the time spent in the light side
of the light/dark apparatus, without any effect on motor activity in the open-field test.
On the other hand, Vale and collaborators [75], and Costa and collaborators [27] did not observe
anxiolytic-like effect for limonene in the elevated plus-maze. In both studies, diazepam, used as the
positive control, increased open arm exploration, which confirms the sensitivity of the procedure.
Molecules 2015, 20 18648

Male mice treated orally (1 and 14 days) with (+)-limonene epoxide reduced the number of marbles
buried in the marble-burying test [83]. No difference between acute and repeated treatments was seen;
suggesting that tolerance did not occur for the anxiolytic-like effect of (+)-limonene epoxide. However,
no measurement of motor activity was done.

2.29.15. Linalool and Linalool Oxide

Linalool is a constituent of certain essential oils such as Lavandula angustifolia, Piper guineense,
and Cymbopogon citratus, all which have shown anxiolytic-like effect. Male mice treated with linalool
(inhalation for 60 min) showed increased time spent in the light side, in the light/dark test (3% linalool),
and increased social interaction (1% linalool), without changes in motor activity. However, linalool also
impaired step down inhibitory avoidance, indicating an amnesic effect. These effects were similar to
diazepam, the positive control [84]. In this line, linalool oxide (inhaled) increased the number of entries
and time spent in the open arms of the elevated plus-maze, and increased the number of crossings, and
the time spent in the light side of the light/dark test [85]. However, Cline and collaborators [105] did not
observe increases in open arms exploration in the elevated plus-maze after linalool administration
(at 125 mg/kg, i.p.), although midazolam increased open arms exploration.
It was shown that linalool interacts with GABA-A receptors and potentiates GABA transmission [106],
although it was also found that linalool did not bind to the [3H]muscimol (GABA-A) site [107]. Linalool
also reduced [3H]MK801 (NMDA glutamate) cortex binding [107]. More recently, Schuwald and
collaborators [55] showed that linalool prevented voltage operated calcium channel activation, similar
(but not identical) to the effect of pregabalin.

2.29.16. Myrtenol

Myrtenol is a compound originating from essential oil of Myrtus communis, a plant used in Brazil to
treat nervous conditions [86]. Moreira and collaborators [86] evaluated the effect of ()-myrtenol in
the elevated plus-maze and light/dark test. Rats treated with myrtenol (acute, i.p.) showed increases in
open arm exploration (number of entries and time spent) in the elevated plus-maze, and time spent in the
light side of the light/dark apparatus. Flumazenil was able to block the effects of myrtenol. The effects of
myrtenol were found at a dose that did not change motor activity in the open field, or in the rota rod tests.
Interestingly, the lower dose tested was the most effective dose, which also reduced the number of closed
arm entries in the elevated plus-maze. Thus, in general, ()-myrtenol exhibited an anxiolytic-like effect,
which is probably mediated by benzodiazepine-like action.

2.29.17. Phytol

Phytol administration (2575 mg/kg, i.p.) in mice (gender not specified) increased open arm exploration
(number of entries and time spent) in the elevated plus-maze, and increased social interaction, while
decreasing the numbers of marbles buried [87]. These effects were seen at doses that did not change the
number of open field crossings, suggesting an anxiolytic-like effect. Moreover, the effect for the elevated
plus-maze, social interaction, and marble burying were blocked by pre-treatment with flumazenil,
indicating a benzodiazepine-like action.
Molecules 2015, 20 18649

2.29.18. Pulegone

Pulegone, a monoterpenic compound, is found in the essential oils of Mentha piperita and
Mentha pulegum L. Silveira and collaborators [88] observed that acute pulegone (i.p.) increased the
percentage of time spent in the open arms for male mice submitted to the elevated plus-maze. The effect
was not blocked by flumazenil pre-treatment, indicating a non-benzodiazepine meditation mechanism.
However, the increase in open arm exploration was seen at a dose that also increased ambulation in the
open-field. Unfortunately, the pulegone molecule is hepatotoxic [88], which precludes its use.

2.29.19. Thymol

Thymol, a main component of certain aromatic plant essential oils, from s (e.g., Origanum vulgare and
Ocimum gratissimum L.), was tested in female quail submitted to mechanical restraint stress [89]. Thymol
was administered as a feed supplement for 2 and 15 days. Mechanical stress (wing restraint) induced
immobility and consequent struggle behavior. Thymol administration reduced struggle latency and
increased struggle bouts, which would be indicative of fear reduction. No effect on motor activity in an
open field test was seen. Thus, the results suggest that thymol supplementation exerts an anxiolytic-like
effect [89].

2.29.20. Vanillin

Vanillin, a component of Vanilla planifolia (the vanilla bean), showed anxiolytic-like effect in male
rats tested in the elevated plus-maze and light-dark tests [90]. Acute and repeated administration of vanillin
(orally) increased the percentage of entries and time spent in the open arms of the elevated plus-maze
and increased the number of entries, time spent, and number of rears in the bright side of light-dark test.
This effect was similar to that observed with diazepam, the positive control.

2.30. General Discussion

Reviewing the results for essential oils in animal anxiety models, clear anxiolytic-like effect was
found for Citrus aurantium, Cymbopogon citratus, Lavendula angustifolia, and Lippia alba. Although
Citrus sinensis and bergamot oil have one pre-clinical study, clinical studies showing an acute anxiolytic
effect in normal volunteers [94,97] strengthen their potential for creating new anxiolytic drugs. Putative
anxiolytic-like effect was seen in: Achillea wilhemsii, Angelica sinensis, Alpinia zerumbet, Celastarus
paniculatus, Citrus janus, Citrus latifolia, Citrus reticulate, Cymbopogon citratus, Copaifera reticulate,
Ducrosia anethifolia, Foeniculum vulgare, Spiranthera odoratissima and Stachys tibetica (only one study),
Litsea cubeba, Ocimum gratissimum, Piper guineense and Propolis (confounded by sedative/depressant
effect, motor decrease or increase), Abies sachalinensis, Chamaecyparis obtuse and Dennettia tripetala
(absence of motor activity measurement). Citrus limon and rose oil had contradictory results. Although
there was one negative result with rose oil [34], other studies of this essential oil observed anxiolytic-like
effect [6466].
It is very interesting that the pre-clinical studies suggested that some essential oils do not act by
the GABA/benzodiazepine mechanism. This might contribute to develop a truly new and different class
of anxiolytics, possibly with a better clinical profile, and avoiding benzodiazepines drawbacks
Molecules 2015, 20 18650

(e.g., withdrawal syndrome or dependence). However, even drugs acting thru the GABA/benzodiazepine
system could be interesting (e.g., partial or selective benzodiazepine site agonists). In this line, the studies
reviewed indicated that essential oils from Achillea wilhemsii, Alpinia zerumbet, Lavendula angustifolia,
Citrus aurantium, and Spiranthera odoratissima did not act thru the GABA/BDZ system.
The elevated plus-maze is the most frequently used animal model, similar to that observed by Tsang
and Ho [6], followed by the light/dark test, and the open-field test. One important point to consider is that
animal models of anxiety may not represent the same anxiety disorder [5]. Recently, there has been
growing evidence that certain animal models of anxiety are related to panic disorder (e.g., escape behavior
in the elevated T-maze), obsessive-compulsive disorder (e.g., marble-burying), or generalized anxiety
(e.g., avoidance in the elevated T maze or elevated plus-maze). Thus, it is important to give attention to
which animal anxiety model is sensitive to which essential oil, giving a better prediction of the oils
putative clinical efficacy for a specific anxiety disorder.
Moreover, considering the probability of false positive (and false-negative) results it is also important
to evaluate a potential new anxiolytic drug in at least two different animal models of anxiety, since different
models are sensitive to different anxiolytic treatments [5,6,87]. There are also criticisms to the use of the
open field test to measure anxiety (increased center visiting or exploration, due to a reduction in thigmotaxis,
as an index of anxiety reduction), since it lacks sensitivity to some anxiolytic treatments such as repeated
antidepressants [108]. Furthermore, amphetamine, while not producing anxiolytic-like effect (on the
contrary, several times it induced anxiogenic effect), increases center exploration (or center preference),
which is considered an indication of increased risk-taking/poor judgment behavior, an index of a mania
state [109]. It is important to note that several studies reviewed included one animal model only. Another
important methodological issue is the use of a dose/concentration-response design (i.e., at least two
different doses/concentrations), to reduce the chance of false-negative and false-positive results.
When considering these results for essential oils, positive results in several animal models (and in different
species), employing a dose/concentration-response design and a lower toxicity profile, are promising for
tests in a clinical setting. Lavender essential oil is a good example, since it revealed positive results in
different animal models (e.g., elevated plus-maze, marble-burying, and the social interaction tests), and
in different species (e.g., rats, mice, and gerbils). It may be noted that most of the studies reviewed used
acute administration, and different routes of administration (e.g., inhaled), which may be not related
(or convenient) for clinical use in patients with generalized anxiety disorder. In this line, for a putative
anxiolytic and chronic use, repeated systemic oral administration must be evaluated. However, it must
also be stressed that acute inhaled essential oil might be used in other clinical situations, and that the
approach is adequate. For example, the studies of Lehrner and collaborators [104] and Kritsidima and
collaborators [110] showed an important use of inhaled essential oil for anxiety reduction in dentistry.
Attention must be given to certain methodological aspects. Motor activity must be appropriately
evaluated, since changes in motor activity (e.g., sedation or muscle relaxation) can interfere with behavior
in animal anxiety models. For example, although some authors proposed the total number of arm entries
as a measure of activity in the elevated plus-maze [111], other authors pointed out that this measure could
be contaminated by anxiety and proposed that the number of closed arm entries is a better index [112]. In the
light/dark test, increase latency to first transition is sometimes taken as an index of motor impairment [113].
Thus, it is recommended that an additional test be included (e.g., open field testing). We note for the anxiety
indexes used, that it would be interesting to standardize the procedures [7]. For example, in the elevated
Molecules 2015, 20 18651

plus-maze test, (the most frequently used animal model to test anxiolytic-like drugs) [5,114] sometimes the
open arm exploration is presented as a percentage of open arm entries, and as a percentage of time spent
in the open arms (the most frequently reported and validated indexes of anxiety in this model). Sometimes
exploration is presented as a raw measure (time in seconds, or the absolute number of entries). It would
also be interesting to incorporate certain ethological measures (e.g., risk assessment), which would make
the model more sensitive to non GABA/BDZ drugs [114].
Another important issue is to use a validated and standardized procedure to better translate pre-clinical
results to the clinical. For example, the standard procedure for the elevated plus-maze employs a 5-min
session [115] while some authors used a 10-min session, which could change the nature of behavior
observed, and could lead to different results. Gender is also a relevant issue, since male and female
behavior can differ in the same animal model, as seen for example in the elevated plus-maze [116].
Standardization must also be applied to open-field testing, which is a very interesting and rich, yet not
well explored animal model. There are many versions of the open-field (circular, square or rectangular;
black, white or transparent; under dim or bright light; motor activity measured manually, by square
crossed, or even automated, using video tracking or beam interruptions), that make it difficult to compare
divergent results. For example, Lamprea and collaborators [117] observed in rats an increasing preference
to stay in the corner given an increasing number of walls (0, 1 or 2).
As noted by Tsang and Ho [6], it is also very important to include adequate positive controls
(e.g., benzodiazepine, buspirone, or pregabalin) in order to avoid false negative results. In this line, the
use of a neutral odor is interesting to control olfactory stimulation, since any new odor can trigger some
behaviors by itself, or occult an aversive odor (e.g., eugenol in dentistry) [118]. Some studies included
this control [37,51]. Regarding positive controls, some authors claimed that the tested essential oil is
more potent or efficient or induced lower side effects than the standard anxiolytic (e.g., diazepam). This
is a complicated issue since only one dose of the standard drug is administered, which does not permit
conclusions in these comparisons (for lack of a dose response curve).
It has been proposed that odor has an effect, activating olfactory receptors or neurons in the vomeronasal
organ, and activating limbic areas, such as the amygdala [6466,82,119]. However, studies with anosmic
mice [52,53], or with intraperitoneal administration (oral administration may still stimulate olfactory cues),
indicate that systemic absorption (essential oil reaching the central nervous system via systemic circulation),
contributes to the anxiolytic-like effects of essential oils.

3. Methodology

Searches were performed in the scientific literature databases: Chemical Abstracts, PubMed, and Web
of Science through December 2014 using the following key words: essential oils (with each individual
essential oil cited in the original articles and reviews), anxiety, animal models, (or each animal model).
The search was restricted to English, and to experimental studies. An additional manual reference search
in all of the articles found by the electronic search was also performed. We only included essential oils
having as a minimum one study suggesting anxiolytic-like effect; essential oils with exclusively negative
results were not included.
Molecules 2015, 20 18652

4. Conclusions

In conclusion, reviewing the literature of essential oils in animal models of anxiety, certain essential
oils exerted anxiolytic-like effect in differing animal models and species; as such the following oils
reveal the stronger potential for development of new treatments for anxiety disorders. Achillea wilhemsii,
Angelica sinensis, Alpinia zerumbet, Celastarus paniculatus, Chamaecyparis obtuse, Citrus aurantium,
Citrus aurantium subsp. bergamia, Citrus janus, Citrus latifolia, Citrus reticulate, Citrus sinensis,
Cymbopogon citratus, Copaifera reticulate, Ducrosia anethifolia, Foeniculum vulgare, Lavendula
angustifolia, Lippia alba, Spiranthera odoratissima and Stachys tibetica (only one study). Some of these
essential oils are particularly interesting: Achillea wilhemsii, Alpinia zerumbet, Lavendula angustifolia,
Citrus aurantium, and Spiranthera odoratissima, since their activity is not likely to be related to the
GABA/BDZ system; and Lavendula angustifolia, Citrus sinensis and Citrus aurantium subsp. bergamia,
since they have demonstrated clinical efficacy in controlled studies. Among the 25 constituents of essential
oils with reported anxiolytic activity, no chemical characteristic was found common to all, except low
molecular weight. It was not possible therefore to establish structure-activity relationships for this collection
of psychoactive compounds.

Acknowledgments

This research was supported by the Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico
(CNPq), the Coordenao de Aperfeioamento de Pessoal de Nvel Superior (CAPES), and the Fundao
de Apoio Pesquisa e Inovao Tecnolgica do Estado de Sergipe (FAPITEC/SE).

Author Contributions

Damio Pergentino de Sousa designed the research and appraisal of the paper; Palloma de Almeida Soares
Hocayen, and Luciana Nalone Andrade reviewed the literature and formatted the text; Roberto Andreatini,
designed the research, analyzed the data and wrote the paper.

Conflicts of Interest

The authors declare no conflicts of interest.

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Sample Availability: Samples of the compounds not are available from the authors.

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