Mesfin et al.

BMC Complementary and Alternative Medicine 2014, 14:310

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RESEARCH ARTICLE Open Access

Evaluation of anxiolytic activity of the essential

oil of the aerial part of Foeniculum vulgare Miller
in mice
Miraf Mesfin1, Kaleab Asres2 and Workineh Shibeshi1*

Abstract
Background: Foeniculum vulgare locally known as ensilal, is an aromatic plant widely cultivated in temperate and
tropical regions. The anti-anxiety activity of the crude extract of F. vulgare has been reported. However, the fraction
responsible for anxiolytic activity is not known and there is no any report on the anti-anxiety activity of the essential oil
of F. vulgare. The objective of study was to evaluate the anxiolytic activity of the essential oil of Foeniculum vulgare Miller.
Methods: Adult Swiss albino male mice were randomly divided into six groups (n = 6). Groups I and II received
Tween 80 (5%, v/v) and diazepam (0.5 mg/kg, ip), respectively, while groups III to V received orally 50, 100, and 200 and
400 mg/kg doses of the essential oil of F. vulgare, respectively. The mice were then individually placed in animal anxiety
models: elevated plus maze (EPM), staircase test (SCT) and open field test (OFT) and evaluated for various parameters.
Results: In EPM test, 100 and 200 mg/kg doses of the essential oil significantly increased percent number of entries
and time spent in open arms compared to control. In SCT these doses also reduced rearing significantly compared
to controls, while only the 200 mg/kg dose significantly increased number of squares crossed at the center in the
OFT test.
Conclusion: The essential oil of F. vulgare was found to exhibit a promising anxiolytic activity.
Keywords: Anxiolytic activity, Foeniculum vulgare, Essential oil, Mice

Background unexplained symptoms, increased utilization of healthcare,

Anxiety disorders are among the most common mental, strongly and independently associated with chronic medical
emotional, and behavioral problems affecting one-eighth illnesses, low levels of quality of life and disability [3,4].
of the total population worldwide, and have become a Pharmacologic treatment of anxiety through the ages has
very important area of research interest in psycho- included different drugs. The first class of drugs developed
pharmacology. Anxiety represents a heterogenous group (barbiturates) was highly effective, unfortunately, the barbi-
of disorders, probably with no single unifying etiology; turates can cause respiratory arrest and have a narrow
various psychodynamic, psychoanalytic, behavioral, cogni- therapeutic index [5]. The benzodiazepines were developed
tive, genetic and biological theories have been proposed to as a safer alternative to barbiturates, however, their benefi-
explain the etiology of anxiety disorders [1]. It is reported cial effects are overshadowed by the emergence of physical
to have increasing prevalence in recent cohorts in many and psychological dependence and withdrawal reactions
countries and to have much earlier ages of onset than [6,7]. Other drugs used for treatment of anxiety having
other commonly occurring chronic conditions [2]. Anxiety unfavorable side-effect profiles include buspirone [8,9],
disorders cause performance impairments on numerous antidepressants [10-12] and beta-blockers [5].
tasks and are associated with high rates of medically Due to adverse effects associated with the currently
available drugs, patients on anxiolytic drugs usually
terminate the treatment before full recovery [13]. In
* Correspondence: workineh.shibeshi@aau.edu.et
1
Department of Pharmacology and clinical Pharmacy, School of Pharmacy,
addition, one-third of patients in controlled studies are
Addis Ababa University, Addis Ababa, Ethiopia
Full list of author information is available at the end of the article

© 2014 Mesfin et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Mesfin et al. BMC Complementary and Alternative Medicine 2014, 14:310 Page 2 of 7
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unresponsive to any one of the medications [14]. Thus, Plant material

there is a critical need for development of newer anxiolytic The fresh aerial parts of F. vulgare were purchased from
agents. In the search for new therapeutic products for local market in Addis Ababa. The plant was identified at
the treatment of neurological disorders, medicinal plant the National Herbarium, College of Natural Sciences,
research, worldwide, has progressed constantly, demon- Addis Ababa University, where a voucher specimen was
strating the pharmacological effectiveness of different deposited (collection number MM001).
plant species in a variety of animal models [15]. Several
essential oils obtained from plants are employed in order Experimental animals
to balance emotions, improve physical and mental well- Adult Swiss albino male mice weighting 25-35 g were
being [16]. Foeniculum vulgare Mill. locally known as obtained from rodent breeding unit of the School of
ensilal or commonly known as fennel, is an aromatic plant Pharmacy, Addis Ababa University. The animals were
widely cultivated in temperate and tropical regions [17]. housed under standard environmental conditions and were
The chemical constituents of F. vulgare include essential allowed free access to tap water and standard laboratory
oil, fatty acid, phenylpropanoids, monoter-penids, sesqui- pellet ad libitum. The ethical handling (33) of mice used
terpenes and coumarins. It also contains triterpenoids, in our study and the experimental protocols used were
tannins, flavonoids, cardiac glycosides, saponins, and other approved by Research and Ethics committee of the School
types of compounds [18,19]. of Pharmacy, Addis Ababa University.
The essential oil of F. vulgare is characterized by the
presence of several components including major compo-
Extraction of essential oil
nents trans-anethole, fenchone, methyl chavicol, eugnol,
About 3 Kg of the fresh leaves of F. vulgare were dis-
limonene, p-anisaldehyde, α-phellandrene, α-pinene, 1,8-
tilled by hydrodistillation for a period of about 3 h using
cineole, γ-terpinene and P-cymene. Compounds such as
Clevenger-type apparatus. The oil was dried over anhyd-
3-methylbutanol, linalool, β-pinene, mycerene, δ-3-carene,
rous sodium sulphate to remove traces of moisture and
camphor, α-terpinol, cis-anethole, thymol are also found
stored in a vial inside a refrigerator at 4°C until use.
in lower concentration [20]. The essential oil of F. vulgare
is generally recognized safe as the toxicity it may cause is
negligible [21]. Acute toxicity study
In traditional medicine, F. vulgare is used to: encourage Acute oral toxicity study was determined by using five
menstruation and lactation, stimulate gastrointestinal female mice weighing 25–30 g. The mice were fasted for
motility, relieve intestinal gas accumulation, improve 3 h prior to the experiment. The mice were administered
eyesight, alleviate productive coughs, ease spasm, pro- using oral gavage a single high dose (2000 mg/kg) of the
mote courage and mental strength, reduce stress and essential oil and were observed for any symptoms of tox-
nervousness and produce calming [22,23]. The medicinal icity continuously for 1 h, intermittently for 4 h, over a
properties of F. vulgare as evidenced by different animal period of 24 h and for 14 days [33].
and clinical studies include, antibacterial and antifungal
[24], antioxidant [25], anti-inflammatory [26], anti-athero- Experimental designs
sclerotic [27], gastroprotective [28], hepatoprotective [29], The mice were randomly divided into six groups (n = 6).
and diuretic [30]. The anti-anxiety activity of the crude The first group received the vehicle (5% Tween 80 in
extract of F. vulgare has been reported [31,32]. However, distilled water), the second group received standard
the fraction responsible for anxiolytic activity is not known drug, diazepam (0.5 mg/kg, Ip). Groups III to VI using
and there is no any report on the anti-anxiety activity of oral gavage received 50, 100, 200 and 400 mg/kg doses
the essential oil of F. vulgare. of the essential oil, respectively. The doses were selected
Thus, the objective of this study was to evaluate the based on the results of the oral acute toxicity study.
anxiolytic activities of essential oil of F. vulgare in animal Taking into account the safety of the essential oil, 1/
models of anxiety. 10th of the maximum dose (2000 mg/kg) given in the
toxicity study limit test was considered as a middle dose
(200 mg/kg). Double, one-fourth and half of the middle
Methods dose were assigned as high dose (400 mg/kg), first low
Chemicals dose (50 mg/kg) and second low dose (100 mg/kg)
The chemicals used in the experiment include diazepam respectively.
(Intlas Pharmaceuticals, India), tween 80 (Research-lab fine After 30 min of diazepam treatment or 60 min essential
Chem Industries, India), anhydrous sodium sulfate (Bio-lab oil/vehicle pretreatment, the mice were individually placed
laboratories Ltd, Israel) and ethyl alcohol (Changshu in animal models. All the tests were carried out at night
Yangyuan Chemical, China). with a minimal amount of background noise. After each
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test, the maze was cleaned with ethyl alcohol to eliminate Results
any olfactory cues to the next animal [34]. Acute toxicity test
After the administration of 2000 mg/Kg dose of the
essential oil of F. vulgare, the animals didn’t show loss of
Elevated plus maze (EPM)
weight, autonomic behavioral changes or other signs of
The test was conducted using apparatus validated by
toxicity. There was also no mortality observed in the
Lister [35]. After each mouse was placed in the centre of
study period, suggesting that the LD50 (median lethal
the maze facing one of the open arms, the number of
oral dose) of the essential oil is higher than 2000 mg/kg
entries made into the open and closed arms and the
when given orally [33].
time spent in them was recorded, using a video camera
for the next five min. From these data, the percentage of
Anxiolytic effects
entries and the percentage of time spent in each arms
In the EPM paradigm (Table 1), the essential oil at
was calculated.
200 mg/kg dose showed significant increase in the number
of entries into the open arms compared to the negative
Staircase test (SCT) control, while all other doses of the oil did not significantly
SCT was carried out as used by Simiand et al. [36]. Each affect the parameter compared. On the other hand, none of
mouse was placed on the floor of the box with its back the doses of the oil or diazepam significantly affected the
to the staircase, then the number of steps climbed and the number of closed arm entries except the 400 mg/kg dose
numbers of rears were recorded over 3 min period using a which significantly decreased the parameter. The time the
video camera placed over head. A step is considered to be mice spent in the open arms was significantly increased in
climbed only if the mouse had placed all four paws on animals treated with 100 and 200 mg/kg doses of the oil
the step, the number of steps descended was not taken compared to the control group. Mice treated with 100 and
into account. 200 mg/kg doses significantly increased the percentage of
open arm entry compared to the vehicle (Figure 1) and at
a dose of 200 mg/kg the oil produced a significant reduc-
Open field test (OFT)
tion in the percentage of time spent in the closed arms
The test was conducted as described by Aragao et al.
(Figure 2).
[37]. Briefly, the mice were placed individually in a cor-
In SCT (Table 2), mice treated with the 100 and
ner square of the OFT apparatus and the total number
200 mg/kg doses of the oil showed significant reduction
of squares crossed at the periphery, number of central
of rearing compared to control group, however, only
squares crossed, total number of squares traveled, and
200 mg/kg dose of the oil showed statistically significant
the time the mouse spent in the center were video
reduction in rearing compared with diazepam. The reduc-
recorded for 5 min.
tion in the number of climbing was produced by the
essential oil only at 400 mg/kg dose compared with
Statistical analysis control, while other doses were not effective.
The data are presented as mean ± S.E.M (standard error In the OFT model, the number of central squares crossed
of mean) of six mice. Statistical analysis of the data was was increased in all groups treated with the essential
performed using one-way analysis of variance (ANOVA) oil, however, statistical significance was reached only in
followed by Tukey posthoc test. Significant differences the groups treated with 200 mg/kg compared to the
were set at P values lower than 0.05. control (Table 3). In mice treated with 400 mg/kg the

Table 1 The behavior of mice treated with the essential oil of Foeniculum vulgare and reference compounds in the
elevated plus maze model
Treatment Dose, Number of entries (counts/5 min) Time spent (sec/5 min)
mg/kg
Open arm Closed arm Total Open arm Closed arm Center
Vehicle - 3.17 ± 1.33 8.50 ± 2.59 11.67 ± 3.66 44.17 ± 20.19 137.83 ± 38.24 118.00 ± 41.25
FV 50 4.83 ± 1.72 6.83 ± 0.75 11.66 ± 1.37 68.00 ± 25.69 117.50 ± 46.78 114.50 ± 50.03
FV 100 7.50 ± 4.59 5.83 ± 3.31 13.66 ± 3.78 109.67 ± 48.64a* 99.83 ± 43.32 90.50 ± 33.59
a* c*
FV 200 11.00 ± 5.48 8.00 ± 4.90 19.00 ± 9.87 112.67 ± 35.62a* 71.33 ± 37.17a* 119.00 ± 36.87
a* (b, d)*
FV 400 4.01 ± 2.10 4.00 ± 1.4 8.00 ± 2.76 87.50 ± 36.32 93.33 ± 35.27 119.17 ± 27.13
Diazepam 0.5 10.83 ± 9.06a* 6.50 ± 2.59 17.50 ± 8.53 c* 123.67 ± 50.67a** 67.33 ± 34.63a* 84.67 ± 44.23
a b c d
Data are presented as mean ± S.E.M, n = 6, *P < 0.05, **P < 0.01, against control, against 200 mg/kg, against 400 mg/kg, against diazepam; FV = Foeniculum
vulgare essential oil.
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Figure 1 Effects of the essential oil of Foeniculum vulgare and controls on percentage of arm entries in the elevated plus maze (EPM)
test in mice. Data are mean ± S.E.M (n = 6); *P < 0.05, **P < 0.01, compared with vehicle. FV = Foeniculum vulgare essential oil.

oil, a significant reduction in the number of squares approach-avoidance conflict and rodents consistently
crossed at the periphery compared to both vehicle and spend greater time in the closed arms when placed in
200 mg/kg dose was noted. Mice treated with 100 and mazes comprising of open and closed arms [40]. Based
200 mg/kg dose of the essential oil spent more time in on these assertions, EPM tests are reliable means of
the central squares compared to the control group. identifying selective anxiolytic effect of drugs and used
as a tool in the investigation of the psychological and
Discussion neuro-chemical basis of anxiety, for screening anxiety-
The present work has evaluated the anxiolytic activity of modulating drugs or mouse genotypes [41]. The EPM
various doses of the essential oil of F. vulgare in mice test has been validated pharmacologically, physiologically
employing three non-conditioned behavioral animal models and behaviorally, and has become one of the most
of anxiety; EPM model, OFT and SCT. These tests are widely used behavioral tests for anxiety [34]. Anxiety is
classic and standard models for screening central nervous induced by a fear due to height in rodents when placed
system actions providing information about anxiety and on the EPM. The ultimate manifestation of anxiety is
psychomotor performance [38]. Further, these models can exhibited by preference to remain at safer places and a
create an anxiety state in normal rodents in a reproducible decrease in the motor activity.
paradigm while minimizing some of the confounding Treatment of mice with the essential oil of F. vulgare
factors of other conditioned assays [39]. resulted in significant alterations on the behavioral
The EPM test is principally based on the behavior that responses measured in the EPM test. Experimental
exposure of animals to an elevated maze alley evokes an animals treated with 100 and 200 mg/kg doses of F.

Figure 2 Effects of the essential oil of Foeniculum vulgare and controls on the percentage of duration in arms in the elevated plus maze
test in mice. Data are expressed as mean ± S.E.M. (n = 6); *P < 0.05, **P < 0.01 as compared to control. FV = Foeniculum vulgare essential oil.
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Table 2 Effect produced by administration of the vulgare (100 and 200 mg/kg) in relieving anxiety in this
essential oil of Foeniculum vulgare and reference model may suggest a possible positive modulation of
compounds to mice in the staircase test the GABA-A/benzodiazepine receptor complex.
Treatment Dose (mg/kg) Number of rearing Number of The SCT for screening anxiolytic activity is a simple and
stairs climbed
rapid procedure for preliminary screening of anxiolytic
Vehicle - 19.67 ± 2.50 16.17 ± 6.11 agents [45]. Step climbing reflects exploratory or locomotor
FV 50 15.00 ± 1.67 d** 18.00 ± 4.19 e** activity, while rearing behavior was a manifestation of
FV 100 13.33 ± 4.93 a* d*
13.34 ± 1.37 anxiety state [46]. The present study showed that exposure
FV 200 8.00 ± 3.03 (a, b, e)**. (c, f)*
11.50 ± 3.56 of the experimental animals to the essential oil of F. vulgare
d** significantly reduced rearing activity at doses (100 and
FV 400 18.33 ± 3.44 9.50 ± 4.09 a*. b**
200 mg/kg) that did not suppress climbing, aligning with
Diazepam 0.5 14.00 ± 3.29(a*.d*) 12.00 ± 4.56
a behavior of an anxiolytic compound in a staircase model
Data are mean ± S.E.M, n = 6, compared with vehicle, to 50 mg/kg, cto
a b

100 mg/kg, dto 200 mg/kg, eto 400 mg/kg, fto diazepam, *P < 0.05, **P < 0.01.
[47]. At a dose of 400 mg/kg, the essential oil produced
FV = Foeniculum vulgare essential oil. a significant reduction in the number of steps ascended
indicating suppression of locomotor activity, which is
vulgare essential oil showed significantly increased interpreted as a sedative, rather than anxiolytic effect in
percentage of number of entries into the open arms. different studies [46,48]. Only GABA receptor complex
This decreased aversion to open arms compared to active agents have been shown to reduce rearing at
control group indicated its anxiolytic activity [40]. The doses that do not reduce climbing in the SCT. Other
decrease in aversion to open arms by the 100 and non-benzodiazepine compounds induce non-specific sup-
200 mg/kg doses of the oil was also verified by the pression of both rearing and climbing behavior [49],
increased percentage of time spent in the open arms strengthening the suggested possible mechanism of the oil.
compared with the negative control. The OFT is a classical animal model used to evaluate
Time spent in the central platform of EPM appears to effects of drugs on anxiety, general motor activity and
be related to decision making and/or risk assessment. exploratory behavior. It uses the normal aversion of rodents
A decreased time spent on the central platform serves to an open, brightly lit area, as the confrontation with the
as indicator of a reduced decision making behavior, a situation induces anxiety behavior in rodents [50]. Rodents
parameter accepted as reliable indicators of anxiety and normally spend more time in the protective corners,
fearfulness [42]. However, neither doses of the essential suggesting that the walls confer anxiety-relieving body
oil nor diazepam altered the parameter significantly contact. When animals are placed in OFT, they express
compared to control. At a dose of 400 mg/kg, however, their anxiety by a decrease in exploratory behavior [51].
the oil showed a significant decrease on the number of Increased entry and the time spent in the center of the
entries in the closed arms. The absence of significant arena in the OFT reflects decreased anxiety altered by
modification in the number of closed arm entries, in anxiolytic property, as measures of central exploration are
the anxiolytic doses of the oil, in the EPM indicated often regarded as anxiety-related indices [52]. Diazepam
that the anxiolytic activity was observed at doses that significantly increased the total number of squares traveled
did not impair motor activity [43]. at the periphery, which may be an increase in exploratory
The EPM test is one of the most popular tests for activity on reduced anxiety. The anxiolytic activity of the
search of new benzodiazepine-like anxiolytic agents [44]. essential oil at the effective doses (100 and 200 mg/kg),
In this context, the activity of the essential oil of F. could not be confirmed by this parameter, as the increase

Table 3 Effect of the essential oil of Foeniculum vulgare and reference compounds on the behavior of mice in the open
field model
Treatment Time spent in Number of squared crossed
central squares
Center Periphery Total
(sec)
Vehicle 2.17 ± 1.33 2.00 ± 1.26 85.17 ± 19.37 87.16 ± 19.31
FV50 3.33 ± 5.92 c* 3.67 ± 7.12 d* 80.83 ± 25.34 84.50 ± 25.99
(a, b)* f**
FV100 10.67 ± 4.32 9.00 ± 7.54 79.33 ± 10.32 88.33 ± 17.81 f**
a* (a,b,e)* (e, f)*
FV200 10.50 ± 4.04 14.50 ± 3.27 87.50 ± 19.67 102.00 ± 21.87 e*
FV400 4.50 ± 5.92 3.34 ± 4.55 d* 53.00 ± 4.94 (a ,d)* 56.33 ± 7.76 d*
a* a* (a, d)*.c**
Diazepam 9.33 ± 5.32 12.83 ± 10.66 122.67 ± 31.6 133.83 ± 34.93(a, c)**

The data are mean ± S.E.M, n = 6, aagainst control, bagainst 50 mg/kg, cagainst 100 mg/kg, dagainst 200 mg/kg, eagainst 400 mg/kg, fagainst diazepam, *P < 0.05
and **P < 0.01. FV = Foeniculum vulgare essential oil.
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in locomotor activity can be used as indice of anxiolytic Competing interests

effect [53]. The decrease in ambulation at the highest The authors declare that they have no competing interests.

dose (400 mg/kg) of the oil might be due to the sedative

property of the oil. Suppression of exploratory behavior is Authors’ contributions
MM conceived the study, designed and conducted all laboratory
an indication of central nervous system depressant activity
experiments; analyzed and interpreted experimental results. KA and WS
[44]. This decline in activity at highest doses of the essen- participated in the proposal, study design and manuscript preparations. All
tial oil in EPM, SCT and OFT can be correlated well with authors read and approved the final manuscript.
each other.
In the present study, the activity of F. vulgare corre- Acknowledgements
sponding to the doses given had seemingly a biphasic The authors are grateful to Ato Melaku Wondafrash, the National Herbarium,
Addis Ababa University for identification of the plant material. One of us
inverted-U (bell) shape, as the administration of the (MM) would like to acknowledge the office of the Vice President for
highest (400 mg/kg) and lowest (50 mg/kg) doses showed Research and Dean of Graduate Studies for providing financial support.
insignificant low effects whereas the 100 mg/kg and
Author details
200 mg/kg dose levels produced significant effects and 1
Department of Pharmacology and clinical Pharmacy, School of Pharmacy,
the peak in mean response was shown at 200 mg/kg. Addis Ababa University, Addis Ababa, Ethiopia. 2Department of
The ineffectiveness of the lowest dose of the oil may be Pharmaceutical Chemistry and Pharmacognosy, School of Pharmacy, Addis
Ababa University, Addis Ababa, Ethiopia.
because it is a sub-threshold level. However, the lack of
effect at the highest dose level may be due to a decreased Received: 6 March 2014 Accepted: 19 August 2014
locomotion which might be due to an interference with Published: 23 August 2014
a normal sensory-motor function or sedative effect. The
sedative activity of the essential oil is also verified by a References
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