Study on anti-anxiety effect of Willughbeia cochinchinesis in scopolamine-administrered mice using open field test

Journal of military pharmaco-medicine no5-2018

STUDY ON ANTI-ANXIETY EFFECT OF WILLUGHBEIA
COCHINCHINENSIS IN SCOPOLAMINC-ADMINISTERED MICE
BY USING OPEN FIELD TEST
Nguyen Tat Dinh*; Cao Tien Duc*; Le Van Quan**
SUMMARY
Objective: To investigate effects of Wullughbeia cochinchinensis to ameliorate disorders in
anxiety-like behaviors of mice. Subjects and methods: In the present study, 50 Swiss mice were
induced anxiety-like behaviors by scopolamine. Behaviors of mice treated with and without
Wullughbeia cochinchinensis at doses of 100 mg/kg, 150 mg/kg and 200 mg/Kg and/or
scopolamine were tested using the open field test. Results: Wullughbeia cochinchinensis at
dose of 150 mg/kg and 200 mg/kg induced a significant increase in time spent, number of
entries and travel distances in the center area of open field in animals with scopolamine-induced
anxiety-like behaviors. Conclusion: Wullughbeia cochinchinensis 150 mg/kg and 200 mg/kg
might ameliorate deficits in anxiety like symptoms in experimental animals.
* Keywords: Anxiety, Mice; Willughbeia cochinchinensis .

INTRODUCTION
Anxiety is one of the common symptoms
in mental disorders such as depression,
phobia, etc. In a recent study, it has
suggested that 33.7% of the population
are affected by an anxiety disorder during
their lifetime [1]. Furthermore, ratio of
females with anxiety disorder is higher
than this of male with this symptom [2]. In
treatment, benzodiazepines have been
suggested to be effective to anxiety
disorders. However, these drugs might
induce drug dependence when they are

used a long time [3]. Thus, it is necessary
to investigate new natural plants or drugs
for treating anxiety disorders.
It has suggested that anxiety-like
disorders might relate to activities of

achetylcholine. Thus, scopolamine, an
antagonist to acetylcholinergic receptors,
might be used to induce anxiety-like
behaviors in experimental animals [4]. In
the present study, we used this animal
model to investigate effects of a new
natural plant, Wullughbeia cochinchinensis
(WC) to meliorate deficit in anxiety-like
behaviors in mice.
SUBJECTS AND METHODS
1. Subjects.
50 Swiss mice (150 - 250 g body
weight) were used in the present study.
Animals were housed in individual cages,
maintained in controlled temperature and
12 h light/dark cycles with free access to
water and food.

*
**
Corresponding author: Le Van Quan (@gmail.com)
Date received: 26/02/2018
Date accepted: 30/05/2018

170


Journal of military pharmaco-medicine no5-2018
Animals were separated randomly into
5 experimental groups, 10 mice for each
group: group 1 (control group): mice were
ip and p.o treated saline; group 2
(scopolamine group): mice were i.p
treated scopolamine 1.5 mg/Kg and p.o
treated saline at 0.1 ml/10 g; group 3,
group 4 and group 5 (WC groups): mice
were i.p injected scopolamine 1.5 mg/Kg
and p.o WC 100 mg/Kg, 150 mg/Kg and
200 mg/Kg, respectively. WC and saline
were orally administered at 60 minutes
and scopolamine and saline were i.p
injected at 30 minutes before the
behavioral task.
The present study was conducted at
Department of Physiology, Vietnam
Military Medical University. All procedures
were performed in accordance with the
Animal Center Guidelines for the Care
and Use of Laboratory Animals at the
Vietnam Military Medical University.
2. Materials.
- WC was isolated by Department of
Pharmacy, Hochiminh City University of
Medicine and Pharmacy and was

supplied in power form. WC power was
dissolved in saline using a magnetic
stirrer.
- Open field box was a square box (40
x 40 x 60 cm), covered with polypropylene
sheets inside the wooden box. In the
present study, the open field was
separated into two areas: a center area
and a periphery zone (figure 1).

Figure 1: Open field box.
3. Methods.
* Open field test:
60 minutes after drug treatments, mice
were placed in the center of an open field
box. Animals were allowed to free explore
inside open field box for 5 minutes.
Behaviors of animals were recorded using
a digital video system. Data was analyzed
offline by ANY-maze software (Stoelting
Co., Wood Dale, IL, USA).
* Research indicators:
To investigate anxiety-like behaviors in
mice, we concentrated to analyze activities
of animals in the center area of the open
field apparatus. These were:
- Time spent in the central zone (s).
- Numbers of entries into the central area.
- Travel distances in the central area (m).
* Data analysis:

Time spent in the central area;
numbers of entries in the central zone and
travel distances in the center area were
analyzed by one-way analysis of variance
(ANOVA) followed by the Tukey’s posthoc test for multiple comparisons, using
SPSS 19.0. Results were considered to
be statistically significant at p < 0.05. All
results were expressed as mean ± SEM.
171


Journal of military pharmaco-medicine no5-2018
RESULTS
1. Time spent in the central area of the open field.

Figure 2: Time spent in the center area.
Time spent in the central area is used to assess exploring abilities as well as
anxiety-like behaviors of mice. In the fig. 2, time spent in the central area of animals
treated by scopolamine was significant shorter than this of animals treated by saline (p
< 0.05). After animals treated by WC, there were gradual increases in time spent in the
central area. However, significant differences were found only in animals treated by
WC at doses of 150 mg/kg and 200 mg/kg (p < 0.05).
2. Numbers of entries to the central area.

Figure 3: Entries to the central area.
172


Journal of military pharmaco-medicine no5-2018
Fig. 3 indicated results of entries of

mice into the central area. Results
showed that number of entries into the
central area of mice treated by
scopolamine was significant lower than
this of mice treated by saline (control
group) (p < 0.01). Furthermore, there
were gradual increases in entries of mice
into central areas following treatments of
WC at doses of 100 mg/kg; 150 mg/kg
and 200 mg/kg. However, there were
significant differences in numbers of
entries into the central area of mice
treated by WC at doses of 150 mg/kg and
200 mg/Kg (p < 0.05).

3. Travel distances in the central
area,
Fig. 3 showed travel distances in the
central areas of mice in experimental
groups. Results showed that mean
travel distance in the central area of
scopolamine-treated mice was significant
shorter than this of saline-treated mice
(p < 0.01). After treatments of WC 60
minutes, travel distances in the central
area of mice increased gradually.
However, travel distances of mice in
the central areas increased significantly
in groups WC 150 and WC 200 only
(p < 0.01).

Figure 4: Travel distances in the central area.
DISCUSION
Open field is one of the common
behavioral tests which are employed to
investigate anxiety-like disorders in rodent
animals. In the open field test, the time
animals spent and number of entries to
the center of arena is often used to

measure anxiety-related behaviors in
mice. In general, due to life characteristics
of mice, they avoid the center of open
field arena. Increases in time spent and
numbers of entries into the central areas
of the open fields might indicate a
decrease in anxiety behaviors [5].
173


Journal of military pharmaco-medicine no5-2018
In the present study, we found evidence
of improving anxiety disorder effect of WC.
These were: treatments of WC at doses
of 150 mg/kg and 200 mg/kg induced
increases in time spent, numbers of
entries and travel distances in the center
of open field arena. These results are
consistent with previous studies which
have concentrated to investigate anxiety

disorders in mice. Anchan et al showed
that GPR30 activation decreases anxiety
in the open field which was expressed by
a greater distance and higher number of
entries into the central area in the open
field [6]. Similarly, by the some way, they
have demonstrated that γ-aminobutyric
acid transporter-1 also is involved in
decrease of anxiety like behaviors in mice
[7]. From above results, our study showed
a direct evidence for effects of WC to
decreasing anxiety like behaviors in mice.
CONCLUSION
In the present study, we investigated
anti-anxiety effect of WC in mice. We
found that: After treatments of WC at
doses of 150 mg/kg and 200 mg/kg
increase in time spent in the center of
arena, greater travel distances and higher
numbers of entries into the central of
open field area. These results provided
new evidences for using new natural

174

plants in treatments for anxiety disorders
in humans.
REFERENCES
1. Bandelow B. Epidemiology of anxiety
st

disorders in the 21 century. Dialogues Clin
Neurosci. 2015, 17 (3), pp.327-335.
2. McLean C.P, Asnaani A, Litz B.T,
Hofmann S.G. Gender differences in anxiety
disorders: Prevalence, course of illness,
comorbidity and burden of illness. J Psychiatr
Res. 2011, 45 (8), pp.1027-1035.
3. Starcevic V. Benzodiazepines for
anxiety disorders: maximizing the benefits and
minimizing the risks. Advances in Psychiatric
Treatment. 2012, 18 (4), pp.250-258.
4. Rodgers R.J, Cao B.J, Dalvi A, Holmes
A. Animal models of anxiety: an ethological
perspective. Braz J Med Biol Res. 1997, 30
(3), pp.289-304.
5. Walsh R.N, Cummins R.A. The openfield test: a critical review. Psychological
Bulletin. 1976, 83, pp.482-504.
6. Anchan D, Clark S, Pollard K,
Vasudevan N. GPR30 activation decreases
anxiety in the open field test but not in the
elevated plus maze test in female mice. Brain
Behav. 2014, 4 (1), pp.51-59.
7. Gong X, Shao Y, Li B, Chen L, Wang C,
Chen Y. γ-aminobutyric acid transporter-1 is
involved in anxiety-like behaviors and
cognitive function in knockout mice. Exp Ther
Med. 2015, 10 (2), pp.653-658.