Willughbeia cochinchinensis ameliorates locomotor disorders in model of alzheimer’s disease mice

Journal of military pharmaco-medicine No7-2017

WILLUGHBEIA COCHINCHINENSIS AMELIORATES
LOCOMOTOR DISORDERS IN MODEL OF
ALZHEIMER’S DISEASE MICE
Can Van Mao*; Tran Hai Anh*; Le Van Quan*
SUMMARY
Objectives: To evaluate the effects of willughbela cochinchinessis (WC) to locomotor
disorders in an Alzheimer's disease (AD) model of mice. Subjects and methods: 50 Swiss mice
were separated randomly into 5 experimental groups, 10 mice for each group. Group 1: Mice
were intraperitoneally injected (i.p) and orally administered (p.o) saline at dose 0.1 mL/10 g;
group 2: mice were injected i.p scopolamin 1.5 mg/kg and p.o saline 0.1 mL/10 g; group 3,
group 4 and group 5: mice were injected i.p 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
scopolamin and saline were injected i.p at 30 minutes before the behavioral task. 60 minutes
after WC injections, mice were placed in an open field for 5 minutes. Behaviors of mice were
observed by a camera and analyzed by Anymaze software. Results: WC at doses 150 mg/kg
and 200 mg/kg reversed scopolamin-induced hyperactivities in mice. Conclusion: These results
provided a basic for developing a new drug to treat patients with AD.
* Keywords: Alzheimer's disease; Willughbela cochinchinessis; Scopolamin; Locomotor
behaviors; Mice.

INTRODUCTION
Alzheimer’s deasease is one form of
dementia in older humans. Mechanism of
this kind disease has been suggested to
be involved in neurodegeneration and
formation of plaques and neurofibrillary
tangles [1]. These changes in the brain
cause behavioral disorders such as
cognitive and memory impairments and

locomotor hyperactivities. In patients with
AD, it has been shown that patients with
AD expressed hyperactivities in the late
afternoon and the evening. These
hyperactivities are termed as sundown
syndrome or sundowing [2, 3]. In animal
models of AD, previous studies have

been demonstrated that animals also
exhibit increased locomotor activities
including hyperactivity, stereotypic behaviors,
and home cage activity disturbances [4].
It has been suggested that disorders of
neurotransmitter systems, especially
cholinergic system and glutamate systems
are associated with abnormal behaviors
of patients with AD [5]. Furthermore,
abnormal activity of the cholinergic system
affects glutamatergic systems [6]. Thus,
it has developed a animal model of AD
by injecting intraperitoneally scopolamin,
a form of anticholiergic drug and used
this model to study effects of new drugs
or natural plants in animal models of AD.

* Military Medical University
Corresponding author: Cao Van Mao ()
Date received: 15/06/2017
Date accepted: 10/08/2017

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Journal of military pharmaco-medicine no7-2017
Recently, we applied this model to evaluate
effects of WC to scopolamin-induced
deficits in cognition and memory in mice.
However, patients and scopolamin induced
animals with AD also showed disorders in
locomotion [2, 3, 7, 8]. Thus, effects of
WC to locomotor disorders should be
evaluated. We conducted the present study
with the aim: To investigate effects of WC
to ameliorate locomotor disorders in an
animal model of AD.
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 12h light/dark
cycles with free access to water and food.
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.

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
scopolamin 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 scopolamin and saline
were i.p injected at 30 minutes before the
behavioral task.
* Open field test:
60 minutes after drug treatments, mice
were placed in the center of a open field
box. Open field box was a square box
(40 × 40 × 60 cm), covered with
polypropylene sheets inside the wooden
box (figure 1). 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).

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.
3. Methods.
* Animal grouping and drug treatments:

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 (scopolamin
group): mice were i.p treated scopolamin

Figure 1: Open field box.
* Research indicators:
In the present study, we analyzed some
research indicators as followed:
- Travel distances (m).
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Journal of military pharmaco-medicine No7-2017
- Average speeds (m/s).
- Ratios of mobile time/immobile time.
* Data analysis:
of

Travel distance, travel speed and ratio
mobile time/immobile time were

analyzed by one-way analysis of variance
(ANOVA) followed by the Tukey’s posthoc test for multiple comparison, using
SPSS 19.0. Results were considered to
be statistically significant at p < 0.05. All
results were expressed as mean ± SEM.

RESULTS

1. Changes in travel distance.

Figure 2: Travel distance of mice.
Figure 2 showed travel distance of mice in the open field test. There was a
significant difference in travel distance of mice in the experimental groups [F(5.49) =
3.82, p = 0.016]. Post hoc test indicated that mean travel distance of mice in the
scopolamin group was significantly longer than this in the control group (Tukey test, p <
0.05). After WC treatment, travel distance of mice decreased gradually from WC 100
mg/kg to WC 200 mg/kg. However, a significant decrease in mean travel distance of
mice was observed in only WC 200 mg/kg group (Tukey test, p < 0.05).
2. Changes in average speed.

Figure 3: Average speed of mice.
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Journal of military pharmaco-medicine no7-2017
Figure 3 showed changes in average speed of mice in the open field test. One way
ANOVA indicated that there was a significant difference in average speed of mice in
experimental groups [F(4.49) = 4.12, p = 0.011]. Post hoc test indicated that there was
a significant increase in average speed of mice in scopolamin group, compared to this
in the control group (Tukey test, p < 0.05). After WC treatments, average speed also
reduced gradually from WC 100 mg/kg to WC 200 mg/kg. However, compared to
average speed of mice in control group, a significant decrease in average speed was
also observed in the WC 200 mg/kg group only (Tukey’s test, p < 0.05).
3. Changes in ratio of mobile time/immobile time.

Figure 4: Ratio of mobile time/immobile time of mice.
Figure 4 showed changes in ratio of
mobile time/immobile time of mice in the

experimental groups. One way ANOVA
indicated that there was a significant
difference in ratio of mobile time/immobile
time of mice between experimental groups
[F(4.49) = 6.11, p = 0.01]. Post hoc test
indicated that mean ratio of mobile
time/immobile time of mice in the
scopolamin group was significantly higher
than this in the control group (Tukey test,
p < 0.05). When mice were treated by WC,
these ratios decreased gradually. Ratios
of mobile time/immobile time of mice in
WC 150 mg/kg group and WC 200 mg/kg
group were significantly lower than those

in the scopolamin group (Tukey test, p <
0.05).
Ratios of mobile time/immobile time of
mice expressed mobile or immobile
tendencies of animals. If this ratio is
higher than 1, it indicates that animals
might tend to be mobile. On the contrary,
it indicates that animals might tend to be
immobile. Results in figure 4 indicated that
WC at doses 150 mg/kg and 200 mg/kg
reversed scopolamin induced increasing
mobile tendencies of mice.
DISCUSSION
Hyperactivity is one of behavioral
disorders in both patients with AD and

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Journal of military pharmaco-medicine No7-2017
animal models of AD. In patients with AD,
it was demonstrated that they display
sleeping and locomotion disorders. These
disorders are more serious in the late
afternoon and evening. Thus, these disorders
were called as sundown syndrome or
sundowning [2, 3]. Locomotor disorders
are observed in animal models of AD,
including the scopolamin-induced Alzheimer
model [7, 8]. In these models, mice exhibit
hyperactivity tendencies during the dark
phase (the active phase of mice) [4].
Thus, in developments of new drugs or
natural plants to treat for AD, study on
locomotor functions of experimental animals
is necessary.
Open field test used widely to investigate
locomotor functions in small animals such
as rats and mice. This test allows to evaluate
many research indicators. Thus, this test
will help us to study more particularly and
exactly the effects of new drugs to
locomotor functions of experimental
animals [9]. In the present study, the open
field test was used to assess effects of WC
to scopolamine- induced hyperactivities of

animals with Alzheimer-like symptoms.
Results showed changes in some
research indicators to indicate that WC
ameliorated disorders in locomotor
functions of animals. These are: WC has
effects to decrease travel distance,
average speed and ratio of mobile
time/immobile time of animals with
scopolamin-induced hyperactivities. These
results along with our previous study’s
results [6] provided a important basic to
apply new plants for treating AD in humans.
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CONCLUSION
In the present study, we used open
field test to investigate effects of WC on
scopolamin induced locomotor disorders
in animals with Alzheimer-like symptoms.
Our results indicated that WC at doses
150 mg/kg and 200 mg/kg reduced
scoplamin-induced hyperactivities in
experimental animals. The reduction of
hyperactivities was expressed by significant
decreases in travel distance, average
speed and ratio of mobile time/immobile
timey. These results provided important
basic for next researches to use WC for
treatment of AD in humans.
ACKNOWLEDGEMENTS

This work was supported by Grant 106YS.05-2013.24 from Vietnam’s National
Foundation for Science and Technology
Development (NAFOSTED).
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