Amelioration of learning and memory deficits by willughbeia cochinchinensis in mice

Journal of military pharmaco-medicine No7-2017

AMELIORATION OF LEARNING AND MEMORY DEFICITS
BY WILLUGHBEIA COCHINCHINENSIS IN MICE
Nguyen Thi Hoa*; Le Van Quan*; Can Van Mao*
SUMMARY
Objectives: To study effects of willughbela cochinchinensis (WC) on learning and memory
deficits in experimental animals. Subjects and methods: 50 Swiss mice were randomly
separated into 5 experimental groups, 10 mice for each group. Group 1: mice were
intraperitoneally (i.p) injected and orally (p.o) administered saline at dose 0.1 mL/10 g; group 2:
mice were injected i.p 1.5 mg/kg scopolamin and p.o 0.1 mL/10 g saline; group 3, group 4 and
group 5: mice were injected i.p 1.5 mg/kg scopolamin and p.o 100 mg/kg, 150 mg/kg and 200
mg/kg WC, respectively. 60 minutes after drug injections, animals performed a passive
avoidance test which includes two phases: training phase: animals were placed in the light
compartment and if they moved to the dark compartment, they were given electrical foot shocks
for 3 seconds; test phase: animals were also placed in the light compartment but they were not
given any electrical foot shocks at the dark compartment. Results: In test phase, mean latency
to entry dark compartment in group 2 was shorter than that in group 1 and the latencies in group
4 and 5 were longer than that in group 2. Conclusion: Our results provided an evidence for
effective treatment of WC in memory deficits animal model.
* Keyword: Willughbela cochinchinensis; Learning and memory deficits; Mice.

INTRODUCTION
Alzheimer’s disease (AD) accounts for
60 - 80% of cases of dementia in older
people [1]. Mechanism of AD has been
suggested
to
be
involved
in

neurodegeneration and formations of
plaques and neurofibrillary tangles in
brains which cause atrophied cortex and
enlarged ventricles [2]. Following these
damages in brains, patients with AD
develop deficits in memory, recognition
and behavioral controlling [3]. If they don’t
receive any treatments, these disorders
will be worse and seriously affect to their
life as well as their families.

Up to date, there is no effective special
treatments for AD and patients with AD
must receive treatments in all their life.
Furthermore, in some cases, effects of
drugs on AD treatments are limited. Thus,
development of new drugs and natural
plants for effective treatments of AD are
necessary.
It has indicated that dementia in patients
with AD relates to disorders in cholinergic
systems. Thus, they used scopolamin,
cholinergic receptor antagonist, to induce
a animal model of AD. WC or “Gui do”
has been used in Vietnamese traditional
medicines used for treatment of dementia

* Vietnam Military Medical University
Corresponding author: Can Van Mao ()
Date received: 10/06/2017

Date accepted: 10/08/2017

42


Journal of military pharmaco-medicine no7-2017
as well as diarrhea, heartburn, and
subcutaneous abscess and as a diuretic.
Our preliminary screening study revealed
that methanolic extracts derived from the
wood of W. cochinchinensis exhibit AChE
(acetylcholinesterase)
and
BChE
(butyrylcholinesterase) strong inhibited
activities which are main mechanism of
actions of drugs for treatments of AD [4].
To provide basics for using WC to treat
AD in humans, we conducted the present
study with the aims: To investigate effects
of WC on deficits in learning and memory
in experimental animals.
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.
* 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.

2. 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 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.
* Passive avoidance test:
Animals were required to perform
passive avoidance test, which includes
two phases:
- Training phase: was conducted at
60 minute after WC treatments on the first
day. Passive avoidance box (Ugo Basile)
was a chamber which contained
2 compartments: light one and dark one.
There was a wall with a door to separate
these compartments (fig.1). The mice
were placed in light compartment and
explore freely for 30 minutes. Then, the
door was raised to allow the mice to enter
the dark compartment. When the mice
entered the dark compartment, the door
was closed and an electronic foot shock
was delivered for 3 seconds. If mice didn’t
entered the dark compartment within
300 seconds, mice were captured and
placed inside the dark compartment and a
foot shock was delivered for 3 seconds.
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Journal of military pharmaco-medicine No7-2017

Figure 1: Apparatus of passive avoidance test.
- Test phase: were conducted on the
second day. Mice were placed in the light
compartment and the door was raised. In
the test phase, when mice entered the
dark compartment, no foot shock was
delivered. Mice’s behaviors were recorded
for 300 seconds by using a digital video
system. If animals didn’t enter the dark
compartment, entry latencies were
measured as 300 seconds and the trials
were over.
* Research indicators:
In the present study, we analyzed
some research indicators, follow as:

- Latencies from beginning of trials to
entrance into the dark compartment. Units
were measured as seconds (s). In the
present study, entry latencies were analyzed
in the training phase and the test phase.
- Average speeds, units were measures
as meter/second (m/s).
* Data analyses:
Entry latencies and average speeds
were analyzed by one-way analysis of
variance (ANOVA) followed by the Tukey’s
post-hoc 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.

RESULTS
1. Differences in entry latencies in the training phase.

Figure 2: Entry latencies in training phase.
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Journal of military pharmaco-medicine no7-2017
Figure 2 showed mean entry latencies of experimental groups in the training phase.
One way ANOVA indicated that there were no significant differences in entry latencies
between experimental groups in the training phase (p > 0.05).
2. Differences in entry latencies in the test phase.

Figure 3: Entry latencies in test phase.
Figure 3 showed differences in entry latencies between experimental groups in the
test phase. One way ANOVA indicated there was a significant main effects of
experimental group [F(4.49) = 4.949; p = 0.002]. Post hoc test indicated that mean
entry latency in the scopolamin group was significantly shorter than this in the control
group (Tukey test, p < 0.05). Contrarily, entry latencies in WC 150 mg/kg WC 200 mg/kg
treated groups were significantly longer than that in the scopolamin treated group
(p < 0.001).
3. Differences in average speeds.

Figure 4: Average speeds in experimental groups.
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Journal of military pharmaco-medicine No7-2017
Figure 4 showed differences in average
speeds between experimental groups.
One way ANOVA indicated that there was
a significant difference in average speeds
between experimental groups [F(4.49)
= 2.991, p = 0.029]. Post hoc test
indicated that mean average speed was
significantly higher than that in the control
group (Tukey test, p < 0.05). After WC
treatments, mean average speeds in the
WC 150 mg/kg group and WC 200 mg/kg
group were significantly lower than that in
the scopolamin group (p < 0.05).
DISCUSSION
Passive avoidance test is used widely
to evaluate learning and memory in
rodents [5] Thus, this behavioral test is
appropriate for the aim of our study. In the
present study, in the training phase, mice
had no experience with dangerous events
(foot shocks) in the dark compartment.
Thus, mice had tendencies to move to the
dark compartment because of their
nocturnal life. These reasons induced,
there was no significant difference in entry
latencies between experimental groups.
In the test phase, learning and memory
abilities of animals were expressed. When
animals had these better abilities, they

would recognize that when they moved to
the dark compartment, they were received
foot shocks. Thus, the longer entry latencies
they present, the better learning and
memory abilities of animals they have. In
the present study, scopolamin induced
deficits in learning and memory abilities of
animals expressed by differences in entry
latencies between the control group and
scopolamin group. Entry latencies of
scopolamin treated mice were shorter
46

than these of saline treated mice. These
results are consisted with previous studies
[6, 7]. Interestingly, in the present study,
we found that after WC treatments, there
were a significant increases in entry
latencies of animals treated by WC at
doses 150 mg/kg and 200 mg/kg, in
compared to that of animals treated by
scopolamin. These results indicated that
WC ameliorated scopolamin-induced
disorders in learning and memory in
experimental animals.
Furthermore, in the present study, we
also found that scopolamin induced
hyperactivities in experimental animals.
These results are consistent with effects
of scopolamin to inhibit acetylcholinesterase

enzyme [8] and also with hyperactivities
of patients with AD caused by disorders in
activities of the cholinergic system [9].
Interestingly, WC treatments also reduced
locomotordisorders of experimental animals.
The present’s results provided a
important base for us to conduct next
steps to apply WC for treatments of
diseases relating to deficits In learning
and memory, such as Alzheimer’s disease.
CONCLUSION
In the present study, we demonstrated
that WC reduced disorders in learning
and memory as well as locomotion in
experimental animals:
- In the test phase of passive
avoidance test, WC at doses 150 mg/kg
and 200 mg/kg increased entry latencies
in animals with scopolamin-induced
deficits in learning and memory.
- WC at the same doses decreased
average speeds of animals with scopolamininduced hyperactivities.


Journal of military pharmaco-medicine no7-2017
ACKNOWLEDGEMENTS
This work was supported by Grant
106-YS.05-2013.24 from Vietnam’s
National Foundation for Science and
Technology Development (NAFOSTED).

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