Effect of Acanthopanax gracilistylus W. W. Smith on olfactory bulbectomized –induced cognitive deficit in mice and its molecular mechanism.
Name of dispatched researcher（派遣研究者名）

Nguyen Thi Phuong

Instructor（指導教官，研究指導者）

Nguyen Minh Khoi

Afﬁliation of Instructor（所属）

Department of Pharmacology andBiochemistry,National Institute ofMedicinal Materials, Vietnam

Host Collaborator（受入研究者）

Kinzo Matsumoto

1. Abstract:
Aim: Acanthopanax gracilistylus W. W. Smith (AG) is commonly used as a tonic to strengthen
the tendons and bones as well as to improve learning and memory in traditional medicine.
However, no scientiﬁc evidence is available to support the cognitive improvement effect of AG.
This study aimed to clarify the effects of alcoholic extract of AG (AG-E) on cognitive deﬁcits in
olfactory bulbectomized (OBX) mice, one of the animal models of Alzheimer’s disease (AD).
Methods: OBX ddY mice were treated with AG-E at a daily dose of 180 mg and 360 mg/kg
via feeding needle for 1 week before and continuously 3 days after OBX. Administration of thereference drug tacrine (2.5 mg/kg/day, i.p.) to OBX mice was started 3 days after OBX. Cognitive
performances of the animals was analyzed using the novel object recognition test (ORT), modiﬁed
Y maze test, fear conditioning test (FC) and tail suspension test (TST) to elucidate non-spatial
short term memory, spatial working memory, long-term memory, and depression-like behavior,respectively.
Results: OBX caused impairment of non-spatial short term memory, spatial working memory,
depression-like behavior and long-term memory. OBX-induced memory deﬁcits were reversed by
AG-E and tacrine.
Discussion: These ﬁndings suggest that AG-E may have a potential to improve cognitive deﬁcits
and depression-like behavior in a model of OBX but further experiments will be required to test
this hypothesis.
2. Introduction
Dementia is one of the psychiatric disorders caused by aging and life style-related diseases.
The most common cause of dementia in the aged people is Alzheimer’s disease (AD). AD is a
progressive brain disorder and results in learning and memory deﬁcit, unusual behavior, and a－ 63 －

decline in thinking. The number of AD patients is increasing dramatically not only in advanced
countries but also in developing countries. Therefore, it is necessary to develop drugs without/mild side effects for prevention and therapy of AD. Medicinal herbs are the best candidate
sources for development of effective AD drugs.
Some species of Acanthopanax such as Acanthopanax senticosus (Rupr. et Maxim.) Harms(Eleutherococcus senticosus Maxim), Acanthopanax divaricatus, Acanthopanax obovatus,
Acanthopanax gracilistylus W. W. Smith (AG) have long been used in Vietnamese traditional
medicine expecting tonic action, improvement of mood and cognitive deﬁcits, immuno-modulation
and anti-tumor activity [1,2]. Acanthopanax gracilistylus W. W. Smith (AG) is also a speciesof Acanthopanax genus that located in Ha Giang province, Vietnam. It is a valuable medicinal
plant described in the Red Data Book of Vietnam for conversation. In traditional medicine, AG iscommonly used as a tonic to strengthen the tendons and bones as well as to improve learning andmemory. It is also effective for the cure of rheumatism and arthralgia [3]. Some lines of evidenceshow that AG has effects on inﬂammatory responses, inhibits human platelet aggregation, andexhibits antioxidant and anti-tumor activity [4, 5]. However, no scientiﬁc evidence is available tosupport the cognitive improvement effect of AG.
In our preliminary study where the in vitro acetylcholinesterase inhibitory activity (AChE)
and neuroprotective effect of ethanol extract from AG were screened, we found that ethanol
extract at dose of 1 mg/ml inhibited the AChE activity by 49.1%. Furthermore, this extract
exhibited potential neuroprotection activity against b-amyloid25-35-induced neuronal cell damage.
Based on these results, this study evaluated the effect of AG on dementia-related behavioral
performance using an animal model of OBX.
3. Materials and Methods
3.1. Plant materials and preparation of crude extract:
AG was collected from Ha Giang Province (Northern mountain of Vietnam). It was identified
by Dr. Pham Thanh Huyen, Department of Medicinal Plant Resources, National Institute ofMedicinal Materials (NIMM, Vietnam). The sterm-bark of AG was dried, grinded into semipowder, and hot extract with 70% ethanol. Ethanol extraction was condensed under reducedpressure. The concentrated extract was then dissolved in water for 24 hrs and rejected residue by
vacuum-ﬁlter. Evaporate water under reduced pressure and dry under vacuum to get the powder.
3.2. Animal
Male ddY mice (Japan SLC Inc., Shizuoka, Japan) were obtained at the age of 9 weeks old andhoused with a 12-h light/dark cycle (light on: 07:30-19:30) at 22 ± 1oC. Food and water wereavailable ad libitum. The animals were habituated to the laboratory animal room for at least 1
week before surgery. The behavioral experiments were performed during the light phase from9:00 to 17:00. The present studies were conducted in accordance with the Guiding Principles
for the Care and Use of Animals and were approved by the Institutional Animal Use and Care

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Committee in the University of Toyama. The animals were divided to the following 5 groups:
１．Group S: Sham (S).
２．Group OBX: OBX (O).
３．Group A: OBX + 0.18 g ethanol 70 % extract of Acanthopanax gracilistylus sterm-bark (A).
４．Group B: OBX + 0.36 g ethanol 70 % extract of Acanthopanax gracilistylus sterm-bark (B).
５．Group T: OBX + 2.5 mg/kg (i.p.) tacrine
3.3. Surgical operation:
OBX of mice was conducted as previously reported (6, 7). Brieﬂy, the mice were anesthetized withsodium pentobarbital then ﬁxed on stereotactic instrument (Narishige, Tokyo, Japan). The skull
covering the bulb was exposed by skin incision; 1 mm burr hole was drilled. The bilateral bulbs
were aspired through a syringe and the cavity of the bulbs was filled with hemostatic gelatin
sponge. After completing the behavioral tests, all the animals were sacriﬁced and the operated
lesion was veriﬁed visually. The data from animal with less than 70 % removal or with no intact
cortex were excluded from the analysis. Sham operation was performed in a similar way without
remove of the bulb. At the end of experiments, the olfactory bulbs of sham group mice were

Fig. 1

conﬁrmed to be intact.
3.4. Drug administration:
Drug treatments and experiment schedule were done according to the schematic in the ﬁgure 1.

Fig. 1 Schematic drawing of experimental schedule

3.5. Behavioral study
Behavioral experiments were usually conducted during a period from 09:00 to 17:00 according to
the previous reports from Dr. Matsumoto’s laboratory, Institute of Natural Medicine, Universityof Toyama.

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3.5.1. Object recognition test (ORT) (6,8).
ORT was conduct 2 weeks after operation days.
ORT consisted of a sample trial and a test trial. Before starting experiment, mice were
habituated with the experimental room for at least 30 – 60 min. In the sample trial, object 1 and
2 are exactly the same. In the test trial, object 1 or 2 was replaced by object 3 which had a shapediffering from object 1. SMART system® (PanLab, Barcelona, Spain) was used to analyze total
time the animals spent to explore each object in the sample and test trials. After each trial, thechamber was cleaned with 70 % ethanol.
3.5.2. Modiﬁed Y maze test (6).
Y-maze test was conducted 2 weeks after ORT. The test consisted of a sample trial and a test
trial. Before starting experiment, mice were habituated with the experimental room for at least
30 – 60 min. Three different cues were set above the middle of each arm. In the sample trial, eachmouse was individually placed in the maze with one of 3 arms closed, and allowed to explore thearms freely for 5 min. In the test trial, each mouse was again placed in the maze with all 3 arms
opened, and allowed to explore the arms freely for 5 min. SMART system® was used to analyze
the total time that mice explore each arm (only in the test trial). The chamber was clean with 70% ethanol and dried after each trial.
3.5.3. Fear conditioning test (FCT) (6,9).
FCT was conducted 1 week after the modified Y-maze test. This test consisted of a training
session (first day), contextual memory test (1 day after the training session) and an auditory
memory test (5 days after training session). Before starting experiment, mice were habituatedwith the experimental room for at least 30 – 60 min. The chamber for fear conditioning consistedof a transparent acrylic chamber and a stainless-steel grid ﬂoor equipped with an electric shock
generator/scrambler, SGS-002R, CS Controller CSS-001R,
and Cycle TimerCMTR (Muromachi Kikai, Co. Ltd., Tokyo, Japan). The apparatus was placedin a sound proof observation box (MC-050/CM, Muromachi Kikai, Co. Ltd., Tokyo, Japan)
through which the auditory tone (2.9 kHz, 80 dB) (SonalertR, Mallory Sonalert Products Inc.,
Indianapolis, IN, USA) was delivered to the animal. The chamber was cleaned with 70 % ethanol
after each session. Animal behavior in the chamber was video-recorded for later analysis.
In the training session, the animals were individually placed in the fear conditioning chamber
and allowed to explore freely for 3 min for habituation and then they received an acoustic tone
and foot shock (0.8 mA, 2s) at a 1-min interval. The contextual memory test and auditorydependent fear memory test were conducted one and 5 days after the training, respectively. In
the contextual fear memory test, the animal was placed in the same chamber to provide thecontextual stimuli and allowed to move freely for 6 min without tone or foot shock. One min after
placing the animal in the chamber, freezing behavior during the 5-min period was recorded. In
the auditory test, the animal was placed in a chamber different from that used in the training－ 66 －

session for 4 min and then received the tone for 2 min to record the freezing behavior. The animal
behavior was video-recorded and analyzed using the SMART system.
3.5.4. Tail suspension test (TST).
TST was used to assess the anti-depressant-like effect of AG extract. The test was conducted 1
week after FCT. The test was conducted as previously reported (Mizuki et al., 2014a,b). Before
starting experiment, the animals were habituated in the experimental room for at least 30 – 60
min. Sixty min after the administration of test drugs, each mouse was individually suspended
50cm above the ﬂoor by the tail with adhesive tape placed approximately 2 cm from the tip of thetail. Immobility was deﬁned as a state with movement speed of no more than 0.05cm2/s using the
SMART system and was recorded for 8min. The performance during the last 6-min period wasanalyzed.
3.7. Data analysis
The data are expressed as the mean ± SE. The data from the behavioral tests and neurochemical
experiments were analyzed by paired or unpaired Student’s t-test or one-way analysis of variance(ANOVA) follow by a post hoc test (Dunett’s method) as appropriate. Differences of P < 0.05
were considered as signiﬁcant. The analysis was conducted using SigmaStat® ver. 3.5 (SYSTAT
software Inc., Richmon, CA, USA)
4. Results
4.1. AG improves OBX-induced non- spatial memory deficit in the novel object recognition test
Using the novel object recognition test, we elucidated the effect of AG on the non-spatial cognitive
performance of OBX mice (Fig. 2). In the sample trial, the times spent exploring each identicalobject (O1 and O2) were not different in each group. However, in the test trial, the sham and
AG groups (A and B group) spent a signiﬁcantly longer time exploring the novel object (O3) than
exploring the familiar one (O1). In this case, tacrine (2.5 mg/kg)-treated OBX mice spent time
exploring the familiar object as long as exploring the novel one. This result differed from theresults reported by others including this laboratory.

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Fig. 2A) Sample trialB) Testtrial

35

40

O2
Time exploring object (seec)

Time exploring object (sec
c)

O1
30
25
20
15
10
5

O1

O３

*

35

**
30

**

25
20
15
10
5
0

0
S

O

A

B

S

T

O

GroupsA

B

T

GroupsFig. 2 Effects of AG on OBX-induced short-term memory deﬁcits in the object recognition test.
Each data column represents the mean ± S.E.M (n=8-9). *P < 0.05 and **P<0.01 vs. time spent
exploring a familiar object (pair t-test).

4.2. AG had the trend on improvement OBX-induced spatial working memory deficits in themodified Y maze test.
Using the modiﬁed Y maze test, we evaluate the effect of AG on spatial working memory on OBX
mice. The results showed on the ﬁgure 3.
Fig. 3

5 min

5 min
30 min60% Time in new arm
m

50

*
40
30
20
10
0
S

O

A

B

T

GroupsFig. 3 Effect of AG on OBX-induced spatial working memory deﬁcit in the modiﬁed Y maze test.
Each data column represents the mean ± S.E.M (n=9). * P < 0.05 vs. S group (t-test).

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In the Fig. 3, total time that the mice in the sham group used in the novel arm are longer thanthat of OBX group. The higher dose of AG (B group) and tacrine group have time in novel arm
are no longer than that of OBX group, though they has the trend on improvement OBX-induced
spatial working memory deﬁcits in the modiﬁed Y maze test. The result of tacrine group was not
the same as theory and others at this laboratory maybe because of quality of tacrine (tacrine vial
was used for long time with many times taken out of low temperature (4 ℃ ), so that in the ORT
tacrine had no any effect even the trend. In the Y-maze test, new tacrine was used but only 4days before the experiment so the result was still not good but it had the trend on increasing thetime spending in the novel arm.
4.3. BM ameliorated OBX induced long term memory deficit in the fear conditioning test
To determine the effects of AG on long-term memory deficits induced by OBX, we used fear
conditioning test (FC). One week after ﬁnishing the modiﬁed Y maze test, the fear conditioning
test was performed. Mice were fear-conditioned to the context and auditory stimuli by electricalfoot shocks as unconditioned stimuli, 1 day and 5 days after, freezing responses to the contextual
and auditory stimuli were recorded, respectively. The results showed in the ﬁgure 4.
As shown in Fig. 4, AG at a higher dose (group B) and tacrine increased freezing time in thecontextual memory test compare to that of the group O indicating that AG and tacrine improved
hippocampus-dependent memory formation. In the auditory memory test, only tacrine increased
the freezing
Fig. 4time compared to the group O.

(A) Contextual memory(B) Auditory memory

Drug treatment

Tone

Drug treatment

60 min.

1 min

60 min.

5 min

2 min

Measurement offreezing behavior

Measurement ofFreezing behavior

100

**

**

Freezing time/5 min (%)

Freezing time/5 min (%)

100
80

3 min

*
60
40
20
10

80

*

60
40
20
10

0

0
S

O

A

B

T

Groups

S

O

A

B

T

GroupsFig. 4 Effect of AG on OBX-induced long-term memory deﬁcit in the fear conditioning test. Eachdata column represents the mean ± S.E.M (n=9). (A) Contextual memory performance; (B)Auditory memory performance were analyzed as described in the text. * P < 0.05 and **P < 0.01
vs. O group (ANOVA followed by post hoc Student-Newman-Keuls test).

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4.4. Effect of AG on depression-like behavior of OBX mice in the TST.
To evaluate the effect of AG on depression-like behavior of OBX mice, we used tail suspension
test (TST). In this experiment, the group O showed a signiﬁcantly longer immobility time than
the group S (ANOVA followed by post hoc Student-Newman-Keuls test). Tacrine had no effect on
depression-likebehavior of OBX mice. In fact, the immobility time of the group T was the sameFig.
5
as that of the group O. The group B tended to reduce immobility time compare with the group O(Fig. 5).

% Immobility time (sec/6 min)

160

*

140
120
100
80
60
40
20
0
S

O

A

B

T

Fig 5 Effect of AG on OBX-induced depression-like behavior in the tail suspension test. Eachdata column represents the mean ± S.E.M (n=9). * P < 0.05 vs. S group (ANOVA followed bypost hoc Student-Newman-Keuls test).

5. Discussion
In this study, we investigated the effects of AG on OBX-induced cognitive deﬁcits and depressionlike behavior. We first elucidated the effect of AD on short-term non-spatial working memory
using the object recognition test. In the sample trial of ORT, no significant difference in total
time spent exploring two identical objects was observed in all groups, indicating that OBX does
not change vision and/ or an ability to visually recognize
objects. In the test trial, sham-operated mice spent more time exploring a novel object, whilevehicle-treated OBX mice failed to discriminate between familiar and novel object. AG treatment
significantly ameliorated OBX-induced deficit of novel object recognition. The administrationof AG and tacrine could attenuate OBX-induced deficits of long-term memory in the fear
conditioning test, especially in the contextual test. Though in the Y-maze test, AG only had trend
on exhibited impaired spatial working memory performance and in the TST, AG group (GroupB) had reduced immobility time induced by OBX. These ﬁndings suggest that AG-E may have apotential to improve cognitive deﬁcits and depression-like behavior in a model of OBX but further
experiments will be required to test this hypothesis.

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