Ameliorative eﬀects of Acanthopanax trifoliatus onlearning and memory deﬁcits in olfactory bulbectomized mice
Name of dispatched researcher（派遣研究者名）

SITHISARN PONGTIP

Instructor（指導教官，研究指導者）

SITHISARN PONGTIP

AfÀliation of Instructor（所属）

Department of Phamacognosy, Faculty of Pharmacy,
Mahidol University

Host Collaborator（受入研究者）

Matsumoto Kinzo

1. Introduction
Acanthopanax is a plant species that possess ginseng-like activities and is known as aginseng-like herb (Huang, 1999, Bucci, 2000). The roots and stem bark of Acanthopanax plants
have also been used as tonics and sedatives, as well as in the treatment of rheumatism and diabetes(Perry and Metzger, 1981a, Perry and Metzger, 1981b). A Thai traditional plant that belongs
to this species is Acanthopanax trifoliatus or phak paem which is used in the folk medicines ofSoutheast Asia as a drug with ginseng-like activity ((Perry and Metzger, 1981a, Perry and Metzger,
1981b, Chi, 1997, Petelot, 1954). Moreover, young leaves and the shoots are popularly consumed
with Northern Thai traditional cuisine as vegetables (Petelot, 1954). From its ethnomedical uses
as a ginseng-like herb, A. trifoliatus has potential for the treatment of degenerative diseases andcognitive dysfunction. Our previous works revealed that extracts from young leaves, root and root
bark exhibited strong in vitro antioxidant activity tested by 1,1-diphenyl-2-picrylhydrazyl (DPPH)
scavenging assay and inhibitory effect to lipid peroxidation of rat brain homogenate tested by
thiobarbituric reactive substances (TBARS) method (Sithisarn and Jarikasem, 2009). Moreover,leaf extract from A. trifoliatus signiÀcantly exhibited anti-inÁammatory and anti-anxiety effects
in animal models (Sithisarn et al., 2009, Sithisarn et al., 2010). Phytochemical investigation by
HPLC-MS suggested that leaf decoction extract contained phenolics and Áavonoids (Sithisarn etal., 2009). Since the extracts from A. trifoliatus showed antioxidant, anti-inÁammatory and antianxiety properties, it is interesting to investigate the efficacy of these extracts on learning andmemory impairments, the possible mechanisms of action in olfactory bulbectomized mice (OBXmice) which was previously reported as animal model of Alzheimer’s disease.
2.

Materials and Methods

2.1.

Materials

2.1.1. Animals
The overall study was conducted according to the experimental protocols described in Fig. 1− 87 −

and 2. Male ddY mice (Japan SLC Inc., Shizuoka, Japan) were obtained at the age of 9 weeks old
and housed with a 12-h light/dark cycle (lights on: 07:30–19:30) at 22 ± 1°C. 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 18:00. The present studies were conducted in accordance with the Guiding Principles (NIH
publication #85-23, revised in 1985) for the Care and Use of Animals and were approved by theInstitutional Animal Use and Care Committee in the University of Toyama.
2.1.2.

Plant extract preparation

The leaves of Acanthopanax trifoliatus were collected from Sunpathong district, Chiang
Mai province, Thailand in 2010. The plant samples were identiÀed by Mr.Winai Supatanakul,
botanist of Thailand Institute of ScientiÀc and Technological Research. The voucher specimens
were deposited at the same place (AT11001). The leaves were cleaned, dried in a hot air oven
(60°C) and powdered with electronic mill (20 mesh sieve) then were boiled with distilled water(1:10 w/v) for 3 h and Àltered. The Àltrate was taken to dryness by lyophilization to yield driedleaf decoction extracts (YD). The extract was previously standardized using high performanceliquid chromatography (HPLC) for quantitative analysis of phenolic and Áavonoid components
and in vitro antioxidant activity tests for analysis of biological activities (Sithisarn andJarikasem, 2009, Sithisarn et al., 2011).
2.2.

Methods

2.2.1.

Surgical operation

OBX of mice was conducted according to a previous report (Hozumi et al., 2003, Yamada etal., 2011). BrieÁy, the mice were anesthetized with sodium pentobarbital (60 mg/kg, i.p.), andfixed on stereotactic instruments (Narishige, Tokyo, Japan). The skull covering the bulbs was
exposed by skin incision, 1% lidocaine solution was used as local anesthetic then a 1mmburr hole
was drilled. The bilateral bulbs were aspired through a syringe and the cavity of the bulbs wasÀlled with hemostatic gelatin sponge. After completing behavioral studies, all the animals weresacriÀced and the operated lesion was veriÀed visually. The data from animals with less than
70% of removal or with no intact cortex was excluded from data analysis. Sham operation was
performed in a similar way without removal of the bulb. At the end of experiments, the olfactory
bulbs of sham group mice were conÀrmed to be intact.
2.2.2.

Drug administration

Tacrine (THA, 2.5 mg/kg, i.p.) was kindly supplied by Eisai Co. Ltd. (Tokyo, Japan) and imipramine(IM, 10 mg/kg, i.p.) were separately dissolved in 0.9% normal saline solution and intraperitoneallyadministered. Sham group of mice and OBX control group mice were orally administered water. Leaf
extract of A. trifoliatus was dissolved in water and given per orally at daily doses of 250–500 mg/kg. On
a behavioral testing day, either THA or YD was administered 1 h before the testing.

− 88 −

2.2.3.

Behavioral study0RGLÀHG<PD]HWHVW
Forty mice were randomly divided into four groups of ten mice. Each group of mice wasorally administrated with distilled water as a control group, THA (2.5 mg/kg, i.p.) was used asa standard drug, and A. trifoliatus leaf extract in 2 different doses of 250 and 500 mg/kg, p.o.,respectively. A modiÀed version of the Y-maze test has been reported by Yamada et al. (2011).
The apparatus used for the modified Y-maze test consists of black polypropylene walls with 3
arms each 40cm long, 12cm wide at the top, 3 cm wide at the bottom, 18cm high. This test is a
two-trial task with a sample phase trial and a test phase trial that are separated by an intertrial interval. In the sample phase trial, each mouse was individually placed in the maze with
one of the 3 arms closed. The animals were allowed to freely explore the other 2 arms for 5 min.
Thirty min after the sample phase trial, the animal was again placed in the maze with all 3
arms opened, and allowed to explore the arms freely. The closed arm that is opened in the test
phase trial is deÀned as the new arm. The animal behavior was video-recorded for later analysis.
Percent time spent in the new arm and numbers of total arm entries were analyzed by Smart®
system.
1RYHOREMHFWUHFRJQLWLRQWHVW 257
This test is based on the tendency of mice to discriminate a familiar from a new object. On
the day before the test, mice were individually habituated to an open-Àeld box (35x 35x50 cm)
for 10 min, and performance of the animals was analyzed automatically by the Smart® system.
The total distance exploring the arena was used to determine the locomotor activity. The ORT
consists of a sample phase trial and a test phase trial. During the sample phase trial, twoobjects of the same material were placed in a symmetric position in the center of the chamber
for 10 min. thirty minute after the sample phase trials, one of the objects was replaced by anovel object, and exploratory behavior was again analyzed for 5 min. After each session, objects
were thoroughly cleaned with 70% ethanol to prevent odor recognition. Exploration of an object
was defined as rearing on the object or sniffing it at a distance of less than 2 cm. Successfulrecognition of a previously explored object was reÁected by preferential exploration of the novel
object. Discrimination of spatial novelty was assessed by comparing the difference between timeof exploration of the novel and familiar.
7DLOVXVSHQVLRQWHVWTail suspension test (TST) is a widely used model for assessing antidepressant effect.
Applied from Shioda et al. (2010), mice were subjected to the short-term inescapable stress of
being suspended by the tail that will develop an immobile posture. Using another group of miceas indicated in protocol#2 (Fig 2), 3 mice were separately suspended 50 cm above the Áoor in achamber by adhesive tape placed approximately 2 cm from the tip of the tail. Immobility time
was recorded during a 8 min period using Smart® system.

− 89 −

2.2.4.

Quantitative real-time polymerase chain reaction (PCR)

To analyze changes in expression levels of choline acetyltransferase (ChAT) and muscarinic
M1 receptor mRNA in the brain, the animals were killed by decapitation after completing the
behavioral studies. The brain was removed immediately and the hippocampus were dissectedout and kept at − 80°C until use. Quantitative PCR was conducted as previously described (Zhaoet al, 2011). Briefly, total RNA was extracted from the hippocampus using Sepazol® (Nacalai
Tesque, Kyoto) according to the manufacturer’s instructions. First-strand cDNA synthesis was
conducted using oligo (dT) primers and M-MLV Reverse Transcriptase® (Invitrogen, Rockville,
MD, USA) in a total volume of 20 µl. The reaction was performed at 25°C for 10 min and heatedat 37°C for 60 min and 98°C for 5 min before cooling to 4°C. DNA corresponding to the RNA wasused as a template for real-time PCR. Quantitative realtime PCR was carried out using FastSYBR Green Master Mix (Applied BioSystems, Foster City, CA, USA) in a StepOne Real-time
PCR System (Applied BioSystems). Melting curve analysis of each gene was performed every
time after ampliÀcation was completed. Standard curves of the log concentration of each gene vs.
cycle threshold were plotted to prove negative linear correlations.
2.2.5.

Neurochemical study

2.2.5.1. ([YLYRPHDVXUHPHQWRIFKROLQHVWHUDVHDFWLYLW\LQWKHEUDLQ
After completing the behavioral experiments, all mice were decapitated and the frontal
cortices were dissected out and kept at í80°C until use. Determination of cholinesterase
activity was performed on the basis of the colorimetric method as previously described (Ellman
et al., 1961). Briefly, the frozen cortex was weighed and homogenized in 10-times volume of
0.1 M phosphate buffer (pH 7.4) containing 1% Triton-X-100. After centrifugation at 15,000 ×
g at 4°C for 20 min, the clear supernatants were collected and served as the enzyme source.
Cholinesterase activity was determined in 10 µl aliquots of homogenates (run as triplicates) in
96-well Áat-bottom microplates. The reaction was started by adding 8 µl of 10 mM 5,5́-dithiobis2-nitrobenzoic acid (DTNB), 16 µl of 7.5 mM acetylthiocholine (ATCI), and 201 µl of 0.1 M
sodium phosphate buffer (pH 8.0). The spectrophotometric absorption at 405 nm during a 3
min incubation period at 25°C was quantitatively measured using a microplate reader (Sunrise
Classic; TECAN Japan, Kawasaki) and expressed as nmol ACh hydrolyzed/min/mg tissue.
,QYLWURPHDVXUHPHQWRIFKROLQHVWHUDVHDFWLYLW\LQWKHEUDLQ
The assay for measuring AChE activity was modiÀed from the assay described by Ellman et al.
(1961) and Ingkaninan et al. (2003). Cholinesterase activity was determined in 10µl aliquots ofhomogenates in 96-well Áat-bottom microplates using frontal cortex supernatants as the enzyme
source. As same as the ex vivo experiment, the reaction was started by adding 8 µl of 10 mM
5,5́-dithiobis-2-nitrobenzoic acid (DTNB), 16 µl of 7.5 mM acetylthiocholine (ATCI), 10 µl of YD or
THA, and 146 µl of 0.1 M sodium phosphate buffer (pH 8.0). The spectrophotometric absorption
at 405 nm during a 3 min incubation period at 25°C was quantitatively measured using a− 90 −

microplate reader (Sunrise Classic; TECAN Japan, Kawasaki). The velocities of the reactions
were measured. Enzyme activity was calculated as a percentage of the velocities compared tothat of the assay using buffer without any inhibitor. Inhibitory activity was calculated from 100
subtracted by the percentage of enzyme activity. Every experiment was done in triplicate.
2.3.

Data analysis
The results were expressed as the mean ± SEM. The data obtained from the behavioral tests

and neurochemical experiments were analyzed by pair and unpaired student’s t-test or oneway analysis of variance (ANOVA) followed by a post hoc multiple comparison test (Dunnett’s
method) as appropriate. Differences of P < 0.05 were considered as signiÀcant. The analysis was
conducted using SigmaStat® ver 3.5 (SYSTAT Software Inc., Richmond, CA, USA).

surgery

10 days

drug administration

7 days14 daysmodified Y-maze test

14 daysORT- decapitation- gene expression- ex vivo AChE activity- in vitro AChE activity

Fig. 1 Schematic drawing of experimental schedule protocol#1
After one week of acclimatization, the ddY mice were randomly divided into 4 groups of 10 mice. All
mice (except mice in sham group) were subjected for OBX surgery. Ten days after the surgery, the drug
administration was started. ModiÀed Y-maze and object recognition tests were performed 1 and 3 weeks
after starting drug administration, respectively. Quantitative real-time polymerase chain reaction andneurochemical studies were done after decapitation of all mice.
surgery

Drug administration







3 days7 days7 days

tail suspension test

decapitation

Fig. 2 Schematic drawing of experimental schedule protocol#2
After one week of acclimatization, the ddY mice were randomly divided into 4 groups of 6 mice. All mice(except mice in sham group) were subjected for OBX surgery. Three days after the surgery, the drug
administration was started. Tail suspension test was performed 7 days after starting drug administration.

− 91 −

3. Results 0RGLÀHG<PD]HWHVW
As shown in Fig. 3, percentage time spent of the vehicle-treated sham mice visiting the new arm(49.30 ± 2.19%) was signiÀcantly higher than the chance level of 36.77 ± 3.05% indicating a preference
for new arms over the familiar arms. OBX mice that were administered THA (2.5 mg/kg, i.p) or YD (250
and 500 mg/kg. p.o.) 1 hour before the experiment spent signiÀcantly longer time exploring new arm
than the vehicle-treated OBX group (50.26 ± 2.75, 46.97 ± 2.18 and 54.13 ± 3.52%, respectively).

100














%Time in new arm

sham


OBX

80

*

60

*

*

500

2.5
THA

40
20
0
Vehicle Vehicle 250
YD*(mg/kg per day)Fig. 3 Evaluation of the modiÀed Y-maze test of OBX-induced spatial working memory deÀcit mice usingreference drug, tacrine or A. trifoliatus leaf extract (YD). Surgical operation-naïve mice (OBX) were orally
administered with distilled water 60 min before sample phase trial while tacrine (2.5 mg/kg) and YD (250
and 500 mg/kg) were administered by intra-peritoneal injection and oral administration, respectively (n=10).
Each data column represents the mean ± S.E.M. *P<0.05 compared with vehicle-treated OBX group (one-way
ANOVA, Dunnett’s method).

3.2.

Novel object recognition test (ORT)As shown in Fig. 4A, there was no signiÀcantly difference in locomotor activity determined as

total distance between vehicle-treated OBX and sham mice groups (6383.07 ± 566.87 and 5834.80± 289.14 cm, respectively). Moreover, daily administration of THA (2.5 mg/kg, i.p.) or YD (250
and 500 mg/kg, p.o.) for 1 week before the experiments showed no effect on locomotor activity (total
distance of 5514.95 ± 299.20, 5688.09 ± 284.13 and 5791.06 ± 196.18 cm, respectively).
In sample phase, no mice groups showed significantly differences in time spent exploringeach identical object. There also no signiÀcant difference in total time spent exploring two objects
between sham and OBX groups (Fig. 4B). In test phase, the sham group spent a significantly
longer time exploring the new object than exploring the familiar one (P<0.05) while the vehicletreated OBX group showed the deficit of the novel object recognition performance as shownin Fig. 4C. THA (2.5 mg/kg, i.p.) and YD (250 and 500 mg/kg, p.o.)- treated mice groups spentsigniÀcantly longer time exploring the new object than exploring the familiar object (P<0.01).

− 92 −

3.3.

Tail suspension test (TST)The duration of immobility in TST was measured 10 days after OBX. The duration of

immobility in vehicle-treated OBX mice signiÀcantly increased (25.18 ± 1.72 s) compared withthat observed in sham-operated group (11.47 ± 2.13 s) (Fig. 5). One week of daily administrationof imipramine (10 mg/kg, i.p.) or YD (500 mg/kg, p.o.) significantly reduced the duration of
immobility (6.48 ± 3.85 and 3.86 ± 2.64 s, respectively) while mice in THA administered group (2.5mg/kg, i.p.) showed no reducing effect to the immobility (17.44 ± 3.75 s).

Ϳ>ŽĐŽŵŽƚŽƌĂĐƚŝǀŝƚǇ
Total distance (cm)

8000

sham

OBX

7000
6000
5000
4000
3000
2000
1000
0

Vehicle Vehicle 250ͿKďũĞĐƚƌĞĐŽŐŶŝƚŝŽŶƚĞƐƚSample phase

100

Test phase
Object O2
OBX

80

60
40
20
0

THA

100

% Time spent% Time spent

80

Object O1
sham

2.5

500
YD*

Vehicle Vehicle 250

500
YD*

2.5
THA

new object
sham*familiar object
OBX

**

**

**

60
40
20
0

Vehicle Vehicle 250

500
YD*

2.5
THA
(mg/kg per day)Fig. 4 Effects of tacrine and YD on object recognition deÀcits in OBX mice in sample phase (B) and test
phase (C) while data of locomotor activities is shown in (A). Each datum represents the mean ± S.E.M. (n=10).
Each data column represents the mean ± S.E.M. *P<0.05 and **P<0.01 vs. time spent exploring a familiar
object (paired t-test).

− 93 −

50%Resting time

sham

OBX

40
30
20

*

*

*

10

0
Vehicle Vehicle 500

2.5
THA

YD*

10
IMI

(mg/kg per day)Fig. 5 Effects of administration with tacrine (2.5 mg/kg, i.p.), imipramine (10 mg/kg, i.p.) and YD (500 mg/
kg, p.o.) on OBX-induced depressive behavior in the TST. Each data column represents the mean ± S.E.M.
*P<0.05 vs. % resting time in vehicle-treated OBX mice (one-way ANOVA, Dunnett’s method).

3.4.

Quantitative real-time polymerase chain reaction (PCR)
It was found that the expression levels of M1 receptor and ChAT mRNAs in the hippocampus

of vehicle-treated OBX mice was significant lower compared with sham-operated group (0.23± 0.04 and 0.41 ±0.03, respectively for M1 receptor mRNA and 0.12 ± 0.02 and 0.56 ±0.28,
respectively for ChAT mRNA). However, the expression levels of M1 receptor and ChAT mRNAs
in hippocampus were up-regulated in OBX mice treated with THA (2.5 mg/kg, i.p.) or YD (250
and 500 mg/kg, p.o.). The expression levels of M1 receptor mRNA in hippocampus in THA andYD (250 and 500 mg/kg, p.o.) were 0.35 ± 0.02, 0.52 ± 0.07 and 1.78 ± 0.64, respectively (Fig. 6A)
while the expression levels of ChAT mRNA in hippocampus in THA and YD (250 and 500 mg/kg,

3.0

sham

OBX

*

2.5

*

2.0
1.5
1.0

*

*

0.5
0
Vehicle Vehicle 250

500
YD*

2.5

M1 receptor mRNA/ β-actin mRNA ratio

ChAT mRNA/ β-actin mRNA ratiop.o.) were 0.59 ± 0.11, 1.15 ± 0.58 and 2.06 ± 0.39, respectively (Fig. 6B).

THA

3.0

sham

OBX

*

2.5
2.0
1.5
1.0
0.5

*

*

*

0
Vehicle Vehicle 250

500
YD*

2.5
THA
(mg/kg per day)Fig. 6 Effects of tacrine and YD on M1 receptor (A) and ChAT (B) mRNA expression levels in the
hippocampus of OBX mice. Each data column represents the mean ± S.E.M. *P<0.05 vs. mRNA expression
levels in vehicle-treated OBX mice (one-way ANOVA, Dunnett’s method).

− 94 −

3.5.

Ex vivo measurement of cholinesterase activity in the brain
The activity of acetylcholinesterase (AChE) in the cerebral cortex was measured in micetreated with YD (500 mg/kg, p.o.) or THA (2.5 mg/kg, i.p.). As shown in Fig. 7, it was foundthat the activity of cortical AChE in YD- and THA-treated groups were significantly reduced
compared with the activity measured in vehicle-treated mice (2.27 ± 0.08 and 2.21 ± 0.21 nmol/
min/mg tissue, respectively).
3.6.

In vitro measurement of cholinesterase activity in the brain
YD (100 µg/ml) and THA (0.05 – 5 µg/ml) were tested for AChE inhibitory activity using

Ellman’s colorimetric method in 96-welled microplate. At concentration of 100 µg/ml, YD showed
low inhibitory activity (5.01 ± 0.37%) while THA showed high inhibitory effect with IC50 less than
50 ng/ml. Inhibitory effects of THA are shown in Table 1.

Ex vivo AChE activity(nmol/min/mg tissue)

5

sham

OBX

4
3

*

*

2
1
0
VehicleVehicle500
YD*

2.5 (mg/kg per day)
THA

Fig. 7 Effects of YD and tacrine on ex vivo acetylcholinesterase activity in OBX mice. *P<0.05 vs.
acetylcholinesterase activity in vehicle-treated OBX mice (one-way ANOVA, Dunnett’s method).

Table 1 In vitro inhibitory effects of THA on acetylcholinesterase activity in the mouse brain homogenate.

− 95 −

Discussion
The effects of Acanthopanax trifoliatus leaf extract (YD) on OBX induced short term
memory deficit were investigated. The results showed that YD treatment could ameliorate
the impairments via restoring the function of the central cholinergic system down-regulated
by OBX. In modified Y-maze test, it was found that OBX operation impaired spatial working
memory performance in mice. Treatments of THA (2.5 mg/kg, i.p.) and YD (250 and 500 mg/
kg, p.o.) ameliorated spatial working memory performance indicated by the increasing of total
time spent exploring new arm. The ameliorative effect of YD on memory deficit was further
studied using novel object recognition test. In sample phase trial, no significant difference intotal time spent exploring two identical objects was observed between sham and OBX groups,
indicating no differences in ability to recognize objects between animals. In test phase, theresults showed that mice in sham group spent more time exploring new object, while vehicle
treated OBX mice showed no total time difference between familiar and new object indicating theimpairment of non-spatial object recognition memory. Administration of YD (250 and 500 mg/
kg, p.o.) and THA (2.5 mg/kg, i.p.) could signiÀcantly ameliorate OBX induced object recognitiondeÀcit suggesting the ability of YD in the therapy of cognitive deÀcits in human in the future.
From the quantitative real-time PCR, the results revealed that mice in vehicle-treated OBX
group exhibited down-regulated expression levels of both ChAT and M1 muscarinic mRNAs in
hippocampus, a brain region responsible for spatial learning and memory performance (Zhaoet al, 2011) indicating the deÀcit of cognitive performance and pathological changes in central
cholinergic systems. Our Àndings agree with previous reports (Hozumi et al, 2003 and Yamada etal, 2011).
Administration of YD (250 and 500 mg/kg, p.o.) and THA (2.5 mg/kg, i.p.) showed upregulate effects to the expression levels of ChAT and muscarinic M1 receptor in OBX micesuggests that YD can protect cognitive function against degeneration of central cholinergic
system caused by OBX via the reversal of hippocampal expression of ChAT and muscarinic
M1 receptor mechanisms. Moreover, the similar action between YD and THA was found in
ex vivo acetylcholinesterase activity test. Indeed, the present results demonstrated that YD
could significantly reduce the activity of AChE as well as THA. However, in vitro test using
hippocampal tissue as an enzyme source revealed that only THA showed strong inhibitory
effect to AChE while YD exhibited low effect. Taken together, the present results suggestthat there could be other mechanisms or pathway affecting the effect of YD to hippocampal
acetylcholinesterase activity. Cholinergic system was reported to implicate in depressive
behavior (Shioda et al, 2003). There was a significant reduction of hippocampal neurogenesis
in OBX mice (Harkin et al, 2003, Jaako-Movits and Zharkovsky, 2005). YD has significantly
antidepressant activity in OBX mice. However, the mechanisms of action are still unknown. The
possible mechanisms could be the effects to AChE and ChAT which are presynaptic markers
of cholinergic neurons or enhancement to neurogenesis in OBX mice. From all the results,
YD showed improving effects on spatial working memory and non-spatial short term memory− 96 −

in OBX mice which could mediate by the up regulation of the expression levels of ChAT andmuscarinic M1 receptor mRNA and the inhibitory effects to acetylcholine esterase. Moreover,YD also showed in vivo antidepressant effect in OBX mice. From these results, it is likely that
YD is beneficial to treatment of depression and improvement cognitive impairment related toAlzheimer’s disease and that the effects of YD may be mediated by improving dysfunction ofcentral cholinergic systems.
Acknowledgements
Author would like to thank Dr. Piyanuch Rojsanga, Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Mahidol University for her kind cooperation. Author would like to thankDr. Siripen Jarikasem, Pharmaceutical and Natural Products Department, Thailand Instituteof ScientiÀc and Technological Research for providing Acanthopanax trifoliatus leaf extract andgiving some advices and also all laboratory members in Department of Medicinal Pharmacology,
Institute of Natural Medicine, University of Toyama. This work was supported by GRANTIN-AID for general research II program, Institute of Natural Medicine, University of Toyama,
Japan.

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