Journal of Ethnopharmacology 132 (2010) 7074

Contents lists available at ScienceDirect

Journal of Ethnopharmacology
j our nal homepage: www. el sevi er . com/ l ocat e/ j et hphar m
Anti-amnesic effect of ChongMyungTang on scopolamine-induced memory
impairments in mice
Mi-Ra Lee
a
, Beom-Sik Yun
a
, Sun-Young Park
b
, Sun-Young Ly
b
, Sung-Nam Kim
c
, Byung-Hee Han
c
,
Chang-Keun Sung
a,
a
Department of Food Science and Technology, Chungnam National University, Daejon 305-764, Republic of Korea
b
Department of Food and Nutrition, Chungnam National University, Daejon 305-764, Republic of Korea
c
Department of Chemistry, Chungnam National University, Daejon 305-764, Republic of Korea
a r t i c l e i n f o
Article history:
Received 17 April 2010
Received in revised form 9 July 2010
Accepted 20 July 2010
Available online 29 July 2010
Keywords:
ChongMyungTang
Scopolamine
Acetylcholinesterase
Choline acetyltransferase
a b s t r a c t
Aim of the study: ChongMyungTang (CMT) consisted of Acorus gramineus Soland, Polygala tenuifolia
Willdenow, and Poria cocos Wolf is one of the traditional Korean herbal medicines used for the therapy
of learning and memory improvement. The present study was investigated the effect of CMT on learning
and memory functions in SCOP-induced memory decits mice.
Materials and methods: The cognitive-enhancing effect of CMT on amnesic mice induced by SCOP was
investigated by assessing the passive avoidance test and the Morris water maze test. In order to conrm
the underlying mechanisms of memory enhancing effects of CMT, activities of AChE, choline acetyltrans-
ferase (ChAT), and antioxidant enzymes were measured.
Results: Administration of CMT signicantly restored memory impairments induced by SCOP in the pas-
sive avoidance test and also reduced escape latency during trial sessions in the Morris water maze test.
The increased AChE activity produced by SCOP was signicantly inhibited by CMT. CMT signicantly
enhanced ChAT activity. Moreover, treatment with CMT to the amnesic mice induced by SCOP consider-
ably decreased malondialdehyde levels and restored activities of superoxide dismutase and catalase to
the control values.
Conclusions: These results suggest that CMT may be useful for the cognitive improvement via regulation
of cholinergic marker enzyme activities and the antioxidant defense system.
2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Alzheimers disease (AD) is the most common neurodegener-
ative disease, leading to dementia characterized by a progressive
decline of cognitive function due to degeneration of the cholin-
ergic nervous system (Cummings and Kaufer, 1996). Cholinergic
decit is a major neuropathological feature that is associated with
memory loss, and closely correlated with the severity of cogni-
tive dysfunction in AD (Hasselmo, 2006). Scopolamine (SCOP), a
blocker of muscarinic acetylcholine (ACh) receptor, induces cogni-
tive decit in various animals and is widely used to evaluate the
antidementia activity of herbal agent on learning ability (Betty et
al., 1986). Cholinergic transmission is terminated mainly by ACh
hydrolysis through the enzyme acetylcholinesterase (AChE) which
is responsible for degradation of ACh to acetate and choline in
the synaptic cleft (Ballard et al., 2005). Inhibition of AChE serves

Corresponding author. Tel.: +82 42 821 6722; fax: +82 42 822 2287.
E-mail address: kchsung@cnu.ac.kr (C.-K. Sung).
as a strategy for the treatment of AD, senile dementia, ataxia and
Parkinsons disease. The drugs approved for the AD therapy act by
counteracting the acetylcholine decit, that is, they try to enhance
the acetylcholine level in the brain (Heinrich and Teoh, 2004).
Although the mechanisms on the anti-amnesic effects of most
herbal extracts andconstituents are not yet fullyunderstood, one or
more of the following components were considered to activate the
central ACh function through inhibition of AChE and activation of
ACh synthesis (Zhang, 2004). Recent studies have pointed out that
AD is associated with inammatory processes. Reactive oxidative
species (ROS) are able to damage cellular constituents and act as
secondary messenger ininammation. The use of antioxidants may
be useful in the treatment of AD (Gilgun-Sherki et al., 2002)
ChongMyungTang(CMT), one of the traditional Koreanherbal
medicines, has been widely used as a remedy for amnesia to
ameliorate learning and memory. It is consisted of 3 herbs, con-
taining Acorus gramineus Soland, Polygala tenuifolia Willdenow,
and Poria cocos Wolf. The extracts of each herbs have been used
as an oriental traditional medicine and reported their pharma-
cological effects as follows. Recent several studies have shown
0378-8741/$ see front matter 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2010.07.041
M.-R. Lee et al. / Journal of Ethnopharmacology 132 (2010) 7074 71
that Acorus gramineus Soland has been used clinically as orien-
tal traditional medicines against stroke, AD or vascular dementia.
Moreover, Acorus gramineus Soland and its major component,
asarone, have a neuroprotective effect against exocitotoxic neural
death and cognitive-enhancing effects on SCOP-induced memory
decit (Hsieh et al., 2000; Cho et al., 2002). On the other hand,
Polygala tenuifolia Willdenow has been used as a treatment for ill-
ness of the brain and for promoting intelligence. In combination
with other herbal drugs, Polygala tenuifolia Willdenow is one of
the major components for treatment of cognitive disorders such
as cerebrovascular diseases, aging, senile dementia including AD
(Chen et al., 2004). Poria cocos Wolf also has long been prescribed
with other herbal medicines for the treatment of cognitive dis-
orders (Shiada et al., 2004). Furthermore, Kim et al. (1999) have
demonstrated that CMT has an inhibitory activity of tumor necrosis
factor- (TNF-) production by mouse astrocyte stimulated with
lipopolysaccharide (LPS) plus substance P. Although each herb of
CMT is pharmacologically effective, the mechanism of therapeu-
tic action against amnesia induced by SCOP has not been well
dened.
The present study evaluated the effect of CMT on learning and
memory functions in SCOP-induced memory decits mice through
in performance on the passive avoidance and Morris water maze
test. Moreover, in order to investigate whether CMT possesses a
central cholinergic activity, we examined activities of cholinergic
enzymes suchas AChEandcholineacetyltransferase(ChAT) inbrain
tissue. Altered brain oxidative injury was investigated by determi-
nation of activities of antioxidant enzymes such as catalase (CAT),
superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and
malondialdehyde (MDA) level.
2. Materials and methods
2.1. Preparation of CMT
The ingredients of CMT were Acorus gramineus Soland (China,
120g), Polygala tenuifolia Willdenow (China, 120g) and Poria cocos
Wolf (North Korea, 120g) and purchased from Geumsan mar-
ketplace. All components were identied by Professor Ki Hwan
Bae from the college of Pharmacy, Chungnam National Univer-
sity where voucher specimens are deposited. Extract of CMT were
prepared by decocting the powdered herbs with distilled water
(100g/L) for 2h. After ltration, the ltrate was concentrated with
a rotary evaporator followed by lyophilized. The yield of the CMT
extract was 21.21% (w/w).
2.2. Animals
Male ICR mice, weighing 2530g, 8 weeks of age were obtained
fromDae Han Bio-Link. Co., Ltd. (Eum-Seong, Korea). Animals were
housed 5 per cage, allowed access to water and food ad libitum
and maintained in a constant temperature 222

C and humidity
5055% and 12h of light/dark cycle (light on at 08:00). All exper-
imental procedures were carried out in accordance with the NIH
Guide for Care (NIHPublication No. 85-23, 1985, revised 1996) and
were approvedbythe Institutional Animal Care andUse Committee
at Chungnam National University.
2.3. Drug administration
Animals were randomly divided into the following groups with
10 mice in each group: Control (0.9% saline), SCOP (scopolamine,
2mg/kg, i.p.), THA(tacrine, 10mg/kg, p.o.) as apositivecontrol, CMT
(50, 100, 200, 300mg/kg, p.o.). Memory impairment was induced
by SCOP (2mg/kg, i.p.) 30min after the administration of each drug
or vehicle solution (0.9% saline). Control animals were adminis-
tratedvehicle solutiononly. SCOP, THA, CMT were dissolvedin0.9%
physiological saline. All drugs were prepared fresh daily.
2.4. Passive avoidance test
Passive avoidance test was carried out using a passive avoid-
ance apparatus (Jungdo Bio & Plant Co. Ltd, Seoul, Korea) in order
to nd the appropriate dose within several doses for in vivo
experiment. This apparatus is comprised of 2 equal compartments
(202020cm) separated by a guillotine door (55cm). For the
acquisition trial, mice were initially placed in the illuminated com-
partment andthedoor betweenthetwocompartments was opened
20s later. The time (step-through latency) taken for a mouse to
enter the dark compartment was measured. Upon entering the
dark compartment, the door was closed and anelectrical foot shock
(0.5mA for 5s) was delivered through the stainless steel rods. On
the second day, the same procedure was followed. Mice were again
placed in the illuminated compartment to test retention. The step-
through latency was recorded and used as a measure of retention
(Wormet al., 1989). If a mouse did not enter the dark compartment
within 300s, it was assumed that the mouse had remembered the
single acquisition trial experience.
2.5. Morris water maze test
The Morris water maze test was carried out in a circular pool
(90cm in diameter and 50cm in height) with a featureless inner
surface. The circular pool was lled to a height of 30cmwith water
containing white paints (231

C). A white plastic platform (9cm

diameter and29cmheight) was submergedinoneof thepool quad-
rants. It was located in the same position on every trial. The rst
day of the experiment was dedicated to swimming training for
60s in the absence of the platform. During the four consecutive
days, the mice were given two trial sessions per day with an inter-
trial interval 20min. Once the mouse found the platform, it was
permitted to remain on it for 10s. If the mouse failed to nd the
platform within 120s, it was placed on the platform for 10s. In
the fth day, mice were individually subjected to a probe trial ses-
sion by removing the platform and mice were allowed to swim for
120s to search for it and the number of platformarea crossing was
determined.
2.6. Biochemical analysis
Following the Morris water maze test, mice were killed. Whole
brain was carefully removed from the skull, and stored at 80

C
until use. The brains were homogenized in a glass Teon homoge-
nizer containing 10 volumes of homogenization buffer (12.5mM
sodium phosphate buffer pH 7.0, 400mM NaCl), and then cen-
trifuged at 1000g for 10min at 4

C. The supernatant so obtained

was used as a source of enzyme for the assay. AChE activity
was determined according to Ellman et al. (1961). Measurement
of MDA contents was performed using the thiobarbituric acid
reactive substances (TBARS) assay (Uchiyama and Mihara, 1978).
ChAT, CAT, SOD, and GSH-Px activities were determined using
the commercial kits (Jiancheng Institute of Biotechnology, Nan-
jing, China) utilizingthefollowingprinciples. Namely, ChATactivity
was determined by the chromogenic reaction with the ACh syn-
thesized. CAT activity was measured by an assay of hydrogen
peroxide based on formation of its stable complex with ammo-
nium molybdated. SOD activity was determined according to
xanthine oxidase method. GSH-Px activity was detected by mea-
suring the reduction of glutathione (GSH) per min on the base of its
catalysis.
72 M.-R. Lee et al. / Journal of Ethnopharmacology 132 (2010) 7074
Fig. 1. Effect of a single administration of CMT on SCOP-induced memory decits
in the passive avoidance task. Acquisition trials were carried out 30min after SCOP
treatment. Retentiontrials were carriedout for 5min24hafter the acquisitiontrials.
Data are expressedas means S.E.M. (n=10). *p<0.05, **p<0.01 versus SCOP group,
##
p<0.01 versus control group.
2.7. Statistical analysis
All the results were expressed as means S.E.M. Data were ana-
lyzed by one-way ANOVA followed by Duncans test for multiple
comparisons. Especially, the escape latency in the Morris water
maze task was analyzed using the two-way ANOVA with repeated
measures. Adifferenceof p<0.05was regardedas beingstatistically
signicant.
3. Results
3.1. Passive avoidance test
To assess whether a single administration of CMT has enhanc-
ing effects against learning and memory impairment, the passive
avoidance test was carried out using SCOP-induced amnesic mice.
As shown in Fig. 1, the step-through latency of SCOP-treated mice
was signicantly shorter than that of the vehicle-treated control
mice (p<0.01). A single oral administration of CMT between the
dose of 50 and300mg/kg reverseddose-dependently of the shorter
step-through latency induced by SCOP. Furthermore, the step-
through latency time of mice treated with 200 and 300mg/kg CMT
was signicantly increased compared to SCOP-treated mice. Addi-
tionally, the dose of 200mg/kg CMT was not only more effective,
but also had a higher potency than that of THA (86.6% versus 43.7%
of control, respectively). Therefore, we chose this dose inthe Morris
water maze test.
3.2. Morris water maze test
The effect of CMT (200mg/kg) on spatial learning was eval-
uated in the Morris water maze test. As shown in Fig. 2A, the
escape latency of the SCOP group was signicantly longer by means
of memory impairment than that of the control group during all
trial sessions [F(1, 79) =65.117, p<0.001]. CMT administration sig-
nicantly recovered the escape latency prolonged by SCOP [F(1,
79) =48.867, p<0.001], as did THA [F(1, 79) =66.171, p<0.001]. On
day 5, the probe trial session was performed by removing the plat-
Fig. 2. Effect of CMT onescape latency inthe training trial sessions (A) andthe probe
trial session (B), number of crossing platform area (C) for the SCOP-induced mem-
ory decits mice. Data are expressed as means S.E.M. (n=10). *p<0.05, **p<0.01,
***p<0.001versus SCOP-treatedgroup.
#
p<0.05,
##
p<0.01,
###
p<0.001versus con-
trol group.
formtoestimatethespatial-workingmemory. Fig. 2BandCshowed
theescapelatencyof theCMT-treatedgroupwas signicantlylower
than that of the SCOP group (p<0.001) and considerably increased
the amount of time crossing over the platform site (p<0.05).
3.3. AChE and ChAT activities
To investigate the underlying mechanism of CMT in enhanc-
ing memory impairment induced by SCOP, activities of cholinergic
marker enzymes were determined following the water maze test.
As shown in Table 1, the SCOP-treated group had the highest AChE
activity (p<0.05, 1.06-fold with respect to control group). How-
ever, the increased AChE activity induced by SCOP was signicantly
inhibited by CMT treatment (p<0.01, 23%of SCOP group). The ChAT
activity did not change by SCOP treatment. However, CMT signi-
M.-R. Lee et al. / Journal of Ethnopharmacology 132 (2010) 7074 73
Table 1
Effect of CMT on activities of AChE and ChAT in mice brain with memory impair-
ments induced by SCOP.
Group AChE (U/mg protein) ChAT (IU/mg protein)
CON 8.51 0.99
ab
3.22 0.31
b
SCOP 9.04 1.03
a
3.15 0.13
b
THA 7.72 0.38
ab
3.37 0.36
b
CMT 6.98 1.53
b**
5.47 0.95
a**
Values are expressed as means S.E.M. (n=10). Values with a common superscript
letter within the same column differ signicantly (p<0.05),
**
p<0.01versus SCOP
group.
Table 2
Effect of CMT on the activities of antioxidant enzymes and MDA level in mice brain
with memory impairment induced by SCOP.
Group CAT SOD GSH-Px MDA
U/mg protein nmol/mg protein
CON 1.80 0.59
a
65.49 4.15
b
75.37 14.9
b
7.44 1.17
c
SCOP 1.25 0.24
b
88.23 3.31
a##
98.40 6.11
a
12.67 1.66
a##
THA 1.32 0.21
ab
64.35 9.30
b**
96.47 19.16
a
7.96 0.41
bc*
CMT 1.76 0.14
a
69.19 7.76
b**
86.16 9.85
ab
9.95 2.50
b
Values are expressed as means S.E.M. (n=10). Values with a common superscript
letter within the same column differ signicantly (p<0.05).
**
p<0.01,
***
p<0.001 versus SCOP.
##
p<0.01,
###
p<0.001 versus control.
cantly increased ChAT activity as compared to the vehicle-treated
control group (p<0.01).
3.4. Antioxidant activities
In order to further elucidate the mechanism of anti-amnesic
activity of CMT, lipid peroxidation and the activities of antioxidant
enzymes in the brain were measured following the Morris water
maze test (Table 2). SCOP treatment signicantly increased SOD,
GSH-Px enzyme activities and MDA levels by 35%, 31% and 70%,
respectively. However, CAT activity was signicantly decreased
by 30% as compared to the vehicle-treated control group. Treat-
ment of amnesic mice with CMT signicantly reduced the elevated
MDA level and SOD activity. GSH-Px activity was reduced without
signicance. The reduction in CAT activity induced by SCOP was
also reversed by treatment with CMT. In addition, the THA-treated
group also signicantly restored the elevated brain MDA level and
SOD activity to a level similar to the control group.
4. Discussion
In the present study, the effect of improving memory decit of
CMT was evaluated using the amnesic mouse model induced by
SCOP. It is well known that SCOP as a cholinergic receptor antag-
onist has been shown to impair learning and memory processing.
Acute and systemic administration of SCOP in young animals pro-
vides the appropriate memory decits related with the cholinergic
decit in ADor senile CNS dysfunction. The SCOP-induced amnesic
model has been widely used to provide a pharmacological model of
memory dysfunction for screening potential cognition enhancing
agents (Ebert and Kirch, 1998).
The cognitive-enhancing activity of CMT on the SCOP-induced
memory impairments in mice was investigated by using passive
avoidance test, Morris water maze test and biochemical assess-
ments. The passive avoidance test is generally used to evaluate the
treatments on the three stages of memory, such as learning acqui-
sition, memory retention, and the retrieval process (Lorenzini et al.,
1996). In this test, it was shown that SCOP also signicantly short-
ened the step-through latency of the retention trial, demonstrating
that the central cholinergic neuronal system plays an important
role in learning acquisition. CMT prolonged dose-dependently the
step-through latency shortened by SCOP. These results suggested
that the anti-amnesic effect of CMT on SCOP-induced memory
impairment may be related to mediation of the cholinergic nervous
system.
In order to conrmthe effects of CMT on other types of memory,
we performed the Morris water maze test on spatial learning. Mice
treated with SCOP showed a more prolonged escape latency than
mice from the control group. CMT and THA groups signicantly
reducedescape latencyprolongedbySCOPduringthe trial sessions.
In the probe test, CMT treatment lowered the escape latency com-
parable tothe control groupandsignicantly increasedthe number
of times of crossing over the platform site. It is important to notice
that the Morris water maze test investigating spatial learning and
memory has been used in detecting changes of the central cholin-
ergic system (Gage and Bjorklund, 1986; DHooge and De Deyn,
2001). If theanimals spent moretimeandswama longer distancein
the pool quadrant where the platform had previously been placed
during the training session, this would indicate that the animals
learned from prior experience with the Morris water maze test,
showing the spatial memory improvement (Blokand et al., 2004).
Therefore, these results suggest that CMT can repair the long-term
memory in SCOP-induced memory impairments.
To elucidate the underlying mechanisms of memory enhancing
effects of CMT, activities of AChE and ChAT as cholinergic markers
were assessed using brain homogenates. Neurochemical studies
suggested that the cholinergic system plays an important role in
learning and memory (Blokland, 1995). In an in vivo study as a
model of dementia treated with SCOP, cholinergic neurotransmis-
sionwas obstructedleadingtoanincreaseof theAChEandimpaired
cognition (Levey, 1996). ChAT, the biosynthetic enzyme for ACh,
is presently the most specic cholinergic marker for checking the
functional state of cholinergic neurons in the CNS and peripheral
nervous system(Oda, 1999). According to the cholinergic hypothe-
sis, memory impairments in patients with senile dementia are due
toaselectiveandirreversibledeciencyinthecholinergic functions
in the brain (Giacobini, 2002). Therefore, cholinesterase inhibitors
and ChAT activators may compensate for reduced ACh levels in
brains with AD disease.
In this study, CMT treatment showed not only signicantly
inhibition of AChE activity, but also promotion of ChAT activity.
However, ChAT activity did not changed by SCOP injection and
it concurs with the results of the previous studies that systemic
injection of SCOP dose not change ChAT activity in the cortex and
hippocampus (Jackson and Soliman, 1995; Hirokawa et al., 1996).
Thus, it could be explained that the anti-amnesic effect of CMT on
SCOP-induced impairment of learning and memory may be related
to modication of cholinergic neuronal systems.
This study evaluated whether such impaired cognition by SCOP
is associated with altered oxidative stress indices. SCOP-treated
mice had elevated MDA level, GSH-Px and SOD activities, but CAT
activity was reduced. Increased MDA levels have been shown to
be an important marker for in vivo lipid peroxidation. Oxidative
stress results from a marked imbalance between free radical pro-
duction and elimination by antioxidant systems. Recently, many
studies have reported the strong positive correlation that memory
impairments in the SCOP-induced amnesic rats show similar pat-
terns of oxidative damage in patients with amnesic mild cognitive
impairment (El-Sherbiny et al., 2003; Fan et al., 2005). Moreover,
many clinical studies have reported that oxidative stress is closely
involved in the pathogenesis of AD (Marcus et al., 1998).
Superoxide radicals are inactivated by SOD into hydrogen per-
oxide, which is converted into water and oxygen by either CAT or
GSH-Px (Blomgren and Hagberg, 2006). Our result shown in the
SCOP group was consistent with the previous studies that were
obtained from Tg2576 or 3xTg-AD mice, in which the activities of
74 M.-R. Lee et al. / Journal of Ethnopharmacology 132 (2010) 7074
SOD and GSH-Px were increased (Apelt et al., 2004; Resende et
al., 2008). Enhanced activity of the antioxidant enzymes SOD and
GSH-Px has also been commonly used as relevant indices of brain
oxidative stress. Moreover, decreased CAT activity by SCOP may
reect the increased susceptibility of the brain to oxidative stress
(Shila et al., 2005).
Treatment of amnesic micewithCMTsignicantlyreducedMDA
levels, and preserved the activities of GSH-Px, SOD, and CAT in a
similar level to that observed in the control group. The results of
the present study indicate that CMT possesses potent antioxidant
activity by scavenging ROS and exerting a protective effect against
oxidative damage induced by SCOP.
In conclusion, the cognitive-enhancing activities of CMT might
result from the regulation of cholinergic marker enzyme activities
such as AChE, ChAT and the antioxidative defense system. These
results suggest that CMT may possibly be used as an effective agent
to prevent cholinergic dysfunctions, such as AD.
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