Brain Research 974 (2003) 193–201

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Research report

Memory enhancing actions of Asiasari radix extracts via activation of

insulin receptor and extracellular signal regulated kinase (ERK) I / II in
rat hippocampus
Yong Han, Sung-Jin Kim*
Department of Pharmacology, School of Dentistry, Kyung Hee University, Seoul 130 -701, South Korea

Accepted 3 March 2003

Abstract

Brain insulin receptor and ERK I / II are suggested to play a role in memory formation. We designed a series of experiments to explore
if Asiasari radix (AR) extracts could display memory enhancing actions possibly via the activation of insulin receptor and ERK I / II in
mice and rats. Methanol extract of AR had significantly increased survival time in the NaNO 2 intoxication assay in mice. Methanol
extract of Asiasari radix (fraction 1) and its subfractions, chloroform-soluble fraction (fraction 2) and chloroform-insoluble, methanol-
soluble fraction (fraction 4) were further tested for memory formation. In eight-arm radial maze experiments, both reference memory
errors and working memory errors were significantly decreased in mice by fractions 1, 2 and 4. In addition, these fractions were also
effective in promoting memory in the passive avoidance test in mice and rats. To gain insight into the mechanism of memory enhancing
effects by Asiasari radix extracts, the activities of hippocampal insulin receptors and ERK I / II were tested in mice and rats. Fraction 1
significantly stimulated tyrosine phosphorylation of the insulin receptor, whereas ERK I / II were stimulated by fractions 1, 2 and 4. These
fractions also inhibited cholinesterase activities in rats. These results suggest that Asiasari radix extracts may exert memory enhancing
effects via activation of insulin receptor and ERK I / II as well as decreasing cholinesterase activity.
 2003 Elsevier Science B.V. All rights reserved.

Theme: Neural basis of behavior

Topic: Learning and memory: pharmacology

Keywords: Memory; Insulin receptor; Extracellular signal-regulated kinase; Cholinesterase; Hippocampus; Asiasari radix

1. Introduction elderly patient infected with Pseudomonas aeruginosa

Asiasari radix is the dried wholeplant, rhizome or root of
a number of Asarum species [36]. Asiasari radix (AR) is a
traditional herbal medicine widely used to treat various
diseases such as aphthous stomatitis, local anesthesia,
headache, toothache and inflammatory diseases including
gingivitis, in Korea and China [34,36]. It has been found
that Asiasari radix inhibits immunoglobulin E production
in experimental models in vitro and in vivo [21]. Recently,
a Korean medicinal plant recipe ‘Ma-Huang-Fu-Zi-Xi-Xin-
Tang’, which is formulated with extractions of several
plants including Asiasari radix, has been shown to improve
C-reactive protein levels and the body temperature of an
*Corresponding author. Fax: 182-2-957-5309.
E-mail address: kimsj@khu.ac.kr (S.-J. Kim).
[19]. It contains essential oils such as methyleugenol,
asarylketone, cineol, safrole, limonen, eucarvone and
acidamide such as N-isobutyl 2, 4, 8, 10-dodecatet-
raenamide, pellitorin and lignans such as asarinin, and
several alkaloids including hygenamine [36].
Insulin receptor in peripheral tissues participates mainly
in glucose metabolism, however its role in the CNS
appears not to be related to glucose metabolism but to
other neuronal activities such as memory [35]. In fact,
insulin receptor is widely distributed in different areas in
the brain and is abundantly present in the hippocampus
[6,7,24]. Hence, the hippocampus is an important target of
insulin action in the brain. Recently, much evidence has
been presented regarding the role of brain insulin or insulin
receptors in memory formation [9]. It has been found that
experimental damage to the neuronal insulin receptor and

0006-8993 / 03 / $ – see front matter  2003 Elsevier Science B.V. All rights reserved.
doi:10.1016 / S0006-8993(03)02580-0
194 Y. Han, S.-J. Kim / Brain Research 974 (2003) 193–201
Alzheimer’s brain cause similar metabolic abnormalities
[15].
An interesting hypothesis has been suggested that
sporadic Alzheimer disease might be the brain type of
non-insulin dependent diabetes mellitus [14]. It has been
suggested that intracerebroventricular insulin enhances
memory in a passive-avoidance task [26]. Insulin receptor
density and tyrosine kinase activity are known to be
significantly decreased in sporadic Alzheimer’s disease
(SDAT) [9]. Interestingly, tyrosine phosphorylation of the
hippocampal insulin receptor has been shown to play an
essential role in spatial memory formation [35]. Taken
together, insulin receptor activators could be used for
memory enhancement in addition to cholinesterase in-
hibitors.
Recently, it has been found that ERK (extracellular
signal-regulated kinase or MAPK) I and II, which are
important downstream signaling mediators of insulin re-
ceptor, are implicated in memory and learning [30,31]. It
has been also demonstrated that rats subjected to avoidance
learning showed significant and specific increases in the
activated forms of ERK I and II in rat hippocampus [3].
Neurodegenerative disorders in the central nervous system
are often accompanied by a decrease in cognition and
memory. In particular, dementia is a serious problem for
modern societies with high populations of elderly people.
Dementia is typically caused by genetic abnormalities,
aging and a variety of environmental factors such as brain
damage, smoking and alcohol, and other complex factors.
The hippocampus of patients suffering from dementia is
severely damaged and this is closely related to the reduc-
tion of acetylcholine levels in the brain [8]. Thus, acetyl-
cholinesterase inhibitors are clinically used in Alzheimer’s
disease to increase acetylcholine levels [4].
As Asiasari radix has been shown to possess beneficial
effects in headache and anxiety [37], which are CNS
effects, we sought to test whether Asiasari radix has any
effect on memory. The potential roles of insulin receptor
and ERK I / II in memory enhancement by Asiasari radix
extracts are explored.
2. Materials and methods
2.1. Materials
Male ICR mice and Sprague–Dawley rats were pur-
chased from Dai-Han Experimental Animals, Seoul, South
Korea. Asiasari radix was purchased from Kyung-Dong
Oriental Medicine Market, Seoul, South Korea. Antibody
against insulin receptor b subunit (Cat. [ 116630) was
purchased from Transduction Laboratories. Antibody
against phospho-p42 / 44 MAP kinase (Cat. [ 9101) was
purchased from Cell Signaling Tech. Anti-phosphotyrosine
antibody (4G10, Cat. [ 05-321) was purchased from
Upstate and Protein A-agarose (Cat. [ sc-2001) was
purchased from Santa Cruz Biotech. Organic solvents such
as methanol and chloroform were molecular biology grade
and purchased from Duksan, Seoul, South Korea. Nitro-
cellulose membrane was purchased from Schleicher &
Schuell. ECL kit was purchased from NEN. Other materi-
als were purchased from Sigma, USA.
2.2. Animals
Mice (20 g) and rats (200 g) were housed eight and four
per cage, respectively. The cages were placed in the
experimental room 24 h before the test for adaptation. The
animals were fed a standard laboratory diet and tap water
ad libitum and kept at 2361 8C with a 12-h light / dark
cycle, with lights on at 7 a.m.
2.3. Preparation of plant extracts
Samples of Asiasari radix were purchased from Kyung-
Dong Oriental Market in Seoul, and were authenticated by
Professor Chang-Soo Yok, Department of Oriental Phar-
macy, College of Pharmacy, Kyung Hee University, Seoul,
South Korea. A voucher specimen (No. 98-07) was
deposited at the herbarium of the Department of Pharma-
cology, School of Dentistry, Kyung Hee University. Ex-
traction and fractionation of Asiasari radix were performed
as described by Harbone [13]. Briefly, Asiasari radix (250
g) was cut into small pieces and extracted with 70%
methanol (750 ml) for 3 h (three times). The resulting
methanol extract was concentrated by a rotary evaporator
and dried in a freeze-dryer, yielding |23 g (fraction 1).
Then 10 g of fraction 1 (Fr. 1) was resuspended with 200
ml MeOH–H 2 O (4:1), adjusted to pH 3 with 2 M H 2 SO 4 ,
further extracted with 200 ml of CHCl 3 (three times) and
concentrated using the rotary evaporator, followed by
freeze-drying to yield 2.4 g (Fr. 2). CHCl 3 insoluble
fractions were adjusted to pH 10 with NH 4 OH and
extracted with equal volume of CHCl 3 –MeOH (3:1)
(twice). The CHCl 3 –MeOH (3:1) soluble fractions were
concentrated and freeze-dried to yield 0.1028 g (Fr. 3).
CHCl 3 –MeOH (3:1) insoluble fractions were mixed with
equal volume of MeOH and subjected to further extraction.
The MeOH soluble fractions were concentrated and freeze-
dried to yield 1.371 g (Fr. 4). The MeOH insoluble
fractions were concentrated and freeze-dried to yield
4.0043 g (Fr. 5).
For subsequent experiments, Asiasari radix extracts
were dissolved in 0.9% NaCl and administered to the
animals. The same volume of the vehicle (0.9% NaCl) was
administered to the control animals.
2.4. NaNO2 intoxication assays
NaNO 2 intoxication assays were carried out as described
previously [28]. Briefly, 60 min after AR extract adminis-
tration (100 mg / kg, i.p.), chemical hypoxia is induced in
 Y. Han, S.-J. Kim / Brain Research 974 (2003) 193–201
mice by injection of sodium nitrite (NaNO 2 , 250 mg / kg,
s.c.) which reduces the oxygen-carrying capacity of the
blood by converting hemoglobin to methemoglobin. Imme-
diately after NaNO 2 injection, the time between injection
of NaNO 2 and cessation of respiration is recorded.
Prolongation time is expressed as minutes.
2.5. Eight-arm radial maze experiments
The eight-arm radial maze tests were performed accord-
ing to the method described previously [16]. An eight-arm
radial maze (SciTech, South Korea), which was designed
according to Ref. [16], was used. Fr. 1, 2 or 4 (10 mg / kg
per day, p.o.) was administered once a day for 5 days and
tested for the eight-arm radial maze. Pre-training for 2 days
(5 min / day) allowed the mice to explore the baited radial
arm maze. During the experimental phase, four of the arms
were baited in a semi-random manner, with a unique
combination of baited arms for each mouse. After each
return to the center from an arm, the doors were closed for
5 s. Training was continued until all baits were consumed
or 15 min had passed. The test involved three parameters
of memory function: (i) working memory error was
defined as a repeated entry into arms that had already been
visited within a trial; (ii) reference memory error was
defined as entry into unbaited arms [10]; and (iii) time
spent in the collection of all baits was also measured.
2.6. Passive avoidance test
The test was basically performed according to the step-
through method described previously [18]. The Gemini
Avoidance System (SD Instruments) was used for this
experiment. The apparatus consists of a two-compartment
acrylic box with a lit compartment connected to a dark
compartment by an automatic guillotine door. Fr. 1, 2 or 4
(10 mg / kg per day, p.o.) was administered once a day for
3 days and tested for the passive avoidance test. Mice were
placed in the lit box for 300 s. The guillotine door was
then open. Mice received a electric shock (0.3 mA, 1 s) as
soon as they entered the dark compartment. The latency
times for entering the dark compartment were measured in
the training test and after 24 h in the retention test. The
maximum entry latency allowed in the retention session
was 500 s.
2.7. Preparation of hippocampal lysate
Male Sprague–Dawley rats or ICR mice were decapi-
tated 60 min after administration of AR extracts (10
mg / kg, p.o.) and the hippocampus was isolated at 4 8C. In
some experiments, AR extracts were administered for 3
consecutive days and then the hippocampus was isolated.
The removed hippocampus was resuspended with an
isolation buffer containing 50 mM Tris HCl, pH 7.4, 1 mM
EDTA, 1 mM EGTA, 150 mM NaCl, 1% Triton X-100,
195
0.5 mM PMSF, 1 mM Na 3 VO 4 , 1 mg / ml of leupeptin and
aprotinin and subjected to homogenization with a Potter-
Elvehjem homogenizer as described by Zhao with some
modification [35]. The insoluble material was removed by
centrifugation for 20 min (10,0003g) at 4 8C and the
supernatants were subjected to protein assay [23] and
stored at 270 8C.
2.8. Immunoprecipitation
Immunoprecipitation was performed as described previ-
ously [20]. Equal amounts of proteins (100 mg) from
hippocampal lysates were allowed to incubate with insulin
receptor b subunit antibody (5 ml; Transduction Lab-
oratories) for 1 h at 4 8C, followed by the addition of
Protein A-agarose (Santa Cruz Biotech.), and the immune
complex was precipitated by centrifugation. The pellets
were washed successively with 1 ml of buffer A (0.01 M
Tris, pH 7.4, 1 M NaCl, 1% Nonidet P-40), buffer B (0.01
M Tris, pH 7.4, 0.1 M NaCl, 0.01 M EDTA, 1% Nonidet
P-40, 0.3% SDS) and buffer C (0.01 M Tris, pH 7.4 and
1% Nonidet P-40). The final pellets were solubilized with
Laemmli buffer containing 100 mM dithiothreitol, boiled
for 5 min, centrifuged in a microcentrifuge, and the
supernatant was subjected to SDS–PAGE and Western blot
analysis with anti-pTyr antibody (4G10, Upstate).
2.9. Western blot analysis
Electrotransfer of proteins from the gels to nitrocellulose
paper was carried out for 1 h at 100 V (constant) as
described previously [32]. The filter papers were preincu-
bated for 1 h at 23 8C with PBS containing 0.1% Tween 20
and 3% bovine serum albumin and washed with PBS
containing 0.1% Tween 20 (three times) for 10 min each.
The blots were probed with pTyr antibody (1:1000; 4G10,
Upstate) or phospho-p44 / 42 MAP kinase antibody
(1:1000; Cell Signaling Tech.) for 1 h at 23 8C. The blots
were then incubated with HRP-conjugated anti-rabbit IgG
(1:3000) for 30 min and washed with PBS containing
Tween 20 (five times) for 10 min each. The detection of
immobilized specific antigens was carried out by ECL
(NEN). The corresponding bands were quantitated by
scanning densitometry.
2.10. Ex vivo acetylcholinesterase assay
Male Sprague–Dawley rats were dosed p.o. (10 mg / kg)
with vehicle or fractions of AR extract. The rats were
decapitated after 60 min, brains rapidly removed, hip-
pocampus and corpora striata dissected free, weighed and
homogenized as described in Section 2.7. Acetylcho-
linesterase activity was measured as described by Ellman
et al. [5]. Briefly, 3 ml of buffer I (100 mM phosphate, pH
8.0), 0.2 ml of 75 mM acetylthiocholine iodide and 0.1 ml
of buffered Ellman’s reagent (DTNB 10 mM, NaHCO 3 15
196 Y. Han, S.-J. Kim / Brain Research 974 (2003) 193–201
mM) were mixed and allowed to incubate for 10 min at
25 8C. Then, 20 ml of enzyme sample or 20 ml of the
isolation buffer was added and absorbance was measured
at 30-s intervals. The percent decreased activity was
calculated by comparison with the enzyme activity of the
control group.
2.11. Acute toxicity test
Acute toxicity (LD 50 ) was calculated by the method of
Litchfield and Wilcoxon [22]. Increasing doses of Asiasari
radix extract (Fr. 1) were administered p.o. to mice (n510)
and mortality rate was observed for 7 days. In addition,
general behaviors following administration of Fr. 1 (100
mg / kg) such as catalepsy, traction, tremor, convulsion,
exopthalmos, piloerection, salivation, lacrimation, defeca-
tion, skin color, pinna reflex, righting reflex, body tone, tail
elevation, eyelid, locomotion, respiratory rate, and death
were observed before and 15, 30, 60, 120 and 240 min
after treatment according to a modified Irwin’s test [17].
2.12. Statistics
All values were expressed as the mean6S.D. Com-
parisons between the control and treated groups were
performed by one-way ANOVA Dunnett’s post hoc test.
3. Results
3.1. NaNO2 intoxication assay
Administration of Fr. 1 (100 mg / kg, i.p.) caused a 47%
increase in survival time (Fig. 1). Subfractions of Fr. 1
were further screened. Each fraction (100 mg / kg) was
administered i.p. While Fr. 3 and 5 did not increase
Fig. 1. Effects of AR extracts on NaNO 2 -induced cessation of respira-
tion. After AR extract (Fr. 1, 2, 3, 4, or 5) i.p. administration, chemical
hypoxia was induced as described in Materials and methods, and the time
to cessation of respiration was measured. Data are expressed as
mean6S.D. (n58). *P,0.05 with respect to control.
survival time significantly, Fr. 2 and 4 increased survival
time by 65 and 35%, respectively. To examine whether
oral administration allows efficient absorption through the
gastrointestinal tract, oral administration of Fr. 2 was
compared to i.p. administration. Since Fr. 2 has the
greatest effect, we examined whether a decreased dose of
Fr. 2 administered p.o. increased survival time. When Fr. 2
(10 mg / kg) was administered orally, it caused a 73%
increase in survival time (Fig. 2). Fr. 1, 2 and 4 were
chosen for further tests since these fractions increased
survival time in the NaNO 2 intoxication assay whereas Fr.
3 and 5 did not (Fig. 1).
3.2. Eight-arm radial maze assay
To test whether fractions of Asiasari radix extract
enhance memory, the eight-arm radial maze assay was
conducted as described in Materials and methods. Fr. 1, 2
or 4 was administered for 5 days (10 mg / kg per day, p.o.)
(Fig. 3). Reference memory error was reduced by 50, 67
and 71% in response to Fr. 1, 2 and 4 administration,
respectively. Similarly, working memory error was re-
duced by 54, 63 and 74% in response to Fr. 1, 2 and 4
administration, respectively. The time taken to acquire all
baits was reduced by 55, 61 and 58% following Fr. 1, 2
and 4 administration, respectively.
3.3. Passive avoidance test
We used both mouse and rat to confirm that Asiasari
radix extract enhances memory. In the case of mouse, there
were no significant differences among AR extract fractions
in the training session. However, in the retention session,
administration of Fr. 1, 2 and 4 increased the step-through
time 8.4-, 4.7- and 2.9-fold, respectively (Fig. 4). Similar-
ly, in the case of rat, there were no significant differences
Fig. 2. Effects of oral administration of AR extracts on NaNO 2 -induced
cessation of respiration. Following p.o. administration of Fr. 2 of AR
extract, chemical hypoxia was induced as described in Materials and
methods, and the time to the cessation of respiration was measured. Data
are expressed as mean6S.D. (n58). *P,0.05 with respect to control.
 Y. Han, S.-J. Kim / Brain Research 974 (2003) 193–201
Fig. 3. Effects of AR extract (Fr. 1, 2 or 4) on memory function in the
eight-arm radial maze test. Following oral administration of each AR
fraction (10 mg / kg) once a day for 5 days, the eight-arm radial maze test
was conducted as described in Materials and methods. Data are expressed
as mean6S.D. (n510). *P,0.05, **P,0.01, ***P,0.001 with respect
to the control in each experimental group.
in the training session, while in the retention session,
administration of Fr. 1, 2 and 4 increased the step-through
time 2.2-, 1.6- and 2.2-fold, respectively (Fig. 5).
Fig. 4. Effects of AR extract (Fr. 1, 2 or 4) on memory formation of
mouse in the passive avoidance test. Following oral administration of
each AR fraction (10 mg / kg) once a day for 3 days, passive avoidance
test was conducted with mice as described in Materials and methods. Data
are expressed as mean6S.D. (n510). ***P,0.001 with respect to the
control after initial training.
197
Fig. 5. Effects of AR extract (Fr. 1, 2 or 4) on memory formation of rat
in the passive avoidance test. Following oral administration of each AR
fraction (10 mg / kg) once a day for 3 days, passive avoidance test was
conducted with rat as described in Materials and methods. Data are
expressed as mean6S.D. (n510). ***P,0.001 with respect to control
after initial training.
3.4. Effect on tyrosine phosphorylation of hippocampal
insulin receptors
Asiasari radix extract (Fr. 1) treatment (10 mg / kg, p.o.)
in mice stimulated tyrosine phosphorylation of insulin
receptor b subunit 3.5-fold, whereas Fr. 2 and 4 had little
effect. However, treatment with Asiasari radix extracts (Fr.
1, 2 and 4; 10 mg / kg, p.o., per day) for 3 consecutive days
had little effect on insulin receptor phosphorylation (Fig.
6). Asiasari radix extract (Fr. 1) treatment (10 mg / kg, p.o.)
in rats stimulated tyrosine phosphorylation of insulin
receptor b subunit 49.1-fold. Fr. 2 stimulated insulin
receptor phosphorylation 6.3-fold in rats while Fr. 4 had
little effect (Fig. 7). Qualitatively, similar patterns of
results were observed in replicate experiments.
3.5. Effect on activation of hippocampal ERK I /II
Asiasari radix extract (Fr. 1, 2 and 4) treatment (10
mg / kg, p.o.) in mice stimulated tyrosine phosphorylation
of hippocampal ERK II 3.1-, 3.7- and 6.6-fold, respective-
ly, while ERK I was stimulated 1.2-, 14- and 27.6-fold in
response to Fr. 1, 2 and 4, respectively (Fig. 8). In
contrast, Asiasari radix extract (Fr. 1, 2 and 4) treatment
(10 mg / kg, p.o., per day) for 3 consecutive days had little
effect on ERK I / II activation (Fig. 8). Asiasari radix
extract (Fr. 1, 2 and 4) treatment (10 mg / kg, p.o.) in rats
stimulated tyrosine phosphorylation of ERK I 3.6-, 4.9-
and 6.4-fold, whereas ERK II activation was stimulated
1.8-, 2.1- and 2.3-fold in response to Fr. 1, 2 and 4,
198 Y. Han, S.-J. Kim / Brain Research 974 (2003) 193–201
Fig. 6. Effects of AR extract (Fr. 1, 2 or 4) on tyrosine phosphorylation
of insulin receptor in mouse hippocampus. In experiment 1, 10 mg / kg of
each AR extract was orally administered and in experiment 2, the same
amount of each dose was administered for 3 consecutive days. Then 60
min after the last dose in both experiments, hippocampal cell lysate was
prepared. Equal amount of protein was incubated with insulin receptor
Ab, followed by precipitation with protein A Sepharose. The immune
complex was subjected to SDS–PAGE and Western blot analysis with
antiphosphotyrosine Ab. Detection of the phosphorylated insulin re-
ceptors was performed using an ECL kit. A representative of two separate
experiments is presented (A). The result from immunoblotting of phos-
phorylated insulin receptor b subunit (A) was subjected to densitometry
(B).
respectively (Fig. 9). Qualitatively, similar patterns of
results were observed in replicate experiments.
3.6. Effect on tyrosine phosphorylation of hippocampal
proteins
Tyrosine phosphorylation of a number of proteins in rat
hippocampus with molecular sizes of |180, 130, 95, 55
and 42 kDa (Fig. 10) was stimulated in response to Fr. 1, 2
or 4.
3.7. Effect on cholinesterase activity
Administration of Fr. 1, 2 and 4 caused a decrease in
hippocampal cholinesterase activity by 39, 24 or 12%,
respectively (Fig. 11).
3.8. Acute toxicity
Increasing concentrations of methanol extract of Asia-
sari radix (Fr. 1; 100, 1000, 3000, 5000, 10,000 mg / kg)
were orally administered to mice. Mortality was observed
for 7 days to measure LD 50 , which was calculated to be
|3400 mg / kg. There were no significant behavioral
Fig. 7. Effects of AR extract (Fr. 1, 2 or 4) on tyrosine phosphorylation
of insulin receptor in rat hippocampus. Each AR extract (10 mg / kg) was
orally administered and after 60 min, hippocampal cell lysate was
prepared. Equal amount of protein was incubated with insulin receptor
Ab, followed by precipitation with protein A Sepharose. The immune
complex was subjected to SDS–PAGE and Western blot analysis with
antiphosphotyrosine Ab. Detection of the phosphorylated insulin re-
ceptors was performed using an ECL kit. A representative of two separate
experiments is presented (A). The result from immunoblotting of phos-
phorylated insulin receptor b subunit (A) was subjected to densitometry
(B).
changes in the mice for 240 min following oral administra-
tion of 100 mg / kg of Asiasari radix (Fr. 1).
4. Discussion
Asiasari radix has been shown to exert a number of
pharmacological actions including mental effects such as
sedation and alleviation of headache; however, its actions
with regard to memory remain to be determined. In the
present study, for the first time, we present evidence that
AR extracts have stimulatory actions on memory formation
with the activation of insulin receptor and ERK I / II.
Manipulation of brain metabolism has shown the benefi-
cial effects of substances that influence learning and
memory. It has been demonstrated that impairment of
cholinergic transmission develops with induction of brain
hypoxia by NaNO 2 [11,12]. Cholinergic antagonists, such
as scopolamine, and hypoxia impair cognitive function
similarly and oxygen deprivation reduces acetylcholine
release and formation [12,27]. Thus, prolongation of
survival time by AR extracts following NaNO 2 administra-
tion could be regarded as an increase of learning or
memory. Fr. 1, 2 and 4 caused a significant increase in
 Y. Han, S.-J. Kim / Brain Research 974 (2003) 193–201
Fig. 8. Effects of AR extract (Fr. 1, 2 or 4) on the ERK I / II activities in
mouse hippocampus. In experiment 1, 10 mg / kg of each AR extract was
orally administered and in experiment 2, the same amount of each dose
was administered for 3 consecutive days. Then 60 min after the last dose
in both experiments, hippocampal cell lysate was prepared. Equal amount
of protein was used for the Western blot analysis with anti-phospho ERK
Ab. Detection of the phosphorylated ERKs was performed using an ECL
kit. A representative of two separate experiments is presented. The result
from immunoblotting of phosphorylated ERK (A) was subjected to
densitometry (B).
survival time following NaNO 2 treatment in mice, sug-
gesting these fractions of AR extract may increase memory
formation possibly by increasing acetylcholine release or
synthesis. Oral as well as intraperitoneal administration
exhibited similar effects in the NaNO 2 assay in mice,
indicating that absorption of AR extract through gastroin-
testinal tract occurs efficiently to exert pharmacological
actions in mice. The potential memory enhancing actions
of AR extracts were further evidenced by standard memory
199
Fig. 9. Effects of AR extract (Fr. 1, 2 or 4) on the ERK I / II activities in
rat hippocampus. Each AR extract (10 mg / kg) was orally administered
and after 60 min hippocampal cell lysate was prepared. Equal amount of
protein was used for the Western blot analysis with anti-phospho ERK
Ab. Detection of the phosphorylated ERKs was performed using an ECL
kit. A representative of two separate experiments is presented (A). The
result from immunoblotting of phosphorylated ERK (A) was subjected to
densitometry (B).
tests such as the eight-arm radial maze and passive
avoidance tests. Since oral administration of Fr. 1 of
Asiasari radix extract exerted considerable effects in the
NaNO 2 assay, this dose was used for all subsequent
experiments. In terms of treatment periods, we chose 3–5
days for the memory test based on information obtained
from the literature, while for molecular studies we used a
short treatment period (60 min) since insulin receptor and
Fig. 10. Effects of AR extract (Fr. 1, 2 or 4) on tyrosine phosphorylation
of rat hippocampal proteins. Each AR extract (10 mg / kg) was orally
administered and after 60 min hippocampal cell lysate was prepared.
Equal amount of protein was used for the Western blot analysis with
anti-phosphotyrosine Ab. Detection of the tyrosine phosphorylated pro-
teins was performed using an ECL kit. A representative of two separate
experiments is presented.
200 Y. Han, S.-J. Kim / Brain Research 974 (2003) 193–201
Fig. 11. Effects of AR extract (Fr. 1, 2 or 4) on cholinesterase activity in
rat hippocampus. Each AR extract (10 mg / kg) was orally administered
and after 60 min hippocampal cell lysate was prepared. Equal amount of
protein was used for the cholinesterase assay as described in the Materials
and methods. Data are expressed as mean6S.D. (n55). *P,0.05, **P,
0.01 with respect to control.
ERK I / II activation occurs during a short period following
stimuli. The eight-arm radial maze test involved three
parameters of memory impairment in mice, which were
working memory error, reference memory error and time
spent to obtain all baits [10]. In all cases, Fr. 1, 2 and 4
showed very similar patterns of decreasing working and
reference errors and time spent collecting all baits, sug-
gesting these fractions significantly increase memory for-
mation. In the passive avoidance test with mouse, adminis-
tration of Fr. 1, 2 and 4 caused a considerable increase in
memory activity, with Fr. 1 being the best, followed by Fr.
2 and Fr. 4. To exclude species difference between mouse
and rat, the passive avoidance tests were also performed
with rats. Administration of Fr. 1, 2 and 4 to rats also
significantly stimulated of memory formation, with Fr. 1
and 4 producing strong enhancing effects, while Fr. 2 had
a moderate effect. It is clear that Fr. 1, 2 and 4 of AR
extracts have memory enhancing effects in mouse as well
as in rat, indicating that species differences are minimal in
terms of memory formation by Asiasari radix extracts.
Though the major activity of insulin receptor is glucose
metabolism, its role in neurological activity is increasingly
being recognised, especially in memory and related pro-
cesses [28,29]. The high concentration of insulin receptor
in hippocampus means it is regarded as the center of
insulin actions in brain [24,25,29], which is further empha-
sised by its active role in memory formation [35]. Studies
to further correlate insulin receptor and memory formation
have been reported; similar metabolic abnormalities have
been observed in experimentally induced damage to neural
insulin receptor and in Alzheimer’s brain. Such studies
further provide proof for an unique relationship between
insulin receptor and memory formation [15]. These ob-
servations lead to the suggestion that Alzheimer’s disease
might be the brain type of non-insulin dependent diabetes
mellitus [14]
To further support the mechanistic view of an insulin–
memory relationship, it is suggested that intracerebroven-
tricular insulin enhances memory in a passive avoidance
task [26]. The involvement of insulin receptor in SDAT
(sporadic Alzheimer’s disease) is also suggested by a
decrease in insulin density and tyrosine kinase activity [9].
However, tyrosine phosphorylation of hippocampal insulin
receptor has been reported to play a role in spatial memory
formation [35].
In the present study, we found that Fr. 1 of Asiasari
radix extract has the ability to stimulate the tyrosine kinase
activity of hippocampal insulin receptor in mouse as well
as in rat. Fr. 2 of the Asiasari radix extract also stimulated
the tyrosine kinase activity of hippocampal insulin receptor
in rat; however, this stimulation was weaker than that of
Fr. 1. The effect of Fr. 2 on mouse hippocampal insulin
receptor was not detected in our assay system. Taken
together, it is logical to speculate that the memory enhanc-
ing activity of Fr. 1 and 2 of the Asiasari radix extract
might be partly due to the activation of insulin receptor
tyrosine kinase.
Recent studies also suggested that ERK I / II play an
important role in memory formation [1,30,31,33]. The data
presented in the present study suggest that ERK I / II
activation by the Asiasari radix extracts (Fr. 1, 2 and 4)
could contribute to the activation of memory formation in
both mouse and rat. It is possible that insulin receptor
activation by the Asiasari radix extracts may subsequently
cause the activation of ERK I / II, which eventually leads to
the enhancement of memory: Fr. 1 or 2 may act this way.
It is also possible that some component of the Asiasari
radix extracts may directly activate ERK I / II without
influencing insulin receptor: Fr. 4 may act through this
mechanism. The activation of insulin receptor and ERK
I / II by the Asiasari radix extracts occurred immediately,
lasting for 60 min after administration; however, adminis-
tration for 3 consecutive days had little effect on the
activation of insulin receptor and ERK I / II. We believe
that early stimulation of insulin receptors and ERK I / II by
Asiasari radix extracts might play a role in the activation
of subsequent signal transduction pathways in the hip-
pocampus, thereby inducing memory enhancement later.
On critical analysis, the patterns of Western blot of insulin
receptor and ERK are more or less same in replicate sets.
Hence, undoubtedly, similar patterns of stimulation by the
extract have been observed.
In addition, the decrease in cholinesterase activity by Fr.
1, 2 and 4 could also contribute to the memory enhancing
action; these results are consistent with the data obtained
from NaNO 2 assays. Considering that acetylcholine re-
ceptors activate ERK in hippocampus [2], an increase in
acetylcholine levels induced by decreased activity of
acetylcholinesterase following Asiasari radix extract treat-
ment may cause activation of acetylcholine receptors,
thereby stimulating ERK. In terms of the active principles
responsible for memory enhancement, it is reasonable to
propose that some terpenoids and phenolic compounds
may be involved in the memory formation process, consi-
dering the chloroform-soluble fractions of the methanol
 Y. Han, S.-J. Kim / Brain Research 974 (2003) 193–201
extract of plant materials are known to be enriched with
terpenoids and phenolic compounds [13]. Some N-oxides
and quaternary alkaloids enriched in Fr. 4 may also play a
role in the memory enhancement. Further studies are
necessary to purify and identify active components respon-
sible for memory enhancement by Asiasari radix extracts.
As Asiasari radix is a natural product and easily
obtainable for the general population, it would be good to
utilize this plant to alleviate symptoms of neurodegenera-
tive diseases, such as Alzheimer’s, and to enhance cogni-
tive function.
References
[1] J.P. Adams, J.D. Sweatt, Molecular psychology: roles for the ERK
MAP kinase cascade in memory, Annu. Rev. Pharmacol. Toxicol.
42 (2002) 135–163.
[2] J.L. Berkeley, J. Gomeza, J. Wess, S.E. Hamilton, N.M. Nathanson,
A.I. Levey, M1 muscarinic acetylcholine receptors activate extracel-
lular signal-regulated kinase in CA1 pyramidal neurons in mouse
hippocampal slices, Mol. Cell. Neurosci. 18 (2001) 512–524.
[3] M. Cammarota, L.R. Bevilagua, P. Ardenghi, G. Paratcha, M. Levi
de Stein, I. Izquierdo, J.H. Medina, Learning-associated activation
of nuclear MAPK, CREB and Elk-1, along with Fos production, in
the rat hippocampus after a one-trial avoidance, Brain Res. Mol.
Brain Res. 76 (2000) 36–46.
[4] P. Camps, D. Munoz-Torrero, Cholinergic drugs in pharmacotherapy
of Alzheimer’s disease, Mini Rev. Med. Chem. 2 (2002) 11–25.
[5] G.L. Ellman, K.D. Courtney, V. Andres, R.M. Featherstone, A new
and rapid colorimetric determination of acetylcholinesterase activity,
Biochem. Pharmacol. 7 (1961) 88–95.
[6] M.L.L.A. Fernandes, M.J.A. Saad, L.A. Velloso, Insulin induces
tyrosine phosphorylation of the insulin receptor and SHC, and
SHC / GRB2 association in cerebellum but not in forebrain cortex of
rats, Brain Res. 826 (1999) 74–82.
[7] F. Folli, S. Ghidella, L. Bonfanti, C.R. Kahn, A. Merighi, The early
intracellular signal pathway for the insulin / insulin-like growth
factor family in the mammalian central nervous system, Mol.
Neurobiol. 13 (1996) 155–183.
[8] U. Freo, G. Pizzolato, M. Dam, C. Ori, L. Battistin, A short review
of cognitive and functional neuroimaging studies of cholinergic
drugs: implications for therapeutic potentials, J. Neural Transm. 109
(2002) 857–870.
[9] L. Frolich, D. Blum-degen, H.G. Bernstein, S. Engelsberger, J.
Humrich, S. Laufer, D. Muschner, A. Thalheimer, A. Turk, S.
Hoyer, R. Zochling, K.W. Boissl, K. Jellinger, P. Piederer, Brain
insulin and insulin receptors in aging and sporadic Alzheimer’s
disease, J. Neural Transm. 105 (1998) 423–438.
[10] S. Gamoh, M. Hashimoto, S. Hossain, S. Masumura, Chronic
administration of docosahexaenoic acid improves the performance
of radial arm maze task in aged rats, Clin. Exp. Pharmacol. Physiol.
28 (2001) 266–270.
[11] G.E. Gibson, J.P. Blass, Impaired synthesis of acetylcholine in brain
accompanying mild hypoxia and hypoglycemia, J. Neurochem. 27
(1976) 37–42.
[12] G.E. Gibson, M. Shimada, J.P. Blass, Alterations in acetylcholine
synthesis and cyclic nucleotides in mild cerebral hypoxia, J.
Neurochem. 31 (1978) 757–760.
[13] J.B. Harbone, in: Phytochemical Methods, A Guide to Modern
Techniques of Plant Analysis, Chapman and Hall, London, 1998, pp.
4–7.
[14] S. Hoyer, Is sporadic Alzheimer disease the brain type of non-
insulin dependent diabetes mellitus? A challenging hypothesis, J.
Neural Transm. 105 (1998) 415–422.
201
[15] S. Hoyer, D. Muller, K. Plaschke, Desensitization of brain insulin
receptor. Effect on glucose / energy and related metabolism, J.
Neural Transm. 44 (Suppl.) (1994) 259–268.
[16] S. Ikonen, P. Riekkinen Jr., Effects of apamin on memory processing
of hippocampal-lesioned mice, Eur. J. Pharmacol. 382 (1999) 151–
156.
[17] S.I. Irwin, in: J.H. Nodine, P.E. Siegler (Eds.), Animal and Clinical
Pharmacologic Techniques in Drug Evaluation, Yearbook Medical
Publishers, Chicago, 1968, pp. 36–54.
[18] M.E. Jarvik, R. Kopp, An improved one-trial passive avoidance
learning situation, Psychol. Rep. 21 (1967) 221–224.
[19] T. Kamel, T. Kondoh, S. Nagura, Y. Toriumi, H. Kumano, H.
Tomioka, Improvement of C-reactive protein levels and body
temperature of an elderly patient infected with Pseudomonas
aeruginosa on treatment with Mao-bushi-saishin-to, J. Altern.
Complement. Med. 6 (2000) 235–239.
[20] S.J. Kim, C.R. Kahn, Insulin stimulates phosphorylation of c-Jun,
c-Fos, and Fos-related proteins in cultured adipocytes, J. Biol.
Chem. 269 (1994) 11887–11892.
[21] H.M. Kim, Y.S. Moon, Asiasari radix inhibits immunoglobulin E
production on experimental models in vitro and in vivo, Immuno-
pharmacol. Immunotoxicol. 21 (1999) 469–481.
[22] J.T. Litchfield, F. Wilcoxon, A simplified method of evaluating
dose-effect experiments, J. Pharmacol. Exp. Ther. 96 (1949) 99–
113.
[23] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein
measurement with the Folin phenol reagent, J. Biol. Chem. 193
(1951) 263–275.
[24] J.L. Marks, J. Maddison, C.J. Eastman, Subcellular localization of
rat brain insulin binding sites, J. Neurochem. 50 (1988) 774–781.
[25] D.S. Olton, J.A. Walker, F.H. Gage, Hippocampal connections and
spatial discrimination, Brain Res. 139 (1978) 295–308.
[26] C.P. Park, R.J. Seeley, S. Craft, S.C. Woods, Intracerebroventricular
insulin enhances memory in a passive avoidance task, Physiol.
Behav. 68 (2000) 509–514.
[27] C. Peterson, G.E. Gibson, 3,4-Diaminopyridine alters acetylcholine
metabolism and behavior during hypoxia, J. Pharmacol. Exp. Ther.
222 (1982) 576–582.
[28] U. Schindler, D.K. Rush, S. Fielding, Nootropic drugs: animal
models for studying effects on cognition, Drug Dev. Res. 4 (1984)
567–576.
[29] R.J. Schulingkamp, T.C. Pagano, D. Hung, R.B. Rafa, Insulin
receptors and insulin action in the brain: review and clinical
implications, Neurosci. Biobehav. Rev. 24 (2000) 855–872.
[30] J.D. Sweat, The neuronal MAP kinase cascade: a biochemical signal
integration system subserving synaptic plasticity and memory, J.
Neurochem. 76 (2001) 1–10.
[31] E. Thiels, E. Klann, Extracellular signal-regulated kinase, synaptic
plasticity, and memory, Rev. Neurosci. 12 (2001) 327–345.
[32] H. Towbin, J. Staehelin, J. Gordon, Electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets: pro-
cedures and some applications, Proc. Natl. Acad. Sci. USA 76
(1979) 4350–4354.
[33] E. Valgent, J. Caboche, P. Vanhoutte, Mitogen-activated protein
kinase / extracellular signal-regulated kinase induced gene regulation
in brain: a molecular substrate for learning and memory?, Mol.
Neurobiol. 23 (2001) 83–99.
[34] Y.S. Wang, in: Pharmacology and Application of Chinese Materia
Medica, People’s Health Publisher, Beijing, 1983, pp. 723–727.
[35] W. Zhao, H. Chen, H. Xu, E. Moore, N. Meiri, M.J. Quon, D.L.
Alkon, Brain insulin receptors and spatial memory, J. Biol. Chem.
274 (1999) 34893–34902.
[36] R.H. Zhou, in: Resource Science of Chinese Medicinal Materials,
China Medical and Pharmaceutical Sciences Press, Beijing, 1993,
pp. 202–211.
[37] Y.P. Zhu (Ed.), Chinese Materia Medica. Chemistry, Pharmacology
and Applications, Harwood Academic Publisher, Amsterdam, 1998,
pp. 66–69.