Journal of Ethnopharmacology 137 (2011) 783–789

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

A modified formulation of Chinese traditional medicine improves memory

impairment and reduces A␤ level in the Tg-APPswe/PS1dE9 mouse model of
Alzheimer’s disease
Songhee Jeon a,∗ , Shambhunath Bose b , Jinyoung Hur c , Kiyoung Jun d , Young-Kyoon Kim e ,
Kyoung Sang Cho f , Byung-Soo Koo g,∗∗
a
Dongguk University Research Institute of Biotechnology, 26, 3-GA, Pil-dong, Chung-gu, Seoul 100-715, Republic of Korea
b
Institute of Medical Research, College of Medicine, Dongguk University, Seoul, Republic of Korea
c
Department of Physiology, Biomedical Science Institute and Medical Research Center for Reactive Oxygen Species, Kyung Hee University School of Medicine, Hoegi-dong, Dongdaemun-
gu, Seoul 130-701, South Korea
d
Hanpoong Pharm.Co., Ltd. 333-24, 1st Palbok-dong, Deokjin-gu, Jeonju-si, Jeollabuk-do 561-841, Republic of Korea
e
Department of Forest Products & Biotechnology, Kookmin University, Seoul 136-702, Republic of Korea
f
Department of Biological Sciences, Konkuk University, Seoul 143-701, Republic of Korea
g
Department of Neuropsychiatry, Graduate School of Oriental Medicine, Dongguk University, Seoul, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Ethnopharmacological relevance: SuHeXiang Wan (SHXW), a Chinese traditional medicine has been used
Received 12 February 2011 orally for the treatment of seizures, infantile convulsion, stroke and so forth. Previously, we reported the
Received in revised form 20 June 2011 effects of modified SHXW essential oil mixture of the fragrance containing herbs on the sedative effect,
Accepted 28 June 2011
anticonvulsant property and antioxidative activity after fragrance inhalation.
Available online 5 July 2011
Materials and Methods: This study was undertaken to evaluate beneficial effects of a modified recipe of
SHXW (termed as KSOP1009) consisting of a ethanol extract of 8 herbs including resin of Liquidambar
Keywords:
orientalis Miller, seed of Myristica fragrans Houtt., rhizome of Cnidium officinale Makino, lumber of San-
Alzheimer’s disease
SuHeXiang Wan
talum album L., fructus of Piper longum L., flower buds of Eugenia caryophyllata Merrill et Perry, pollen
Salvia miltiorrhiza Bunge of Typha orientalis Presl., and root of Salvia miltiorrhiza Bunge in the neurodegenerative diseases such as
Tanshinone IIA Alzheimer’s disease (AD). The transgenic mice of AD, Tg-APPswe/PS1dE9, were fed KSOP1009 or as a posi-
Eugenol tive control, donepezil for 3 months from 4.5 months of age. Behavioral, immunological and ELISA analyses
were used to assess memory impairment, A␤ accumulation and plaque deposition in the brain. Other
in vitro works were performed to examine whether KSOP1009 inhibits the A␤1–42 -induced neurotoxicity
in human neuroblastoma cell line, SH-SY5Y cells.
Results: Intake of KSOP1009 improved the A␤-induced memory impairment and suppressed A␤ levels
and plaque deposition in the brain of Tg-APPswe/PS1dE9 mice as much as that of donepezil treatment.
KSOP1009 prevented the down-regulation of phospho-CREB and increased AKT phosphorylation in the
AD-like brains. Moreover, KSOP1009 suppresses A␤-induced apoptosis and ROS production in SH-SY5Y
cells.
Conclusion: The present study suggests that KSOP1009 may develop as a therapeutic drug for treatment
of AD patients.
© 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Alzheimer’s disease (AD) is the most prevalent cause of demen-

∗ Corresponding author at: Dongguk University Research Institute of Biotechnol-
ogy, 27-3, Phildong 3, Joong-gu, Seoul 100-715, Republic of Korea. Tel.: +82 2 2260
8535; fax: +82 2 2271 3489.
∗∗ Corresponding author at: Department of Neuropsychiatry, Dongguk University
International Hospital, 814, Siksa-dong, Ilsandong-gu, Goyang-si, Gyeonggi-do 410-
773, Republic of Korea. Tel.: +82 31 961 0001; fax: +82 31 961 9009.
E-mail addresses: jsong0304@dongguk.edu (S. Jeon), koobs@dongguk.ac.kr
(B.-S. Koo).
tia and is characterized by loss of memory and cognition as
well as behavioral and occupational instability in old age. One
of the pathological characteristics of AD is the progressive depo-
sition of insoluble amyloid ␤ protein (A␤) as a form of senile
plaques (Wirths et al., 2004). Moreover, genetic mutations in ␤-
amyloid precursor protein (APP), presenilin-1 (PS1), and PS2 (Kim
and Tanzi, 1997; Selkoe, 1998) produce familial causes of AD
(Kowalska et al., 2004). However, most AD cases are caused by

0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2011.06.046
784 S. Jeon et al. / Journal of Ethnopharmacology 137 (2011) 783–789
non-genetic environmental factors (Ferri et al., 2005). Previous
studies have reported that abnormal metabolism of APP is crit-
ical for AD pathogenesis (Hardy and Higgins, 1992; Lee et al.,
2007).
Several investigations have revealed the presence of oxidation
products of proteins, lipids, and DNA in postmortem tissue from
AD patients, which is indicative of increased oxidative stress
(Subbarao et al., 1990; Smith et al., 1997; Butterfield and Boyd-
Kimball, 2004). In addition, A␤ promotes oxidative stress and
lipid peroxidation in synaptosomes and neuronal cultures (Keller
et al., 1997; Mark et al., 1997; Yatin et al., 1999). Consistently,
increased lipoperoxidation has been observed in an animal model
of Alzheimer amyloidosis (Pratico et al., 2001). Altogether, these
studies suggest that A␤ plays a central role in the pathogenesis of
AD as a mediator of oxidative stress.
So far, several drug candidates have been developed to
cure AD (Mangialasche et al., 2010). For example, given the
characteristics of AD, loss of cholinergic neurons and reduced
activity of choline acetyltransferase in the cerebral cortex and
hippocampus, acetylcholinesterase inhibitors, such as donepezil
hydrochloride, have been used as a drug to reduce the symp-
toms of AD (Dong et al., 2005). However, neuroprotective activity
of current drugs for AD is still debated (Mangialasche et al.,
2010).
SuHeXiang Wan (SHXW), a Chinese traditional medicinal pre-
scription (Storax Pill), consists of 15 crude herbs. This prescription
has been used orally for the treatment of seizures, infantile con-
vulsion, Qi (spirit) obstruction, sudden loss of consciousness,
stroke and so forth (Bensky and Gamble, 1986). Previously, we
reported the effects of modified SHXW essential oil mixture of
the fragrance containing herbs on the sedative effect, anticonvul-
sant property and antioxidative activity after fragrance inhalation
(Koo et al., 2004) and oral administration of SHXW reduced the
stress-hormone levels increased by immobilization stress (Kim and
Jeong, 1993). Recently, we examined the effect of modified SHXW
(KSOP1009) on the AD-like phenotypes in Drosophila AD mod-
els, which expressing human A␤42 in their neurons. KSOP1009
intake ameliorated the neurological phenotypes of these mod-
els, and reduced JNK activation and apoptosis (Hong et al., 2011).
In the present study, we investigated the effect of KSOP1009 on
the AD-like phenotypes in Tg-APPswe/PS1dE9 transgenic mouse
model.
2. Materials and methods
2.1. Preparation of KSOP1009 and control of standard biomarkers
As some materials of original prescription of SHXW are pro-
hibited by Korea Food and Drug Administration because of their
toxicity, or are hardly obtainable, we used a modified version of
SHXW, KSOP1009. To prepare the extract of KSOP1009, a total
of 13 kg of the mixture, such as resin of Liquidambar orientalis
Miller (Hamamelidaceae) (voucher number: 2009.05, 411 g), seed
of Myristica fragrans Houtt. (Myristicaceae) (2009.06, 1642 g), rhi-
zome of Cnidium officinale Makino (Umbelliferae) (2009.07, 2189 g),
lumber of Santalum album L. (Santalaceae) (2009.08, 821 g), fructus
of Piper longum L. (Piperaceae) (2009.09, 2737 g), flower buds of
Eugenia caryophyllata Merrill et Perry (Myrtaceae) (2009.10, 821 g),
pollen of Typha orientalis Presl. (Typhaceae) (2009.11, 1095 g), and
root of Salvia miltiorrhiza Bunge (Lamiaceae) (2009.12, 3284 g),
was pulverized and extracted twice with 10 vol. of 30% ethanol at
85–90 ◦ C with reflux condenser for 3 h, then filtered with 50 ␮m
filter and concentrated by vacuum evaporation at 60 ◦ C. The final
yield from the whole procedure was approximately 2.4–3.6 kg of
dried mixture (average yield rate is 23.08%). All medical herbs used
were purchased from Dong Yang herb Pharm. Co. (Seoul, Korea).
Each voucher specimen was identified by Prof. Young-Kyoon Kim,
College of Forest Science, Kookmin University and deposited in the
herbarium of the College. The dried material was stored at −80 ◦ C
until use. For cell culture works, each plant was extracted sepa-
rately with 30% ethanol and concentrated by vacuum evaporation
at 60 ◦ C.
We chosen tanshinone IIA, eugenol and cinnamic acid as stan-
dard molecular markers for root of Salvia miltiorrhiza Bunge, flower
buds of Eugenia caryophyllata Merrill et Perry and resin of Liq-
uidambar orientalis Miller, respectively. The amount of each marker
in KSOP1009 was determined using high performance liquid chro-
matography (HPLC) (Waters Alliance 2695, detector, Waters 2996
Photodiode Array Detector Waters micromass ZQ, USA) equipped
with column (XTerra® C18 5 ␮m 4.6 mm × 150 mm) by eluting with
50% methanol and 50% water. The column temperature was set
at 20 ◦ C, at 1 ml/min and sample injection volume was 10 ␮l the
detection wavelength was set at 285 nm. The content of tanshi-
none IIA, eugenol and cinnamic acid in 1 g of KSOP1009 extract
was detected to be 0.0533 mg (0.00533%), 0.659 mg (0.0659%), and
0.752 mg (0.0752%), respectively. In order to ensure the safety of
KSOP1009 formulation, single- as well as repeated-dose toxicity
tests for a period of 13 weeks were performed at pre-clinical level
by a GLP certification authority (MedVill. Co. LTD, Seoul, Korea).
General toxicity was not found in the rats treated with KSOP1009
at a dose of 1000 mg/kg.
2.2. Cell culture works
SH-SY5Y cells were obtained from the American Type Culture
Collection (Rockville, MD, USA) and cultured in DMEM contain-
ing 10% of FBS and 1% of antibiotics (Hyclone Laboratories Inc.,
Logan, UT, USA). To investigate the effect of KSOP1009 or each
component in cell viability, cells were pre-treated with it for 1 h
and stressed with 25 ␮M A␤1–42 (Biosource International, Camar-
illo, CA, USA) for a further 23 h. Treated cells were incubated with
1 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) (Sigma) at 37 ◦ C in a CO2 incubator for 3 h. MTT
medium was carefully aspirated and the formazan dye was eluted
using DMSO. The plate was shaken and measured using a spec-
trophotometer (Versamax microplate reader, Molecular Device;
Sunnyvale, CA, USA) at a wavelength of 570 nm.
2.3. Determination of intracellular ROS
Intracellular reactive oxygen sepsis (ROS) generation was
measured using a fluorometer. The ROS-sensitive dye, 2,7-
dichlorodihydrofluorescein diacetate (H2 DCF-DA) (Sigma–Aldrich,
St. Louis, MO, USA). H2 DCF-DA, was passively entered, converted to
dichlorofluorescin diacetate (DCFH), reacted with ROS and formed
the fluorescent product, dichlorofluorescin (DCF). The treated cells
were incubated with 20 ␮M H2 DCF-DA for 30 min. The fluorescence
intensity was measured at 480 nm excitation and 530 nm emission
using a fluorescence microplate reader (SpectraMax Gemini EM,
Molecular Device; Sunnyvale, CA, USA).
2.4. AD murine model
Tg-APPswe/PS1dE9 transgenic mice overexpressing human
mutated APP and PS1 (APPswe/PS1dE9) were initially purchased
from the Jackson Laboratory (Bar Harbor, ME, USA) and bred
to maintain in C57BL6 × C3H F1 hybrid as described previously
(Jankowsky et al., 2001). The animals were housed under laboratory
conditions at a controlled temperature (20 ± 2 ◦ C) and maintained
under light–dark cycles, 12 h of each (from 07:00 to 19:00 h)
with food and water made available ad libitum. The experimental
 S. Jeon et al. / Journal of Ethnopharmacology 137 (2011) 783–789
procedures were performed in accordance with the animal care
guidelines of the National Institute of Health (NIH) and carried
out with a prior approval from the Institutional Animal Ethical
Committee. Tg-APPswe/PS1dE9 mice at 4.5 months of age were
randomized into control (n = 6), KSOP1009 (n = 9) and donepezil
hydrochloride (Eisai Korea, Inc.) (n = 6) groups as positive con-
trol showing protective effect against cytotoxicity of A␤1–42 . Lab
chow containing KSOP1009 or donepezil was prepared for a mouse
to have 124 mg/kg/day or 1.036 mg/kg/day, which is equivalent
to 600 mg/60 kg/day or 5 mg/60 kg/day, respectively, for human
according to the translation formula of Reagan-Shaw et al. (2008)
and was gamma-irradiated. The mice were fed lab chow contain-
ing KSOP1009 or donepezil for 3 months beginning at 4.5 months
of age.
2.5. Step-through passive-avoidance test
The apparatus (AP model; O’Hara Co., Tokyo, Japan) for the step
through passive-avoidance test consisted of two compartments,
illuminated compartment [100 mm × 120 mm × 100 mm; light at
the top of compartment (27 W, 3000 lx)] and dark compartment
(100 mm × 170 mm × 100 mm). The compartments were separated
by a guillotine door. During the learning stage, a mouse was placed
in the illuminated safe compartment. As the compartment was lit,
the mouse stepped through the opened guillotine door into the
dark compartment. The time spent in the illuminated compartment
was defined as the latency time. Three seconds after the mouse
entered the dark compartment, a foot shock (0.3 mA, 50 V, 50 Hz
ac, for 3 s) was delivered to the floor grids in the dark compart-
ment. The mouse could escape from the shock only by stepping
back to the safe illuminated compartment. Such acquisition tri-
als during the learning stage were carried out at 7.5 months of
age. It was judged as learning avoidance from foot-shock if the
mouse remained in the illuminated compartment for 300 s after
being placed there. The retention trials were carried out at 24 h
after training trial to evaluate the retention of avoidance memory.
The latency time was measured for up to 300 s without delivering
foot shock. It was judged that the mouse retained the avoidance
memory when it stayed in the illuminated safe compartment for
300 s.
2.6. Morris water maze test
This behavioral test was performed at 7.4 months of age and we
performed this test as the method described by Morris (1984). Maze
testing was performed by the SMART-CS (Panlab, Barcelona, Spain)
program and equipment. A circular plastic pool (height: 35 cm,
diameter: 100 cm) was filled with milky water kept at 22–25 ◦ C.
An escape platform (height: 14.5 cm, diameter: 4.5 cm) was sub-
merged 0.5–1 cm below the surface of the water in position. On
training trials, the mice were placed in a pool of water and allowed
to remain on the platform for 10 s and were then returned to the
home cage during the second-trial interval. The mice that did not
find the platform within 120 s were placed on the platform for 10 s
at the end of trial. A video camera was mounted on the ceiling above
the pool and connected to a video recorder and tracking device
(S-MART; Pan-Lab, Barcelona, Spain), permitting on- and offline
automated tracking of the path taken by the mouse.
2.7. Measurement of Aˇ1–40 and Aˇ1–42
The brains were immediately removed and frozen. Tissues were
stored at −80 ◦ C until use. Each brain was homogenized in 8 vol.
(w/v) of cold 5 M guanidine HCl/50 mM Tris–HCl and mixed at room
temp for 4 h. Dilutions of the extracts were made in Dulbecco’s
phosphate-buffered saline containing 5% BSA and 0.03% Tween 20
785
(pH 7.4) supplemented with 1× protease inhibitor cocktail (Roche,
Germany). Following centrifugation at 16,000 × g for 20 min at 4 ◦ C,
aliquots were diluted with sample buffer provided by the manu-
facturer and used for the measurement of either A␤1–40 or A␤1–42
levels by enzyme-linked immunosorbent assay (ELISA) (Invitrogen,
USA).
2.8. Immunohistological and microscopic works
For immunohistochemistry, the right hemisphere was post-
fixedwith 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4)
at 4 ◦ C overnight, and coronally cut into 40-m-thick sections with a
vibratome (Leica VT 1000S; Leica Instruments, Nussloch, Germany).
Free-floating sections were blocked by 5% normal goat serum, 2%
BSA, and 2% FBS. A biotinylated HRP system was used for color
development. Anti-A␤ antibody Bam-10 (A5213) (Sigma, St. Louis,
MO, USA). Microscopic studies were carried out using an Oympus
BX 51 microscope equipped with a DP71 camera and DP-B soft-
ware (Olympus Co., Tokyo, Japan). For the quantification of plaque
levels, microscopic images of anti-A␤ antibody (Bam-10)-stained
superior prefrontal cortex was captured. The numbers of plaques
in the region were measured using TOMORO ScopeEye 3.6 program
(Techsan Community, Seoul, Korea).
2.9. Western blot analysis
For western blotting, the right hemisphere was dissected into
cortex and hippocampus. Tissues were homogenized in lysis buffer
containing 50 mM Tris-base (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1%
glycerol, 10 mM NaF, 10 mM Na-pyrophosphate, 1% NP-40 and pro-
tease inhibitors (0.1 mM phenylmethylsulfonylfluoride, 5 ␮g/ml
aprotinin, and 5 ␮g/ml leupeptin). Thirty ␮g of cell lysates were
electrophoresed in 10% SDS–polyacrylamide gels and transferred
to nitrocellulose membranes which were then incubated with
anti-phospho-CREB, anti-CREB, anti-phospho-MAPK, anti-MAPK,
anti-phospho-AKT, and anti-AKT (Cell Signaling Technology, Bev-
erly, MA, USA) for 16 h at 4 ◦ C. After washing with TBS-T (0.05%),
the blots were incubated with horseradish peroxidase-conjugated
anti-rabbit or anti-mouse IgG, and the bands were visualized using
the ECL system (Thermo Fisher Scienctific, USA). Band images were
obtained by using Molecular Imager ChemiDoc XRS+ (Bio-Rad, Her-
cules, CA, USA) and band intensity was analyzed by Image LabTM
software version 2.0.1 (Bio-Rad).
2.10. Statistical analysis
All values are expressed as means ± SEM. Data were analyzed
by one-way ANOVA, and then differences among means were ana-
lyzed using Dunnett’s test or Tukey–Kramer’s multiple comparison
test. Differences were considered significant at p < 0.05.
3. Results
The viability of SH-SY5Y cells was determined by MTT assay and
the results are presented in Fig. 1. KSOP1009 had no negative effect
on the cell viability in SH-SY5Y cell (data not shown). In Fig. 1A,
A␤1–42 -treated SH-SY5Y cells had significantly lower cell viability
than the control group. However, the cell viability in 10 ␮g/ml or
100 ␮g/ml of KSOP1009 treated groups was much higher than the
only A␤1–42 -treated group in SH-SY5Y cells (Fig. 1A). To investi-
gate the effect of KSOP1009 on the A␤1–42 induced reactive oxygen
species (ROS) overproduction in SH-SY5Y cells, we used DCF-DA.
A␤1–42 treated group increased ROS level by 3 times compared
to that of control group, while treatment of 10 or 100 ␮g/ml of
KSOP1009 with A␤1–42 decreased ROS generation to the basal level
(Fig. 1B). To compare KSOP1009 mixture with single component,
786 S. Jeon et al. / Journal of Ethnopharmacology 137 (2011) 783–789

Fig. 1. Effects of KSOP1009 on A␤1–42 -induced cell viability and ROS generation in SH-SY5Y cells. (A) Cells were treated 25 ␮M of A␤ with or without KSOP1009 (10 or
100 ␮g/ml) for 24 h. Cell viability was then determined by MTT assay. (B) ROS generation was measured by the fluorescence intensity of DCF after 25 ␮M A␤1–42 stimulation
for 1 h with or without KSOP1009 (10 or 100 ␮g/ml). (C) Cells were treated 25 ␮M of A␤ with or without each component (10 ␮g/ml) or KSOP1009 (10 ␮g/ml) for 24 h. Cell
viability was then determined by MTT assay. Values are presented as mean ± SEM of four determinations. *p < 0.01 vs. A␤ alone.

10 ␮g/ml of each component was treated into the cells with or
without 25 ␮M of A␤1–42 . As shown in Fig. 1C, KSOP1009 was most
effective on the protection of A␤-induced cell death.
To demonstrate the in vivo effects of KSOP1009, Tg-
APPswe/PS1dE9 mice were fed lab chow containing KSOP1009 at
dose of 124 mg/kg/day and donepezil at dose of 1.036 mg/kg/day,
as a positive control for 3 months beginning at 4.5 months of age.
Body weight and food intake amount were measured every two
weeks, but no significant difference between animal groups was
found (data not shown).
Next, we examined whether KSOP1009 could attenuate cog-
nitive deficits in AD-like animal model, Tg-APPswe/PS1dE9
transgenic mice. We first performed passive avoidance test for
memory testing at 7.5 months of age. At the learning trial, mice
of all groups entered the dark compartment, and there were no
significant differences among the animals. However, in the test-
ing trial, the Tg mice which received KSOP1009 or donepezil
treatment showed a significantly increased step-through latency
compared to that of control group (p < 0.05) (Fig. 2A). In the water
maze test, the Tg mice which received KSOP1009 or donepezil
treatment exhibited progressively decreased escape latency by
the training (4 days after training; 2 times/day, total of 8 times
training (p < 0.05) (Fig. 2B). These results indicated that treatment
of KSOP1009 remarkably improved the Tg-APPswe/PS1dE9 mice
memory impairment.
Then, we measured the levels of total A␤ peptides by using
ELISA analysis in the prefrontal cortex homogenates. The levels of
A␤1–40 and A␤1–42 were significantly reduced in the KSOP1009 or
donepezil treated Tg-APPswe/PS1dE9 mice compared with that of
no treated mice (Fig. 3A and B). Consistent with the ELISA data, anti-
A␤1–42 antibody-stained brain sections showed that KSOP1009 and
donepezil significantly reduced plaque deposition in the prefrontal
cortex of Tg-APPswe/PS1dE9 mice (Fig. 3C and D).
In order to examine the mechanisms of action of KSOP1009
on the neuroprotective effect, we checked the phosphorylation
level of CREB and MAPK in the cortex and hippocampus of Tg-
APPswe/PS1dE9 mice. As shown in Fig. 4, the phosphorylation of
CREB was notably increased in the cortex and hippocampus of
KSOP1009 treated mice, and also donepezil treated mice. However,
MAPK phosphorylation was not changed in the both drug treated
mcie. Next, in order to demonstrate the upstream signaling path-
way for CREB phosphorylation, we examined the phosphorylation
level of AKT in the brain. In the KSOP1009 or denepezil treated mice,
AKT phosphorylation was significantly increased (Fig. 5). These sug-
gest that KSOP1009 induces AKT and CREB phosphorylation in the
brain.
4. Discussion
In the present study, we demonstrated that treatment of
KSOP1009, a modified recipe of traditional SHXW, significantly
improves memory impairments, A␤ levels and plaque deposi-
tion in the brain of Tg-APPswe/PS1dE9 mice. Moreover, KSOP1009
increased CREB and AKT phosphorylation. In addition, KSOP1009
reduced A␤1–42 -induced apoptotic cell death and ROS generation
in vitro, SH-SY5Y cells.
SHWX, a Chinese traditional medicinal prescription has been
used orally for the treatment of seizures, sudden loss of conscious-
ness, stroke and so forth (Bensky and Gamble, 1986). Here, we
modified the traditional SHXW consisting of 8 medicinal plants to
increase therapeutic effect against AD-like pathogenesis. Among
the mixture, methanolic extracts of Cnidium officinale has been
reported that it has scavenging activities of free radicals with an
additional antioxidant capacity (Ramalingam and Yong-Ki, 2010)
and Tanshinone IIA is one of the key components of Salvia mil-
tiorrhiza Bunge attenuate A␤-induced neuronal damage and the
impairment of long-term potentiation induced by hydrogen perox-
ide (Liu et al., 2010; Wang et al., 2011). In this study, we compared
KSOP1009 with each component and demonstrated that KSOP1009
is most effective on the protection of A␤-induced cell death. How-
ever, each component also showed the neuroprotective effect on
the AD-like neuronal cells. Thus, these results suggest that the effect
of the composite herb medicine might be better than a single one.
 S. Jeon et al. / Journal of Ethnopharmacology 137 (2011) 783–789 787

Fig. 2. Administration of KSOP1009 improved the memory impairment of Tg-APPswe/PS1dE9 mice. (A) For the passive avoidance performance test, mice were trained one
time and then the mice were returned to their home cage. One day after training, passive avoidance performance was tested. The latency period was measured as described
in Section 2. (B) For the water maze test, the animals were trained for 1 day and then, the memory function was determined by the escape latencies (s) for 4 days as described
in Section 2. The data are expressed as mean ± SEM with n = 6 mice group. Control mice were not given any other treatment KSOP1009: KSOP1009 extract treated group,
donepezil: donepezil treated group. *p < 0.05 vs. control.

CREB functions as a transcription factor following Ser-133
phosphorylation, and is a downstream substrate and effecter of
numerous signal transduction pathways. A wide range of neu-
romodulators and neurotransmitters could converge on CREB,
via various kinase such as protein kinase C (PKC), protein
kinase A (PKA), mitogen activated protein kinases (MAPKs) and
PI3K/AKT/GSK3␤ pathways in neurons (Adams and Sweatt, 2002;
Carlezon et al., 2005). Thus, status of CREB phosphorylation may
serve as a neural activity marker. In the brains of AD patients, CREB
phosphorylation level was reduced and the CREB-related intracel-
lular signaling pathway was disturbed (Yamamoto-Sasaki et al.,
1999). Phosphorylation level of CREB was also decreased in the
transgenic mice expressing mutant forms of human APP (Palop
et al., 2003), and the C terminal fragment of APP (Lee et al., 2006).
Moreover, inhibition of CREB activity by antisense oligodeoxynu-
cleotides or by a targeted mutation impaired long-term memory
formation (Bourtchuladze et al., 1994; Guzowski and McGaugh,
1997), whereas enhanced CREB activity in the brain produced a
beneficial effect on long-term memory (Josselyn et al., 2001). In
agreement with these findings, KSOP1009 increased more CREB
phosphorylation level in the brain of Tg-APPswe/PS1dE9 mice
than that of donepezil. Furthermore, as an upstream kinase of
CREB, AKT phosphorylation was increased by KSOP1009 treatment
but not MAPKs. Thus, these results suggest that KSOP1009 may
strengthen the neuronal activity in the AD-like brain via PI3K/AKT
and CREB phosphorylation. However, it is remained to be elucidated
whether the enhanced expression of phospho-CREB and AKT in the
brain with KSOP1009 results from its therapeutic effect against AD

Fig. 3. Administration of KSOP1009 reduced A␤1–42 and A␤1–40 levels and plaque deposition in the brain of Tg-APPswe/PS1dE9 mice. (A and B) ELISA analyses showing
that the amounts of A␤1–40 (A) and A␤1–42 (B) levels in the prefrontal cortex of Tg-APPswe/PS1dE9 mice at 7.5 months of age. Photomicrographs showing anti-A␤ antibody
(Bam-10)-stained parietal cortex (C) of Tg-APPswe/PS1dE9 mice. Scale bar, 200 ␮m. (D) Quantification of plaque numbers in the parietal cortex of Tg-APPswe/PS1dE9 mice.
Microscopic images of anti-A␤ antibody-stained sections were converted into digital images and plaque numbers were counted using the TOMORO ScopeEye 3.6 program.
Data are presented as the means ± SEM (n = 6 animals). Control mice were not given any other treatment KSOP1009: KSOP1009 extract treated group, donepezil: donepezil
treated group. *p < 0.05 vs. control.
788 S. Jeon et al. / Journal of Ethnopharmacology 137 (2011) 783–789

Fig. 4. Administration of KSOP1009 recovered CREB phosphorylation in the brain of Tg-APPswe/PS1dE9 mice. The cortical and hippocampal lysates were electrophoresed in
10% SDS-PAGE and inmmunoblotted with each antibody. The intensity of the protein bands was quantitated by densitometry and the phosphorylated form was normalized
vs. total form. The data are expressed as mean ± SEM with n = 6 mice group. Control mice were not given any other treatment KSOP1009: KSOP1009 extract treated group,
donepezil: donepezil treated group. *p < 0.05 vs. control.

Fig. 5. Administration of KSOP1009 increased AKT phosphorylation in the brain of Tg-APPswe/PS1dE9 mice. The cortical and hippocampal lysates were electrophoresed in
10% SDS-PAGE and inmmunoblotted with each antibody. The intensity of the protein bands was quantitated by densitometry and the phosphorylated form was normalized
vs. total form. The data are expressed as mean ± SEM with n = 6 mice group. Control mice were not given any other treatment KSOP1009: KSOP1009 extract treated group,
donepezil: donepezil treated group. *p < 0.05 vs. control.

pathogenesis or is produced secondly by the mechanism by which
KSOP1009 enhances the general neural activity in the brain.
Many studies have shown that A␤ overexpression in neurons
results in apoptosis via JNK activation, and that inhibition of JNK
activity protects cells from A␤42 toxicity (Borsello and Forloni,
2007; Braithwaite et al., 2010). Recently, we also demonstrated
that KSOP1009 suppresses the cytotoxicity of A␤ via inhibition of
the hyperactivation of JNK in the AD-like Drosophila model (Hong
et al., 2011). However, in this study, there was no difference on the
phosphorylation of JNK in the brain between each group (data not
shown), suggesting that mechanisms of action of KSOP1009 may
be different between mammals and insects. Indeed, the same gene
shows different response in mammals and insects (Balamurugan
et al., 2004; Godenschwege et al., 2004).
Reactive oxygen species (ROS) are a cause leading to the pathol-
ogy of AD. Various natural compounds show anti-ROS effects, which
are effective in inhibiting AD-like pathology in AD model animals
(Russo et al., 2003; Frank and Gupta, 2005). Curcumin and ginkgo
biloba extract, which have beneficial effects on AD-like brains, carry
anti-ROS function (Balasubramanyam et al., 2003; Smith and Luo,
2003). In the present study, KSOP1009 suppressed the cytotoxicity
of SH-SY5Y neuroblastoma cells induced by A␤1–42 via suppress-
ing ROS generation. Though it is preliminary, the present study
provides evidence that KSOP1009 may protect AD pathogenesis
through antioxidant property in the Tg-APPswe/PS1dE9 mice.
Taken together, our results demonstrate that KSOP1009 sup-
presses A␤-induced apoptosis and ROS production in SH-SY5Y cells.
Moreover, intake of KSOP1009 improved the A␤-induced mem-
ory impairment and suppressed A␤ levels and plaque deposition in
the brain of Tg-APPswe/PS1dE9 mice as much as that of donepezil
treatment. Thus, these suggest that KSOP1009 may develop as a
therapeutic drug for treatment of AD patients.
Acknowledgement
This study was supported by a grant of the Oriental Medicine
R&D Project, Ministry for Health & Welfare & Family Affairs, Repub-
lic of Korea (B090068).
References
Adams, J.P., Sweatt, J.D., 2002. Molecular psychology: roles for the ERK MAP kinase
cascade in memory. Annual Review of Pharmacoloy and Toxicology 42, 135–163.
Balamurugan, K., Egli, D., Selvaraj, A., Zhang, B., Georgiev, O., Schaffner, W., 2004.
Metal-responsive transcription factor (MTF-1) and heavy metal stress response
 S. Jeon et al. / Journal of Ethnopharmacology 137 (2011) 783–789
in Drosophila and mammalian cells: a functional comparison. Biological Chem-
istry 385, 597–603.
Balasubramanyam, M., Koteswari, A.A., Kumar, R.S., Monickaraj, S.F., Maheswari,
J.U., Mohan, V., 2003. Curcumin-induced inhibition of cellular reactive oxygen
species generation: novel therapeutic implications. Journal of Bioscience 28,
715–721.
Bensky, D., Gamble, A., 1986. Chinese Herbal Medicine (Materia Medica). Eastland
Press, Seattle, p. 592.
Borsello, T., Forloni, G., 2007. JNK signalling: a possible target to prevent neurode-
generation. Current Pharmaceutical Design 13, 1875–1886.
Bourtchuladze, R., Frenguelli, B., Blendy, J., Cioffi, D., Schutz, G., Silva, A.J., 1994.
Deficient long-term memory in mice with a targeted mutation of the cAMP-
responsive element-binding protein. Cell 79, 59–68.
Braithwaite, S.P., Schmid, R.S., He, D.N., Sung, M.L., Cho, S., Resnick, L., Monaghan,
M.M., Hirst, W.D., Essrich, C., Reinhart, P.H., Lo, D.C., 2010. Inhibition of c-Jun
kinase provides neuroprotection in a model of Alzheimer’s disease. Neurobiol-
ogy of Disease 39, 311–317.
Butterfield, D.A., Boyd-Kimball, D., 2004. Amyloid ␤-peptide(1–42) contributes to
the oxidative stress and neurodegeneration found in Alzheimer disease brain.
Brain Pathology 14, 426–432.
Carlezon Jr., W.A., Duman, R.S., Nestler, E.J., 2005. The many faces of CREB. Trends in
Neuroscience 28, 436–445.
Dong, H., Csernansky, C.A., Martin, M.V., Bertchume, A., Vallera, D., Csernansky,
J.G., 2005. Acetylcholinesterase inhibitors ameliorate behavioral deficits in
the Tg2576 mouse model of Alzheimer’s disease. Psychopharmacology 181,
145–152.
Ferri, C.P., Prince, M., Brayne, C., et al., 2005. Global prevalence of dementia: a Delphi
consensus study. Lancet 366, 2112–2117.
Frank, B., Gupta, S., 2005. A review of antioxidants and Alzheimer’s disease. Annals
of Clinical Psychiatry 17, 269–286.
Godenschwege, T.A., Reisch, D., Diegelmann, S., Eberle, K., Funk, N., Heisenberg, M.,
Hoppe, V., Hoppe, J., Klagges, B.R., Martin, J.R., Nikitina, E.A., Putz, G., Reifegerste,
R., Reisch, N., Rister, J., Schaupp, M., Scholz, H., Schwärzel, M., Werner, U., Zars,
T.D., Buchner, S., Buchner, E., 2004. Flies lacking all synapsins are unexpectedly
healthy but are impaired in complex behaviour. European Journal of Neuro-
science 20, 611–622.
Guzowski, J.F., McGaugh, J.L., 1997. Antisense oligodeoxynucleotide-mediated dis-
ruption of hippocampal cAMP response element binding protein levels impairs
consolidation of memory for water maze training. Proceedings of the National
Academy of Sciences of the United States of America 94, 2693–2698.
Hardy, J.A., Higgins, G.A., 1992. Alzheimer’s disease: the amyloid cascade hypothesis.
Science 256, 184–185.
Hong, Y.K., Park, S.H., Lee, S., Hwang, S., Lee, M.J., Kim, D., Lee, J.H., Han, S.Y., Kim, S.T.,
Kim, Y.K., Jeon, S., Koo, B.S., Cho, K.S., 2011. Neuroprotective effect of SuHeXiang
Wan in Drosophila models of Alzheimer’s disease. Journal of Ethnopharmacology
34, 1028–1032.
Jankowsky, J.L., Slunt, H.H., Ratovitski, T., Jenkins, N.A., Copeland, N.G., Borchelt,
D.R., 2001. Co-expression of multiple transgenes in mouse CNS: a comparison
of strategies. Biomolecular Engineering 17, 157–165.
Josselyn, S.A., Shi, C., Carlezon Jr., W.A., Neve, R.L., Nestler, E.J., Davis, M., 2001.
Long-term memory is facilitated by cAMP response element-binding protein
overexpression in the amygdala. Journal of Neuroscience 21, 2404–2412.
Keller, J.N., Pang, Z., Geddes, J.W., Begley, J.G., Germeyer, A., Waeg, G., Mattson, M.P.,
1997. Impairment of glucose and glutamate transport and induction of mito-
chondrial oxidative stress and dysfunction in synaptosomes by ␤-peptide: role
of the lipid peroxidation product 4-hydroxinonenal. Journal of Neurochemistry
69, 273–284.
Kim, K.D., Jeong, D.G., 1993. Experimental study on the change of stress-related
hormone contents by Kyogamdan and Sohaphyangwon. Journal of Oriental Neu-
ropsychiatry 3, 121–134.
Kim, T.W., Tanzi, R.E., 1997. Presenilins and Alzheimer’s disease. Current Opinion
Neurobiology 7, 683–688.
789
Koo, B.S., Lee, S.I., Ha, J.H., Lee, D.U., 2004. Inhibitory effects of the essential oil from
SuHeXiang Wan on the central nervous system after inhalation. Biological and
Pharmaceutical Bulletin 27, 515–519.
Kowalska, A., Pruchnik-Wolinska, D., Florczak, J., et al., 2004. Genetic study of familial
cases of Alzheimer’s disease. Acta Biochimica Polonica 51, 245–252.
Lee, H.G., Zhu, X., Castellani, R.J., Nunomura, A., Perry, G., Smith, M.A., 2007. Amy-
loidbeta in Alzheimer disease: the null versus the alternate hypothesis. Journal
of Pharmacology and Experimental Therapeutics 321, 823–829.
Lee, K.W., Im, J.Y., Song, J.S., Lee, S.H., Lee, H.J., Ha, H.Y., Koh, J.Y., Gwag, B.J., Yang, S.D.,
Paik, S.G., Han, P.L., 2006. Progressive neuronal loss and behavioral impairments
of transgenic C57BL/6 inbred mice expressing the carboxy terminus of amyloid
precursor protein. Neurobiology of Disease 22, 10–24.
Liu, T., Jin, H., Sun, Q.R., Xu, J.H., Hu, H.T., 2010. The neuroprotective effects of
tanshinone IIA on ␤-amyloid-induced toxicity in rat cortical neurons. Neu-
ropharmacology 59, 595–604.
Mangialasche, F., Solomon, A., Winblad, B., Mecocci, P., Kivipelto, M., 2010.
Alzheimer’s disease: clinical trials and drug development. The Lancet Neurology
9, 702–716.
Mark, R.J., Geddes, J.W., Uchida, K., Mattson, M.P., 1997. Amyloid ␤ peptide impairs
glucose transport in hippocampal and cortical neurons: involvement of mem-
brane lipid peroxidation. Journal of Neuroscience 17, 1646–1654.
Morris, R., 1984. Developments of water-maze procedure for studying spatial learn-
ing in the rat. Journal of Neuroscience Methods 11, 47–60.
Palop, J.J., Jones, B., Kekonius, L., Chin, J., Yu, G.Q., 2003. Neuronal depletion of
calcium-dependent proteins in the dentate gyrus is tightly linked to Alzheimer’s
disease-related cognitive deficits. Proceedings of the National Academy of Sci-
ences of the United States of America 100, 9572–9577.
Pratico, D., Uryu, K., Leight, S., Trojanowski, J.Q., Lee, V.M., 2001. Increased lipid per-
oxidation precedes amyloid plaque formation in an animal model of Alzheimer
amyloidosis. Journal of Neuroscience 21, 4183–4187.
Ramalingam, M., Yong-Ki, P., 2010. Free radical scavenging activities of Cnidium
officinale Makino and Ligusticum chuanxiong Hort. methanolic extracts. Phar-
macognosy Magazine 6, 323–330.
Reagan-Shaw, S., Nihal, M., Ahmad, N., 2008. Dose translation from animal to human
studies revisited. The FASEB Journal 22, 659–661.
Russo, A., Palumbo, M., Aliano, C., Lempereur, L., Scoto, G., Renis, M., 2003. Red
wine micronutrients as protective agents in Alzheimer-like induced insult. Life
Science 72, 2369–2379.
Selkoe, D.J., 1998. The cell biology of beta-amyloid precursor protein and presenilin
in Alzheimer’s disease. Trends in Cell Biology 8, 447–453.
Smith, M.A., Harris, P.L., Sayre, L.M., Perry, G., 1997. Iron accumulation in Alzheimer
disease is a source of redox-generated free radicals. Proceedings of the National
Academy of Sciences of the United States of America 94, 9866–9868.
Smith, J.V., Luo, Y., 2003. Elevation of oxidative free radicals in Alzheimer’s dis-
ease models can be attenuated by Ginkgo biloba extract EGb 761. Journal of
Alzheimers Disease 5, 287–300.
Subbarao, K.V., Richardson, J.S., Ang, L.C., 1990. Autopsy samples of Alzheimer’s
cortex show increased peroxidation in vitro. Journal of Neurochemistry 55,
342–345.
Wang, W., Zheng, L.L., Wang, F., Hu, Z.L., Wu, W.N., Gu, J., Chen, J.G., 2011. Tan-
shinone IIA attenuates neuronal damage and the impairment of long-term
potentiation induced by hydrogen peroxide. Journal of Ethnopharmacology 134,
147–155.
Wirths, O., Multhaup, G., Bayer, T.A., 2004. A modified beta-amyloid hypothesis:
intraneuronal accumulation of the beta-amyloid peptide—the first step of a fatal
cascade. Journal of Neurochemistry 91, 513–520.
Yamamoto-Sasaki, M., Ozawa, H., Saito, T., Rösler, M., Riederer, P., 1999. Impaired
phosphorylation of cyclic AMP response element binding protein in the hip-
pocampus of dementia of the Alzheimer type. Brain Research 824, 300–303.
Yatin, S.M., Varadarajan, S., Link, C.D., Butterfield, D.A., 1999. In vitro and in vivo
oxidative stress associated with Alzheimer’s amyloid ␤ peptide (1–42). Neuro-
biology of Aging 20, 325–330.