Ameliorative effects of Thunbergia laurifolia on cognitiveGHÀFLWDQGGHSUHVVLRQLQROIDFWRU\EXOEHFWRPL]HGPLFH
Name of dispatched researcher（派遣研究者名）

Rojsanga Piyanuch

Instructor（指導教官，研究指導者）

Rojsanga PiyanuchAfÀliation of Instructor（所属）

Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Mahidol University, Thailand

Host Collaborator（受入研究者）

Matsumoto Kinzo

1. Introduction
Recently, dementia has been one of the world’s important public health problems.
Alzheimer's disease (AD) is the most common form of dementia, a neurodegenerative diseasecharacterized by progressive memory loss and cognitive impairment [1]. AD is pathologicallycharacterized by extracellular deposition of senile plaques; formation of intracellular
neuroÀbrillary tangles; and lesions of cholinergic neurons together with synaptic alterations in
cerebral cortex, hippocampus, and other brain regions[2]. However, current approved treatments
including cholinesterase inhibitors and agonists of N-methyl-D-aspartate receptors (NMDA) do
not terminate the progression of the disease, and have provided marginal therapeutic beneÀts [3].
Therefore, it is very essential to discover novel and effective medications for AD.
Thunbergia laulifolia Lindl., commonly known as Rang Jurd (RJ), belongs to thebotanical family of Acanthaceae. The aqueous preparations of leaves and root have been usedin Thai traditional medicine as anti-inflammatory and antipyretic agents as well as antidote
for detoxification of poison including insecticide, ethyl alcohol, arsenic and strychnine [4, 5].
Preparation of the extracts of dried TL leaves with boiling water was found to contain apigenin
and its glycosides, phenolic acids such as caffeic acid, gallic acid and protocatechuic acid [6].
In mice, RJ leaf extract alleviated adverse effects of lead on learning deÀcit and memory loss,
evaluated with water maze swimming test [7].
Several studies suggest that impairment of the olfactory system leads to impaired memoryand cognitive function in rodent. For example, impaired performance on learning and memorytasks, such as passive avoidance, modiÀed Y-maze and object recognition, was observed in olfactorybulbectomized (OBX) mice and rats [8-10]. In addition, elevated levels of ȕ-amyloid peptide [11] anddegeneration of cholinergic neurons [12] are observed in the brain of OBX mice. Interestingly, theimpairment of the olfactory system is not only observed in the early stages of Alzheimer’s disease(AD) [13, 14], but also in a transgenic AD model of mice, demonstrating that OBX provides abeneÀcial animal model of AD that is independent from transgenic animal models.

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2. Materials and Methods
2.1 Animals
The overall study was conducted according to the experimental protocols described in Fig.
1 and 2. Male ddY mice (Japan SLC Inc., Shizuoka, Japan) were obtained at the age of 9 weeks
old and housed with a 12-h light/dark cycle (lights on: 07:30–19:30) at 22 ± 1 ℃. Food and water
were available ad libitum. The animals were habituated to the laboratory animal room for atleast 1 week before surgery. The behavioral experiments were performed during the light phasefrom 9:00 to 18:00. The present studies were conducted in accordance with the Guiding Principles(NIH publication #85-23, revised in 1985) for the Care and Use of Animals and were approved by
the Institutional Animal Use and Care Committee in the University of Toyama.
2.2 Plant extract preparation
The leaves of Thunbergia laulifolia were collected from Wangnamyen district, Sa kaeo
province, Thailand in 2011. The plant samples were identiÀed by Mr. Pinit Chinsoi, Pharmacist,
Wangnamyen Hospital, Sa Kaeo province, Thailand. The voucher specimens were deposited at
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University, Thailand(RJ01001). The leaves were cleaned, dried in a hot air oven (60℃) for 6 h, and powdered withelectronic mill (20 mesh sieve) then were boiled with distilled water (1:10 w/v) for 8 h and Àltered.
The Àltrate was taken to dryness by lyophilization to yield dried leaf decoction extracts (RJ).
2.3 Surgical operation
OBX of mice was conducted according to a previous report. Briefly, the mice wereanesthetized with sodium pentobarbital (60 mg/kg, i.p.), and Àxed on stereotactic instruments(Narishige, Tokyo, Japan). The skull covering the bulbs was exposed by skin incision, 1%
lidocaine solution was used as local anesthetic then a 1mm burr hole was drilled. The bilateralbulbs were aspired through a syringe and the cavity of the bulbs was filled with hemostatic
gelatin sponge. After completing behavioral studies, all the animals were sacrificed and theoperated lesion was veriÀed visually. The data from animals with less than 70% of removal orwith not intact cortex was excluded from data analysis. Sham operation was performed in asimilar way without removal of the bulb. At the end of experiments, the olfactory bulbs of shamgroup mice were conÀrmed to be intact.
2.4 Drug administration
Tacrine (THA, 2.5 mg/kg, i.p.) was kindly supplied by Eisai Co. Ltd. (Tokyo, Japan) andImipramine (IM, 10 mg/kg, i.p.) were separately dissolved in 0.9% normal saline solution andintraperitoneally administered. Sham group of mice and OBX control group mice were orally
administered water. Leaf extract of T. laulifolia was dissolved in water and given per orally at
daily doses of 250–500 mg/kg. On a behavioral testing day, either THA or RJ was administered 1
h before the testing.

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2.5 Behavioral study
2.5.1 Locomotor activity
Ten days after the surgical operation, the animals underwent administration of test drugs
once daily for a week. One hour after the last administration, locomotor activity of each animalin the open Àeld (35x 35x50 cm) was video-recorded for later analysis. The activity during a 10min observation period was analyzed using Smart® system (PanLab, S.L., Barcelona, Spain). The
total distance exploring the arena was used to determine the locomotor activity.
0RGLÀHG<PD]HWHVW
Forty mice were randomly divided into four groups of ten mice. Each group of mice wasorally administrated with distilled water as a control group, tacrine (2.5 mg/kg, i.p.) was used asa standard drug, and T. laulifolia leaf extract in 2 different concentrations of 250 and 500 mg/
kg, p.o., respectively. A modiÀed version of the Y-maze test has been reported by Yamada et al.
[15]. The apparatus used for the modiÀed Y-maze test consists of black polypropylene walls with
3 arms each 40cm long, 12cm wide at the top, 3 cm wide at the bottom, 18cm high. This test isa two-trial task with a sample phase trial and a test phase trial that are separated by an intertrial interval. In the sample phase trial, each mouse was individually placed in the maze with
one of the 3 arms closed. The animals were allowed to freely explore the other 2 arms for 5 min.
Thirty min after the sample phase trial, the animal was again placed in the maze with all 3
arms opened, and allowed to explore the arms freely. The closed arm that is opened in the test
phase trial is deÀned as the new arm. The animal behavior was video-recorded for later analysis.
Percent time spent in the new arm and numbers of total arm entries were analyzed by Smart®
system.
1RYHOREMHFWUHFRJQLWLRQWHVW 257
This test is based on the tendency of mice to discriminate a familiar from a new object. Mice
were individually habituated to an open-Àeld box (35x 35x50 cm) for 10 min on the day before
the experiment. The ORT consists of a sample phase trial and a test phase trial. During thesample phase trial, two objects of the same material were placed in a symmetric position in thecenter of the chamber for 10 min. thirty minute after the sample phase trials, one of the objects
was replaced by a novel object, and exploratory behavior was again analyzed for 5 min. Aftereach session, objects were thoroughly cleaned with 70% ethanol to prevent odor recognition.
Exploration of an object was deÀned as rearing on the object or snifÀng it at a distance of lessthan 2 cm. Successful recognition of a previously explored object was reÁected by preferential
exploration of the novel object. Discrimination of spatial novelty was assessed by comparing thedifference between time of exploration of the novel and familiar7DLOVXVSHQVLRQWHVW 767
Tail suspension test (TST) is a widely used model for assessing antidepressant effect. Mice

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were subjected to the short-term inescapable stress of being suspended by the tail that willdevelop an immobile posture. Using another group of mice as indicated in protocol#2 (Fig 2),
three mice were separately suspended 50 cm above the floor in a chamber by adhesive tapeplaced approximately 2 cm from the tip of the tail. Immobility time was recorded during a 8 min
period using Smart® system.
2.6 Neurochemical study([YLYRPHDVXUHPHQWRIFKROLQHVWHUDVHDFWLYLW\LQWKHEUDLQ
After completing the behavioral experiments, the OBX mice treated with dailyadministrations of tacrine and RJ were decapitated and the frontal cortices were dissected out
and kept at í80 ƕC until use. Determination of cholinesterase activity was performed on the basisof the colorimetric method. BrieÁy, the frozen cortex was weighed and homogenized in 10-times
volume of 0.1M phosphate buffer (pH 7.4) containing 1% Triton-X-100. After centrifugation at
15,000 × g at 4 ƕC for 20 min, the clear supernatants were collected and served as the enzyme
source. Cholinesterase activity was determined in 96-well Áat-bottom microplates. The reaction
was started by adding 20 µl of 10mM 5,5́-dithiobis-(2-nitrobenzoic acid), 20 µl of 30mM
acetylthiocholine, and 160 µl of phosphate buffer. The spectrophotometric absorption at 405
nm during a 3 min incubation period at 25 ƕC was quantitatively measured using a microplate
reader (Sunrise Classic; TECAN Japan, Kawasaki) and expressed as nmol ACh hydrolyzed/min/mg tissue.
,QYLWURPHDVXUHPHQWRIFKROLQHVWHUDVHDFWLYLW\LQWKHEUDLQ
Cholinesterase activity was determined in 96-well flat-bottom microplates using frontal
cortex supernatants as the enzyme source. As same as the ex vivo experiment, the reaction
was started by adding 20 µl of 10mM 5,5́-dithiobis-(2-nitrobenzoic acid), 20 µl of 30 mM
acetylthiocholine, 50 µl of RJ or tacrine, and 160 µl of phosphate buffer. The spectrophotometricabsorption at 405 nm during a 3 min incubation period at 25 ƕC was quantitatively measured
using a microplate reader (Sunrise Classic; TECAN, Kawasaki, Japan). The velocities of thereactions were measured. Enzyme activity was calculated as a percentage of the velocities
compared to that of the assay using buffer without any inhibitor. Inhibitory activity wascalculated from 100 subtracted by the percentage of enzyme activity. Every experiment was donein triplicate.
4XDQWLWDWLYHUHDOWLPHSRO\PHUDVHFKDLQUHDFWLRQ 3&5
To analyze changes in expression levels of choline acetyltransferase (ChAT) and muscarinic
M1 receptor mRNA in the brain, the animals were killed by decapitation after completing the
behavioral studies. The brain was removed immediately and the hippocampus were dissectedout and kept at − 80°C until use. Quantitative PCR was conducted as previously described [16].
BrieÁy, total RNA was extracted from the hippocampus using Sepazol® (Nacalai Tesque, Kyoto)− 78 −

according to the manufacturer’s instructions. First-strand cDNA synthesis was conducted using
oligo (dT) primers and M-MLV Reverse Transcriptase® (Invitrogen, Rockville, MD, USA) in a
total volume of 20 ȝl. The reaction was performed at 25°C for 10 min and heated at 37°C for
60 min and 98°C for 5 min before cooling to 4°C. DNA corresponding to the RNA was used as atemplate for real-time PCR. Quantitative realtime PCR was carried out using Fast SYBR Green
Master Mix (Applied BioSystems, Foster City, CA, USA) in a StepOne Real-time PCR System(Applied BioSystems). The following primer sets used in this study were synthesized by Nippon
EGT Co. (Toyama): ChAT mRNA (NM_009891): 5 -CCTGTACAAGCTTCTAGCTGTGAG-3(forward) and 5 -GTAGCTAAGCACACCAGAGATGAG-3 (reverse); M1 mRNA (M16406): 5-ACTGTCTTGGCACCAGGAAA-3 (forward) and 5 -TGCTAGGCCAATCATCAGAG-3 (reverse)and ȕ-actin mRNA (NM_007393): 5 -CATCCGTAAAGACCTCTATGCCAAC-3 (forward) and 5-ATGGAGCCACCGATCCACA-3 (reverse). Melting curve analysis of each gene was performed
every time after ampliÀcation was completed. Standard curves of the log concentration of eachgene vs. cycle threshold were plotted to prove negative linear correlations.
2.7 Data analysis
The results were expressed as the mean

SEM. The data obtained from the behavioral tests

and neurochemical experiments were analyzed by pair and unpaired student’s t -test or oneway analysis of variance (ANOVA) followed by a post hoc multiple comparison test (Dunnett’s
method) as appropriate. Differences of 3 < 0.05 were considered as signiÀcant. The analysis was
conducted using SigmaStat® ver 3.5 (SYSTAT Software Inc., Richmond, CA, USA).

surgery

drug administration

10 days

7 days14 days

14 days

modified Y-maze testORT- decapitation- gene expression- ex vivo AChE activity- in vitro AChE activity

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

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surgery

Drug administration

7 days3 days

7 days

tail suspension test

decapitation

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

Results
3.1 The effect of RJ and Tacrine on OBX-induced elevation of motor activity

OperationDaily treatment

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Table 1 ::97HC:)!5B8H57F=B9CB@C7CACHCF57H=J=HMC:&/A=79
The sham-operated or olfactory bulbectomized (OBX) mice received daily administration ofvehicle (distilled water), tacrine or RJ according to protocol #1. Each datum represents themean ± S.E.M. from 10 mice in each group.

As summarized in Table 1, there was no significantly difference in locomotor activity
determined as total distance between vehicle-treated OBX and sham mice groups. Moreover,daily administration of tacrine (2.5 mg/kg, i.p.) or RJ (250 and 500 mg/kg, p.o.) for 1 week before
the experiments showed no effect on locomotor activity.
5-KDGQRHIIHFWRQ2%;LQGXFHGVSDWLDOZRUNLQJPHPRU\GHÀFLWVLQWKHPRGLÀHGY-maze test
As shown in Fig. 3, percentage time spent exploring the new arm of vehicle-treated shammice (49.30 ± 2.19%) was signiÀcantly higher than the chance level of 33.09 ± 3.05% indicatinga preference for new arms over the familiar arms. The tacrine-treated OBX mice(2.5 mg/kg,
i.p.) spent signiÀcantly longer time visiting new arm than the vehicle-treated OBX group (50.26± 2.75),while RJ treated groups (250 and 500 mg/kg. p.o.) did not spend significantly longer− 80 −

time visiting new arm than the vehicle-treated OBX group (40.12 ± 1.75% and 43.20 ± 2.41% ,respectively).

A)closed arm

new arm

30 mins

WSample phase(5 min)Test phase(5 min)

B)

100

Sham

% Time in new arm

m
r 80
a
w
e 60
n
in
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m
i 40
T
% 20
0

OBX

*

*

Veh.

Veh.

250

500
RJ

2.5

(mg/kg)

THA

Fig. 3 Evaluation of the modiÀed Y-maze test of OBX-induced spatial working memory deÀcit mice using
tacrine or T. laulifolia leaf extract (RJ). A: Schematic illustration of an experimental protocol for themodiÀed Y-maze test. B: Sham-operated mice were orally administered with distilled water 60 min before
sample phase trial while tacrine (2.5 mg/kg) and RJ (250 and 500 mg/kg) were administered by intraperitoneal injection and oral administration, respectively (n=10). Each data column represents the mean ±S.E.M. 3<0.05 compared with vehicle-treated OBX group (one-way ANOVA, Dunnett’s method).

5-DQGWDFULQHLPSURYHV2%;LQGXFHGQRQVSDWLDOPHPRU\GHÀFLWVLQWKHQRYHOREject recognition test
In sample phase, no mice groups showed significantly differences in time spent exploringeach identical object. There also no signiÀcant difference in total time spent exploring two objects
between sham and OBX groups (Fig. 4 (a)). In test phase, the sham group spent a signiÀcantly
longer time exploring the new object than the familiar one (3<0.05) while the vehicle-treated
OBX group showed the deÀcit of the novel object recognition performance as shown in Fig. 4 (b).
Tacrine (2.5 mg/kg, i.p.) and RJ (250 and 500 mg/kg, p.o.)-treated mice groups spent signiÀcantly
longer time exploring the new object than the familiar object (3<0.01).

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A)

s
30 min)2

2

Test phase(5 min)Sample phase(5 min)B)W11) Sample phase trial

2) Test phase trial
New Object (N)Object O1Familiar Object (F)Object O2
100

Sham

100

OBX

20
0

Sham

OBX

t 80
n
e
p 60
s
e
im
T 40
%

% Time Spent% Time Spent

t 80
n
e
p 60
s
e
im
T 40
%

Veh.

Veh.

250

500
RJ

20
0

2.5

Veh.

Veh.

250

THA

500
RJ

2.5 (mg/kg)
THA
Fig. 4 Effects of tacrine and RJ on object recognition deÀcits in OBX mice in sample phase and test phase.
A: Schematic illustration of the experimental protocol for object recognition test. B: Time each mouse spent
exploring objects in the sample phase (B1) and test phase (B2) were recorded using PanLab system. Eachdatum represents the mean ± S.E.M. (n=10). Each data column represents the mean ± S.E.M.

3.4 RJ improved OBX-induced depression in tail suspension test
As shown in Fig. 5, the duration of immobility in TST was measured 10 days after OBX. The
duration of immobility in vehicle-treated OBX mice signiÀcantly increased (25.18 ± 1.72 s) comparedwith that observed in sham-operated group (11.47 ± 2.13 s). One week of daily administrationof imipramine (10 mg/kg, i.p.) or RJ (500 mg/kg, p.o.) significantly reduced the duration of
immobility (6.48 ± 3.85 and 3.16 ± 1.94 s, respectively) while tacrine-treated group (2.5 mg/kg, i.p.)
showed no reducing effect on the immobility (17.44 ± 3.75 s).

Sham

e
m
ti 40
g
n
ti
s
e
R
% 20

*

% Resting time

60

OBX

*

*
0
Veh.

Veh.

500
RJ

2.5
THA

10 (mg/kg)
IMP

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

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3.5 Effects of RJ on expressions of cholinergic marker genes
The expression levels of muscarinic M1 and ChAT mRNA in the hippocampus of vehicletreated OBX mice were signiÀcantly lower compared with sham-operated group (0.23 ± 0.04 and0.41 ± 0.03, respectively for muscarinic M1 mRNA and 0.12 ± 0.02 and 0.56 ± 0.28, respectively
for ChAT mRNA). Nevertheless, the expression levels of M1 and ChAT mRNA were up-regulated
in OBX mice treated with tacrine (2.5 mg/kg, i.p.) or RJ (250 and 500 mg/kg, p.o.). The expression
levels of M1 mRNA of tacrine and RJ (250 and 500 mg/kg, p.o.) treated groups were 0.35 ± 0.02,
0.38 ± 0.01 and 0.48 ± 0.04, respectively (Fig. 6 (A)) while the expression levels of ChAT mRNAof tacrine and RJ (250 and 500 mg/kg, p.o.) were 0.59 ± 0.11, 0.17 ± 0.01 and 0.24 ± 0.03,
respectively (Fig. 6 (B)).

M1 receptor/ȕ-actinmRNA ratio

1.0
0.8
0.6
0.4
0.2
0
1.0

ChAT/ȕ-actinmRNA ratio

0.8
0.6
0.4
0.2
0

Fig. 6 Effects of tacrine and RJ on M1 (A) and ChAT (B) mRNA expression levels in hippocampus of OBX
mice. Each data column represents the mean ± S.E.M. *P<0.05 vs. mRNA expression levels in vehicletreated OBX mice (one-way ANOVA, Dunnett’s method).

3.6 Effect of RJ on AChE activity
RJ (100 μg/ml) and tacrine (0.05 – 5 μg/ml) were tested for AChE inhibitory activity using
Ellman’s colorimetric method in 96-welled microplate. At concentration of 100 μg/ml, RJ showed
low inhibitory activity (15.50 ± 4.29 %) while tacrine showed high inhibitory effect with IC50 less

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than 50 ng/ml (Table 2). For ex vivo measurement of cholinesterase activity in the brain, theactivity of cortical AChE in tacrine-treated groups was signiÀcantly reduced compared with theactivity measured in vehicle-treated OBX mice (2.21 ± 0.21 nmol/min/mg tissue, respectively).
However, the activity of cortical AChE in OBX mice treated with RJ (500 mg/kg, p.o.) was not
signiÀcantly different compared with the activity of vehicle-treated OBX mice.
7DEOH In vitro inhibitory effects of tacrine on acetylcholinesterase activity
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Ex vivo AChE activity

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

Discussion
The effects of Thunbergia laulifolia leaf extract (RJ) on OBX induced short term memory
deficit were investigated. The results revealed that RJ treatment could ameliorate theimpairments via restoring the function of the central cholinergic system down-regulated by
OBX. In this study, a modified version of the Y-maze test and novel object recognition test
(ORT) were employed to elucidate short-term spatial working memory and non-spatial working
memory, respectively. Previous reports have shown that the modiÀed Y-maze test and ORT are
the experimental paradigms appropriate to evaluate anti-dementia activities of drugs including
natural products [15, 17]. In modified Y-maze test, treatment of tacrine ameliorated spatial
working memory performance indicated by the increasing of total time spent exploring new arm.
However, treatment of RJ did not improve spatial working memory deficit induced by OBX.
Furthermore, the effect of RJ on memory deÀcit using novel object recognition test was studied.

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In sample phase trial, no signiÀcant difference in total time spent exploring two identical objects
was observed between sham and OBX groups, indicating no differences in ability to recognize
objects between animals. In test phase, the results showed that sham-operated mice spent
more time exploring new object, while vehicle treated OBX mice showed no total time difference
between familiar and new object indicating the impairment of non-spatial object recognition
memory. Administration of RJ and tacrine could significantly ameliorate OBX induced object
recognition deÀcit suggesting the ability of RJ in the therapy of cognitive deÀcits in human in the
future.
The therapeutic potential of RJ for cognitive deficit and possible involvement of central
cholinergic systems in its mechanisms of action were next examined. The results revealed thatmice in vehicle-treated OBX group exhibited down-regulated expression levels of both ChAT
and M1 muscarinic mRNAs in hippocampus, a brain region responsible for spatial learning andmemory performance indicating the deÀcit of cognitive performance and pathological changes incentral cholinergic systems conÀrming the results from previous reports [12, 15]. Administrationof RJ and tacrine increased the expression levels of ChAT and muscarinic M1 receptor mRNA in
OBX mice However, the RJ administration did not reduced the activity of cortical AChE in OBX
mice. The results suggest that RJ can protect cognitive function against degeneration of central
cholinergic system caused by OBX via the reversal of expression level of ChAT and muscarinic
M1 receptor mRNA in hippocampus.
The depression model induced by bilateral olfactory bulbectomy (OBX) has been validated
in rats as a good model of depression characterized by behavioural, neuroendocrine andneurochemical changes similar to human depression [18]. Moreover, the cholinergic neuronal
activity does not only involve in cognitive function, but also in the depressive behavior. Therefore,
the anti-depressive like effect of RJ was investigated using TST. RJ processed significantly
antidepressant activity in OBX mice. However, the mechanisms of action are still unknown.
In conclusion, RJ showed improving effects on non-spatial short term memory in OBX mice
which could mediate by the up regulation of the expression levels of ChAT and muscarinic M1
receptor mRNA. Moreover, RJ also showed in vivo antidepressant effect in OBX mice. The results
suggest the beneÀcial of RJ in treatment of depression and improvement cognitive impairment
related to Alzheimer’s disease by improving dysfunction of central cholinergic systems.
Acknowledgements
The Author would like to thank Dr. Pongtip Sithisarn, Department of Pharmacognosy, Facultyof Pharmacy, Mahidol University for her kind cooperation. Author would like to thank alllaboratory members in Department of Medicinal Pharmacology, Institute of Natural Medicine,
University of Toyama. This work was supported by GRANT-IN-AID for general research II
program, Institute of Natural Medicine, University of Toyama, Japan.

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