Journal of Ethnopharmacology 119 (2008) 7480

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Journal of Ethnopharmacology
j our nal homepage: www. el sevi er . com/ l ocat e/ j et hphar m
Cholinesterase inhibitory and anti-amnesic activity of
alkaloids from Corydalis turtschaninovii
Tran Manh Hung
a
, MinKyun Na
a
, Nguyen Tien Dat
a
, Tran Minh Ngoc
a
, UiJoung Youn
a
,
Hong Jin Kim
a
, Byung-Sun Min
b
, JongPill Lee
c
, KiHwan Bae
a,
a
College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea
b
College of Pharmacy, Catholic University of Daegu, Gyeongsan 712-702, Republic of Korea
c
Korea Food and Drug Administration, Seoul 122-704, Republic of Korea
a r t i c l e i n f o
Article history:
Received 14 February 2008
Received in revised form 7 May 2008
Accepted 29 May 2008
Available online 13 June 2008
Keywords:
Corydalis turtschaninovii
Oxoglaucidaline
Pseudodehydrocorydaline
Pseudoberberine
Acetylcholinesterase
Amnesic
a b s t r a c t
Inthe course of screening plants usedinKoreanfolk medicine as memory enhancers, a 70%ethanol extract
of tuber from Corydalis turtschaninovii Besser (Papaveraceae) showed signicant acetylcholinesterase
(AChE) inhibitory activity. Repeated column chromatography led to the isolation of a new aporphine
alkaloid, oxoglaucidaline (9), and a new protoberberine, pseudodehydrocorydaline (13) together with 14
known compounds (18, 1012, and 1416). The chemical structures of isolated compounds were elu-
cidated base on extensive 1D and 2D NMR spectroscopic data. Compounds 116 were investigated in
vitro for their anti-cholinesterase activity using the mice cortex AChE enzyme. In further study, the anti-
amnesic activities of pseudoberberine (16) in mice on the learning and memory impairments induced by
scopolamine (1.0mg/kg, i.p.) were examined. This alkaloid (5.0mg/kg, p.o.) administration signicantly
reversed cognitive impairments in mice by passive avoidance test (P<0.05). It also reduced escape laten-
cies in training trials and prolonged swimming times in the target quadrant during the probe trial in the
water maze task (P<0.05). These results indicated that Corydalis turtschaninovii due to its alkaloids have
anti-cholinesterase activity and pseudoberberine and other alkaloids have anti-amnesic activities that
may be useful for cognitive impairment treatment.
2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Alzheimers disease (AD) is the most common age-related
neurodegenerative disease with many cognitive and neuropsy-
chiatric manifestations that result in progressive disability and
eventual incapacitation. A decrease of acetylcholine in the brain
of patients with AD appears to be a critical element in pro-
ducing dementia (Becker et al., 1988). Loss of cholinergic cells,
particularly in the basal forebrain, is accompanied by loss of
the neurotransmitter acetylcholine. One approach is to inacti-
vate acetylcholinesterase (AChE), the enzyme that cleaves synaptic
acetylcholine and terminates neuronal signaling. AChE inhibitors
increase the availability of acetylcholine in central cholinergic
synapses and are the most promising currently available drugs for
the treatment of AD(Giacobini, 2000). AChEinhibitors fromgeneral
chemical classes such as physostigmine, tacrine, donepezil, galan-
thamine, huperzine A and heptylphysostigmine have been tested
for the symptomatic treatment of AD. Although there have been a

Corresponding author. Tel.: +82 42 821 5925; fax: +82 42 823 6566.
E-mail address: baekh@cnu.ac.kr (K. Bae).
number of reports on the designing and development of synthetic
AChE inhibitors, that were necessary for other studies, which have
been reported the AChE inhibitors derived from medicinal plants
(Oh et al., 2004; Houghton et al., 2006).
In our screening program to search for AChE inhibitors
from plants, a 70% ethanolic extract of Corydalis turtschani-
novii Besser forma yanhusuo (Papaveraceae) exhibited signicant
AChE inhibitory activity (72.5% at 100g/mL). Previously reports
showed that several species of the genera Corydalis have
been used in the treatment of memory dysfunction in folk
medicine (Orhan et al., 2004; Houghton et al., 2006). Cory-
dalis turtschaninovii have been used in traditional medicine for
the treatment of gastric, duodenal ulcer, cardiac arrhythmia dis-
ease (Kamigauchi and Iwasa, 1994), rheumatism, dysmenorrheal
(Tang and Eisenbrand, 1992), and memory dysfunction (Orhan
et al., 2004; Houghton et al., 2006). This tuber contains sev-
eral pharmacologically important alkaloids (Sagara et al., 1985;
Matsuda et al., 1988; Saito et al., 2004). In this paper, we
report the isolation and identication of two new quaternary,
together with several alkaloids with regard to active principles
in AChE activity, and anti-amnesic effect of pseudoberberine in
mice.
0378-8741/$ see front matter 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2008.05.041
T.M. Hung et al. / Journal of Ethnopharmacology 119 (2008) 7480 75
2. Materials and methods
2.1. Plant material
Thetuber of Corydalis turtschaninovii werepurchasedat Yuseong
oriental herbarium market in Daejeon, Korea on September 2006.
The plant was botanically identied by Prof. KiHwan Bae, College
of Pharmacy, Chungnam National University where the voucher
specimens was deposited (CNU-00124).
2.2. Extraction and isolation
The tuber of Corydalis turtschaninovii (2.0kg) were dried and
extracted with 70% EtOH. The solution was evaporated and residue
(110g) was diluted with 3% aqueous HCl to give acidic solution and
insoluble part. The acidic H
2
O layer was then basied with aque-
ous NH
4
OH (pH 9) to produce free bases, and then extracted with
CH
2
Cl
2
, resulting in organic phase (Part A) and aqueous phase (Part
B). Part I was subjected to a silica gel column using step gradient
elutionwithCHCl
3
MeOH(100:1, 80:1, 50:1, 30:1, 15:1, 5:1and1:1,
6000mL each) to afford seven fractions (A1A7). Fraction A2 was
chromatographed further on a silica gel column using step gradient
elution with hexaneacetone (15:1 and 10:1) followed by prepara-
tive TLC (silica gel F
254
plates) to successively give 1 (190mg) and
2 (86mg). Fraction A3 was separated on a silica gel column using
step gradient elution with hexaneEtOAc (10:1 and 5:1) to give ve
sub-fractions (A3.1A3.5). Compounds 3 (445mg) and 4 (50mg)
were isolated fromfraction A3.3 and A3.4 by column chromatogra-
phy using same solvent system as hexaneEtOAc (8:1), separately.
Fraction A4 was subjected to a Sephadex LH-20 columns eluted
with 100% MeOH to afford compounds 5 (5.2mg), and 6 (20.8mg).
Fraction A4.3 was subjected to a YMC gel column and eluted with
MeOHH
2
Ofolic acid (3:1:0.05) to afford compound 7 (6.2mg),
8 (4.5mg), and 9 (4.8mg). Fraction A5 was subjected to a silica
gel column and eluted with CHCl
3
EtOAcMeOH(20:5:1) to afford
compounds 10 (23.1mg), 11 (195.4mg), and 12 (40.3mg). Part
B, aqueous phase, was subjected to Dianion column chromatog-
raphy using step gradient elution with H
2
OMeOH (H
2
O30%
MeOH, 4070% MeOH, and 80100% MeOH) to give three fractions
(B1B3). Fraction B2 was applied to MCI gel column and eluted
with H
2
OMeOH (1:1) to give B2.1B2.5. B2.2 was re-subjected to
MCI gel CHP-20 column chromatography using 2% AcOHMeOH to
give compounds 13 (5mg), and 14 (420mg). Compound 15 (38mg)
was obtained from sub-fraction B2.3 by crystallization in MeOH.
Fraction B2.4 was ltered and applied to MPLC system: ODSS50
column (11mm300mm), UV 245nm, ow rate 2mL/min, using
H
2
OMeOHNH
4
OH (1:4:0.05) to afford compound 16 (39mg).
2.2.1. Oxoglaucidaline (9)
Brown powder; mp 162164

C; UV (CHCl
3
)
max
: 233 and
290nm; IR (KBr)
max
: 2931, 1754, 1610, 1508, 1450, 1250, 1190
and 1040cm
1
; HREI-MS: m/z 383.1328 [M+H]
+
(calculated for
C
21
H
20
NO
6
);
1
H (300MHz, CDCl
3
) and
13
C (75MHz, CDCl
3
) are
shown in Table 1.
2.2.2. Pseudodehydrocorydaline (13)
Yellow powder; mp 187189

C; UV (MeOH)
max
: 287 and
228nm; IR (KBr)
max
: 2918, 2805, 1605, 1535 and 1506cm
1
;
HREI-MS: m/z 366.1705 [M]
+
(calculated for C
22
H
24
NO
4
);
1
H
(300MHz, CD
3
OD) and
13
C (75MHz, CD
3
OD) are shown in Table 2.
2.3. Animals
Adult SpragueDawleymale rats, weighing200250g, andmale
ICR mice, weighing 2025g, were purchased from the Daehan
Table 1
1
H and
13
C NMR assignments of compound 9 (in CD
3
OD)
Position H
a

C
b
1 152.7
2 149.5
3 7.25 (1H, s) 108.9
3a 121.6
4 156.7
5 7.42 (1H, s) 113.9
6a 130.8
7 175.8
7a 128.0
8 8.23 (1H, s) 112.6
9 154.1
10 150.1
11 9.11 (1H, s) 108.9
11a 124.1
12 118.8
12a 124.5
1-OCH3
4.09 (3H, s) 56.1
2-OCH
3
4.11 (3H, s) 60.7
9-OCH
3
4.13 (3H, s) 56.7
10-OCH
3
4.10 (3H, s) 56.1
NCH
3
3.85 (3H, s) 30.7
a
300MHz.
b
75MHz.
BiolinkCo., Ltd. Animals werehoused5or 6per cage, allowedaccess
to water and food ad libitum, and maintained in a constant tem-
perature (231

C) and humidity (6010%) environment under a

12hlight/dark cycle (light on07.3019.30h). Animal treatment and
maintenance were carried out in accordance with the Principle of
Laboratory Animal Care (NIH Publication No. 85-23, revised 1985)
and the Animal Care and Use Guidelines of Chungnam National
University, Korea.
2.4. Rat brain cortex AChE activity
Rats were decapitated; the cortex was rapidly dissected from
the brain on ice and then weighed and homogenized in ve vol-
umes of cold75mMsodiumphosphatebuffer, pH7.4. Homogenates
were centrifuged at 13,000g for 30min at 4

C; supernatants
Table 2
1
H and
13
C NMR assignments of compound 13 (in CDCl
3
)
Position H
a

C
b
1 7.39 (1H, s) 112.1
2 146.3
3 151.9
4 7.13 (1H, s) 115.9
4a 133.4
5 3.19 (2H, t, 5.8) 28.8
6 4.83 (2H, t, 5.8) 58.8
8 9.77 (1H, s) 145.0
8a 120.9
9 8.15 (1H, s) 127.3
10 149.4
11 153.0
12 8.15 (1H, s) 122.1
12a 131.7
13 122.7
14 138.2
14a 135.4
2-OMe 3.96 (3H, s) 56.8
3-OMe 4.12 (3H, s) 57.2
10-OMe 3.92 (3H, s) 62.7
11-OMe 4.21 (3H, s) 57.6
13-Me 3.03 (3H, s) 18.3
a
300MHz.
b
75MHz.
76 T.M. Hung et al. / Journal of Ethnopharmacology 119 (2008) 7480
selected as AChE sources were divided into aliquots and stored at
20

C (Zhao and Tang, 2002). AChE activity was measured by the

principle of Ellman method with some modications (Ellman et
al., 1961). The tested compound was initially dissolved in 0.02%
dimethyl sulfoxide (DMSO) and diluted to various concentra-
tions in sodium phosphate buffer (100mM pH 8.0) immediately
before use. An aliquot of diluted compound solution was then
mixed with sodium phosphate buffer (100mM pH 8.0), acetylth-
iocholine iodide solution (75mM) and Ellmans reagent (10mM
5,5

-dithio-bis[2-nitrobenzoic acid] and 15mM sodium bicarbon-

ate) and reacted at room temperature for 30min. Absorbance was
measured at 410nm immediately after adding the enzyme source
to the reaction mixtures using a spectrophotometer (Shimadzu
UV-1240, Tokyo, Japan). Readings were taken at 30s intervals
for 5min. The concentration of compound required to inhibit
AChE activity by 50% (IC
50
) was calculated using an enzyme
inhibition dose response curve. Tacrine was used as a positive
control.
2.5. Passive avoidance task
Passive avoidance task was carried out in identical illumi-
nated and non-illuminated boxes. The illuminated compartment
(20cm20cm20cm) contained a 100W bulb, and the oor of
non-illuminated compartment (20cm20cm20cm) was com-
posed of 2mm stainless steel rods spaced 1cm apart. These
compartments were separated by a guillotine door (5cm5cm).
For acquisition trial, mice were initially placed in the illuminated
compartment and the door between the two compartments was
opened 10s later. When mice entered the dark compartment, the
door automaticallyclosedandanelectrical foot shock(0.5mA) of 3s
durations was delivered through the stainless steel rods. One hour
before the acquisition trial, mice were administered pseudoberber-
ine (2.5, 5.0, 10.0 and 20.0mg/kg, p.o.) or tacrine (10.0mg/kg, p.o).
After a delay of 60min, amnesia was induced in mice with scopo-
lamine (1.0mg/kg, i.p.) givensubcutaneously. Control animals were
administered 10% Tween 80 solution only. Twenty-four hours after
acquisition trial, the mice were again placed in the illuminated
compartment for the retention trials. The time taken for a mouse
to enter the dark compartment after door opening was measured
as latency times in both acquisition and retention trials. If the mice
stayed in the light compartment for 180s, it was concluded that the
mice had memorized the passive avoidance training after training
trial (Park et al., 1996).
2.6. Morris water maze task
Aspatial test was performedbythemethodof Morris withminor
modication. The water maze is a circular pool (90cm in diameter
and 45cmin height) with a featureless inner surface. The pool was
lled to a depth of 30cm with water containing 500mL of milk
(201

C). The pool was divided into four quadrants of equal area.
A white platform (6cm in diameter and 29cm in height) was then
placed in one of the pool quadrants. The rst experimental day was
dedicated to swimming training for 60s without the submerged
platform. During the ve subsequent days, the mice were given
two daily trials with an inter-trial interval of 30min in the pres-
ence of the platform in place. When a mouse located the platform,
it was permitted to remain on it for 10s. If the mouse did not locate
the platform within 120s, it was placed on the platform for 10s.
The animal was taken to its home cage and was allowed to dry
up under an infrared lamp after each trial (Kim et al., 2006). Dur-
ing each trial session, the time taken to nd the hidden platform
(latency) was recorded. One day after the last training trial sessions,
mice were subjected to a probe trial session in which the platform
was removed from the pool, allowing the mice to swim for 120s to
search for it. A record was kept of the swimming time in the pool
quadrant where the platform had previously been placed. Pseu-
doberberine (5.0mg/kg, p.o.) or tacrine (10.0mg/kg, p.o.) was given
1h before the rst trial session at every consecutive day. Memory
impairment was induced in mice with scopolamine (1.0mg/kg, i.p.)
at 60min after treatment of test samples. Control group received
10% Tween 80 solution only.
2.7. Statistical analysis
The results are expressed as mean value S.D. Statistical analy-
sis was performedusingone-wayANOVA. AP<0.05was considered
statistically signicant.
3. Results and discussion
3.1. Identication of active constituents
Repeated column chromatography led to the isolation of 16
compounds (116). All isolated compounds show positive with
Dragendoffs reagent. The UV, IR, and other physico-chemical data
showed that their chemical structures are belonging to isoquino-
line alkaloid. Based on the mass,
1
H, and
13
C NMR spectra, the
isolated alkaloids can be divided to two main skeletons: protober-
berine and aporphine skeleton. By comparing spectral data with
those reported in the literatures and authentic samples, the known
compounds were identied as corydaline (1) (Lee et al., 2001),
xylopinine (2) (Sasagawa et al., 1969), stylopine (3) (Kiryakov et
al., 1982), protopine (4) (Weiguang et al., 1987), oxypseudopalma-
tine (5), tetrahydropalmatine (6), epiberberine (7), palmatine (8)
(Patraet al., 1987), corytenchine(10) (Sasagawaet al., 1969), berber-
ine (14), pseudocoptisine (15), and pseudoberberine (16) (Lee et
al., 2001), which have basic skeleton as protoberberine (Shamma,
1972). Meanwhile oxoglaucine (11), and glaucine (12) (Saito et al.,
2004) have aporphine skeleton. All chemical structure of isolated
compounds is shown in Fig. 1.
Compound 9 was obtained as a brown amorphous powder and
was positive toDragendoffs reagent. The HREI-MS revealeda [M]
+
at m/z 383.1328(calc. 383.1331) correspondingtothemolecular for-
mula C
21
H
20
NO
6
. The UV absorptions at 233 and 290nm, revealed
9 as a 1,2-disubstituted N-methylcarbonyl aporphine (Shamma,
1972). The IR spectrum showed absorption bands centered at
2931cm
1
(methoxygroups), 1610and1508cm
1
(aromatic rings),
and 1654cm
1
(carbonyl group). The
1
H NMR spectrum showed
four single signals at 4.09 (3H, s), 4.11 (3H, s), 4.13 (3H, s), and 4.10
(3H, s), whichrevealedtothepresenceof four methoxygroups. Four
single signals were observed at 7.25 (1H, s, H-3), 7.42 (1H, s, H-4),
8.23 (1H, s, H-8), and 9.11 (1H, s, H-11), which were assignable to
aromatic protons. In addition, the
1
HNMRspectrumalso contained
one N-methyl protonsignal at 3.85(3H, s). The
13
CNMRcombined
with DEPT experiments showed the presence of 21 carbon atoms
consisting of four methine signals as aromatic carbons at 108.9(C-
3), 113.9 (C-4), 112.6 (C-8) and 108.9 (C-11), four methoxy signals at
56.1 (1-OCH
3
), 60.7 (2-OCH
3
), 56.7 (9-OCH
3
), and 56.1 (10-OCH
3
),
one nitrogen bonding methyl at 30.7, and 12 quaternary carbons,
one of them at 175.8 was characteristic of a carbonyl group, and
one at 156.7 was characteristic of aromatic carbon with hydroxyl
group bonded. The
1
H and
13
C NMR features were closely similar
to those of oxoglaucine (Saito et al., 2004), and corunnine (Ribas et
al., 1971) except for the change of a hydroxyl group that related to
the aporphine skeleton. In particular, HMQC spectrum showed the
following connections H-3/C-3, H-5/C-5, H-8/C-8 and H-11/C-11.
The HMBC spectrumconrmed the correlations between N-methyl
T.M. Hung et al. / Journal of Ethnopharmacology 119 (2008) 7480 77
Fig. 1. Structures of isolated compounds, protoberberine (18, 10, and 1316) and aporphine skeleton (9, 11, and 12).
proton (
H
3.85, s) and C-6a (
C
130.8) and C-5 (
C
113.9). The corre-
lation between H-5 (
H
7.42, s) and C-4 (
C
156.7) could be assigned
for oxygenatedaromatic carbonsuggesting the locationof hydroxyl
group on C-4 (Fig. 2). Based on the above analysis, the structure of
compound 9 was elucidated as oxoglaucidaline.
Compound 13 was isolated as a yellowpowder and was positive
to Dragendoffs reagent. The molecular formula of 2was deducedto
be C
22
H
24
NO
4
on the basic of the peak at m/z 366.1705 [M]
+
in the
HREI-MS. The IR spectrumshowed absorption bands at 1605, and
1535cm
1
, characteristic of aromatic groups. The
1
H NMR spec-
trumrevealed proton signals at 4.28 (3H, s), 4.07 (3H, s), 4.00 (3H,
s), and 3.95 (3H, s) indicating the presence of four methoxy groups.
Two 2H-triplet signals at 4.82 (2H, t, J =5.8Hz, H-6), and 3.19 (2H,
t, J =5.8Hz, H-5) were assigned to protons of [ CH
2
CH
2
] bonded
with an aryl moiety and a nitrogen atom. Four aromatic proton sig-
nals were observed at 7.39 (1H, s, H-1), 7.13 (1H, s, H-4), 8.15 (1H,
s, H-9) and 8.14 (1H, s, H-12) assignable to aromatic protons. In
addition, a downeld singlet signal at 9.77 (1H, s, H-8) indicated
that is other aromatic proton bonded with a nitrogen atom and a
signal at 3.03 (3H, s, 13-Me) due to a methyl group bonded with
78 T.M. Hung et al. / Journal of Ethnopharmacology 119 (2008) 7480
Fig. 2. Selected HC long-range correlations in HMBC and
1
H
1
H correlation in COSY spectrum of 9 and 13.
aromatic ring. The
13
C NMR spectrum revealed the presence of 22
carbon atoms including benzene rings, methyl, methoxy, methy-
lene groups. The
1
H and
13
C NMR features were closely similar to
those of dehydrocorydaline (Miyazaki et al., 1975) except for the
change of one methoxy group from C-9 (
H
8.15,
C
127.3) to C-11
(
C
153.0) in tetrahydroprotoberberine skeleton (Lee et al., 2001).
The structure deductionof 13was further conrmedby the analysis
of HMBC and COSYspectra (Fig. 2). Accordingly, this compound was
elucidated as pseudodehydrocorydaline, this is the rst occurrence
of 13 from a natural source.
3.2. AChE inhibitory activity
For investigation of anti-cholinesterase activity of isolated alka-
loids, mouse brain cortex was selected as a source of AChE enzyme
Table 3
Inhibition of compounds on mouse cortex AChE
Compound IC
50
(M)
a
1 30.71.5
*
2 38.11.8
*
3 15.81.2
*
4 14.50.5
*
5 >50
6 41.32.2
*
7 6.50.5
*
8 10.40.4
*
9 27.11.8
*
10 >50
11 48.71.8
*
12 >50
13 8.40.5
*
14 4.70.2
*
15 4.30.3
*
16 4.50.2
*
Tacrine
b
0.170.02
a
Results are the mean of three replications.
b
Reference control.
*
P<0.05, compared with tacrine.
(Zhao and Tang, 2002), and the results (IC
50
) were summarized in
Table 3. Of these, compounds 1, 2, 5, 6, and 1012 showed weak
inhibitory activity with IC
50
values over than 30.0M. Compound
3, 4, 8, and 9 showed moderate inhibitory activity with IC
50
values
ranging from 10.4 to 27.1M, whereas, compounds 7, and 1316
exhibited potent inhibitory activity with the IC
50
values less than
6.5M. With the same AChE source, tacrine, which was used as
positive control, exhibited inhibitory activity with IC
50
value of
170.2nM.
3.3. Effect of pseudoberberine on passive avoidance
Since such AChE inhibitors have been used to reduce the
cholinergic decit in AD, and based on the inhibition effect of
pseudoberberine (16), one of the most active components, we
assessed whether pseudoberberine, improves memory function by
passive avoidance learning, which is largely dependent on long-
term memory. When placed into the bright side of a step-through
box, mice quickly entered the dark compartment. Mice were condi-
tioned using a mild foot shock after entering the dark compartment
and hesitated to re-enter the dark compartment when tested
24h later. By using this shuttle box system, saline-treated con-
trol mice stay in the light compartment for 18010s after the
passive avoidance training. Meanwhile, the mice given scopo-
lamine (1.0mg/kg body weight) stayed in the light compartment
for only 213s before moving into the dark compartment. Thus,
the step-through latency of the scopolamine-treated mice was sig-
nicantly shorter than that of vehicle-treated control mice (Fig. 3,
P<0.05). In tacrine plus scopolamine-treated mice, step-through
latencywas signicantlygreater thanfor scopolamine-treatedmice
(1526s vs. 223s). Moreover, the reduction in step-through
latency induced by scopolamine was signicantly reversed by
pseudoberberine 5.0 and 10.0mg/kg, p.o (P<0.05) at 1188 and
13010s). However, at doses of 2.5 and 20.0mg/kg, this com-
pound did not exhibit a much signicant effect. Latency time
during the acquisition trial was not affected by any drug treatment
(Fig. 3).
T.M. Hung et al. / Journal of Ethnopharmacology 119 (2008) 7480 79
Fig. 3. Effect of a single administration of pseudoberberine (16) on scopolamine-
induced memory decits as determined by the passive avoidance task. At 60min
before acquisition trials, pseudocoptisine (2.5, 5.0, 10.0 or 20.0mg/kg, p.o.), tacrine
(10.0mg/kg, p.o.) or vehicle (same volume of 10% Tween 80) solution was admin-
istered to mice. Memory impairment was induced by administering scopolamine
(1.0mg/kg, i.p.). Five different animals were used per treatment group. Acquisi-
tion trials were carried out 30min after a single scopolamine treatment. 24h after
the acquisition trials, retention trials were carried out for 3min. Data represent
means S.E.M. (
#
P<0.05vs. vehicle control group; *P<0.05vs. scopolamine-treated
group).
3.4. Effect of pseudoberberine on Morris water maze
The effect of pseudoberberine (16) (5.0mg/kg, p.o.) on spa-
tial learning (long-term or short-term memory) was evaluated
using the Morris water maze test. The saline-treated control
group rapidly learned the location of the platform (Fig. 4A).
The scopolamine-treated animals exhibited longer escape laten-
cies (the time taken to nd the platform) throughout training
days than the vehicle-treated controls (P<0.001). Pseudoberber-
ine (5.0mg/kg) signicantly attenuated the effects of scopolamine
on escape latency (P<0.05), as did tacrine (P<0.05). On the day
of probe testing, a signicant group effect was observed on swim-
ming times in the probe test (Fig. 4B). Swimming times within the
platform quadrant for the scopolamine-treated group were signif-
icantly lower than those of vehicle-treated control group animals
(Fig. 4B, P<0.05). Moreover, the shorter swimming times within
the platform quadrant induced by scopolamine were signicantly
reversed by pseudocoptisine or tacrine (P<0.05).
4. Discussion
As mentioned earlier, AChE inhibitors increased the availabil-
ity of acetylcholine in central cholinergic synapses and are the
most promising currently available drugs for the treatment of AD
(Giacobini, 1990; LeDoux, 1993). Scopolamine, which is a mus-
carinic receptor antagonist and can pass through bloodbrain
barrier causes amnesia in animals by blocking cholinergic neu-
rotransmission. Cholinergic interneurons in the striatum are an
even richer source of AChE and would also be affected strongly
by such enzyme inhibitors. In some species of Papaveraceae fam-
ily, AChE inhibitory activity has been detected and traced to the
benzylisoquinoline alkaloids present like berberine (Kuznetsova
et al., 2002), corynoline, protopine, palmatine (Ito et al., 1990;
Kim et al., 2004; Houghton et al., 2006), corydaline and bulbocap-
nine (Adsersen et al., 2007). The in vitro activity of berberine and
protopine showed to be translated into effects since it improved
scopolamine-induced amnesia in rats (Peng et al., 1997; Kim et al.,
1999). Natural drugs fromplant species used as memory enhancers
were identied and furthermore Corydalis species were selected
as several species of this genera have been used in treatment of
memory dysfunction in folk medicines. In this study, some iso-
lated alkaloids from Corydalis turtschaninovii can manifest AChE
inhibitory activity, although they are less effective than tacrine.
However, those compounds were puried from a natural medic-
inal plant used as a folk medicine for many years, and the low
molecular mass material can easily reach the site of action fol-
lowing oral administration, since it crosses the bloodbrain barrier,
which is the tight function controlling the transport of the mate-
rial in to the brain (Broadwell and Sofroniew, 1993). In addition,
through our in vivo experiments, pseudoberberine, one of the most
active compound with the treatment of 5.0mg/kg could reverse
the decits produced by scopolamine treatment in comparison
with the vehicle-treated group on passive avoidance task and sig-
nicantly shortened the escape latency, improved the swimming
Fig. 4. Effect of pseudoberberine (16) on latency time during the training trial ses-
sions (A), and on swimming time (B) during the probe trial session of the Morris
water maze task in scopolamine-induced memory decits mice. At 60min before
trainingtrial sessions, pseudoberberine(5.0mg/kg, p.o.), tacrine(10.0mg/kg, p.o.) or
vehicle (same volume of 10%Tween 80) solution was administered to mice. Memory
impairment was induced by scopolamine treatment (1.0mg/kg, i.p.). Five different
animals were used per treatment group. The training trial and probe trial sessions
were performed as described in Section 2. Data represent means S.E.M. (
#
P<0.05
vs. vehicle control group; *P<0.05 vs. scopolamine-treated group).
80 T.M. Hung et al. / Journal of Ethnopharmacology 119 (2008) 7480
time within the zone of platform on water maze task. The passive
avoidance is generally accepted as an indicator of long-term mem-
ory in animals (LeDoux, 1993), and the water maze-learning task
was used to assess hippocampal-dependent spatial learning abil-
ity (Morris, 1984). Pseudoberberine could inhibit AChE activity in
a dose-dependent manner (IC
50
=4.5M), these suggest that abil-
ity to alleviate scopolamine-induced memory impairment or the
anti-amnesic activity of this alkaloid is, in part, mediated by the
AchE inhibition. It would be interesting to indicate that quaternary
nitrogen is necessary for strong AChE inhibitory activity in alka-
loids possessing the benzylisoquinoline skeleton from Corydalis
species (Houghton et al., 2006; Hung et al., 2008). Interestingly,
the anti-cholinesterase activity of the compounds having both of
aromatic methylenedioxy groups and quaternary atom of nitro-
gen are somewhat stronger than that of remaining compounds.
It is worthy to suggest that aromatic methylenedioxy maybe also
important factors for AChE inhibitory activity. Thus, in accordance
with our previous result (Hung et al., 2008), pseudoberberine and
some other constituents onCorydalis turtschaninovii are able toplay
an important role on the treatment of memory dysfunction of this
plant intraditional medicine. However, more studies shouldbe pre-
served if those compounds may be offered as a useful therapeutic
choice in the prevention of AD.
Acknowledgments
This study was partially supported by a grant from the Korean
Food and Drug Administration (2007). We are grateful to KBSI for
measuring 1D, 2D NMR and MS spectra.
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