Journal of Ethnopharmacology 119 (2008) 74–80

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Journal of Ethnopharmacology

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Cholinesterase inhibitory and anti-amnesic activity ofalkaloids from Corydalis turtschaninovii

Tran Manh Hunga, MinKyun Naa, Nguyen Tien Data, Tran Minh Ngoca, UiJoung Youna,Hong Jin Kima, Byung-Sun Minb, JongPill Leec, KiHwan Baea,∗

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 Koreac

planturtsch. Rep

(9), an10–1

1Dsteradober.p.) wmentprolo. The

ty ane imp

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 2008Received in revised form 7 May 2008Accepted 29 May 2008Available online 13 June 2008

Keywords:Corydalis turtschaninoviiOxoglaucidalinePseudodehydrocorydalinePseudoberberineAcetylcholinesteraseAmnesic

a b s t r a c t

In the course of screeningof tuber from Corydalis t(AChE) inhibitory activityalkaloid, oxoglaucidalineknown compounds (1–8,cidated base on extensivevitro for their anti-cholineamnesic activities of pseuscopolamine (1.0 mg/kg, ireversed cognitive impaircies in training trials andwater maze task (P < 0.05)anti-cholinesterase activimay be useful for cognitiv

1. Introduction

Alzheimer’s disease (AD) is the most common age-relatedneurodegenerative disease with many cognitive and neuropsy-chiatric manifestations that result in progressive disability andeventual incapacitation. A decrease of acetylcholine in the brainof 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 ofthe neurotransmitter acetylcholine. One approach is to inacti-vate acetylcholinesterase (AChE), the enzyme that cleaves synapticacetylcholine and terminates neuronal signaling. AChE inhibitorsincrease the availability of acetylcholine in central cholinergicsynapses and are the most promising currently available drugs forthe treatment of AD (Giacobini, 2000). AChE inhibitors from generalchemical classes such as physostigmine, tacrine, donepezil, galan-thamine, huperzine A and heptylphysostigmine have been testedfor 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).

0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.jep.2008.05.041

s used in Korean folk medicine as memory enhancers, a 70% ethanol extractaninovii Besser (Papaveraceae) showed significant acetylcholinesteraseeated column chromatography led to the isolation of a new aporphined a new protoberberine, pseudodehydrocorydaline (13) together with 14

2, and 14–16). The chemical structures of isolated compounds were elu-and 2D NMR spectroscopic data. Compounds 1–16 were investigated inse activity using the mice cortex AChE enzyme. In further study, the anti-berine (16) in mice on the learning and memory impairments induced byere examined. This alkaloid (5.0 mg/kg, p.o.) administration significantlys in mice by passive avoidance test (P < 0.05). It also reduced escape laten-nged swimming times in the target quadrant during the probe trial in these results indicated that Corydalis turtschaninovii due to its alkaloids haved pseudoberberine and other alkaloids have anti-amnesic activities thatairment treatment.

© 2008 Elsevier Ireland Ltd. All rights reserved.

number of reports on the designing and development of synthetic

AChE inhibitors, that were necessary for other studies, which havebeen reported the AChE inhibitors derived from medicinal plants(Oh et al., 2004; Houghton et al., 2006).

In our screening program to search for AChE inhibitorsfrom plants, a 70% ethanolic extract of Corydalis turtschani-novii Besser forma yanhusuo (Papaveraceae) exhibited significantAChE inhibitory activity (72.5% at 100 �g/mL). Previously reportsshowed that several species of the genera Corydalis havebeen used in the treatment of memory dysfunction in folkmedicine (Orhan et al., 2004; Houghton et al., 2006). Cory-dalis turtschaninovii have been used in traditional medicine forthe treatment of gastric, duodenal ulcer, cardiac arrhythmia dis-ease (Kamigauchi and Iwasa, 1994), rheumatism, dysmenorrheal(Tang and Eisenbrand, 1992), and memory dysfunction (Orhanet 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, wereport the isolation and identification of two new quaternary,together with several alkaloids with regard to active principlesin AChE activity, and anti-amnesic effect of pseudoberberine inmice.

nopharmacology 119 (2008) 74–80 75

Table 11H and 13C NMR assignments of compound 9 (in CD3OD)

Position ıHa ıC

b

10-OCH3 4.10 (3H, s) 56.1N–CH3 3.85 (3H, s) 30.7

a 300 MHz.b 75 MHz.

Biolink Co., Ltd. Animals were housed 5 or 6 per cage, allowed accessto water and food ad libitum, and maintained in a constant tem-perature (23 ± 1 ◦C) and humidity (60 ± 10%) environment under a12 h light/dark cycle (light on 07.30–19.30 h). Animal treatment andmaintenance were carried out in accordance with the Principle ofLaboratory Animal Care (NIH Publication No. 85-23, revised 1985)and the Animal Care and Use Guidelines of Chungnam NationalUniversity, Korea.

2.4. Rat brain cortex AChE activity

Rats were decapitated; the cortex was rapidly dissected fromthe brain on ice and then weighed and homogenized in five vol-umes of cold 75 mM sodium phosphate buffer, pH 7.4. Homogenateswere centrifuged at 13,000 × g for 30 min at 4 ◦C; supernatants

Table 21H and 13C NMR assignments of compound 13 (in CDCl3)

a b

T.M. Hung et al. / Journal of Eth

2. Materials and methods

2.1. Plant material

The tuber of Corydalis turtschaninovii were purchased at Yuseongoriental herbarium market in Daejeon, Korea on September 2006.The plant was botanically identified by Prof. KiHwan Bae, Collegeof Pharmacy, Chungnam National University where the voucherspecimens was deposited (CNU-00124).

2.2. Extraction and isolation

The tuber of Corydalis turtschaninovii (2.0 kg) were dried andextracted with 70% EtOH. The solution was evaporated and residue(110 g) was diluted with 3% aqueous HCl to give acidic solution andinsoluble part. The acidic H2O layer was then basified with aque-ous NH4OH (pH 9) to produce free bases, and then extracted withCH2Cl2, resulting in organic phase (Part A) and aqueous phase (PartB). Part I was subjected to a silica gel column using step gradientelution with CHCl3–MeOH (100:1, 80:1, 50:1, 30:1, 15:1, 5:1 and 1:1,6000 mL each) to afford seven fractions (A1–A7). Fraction A2 waschromatographed further on a silica gel column using step gradientelution with hexane–acetone (15:1 and 10:1) followed by prepara-tive TLC (silica gel F254 plates) to successively give 1 (190 mg) and2 (86 mg). Fraction A3 was separated on a silica gel column usingstep gradient elution with hexane–EtOAc (10:1 and 5:1) to give fivesub-fractions (A3.1–A3.5). Compounds 3 (445 mg) and 4 (50 mg)were isolated from fraction A3.3 and A3.4 by column chromatogra-phy using same solvent system as hexane–EtOAc (8:1), separately.Fraction A4 was subjected to a Sephadex LH-20 columns elutedwith 100% MeOH to afford compounds 5 (5.2 mg), and 6 (20.8 mg).Fraction A4.3 was subjected to a YMC gel column and eluted withMeOH–H2O–folic acid (3:1:0.05) to afford compound 7 (6.2 mg),8 (4.5 mg), and 9 (4.8 mg). Fraction A5 was subjected to a silicagel column and eluted with CHCl3–EtOAc–MeOH (20:5:1) to affordcompounds 10 (23.1 mg), 11 (195.4 mg), and 12 (40.3 mg). PartB, aqueous phase, was subjected to Dianion column chromatog-raphy using step gradient elution with H2O–MeOH (H2O–30%MeOH, 40–70% MeOH, and 80–100% MeOH) to give three fractions(B1–B3). Fraction B2 was applied to MCI gel column and elutedwith H2O–MeOH (1:1) to give B2.1–B2.5. B2.2 was re-subjected toMCI gel CHP-20 column chromatography using 2% AcOH–MeOH togive compounds 13 (5 mg), and 14 (420 mg). Compound 15 (38 mg)was obtained from sub-fraction B2.3 by crystallization in MeOH.

Fraction B2.4 was filtered and applied to MPLC system: ODS–S–50column (11 mm × 300 mm), UV 245 nm, flow rate 2 mL/min, usingH2O–MeOH–NH4OH (1:4:0.05) to afford compound 16 (39 mg).

2.2.1. Oxoglaucidaline (9)Brown powder; mp 162–164 ◦C; UV (CHCl3) �max: 233 and

290 nm; IR (KBr) �max: 2931, 1754, 1610, 1508, 1450, 1250, 1190and 1040 cm−1; HR–EI-MS: m/z 383.1328 [M+H]+ (calculated forC21H20NO6); 1H (300 MHz, CDCl3) and 13C (75 MHz, CDCl3) areshown in Table 1.

2.2.2. Pseudodehydrocorydaline (13)Yellow powder; mp 187–189 ◦C; UV (MeOH) �max: 287 and

228 nm; IR (KBr) �max: 2918, 2805, 1605, 1535 and 1506 cm−1;HR–EI-MS: m/z 366.1705 [M]+ (calculated for C22H24NO4); 1H(300 MHz, CD3OD) and 13C (75 MHz, CD3OD) are shown in Table 2.

2.3. Animals

Adult Sprague–Dawley male rats, weighing 200–250 g, and maleICR mice, weighing 20–25 g, were purchased from the Daehan

1 152.72 149.53 7.25 (1H, s) 108.93a 121.64 156.75 7.42 (1H, s) 113.96a 130.87 175.87a 128.08 8.23 (1H, s) 112.69 154.110 150.111 9.11 (1H, s) 108.911a 124.112 118.812a 124.51-OCH3 4.09 (3H, s) 56.12-OCH3 4.11 (3H, s) 60.79-OCH3 4.13 (3H, s) 56.7

Position ıH ıC

1 7.39 (1H, s) 112.12 146.33 151.94 7.13 (1H, s) 115.94a 133.45 3.19 (2H, t, 5.8) 28.86 4.83 (2H, t, 5.8) 58.88 9.77 (1H, s) 145.08a 120.99 8.15 (1H, s) 127.310 149.411 153.012 8.15 (1H, s) 122.112a 131.713 122.714 138.214a 135.42-OMe 3.96 (3H, s) 56.83-OMe 4.12 (3H, s) 57.210-OMe 3.92 (3H, s) 62.711-OMe 4.21 (3H, s) 57.613-Me 3.03 (3H, s) 18.3

a 300 MHz.b 75 MHz.

noph

76 T.M. Hung et al. / Journal of Eth

selected as AChE sources were divided into aliquots and stored at−20 ◦C (Zhao and Tang, 2002). AChE activity was measured by theprinciple of Ellman method with some modifications (Ellman etal., 1961). The tested compound was initially dissolved in 0.02%dimethyl sulfoxide (DMSO) and diluted to various concentra-tions in sodium phosphate buffer (100 mM pH 8.0) immediatelybefore use. An aliquot of diluted compound solution was thenmixed with sodium phosphate buffer (100 mM pH 8.0), acetylth-iocholine iodide solution (75 mM) and Ellman’s reagent (10 mM5,5′-dithio-bis[2-nitrobenzoic acid] and 15 mM sodium bicarbon-ate) and reacted at room temperature for 30 min. Absorbance wasmeasured at 410 nm immediately after adding the enzyme sourceto the reaction mixtures using a spectrophotometer (ShimadzuUV-1240, Tokyo, Japan). Readings were taken at 30 s intervalsfor 5 min. The concentration of compound required to inhibitAChE activity by 50% (IC50) was calculated using an enzymeinhibition dose response curve. Tacrine was used as a positivecontrol.

2.5. Passive avoidance task

Passive avoidance task was carried out in identical illumi-nated and non-illuminated boxes. The illuminated compartment(20 cm × 20 cm × 20 cm) contained a 100 W bulb, and the floor ofnon-illuminated compartment (20 cm × 20 cm × 20 cm) was com-posed of 2 mm stainless steel rods spaced 1 cm apart. Thesecompartments were separated by a guillotine door (5 cm × 5 cm).For acquisition trial, mice were initially placed in the illuminatedcompartment and the door between the two compartments wasopened 10 s later. When mice entered the dark compartment, thedoor automatically closed and an electrical foot shock (0.5 mA) of 3 sdurations was delivered through the stainless steel rods. One hourbefore the acquisition trial, mice were administered pseudoberber-ine (2.5, 5.0, 10.0 and 20.0 mg/kg, p.o.) or tacrine (10.0 mg/kg, p.o).After a delay of 60 min, amnesia was induced in mice with scopo-lamine (1.0 mg/kg, i.p.) given subcutaneously. Control animals wereadministered 10% Tween 80 solution only. Twenty-four hours afteracquisition trial, the mice were again placed in the illuminatedcompartment for the retention trials. The time taken for a mouseto enter the dark compartment after door opening was measuredas latency times in both acquisition and retention trials. If the micestayed in the light compartment for 180 s, it was concluded that themice had memorized the passive avoidance training after trainingtrial (Park et al., 1996).

2.6. Morris water maze task

A spatial test was performed by the method of Morris with minormodification. The water maze is a circular pool (90 cm in diameterand 45 cm in height) with a featureless inner surface. The pool wasfilled to a depth of 30 cm with water containing 500 mL of milk(20 ± 1 ◦C). The pool was divided into four quadrants of equal area.A white platform (6 cm in diameter and 29 cm in height) was thenplaced in one of the pool quadrants. The first experimental day wasdedicated to swimming training for 60 s without the submergedplatform. During the five subsequent days, the mice were giventwo daily trials with an inter-trial interval of 30 min in the pres-ence of the platform in place. When a mouse located the platform,it was permitted to remain on it for 10 s. If the mouse did not locatethe platform within 120 s, it was placed on the platform for 10 s.The animal was taken to its home cage and was allowed to dryup under an infrared lamp after each trial (Kim et al., 2006). Dur-ing each trial session, the time taken to find 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

armacology 119 (2008) 74–80

was removed from the pool, allowing the mice to swim for 120 s tosearch for it. A record was kept of the swimming time in the poolquadrant where the platform had previously been placed. Pseu-doberberine (5.0 mg/kg, p.o.) or tacrine (10.0 mg/kg, p.o.) was given1 h before the first trial session at every consecutive day. Memoryimpairment was induced in mice with scopolamine (1.0 mg/kg, i.p.)at 60 min after treatment of test samples. Control group received10% Tween 80 solution only.

2.7. Statistical analysis

The results are expressed as mean value ± S.D. Statistical analy-sis was performed using one-way ANOVA. A P < 0.05 was consideredstatistically significant.

3. Results and discussion

3.1. Identification of active constituents

Repeated column chromatography led to the isolation of 16compounds (1–16). All isolated compounds show positive withDragendoff’s reagent. The UV, IR, and other physico-chemical datashowed that their chemical structures are belonging to isoquino-line alkaloid. Based on the mass, 1H, and 13C NMR spectra, theisolated alkaloids can be divided to two main skeletons: protober-berine and aporphine skeleton. By comparing spectral data withthose reported in the literatures and authentic samples, the knowncompounds were identified as corydaline (1) (Lee et al., 2001),xylopinine (2) (Sasagawa et al., 1969), stylopine (3) (Kiryakov etal., 1982), protopine (4) (Weiguang et al., 1987), oxypseudopalma-tine (5), tetrahydropalmatine (6), epiberberine (7), palmatine (8)(Patra et al., 1987), corytenchine (10) (Sasagawa et al., 1969), berber-ine (14), pseudocoptisine (15), and pseudoberberine (16) (Lee etal., 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 isolatedcompounds is shown in Fig. 1.

Compound 9 was obtained as a brown amorphous powder andwas positive to Dragendoff’s reagent. The HR–EI-MS revealed a [M]+

at m/z 383.1328 (calc. 383.1331) corresponding to the molecular for-mula C21H20NO6. The UV absorptions at 233 and 290 nm, revealed9 as a 1,2-disubstituted N-methylcarbonyl aporphine (Shamma,1972). The IR spectrum showed absorption bands centered at

2931 cm−1 (methoxy groups), 1610 and 1508 cm−1 (aromatic rings),and 1654 cm−1 (carbonyl group). The 1H NMR spectrum showedfour single signals at ı 4.09 (3H, s), 4.11 (3H, s), 4.13 (3H, s), and 4.10(3H, s), which revealed to the presence of four methoxy groups. Foursingle signals were observed at ı 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 toaromatic protons. In addition, the 1H NMR spectrum also containedone N-methyl proton signal at ı 3.85 (3H, s). The 13C NMR combinedwith DEPT experiments showed the presence of 21 carbon atomsconsisting of four methine signals as aromatic carbons at ı 108.9 (C-3), 113.9 (C-4), 112.6 (C-8) and 108.9 (C-11), four methoxy signals atı 56.1 (1-OCH3), 60.7 (2-OCH3), 56.7 (9-OCH3), and 56.1 (10-OCH3),one nitrogen bonding methyl at ı 30.7, and 12 quaternary carbons,one of them at ı 175.8 was characteristic of a carbonyl group, andone at ı 156.7 was characteristic of aromatic carbon with hydroxylgroup bonded. The 1H and 13C NMR features were closely similarto those of oxoglaucine (Saito et al., 2004), and corunnine (Ribas etal., 1971) except for the change of a hydroxyl group that related tothe aporphine skeleton. In particular, HMQC spectrum showed thefollowing connections H-3/C-3, H-5/C-5, H-8/C-8 and H-11/C-11.The HMBC spectrum confirmed the correlations between N-methyl

T.M. Hung et al. / Journal of Ethnopharmacology 119 (2008) 74–80 77

Fig. 1. Structures of isolated compounds, protoberberine (1–8

proton (ıH 3.85, s) and C-6a (ıC 130.8) and C-5 (ıC 113.9). The corre-lation between H-5 (ıH 7.42, s) and C-4 (ıC 156.7) could be assignedfor oxygenated aromatic carbon suggesting the location of hydroxylgroup on C-4 (Fig. 2). Based on the above analysis, the structure ofcompound 9 was elucidated as oxoglaucidaline.

Compound 13 was isolated as a yellow powder and was positiveto Dragendoff’s reagent. The molecular formula of 2 was deduced tobe C22H24NO4 on the basic of the peak at m/z 366.1705 [M]+ in theHR–EI-MS. The IR spectrum showed absorption bands at 1605, and1535 cm−1, characteristic of aromatic groups. The 1H NMR spec-

, 10, and 13–16) and aporphine skeleton (9, 11, and 12).

trum revealed proton signals at ı 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 ı 4.82 (2H, t, J = 5.8 Hz, H-6), and 3.19 (2H,t, J = 5.8 Hz, H-5) were assigned to protons of [ CH2 CH2 ] bondedwith an aryl moiety and a nitrogen atom. Four aromatic proton sig-nals were observed at ı 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. Inaddition, a downfield singlet signal at ı 9.77 (1H, s, H-8) indicatedthat is other aromatic proton bonded with a nitrogen atom and asignal at ı 3.03 (3H, s, 13-Me) due to a methyl group bonded with

78 T.M. Hung et al. / Journal of Ethnopharmacology 119 (2008) 74–80

Fig. 2. Selected H–C long-range correlations in HMBC an

aromatic ring. The 13C NMR spectrum revealed the presence of 22carbon atoms including benzene rings, methyl, methoxy, methy-lene groups. The 1H and 13C NMR features were closely similar tothose of dehydrocorydaline (Miyazaki et al., 1975) except for thechange of one methoxy group from C-9 (ıH 8.15, ıC 127.3) to C-11(ıC 153.0) in tetrahydroprotoberberine skeleton (Lee et al., 2001).The structure deduction of 13 was further confirmed by the analysisof HMBC and COSY spectra (Fig. 2). Accordingly, this compound waselucidated as pseudodehydrocorydaline, this is the first occurrenceof 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 3Inhibition of compounds on mouse cortex AChE

Compound IC50 (�M)a

1 30.7 ± 1.5*

2 38.1 ± 1.8*

3 15.8 ± 1.2*

4 14.5 ± 0.5*

5 >506 41.3 ± 2.2*

7 6.5 ± 0.5*

8 10.4 ± 0.4*

9 27.1 ± 1.8*

10 >5011 48.7 ± 1.8*

12 >5013 8.4 ± 0.5*

14 4.7 ± 0.2*

15 4.3 ± 0.3*

16 4.5 ± 0.2*

Tacrineb 0.17 ± 0.02

a Results are the mean of three replications.b Reference control.* P < 0.05, compared with tacrine.

d 1H–1H correlation in COSY spectrum of 9 and 13.

(Zhao and Tang, 2002), and the results (IC50) were summarized inTable 3. Of these, compounds 1, 2, 5, 6, and 10–12 showed weakinhibitory activity with IC50 values over than 30.0 �M. Compound3, 4, 8, and 9 showed moderate inhibitory activity with IC50 valuesranging from 10.4 to 27.1 �M, whereas, compounds 7, and 13–16exhibited potent inhibitory activity with the IC50 values less than6.5 �M. With the same AChE source, tacrine, which was used aspositive control, exhibited inhibitory activity with IC50 value of170.2 nM.

3.3. Effect of pseudoberberine on passive avoidance

Since such AChE inhibitors have been used to reduce thecholinergic deficit in AD, and based on the inhibition effect ofpseudoberberine (16), one of the most active components, weassessed 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-throughbox, mice quickly entered the dark compartment. Mice were condi-tioned using a mild foot shock after entering the dark compartmentand hesitated to re-enter the dark compartment when tested24 h later. By using this shuttle box system, saline-treated con-trol mice stay in the light compartment for 180 ± 10 s after thepassive avoidance training. Meanwhile, the mice given scopo-lamine (1.0 mg/kg body weight) stayed in the light compartmentfor only 21 ± 3 s before moving into the dark compartment. Thus,the step-through latency of the scopolamine-treated mice was sig-nificantly shorter than that of vehicle-treated control mice (Fig. 3,P < 0.05). In tacrine plus scopolamine-treated mice, step-throughlatency was significantly greater than for scopolamine-treated mice(152 ± 6 s vs. 22 ± 3 s). Moreover, the reduction in step-throughlatency induced by scopolamine was significantly reversed bypseudoberberine 5.0 and 10.0 mg/kg, p.o (P < 0.05) at 118 ± 8 and130 ± 10 s). However, at doses of 2.5 and 20.0 mg/kg, this com-pound did not exhibit a much significant effect. Latency timeduring the acquisition trial was not affected by any drug treatment(Fig. 3).

T.M. Hung et al. / Journal of Ethnopharmacology 119 (2008) 74–80 79

protopine showed to be translated into effects since it improvedscopolamine-induced amnesia in rats (Peng et al., 1997; Kim et al.,1999). Natural drugs from plant species used as memory enhancerswere identified and furthermore Corydalis species were selectedas several species of this genera have been used in treatment ofmemory dysfunction in folk medicines. In this study, some iso-lated alkaloids from Corydalis turtschaninovii can manifest AChEinhibitory activity, although they are less effective than tacrine.However, those compounds were purified from a natural medic-inal plant used as a folk medicine for many years, and the lowmolecular mass material can easily reach the site of action fol-lowing oral administration, since it crosses the blood–brain 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 mostactive compound with the treatment of 5.0 mg/kg could reversethe deficits produced by scopolamine treatment in comparisonwith the vehicle-treated group on passive avoidance task and sig-nificantly shortened the escape latency, improved the swimming

Fig. 3. Effect of a single administration of pseudoberberine (16) on scopolamine-induced memory deficits as determined by the passive avoidance task. At 60 minbefore acquisition trials, pseudocoptisine (2.5, 5.0, 10.0 or 20.0 mg/kg, p.o.), tacrine(10.0 mg/kg, p.o.) or vehicle (same volume of 10% Tween 80) solution was admin-istered to mice. Memory impairment was induced by administering scopolamine(1.0 mg/kg, i.p.). Five different animals were used per treatment group. Acquisi-tion trials were carried out 30 min after a single scopolamine treatment. 24 h afterthe acquisition trials, retention trials were carried out for 3 min. Data representmeans ± S.E.M. (#P < 0.05 vs. vehicle control group; *P < 0.05 vs. scopolamine-treatedgroup).

3.4. Effect of pseudoberberine on Morris water maze

The effect of pseudoberberine (16) (5.0 mg/kg, p.o.) on spa-tial learning (long-term or short-term memory) was evaluatedusing the Morris water maze test. The saline-treated controlgroup rapidly learned the location of the platform (Fig. 4A).The scopolamine-treated animals exhibited longer escape laten-cies (the time taken to find the platform) throughout trainingdays than the vehicle-treated controls (P < 0.001). Pseudoberber-ine (5.0 mg/kg) significantly attenuated the effects of scopolamineon escape latency (P < 0.05), as did tacrine (P < 0.05). On the dayof probe testing, a significant group effect was observed on swim-ming times in the probe test (Fig. 4B). Swimming times within theplatform 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 withinthe platform quadrant induced by scopolamine were significantlyreversed 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 themost 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 blood–brainbarrier causes amnesia in animals by blocking cholinergic neu-rotransmission. Cholinergic interneurons in the striatum are aneven richer source of AChE and would also be affected stronglyby such enzyme inhibitors. In some species of Papaveraceae fam-ily, AChE inhibitory activity has been detected and traced to thebenzylisoquinoline alkaloids present like berberine (Kuznetsovaet 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

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 Morriswater maze task in scopolamine-induced memory deficits mice. At 60 min beforetraining trial sessions, pseudoberberine (5.0 mg/kg, p.o.), tacrine (10.0 mg/kg, p.o.) orvehicle (same volume of 10% Tween 80) solution was administered to mice. Memoryimpairment was induced by scopolamine treatment (1.0 mg/kg, i.p.). Five differentanimals were used per treatment group. The training trial and probe trial sessionswere performed as described in Section 2. Data represent means ± S.E.M. (#P < 0.05vs. vehicle control group; *P < 0.05 vs. scopolamine-treated group).

noph

ine on scopolamine-induced amnesia in rats. Japanese Journal of Pharmacology

80 T.M. Hung et al. / Journal of Eth

time within the zone of platform on water maze task. The passiveavoidance is generally accepted as an indicator of long-term mem-ory in animals (LeDoux, 1993), and the water maze-learning taskwas used to assess hippocampal-dependent spatial learning abil-ity (Morris, 1984). Pseudoberberine could inhibit AChE activity ina dose-dependent manner (IC50 = 4.5 �M), these suggest that abil-ity to alleviate scopolamine-induced memory impairment or theanti-amnesic activity of this alkaloid is, in part, mediated by theAchE inhibition. It would be interesting to indicate that quaternarynitrogen is necessary for strong AChE inhibitory activity in alka-loids possessing the benzylisoquinoline skeleton from Corydalisspecies (Houghton et al., 2006; Hung et al., 2008). Interestingly,the anti-cholinesterase activity of the compounds having both ofaromatic 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 alsoimportant factors for AChE inhibitory activity. Thus, in accordancewith our previous result (Hung et al., 2008), pseudoberberine andsome other constituents on Corydalis turtschaninovii are able to playan important role on the treatment of memory dysfunction of thisplant in traditional medicine. However, more studies should be pre-served if those compounds may be offered as a useful therapeuticchoice in the prevention of AD.

Acknowledgments

This study was partially supported by a grant from the KoreanFood and Drug Administration (2007). We are grateful to KBSI formeasuring 1D, 2D NMR and MS spectra.

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