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Journal of Ethnopharmacology 113 (2007) 179–182

Ethnopharmacological communication

Acetylcholinesterase and butyrylcholinesterase inhibitorycompounds from Corydalis cava Schweigg. & Kort

Anne Adsersen ∗, Anne Kjølbye, Ole Dall, Anna K. JagerDepartment of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen,

2 Universitetsparken, Copenhagen O 2100, Denmark

Received 22 January 2007; received in revised form 16 April 2007; accepted 1 May 2007Available online 6 May 2007

bstract

In the course of screening plants used in Danish folk medicine as memory enhancers, a crude methanolic extract of tubers from Corydalisava showed significant acetylcholinesterase inhibitory activity in a dose-dependent manner. Activity guided fractionation of the methanolicxtract resulted in the isolation of three alkaloids, bulbocapnine (1), corydaline (2) and corydine (3) as active constituents. Bulbocapnine inhibitedcetylcholinesterase as well as butyrylcholinesterase in a dose-dependent manner with IC values of 40 ± 2 �M and 83 ± 3 �M, respectively.

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orydaline inhibited acetylcholinesterase in a dose-dependent manner with an IC50 value of 15 ± 3 �M and corydine inhibited butyrylcholinesterasen a dose-dependent manner with an IC50 value of 52 ± 4 �M. Corydaline was considered inactive against butyrylcholinesterase and corydine againstcetylcholinesterase, due to IC50 > 100 �M.

2007 Elsevier Ireland Ltd. All rights reserved.

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eywords: Corydalis cava; Tuber; Bulbocapnine; Corydaline; Corydine; Acety

. Introduction

Alzheimer’s disease (AD) is the most predominant cause ofementia in the elderly. According to the cholinergic hypoth-sis memory impairments in patients suffering from AD is aesult of decreased levels of the neurotransmitter acethylcholinACh) in the cortex (Lahiri et al., 2002). In the healthy braincethylcholinesterase (AChE) is the most important enzyme reg-lating the level of ACh while butyrylcholinesterase (BChE)lays a minor role. In patients with AD the level of AChE activ-ty declines and the activity of BChE increases and the ratioetween BChE and AChE can change from 0.6 in the normalrain to as high as 11 in cortical areas affected by the diseaseGreig et al., 2002). Currently cholinesterase inhibition is theost used treatment for the symptoms of AD and AChE as well

s BChE is therapeutic targets for improving the cholinergic

eficit (Greig et al., 2002).

Several species of the genera Corydalis have been used inhe treatment of memory dysfunction in folk medicine (Orhan

∗ Corresponding author. Tel.: +45 3530 6295; fax: +45 3530 6041.E-mail address: aad@farma.ku.dk (A. Adsersen).

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378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2007.05.006

inesterase inhibiton; Butyrylcholinesterase inhibition

t al., 2004; Houghton et al., 2006). In a screening of plantssed in Danish folk medicine as memory enhancers tubers fromorydalis cava showed significant AChE inhibitory activity

Adsersen et al., 2006) and were for these reason chosen forurther studies.

. Material and methods

.1. Plant material

The tubers from Corydalis cava Schweigg. & Kort. (Papaver-ceae) were collected in Utterslev Mose, Copenhagen in May005. The tubers were washed, cut and dried at 40 ◦C. Aoucher specimen is deposited in the Department of Medici-al Chemistry, Faculty of Pharmaceutical Sciences, Universityf Copenhagen.

.2. Cholinesterase inhibition assays

In the bioguided isolation AChE and BChE inhibitory activityas detected by a thin-layer chromatography (TLC) bioau-

ographic assay (Rhee et al., 2001; Risa et al., 2004) andhe concentration of the isolated compounds that inhibited the

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saoeluted with methanol yielded corydine (5.8 mg).

The structures of the known alkaloids (Fig. 1) were confirmedfrom their 1H NMR spectra.

80 A. Adsersen et al. / Journal of Eth

nzyme activity by 50% (IC50) was determined by a microtitrelate assay based on Ellman’s method (Rhee et al., 2001; Orhant al., 2004). Galanthamine hydrobromide was used as a positiveontrol.

.2.1. TLC bioautographic assayAcetylcholinesterase from Electric eel (type VI-S), butyryl-

holinesterase from equine serum, acetylthiocholine iodideATCI), S-butyrylthiocholine chloride (BTCCl) and 5,5′-dithio-is(2-nitrobenzoic acid) (DTNB) was purchased from Sigma.xtracts were applied to TLC plates and after developing, theLC plate was sprayed with 5 mM ATCI and 5 mM DTNB

n 50 mM Tris–HCl, pH 8 until the silica was saturated withhe solvent. The plate was then sprayed with 3 U/ml AChEissolved in 50 mM Tris–HCl, pH 8 at 37 ◦C. After a few min-tes a yellow background appeared, with white spots for AChEnhibiting compounds. False-positive reactions were eliminatedy the method of Rhee et al. (2003). A TLC plate identical tohe one in the TLC assay was prepared. The developed TLClate was sprayed with 5 mM DTNB in 50 mM Tris–HCl, pH. Then it was sprayed with 5 mM ATCI and 3 U/ml AChE dis-olved in 50 mM Tris–HCl, pH 8 at 37 ◦C. After a few minutesyellow background appeared; occurrence of white spots indi-

ated false positive reactions. In the test for BChE inhibitoryctivity ATCI was replaced with BTCCl and AChE withChE.

.2.2. Microtitre plate assayIn the 96-well plates, 25 �l substrate, 15 mM ATCI or BTCCl

n Millipore water, 125 �l 3 mM DTNB in buffer C (50 mMris–HCl, pH 8, 0.1 M NaCl, 0.02 M MgCl2·6H2O), 72.5 �luffer B (50 mM Tris–HCl, pH 8, 0.1% bovine serum albumin)nd 2.5 �l test compound solution dissolved in DMSO weredded and the absorbance was measured five times at 405 nmvery 13 s in a Labsystems Multiscan EX type 355 plate reader.hen 25 �l 0.22 U/ml AChE or 0.1 U/ml BChE in buffer B weredded to the wells and the absorbance was measured again eightimes at 405 nm every 13 s. The reaction rate was calculated by

ultiskan EX software version 1.0 and Microsoft Excel. Anyncrease in absorbance due to the spontaneous hydrolysis of sub-trate was corrected by subtracting the rate of the reaction beforedding the enzyme. The percentage inhibition was calculated byomparing the rates for the samples to the blank (2.5 �l DMSOnstead of test compound solution). The experiment was done inriplicate.

.3. Extraction and isolation

The dried powdered tubers of Corydalis cava (37 g) wasxtracted with MeOH (3 × 400 ml) in an ultrasonic bath, which,pon removal of the solvent in vacuum at 40 ◦C, yielded.3 g crude extract. The crude extract was suspended in 1Nydrochloric acid and partitioned successively with heptane.

he HCl fraction was alkalised with 25% NH3 and extractedith 3 × 250 ml EtOAc (Kim et al., 2004). The EtOAc extractas evaporated to dryness (1.6 g), subjected to vacuum liq-id chromatography with mixtures of CH2Cl2–EtOAc and

armacology 113 (2007) 179–182

tOAc–MeOH in order of increasing polarity (100:0–0:100)o give twenty-one 100 ml fractions. Fractions were pooledccording to their similarity in Rf values on thin-layer chro-atography to give four subfractions, which were tested

or their inhibition of AChE. Subfractions 1, 3 and 4 werective.

Subfraction 1 (466 mg) was further chromatographedn a silica gel column (60 g; 2 × 50 cm) using a gradi-nt of hexane:acetone (100:0–0:100) to give 100 fractions.rom the combined fractions 1–20 crystals precipitated andecrystallisation with EtOAc:(CH3)2CO yielded 85 mg cor-daline.

Recrystallisation of the crystalline material formed by evapo-ating subfraction 3 (480 mg) using MeOH–EtOAc (1:1) yieldedulbocapnine (150 mg).

Subfraction 4 (152 mg) was further chromatographed on ailica gel column (21 g; 2 × 50 cm) using a gradient of hex-ne:acetone (9:1–5:5) to give 114 fractions. Further purificationf the combined fractions 82–108 (9.2 mg) on Sephadex LH-20

Fig. 1. Compounds isolated from Corydalis cava Schweigg. & Kort.

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. Results and discussion

Three benzylisoquinoline alkaloids bulbocapnine (1), cory-aline (2) and corydine (3), known from Corydalis cava (Slavıknd Slavıkova, 1979), were isolated by activity guided frac-ionation and their structures were identified by comparison ofheir 1H NMR spectral data with those reported in the literatureShamma and Salgar, 1973; Halbsguth et al., 2003; de Wet et al.,005). The isolated alkaloids were tested for AChE and BChEnhibitory activity (Table 1).

Corydaline was the most active compound inhibiting AChEn a dose-dependent manner with IC50 value of 15 �M ± 3.

iyazawa et al. (1998) demonstrated less AChE inhibitoryctivity of corydaline isolated from Corydalis bulbosa with8.2% inhibition at 1.0 mM using a crude extract from theeads of Drosophila melanogaster as enzyme source. Coryda-ine belongs to the tetrahydroberberine skeletal type of alkaloidsnd corynoxidine, an alkaloid of the same type, has been isolatedrom Corydalis speciosa, and was shown to display much weakernhibiton of AChE with an IC50 value of 89.0 �M (Kim et al.,004). Corydaline was considered inactive against BChE due toC50 > 100 �M. Kim et al. (1999) isolated a protopine skeletalype alkaloid protopine from Corydalis ternata, determined theC50 value for AChE inhibition to 50 �M and showed that miceretreated with protopine exhibited diminished scopolaminenduced dementia in a passive avoidance task. No inhibitoryctivity at a concentration of 100 �M was demonstrated againstChE. Bulbocapnine inhibited AChE as well as BChE in aose-dependent manner with IC50 values of 40 ± 2 �M and3 ± 3 �M, respectively, and corydine inhibited BChE in a dose-ependent manner with an IC50 value of 52 ± 4 �M. The IC50alues of the positive control, galanthamine hydrobromide, were.4 ± 0.2 �M and 4 ± 1.4 �M for inhibition of AChE and BChE,espectively. Bulbocapnine and corydine belongs to the apor-hine skeletal type of alkaloids, a group of benzylisoquinolinelkaloids, to our best knowledge, not proven to show AChE orChE inhibitory activity

Previous studies concerning cholinesterase inhibiting activ-ty of benzylisoquinoline alkaloids have shown that the mostctive alkaloids are found within compounds with a quarte-

ary nitrogen atom. Quartenary protoberberines palmatine anderberine were isolated from Corydalis speciosa with IC50 val-es of 5.8 �M and 3.3 �M, respectively (Kim et al., 2004),nd berberine was isolated from Corydalis ternata with an

able 1n vitro AChE and BChE inhibitory activity of compounds 1–3

ompound IC50 (�M)a

AChE BChE

ulbocapnine (1) 40 ± 2 83 ± 3orydaline (2) 15 ± 3 >100b

orydine (3) >100b 52 ± 4

alantaminec 1.4 ± 0.2 4.0 ± 1.4

a Results are the mean of three replications.b Considered inactive.c Reference compound.

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armacology 113 (2007) 179–182 181

C90 value of 2.5 �M (Hwang et al., 1996). Ulrichova et al.1983) studied the AChE inhibitory effect of 7 quartenaryrotoberberine alkaloids and 6 quartenary benzophenanthri-ine alkaloids. The IC50 values for the protoberberines wererom 1 �M (berberine) to 34 �M (protoberberine) and for theenzophenanthridine alkaloids from 9.4 �M (chelerythrine) to0 �M (chelirubine). Corynoline, another benzophenanthridinelkaloid isolated from Corydalis incisa, inhibited AChE withn IC50 value of 30.6 �M (Kim, 2002). Kuznetsova et al.2002) examined the inhibitory effect of berberine and the ben-ophenanthridine alkaloids sanguinarine and chelidonine onuman AChE and BChE. Sanguinarine and berberine, whichn contrast to chelidonine, contain a quarternary atom of nitro-en, produced the strongest inhibitory activity on both enzymesnd it was unequivocally demonstrated that the action of san-uinarine and berberine on BChE was weaker than the actionn AChE; this is in accordance with our result for bulbocapnine.

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