Journal of Ethnopharmacology 91 (2004) 57–60

Acetylcholinesterase and butyrylcholinesterase inhibitory

activity of some Turkish medicinal plants
I. Orhan a,∗ , B. Şener a , M.I. Choudhary b , A. Khalid b
a Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey
b H.E.J. Research Institute of Chemistry, University of Karachi, 75270 Karachi, Pakistan

Received 12 February 2003; received in revised form 23 October 2003; accepted 21 November 2003

Abstract

The chloroform:medianol (1:1) extracts of a number of the plant species belonging to eight families, namely Corydalis solida (L.) Swartz
subsp. solida and Glaucium corniculatum (L.) J. H. Rudolph (Papaveraceae), Rhododendron ponticum L. subsp. ponticum and Rhododendron
luteum Sweet. (Ericaceae), Buxus sempervirens L. (Buxaceae), Vicia faba L. (Fabaceae), Robinia pseudoacacia L. (Caeselpiniaceae), Tribulus
terrestris L. and Zygophyllum fabago L. (Zygophyllaceae), Lycopodium clavatum L. (Lycopodiaceae), Fumaria vaillantii Lois., Fumaria
capreolata L., Fumaria kralikii Jordan, Fumaria asepala Boiss., Fumaria densiflora DC., Fumaria flabellata L., Fumaria petteri Reichb.
subsp. thuretii (Boiss.) Pugsley, Fumaria macrocarpa Boiss. ex Hausskn., Fumaria cilicica Hauskkn., Fumaria parviflora Lam. and Fumaria
judaica Boiss. (Fumariaceae) were screened for their anticholinesterase activity on acetylcholinesterase (AChE) and butyrylcholinesterase
(BChE) enzymes by in vitro Ellman method at 10 ␮g/ml and 1 mg/ml concentrations. The extracts did not show any noticeable inhibitory
activity against both of the enzymes at 10 ␮g/ml. The extracts of Rhododendron ponticum subsp. ponticum, Rhododendron luteum, Corydalis
solida subsp. solida, Glaucium corniculatum, and Buxus sempervirens showed remarkable inhibitory activity above 50% inhibition rate on
AChE at 1 mg/ml. Among them, Rhododendron ponticum subsp. ponticum, Corydalis solida subsp. solida and Buxus sempervirens were the
most active extracts against BChE having 95.46±1.03%, 93.08±0.97%, and 93.45±0.88% inhibition rates, respectively. Among the extracts
screened, all of the Fumaria extracts displayed highly potent inhibition against both of the enzymes at 1 mg/ml concentration compared to
the standard.
© 2003 Elsevier Ireland Ltd. All rights reserved.

Keywords: Anticholinesterase activity; Acetylcholinesterase; Butyrylcholinesterase; Alzheimer’s Disease; Ellman method

1. Introduction pathology of AD (Hebert et al., 1995). Both enzymes are

Alzheimer’s Disease (AD) is a chronic neurological dis-
order characterized by memory impairment, cognitive dys-
function, behavioral disturbances, and deficits in activities
of daily living (Jann, 1998; Adams et al., 1984; Aisen and
Davis, 1997). AD has been found to be associated with a
cholinergic deficit in the post-mortem brain characterized
by a significant decrease in acetylcholine amount (Bachman
et al., 1992; Terry, 1983). AD has become a major problem,
particularly in developed countries due to increasing old-age
population with a high life quality.
Acetylcholine is a neurotransmitter inhibited primarily
by acetylcholinesterase (AChE) and secondly by butyryl-
cholinesterase (BChE), considered to play a role in the
∗ Corresponding author. Tel.: +90-312-2126645x1317;
fax: +90-312-2133921.
E-mail address: iorhan@gazi.edu.tr (I. Orhan).
present in the brain and are detected among neurofibrillary
tangles and neuritic plaques (Beard et al., 1995). Despite
the unknown etiology of AD, elevation of acetylcholine
amount through AChE enzyme inhibition has been accepted
as the most effective treatment strategy against AD (Arnold
and Kumar, 1993). Therefore, AChE and BChE inhibitors
have become the remarkable alternatives in treatment of
AD. However, the present drugs (tacrin, rivastigmin and
donepezil) with AChE inhibitory activity possess some side
effects and are effective only against the mild type of AD
and there has been no drug available with BChE inhibitory
activity to present, yet (Schneider, 2001). Consequently, it
is compulsory to develop new drugs in order to combat AD.
The history of drug discovery showed that plants are
highly rich sources in the search for new active compounds
and they have become a challenge to modern pharmaceu-
tical industry. Many synthetic drugs owe their origin to
plant-based complementary medicine. Since AD, one of the

0378-8741/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2003.11.016
58 I. Orhan et al. / Journal of Ethnopharmacology 91 (2004) 57–60

most common cause of death worldwide, has become a Table 1

threaten to public health, new treatment strategies based on Turkish medicinal plants selected for anticholinesterase activity screening
medicinal plants have been focused. Plant species Family Collection site
Since the plants have been used in treatment of mem- Corydalis solida Papaveraceae Ankara-Kizilcahamam
ory dysfunction in some folk medicines since centuries, subsp. solida
the present study was undertaken to evaluate the anti- Glaucium corniculatum Papaveraceae Denizli vicinity
cholinesterase potential of a number of selected Turkish Rhododendron ponticum Ericaceae Bolu vicinity
medicinal plants with various ethnobotanical uses, aiming to subsp. ponticum
Rhododendron luteum Ericaceae Bolu vicinity
discover new candidates for anticholinesterase compounds Buxus sempervirens Buxaceae Ankara
(Orhan, 2002). The inhibitory activity of the plant extracts Vicia faba Fabaceae Ankara-Lalahan
prepared with chloroform:methanol (1:1) against AChE Robinia pseudoacacia Caeselpiniaceae Ankara
and BChE were determined by in vitro Ellman method at Tribulus terrestris Zygophyllaceae Yalova vicinity
10 ␮g/ml and 1 mg/ml doses. Zygophyllum fabago Zygophyllaceae Ankara
Lycopodium clavatum Lycopodiaceae Trabzon-Yomra
Fumaria vaillantii Fumariaceae Ankara-Lalahan
Fumaria capreolata Fumariaceae Antalya-Aspendos
2. Materials and methods Fumaria kralikii Fumariaceae Izmir-Çeşme
Fumaria asepala Fumariaceae Ankara-Sivrihisar
2.1. Plant materials Fumaria densiflora Fumariaceae Bursa-Gemlik
Fumaria flabellata Fumariaceae Aydin-Kusadasi
Fumaria petteri Fumariaceae Bursa-Gemlik
Collection sites of the samples of the plants, Corydalis subsp. thuretii
solida (L.) Swartz subsp. solida and Glaucium cornicu- Fumaria macrocarpa Fumariaceae Izmir-Bornova
latum (L.) J.H. Rudolph (Papaveraceae), Rhododendron Fumaria cilicica Fumariaceae Ankara-Beynam
ponticum L. subsp. ponticum and Rhododendron luteum Fumaria parviflora Fumariaceae Ankara
Fumaria judaica Fumariaceae Izmir-Urla
Sweet. (Ericaceae), Buxus sempervirens L. (Buxaceae),
Vicia faba L. (Fabaceae), Robinia pseudoacacia L. (Cae-
selpiniaceae), Tribulus terrestris L. and Zygophyllum fabago
L. (Zygophyllaceae), Fumaria vaillantii Lois., Fumaria hibition was determined spectrophotometrically using
capreolata L., Fumaria kralikii Jordan, Fumaria asepala acetylthiocholine as substrate by modifying the method
Boiss., Fumaria densiflora DC., Fumaria flabellata L., of Ellman (Ellman, 1958; Ellman et al., 1961). In this
Fumaria petteri Reichb. subsp. thuretii (Boiss.) Pugsley, method, 140 ␮l of 0.1 mM sodium phosphate buffer (pH
Fumaria macrocarpa Boiss. ex Hausskn., Fumaria cilicica 8.0), 20 ␮l enzyme preparation and 20 ␮l test compound
Hauskkn., Fumaria parviflora Lam. and Fumaria judaica solution dissolved in methanol were mixed and incu-
Boiss. (Fumariaceae) are listed in Table 1. Voucher speci- bated for 30 min. 10 ␮l of DTNB was added and the
mens are kept in the Herbarium of Faculty of Pharmacy of reaction was then started by adding 10 ␮l of acetylth-
Gazi University, Ankara, Turkey. iocholine. Ten microliters of butyrylthiocholine chloride
was used as a substrate to assay butyrylcholinesterase en-
2.2. Extraction zyme, while all the other reagents and conditions were
the same. The hydrolysis of acetylthiocholine or butyrylth-
Fresh samples of the plant materials were air-dried and iocholine was determined by monitoring the formation
powdered in a grinder. 50 g of each samples was extracted of the yellow 5-thio-2-nitrobenzoate anion as a result of
with chloroform:methanol (1:1) (200 ml×3). After filtration, the reaction with DTNB with thiocholines, catalyzed by
organic layers were distilled in vacuo until dryness. The enzymes at a wavelength of 412 nm. Methanol was used
crude extracts obtained were used in the anticholinesterase as negative control. Inhibition percentage was calculated
assays. according to Michaelis–Menten model by using “EZ-Fit.
Enzyme Inhibition Kinetic Analysis (EZ-Fit:Enzyme Ki-
2.3. Anticholinesterase assays netics MS Windows Software, Perrella Scientific, Inc.,
Amshert, USA)” program. Galanthamine dissolved in
Electric eel acetylcholinesterase (EC 3.1.1.7, type-VI-S), methanol was used as standard drug at 10 ␮g and 1 mg/ml
horse butyrylcholinesterase (EC 3.1.1.8), acetylthiocholine concentrations.
iodide, butyrylthiocholin chloride, and 5,5 -dithio-bis-nitro-
benzoic acid (DTNB) were purchased from the Sigma (St. 2.4. Statistical method
Louis, MO). Buffers and other chemicals were of extra pure
analytical grade. All the other reagents and conditions were The assays were conducted in triplicate and all tabulated
same as described in previous publication (Rahman et al., results were expressed as means ± S.E.M., and were com-
2000). Galanthamine (Reminyl® Johnson & Johnson) pared using Student’s t-test. A P value of less than 0.05 was
was used as the standard drug. Acetylcholinesterase in- considered significant.
 I. Orhan et al. / Journal of Ethnopharmacology 91 (2004) 57–60 59

Table 2
Anticholinesterase activity of the plant extracts against AChE and BChE
Plant species Inhibition (%)

AChE (10 ␮g/ml) BChE (10 ␮g/ml) AChE (1 mg/ml) BChE (1 mg/ml)

Corydalis solida subsp. solida 8.05 ± 0.98 29.08 ± 0.88∗∗∗ 87.56 ± 1.24∗∗∗ 93.18 ± 0.89∗∗∗
Glaucium corniculatum 9.34 ± 0.88 0 86.55 ± 0.67∗∗∗ 81.45 ± 0.74∗
Rhododendron ponticum subsp. ponticum 8.76 ± 0.65 5.06 ± 1.64 93.03 ± 1.12∗∗∗ 95.23 ± l.28∗∗∗
Rhododendron luteum 1.87 ± 0.32 6.012 ± 0.15 76.32 ± 0.58∗∗∗ 69.14 ± 1.89∗∗
Buxus sempervirens 4.09 ± 0.66 22.36 ± 0.98∗∗∗ 61.76 ± 0.76∗∗∗ 93.64 ± 0.87∗∗∗
Vicia faba 1.03 ± 0.56 0 45.23 ± 1.03∗∗∗ 55.85 ± 0.48
Robinia pseudoacacia 0 6.67 ± 1.15 26.32 ± 0.82 31.47 ± 0.99
Tribulus terrestris 0 17.06 ± 0.55∗∗∗ 37.89 ± 0.77 78.32 ± 1.27∗∗
Zygophyllum fabago 0 6.08 ± 0.87∗∗ 13.25 ± 0.45 78.37 ± 0.95∗∗
Lycopodium clavatum 1.56 ± 0.67 11.78 ± 0.31∗∗∗ 49.85 ± 1.33∗∗∗ 71.05 ± 0.25∗∗
Fumaria vaillantii 12.00 ± 1.07∗∗ 6.43 ± 0.77 94.23 ± 0.47∗∗∗ 99.32 ± 0.25∗∗∗
Fumaria capreolata 33.99 ± 0.68∗∗∗ 13.44 ± 0.32∗∗∗ 96.89 ± 0.17∗∗∗ 89.24 ± 0.83∗∗∗
Fumaria kralikii 5.09 ± 0.99∗∗ 0 84.98 ± 1.07∗∗∗ 75.43 ± 0.98∗∗∗
Fumaria asepala 9.76 ± 0.89∗∗ 14.65 ± 1.61∗∗∗ 91.99 ± 0.70∗∗∗ 93.12 ± 0.28∗∗∗
Fumaria densiflora 17.43 ± 0.73∗∗∗ 0 93.42 ± 0.92∗∗∗ 85.66 ± 1.24∗∗∗
Fumaria flabellata 23.05 ± 0.11∗∗∗ 2.51 ± 0.40 92.14 ± 1.01∗∗∗ 87.91 ± 0.61∗∗∗
Fumaria petteri subsp. thuretii 15.33 ± 0.67∗∗∗ 4.00 ± 0.97 89.45 ± 0.86∗∗∗ 87.32 ± 0.76∗∗∗
Fumaria macrocarpa 25.02 ± 0.35∗∗∗ 16.62 ± 0.43∗∗∗ 93.43 ± 0.64∗∗∗ 88.74 ± 0.34∗∗∗
Fumaria cilicica 13.09 ± 0.13∗∗ 2.01 ± 0.55 88.03 ± 0.65∗∗∗ 80.03 ± 0.28∗∗∗
Fumaria parviflora 14.05 ± 0.78∗∗ 10.76 ± 0.33∗ 87.02 ± 0.31∗∗∗ 87.09 ± 1.45∗∗∗
Fumaria judaica 54.44 ± 0.56∗∗∗ 3.00 ± 0.12 96.47 ± 0.63∗∗∗ 98.43 ± 0.39∗∗∗
Standard
Galanthamine 5.08 ± 0.34 11.23 ± 0.87 48.80 ± 0.31 80.31 ± 1.14
Values were expressed as mean ± S.E.M. (n = 6).
P > 0.05.
∗ P < 0.05.
∗∗ p < 0.01.
∗∗∗ P < 0.001.

3. Results On the other hand, while the plant extracts appeared

AChE and BChE inhibitory activities of the plant ex-
tracts aforementioned are summarized in Table 2. All of
the extracts dissolved in methanol possessed some AChE
and BChE inhibitory activity at 1 mg/ml concentration. All
of the Fumaria species studied showed the most potent
inhibitory activity against AChE as well as Rhododendron
ponticum subsp. ponticum (93.03 ± 1.12%), Corydalis sol-
ida subsp. solida (87.56 ± 1.24%), Glaucium corniculatum
(86.55 ± 0.67%), Rhododendron luteum (76.32 ± 0.58%),
and Buxus sempervirens (61.76 ± 0.76%). The rest of
the extracts had activity below 50%. As to BChE, except
Robinia pseudoacacia (31.54 ± 0.78%) and Vicia faba
(55.85 ± 0.48%), all of the extracts displayed highly po-
tent inhibitory activity compared to the standard which
exhibited 80.31% inhibition on BChE. Among the plants
screened, the most active extracts against both of the en-
zymes belonged to all of the Fumaria species as well as
Rhododendron ponticum subsp. ponticum, Corydalis solida
subsp. solida, Glaucium corniculatum. Some of the extracts
such as Buxus sempervirens, Tribulus terrestris and Zygo-
phyllum fabago, which had lower activity against AChE,
exhibited much higher activity against BChE. This may
suggest that these extracts might be interacting with the
enzymes in different mechanisms.
to possess insignificant activity against both enzymes at
10 ␮g/ml, only the extract of Fumaria judaica displayed
a noticeable inhibition (54.44 ± 0.56%) against AChE
(Table 2).
4. Discussion
In addition to well-reputed plant extracts for the treatment
of AD such as Gingko biloba, Huperzia serrata and Panax
ginseng, a number of plants (listed in Table 2) have been
identified to be effective in inhibition of AChE and BChE
enzymes which are considered to be related to the mecha-
nism of memory dysfunction in this study.
In the light of these findings, we can conclude that most
of the plant extracts screened herein showed inhibitory
activity against both of the enzymes in dose-dependent
manner and they could be considered for further studies
in the treatment of AD. In particular, the species belong-
ing to Fumariaceae, Papaveraceae and Ericaceae families
had highest activity ranging between 99.32 and 75.43% at
1 mg/ml concentration against both enzymes. Since most
of the acetylcholinesterase inhibitors are known to contain
nitrogen, the higher activity of these extracts may be due
to their rich alkaloidal content. Further works related to the
60 I. Orhan et al. / Journal of Ethnopharmacology 91 (2004) 57–60
isolation of the active constituents through bioassay-directed
fractionation are in progress in our laboratory.
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