Acetylcholinesterase and butyrylcholinesterase inhibitory activity of some Turkish medicinal plants

Journal of Ethnopharmacology 91 (2004) 57–60

Acetylcholinesterase and butyrylcholinesterase inhibitory activity of some Turkish medicinal plants a , M.I. Choudhary b , A. Khalid b I. Orhan a,∗ , B. Sener ¸ 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 Alzheimer’s Disease (AD) is a chronic neurological disorder characterized by memory impairment, cognitive dysfunction, 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 butyrylcholinesterase (BChE), considered to play a role in the ∗ Corresponding author. Tel.: +90-312-2126645x1317; fax: +90-312-2133921. E-mail address: [email protected] (I. Orhan).

pathology of AD (Hebert et al., 1995). Both enzymes are 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 pharmaceutical industry. Many synthetic drugs owe their origin to plant-based complementary medicine. Since AD, one of the

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most common cause of death worldwide, has become a threaten to public health, new treatment strategies based on medicinal plants have been focused. Since the plants have been used in treatment of memory dysfunction in some folk medicines since centuries, the present study was undertaken to evaluate the anticholinesterase potential of a number of selected Turkish medicinal plants with various ethnobotanical uses, aiming to discover new candidates for anticholinesterase compounds (Orhan, 2002). The inhibitory activity of the plant extracts prepared with chloroform:methanol (1:1) against AChE and BChE were determined by in vitro Ellman method at 10 ␮g/ml and 1 mg/ml doses.

2. Materials and methods 2.1. Plant materials Collection sites of the samples of the plants, 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), 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) are listed in Table 1. Voucher specimens are kept in the Herbarium of Faculty of Pharmacy of Gazi University, Ankara, Turkey. 2.2. Extraction Fresh samples of the plant materials were air-dried and powdered in a grinder. 50 g of each samples was extracted with chloroform:methanol (1:1) (200 ml×3). After filtration, organic layers were distilled in vacuo until dryness. The crude extracts obtained were used in the anticholinesterase assays. 2.3. Anticholinesterase assays Electric eel acetylcholinesterase (EC 3.1.1.7, type-VI-S), horse butyrylcholinesterase (EC 3.1.1.8), acetylthiocholine iodide, butyrylthiocholin chloride, and 5,5 -dithio-bis-nitrobenzoic acid (DTNB) were purchased from the Sigma (St. Louis, MO). Buffers and other chemicals were of extra pure analytical grade. All the other reagents and conditions were same as described in previous publication (Rahman et al., 2000). Galanthamine (Reminyl® Johnson & Johnson) was used as the standard drug. Acetylcholinesterase in-

Table 1 Turkish medicinal plants selected for anticholinesterase activity screening Plant species

Family

Collection site

Corydalis solida subsp. solida Glaucium corniculatum Rhododendron ponticum subsp. ponticum Rhododendron luteum Buxus sempervirens Vicia faba Robinia pseudoacacia Tribulus terrestris Zygophyllum fabago Lycopodium clavatum Fumaria vaillantii Fumaria capreolata Fumaria kralikii Fumaria asepala Fumaria densiflora Fumaria flabellata Fumaria petteri subsp. thuretii Fumaria macrocarpa Fumaria cilicica Fumaria parviflora Fumaria judaica

Papaveraceae

Ankara-Kizilcahamam

Papaveraceae Ericaceae

Denizli vicinity Bolu vicinity

Ericaceae Buxaceae Fabaceae Caeselpiniaceae Zygophyllaceae Zygophyllaceae Lycopodiaceae Fumariaceae Fumariaceae Fumariaceae Fumariaceae Fumariaceae Fumariaceae Fumariaceae

Bolu vicinity Ankara Ankara-Lalahan Ankara Yalova vicinity Ankara Trabzon-Yomra Ankara-Lalahan Antalya-Aspendos Izmir-Çe¸sme Ankara-Sivrihisar Bursa-Gemlik Aydin-Kusadasi Bursa-Gemlik

Fumariaceae Fumariaceae Fumariaceae Fumariaceae

Izmir-Bornova Ankara-Beynam Ankara Izmir-Urla

hibition was determined spectrophotometrically using acetylthiocholine as substrate by modifying the method of Ellman (Ellman, 1958; Ellman et al., 1961). In this method, 140 ␮l of 0.1 mM sodium phosphate buffer (pH 8.0), 20 ␮l enzyme preparation and 20 ␮l test compound solution dissolved in methanol were mixed and incubated for 30 min. 10 ␮l of DTNB was added and the reaction was then started by adding 10 ␮l of acetylthiocholine. Ten microliters of butyrylthiocholine chloride was used as a substrate to assay butyrylcholinesterase enzyme, while all the other reagents and conditions were the same. The hydrolysis of acetylthiocholine or butyrylthiocholine was determined by monitoring the formation of the yellow 5-thio-2-nitrobenzoate anion as a result of the reaction with DTNB with thiocholines, catalyzed by enzymes at a wavelength of 412 nm. Methanol was used as negative control. Inhibition percentage was calculated according to Michaelis–Menten model by using “EZ-Fit. Enzyme Inhibition Kinetic Analysis (EZ-Fit:Enzyme Kinetics MS Windows Software, Perrella Scientific, Inc., Amshert, USA)” program. Galanthamine dissolved in methanol was used as standard drug at 10 ␮g and 1 mg/ml concentrations. 2.4. Statistical method The assays were conducted in triplicate and all tabulated results were expressed as means ± S.E.M., and were compared using Student’s t-test. A P value of less than 0.05 was considered significant.

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Table 2 Anticholinesterase activity of the plant extracts against AChE and BChE Plant species

Inhibition (%) AChE (10 ␮g/ml)

Corydalis solida subsp. solida Glaucium corniculatum Rhododendron ponticum subsp. ponticum Rhododendron luteum Buxus sempervirens Vicia faba Robinia pseudoacacia Tribulus terrestris Zygophyllum fabago Lycopodium clavatum Fumaria vaillantii Fumaria capreolata Fumaria kralikii Fumaria asepala Fumaria densiflora Fumaria flabellata Fumaria petteri subsp. thuretii Fumaria macrocarpa Fumaria cilicica Fumaria parviflora Fumaria judaica Standard Galanthamine

8.05 9.34 8.76 1.87 4.09 1.03 0 0 0 1.56 12.00 33.99 5.09 9.76 17.43 23.05 15.33 25.02 13.09 14.05 54.44

± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ± ± ±

0.98 0.88 0.65 0.32 0.66 0.56

0.67 1.07∗∗ 0.68∗∗∗ 0.99∗∗ 0.89∗∗ 0.73∗∗∗ 0.11∗∗∗ 0.67∗∗∗ 0.35∗∗∗ 0.13∗∗ 0.78∗∗ 0.56∗∗∗

5.08 ± 0.34

BChE (10 ␮g/ml) 29.08 0 5.06 6.012 22.36 0 6.67 17.06 6.08 11.78 6.43 13.44 0 14.65 0 2.51 4.00 16.62 2.01 10.76 3.00

± 0.88∗∗∗ ± 1.64 ± 0.15 ± 0.98∗∗∗ ± ± ± ± ± ±

1.15 0.55∗∗∗ 0.87∗∗ 0.31∗∗∗ 0.77 0.32∗∗∗

± 1.61∗∗∗ ± ± ± ± ± ±

0.40 0.97 0.43∗∗∗ 0.55 0.33∗ 0.12

11.23 ± 0.87

AChE (1 mg/ml) 87.56 86.55 93.03 76.32 61.76 45.23 26.32 37.89 13.25 49.85 94.23 96.89 84.98 91.99 93.42 92.14 89.45 93.43 88.03 87.02 96.47

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.24∗∗∗ 0.67∗∗∗ 1.12∗∗∗ 0.58∗∗∗ 0.76∗∗∗ 1.03∗∗∗ 0.82 0.77 0.45 1.33∗∗∗ 0.47∗∗∗ 0.17∗∗∗ 1.07∗∗∗ 0.70∗∗∗ 0.92∗∗∗ 1.01∗∗∗ 0.86∗∗∗ 0.64∗∗∗ 0.65∗∗∗ 0.31∗∗∗ 0.63∗∗∗

48.80 ± 0.31

BChE (1 mg/ml) 93.18 81.45 95.23 69.14 93.64 55.85 31.47 78.32 78.37 71.05 99.32 89.24 75.43 93.12 85.66 87.91 87.32 88.74 80.03 87.09 98.43

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.89∗∗∗ 0.74∗ l.28∗∗∗ 1.89∗∗ 0.87∗∗∗ 0.48 0.99 1.27∗∗ 0.95∗∗ 0.25∗∗ 0.25∗∗∗ 0.83∗∗∗ 0.98∗∗∗ 0.28∗∗∗ 1.24∗∗∗ 0.61∗∗∗ 0.76∗∗∗ 0.34∗∗∗ 0.28∗∗∗ 1.45∗∗∗ 0.39∗∗∗

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 AChE and BChE inhibitory activities of the plant extracts 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 solida 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 potent 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 enzymes 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 Zygophyllum 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.

On the other hand, while the plant extracts appeared 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 mechanism 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 belonging 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

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