Acta Biologica Hungarica 65(4), pp. 396–404 (2014) DOI: 10.1556/ABiol.65.2014.4.4

COMPARISON OF ANTI-ACETYLCHOLINESTERASE ACTIVITY OF AND LEAF EXTRACTS OF CANDIDA MATHEW & T. BAYTOP

M. Z. HAZNEDAROGLU1and G. GOKCE2

1Department of Pharmaceutical Botany, Faculty of Pharmacy, Ege University, 35100, Izmir, Turkey 2Department of Pharmacology, Faculty of Pharmacy, Ege University, 35100, Izmir, Turkey

(Received: July 3, 2013; accepted: April 16, 2014)

Studies on Alzheimer’s disease have been highlighted due to increasing prevalence of this disease. Oldest hypothesis about the pathophysiology strengthens the research of cholinesterase inhibitors for treatment. are well known for alkaloids showing cholinesterase inhibiting activity. Among them, Sternbergia species gained attention as a source of metabolites of these alkaloid contents. Studies have focused mainly on the of these plants. In this study the potential acetylcholinesterase (AChE) inhibitory activity of endemic Sternbergia candida Mathew & T. Baytop (Sc) species was evaluated in both bulbs and leaves in comparison with lycorine.ˆ- form leaf extracts of the show AChE inhibitory (AChEI) activity. Among the leaf extracts metha- nolic extract was much more potent than chloroform extract by means of AChE inhibition. Although IC50 values for methanolic extract was found to be lower than reference drug lycorine; this value of inhibition b Š inhibitory activity could be considered using leaves of the plant.

Keywords: Sternbergia candida – lycorine – AchE inhibitory activity – bulbs – leaves

INTRODUCTION

Plants have been used as medications throughout the history of humanity; they are still being commonly used in developing countries while their usage is also increasing rapidly in developed countries [32]. Thus medicinal plants are still being widely stud- ied in contemporary pharmaceutical sciences. As the proportion of elderly in popula- tion increases, screening of herbal drugs which have been used for the treatment of cognitive disorders has been highlighted [2]. Cognitive impairment in the elderly is one of the facts that causes malfunction in the daily life. Alzheimer’s disease (AD) is the most common reason of dementia [13]. The pathophysiology of AD and new treatment strategies for the pathogenesis are still being investigated. A well-known pathophysiological change in cognitive decline is the cholinergic dysfunction. Acetylcholine (ACh) is a key neurotransmitter in cog- nition. That is why the agents, which may enhance the cholinergic transmission are

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0236-5383/$ 20.00 © 2014 Akadémiai Kiadó, Budapest AChEI activity of leaves and bulbs of S. candida 397 thought to enhance the cognition, too. The cholinergic function could be enhanced by stimulation of cholinergic receptors or prolonging the availability of the neurotrans- mitter ACh at cholinergic synapses. The latter can be achieved by using acetylcho- linesterase inhibitors that block this key enzyme in the breakdown of ACh [27]. For treatment, oldest hypothesis strengthens the search of cholinesterase inhibitors [14, 21–23, 29]. Cholinesterase inhibitors such as tacrine, donepezil, rivastigmine and galanthamine, are currently being used as registered drugs. However, common side effects of these drugs, such as hepatotoxicity, gastrointestinal disturbance and hypo- tension have limited their use [14, 25]. Therefore, search for new biologically active alkaloids showing ACh inhibitory (AChI) activity is an area of growing interest. As other members of Amaryllidaceae family, Sternbergia species are known to be rich in alkaloid and phenolic content [20, 22]. These alkaloids with various chemical structures show a wide range of biological activities. In previous studies the genus has !"\! [6, 8], antioxidant, antimicrobial [18, 31] and analgesic [28]. Besides the genus has Š$ 69! @

MATERIALS AND METHODS Chemicals

All reagents used were of analytical grade. Chloroform, methanol, ethyl acetate, hydrochloric acid were purchased from Riedel de Haen; ammonium hydroxide, fast blue B salt (FBS) was purchased from Merck; acetylcholinesterase (AChE) and ! @" ! 9!9a""ƒ*"- trobenzoic acid) (DTNB), Tris-HCl, bovine serum albumin (BSA), DMSO were purchased from Sigma. Two different buffer systems were used. Buffer A: 50 mM Tris-HCl, pH 7.9 containing 0.1% BSA; Buffer B: 50 mM Tris-HCl, pH 7.9 contain- ing 0.1 M NaCl and 0.02 M MgCl2^{ 2O. Lycorine was a kind gift from Prof. Dr. Ulvi Zeybek (Ege University, Turkey).

Acta Biologica Hungarica 65, 2014 398 M. Z. HAZNEDAROGLU and G. GOKCE

Plant material

Sternbergia candida !% the Department of Pharmaceutical Botany, Faculty of Pharmacy, Ege University, $|z{™ z!b Science, Ege University. Voucher specimens are kept at IZEF Herbarium (IZEF5798). Leaves and bulbs were dried in controlled room conditions at 25 °C under ventilation. Dried plant material was homogenized using a blender.

Preparation of extract

Extraction was performed on bulbs and leaves (50 g) by sonication with 500 ml chlo- roform (CHCl35! 45 °C and CHCl3 extract was obtained. The same procedure was performed with ethyl acetate (EtOAc) and methanol (MeOH). Extraction yields for bulbs were 0.74% with CHCl3, 0.24% with EtOAc, 2.86% with MeOH. Extraction yields for leaves were 3.70% with CHCl3, 0.56% with EtOAc and 9.12% with MeOH. All extracts were examined by TLC on silica plates by use of adequate mobile phases for compounds of different polarity. Detection was performed with Dragendroff reagent to check the chemical composition of the extracts. Alkaloids were detected in CHCl3 and MeOH extracts however no alkaloids were found in EtOAc extracts. Therefore, alkaloid enrichment extraction was carried out with CHCl3 and MeOH extracts. In order to perform alkaloid enrichment, CHCl3 and MeOH (1 g) extracts were dissolved in 1% hydrochloric acid (HCl) and partitioned with 100 ml CHCl3. The obtained aqueous phase was adjusted to pH 9 with ammonium hydroxide (NH4OH); partitioned by CHCl3. Thereafter, CHCl3 was evaporated under vacuum. Extraction yields following alkaloid enrichment for bulb extracts were 10.3% with CHCl3 (SCBC), 3% with MeOH (SCBM). Extraction yields following alkaloid enrichment for leaf extracts were 9.3% with CHCl3 (SCLC), 0.5% with MeOH (SCLM).

TLC bioautography assay

TLC bioautography-screening assay was performed for AChEI activity based on Ellman’s method [2, 12, 19]. Lycorine was used as a positive control. All extracts and q@€@<<)q*9 )q<b*9Wq (20 cm × 20 cm), 1.5 cm from the bottom edge. One dimensional development condi- tions were established at 25 °C; with the mobile phase CHCl3 : MeOH : NH4OH, (75 : 24.5 : 0.5). The plate was dried with hair dryer for complete removal of solvent and then sprayed with enzyme stock solution (6.7 U/mL in buffer A) and thoroughly b|!\

Acta Biologica Hungarica 65, 2014 AChEI activity of leaves and bulbs of S. candida 399 a plastic tank (26 × 36 cm) containing 350 ml water at 1 cm height. The plate was placed above water, so that water did not come directly into contact with the plate but the atmosphere was kept humid. The incubation was performed at 40 °C for 20 min. For detection of the enzyme, solutions of 1-naphthyl acetate (250 mg) in ethanol (100 mL) and of Fast Blue B salt (400 mg) in distilled water (160 mL) were prepared immediately before use (in order to prevent decomposition). After incubation of the TLC plate, 1 mL of the naphthyl acetate solution and 4 ml of the Fast Blue B salt solution were mixed and sprayed onto the plate to give a pink-purple coloration after 1–2 min [19].

Microplate assay

The AChEI activities of the samples were measured by a quantitative colorimetric ŠR6*!Q!@*!*W=$‰"!*9)q@9 $!@*9)qQz'ƒ9<"{!{+‰<@ M NaCl and 0.02 M MgCl2^{ 2‹5!9<)qƒ9<"{!{+‰ <@« X5 *9 )q ƒ zX‹ buffer A to a concentration of 1 mg/mL) were mixed and the absorbance was meas- W<9@9*9)qŠƒ<**˜€q5 was added and the plate was incubated at 25 °C for 10 min. Then the absorbance was measured again eight times for every 15 seconds. Each assay was repeated three times. To avoid any increase in absorbance due to the color of the extracts or sponta- neous hydrolysis of substrate, the absorbance before addition of the enzyme was subtracted from the absorbance after adding the enzyme. The assay was validated by measurement of lycorine as a positive control. The percentage of inhibition was cal- culated by comparing the absorbance of sample to blank (10% DMSO in buffer A).

Statistical analysis

All data were expressed as means ± SD. ANOVA followed by Tukey’s Multiple Comparison test was performed using GraphPad Prism (GraphPad Software, Version 3.02, San Diego, California, USA). A value of p¼<<9

RESULTS

Results of TLC bioautography assay and detection with Dragendorff reagent of extracts with reference compound are presented in Figure 1. TLC bioassay is a rapid method for detection of enzyme inhibition. Sternbergia candida extracts were tested for their anticholinesterase activity. TLC bioautography of tested extracts showed active spots as white bands on pink-purple background. The Rf values were identical for lycorine and tested extracts (0.57). Methanolic extract of leaves (SCLM) showed

Acta Biologica Hungarica 65, 2014 400 M. Z. HAZNEDAROGLU and G. GOKCE

Fig. 1. Detection of extracts and reference compound (lycorine) with Dragendorff reagent and TLC- bioautography. SCBC: Sternbergia candida bulb extracted with CHCl3; SCLC: Sternbergia candida leaves extracted with CHCl3; SCBM: Sternbergia candida bulb extracted with MeOH; SCLM: Sternbergia candida leaves extracted with MeOH

Acta Biologica Hungarica 65, 2014 AChEI activity of leaves and bulbs of S. candida 401

Fig. 2. AChEI activity of extracts and reference compound. SCBC: Sternbergia candida bulb extracted with CHCl3; SCLC: Sternbergia candida leaves extracted with CHCl3; SCBM: Sternbergia candida bulb extracted with MeOH; SCLM: Sternbergia candida leaves extracted with MeOH

Fig. 3. IC50 values for AChE inhibition. SCBC: Sternbergia candida bulb extracted with CHCl3; SCLC: Sternbergia candida leaves extracted with CHCl3; SCBM: Sternbergia candida bulb extracted with MeOH; SCLM: Sternbergia candida leaves extracted with MeOH. All data are expressed as means ± SD. Comparisons were made by one-way analysis of variance (ANOVA) followed by Tukey’s Multiple Comparison Test. A value of p < <<9 ZZZp < 0.001; SCBM vs Lycorine (n = 3) and +p < 0.001; SCLC vs Lycorine (n = 3) four active spots. Besides, methanolic extract of bulbs (SCBM) and chloroform extract of leaves (SCBC) showed three active spots. Lycorine showed one active spot. The TLC assay demonstrated that tested Sternbergia candida extracts have AChE inhibitory activity. The extracts of Sternbergia candida were tested for their in vitro anticholinesterase activity at 0.016, 0.0313, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/ml concentrations with microplate assay. As shown in Fig. 2, the most potent inhibition was obtained by methanolic bulb extract (SCBM). At concentration level of 1 mg/ml, degree of ace-

Acta Biologica Hungarica 65, 2014 402 M. Z. HAZNEDAROGLU and G. GOKCE tylcholinesterase inhibition for the tested extracts was as follows; SCBM > SCLM > SCBC > Lycorine > SCLC. IC50 values of the tested plant extracts as well as the reference drug, lycorine, are shown in Figure 3. All the plant extracts were found to be more potent than lycorine, except for methanolic leave extract (SCLC) which showed relatively weak inhibitory activity against acetylcholinesterase. In particular, the highest level of AChE inhibi- tion was achieved by SCBM, which was approximately six times higher than that of lycorine. Also, when compared to lycorine, a higher degree of AChE inhibition was obtained by SCBC and SCLM (16% and 36%, respectively).

DISCUSSION

$!Sternbergia candida Š$ of Alzheimer’s disease, Amaryllidaceae plants gained considerable attention by means of their AChE inhibiting potential. Plants of the Amaryllidaceae family, com- prising ca. 75 genera and about 1100 species, were widely studied and to date, more than 500 exclusive and structurally diverse alkaloids, namely Amaryllidaceae alka- loids, have been isolated from different plants of this family [16]. Among these, alkaloid compositions of Crinum bulbispermum Milne-Redhead & Schweick, Boophane disticha (L. f.), Hippeastrum morelianum Lem., Lycoris radiata (L’Herit.) Herb., Nerine bowdenii Watson, Zephyranthes robusta Baker, Chlidanthus fragrans Herb., Lindl. [16], Ammocharis coranica (Ker-Gawl.) Herb. [11], Galanthus rizehensis Stern [26] have been investigated. Alkaloids such as ! ! ! ! @"‹"! ! ! crinine, and 1-epideacetylbowdensine, 6a-hydroxycrinamine, candimine, tazettine, pretazettine, 3-epimacronine, haemanthamine, hamayne, and trisphaeridine, vittatine, galanthine, tazetine, lycoramine, nerbowdine, incartine N-oxide and lycorine N-oxide 6@@!@!*=$- ies were mostly performed using bulbs. There are only a few studies regarding the use of leaves as a source of Amaryllidaceae alkaloids. Most recently, alkaloids isolated from the leaves of Leucojum aestivum, N-(14-methylallyl) norgalanthamine (IC50: 0.18 μM) and N-allylnorgalanthamine 45 (IC50: 0.16 μM) have been found to show more potent AChE inhibitory activity than the approved drug galanthamine (IC50: 1.82 μM) [4]. In addition, Torras-Claveria et al. [30], demonstrated that total alkaloid contents of leaf and fruit extracts of Pancaratium canariense - cantly higher than those in bulb extracts. Moreover, in our previous study, lycorine was detected in methanolic leaf extract of Sternbergia candida [15]. Taking together, these reports suggest that leaves of Amaryllidaceae plants may provide a useful source for alkaloids showing AChEI activity. Since most of the species of Amaryl- lidaceae are on the edge of extinction, preservation of the bulbs gains more importance in means of sustainability. In the present study, methanolic bulb extract of Sternbergia candida (SCBM) showed the most potent AChEI activity (IC50: 103.6 μg/mL).

Acta Biologica Hungarica 65, 2014 AChEI activity of leaves and bulbs of S. candida 403

Nevertheless, AChEI activity of methanolic leaf extract (SCLM) was 74% higher than the reference drug lycorine. Further studies are needed to incorporate comprehensive phytochemical analysis of the extract and its potential in vivo activity.

ACKNOWLEDGEMENTS

The authors are thankful to Ernst Mach Grants and OEAD for the supports; Prof. Dr. Ulvi Zeybek and Prof. Dr. Levent Ustunes for facilities; Dr. Bio. Hasan Yildirim, Burak Isman and Ali Es for kind helps in collecting the plant.

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Acta Biologica Hungarica 65, 2014