Acta Biologica Hungarica 65(4), pp. 396–404 (2014) DOI: 10.1556/ABiol.65.2014.4.4
COMPARISON OF ANTI-ACETYLCHOLINESTERASE ACTIVITY OF BULB AND LEAF EXTRACTS OF STERNBERGIA CANDIDA MATHEW & T. BAYTOP
M. Z. HAZNEDAROGLU1 and 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. Amaryllidaceae plants 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 bulbs 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. - form leaf extracts of the plant 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 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
Z \ " ? | % | ]
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 $ 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" "*" - 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 @ @<<