Acetylcholinesterase Inhibitory Effects of the Bulb of Ammocharis Coranica (Amaryllidaceae) and Its Active Constituent Lycorine

South African Journal of Botany 85 (2013) 44–47

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South African Journal of Botany

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Acetylcholinesterase inhibitory effects of the bulb of Ammocharis coranica (Amaryllidaceae) and its active constituent lycorine

I.L. Elisha a,b, E.E. Elgorashi a,1, A.A. Hussein c, G. Duncan d, J.N. Eloff a,⁎ a Phytomedicine Programme, Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, Onderstepoort 0110, South Africa b Drug Development Section, Biochemistry Division, National Veterinary Research Institute, Vom, Nigeria c Department of Chemistry, University of Western Cape, Modderdam Road, Bellville 7535, South Africa d Kirstenbosch National Botanical Garden, Private Bag X7, Claremont, Cape Town, South Africa article info abstract

Article history: Ammocharis coranica (Ker-Gawl.) Herb. (Amaryllidaceae) is used in southern Africa for the treatment of mental Received 28 June 2012 illnesses. The ethanol extracts of the bulb of A. coranica and its total alkaloids rich fractions were screened for in- Received in revised form 16 November 2012 hibition of acetylcholinesterase enzyme (AChE), which is implicated in the pathophysiology of Alzheimer's dis- Accepted 28 November 2012 ease. The ethanolic extracts signiﬁcantly inhibited AChE with IC value of 14.3±0.50 μg/ml. The basic ethyl Available online 7 January 2013 50 acetate and butanol fractions of the crude extracts were the most active against AChE with IC50 values of μ Edited by J Van Staden 43.1±1.22 and 0.05±0.02 g/ml respectively. Bioassay-guided fractionation of the basic fractions led to the isolation of lycorine and 24-methylenecycloartan-3β-ol. Lycorine which was isolated from both butanol and ethyl μ β Keywords: acetate fractions had IC50 of 29.3±3.15 g/ml, while 24-methylenecycloartan-3 -ol was not active. Acetylcholinesterase inhibitors © 2012 SAAB. Published by Elsevier B.V. All rights reserved. Bulbs Ammocharis coranica Ethanolic crude extract

1. Introduction aggression, depression, and wandering. Caregivers find these features difficult to cope with, and often lead to the hospitalisation of the patient Alzheimer's disease (AD) is a progressive neurodegenerative disor- (Esiri, 1996). der characterised by dementia, behavioural abnormalities and death. The “cholinergic hypothesis of AD” was postulated on the basis of It is the leading cause of dementia in elderly people and with the the negative cognitive effect of cholinergic antagonists and the posi- proportion of elderly people in the population increasing steadily, the tive effect of muscarinic agonists on memory in humans (Guillou et burden of the disease, both to caregivers and national economies, is al., 2000), and became the neurobiological incentive for a treatment expected to become substantially greater over the next 2 to 3 decades aiming at the improvement of cholinergic function in AD. Inhibitors (Francis et al., 1999; Maslow, 2008). of acetylcholinesterase (AChE), the enzyme involved in the metabolic The brain regions associated with higher mental functions, such as hydrolysis of acetylcholine (ACh) at cholinergic synapses, promote the neocortex and hippocampus, are mostly affected by the characteris- increase in the concentration and duration of action of synaptic ACh tic pathology of AD (Cummings, 2004). These abnormal pathologic fea- (Rollinger et al., 2004), thus serving as treatment strategy for AD, se- tures include, extracellular deposits of β-amyloid derived from amyloid nile dementia, ataxia, myasthenia gravis and Parkinson's disease precursor protein (APP) in senile plaques, intracellular formation of (Houghton et al., 2006). Acetylcholinesterase (AChE) inhibitors from neurofibrillary tangles containing hyperphosphorylated form of a mi- general chemical classes such as galanthamine, an Amaryllidaceae al- crotubule associated protein, tau and the loss of neuronal synapses kaloid isolated from the plant Galanthus woronowii (Heinrich and and pyramidal neurons, as well as a decrease in levels of the neurotrans- Teoh, 2004) and other ACHE inhibitors have been used for the symp- mitter acetylcholine (ACh) by nearly 90% (Cummings, 2004). These tomatic treatment of AD (Cummings, 2004). The non-selectivity of changes result in the development of the typical symptoms of AD these drugs, their limited efficacy, and poor bioavailability, adverse characterised by gross and progressive damage of cognitive function, cholinergic side effects in the periphery, such as nausea, vomiting, di- often accompanied by behavioural disturbances such as memory loss, arrhoea, dizziness, and hepatotoxicity are among the several limita- tions to their therapeutic success (Burns and Iliffe, 2009) hence the continuous search for better AChE inhibitors from natural sources in- ⁎ Corresponding author. Tel.: +27 125298244; fax: +27 5298304. cluding plants (Hostettmann et al., 2006; Houghton et al., 2006). E-mail address: [email protected] (J.N. Eloff). 1 Present address: Toxicology & Ethnoveterinary Medicine, ARC-Onderstepoort Ammocharis coranica (Ker-Gawl.) Herb., Amaryllidaceae, is a bulbous Veterinary Institute, Private Bag X05, Onderstepoort 0110, South Africa. plant widely distributed in the summer-rainfall regions of southern

0254-6299/$ – see front matter © 2012 SAAB. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.sajb.2012.11.008 I.L. Elisha et al. / South African Journal of Botany 85 (2013) 44–47 45

Africa (Snijman and Linder, 1996). Medicinally, the bulbs of A. coranica 1 mM of DTNB in buffer A was used, while 3 mM DTNB was used for are used as cure for certain conditions resulting from witchcraft and di- the microplate assay. In the 96 well plates, 25 μl of 15 mM ATCI in abolic spells (Hulme, 1954) and a remedy for mental illnesses (Stafford water, 125 μl DTNB (3 mM) in buffer C, 50 μl buffer B and 25 μlofseri- et al., 2008). Despite the extensive chemical investigation of A. coranica ally diluted samples (two-fold for extracts, ten-fold for pure compounds (Koorbanally et al., 2000; Mason et al., 1955) there is no report on the and Physostigmine) were added and the absorbance was measured, in a effect of the bulbs of A. coranica on AChE activity. This project aimed at microplate reader, at 405 nm every 30 s three times, at 30 °C. Finally, screening the ethanolic crude extracts of the bulb of A. coranica and 25 μl of 0.2 U/ml of enzyme solution was added in and the absorbance their fractions for AChE inhibitory activity using TLC bioautographic was measured again after 3, 5, and 10 min respectively. The assays and microplate assays and isolating the bioactive compounds there were conducted in triplicate. Any increase in absorbance due to the from. spontaneous hydrolysis of the substrate was corrected by subtracting the average absorbance before adding the enzyme from the absorbance 2. Materials and methods after adding the enzyme. The percentage inhibition was calculated by comparing the inhibition of the sample to the blank (10% methanol in

2.1. Plant material buffer A). The inhibitory concentration of 50% of AChE (IC50) value was calculated from at least four different concentrations of the sample The bulbs of A. coranica were collected from Bethelsdorp in the using the Microsoft Excel statistical package. Eastern Cape region of South Africa and authenticated by Mr Graham Duncan. A voucher specimen, No. 39/69 was kept in the herbarium 2.3.2. Bioautographic assay for acetylcholinesterase inhibition of the Kirstenbosch National Botanical Garden. The collected plant The TLC bioautographic assay was carried out as adopted from material was dried in an oven at 40 °C for several days and powdered Rhee et al. (2001). The dried crude extract of the bulbs of A. coranica before extraction. was dissolved in methanol (MeOH) to a concentration of 10 mg/ml,

of which 3 μl was spotted on TLC plate (Silica gel 60 F254, Merck) 2.2. Extraction and bioassay guided fractionation and developed using chloroform:methanol (4:1) as the eluent. Physo- stigmine (3 μl of 0.01 M) was used as a positive control. Thereafter, The dried, powdered plant materials (210 g) were extracted using enzyme inhibiting activity of the samples was detected by spraying 96% ethanol (2×2 l) by shaking for 48 h. The supernatant was filtered a mixture of the substrate (1 mM ATCI) and the dye (1 mM DTNB) using Whatman No. 1 filter paper and concentrated to dryness under until the silica was saturated with the solution. The plates were allowed reduced pressure to yield 16 g of crude extract. The crude extract was to dry for 3–5 min, before 3 units/ml enzyme solutions were sprayed treated with 6% acetic acid (300 ml) and then basified with ammonia on the plates. A yellow background appeared, while white spots for to pH 9.5, after removal of acidic and neutral material with dichlo- inhibiting compounds became visible after 5 min. The TLC plates were romethane (200 ml×3). The basified solution was extracted with scanned or photographed within 15 min before the reaction colour ethyl acetate and butanol (200 ml×3) respectively and the organic disappeared. phases evaporated to dryness. The ethyl acetate fraction (2.08 g) was subjected to column chro- 2.3.3. Test for false positive results in the bioautographic method matography (20×5 cm) on silica gel 60 (200 g, Merck), eluted with The false positive reactions were eliminated using a method de- 100% dichloromethane enriched gradually with 10% methanol up to veloped by Rhee et al. (2003). The TLC plate was prepared as de- 100% methanol. Nine fractions (A–I) were collected based on UV ab- scribed above and sprayed with 1 mM DTNB in buffer A. The plate sorption, Dragendorff's test and AChE bioautographic assays. Fraction was allowed to dry (3–5 min) and then sprayed with thiocholine, E (tubes 68–78) was dissolved in dichloromethane:methanol (1:1) the product formed when 1 mM ATCI in 3 units/ml enzyme stock so- and allowed to crystallise at room temperature to give compound 1. lution was incubated at 37 °C for 20 min. The appearance of a yellow Sub-fraction C (tubes 19–35) was dissolved in dichloromethane: background or white spot was scanned or photographed and saved. methanol (1:1). This was then applied on a preparative TLC plate and eluted in hexane:ethyl acetate (2:1). The target compound was 3. Results and discussions scraped off the plate and the silica gel eluted with dichloromethane to give compound 2. Table 1 shows the IC50 values for AChE inhibitory activity of the eth- The butanol fraction was subjected to column chromatography anol extracts of A. coranica, the different fractions obtained using acid– (10×5 cm) on 100 g silica gel 60 (Merck) eluted with 100% dichlo- base fractionation and the isolated compounds there from. The ethanol romethane and then with dichloromethane enriched gradually with crude extracts of A. coranica had an IC50 value of 14.3±0.50 μg/ml. 20% methanol up to 100% methanol. Fractions 7–11 were combined However, butanol and ethyl acetate fractions had the highest AChE and allowed to crystallise and give more of compound 1. inhibitory effect among the fractions tested with IC50 values of 0.05± 0.02 and 43.1±1.22 μg/ml respectively, which had been chosen for 2.3. Acetylcholinesterase enzyme inhibitory assay the isolation of compounds with AChE inhibitory activity. A number of Amaryllidaceae species have also shown AChE inhibitory activity in- 2.3.1. Microtitre plate assay for acetylcholinesterase inhibition cluding Narcissus species (Coelho and Birks, 2001), Crinum macowanii, Inhibition of acetylcholinesterase by the crude extract and, acid– base fractions and the isolated compounds was determined using the Table 1 microplate assay (Rhee et al., 2001). Acetylcholinesterase (AChE) from Acetylcholinesterase inhibitory activity of the ethanolic crude extracts, butanol, ethyl electric eel (type VI-s, lyophilised powder), the substrate, Acetylthio- acetate fractions and lycorine isolated from the bulbs of Ammocharis coranica,expressed choline iodide (ACTI), Ellman's reagent, 5.5′-Dithiobis-(2-nitrobenzoic as IC50. acid) (DTNB) and Physostigmine, the reference compound were pur- a Sample IC50 (μg/ml) chased from Sigma (South Africa). The following buffers were used, – – Ethanolic crude extract 14.3±0.50 Buffer A: 50 mM Tris HCl, pH 8; buffer B: 50 mM Tris HCl, pH 8, Butanol fraction 0.05±0.02 containing 0.1% bovine serum albumin (BSA); buffer C: 50 mM Tris– Ethyl acetate fraction 43.1±1.22 HCl, pH 8, containing 0.1 M NaCl and 0.02 M MgCl2.6H2O(ATCI)inbuff- Lycorine 29.3±3.15 er A and 15 mM in Millipore water were used for TLC bioautographic Physostigmine (reference compound) 1.51 and microplate assay respectively. For the TLC bioautographic assay, a Expressed as mean±standard error mean (SEM). 46 I.L. Elisha et al. / South African Journal of Botany 85 (2013) 44–47

Crinum campanulatum, Crinum graminicola, Crinum variabile and Nerine that the IC50 of lycorine isolated from C. macowanii was 213 μM while bowdenii (Jäger et al, 2004; Rhee et al., 2003; Risa et al., 2004). Houghton et al. (2004) established an IC50 of 450 μM, for lycorine Bioassay-guided fractionation of the butanol and ethyl acetate isolated Crinum jagus and Crinum glaucum from south-western part of fractions led to the isolation of two compounds lycorine (Fig. 1)and Nigeria. According to López et al. (2002) and McNulty et al. (2010) 1 24-methylenecycloartan-3β-ol (Fig. 1). The H NMR (DMSO-d6)spec- lycorine was not active, however, McNulty and co-workers tested the tra of lycorine revealed peaks of H-8, H-11 at 6.79, 6.66, signal of a alkaloid at a maximum concentration of 10 μM which is not expected methylenedioxy group (CH2-12) at 5.94, signals of H-1, H-2, and H-3 to exhibit any activity while information on the concentration used by appear at 4.90, 4.26 and 5.35 respectively; in addition to the signals of López et al. (2002) is lacking. The discrepancies in the IC50 of lycorine three methylene groups of H-4, H-5, and H-7 between 4.00 and 2.20. are obvious and could be attributed to differences in the method and 13 The C NMR (DMSO-d6) exhibited 16 carbons, four of them were conditions adopted in the independent experiments, and the poor solu- methylene groups at 100.6, 56.7, 53.3 and 28.1 of C-12, C-7, C-5, C-4 bility of lycorine in most organic solvents including methanol. Although and seven methane groups at 118.5, 107.1, 105.1, 71.7, 70.2, 60.8 the calculated values of the three independent IC50 of lycorine varied, and 40.2 of C-3, C-8, C-11c and C-11b data are in agreement with previ- the most consistent deduction from the three experiments is that ously published data for lycorine (Evidente et al., 1983; Schultz et lycorine has appreciable inhibitory activity against AChE. In our experi- al., 1996; Toriizuka et al., 2008) and 24-methylenecycloartan-3β-ol ment physostigmine (positive control) is about 68 times a stronger in- (Koorbanally et al., 2000; Mesquita et al., 2008; Teresa et al., 1987). hibitor of AChE than lycorine. The fact that lycorine is toxic (Ghosal et

The NMR (CDCl3) data of 24-methylenecycloartan-3β-ol revealed in al., 1985; Lin et al., 1995) and has weak inhibitory effect on AChE the 1H NMR spectra signals assigned to H-3 proton at 3.19, two proton (Elgorashi et al., 2004; Houghton et al., 2004) makes it a less suitable singlet of CH2-28 at 4.69, 4.64, high field signals at 0.13, 0.36 of CH2-19, candidate for the development of an AD drug. in addition to unresolved seven methyl group signals around 1.00 ppm. The second compound, 24-methylenecycloartan-3β-ol, has been The 13C NMR data confirmed the presence of the terminal double bond isolated previously from A. coranica (Koorbanally et al., 2000). The at 157.3, 106.3, and the hydroxylated C-3 at 76.5. The remaining data compound did not exhibit acetylcholinesterase inhibitory activity. could not be resolved due to the compactness of the spectra. The negative values indicate that the compound had no inhibitory ac- Lycorine is the most widely distributed alkaloid in the family tivity against AChE at the concentration used in the microplate assay, Amaryllidaceae and has been isolated from almost every member of although it showed zone of inhibition when screened using the thin the family including A. coranica (Koorbanally et al., 2000). Lycorine layer chromatography bioautographic assay. This is however, the also had a wide range of biological activities including antifungal first report of the test of this compound for AChE inhibitory activity. (Evidente et al., 2004), antiviral (Ieven et al, 1982), antimalarial Lycorine is the only alkaloid isolated in this study from the most (Campbell et al., 1998) activities and toxic effects both in vitro and active butanol fraction of A. coranica. Previous studies, however, in vivo (Likhitwitayawuid et al., 1993; Lin et al., 1995; Liu et al., 2007). highlighted the presence of the alkaloids lycorine, caranine, acetyl-

In our experiment the IC50 of lycorine was 29.4±3.15 μg/ml (102± caranine, crinamine, hippadine, 6α-hydroxypowelline, 1-O-acetyl- 7.8 μM). Lycorine isolated from other Amaryllidaceae plants has also 9-O-demethylpluviine, buphandrine, ambelline, epivittatine and been tested for AChE inhibitory activity. Elgorashi et al. (2004) found 1-O-acetyllycorine in extracts from A. coranica (Koorbanally et al., 2000; Mason et al., 1955). Given the low ACHE inhibitory activity of lycorine, it is possible that the potency of the butanol fraction OH may have resulted from some these alkaloids, especially 1-O- acetyllycorine, one of the most potent ACHE inhibitors. HO 2 3 In conclusion, the ethanolic crude extract of the bulbs of A. 1 coranica like other members of the Amaryllidaceae family showed 11 3a AChE inhibitory activity. The preliminary thin layer chromatography O 10 11a bioautographic assay investigation of the ethanolic crude extract of 11b 4 11c the bulbs of A. coranica showed several white spots of inhibition, con- 12 ﬁrmed not to be false positives. Lycorine is again established to inhibit N 5 O 9 7a acetylcholinesterase, while 24-methylenecycloartan-3β-ol is not ac- 8 7 tive. The high acetylcholinesterase inhibitory activity of the butanol fraction could be attributed to the synergistic action of its constitu- 1 ents and more research should be carried out on this fraction due to the promising acetylcholinesterase inhibitory activity.

21 28 22 Acknowledgements 18 12 20 The Project was ﬁnancially supported by the University of Pretoria 23 24 26 and the National Research Foundation (NRF), South Africa. 11 17 19 13 25 16 1 14 27 Appendix A. Supplementary data 2 9 15 10 8 Supplementary data to this article can be found online at http:// 3 31 4 7 dx.doi.org/10.1016/j.sajb.2012.11.008. HO 5 6 29 30 References Burns, A., Iliffe, S., 2009. Alzheimer's disease. British Medical Journal 338 (b158), 467–471. 2 Campbell, W.E., Nair, J.J., Gammon, D.W., Bastida, J., Codina, C., Viladomat, F., Smith, P.J., Albrecht, C.F., 1998. Cytotoxic and antimalarial alkaloids from Brunsvigia littoralis. Fig. 1. The chemical structure of lycorine 1 and 24-methylenecycloartan-3B-ol 2. Planta Medica 64, 91–93. I.L. Elisha et al. / South African Journal of Botany 85 (2013) 44–47 47

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