PHYTOCHEMISTRY Phytochemistry 65 (2004) 2893–2896 www.elsevier.com/locate/phytochem
Choline esterase inhibitory properties of alkaloids from two Nigerian Crinum species Peter J. Houghton a
a,*
, Joseph M. Agbedahunsi b, Aderonke Adegbulugbe
a
Pharmacognosy Research Laboratories, Department of Pharmacy, KingÕs College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NN, UK b Drug Research and Production Unit, Faculty of Pharmacy, Obafemi Awolowo University, Ile Ife, Nigeria Received 1 April 2004; received in revised form 16 July 2004 Available online 1 October 2004 Dedicated to Prof. Dr. Kurt Hostettmann on the occasion of his 60th birthday
Abstract The bulbs of Crinum jagus and Crinum glaucum are used in traditional medicine in southern Nigeria for memory loss and other mental symptoms associated with ageing. Alkaloidal extracts of bulbs from each species showed inhibition of acetylcholinesterase, an activity exploited therapeutically to raise the depressed levels of acetylcholine in the brain associated with AlzheimerÕs disease. Using the in situ bioautographic test method for enzyme inhibition, a number of alkaloids were isolated and their activity quantified using the Ellman spectrophotometric test. The most active alkaloids isolated were hamayne (IC50 250 lM) and lycorine (IC50 450 lM) whilst other alkaloids were comparatively inactive with haemanthamane giving 3% inhibition and crinamine giving 4.4% inhibition at 50 mg ml1 (174 lM). These contrast with the positive control physostigmine which gave IC50 of 0.25 lM. Cholinesterase activity appears to be associated with the presence of two free hydroxy groups in this structural type of Amaryllidaceae alkaloid. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Crinum glaucum; Crinum jagus; Amaryllidaceae; Alkaloids; Acetylcholinesterase inhibition; AlzheimerÕs disease
1. Introduction Acetylcholine (ACh) is found in the synapse of the cerebral cortex and a deficiency in the cerebral cortex is one of the major features seen in sufferers of AlzheimerÕs disease (Bierer et al., 1995). Therapeutic agents in use for symptomatic treatment of AlzheimerÕs disease boost levels of ACh by inhibition of acetylcholinesterase (AChE), and compounds with this effect are known to occur in plants with a tradition of being used for failing memory and other cognitive decline associated with old age (Howes et al., 2003a,b). Galanthamine, an alkaloid obtained from Galanthus and Narcissus *
Corresponding author. Tel.: +44 207 848 4775; fax: +44 207 848 4800. E-mail address:
[email protected] (P.J. Houghton). 0031-9422/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2004.08.052
species (Amaryllidaceae), has been recently introduced into clinical use and has the added advantage of stimulating nicotinic receptors as well as inhibiting AChE (Pearson, 2001). Several other members of the Amaryllidaceae, and also their constituent alkaloids, have been tested for AChE inhibition but West African species have been comparatively neglected. When local traditional healers in southwest Nigeria were questioned by one of the authors (A.A) about which plant species were used to help memory loss in old age, two Crinum species, Crinum glaucum A. Chev. and Crinum jagus, (Thomps.) Dandy were frequently mentioned. It therefore appeared logical to test extracts for AChE inhibitory activity. Crinum species are known to produce alkaloids such as hamayne (Adesanya et al., 1992) but few of these compounds have been tested for their biological effects.
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2. Results and discussion Results for the AChE inhibitory properties of the crude methanol extract of the bulbs of the two species, and the fractions showing AChE inhibitory zones on the TLC plates are shown in Table 1. The activity is associated with the alkaloids since it was shown only by those fractions expected to contain alkaloids and this was confirmed by spraying with DragendorffÕs reagent. The isolated alkaloid 1 was identified as haemanthamine by comparison of its spectral properties with those reported in the literature (Bastida et al., 1987). Similarly 2 was identified as hamayne (Ochi et al., 1976;Adesanya et al., 1992), 3 as crinamine (Frahm et al., 1985;Viladomat et al., 1994) and 4 as lycorine (Evidente et al., 1983). OR OH O N
O 1 2 3
R = β-OCH3 R = β-OH R = α-OCH3 OH
The inhibitory activity against AChE of the four isolated alkaloids is shown in Table 2. Although related alkaloids have been previously tested for this activity, using the same assay method (Lopez et al., 2002), the AChE inhibitory effects of the alkaloids isolated in this study have not been previously reported. It can be seen that hamayne, 2 and lycorine, 4 are the only alkaloids with appreciable activity and for which the IC50 values were determined. These alkaloids appear to be weaker than the positive control physostigmine by three orders of magnitude and galanthamine is 250 times stronger than hamayne (Lopez et al., 2002). Hamayne is distinct from the other alkaloids in having two OH groups and it is noteworthy that 11-hydroxygalanthamine, which also has two OH groups in similar positions on the ring system is also quite active, although it is of the same order of magnitude as galanthamine itself (Lopez et al., 2002). Although the occurrence of two active alkaloids, hamayne, 2 and lycorine, 4, in the C. glaucum extract could explain the activity of the extract, and thereby provide an explanation for its reputed use for helping memory, it is more difficult to correlate the activity of the methanolic extract of C. jagus, since the major alkaloid present, haemanthamine, 1, displayed only weak activity. Minor constituents were observed in the extract when it was examined by TLC, but amounts were insufficient for isolation and testing, so it could be that some of these have potent AChE inhibitory effects.
HO
3. Experimental
H O
3.1. Plant Material
N
O 4
Previous work has shown the presence in C. jagus of hamayne, 2, crinamine, 3, lycorine, 4, pseudolycorine, and 6-hydroxycrinamine (Adesanya et al., 1992) so the isolation of a single major alkaloid, haemanthamine, 1, reported here is of interest and this is the first report of its occurrence in this species. This is the first report of the isolation of alkaloids from C. glaucum and it can be seen that the alkaloidal profile is similar to that in C. jagus reported by previous studies (Adesanya et al., 1992). Table 1 Inhibitory activity on acetylcholinesterase of extracts and fractions of C. jagus and C. glaucum Extract
%Inhibition at 100 lg ml1
JM GM JMAA GMAA
45.1 40.8 46.2 51.5
JM, methanolic extract of C. jagus; GM, methanolic extract of C. glaucum.
C. jagus (bulb, 1.2 kg wet weight) was collected in March 2003 by the Opa stream on the Campus of Obafemi Awolowo University, Ile Ife Nigeria. by Mr. Oladele of the Department of Pharmacognosy and was identified by Mr. G. Daramola of the Botany Department, Obafemi Awolowo University, Ile Ife, Nigeria. Voucher specimen (# IFE 8701) is deposited in the herbarium of the Department of Botany, Obafemi Awolowo University. C. glaucum (bulb, 700 g wet weight) was collected from Ilogbo Ekiti, identification was done by Dr. E.O. Ayodele, Botany Department, University of Table 2 Inhibitory activity on acetylcholinesterase of compounds from C. jagus and C. glaucum Compound
%Inhibition at 50 mg ml1
IC50 (lM)
Haemanthamine, 1 Hamayne, 2 Crinamine, 3 Lycorine, 4 Physostigmine (positive control)
3.3 100 4.4 18 nd
nd 250 nd 450 0.25
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Ibadan, Nigeria and a voucher specimen (# IFE 8699) is deposited in the herbarium of the Department of Botany, Obafemi Awolowo University. The bulb of C. jagus was sliced and oven dried at 60 °C due to the high water content whilst the bulb of C. glaucum was extracted fresh. 3.2. Extraction and isolation of alkaloids About 500 g (dry weight) of powdered sliced and dried C. jagus was extracted with methanol using Soxhlet apparatus for 48 h. The extract was filtered and concentrated in vacuo at 50 °C to dryness [115 g (20%) yield]. About 700 g fresh weight of C. glaucum bulb was macerated and extracted with methanol for 48 h. The mixture was filtered under vacuum and the filtrate concentrated to dryness in vacuo at 45 °C to give 36.19 g of extract [5.17% yield]. About 10 g of the methanolic extract of C. jagus and C. glaucum (JM and GM) were separately partitioned between H2O and CHCl3 to yield the CHCl3 fractions JMC and CMC, respectively. JMC and CMC were each extracted with 5% H2SO4 and the combined acid extracts for each species were basified with 3 M aqueous Na2CO3 (pH 9.2) and extracted with CHCl3. The combined CHCl3 layers in each case were washed with H2O and then taken down to dryness in vacuo to yield JMCA and GMCA for C. jagus and C. glaucum, respectively. The CHCl3 layer remaining after the fractionation of JMC with dilute acid was washed with H2O and taken to dryness in vacuo to give JMCC, GMCC being similarly obtained from GMC. The aqueous portions of the initial fractionation of JM and GM were separately acidified with H2 SO4 to make a 1% solution (pH 2.5) and partitioned with CHCl3, the CHCl3 fraction was further extracted with 1% H2SO4 and the acid layers combined. The combined acid layers were further partitioned with CHCl3. The acidic aqueous layer was basified with 3 M Na2CO3 (pH 9.2) and extracted with CHCl3. The combined CHCl3 layers for each plant were washed with H2O and then taken down to dryness in vacuo to yield JMAA and GMAA for C. jagus and C. glaucum, respectively. When JMAA and JMCA were monitored by TLC [CHCl3:MeOH (9:1)] the zone patterns were seen to be identical so the two layers were combined as JMAA. Similarly GMAA and GMCA showed identical profiles and were combined as GMAA. About 500 mg JMAA was dissolved in CHCl3 and pre-absorbed in a small amount of diatomaceous earth (Isolute HM-N). The blend was added to the top of silica gel Isolute SPE Column [20 g, Lot. # 1335102JA from International Sorbent Technology IST], previously wetted with CHCl3. Flash chromatography [Flash Master Parallel (Jones Chromatography)] was carried out with a step gradient CHCl3:MeOH in ratio 19:1, 9:1, 4:1, 7:3, 3:2 and 1:1 (450 ml each) successively (flow rate
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90 ml min1, collecting 30 ml fractions). The fractions were monitored using silica TLC [CHCl3:MeOH (9:1)] and like fractions bulked together to give seven subfractions J1–J7 (3.9, 9.0, 18.3, 103.1, 29.0, 137.5 and 82.2 mg, respectively). Fraction J4 showed AChE inhibitory activity on the TLC detection system and so 100 mg J4 was further purified on silica Prep. TLC [CHCl3:MeOH (9:1)] to obtain 1 (2.3 mg). Other alkaloids were present in the fraction but only in small amounts. Separation of C. glaucum fraction GMAA by flash chromatography, using the same system as for JMAA from C. jagus, yielded seven sub-fractions G1–G7 (17.5, 9.5, 10.8, 19.1, 75.0, 170.9 and 24.3 mg, respectively). Fractions G5 and G6 alone gave inhibitory zones on the TLC AChE inhibition test so were subjected to silica PTLC [CHCl3:MeOH (3:2)]. G5 yielded 2 (28 mg) and G6 gave 3 (72 mg) and 4 (11 mg) using the same solvent system. 3.3. Acetylcholine esterase inhibition assay methods AChE inhibition was assessed by modifications of the Ellman method, which is based on the reaction of released thiocholine to give a coloured product with a chromogenic reagent. The spectrophotometric method (Perry et al., 2000) was used to determine the activity of fractions and isolated compounds showing activity by preliminary TLC screening as outlined below. Acetylcholinesterase (40 ll of 0.86 U ml1 buffer pH 8) and extract or compound solution (20 ll) were added to 2.0 ml phosphate buffer (pH 8) and incubated at 4 °C for 20 min. The reaction was started by adding dithionitrobenzene (DTNB) (20 ll of 0.05 mM phosphate buffer pH 7) and acetylthiocholine (20 ll of 0.06 mM phosphate buffer pH 7) in phosphate buffer pH 7; 20 ll at 37 °C for 20 min. The reaction was halted by placing the assay solution tubes in a bath at 4 °C and adding physostigmine (20 ll 0.018 mM in phosphate buffer pH 7). The production of the yellow anion was recorded for 10 min on a Shimadzu UV/Vis 2101 double beam spectrophotometer at 412 nm. Blanks were used of reagents without extract and a positive control was set up which was the same as the blank except that physostigmine (20 ll 0.018 mM in buffer pH 7) was added. The inhibition rate (%) was calculated by the equation. Inhibition% ¼ ðBlank Blank positive controlÞ ðExperiment Experiment controlÞ= ðBlank Blank positive controlÞ 100 An in situ TLC autobiographic method was also employed using the method outlined by Rhee et al. (2001a) and was used for preliminary screening of extracts and fractions for compounds with AChE inhibitory effects. After the development of the TLC plate in appropriate
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solvent systems the dried plate was sprayed with AChE in bovine serum albumin [Sigma, Product # C-5021] and then was left in moist atmosphere (incubator) for 20 min at 37 °C. The plate was sprayed with ATCI [Sigma A5751] solution (45 mg/2.5 ml H2O) and the plate was allowed to dry in a stream of cold air. It was then sprayed with DTNB solution (100 mg/5 ml phosphate buffer pH 7). Eserine (Physostigmine) [Sigma Lot. 100K5002] and galanthamine [Sigma] were co-chromatographed as standard AChE inhibitors. Any compound inhibiting hydrolysis showed as a white spot against yellow background. A duplicate plate according to the method cited by Rhee et al. (2001b) was run to detect false positive effects due to interaction with the components of the extract and the chromogenic reagents. Acknowledgements Mrs. J. Hawkes (University of London Intercollegiate NMR Service), F. Cakebread and R. Tye (ULIRS Mass Spectrometry) from Kings College London are acknowledged for NMR and MS measurements. Dr. Agbedahunsi was supported by a Royal Society Developing World Short Term Visit Award. References Adesanya, S.A., Olugbade, T.A., Odebiyi, O.O., Aladesanmi, J.A., 1992. Antibacterial alkaloids in Crinum jagus. Int. J. Pharmacognosy 30, 303–307. Bastida, J., Viladomat, F., Llabres, J.M., Codina, C., Fleiz, M., Rubiralta, M., 1987. Alkaloids from Narcissus confusus. Phytochemistry 26, 1519–1524. Bierer, L.M., Haroutunian, V., Gabriel, S., Knott, P.J., Carlin, L.S., Purohit, D.P., Perl, D.P., Schmeidler, J., Kanof, P., Davis, K.L.,
1995. Neurochemical correlates of dementia severity in AlzheimerÕs disease – relative importance of the cholinergic deficits. J. Neurochem. 64, 749–760. Evidente, A., Cicala, M.R., Giudicianni, I., Randazzo, G., Riccio, R., 1983. 1H and 13C NMR analysis of lycorine and a-dihydrolycorine. Phytochemistry 22, 581–584. Frahm, A.W., Ali, A.A., Ramadan, M.A., 1985. C-13 Nuclear magnetic resonance spectra of Amaryllidaceae alkaloids – 1. Alkaloids with the crinane skeleton. Magn. Reson. Chem. 23, 804–808. Howes, M.-J.R., Perry, N.S.L., Houghton, P.J., 2003a. Plants with traditional uses and activities, relevant to the management of AlzheimerÕs disease and other cognitive disorders. Phytother. Res. 17, 1–18. Howes, M.-J.R., Houghton, P.J., 2003b. Plants used in Chinese and Indian traditional medicine for improvement of memory and cognitive function. Pharmacol. Biochem. Behav. 75, 513– 527. Lopez, S., Bastida, J., Viladomat, F., Codina, C., 2002. Acetylcholinesterase inhibitory activity of some Amaryllidaceae alkaloids and Narcissus extracts. Life Sci. 71, 2521–2529. Ochi, M., Otsuki, H., Nagao, K., 1976. Structure of hamayne, a new alkaloid from Crinum asiaticum L. var Japonicum Baker. Bull. Chem. Soc. Jpn. 49, 3363–3364. Pearson, V.E., 2001. Galantamine: a new Alzheimer drug with a past life. Ann. Pharmacother. 35, 1406–1413. Perry, N.S.L., Houghton, P.J., Theobald, A.E., Jenner, P., Perry, E.K., 2000. In-vitro inhibition of human erythrocyte acetylcholine esterase by Salvia lavandulaefolia essential oil and constituent terpenes. J. Pharm. Pharmacol. 52, 895–902. Rhee, I.K., van de Meent, M., Ingkaninan, K., Verpoorte, R., 2001a. Screening for acetylcholinesterase inhibitors from Amaryllidaceae using silica gel thin-layer chromatography in combination with bioactivity staining. J. Chromatogr. A 915, 217– 223. Rhee, I.K., van Rijn, R.M., Verpoorte, R., 2001b. Qualitative determination of false-positive effects in the acetylcholinesterase assay using thin layer chromatography. Phytochem. Anal. 14, 127– 131. Viladomat, R.F., Bastida, J., Codina, C., Campbell, W.E., Mathee, S., 1994. Alkaloids from Brunsvigia josephinae. Phytochemistry 35, 809–812.