AMERICAN JOURNAL OF SCIENTIFIC AND INDUSTRIAL RESEARCH © 2013, Science Huβ, http://www.scihub.org/AJSIR ISSN: 2153-649X, doi:10.5251/ajsir.2013.4.2.226.230 Isolation, characterisation and anti-cholinesterase activities of Physostigma venenosum (Calabar bean) 1John Bull E.O. and 2Ikpa, C.B.C.* 1Department of Chemistry, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria. 2Department of Chemistry, Imo State University, Owerri, Imo State, Nigeria.
ABSTRACT
Chemical investigation of the anticholenestrases activity of the seeds of physostigma venenosum (ordeal or calabar bean, esere bean or calabar bohme) resulted in the isolation of sangainarine N-diglycoside. The structure of the compound was established using NMR spectroscopy of (1H, 13C, COSY, DEPT and HSQC) in combination with IR and MS spectral data. The seed of the plant was extracted by percolation using ethanol. The extract was partitioned to obtain chloroform, water, methanol, and pet-ether fractions. The chloroform fraction was discovered as the most active fraction in anticholinesterase activity. The compound displayed a very high anticholinesterase activity (99.5%) in an in vitro test. The result did not support the use of physostigma venenosum as an ordeal poison by the Calabar people of Nigeria to justify person accused of witch craft.
Keywords: Physostigma venenosum, Anticholinesterase, Huperzin ‘A’, Enzyme assay
INTRODUCTION alkaloid extracted from physostigma venenosium have higher cholinesterase inhibitory activities when Most micro organisms and pest have developed compared to other monoterpenes (Pery 2001). In our resistance to the existing synthetic drugs and research for the antichollinesterase agents from pesticide. For these reasons, the continuing search natural source, the dark brownish oval shape bean for new drugs and pesticides have been most physostigma venenosum, commonly known as intensive from plants such as the marine plants called (ordeal poison, esere, feve de calaber, ekwuru or physostigma venenosum [calabar bohme (Rehm, Calabar bean) was selected because of its use by the 1994)]. native Calabar to justify persons accused of Physostigma venenosum is a woody climber growing witchcraft or other crimes. Most anticholinesterase on the bank of streams/rivers in the tropical West are toxins which are natural poisons that include the Africa (Louis et al 1997). most toxic substances known (Koelle 1975). The bean seems to confine in its habitat to a limited The study involves the isolation, structural elucidation area around the Gulf of Guinea and particularly about and characterization of the bioactive constituents in the mouth of the Calabar river, hence its common the plant and consequently evaluates the anti- name “Calabar bean (Daniel 1846). The seed of cholinesterase activity of the partitioned fractions in physostigma which was used by the native of old comparism to a standard poison Huperzine “A” for Calabar as an ordeal poison to justify persons possible development of new drugs and pesticides. accused of witchcraft or other crimes (Balfour 1860) MATERIALS AND METHODS are also used internally to alloy tension (Laura 2003). It is also used to reduce tension caused by irritation, General Experimental Procedure: IR spectrum was to treat tetanus, epilepsy, convulsion, glaucoma, determined on cm-1 Peckinelmer model 760 Canada. photophobia, ulcer, ocula, tension caused by Mass spectrum which was the report of mass divided m/ atropine, phatom tumor etc (Finely 1919). The extract by charge ( 2) (JohnBull 2000) was recorded by of the Calabar bean is an alkaloid that has been used advanced chemistry development elucidator at A1-R since mid 19th century for Ophthalmologic treatment 100 (1.871) Sb (1,65.00); CM (85:114). The NMR of (Nhi-ha et al 2003). Phytochemicals like tanines, 1H and 13C were recorded at double frequency of 1 13 alkaloids, phenols, saponines and flavonoids have Bruker ( H=300.131mHz, C F1=75.475MHz and been isolated from physostigma venenosum. The F2=300.131MHz) in CDCl3 using tetramethylsilane Am. J. Sci. Ind. Res., 2013, 4(2): 226-230
(TMS) as internal standard. The cossy spectrum The column was packed with silica gel and the used to record proton- proton coupling and the sample slurry was added on top of the silica gel and hetero-nuclear single quantum coherence (HSQC) eluted with chloroform and pet-ether in the ratio of used to correlate proton and carbon peaks for direct 0:10, 5:95, 10:90 to 100:0mills mixture respectively. bonded C-H pairs were recorded by Bruker at Bruker The eluted fractions labeled in roman numerals I to Biospin India. Column chromatography was carried XIV were analyzed with TLC and the brownish oily out at Michael Okpara University of Agriculture fraction X (0.45g) gave one spot on the TLC with Rf Umudike with silica gel (60-120mesh) to monitor the (0.67). preparation separations, analytical thin layers The pure compound (compound X with one spot) was chromatography (TLC) was carried out at room further analyzed to be Sangainarine N-diglycoside temperature. The TLC plate was prepared using TLC using Peckinelmer model 760 Canada IR. The IR plate (glass of 5×15cm) coated with homogenous spectrum showed bands at 3093.6cm-1 (Ar-c), mixture of TLC silica gel Merck grade 60A with 2925.2cm-1(C-H) stretching, 2854(C-H) aliphatic, gypsium binder, ethanol, methanol, petroleum ether 2541(C=N).1676.3(C=O), 1589.4(aromatic ring (pet ether), chloroform, ethyl acetate, acetone and conjugated) and 1573.3 (conjugated ring). azo dye were all analytical grade and were procured from Merck. The liver for the enzyme assay was At finger print region the following bands were procured from a healthy female sheep bought at characterized 1420 (C-H) aromatic bending, 1300 (N- Ndioru market Ikwuano Umuahia, Abia State. O Ar) and 850 (C-H) aromatic. PLANT MATERIALS Advance chemistry elucidator mass of m/z 845 (m-2) calculated for C H O N (m/z 643 with base peak Matured seed of physostigma venenosum (Calabar 42 55 17 of m/z 637). bean) where purchased from Ariara International 1 13 market Aba in Abia state on 14th July2006. Brucker multinuclear NMR experiments of H, C, cosy of 1H-1H and two dimensional NMR HSQC of Authentication of the plant was done by Dr. A. 1H-13C recorded at f1 300MHz and f2 300MHz using Nmeregini of Taxonomy section, Forestry TMS as internal standard were used. (Table1) Department of Michael Okpara University of Agriculture Umudike Nigeria. Enzyme assay: The in vitro cholinesterase inhibition was carried out by colorimetric method (Keite et al, Extraction and isolation of plant materials: 1996 and JohnBull et al, 2000) in which 10mg, 20mg Matured seed samples weighing 4.12kg, were and 30mg of isolated fractions were dissolved in 1ml collected, peeled and dried under shade for three acetone respectively. months in the laboratory bench of Chemistry Department of Michael Okpara University of The sheep liver used for the test was procured from Agriculture, Umudike. a healthy female sheep bought from Ndioru market. The dry seed sample was grounded to powdery form The killing and procurement of the sheep’s liver was done within five minutes, the liver was immediately with new mortar and pestle. The powdered seed 0 sample 3.2kg was percolated with 4 liters of placed at a temperature of 0 C in a thermo flask of redistilled ethanol (98%) for one week, the extract ice. was filtered and concentrated with rota evaporator at 5g of the liver was grinded, dispensed in 5mills of 350C. distilled water to get a homogenous liver solution. 1ml The concentrated ethanol extract was kept to dry for of liver homogenate was pre-incubated with 10mg, 20mg and 30mg of test compound of each sample two days to obtain a dark brown gummy crude extract 0 of 3.86g, a portion of 26.33g of crude extract was fraction for 15 minutes at 37 C in a thermostatic partitioned between chloroform and water . water bath. A chloroform soluble fraction 8.87g was obtained, 10mg, 20mg and 30mg of Huperzine ‘A’ (control 4.41g of the chloroform fraction were then partitioned standard) was treated with 1ml of sheep liver between petroleum ether (60-800C) and aqueous homogenate in the same manner. methanol to obtain 2.13g of methanol fraction and After incubating, 1ml of water followed by 1ml of 2.27g of pet-ether fraction. 0.01molar ethyl acetate and 1ml of 0.2% of green azo 4.16g of the chloroform fraction was dissolved in 30 dye were added to the mixtures and pre incubated for mills of chloroform and mixed with 25g of silica gel to another one minute. make the slurry for the column chromatography.
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The reaction was stopped at exactly one minute with 13C NMR spectrum showed 42 signals consisting of 4mls of glacial acetic acid. The reading of the color four aromatic quantinary carbon at [C:128.80, change was taken at 540nm color absorbance with 130.98, 132.44 and 152.02] one quantinary Carbonyl spectrophotometer in the chemistry laboratory of carbon at [C:200.00] and four quantinary oxygen Michael Okpara University of Agriculture Umudike. bearing carbon at [C:182.00,165.00,167.68 and The reading was normalized to 100% by subtracting 152.85]. Methine conjugated carbon are shown at from one. The percentage inhibition was calculated chemical shift of [C: 114.00, 85.03, 80.20, 77.44, using the formula 98.00 and 116.38]. Methine carbon bearing an % ChE inhibition=C-E/C × 100/1 oxygen at [C:122.50] and methine carbon of C = Control absorbance heterocyclic N-bridge at [C:98.88 and 112.00]. Two aromatic methylene carbon were observed at E = Sample absorbance [C:60.50 and 66.92] and two oxygen bearing ChE - Cholinesterase (Table 2) methylene carbon at [C:68.16 and 76.60]. Methyl STATISTICAL ANALYSIS carbon was observed at [C:23.74] while methoxy at All measurements were replicated three times. [C:28.92]. The signals for the sugar moity diffuse at The results were based on the mean ± standard the chemical shift of [C:29.57 to 38.72]. (Table I) I 13 deviation of the triplicate sample measurement. Table I: H and C NMR DATA OF COMPOUND X The enzyme assay test was normalized to 100% by Assignign 13c 1h M Int subtracting from one. ment value The percentage inhibition was calculated using the 1 68.16 3.018 s 2H formula 2 165.00 % ChE inhibition = C-E/C × 100/1 3 182.00 C = Control absorbance 4 200.00 5 114.02 7.723 s 1H E = Sample absorbance. 6 128.80 7 130.98 RESULTS 8 85.03 7.527 t 1H Characterization: Compound X labeled 1K-B was a 9 60.50 2.643 d/d 2H light brown gummy substance with Rf of 0.67. Its 10 80.20 7.263 M 1H molecular composition was C42H55O17N as 11 80.30 7.516 d/d 1H determined by mass M/Z 847(m-2) calculated 12 77.44 4.234 m 1H 845,base peak of 657 m/z, IR, NMR of 1H, 13C, 13 98.00 7.534 d 1H COSY and HSQC. 14 132.44 1H The IR spectrum of 1K-B measured in cm-1 indicates 15 152.02 1H absorption at 3093.6 for aromatic carbon (Ar-c) 16 98.88 7.705 s 1H 2925.2(C-H) stretching, 2854(C-H) aliphatic, 17 112.00 7.546 m 1H indicating pressure of the CH3,CH2.1796(C=O) sand 18 116.38 7.693 d/d 1H witching, 1676.3(C=O), 1589.4(C=C-C=C) aromatic 19 122.50 7.712 q 1H ring conjugated, and 1573.3 conjugated ring. At 20 167.78 finger print region we have 1420(C-H) aromatic 21 152.85 bending, 1300 (N-O) aromatic and 850 substituted C- 22 66.92 2.742 s 2H 1 H of the aromatic ring. H NMR showed presence of 23 76.60 2.894 s 2H methine hydrogen at [H:7.723(s), 7.527(t)7.263(m), 24 28.93 2.034 s 3H 7.516(d/d)4.234(m), 7.534(d), 7.705(s), 7.546(m), 7.693(d/d), 25 23.74 1.569 d 3H 7.712(q) with integral values of one hydrogen each. Sugar 29.37 to 4,7000 to Methylene protons were shown at 38.72 6.818 (H:3.018(s),2.643(d/d), 2.743(s), 2.894(s)]. Methyl proton was shown at [H:1.567(d)] and methoxy proton at COSSY spectra shows coupling at [H:4.234 and [H:2.037 ] while chemical shifts for sugar moity (m) (s) 1.569 ] of H=12 and H=25 respectively. were found at the dwarf peaks of [H:4.700- (d) H:6.818]. 228
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In HQSC correlation was observed at [C:28.93 and (ChE) inhibition shows that chloroform fraction IfA
H:2.034(s)] of C=24 and H=24 respectively and at indicates the best inhibition at all concentration A [C:23:74 and H:1.56 ] of C=25 and H=25 followed by the methanol fraction (2f ). The ethanol (d) o respectively combining the MS, NMR of 1H , 13C, fraction (F ) shows a very low inhibition at lower COSSY, HQSC and IR spectra compound X is concentration but at higher concentration it gave a identified as Sangainarine N-diglycoside (Fig. 1) good percentage of cholinesterase inhibition. Enzyme essay result: Table II shows fractions with various degree of inhibition as expressed in terms of Huperzin ‘A’ units (HAU). The order of cholinesterase
Table II: Percentage of Cholinesterase inhibition of crude fractions compared with Huperzin ‘A’ Unit Factions 10Mg/Ml 20Mg/Ml 30Mg/Ml Remark Ethanol (Fo) 28.020.6 69.130.5 94.460.01 Poor inhibition at low concentration but good inhibition at high concentration Chloroform (IFA) 65.410.11 70.307 99.610.001 Good inhibition at all concentration Methanol (2fA) 64.7 0.1 69.340.65 95.500.02 Good inhibition at all concentration Pet-ether (2fB) 5.501 18.540.2 22.970.05 Poor inhibition at all concentration Result based on mean standard deviation of triplicate sample measurement Fig. 1
4
1 3 5 25 CH3 6 12 2 7 11 13 24
OCH3 8 14 19 10 18 9 15 20
1 6 23 (b) 17 21 H N H3 CO 22 OCH3
H H H OCH H 3 (a) OCH H 3 H OCH 3 H H OCH OCH 3 3 H
Compound X = Sangainarine - N-diglycoside
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REFERENCES The pet-ether fraction at (2fB) at all concentration Balfour (1860)Description Of The Plant Which Produces exhibits a very poor inhibition of 5.50% at 10mg, The Ordeal Bean of Calabar. Trans Roy. Sec. edition 18.54% at 20mg and 22.9% at 30mg so it cannot vol. XXII pp 305-312. show a significant effect in vivo (Koelle G.B., 1975) Daniel F.W- (1846) The Native of old Calabar. New phillos Journal 5: 159. The cholinesterase inhibition of chloroform fraction (IFA) and methanol fraction (2FA) shows a good Finley Ellingwood NM. D. (1919) by America Material comparism with the percentage ChE inhibition of Medical, therapeutic and pharmacognosy. standard Huperzin ‘A’ unit. The very high percentage o A A Higgins, J.P. and Flicker, L. (2000). Lecithin for dementia inhibition of the crude fraction of F , IF and 2F and cognitive impairment. Cochrane Database indicates that these fractions can accelerate the System. Rev (2): CD 001015; 102. synthesis of acetylcholin in the brain (Huggins, et al, JohnBull E.O. and Naidu M.S.R. (2000) In Search of new 2000). The accumulation of acetylcholine in the brain Organophosphorus Pesticides and Insecticides, Part which causes a log-jam of the body nervous system, III Synthesis and Anticholinestrase Studies of new 3- resulting to mental confusion, shortness of breath, (Subs)-quinoxylPyridoxy/eyclicamino-2-3-dihydro-2-(4- convulsion, coma, and death (Rahimi et al, 2000). chlorophenyl)-l H-napth [l,2-e] [1,2,3] This explains the constant observation of dead Oxazaphosphorine 3-sulphitcs. Phosphorus, sulfur, insects (House flies, Drosophila and Cockroaches) in silicon and Related Elements. U.S.A. 167, 9-20; Chem. uncovered or unwashed beakers used during the Abstract 135 (2001) 122SS8q. investigation. Koelle G.B. (1975) Drugs Acting at Synaptic and The high level of enzyme inhibition of the seed crude Neuroeffector Functional Sites, in Goodman L.S, Gilman A (edition). The pharmacological Basis of fraction explain why it is used to allay tension induced therapeutics 5th ed, New York, Macmillan Publishing externally (Laura, 2003) and why it quickly causes Co Inc. pp. 404 - 476. vomiting, purging and death in ordeal test to justify persons accused of witchcraft and other crimes (Nhi- Laura Spinney (2003); Histories, New Scientist ha et al, 2003). Encyclopeadia Britannica. Eleventh edition. “The killer bean of Calabar”. P. 482. CONCLUSION Louis .S Goodman and Alfred Gilman (1997) The The plant physostigma venenosum otherwise called Pharmacological Basis of esere in Calabar land or ordeal poison in English is Therapeutics. Fifth edition, Macmillan Publishing Co. confirmed to have secondary metabolites of high anti- Inc New York pp. 445-465. cholinesterase activity. The results of the enzyme Nhi-Ha, T Jennifer, H Subhajoy, M and Tristine, Y. (2003): assay technique of fraction IFA gave a very good Efficacy or Cholinesterase Inhibitors in The Treatment comparism with Huperzin ‘A’ – a synthetic anti- of Neuropsychiatry Symptoms and Functional cholinesterase agent. Impairment in Alzheimer disease JAMA 289 (2): 210 - 216. The isolated compounds which its structure were Perry N, Court G, Bi det N, court J, penny E, (2001). proposed with the physical parameters of 1R, MS, European. Herbs with Cholinergic Activities, potential NMR spectra of 13C and 1H, 1H-1H, COSSY, and 1 13 in dementia therapy, international journal geriatric HSQC ( H- C COSY) showed that an alkaloid linked psychiatry 11: 1063 - 9. to two pyrose sugar called Sangainarine N- diglycoside was isolated from compound X. Rahimi, R, Nokofa S, Abdollahi M. (2000); Morbidity and mortality in acute organophosphate poisoned patients treated by oxime. Meta-analysis of chemical trial Journal of human explo. Toxicol 3. 157-16.
Rehm S. (1994); Multilingual Dictionary of Agronomic Plants (Longman Publisher) CRC Press USA, p. 2064.
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