AMERICAN JOURNAL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

2013, Science Hu, 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)
1

John Bull E.O. and 2Ikpa, C.B.C.*

Department 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
1
N-diglycoside. The structure of the compound was established using NMR spectroscopy of ( H,
13
C, 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
Most micro organisms and pest have developed
resistance to the existing synthetic drugs and
pesticide. For these reasons, the continuing search
for new drugs and pesticides have been most
intensive from plants such as the marine plants called
physostigma venenosum [calabar bohme (Rehm,
1994)].
Physostigma venenosum is a woody climber growing
on the bank of streams/rivers in the tropical West
Africa (Louis et al 1997).
The bean seems to confine in its habitat to a limited
area around the Gulf of Guinea and particularly about
the mouth of the Calabar river, hence its common
name Calabar bean (Daniel 1846). The seed of
physostigma which was used by the native of old
Calabar as an ordeal poison to justify persons
accused of witchcraft or other crimes (Balfour 1860)
are also used internally to alloy tension (Laura 2003).
It is also used to reduce tension caused by irritation,
to treat tetanus, epilepsy, convulsion, glaucoma,
photophobia, ulcer, ocula, tension caused by
atropine, phatom tumor etc (Finely 1919). The extract
of the Calabar bean is an alkaloid that has been used
th
since mid 19 century for Ophthalmologic treatment
(Nhi-ha et al 2003). Phytochemicals like tanines,
alkaloids, phenols, saponines and flavonoids have
been isolated from physostigma venenosum. The

alkaloid extracted from physostigma venenosium

have higher cholinesterase inhibitory activities when
compared to other monoterpenes (Pery 2001). In our
research for the antichollinesterase agents from
natural source, the dark brownish oval shape bean
physostigma
venenosum, commonly known as
(ordeal poison, esere, feve de calaber, ekwuru or
Calabar bean) was selected because of its use by the
native Calabar to justify persons accused of
witchcraft or other crimes. Most anticholinesterase
are toxins which are natural poisons that include the
most toxic substances known (Koelle 1975).
The study involves the isolation, structural elucidation
and characterization of the bioactive constituents in
the plant and consequently evaluates the anticholinesterase activity of the partitioned fractions in
comparism to a standard poison Huperzine A for
possible development of new drugs and pesticides.
MATERIALS AND METHODS
General Experimental Procedure: IR spectrum was
-1
determined on cm Peckinelmer model 760 Canada.
Mass spectrum which was the report of mass divided
m/
by charge ( 2) (JohnBull 2000) was recorded by
advanced chemistry development elucidator at A1-R
100 (1.871) Sb (1,65.00); CM (85:114). The NMR of
1
13
H and C were recorded at double frequency of
1
13
Bruker ( H=300.131mHz,
C F1=75.475MHz and
F2=300.131MHz) in CDCl3 using tetramethylsilane

Am. J. Sci. Ind. Res., 2013, 4(2): 226-230

(TMS) as internal standard. The cossy spectrum

used to record proton- proton coupling and the
hetero-nuclear single quantum coherence (HSQC)
used to correlate proton and carbon peaks for direct
bonded C-H pairs were recorded by Bruker at Bruker
Biospin India. Column chromatography was carried
out at Michael Okpara University of Agriculture
Umudike with silica gel (60-120mesh) to monitor the
preparation separations, analytical thin layers
chromatography (TLC) was carried out at room
temperature. The TLC plate was prepared using TLC
plate (glass of 515cm) coated with homogenous
mixture of TLC silica gel Merck grade 60A with
gypsium binder, ethanol, methanol, petroleum ether
(pet ether), chloroform, ethyl acetate, acetone and
azo dye were all analytical grade and were procured
from Merck. The liver for the enzyme assay was
procured from a healthy female sheep bought at
Ndioru market Ikwuano Umuahia, Abia State.
PLANT MATERIALS
Matured seed of physostigma venenosum (Calabar
bean) where purchased from Ariara International
th
market Aba in Abia state on 14 July2006.
Authentication of the plant was done by Dr. A.
Nmeregini
of
Taxonomy
section,
Forestry
Department of Michael Okpara University of
Agriculture Umudike Nigeria.
Extraction and isolation of plant materials:
Matured seed samples weighing 4.12kg, were
collected, peeled and dried under shade for three
months in the laboratory bench of Chemistry
Department of Michael Okpara University of
Agriculture, Umudike.
The dry seed sample was grounded to powdery form
with new mortar and pestle. The powdered seed
sample 3.2kg was percolated with 4 liters of
redistilled ethanol (98%) for one week, the extract
was filtered and concentrated with rota evaporator at
0
35 C.
The concentrated ethanol extract was kept to dry for
two days to obtain a dark brown gummy crude extract
of 3.86g, a portion of 26.33g of crude extract was
partitioned between chloroform and water .
A chloroform soluble fraction 8.87g was obtained,
4.41g of the chloroform fraction were then partitioned
0
between petroleum ether (60-80 C) and aqueous
methanol to obtain 2.13g of methanol fraction and
2.27g of pet-ether fraction.
4.16g of the chloroform fraction was dissolved in 30
mills of chloroform and mixed with 25g of silica gel to
make the slurry for the column chromatography.

The column was packed with silica gel and the

sample slurry was added on top of the silica gel and
eluted with chloroform and pet-ether in the ratio of
0:10, 5:95, 10:90 to 100:0mills mixture respectively.
The eluted fractions labeled in roman numerals I to
XIV were analyzed with TLC and the brownish oily
fraction X (0.45g) gave one spot on the TLC with Rf
(0.67).
The pure compound (compound X with one spot) was
further analyzed to be Sangainarine N-diglycoside
using Peckinelmer model 760 Canada IR. The IR
-1
spectrum showed bands at 3093.6cm
(Ar-c),
-1
2925.2cm (C-H) stretching, 2854(C-H) aliphatic,
2541(C=N).1676.3(C=O),
1589.4(aromatic
ring
conjugated) and 1573.3 (conjugated ring).
At finger print region the following bands were
characterized 1420 (C-H) aromatic bending, 1300 (NO Ar) and 850 (C-H) aromatic.
Advance chemistry elucidator mass of m/z 845 (m-2)
calculated for C42H55O17N (m/z 643 with base peak
of m/z 637).
1
13
Brucker multinuclear NMR experiments of H, C,
1
1
cosy of H- H and two dimensional NMR HSQC of
1
13
H- C recorded at f1 300MHz and f2 300MHz using
TMS as internal standard were used. (Table1)
Enzyme assay: The in vitro cholinesterase inhibition
was carried out by colorimetric method (Keite et al,
1996 and JohnBull et al, 2000) in which 10mg, 20mg
and 30mg of isolated fractions were dissolved in 1ml
acetone respectively.
The sheep liver used for the test was procured from
a healthy female sheep bought from Ndioru market.
The killing and procurement of the sheeps liver was
done within five minutes, the liver was immediately
0
placed at a temperature of 0 C in a thermo flask of
ice.
5g of the liver was grinded, dispensed in 5mills of
distilled water to get a homogenous liver solution. 1ml
of liver homogenate was pre-incubated with 10mg,
20mg and 30mg of test compound of each sample
0
fraction for 15 minutes at 37 C in a thermostatic
water bath.
10mg, 20mg and 30mg of Huperzine A (control
standard) was treated with 1ml of sheep liver
homogenate in the same manner.
After incubating, 1ml of water followed by 1ml of
0.01molar ethyl acetate and 1ml of 0.2% of green azo
dye were added to the mixtures and pre incubated for
another one minute.

227

Am. J. Sci. Ind. Res., 2013, 4(2): 226-230

13

The reaction was stopped at exactly one minute with

4mls of glacial acetic acid. The reading of the color
change was taken at 540nm color absorbance with
spectrophotometer in the chemistry laboratory of
Michael Okpara University of Agriculture Umudike.
The reading was normalized to 100% by subtracting
from one. The percentage inhibition was calculated
using the formula
% ChE inhibition=C-E/C 100/1
C = Control absorbance
E = Sample absorbance
ChE - Cholinesterase (Table 2)
STATISTICAL ANALYSIS
All measurements were replicated three times.
The results were based on the mean standard
deviation of the triplicate sample measurement.
The enzyme assay test was normalized to 100% by
subtracting from one.
The percentage inhibition was calculated using the
formula
% ChE inhibition = C-E/C 100/1
C = Control absorbance
E = Sample absorbance.

C NMR spectrum showed 42 signals consisting of

four aromatic quantinary carbon at [C:128.80,
130.98, 132.44 and 152.02] one quantinary Carbonyl
carbon at [C:200.00] and four quantinary oxygen
bearing carbon at [C:182.00,165.00,167.68 and
152.85]. Methine conjugated carbon are shown at
chemical shift of [C: 114.00, 85.03, 80.20, 77.44,
98.00 and 116.38]. Methine carbon bearing an
oxygen at [C:122.50] and methine carbon of
heterocyclic N-bridge at [C:98.88 and 112.00]. Two
aromatic methylene carbon were observed at
[C:60.50 and 66.92] and two oxygen bearing
methylene carbon at [C:68.16 and 76.60]. Methyl
carbon was observed at [C:23.74] while methoxy at
[C:28.92]. The signals for the sugar moity diffuse at
the chemical shift of [C:29.57 to 38.72]. (Table I)
I
13
Table I: H and C NMR DATA OF COMPOUND X
Assignign
ment
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Sugar

RESULTS
Characterization: Compound X labeled 1K-B was a
light brown gummy substance with Rf of 0.67. Its
molecular
composition
was
C42H55O17N
as
determined by mass M/Z 847(m-2) calculated
1
13
845,base peak of 657 m/z, IR, NMR of H, C,
COSY and HSQC.
-1
The IR spectrum of 1K-B measured in cm indicates
absorption at 3093.6 for aromatic carbon (Ar-c)
2925.2(C-H)
stretching,
2854(C-H)
aliphatic,
indicating pressure of the CH3,CH2.1796(C=O) sand
witching, 1676.3(C=O), 1589.4(C=C-C=C) aromatic
ring conjugated, and 1573.3 conjugated ring. At
finger print region we have 1420(C-H) aromatic
bending, 1300 (N-O) aromatic and 850 substituted C1
H of the aromatic ring. H NMR showed presence of
methine hydrogen at [H:7.723(s), 7.527(t)7.263(m),
7.516(d/d)4.234(m), 7.534(d), 7.705(s), 7.546(m), 7.693(d/d),
7.712(q) with integral values of one hydrogen each.
Methylene
protons
were
shown
at
(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
[H:2.037(s)] while chemical shifts for sugar moity
were found at the dwarf peaks of [H:4.700H:6.818].

13c

1h

68.16
165.00
182.00
200.00
114.02
128.80
130.98
85.03
60.50
80.20
80.30
77.44
98.00
132.44
152.02
98.88
112.00
116.38
122.50
167.78
152.85
66.92
76.60
28.93
23.74
29.37 to
38.72

3.018

Int
value
2H

7.723

1H

7.527
2.643
7.263
7.516
4.234
7.534

t
d/d
M
d/d
m
d

7.705
7.546
7.693
7.712

s
m
d/d
q

1H
2H
1H
1H
1H
1H
1H
1H
1H
1H
1H
1H

2.742
2.894
2.034
1.569
4,7000 to
6.818

s
s
s
d

2H
2H
3H
3H

COSSY spectra shows coupling at [H:4.234(m) and

1.569(d)] of H=12 and H=25 respectively.

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Am. J. Sci. Ind. Res., 2013, 4(2): 226-230

(ChE) inhibition shows that chloroform fraction If

indicates the best inhibition at all concentration
A
followed by the methanol fraction (2f ). The ethanol
o
fraction (F ) shows a very low inhibition at lower
concentration but at higher concentration it gave a
good percentage of cholinesterase inhibition.

In HQSC correlation was observed at [C:28.93 and

H:2.034(s)] of C=24 and H=24 respectively and at
[C:23:74 and H:1.56(d)] of C=25 and H=25
1
13
respectively combining the MS, NMR of H , C,
COSSY, HQSC and IR spectra compound X is
identified as Sangainarine N-diglycoside (Fig. 1)
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
o

Ethanol (F )
A

Chloroform (IF )
A

Methanol (2f )
B

Pet-ether (2f )

10Mg/Ml

20Mg/Ml

30Mg/Ml

Remark

28.020.6

69.130.5

94.460.01

Poor inhibition at low concentration but

good inhibition at high concentration

65.410.11

70.307

99.610.001

Good inhibition at all concentration

64.7 0.1

69.340.65

95.500.02

Good inhibition at all concentration

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

25
CH3
12

11

13

24
OCH3
14

19

10

18

15

20

23

1 6

(b)
H3 CO

17

22

OCH3

OCH3
OCH3

OCH3

21

Com pound X

H
H

(a)

H
OCH3

OCH3

Sangainarine - N-diglycoside

229

Am. J. Sci. Ind. Res., 2013, 4(2): 226-230

REFERENCES
B

Balfour (1860)Description Of The Plant Which Produces

The Ordeal Bean of Calabar. Trans Roy. Sec. edition
vol. XXII pp 305-312.

The pet-ether fraction at (2f ) at all concentration

exhibits a very poor inhibition of 5.50% at 10mg,
18.54% at 20mg and 22.9% at 30mg so it cannot
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

A
A
(IF ) and methanol fraction (2F ) shows a good
comparism with the percentage ChE inhibition of
standard Huperzin A unit. The very high percentage
o
A
A
inhibition of the crude fraction of F , IF and 2F
indicates that these fractions can accelerate the
synthesis of acetylcholin in the brain (Huggins, et al,
2000). The accumulation of acetylcholine in the brain
which causes a log-jam of the body nervous system,
resulting to mental confusion, shortness of breath,
convulsion, coma, and death (Rahimi et al, 2000).
This explains the constant observation of dead
insects (House flies, Drosophila and Cockroaches) in
uncovered or unwashed beakers used during the
investigation.

Finley Ellingwood NM. D. (1919) by America Material

Medical, therapeutic and pharmacognosy.
Higgins, J.P. and Flicker, L. (2000). Lecithin for dementia
and cognitive impairment. Cochrane Database
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Organophosphorus Pesticides and Insecticides, Part
III Synthesis and Anticholinestrase Studies of new 3(Subs)-quinoxylPyridoxy/eyclicamino-2-3-dihydro-2-(4chlorophenyl)-l
H-napth
[l,2-e]
[1,2,3]
Oxazaphosphorine 3-sulphitcs. Phosphorus, sulfur,
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Abstract 135 (2001) 122SS8q.
Koelle G.B. (1975) Drugs
Neuroeffector Functional
Gilman A (edition). The
therapeutics 5th ed, New
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The high level of enzyme inhibition of the seed crude

fraction explain why it is used to allay tension induced
externally (Laura, 2003) and why it quickly causes
vomiting, purging and death in ordeal test to justify
persons accused of witchcraft and other crimes (Nhiha et al, 2003).

Acting at Synaptic and

Sites, in Goodman L.S,
pharmacological Basis of
York, Macmillan Publishing

Laura Spinney (2003); Histories, New Scientist

Encyclopeadia Britannica. Eleventh edition. The killer
bean of Calabar. P. 482.

CONCLUSION

Louis .S Goodman and Alfred Gilman (1997) The

Pharmacological
Basis
of
Therapeutics. Fifth edition, Macmillan Publishing Co.
Inc New York pp. 445-465.

The plant physostigma venenosum otherwise called

esere in Calabar land or ordeal poison in English is
confirmed to have secondary metabolites of high anticholinesterase activity. The results of the enzyme
A
assay technique of fraction IF gave a very good
comparism with Huperzin A a synthetic anticholinesterase agent.

Nhi-Ha, T Jennifer, H Subhajoy, M and Tristine, Y. (2003):

Efficacy or Cholinesterase Inhibitors in The Treatment
of Neuropsychiatry Symptoms and Functional
Impairment in Alzheimer disease JAMA 289 (2): 210 216.

The isolated compounds which its structure were

proposed with the physical parameters of 1R, MS,
13
1
1
1
NMR spectra of C and H, H- H, COSSY, and
1
13
HSQC ( H- C COSY) showed that an alkaloid linked
to two pyrose sugar called Sangainarine Ndiglycoside was isolated from compound X.

Perry N, Court G, Bi det N, court J, penny E, (2001).

European. Herbs with Cholinergic Activities, potential
in dementia therapy, international journal geriatric
psychiatry 11: 1063 - 9.
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.

230