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Tropical Journal of Pharmaceutical Research
Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, Nigeria
ISSN: 1596-5996 EISSN: 1596-9827
Vol. 8, Num. 6, 2009, pp. 515-519
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Tropical Journal of Pharmaceutical Research, Vol. 8, No. 6, December, 2009,
pp. 515-519
Research Article
Antidiarrhoeal
Activity of Chromatographic Fractions of Stereospermum kunthianum Cham
Sandrine Petit (Bignoniaceae) Stem Bark
FP Ching1*, SO
Okpo2, A Falodun3 and EKI Omogbai2
1Department of
Pharmacology, Faculty of Basic Medical Sciences, College of Health Sciences,
Niger Delta University, Wilberforce Island, Yenagoa, Departments of 2Pharmacology & Toxicology,
and 3Pharmaceutical Chemistry, Faculty of
Pharmacy, University of Benin, Benin City, Nigeria
*Corresponding author: E-mail: fidelching@yahoo.ca
Received: 9 May 2009
Revised
accepted: 11 September 2009
Code Number: pr09066
Abstract
Purpose: The present study was undertaken in order to evaluate
the antidiarrhoeal activity of three chromatographic fractions (L, S and Y) of
Stereospermum kunthianum stem bark in mice.
Methods: Vacuum
liquid/column chromatography (VLC/ CC) were
used to obtain three fractions (L,S and Y) of Stereospermum kunthianum stem
bark fractions. The antidiarrhoeal activities of these fractions at doses
ranging from 100 – 400 mg/kg were evaluated in diarrhoea episodes induced by
castor oil in mice. The controls were given distilled water (10 ml/kg) while
10 mg/kg of morphine was used as the reference drug (positive control).
Results: Pretreatment
of mice with 100, 200 and 400 mg/kg of fraction L significantly (p < 0.05) delayed the onset of diarrhoea compared
with the distilled water-treated mice. Fraction L at the dose of 400 mg/kg
significantly reduced the number of wet stools in the treated mice. Fraction S
at the doses of 200 and 400 mg/kg dose-dependently and significantly delayed
the onset of diarrhoea in the treated mice. At the dose of 400 mg/kg, fraction
S significantly reduced the number of wet stools and total number of stools
as well as the total weight of wet and total weight of stools, compared to
the
distilled water-treated mice. Fraction Y at the three doses significantly
delayed the onset of diarrhoea in the treated mice.
Conclusion: Vacuum
liquid/column chromatography (VLC/CC) derived fractions L, S and Y of Stereospermum
kunthianum possessed antidiarrhoeal
activity but to varying degrees. This study lends further credence to the ethnomedicinal
use of the plant for the treatment of diarrhoea.
Keywords: Antidiarrhoeal
activity, chromatographic fractions, Stereospermum kunthianum, stools
INTRODUCTION
Diarrhoea
is a public health problem in developing countries. Acute diarrhoea is the
leading cause of morbidity and mortality amongst children in developing
countries [1]. Many rural dwellers in the world depend largely on medicinal
herbs for the treatment of diarrhoeal conditions because these herbs are
readily available, affordable and are an indispensable component of traditional
medicine practice. Additionally, the existing antidiarrhoeal drugs are either
not available or are not affordable by many rural dwellers. Stereospermum
kunthianum,Cham, Sandrine Petit (Bignoniaceae) is known as sansami
and golombi amongst the Hausas and Kanuris of northern Nigeria,
respectively, umana amongst the Tivs of the Middle Belt of Nigeria, ayada
amongst the Yorubas of southwest Nigeria, and alakiriti amongst the
Igbos of southeast Nigeria. The plant is found in several countries in Africa.
Stereospermum
kunthianum is used in the management
of diarrhoea in some rural communities in Nigeria [2]. The efficacy of its
water extract in human complement system fixation in-vitro has been
reported [3]. The antiplasmodial activity of naphthoquinones and one
anthraquinone from the lipophilic extract of the root bark of the plant has
also been reported [4]. We recently reported the antidiarrhoeal activity of the
aqueous extract of its stem bark in in-vivo experimentally-induced
diarrhoeal models using mice and rats [5]. Also, the analgesic activity of the
aqueous extract of the stem bark has been investigated [6]. In the present
study, we report the antidiarrhoeal activity of the vacuum liquid/column
chromatographic fractions of S. kunthianum stem bark.
EXPERIMENTAL
Plant
material
The
fresh stem bark of S. kunthianum was collected in March 2006 in Ogun State, Nigeria. Botanical authentification was by a taxonomist, Mr. Usang Felix, of
the Forest Research Institute of Nigeria (FRIN), Ibadan. A voucher specimen
(no. FHI 107277) was deposited in the herbarium of the same institute (FRIN) for
future reference.
Preparation
of plant material and extraction
The
stem bark was carefully separated from the woody part, cut into small bits,
shade-dried and pulverised using a grinder (Lab. mill, Christy and Norris Ltd, England). The powdered stem bark (500 g) was
macerated with methanol (4 x 5 L for 48 h) at room temperature. It was filtered
and the filtrate evaporated to dryness at reduced pressure using a rotary
evaporator to obtain a dark brownish residue (80 g). The extract obtained was
stored in a closed container in the refrigerator at 4 oC until
required.
Chromatography
The
extract (80 g) was subjected to vacuum liquid chromatography (VCC) over silica
gel F254 using different gradient solvent systems of n-C6H14
: CHCl3 and CHCl3 : CH3OH, with 100 % CH3OH
as eluting solvents to obtain 19 fractions. These were subjected to repeated
thin layer chromatography (TLC) using CH3OH: CHCl3 (3:1)
as solvent system. Fractions with similar spot characteristics and Rf –values
were bulked to obtain 3 fractions ( A, 27.36 g; B, 41.40 g and C, 10.23 g).
Each of these fractions (A, B and C) was further subjected to column
chromatography (CC) as previously described by Pettelier et al [7] and
Braithwaite and Smith [8]. The column was eluted with solvents of increasing
polarity consisting of n-C6H14 : CHCl3 (9:1)
and CHCl3: CH3OH (1:4) mixtures. The column
chromatography of fractions A, B and C yielded 99, 75 and 85 fractions,
respectively. These fractions were subjected to thin layer chromatography (TLC)
and subsequently bulked based on the Rf values to obtain 5, 6, and 4
fractions, respectively, with yields as follows: A ( J, 0.21 g; K, 1.24 g; L,
9.20 g; M,5.39 g; N, 1.17 g); B(Q, 1.08 g; R, 1.70 g; S, 7.39 g; T, 6.82 g; V,
6.70 g; W, 6.19 g), and C(U, 0.25 g; X, 0.50 g; Y, 3.36 g; Z, 2.30g).
Fractions L, S and Y were selected for antidiarrhoeal evaluation based on their
relative abundance.
Animals
Swiss
mice (25 - 30 g) of either sex were used. The animals were maintained under
standard laboratory conditions including 12 h light and dark cycles ,
temperature (28 ± 1 oC), and free access to standard chow (Bendel
Feeds and Flour Mill Plc, Ewu, Nigeria) and tap water. The experimental
protocols were approved by institutional Committee on the Care and Use of Animals
in Experiments.
Castor
oil-induced diarrhoea in mice
The
method employed was previously described by Izzo et al [9]. Swiss mice
(5 per group) were administered with distilled water (10 ml/kg, p.o.), morphine
(10 mg/kg, s.c.) or 100, 200, 400 mg/kg of
each of the fractions L, S and Y thirty minutes before oral administration of
castor oil (0.2 ml per mouse). The mice were placed singly under a glass
funnel, the floor of which was lined with weighed filter paper (Whatmann No.1)
and observed for 4 h. The parameters observed include the onset of diarrhoea
stool (first stool that left a halo on the filter paper), number of wet stools,
weight of wet stools, total number of stools and total weight of faecal output.
Statistical
analysis
Data
were expressed as mean ± SEM and analyzed using the unpaired Student’s t-test.
Results were considered significant at p < 0.05 or better.
RESULTS
Pretreatment
of mice with 100 and 200 mg/kg of fraction L of S. kunthianum stem bark
significantly (p < 0.05) delayed the onset of diarrhoea compared with the
distilled water-treated mice (see Table 1). The dose of 400 mg/kg of the
fraction was most efficacious in delaying the onset of diarrhea (p<0.001)
and also significantly reduced the number of wet stools compared to distilled
water-treated mice.
Fraction
S of the extract at doses of 200 and 400 mg/kg significantly (p < 0.0001)
delayed the onset of diarrhoea compared to the distilled water-treated mice
(see Table 2). At the dose of 400 mg/kg, fraction S significantly (p <
0.001) reduced the number wet stools, total number of stools, total weight of
wet stools as well as the total weight of stools, compared to the distilled
water-treated mice (Table 2).
Fraction
Y at the doses of 100, 200 and 400 mg/kg significantly (p < 0.001) delayed
the onset of diarrhoea compared to the distilled water-treated mice (see Table
3).
DISCUSSION
In
diarrhoea, bowel function is disturbed with consequent increase in bowel
contractility, excessive intestinal secretion of water and electrolytes, and
decreased intestinal reabsorption [10]. Ricinoleic acid, the active principle
in castor oil, causes changes in mucosal cell layer permeability, electrolyte
transport and intestinal peristalsis, leading to hypersecretory response and
diarrhoea [11]. It also causes inflammatory response in the mucosa, leading to
prostaglandin release, which results in an increase in the net secretion of
water and electrolytes into the small intestine [12]. Stereospermum
kunthianum stem bark fractions L, S and Y, at the tested doses,
significantly inhibited and delayed the onset of diarrhoea in mice with the
maximum effect observed at a dose of 400 mg/kg body weight. Fractions L and S,
at a dose of 400 mg/kg, significantly reduced the number of diarrhoeal stools but
fraction S, in addition, reduced the total number of stools, total weight of
wet stools as well as the total weight of stools. Some of these effects were
comparable to those produced
Table 1: Effect of fraction L of Stereospermum kunthianum
stem bark on castor oil-induced diarrhoea in mice
Treatment
|
Onset of
diarrhoea
(min)
|
Number of
wet stools
|
Total number
of stools
|
Total weight
of wet stools
(g)
|
Total weight
of stools
(g)
|
Distilled water (10 ml/kg)
|
63.67±4.70
|
6.11±0.70
|
8.22±0.92
|
0.65±0.10
|
0.60±0.12
|
S. kunthianum (100 mg/kg)
|
82.40±3.05*
|
4.60±0.90
|
7.80±0.11
|
0.60±0.18
|
0.56±0.18
|
S. kunthianum (200 mg/kg)
|
85.20±2.68*
|
4.40±0.71
|
7.60±0.24
|
0.60±0.10
|
0.54±0.13
|
S. kunthianum (400 mg/kg)
|
109.40±5.30**
|
4.00±0.20**
|
6.60±0.63
|
0.40±0.09
|
0.32±0.07
|
Morphine (10 mg/kg)
|
201.83±8.90***
|
1.50±0.67**
|
2.00±1.00**
|
0.34±0.19
|
0.35±0.20
|
Values are mean ± SEM of at
least five experiments; *P<0.05, **p<0.001, ***p<0.0001
significantly different from distilled water-treated animals; unpaired
Student’s t-test.
Table
2: Effect of fraction S of Stereospermum
kunthianum stem bark on castor oil-induced diarrhoea in mice
Treatment
|
Onset of
diarrhoea
(min)
|
Number of
wet stools
|
Total number
of stools
|
Total weight
of wet stools
(g)
|
Total weight
of stools
(g)
|
Distilled water (10 ml/kg)
|
63.67±4.70
|
6.11±0.70
|
8.22±0.92
|
0.65±0.10
|
0.60±0.12
|
S .kunthianum (100 mg/kg)
|
84.00±2.45
|
6.00±0.91
|
8.00±0.98
|
0.58±0.14
|
0.58±0.14
|
S .kunthianum (200 mg/kg)
|
131.20±5.11***
|
5.60±2.04
|
6.20±1.85
|
0.54±0.18
|
0.54±0.18
|
S .kunthianum (400 mg/kg)
|
196.20±4.90***
|
1.20±0.80**
|
2.40±1.60**
|
0.11±0.07**
|
0.19±0.11**
|
Morphine (10 mg/kg)
|
201.83±8.90***
|
1.50±0.67**
|
2.00±1.00**
|
0.34±0.19
|
0.35±0.20
|
Values are mean ± SEM of at
least five experiments; **p<0.001, **p<0.0001 significantly different
from distilled water-treated animals; unpaired Student’s t-test.
Table
3: Effect of fraction Y of Stereospermum
kunthianum stem bark on castor oil-induced diarrhoea in mice
Treatment
|
Onset of
diarrhoea
(min)
|
Number of
wet stools
|
Total number
of stools
|
Total weight
of wet stools
(g)
|
Total weight
of stools(g)
|
Distilled water (10 ml/kg)
|
63.67±4.70
|
6.11±0.70
|
8.22±0.92
|
0.65±0.10
|
0.60±0.12
|
S. kunthianum (100 mg/kg)
|
93.00±2.37**
|
4.80±0.86
|
5.80±0.49
|
0.49±0.09
|
0.55±0.06
|
S. kunthianum. (200 mg/kg)
|
97.80±4.96**
|
3.80±1.07
|
5.20±0.97
|
0.44±0.13
|
0.54±0.12
|
S. kunthianum. (400 mg/kg)
|
149.60±5.50***
|
3.40±1.57
|
3.60±1.57
|
0.49±0.23
|
0.51±0.23
|
Morphine (10 mg/kg)
|
201.83±8.90***
|
1.50±0.67**
|
2.00±1.0**
|
0.34±0.19
|
0.35±0.20
|
Values
are mean ± SEM of at least five experiments; **p < 0.001, **p < 0.0001
significantly different from distilled water treated animals; unpaired
Student’s t-test.
by
morphine (10 mg/kg). Castor oil-induced diarrhoea is related to the release of
prostaglandin by colonic cells [13,14]. Delay in castor oil-induced
diarrhoea and inhibition of intestinal fluid secretion is known to characterize
non-steroidal antiinflammatory drugs [12] which are inhibitors of prostaglandin
synthesis. Therefore, the antidiarrhoeal action exerted by the fractions of Stereospermum
kunthianum in the present study may partly be due to possible inhibition of
prostaglandin biosynthesis or release.
CONCLUSION
Chromatography
(VLC/CC) of the methanol extract of S. kunthianum produced three
fractions L, S and Y which possessed antidiarrhoeal activity to varying
degrees, possibly suggesting that the fractions may contain different chemical
constituents. The findings of the present study corroborate our earlier claims
on the antidiarrhoeal activity of the aqueous stem bark of S. kunthianum [5].
These results further support the ethnomedicinal use of the plant in the
treatment of diarrhoea.
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© Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin
City, 300001 Nigeria.
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