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Received:
Accepted:
Available Online:
8 February 2012
14 March 2012
20 March 2012
Antidiarrhea
Castor oil
Enteropooling
Gastrointestinal transit
Pedaliaceae
Number of Tables:
4
Number of Refs:
39
YMT
e-mail: tomuyak@yahoo.com
The antidiarrheal effects of the aqueous leaf extract of
at 25, 50
and 100 mg/kg body weight was evaluated in female rats using
gastrointestinal transit, diarrhea and enteropooling induced by castor oil
models. The extract was positive for alkaloids, saponins, flavonoids and
phenolics. The 25 mg/kg body weight of the extract significantly (p<0.05)
prolonged the onset time of diarrhea, decreased the fecal parameters (number,
water content, fresh weight, total number of wet feaces) with no episode in the
animals treated with 50 and 100 mg/kg body weight. The activity of small
intestine Na+-K+ ATPase increased (p<0.05) while the nitric oxide, volume and
mass of intestinal fluid as well as the distance travelled by the charcoal meal
decreased. The patterns of changes were similar to the reference drugs.
Overall, the antidiarrheal activity of the aqueous leaf extract of
may be due to alkaloids, phenolics, flavonoids and saponins present
in the extract.
Diarrhea is a disease in which waste matter most often
in liquid form is emptied from the bowels much more
frequently than normal. Diarrheal diseases are a major
health concern in developing countries with an estimate
of about 1.8 million deaths per annum (WHO, 2004).
The disease may be caused by a wide array of agents
such as entero-pathogenic microorganisms (
,
,
,
and
), alcohol, irritable bowel
syndrome, bile salts, hormones, secretory tumors and
intoxication (Anne and Geboes, 2002; Gerald et al., 2007;
Brijesh et al., 2011).
Despite improvements in public health and economic
well being, diarrhea remains an important clinical
problem in developed and developing countries
(Casburn-Jones and Farthing, 2004). Generally, the
treatment of diarrheal is non-specific, and is usually
aimed at reducing the discomfort and inconvenience of
frequent bowel movements (Brunton, 2008; Suleiman et
al., 2008). These approaches include maintenance of
fluid and electrolyte balance, use of anti-infective
agents, antidiarrheal agents, and most recently
probiotics or microbial components which have a value
in the treatment of rotavirus infections and post
antibiotic diarrhea (Marcos and DuPont, 2007). In order
to overcome the menace of diarrhea in developing
Bangladesh J Pharmacol 2012; 7: 14-20
countries, especially the discomfort and inconvenience
of frequent bowel movements, the World Health
Organization (WHO, 2004) has introduced a program
for diarrheal control, which involves the use of
traditional herbal medicines due partly to their
economical viability, accessibility and ancestral
experience and perceived efficacy. A medicinal plant
widely claimed to be effective in the management of
diarrhea in Nigeria is
(Pedaliaceae) is found in tropical
Africa like the open savanna woodlands across the
region from Senegal to Northern and Southern part of
Nigeria. It is known by various names such as Eku
(Yoruba-Western Nigeria), Tchaba-laba (Guinea
Bissau), Lalu-caminho (Senegal) and (False SesameEnglish) (Adegoke
1968). It is an erect or sub-erect
herb of about 60 cm tall. The fruits are laterally
flattened capsule with slender horns. The colour of the
flower varies from pink, lilac, lip and throat cream with
dark lines. The locally acclaimed medicinal uses of
include antidiarrheal, antimalarial antidiabetic and anti-inflammatory. Only a very few
scientific studies are available on
. For
example, Fasakin (2004) reported on the proximate
compositions of the leaves and seeds of the plants.
Despite the aforementioned claim of
leaves as an antidiarrheal agent, there has not been any
scientific report, at least to the knowledge of the authors
that has either substantiated or refuted this claim.
Therefore, this study sets out to provide information on
the acclaimed antidiarrheal activity of aqueous leaf
extract of
with a view to ascertaining the
claim.
The plant was obtained from a vegetable
seller at a market (Ipata) in Ilorin, Nigeria. It was
authenticated at the Herbarium Unit of the Department
of Plant Biology, University of Ilorin, Ilorin, Nigeria
where a voucher specimen (I.U. 011) was deposited.
Loperamide hydrochloride,
atropine sulfate, and castor oil were products of Richy
Gold International Ltd., Nigeria, Laborate
Pharmaceuticals, Punjab, India, and Bells Sons and Co.
(Druggist) Ltd., Southport, England, respectively.
Adenosine 5’-triphosphate (disodium salt) was a
product of Sigma Chemical Co, St. Louis, MO, USA.
Nitric oxide assay kit was a product of Assay Designs
Stressgen, Ann Arbor, MI, USA.
Healthy, female albino rats (
)
weighing 137.40 ± 4.04 g obtained from the Animal
House of the Department of Biochemistry, University of
Ilorin, Nigeria were used for this study. All the animals
were housed in clean aluminum cages placed in a wellventilated house conditions (temperature 25ºC,
photoperiod 12 hours natural light, 12 hours dark and
humidity 45-50%). The animals were allowed free
access to rat feeds (Premier Feed Mill Co. Ltd., Ibadan,
Nigeria) and clean tap water except when fasting was
required during the study. The cages were cleaned of
wastes on a daily basis. This study was carried out
according to the guidelines of European Convention for
the Protection of Vertebrate Animals and Other
Scientific Purposes- ETS-123 (2005).
The leaves of
were
separated from the stem, washed under running tap
and oven dried (Uniscope Laboratory Oven, SM9053,
Surgifriend Medicals, England) at 40ºC for 48 hours.
The dried materials were pulverized using an electric
blender (Mikachi MX 1830, Shangai, China) and stored
in an air-tight container prior to extraction. A portion
(30 g) of the powder was extracted in 1500 ml of cold
distilled water for 48 hours. The extract was filtered
(Whatman No. 1 filter paper) and the resulting filtrate
evaporated to dryness on a water bath (Uniscope
Laboratory Water Bath SM801A, Surgifriend Medicals,
England) to give a yield of 15 g which correspond to a
percentage yield of 50%. This was reconstituted
separately in distilled water to give the required doses
of 25, 50 and 100 mg/kg body weight used in the
present study. The doses of 25 and 50 correspond
respectively, to “a pinch” and “a spoon” of the plant
powder estimated to be consumed as a remedy for an
adult 70 kg man. The 100 mg/kg body weight dose
which is a quadruple-fold of the least dose was used to
account for cases of ‘abuse’ by the user.
Preliminary chemical tests were
carried out on the extract to detect the presence of
alkaloids, steroids, saponins, phenolics, flavonoids,
cardiac glycosides, tannins, cardenolides and dienolides
according to the procedures described by Sofowora
(1993).
!
Diarrhea was induced
in the rats using a modified method of Sunil et al (2001).
The test animals were fasted (without food, but water)
for 18 hours prior to the commencement of the
experiment. Each animal was placed in a cage, the floor
of which was lined with blotting paper. Animals in the
first group (negative control) were orally administered
with 1 mL of distilled water while those in the second,
third and fourth groups were respectively administered
with the same volume (1 mL) of the extract
corresponding to 25, 50 and 100 mg/kg body weight.
"
Bangladesh J Pharmacol 2012; 7: 14-20
The fifth group (positive control), was orally
administered with same volume (1 mL) of loperamide
hydrochloride preparation corresponding to 2.5 mg/kg
body weight. At 30 min post treatment, each animal
was again administered orally with 1 mL of castor oil
and the time between the administration of the oil and
the appearance of the first diarrheal drop was noted.
The severity of diarrhea was accessed every hour for a
period of 6 hours by monitoring the diarrheal drops on
the pre-weighed blotting paper placed beneath the
individual rat cages. The total number of feces,
diarrheal feces and total weight of feces excreted were
expressed as average of six determinations and
compared with the control groups. The percentage
inhibition of diarrheal defecation in each group was
also computed. At the end of the 6 hours of monitoring
the diarrheal drops, the animals were sacrificed and
small intestine homogenates prepared according to the
procedure described by Akanji and Yakubu (2000). The
assay of both the activity of Na+-K+ ATPase and nitric
oxide concentration in the small intestine homogenates
was done using the protocol described by Bewaji et al
(1985) and Nathan (1992) respectively.
!
Intraluminal fluid was determined
as described by Havagiray et al (2004). Briefly, fasted
animals as previously described were randomly
selected into five groups of six animals each. Animals in
the negative control group received 1 mL of distilled
water orally while those in the positive control group
were orally administered with 1 mL of atropine
sulphate corresponding to 0.6 mg/kg body weight.
Animals in the test groups were administered with
same volume of the extract corresponding to the doses
of 25, 50 and 100 mg/kg body weight. Immediately
after the administration, 1 ml of castor oil was also
administered orally to each rat in all the groups. After
30 min, the rats were sacrificed using the procedure
previously described by Akanji and Yakubu (2000). The
small intestine was excised and the intestinal content
was squeezed quantitatively into a measuring cylinder.
The volume and mass of the intestinal content were
obtained and the inhibition of intestinal content was
also computed.
"
The method described by
Gerald et al (2007) was adopted for the determination of
the effect of the extract on gastrointestinal transit in the
rats. Fasted animals (as previously described) were
assigned into five groups of six rats each. The animals
in the negative control group received 1 mL of distilled
water orally while those in the positive control received
1 mL of atropine sulphate intramuscularly. Animals in
the third, fourth and fifth groups received equal
volume of the extract corresponding to 25, 50 and 100
mg/kg body weight. After 30 minutes, all the animals
were again administered orally with 1 ml of charcoal
meal (10% charcoal suspension in 5% agarose agar). At
30 min post administration of the charcoal meal, all the
animals were sacrificed using the procedure described
by Akanji and Yakubu (2000). The small intestine was
removed and afterwards, the length of the small
intestine and the distance travelled by charcoal meal
through the organ was measured. The distance was
expressed as a percentage of the length of the small
intestine.
Data were expressed as the means ±
SEM of 6 replicates. Statistical analysis was performed
using One-way Analysis of Variance (ANOVA) and
complemented with Student’s t-test. The values were
considered statistically significant at < 0.05.
The aqueous leaf extract of
was positive
for alkaloids, saponins, flavonoids and phenolics while
tannins, cardiac glycosides, steroids, cardenolides and
dienolides were not detected (
).
!
Alkaloids
Present
Saponins
Present
Tannins
Not detected
Flavonoids
Present
Cardiac glycosides
Not detected
Steroids
Not detected
Phenolics
Present
Cardenolides and dienolides
Not detected
The 25 mg/kg body weight of the extract significantly
(p<0.05) prolonged the onset time of diarrhea while
there was no episode of diarrhea in the 50 and 100 mg/
kg body weight extract treated animals (
).
Compared with the distilled water treated animals, the
extract significantly (p<0.05) decreased the number of
feces in a dose related manner. While the total number
of wet feces, fresh weight of feces and water content of
feces decreased significantly in the animals
administered with 25 mg/kg body weight of the extract
in a manner similar to the loperamide treated animals,
2
Bangladesh J Pharmacol 2012; 7: 14-20
$
!
Parameter/doses
Onset time (mins)
%
Loperamide (mg/
kg body weight)
Water
2.5
0
&
Plant extract (mg/kg body weight)
25
Nil
Total number of feces
2.50 ±
Number of wet feces
2.00 ± 0.00b
4.50 ± 0.55a
2.00 ± 0.00b
Nil
Nil
Fresh weight of feces (g)
1.32 ± 0.02b
1.70 ± 0.00a
1.03 ± 0.00c
Nil
Nil
Water content of feces (mL)
0.62 ± 0.02b
1.27 ± 0.03a
0.55 ± 0.00c
Nil
Nil
Inhibition of defecation (%)
55.56
0
55.56
100
Small intestine
ATPase
activity (µmol Pi/mg protein/
hour)
1322.74 ±
Small intestine nitric oxide concentration (µmol/L)
952.84 ±
88.21 ± 8.01a
15.09b
274. 36 ± 7.11b
194.50 ±
Nil
0.55b
12.22a
8.50 ±
0.55a
6.00 ±
4.93c
100
233 ±
Na+-K+
63.50 ±
1.64a
50
8.76b
0.00c
1210.09 ±
14.44c
86.09 ± 11.07a
2.00 ±
0.00d
1330.05 ±
1.50 ± 0.00e
100
11.88a
87.17 ± 8.10a
1509.07 ± 19.72d
89.00 ± 7.19a
Values are mean ± SD (n = 6); Values carrying different superscript along each rows are significant (p<0.05) different from each other
$
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(%+
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+
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Values are mean ± SD (n = 6). Values carrying different superscript along each rows are significant (P<0.05) different from each other
there was no episode in the animals administered with
the 50 and 100 mg/kg body weight of the extract.
Although there was increase in the computed
percentage inhibition of defecation in all the treated
groups when compared with the distilled water
administered animal, it is worthy of note that the 50
and 100 mg/kg body weight of the extract produced
100% inhibition of defecation. The activity of Na+-K+
ATPase activity in the small intestine also increased
dose dependently in the extract treated animals in a
manner similar to the positive control animals. In
contrast, the concentration of nitric oxide was reduced
significantly by the extract in this study in a similar
fashion to the reference drug (
).
The extract significantly decreased the volume and
mass of intestinal fluid of castor oil-induced
enteropooling in rats. While the reduction in the mass
of intestinal fluid at 50 and 100 mg/kg body weight of
the extract was more than the atropine sulphate, it was
only the 100 mg/kg body weight of the extract that
reduced the volume of the intestinal fluid more than the
reference drug treated animals. Generally, the
inhibition of intestinal fluid was higher in the extract
and atropine sulphate treated animals (
).
Although, the length of the small intestine in all the
experimental animals was not significantly different
from each other, the extract significantly reduced the
distance travelled by the charcoal meal. These values
were lower in the extract and atropine sulphate treated
animals than in the distilled water control animals
(
#).
5
Bangladesh J Pharmacol 2012; 7: 14-20
# $
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7
The age long use of herbal medicines in the treatment of
diarrheal disease is a common practice in many
countries across the globe including Nigeria. Therefore,
the need to substantiate or otherwise the folkloric claim
of
as an antidiarrheal agent using several
models of diarrhea cannot be overemphasized. The
results shows that there has been statistically significant
reduction not only on the onset of diarrhea but also on
its severity as revealed by the castor oil-induced
diarrhea and enteropooling as well as charcoal meal
gastrointestinal transit models in the present study.
Castor oil has been widely used in diarrhea studies
because it is capable of causing the body through its
metabolite, ricinoleic acid, to produce autocoids and
prostaglandins which are known inducers of diarrhea
in animals (Greenbargena et al., 1978). Ricinoleic acid
initiates diarrhea via several mechanisms such as: i.
causing irritation and inflammation of the intestinal
mucosa, leading to the release of prostaglanding which
stimulates motility and secretory diarrhea (Pierce et al.,
1971; Mbagwu and Adeyemi, 2008); ii. affecting
electrolyte transports (by reducing active Na+ and K+
absorption) and smooth muscle contractility in the
intestine via decreasing or inhibiting the activity of Na+K+ ATPase in the small intestine and colon (Palombo,
2006); iii. increasing the volume of intestinal content by
preventing the reabsorption of water; iv. interfering
with oxidative metabolism and thus an effect on
adenylate cyclase or mucosal adenosine 3’, 5’-cyclic
monophosphate content; and being cytotoxic to
intestinal epithelial cells and causing histological
abnormalities and mucosal permeability (Mascolo et al.,
1993). These sequences of events may be related to the
release of eicosanoids, prostaglandins, nitric oxide,
platelet activating factor, cAMP and tachykinins by the
intestinal mucosal, which consequentially could give
rise to diarrhea.
Therefore, the significantly (p<0.05) prolonged time of
induction of diarrhea, decreased frequency of stool and
fecal parameters (total number of feces, fresh weight,
water content and number of wet feces) following the
administration of the extract suggest antidiarheal
activity at this dose. This assertion was further
corroborated with the increased inhibition of
defecation. The same percentage of inhibition of
defecation in the 25 mg/kg body weight of the extract
and loperamide hydrochloride suggest that the
antidiarrheal activity of the extract may proceed via the
same mechanism as that of the reference drug,
loperamide hydrochloride. The clinical effect of the
extract as antidiarrheal agent was demonstrated at 50
and 100 mg/kg body weight where the typical
parameters of diarrhea did not manifest in the animals.
The extract might have exerted its antidiarrheal activity
via secretory mechanism as evident from reduction in
total number of wet faeces. Furthermore, this
antidiarrheal activity could have resulted from the
inhibitory activity of aqueous leaf extract of
on prostanglandins synthesis, nitric oxide
and platelet activating factors production, as inhibitors
of prostaglandins and nitric oxide syntheses are known
to delay diarrhea induced by castor oil (Capasso et al.,
1994; Adzu et al., 2003; Tangpu and Yadav, 2004).
Similar effects were reported in several studies by
Qnais et al (2005), Akindele and Adeyemi (2006) and
Appidi et al (2010) following the administration of
aqueous leaf extracts of #
$
and %
, respectively.
Castor oil, the inducer of diarrhea in animals decrease
or inhibit the activity of Na+-K+ ATPase in the small
intestine and colon and thus affect electrolyte transports
3
Bangladesh J Pharmacol 2012; 7: 14-20
by reducing active Na+ and K+ absorption (Palombo,
2006). Similarly, study by Capasso et al (1994) have
implicated elevated nitric oxide in the pathogenesis of
diarrhea, a disease which was prevented by the
intraperitoneal injection of nitric oxide synthase
inhibitor, NG-nitro-L-arginine methyl ester (2.5–50 mg/
kg twice) to rats. Therefore, the increase in the activity
of Na+-K+ ATPase as well as decrease in the
concentration of nitric oxide in the small intestine of
extract treated animals may be one of the mechanisms
by which the extract exhibits its antidiarrheal effect.
The accumulation of intestinal fluids may be a resultant
clinical effect of bowel function disturbance, in which
case, there is impaired intestinal absorption, excessive
intestinal secretion of water and electrolytes, and a
rapid bowel transit (Gurgel et al., 2001; Mbagwu and
Adeyemi, 2008). The reduction in the parameters of
enteropooling and consequent increase in the
percentage inhibition of intestinal content of the
animals suggest that the extract might have inhibited or
reduced the massive secretion of water into the
intestinal lumen. It is possible that the aqueous leaf
extract of
may be explored in managing
secretory diarrhea. This anti-enteropooling effect of
could be due to the presence of flavonoids in
the extract, as the phytochemical have been reported to
inhibit intestinal motility and hydroelectrolytic
secretion (Perez et al., 2005).
Atropine sulfate is known to produce an anticholinergic
effect on intestinal transit whereas activated charcoal
can prevent the absorption of drugs and other
chemicals into the body by absorbing them on the
surface of the charcoal particles (Venkatesan et al.,
2005). Thus, the suppression or reduction in the
intestinal propulsive movement of the charcoal meal by
all the doses of the extract in the present study suggest
among others that the extract was able to increase the
time for absorption of water and electrolytes in a
manner similar to the reference drug, atropine sulfate
(Teke et al., 2007). It may also indicate a reduction in
peristaltic activity and ultimately reduction in the
gastrointestinal motility (Nwiniyi et al., 2004). This
effect which suggests antidiarrheal activity may be
attributed to the flavonoids since it has been reported to
be able to inhibit fluid secretion in the small intestine
thereby reducing the rate of flow in the gut. The extract
appears to have acted on all parts of the intestine
producing inhibitory effect on both the gastrointestinal
propulsion and fluid secretion. The findings in this
study are similar to the report by Maridass (2011)
following the administration of 500 mg/kg body weight
of ethanolic tuber extract of
to castor
oil-induced diarrheal rats.
Previous studies have implicated a wide array of
phytochemicals with antidiarrheal activity. These
include tannins, alkaloids, saponins, flavonoids, sterols,
terpenoids and reducing sugars (Galvez et al., 1993;
Mukherjee et al., 1998; Otshudi et al., 2000; Shoba, 2001;
Havagiray et al., 2004; Venkatesan et al., 2005).
Flavonoids and saponins are known to inhibit the
release of autocoids and prostaglandins thereby
reducing the motility and secretion induced by castor
oil (Veiga et al., 2001; Perez et al., 2005). Because many
of these compounds might have antidiarrheal effects, it
is difficult to suggest which of them is responsible for
the desired effect. However, we suggest that alkaloids,
saponins and flavonoids present in the extract of
might be responsible for its antidiarrheal
activity.
In conclusion, aqueous leaf extract of
has
antidiarrheal activity made possible by the alkaloids,
phenolics, flavonoids and saponins via reduction or
inhibition of typical indices of diarrhea such as the fecal
parameters, enteropooling, gastrointestinal motility and
stimulation/enhancement of Na+-K+ ATPase activity
and reduction in the nitric oxide concentration of the
small intestine.
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