WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Diby et al. World Journal of Pharmacy and Pharmaceutical Sciences

S JIF Impact Factor 6.041

Volume 5, Issue 9, 387-397 Research Article ISSN 2278 – 4357

ANTIDIARRHOEAL PROPERTIES OF THE ETHYL ACETATE

EXTRACT OF THE STEM BARK OF SPONDIAS MOMBIN L.
(ANACARDIACEAE) IN RATS

Yao Bernard Diby*, Mama Kone, Moussa Gbogbo and Angoué Paul Yapo

Laboratory of Physiology, Pharmacology and Phytothérapy, UFR SN, University of Nangui

Abrogoua, PO Box 801 Abidjan 02, Côte d’Ivoire.

ABSTRACT
Article Received on
12 July 2016, The stem bark of Spondias mombin L. are used frequently in Côte
Revised on 04 Aug. 2016,
Accepted on 25 Aug. 2016
d’Ivoire as a traditional medicine for the treatment of diarrhoea. In this
DOI: 10.20959/wjpps20169-7673 study, the antidiarrhoeal properties of a ethyl acetate extract from the
stem bark of Spondias mombin L were evaluated in a castor oil-
induced diarrhoea model in rat. Ethyl acetate extract at 250, 500 and
*Corresponding Author
1000 mg/kg body weight reduced the number, volume and weight of
Dr. Yao Bernard Diby
Laboratory of Physiology, diarrhoeal faeces. To understand the mechanism of antidiarrhoeal
Pharmacology and activity of ethyl acetate extract, its effect was evaluated on intestinal
Phytothérapy, UFR SN, transit, castor oil-induced fluid accumulation and electrolyte
University of Nangui
concentration in the intestinal fluid. Ethyl acetate extract produced a
Abrogoua, PO Box 801
decrease in intestinal transit and an increase the absorption of the fluid
Abidjan 02, Côte d’Ivoire.
secretion and electrolyte. This effect was similar to the loperamide, the
standard agent. No mortality or visible signs of general weakness were observed in the rats
following administration of ethyl acetate extract in doses up to 5000 mg/kg body weight in an
acute toxicity study. The results indicate that the ethyl acetate extract of Spondias mombin L.
possesses antidiarrhoeal properties against diarrhoea and may explain the use in traditional
medicine.

KEY WORDS: Spondias mombin, antidiarrhoeal effect, ethyl acetate extract, castor oil, rat.

INTRODUCTION
Diarrhoea diseases may be defined an increase in the frequency of wet stool, bowel
movement and abdominal pain.[1] It constitutes a serious health problem for children under

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Diby et al. World Journal of Pharmacy and Pharmaceutical Sciences

the age of 5 and up to 17% of children admitted in the paediatries ward die of diarroea. [2]
According to World Health Organisation (WHO), diarrhoea cause 4 to 5 millions deaths
through out the world.[3] Côte d’Ivoire is no exception to the rule, where diarrhoea is still a
public health problem whose scale is growing. Its prevalence rate nationally is around 26-
27% in Abidjan since the advent of HIV/AIDS and its corollary of opportunistic diseases.[4,5]
The incidence of diarrhoea diseases still remains high despite the efforts of many goverments
and international organizations to curb it. To combat diarrhoea disease, WHO recommends
the oral rehydratation solution (SRO), the antibiotics, the intestinal disinfectant and equally
the medicinal plants. The medicinal plants constitute alternative source of drugs
antidiarrhoeal for population from development countries because its cost less expensive and
easy to found. Many crude plants extracts with antidiarrhoeal activity are used in
development countries and Spondias mombin L. is one of these. Spondias mombin L. in a
fructiferous tree that belongs to the family Anacardiaceae. It is readily common in Brazil,
Nigeria and several other tropical forest. The stem bark of Spondias mombin L. has been
reported for its antibacterial activity; antiviral activity; antimicrobial activity; antidiarrhoeal
activity and alkaloids, proanthocyanins and saponins have been isolated from stem bark of
this plant.[6,7,8,9,10,11] Stem bark of Spondias mombin L. is traditionally used to treat diarrhoea,
dysentery, disorders troubles etc. [12] The present study is aimed at the evaluation of possible
antidiarrhoeal activity of the stem bark of Spondias mombin L. in established animal model.

MATERIALS AND METHODS

Collection of plant material
The stem bark of Spondias mombin L. (Anacardiaceae) was collected in Kokumbo town is
located central in Côte d’Ivoire in january 2008. The plant was authenticated by professor
Aké-Assi a plant taxonomist of the Department of botanic at the university Felix Houphouet
Boigny of Cocody, Abidjan, Côte d’Ivoire. Voucher specimen N° 15778 was deposited at our
institute for future reference. The stem bark then air-dried at room temperature. The dry stem
barks were ground into a fine powder.

Preparation of extract
About 50 g of the powder were macerated with 1000 mL of distilled water for 24 hours. The
extract was then filtered using clean cotton wool and wattman paper N°1. The product was
filtered and freeze-dried. It gave a yield of 15.6% w/w. then, the aqueous extract was further
fractionated medium polar component using ethyl-acetate. To realize the ethyl acetate

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fraction, 10 g of aqueous extract were dissolved in the mixture constituted 150 mL of distilled
water and 150 mL of ethyl acetate. The mixture was macerated about 24 hours and separated.
We were obtained two phases (organic phase and aqueous phase). The organic phase was
evaporated to dryness in an oven set at 45°C until a constant weight was obtained. The yield
was 34% with reference to the aqueous extract. [13] The ethyl acetate extract (EAE) obtained
was stored in closed containers in the refrigerator at -5°C till required.

Animals
Albinos Wistar rats weighing 150-200 g of both sexes were selected for the experiments.
Animals were allowed to be acclimatise for a period of one week in our laboratory
environment prior to the study. Animals were housed in standard metal cages, maintained
under standard laboratory conditions (12:12 hour light and dark sequence, at an ambient
temperature of 25±2°C). The animals were fed with a diet of commercial pellets
(IVOGRAIN, Abidjan, Côte d’Ivoire) and water ad libitum. All experiments were performed
for animal care guidelines.

Drugs and chemicals

Atropine sulphate and loperamide (standard reference antidiarrhoeal drugs); castor oil
(laxative agent); charcoal meal (10% activited charcoal in 5% gum acacia) and distilled water
used.

Acute toxicity testing

Thirty rats were divided in six groups of five animals. The EAE of Spondias mombin L. was
administrated orally at various doses of 1000, 2000, 3000, 4000 and 5000 mg/kg body weight
to the animal groups. The control group received distilled water. After treatment with the
extract, the animals were fasted for 12hours but they have access to water and observed for
clinical signs over a period of 14 days.

Castor oil-induced diarrhea in rats

Rats were fasted for 12 hours prior to the commencement of the experiment and randomly
divided into five groups of six rats including 3 males and 3 females. All groups received
castor oil at a dose of 2 mL/animal orally (P.O.). [14] One hour after castor oil administration,
the first group (control group) received distilled water using orogastric cannulla, the second,
third and fourth groups received the EAE 250, 500 and 1000 mg/mL body weight,
respectively. The fifth group received the standard drug, loperamide (5 mg/kg orally). After

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this administration, the animals were placed separately in metabolic cages with adsorbent
paper, which was changed every hour. During an observation period of 5 hours, the total
number of faeces and diarrhoea faeces excreted and by the animal were recorded. The total
number of diarrhoea faeces of the control group was considered 100%. The results were
expressed as a percentage of inhibition of diarrhoea.[15]

Percentage of wet faeces inhibition= (N0-N1/N0) x 100.

N0=number wet faeces in control group
N1=number wet faeces in test group.
Severity of diarrhea= (diarrhoeal faeces/total faeces) x 100.

Gastrointestinal motility test

To evaluate the effect of the EAE on the gastrointestinal motility, thirty (30) animals were
randomly divided into 5 groups of 6 rats each. All animals were fasted for 12 hours before the
experiment but they were allowed access to water. The first group (the control group) was
orally administered the distilled water. The second, third and fourth group orally received
EAE at doses of 250, 500 and 1000 mg/kg body weight respectively. The fifth group received
the reference drug, atropine sulphate (1 mg/kg body weight orally). Thirty minutes later each
animal was given 1 mL of charcoal meal (10% activated charcoal in 5% gum acacia orally).
All animals of each group were sacrificed thirty minutes after administration of charcoal meal
and the length of the intestine (pyloric sphincter to caecum) as well as the distance travelled
by charcoal as a extract of that length was measured. [16] The charcoal movement in the
intestine was expressed as a percentage.

Percentage of transit inhibition= (N0-N1/N0) x 100.

N0=total length of intestine
N1=charcoal distance of test group.

Castor oil induced fluid accumulation and electrolyte secretion

Intaluminal fluid was determined by the method and modified by.[17, 18] The rats were fasted
for 24 hours but allowed access to water. All animals in experiments were divided into five
groups of six animals each and they were pre-treated with distilled water (2 mL/100g), the
standard drug loperamide (5 mg/kg body weight) and EAE (250, 500 and 1000 mg/kg
followed by castor oil (2mL/rat) one hour later. One hour later, the rats were anaesthetized
with chloroform and sacrificed. The small intestine was removed after tying the ends with

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thread and weight. The intestinal contents were collected by milking into a graduated tube
and their volume was measured. The intestine was reweighed and the difference between full
and empty intestine was calculated. The Na+, K+, Cl- and Ca2+concentration were analysed in
the intraluminal fluid using flame photometry CL361 (Elico, Inde).[19]

Statistical analysis
Data obtained are presented as means± standard error of mean (S.E.M.) for the number of
animals in each (n= 6). The differences between the data obtained from test animal group and
the data obtained from untreated animal groups, were subjected to one-way analysis of
variance (ANOVA; 95 % confidence interval), followed by Dunnett and Turkey tests. Values
with p<0.05 compared with the control group were considered to be statistically significant.

RESULTS
Acute toxicity study
Oral administration of EAE of Spondias mombin L. produced no visible signs of toxicity in
the animals receiving up to a dose 5000 mg/kg body weight. These observations suggest that
the extract of the stem bark of Spondias mombin L. is safe or no toxic to rats.

Effect of Spondias mombin L. ethyl acetate extract. on castor oil-induced diarrhea

In this study of the diarrhoea experiment induced by the castor-oil, the rats that did received
the distilled water, showed the serious diarrhoea signs such as frequent, water and a lot of
defecations. Spondias mombin L. EAE, in the doses of 250, 500 and 1000 mg/kg induced a
significant (p<0.05-0.001) antidiarrhoeal effect against castor-oil induced diarrhoea for up to
5 hours. The total number of diarrhoea defecation of each is (14.67±1.4 at the dose 250
mg/kg; 12.83±1.19 at the dose 500 mg/kg and 8.5±0.92 at the dose 1000 mg/kg) as compared
to the castor-oil treated control group. The percentages of inhibition of castor-oil induced
diarrhoea in the different doses were 26.02 %; 35.30 % and 57.13 % respectively at 250, 500
and 1000 mg/kg. The antidiarrhoeal effect of EAE was similar to that loperamide (5 mg/kg)
which produced an inhibition of 64.69 % (table 1).

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Table 1: Effects of an ethyl acetate extract of Spondias mombin L. on castor oil-induced

diarrhea in rats
Total number Total number of Percentage of Severity
Treatments Doses
of faeces diarrhoeal inhibition (%) diarrhoeal (%)
Distilled water 2 mL/100g 20.4±1.02 19.83±1.13 - 97.20
1,2,b,
EAE 250 mg/kg 17.83±0.94 14.67±1.4 * 26.02 82.27
EAE 500 mg/kg 15.33±1.34 12.83±1.192,b,** 35.30 83.69
a,
EAE 1000 mg/kg 11.30±1.34 8.5±0.92 *** 57.13 75.22
Loperamide 5 mg/kg 9.5±0.95 7.0±1.03*** 64.69 73.68
Values are means ± SEM (n = 6); * P <0.05; ** P <0.01; *** p <0.001 compared to the
negative control group (ANOVA 1 + Dunnett test); a: p <0.05; b: p <0.001 compared to
loperamide (ANOVA 1 + Dunnett's test); 1: p <0.01; 2: p <0.001 compared with EAE to
1000 mg/kg (ANOVA 1+Tukey's test).

Effect of Spondias mombin L. ethyl acetate extract on gastrointestinal motility in Wistar

rats
The EAE of Spondias mombin L. significantly decreased the propulsion of charcoal meal in
rat gastrointestinal tracts by 75.8±3.56; 58.5±3.04; 45.5±3.00%respectively, at 250, 500 and
1000 mg/kg compared to the control group (86.02±4.08 %). A similar reduction in the
gastrointestinal transit of charcoal meal in rats achieved with atropine sulphate
(40.5±3.56 %). Results are shown in table 2.

Table 2: Effects of an ethyl acetate extract of Spondias mombin L. on the intestinal

transit of charcoal meal in rats
Total distance Distance travelled Inhibition of
Treatments Doses
of intestine (cm) by charcoal (%) mobility (%)
Distilled water 2 mL/100g 113,5±5.05 86.02±4.08 75.78
EAE 250 mg/kg 106.8±4.01 75.8±3.56a,* 70.97
EAE 500 mg/kg 110.2±4.75 58.5±3.04** 53.08
EAE 1000 mg/kg 107.3±3.00 45.5±3.00*** 42.40
Atropine 5 mg/kg 109.6±4.85 40.5±3.56*** 36.95

Values are mean ± SEM (n = 6); *: P <0.05; **: P <0.01; *** p <0.001 compared to the
negative control group (ANOVA 1 + Dunnett test); a: p <0.05 compared to atropine
(ANOVA 1 + Dunnett's test).

Effect of Spondias mombin L. ethyl acetate extract on intestinal fluid accumulation in

rats and electrolyte secretion
The results of the volume and weight of intestinal fluid test were reported in table 3. Both
doses of the extract (500 and 1000 mg/kg) decreased respectively the intestinal water

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secretion by 1.48±0.24 and 1.23±0.26 mL. A reduction similar in the weight of the intestinal
content was also observed following treatment with all doses of extract. Concerning, the
electrolyte (Na+, K+, Cl- and Ca++) secretion, they were equally decreased in dose dependent
and the different values were found in the table 4.

Table 3: Effects of an ethyl acetate extract of Spondias mombin L. on volume and weight
of intestinal content
Volume of intestinal Weight of intestinal
Treatments Doses
content (mL) content (g)
Distilled water 2mL/100g 2.1±0.25 2.51±0.32
EAE 250 mg/kg 1.93±0.30a 2.0±0.28c**
EAE 500 mg/kg 1.48±0.241,c,*** 1.73±0.23a***
EAE 1000 mg/kg 1.23±0.263*** 1.43±0.242***
Loperamide 5 mg/kg 1.1±0.15*** 1.28±0.13***

Values are mean ± SEM (n = 6); **: P <0.01 *** p <0.001 compared to the negative control
group (ANOVA 1 + Dunnett test); a: p <0.05; b: p <0.01 c: p <0.001 compared to loperamide
(ANOVA 1 + Dunnett's test); 1: p <0.05; 2: p <0.01 and 3: p <0.001 compared with EAE to
250 mg/kg (ANOVA 1+Tukey's test).

Table 4: Effects of an ethyl acetate extract of Spondias mombin L on intestinal ions

secretion
Electrolytes
Treatments Doses Na+ (mg/L) K+ (mg/L) Cl- (mg/L) Ca++ (mg/L)
Distilled water 2 ml/100 g 6,02±0.9 1.02±0.08 29.9±1.25 0.84±0.03
EAE 250 mg/kg 4.95±0.73c 0.92±0.05c 25.55±1.21c 0.75±0.02c
EAE 500 mg/kg 4.5±0.49b 0.65±0.083,c,** 20.27±1.293,c,** 0.45±0.023,C,***
EAE 1000 mg/kg 3.3±0.041,3,* 0.42±0.042,3,a,*** 16.78±1.012,3,c,*** 0.35±0.022,3,c,***
EAE 5 mg/kg 3.1±0.53*** 0.3±0.08*** 12.52±0.66*** 0.27±0.02***

Values are mean ± SEM (n = 6); *: P <0.05; **: P <0.01 *** p <0.001 compared to the
negative control group (ANOVA 1 + Dunnett test); a: p <0.05; b: p <0.01 c: p <0.001
compared to loperamide (ANOVA 1 + Dunnett's test); 1: p <0.05 and 2: p <0.01 compared
with EAE to 500 mg/kg and 3: p <0.001 compared to 250 mg/kg (ANOVA 1+ Tukey's test).

DISCUSSION
Acute toxicity studies in rats showed that no mortality was recorded in any of the group even
at 5000 mg/kg dose. All the animals were alive and active during the observation period. The
oral median lethal dose of Spondias mombin L. was therefore estimated to be greater than
5000 mg/kg body weight in rat. Diarrhoea may be characterized by imbalance between the

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absorption and secretory mechanisms in the intestine tract accompanied the fluid in the
faeces. Diarrhoea can be induce experimentally with different agents. In our study, castor oil
is used to induce diarrhoea. In fact, castor oil increases peristaltic activity and produced
permeability changes in the intestinal mucosal membrane to electrolytes and water, an effect
associated with prostaglandin release.[20] Prostaglandin is also a major cause of arachidonic
acid-induced diarrhoea.[21] The results of our study showed that an oral administration of the
stem bark ethyl acetate extract of Spondias mombin L. produced significant and dose
dependant decrease in the severity of diarrhoea in terms of reduction in the rate of defecation
and consistency of faeces in rats. The percentage inhibition for the number of wet faeces as
well as mass indicates the presence of antidiarrhoeal activity in extract as compared with that
of control group. This effect was similar with that of loperamide (standard drug) at high dose
of 1000 mg/kg. Indeed, it is well documented that loperamide antagonizes diarrhoea induced
by castor oil, prostaglandins or cholera toxin. [22,23,24] These actions are to its antimotility and
antisecretory properties.[25] So, it is likely that the ethyl acetate extract of Spondias mombin
L. to act by the same mechanism as loperamide. In the other hand, our results indicated that
ethyl acetate extract of Spondias mombin L. also significantly reduced intestinal transit as
observed by the decrease in transit motility of charcoal meal. This may be due to the fact that
the extract may increase the reabsorption of water by decreasing intestinal motility as
observed in the decrease of intestinal transit by charcoal meal. The extract also led to a
marked reduction in the weight and volume of the intestinal contents on castor oil-induced
enteropooling. Above observations suggest that the extract in graded doses reduce diarrhea
by inhibiting peristalsis, gastrointestinal motility and castor oil-induced enteropooling. At
last, the phytochemical screening of the ethyl acetate stem bark extract of Spondias mombin
L. reveals the presence alkaloids, tannins and flavonoids.[26] These observed components
could be responsible for the antidiarrhoeal activity of the ethyl acetate extract of stem bark
from Spondias mombin L. Our results are agreement with many studies in the literature. It
was reported that flavonoids, tannins, and alkaloids were responsible for the antidiarrhoeal
properties of certains plants such as Mangifera indica, Momordica charanta and Calotropis
gigantea.[27,28,29] Indeed, the antidiarrhoeal activity of the flavonoids is known by the
antispasmodic effects, delaying gastrointestinal transit, suppression water absorption or
reduction electrolyte secretion.[30] Tannins are known for their antidiarrhoeal activity
(Kokate, 1988). Infact, tannins are responsible for the denaturation of proteins and from
protein tannate, which makes the intestinal mucosa more resistant and reduces secretion. [31]
Alcaloids, by their antidiarrhoeal property, reduce gastrointestinal motility and inhibit

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acetylcholine secretion.[32] The antidiarrhoeal activity of ethyl acetate extract of the stem bark
of Spondias mombin L. could therefore be due to the presence of tannins, Alcaloids and
flavonoids.

CONCLUSION
The result of the present study suggests that the ethyl acetate stem bark extract of Spondias
mombin possesses significant antidiarrhoeal activity due to its effect on reduction of number
of diarrhea faeces, delayed in gastrointestinal propulsion and inhibition of fluid accumulation
in the intestine tract of rats. The inhibitory effect of the extract justified the use of the plant as
a antidiarrhoeal agent in folk medicine.

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