Chapter 7 : Anti-diarrheal activity

Chapter 7 : anti-diarrheal activity

Evaluation of anti-diarrheal activity by castor oil induced diarrhea method

7.1. Introduction
Intestinal diseases are one of the main causes of death of infants particularly in developing
countries [59]. Diarrheal disease has long been recognized as a leading cause of morbidity and

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 Chapter 7 : Anti-diarrheal activity

[60]
mortality and major cause of sickness and death among young children in developing
countries. It thus becomes important to identify and evaluate commonly available natural
drugs alternative to currently used anti-diarrheal drugs.

7.2. Principle
The anti-diarrheal activity of the crude methanolic extract of Grewia nervosa was evaluated
[61]
using the model of castor oil induced diarrhea in mice . According to this model eachmice
was fed 1ml of highly pure analytical grade of castor oil which would induce diarrhea.Fifty
mice were taken and divided into 4 groups. They were Group I, Group II, Group III, Group
IV, Group V, Group VI, Group VII, Group VIII, Group IX, and Group X. Group I was used
as a control. Group II wasused as a positive standard. Loperamide was used for the purpose.
Group III, Group IV were the methanolic extract 200mg/kg and 400mg/kg respectively,
Group V and VI were the chloroform fraction extract at 200mg/kg and 400mg/kg, Group VII
and VIII were the Dichloromethane fraction extract at 200mg/kg and 400mg/kg and Group
IX and Group X were the Pet- ether fraction extract at 200mg/kg and 400mg/kg respectively.
Each mouse was fed with the test samples. Then thirty minutes later each mouse was given
1ml of castor oil to induce diarrhea. The mice were kept under observation for the next four
hours. For each individual mouse the number of defecation was recorded. The observation of
the experimental groups was compared against that of the control to evaluate the anti-
diarrheal activity of the sample.

7.3. Experimental Design

Fifty experimental animals were randomly selected and divided into four groups denoted as
group-I, group-II, group-III, group IV, group V, group VI, group VII, group VIII, group IX
and group X consisting of 5 mice in each group. Each group received a particular treatment
i.e. control, standard and the methanolice extract, chloroform fraction extract,
Dichloromethane extract and pet-ether extract at 200mg/kg and 400mg/kg respectively. Prior
to any treatment, each mouse was weighed properly and the doses of the test samples and
control materials were adjusted accordingly.

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 Chapter 7 : Anti-diarrheal activity

Figure 7.1: Swiss albino mice

7.4. Preparation of Test Material

7.4.1. Preparation of Test Samples

In order to administer the extract at doses of 400 mg/kg and 200 mg/kg body weight of mice,
120 mg and 60mg of the extract were measured and triturated in unidirectional way by
adding of small amount of Tween-80 (a suspending agent). After proper mixing of extract
and suspending agent, normal saline was slowly added. The final volume of the suspension
was made up to 3.0 ml. To stabilize the suspension, it was stirred well by vortex mixture.

7.4.2. Preparation of standard solution

For the preparation of loperamide at the dose of 50 mg/kg body-weight, 12.5 mg of
loperamide was measured and a suspension of 2.5 ml was prepared.

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 Chapter 7 : Anti-diarrheal activity

Table 7.1: Test samples used in the evaluation of anti-diarrheal effect of crude leaf extract of Grewia
nervosa.
Test samples Group Purpose Dose (mg/kg) Route of
Administration
1%Tween 80 I Control 0.10 ml/10 gm Oral
In normal Group of body
saline weight
Loperamide II Standard 50 Oral
Group
ME III Test sample 200 Oral
ME IV Test sample 400 Oral
CHF V Test sample 200 Oral
CHF VI Test sample 400 Oral
DCMF VII Test sample 200 Oral
DCMF VIII Test sample 400 Oral
PETF IX Test sample 200 Oral
PETF X Test sample 400 Oral
Castor oil All Inducing 0.5ml Oral
Diarrhea
Note: ME200 = Methanolic extract of Grewia nervosa at 200mg/kg of body weight; ME400 =
Methanolic extract of Grewia nervosa at 400mg/kg of body weight.

7.5. Methodology

Test samples and control were given orally by means of a feeding needle to the mice at zero
hour. Mice of Group I received only 1% Tween 80. Mice of Group II received Loperamide at
a dose of 50 mg/kg. Group III and IV, Group V and VI, Group VII and VIII and Group IX
and X receive the methanolice extract, chloroform fraction extract, Dichloromethane extract
and pet-ether extract at a dose of 200 mg/kg and 400mg/kg respectively. A thirty minutes
interval was given to ensure proper absorption of the administered substances. Then 0.5ml of
castor oil was given to each mouse for inducing diarrhea. Each mouse was marked inthe tail
using a permanent marker and kept in a separate container which was pre-cleaned. Each of
the mice was observed for four hours.

Each time a mouse had given stool was recorded. The average of total number of defecation
given by the test group and the average of total number of defecation given by the control

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 Chapter 7 : Anti-diarrheal activity

group was compared. The statistical significance of the data was calculated to evaluate the
anti-diarrheal activity of the test sample.
Calculation of defecation was measured by the following equation
% of inhibition of defecation = (1- B/A) × 100
A = Mean number of defecation by castor oil
B = Mean number of defecation by drug or extract
Each mouse of all groups was observed for consistency of facial matter and frequency
defecation.

7.6. Statistical Analysis

Results of the study were represented by mean±SEM (Standard Error Mean). Data were
analyzed by one-way ANOVA followed by Dunnett’s t test.

7.7. Results
The methanolic extract and different fraction of Grewia nervosa were subjected to screening
for antidiarrheal activity by castor oil induced diarrhea method. The total number of
defecation for each mouse was taken up to three hours and then the data evaluated
statistically to find its significance.

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 Chapter 7 : Anti-diarrheal activity

Table 7.2: The average number of defecation for methanolic leaves extract of each mouse for each
hour.

Group No. No of defecation by Total no of Average no of

of each mice in different defecation defecation
mice hour
1st 2nd 3rd 4th
M-1
8 5 5 4 22
Control M-2
7 5 4 3 19
M-3 16.8
4 6 3 3 16
M-4
5 4 3 3 15
M-5 4 5 1 2 12
M-1
1 1 1 1 4
M-2
0 1 1 3 5
Standar M-3 4.8
0 2 2 2 6
d M-4
1 1 2 0 4
M-5
0 1 2 2 5
M-1
5 2 3 1 11
M-2
5 4 2 3 14
ME200 M-3 12.6
4 3 2 2 11
M-4
5 3 5 3 16
M-5 4 3 3 1 11
M-1 3 5 4 5 17
M-2 6 5 4 4 19
ME400 M-3 4 4 3 1 12 14
M-4 3 2 1 2 8
M-5 4 6 2 2 14
Note: ME200 = Methanolic leaves extract of G. nervosa at 200mg/kg of body weight; ME400 =
Methanolic extract of G.nervosa at 400mg/kg of body weight.

Table 7.3: The average number of defecation for Chloroform and Dichloromethane fraction extract of
each mouse for each hour.

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 Chapter 7 : Anti-diarrheal activity

Group No. No of defecation by Total no of Average no of

of each mice in different defecation defecation
mice hour
1st 2nd 3rd 4th
M-1
4 3 3 1 11
CHF200 M-2
5 3 3 2 13
M-3
5 4 2 1 12 11.4
M-4
4 4 2 2 12
M-5 3 3 2 1 9
M-1
5 3 2 1 11
M-2
4 2 3 1 10
M-3 11.8
CHF400 4 4 2 2 12
M-4
4 3 2 2 11
M-5
5 4 3 3 15
M-1
2 0 0 0 2
M-2
3 2 1 1 7
DCM200 M-3 5.4
2 2 1 0 5
M-4
0 1 1 2 4
M-5 4 2 2 1 9
M-1 3 1 2 0 6
M-2 3 2 0 0 5
DCM400 M-3 2 1 1 0 4 5
M-4 3 2 0 0 5
M-5 2 2 1 0 5
Note: CHF200 = Chloroform fraction of methanolic extract of G.nervosa at 200mg/kg of body
weight; CHF400 = Chloroform fraction of methanolic extract of G.nervosa at 400mg/kg of body
weight. DCM=Dichloromethane fraction of methanolic extract.

Table 7.4: The average number of defecation for Pet-ether fraction extract each mouse for each hour.

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 Chapter 7 : Anti-diarrheal activity

Group No. No of defecation by Total no of Average no of

of each mice in different Defecation defecation
mice hour
1st 2nd 3rd 4th
M-1
2 1 1 0 4
PETF200 M-2
3 2 1 0 6
M-3
2 1 1 1 5 5.8
M-4
2 1 1 1 5
M-5 3 2 2 2 9
M-1
3 2 1 0 6
M-2
3 1 0 0 4
M-3 5.6
PET400 0 3 1 1 5
M-4
3 2 2 0 7
M-5
3 3 0 0 6
Note: PET200 = Pet-ether fraction of leaves extract of G.nervosa at200mg/kg of body weight;
PET400 =pet-ether fraction of leaves extract of G.nervosa at400mg/kg of body weight;

Table 7.5: Evaluation of antidiarrheal activity of crude extract of G.nervosa

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 Chapter 7 : Anti-diarrheal activity

Mean ± SEM
1st hr 2nd hr 3rd hr 4th hr % Total
Animal Dose
(% of (% of (% of of 0f (% of
Group Mg/kg
Inhibition) Inhibition) inhibition) inhibition) inhibition)
Control 5.6 ± 0.81 5 ± 0.32 3.2 ± 0.66 3 ± 0.32 16.8 ± 1.7
Standard 10 0.4 ± 0.24 1.2 ± 0.2 1.6 ± 0.24 1.6 ± 0.51 4.8 ± 0.37
(93.5)*** (76)*** (50)* (46.67) (69.93)***
ME 200 4.6 ± 0.24 3 ± 0.32 3.0 ±0.54 2 ± 0.44 12.6 ± 1.02
(13.21) (46.42)** (6.25) (33.33) (21.84)*
ME 400 4 ± 0.54 4.4 ± 0.68 2.8 ± 0.58 2.8 ± 0.73 15 ±1.92
(23.36) (7.14) (12.5) (6.66) (15.55)
CHF 200 4.2 ± 0.37 3.4 ±0.24 2.4 ±0.24 1.4± 0.24 11.4 ± 0.67
(25) (32)* (25) (53.33)* (30.32)**
CHF 400 4.4 ± 0.24 3.2 ± 0.37 2.4 ± 1.8 ± 0.37 11.8 ± 0.86
(21.43) (36)** 0.24(25) (40) (24.81)*
DCMF 200 2.2 ± 0.66 1.4 ± 0.4 1 ± 0.31 0.8 ± 0.37 5.4 ± 1.2
(16.71)*** (72)*** (68.75)** (73.33)** (64.23)***
DCMF 400 2.6 ± 0.24 1.6 ± 0.2 0.8 ±0.37 0.00 ± 0.00 5.0 ± 0.32
(53.57)*** (68)*** (75)*** (100)*** (69.28)***
PETF 200 2.4 ± 0.24 1.4 ± 0.24 1.2 ± 0.20 0.8 ±0.37 5.8 ± 0.86
(46.43)*** (72)*** (62.5)** (73.33)** (62.13)***
PETF 400 2.4 ± 0.6 2.2 ±0.37 0.8 ± 0.37 0.2 ± 0.20 5.6 ± 0.50
(57.14)*** (56)*** (75)*** (93.33)*** (64.75)***

Note: Each value represents the mean ± SEM. (n= 5). One- way ANOVA followed by Dunnettttest.
*P<0.05 compared with control. ME = Methanolic leaves Extract, CHF = Chloroform fraction extract,
DCMF=Dichloromethane fraction of leaves, PET=Pet-ether fraction.

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 Chapter 7 : Anti-diarrheal activity

% Of Inhibition
80
% of inhibition compared with control

70
60
50
40 % Of Inhibition
30
20
10
0
d 0 0 0 0 0 0 0 0
d ar F20 F40 l20 l40 F20 F40 20 F40
an E E F F F
St M M Ch Ch M M PE PE
DC DC

Figure 7.2: Comparison of inhibition of total defecation given by mice at four hour for different
fraction of G.nervosa.

Primary data showed that the dichloromethane & pet ether extract induced a significant
decrease in the total number of defecation in 4 hour 69.28%& 64.75 (***p<0.001) for
400mg/kg and 64.23 & 62.13(***p< 0.001) for 200mg/kg respectively when compared to
the control. The chloroform extract decrease defecation at 30.32% (**P<0.001) (Table 4.3)

The above result shows methanolic extract and different fraction of G.nervosa shows
antidiarrheal effet .

7.7.1. Discussion

It is known that the active component of castor oil is the ricinoleic acid, which is liberated
from the action of lipases on castor oil. The ricinoleic acid produces irritating and
inflammatory actions on the intestinal mucosa leading to the release of prostaglandins
(Yoshio et al., 1999). This condition induces an increase in the permeability of the mucosal
cells and changes in electrolyte transport, which results in a hyper-secretory response
(decreasing Na+ and K+ absorption), stimulating peristaltic activity and diarrhea (Zavala et
al., 1998). Inhibitors of prostaglandin synthesis are known to delay diarrhea induced with

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 Chapter 7 : Anti-diarrheal activity

castor oil (Sunil et al., 2001). These results suggest that the antidiarrheal effect of both
alcohol and aqueous extracts may be due to the inhibition of prostaglandin biosynthesis

Anti-dysenteric and antidiarrheal properties of medicinal plants were found to be due to

tannins, alkaloids, saponins, flavonoids, sterols and/or triterpenes and reducing sugars
(Havagiray et al., 2004). Alkaloids (Banerjee et al., 1990) and tannins (Lake, 2010) have
been detected in extract of G.nervosa. These constituents may be responsible for
antidiarrhoeal activity.

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