A STUDY ON ANTI-DIARRHOEAL ACTIVITY OF FRUIT EXTRACTS OF CARICA PAPAYA (CARICACEAE) LINN. IN RATS

Pharma Admission

pharma admission

 

1.      Preliminary Phytochemical investigations6, 7, 8:
The preliminary phytochemical investigations were carried out with alcoholic and aqueous extracts of fruit of C. papaya for qualitative identification of phytochemical constituents present with each extract. All the chemicals and reagents used were of analytical grade.

2.      Pharmacological activities
Experimental Animals:

Albino rats (Wistar strain) of either sex weighing between 150-200 g and Albino mice of either sex weighting between  20-30 g were procured from National Centre for Laboratory Animal sciences, C/0 Shri Venkateswara Enterprises Bangalore for experimental purpose. After procuring, all the animals were acclimatized for 7 days under standard husbandry condition as:
Room temperature               -          26 ± 20 C
Relative humidity                -          45-55%
Light/ dark cycle                 -          12:12 h

The animals were fed with synthetic standard diet Amrut Laboratories Pranava Agro Industries Ltd. Sangli. Water was allowed ad libitum and strict hygienic conditions were maintained. After obtaining prior permission from Institutional Animal Ethical Committee (IAEC) of V. L. College of Pharmacy Raichur (Karnataka), all animal studies were performed as per rules and regulations in accordance to guidelines of CPCSEA. [Registration number 557/02/c/CPCSEA 18th February, 2002] and all the procedures were followed.

A) Determination of oral acute toxicity (LD50)9:
The acute oral toxicity of fruit extracts of C. papaya was determined by using female albino mice (16-22g) those maintained under standard husbandry conditions. The animals were fasted (4 h prior to the experiment and Up and Down procedure (OECD Guideline No. 425) method of CPCSEA was adopted for acute toxicity studies. Animals were administered with single dose of extract and observed for its mortality during 48 h study period (short term) toxicity. Based on the short term profile of drug the doses for the next animals were determined as per OECD Guideline No 425. All the animals were observed for long term toxicity (7 days). The LD50 of the test extracts were calculated using AOT- 425 software provided by Environmental protection agency, USA. 1/25th, 1/10th and 1/5th doses of the LD50 doses were taken as effective doses (low, medium, high) Therapeutic dose for the present study.

B) Determination of Anti- Diarrhoeal activity:
1. Castor oil-induced diarrhoea:

The method was described by Awouters10 was followed with modification as described by Majumdar11. In the present study albino rats of either sex weighing 160-190 g were used. They were divided into 8 groups of each containing six animals. They were fasted for 18-24 hrs prior to the test with free access to water.
Group 1          Control (10 ml/kg of 5% w/v of gum acacia p.o)
Group 2          Toxicant  (Castor oil 1ml/100g p.o).                           
Group 3          Standard   (Loperamide 3 mg/kg p.o)
Group 4          ALEFCP   (low dose, {100 mg/kg} p.o)
Group 5          ALEFCP   (medium dose, {200 mg/kg} p.o)
Group 6          ALEFCP   (high dose, {400 mg/kg} p.o)
Group 7          AQEFCP  (low dose, {100 mg/kg} p.o)
Group 8          AQEFCP (medium dose, {200 mg/kg} p.o)
Group 9          AQEFCP  (high dose, {400 mg/kg} p.o)
ALEFCP-   Alcoholic extract of fruit of C. papaya.
AQEFCP-   Aqueous extract of fruit of C. papaya.

1 hour after the above treatment all the groups were received with castor oil (1ml/100g p.o).  Each rat was then housed separately in cage over clean filter paper. Then diarrhoea was observed for a period of 4 h. During this period, number and wet weight of diarrhoeal dropping were noted. Using mean weight of stools, percentage of diarrhoea and percentage protection was calculated. Antidiarrhoeal activity was determined in terms of percentage protection.

 The percentage protection was calculated by the following formula:

%Protection= {Total weight of stool in control animals –    total weight of stool drug treated animals} X100/ Total weight of stool in control animals.

Data analysis:
The values were expressed as mean ± SD from 6 animals. The results were subjected to statistical analysis by using ANOVA followed by Dunnett,s’ -test  to calculate the significant difference if any among the groups. P<0.05 was considered as significant.

2. Magnesium sulphate induced diarrhoea12:
Group 1 Control (10 ml/kg of 5% w/v of gum acacia p.o)
Group 2 Toxicant control (Magnesium sulphate 2 g/kg p.o)
Group 3 Standard (Loperamide 3 mg/kg)
Group 4 ALEFCP   (low dose, {100 mg/kg} p.o)
Group 5 ALEFCP   (medium dose, {200 mg/kg} p.o)
Group 6 ALEFCP   (high dose, {400 mg/kg} p.o)
Group 7 AQEFCP   (low dose, {100 mg/kg} p.o)
Group 8 AQEFCP   (medium dose, {200 mg/kg} p.o)
Group 9 AQEFCP   (high dose, {400 mg/kg} p.o)

1 hafterthe above treatment all the groups were received with Magnesium sulphate 2g/kg and eachrat then housed separately in cages over clean filter paper. Then diarrhoea was observed for a period of 4 h. During this period, number and wet weight of diarrhoeal droppings were noted. Using mean weight of stools, percentages of diarrhoea and percentage protection were calculated. Anti-diarrhoeal activity was determined in terms of percentage protection.

The percentage protection was calculated by the following formula.
Percentage protection = {Total weight of stool
in control animals   – total weight of stool in  drug treated animals}   X 100/
             Total weight of stool in control animals.

Data analysis:
The values were expressed as mean ± SD from 6 animals. The results were subjected to statistical analysis by using ANOVA followed by Dunnett’s-‘t’ -test to calculate the significant difference if any among the groups. P<0.05 was considered as significant.

4. RESULTS
§  Preliminary Phytochemical screening
Alcoholic and aqueous extracts of fruit of C. papaya were subjected for phytochemical screening and were found to contain sterols, flavanoids and triterpenes in alcoholic extracts and triterpenes, flavonoids in aqueous extracts of C. papaya.

§  Pharmacological activities Acute oral toxicity study:
Alcoholic and aqueous extracts of C. papaya fruit were administered orally to different groups of mice at different dose levels. It was found that even up to the dose level of 2000 mg/kg body weight both extracts did not produced any behavioral symptoms or mortality.

Evaluation of Antidiarrhoeal activity:
Castor oil induced diarrhoea
:
When compared to normal control animals in castor oil induced diarrhoeal model animals treated with castor oil have shown with a significant increase in diarrhoea and faecal weight was significantly increased by 65.33%.
Standard drug (Loperamide) has offered significant protection against castor oil induced diarrhoea, and diarrhoea was inhibited by 57.64%
The fruit extracts of C. papaya atlow, medium and high doses (100, 200 and 400 mg/kg) too have reduced the castor oil induced diarrhoea. The percentage inhibition recorded with ALEFCP was found to be 27.39%, 31.04%, and 55.44% and with AQEFCP was found to be 15.52%, 33.26% and 55.84% with low, medium and high doses respectively.

Table 4.1 (i) Anti-diarrhoeal activity of fruit extracts of C. papaya on Castor oil induced diarrhoea in rats.

   

S.No

Animals

Faecal Weight (g)  mean± SD

Normal
Control
(vehicle)

Toxicant
Control
( Castor oil
1ml /
100g p.o)

Standard 
(Loperamide
3mg /
kg p.o)

ALEFCP
(Low)
100
mg/kg

ALEFCP
(medium)
200
mg/kg

ALEFCP
(high)
400
mg/kg

AQEFCP
(Low)
100
mg/kg

AQEFCP
(medium)
200
mg/kg

AQEFCP
(high)
400
mg/kg

1

H

5

8.2

2.20

7.65

5.00

3.20

6.40

5.50

3.0

2

B

4.5

7.4

3.05

7.20

6.60

4.00

7.00

4.60

4.0

3

T

6

8.0

4.24

6.20

4.40

3.60

7.50

6.00

3.6

4

HB

4

9.2

2.60

7.00

5.80

4.00

7.00

6.20

3.5

5

HT

5.5

7.3

5.50

7.50

7.40

3.80

6.00

5.80

3.8

6

BT

5

9.5

3.42

6.60

5.00

3.50

8.00

5.00

4.0

mean

5

8.26

3.5

6.65

6.41

1.49

6.98

5.66

2.08

SD

±0.70

±0.91

±1.20

±0.59

±1.21

±0.27

±0.72

±0.94

±0.30

ALEFCP- Alcoholic extract of fruit of C. papaya.
AQEFCP-Aqueous extract of fruit of C. papaya.

Table 4.1 (ii) Anti-diarrhoeal activity of fruit extracts of C. papaya on Castor oil induced diarrhoea in rats.

Groups

Treatment &dose

weight of stools after

4 h(g) mean ± SD

% change

1

Normal control

(5% gum acacia 10 ml/kg)

5±0.7071

——

2

Toxicant control

(Castor oil 1ml /100 g p.o)

8.266±0.911

65.33%á

3

Standard

(Loperamide 3 mg/kg p.o)

3.501±1.205**

57.64%â

4

ALEFCP (100 mg/kg p.o)

7.02±0.54*

27.39%â

5

ALEFCP (200 mg/kg p.o)

5.7±1.13 **

31.04%â

6

ALEFCP (400 mg/kg p.o)

3.68±0.31**

55.44%â

7

AQEFCP(100 mg/kg p.o)

6.983±0.722*

15.52%â

8

AQEFCP (200mg/kg p.o)

5.51±0.61**

33.26%â

9

AQEFCP(400 mg/kg p.o)

3.65±0.37**

55.84%â

                                        F                                    46.56
One way ANOVA     
                                       df                                    31.44

ALEFCP- Alcoholic extract of fruit of C. papaya
AQEFCP-Aqueous extract of fruit of C. papaya.
n= 6 Significant at P<0.05*,P <0.01** and ns-not significant vs. control group.

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