About Authors:
S. Mahboob Ahmad, T.S Gouda
V.L. College of pharmacy,
Raichur, Karnataka-58410
*syed.ahmad007@yahoo.com
Abstract:
The alcoholic and aqueous extracts of fruit of Carica papaya (Caricaceae)was investigated for anti-diarrhoealactivity in albino Wistar rats. The alcoholic and aqueous extracts was administered by the oral route at a concentration of 100, 200 and 400 mg/kg showed the significant dose dependent anti-diarrhoeal activity in Castor oil induced diarrhea and Magnesium sulphate induced diarrhea. Anti-diarrhoeal activity of the tested extracts was comparable with that of the standard drug Loperamide 3 mg/kg. The results lend support to the traditional use of C. papaya in the treatment of diarrhoeal diseases.
Reference Id: PHARMATUTOR-ART-1372
1. INTRODUCTION
Diarrhoea is associated with an increased frequency of bowel movements with the production of soft or Watery stools. It may be defined as the passage of more than 300ml of liquid faeces in 24 hours. This results in fluid and electrolytes loss that may lead ultimately to death, particularly in children1. Pain, urgency, perianal discomfort and incontinenence often accompany it. Low-volume, painful, bloody diarrhea is known as dysentery2.
Since stool weight is largely determined by stool, most cases of diarrhoea result from disorders of intestinal water and electrolyte transport. From a mechanisteic perspective, diarrhoea can be caused by an increased osmotic load within the intestine (resulting in retention of water with in the lumen) excessive secretion of electrolytes and water into the intestinal lumen; exudation of protein and fluid from the mucosa, and altered intestinal motility, resulting in rapid transit. In most instances simultaneous effects of multiple processes are, leading to a net increase in stool volume and weight accompanied by changes in percent water content.3
Diarrhoea may be due to a specific disease of the intestine or secondary to a disease outside the intestines. For instance, bacillary dysentery directly affects the gut, while diabetes mellitus causes a neuropathic diarrhoeal episode. Diarrhoea can be divided in to acute or chronic forms. Infectious diarrhea is often acute; diabetic diarrhoea has the pathophysiologic causes that help in the identification of specific treatments4.
2. OBJECTIVE
Since no reports are available regarding the anti-diarrhoeal activity with fruit extracts of C. papaya, it is aimed to validate the ethnical medicinal uses of the plant for the activity mentioned above.
The present work is planned with the following objectives:
1. To prepare alcoholic and aqueous extracts with fruit ofC. papaya.
2. Phytochemical investigation
3. To assess the acute toxicity of both the extracts in mice as animal model [OECD guidelines (AOT-425)].
4. To assess the anti-diarrhoeal activity of alcoholic and aqueous extracts by using different models in rats like;
· Castor oil induced diarrhoea.
· Magnesium sulphate induced diarrhoea.
3. METHADOLOGY
Plant Description:
Family:Caricaceae.
Distribution: Throughout India and found near houses and temples.
Parts used:Fruits.
Medicinal uses5:
The fruit extracts ofC. papayahave been reported to possessstomachic, digestive, carminative, diureticgalactagogue. Useful in bleeding piles, haemoptysis, dysentery and chronic diarrhea.
Materials and methods:
Drugs and chemicals: Castor oilwas obtained from Yogesh Pharmacy, Nanded, India. The solvents and other chemicals was used were of analytical grade.
Plant material and extracts: Fruits of C. papaya fruitsis obtained from fields of Sultanpur, Uttar Pradesh, India; the fruits were cut into small pieces and shade dried at room temperature.
A. Preparation of alcoholic extract:
The fruit powder waspacked in a soxhlet apparatus and extracted with 95% alcohol for 18 h. Appearance of colourless solvent in the siphon tube was taken as the termination of extraction. The extract was then transferred in to the previously weighed empty beaker and evaporated to a thick paste on the water bath, maintained at <50ºc to get alcoholic extract. The extract was finally air dried thoroughly to remove all traces of the solvent and the percentage yield was calculated.
B. Preparation of aqueous extract:
About 100 g of powder was taken in a Round bottom flask (2000 ml) and macerated with 500 ml of distilled water and 10 ml of chloroform (preservative) for 24 h. with occasional shaking for every hour in a closed vessel. Then the marc wasremoved by filtering the extract, and then it was concentrated on a water bath at 50ºc.
The two extracts were examined for their colour and consistency. Their percentage yield was calculated with reference to air dried sample and was stored in air tight containers in a re- frigerator below.
Both the fruit extracts (alcoholic and aqueous) of C. papaya were subjected to the following investigations:
1. Preliminary Phytochemical investigations.
2. Pharmacological activities.
a. Toxicity studies (LD50).
b. Antidiarrhoeal activity.
* Castor oil induced diarrhoea.
* Magnesium sulphate induced diarrhoea.
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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 |
Toxicant |
Standard |
ALEFCP |
ALEFCP |
ALEFCP |
AQEFCP |
AQEFCP |
AQEFCP |
||
|
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 |
|||
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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fig 4.1. Effect of ALEFCP and AQEFCP on Castor oil induced diarrhoea in rats
ALEFCP- Alcoholic extract of fruit of C. papaya
AQEFCP- Aqueous extract of fruit of C. papaya
2.Magnesium sulphate induced diarrhoea:
In Magnesium sulphate induced diarrhoea the faecal weight was found to be increased to 74% after administration of magnesium sulphate when compared to normal control animals.
Standard drug has shown significant protection in the castor oil induced diarrhoea .The percentage inhibition was found to be 59.75%.
The fruit extracts of C. papaya low, medium and high dose too have significantly prevented the castor oil induced diarrhoea. The percentage inhibition of ALEFCP was found to be 12.83% (low dose), 29.11% (medium dose), 51.91% (high dose) and the percentage inhibition of AQEFCP was found to be 17.6% (low dose), 25.28% (medium dose) and 55.55% (high dose).
Table 4.2 (i) Anti diarrhoeal activity of fruit extracts of C. papaya
Magnesium sulphate induced diarrhoea in rats.
|
S.No |
Animals |
Faecal Weight (g) mean± SD |
||||||||
|
Normal Control (vehicle) |
Toxicant Control (magnesium sulphate 2 |
Standard (Loperamide 3 |
ALEFCP (Low) 100 |
ALEFCP (medium) 200 |
ALEFCP (high) 400 |
AQEFCP (Low) 100 |
AQEFCP (medium) 200 |
AQEFCP (high) 400 |
||
|
1 |
H |
5 |
8.20 |
2.20 |
7.0 |
5.6 |
4.00 |
7.0 |
6.5 |
3.00 |
|
2 |
B |
4.5 |
7.40 |
3.05 |
8.0 |
6.5 |
4.60 |
8.0 |
6.0 |
3.50 |
|
3 |
T |
6 |
8.00 |
4.24 |
7.0 |
6.2 |
4.50 |
7.0 |
7.0 |
4.00 |
|
4 |
HB |
4 |
9.20 |
2.60 |
8.0 |
6.0 |
4.00 |
7.0 |
6.0 |
4.50 |
|
5 |
HT |
5.5 |
7.30 |
5.50 |
7.5 |
6.3 |
3.50 |
7.0 |
7.0 |
4.20 |
|
6 |
BT |
5 |
9.50 |
3.42 |
8.0 |
6.4 |
4.50 |
7.0 |
6.5 |
4.00 |
|
mean |
5 |
8.43 |
3.5 |
7.58 |
6.16 |
4.18 |
7.16 |
6.5 |
3.86 |
|
|
S.D |
±0.70 |
±0.91 |
±1.20 |
±0.49 |
±0.32 |
±0.42 |
±0.40 |
±0.44 |
±0.53 |
|
ALEFCP - Alcoholic extract of fruit of C. papaya.
AQEFCP-Aqueous extract of fruit of C. papaya.
Table 4.2 (ii) Anti diarrhoeal activity of fruit extracts of C. papaya on
Magnesium sulphate 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 (Magnesium sulphate 2 g/kg p.o) |
8.43 ±0.79 |
74% á |
|
3 |
Standard (Loperamide 3 mg/kg p.o) |
3.50±1.20** |
59.75â |
|
4 |
ALEFCP (100 mg/kg p.o) |
7.58±0.49ns |
12.83%â |
|
5 |
ALEFCP (200 mg/kg p.o) |
6.16±0.32** |
29.11%â |
|
6 |
ALEFCP (400 mg/kg p.o) |
4.18±0.0.42** |
51.91%â |
|
7 |
AQEFCP (100 mg/kg p.o) |
7.16±0.40* |
17.6%â |
|
8 |
AQEFCP (200 mg/kg p.o) |
6.5±0.44** |
25.28%â |
|
9 |
AQEFCP (400 mg/kg p.o) |
3.86±0.53** |
55.55%â |
|
F 41.5 |
|||
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
fig 4.2. Effect of ALEFCP and AQEFCP on Magnesium sulphate induced diarrhoea in rats
ALEFCP- Alcoholic extract of fruit of C. papaya.
AQEFCP - Aqueous extract of fruit of C. papaya.
5. DISCUSSION
The fruit extracts ofC. papayahave been reported to possessstomachic, digestive, carminative, diureticgalactagogue. Useful in bleeding piles, haemoptysis, dysentery and chronic diarrhoea. During acute toxicity studies the extracts (alcoholic and aqueous) of C. papaya were found non toxic and they did not induced any toxic effect / or mortality even up to the dose level of 2000 mg/kg. Phytochemical studies with these fruit extracts revealed the presence ofsterols, flavonoids and triterpenes in both the extracts.
Antidiarrhoeal activity
Diarrhoea is one of the leading causes of death in developing countries. The inhibition of experimental diarrhoea and the reduction in faecal output by a substance are the basis of the pharmacological evaluation of a potential antidiarrhoeal agent. Many antidiarrhoeals act by reducing the gastrointestinal motility and or the secretions. It is well known that ricinoleic acid, an active component of castor oil, induces changes in mucosal permeability, electrolyte transport and intestinal peristalsis, leading to hyper secretory response and diarrhoea. Ricinoleic acid causes irritation and inflammation of the intestinal mucosa, leading to prostaglandin release, which causes an increase in the net secretion of water and electrolytes into the small intestine.Inhibitors of prostaglandin biosynthesis delay castor oil induced diarrhoea. It has been shown that PG-E type causes diarrhoea in experimental animals as well as in human beings. The mechanism has been associated with duel effects on gastrointestinal motility as well as on water and electrolyte transport.PG-E 2 also inhibits the absorption of glucose, a major stimulus to the intestinal absorption of water and electrolytes.The antidiarrhoeal activity of the alcoholic and aqueous extracts was comparable to the standard drugs Loperamide. The antidiarrhoeal activity of flavonoids has been ascribed to their ability to inhibit intestinal motility and hydro-electrolytic secretion,which are known to be altered in this intestinal condition. In vitro and in-vivo experiments have shown that flavonoids are able to inhibit the intestinal secretary response induced by PG-E2.
On the other hand, magnesium sulphate has been reported to induce diarrhoea by increasing the volume of intestinal content through prevention from reabsorption of water. Magnesium sulphate induced diarrhoea has been demonstrated by its osmotic properties and cholecystokinin production. The aqueous and alcoholic extracts were also found to alleviate diarrhoea in this model. The extracts offered an increased absorption of water and electrolyte through the gastrointestinal tract. The present study indicates that the aqueous and alcoholic extracts reduced diarrhoea by increasing reabsorption of electrolytes and water or by inhibiting intestinal accumulation of fluid.
6. CONCLUSION
Ø The preliminary phytochemical screening of both alcoholic(ALEFCP) and aqueous (AQEFCP) extracts of fruit of C. papaya revealed the presence ofsterols, flavonoids and triterpenes.
Ø The crude extracts during their acute toxicity studies have not produced any lethal effect even upto the maximum dose level of 2000 mg/kg.
Ø The anti diarrhoeal activity of fruit extracts (ALEFCP and AQEFCP) of C. papaya was evaluated by castor oil and magnesium sulphate induced diarrhoea in rats.
Ø The both medium dose (200 mg/kg) and high dose (400 mg/kg) but not low dose (100 mg/kg) of the extracts have showed a significantinhibitory activity against castor oil induced diarrhoea byincreasing reabsorption of electrolytes and water and in magnesium sulphate induced diarrhoea by inhibiting intestinalaccumulation of fluid in rats.
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