Evaluation of Antidiarrhoeal Activity of Hydroalcoholic Extract of Ageratum Conyzoides Linn

 

Mangesh V Tote*, Ashutoshpal Jain, Nitin B Mahire, Vaishali R Undale and Ashok V Bhosale

SGRS. College of Pharmacy, Saswad, Pune. (MS), India 412301.

 

ABSTRACT

The leaves of Ageratum conyzoides are used in traditional medicine for the treatment of diarrhoea. Thus the hydroalcoholic extract of leaves Ageratum conyzoides (Asteraceae) was investigated for its antidiarrhoeal property to substantiate folkloric claim. The hydroalcoholic extract of Ageratum conyzoides, at graded dose (250 and 500 mg/kg body weight) was investigated for antidiarrhoeal activity in castor oil and magnesium sulphate induced diarrhoea. Results were comparable to that of standard drug loperamide (3 mg/kg body weight). A single oral dose of Ageratum conyzoides extract of 500 mg/kg body weight produced a significant decrease in the severity of diarrhoea. To understand the mechanism of its antidiarrhoeal activity, its effect was further evaluated on intestinal transit and castor oil induced intestinal fluid accumulation (enteropooling). Extract produced profound decrease in intestinal transit (39.66-58.17%) and significantly inhibited castor oil induced enter pooling comparable to that of intraperitonial injection of standard drug atropine sulphate. The results showed that the hydroalcoholic extract of Ageratum conyzoides have a significant antidiarrhoeal activity and supports its traditional uses in herbal medicine.

 


Keywords: Ageratum conyzoides, antidiarrhoeal activity, castor oil, atropine sulphate.

 

INTRODUCTION

Diarrhoea is a symptom marked by a rapid passage of faecal material through the gastrointestinal tract and a frequent passage of semisolid or liquid faeces. Prolong diarrhoea, whatever the cause, lead to electrolyte loss, the correction of which is of utmost importance1. Diarrhoeal diseases constitute a major cause of morbidity and mortality worldwide; specially in developing countries. More than 5 million children under the age of 5 years die every year because of diarrhoea2. Therefore, the search for safe and more effective agents has continued to be important areas of active research. Since ancient times, diarrhoea has been treated orally with several medicinal plants or their extracts based on folklore medicines.

 

Ageratum conyzoides linn. (Asteraceae) is an annual branching herb which grows to approximately 1 m in height. The stems and leaves are covered with fine white hairs. The leaves are ovate and up to 7.5 cm long. The flowers are purple to white, less than 6 mm across and arranged in close terminal inflorescences. The plant grows commonly in the proximity of habitation, thrives in any garden soil and is very common in waste places and on ruined sites3. A large number of pharmacological activities have been attributed to the essential oil of A. conyzoides; it includes Anti-inflammatory, analgesic and antipyretic activity4, Anti-inflammatory and antioxidant Activity5, Bronchodilating and uterine relaxant activity6, Gastroprotective activity7, Antibacterial activity and wound healing property8. Literature data indicate its efficacy in alleviating pain caused by human arthritis or experimentally induced9.

 

Ageratum is widely utilized in traditional medicine systems where ever it grows. In Brazil an infusion is prepared with the leaves or the entire plant and employed to treat colic, colds and fevers, diarrhoea, rheumatism, spasms, and as a tonic. In addition to its popular use for skin diseases and


 

Observations:

Table 1- Effect of the Hydroalcoholic extract of ageratum conyzoides on Castor oil induced Diarrhoea.

Groups

Onset of Diarrhea (min)

Number of wet stool

Total number of Stools(g)

Weight of

wet Stools(g)

Total weight of Stools(g)

Proctection (%)

Castor oil (1ml) +

Vehicle ( 1%Tween 80)

47.5±1.25

8.5±0.42

12.50±0.76

1.92±0.04

3.53±0.30

0.00

Loparamide (50mg/kg) + castor oil (1ml)

118±1.23**

1.5±0.42*

3.16±0.47**

0.46±0.14**

0.86±0.05**

83.33

HA extract (250 mg/kg) + Castor oil (1ml)

82.5±1.33**

3.66±0.33*

7.83±0.76*

0.78±0.06**

2.33±0.34**

33.34

HA extract (500mg/kg) + castor oil(1ml)

110.33±2.21**

2.83±0.47*

6.00±0.14**

0.64±0.14**

1.13±0.04**

66.67

Values are expressed as mean ± S.E.M (n=6). **p≤0.01,*p≤0.001 when compared with control.

 

Table 2-Effect of the hydroalcoholic extract of ageratum conyzoides on magnesium sulphate induced Diarrhoea

Group

Onset of Diarrhoea

Total number of diarrhoeal faeces (g)

Inhibition (%)

Total weight

of faeces(g)

Inhibition (%)

Magnesium sulphate (1ml) + Vehicle

48±1.82

12.34±2.41

0.00

4.36±0.36

0.00

Loparamide(50mg/kg) +Magnesium sulphate (1ml)

121±1.16*

3.78±1.85**

72.46**

0.94±0.24**

71.83

HEAC (250mg/kg) + Magnesium sulphate (1ml)

96±1.18*

5.00±2.46**

54.98**

2.14±0.25**

70.11

HEAC (500mg/kg) + Magnesium sulphate (1ml)

106±1.20*

8.00±3.42**

41.26*

1.40±0.16**

65.22

Values are expressed as mean ± S.E.M (n=6). **p≤0.01,*p≤0.05 when compared with control.

 


 

wound healing in Nigeria, a decoction of the plant is taken internally to treat diarrhoea and to relieve pain associated with navel in children10.

 

The present study was undertaken to evaluate the antidiarrhoeal potential of hydroalcoholic extract of leaves of Ageratum conyzoides in different experimental models of diarrhoea.

 

MATERIALS AND METHODS:

Plant material:

Aerial parts of Ageratum conyzoides linn. were collected in month of September  from the Empress botanical garden of Pune, India. The plant was authenticated by Botanical survey of India, Pune (Voucher specimen BB680414).

 

Preparation of extract:

Air-dried and powdered leaves of A. conyzoides (100 g) were extracted with 1500 ml of Ethanol: water (70:30 v/v) at room temperature for 48h using Soxhlet apparatus. After extraction, the dark green solution was concentrated to dryness and kept in a freezer. A fresh dilution of dried extract in vehicle (1% Tween 80) was prepared on the day of the experiments, and the employed doses were expressed relative to dried extract.

 

Animals:

Wistar albino rat (180-200 g) and Swiss albino mice (20–25 g) of either sex were kept under standard environmental conditions (i.e.12:12 hour light and dark sequence; at an ambient temperature of 25±20C). They were housed in cages and fed with standard pellet diet and water ad libitum. For experimentation, the animals were fasted overnight and 5–10 animals were included in each group. The principles of Laboratory Animal Care (NIH, 1985) were followed and instructions given by our institutional animal ethical committee were maintained throughout the experiment.

 

Chemicals and Reagents:

Atropine sulphate and loperamide (standard reference antidiarrhoeal drugs), castor oil (laxative agents), charcoal meal (10% activated charcoal in 5% gum acacia) and vehicle (1% v/v Tween 80 in distilled water) were used.

 

Castor oil induced diarrhea:

The animals were screened initially by giving 0.5 ml of castor oil and only those showing diarrhoea were selected for the final experiment. The animals were divided into control, positive control and test groups containing six mice in each group. Control group received vehicle (1% Tween 80 in water) at a dose of 10 ml/kg body weight orally. The positive control group received loperamide at the dose of 3 mg/kg orally, and test groups received the hydroalcoholic extract of Ageratum conyzoides (HEAC) at the doses of 250 and 500 mg/kg body weight orally. Each animal was placed in an individual cage, the floor of which was lined with blotting paper. The floor lining was changed every hour. Diarrhoea was induced by oral administration of 0.5 ml castor oil to each mouse, 30 min after the above treatments. During an observation period of 4 h, the total number of faecal output and the number of diarrhoeic faeces excreted by the animals were recorded. The results were expressed as percentage of protection of diarrhoea11.

 

Magnesium sulphate-induced diarrhea:

A similar protocol as for castor oil-induced diarrhoea was followed. Diarrhoea was induced by oral administration of magnesium sulphate at the dose of 2 g/kg to the animals 30 min after pre-treatment with vehicle (1% Tween 80 in water, 10 ml/kg, p.o) to the control group, loperamide (3 mg/kg) to the positive control group, and the hydroalcoholic extract at the doses of 250 and 500 mg/kg to the test groups. All the administrations were carried out through oral route12.

 

Gastrointestinal motility test:

This experiment was done by using charcoal meal as a diet marker. The rats were divided into five groups of six animals each and fasted for eighteen hours before the experiment. The first group (the control group) was orally administered the vehicle (1% v/v Tween 80 in distilled water). The second and third groups orally received hydroalcoholic extract at doses of 250 and 500 mg/kg body weight respectively. The fourth group received the standard drug, atropine sulphate (3mg/kg body weight i.p). Thirty minutes later each animal was given 1 ml of charcoal meal (10% activated charcoal in 5% gum acacia) orally. Each animal was sacrificed thirty minutes after administration of charcoal meal. The distance covered by the charcoal meal in the intestine was expressed as a percentage of the total distance traveled from the pylorus to the caecum13.

 

Castor oil-induced enteropooling:

Overnight fasted rats were divided into four groups of six animals each. Group 1 which received normal saline (2 ml/kg i.p) served as the control group. Group 2 received atropine (3 mg/kg i.p) and groups 3 and 4 received hydroalcoholic extract of Ageratum conyzoides 250, 500 mg/kg i.p, respectively, one hour before the oral administration of castor oil (1 ml). Two hours later, the rats were sacrificed; the small intestine was removed after tying the ends with threads and weighed. The intestinal content was collected by milking into a graduated cylinder and their volume was measured. The intestine was reweighed and the difference between the full and empty intestine was calculated14.

 

Preliminary phytochemical screening:

Analysis of the hydroalcoholic extract was carried out for various constituents like alkaloids, saponins, flavonoides, tannins, terpenoides/steroids, quinines, polyphenols, anthocyans, amino acids, glucides.etc15.

 

Acute toxicity studies:

Healthy adult albino mice of either sex, starved overnight were divided into six groups ( n = 6) were orally fed with the hydroalcoholic extract of A. conyzoides in increasing dose levels of 100, 500, 1000, 3000, 6000 and 10,000 mg/kg body weight16. The mice were observed continuously for 2 h for behavioral, neurological and autonomic profiles and after a period of 24 and 72 h for any lethality or death17.

 

Statistical analysis:

Data were analyzed by one-way ANOVA followed by Dunnett’s t-test using computerized GraphPad Prism 5.00.288.

 

RESULTS:

In the castor oil-induced diarrhoeal mice, the hydroalcoholic extract of the Ageratum conyzoides, at the doses of 250 and 500 mg/kg, reduced the total number of faeces as well as of diarrhoeic faeces in a dose dependent manner, and the results were statistically significant (Table 1). The percentage inhibition for the number of wet faeces as well as wet mass indicates the presence of antidiarrhoeal activity in extract as compared with that of control group. In the magnesium sulphate-induced diarrhoeal model in mice, the hydroalcoholic extract at the above dose levels significantly (P < 0.01, P < 0.001) reduced the extent of diarrhoea in test animals (Table 2). Both the doses were shown to reduce the total number of faeces and wet faeces when compared to the control. In the gastrointestinal motility test, the extract (HEAC), at the dose of 500 mg/kg, retarded the intestinal transit of charcoal meal in mice as compared to the control (Table 3). The hydroalcoholic extract showed the inhibition of 58.17% and 39.69% at the dose range of 250-500 mg/kg body weight on charcoal-induced gut transit changes. The HEAC significantly inhibited castor oil induced enteropooling in terms of volume and weight of intestinal content comparable to that of intraperitoneal injection of standard drug atropine sulphate at doses of 0.1 mg/kg body weight as indicated in Tables 4. Preliminary phytochemical analysis revealed the presence of alkaloids, tannins, steroids, triterpenoid, saponins and flavonoids. The specific roles of these constituent in the antidiarrhoeal effect of the extract have not been studied. It is possible, that the astringent properties of tannins may enhance the antidiarrhoeal effect of the extract. Acute toxicity studies carried out determined the extract to be safe up to a dose of 10,000 mg/kg. No visible signs of delayed toxicity were observed within 7 days in surviving mice. The LD50 of hydroalcoholic extract was previously determined by the method of Miller and Tainter (1944) and the very high LD50 of the ethnolic extract of Ageratum conyzoides which was observed to be 10.1 g/kg shows that the extract is not toxic18.

 

Table 3- Effect of hydroalcoholic extract of A.conyzoides on charcoal-induced gut transit changes.

Group

Distance travelled by charcoal meal

Inhibition (%)

Vehicle (1% Tween 80)

65.36 ± 2.34

0.00%

Atropin sulphate (3 mg/kg)

28 ± 1.56**

25.47%

HEAC (250mg/kg)

41.74 ± 2.46**

39.66%

HEAC (500 mg/kg)

53.68 ± 2.70**

58.17%

Values are expressed as mean ±S.E.M (n=6) ** p≤ 0.01 when compared with vehicle-control.

 

DISCUSSION:

Diarrhoea can be defined as an abnormal increase in stool frequency, weight, or liquidity. Diarrhoea has many diverse causes but can be classified mechanistically into malabsorptive, maldigestive, or secretory processes; inflammatory states; and deranged intestinal motility19. Castor oil causes diarrhoea due to its active metabolite, ricinoleic acid 15, 16, which stimulates peristaltic activity in the small intestine, leading to changes in the electrolyte permeability of the intestinal mucosa. Its action also stimulates the release of endogenous prostaglandin. The liberation of ricinoleic acid results in irritation and inflammation of intestinal mucosa leading to release of prostaglandin20. Another possibility, though the basis is not derived from this investigation, is the inhibition of prostaglandin formation since castor oil-induced diarrhoea is related to the release of prostaglandins by the colonic cells 21. Also, the delay of castor oil-induced diarrhoea and inhibition of intestinal fluid secretion have been shown to be characteristic of non-steroidal anti-inflammatory drugs22. Abena et.al showed the effectiveness of the extract in inhibiting carrageenin induced inflammation in which prostaglandins have been implicated as mediators. Antimicrobial activity revealed that the hydroalcoholic extract of Ageratum conyzoides possesses a wide range of antibacterial activity against a number of pathogenic bacterial strains that cause diarrhoea and dysentery23.

 

Going by the results obtained in the castor oil induced diarrhoea test, the hydroalcoholic extract of Ageratum conyzoides caused a significant delay in the onset of copious diarrhoea, decreased the frequency of purging (reduction of number of wet stools), weight of wet stools and severity of diarrhoea (diarrhoea score). The results were comparable to that of the standard drug loperamide (3 mg/kg) with regard to the severity of diarrhoea. On the other hand, magnesium sulphate has been reported to induce diarrhoea by increasing the volume of intestinal content through prevention of reabsorption of water. It has also been demonstrated that it promotes the liberation of cholecystokinin from the duodenal mucosa, which increases the secretion and motility of small intestine and thereby prevents the reabsorption of sodium chloride and


 

Table 4- Effect of hydroalcoholic extract of A.conyzoides on castor oil enteropooling.

Group

Volume of intestinal content (ml)

Weight of intestinal content (g)

Normal saline + castor oil (1ml)

5.30 ± 0.24

4.96 ± 0.52

Atropine sulphate + castor oil (1ml)

2.46 ± 0.27

1.98 ± 0.43

HEAC (250mg/kg) + castor oil (1ml)

1.84 ± 0.16

2.19 ± 0.25

HEAC (500mg/kg) + castor oil (1ml)

2.16 ± 0.32

2.62 ± 0.45

 

Values are expressed as mean ±S.E.M (n=6).

 


water 24. The hydroalcoholic extract was found to alleviate the diarrhoeic condition in this model. The extract offered an increased absorption of water and electrolyte from the gastrointestinal tract.  Since the extract (HAE) delayed the gastrointestinal transit in mice as compared to the control, it might have antimotility property. The delay in the gastrointestinal transit prompted by the extract the might have contributed, at least to some extent, to their antidiarrhoeal activity by allowing a greater time for absorption. In enteropooling test, the extract reduced both the weight and volume of intestinal content but results were not significant. Above observations suggest that the extract in graded doses reduce diarrhoea by inhibiting peristalsis, gastrointestinal motility and castor oil induced enteropooling.  

 

CONCLUSION:

The hydroalcoholic extract of Ageratum conyzoides linn. showed anti-diarrhoeal activity in a number of models of diarrhoeic conditions in test animals. It is also reasonable to believe that the extract might be effective in inflammatory diarrhoea, secretary diarrhoea and infectious diarrhoea. These pharmacological characteristics of A. conyzoides found in the present paper could explain the popular use of the plant to alleviate the diarrhoea. Further work is necessary to isolate active principles and elucidate the actual mechanism involved in the antidiarrhoeal activity of this plant.

 

ACKNOWLEDGEMENT:

The authors are thankful to Dr. S.C. Majumdar, Scientist-D, Botanical survey of India (BSI), Pune for authenticating the collected plant material; Poona District Education Association for providing us laboratory facilities to carry out this research work.

 

REFERENCES:

1.       Barar FSK. Essential of pharmacotherapeutics. S.Chand and Company Ltd New Delhi. 2003; 3rd ed: pp.540.

2.       Tripathi KD. Essential of medical pharmacology. Jaypee Brothers, New Delhi. 2003; 5th ed: pp. 615-623.

3.       Marks MK, Nwachuku AC. Weed Res. 1986; 26: 151.

4.       Abena AA, Ouamba JM and Keita A. Antiinflammatory, analgesic and antipyretic activities of essential oil of Agereatum conyzoides. Phytoter. Res. 1996; 10: S164–S165.

5.       Galati et.al. Antiinflammatory and antioxidant activity of Agerarum conyzoides. Pharmaceutical Biology 1999; 39: 336-339

6.       Achola KJ and Munenge RW. Bronchodilating and uterine activities of Ageratum conyzoides extracts. Pharmaceutical Biology 1998; 36(2): 93–96.

7.       Shirwaikar et.al. The gastroprotective activity of the ethanol extract of Ageratum conyzoides. Journal of Ethnopharmacology. 2003; 86: 117–121

8.       Chaha et.al. Antibacterial and wound healing properties of methanolic extracts of some Nigerian medicinal plants. Journal of Ethnopharmacology. 2006; 104: 164–167

9.       Magalhaes et.al. Analgesic and antiinflammatory activities of Ageratum conyzoides in rats. Phytother. Res. 1997; 11: 183–188.

10.     Okunade AL. Ph.D Thesis, University of Ibadan, Nigeria. 1981; 84

11.     Shoba FG and Thomas M. Study of antidiarrhoeal activity of four medicinal plants in castor oil induced diarrhoea. Journal of Ethnopharmacology 2001; 76: 73–76.

12.     Doherty SS. Inhibition of arachinodic acid release, mechanism by which glucocorticoids inhibit endotoxin induced diarrhoea. British Journal of Pharmacology. 1981; 73: 549-554.

13.     Mandal et.al. Antidiarrhoeal evaluation of Ficus racemosa Linn.leaf extract. Joural of Natural product Sciences. 1997; 3(2): 100-103.

14.     Boominathan et.al. Studies on antidiarrhoeal activity of Ionodium suffruticosam ging. (violaceae) extract in rats. Recent Progress in Medicinal Plants (Phytotherapeutics) 2005; 10: 375-380.

15.     Odebiyi OO, Sofowora EA, Phytochemical screening of Nigerian medicinal plants II. Lloydia. 1978; 41: 234–236.

16.     Ghosh MN. Fundamentals of experimental pharmacology. Scientific book agency, Calcutta, 1984; pp. 153.

17.     Turner MA. Screening methods in pharmacology. Academic press, New York. 1965:

18.     Igboasoiyi et.al. Studies on the Toxicity of Ageratum conyzoides. Journal of pharmacology Toxicology 2007; 2(8): 743-747

19.     Gabriel Garcia. Gastrointestinal Disorders. In Melmon and Morrelli’s. Clinical Pharmacology, Basic principles in therapeutics, Edited by Melmon KL. and Morrelli HF. Mcgraw-Hill, New York 1992; 3rd ed: pp.222

20.     Awounters et.al. Delay of castor oil diarrhoea in rats: a new way to evaluate inhibition of prostaglandin biosynthesis. Journal of Pharmacy and Pharmacology. 1978; 30: 41–45

21.     Robert et.al. Enteropooling assay: a test for diarrhoea produced by prostaglandins. Prostaglandins. 1976; 11: 809–814.

22.     Singh S, Majumadar  DK and Rehan  HMS. Evaluation of anti-inflammatory potential of fixed oil of Ocimum sanctum (Holybasil) and its possible mechanism of action. Journal of Ethnopharmacology. 1996; 54; 19–26.

23.     Chaha et.al. Antibacterial and wound healing properties of methanolic extracts of some Nigerian medicinal plants. Journal of Ethnopharmacology. 2006; 104: 164–167.

24.     Galvez et.al. Antidiarrhoeic activity of Euphorbia hirta extract and isolation of an active flavonoid constituent. Planta Medica. 1993; 59: 333- 336.

 

 

Received on 21.04.2009

Accepted on 10.05.2009

© A&V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 1(1): July.-Aug. 2009, 26-29