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.
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
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
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:
2.
Tripathi KD. Essential of medical pharmacology. Jaypee Brothers,
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.
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,
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.
17.
Turner MA. Screening methods in pharmacology.
Academic press,
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,
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