Diallo et al

Trop J Pharm Res, October 2010; 9 (5): 463

Tropical Journal of Pharmaceutical Research October 2010; 9 (5): 463-467
Pharmacotherapy Group,
Faculty of Pharmacy, University of Benin,
Benin City, 300001 Nigeria.
All rights reserved.

Available online at http://www.tjpr.org
Research Article


Acute and Sub-chronic (28-day) Oral Toxicity Studies
of Hydroalcohol Leaf Extract of Ageratum conyzoides
L (Asteraceae)

Aboudoulatif Diallo
1
, Kwashie Eklu-Gadegkeku
1
, Amegnona
Agbonon
1
, Kodjo Aklikokou
1
, Edmond E Creppy
2
, Messanvi
Gbeassor
1
1
Department of Animal Physiology, Faculty of Sciences, University of Lome, Togo,
2
Department of Toxicology,
Laboratory of Toxicology and Applied Hygiene, University Victor Segalen Bordeaux 2. Bordeaux, France


Abstract


Purpose: Ageratum conyzoides is an annual herbaceous plant commonly used in African traditional
medicine as a purgative, antipyretic, anti-ulcer and wound dressing agent. The objective of this study
was to investigate the acute and sub-chronic toxicity of A. conyzoides leaves in Wistar rats.
Methods: In the acute test, the limit test dose of 5000 mg/kg was administered to Wistar rats and then
observed individually 1 h post-dosing, and at least once daily for 14 days. Sub-chronic toxicity was
evaluated after administering daily oral doses of 500 and 1000 mg/kg body wt., for 28 days to the rats,
Biochemical and haematological assessments as well as body and relative organ weights of the rats
were carried out
Results: The limit dose of 5000 mg/kg did not cause any mortality or signs of acute toxicity in the rats
tested during the observation period. In the sub-chronic tests, the results did not show any treatment
related abnormalities in terms of haematological and biochemical parameters. However, urea was
significantly (p < 0.05) lower in the group treated with 500 mg/kg of A. conyzoides extract. The weekly
body and organ weight of the rats showed no significant differences between the control and the rats
treated with the extract except for liver where there was a significant increase (p < 0.05) in rats that
received 1000 mg/kg, i.e., 3 0.2 g as against 2.5 0.1 g for the control.
Conclusion: Our results suggest that the hydroalcohol extract of A. conyzoides is relatively safe when
administered orally in rats.

Keywords: Ageratum conyzoides, Acute and sub-chronic toxicity, Biochemical parameters,
Haematological analysis, Wistar rats.


Received: 27 January 2010 Revised accepted: 18 July 2010






*Corresponding author: E-mail: aboudoulatif@yahoo.fr; Tel: +228-3203332. Fax: +228-2218595
Diallo et al
Trop J Pharm Res, October 2010; 9 (5): 464
INTRODUCTION

Ageratum conyzoides is an erect, annual
herbaceous plant. It is known to possess a
broad spectrum of pharmacological and
therapeutic properties [1]. In African
traditional medicine, A. conyzoides has been
used as a purgative, antipyretic, anti-ulcer
and wound dressing agent [2]. In Togo, it is
used to treat fever, measles and snake bites

[3] while in Nigeria this plant is used for skin
diseases, wound healing, diarrhoea, pain
associated with navel in children [4], and in
the treatment of HIV/AIDS [5].

Phytochemical investigations on A.
conyzoides have identified a number of
secondary metabolites such us flavonoids,
coumarins and alkaloidal compounds [6-8].
Horie et al [7] reported the presence of
hexametoxyflavone and 1-2 benzopirone
while Moore et al [8] identified several
alkaloids including 1.2-desifropirrolizidinic
and licopsamine which are hepatotoxic [8].
Other compounds such as tannins have also
been detected [9,10]. However, the toxicity,
including hepatotoxicity, of this plant has not
been extensively studied [4,11]. In view of the
presence of hepatotoxic alkaloids in A.
conyzoides and the lack of toxicity study on
the leaf extract, the objective of this study
was to investigate the acute and sub-chronic
toxicity of A. conyzoides leaves in Wistar rats.

EXPERIMENTAL

Collection and extraction of plant
materials

A. conyzoides was collected from Djagbl,
Togo, in July 2007. It was identified by Prof
Kouami Kokou from the Botany Department
of University of Lome, Lome, Togo and a
voucher specimen was kept in the herbarium
of the Laboratory of Botany and Plant
Ecology, Faculty of Science, University of
Lome with reference no. 212 of Tchala.

The leaves of the plant were air-dried to a
constant weight and ground to a coarse
powder. The dried powder (250 g) was
soaked in 2 L of ethanol-water (90:10) for 72
h with occasional agitation. The solution was
filtered and evaporated using a rotary
evaporator, giving a dry extract yield of 15.9
%. Appropriate concentrations of the extract
were made in 2 % aqueous Tween 80 and
used in the animal experiments.

Animals

Wistar rats of either sex (150 - 200 g),
provided by the Department of Animal
Physiology, were used. They were housed in
a standard environmental condition and fed
with rodent standard diets and water ad
libitum. Animal care and handling conformed
to accepted guidelines [12,13]. Ethical
approval was obtained from the institutional
Ethical Committee for Teaching and
Research (ref no. CNCB- CEER 2801/2010).

Acute toxicity test

The limit test dose of 5000 mg/kg was used
as stipulated in Organization for Economic
Cooperation Development (OECD) guidelines
[12]. Three female rats, each sequentially
dosed at intervals of 48 h, were used for the
test. The animals were observed individually
for acute toxicity signs and behavioural
changes 1 h post-dosing, and at least once
daily for 14 days.

Sub-chronic toxicity test

Repeat-dose oral toxicity study was carried
out according to OECD guideline 407 [13].
The animals were divided into three groups of
8 animals each (4 males and 4 females).
Group 1 received 10 ml/kg body weight of
distilled water and served as control. Groups
2 and 3 received extract doses of 500 and
1000 mg/kg body wt, respectively. The
extract was administered daily for 28 days
the same time daily and observed at least
twice daily for morbidity and mortality. Body
weights of the animals were evaluated
weekly.

Diallo et al
Trop J Pharm Res, October 2010; 9 (5): 465
On the 29
th
day, after an overnight fast, the
rats were anaesthetized with ether and blood
sample for haematological and biochemical
analysis were collected into tubes with and
without EDTA, respectively. Haemoglobin,
haematocrit, red blood cell count, white blood
cell count, mean corpuscular haemoglobin
concentration (MCHC), mean corpuscular
haemoglobin (MCH), mean corpuscular
volume (MCV) and platelet count were
determined using an automatic counter
(Sysmex K21, Tokyo, Japan). Biochemical
analysis was performed on serum obtained
after centrifugation of total blood (without
anticoagulant) at 2500 rpm for 15 min.
Standardized diagnostic kits (Labkit

) were
used for spectrophotometric determination of
the following biochemical parameters: alanine
aminotransferase (ALT), aspartate amino-
transferase (AST), creatinine, alkaline
phosphatase, glucose, total proteins and
urea.

Necroscopy of all animals was carried and
the organ weights (heart, liver, kidney and
spleen) were recorded.

Statistical analysis

The results are expressed as mean
standard error of the mean (SEM). Statistical
analysis was performed by one-way analysis
of variance (ANOVA) with Tukey test to
evaluate significant differences between
groups. Values of p < 0.05 were considered
significant. All statistical analyses were
carried out using the Instat statistical package
(Graph Pad Software, Inc., USA).

RESULTS

Acute toxicity

The limit dose of 5 g/kg did not cause
mortality or any sign of acute toxicity in the
three rats dosed for a short period (48 h) and
long period (14 days).



Sub-chronic toxicity

No behavioural changes and death were
observed at the end of the treatment.
Similarly, no significant differences in body
weight were observed between control and
treated groups during this period (see Table
1).

Table 1: Mean body weight of rats after 28 days
treatment with hydroalcohol leaf extract of A.
conyzoides

Mean body weight (g, SEM)
Control Extract
Week
500 mg/kg
1000
mg/kg
0 132 4.8 135 5.2 129 4.5
1 139 5.4 139 4.1 133 4.2
2 142 5.1 144 3.9 137 4.7
3 148 4.8 151 4.4 143 5.4
4 152 5.5 156 5.2 147 6.4


Fig 2 shows that the weight of liver in the
group treated with 1000 mg/kg significantly
higher (p < 0.05) than that of the control
group. For other organs and at both doses
tested, there were no changes in organ
weight

Table 2: Mean organ weight of rats after 28 days
treatment with hydroalcohol leaf extract of A.
conyzoides

Mean weight (g, SEM)
Control Extract dose
Organ
500 mg/kg 1000 mg/kg
Heart
0.340.02(8) 0.330.01(8) 0.340.01(7)
Liver
2.500.12(8) 2.700.11(8) 3.000.15(7)*
Spleen 0.170.02(8) 0.180.018(8) 0.230.03(7)
Kidney
0.550.08(8) 0.540.020(8) 0.600.03(7)
Testis 1.300.08(4) 1.000.26(4) 1.000.23(4)

No. of rats/group indicated in parenthesis; * p <0.05,
control group vs extract
Diallo et al
Trop J Pharm Res, October 2010; 9 (5): 466
Tables 3 and 4 show the haematological and
biochemical parameters for the rats after sub-
chronic toxicity dosing

Table 3: Haematological parameters for rats after 28
days treatment with hydroalcoholic extract of A.
conyzoides leaves.


Control
Extract dose
Parameter

500
mg/kg
1000
mg/kg
WBC (10
3
/l) 9.70.9 (8) 9.90.9 (7) 8.71.3 (7)
RBC (10
6
/l) 7.40.1 (8) 6.90.4 (7) 7.00.2 (7)
Haemoglobin
(g/dL)
14.00.4 (8) 14.00.4 (7) 13.00.4 (7)
Haematocrit
(%)
42.00.9 (8) 40.02.2 (7) 41.01.4 (7)
MCV (fl) 57.00.6 (8) 57.00.6 (7) 56.00.9 (7)
MCH (pg) 18.00.4 (8) 20.01.5 (7) 19.00.4 (7)
MCHC (%) 32.00.6 (8) 35.02.4 (7) 33.00.8 (7)
Platelet
(10
3
/L)
92645 (8)

80164 (7)

96643 (7)

No. of rats/group indicated in parenthesis

Table 4: Biochemical parameters for rats
after 28 days treatment with hydroalcohol
extract of A. conyzoides leaves.

Extract dose
Parameter

Control
500
mg/kg
1000
mg/kg
ASAT (U/L) 24125 (8) 20625 (8) 22916 (7)
ALAT (U/L) 6911 (8) 665 (8) 607 (7)
Total proteins
(g/dL)
6.02.2 (8) 6.21.8 (8) 6.51.4 (7)
Creatinine
(mg/dL)
8.50.7 (8) 7.90.4 (8) 7.40.9 (7)
Urea (mg/dL) 454 (8) 292 (8)* 354 (7)
Alkaline
phosphatase
(U/L)
27352 (8) 21545 (8) 22556 (7)

No. of rats/group indicated in parenthesis;
*Statistically significant (p <0.05, control group vs
extract)

Only urea showed any significant change (p
<0.05) being lower in the group treated with
500 mg/kg of the extract than in the control.
Other biochemical and haematological
parameters were similar for the two groups.

DISCUSSION

The results of the acute toxicity study indicate
that the LD
50
of the extract of A. conyzoides
extract is more than 5000 mg/kg. The limit
test is primarily used in situations where the
investigator has information indicating that
the test material is likely to be non-toxic or of
low toxicity [12]. This finding, therefore,
suggests that the extract at the limit dose
tested is essentially non-toxic and safe in oral
formulation. This result is in line with previous
data from Moura et al [11] who reported that
A. conyzoides LD
50
in mice is more than
10,000 mg/kg.

After 28 days of treatment, there were no
treatment-related changes in haematological
parameters between control and treated
groups, indicating that the extract was not
toxic to circulating red cells, nor interfered
with their production and that of platelets. The
haematopoietic system is one of the most
sensitive targets of toxic compounds and is
an important index of physiological and
pathological status in man and animals [14].
In addition, most of the biochemical
parameters were not also altered by the
extract. The lack of significant alterations in
the levels of ALT, AST, alkaline phosphatase,
glucose and creatinine, which are good
indicators of liver and kidney functions,
suggests that sub-chronic administration of
extract neither altered hepatocytes and
kidneys of rats nor the normal metabolism of
the animals. The extract produced significant
decrease in urea levels at the lower dose
(500 mg/kg); the reason is not exactly known
but one possibility could be that the different
active principles present in the extract are
acting differently at the doses tested and
moreover, some drugs are known to
decrease blood urea levels [15].

The change in the relative weight of the liver
and the presence of several alkaloids,
including 1.2-desifropirrolizidinic and licopsa-
Diallo et al
Trop J Pharm Res, October 2010; 9 (5): 467
mine, which can have hepatotoxic activity
[13]

indicate that the extract might have toxic
potential on liver with increasing dose.
However, it could be argued that these
changes may not be toxicologically
significant, as they were not corroborated by
the biochemical findings (ALT, AST, alkaline
phosphatase and glucose). Further
histological study and more specific assays of
toxicity could furnish more information
regarding the hepatotoxicity of the extract.

CONCLUSION

Our results have demonstrated that the
hydroalcohol leaf extract of A. conyzoides is
relatively safe when administered orally in
rats.

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