J Pharm Pharmaceut Sci (www.cspscanada.org) 8(2):370-373, 2005
Acute and subacute toxicity of the hydroalcoholic extract from Wedelia paludosa (Acmela brasiliensis) (Asteraceae) in mice
Cristiani
Bürger, Doris Raquel Fischer, Dórys
Angela Cordenunzzi, Anna Paula de Borba Batschauer, Valdir Cechinel Filho, Adair
Roberto dos Santos Soares
Núcleo de
Investigações Químico-Farmacêuticas (NIQFAR), Centro de Ciências da Saúde
(CCS), Universidade do Vale do Itajaí (UNIVALI),
Received June 6, 2005, Revised June 25, 2005, Accepted June 27, 2005, Published August 19, 2005
Corresponding author: Cristiani Bürger: Núcleo de
Investigações Químico-Farmacêuticas (NIQFAR), CCS, Universidade do Vale do
Itajaí (UNIVALI). R. Uruguai, 458, CP 360, 88302-202, Itajaí, SC, Brazil. cristiani@univali.br
Abstract PURPOSE.
The present study
was carried out to evaluated acute and subacute toxicity of a hydroalcoholic
extract from aerial parts of Wedelia
paludosa (Asteraceae). METHODS.
Toxicity of W. paludosa was evaluated
in Swiss mice after ingestions of the
extract during one day (acute model) and during 15 days (subacute model). RESULTS.
The results showed that the LD50 of the extract is higher than 4000
mg/kg and the subacute treatment did not shows any change in corporal weight
and hematological parameters. However, a change in liver weight but not in
hepatic enzymes was observed. This suggests that the liver function is not
altered by Wedelia paludosa in this
study. Some changes in the creatinine content were observed, but could not be related
with the extract dose. CONCLUSIONS.
The results suggest that the plant seems to be destituted of toxic effects in
mice.
Wedelia
paludosa DC, recently reclassified as Acmela
brasiliensis (Asteraceae), is an
ornamental plant, being widely used in natural medicine in South Brazil. This
plant is used for the treatment of several ailments, including respiratory
infections and pain (0, 0). Our research group has previously investigated the
phytochemical and pharmacological properties of this plant. In this context,
its analgesic (0, 0), antimicrobial and antidiabetic effects (0), were described and related with the presence of
terpenes and flavonoids, including kaurenoic acid and luteolin (0, 0, 0). Other authors showed that kaurane diterpenes from W. paludosa display trypanosomicidal [FJ3] activity (0) and smooth muscle relaxant effect (0). Due to the widespread use of this plant by the
rural communities to treat several diseases, the objective of the present study
was to obtain data on the safety of the crude extract. The acute and subacute
oral toxicity of the hydroalcoholic extract from aerial parts of this plant in
mice was assessed. The changes in selected biochemical and hematological
parameters were also determined.
The aerial parts of Wedelia paludosa DC were collected next
to NIQFAR/UNIVALI, in the town of Itajaí, in the State of Santa Catarina. The
plant material was authenticated by Dr. Ademir Reis (Department of Botany,
UFSC, Florianópolis) and a voucher specimen was deposited at the Barbosa
Rodrigues Herbarium (Itajaí), under number VC Filho 002. The aerial parts of
the plant were air-dried, cut into small pieces and macerated with 50 % ethanol
(w/w) at room temperature for 15 days. After filtration, the solvent was
removed under reduced pressure and the hydroalcoholic extract was then
obtained.
Acute toxicity
The toxicity study as
carried out using female and male Swiss mice (25-35 g). Animals were kept in a
temperature-controlled environment (23 ± 2ºC) with a 12 h light-dark
cycle and food and water were freely available. Ethics Committee of UNIVALI
approved the protocol for these experiments under number 314/2004. The animals
were divided into one control group and five treated groups, each group
consisting of ten animals. The control group received saline and each treated
group received the hydroalcoholic extract in a dose of 100, 500, 1000, 2000 and
4000 mg/kg by gavage. These doses were choose because were 10-100 times higher
than effective doses in other studies. The animals were observed continuously
for 3 h ,and then they were observed each hour during 24 h after administering
the extract to observe any changes in general behavior or other physiological
activities. At the end of the experiment animals were sacrificed by cervical
displacement.
Female Swiss mice, 25-35 g,
were kept in a temperature-controlled environment (23 ± 2ºC) with a 12 h light-dark
cycle. Food and water were freely available and were recorded each 3 days. The
animals were divided into one control group and four treated groups (500, 1000,
2000 and 4000 mg/kg), each group consisting of ten animals. The control group
received saline and each treated group received the hydroalcoholic extract by
gavage for 15 days (once a day) . The animals were weighed each 3 days. At the
end of the experiment, blood was collected from the orbital sinus under ether
anesthesia for biochemical and hematological analysis. After the blood
collection, the animals were sacrificed by cervical displacement and selected
organs (liver, heart, spleen, left kidney and left lung) were removed for
macroscopic analysis. The biochemical parameters evaluated included creatinine,
calcium, inorganic phosphorus, alanine aminotransferase (ALT), aspartate
aminotransferase (AST), gamma-glutamyl transferase and serum alkaline
phosphatase and were assessed using commercial kits. The hematology parameters
were determined for the control and 1000 mg/kg groups and included red
blood-cell count, hematocrit and leukocyte count.
The results are presented as
mean ± s.d. and the
statistical significance between the groups was analyzed by means of an
analysis of variance followed by Dunnett's multiple comparison test. P values
less than 0.05 were considered as significant.
The use of herbal
preparations as in the treatment of diseases is very common in the rural
communities of Brazil. Wedelia paludosa is
frequently used for the treatment of infections, pathologies of the respiratory
tract, fever and others diseases involving inflammation and pain (0, 0). The importance of this plant in folk medicine as
well as its promising pharmacological properties verified in our laboratories (0, 0, 0, 0, 0), make studies about its toxicity very important.
Oral
administration of the hydroalcoholic extract of W. paludosa in doses from 100 to 4000 mg/kg did not produce
significant changes in behavior, breathing, cutaneous effects, sensory nervous
system responses, and gastrointestinal effects in male and female mice. These
effects are observed during the experimental period (24 h). During 24 h of the
experiment, no deaths occurred in any of the groups. These results showed that
in single dose, there are no adverse effects of W. paludosa, indicating that the medium lethal dose (LD50)
is higher than 4000 mg/kg for male and female mice.
The results
showed that a hydroalcoholic extract from Wedelia
paludosa is safe in oral administration in rodents. The doses used in this
study were 10 - 100 times higher than those used in other experimental
pharmacological studies, such as: decrease of blood glucose levels in
alloxan-induced diabetic rats (300 mg/kg) (0); inhibition of pain caused by intraperitoneal
administration of extract on writing test in mice (1mg/kg of roots extract and 3 mg/kg of
leaves and stem extracts (0).
The treatment
with the extract did not decrease the water and food consumption (data not
show). The body weight (table 1) of the animals treated with hydroalcoholic
extract once a day during 15 days (subacute treatment) did not show any
significant change when compared with the control group, although had a
tendency to decrease body weight (2000 and 4000 mg/kg). This decrease can be
associate with the decrease of liver weight at the doses of 2000 and 4000 mg/kg
in comparison with the control group without any concomitant alteration in the
activity of alanine aminotransferase, aspartate aminotransferase, and
gama-glutammyl transferase. Estimation of the serum activity gamma-glutammyl
transferase, alanine aminotransferase and aspartate aminotransferase is one of
the most widely used means of measuring hepatocellular injury (0). The macroscopic analysis of the target organs of
the treated animals (liver, lung, heart, spleen and left kidney) did not show
significant changes in color and texture when compared with the control group.
The results of organs weight are summarized in table 1.
During the
experimental period, there were no treatment-related effects on the
hematological parameters evaluated (table 2). On the other hand, alterations in
the serum concentration of creatinine were observed (table 3). As the
macroscopic appearance and weight of the kidney was not altered, hence, the
possibility of renal injuries could not be confirmed.
Table1. Effect of oral administration of W. paludosa extract on body and organs weight.
|
Dose (mg/kg): |
Control |
500 |
1000 |
2000 |
4000 |
|
Body (g) |
34.1 ± 3.17 |
34.7 ± 1.90 |
36.0 ± 3.77 |
32.0 ± 3.8 |
33.0 ± 2.77 |
|
Liver (g) |
1.559 ± 0.200 |
1.415 ± 0.140 |
1.468 ± 0.225 |
1.3 ± 0.177* |
1.12 ± 0.176** |
|
Heart (g) |
0.144 ± 0.018 |
0.151 ± 0.017 |
0.163 ± 0.037 |
0.155 ± 0.028 |
0.12 ± 0.028 |
|
Left lung (g) |
0.213 ± 0.037 |
0.191 ± 0.016 |
0.235 ± 0.040 |
0.242 ± 0.064 |
0.18 ± 0.040 |
|
Spleen (g) |
0.186 ± 0.092 |
0.156 ± 0.031 |
0.165 ± 0.051 |
0.126 ± 0.013 |
0.14 ± 0.0296 |
|
Left kidney (g) |
0.165 ± 0.021 |
0.157 ± 0.017 |
0.175 ± 0.025 |
0.171 ± 0.027 |
0.14 ± 0.0237 |
Mean
values of 10 animals ± S.D. *p<0.05;
**p< 0.01 vs. control group (Dunnett's test). Control group
received saline. No significant difference was observed in any parameter,
except in liver (2000 and 4000 mg/kg).
Table 2. Hematological parameters after 15 days treatment with the W. paludosa extract.
|
Parameter |
Control |
1000 mg/kg |
4000 mg/kg |
|
Red blood cell (mm3) |
9.043 ± 0.370 |
8.095 ± 0.451 |
8.81 ± 0.129 |
|
Hematocrit (%) |
46.1 ± 4.63 |
44.1 ± 1.524 |
47.049 ± 2.63 |
|
Leukocyte (x106/mL) |
7.550 ± 2.192 |
8.730 ±2.493 |
7.14 ± 1.689 |
Values
are mean of 10 animals ± S.D. (Dunnett's test).
No
significant difference was observed in any parameter.
Table 3. Effect of treatment with W. paludosa extract on biochemical parameters.
|
Dose (mg/kg) |
Control (n=9) |
500 (n=10) |
1000 (n=9) |
2000 (n=8) |
4000 (n=7) |
|
Creatinine (mg/dL) |
0.259 ± 0.050 |
0.285 ± 0.04 |
0.159 ± 0.04** |
0.215 ± 0.06 |
0.248 ± 0.025 |
|
Calcium (mg/dL) |
7.489 ± 0.528 |
7.976 ± 0.91 |
7.754 ± 0.322 |
8.154 ± 0.382 |
8.317± 0.807 |
|
Inorganic phosphorus (mg/dL) |
6.650 ± 0.547 |
6.210 ± 0.62 |
6.770 ± 0.641 |
6.410 ± 1.162 |
6.836 ± 0.671 |
|
ALT (U/L) |
111.9 ± 21.20 |
113.9 ± 10.5 |
91.99 ± 19.89 |
98.29 ± 31.77 |
90.51± 15.25 |
|
AST (U/L) |
54.65 ± 10.15 |
51.57 ± 8.61 |
52.38 ± 8.072 |
48.88 ± 8.059 |
53.67 ± 12.44 |
|
Gamma-glutamyl transferase (U/L) |
2.133 ± 1.920 |
2.780 ± 1.83 |
2.03 ± 1.481 |
2.401 ± 2.81 |
3.42 ± 1.616 |
Values
are mean ± S.D. **p< 0.01 vs. control group (Dunnett's test).
ALT –
alanine aminotransferase
AST –
aspartate aminotransferase
Administration of
hydroalcoholic extract of W. paludosa
in mice not induced any changes in these enzymes, although the liver weight
decreased. This observation could indicate that liver function is preserved by
oral administration of W. paludosa.
Its important to mention that studies on other
species of the genus Wedelia showed
that: i) W. calendulacea has
protective activity against liver
injury in vivo (0); ii) W. glauca
caused liver necrosis in rats (0), sheep and cattle (0); iii) W.
chinensis has an hepatoprotective effect against liver injury (0).
In summary, our
study demonstrated that W. paludosa seems
to be destitute of toxic effects, which could be compromise the medicinal use
of this plant in folk medicine. However, further studies are necessary, such as
histological and morphological experiments, to confirm this evidence.
The authors are grateful to
CNPq (Brazil) for financial support.
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