Journal of Ethnopharmacology 144 (2012) 802–805
Contents lists available at SciVerse ScienceDirect
Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep
Ethnopharmacological communication
Anti-inflammatory effects and acute toxicity of hydroethanolic extract
of Jacaranda decurrens roots in adult male rats
Joyce Alencar Santos a, Aline Arruda a, Magaiver Andrade Silva a, Claudia Andrea Lima Cardoso b,
Maria do Carmo Vieira c, Cândida Aparecida Leite Kassuya a, Arielle Cristina Arena d,n
a
School of Health Sciences, Federal University of Grande Dourados, Dourados-MS, Brazil
Mato Grosso do Sul State University, Dourados-MS, Brazil
c
School of Agrarian Sciences, Federal University of Grande Dourados, Dourados-MS, Brazil
d
~ Junior, S/N, Caixa Postal 510, CEP 18618970,
Department of Morphology, Institute of Biosciences of Botucatu, Sa~ o Paulo State University (UNESP), Distrito de Rubiao
Botucatu-SP, Brazil
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 June 2012
Received in revised form
14 September 2012
Accepted 12 October 2012
Available online 23 October 2012
Ethnopharmacological relevance: Jacaranda decurrens subsp. symmetrifoliolata Farias and Proenc- a
(Bignoniaceae) is a species traditionally used for the treatment of inflammatory diseases. However,
until this moment, there is no scientific evidence of these effects.
Aim of study: To evaluate the anti-inflammatory effects of hydroethanolic root extract of Jacaranda
decurrens in rats and to determine the safe of this plant after acute exposure.
Materials and methods: The acute toxicity of Jacaranda decurrens root extract (EJD) was evaluated by
oral administration to male rats as single doses of 0; 500; 1000 or 2000 mg/kg body weight. General
behavior and toxic symptoms were observed for 14 days. The anti-inflammatory activity was evaluated
in carrageenan-induced inflammatory paw edema and myeloperoxidase activity in male rats.
Results: No signs of acute toxicity were observed, indicating that the LD50 is greater than 2000 mg/kg.
EJD (100 and 300 mg/kg) significantly reduced edema formation and at higher dose, the reduction was
similar to dexamethasone. A significant decrease in myeloperoxidase activity was also observed.
Conclusions: The present study shows that Jacaranda decurrens extract has anti-inflammatory properties in rats without causing acute toxicity. These properties observed may be due to the presence of
bioactive constituents such as ursolic acid.
& 2012 Elsevier Ireland Ltd. Open access under the Elsevier OA license.
Keywords:
Jacaranda decurrens
Inflammation
Paw edema
Carrageenan
Toxicity
1. Introduction
Steroidal or nonsteroidal anti-inflammatory drugs (NSAIDs)
have a number of adverse effects (Batlouni, 2010) and, there is
considerable interest in identifying new anti-inflammatory agents
obtained from plants used in popular medicine.
Jacaranda decurrens subsp. symmetrifoliolata Farias and
Proenc- a (Bignoniaceae), traditional known as ‘‘carobinha-docampo’’, ‘‘carobinha’’ or ‘‘caroba’’, is an endemic species found in
the Brazilian states of Goias, Mato Grosso, Mato Grosso do Sul,
Minas Gerais and Sa~ o Paulo (Bertoni et al., 2010). According to
folk medicine, the leaves and/or the roots were prepared in form
of infusion, decoction and ‘‘garrafadas’’ against inflammatory
diseases and infections (Tresvenzol et al., 2006).
Pharmacological evaluations have revealed that species from
Jacaranda genus possess antioxidants (Carvalho et al., 2009),
antimicrobials (Zatta et al., 2009), chemopreventives properties
n
Corresponding author. Tel.: þ55 14 3880 0495.
E-mail addresses: ariellearena@ibb.unesp.br,
ariellearena@yahoo.com (A.C. Arena).
0378-8741 & 2012 Elsevier Ireland Ltd. Open access under the Elsevier OA license.
http://dx.doi.org/10.1016/j.jep.2012.10.024
(Subbaramaiah et al., 2000). It is suggested that the responsible
for these activities are compounds such as ursolic acid. Phytochemical analyses of the leaves of Jacaranda decurrens indicated
the presence of triterpenes, ursolic acid, oleanolic, flavonoid,
saponins and coumarins (Carvalho et al., 2009; Zatta et al., 2009).
Due to the use of Jacaranda decurrens roots in folk medicine to
combat inflammatory diseases, without scientific evidence of this
potential therapeutic application, the aim of the study was to
evaluate the anti-inflammatory effects of hydroethanolic extract
of Jacaranda decurrens (EJD) roots in male rats and to determine
the toxicity of this plant after acute exposure.
2. Materials and methods
2.1. Plant material, preparation and isolation of extract
Jacaranda decurrens subsp. symmetrifoliolata roots were
collected (April 2010) in the Medicinal Plant Garden of the Federal
University of Grande Dourados. A voucher specimen was identified by Dr. Rosana Farias Singer and deposited (register: W.G.
J.A. Santos et al. / Journal of Ethnopharmacology 144 (2012) 802–805
Garcia 14.008) in the Herbarium of the Department of Botany at
the Biology Institute of the State University of Campinas.
The dry roots (560 g) of Jacaranda decurrens were extracted with 0.7 L of ethanol:water (70:30) at room temperature.
Extracts were united, filtered and concentrated under vacuum
and lyophilizer. During the treatment, the extract was dissolved
in a hydroethanolic solution.
The EJD (1.14 g) was dissolved in water (0.2 L) and fractionated
by XAD-2 (Supelco, Bellefonte, PA, USA) resin column chromatography (30 3 cm) eluted with water (0.3 L), followed by methanol (0.2 L) and with ethyl acetate (0.2 L). The methanolic fraction
(0.59 g) was dissolved in methanol (10 mL) and fractionated by
Sephadex LH-20 (Amersham Pharmacia Biotech, Uppsala, Sweden),
column chromatography (100 3 cm) eluted with methanol
(0.3 mL/min). In total, 29 fractions of 5 mL were collected.
The fractions were combined according to their behavior by
thin-layer chromatography (silica gel plates, ethyl acetate/npropanol/water, 140:8:80 by volume, upper phase). Fractions 17
(12.3 mg) resulted in the isolation of the compound oleanolic acid
(3.9 mg). Fractions 18 was purified using polyvinypolypyrrolidone
(Sigma, St. Louis, MO, USA) column chromatography (10 1 cm)
eluted with methanol, leading to the identification of the compounds oleanolic and ursolic acids (9.8 mg).
2.2. Animals
Adult male Wistar rats (90 days old, weighing approximately
340 g) from the Federal University of Mato Grosso do Sul, were
maintained under controlled temperature (231 C), with a constant
12 h light–dark cycle and free access to food and water. The
experimental procedures were in accordance with the Ethical
Principles in Animal Research and approved by the Committee for
Ethics in Animal Experimentation at the University Center of
Grande Dourados (Protocol no. 334/10).
2.3. Acute oral toxicity
The acute toxicity studies were conducted using the OECD
(Organization for Economic Cooperation and Development)—Guideline 425 and ANVISA guidelines (Brazilian Health Surveillance
Agency) (Brazilian Health Surveillance Agency (ANVISA), 2010;
Organisation for Economic Co-operation and Development
(OECD), 2008). After 12 h of fasting, the animals were divided
into four groups. The treatments were performed by single oral
administration at doses 0; 500; 1000 or 2000 mg/kg of body
weight of EJD. Animals were observed for signs of toxicity during
the first 0.5, 1, 2, 4, 8, and 12 h and at every 24 h for 14 days each.
Behaviors parameters, death, the weight, the amount of water and
feed were analyzed.
After 14 days of treatment, the animals were weighed and
anesthetized (ketamine and xylazine, 25 and 10 mg/kg, respectively). Next, blood samples were collected from the renal vein,
with and without anticoagulant (Heparin sodium, Cristália). The
blood samples were used to determine the hematology parameters (total and differential leukocyte count, hematocrit, hemoglobin and erythrocyte count), and the non-anticoagulated serum
samples were used for biochemical analysis (aspartate
aminotransferase—AST, alanine aminotransferase—ALT, gammaglutamyl transferase—d-GT, creatinine and urea) (Balani et al.,
2011; Organisation for Economic Co-operation and Development
(OECD), 2008). The biochemical parameters were determined
using the semi-automatic Bioplus Bio200 equipment (Gold
Analysis kits).
After that, the animals were euthanised and the vital organs
(liver, lung and right kidney) were removed, weighed (absolute and
relative to body weight) were determined. For the histopathological
803
evaluation of these organs, the samples were fixed in 10% buffered
formalin and processed for histological study by light microscopy.
The parameters investigated were: reversible (degeneration) and
irreversible cell damage (necrosis and apoptosis), leukocyte infiltration, congestion, extravasation of blood and fibrosis.
2.4. Carrageenan-induced rat paw edema
Different groups of rats were orally treated with EJD (100 and
300 mg/kg), or vehicle. Another group was treated subcutaneously with dexamethasone (1 mg/kg). After 1 h, the animals
received a solution of 50 mL carrageenan injection (300 mg/paw)
in one of the hind paws. The other paw received the same volume
of sterile 0.9% saline. The thickness of the paw edema was
measured using a digital micrometer, before the treatment and
at 0.5; 1; 2 and 4 h after the carrageenan. Results were expressed
as micrometer and the difference between basal and postinjection values quantified as edema (Kassuya et al., 2009).
2.5. Determination of myeloperoxidase activity
In this experiment, a carrageenan non-injected group was
inserted and was called as naı̈ve (N) group. Six hours after
carrageenan, the skins of paws were removed after euthanasia
and the methodology of De Young et al. (1989) was performed
with modifications. The tissue was homogenized in phosphate
buffer (80 mM, pH 5.4) containing hexadecyltrimethylammonium
bromide (0.5%). The homogenate was centrifuged at 12,000g
(4 1C, 20 min). Thirty microliter of each supernatant were mixed
with 100 ml of buffer (80 mM), 85 ml of phosphate buffer (0.22 M)
and 15 ml of H2O2 (0.017%) in a 96-well plate. The reaction was
initiated with 20 ml of 3,3,3-tetramethylbenzidine (dissolved in
N,N-dimethylformamide). The plate was kept at 37 1C for 3 min
and the reaction stopped by adding sodium acetate (30 ml, 1.46 M,
pH 3.0). The enzymatic activity was determined by measuring the
optical density (630 nm) and expressed as OD/mg of protein.
Protein levels were measured in the supernatants (Bradford,
1976) using a mixture of each sample with Bradford’s reactant
and after the absorbance was measured.
2.6. Statistical analyses
Data are presented as mean7SEM. Difference between groups
was evaluated by analyses of variance (one-way ANOVA) followed
by Newman–Keuls test. Statistical differences were considered to
be significant at P o0.05.
3. Results and discussion
Jacaranda decurrens roots are empirically used for the treatment of inflammatory disorders (Nunes et al., 2003); however,
its toxic and pharmacological effects on inflammatory experimental models have not been scientifically investigated. To our
knowledge, the present study represents the first research into
the anti-inflammatory effects and toxicity study of extract of
Jacaranda decurrens roots. Several pharmaceutical products currently used to treat inflammation are not completely efficient in
chronic disease and produce many adverse side effects. Therefore,
it is necessary to develop more effective agents that are also
less toxic.
No changes in food consumption, water uptake or behavior
(irritability, contortion, tremors, convulsions, lacrimation and
piloerection) were observed in the animals after acute treatment with EJD. Additionally, the absolute and relative weight
of the vital organs, the hematological parameters, biochemical
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J.A. Santos et al. / Journal of Ethnopharmacology 144 (2012) 802–805
parameters (Table 1) and the histopathological analysis of the
vital organs (data not shown) showed no statistically significant
differences in any of the doses tested. These results confirm those
reported by Zatta et al. (2009), which used the leaf ethanol extract
of Jacaranda decurrens at doses of 2000 and 5000 mg/kg and also
did not observe any signs of toxicity. Thus, it is suggested that the
oral lethal dose (LD50) of Jacaranda decurrens is greater than
2000 mg/kg, and can be classified as a low toxicity extract
according to Organisation for Economic Co-operation and Development (OECD) (2008).
Table 1
Body weight, relative organs weights and hematological and biochemical parameters of animals exposed to Jacaranda decurrens in the acute toxicity study.
Parameter
Control (vehicle)
500 mg/kg
1000 mg/kg
2000 mg/kg
Body weight (g)
Liver (g/100 g)
Lung (g/100 g)
Creatinine (mg/dL)
Urea (mg/dL)
AST (U/L)
ALT (U/L)
d-GT (U/L)
Erythrocyte count ( 106) mm3
Hematocrit (%)
Platelet count ( 103/lL)
Lynphocytes (%)
Eosinophils (%)
Monocyte (%)
Neutrophils (%)
370.00 714.28
2.71 70.08
0.44 70.16
0.90 70.10
35.50 71.20
96.50 721.20
20.90 72.20
11.50 71.30
8.50 70.30
38.20 73.20
1048.00 724.00
73.10 74.10
1.10 70.10
3.60 70.30
22.20 73.20
388.00 7 19.21
2.70 7 0.07
0.47 7 0.03
1.10 7 0.00
35.50 7 1.20
104.20 7 16.90
23.40 7 3.90
11.80 7 2.40
8.60 7 0.40
40.107 3.30
1034.00 7 36.00
75.80 7 2.30
1.10 7 0.20
4.10 7 0.40
19.0 7 1.20
402.007 9.23
2.66 7 0.17
0.457 0.02
1.007 0.10
35.10 7 0.90
113.70 7 19.20
19.50 7 2.40
13.20 7 1.80
8.607 1.10
43.60 7 1.20
1053.00 7 36.00
78.50 7 1.20
1.207 0.20
3.307 0.30
19.40 7 4.20
391.00 7 13.21
2.73 7 0.15
0.44 7 0.02
1.007 0.00
35.00 7 0.80
108.30 7 16.20
22.70 7 2.20
12.20 7 1.30
8.70 7 0.50
42.90 7 0.50
1089.00 7 45.00
76.70 7 2.10
1.20 7 0.20
3.10 7 0.20
21.00 7 1.50
Values expressed as mean 7 SEM, n¼8 animals/group. P 40.05 by ANOVA.
Fig. 1. Effect of Jacaranda decurrens on carrageenan-induced paw edema in rats. Animals received subcutaneously EJD (100 or 300 mg/kg), vehicle (V) or dexamethasone
(Dex—1 mg/kg) and after 1 h, an intraplantar injection of carrageenan (300 mg/paw) was performed. In (A), the time-course of the inhibition induced by EJD and
dexamethasone is shown. In (B), bars show the effect of different doses of EJD and Dex in paw edema (mm) 2 h after carrageenan injection. In (C), bars show the effect of
different doses of EJD and Dex in activity of MPO (D.O) 6 h after carrageenan injection. Each bar represents the mean 7 SEM of 6 animals. # carrageenan naı̈ve (N) group.
*Po 0.05, **P o0.01, ***P o 0.001, compared with the vehicle-treated group. Difference between groups were analyzed by analysis of variance (one-way ANOVA) followed
by Newman–Keuls test.
J.A. Santos et al. / Journal of Ethnopharmacology 144 (2012) 802–805
Acute inflammation is characterized by edema, fever, redness
and pain. The edema is an effective measure of inflammation and
is useful for quantifying induced cutaneous inflammation (Cabrini
et al., 2011). Carrageenan induced edema is a biphasic model with
multiple mediators acting in sequence to produce an inflammatory response. In the early phase (0–1 h) the release of histamine,
serotonin and bradykinin occurs. The later stage (1–6 h)
is correlated with an increased production of prostaglandins,
COX-2 activation and NO release in the inflammatory response
(Di Rosa et al., 1971).
It was observed that EJD caused a reduction in paw edema
induced by carrageenan. Fig. 1 shows that EJD at doses of 100 and
300 mg/kg (P o0.05) and (P o0.001) significantly reduced the
edema of the hind paw after 2 h, and the 300 mg/kg extract
showed the same decrease as the group treated with dexamethasone, and after 4 h it was observed that the groups treated with
EJD showed a statistical significant reduction when compared to
controls. According to the results of this study, it can be seen that
EJD appears to act mainly on the initial phase of the carrageenan
induced inflammatory response. This activity is dose-dependent,
because with the increasing dose there was increased edema
inhibition.
Myeloperoxidase is effective in killing microorganisms and is
abundant in primary azurophilic granules of neutrophils and
monocytes after the activation by a variety of stimuli. Because
of these characteristics, it has been used to quantify tissue MPO as
an inflammatory marker (Kassuya et al., 2009). As for the treatment with EJD, only the group treated with 300 mg/kg showed a
significant decrease in MPO activity, compared to the control
group, as well as the positive control, dexamethasone (Fig. 1). The
protocol used for measuring MPO evaluates the presence of active
enzyme in the tissue, thus, a treatment that can reduce this
parameter can act in two ways: by inhibiting the migration of
neutrophils into the inflammatory site, or by preventing the
enzyme activity (Uzkeser et al., 2012).
Analyses performed using high performance liquid chromatography (HPLC) indicated the presence of ursolic acid in fractions
of the Jacaranda decurrens extract (Carvalho et al., 2009),
a compound whose molecule is similar to dexamethasone.
In the present study, the oleanolic and ursolic acids were isolated
of the EJD roots. The identification of these compounds was
achieved by the experimental data (infrared, nuclear magnetic
resonance, mass spectrometry). Their structures were also confirmed by comparing with the previously reported respective
literature data (Mahato and Kundu, 1994; Taketa et al., 2004).
Thus, ursolic acid could explain the anti-inflammatory action of
the Jacaranda decurrens extract in the adult male rats in this study
(Subbaramaiah et al., 2000).
The present study shows that Jacaranda decurrens extract has
anti-inflammatory properties in rats without causing acute toxicity. The properties observed may be due to the presence of
bioactive components such as ursolic acid.
805
Acknowledgments
The authors thank CAPES (Coordination for the Improvement
of Higher Education Personnel) and FUNDECT (Foundation to
Support the Development of Education, Science and Technology
of the State of Mato Grosso do Sul) for the financial assistance.
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