Vol. 8(47), pp.

1180-1185, 22 December, 2014

DOI: 10.5897/AJPP2014.4167
Article Number: 062720649226
ISSN 1996-0816
African Journal of Pharmacy
Copyright 2014 Author(s) retain the and Pharmacology
copyright of this article
http://www.academicjournals.org/AJPP

Full Length Research Paper

Evaluation of anti-inflammatory and antinociceptive

activities of the Austroplenckia populnea extract in
topical formulations
Larissa Dias Menezes, Gbio Oliveira Gomes, Jos Antnio Paixo da Silva Neto, Mariana
Laundry de Mesquita, Vania Moraes Ferreira and Mnica Valero da Silva*
Department of Pharmaceutical Sciences, Faculty of Health Sciences, University of Braslia (UnB), Darcy Ribeiro
University Campus, DF 70910-900, Brazil.
Received 3 September, 2014; Accepted 1 December, 2014

Austroplenckia populnea (Reiss.) Lundell is a Brazilian Cerrado plant that has been widely used in folk
medicine and has many therapeutic properties. The aim of this study is to evaluate the anti-
inflammatory and antinociceptive properties of a raw hydroalcoholic wood extract of A. populnea in
semi-solid formulations administered topically in rats. The extract of A. populnea was administrated
topically in male and female Wistar rats using cream and gel (4.0 and 8.0%) in the paw edema test
induced by carrageenan and in the formalin test. Creams remained stable, and the gels showed
syneresis. In the paw edema test induced by carrageenan, the gels and creams showed a significant
effect compared to the control group. Only 4.0% cream showed swelling inhibition at the 3rd hour
compared to the Piroxicam gel. Likewise, in the formalin test, the 4.0% cream had an effect similar to the
methyl salicylate, showing a significant anti-inflammatory effect. Based in these results, there was
antinociceptive effect with 4.0% cream. We conclude that topical preparations with extract of A.
populnea may generate new methods for the treatment of local inflammation.

Key words: Analgesia, inflammation, topical administration, formalin test.

INTRODUCTION

The use of medicinal plants by the world population has
contributed to the cure of various diseases and has the
potential for many additional applications, compared with
the rigor displayed by alternative substances synthesized
in laboratory (Cordell and Colvard, 2012; Shaw et al.,
2012; Wang et al., 2013). Austroplenckia populnea
(Reiss.) Lundell is a Brazilian Cerrado plant that is a
potential candidate for use in alternative medicine. It is
known as marmelinho do campo, mangabeira brava,
mangabarana, Maria mole or vime and it is used in

*Corresponding author. E-mail: mvalero2@unb.br. Tel: +55 (61) 8111-6758.

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License

Brazilian folk medicine to treat dysenteries and especially
inflammatory disorders, such as rheumatism (Andrade et
al., 2006). It belongs to the botanical family Celastraceae,
which includes several other plant species that have
been widely used in folk medicine and have ant-
iulcerogenic, analgesic, anti-inflammatory, antitumor and
other properties (Andrade et al., 2006, 2007, 2008).
Phytochemical studies have revealed several com-
pounds, among them the pentacyclic triterpenes, which
could be responsible for the biological activities of this
species, particularly their anti-inflammatory and analgesic
activities (Andrade et al., 2006, 2007; De Sousa et al.,
2006, 1990).
Pharmaceutical creams are semi-solid preparations
that contain one or more medicinal agents dissolved or
dispersed in oil/water (O/W) or water/oil (W/O) emulsions.
They are primarily used in products for topical application
on the skin and in products for rectal or vaginal applica-
tion (Allen et al., 2010). Various strategies have been
proposed to achieve efficient drug delivery. Hydrogels
have been chosen for use in the medical and
pharmaceutical industries due to their biocompatibility
and similarity to natural tissue (Bonacucina et al., 2006).
This study was performed to evaluate the anti-
inflammatory and antinociceptive activities of the
hydroalcoholic wood extract of A. populnea in semi-solid
formulations administered topically in rats.
MATERIALS AD METHODS
Preparations of the raw hydroalcoholic A. populnea extract
(RHAPE)
A. populnea was collected in the Cerrado area of Brazlndia,
Gois, Brazil in June 2007. The wood portion was used to prepare
the extract and was allowed to dry at room temperature for one
week before being pulverized using a knife mill. Subsequently,
1300 g of material was macerated with 5 L of aqueous ethanol
(Vetec 96% v/v) for 12 days, also at a room temperature (252C).
The crude extract (103 g) was obtained after the evaporation of the
solvent under reduced pressure (600 mmHg) at 40C. A voucher
specimen was deposited in the herbarium of the University of
Brasilia under the number J. Elias de Paula (UB) 3747.
Laboratory animals
Wistar Rattus norvegicus (n = 84) weighing between 230 and 280 g
were used. The animals were kept under controlled environmental
conditions, with food and water ad libitum, which was withdrawn 12
h before the test. All experimental procedures were approved by
the Ethics Committee on Animal Use, Institute of Biology, under
protocol number 46510/2010.
Preparation of formulations
The O/W creams were prepared by a phase inversion technique.
The technique consisted of weighing the aqueous and oily
excipients separately, heating the two phases to 70C and adding
Menezes et al. 1181
the aqueous phase of the oil with stirring at 300 rpm in a
mechanical stirrer (Fisatom, Model 175, So Paulo, Brazil). When
the mixture reached 50C, it was added to the raw extract dissolved
in ethyl alcohol (Allen et al., 2010; Lachman et al., 2009). The
preparation was cooled to 30C while being stirred and was then
added to a solution of sodium lauryl sulfate (SLS) in distilled water.
The final pH value of the O/W cream was verified. The carbopol
940 was hydrated in cold water 24 h before the preparation of the
hydrogels. After hydration, the following were added with stirring at
200 rpm: propylene glycol, glycerin, disodium EDTA, preservatives,
RHAPE diluted in ethyl alcohol, SLS diluted in 20 ml of distilled
water and triethanolamine. For the last step, the pH of the
preparation was verified.
Stability test
The samples of the formulations were subjected to accelerated
stability testing for 180 days. The formulations were fractioned in
duplication of 20 g, being conditioned inside plastic pots, after 24 h.
The samples were stored at temperatures of 42C, 402C/75%
relative humidity (RH) and room temperature (252C) for a
predetermined number of days (1, 15, 30, 60, 90, 120, 150 and
180). Centrifugation was carried out with duplicated samples of 10
g of each cream at 1575 g for 30 min in a centrifuge (Centribio,
Model 80-2B, Belo Horizonte, Brazil). The pH of the preparations
stored at ambient temperature was verified by employing a digital
potentiometer (Gehaka, Model 1500, So Paulo, Brazil). The
freeze-thaw test was carried out with duplicated samples of 30 g of
gel for 24 h at -5C and 24 h at 45C for 12 days, for a total of 6
cycles. The color, odor and physical appearance were evaluated
during stability test (Allen et al., 2010).
Paw edema test induced by carrageenan
One hour before applying the carrageenan, 0.2 g of each treatment
(4.0% cream, 8.0% cream, 4.0% gel, and 8.0% gel) was rubbed 50
times on right paw of each animal to help the penetration of
formulation through the skin. A preparation was applied in the same
amount to animals in the control groups (gel base or cream base
without extract) and one reference group using 0.5% Piroxicam gel
for a total of 7 groups of six animals. One hour after treatment, 0.1
ml of 1.0% carrageenan solution (w/v) (Sigma Co., USA) was
administered into the subplantar region of the right hind paw and an
equal volume of 0.9% saline was administered to the left hind paw.
Immediately after the carrageenan injection, the paw volume was
measured using fluid displacement in a calibrated plastic tube.
Measurements were taken at 1, 2, 3, 4 and 5 h after the injection of
carrageenan. When the rat paw was placed in a calibrated plastic
tube, the displacement volume permitted an evaluation of the
swelling. The average of three readings for each paw was
calculated by subtracting the size of the right paw from that of the
left paw (Calvo, 2006; Semnani et al., 2004; Sleyman et al., 2003;
Winter et al., 1962).
Formalin test
One hour prior to formalin administration, 0.2 g of treatments (4.0%
cream, 8.0% cream, 4.0% gel, and 8.0% gel) was administered to
the dorsal surface of the right paw of each animal by gently rubbing
with a finger 50 times. Rats from the control group received only gel
base and cream base (without extract). Methyl salicylate ointment
was applied in the same manner as in the reference group. Seven
groups of 6 animals were used.
1182 Afr. J. Pharm. Pharmacol.

Table 1. Effect of topical administration of O/W creams with RHAPE on the paw edema test induced by carrageenan (n = 6/treatment).

Treatments/Paw volume (ml) at time after

0h 1h 2h 3h 4h 5h
carrageenan injection
Cream base 1.520.17 1.540.25 1.720.21 1.700.12 1.660.25 1.560.32
4.0% Cream 1.050.11* 1.190.07* 1.350,21* 1.100.11* 1.410.21* 1.470.08
8.0% Cream 1.070.09* 1.240.14* 1.390.16* 1.300.07* 1.600.26 1.620.23
Piroxicam gel 0.5% 1.350.22 1.330.20 1.480.18* 1.490.11*# 1.460.13 1.520.14
#
*P < 0.05 represents a significant difference compared to the control (Cream base). P < 0.05 represents a significant difference compared to
the 4.0% cream

Table 2. Effect of topical administration of gels with RHAPE on the paw edema test induced by carrageenan (n = 6/treatment).

Treatments/Paw volume (ml) at time after

0h 1h 2h 3h 4h 5h
carrageenan injection
Gel base 1.630.17 1.880.13 2.050.30 2.10.22 2.10.41 2.10.30
4.0% Gel 1.110.29* 1.130.21* 1.260.20* 1.490.26* 1.520.26* 1.490.28*
8.0% Gel 1.050.14* 1.340.17* 1.290.18* 1.430.27* 1.360.21* 1.450.17*
Piroxicam gel 0.5% 1.350.22* 1.330.20* 1.480.18* 1.490.11* 1.460.13* 1.520.14*
*P < 0.05 represents a significant difference compared to the control.

The procedures and doses were based on experiments from our
research group and other laboratories (Calvo, 2006; Semnani et al.,
2004; Silva et al., 2014). After injection of 20 l of formalin 2.5%
into the subplantar region of the right paw, the rat was observed for
30 min, at intervals of 0 to 5 min (neurogenic phase) and 15 to 30
min (inflammatory phase), during which the recorded time interval
that passed before licking or biting the right paw began was
recorded (Semnani et al., 2004; Silva et al., 2014).
Statistical analysis
All values are expressed as the means standard error of mean
(SEM) for six animals in each experimental group. A statistical
comparison of results was performed using one way analysis of
variance (ANOVA), followed by Newman-Keuls post-hoc multiple
comparison test. The acceptable level of significance of the tests
was P 0.05. All tests were performed using the Statistical
software package (StatSoft Inc., Tulsa, OK, USA).
RESULTS
The formulations of the O/W cream with the RHAPE were
stable at different temperatures during the evaluation
period of 180 days. The gel-type formulations were stable
at room temperature and 42C, while a temperature of
402C/75% RH caused instability. The average pH over
the 180 days period for 4.0% cream was 5.5, while 8.0%
cream had an average pH of 5.11. These changes were
not statistically significant, as the initial pH values for 4.0
and 8.0% creams were 5.65 and 5.61, respectively. No
changes were observed macroscopically or in the
centrifugation test of the O/W creams. With regard to the
gels, the pH showed no significant change. However, at a
temperature of 402C/75% RH, both gels 4.0 and 8.0%
showed syneresis from the 90 and 60th days of
observation, respectively. In the freeze-thaw test both
gels showed syneresis at the end of the test period on
the 12th cycle. Even though, gels and hydrogels
generally have been considered good candidates for oral,
rectal, ocular, dermal and subcutaneous administration
(Bonacucina et al., 2006), probably in this present
situation there was incompatibility between the hydrogel
and extract when temperature changes were applied.
Table 1 shows the results of the paw edema test for the
O/W creams following hourly paw volume assessment for
5 h. At the 0, 1, 2 and 3 h time points, there was a
reduction in paw volume following administration of 4.0
and 8.0% creams when compared with the cream base.
However, the Piroxicam gel significantly reduced edema
when compared with the control group (cream base) with
2 and 3 h. At the 3 h time point, the 4.0% cream had
significantly reduced swelling by 54.43%, but the 8.0%
cream showed negative value, indicating that was not
able to inhibit swelling. Therefore, only 4.0% cream
showed inhibition of swelling in the 3rd h compared to the
Piroxicam gel (12.06%).
Table 2 presents the values of the paw edema test
when the gels with RHAPE were administered for 5 h
evaluations. The 4.0 and 8.0% gels and Piroxicam gel
significantly reduced edema when compared with the
control group (gel base) over 5 h. The 4.0% gel was more
efficient during the initial period of the treatment until the
2 h assessment when compared with control, with an

250
200
LICKING (sec)
Licking (s)
150
100
50
*
0
0-5 min
Figure 1. Effect of RHAPE on the reaction time of licking the formalin-induced paw (n = 6/group) when
used as a creams formulation. *P0.05 represents a significant difference compared to control animals
treated with cream base.
inhibition of 87.32% at the 1 h time point and 47.34% at
the 2 h time point; however, these values were lower than
those from the Piroxicam gel (112.87 and 64.75%
inhibition, respectively).
The results obtained in the formalin test (Figures 1 and
2) demonstrated that treatment with 4.0% cream invoked
a better antinociceptive and anti-inflammatory response
when compared with the methyl salicylate ointment in the
late phase of inflammation origin (15 to 30 min). The gels
did not show a significant response at any stage.
DISCUSSION
The results of the stability study showed that the cream-
type formulations were more stable than the gels. Despite
the unfavorable results from the stability tests of the gels,
they were evaluated together with the O/W creams for
anti-inflammatory and analgesic activities to determine if
the pharmaceutical base would affect the pharmaco-
dynamic properties. In this case, we deduced that the
pharmaceutical base was important during administration
of extract because cream formulation has two phases, oil
and water, and the skin has lipophilic and hydrophilic
characteristics. So, the cream formulation was better than
gel formulation for penetration of extract through the skin.
The edema induced by carrageenan occurs in two
phases, as various mediators act in sequence to produce
an anti-inflammatory response. In the early phase (0 to 1
h), it is possible to observe the release of various
chemical mediators such as histamine, serotonin and
bradykinin. The inflammatory response is correlated with
Menezes et al. 1183
Base cream
Methyl Salicylate
4% Cream
8% Cream
* *
15-30 min
the subsequent phase (1 to 6 h) and is characterized by
increased production of prostaglandins, cyclooxygenase-
2 (COX-2) activation, and the release of nitric oxide
(Gorzalczany et al., 2011). Carrageenan causes an
inflammatory process mediated by prostaglandin,
reaching peak levels within 2 to 3 h after application
(Arawwawala et al., 2010). Because inflammation
mediated by the release of prostaglandin occurs between
the 2nd and 3rd hour, it is relevant to assess the
possibility of reducing edema with RHAPE treatments.
Inhibition of inflammation with the treatments
investigated was significantly observed, as 4.0% cream
showed significant reduction at the 3 h time point that
was greater than that of Piroxicam gel, as previously
mentioned. The fact that the formulation with a lower
concentration of extract had a greater anti-inflammatory
effect than the formulation with higher concentration
indicates that the maximum effect would have been
reached by the 4.0% cream. Our hypothesis is that this
probably could have been the result of a saturation of
likely targets of action of the substances under study.
The formalin test is frequently used because it is an
antinociceptive model sensitive and reliable to evaluate
various classes of analgesic drugs. The test can also be
used to identify the potential mechanism for the
antinociceptive effect of a particular analgesic. Centrally
acting drugs, such as opioids, inhibit the nociceptive and
the inflammatory phases equally. Peripherally acting
drugs, such as aspirin, indomethacin and
dexamethasone, on the other hand, only inhibit the late
stage, because this inflammatory phase can be inhibited
by anti-inflammatory agents (Calvo, 2006; Wang et al.,
1184 Afr. J. Pharm. Pharmacol.

250
Base Gel
M ethyl Salicylate
(sec) 200 4% Gel
(s)

8% Gel
Licking

150
*
LICKING

100

50
*
0
0-5 min 15-30 min
Figure 2. Effect of RHAPE on the reaction time of licking the formalin-induced paw (n = 6/group)
when used as a gels formulation. *P0.05 represents a significant difference compared to control
animals treated with gel base.

2011). The neurogenic phase (0 to 5 min) is the pain Conclusion

process which activates the channels; the late stage
involves the production of nociceptive mediators of
inflammation and occurs within 15 min of formalin
application (late stage). Substance P and bradykinin act
as mediators of the early phase, while histamine,
serotonin, prostaglandins and bradykinin are involved in
the nociceptive response in the late phase (Nonato et al.,
2011). Based on the result of the formalin test, the 4.0%
cream has a similar effect as the methyl salicylate,
suggesting a significant reduction of production of
nociceptive mediators of inflammation involved in the
response observed in Figure 1.
The anti-inflammatory response observed with topical
administration of RHAPE supports previous research
showing an anti-inflammatory effect following oral
administration of the hydroalcoholic wood A. populnea
extract in rats (Andrade et al., 2007).
It was observed that this plant can be an alternative in
phytotherapeutical medicine to treated inflammation and
pain because our results had similar effects when drugs
like piroxicam and methyl salicylate were used as control
in paw edema and formalin test, respectively. The
inflammation is a complex process, which is frequently
associated with pain; so if a medicinal plant has anti-
inflammatory and antinociceptive activities, it is essential
for study and development of an herbal medicine.
Considering that there are only a few preliminary data
reported in the literature regarding the anti-inflammatory
properties of A. populnea preparations, these results
collaborate to increase interest to study more about this
plant and its anti-inflammatory activity using other routes
of administration and types of pharmaceutical formulations.
The results of this study serve as a starting point for
further investigations related to the effectiveness of
topical preparations of RHAPE in treating localized
inflammation. The formulations of O/W cream were stable
over 180 days; moreover, the in vivo tests with 4.0%
cream showed significant anti-inflammatory action
relative to the control. It was also observed that the
pharmaceutical base influenced the effectiveness of
RHAPE, as the cream was superior to the gel base with
this extract and at this concentration. Then, we concluded
that this herbal extract has activity in pharmaceutical
base like O/W cream to topical administration. So, this
study contributed with other treatment alternatives in
case of inflammation.
Competing interests
The authors declare that they have no competing
interests.
ACKNOWLEDGEMENT
The authors are grateful to Dr. Laila S. Espindola for
collaboration and technical support.
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