Journal of Ethnopharmacology 173 (2015) 127–133

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Antinociceptive and anti-inflammatory effects of Brazilian red propolis

extract and formononetin in rodents
Rodrigo Lima Cavendish a, Jandson de Souza Santos b, Reinaldo Belo Neto a,
Ailma Oliveira Paixão a, Juciele Valéria Oliveira a, Edilson Divino de Araujo c,
Andresa Aparecida Berretta e Silva d, Sara Maria Thomazzi b,n, Juliana Cordeiro Cardoso a,
Margarete Zanardo Gomes a
a
Instituto de Tecnologia e Pesquisa (ITP), Universidade Tiradentes, Av. Murilo Dantas, 300, Farolândia, CEP 49032-490 Aracaju, Sergipe, Brazil
b
Departamento de Fisiologia, Universidade Federal de Sergipe, Av. Marechal Rondon, Cidade Universitária, CEP 49100-000 São Cristóvão, Sergipe, Brazil
c
Departamento de Biologia, Universidade Federal de Sergipe, Av. Marechal Rondon, Cidade Universitária, CEP 49100-000 São Cristóvão, Sergipe, Brazil
d
Universidade de São Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Av. do Café, CEP 14049-900 Ribeirão Preto, São Paulo, Brazil

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Propolis has been used as a folk medicine for centuries around the
Received 9 February 2015 world due to its wide spectrum of biological activities. The red propolis, a new Brazilian variety of this
Received in revised form apimaterial, has presented an unusual chemical composition, including isoflavones such as formononetin
20 June 2015
and biochanin A. Since both the green and red varieties of propolis are traditionally used as medicine and
Accepted 16 July 2015
Available online 17 July 2015
commercialized with no label differentiation, the study of the activities of red propolis extract has be-
come important in order to clarify whether this product has the same activities as commercial ones. In
Keywords: this work, we demonstrated the potential action of the hydroalcoholic extract of red propolis (HERP) and
Propolis its biomarker, formononetin, as antinociceptive and anti-inflammatory drugs on experimental models.
Analgesics
Materials and methods: The HERP was chemically characterised by HPLC/DAD analyses. The biological
Flavonoids
activities of the HERP (3, 10, and 30 mg/kg) and formononetin (10 mg/kg) were evaluated using the
Formononetin
Inflammation. antinociceptive (acetic acid, formalin, and glutamate injections) and anti-inflammatory (carrageenan-
induced hindpaw oedema and peritonitis) models in mice after oral administration. The open field test
was also performed.
Results: Formononetin, one of the main biomarker of red propolis, was identified in the HERP (21.62 mg/
g). Pretreatment with the HERP (10 and 30 mg/kg) and formononetin (10 mg/kg) produced reduction
(P o0.001) in the number of abdominal writhes, but the HERP was more effective (Po 0.001) than for-
mononetin. In the formalin test, all HERP doses (3, 10, and 30 mg/kg, Po 0.001) inhibited the late phase
(inflammatory pain) of formalin-induced licking, but the inhibition of neurogenic pain was observed only
when the higher doses (10 and 30 mg/kg; P o 0.05) were used. Formononetin caused inhibition
(P o0.001) only in the second phase of formalin-induced nociception similarly at all HERP doses in the
same phase of the test. The responses in glutamate-induced model presented crescent inhibition
(P o0.05) with 10 and 30 mg/kg of HERP. Also, formononetin inhibited (Po 0.001) the nociception in-
duced by glutamate similarly to 30 mg/kg of HERP. There were no significant differences in the open field
test after HERP administration, but formononetin decrease the spontaneous motor behaviour. Regarding
the anti-inflammatory assessment, the HERP (10 and 30 mg/kg, P o0.05) and formononetin (P o0.001)
treatments caused a significant inhibition of the oedema response. All doses of HERP (3, 10, and 30 mg/
kg, Po 0.05) and formononetin (Po 0.001) also inhibited the carrageenan-induced leukocyte migration.
In both cases, the results for the HERP at 30 mg/kg and formononetin were similar.

Abbreviations: ASA, acetylsalicylic acid; CI 95%, 95% confidence interval; Dexa, dexamethasone; ED50, median effective dose; FOR, formononetin; HERP, hydroalcoholic
extract of red propolis; IL, interleukin; Morph, morphine; NF-κB, nuclear factor-κB; NMDA, N-methyl-D-aspartate; NO, nitric oxide; NSAID, nonsteroidal anti-inflammatory
drug; ROS, reactive oxidative species; TNF, tumor necrosis factor; TRPA1, transient receptor potential ankyrin 1; TRPV1, transient receptor potential vanilloid 1
n
Correspondence to: Department of Physiology, Center of Biological and Health Sciences Federal University of Sergipe, 49100-000 São Cristóvão, SE,
Brazil. Fax: þ 55 79 21056474.
E-mail address: sthomazzi@gmail.com (S. Maria Thomazzi).

http://dx.doi.org/10.1016/j.jep.2015.07.022
0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
128 R. Lima Cavendish et al. / Journal of Ethnopharmacology 173 (2015) 127–133
Conclusions: The HERP and formononetin presented significant anti-inflammatory activity. Moreover, the
HERP presented antinociceptive action on inflammatory and neurogenic pain without motor side effects,
possibly due to the action of other constituents present in the extract. These results, together, support the
popular usage of this natural product.
1. Introduction
Pain states are related to a series of diseases and tissue injury,
and usually appear as a sign of inflammatory conditions. Non-
steroidal anti-inflammatory drugs (NSAIDs) are commonly used to
treat pain and inflammation, but their long-term use is closely
associated with complications of the gastrointestinal tract, in-
cluding peptic ulcers (Tielemans et al., 2014). Therefore, new
therapies for alleviating inflammatory response, pain and to
avoiding side effects are required.
Natural products are, in turn, potential sources for new thera-
pies. For example, propolis is a bee product that has been widely
used from early times in folk medicine as an anti-inflammatory
and antibacterial medicine (Toreti et al., 2013).
Propolis extracts have received great interest in medicine and
various biological actions have been demonstrated, like anti-
oxidant (Frozza et al., 2013), antinociceptive (Franchin et al., 2012),
and anti-inflammatory (Búfalo et al., 2013; Szliszka et al., 2013).
These activities are linked to its bioactive compounds and it is
noteworthy that propolis from different geographic regions can
vary significantly in its chemical composition (López et al., 2014;
Piccinelli et al., 2011; Sawaya et al., 2011).
In the Northeast region of Brazil, the Apis mellifera (Apidae)
bees produce red propolis from the botanical origin described as
Dalbergia ecastophyllum (L.) Taub., Fabaceae (Daugsch et al., 2008).
This red propolis presents new chemical substances (triterpenoids,
isoflavonoids, prenylated benzophenones, and naphthoquinone
epoxide) when compared to other samples (López et al., 2014;
Morsy et al., 2013; Righi et al., 2011).
Although red propolis extracts and its chemical markers have
demonstrated several biological properties, such as healing activ-
ity (Albuquerque-Júnior et al., 2009; de Almeida et al., 2013), cy-
totoxic activity toward tumour cells (Awale et al., 2008), anti-
microbial (Righi et al., 2011), and potent antioxidant (Franchi et al.,
2012; Righi et al., 2011) actions, the possible antinociceptive and
anti-inflammatory effects of this product have not been explored
so far.
This work aimed to assess the actions of hydroalcoholic extract
of red propolis (HERP) and its main biomarker, formononetin, in
experimental models of chemical-induced nociception and in-
flammatory responses.
2. Materials and methods
2.1. Drugs and reagents
The following drugs and reagents were used: acetylsalicylic
acid (ASA), carrageenan, dexamethasone, L-glutamic acid hydro-
chloride, morphine hydrochloride, and formononetin, all obtained
from Sigma Chemical Co. (St. Louis, MO, USA). Acetic acid was from
Merck (Damstadt, Germany), formalin was obtained from Baker
(Santo Amaro, SP, Brazil), and haloperidol was purchased from
Janssen-Cilag Pharmaceuticals (São Paulo, SP, Brazil). All of the
other reagents used were of analytical grade. The substances were
dissolved in 0.2% Tween 80 in saline solution.
& 2015 Elsevier Ireland Ltd. All rights reserved.
2.2. Red propolis collection and preparation of the hydroalcoholic
extract
Red propolis was collected at Brejo Grande/Sergipe/Brazil
(10°28'25''S, 36°26'12''W). The extraction was performed using
propolis samples (2 g) and 70% ethanol (25 mL) at room tem-
perature for 1 h in an ultrasound bath. After extraction, the mix-
ture was filtered, and the solvent was evaporated to produce the
HERP. The yield of the extraction process was estimated based on
the percentage of dry mass obtained, which was calculated with
reference to the initial mass of propolis before extraction.
2.3. High performance liquid chromatography-diode array detection
analysis
The quantification of formononetin in the HERP was de-
termined using HPLC analysis. A reverse-phase column C18 (Shi-
madzu Shim-Pack CLC-ODS (M) 250  4.6 mm2, 5 μm particle size)
with a diode array detector (Shimadzu Co. mod SPC-M20A) was
used for chromatography analysis. The HERP was homogenised in
methanol to a final concentration of 2.5 mg/mL. Ten μL of sample
was injected into the HPLC system and the gradient started from
5% to 70% solvent A over a period of 170 min. The elution was
performed in a linear gradient using solvent A (0.1% formic acid
(v/v) in acetonitrile) and solvent B (0.1% formic acid (v/v) in water).
The elution rate was 0.8 mL/min and the detection was recorded at
275 nm. The authentic standard of formononetin was used to
prepare the standard curve.
2.4. Animals
Young adults Swiss mice (20–30 g) and Wistar rats (120–180 g)
of both sexes were obtained from the Central Biotery of the Federal
University of Sergipe (São Cristóvão, Brazil) and Central Biotery of
the Tiradentes University (Aracaju, Brazil), respectively. Animals
were maintained at controlled room temperature (21 72 °C) with
s
free access to food (Purina ) and water, under a 12 h light/dark
cycle. The experiments were performed after approval of the
protocols by the Institutional Ethics Committee (011213 and
250608) of the Tiradentes University (Aracaju, Brazil) and were
carried out in accordance with the current guidelines for the care
of laboratory animals. The protocols used in the antinociceptive
study were conducted in accordance with the ethical guidelines
for investigations of experimental pain in conscious animals
(Zimmermann, 1983).
2.5. Acetic acid-induced abdominal constriction test
Abdominal writhes were induced by intraperitoneal (i.p.) in-
jection of acetic acid (0.6%, 0.1 mL/10 g) in mice (Koster et al.,
1959). Animals were pretreated orally (p.o.) with HERP (3, 10, or
30 mg/kg), formononetin (10 mg/kg), acetylsalicylic acid (ASA,
300 mg/kg), or vehicle (0.2% Tween 80 in saline solution, 0.1 mL/
10 g) 60 min before initiating the algesic stimulation (n ¼6/group).
The abdominal writhes were observed for a period of 20 min and
began 5 min after injection of the nociceptive agent.
 R. Lima Cavendish et al. / Journal of Ethnopharmacology 173 (2015) 127–133
2.6. Formalin-induced paw licking test
Mice were pretreated with the HERP (3, 10, or 30 mg/kg, p.o.,
60 min before), formononetin (10 mg/kg, p.o., 60 min before),
morphine (3 mg/kg, i.p., 30 min before), ASA (300 mg/kg, p.o.,
60 min before), or vehicle (0.2% Tween 80 in saline solution,
0.1 mL/10 g, p.o., 60 min before). An intraplantar injection of 2%
formalin solution (20 μL) was given to the right hindpaw of the
animal (n ¼6/group). The time that the animal spent licking or
biting its paw was measured during the first phase (0–5 min) and
the second-phase (15–30 min) of the test (Hunskaar and Hole,
1987).
2.7. Glutamate-induced nociception
Mice (n ¼6/group) were treated with HERP (3, 10, or 30 mg/kg,
p.o., 60 min before), formononetin (10 mg/kg, p.o., 60 min before),
morphine (3 mg/kg, i.p., 30 min before), or vehicle (0.2% Tween 80
in saline solution, 0.1 mL/10 g, p.o., 60 min before). After, a volume
of 20 μL of glutamate (20 μmol/paw) was injected intraplantarly in
the ventral surface of the right hind paw. Animals were observed
individually for 15 min after glutamate injection. The amount of
time spent licking the injected paw was recorded with a chron-
ometer and was considered indicative of nociception (Beirith et al.,
2002).
2.8. The open field test
The open-field test was performed in order to evaluate the
motor and emotional states (Prut and Belzung, 2003). Mice (n ¼5–
8/group) were orally treated with HERP (30 mg/kg), formononetin
(10 mg/kg), or vehicle (0.2% Tween 80 in saline solution, 0.1 mL/
10 g) 30 min before being introduced to the open field apparatus.
The haloperidol (0.2 mg/kg) was given intraperitoneally (n ¼5).
The equipment was made of white-coloured wood, and consisted
of a quadrilateral with a rectangular area of 4830.25 cm2 and walls
that were 34.5 cm high, with the base subdivided into sixteen
quadrants (O’Leary et al., 2013). The parameters evaluated during a
5 min period were: (i) locomotion or crossings (number of line
crosses), (ii) rearings (the number of times the mouse stood on its
hind legs), and (iii) grooming (the number of times the mouse ″
washed″ itself by licking during the observation period) (Whimbey
and Denenberg, 1967).
2.9. Assessment of carrageenan-induced inflammatory response in
the rat hindpaw
The anti-inflammatory effect of HERP and formononetin was
measured using the carrageenan-induced hindpaw oedema model
(Mendes et al., 2010; Winter et al., 1962). Briefly, carrageenan
suspension (1%, 0.1 mL) was administered into the subplantar re-
gion of the right hindpaw of rats, previously treated (60 min be-
fore, n ¼6/group) with HERP (3, 10, or 30 mg/kg, p.o.), for-
mononetin (10 mg/kg, p.o.), dexamethasone (2 mg/kg, s.c.), or
vehicle (0.2% Tween 80, p.o.). Paw oedema was measured ple-
thysmographically (model 7150, Ugo Basile, Varese, Italy), at 1, 2, 3,
and 4 h after the carrageenan. The data obtained were expressed
in mL. The percentage of inhibition was calculated based on the
area under the time–course curves (AUC0–4 h) using the trapezoi-
dal rule.
2.10. 2.Carrageenan-induced peritonitis
Peritonitis was induced by injecting carrageenan (1%, 250 mL)
into the peritoneal cavity (i.p.) of mice (n¼ 6/group) treated
60 min before with HERP (3, 10, or 30 mg/kg, p.o.), formononetin
129
(10 mg/kg, p.o.), dexamethasone (2 mg/kg, s.c.), or vehicle (0.2%
Tween 80, p.o.) (Mendes et al., 2010). Following 4 h of peritonitis
induction, the mice were euthanized, the peritoneal cavity was
washed with saline (3 mL) containing ethylenediaminetetraacetic
acid (EDTA, 1 mM), and total cells were counted in a Neubauer
chamber. The results were expressed as the number of
leukocytes/mL.
2.11. Statistical analysis
Data are presented as median effective dose (ED50) value with
it 95% confidence interval (CI 95%) obtained by nonlinear regres-
sion, or as mean 7SEM of n animals/group calculated using the
GRAPHPAD program (Intuitive Software for Science, San Diego,
CA). Statistical evaluation of the data was performed using one-
way analysis of variance (ANOVA) followed by Bonferroni’s test.
Values of Po 0.05 were considered statistically significant.
3. Results
3.1. Sample characterisation
The yield of HERP extraction was 33.9% (w/w). Fig. 1 shows the
chromatograms for formononetin, which was used as a standard
(A), and HERP (B). Under the chromatographic conditions em-
ployed, formononetin eluted with a retention time of approxi-
mately 85.5 min (Fig. 1). The content of formononetin observed in
the HERP was 21.62 mg/g (2.16% w/w).
3.2. Antinociceptive activity
The writhes evoked by injection of acetic acid were markedly
reduced, in a dose-related manner, by the pretreatment with HERP
at doses of 10 and 30 mg/kg (64.1 and 86.7%, respectively,
Po 0.001; Fig. 2). The calculated ED50 for the HERP was 6.85 mg/kg
(CI 95%: 5.54–8.46 mg/kg). Formononetin (FOR, 10 mg/kg) and ASA
(300 mg/kg) also significantly inhibited (35.8 and 80.8%, respec-
tively, P o0.001) the writhes induced by acetic acid (Fig. 2). In
addition, the antinociceptive effect of the HERP, at 10 and 30 mg/
kg, was greater (Po 0.001) than the effect of formononetin.
The intraplantar injection of the formalin solution produced
nociceptive behaviour in both the first and second phases
(74.5 75.9 and 86.7 78.5 s, respectively; Fig. 3). The HERP at 10
and 30 mg/kg produced marked inhibition of formalin-induced
neurogenic (36.2 and 66.9%, respectively, Po 0.05) and in-
flammatory (76.4 and 82.3%, respectively, P o0.001) phases
(Fig. 3). Similarly, morphine (Morph, 3 mg/kg) caused significant
inhibition of 63.4% and 90.5% of formalin-induced nociceptive
behaviour in the first and second phases, respectively (P o0.001;
Fig. 3). Formononetin (FOR, 10 mg/kg) and HERP at 3 mg/kg caused
inhibition only in the second phase of formalin-induced nocicep-
tion (68.4 and 74.9%, respectively; P o0.001); the same profile was
observed for ASA (80.2%; P o0.001; Fig. 3). In addition, the anti-
nociceptive effect of the HERP at 30 mg/kg was greater (P o0.001)
than the effect of formononetin in the first phase of formalin-in-
duced nociception.
The oral administration of the HERP produced attenuation of
the glutamate-induced nociception at 10 and 30 mg/kg (37.7 and
71.0%, respectively, P o0.05; Fig. 4). Formononetin (FOR, 10 mg/kg)
and morphine (Morph, 3 mg/kg) also inhibited (89.0 and 73.0%,
respectively, P o0.001) the nociception induced by glutamate
(Fig. 4). The antinociceptive effect of formononetin was higher
(P o0.01) than the HERP at 3 and 10 mg/kg.
The treatment with HERP (30 mg/kg) failed to significantly
change any of the parameters evaluated compared with the
130 R. Lima Cavendish et al. / Journal of Ethnopharmacology 173 (2015) 127–133

Fig. 1. (A) Representative chromatogram of formononetin standard (peak 1; Rt ¼85.5 min); and (B) Representative chromatogram of the dry extract of red propolis. The
chromatographic conditions used were: column C18 Shim-Pack CLC-ODS, the mobile-phase consisting of formic acid 0.1% (v/v) in acetonitrile (solvent A) and formic acid 0.1%
(v/v) in water (solvent B), flow rate of 0.8 mL/min, and detection at 275 nm; Rt-retention time.

Fig. 2. The effect of the hydroalcoholic extract of red propolis (HERP) and for-
mononetin on acetic acid-induced visceral nociception. Mice were pretreated with
vehicle, HERP (3, 10, or 30 mg/kg), formononetin (FOR, 10 mg/kg), or acetylsalicylic
acid (ASA, 300 mg/kg) before acetic acid injection. The results are presented as the
means 7 SEM (n¼ 6/group). ANOVA followed by Bonferroni’s test. ***P o 0.001 vs.
the vehicle group, and ###P o 0.001 vs. formononetin group.
Fig. 3. The effect of the hydroalcoholic extract of red propolis (HERP) and for-
mononetin on formalin-induced nociception. Mice were pretreated with vehicle,
HERP (3, 10, or 30 mg/kg), formononetin (FOR, 10 mg/kg), acetylsalicylic acid (ASA,
300 mg/kg), or morphine (Morph, 3 mg/kg) before formalin injection. The results
are presented as the means 7 SEM (n ¼6/group). ANOVA followed by Bonferroni’s
test. *Po 0.05 and ***Po 0.001 vs. the respective vehicle group; and ###Po 0.001
vs. formononetin group in the first phase.

vehicle group. The formononetin (10 mg/kg), in turn, decreased
the motor parameters: the mean of crossings decreased compared
to vehicle group and the rearing decreased in comparison to ve-
hicle and HERP groups. The haloperidol (0.2 mg/kg) decreased
crossing, rearing, and grooming when compared to both vehicle
and HERP groups (Table 1).
3.3. Anti-inflammatory activity
Based on AUC0–4 h values, the HERP at 3 mg/kg did not affect
the carrageenan-induced paw oedema. In contrast, higher doses of
the HERP, 10 and 30 mg/kg, caused a 23.4 and 46.7% inhibition
(P o0.05), respectively, of the oedema response compared to ve-
hicle-treated group (2.6 7 0.2 mL  h). Formononetin (FOR, 10 mg/
kg) and dexamethasone (Dexa, 2 mg/kg) caused an inhibition of
43.7 and 74.7%, respectively (P o0.001) (Fig. 5).
The carrageenan injection in control animals induced leukocyte
migration into the peritoneal cavity after 4 h. The HERP at 3, 10,
and 30 mg/kg significantly inhibited (23.0, 25.9, and 50.7%,
respectively, Po 0.05) this response (Fig. 6). Formononetin (FOR,
10 mg/kg) and dexamethasone (Dexa, 2 mg/kg) injection also in-
hibited (47.6 and 84.9%, respectively, Po 0.001) the carrageenan-
induced leukocyte migration (Fig. 6).
4. Discussion
In the chemical analysis, we identified and quantified the iso-
flavonoid formononetin as a major chromatographic peak. This
finding is consistent with previous works, since the chemical com-
position of Brazilian red propolis has been found to be different to
other varieties (Alencar et al., 2007; Righi et al., 2011) and presents
formononetin as one of the main components and an important
marker for this Brazilian red propolis (López et al., 2014).
In the present work, the antinociceptive and anti-inflammatory
activities of HERP and formononetin were evaluated using che-
mical stimuli to assess their central and/or peripheral action. We
demonstrated that the HERP and formononetin were capable of
 R. Lima Cavendish et al. / Journal of Ethnopharmacology 173 (2015) 127–133
Fig. 4. The effect of the hydroalcoholic extract of red propolis (HERP) and for-
mononetin on glutamate-induced nociception. Mice were pretreated with vehicle,
HERP (3, 10, or 30 mg/kg), formononetin (FOR, 10 mg/kg), or morphine (Morph,
3 mg/kg) before glutamate injection. The results are presented as the means 7SEM
(n¼ 6/group). ANOVA followed by Bonferroni’s test. *P o 0.05 and ***P o 0.001 vs.
the vehicle group, and ##Po 0.01 vs. formononetin group.
Table 1
The effects of the hydroalcoholic extract of red propolis (HERP) and formononetin
on the motor and emotional states in mice.
Treatment Crossing Rearing Grooming
Vehicle (p.o.) 142.007 6.15 32.08 7 3.22 2.86 70.69
Haloperidol (i.p.) 75.50 7 5.17nnn,## 10.75 7 4.07n,# 1.25 70.25n,#
HERP (p.o.) 144.80 7 6.81 33.80 7 2.61 3.25 71.11
Formononetin (p.o.) 105.50 7 11.55n 12.25 7 4.60nn,# 1.50 70.19
Results as mean 7SEM (n¼ 5–8/group). Statistical comparison was performed
using ANOVA followed by the Bonferroni’s test.
n
Po 0.05
nn
Po 0.01
nnn
Po 0.001 vs. vehicle group.
#
Po 0.05
##
P o0.01 vs. HERP group.
showing antinociceptive and anti-inflammatory effects in animal
models. Also, although both HERP and formononetin presented
anti-inflammatory activity, the mechanism of antinociception may
differ.
The HERP reduced the acetic acid-induced abdominal con-
strictions in a dose-dependent manner. The reduction was also
observed after formononetin and ASA treatments compared to
vehicle. These results suggest that HERP and formononetin can
decrease inflammatory response, since this test is considered a
model of visceral pain (Collier et al., 1968) and has been used to
evaluate the peripheral antinociceptive activity of compounds.
However, we can see that the antinociceptive action of for-
mononetin (10 mg/kg) was lower than the HERP (10 and 30 mg/
kg) in the abdominal constrictions model. Possibly, this fact is due
to antinociceptive action of other constituents present in the HERP
that have peripheral antinociceptive/anti-inflammatory effects. In
addition, we observed that both the HERP as formononetin at
10 mg/kg produced antinociceptive effect, but this effect after
administration of the HERP was significantly higher than for-
mononetin, corroborating this hypothesis. In this model, the pain
is generated indirectly via endogenous mediators (Serhan and
Haeggström, 2010) such as histamine, bradykinin, serotonin,
prostaglandins (Duarte et al., 1988), and cytokines [tumour ne-
crosis factor (TNF)-α, interleukin (IL)-1β, IL-8] by residential mac-
rophages and mast cells (Ribeiro et al., 2000). These mediators are
capable of increasing vascular permeability, reducing the thresh-
old of nociception, and stimulating the nerve endings of primary
afferent nociceptive fibres (Martinez et al., 1999), through the
131
Fig. 5. The effect of the hydroalcoholic extract of red propolis (HERP) and for-
mononetin on rat paw oedema. Rats were pretreated with vehicle, HERP (3, 10, or
30 mg/kg), formononetin (FOR, 10 mg/kg), or dexamethasone (Dexa, 2 mg/kg) be-
fore a carrageenan injection. The results are presented as the means 7 SEM (n¼ 6/
group) of area under the curves (AUC0–4 h, in mL  h) of the paw oedema after
injection of carrageenan. ANOVA followed by Bonferroni’s test. *Po 0.05 and
***Po 0.001 vs. the vehicle group.
Fig. 6. The effect of the hydroalcoholic extract of red propolis (HERP) and for-
mononetin on leukocyte migration. Mice were pretreated with vehicle, HERP (3, 10,
or 30 mg/kg), formononetin (FOR, 10 mg/kg), or dexamethasone (Dexa, 2 mg/kg)
before carrageenan-induced peritonitis. The results are presented as the mean-
s7 SEM (n¼6/group). ANOVA followed by Bonferroni’s test. *Po 0.05, **Po 0.01,
and ***Po 0.001 vs. the vehicle group.
activation of ion channels that are sensitive to acids and also the
transient receptor potential vanilloid 1 (TRPV1) cation channels
(Premkumar and Abooj, 2013).
In agreement, HERP and formononetin produced marked in-
hibition of formalin-induced inflammatory (second) phase and
also decreased the inflammatory oedema response and carragee-
nan-induced leukocyte migration. Other studies confirmed the
anti-inflammatory activity of red propolis samples. For example,
pharmacological evaluation of Cuban red propolis (Ledón et al.,
1997), at a similar dosage, showed anti-inflammatory activity in
the cotton-pellet granuloma assay in rats, in croton oil-induced
oedema in mice, in the peritoneal capillary permeability test in
mice, and in the model of acetic acid-induced writings. Also, the
isolated isoflavonoids vestiol and neovestiol, as well as the etha-
nolic extract of Brazilian red propolis, exhibited anti-inflammatory
action by inhibiting neutrophil migration (Bueno-Silva et al.,
2013), and the incorporation of Brazilian red propolis into col-
lagen-based dressing films decreased the inflammatory severity
and improved dermal burn healing (de Almeida et al., 2013).
The red propolis contains considerable amounts of flavonoids
(Campo Fernández et al., 2008; Righi et al., 2013) and these
compounds possess anti-inflammatory activities (Rathee et al.,
132 R. Lima Cavendish et al. / Journal of Ethnopharmacology 173 (2015) 127–133
2009). For example, formononetin reduced the action of IL-1β and
nuclear factor κB (NF-κB) in vitro (Wang et al., 2012). Also, the anti-
inflammatory and antioxidant activities of formononetin pro-
moted neuron and lung protective effects in vivo, by means of
decreasing the levels of TNF-α and IL-6 (Li et al., 2014) and im-
proving superoxidase dismutase activity (Ma et al., 2013).
The formalin-induced paw licking test was used to evaluate
both the neurogenic (nociceptive) and inflammatory pain re-
sponses. While the first phase is caused by activation of nociceptor
ion channel transient receptor potential ankyrin 1 (TRPA1)
(McNamara et al., 2007), the second is mediated by a combination
of peripheral input and sensitisation of spinal cord neurons
(Hunskaar and Hole, 1987). Moreover, whereas the writhing test is
sensitive to several drugs with both peripheral and central actions
(Loux et al., 1978), in the formalin test, the drugs with central
action block both phases, while peripherally acting drugs, such as
NSAIDs (including ASA), cause inhibition of the second stage only
(Malmberg and Yaksh, 1992; Shibata et al., 1989).
Both HERP and morphine decreased the response of the first
and second stages of the formalin test, while ASA and for-
mononetin decreased the second phase only. These data indicate
that HERP acts in both the central and peripheral nociception apart
from inflammatory response, possibly due to a synergistic action
of formononetin and other constituents present in the HERP. This
similarity with the morphine effect was also observed by others
(Ledón et al., 1997); the ethanolic extract of red propolis was ef-
fective in the hot plate test in mice.
While the effect of HERP and formononetin in the second phase
may be explained by anti-inflammatory actions, the action of HERP
in the neurogenic nociceptive response remains to be elucidated.
We evaluated the possible influence of HERP in the nociceptive
transmission mediated by glutamate. It is the major excitatory
neurotransmitter in the central nervous system and is involved in
the primary afferent nociceptive transmission, in both the initia-
tion and maintenance of the response (Beirith et al., 2002).
Moreover, several studies have shown that glutamate is important
for the spinal and supraspinal transmissions (Fundytus, 2001), as
well as the spinal TRPA1 participation in the enhancement of
glutamatergic nociceptive response (Klafke et al., 2012).
In this study, we observed that both HERP and formononetin
produced an antinociceptive effect on the glutamate-induced paw
licking response. The action of glutamate involves metabotropic and
ionotropic receptors [N-methyl-D-aspartate (NMDA) and non-NMDA]
and depends on activation of the L-arginine/nitric oxide (NO) path-
way (Beirith et al., 2002). NO and other reactive oxygen species (ROS)
have been implicated in tissue damage and inflammatory and noci-
ceptive responses (Heine et al., 2011). Also, ROS enhance excitatory
synaptic transmission in rat spinal dorsal horn neurons by activating
TRPA1 and TRPV1 channels (Nishio et al., 2013). The HERP and its
chemical markers (flavonoids, acids, pigments) are able to scavenge
NO (Frozza et al., 2013; Righi et al., 2011) and they could attenuate
the inflammatory processes and/or modify glutamatergic transmis-
sion. So, the effects described for HERP and mainly formononetin in
the glutamate response can be related to their antioxidant activity.
Interestingly, although a higher dose was required for HERP
(30 mg/kg) to show similar effects to morphine dose (3 mg/kg) in the
formalin (first phase) and glutamate tests, the HERP produced no
adverse effects on the motor and emotional states of the animals, as
seen in the open field test. It can represent an advantage with respect
to opioids for the treatment of neuropathic pain. In a recent study, da
Silva et al. (2015) evaluated the acute and sub-acute toxicities of the
HERP and they observed that this extract administered orally in rats
shows no lethal effects at 300 mg/kg, indicating that the HERP has a
LD50 above this value. Toxic signals were observed only at doses higher
than 100 mg/kg in sub-acute study, while 10 mg/kg showed to be safe.
Yet, some extracts from different types of propolis were able to in-
crease the rat activity in the open field tests (Reis et al., 2014; Zárraga-
Galindo et al., 2011), but this difference from our results can be ex-
plained by the distinct chemical profiles of propolis samples.
Formononetin decreased motor parameters in the open field
test. Then, it cannot be discarded the hypothesis of a central action
of formononetin. Indeed, the formononetin systemically admini-
strated (i.p. and intravenous) reached the nervous system and
protected it against ischemia/reperfusion (Liang et al., 2014; Zhu
et al., 2014) and traumatic (Li et al., 2014) brain injuries. Because
the formononetin decreased the spontaneous activity in the open
field test, the antinociceptive action of this substance could be
attributed, at least in part, by a sedative effect. Also, formononetin
failed to produce a neurogenic mediated antinociceptive response.
Thus, this action of HERP is probably due to the presence of other
bioactive components or their combination and synergy.
For example, isolated flavonoids such as 3′,4′-dihydroxy-4-
methoxydalbergione, 4-methoxydalbergion, cearoin, and chrysin
(that are founded in species of Dalbergia) (Chan et al., 1998) in-
hibited the IL-33-induced mRNA expression of inflammatory genes
including IL-6, TNF-α, and IL-13 in bone marrow-derived mast cells
and the activation of NF-κB (Funakoshi-Tago et al., 2015). Other
flavonoids such as medicarpin and isoliquiritigenin - which are
also present in the Brazilian red propolis (Inui et al., 2014; Righi
et al., 2011) - had no effect on the IL-33 signalling pathway or
cytokine expression (Funakoshi-Tago et al., 2015).
Other compounds were also identified in red propolis samples,
such as daidzein, quercetin (Alencar et al., 2007), pinocembrin,
and biochanin A (Bueno-Silva et al., 2013; Inui et al., 2014; López
et al., 2014; Righi et al., 2011). Between them, the flavonoid
quercetin has been implied in neurogenic nociception - and in
chronic painful peripheral neuropathy - through mechanisms that
involve interactions with L-arginine-NO, serotonin, and GABAergic
system (Azevedo et al., 2013; Filho et al., 2008).
Taken together, the results indicate that formononetin and HERP
present anti-inflammatory activity that can be associated with their
antioxidant properties and/or their interactions with inflammatory
mediators. Moreover, HERP presented also antinociceptive action on
neurogenic and inflammatory pain without motor and emotional
side effects in rats. It supports the popular use of red propolis as an
alternative to treat inflammatory-painful-infectious diseases and
points to a promising new therapeutic strategy for neuropathic pain.
Acknowledgements
This work was supported by FAPITEC/SE (Fundação de Apoio à
Pesquisa e à Inovação Tecnológica do Estado de Sergipe
(019.203.01157/2011-9). AABS and SMT (306484/2012-9) are re-
cipients of CNPq, Brazil productivity Grants. RLC received scho-
larship from FAPITEC/SE.
References
Albuquerque-Júnior, R.L.C., Barreto, A.L.S., Pires, J.A., Reis, F.P., Lima, S.O., Ribeiro, M.
A.G., Cardoso, J.C., 2009. Effect of bovine type-I collagen-based films containing
red propolis on dermal healing in rodent model. Int. J. Morphol. 27, 1105–1110.
Alencar, S.M., Oldoni, T.L., Castro, M.L., Cabral, I.S., Costa-Neto, C.M., Cury, J.A., Ro-
salen, P.L., Ikegaki, M., 2007. Chemical composition and biological activity of a
new type of Brazilian propolis: red propolis. J. Ethnopharmacol. 113, 278–283.
Awale, S., Li, F., Onozuka, H., Esurni, H., Tezuka, Y., Kadota, S., 2008. Constituents of
Brazilian red propolis and their preferential cytotoxic activity against human
pancreatic PANC-1 cancer cell line in nutrient-deprived condition. Bioorg. Med.
Chem. 16, 181–189.
Azevedo, M.I., Pereira, A.F., Nogueira, R.B., Rolim, F.E., Brito, G.A., Wong, D.V., Lima-
Júnior, R.C., de Albuquerque Ribeiro, R., Vale, M.L., 2013. The antioxidant effects
of the flavonoids rutin and quercetin inhibit oxaliplatin-induced chronic
 R. Lima Cavendish et al. / Journal of Ethnopharmacology 173 (2015) 127–133
painful peripheral neuropathy. Mol. Pain 9, 53.
Beirith, A., Santos, A.R.S., Calixto, J.B., 2002. Mechanisms underlying the nociception
and paw oedema caused by injection of glutamate into the mouse paw. Brain
Res. 924, 219–228.
Bueno-Silva, B., Alencar, S.M., Koo, H., Ikegaki, M., Silva, G.V., Napimoga, M.H., Rosalen,
P.L., 2013. Anti-inflammatory and antimicrobial evaluation of neovestitol and ves-
titol isolated from Brazilian red propolis. J. Agric. Food Chem. 61, 4546–4550.
Búfalo, M.C., Ferreira, I., Costa, G., Francisco, V., Liberal, J., Cruz, M.T., Lopes, M.C.,
Batista, M.T., Sforcin, J.M., 2013. Propolis and its constituent caffeic acid sup-
press LPS-stimulated pro-inflammatory response by blocking NF-κB and MAPK
activation in macrophages. J. Ethnopharmacol. 149, 84–92.
Campo Fernández, M., Cuesta-Rubio, O., Rosado Perez, A., Montes De Oca Porto, R.,
Márquez Hernández, I., Piccinelli, A.L., Rastrelli, L., 2008. GC–MS determination
of isoflavonoids in seven red Cuban propolis samples. J. Agric. Food Chem. 56,
9927–9932.
Chan, S.C., Chang, Y.S., Wang, J.P., Chen, S.C., Kuo, S.C., 1998. Three new flavonoids
and antiallergic, anti-inflammatory constituents from the heartwood of Dal-
bergia odorifera. Planta Med. 64, 153–518.
Collier, H.O., Kinneen, L.C., Johnson, C.A., Schneider, C., 1968. The abdominal con-
striction response and its suppression by analgesic drugs in the mouse. Brit. J.
Pharmacol. 32, 295–310.
da Silva, R.O., Andrade, V.M, Bullé Rêgo, E.S., Azevedo Dória, G.A., Santos Lima, B.D.,
de Souza Araújo, A.A., de Albuquerque Júnior, R.L., Cordeiro Cardoso, J., Zanardo
Gomes, M., 2015. Acute and sub-acute oral toxicity of Brazilian red propolis in
rats. J. Ethnopharmacol. 170, 66–71. http://dx.doi.org/10.1016/j.jep.2015.05.009.
Daugsch, A., Moraes, C.S., Fort, P., Park, Y.K., 2008. Brazilian red propolis—chemical
composition and botanical origin. Evid. Based Complement. Alternat. Med. 5,
435–441.
de Almeida, E.B., Cordeiro Cardoso, J., Karla de Lima, A., de Oliveira, N.L., de Pontes-
Filho, N.T., Oliveira Lima, S., Leal Souza, I.C., de Albuquerque-Júnior, R.L., 2013.
The incorporation of Brazilian propolis into collagen-based dressing films im-
proves dermal burn healing. J. Ethnopharmacol. 147, 419–425.
Duarte, I.D., Nakamura, M., Ferreira, S.H., 1988. Participation of the sympathetic system
in acetic acid-induced writhing in mice. Braz. J. Med. Biol. Res 21, 341–343.
Filho, A.W., Filho, V.C., Olinger, L., de Souza, M.M., 2008. Quercetin: further in-
vestigation of its antinociceptive properties and mechanisms of action. Arch.
Pharm. Res. 31, 713–721.
Franchi Jr., G.C., Moraes, C.S., Toreti, V.C., Daugsch, A., Nowill, A.E., Park, Y.K., 2012.
Comparison of effects of the ethanolic extracts of brazilian propolis on human
leukemic cells as assessed with the MTT assay. Evid. Based Complement. Al-
ternat. Med. 2012, 918956.
Franchin, M., da Cunha, M.G., Denny, C., Napimoga, M.H., Cunha, T.M., Koo, H., de
Alencar, S.M., Ikegaki, M., Rosalen, P.L., 2012. Geopropolis from Melipona scu-
tellaris decreases the mechanical inflammatory hypernociception by inhibiting
the production of IL-1β and TNF-α. J. Ethnopharmacol. 143, 709–715.
Frozza, C.O., Garcia, C.S., Gambato, G., de Souza, M.D., Salvador, M., Moura, S., Pa-
dilha, F.F., Seixas, F.K., Collares, T., Borsuk, S., Dellagostin, O.A., Henriques, J.A.,
Roesch-Ely, M., 2013. Chemical characterization, antioxidant and cytotoxic ac-
tivities of Brazilian red propolis. Food Chem. Toxicol. 52, 137–142.
Funakoshi-Tago, M., Okamoto, K., Izumi, R., Tago, K., Yanagisawa, K., Narukawa, Y.,
Kiuchi, F., Kasahara, T., Tamura, H., 2015. Anti-inflammatory activity of flavo-
noids in Nepalese propolis is attributed to inhibition of the IL-33 signaling
pathway. Int. Immunopharmacol. 25, 189–198.
Fundytus, M.E., 2001. Glutamate receptors and nociception: implications for the
drug treatment of pain. CNS Drugs 15, 29–58.
Heine, S., Michalakis, S., Kallenborn-Gerhardt, W., Lu, R., Lim, H.Y., Weiland, J., Del
Turco, D., Deller, T., Tegeder, I., Biel, M., Geisslinger, G., Schmidtko, A., 2011.
CNGA3: a target of spinal nitric oxide/cGMP signaling and modulator of in-
flammatory pain hypersensitivity. J. Neurosci. 31, 11184–11192.
Hunskaar, S., Hole, K., 1987. The formalin test in mice: dissociation between in-
flammatory and non-inflammatory pain. Pain 30, 103–114.
Inui, S., Hatano, A., Yoshino, M., Hosoya, T., Shimamura, Y., Masuda, S., Ahn, M.R.,
Tazawa, S., Araki, Y., Kumazawa, S., 2014. Identification of the phenolic com-
pounds contributing to antibacterial activity in ethanol extracts of Brazilian red
propolis. Nat. Prod. Res. 28, 1293–1296.
Klafke, J.Z., da Silva, M.A., Trevisan, G., Rossato, M.F., da Silva, C.R., Guerra, G.P.,
Villarinho, J.G., Rigo, F.K., Dalmolin, G.D., Gomez, M.V., Rubin, M.A., Ferreira, J.,
2012. Involvement of the glutamatergic system in the nociception induced
intrathecally for a TRPA1 agonist in rats. Neuroscience 222, 136–146.
Koster, R., Anderson, N., Debber, E.J., 1959. Acetic acid for analgesic screening. Fed.
Proc. 18, 418–420.
Ledón, N., Casaco, A., Gonzalez, R., Merino, N., Gonzalez, A., Tolon, Z., 1997. Anti-
psoriatic, anti-inflammatory, and analgesic effects of an extract of red propolis.
Acta Pharmacol. Sin. 18, 274–276.
Li, Z., Dong, X., Zhang, J., Zeng, G., Zhao, H., Liu, Y., Qiu, R., Mo, L., Ye, Y., 2014.
Formononetin protects TBI rats against neurological lesions and the underlying
mechanism. J. Neurol. Sci. 15, 112–117.
Liang, K., Ye, Y., Wang, Y., Zhang, J., Li, C., 2014. Formononetin mediates neuroprotection
against cerebral ischemia/reperfusion in rats via downregulation of the Bax/Bcl-2
ratio and upregulation PI3K/Akt signaling pathway. J. Neurol. Sci. 344, 100–104.
López, B.G., Schmidt, E.M., Eberlin, M.N., Sawaya, A.C., 2014. Phytochemical markers
of different types of red propolis. Food Chem. 146, 174–180.
Loux, J.J., Smith, S., Salem, H., 1978. Comparative analgesic testing of various
compounds in mice using writhing techniques. Arzneimittelforschung 28,
1644–1647.
Ma, Z., Ji, W., Fu, Q., Ma, S., 2013. Formononetin inhibited the inflammation of LPS-
133
induced acute lung injury in mice associated with induction of PPAR gamma
expression. Inflammation 36, 1560–1566.
Malmberg, A.B., Yaksh, T.L., 1992. Antinociceptive actions of spinal nonsteroidal
anti-inflammatory agents on the formalin test in the rat. J. Pharmacol. Exp.
Ther. 263, 136–146.
Martinez, V., Thakur, S., Mogil, J.S., Taché, Y., Mayer, E.A., 1999. Differential effects of
chemical and mechanical colonic irritation on behavioral pain response to in-
traperitoneal acetic in mice. Pain 81, 16–18.
McNamara, C.R., Mandel-Brehm, J., Bautista, D.M., Siemens, J., Deranian, K.L., Zhao,
M., Hayward, N.J., Chong, J.A., Julius, D., Moran, M.M., Fanger, C.M., 2007. TRPA1
mediates formalin-induced pain. Proc. Natl. Acad. Sci. USA 104, 13525–13530.
Mendes, S.S., Bomfim, R.R., Jesus, H.C.R., Alves, P.B., Blank, A.F., Estevam, C.S., An-
toniolli, A.R., Thomazzi, S.M., 2010. Evaluation of the analgesic and anti-in-
flammatory effects of the essential oil of Lippia gracilis leaves. J. Ethno-
pharmacol. 129, 391–397.
Morsy, A.S., Abdalla, A.L., Soltan, Y.A., Sallam, S.M., El-Azrak Kel, D., Louvandini, H.,
Alencar, S.M., 2013. Effect of Brazilian red propolis administration on hemato-
logical, biochemical variables and parasitic response of Santa Inês ewes during
and after flushing period. Trop. Anim. Health Prod. 45, 1609–1618.
Nishio, N., Taniguchi, W., Sugimura, Y.K., Yamanaka, M., Kiyoyuki, Y., Yamada, H.,
Miyazaki, N., Yoshida, M., Nakatsuka, T., 2013. Reactive oxygen species enhance
excitatory synaptic transmission in rat spinal dorsal horn neurons by activating
TRPA1 and TRPV1 channels. Neuroscience 5, 201–212.
O’Leary, T.P., Gunn, R.K., Brown, R.E., 2013. What are we measuring when we test
strain differences in anxiety in mice? Behav. Genet. 43 (1), 34–50.
Piccinelli, A.L., Lotti, C., Campone, L., Cuesta-Rubio, O., Campo Fernandez, M., Ras-
trelli, L., 2011. Cuban and Brazilian red propolis: botanical origin and com-
parative analysis by high-performance liquid chromatography-photodiode ar-
ray detection/electrospray ionization tandem mass spectrometry. J. Agric. Food
Chem. 59, 6484–6491.
Premkumar, L.S., Abooj, M., 2013. TRP channels and analgesia. Life Sci. 92, 415–424.
Prut, L., Belzung, C., 2003. The open field as a paradigm to measure the effects of
drugs on anxiety-like behaviors: a review. Eur. J. Pharmacol. 463, 3–33.
Rathee, P., Chaudhary, H., Rathee, S., Rathee, D., Kumar, V., Kohli, K., 2009. Me-
chanism of action of flavonoids as anti-inflammatory agents: a review. In-
flamm. Allergy Drug Targets 8, 229–235.
Reis, J.S., Oliveira, G.B., Monteiro, M.C., Machado, C.S., Torres, Y.R., Prediger, R.D.,
Maia, C.S., 2014. Antidepressant- and anxiolytic-like activities of an oil extract
of propolis in rats. Phytomedicine 21, 1466–1472.
Ribeiro, R.A., Vale, M.L., Thomazzi, S.M., Paschoalato, A.B., Poole, S., Ferreira, S.H.,
Cunha, F.Q., 2000. Involvement of resident macrophages and mast cells in the
writhing nociceptive response induced by zymosan and acetic acid in mice. Eur.
J. Pharmacol. 387, 111–118.
Righi, A.A., Alves, T.R., Negri, G., Marques, L.M., Breyer, H., Salatino, A., 2011. Bra-
zilian red propolis: unreported substances, antioxidant and antimicrobial ac-
tivities. J. Sci. Food Agric. 91, 2363–2370.
Righi, A.A., Negri, G., Salatino, A., 2013. Comparative chemistry of propolis from
eight brazilian localities. Evid. Based Complement. Alternat. Med. 2013, 267878.
Sawaya, A.C., Barbosa da Silva, C.I., Marcucci, M.C., 2011. Analytical methods applied
to diverse types of Brazilian propolis. Chem. Cent. J. 1, 5–27.
Serhan, C.N., Haeggström, J.Z., 2010. Lipid mediators in acute inflammation and
resolution: eicosanoids, PAF, resolvins, and proteins. In: Serhan, C.N., Ward, P.A.,
Gilroy, D.W. (Eds.), Fundamentals of Inflammation. Cambridge University Press,
United Kingdom, pp. 153–174.
Shibata, M., Ohkubo, T., Takahashi, H., Inoki, R., 1989. Modified formalin test:
characteristic biphasic pain response. Pain 38, 347–352.
Szliszka, E., Kucharska, A.Z., Sokół-Łętowska, A., Mertas, A., Czuba, Z.P., Król, W.,
2013. Chemical composition and anti-inflammatory effect of ethanolic extract
of Brazilian Green Propolis on activated J774A.1 macrophages. Evid. Based
Complement. Alternat. Med. 2013, 976415.
Tielemans, M.M., van Rossum, L.G., Eikendal, T., Focks, J.J., Laheij, R.J., Jansen, J.B.,
van Oijen, M.G., 2014. Gastrointestinal symptoms in NSAID users in an ‘average
risk population’: results of a large population-based study in randomly selected
Dutch inhabitants. Int. J. Clin. Pract. 68, 512–519.
Toreti, V.C., Sato, H.H., Pastore, G.M., Park, Y.K., 2013. Recent progress of propolis for
its biological and chemical compositions and its botanical origin. Evid. Based
Complement. Alternat. Med. 2013, 697390.
Wang, Y., Zhu, Y., Gao, L., Yin, H., Xie, Z., Wang, D., Zhu, Z., Han, X., 2012. For-
mononetin attenuates IL-1β-induced apoptosis and NF-κB activation in INS-1
cells. Molecules 17, 10052–10064.
Whimbey, A.E., Denenberg, V.H., 1967. Two independent behavioral dimensions in
open-field performance. J. Comp. Physiol. Psychol. 63, 500–504.
Winter, C.A., Risley, E.A., Nuss, G.W., 1962. Carrageenin-induced edema in hind paw
of the rat as an assay for anti-inflammatory drugs. Proc. Soc. Exp. Biol. Med 111,
544–547.
Zárraga-Galindo, N., Vergara-Aragón, P., Rosales-Meléndez, S., Ibarra-Guerrero, P.,
Domínguez-Marrufo, L.E., Oviedo-García, R.E., Hernández-Ramírez, H., Her-
nández-Téllez, B., López-Martínez, I.E., Sánchez-Cervantes, I., Vázquez-García,
M., Santiago, J., 2011. Effects of bee products on pentylenetetrazole-induced
seizures in the rat. Proc. West. Pharmacol. Soc. 54, 33–40.
Zhu, H., Zou, L., Tian, J., Lin, F., He, J., Hou, J., 2014. Protective effects of sulphonated
formononetin in a rat model of cerebral ischemia and reperfusion injury.
Planta. Med. 80, 262–268.
Zimmermann, M., 1983. Ethical guidelines for investigations of experimental pain
in conscious animals. Pain 16, 109–110.