Journal of Ethnopharmacology 155 (2014) 1118–1124

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

Research Paper

Curine inhibits mast cell-dependent responses in mice

Jaime Ribeiro-Filho a, Fagner Carvalho Leite d, Hermann Ferreira Costa d,
Andrea Surrage Calheiros a, Rafael Carvalho Torres b, Carolina Trindade de Azevedo b,
Marco Aurélio Martins b, Celidarque da Silva Dias c, Patrícia T. Bozza a,n,1,
Márcia Regina Piuvezam d,1
a
Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, 21040-360 Rio de Janeiro, RJ, Brazil
b
Laboratório de Inflamação, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil
c
Laboratório de Fitoquímica, Departamento de Ciências Farmacêuticas, UFPB, João Pessoa, Paraíba, Brazil
d
Laboratório de Imunofarmacologia, Departamento de Fisiologia e Patologia, UFPB, João Pessoa, Paraíba, Brazil

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

Article history: Ethnopharmacological relevance: Curine is a bisbenzylisoquinoline alkaloid and the major constituent
Received 18 March 2014 isolated from Chondrodendron platyphyllum, a plant that is used to treat inflammatory diseases in
Received in revised form Brazilian folk medicine. This study investigates the effectiveness of curine on mast cell-dependent
29 May 2014
responses in mice.
Accepted 15 June 2014
Available online 23 June 2014
Materials and methods: To induce mast cell-dependent responses, Swiss mice were subcutaneously
sensitized with ovalbumin (OVA—12 μg/mouse) and Al(OH)3 in a 0.9% NaCl solution. Fifteen days later,
Keywords: the animals were challenged with OVA through different pathways. Alternatively, the animals were
Curine injected with compound 48/80 or histamine, and several parameters, including anaphylaxis, itching,
Chondrodentron platyphyllum edema and inflammatory mediator production, were analyzed. Promethazine, cromoglycate, and
Anti-allergic
verapamil were used as control drugs, and all of the treatments were performed 1 h before the
Mast cell
challenges.
Lipid mediators
Results: Curine pre-treatment significantly inhibited the scratching behavior and the paw edema
induced by either compound 48/80 or OVA, and this protective effect was comparable in magnitude
with those associated with treatment with either cromoglycate or verapamil. In contrast, curine was
a weak inhibitor of histamine-induced paw edema, which was completely inhibited by promethazine.
Curine and verapamil significantly inhibited pleural protein extravasations and prostaglandin D2 (PGD2)
and cysteinyl leukotrienes (CysLTs) production following allergen-induced pleurisy. Furthermore, like
verapamil, curine inhibited the anaphylactic shock caused by either compound 48/80 or an allergen.
In in vitro settings, these treatments also inhibited degranulation as well as PGD2 and CysLT production
through IgE-dependent activation of the mast cell lineage RBL-2H3.
Conclusion: Curine significantly inhibited immediate allergic reactions through mechanisms more
related to mast cell stabilization and activation inhibition than interference with the pro-inflammatory
effects of mast cell products. These findings are in line with the hypothesis that the alkaloid curine may
be beneficial for the treatment of allergic disorders.
& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Allergic diseases are an important public health problem,
affecting approximately 20% of the world population (Edwards et
Abbreviations: AHR, airway hyper-responsiveness; BBA, bisbenzylisoquinoline
alkaloid; β-hex, beta-hexosaminidase; COX, cyclooxygenase; CysLT, cysteinyl leu-
kotriene; DNP, dinitrophenol; IgE, Immunoglobulin E; IL-(), interleukin-(); OVA,
ovalbumin; PG (), prostaglandin
n
Corresponding author. Tel.: þ 5521 25621767.
E-mail address: pbozza@ioc.fiocruz.br (P.T. Bozza).
1
These senior authors contributed equally.
al., 2009). The most common allergic diseases include asthma,
rhinoconjunctivitis, sinusitis, food allergy, atopic dermatitis,
angioedema, urticaria, anaphylaxis and allergy to drugs and
insects (Holgate and Polosa, 2008). The allergic reactions are
triggered by stimuli that lead to mast cell activation, causing
degranulation; the release of pre-formed mediators, such as
histamine and serotonin; and the synthesis of cytokines and lipid
mediators, including prostaglandin D2 (PGD2) and cysteinil leuko-
trienes (CysLTs) through specific signaling pathways (Cockcroft et
al., 2007; Gould and Sutton, 2008). Under allergic conditions, mast
cell activation is triggered by the IgE–FcεRI complex (Galli et al.,
1999; Gould and Sutton, 2008). It has been demonstrated that

http://dx.doi.org/10.1016/j.jep.2014.06.041
0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
 J. Ribeiro-Filho et al. / Journal of Ethnopharmacology 155 (2014) 1118–1124
mast cells can be activated by synthetic compounds, such as
calcium channel agonists and compound 48/80, which is an
important experimental tool in allergy research (Tatemoto et al.,
2006). Because histamine is a major mediator in immediate
allergic responses, histamine receptor antagonists and mast cell
stabilizers have been used to treat many allergic symptoms,
including edema and itch. However, there are numerous condi-
tions for which they are not effective (Cook et al., 2002; Kim,
2012), justifying the search for substances that can be used as
molecular templates in drug discovery for allergy.
Curine is a bisbenzylisoquinoline alkaloid (BBA) and the major
constituent of the root bark of Chondrodendron platyphyllum
(Menispermaceae). This plant is popularly known as “abútua”
and has been used in Brazilian folk medicine to treat inflammatory
conditions (Correa, 1984; Gotfredsen, 2013). Earlier reports
demonstrated that curine has vasodilator effects associated with
the inhibition of calcium influx, possibly through a direct blockade
of L-type Ca2 þ channels (Dias et al., 2002; Medeiros et al., 2011).
We have demonstrated the anti-allergic properties of curine
administered orally using a mouse model of allergic asthma. In
these experiments, curine significantly inhibited eosinophilic
inflammation, eosinophil lipid body formation, cytokine produc-
tion and airway hyper-responsiveness (AHR) in vivo. Verapamil,
a calcium channel antagonist, had similar anti-allergic properties,
and curine pre-treatment inhibited the calcium-induced tracheal
contractile response ex-vivo, suggesting that the mechanism by
which curine exerts its effects is through the inhibition of a
calcium-dependent response. Importantly, oral treatment with
curine for 7 consecutive days in doses up to 10-fold higher than
its median effective dose (ED50) did not induce evident toxicity
(Ribeiro-Filho et al., 2013).
Additionally, we have demonstrated that curine has anti-
inflammatory and analgesic effects due to its inhibition of edema
and vascular permeability and its inhibition of the nociceptive
responses associated with an anti-inflammatory but without
neurogenic mechanisms. Importantly, curine inhibited PGE2 pro-
duction in vitro, without affecting COX-2 expression. Moreover, the
effects of curine treatment were similar to those of indomethacin
(a non-steroidal anti-inflammatory drug) but were different from
morphine (a central acting analgesic drug), suggesting that the
analgesic effects of curine do not result from a direct inhibitory
effect on neuronal activation but that they depend on anti-
inflammatory mechanisms that, at least in part, result from the
inhibition of PGE2 production (Leite et al., 2014).
Despite the consistent anti-inflammatory and anti-allergic
properties of curine, the effects of this compound on mast cell-
dependent responses remain to be elucidated. Therefore, this work
aims to characterize the effects of curine on mast cell-dependent
responses in mice.
2. Methods
2.1. Curine purification
Chondrodendron platyphyllum Hill St. (Miers) was collected in
Santa Rita, Paraíba, Brazil. The specimen voucher was deposited in
the Herbarium of Prof. Lauro Pires Xavier (UFPB - João Pessoa,
Brazil), number 3631-P, and identified by Prof. Dr. Maria de Fatima
Agra. The Chondrodendron platyphyllum bark was dried and
pulverized in a HARLEY type grinder and then extracted under
exhaustive percolation with 95% ethanol for 3–4 days. The extract
was concentrated under vacuum at temperatures ranging from
501C to 601C to obtain the crude ethanol extract. This extract was
then dissolved in 3% HCl, filtered through Celite (545 Fischer
Scientific) and submitted to CHCl3 extraction alternated with
1119
NH4OH (pH 8) basification. After washing with water and MgSO4,
the solvent was evaporated, and this CHCl3 extract became the
total tertiary alkaloid fraction (TTA). The TTA was separated using
column chromatography (60  600 mm2) on aluminum oxide
using a step gradient of hexane, hexane-CH2Cl2 and CH2Cl2:MeOH.
Fractions of 50 mL were collected and monitored using TLC with a
step gradient of CHCl3–MeOH 100:0 (3 L) and 97:3 (2 L). Fractions
of 50 mL were collected for each system (hexane (100%), hexane:
CH2Cl2 (1:1), CH2Cl2 (100%), CH2Cl2:MeOH (99:1), CH2Cl2:MeOH
(8.5:1.5) and CH2Cl2:MeOH (2.5:7.5)), yielding 60 fractions. Frac-
tions 52–60 monitored using TLC (system 6) contained curine
(0.031% of dry plant; purity 4 98%) that achieved fusion at 215 1C
[α]D–2251C (MeOH, c¼ 0.04). The structure was established using
spectroscopic NMR 13C and NMR 1H (CDCl3, 400 MHz) data
analysis (Leite et al., 2014), which demonstrated that the product
was curine when compared to the literature data (Mambu et al.,
2000). The curine solution was prepared using 1 mg of crystalline
powder, 50 μL of 1 N HCl and 500 μL of distilled water. The pH was
adjusted to 7–8 with 1 N NaOH. The volume was brought up to
1 mL with phosphate buffered saline (PBS).
2.2. Animals
Male Swiss mice weighing 25–30 g were obtained from the
breeding units at the Oswaldo Cruz Foundation and Federal
University of Paraiba. The animals were maintained with food
and water ad libitum in a room with a temperature ranging from
22 to 24 1C and a 12 h light/dark cycle. This study was carried out
in accordance with the recommendations of the Brazilian National
Council for the Control of Animal Experimentation (CONCEA). The
protocols were approved by the Animal Welfare Committee of the
Oswaldo Cruz Foundation (CEUA/FIOCRUZ protocol # L-002/08)
and by the Ethical Committee for Experimental Animal (CEPA No.
0504/08, UFPB). Groups of 6–10 animals were used in each
experiment.
2.3. Treatments
For the in vivo experiments, the animals were orally (p.o.) or
subcutaneously (s.c.) pre-treated with curine (2.5 mg/kg). Alter-
natively, the groups of mice were treated with either an intraper-
itoneal (i.p.) injection of sodium cromoglycate (10 mg/kg), an
intramuscular (i.m.) injection of promethazine (10 mg/kg) or an
oral (p.o.) administration of verapamil (2.5 mg/kg), all of which
were used as reference drugs. PBS (p.o.) was given as a negative
control. All treatments were performed 1 h before the allergic
challenge. For the in vitro experiments, RBL-2H3 cells were
exposed to curine (1 or 10 μM) or vehicle 1 h before the stimulus.
The dose of curine (2.5 mg/kg) was chosen based on the data
obtained from a prior study (Ribeiro-Filho et al., 2013). Of note, the
curine concentrations used in the in vivo experiments did not
affect cell viability (viability 4 95%).
2.4. Induction and evaluation of the scratching behavior (itching)
This protocol was used to evaluate the potential of curine in
modulating allergic itching. For this purpose, Swiss mice were
treated subcutaneously (s.c.) with curine. One hour later, the
animals received an intradermal injection of compound 48/80
(10 μg) dissolved in PBS (20 μL) in the rostral part of the back.
Immediately after the injection, the animals were placed under
observation in individual boxes and their behavior was monitored
for 60 min. The results are expressed as the number of times that
the animals scratched the injection site with the hind paw over
60 min (number of scratchings) (Inagaki et al., 2002).
1120 J. Ribeiro-Filho et al. / Journal of Ethnopharmacology 155 (2014) 1118–1124
2.5. The anaphylactic shock reaction
The anaphylactic shock reaction was induced as previously
described by Kim et al. (2004). Briefly, Swiss mice were orally pre-
treated with curine, and 1 h later, they were intraperitoneally (i.p.)
challenged with compound 48/80 (8 μg/g body weight). Alterna-
tively, Swiss mice were subcutaneously (s.c.) sensitized with
ovalbumin (OVA—12 μg/mouse) and Al(OH)3 in a 0.9% NaCl solu-
tion (saline 0.2 mL). Fifteen days later, the animals were intrave-
nously (i.v.) challenged with OVA (200 μg/mouse) dissolved in
saline (0.1 mL). In another set of experiments, the mice received
a sub-lethal dose of OVA (60 μg/mouse, s.c.). Immediately after the
challenges, the animals were placed in individual boxes and their
mortality was monitored for 60 min. The results are expressed as
percent survival at 10, 30 and 60 min.
2.6. Paw edema induced by compound 48/80, histamine or OVA
One hour after the treatments, paw edema was induced as
previously described by Henriques et al. (1987). Briefly, Swiss mice
were injected in the left paw with a solution containing compound
48/80 (100 ng/paw) dissolved in PBS (20 μL). The right paw
received the same volume of PBS and was used as a control. The
diameter of each paw was measured using a caliper 30, 60 and
120 min after stimulation with 48/80. In another series of experi-
ments, Swiss mice were injected with histamine (100 μg/paw)
dissolved in PBS (20 μL) in the left paw. Again, the right paw
received the same volume of PBS and was used as a control. The
diameter of each paw was measured with a plethysmometer 30
and 60 min after stimulation with histamine. Alternatively, OVA-
sensitized mice were injected with OVA (12 μg/paw) dissolved in
PBS (20 μL) in the left paw 15 days after allergic sensitization. In all
of the experiments, the difference in the diameter of the left and
the right paw was expressed as edema.
2.7. Allergic pleurisy
Swiss mice were sensitized as described in Section 2.5. Fifteen
days later, the orally pre-treated animals received an intrapleural
injection of OVA (12 μg/mouse) in 0.1 mL saline. Four hours after
the provocation, the pleural cavities were washed with 0.5 mL of
saline using a sterile syringe. The pleural washes were centrifuged
at 800g for 5 min, and the supernatants were collected. The total
protein concentration of the samples was determined using a kit
for determination of total proteins (BCA, from Pierce, Rockford, IL)
and was used as a parameter of pleural edema. The concentrations
of PGD2 and CysLT in the supernatants were analyzed using
DuoSet kits, according to the manufacturer's instructions (R & D
Systems).
2.8. Cell culture
The RBL-2H3 cell line is a basophilic leukemia cell line isolated
from Wistar rat basophilic cells maintained as tumors. These cells
have high-affinity IgE receptors that can be activated to secrete
beta-hexosaminidase (β-hex) and lipid mediators (Okuyama et al.,
2005). The RBL-2H3 cells were maintained in complete (15% fetal
bovine serum, penicillin 100 IU/ml, streptomycin 0.1 mg/ml) Dul-
becco's modified Eagle's medium (DMEM) at 37 1C in a 5% CO2
atmosphere.
2.8.1. RBL-2H3 cells degranulation assay
The degranulation of the RBL-2H3 cells was evaluated by
measuring the release of β-hex (Han et al., 2009). Briefly, after
confluence, cultured RBL-2H3 cells were dissociated with 0.25%
(w/v) Trypsin—0.53 mM EDTA solution and plated in a 48-well
plate (1.25  105 cells/well). The cells were sensitized with dini-
trophenol (DNP)-specific IgE (1 μg/mL) for 20 h. After this period,
the growth medium was replaced with Tyrode assay buffer
(119 mM NaCl, 4.74 mM KCl, 2.5 mM CaCl2, 1.19 mM MgSO4,
10 mM 4-2-hydroxyethyl-1-piperazineethane sulfonic acid (HEPES),
5 mM glucose, and 0.1% (w/v) BSA, pH 7.3), and the cells were treated
with curine or verapamil (1 or 10 μM) for 1 h. RBL-2H3 cell
treatment was followed by stimulation with DNP–BSA (10 ng/mL)
for 45 min, and the β-hex content was expressed using the percen-
tage amount of the enzyme released into the supernatant. The β-hex
reaction (37 1C, 50 min) with1 mM p-nitrophenyl N-acetyl-β-D-glu-
cosamide in 0.1 M sodium citrate buffer (pH 4.5) was determined
using a spectrophotometer at 405 nm after stopping the reaction
with 0.2 M glycine.
2.8.2. Determination of lipid mediator production
RBL-2H3 cells were plated in 24-well plates (1.25  105 cells/
well) and sensitized with dinitrophenol (DNP)-specific IgE (1 μg/mL)
for 20 h in complete DMEM. After sensitization, the cells were treated
with curine or verapamil (1 or 10 μM) for 1 h in DMEM without
serum. Lipid mediator production was determined in supernatants
collected 1 h after stimulation with DNP–BSA (10 ng/mL). The con-
centrations of PGD2 and CysLT in the supernatants were quantified
using DuoSet kits according to the manufacturer's instructions (R & D
Systems).
2.9. Statistical analysis
The data were analyzed using a one-way ANOVA followed by
Tukey's test, and the survival data were analyzed using the Log-
rank (Mantel-Cox) Test with GraphPad Prism software (GraphPad,
San Diego, CA). The values are expressed as the means 7S.E.M. The
differences with p o0.05 were considered significant.
3. Results
3.1. Curine inhibits the scratching behavior induced by
compound 48/80
The intradermal injection of compound 48/80 significantly
induced scratching behavior in non-treated mice. As shown in
Fig. 1, pre-treatment with curine significantly reduced the number
of scratches in the challenged mice compared with animals that
did not receive treatment, indicating that curine has an inhibitory
effect on mast cell-dependent itch.
3.2. The effects of curine on edema formation
Swiss mice challenged with compound 48/80 developed sig-
nificant paw edema at 30, 60 and 120 min (Fig. 2). The group of
animals pre-treated with curine had significantly reduced edema
formation compared with the non-treated group at all of the times
analyzed (Fig. 2A). The groups of animals treated with cromogly-
cate, a mast cell stabilizer drug, and promethazine, an antagonist
of the histamine receptor 1 (H1), showed a significant inhibition of
the paw edema induced by compound 48/80, and there were no
differences between curine and cromoglycate treatments, demon-
strating that mast cells participate predominantly in the mechan-
ism of paw edema formation by releasing histamine and
suggesting that curine plays a modulator effect in this process.
As shown in Fig. 2B, the injection of histamine into the paw
induced significant edema formation in untreated mice. The edema
formation was partially inhibited by the curine pretreatment,
 J. Ribeiro-Filho et al. / Journal of Ethnopharmacology 155 (2014) 1118–1124 1121

whereas the promethazine pretreatment completely inhibited this
response suggesting that curine might inhibit the allergic reaction by
modulating mast cell degranulation instead of blocking histamine
action at the allergic site. Because we have demonstrated that the
effects of curine are associated with calcium influx inhibition and
that curine and verapamil have similar anti-allergic effects, we
analyzed the effect of these drugs on allergic edema. As shown in
Fig. 2B, curine and verapamil significantly inhibited the edema and
there were no differences between the two treatments, suggesting
that the effects of curine on mast cell degranulation may result from
the inhibition of a calcium-dependent response.
3.3. Curine and verapamil inhibit mediator production in a mouse
model of allergic pleurisy
An allergic challenge with OVA in actively sensitized mice
induced a significant increase in the total protein and the CysLT
and PGD2 concentrations in the supernatants of the pleural
washes at 4 h (Fig. 3 A–C), and these increases were significantly
decreased by oral pretreatment with curine. Interestingly, verapa-
mil administered with the same conditions (dose, time and path-
way) had similar effects. Because, at the time point that the
analyses were performed, mast cell is the main source of PGD2
and CysLT, the data suggest that curine and verapamil inhibit the
production of these mediators by modulating mast cell activation.
3.4. Curine and verapamil inhibit mast cell activation in vitro
Antigen-stimulated RBL-2H3 cells have been used as the
principal model of mast cell activation in immediate allergic
reactions. The level of activation of this cell lineage is directly
associated with the level of cell degranulation, which can be
determined by measuring hexosaminidase release, a well-
established granule marker (Hwang et al., 2013), as well as by
analyzing the production of degranulation-independent media-
tors, such as prostaglandin and leukotrienes. As shown in Fig. 4,
RBL-2H3 cells sensitized with DNP–BSA and challenged with IgE
anti-DNP produced a significant release of β-hex (A) and increased
concentrations of CysLT (B) and PGD2 (C). Curine and verapamil
significantly, but only partially, inhibited degranulation. However,
the treatments produced significantly more expressive inhibition
of PGD2 and CysLT production indicating that curine inhibits mast
cell activation by modulating both degranulation and mediator
production, through mechanisms that depend, at least in part, on
calcium influx inhibition.

3.5. The effect of curine on the anaphylactic shock reaction

Fig. 1. The effect of curine on scratching behavior. Swiss mice (n¼7–10) were
subcutaneously treated with curine (2.5 mg/kg) 1 h before an intradermal chal-
lenge with compound 48/80. The animals were placed under observation in
individual boxes, and their behavior was monitored for 60 min. (A) The chemical
structure of curine; (B) the number of scratchings. The data are expressed as the
number of times that the animals scratched the injection site with its hind paw
over 60 min (number of scratchings). These results are expressed as the mean 7 -
SEM. þ Significantly different (p o 0.05) from the unchallenged group; n signifi-
cantly different from the untreated, 48/80-challenged group.
The intraperitoneal administration of compound 48/80 in non-
treated mice rapidly induced the development of an anaphylactic
shock reaction followed by 40% and 100% mortality at 10 and
30 min, respectively (Fig. 5A). Interestingly, mice orally pretreated
with curine presented no mortality at 10 min, and the total
mortality at 60 min was 20% lower than that in mice challenged
with 48/80 but that did not receive treatment. Similarly, the
systemic administration of OVA (200 μg/mouse) in sensitized mice

Fig. 2. The effects of curine on allergic edema formation. Swiss mice (n¼7–10) were orally treated with curine (2.5 mg/kg), and 1 h later they were injected in the left hind
paw with compound 48/80 (100 ng/paw) or histamine (100 mg/paw) dissolved in PBS (20 μL). The allergen-sensitized mice were injected with OVA (12 μg/paw) 1 h after the
treatments. The right paw received the same volume of PBS and was used as a control. Cromoglycate (10 mg/kg), promethazine (10 mg/kg) and verapamil (2.5 mg/kg) were
used as control drugs. The diameter of each paw was measured using a plethysmometer 30 and 60 or 120 min after the challenge. The difference between the diameter of the
left and right paws was expressed as edema. (A) Compound 48/80-induced paw edema; (B) histamine-induced paw edema; (C) OVA-induced paw edema. These results are
expressed as the mean7 SEM. þ Significantly different (po 0.05) from the unchallenged group; n significantly different from the untreated, challenged groups.
1122 J. Ribeiro-Filho et al. / Journal of Ethnopharmacology 155 (2014) 1118–1124

Fig. 3. The effects of curine and verapamil on acute allergic pleurisy. OVA-sensitized Swiss mice (n¼ 6–8) were orally treated with curine (2.5 mg/kg) or verapamil (2.5 mg/kg)
1 h before an allergic challenge. Four hours after the challenge, the pleural lavage was collected and the concentration of protein and lipid mediators in the supernatants was
analyzed using BCA kits and ELISA, respectively. (A) The concentration of total proteins; (B) the concentration of CysLT; (C) the concentration of PGD2. These results are expressed
as the mean7SEM of at least 6 animals. þ Significantly different (po0.05) from the unchallenged group; n significantly different from the untreated, OVA-challenged group.

Fig. 4. The effects of curine and verapamil on mast cell activation. RBL-2H3 cells (1.25  105 cells/well) were sensitized with IgE anti-DNP (1 μg/mL), incubated for 20 h,
treated with curine (1–10 μM) or verapamil (1–10 μM) and stimulated with DNP–BSA (10 ng/ml) 45 min later. The β-hexosaminidase levels were quantified 45 min after
stimulus using a spectrophotometer at 405 nm. The mediator concentrations in the supernatants were analyzed 1 h after stimulus. (A) β-hexosaminidase release; (B) the
concentration of CysLT; (C) the concentrations of PGD2. These results are expressed as the mean 7 SEM. þ Significantly different (po 0.05) from the unchallenged group;
n
significantly different from the untreated, DNP–BSA challenged group.

induced the development of an anaphylactic shock reaction
followed by 60% and 100% mortality at 10 and 30 min, respectively
(Fig. 5B). However, curine or verapamil treated mice presented no
mortality at 10 min, and the total mortality at 60 min was 29%
lower than in untreated mice. Additionally, when OVA was given
subcutaneously at a lower dose (60 μg/mouse), the untreated
group had 62.5% mortality, and the curine and verapamil treated
groups had mortality rates of 37.5% and 28.5%, respectively, at both
30 and 60 min (Fig. 5C), suggesting that these substances may
modulate the anaphylactic shock reaction in mice.
4. Discussion
Allergic reactions are associated with the activation of mast
cells and the release of inflammatory mediators. In most cases, this
process relies on signaling through the FcεRI–IgE complex (Liew et
al., 2010). However, it has been shown that other substances,
including compound 48/80, can directly activate mast cells, thus
inducing degranulation. Compound 48/80 is known to trigger mast
cell activation by binding to G proteins in the cytoplasm and
initiating biochemical cascades that result in increased intracel-
lular calcium and the release of inflammatory mediators (Tatemoto
et al., 2006). The pathophysiological features of allergic diseases
are directly influenced by the environment of the organ in which
they occur. Thus, itching is a key feature of most skin allergies
(Buddenkotte and Steinhoff, 2010) and, in this context, results
primarily from the stimulation of specific receptors in neurons by
a wide range of mediators (Greaves, 2010; Garibyan et al., 2013).
The severe itch is a major problem because it induces scratching
behavior, thus resulting in the formation of skin lesions (Wahlgren,
1991). Additionally, systemic allergic reactions may result in a
severe clinical condition known as anaphylactic shock (Kemp and
Lockey, 2002), which is characterized by shortness of breath,
hypotension, cardiac arrhythmia, vomiting, urticaria, headache
and unconsciousness and can result in death (Wade et al., 1989).
In the present study, we demonstrated the effectiveness of
curine in inhibiting immediate allergic responses in mice. Oral
curine pre-treatment significantly inhibited the scratching beha-
vior induced by compound 48/80. As previously described, it has
long been established that histamine is an important mediator of
itch (Lewis, 1927) acting mainly through receptor H1. However,
some studies showed that the H4 receptor is also involved in
histamine-mediated itch (Bell et al., 2004). Although numerous
studies suggest that in allergic reactions, itch is mostly dependent
on histamine release by mast cells, these cells can also release
other mediators, such as adenosine triphosphate (ATP), tryptase,
CysLTs, leukotriene B4 (LTB4), IL-31 and prostaglandins, which
directly activate nerve fibers. In turn, the activated nerve fibers
may release neuropeptides, such as substance P, which has
recently been implicated in allergic responses, including modulat-
ing scratching behavior (Hossen et al., 2006; Nakanishi and
 J. Ribeiro-Filho et al. / Journal of Ethnopharmacology 155 (2014) 1118–1124
Fig. 5. The effect of curine on the anaphylactic shock. Swiss mice (n¼ 7–10) were
orally treated with curine (2.5 mg/kg) or verapamil (2.5 mg/kg) 1 h before the
challenge. Their mortality was monitored for 60 min. (A) Compound 48/80-induced
anaphylactic shock; (B) anaphylactic shock induced by systemically administered-
OVA (200 μg/mouse); (C) anaphylactic shock induced by subcutaneously adminis-
tered-OVA (60 μg/mouse). The data were analyzed using the Log-rank (Mantel-Cox)
Test and expressed as percent survival after the challenge. þ Significantly different
(po 0.05) from the unchallenged group; n significantly different from the untreated
and challenged group.
Furuno, 2008). Therefore, the modulation of scratching behavior
by curine suggests that this alkaloid inhibits mast cell-dependent
responses in mice.
Edema is another important feature of allergic inflammation
(Ash and Schild, 1997). Thus, to investigate the effects of curine on
edema formation as well as to address the possible mechanisms
involved in curine's effects in this model, we compared the effects
of curine treatment with cromoglycate, a mast cell stabilizer, and
promethazine, an antagonist of receptor H1. Using models of
mouse paw edema induced by compound 48/80 or histamine
administration, we demonstrated that curine had an anti-allergic
effect similar to cromoglycate but different from promethazine,
suggesting that curine's effects may result from the inhibition of
mast cell degranulation, instead of the inhibition of histamine at
allergic sites. Using a mouse model of allergic asthma, we
previously demonstrated that curine and verapamil, a calcium
channel antagonist, had similar anti-allergic effects that are
associated with calcium influx inhibition (Ribeiro-Filho et al.,
2013). Here, we demonstrated that curine and verapamil signifi-
cantly inhibit allergen-triggered edema and that there were no
1123
differences between these treatments, corroborating previously
described data.
To better elucidate the effects of curine on immediate allergic
responses in mice, we used a model of acute allergic pleurisy,
induced by sensitization and challenge with OVA. We demon-
strated that curine and verapamil decreased the concentrations of
the proteins PGD2 and CysLT in the pleural washes of OVA-
stimulated mice. Because previous studies have shown that mast
cells are a major source of CysLTs and PGD2 in the acute phase of
allergy (Boyce, 2003), these data suggest that curine and verapamil
inhibited the production of these mediators, at least in part,
through a direct effect on mast cell activation.
To confirm our hypothesis, we investigated the effects of curine
pre-treatment in RBL-2H3 cells stimulated in vitro with IgE anti-
DNP. This is a well-established model for evaluating the effect of
drugs on mast cell activation (Paumet et al., 2000; Blank et al.,
2002; Hemmerling et al., 2010). We demonstrated that curine and
verapamil partially inhibited mast cell degranulation. However,
the treatments produced significantly more expressive inhibition
of PGD2 and CysLT production, indicating that curine inhibits mast
cell activation by modulating both degranulation and mediator
production.
Systemic anaphylaxis is a severe allergic reaction that results
from a sudden release of mediators primarily from mast cells
(Kemp and Lockey, 2002). Because anaphylaxis is one of the most
potent immune reactions, it is a difficult condition to control and,
as such, frequently causes death. We demonstrated that a single
oral pre-treatment with curine delayed death by anaphylactic
shock in mice stimulated with either OVA or compound 48/80.
Additionally, using a lower dose of OVA (of which the untreated
group presented mortality o 100%), we found that curine reduced
the total mortality, and similar results were obtained with ver-
apamil administered under the same conditions. These data
suggest that both curine and verapamil modulate the anaphylactic
shock reaction in mice. Importantly, this is the first work to
demonstrate the anti-anaphylactic effect of verapamil in
this model.
Previous studies have demonstrated the anti-allergic effects of
natural products in mast cell-dependent responses both in vivo
and in vitro. Hossen et al. (2006) demonstrated that treatment
with caffeic acid, a phenolic compound widely distributed in
plants such as coffee, inhibited scratching behavior, decreased
vascular permeability and reduced histamine release after chal-
lenge with compound 48/80. Similar results were obtained with
propolis extract, which is rich in phenolic compounds, including
caffeic acid and flavonoids (Shinmei et al., 2004). The ethanol
extracts of Gleditsia sinensis (Leguminosae) and Plumbago zeylanica
(Plumbaginaceae) inhibited the anaphylactic shock reaction and
affected mast cell activation (Dai et al., 2002), and the compound
paeonol inhibited the anaphylactic shock reaction through
mechanisms dependent on the inhibition of histamine release
and tumor necrosis factor (TNF)-α production (Kim et al., 2004).
Interestingly, Bezerra-Santos et al. (2005) demonstrated that an
ethanol extract of Cyssampelos sympodialis (Menispermaceae)
significantly inhibited the OVA-induced but not the 48/80-induced
anaphylactic shock reaction in mice. Finally, Costa et al. (2008)
demonstrated that warifteine, a BBA isolated from Cyssampelos
sympodialis, significantly inhibited the scratching behavior
induced by compound 48/80 in addition to reducing allergen-
triggered mast cell activation in vitro corroborating the role of BBA
in modulating immediate allergic responses.
As previously described, using a mouse model of allergic
asthma, we demonstrated that oral pretreatment with curine
significantly inhibited eosinophilic inflammation and airway
hyper-responsiveness through mechanisms that involve the inhi-
bition of IL-13 and eotaxin production and the inhibition of
1124 J. Ribeiro-Filho et al. / Journal of Ethnopharmacology 155 (2014) 1118–1124
calcium influx (Ribeiro-Filho et al., 2013). Additionally, using the
same model, we demonstrated that curine and verapamil signifi-
cantly inhibited the production of CysLT in the BAL (not shown),
corroborating the data presented in this work. Because mast cells
produce mediators that play key roles in asthma pathogenesis,
including CysLT, eotaxin and IL-13 (Shakoory et al., 2004; Hong et
al., 2013), the new data provided in this work suggest that the
inhibitory effect that curine plays on mast cell activation may
directly contribute to the anti-asthmatic effects of this alkaloid.
In conclusion, our results demonstrate that curine inhibits mast
cell-dependent responses by modulating mast cell activation
through mechanisms that may involve the inhibition of a calcium
dependent response. This finding supports previous data suggest-
ing that curine has the potential to be developed as a novel anti-
allergic drug.
Acknowledgments
This work was supported by PRONEX/MCT, CNPq, FAPERJ and
INCT-Cancer. The authors thank Edson Fernandes de Assis and
Josenilson Feitosa de Lima for their technical assistance.
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