Chemico-Biological Interactions 348 (2021) 109656

Contents lists available at ScienceDirect

Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

Investigation of the relaxing effect of a camphor nanoemulsion on rat

isolated trachea
Maíra M. Freitas a, Pedro M. Cavalcante a, Luiz A.M.S. Duarte-Filho a, Cicero A.F. Macedo b,
Mariana C. Brito b, Pedro M.N. Menezes c, Thiago F. Ribeiro c, Sâmara M. Costa d, Bárbara A.
G. Carvalho d, Fernanda P.R.A. Ribeiro e, Marigilson P.S. Moura f, Fabricio S. Silva f, Luciano A.
A. Ribeiro f, *
a
Programa de Pós-Graduação em Biociências (PPGB), Universidade Federal do Vale do São Francisco (UNIVASF), Campus Centro, Av. José de Sá Maniçoba, S/N, Cx.
Postal 252, CEP: 56.304-205, Petrolina-PE, Brazil
b
Programa de Pós-graduação em Biotecnologia, Universidade Estadual de Feira de Santana (UEFS), Av. Transnordestina, S/N, Novo Horizonte, CEP: 44036-900, Feira
de Santana-Ba, Brazil
c
Rede Nordeste de Biotecnologia (RENORBIO), Universidade Federal Rural de Pernambuco (UFRPE), Rua Dom Manuel de Medeiros, S/N, Dois Irmãos, CEP: 52171-
900, Recife-PE, Brazil
d
Curso de Graduação em Farmácia, Universidade Federal do Vale do São Francisco (UNIVASF), Campus Centro, Av. José de Sá Maniçoba, S/N, Cx. Postal 252, CEP:
56.304-205, Petrolina-PE, Brazil
e
Colegiado de Ciências Farmacêuticas (CFARM), Laboratório de Farmacologia Experimental (LAFEX), Universidade Federal do Vale do São Francisco (UNIVASF),
Campus Centro, Av. José de Sá Maniçoba, S/N, Cx. Postal 252, CEP: 56.304-205, Petrolina-PE, Brazil
f
Programa de Pós-Graduação em Biociências (PPGB), Colegiado de Ciências Farmacêuticas (CFARM), Universidade Federal do Vale do São Francisco (UNIVASF),
Campus Centro, Av. José de Sá Maniçoba, S/N, Cx. Postal 252, CEP: 56.304-205, Petrolina-PE, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Asthma is a chronic inflammatory disease that targeting lower airways, being characterized by bronchial smooth
Camphor muscle hyper responsiveness and mucus hypersecretion. Asthma is considered the most common respiratory
Nanoemulsion disease in the world, affecting approximately 235 million individuals. The main therapy sometimes fails to
Spasmolytic effect
establish clinical improvement in patients, which leads to a constant search for new alternatives. Camphor is a
transparent solid monoterpene with a strong aroma, which due to its high lipophilicity is insoluble in water.
Nanostructured carrier systems have shown promise as a delivery system for lipophilic compounds such as
monoterpenes. Therefore, the objective of this work was to evaluate the relaxant effect of nanoemulsified
camphor (NEC), as well as the mechanism of action of that monoterpene, in isolated rat trachea. The results
obtained demonstrated that NEC promote relaxation of the isolated rat trachea when smooth muscle contraction
was induced by both carbachol (CCh) and KCl, presenting a pCE50 of 2.25 ± 0.27 and 3.30 ± 0.07, respectively.
In the presence of dexamethasone (DEXA), tetraethylammonium (TEA), glibenclamide (GLIB), 1H-[1,2,4]-oxa­
diazole-[4,3,-a]-quinoxaline-1-one (ODQ) and ruthenium red (RR) there was a significant difference in at least
one of the evaluated pharmacological parameters, such as concentration-response curves shape, Emax or pCE50.
As conclusion, NEC may be involved with β-adrenergic receptors, channels for K+ sensitive to ATP (KATP) or
Channels for K+ opened by Ca2+ (KCa), increase in prostanoids and with receptor channel with transient po­
tential (TRPv). In conclusion, β-adrenergic receptors, prostanoids, nitric oxide (NO), ATP-sensitive K+ channels
(KATP), Ca2+-opened K+ channels (KCa), and transient receptor potential cation channel subfamily V (TRPV) are
involved in the relaxing effect of NEC.
In addition, the mechanism of action of NEC may be involved with the signal transduction pathway Nitric
Oxide/soluble guanylyl cyclase/cGMP/cGMP-activated protein kinase. NEC, therefore, demonstrates spasmolytic
activity when presenting tracheal relaxation compared to CCh and KCl contracturants.

* Corresponding author. Universidade Federal do Vale do São Francisco (UNIVASF), Colegiado de Ciências Farmacêuticas (CFARM), Av. José de Sá Maniçoba, s/n,
Centro, CEP 56304-205, Caixa Postal 252, Petrolina-PE, Brazil.
E-mail address: [email protected] (L.A.A. Ribeiro).

https://doi.org/10.1016/j.cbi.2021.109656
Received 27 July 2020; Received in revised form 15 July 2021; Accepted 9 September 2021
Available online 11 September 2021
0009-2797/© 2021 Elsevier B.V. This article is made available under the Elsevier license (http://www.elsevier.com/open-access/userlicense/1.0/).
M.M. Freitas et al. Chemico-Biological Interactions 348 (2021) 109656

1. Introduction CEUA-UNIVASF, the trachea removed, and cleaned of all connective and
adipose tissue. After that, the organ was divided into segments con­
Asthma is a chronic obstructive inflammatory disease of the lower taining 4 to 5 cartilaginous rings and put in organ-bath chambers,
airways, being the most common respiratory disease in the world, mounted between two L-shaped stainless-steel hooks, one fixed in place
affecting approximately 235 million individuals [1]. It is characterized and the other attached to a force transducer, which was coupled to an
by symptoms that vary according to the severity of the disease, which signal amplifier connected to a A/D converter in a PC (Insight, Brazil).
may include wheezing, dry cough and shortness of breath, and may lead The rings were immersed in 10 ml of Krebs PSS in the organs bath
to bronchospasm with obstruction of the air flow that may require apparatus at 37 ◦ C and bubbled with carbogenic mixture. The tracheal
hospitalization of the individual [2]. Treatment may include rings were allowed to stabilize for 60 min at a preload tension of 1 gF
non-pharmacological and pharmacological measures, the most common (baseline). During this resting period the Krebs PSS was renewed every
being the use of corticosteroids and bronchodilators [3]. However, the 15 min to avoid accumulation of metabolites. The Krebs PSS was
search for new drugs, which can assist in the treatment of asthma and composed (in mM) by NaCl (118), KCl (4.6), MgSO4.7H2O (5.7),
other COPD, is an ongoing objective. NaH2PO4.H2O (1.1), CaCl2 (2.5), NaHCO3 (25) and glucose (11)
An example of that is the monoterpene eucalyptol (1,8-cineol), continuously bubbled with a 95% O2 and 5% CO2 carbogenic gas
present in Soledum®, which is indicated for the symptomatic treatment mixture.
of bronchitis and colds, acting as a decongestant and anti-inflammatory
[4]. 2.4. General pharmacological protocol
Camphor, another monoterpene, which is present in essential oils of
several plant species, is present as an active ingredient in some topical After a 60 min stabilization period, the tracheal rings were con­
drug formulations such as Vick VapoRub®, which is used as a decon­ tracted by CCh (1 μM) or KCl (60 mM) and the isometric tension was
gestant in colds, and Gelol®, which is used in rheumatism, neuralgia, recorded. When a stable contraction was attained (plateau was reached
torticollis, bruising and muscle pain [5]. in approximately 15–20 min) the nanoemulsion loaded with camphor
One of the main obstacles in the use of nonpolar molecules, such as (NEC) was cumulatively add to the organ-chamber (range of 10− 6 to
monoterpenes, would be the low solubility in water, making it difficult 10− 3 M) and its effect was measured. In parallel experiments, the
to dissolve in biological fluids [6]. In this context, nanostructured sys­ nanoemulsion alone (NE), i.e. without camphor, was added to the organ-
tems have shown promise, lipid nanoemulsions, oil in water, have been chamber in volumes equivalents to those in cumulative concentrations
used as drug delivery systems, capable of encapsulating, protecting and of NEC and its effect was measured. The relaxant effect induced by NEC
releasing lipophilic bioactive components [7,8]. or NE was expressed as the reverse percentage of initial contraction force
In view of the possible pharmacological potential of monoterpene elicited by CCh or KCl (100% of relaxation when baseline was reached).
camphor in the treatment of diseases such as asthma, the purpose of this In addition, some experiments were carried out to verify the effect of
work is to use this compound incorporated in nanoemulsions, in an NEC on the organ’s basal tone (1 gF of preload tension). Therefore, after
attempt to improve its bioavailability and consequently enhance its the organ viability test, which resulted in a full contractile response to
effect. CCh, with subsequent agonist washout and return to basal tone, the NEC
was added cumulatively to the organ bath and its effect was measured.
2. Material E methods
2.5. Effect of NEC on contractions induced by Ca2+ in a high K+ medium
2.1. Nanoemulsion
To assess the effect of NEC on contractions induced by the influx of
The (±) -Camphor (1,7,7-Trimethylbicyclo [2.2.1] heptan-2-one), Ca2+ through voltage-opened calcium channels (CaV), the trachea rings
with 96% purity, used in the preparation of the nanoemulsions was were exposed to a high external K+ concentration (60 mM) and a
purchased from Sigma-Aldrich Brasil Ltda. The nanoemulsion contain­ concentration-response curve to CaCl2 (10 mM–120 mM) was built [9],
ing camphor, which were prepared by the direct emulsification method both in the absence and in the presence of NEC concentrations (10− 3, 3
[8], with some modifications, was supplied by the Pharmacotechnics × 10− 3 and 9 × 10− 3 M).
Laboratory of the Universidade Federal do Vale do São Francisco
(UNIVASF), under the supervision of Professor Marigilson P. de S. Moura 2.6. Investigation of the mechanism of action of NEC in a trachea pre-
(Ph.D.). contracted by CCh

2.2. Animals The participation of potassium channels in the relaxing effect of NEC
was investigated through the use of potassium channel blockers. The
Male rats (Rattus norvegicus), with 6–8 weeks of age and weight general protocol was followed as described above, with the effect of NEC
ranging from 250 to 300 g, were used in this study. All animals were measured in the absence (control) and in the presence of: cesium chlo­
supplied by the Central Bioterium of UNIVASF, housed following a 12 h ride (CsCl, 5 mM), a non-selective K+ channel blocker [9]; tetraethy­
light-dark cycle (lights on: 06:00–18:00 h) under controlled temperature lammonium (TEA, 5 mM), a Ca2+-activated K+ channels (KCa) blocker
conditions (21±1 ◦ C) and air circulation, fed with standard chow [10]; glibenclamide (GLIB, 1 μM), an ATP-sensitive potassium channel
(PURINA®, Brazil) and tap water ad libitum. This work was carried out (KATP) blocker [11]; and 4-aminopyridine (4-AP, 2 mM), a voltage-gated
after approval (protocol #0003/080716) by the Ethics Committee on K+ channel (KV) blocker [12].
the Use of Animals (CEUA-UNIVASF). All animal experiments were In another set of experiments, the participation of the nitric oxide
carried out in compliance with the National Council for the Control of (NO) pathway in the relaxing effect of NEC was investigated. For this
Animal Experimentation (CONCEA) and are also in accordance with the purpose, the following inhibitors were used: Nω-nitro-L-arginine methyl
ARRIVE guidelines and carried out in accordance with the UK Animals ester (L-NAME, 10 μM), a nitric oxide synthase (NOS) inhibitor [13];
(Scientific Procedures) Act, 1986 and associated guidelines, EU Direc­ methylene blue (MB, 1 μM), soluble guanylyl cyclase (sGC) inhibitor
tive 2010/63/EU for animal experiments. [14]; 1H-[1,2,4] oxadiazole-[4,3,-a]-quinoxaline-1-one (ODQ, 10 mM),
a sGC inhibitor [15].
2.3. Organ preparation For the investigation of other cell signaling pathways that could have
a role in the relaxing effect of NEC, other inhibitors have been used, such
The rats were euthanized according to the methods approved by as: indomethacin (INDO, 10 μM), a non-selective cyclooxygenase (COX)

2
M.M. Freitas et al. Chemico-Biological Interactions 348 (2021) 109656

inhibitor [16]; dexamethasone (DEXA, 10 μM), a phospholipase A2 in­

hibitor (PLA2) [17]; ruthenium red (RR, 20 μM), a non-selective tran­
sient receptor potential channels (TRPC) blocker [19].
All inhibitors and blockers were added to the organ-bath chamber 20
min before a second CCh-induced contraction and when the sustained
tonic phase of this contraction became stable, NEC was added cumula­
tively (10− 6 to 10− 3 M) and its effect was measured and compared with a
control.

2.7. Data analyses

All values were expressed as mean ± standard error of the mean

(X±SEM) relative to the maximum contraction force (gF) developed by
the organ in response to addition of a single dose of KCl (60 mM). The
concentration-response curves were adjusted using a non-linear
regression enabling calculation of pEC50 (-log[EC50] M) and Emax
(maximum effect) values. The hypothesis of difference between the
means of the groups was compared using the unpaired Student’s t-test or
the two-way analysis of variance (ANOVA two-way) with repeated
measures, when necessary, with the differences considered significant
when the calculated value of “p” was less than 0.05 (p < 0.05).

3. Results

Fig. 2. Effect of nanoemulsified camphor (NEC, ■) and nanoemulsion alone

NEC relaxed the isolated rat trachea in a concentration-dependent
(NE, x̂) on the isolated rat trachea contracted by CCh and NEC upon basal tone
manner, both when the contractions were induced by CCh (pEC50 = (●). The symbols and error bars represent the mean and SEM, respectively. *p
2.22 ± 0.27, with Emax = 94.3 ± 4.6%) and by KCl (pEC50 = 3.12 ± 0.12 < 0.05 (Two-way ANOVA, followed by Sidak’s multiple comparisons test: NEC
with Emax = 111.5 ± 4.9%), with no significant differences between the on contracted trachea vs. NEC basal/NE).
pEC50 and Emax values (Fig. 1). Since there were no significant differ­
ences between NEC relaxation regardless the agent used to contract this cumulative addition of CaCl2 in a depolarizing high extracellular K+
organ, we chose to work from now on only with CCh as agonist. medium, it was observed that NEC was able to inhibit these contractions
In addition, it was possible to observe that the nanoemulsion by itself in a concentration-dependent manner by right-shifting the CaCl2
(NE) had no effect on the trachea contracted by CCh, as well as NEC does concentration-response curves with a reduction in Emax to CaCl2 (Fig. 3).
not have a significant basal effect, considering its cumulative addition to The Emax for CaCl2, in the three NEC concentrations, were 113.9 ± 5.7%,
the organ in its basal tone, only possessed effect when the organ was pre- 65.4 ± 6.3% and 7.5 ± 1.6%, respectively, and these values were
contracted (Fig. 2).
In a protocol designed to investigate whether NEC (10− 3, 3 × 10− 3 or
9 × 10− 3 M) would be able to inhibit the contractions induced by the

Fig. 3. Effect of nanoemulsified camphor (NEC) in the concentrations of 10− 3

M (□), 3 × 10− 3 M (▴) and 9 × 10− 3 M (△). against the contractions induced
Fig. 1. Effect of nanoemulsified camphor (NEC) on the isolated rat trachea by the addition of CaCl2 in a high K+ depolarizing medium (■, control). The
contracted by CCh (■) or KCl (□). The symbols and error bars represent the symbols and error bars represent the mean and SEM, respectively. *p < 0.05
mean and SEM, respectively. *p < 0.05 (Two-way ANOVA, followed by Sidak’s (Two-way ANOVA, followed by Sidak’s multiple comparisons test: control vs.
multiple comparisons test: CCh vs. KCl). NEC 10− 3, 3 × 10− 3 and 9 × 10− 3 M).

3
M.M. Freitas et al. Chemico-Biological Interactions 348 (2021) 109656

significantly different (p < 0.05) from the control (148.9 ± 9.0%).

To access channels for potassium role in the relaxing effect of NEC,
that effect was compared in the absence and presence of potassium
channels blockers, such as CsCl, GLIB, 4-AP and TEA. However, there
were no significant differences between the pEC50 and Emax values when
compared to the control (pEC50 = 2.7 ± 0.3, with Emax = 94.3 ± 4.6%).
The pEC50 and Emax values in the presence of these blockers were, 2,8 ±
0.2 and 83.4 ± 6.3% for CsCl, 2.8 ± 0.1 and 71.9 ± 5.8% for GLIB, 2.8 ±
0.1 and 94.4 ± 5.1% for 4-AP, 3.1 ± 0.1 and 90.1 ± 7.3% for TEA
(Fig. 4). We have also observed that there was a transient contractive
effect of NEC, between concentrations from 10− 6 to 3 × 10− 5 M, in the
presence of CsCl, GLIB and TEA, which was following the usual relaxing
effect of NEC in concentrations from 10− 4 M and beyond.
With regard to the participation of the NO-sGC-cGMP-PKG pathway
in the relaxing effect of NEC, that effect was compared in the absence
and presence of some key inhibitors, such as L-NAME, MB and ODQ.
However, again, there were no significant differences between the pEC50
and Emax values when compared to the control (pEC50 = 2.7 ± 0.3, with
Emax = 94.3 ± 4.6%). The pEC50 and Emax values in the presence of these
inhibitors were, 2,5 ± 0.3 and 99.1 ± 7.7% for L-NAME, 2.3 ± 0.3 and
105.3 ± 11.3% for MB, 2.7 ± 0.3 and 83.5 ± 3.0% for ODQ (Fig. 5). Once
again, from 10− 6 to 10− 4 M of NEC, we could observe a transient
contractive effect in the presence of ODQ.
To investigate the participation of prostanoids in the relaxing effect
of NEC, INDO and DEXA were used. The pEC50 values in the presence of Fig. 5. Effect of nanoemulsified camphor (NEC) on the isolated rat trachea
these inhibitors were, 2,6 ± 0.3 with Emax = 95.5 ± 12.3% for INDO and contracted by CCh in the absence (■) and in the presence of L-NAME (⋄), MB (x̂)
2.1 ± 0.3 with Emax = 81.7 ± 4.3% for DEXA. When comparing these or ODQ (◆). The symbols and error bars represent the mean and SEM,
values with those of the control group (pEC50 = 2.7 ± 0.3, with Emax = respectively.
94.3 ± 4.6%) with no significant differences between them. We can
observed that there were a transient contractive effect of NEC, between
concentrations from 10− 6 to 3 × 10− 5 M, only in the presence of DEXA,
which was following the usual relaxing effect (Fig. 6).
A similar result was obtained in the presence of RR, where between
concentrations from 10− 6 to 3 × 10− 4 M of NEC there was a transient
contractile effect. Despite this, there were no significant differences
between the pEC50 and Emax values, which were pEC50 = 2.7 ± 0.3 and
Emax = 94.3 ± 4.6% for the control group and pEC50 = 2.9 ± 0.04 and

Fig. 6. Effect of nanoemulsified camphor (NEC) on the isolated rat trachea

contracted by CCh in the absence (■) and in the presence of INDO (x̂) or DEXA
(▴). The symbols and error bars represent the mean and SEM, respectively. *p
< 0.05 (Two-way ANOVA, followed by Sidak’s multiple comparisons test:
absence vs. DEXA).

Emax = 86.0 ± 2.0% for the RR group (Fig. 7).

4. Discussion
Fig. 4. Effect of nanoemulsified camphor (NEC) on the isolated rat trachea
contracted by CCh in the absence (■) and in the presence of CsCl (□), GLIB (▾), In this work it was possible to observe a relaxing effect for the
4-AP (▽) or TEA (△). The symbols and error bars represent the mean and
camphor in isolated rat trachea, which was only possible through the use
SEM, respectively.

4
M.M. Freitas et al. Chemico-Biological Interactions 348 (2021) 109656

allows us to suppose that the potassium channels, for example, are

extremely important in the relaxing action mechanism for several sub­
stances on the smooth muscle [24,25]. Potassium channels are espe­
cially important in controlling Ca2+ influx through CaV when smooth
muscle contraction is evoked by agonists using the pharmacomechanical
contraction pathway, such as acetylcholine (ACh) or its analog CCh [26,
27], for this reason, the study of the mechanism of action for NEC
continued using only protocols using CCh as a contractile agonist.
In this work, NEC was able to relax the rat trachea contracted by CCh,
even in the presence of several channel K+ channels blockers, such as
Cs+ (non-selective K+ channels blocker), TEA, (a Ca2+-activated K+
channels blocker, the KCa), glibenclamide (ATP sensitive K+ channels
blocker, the KATP) and 4-AP (voltage-gated K+ channels blocker, the KV).
This leads us to the conclusion that, at least, KCa, KATP and KV are not
involved in the relaxing effect of camphor. However, it was observed
that there was a transient contractive effect of NEC, between concen­
trations from 10− 6 to 3 × 10− 5 M, in the presence of CsCl, GLIB and TEA,
which was following the usual relaxing effect of NEC in concentrations
from 10− 4 M and beyond. This data implies that, somehow, the K+
channels have a role in the initial effect of the camphor, but without
changing the parameters of pEC50 and Emax in relation to the control
group.
The free radical nitric oxide (NO) is an endogenous molecule
involved in several physiological and pathophysiological processes [28].
Fig. 7. Effect of nanoemulsified camphor (NEC) on the isolated rat trachea Nitric oxide synthase (NOS) is an enzyme that catalyzes the production
contracted by CCh in the absence (■) and in the presence of RR (⋄). The of NO from L-arginine and oxygen. Generally, NO is produced in various
symbols and error bars represent the mean and SEM, respectively. *p < 0.05 tissues, including the airway epithelium and is of great importance for
(Two-way ANOVA, followed by Sidak’s multiple comparisons test: absence the relaxation of bronchial smooth muscle. Its effects depend largely on
vs. RR). the activation of soluble guanylyl cyclase (GCs), on the increase in
production of cyclic guanylyl monophosphate (cGMP), activation of
of a nanostructured carrier system. cGMP-dependent protein kinase (PKG) and consequent phosphorylation
Most monoterpenes are hydrophobic, which presents challenges in of target proteins [29].
the formulation of medicines containing them, often making it impos­ An analogue of L-arginine, known as L-NAME, works by reducing NO
sible to continue the development of pharmaceutical forms. A current synthesis by acting as a false substrate for NOS isoforms, promoting the
approach involves the use of nanoparticles, which encapsulate the reduction of GC activation, cGMP production, PKG activation and
lipophilic compound generating a nanostructured matrix, which can be smooth muscle relaxation [30]. Two other pharmacological tools that
combined with other adjuncts to generate a stable formulation [18]. are useful in the study of the NO-sGC-cGMP-PKG signaling pathway are
Nanoemulsions based on lipids have the main advantages of methylene blue (MB), a non-selective cGC inhibitor, and ODQ, a selec­
increasing the solubility of lipophilic active compound, as well as tive cGC inhibitor. In this work, we use L-NAME as a tool to investigate
improving the bioavailability of these molecules, since the presence of the NO pathway in the relaxing effect of NEC [31,32].
lipids protects the active compound from changes due to the body’s In the data presented here, it can be seen that NEC was able to relax
digestive mechanisms [19]. the rat tracheal rings, even in the presence of L-NAME, MB or ODQ, with
Some successful examples can be observed, as in the case of ostrich pEC50 and Emax values compatible with the control situation, with no
oil, which has an anti-inflammatory potential in rats, which can be significant differences, which in theory discards the participation of NO
enhanced when it was loaded in nanoemulsions. Another study evalu­ in the relaxing effect of camphor. Nevertheless, in the presence of ODQ
ated the production of an aerosol containing curcuminoids (insoluble in there was a transient contraction in lowers concentrations of NEC, which
water) using two different formulation technologies (nanoelmusons and we cannot explain.
microsuspension) [20]. Subsequently, it was observed that the aerosol Nowadays, it is known that the epithelium has a paracrine role,
based on the nanoemulsion had a superior response to the formulation producing and releasing substances with the ability to modulate the
based on microsuspension [21]. contraction/relaxation of the airway smooth muscle [33]. However, in
In this work, NEC promoted relaxation of the tracheal smooth mus­ this study, it was observed that the parameters of potency and efficiency
cle, regardless of whether the contractions were induced by KCl or CCh. (pEC50 and Emax) for NEC were not altered both in the presence of
The agonists used in the work induce contraction from different mech­ indomethacin, a non-selective cyclooxygenase (COX) inhibitor, and in
anisms, one by electromechanical coupling (KCl) and the other by the presence of dexamethasone, an inhibitor of phospholipase A2
pharmacomechanical coupling (CCh). Contractile induction through the (PLA2), what makes it believe that there would be no participation of
use of high concentrations of extracellular K+ causes a depolarization of prostanoids in the relaxing effect of the camphor.
the membrane, opening channels for calcium opened by voltage (CaV), There are reports that the activation of transient potential receptor
with the consequent increase in the influx of Ca2+, thus promoting (TRP) channels, more properly TRPV1-type, causes the release of pro-
muscle contraction [22]. In this work, NEC was able to reduce the inflammatory cytokines from the bronchial epithelial cells [34].
contractions induced by the cumulative addition of Ca2+ in depolarizing Camphor is known to interact strongly with TRP channels, especially
medium (high K+), in a concentration-dependent manner, which is TRPV1 and TRPV3 [35], thereby testing the hypothesis of the partici­
consistent with what is expected for substances that somehow reduce pation of these channels in their relaxing effect would be logical step.
the influx of Ca2+ through of the CaV [23,26]. Despite this, the presence of ruthenium red, a non-selective blocker of
The control of bronchial smooth muscle contractility is highly TRP channels, did not change either the pEC50 value or the NEC Emax.
dependent on the level of K+ efflux through the plasma membrane, However, once again, in the presence of RR there was a transient
which alters the sensitivity of this tissue to contractile agonists. This contraction in lowers concentrations of NEC.

5
M.M. Freitas et al. Chemico-Biological Interactions 348 (2021) 109656

5. Conclusion [7] D.F. Driscoll, Lipid injectable emulsions: pharmacopeial and safety issues, Pharm.
Res. 23 (1959) (2006).
[8] D.J. Mcclements, Nanoemulsions versus microemulsions: terminology, differences
It is concluded that the nanoemulsified camphor (NEC), a nano­ and similarities, Soft Matter 8 (2012) 1719–1729. Cambridge.
structured carrier system, was useful for the in vitro study of the [9] C. Solans, et al., Nanoemulsions. Current opinion in Colloid e Interface Science,
camphor monoterpene, due to the improvement in the solubility of this Kidlington 10 (2005) 102–110.
[10] T.B. Vasconcelos, et al., β-Citronellol, an alcoholic monoterpene with inhibitory
compound, which allowed its study. In addition, it was possible to properties on the contractility of rat trachea, Braz. J. Med. Biol. Res. 49 (2) (2016)
determine that the camphor has a relaxing effect on the smooth muscle 1–9.
of the rat trachea with a still unknown mechanism of action. This [11] R. Latorre, et al., Varieties of calcium-activated potassium channels, Annu. Rev.
Physiol. 51 (1989) 385–399.
mechanism of action probably involves multiple targets, where their [12] L.J. Janssen, et al., Muscarinic excitation-contraction coupling mechanisms in
individual contributions to the relaxing effect could not be detected, but tracheal and bronchial smooth muscles, J. Appl. Physiol. 91 (2001) 1142–1151.
the sum of their contributions may explain the final effect of the [13] S.J. Hughest, m. Hollingsworth, Relaxin as a relaxant of the isolated rat uterus:
comparison with its mechanism of action, In Vivo General Pharmacology 29 (5)
camphor. Further studies are needed to determine precisely what is the (1997) 829–833.
mechanism of action of this monoterpene in smooth muscle. [14] L. Li, et al., Bradykinin, lemakalim and sodium nitroprusside relax the mouse
trachea in vitro by different mechanisms, Life Sci. 7 (1997) 67–73.
[15] S. Estrada-Soto, et al., Relaxant effects of Artemisia ludoviciana on isolated rat
Authors statement smooth muscle tissues, J. Ethnopharmacol. 139 (2) (2012) 513–518.
[16] E.A. Ko, et al., Physiological roles of K+ channels in vascular smooth muscle cells,
This work was carried out after approval (protocol #0003/080716) J. Smooth Muscle Res. 44 (2) (2008) 65–81.
[17] S. Estrada-Soto, et al., Relaxant effects of Artemisia ludoviciana on isolated rat
by the Ethics Committee on the Use of Animals (CEUA-UNIVASF). All smooth muscle tissues, J. Ethnopharmacol. 139 (2) (2012) 513–518.
animal experiments were carried out in compliance with the National [18] F. Matsuda, et al., Comparative effect of amrinone, aminophylline and diltiazem on
Council for the Control of Animal Experimentation (CONCEA) and are rat airway smooth muscle, Acta Anaesthesiologica Scandinavica. Japan 44 (6)
(2000) 763–766.
also in accordance with the ARRIVE guidelines and carried out in [19] V. Schlemper, et al., Mechanisms underlying the relaxation response induced by
accordance with the UK Animals (Scientific Procedures) Act, 1986 and bradykinin in the epithelium-intact Guinea-pig trachea in vitro, Br. J. Pharmacol.
associated guidelines, EU Directive 2010/63/EU for animal 145 (6) (2005) 740–750.
[20] E.A. Townsend, et al., Effects of ginger and its constituents on airway smooth
experiments.
muscle relaxation and calcium regulation, Am. J. Respir. Cell Mol. Biol. 48 (2)
(2013) 157–163.
[21] Abu Zayed Md Badruddoza, et al., Low energy nanoemulsions as templates for the
Declaration of competing interest formulation of hydrophobic drugs, Adv. Ther. (2018).
[22] S.H. Jeong, J.H. Park, K. Park, Formulation Issues Around Lipid-Based Oral and
The authors declare that they have no known competing financial Parenteral Delivery Systems: Role of Lipid Excipients in Modifying Oral and
Parenteral Drug Delivery, Wiley-Interscience, Hoboken, 2007.
interests or personal relationships that could have appeared to influence [23] M. Saad, Alshahrani. Preparation, characterization and in vivo anti-inflammatory
the work reported in this paper. studies of ostrich oil based nanoemulsion, J. Oleo Sci. 68 (3) (2019) 203–208.
[24] Y. Al Ayoub, et al., Development and evaluation of nanoemulsion and
microsuspension formulations of curcuminoids for lung delivery with a novel
Acknowledgment approach to understanding the aerosol performance of nanoparticles, Int. J. Pharm.
557 (2019) 254–263.
This study was financed in part by the Coordenação de Aperfeiçoa­ [25] T. Godfraind, R. Miller, M. Wibo, Calcium antagonism and calcium entry blockade,
Pharmacol. Rev. 38 (4) (1986) 321–416.
mento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. [26] A.P. Somlyo, A.V. Somlyo, Signal transduction and regulation in smooth muscle,
Nature 372 (6503) (1994) 231.
References [27] Mark T. Nelson, et al., Noradrenaline contracts arteries by activating voltage-
dependent calcium channels, Nature 336 (6197) (1988) 382.
[28] J.L. Black, P.J. Barnes, Potassium channels and airway function: new therapeutic
[1] World Health Organization, WHO. Asthma fact sheet, Available in: https://www. prospects, Thorax 45 (1990) 213–218.
who.int/en/news-room/fact-sheets/detail/asthma, 2017. July 12th, 2021. [29] S. Hirota, P. Helli, L.J. Janssen, Ionic mechanisms and Ca2+ handling in airway
[2] Sociedade Brasileira De Pneumologia E Tisiologia (Sbpt), Diretrizes da Sociedade smooth muscle, Eur. Respir. J. 30 (2007) 114–133.
Brasileira de Pneumologia e Tisiologia para o manejo da asma, J. Bras. Pneumol. [30] G. Pelaia, et al., Potential role of potassium channel openers in the treatment of
38 (1) (2012) S1–S46. Available in: http://www.jornaldepneumologia.com.br/ asthma and chronic obstructive pulmonary disease, Life Sci. 70 (2002) 977–990.
pdf/suple_200_70_38_completo_versao_corrigida_04-09-12.pdf. September 12th, [31] E.R. Derbyshire, M.A. Marletta, SCHMIDT, H.H.H.W. et al., Biochemistry of soluble
2021. guanylate cyclase, in: cGMP: Generators, Effectors and Therapeutic Implications,
[3] Brasil Ministério da Saúde Secretaria de atenção à saúde, Portaria nº 1.317, de 25 Handbook of Experimental Pharmacology, vol. 191, Editora Springer-Verlag
de novembro de 2013. Aprova o protocolo clínico e diretrizes terapêuticas da asma. Berlin Heidelberg, 2009, p. 583.
Brasília-DF, Available in: http://bvsms.saude.gov.br/bvs/saudelegis/sas/2013/p [32] P.R.B. Evora, Methylene blue is a guanylate cyclase inhibitor that does not interfere
rt1317_25_11_2013.html, 2013. September 12th, 2021. with nitric oxide synthesis, Tex. Heart Inst. J. 43 (1) (2016).
[4] U.R. Juergens, et al., Anti-inflammatory activity of 1.8-cineol (eucalyptol) in [33] Y.C. Ruan, W. Zhou, H.C.C. Chan, Regulation of smooth muscle contraction by the
bronchial asthma: a double-blind placebo-controlled trial, Respir. Med. 97 (2003) epithelium: role of prostaglandins, Physiology 26 (2011).
250–256. [34] M.S. Grace, et al., Transient receptor potential (TRP) channels in the airway: role in
[5] R.L. Barkin, The pharmacology of topical analgesics, Postgrad. Med. 125 (4 Suppl airway disease, Br. J. Pharmacol. 171 (10) (2014) 2593–2607. London.
1) (2013) 7–18. [35] M. Saad, Alshahrani. Preparation, characterization and in vivo anti-inflammatory
[6] K. Cerpnjak, et al., Lipid-based systems as a promising approach for enhancing the studies of ostrich oil based nanoemulsion, J. Oleo Sci. 68 (3) (2019) 203–208.
bioavailability of poorly water-soluble drugs, Acta Pharm. 63 (2013) 427–445.