2018_Magalhaes_Orofacial antinociceptive effect of Mimosa tenuiflora

Prévia do material em texto

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/321288481
Orofacial antinociceptive effect of Mimosa tenuiflora (Willd.) Poiret
Article  in  Biomedicine & Pharmacotherapy · November 2017
DOI: 10.1016/j.biopha.2017.11.001
CITATIONS
5
READS
124
25 authors, including:
Some of the authors of this publication are also working on these related projects:
Development and pharmacological study of pharmaceutical preparations containing cyclodextrins View project
Antioxidant and pharmacological screening of natural product View project
Francisco Ernani Alves Magalhães
Universidade Estadual do Ceará
34 PUBLICATIONS   67 CITATIONS   
SEE PROFILE
Angelo Silva
Universidade de Fortaleza
12 PUBLICATIONS   39 CITATIONS   
SEE PROFILE
Maria Izabel Florindo Guedes
Universidade Estadual do Ceará
131 PUBLICATIONS   905 CITATIONS   
SEE PROFILE
Sacha Aubrey A. R. Santos
19 PUBLICATIONS   95 CITATIONS   
SEE PROFILE
All content following this page was uploaded by Francisco Ernani Alves Magalhães on 25 November 2017.
The user has requested enhancement of the downloaded file.
https://www.researchgate.net/publication/321288481_Orofacial_antinociceptive_effect_of_Mimosa_tenuiflora_Willd_Poiret?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_2&_esc=publicationCoverPdf
https://www.researchgate.net/publication/321288481_Orofacial_antinociceptive_effect_of_Mimosa_tenuiflora_Willd_Poiret?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_3&_esc=publicationCoverPdf
https://www.researchgate.net/project/Development-and-pharmacological-study-of-pharmaceutical-preparations-containing-cyclodextrins?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_9&_esc=publicationCoverPdf
https://www.researchgate.net/project/Antioxidant-and-pharmacological-screening-of-natural-product?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_9&_esc=publicationCoverPdf
https://www.researchgate.net/?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_1&_esc=publicationCoverPdf
https://www.researchgate.net/profile/Francisco_Ernani_Magalhaes?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_4&_esc=publicationCoverPdf
https://www.researchgate.net/profile/Francisco_Ernani_Magalhaes?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_5&_esc=publicationCoverPdf
https://www.researchgate.net/institution/Universidade_Estadual_do_Ceara?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_6&_esc=publicationCoverPdf
https://www.researchgate.net/profile/Francisco_Ernani_Magalhaes?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_7&_esc=publicationCoverPdf
https://www.researchgate.net/profile/Angelo_Silva2?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_4&_esc=publicationCoverPdf
https://www.researchgate.net/profile/Angelo_Silva2?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_5&_esc=publicationCoverPdf
https://www.researchgate.net/institution/Universidade_de_Fortaleza?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_6&_esc=publicationCoverPdf
https://www.researchgate.net/profile/Angelo_Silva2?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_7&_esc=publicationCoverPdf
https://www.researchgate.net/profile/Maria_Izabel_Florindo_Guedes?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_4&_esc=publicationCoverPdf
https://www.researchgate.net/profile/Maria_Izabel_Florindo_Guedes?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_5&_esc=publicationCoverPdf
https://www.researchgate.net/institution/Universidade_Estadual_do_Ceara?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_6&_esc=publicationCoverPdf
https://www.researchgate.net/profile/Maria_Izabel_Florindo_Guedes?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_7&_esc=publicationCoverPdf
https://www.researchgate.net/profile/Sacha_Santos?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_4&_esc=publicationCoverPdf
https://www.researchgate.net/profile/Sacha_Santos?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_5&_esc=publicationCoverPdf
https://www.researchgate.net/profile/Sacha_Santos?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_7&_esc=publicationCoverPdf
https://www.researchgate.net/profile/Francisco_Ernani_Magalhaes?enrichId=rgreq-8d0fbe70df3d159e402f00d94d076729-XXX&enrichSource=Y292ZXJQYWdlOzMyMTI4ODQ4MTtBUzo1NjQ0MjMzMjYwODkyMTZAMTUxMTU4MDQxNTQ2MA%3D%3D&el=1_x_10&_esc=publicationCoverPdf
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
Original article
Orofacial antinociceptive effect of Mimosa tenuiflora (Willd.) Poiret
Francisco Ernani A. Magalhãesa,b,c, Francisco Lucas A. Batistaa, Ohanna F. Serpaa,
Luiz F. Wemmenson G. Mouraa, Maria da Conceição L. Limaa, Ana Raquel A. da Silvab,
Maria Izabel F. Guedesb, Sacha Aubrey A.R. Santosc, Breytiner A. de Oliveirac,
Andressa B. Nogueirac, Talita M. Barbosac, Dayse Karine R. Holandac,
Marina B.M.V. Damascenoc, José de Maria A. de Melo Júniorc, Lana Karine V. Barrosoc,
Adriana R. Camposc,⁎
a Laboratory of Bioprospecting of Natural Products and Biotechnology, Ceará State University, Department of Chemistry, CECITEC, Tauá, Ceará, Brazil
b Laboratory of Biotechnology and Molecular Biology, Ceará State University, Department of Nutrition, Fortaleza, Ceará, Brazil
c Experimental Biology Center, University of Fortaleza, Fortaleza, Ceará, Brazil
A R T I C L E I N F O
Keywords:
Mimosa tenuiflora
Antioxidant
Orofacial pain
Antinociception
A B S T R A C T
Mimosa tenuiflora (Willd.) Poiret, popularly known in Brazil as “jurema-preta” is widely used against bronchitis,
fever, headache and inflammation. Its antioxidant, anti-inflammatory and antinociceptive potential has already
been reported. To assess the orofacial antinociceptive effect of M. tenuiflora, ethanolic extracts of M. tenuiflora
(leaves, twigs, barks and roots) were submitted to in vitro tests of antioxidant activity. The extract with the
highest antioxidant potential was partitioned and subjected to preliminary chemical prospecting, GC–MS,
measurement of phenolic content and cytotoxicity tests of the fractionwith the highest antioxidant activity. The
nontoxic fraction with the highest antioxidant activity (FATEM) was subjected to tests of acute and chronic
orofacial nociception and locomotor activity. The possible mechanisms of neuromodulation were also assessed.
The EtOAc fraction, obtained from the ethanolic extract of M. tenuiflora barks, was the one with the highest
antioxidant potential and nontoxic (FATEM), and Benzyloxyamine was the major constituent (34.27%). FATEM
did not alter the locomotor system of mice and reduced significantly the orofacial nociceptive behavior induced
by formalin, glutamate, capsaicin, cinnamaldehyde or acidic saline compared to the control group. FATEM also
inhibited formalin- or mustard oil-induced temporomandibular nociception. In addition, it also reduced mustard
oil-induced orofacial muscle nociception. However, FATEM did not alter hypertonic saline-induced corneal
nociception. Neuropathic nociception was reversed by treatment with FATEM. The antinociceptive effect of
FATEM was inhibited by naloxone, L-NAME and glibenclamide. FATEM has pharmacological potential for the
treatment of acute and neuropathic orofacial pain and this effect is modulated by the opioid system, nitric oxide
and ATP-sensitive potassium channels. These results lead us to studies of isolation and characterization of
bioactive principles.
1. Introduction
Pain in the oral and craniofacial system represents a medical and
social problem. It is derived from many unique target tissues, such as
the meninges, cornea, tooth pulp, oral/nasal mucosa and tempor-
omandibular joint and thus has several unique physiologic character-
istics compared with the nociceptive system. Thus, the treatment of
orofacial pain conditions is a significant health inssue and a challenge
for the pharmaceutical industry [1].
Trigeminal Neuralgia (TN) and Temporomandibular Disorder
(TMD) represent the worst types of orofacial pain and some of the most
common. TN is usually treated with anticonvulsants. About 75% of
patients need neurosurgery at some time in their history of the disease.
Surgical procedures are divided into percutaneous procedures and
surgeries, with high success rates. TMD is a general term for mastica-
tory musculoskeletal pains with multiple etiology. It is the main cause
of chronic orofacial pain and is associated with psychosocial, cognitive-
behavioral and emotional factors. Treatment Includes drug therapy,
physiotherapy and surgery [2].
Currently, there is a gap in knowledge concerning the drug regimen
https://doi.org/10.1016/j.biopha.2017.11.001
Received 13 August 2017; Received in revised form 17 October 2017; Accepted 3 November 2017
⁎ Corresponding author at: Universidade de Fortaleza, Núcleo de Biologia Experimental (NUBEX), Av. Washington Soares, 1321, CEP 60811-905, Edson Queiroz, Fortaleza, Ceará,
Brazil.
E-mail address: adrirolim@unifor.br (A.R. Campos).
Biomedicine & Pharmacotherapy 97 (2018) 1575–1585
0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.
T
http://www.sciencedirect.com/science/journal/07533322
https://www.elsevier.com/locate/biopha
https://doi.org/10.1016/j.biopha.2017.11.001
https://doi.org/10.1016/j.biopha.2017.11.001
mailto:adrirolim@unifor.br
https://doi.org/10.1016/j.biopha.2017.11.001
http://crossmark.crossref.org/dialog/?doi=10.1016/j.biopha.2017.11.001&domain=pdf
for the treatment of pain. There are no ideal analgesic and/or anti-in-
flammatory drugs available, i.e., drugs that will not cause potential side
effects [3].
An alternative solution to this problem has been presented by sev-
eral studies that have suggested the use of extracts of medicinal plants
rich in antioxidant properties due to their antinociceptive activities [4]
against acute and/or chronic and/or neuropathic orofacial pain, which
may occur due to factors such as the scavenging of reactive oxygen
species (ROS), which are formed and activate channels of pain mod-
ulation during nociception [5], as well as due to inhibition of the ROS
generating oxidases and/or direct inhibition of enzyme that catalyzes
oxidation of cellular components [4]. These activities are generally
mediated by phenolic compounds such as phenols, flavonoids, tannins,
among others [6].
It should be noted that plants have also been used as sources of
secondary metabolites for the treatment of many diseases caused by
free radicals, such as atherosclerosis, arthritis, cardiovascular disorders,
Alzheimer’s disease, and cancer [7]. Thus, the search for new plants
sources of natural antioxidants with antinociceptive activity has be-
come a challenge.
Mimosa tenuiflora (Willd.) Poiret, popularly known in Brazil as
“jurema-preta”, has been reported as a promising source of compounds
with antioxidant, anti-inflammatory and antinociceptive potential
[8,9]. It is commonly found in tropical forests in Mexico, Venezuela and
Brazil [10]. In Northeastern Brazil, it is a common tree rich in several
secondary metabolites (phenols, saponins, tannins and flavonoids) that
has been widely used against bronchitis, cough, fever, headache, and
skin ulcers [11]). Aqueous and ethanolic extracts of the bark exhibit
anti-inflammatory activity [1,10].
Given the above, the present study reports the antinociceptive effect
of M. tenuiflora (Willd.) Poiret on orofacial pain and its possible me-
chanism of action.
2. Material and methods
2.1. Botanical material
Mimosa tenuiflora (Willd.) Poiret was collected in August 2015 in the
micro-region of the Inhamuns (Tauá, Ceará, Northeastern Brazil),
(040°18′05.4″ W; 06°01′03.6″ S), using the methodology described by
Cartaxo et al. [8]. The collection was approved by the Instituto Brasi-
leiro do Meio Ambiente e dos Recursos Naturais Renováveis – IBAMA-
SISBIO under approval No. 29145-4. After collection, the species was
identified and deposited in the Dárdano de Andrade-Lima Herbarium of
the Universidade Regional do Cariri (URCA), exsiccate No. 6675.
2.2. Animals
Swiss mice (20 and 25 g) and Wistar rats (250 and 300 g) were
obtained from the Núcleo de Biologia Experimental (NUBEX) of the
Universidade de Fortaleza (UNIFOR), Ceará, Northeastern Brazil. The
animals were housed in appropriate cages and kept at room tempera-
ture of 22–24 °C on a 12:12 h light:dark cycle. They received standard
feed (Purina, São Paulo, Brazil) and water ad libitum. All protocols were
in strict compliance with the standards established by Brazil’s National
Council on Animal Experimentation Control and received approval
from the Committee on Animal Research and Ethics of UNIFOR
(Aproval No. 0132015).
2.3. Drugs and reagents
The following drugs and reagents were used in the study: 96%
ethanol (Ciclo Farma). Hexane, Dichloromethane, Acetic acid,
Folin–Ciocalteu, and ethyl acetate were purchased from Dinamica.
Ferric chloride, Sodium Chloride, and Dimethyl sulfoxide were pur-
chased from Synth. Formaldehyde was purchased from Dinamica.
Saline was purchased from Arboreto. Naloxone was purchased from
Tocris Bioscience. Gallic acid, amiloride, capsaicin, camphor, cynna-
maldehyde, glutamate, glibenclamide, Ketamine, mustard oil (allyl
isothiocyanate), quercetin, ruthenium red, 3-(4,5-Dimethylthiazol-2-
yl)-2,5-Diphenyltetrazolium Bromide (MTT), 2,2-diphenyl-1-picrylhy-
drazyl (DPPH), 2,2′-„azino-bis(3-ethylbenzothiazoline-6-sul-
phonic acid) (ABTS), xylazine, and L-NAME were purchased from
Sigma (USA).
2.4. Preparation of extracts and bioguided fractionation of antioxidant
activity
Crude extracts were obtained from leaves, twigs, barks and roots of
M. tenuiflora (Willd.) Poiret using 96% ethanol [12]. The plant extract
with the highest antioxidant potential was submitted to fractionation by
liquid-liquid partition, as described by Seito et al. [13], using solvents
of different polarities, such as: a) hexane (AR), b) dichloromethane
(AR), and c) ethyl acetate (AR). Thus, three fractions were obtained: a)
hexane fraction (Hex-F); b) dichloromethane fraction (Dic-F); c) ethyl
acetate fraction (EtOAc-F); and d) hydroalcoholicfraction (Hyd-F), a
residual fraction containing distilled water (2%).
2.5. Analysis of antioxidant activity
2.5.1. Thin layer chromatography (TLC) analysis of antioxidant activity
The extracts were analyzed by TLC using quercetin and gallic acid as
reference standards as suggested by Hidalgo et al. [14]. The plates were
eluted with methanol and nebulized with FeCl3 or DPPH solutions. The
plates were observed until the appearance of dark blue spots (phenolic
compounds) or yellow spots on a purple background (antioxidant
compounds).
2.5.2. In vitro antioxidant activity
Solutions of DPPH (3.9mL, 6.5× 10–5 M) [15] or ABTS (5mL;
7mM) [16] were added to the solutions containing the samples
(10–10,000 μg/mL). The tests were performed in triplicate. The absor-
bance values were measured spectrophotometrically at 515 and
734 nm, respectively. The antioxidant capacity was compared with the
standard, i.e., quercetin. EC50 was determined and Pearson’s correlation
(r) was used to evaluate the relationship between the two methods [17].
2.6. Chemical prospecting
2.6.1. Measurement of the potential of hydrogen (pH)
The pH of the fractions of the extract of M. tenuiflora with the
highest antioxidant potential was measured using a potentiometer. The
pH of the distilled water was used as a standard of comparison (n= 3)
[18].
2.6.2. Preliminary phytochemical screening
The extracts were subjected to preliminary phytochemical screening
to detect the main classes of secondary metabolites through chemical
reactions that result in changes in color and/or formation of pre-
cipitates which are specific to each class of substances [12].
2.6.3. Estimation of total phenolic (TP) content
Folin–Ciocalteu reagent [19] was used with a gallic acid standard
curve (1–500 μg/mL). The value obtained from the equation was
C=0.0009 A, where C is the concentration of gallic acid, A is the ab-
sorbance at 750 nm and the correlation coefficient R= 0.9916. The
total phenolic content was expressed in mg GAE (gallic acid equiva-
lents) per 100 g of extract. The results were presented as mean ±
standard deviation (SD). Pearson’s correlation (r) was used to assess the
relationship between total phenolic contents and antioxidant activities
[17].
F.E.A. Magalhães et al. Biomedicine & Pharmacotherapy 97 (2018) 1575–1585
1576
2.6.4. Gas chromatography–mass spectrometry (GC–MS) analysis
The fraction of M. tenuiflora with the highest antioxidant potential
(FATEM) was subjected to silylation reaction [20]. The reaction
medium was injected into a Shimadzu GCMS-QP2010 sE quadrupole
gas chromatograph-mass spectrometer (GC–MS) using a Rtx®-5 MS
capillary column (30m×0.25mm×0.25 μm) for the relative quanti-
fication of the constituents present in the sample. GC–MS conditions
were: 70 eV, helium (carrier gas) at 13.6 mLmin-1 and pressure of
53.5 kPa. The temperature program was as follows: 100 °C (3min) to
310 °C (3.5 °C/min). The compounds in the sample were identified by
comparing them with those published in the mass spectral library
(NIST). Mass spectra were recorded using a scan range of 10–1000 m/z.
2.7. Toxicity
2.7.1. Non-specific toxicity to Artemia salina L.
Sample concentrations (100, 500 and 1000 μg/mL) were tested in
triplicate. The surviving nauplii were counted after 24 h and the per-
centage of dead nauplii was determined by calculating the concentra-
tion to kill 50% (LC50) using Probit analysis with 95% confidence in-
tervals [21]. The toxicity potential of the samples was classified into: a)
Nontoxic (LC50 > 1000 μg/mL); b) Toxic (LC50≤ 1000 μg/mL).
2.7.2. Cytoxicity to Vero cells
The fraction of M. tenuiflora with the highest antioxidant potential
(FATEM) was subjected to the test of specific cytotoxicity to Vero cells
(African green monkey kidney). The MTT colorimetric method was
used [22]. Solutions (1000–62.5 μg/mL) were used and the percentage
of cell viability was calculated. The concentration capable of reducing
viability to 50% compared to cell control (CC50) was obtained by
analyzing the non-linear regression of the concentration-effect curve.
The cytotoxicity potential (CTP) of the sample was classified according
to Fadeyi et al. [23] into (a) Toxic (CC50≤ 30 μg/mL), b) Nontoxic
(CC50 > 30 μg/mL).
2.8. Orofacial antinociceptive activity
Acute nociception tests were performed with three doses of FATEM
(12.5; 25 or 50mg/kg). Neuromodulation mechanisms, tempor-
omandibular joint nociception, craniofacial nociception, and chronic
nociception (IONX) were assessed using the 25mg/kg dose since it
presented the best effect in the acute nociception tests (see the Results
Section).
2.8.1. Formalin-induced orofacial nociception
Orofacial nociception was induced in mice (n= 6/group) by sub-
cutaneous injection of 20 μL of 2% formalin [24] into the right vibrissal
pad (perinasal area) using a 27-gauge needle. Orofacial nociception was
quantified by measuring the time that the mice spent rubbing the in-
jection site (Face rubbing, FR) with the fore or hind paw for 0–5min
and 15–30min after induction. Treatments with vehicle (Control: 3%
DMSO, i.p.) or FATEM (12.5; 25 or 50mg/kg, i.p.) were performed
30min prior to formalin injection (n= 6/group). A Naive group was
included.
In subsequent experiments, the animals were pretreated in-
traperitoneally with the respective antagonists Naloxone (2mg/kg)
[25], L-NAME (10mg/kg) [25] or Glibenclamide (2mg/kg) [26],
30 min prior to administration of FATEM (25mg/kg) to check for the
possible involvement of opioid and nitrergic systems and ATP-sensitive
potassium (KATP) channels.
2.8.2. Glutamate-induced orofacial nociception
Using the method described above, orofacial nociception was in-
duced with glutamate (40 μL, 25 μM) [27] to assess the effect of the test
drug. Face rubbing was observed individually during 15 min. A Naive
group was included.
2.8.3. Capsaicin-induced orofacial nociception
Using the method described above, orofacial nociception was in-
duced with capsaicin (20 μL, 2.5 μg, s.c.) [28] dissolved in ethanol, PBS
and distilled water (1:1:8) to assess the effect of the test drug. Face
rubbing was observed individually during 10–20min. A Naive group
was included.
In a subsequent experiment, ruthenium red (3 mg/kg; s.c.) [29] was
administered 30min prior to FATEM (25 mg/kg) to check for possible
effects on the TRPV1 channel.
2.8.4. Cinnamaldehyde-induced orofacial nociception
Using the method described above, orofacial nociception was in-
duced with cinnamaldehyde (20 μL, 0.66 μM, s.c.) [30] to assess the
effect of the test drug. Face rubbing was observed individually during
0–5min. A Naive group was included.
In a subsequent experiment, camphor (7.6 mg/kg; s.c.) [31] was
administered 30min prior to FATEM (25mg/kg) to check for possible
effects on the TRPA1 channel.
2.8.5. Acidic saline-induced orofacial nociception
Using the method described above, orofacial nociception was in-
duced with acidic saline (20 μL; 2% acetic acid, pH 1.98; s.c.) [32] to
assess the effect of the test drug. Face rubbing was analyzed during
0–20min. A Naive group was included.
In a subsequent experiment, amiloride (100mg/kg; s.c.) [32] was
administered 30min prior to FATEM (25mg/kg) to check for possible
effects on ASICs.
2.8.6. Hypertonic saline-induced corneal nociception
Corneal nociception was induced in mice by applying 20 μL of hy-
pertonic saline (5M NaCl) [33] onto the left eye cornea. Corneal no-
ciception was quantified by measuring the number of times that the
mice cleaned the eye with the fore or hind paw for 30 s. The animals
(n= 6/group) were treated with vehicle (Control-3% DMSO, 10mL/kg;
i.p.) or FATEM (12.5; 25 or 50mg/kg; i.p.) 30min prior to induction. A
Naive group was included.
2.8.7. Formalin- or mustard oil-induced temporomandibular joint (TMJ)
nociception
Formalin (2%, 20 μL) [34] or mustard oil (2.5%, 50 μL) [35] were
used as noxious agents applied to the left temporomandibular joint of
rats. The animals (n= 6/group) were treated with vehicle (Control–
3% DMSO 10mL/kg, i.p.) or FATEM (25mg/kg; i.p.) 30min prior to
induction. Nociception was quantified by measuring the time that the
mice spent rubbing the injection site (Face rubbing, FR) with the fore or
hind paw and head flinching (HF) for 36min after induction recorded
12 times at 3min intervals. A group of animals treated with vehicle and
saline in the TMJ (Sham) and a Naive group were also assessed.
2.8.8. Mustard oil-induced craniofacial nociception
Mustard oil (20%, 20 μL) was injected in the mid-region of the left
masseter muscle of rats [36]. The animals (n= 6/group) were treated
with vehicle (Control- 3% DMSO, 10mL/kg, i.p.) or FATEM (25mg/kg;
i.p.) 30min prior to induction. Craniofacial nociception was quantified
by measuring the time that the rats spent shaking hind paws within
4min recorded 8 times at 30-s intervals. A group of animals treated
with vehicle and saline (Sham) and a Naive group were also assessed.
2.8.9. Assessment of mechanical sensitivity after infraorbital nerve
transection
Rats (n=6/group) were anesthetized with ketamine and xylazine
(100 and 10mg/kg, respectively, i.p.) [29] prior to infraorbital nerve
(ION) exposure at the infraorbital foramen using an intraoral incision
(2mm) made in the oral mucosa of the left fronto-lateral maxillary
vestibulum. The ION was cut and removed from the maxillary bone
(IONX) without damaging adjacent nerves. Subsequently, the animals
F.E.A. Magalhães et al. Biomedicine & Pharmacotherapy 97 (2018) 1575–1585
1577
were returned to their cages and fed with chow mash.
For the assessment of mechanical sensitivity, the animals were
monitored daily postoperatively with von Frey filaments. The rats were
acclimatized, trained for 3 days prior to the surgery to obtain baseline
values and then tested on postoperative days 1, 3, 5, 7, 10, 14 and 21.
The animals were treated daily with a single dose of FATEM (25mg/kg)
or saline (10mL/kg) intraperitoneally shortly after surgery. Naive and
Sham-operated groups were also assessed. Facial mechanical sensitivity
was evaluated as the animals’ head-withdrawal thresholds which were
obtained by slowly applying graded von Frey filament hairs to the ip-
silateral facial skin area 2mm below the lower lip. Escape responses to
the mechanical stimulation were demonstrated as a sudden backward
withdrawal movement of the head. The head-withdrawal threshold
(HWT) was defined as the lowest filament intensity that evoked three or
more escapes out of five stimulation trials with intervals of more than
10 s [37].
2.9. Assessment of locomotor activity
Mice (n=6/group) were treated with vehicle (3% DMSO 10mL/
kg, i.p.) or FATEM (12.5; 25 or 50mg/kg; i.p.). After 30min of treat-
ment, the animals were positioned at the center of the open field arena
(40× 40×40 cm) and the total number of line crossings was recorded
over a 5-min period. A Naive group was also assessed.
2.10. Statistical analysis
The results are presented as mean ± S.D or mean ± S.E.M. The
statistical analysis consisted of one-way analysis of variance (ANOVA),
followed by the Tukey or Bonferroni post-hoc tests for multiple com-
parisons. The level of statistical significance was set at 5% (p < 0.05).
3. Results
3.1. Yields and antioxidant activity of ethanolic extracts
Of the four extracts obtained, the ethanolic extract of the M. tenui-
flora barks (EtEMtB) was the one with the highest extraction yield
(28.58%), resulting in 75.01 g. The test of antioxidant activity by TLC
identified phenolic compounds in all the extracts. They presented ra-
dical scavenger activity against DPPH. EtEMtB showed the lowest EC50
values against DPPH (EC50= 132.99 ± 0.04 μg/mL) and ABTS
(EC50= 189.14 ± 0.19 μg/mL) radicals and was therefore chosen for
fractionation.
3.2. Fractionation of EtEMtB and in vitro antioxidant activity of obtained
fractions
A total of 55.28 g of the EtEMtB was partitioned. Four fractions
(EtEMtB-F) were obtained: hexane (Hex-F), dichloromethane (Dic-F), ethyl
acetate (EtOAc-F) and hydroalcoholic (Hyd-F). The fractionation yield (FY)
ranged 6.19% (Hex-F) to 50.01% (Hyd-F). Of the four EtEMtB-F tested,
only the EtOAc-F presented good antioxidant activity in both methods,
with the lowest EC50 values against DPPH (EC50=141.20 ± 0.023 μg/
mL) and ABTS (EC50=273.00 ± 0.084 μg/mL). Pearson’s correlation
coefficient (r) for the study with the EtEMtB-F indicated that the methods
are 99.85% correlated (r=0.9985). Given these results, the EtOAc-F was
selected as the fraction with the highest antioxidant potential and was then
named FATEM.
3.3. Chemical prospecting of obtained fractions
3.3.1. Measurement of the potential of hydrogen and preliminary
phytochemical screening
FATEM presented the highest pH value (4.94). The preliminary
phytochemical screening found phenolic compounds in all the EtEMtB-
F. These results led to the determination of phenolic content.
3.3.2. Total phenolic (TP) content
Total phenolic content varied significantly between the EtEMtB-F.
However, FATEM (EtOAc-F) presented the highest total phenolic con-
tent value (429.63 ± 0.002mg GAE/g). The total phenolic content of
the EtEMtB-F is 92.72% correlated with the antioxidant activity by the
DPPH method (r= 0.9272) and 97.90% by the ABTS method
(r = 0.9790).
3.3.3. Gas chromatography–mass spectrometry (GC–MS) analysis of
FATEM
According to the GC–MS library (NIST08), FATEM is composed of
20 chemical compounds (Fig. 1), five of which are major (Table 1):
Benzyloxyamine (34.27%); 3-Fluorophenol, diisopropylsilyl ether
(14.7%); 1,2-Bis(trimethylsiloxy)ethane (21.83%); Trimethylsilyl ether
Fig. 1. GC–MS Chromatogram of FATEM.
F.E.A. Magalhães et al. Biomedicine & Pharmacotherapy 97 (2018) 1575–1585
1578
of glycerol (13.53%); and oleic acid (5.25%).
3.4. Toxicity
Only the hydroalcoholic fraction (Hyd-F) killed 50% of the nauplii
(LC50= 793.70 μg/mL). FATEM was not toxic to A. salina L.
(LC50 ˃ 1000.00 μg/mL) or Vero cells (CC50 = 512.6 μg/mL).
3.5. Antinociceptive activity
3.5.1. Formalin-induced orofacial nociception
FATEM (25 or 50mg/kg, i.p.) reduced (p < 0.001 or p < 0.01 vs
control) face rubbing behavior in both phases of the formalin-induced
orofacial nociception test (Fig. 2A and B). The dose of 12.5 mg/kg (i.p.)
caused a significant reduction (p < 0.01 vs control) only in the 1st
phase (Fig. 2A). The antinociceptive effect of FATEM was partially
prevented by Naloxone (Fig. 3A and B), L-NAME (Fig. 4A and B) and
Glibenclamide (Fig. 5A and B).
3.5.2. Orofacial nociception induced by glutamate, capsaicin,
cinnamaldehyde and acidic saline
Pretreatment with FATEM (12.5; 25 or 50mg/kg; i.p.) reduced
(p < 0.001 vs control) face rubbing behavior induced by glutamate,
capsaicin, or acidic saline (Figs. 6–8). However, only the doses of 25
and 50mg/kg caused a significant reduction (p < 0.01 and p < 0.001
vs control, respectively) in the nociceptive behavior induced by cinna-
maldehyde (Fig. 9). Ruthenium red (TRPV1 antagonist), camphor
(TRPA1 antagonist) or amiloride (ASIC antagonist) did not alter the
antinociceptive effect of FATEM (Figs. 10–12). Such groups did not
differ significantly (p > 0.05) when compared with the FATEM group,
which rules out the involvement of such channels in the mechanism of
the antinociceptive action of FATEM.
3.5.3. Hypertonic saline-induced corneal nociception
FATEM did not prevent hypertonic saline-induced eye rubbing be-
havior (Fig. 13).
3.5.4. Formalin- or mustard oil-induced temporomandibular joint (TMJ)
nociception
Injections of formalin or mustard oil in the TMJ induced nociceptive
Table 1
Chemical constituents of FATEM according to GC–MS analysis (NIST08).
Classification RT (min) Occurrence (%) Compounds
1 5.073 34.27 Benzyloxyamine, N,N-Bis(trimethylsilyl)
2 8.575 21.83 3-Fluorophenol, diisopropylsilyl ether
3 6.03 14.27 1,2-Bis(trimethylsiloxy) ethane
4 10.74 13.53 Trimethylsilyl ether of glycerol
5 25.07 5.25 Oleic acid
6 6.335 1.47 Acetic acid, [(trimethylsilyl)oxy]-, trimethylsilyl ester
7 8.994 1.13 Butane,1,2,3-tris(trimethylsiloxy)-
8 6.924 1.06 Hexamethyldisiloxane
9 36.459 0.85 22-tricosanoic acid
10 22.568 0.78 N-Hexadecanoic acid
11 42.690 0.75 Benzoic acid, 2,6-bis[(trimethylsilyl)oxy]- trimethylsilyl ester
12 23.135 0.73 Oxalic acid di-(bis 2,2,6,6-tetramethyl, 1-oxylpiperidyl)
13 38.694 0.71 Ethyl 9-hexadecenoate
14 20.521 0.63 β-D-Galactofuranose acetate 2,3,5,6-Tetrakis
15 8.765 0.45 2-Methylthio-5-nitroanisole
16 25.381 0.41 Octadecanoic acid, 2-hydroxy-1,3-propanediyl ester
17 21.043 0.40 Inositol, 1,2,3,4,5,6-hexakis-O-(trimethylsilyl)
18 5.583 0.39 Pentane, 2-methyl-4-keto-2-trimethylsiloxy
19 22.615 0.37 β-D-Glucopyranose, 6-O-methyl-1,2,3,4-Tetrakis
20 20.803 0.32 Inositol, 1,2,3,4,5,6-hexakis-O-(trimethylsilyl)
RT – retention time.
Control 12,5 25 50 Naive
0
20
40
60
80
**
*** ***
***
______________
91.56%
59.91%64.14%
53.80%
A
i
Control 12,5 25 50 Naive
0
50
100
150
200
250
** **
***
______________
92.25%
65.83%62.84%
41.06%
B
)s(
gnibbur
ecaF
Fig. 2. Effect of FATEM (12.5; 25 or 50 mg/kg, i.p.) on formalin-induced orofacial nociception in mice. Each column represents the mean ± S.E.M (n = 6/group). Numbers above the
bars indicate the percentage of analgesia. One-way ANOVA with post-hoc Tukey’s test (**p < 0.01 and ***p < 0.001 vs control). Control: vehicle (3% DMSO). Naive: untreated group.
F.E.A. Magalhães et al. Biomedicine & Pharmacotherapy 97 (2018) 1575–1585
1579
behaviors in mice characterized by face rubbing (A and C) and head
withdrawal (B and D), as shown in Table 2. These behaviors were re-
duced by pretreatment with FATEM (25mg/kg). These tests were per-
formed with this single dose of FATEM because it presented the best
antinociceptive effect in the previous tests.
3.5.5. Mustard oil-induced craniofacial nociception
FATEM (25mg/kg) also reduced (p < 0.01 vs control) hind paw
shaking behavior after nociception induced by mustard oil injected into
the masseter muscle of rats (Fig. 14).
3.5.6. Assessment of mechanical sensitivity after infraorbital nerve
transection
FATEM (25mg/kg) significantly reduced (p < 0.05 vs control)
neuropathic pain at days 1, 10 and 14 postoperatively (Fig. 15).
3.6. Assessment of locomotor activity
FATEM (12.5; 25 or 50mg/kg) did not alter the locomotor activity
of the animals in the open field test (Fig. 16).
4. Discussion
Although the antinociceptive potential of Mimosa tenuiflora [10] has
already been investigated, the present research is the first to report the
assessment of M. tenuiflora capacity to attenuate orofacial pain in ex-
perimental animal models.
A bioguided assay detected the presence of phenolic compounds in
the ethanolic extracts of M. tenuiflora. These compounds are DPPH re-
ducers. After bioguided fractionation, the EtOAc fraction of the etha-
nolic extract of M. tenuiflora barks (FATEM) presented the highest an-
tioxidant action against DPPH and ABTS radicals.
According to Sousa et al. [38], the antioxidant activity of plants may
be associated with several groups of phenolic compounds present in
plant extracts. Thus, because FATEM presented the highest total phe-
nolic content we believe that such antioxidant action is due to the
presence of these compounds. The results of the antioxidant action
suggest that the phenolic compounds present in FATEM may be
C F N N + F
0
50
100
150
*** ***
***
Treatments i.p: C - Control (DMSO 3%; 10 mL/Kg);
____________________________
81.26%
66.47%68.05%
A
C F N N + F
0
100
200
300
**
________________________________________________________
Treatments i.p: C - Control (DMSO 3%; 10 mL/Kg);
26.91%22.30%
56.65%
B
)s(
gnibbur
ecaF
Fig. 3. Effect of Naloxone (A and B) on the antinociceptive activity of FATEM (25mg/kg, i.p.) in the formalin-induced orofacial nociception model. Each column represents the
mean ± S.E.M (n = 6/group). Numbers above the bars indicate the percentage of analgesia. One-way ANOVA with post-hoc Tukey’s test (**p < 0.01 and ***p < 0.001 vs control).
Control: vehicle (3% DMSO).
C F L-N L-N + F
0
20
40
60
80
**
____________________________
A
33.76%
26.58%
74.47%
C F L-N L-N + F
0
20
40
60
80
100
***
**
____________________________
B
56.76%
19.13%
64.66%
)s(
gnibbur
ecaF
Fig. 4. Effect of L-NAME (A and B) on the antinociceptive activity of FATEM (25mg/kg, i.p.) in the formalin-induced orofacial nociception model. Each column represents the
mean ± S.E.M (n = 6/group). Numbers above the bars indicate the percentage of analgesia. One-way ANOVA with post-hoc Tukey’s test (**p < 0.01 and ***p < 0.001 vs control).
Control: vehicle (3% DMSO).
F.E.A. Magalhães et al. Biomedicine & Pharmacotherapy 97 (2018) 1575–1585
1580
hydrophilic and/or lipophilic because they have reduced both radicals
[39].
Benzyloxyamine was the major constituent (34.27%) of FATEM.
Reports on the detection of this constituent as a natural product were
not found in the literature. It is worth mentioning that compounds
derived from benzyloxyamines have been found to have anti-in-
flammatory, anticancer, anorexigenic and antimalarial effects [40].
C F G G + F
0
20
40
60
***
*
# #
____________________________
A
28.72%23.61%
66.18%
C F G G + F
0
50
100
150
200
***
*
____________________________
47.22%33.91%
80.24%
B
)s(
gnibbur
ecaF
Fig. 5. Effect of Glibenclamide (A and B) on the antinociceptive activity of FATEM (25mg/kg, i.p.) in the formalin-induced orofacial nociception model. Each column represents the
mean ± S.E.M (n = 6/group). Numbers above the bars indicate the percentage of analgesia. One-way ANOVA with post-hoc Tukey’s test (*p < 0.05 and ***p < 0.001 vs control;
##p < 0.01 vs FATEM). Control: vehicle (3% DMSO).
Control 12,5 25 50 Naive
0
20
40
60
______________
*** ***
***
***
96.60%
72.45% 78.32%
76.17%
Fig. 6. Effect of FATEM (12.5; 25 or 50mg/kg, i.p.) on glutamate-induced orofacial no-
ciception in mice. Each column represents the mean ± S.E.M (n= 6/group). Numbers
above the bars indicate the percentage of analgesia. One-way ANOVA with post-hoc
Tukey’s test (***p < 0.001 vs control). Control: vehicle (3% DMSO). Naive: untreated
group.
Control 12,5 25 50 Naive
0
10
20
30
***
***
***
***
______________
96.76%
77.85%
86.47%
75.13%
gnibbu
Fig. 7. Effect of FATEM (12.5; 25 or 50mg/kg, i.p.) on capsaicin-induced orofacial no-
ciception in mice. Each column represents the mean ± S.E.M (n= 6/group). Numbers
above the bars indicate the percentage of analgesia. One-way ANOVA with post-hoc
Tukey’s test (***p < 0.001 vs control). Control: vehicle (3% DMSO). Naive: untreated
group.
Fig. 8. Effect of FATEM (12.5; 25 or 50mg/kg, i.p.) on acid saline-induced orofacial
nociception in mice. Each column represents the mean ± S.E.M (n=6/group). Numbers
above the bars indicate the percentage of analgesia. One-way ANOVA with post-hoc
Tukey’s test (***p < 0.001 vs control). Control: vehicle (3% DMSO). Naive: untreated
group.
Control 12,5 25 50
0
10
20
30
***
**
***
***
______________
89.17%
18.31%
56.02%
78.30%
Fig. 9. Effect of FATEM (12.5; 25 or 50mg/kg, i.p.) on cinnamaldehyde-induced orofacial
nociception in mice. Each column represents the mean ± S.E.M (n=6/group). Numbers
above the bars indicate the percentage of analgesia. One-way ANOVA with post-hoc
Tukey’s test (**p < 0.01; ***p < 0.001 vs control). Control: vehicle (3% DMSO). Naive:
untreated group.
F.E.A. Magalhães et al. Biomedicine & Pharmacotherapy 97 (2018) 1575–1585
1581
Nikodijevic et al. [41] report that benzyloxyamine acts as an inhibitor
of dopamine beta-oxidase. Thus, we believe that Benzyloxyamine may
be responsible for the anti-inflammatory potential of FATEM because
benzyloxyamines are inhibitors of histidine decarboxylase [40].
3-Fluorophenol, diisopropylsilyl ether was detected as the second
major constituent of FATEM (14.7%). The compounds derived from
fluorophenol are of pharmaceutical importance because they are pre-
cursors of antidiabetic, gastroprotective and antiepilepticdrugs; in
addition, they are used by the agrochemical industry in the production
of fungicides [42]. According to Chatzopoulou et al. [43], fluorophenol
derivatives have anti-inflammatory activity as they act as inhibitors of
oxidative enzymes in inflammatory processes caused by diabetes,
asthma and sepsis and in inflammation-mediated neoplasms such as
colon, lung, breast, and prostate cancers. Thus, we believe that the 3-
fluorophenol present in FATEM may also act as an anti-inflammatory
agent in the tests used.
Trimethylsilyl ether of glycerol (13.53%) and oleic acid (5.25%)
were detected as the fourth and fifth major constituents of FATEM.
Derivatives of these compounds are found to be antioxidants used in the
chemical, food, and pharmaceutical industries, as well as in human
medicine [44]. Figueroa et al. [45] report that fatty acids present acute
and chronic antinociceptive action and anti-inflammatory responses.
Thus, we believe that these constituents present in FATEM are also
responsible for the antioxidant and antinociceptive potential presented
in this study.
The absence of toxicity points to the potential phytotherapeutic use
of FATEM as samples nontoxic to A. salina and Vero cells will not be
toxic to humans [21,23].
Sensory information from the face is processed by the trigeminal
nerve, which is also related to motor activities such as biting, chewing
and swallowing. This nerve is divided into three main branches (oph-
thalmic nerve, maxillary nerve, and mandibular nerve) [46]. The an-
tinociceptive effect of FATEM was assessed in animal models involving
the three branches of the trigeminal nerve. FATEM prevented the no-
ciception caused by chemical stimulation of the maxillary and man-
dibular nerves.
FATEM was effective in attenuating acute neurogenic and tonic
inflammatory phases of the formalin response. The antinociceptive ef-
fect of FATEM was partially prevented by Naloxone, an opioid an-
tagonist, only in the 2nd phase (Fig. 3B), thus suggesting a potential
direct effect on the inflammatory process, as reported by Hunskaar and
Hole [47] and Clavelou et al. [48]. According to Capuano et al. [49],
naloxone also inhibited the peripheral antinociceptive effect of an-
algesic drugs in an in vivo model of trigenimal nociception. In the
present study, it did not inhibit the action of FATEM in the first phase of
the formalin test (Fig. 3A). However, Berg et al. [50] state that although
peripheral opioid receptors are expressed by primary afferents, they are
“inactive” under normal basal conditions and their functional activa-
tion occurs during inflammation.
Systemic administration of L-NAME, an inhibitor of nitric oxide
(NO) synthesis, inhibited the antinociceptive effect of FATEM only in
the first phase of the formalin test (Fig. 4A), thus suggesting its po-
tential direct effect on peripheral nociceptors [51]. Moore et al. [52]
reported that L-NAME produces both first- and second-phase anti-
nociception in the formalin test, possibly by a direct effect within the
central nervous system. However, the present results suggest that the
Fig. 10. Effect of Ruthenium Red on the antinociceptive activity of FATEM (25mg/kg,
i.p.) in the capsaicin-induced orofacial nociception model in mice. Each column re-
presents the mean ± S.E.M (n= 6/group). Numbers above the bars indicate the per-
centage of analgesia. One-way ANOVA with post-hoc Tukey’s test (**p < 0.01 and
***p < 0.001 vs control). Control: vehicle (3% DMSO).
Fig. 11. Effect of camphor on the antinociceptive activity of FATEM (25mg/kg, i.p.) in
the cinnamaldehyde-induced orofacial nociception model in mice. Each column re-
presents the mean ± S.E.M (n= 6/group). Numbers above the bars indicate the per-
centage of analgesia. One-way ANOVA with post-hoc Tukey’s test (**p < 0.01vs control).
Control: vehicle (3% DMSO).
Control F-25 A A + F-25
0
50
100
150
**
***
**
___________________
69.99%
83.47%
63.66%
Fig. 12. Effect of Amiloride on the antinociceptive activity of FATEM (25mg/kg, i.p.) in
the acid saline-induced orofacial nociception model in mice. Each column represents the
mean ± S.E.M (n= 6/group). Numbers above the bars indicate the percentage of an-
algesia. One-way ANOVA with post-hoc Tukey’s test (**p < 0.01 and ***p < 0.001 vs
control). Control: vehicle (3% DMSO).
Control 12,5 25 50 Naive
0
5
10
______________
100%
26.25% 21.34%
3.33%
***
Fig. 13. Effect of FATEM (12.5; 25 or 50mg/kg, i.p.) in the hypertonic saline-induced
corneal nociception model in mice. Each column represents the mean ± S.E.M (n= 6/
group). Numbers above the bars indicate the percentage of analgesia. One-way ANOVA
with post-hoc Tukey’s test (***p < 0.001 vs control). Control: vehicle (3% DMSO). Naive:
untreated group.
F.E.A. Magalhães et al. Biomedicine & Pharmacotherapy 97 (2018) 1575–1585
1582
peripheral mechanism may also contribute in part to the effect of sys-
temic L-NAME. Further, the dose of L-NAME did not result in complete
suppression of formalin-induced orofacial nociceptive responses in the
second phase (Fig. 4B), suggesting that NO is an important but not
exclusive factor in the induction of the second-phase orofacial noci-
ception.
The antinociceptive effect of FATEM also was partially prevented by
Glibenclamide, K+-ATP channel antagonist, thus suggesting the parti-
cipation of these systems in its action. Drugs that modulate anti-
nociception through the opioid system are also involved in the blockade
of nitric oxide synthesis and K+-ATP channels [53,54].
Nociception induced by glutamate (Fig. 6), capsaicin (Fig. 7) or
acidic saline (Fig. 8) was also attenuated by FATEM in mice, suggesting
an antagonistic action on glutamatergic, vanilloid and ASIC receptors.
This non-specific effect may be due to phenolic compounds with anti-
oxidant activity present in the fraction, as reported by Jaios et al. [55].
In addition, it is important to emphasize the involvement of TRP
channels, particularly the TRPV1 receptor and its interrelationship with
the glutamatergic system. According to Dal Bó et al. [56], the activation
of TRPV1 induces the release of glutamate; therefore, blocking this
receptor may reduce the nociception induced by this amino acid.
The results of the present study also suggest that the effect of
FATEM on glutamate-induced nociception may occur, at least in part,
due to the reduction of glutamate release via TRPV1 receptor blockade.
However, the antinociceptive effect of FATEM was not blocked by ru-
thenium red (TRPV1 channels antagonist; Fig. 10) or by amiloride
(ASICs antagonist; Fig. 12). Thus, further studies are needed to detect
the possible neuromodulatory effects of FATEM.
FATEM inhibited the nociceptive behavior of mice induced by for-
malin (Fig. 2A) or cinnamaldehyde (Fig. 6) – both are TRPA1 agonists.
However, this effect was not reversed by camphor (TRPA1 antagonist)
(Fig. 11), which suggests that the orofacial antinociceptive effect of
FATEM is not related to the TRPA1 receptor.
As reported previously, FATEM does not appear to act on TRPV1
channels; therefore, it did not prevent hypertonic saline-induced eye
rubbing behavior, which is related to the activation of TRPV1 receptors
in the trigeminal subnucleus caudalis [57].
Temporomandibular disorders are musculoskeletal pain conditions
characterized by pain in the temporomandibular joint (TMJ) and/or
masticatory muscles. They are the most common cause of chronic facial
pain conditions [58]. Mustard oil (MO) [58] and formalin [34] have
been used to activate nociceptors in a variety of animal models. In the
present study, TMJ or craniofacial nociception induced by formalin or
mustard oil was prevented by FATEM (25 mg/kg) (Table 2), thus sug-
gesting its use for the treatment of these disorders.
Trigeminal neuralgia is known as one of the most painful diseases in
humans and is characterized by recurrent episodes of stabbing or
electric shock-like pain localized to one side of the face, including the
oral cavity [59]. This disorder wasassessed in the present study using
an animal model of neuropathic orofacial pain after chronic constric-
tion of the infraorbital nerve. FATEM (25mg/kg) increased the
threshold of responsiveness to mechanical stimulation (Fig. 15).
Table 2
Effect of FATEM on formalin- and mustard oil-induced TMJ nociception in mice.
Group (Dose) Formalin Mustard oil
A-Face Rubbing (s) (A%) B-Head Flinching (s) (A%) C-Face Rubbing (s) (A%) D-Head Flinching (s) (A%)
Control 43.60 ± 13.96 (0.00%) 52.67 ± 13.41 (0.00%) 15.50 ± 3.94 (0.00%) 33.83 ± 3.54 (0.00%)
Naive 2.33 ± 0.56**(94.66%) 2.33 ± 0.92***(95.58%) 1.67 ± 0.61**(89.23%) 3.67 ± 0.76***89.15%
Sham 4.83 ± 1.08**(88.92%) 9.83 ± 3.82**(81.34%) 3.50 ± 1.06**(77.42%) 6.50 ± 1.61***(80.79%)
FATEM(25mg/kg) 9.67 ± 3.49**(77.81%) 19.00 ± 3.28*(63.93%) 3.33 ± 1.36**(78.52%) 8.33 ± 2.19***(75.38%)
A% – Percentage of analgesia [A%= FR% or HF% (Control) – FR% or HF% (Sample)], where FR – Face rubbing; HF – head flinching. Each column represents the mean ± S.E.M (n = 6/
group). Analysis of Variance ANOVA with post-hoc Tukey’s test (*p < 0.05; **p < 0.01; ***p < 0.001 vs control). Control: vehicle (3% DMSO). Naive: untreated group. Sham: treated
with vehicle and saline in the TMJ.
Control 25 Sham Naive
0
10
20
30
**
***
***
100%
71.70%
93.97%
_____
Fig. 14. Effect of FATEM (25mg/kg, i.p.) in the mustard oil-induced craniofacial noci-
ception model in rats. Each column represents the mean ± S.E.M (n= 6/group).
Numbers above the bars indicate the percentage of analgesia. One-way ANOVA with post-
hoc Tukey’s test (**p < 0.01; ***p < 0.001 vs control). Control: vehicle (3% DMSO).
Naive: untreated group. Sham: treated with vehicle and saline in the masseter muscle.
BA
SE 1P
O
3P
O
5P
O
7P
O
10
PO
14
PO
21
PO
60
80
100
120
CONTROLE
SHAM
NAIVE
*
* *s
Fig. 15. Effect of FATEM (25mg/kg, i.p.) on neuropathic nociception induced by infra-
orbital nerve transection in rats. PO=Postoperatively. Two-way ANOVA with post-hoc
Bonferroni correction (*p < 0.05 vs control). Control: vehicle (0.9% saline). Naive: un-
treated and unoperated group. Sham: Fake operation and treatment with saline.
Fig. 16. Effect of FATEM (12.5; 25 or 50mg/kg, i.p.) on locomotor activity of mice
(n=6/group) in the open field test. Each column represents the mean ± S.E.M (n=6/
group). One-way ANOVA with post-hoc Tukey’s test. Control: vehicle (3% DMSO). Naive:
untreated group.
F.E.A. Magalhães et al. Biomedicine & Pharmacotherapy 97 (2018) 1575–1585
1583
The antinociceptive effect of FATEM may be related to the presence
of antioxidant compounds in the fraction. These compounds can elim-
inate free radicals that are produced in nociceptive processes and which
are responsible for modulating pain during these processes [4,6]. Stu-
dies have shown that oxidative stress modifies experimental nocicep-
tion [60–62] and antioxidants ameliorate nociception [63,64]. The
antinociceptive potential of FATEM in the present study is consistent
with earlier reports on plant-derived substances [65–67] with anti-
oxidant properties. Therefore, studying the pain modulation effects of
antioxidants is considered an emerging area of interest [68].
The fact that FATEM did not interfere with locomotor activities in
the open-field test rules out non-specific FATEM-induced muscle re-
laxation and supports the nociceptive test results of the present study.
In conclusion, the present study showed the potential pharmacolo-
gical relevance of FATEM as an inhibitor of orofacial nociception. Our
findings suggest that the antinociceptive effect of FATEM on acute and
neuropathic orofacial pain seems to be modulated, in part, by the
opioid system, nitric oxide and ATP-sensitive potassium channels.
These results encourage the continuation of the study in order to isolate
and characterize the active agent, which may lead to the development
of a new drug for the treatment of orofacial pain.
Conflict of interest
We declare that we have no conflict of interest.
Acknowledgements
We would like to thank CNPq, Funcap and Capes for financial
support, the Medical Sciences Graduate Program of the University of
Fortaleza, the Edson Queiroz Foundation for infrastructure, and the
Ceará State University for the release of Prof. Dr. Francisco Ernani Alves
Magalhães for the development of his postdoctoral training.
References
[1] D.A. Bereiter, K.M. Hargreaves, J.W. Hu, Trigeminal mechanisms of nociception:
Peripheral and Brainstem organization, in: M.C.B.A. Bushnell (Ed.), The Senses, A
Comprehensive Reference, Academic Press, San Diego, 2008, pp. 435–460.
[2] A.R. Castro, S.R.D.T. Siqueira, D.M.N. Perissionotti, M.J. Teixeira, J.T.T. Siqueira,
Emotional aspects of chronic orofacial pain and surgical treatment, Int. J. Surg. 7
(2009) 196–199.
[3] H.P. Rang, M.M. Dale, J.M. Ritter, R.J. Flower, G. Henderson, Farmacologia, 7ª ed.,
Elsevier, Rio de Janeiro, 2012.
[4] S.O. Onoja, M.I. Ezeja, Y.N. Omeh, B.C. Onwukwe, Antioxidant, anti-inflammatory
and antinociceptive activities of methanolic extract of Justicia secunda Vahl leaf,
Alexandria J. Med. 53 (2017) 207–213.
[5] D. Salvemini, T.M. Doyle, S. Cuzzocrea, Superoxide, peroxynitrite and oxidative/
nitrative stress in inflammation, Biochem. Soc. Trans. 34 (5) (2006) 965–970.
[6] A.S. Rabelo, I.D. Oliveira, A.G. Guimarães, J.S.S. Quitans, A.P.N. Prata, D.P. Gelain,
E.M. Venceslau, J.P.A. Santos, L.J. Quintans-Júnior, L.R. Bonjardim, A. Barison,
F.R. Campos, A.D.C. Santos, P.C.L. Nogueira, E.V. Costa, V.R.S. Moraes,
A.A.S. Araújo, Antinociceptive, anti-inflammatory and antioxidant activities of
aqueous extract from Remirea maritima (Cyperaceae), J. Ethnopharmacol. 145
(2013) 11–17.
[7] K. Bouterfas, Z. Mehdadi, M.M. Elaoufi, A. Latreche, W. Benchiha, Antioxidant
activity and total phenolic and flavonoids content variations of leaves extracts of
white Horehound (Marrubium vulgare Linné) from three geographical origins, Ann.
Pharm. Françaises 74 (6) (2016) 453–462.
[8] S.L. Cartaxo, M.M.A. Souza, U.P. de Albuquerque, Medicinal plants with biopros-
pecting potential used in semi-arid Northeastern Brazil, J. Ethnopharmacol. 131 (2)
(2010) 326–342.
[9] D.C. Paiva, C.A. dos Santos, J.C. Diniz, F.A. Viana, S.M. Thomazzi, D.A. Falcão,
Anti-inflammatory and antinociceptive effects of hydroalcoholic extract from
Pseudobombax marginatum inner bark from Caatinga Potiguar, J. Ethnopharmacol.
149 (2013) 416–442.
[10] M.P. Cruz, C.M.F. Andrade, K.O. Silva, E.P. de Souza, R. Yatsuda, L.M. Marques,
J.P. David, M.H. Napimoga, T. Clemente-Napimoga, Antinoceptive and anti-in-
flammatory activities of the ethanolic extract, fractions and flavones isolated from
Mimosa tenuiflora (Willd.) Poir (Leguminosae), PLoS One 11 (3) (2016) 1–29.
[11] M.J.D. Silva, L.H. Endo, A.L. Tranches Dias, A.G. Silva, M.H. Santos, M.A. Silva,
Avaliação da atividade antioxidante e antimicrobiana dos extratos e frações
orgânicas de Mimosa caesalpiniifolia Benth. (Mimosaceae), Rev Ciênc Farm Básica
Apl. 33 (2) (2012) 267–274.
[12] F.J.A. Matos, Introduction to Experimental Phytochemical. Editions UFC, (1997)
Fortaleza, Brazil.
[13] L.N. Seito, J.M. Sforcin, J.K. Bastos, L.C. Di Stasi, Zeyheria montana Mart.
(Bignoniaceae) as source of antioxidant and immunomodulatory compounds with
beneficial effects on intestinal inflammation, J. Pharm. Pharmacol. 67 (2014)
597–604.
[14] P.S.P. Hidalgo, R.C.S. Nunomura, S.M. Nunomura, Plantas oleaginosas amazônicas:
química e atividade antioxidante de patauá (Oenocarpus bataua Mart.), Revista
Virtual de Química 8 (1) (2016) 130–140.
[15] L. Mosca, C. Marco, F. Visioli, C. Cannella, Enzymatic assay for the determination of
olive oil polyphenol content: assay conditions and validation of the method, J.
Agric. Food Chem. 48 (2000) 297–301.
[16] A. Campos, E. Lissi, Evaluation of the antioxidant capacity of herbal teas by a
procedure based on the bleaching of ABTS radical cations, Bol. Soc. Chil. Quim. 40
(1995) 375–381.
[17] J. Sauro, J.R. Lewis, Quantifying the User Experience.Practical Statistics for User
Research, 2ª ed., Morgan Kaufmann, Cambridge, United States, 2016.
[18] Farmacopeia Brasileira, v.1 (2010): métodos gerais, 5ª ed., ANVISA; Fundação
Oswaldo Cruz, Brasília, 2010.
[19] K. Slinkard, V.L. Singleton, Total phenol analysis: automation and comparison with
manual methods, Am. J. Enol. Viticult. 28 (1977) 49–55.
[20] J.P. Karmeling, J.F.G. Vliegenthart, C. Verluis, R. Schauer, Identification of O-
acetylated N-acylneuraminic acids by mass spectrometry, Carbohydr. Res. 41
(1975) 7–17.
[21] B.N. Meyer, N.R. Ferrigni, J.E. Putnan, L.B. Jacobsen, D.E. Nichols, J. McLaughlin,
Brine shrimp: a convenient general bioassay for active plant constituents, J. Med.
Plants Res. 45 (1982) 31–34.
[22] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1–2) (1983)
55–63.
[23] S.A. Fadeyi, O.O. Fadeyi, A.A. Adejumos, C. Okoro, E.L. Myles, In vitro anticancer
screening of 24 locally used Nigerian medicinal plants, BMC Complement. Altern.
Med. 13 (2013) 79–87.
[24] P. Luccarini, A. Childeric, A.M. Gaydier, D. Voisin, R. Dallel, The orofacial formalin
test in the mouse: a behavioral model for studying physiology and modulation of
trigeminal nociception, J. Pain 7 (12) (2006) 908–914.
[25] M.B.M.V. Damasceno, J.M.A. de Melo Júnior, S.A.A.R. Santos, L.T.M. Melo,
L.H.I. Leite, A.E. Vieira-Neto, R.A. Moreira, A.C.O. Monteiro-Moreira, A.R. Campos,
Frutalin reduces acute and neuropathic nociceptive behaviours in rodent models of
orofacial pain, Chem. Biol. Interact. 256 (2016) 9–15.
[26] L.H.I. Leite, G.O. Leite, T.S. Coutinho, S.D.G. de Sousa, R.S. Sampaio, J.G.M. da
Costa, I.R.A de Menezes, A.R. Campos, Topical antinociceptive effect of
Vanillosmopsis arbórea baker on acute corneal pain in mice, Evid. Based
Complement. Altern. Med. 2014 (2014) 1–6.
[27] L.J. Quitans-Júnior, M.S. Melo, D.P. Sousa, A.A.S. Araújo, A.C.S. Onofre,
D.P. Gelain, J.C.R. Gonçalves, A.A.M. Araújo, J.R.G.S. Almeida, L.R. Bonjardim,
Antinociceptive effects of citronella in formalin, capsaicin, and glutamate – induced
orofacial nociception in rodents and its action on nerve excitability, J. Orofacial
Pain 24 (3) (2010) 305–312.
[28] T. Pelisser, J. Pajot, R. Dallel, The orofacial capsaicin test in rats: effects of different
capsaicin concentrations and morphine, Pain 96 (2002) 81–87.
[29] J.M.A. de Melo Júnior, M.B.M.V. Damasceno, S.A.A.R. Santos, T.M. Barbosa,
J.R.C. Araújo, A.E. Vieira-Neto, D.V.T. Wong, R.C.P. Lima-Júnior, A.R. Campos,
Acute and neuropathic orofacial antinociceptive effect of eucalyptol,
Inflammopharmacol 25 (2) (2017) 247–254.
[30] E.C.O. Nomura, M.R.A. Rodrigues, C.F. da Silva, L.A. Hamm, A.M. Nascimento,
L.M. de Souza, T.R. Cipriani, C.H. Baggio, M.F.P. Werner, Antinociceptive effects of
ethanolic extract from the flowers of Acmella oleracea (L.) R.K. Jansen in mice, J.
Ethnopharmacol. 150 (2013) 583–589.
[31] M.M. Córdova, M.F. Werner, M.D. Silva, A.P. Ruani, M.G. Pizzolatti, A.R.S. Santos,
Further antinociceptive effects of myricitrin in chemical models of overt nocicep-
tion in mice, Neurosci. Lett. 495 (3) (2011) 173–177.
[32] C.H. Baggio, C.S. Freitas, R. Marcon, M.F.P. Werner, G.A. Rae, F.R. Smiderle,
G.L. Sassaki, M. Iacomini, M.C.A. Marques, A.R.S. Santos, Antinociception of beta-D-
glucan from Pleurotus pulmonarius is possibly related to protein kinase C inhibition,
Int. J. Biol. Macromol. 50 (2012) 872–877.
[33] R. Farazifard, F. Safapour, V. Sheibani, M. Javan, Eyewiping test: a sensitive animal
model for acute trigeminal pain studies, Brain Res. Brain Res. Protoc. 16 (2005)
44–49.
[34] R.C. Roveroni, C.A. Parada, M.C.F.A. Veiga, C.H. Tambeli, Development of a be-
havioral model of TMJ pain in rats: the TMJ formalin test, Pain (Amsterdam) 94 (2)
(2001) 185–191.
[35] Q. Zhou, H. Imbe, R. Dubner, K. Ren, Persistent Fos protein expression after or-
ofacial deep or cutaneous tissue inflammation in rats: implications for persistent
orofacial pain, J. Comp. Neurol. 412 (1999) 276–291.
[36] J.Y. Ro, N. Capra, R. Masri, Development of a behavioral assessment of craniofacial
muscle pain in lightly anesthetized rats, Pain 104 (2003) 179–185.
[37] N. Kumar, P.S. Cherkas, V. Varathan, M. Miyamoto, C.Y. Chiang, J.O. Dostrovsky,
B.J. Sessle, T.J. Coderre, Systemic pregabalin attenuates facial hypersensitivity and
noxious stimulus-evoked release of glutamate in medullary dorsal horn in a rodent
model of trigeminal neuropathic pain, Neurochem. Int. 62 (2013) 831–835.
[38] E.O. Sousa, C.M.B.A. Miranda, C.B. Nobre, A.A. Boligon, M.L. Athayde,
J.G.M. Costa, Phytochemical analysis and antioxidant activities of Lantana camara
and Lantana montevidensis extracts, Ind. Crops Prod. 70 (2015) 7–15.
[39] A.R.A. da Silva, S.M. de Morais, M.M.M. Marques, D.F. de Oliveira, C.C. Barros,
R.R. de Almeida, I.G.P. Vieira, M.I.F. Guedes, Chemical composition, antioxidant
and antibacterial activities of two Spondias species from Northeastern Brazil,
F.E.A. Magalhães et al. Biomedicine & Pharmacotherapy 97 (2018) 1575–1585
1584
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0005
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0005
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0005
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0010
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0010
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0010
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0015
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0015
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0020
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0020
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0020
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0025
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0025
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0030
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0030
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0030
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0030
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0030
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0030
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0035
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0035
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0035
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0035
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0040
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0040
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0040
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0045
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0045
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0045
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0045
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0050
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0050
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0050
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0050
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0055
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0055
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0055
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0055
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0060
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0060
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0065
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0065
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0065
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0065
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0070
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0070
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0070
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0075
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0075
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0075http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0080
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0080
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0080
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0085
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0085
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0090
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0090
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0095
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0095
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0100
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0100
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0100
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0105
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0105
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0105
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0110
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0110
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0110
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0115
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0115
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0115
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0120
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0120
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0120
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0125
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0125
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0125
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0125
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0130
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0130
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0130
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0130
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0135
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0135
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0135
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0135
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0135
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0140
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0140
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0145
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0145
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0145
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0145
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0150
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0150
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0150
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0150
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0155
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0155
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0155
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0160
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0160
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0160
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0160
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0165
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0165
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0165
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0170
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0170
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0170
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0175
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0175
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0175
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0180
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0180
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0185
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0185
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0185
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0185
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0190
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0190
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0190
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0195
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0195
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0195
Pharm. Biol. 50 (6) (2012) 740–746.
[40] G.H. Hamor, D.M. Breslow, G.W. Fisch, Benzyloxyamines as possible inhibitors of
histamine biosynthesis, J. Pharm. Sci. 59 (12) (1970) 1752–1756.
[41] B. Nikodijevic, C.R. Creveling, S. Udenfriend, Inhibition of dopamine beta-oxidase
in vivo by benzyloxiamine and benzylhydrazine analogs, J. Pharmacol. Exp. Ther.
140 (2) (1963) 224–228.
[42] C. Mercier, P. Youmans, 4-Fluorophenol: a key intermediate for agrochemical and
pharmaceuticals, Ind. Chem. Library 8 (1999) 293–300.
[43] A. Chatzopoulou, A. Patsilinakos, T. Vallianatou, M.S. Prnova, S. Zakelj, R. Ragno,
M. Stefek, A. Kristl, A. Tsantili-Kakoulidou, J. Demo, Decreasing acidity in a series
of aldose reductase inhibitors: 2-fluoro-4-(1H-pyrrol-1-yl)phenol as a scaffold for
improved membrane permeation, Bioorg. Med. Chem. 22 (2014) 2194–2207.
[44] M. Kozlowska, E. Gruczyńska, I. Ścibisz, M. Rudzińska, Fatty acids and sterols
composition, and antioxidant activity of oils extracted from plant seeds, Food
Chem. 5 (213) (2016) 450–456.
[45] J.D. Figueroa, K. Cordero, M. Serrano-Illan, A. Almeyda, K. Baldeosingh,
F.G. Almaguel, M. de Leon, Metabolomics uncovers dietary ômega-3 fatty acid-
derived metabolites implicated in antinociceptive responses after experimental
spinal cord injury, Neuroscience 255 (2013) 1–18.
[46] A.C.B. de Lima, M.S. Paixão, M. Melo, M.T. de Santana, N.P. Damascena, A.S. Dias,
Y.C.B.S. Porto, X.A. Fernandes, C.C.S. Santos, C.A. Lima, L. Quitans-Júnior,
C.S. Estevam, B.S. Araújo, Orofacial antinociceptive effect and antioxidant prop-
erties of the hydroethanol extract of Hyptis fruticosa salmz ex Benth, J.
Ethnopharmacol. 146 (2013) 192–197.
[47] S. Hunskaar, K. Hole, The formalin test in mice: dissociation between inflammatory
and non-inflammatory pain, Pain 30 (1987) 103–114.
[48] P. Clavelou, R. Dallel, T. Orliaguet, A. Woda, P. Raboisson, The orofacial formalin
test in rats: effects of different formalin concentrations, Pain 62 (1995) 295–301.
[49] A. Capuano, A. de Corato, M. Treglia, G. Tringali, D. Currò, C.D. Russo, P. Navarra,
Peripheral antinociceptive effects of low doses of naloxone in an in vivo and in vitro
model of trigeminal nociception, Neuropharmacology 58 (2010) 784–792.
[50] K.A. Berg, A.M. Patwardhan, T.A. Sanchez, Y.M. Silva, K.M. Hargreaves,
W.P. Clarke, Rapid modulation of micro-opioid receptor signaling in primary sen-
sory neurons, J. Pharmacol. Exp. Ther. 321 (2007) 839–847.
[51] P.K. Janicki, M. Jeske-Janicka, Relevance of nitric oxide in pain mechanisms and
pain management, Curr. Rev. Pain 2 (1998) 211–221.
[52] P.K. Moore, A.O. Oluyomi, R.C. Babbedge, P. Wallace, S.L. Hart, L-NG-nitroarginine
methyl ester exhibits antinociceptive activity in the mouse, Br. J. Pharmacol. 102
(1991) 198–202.
[53] G.G. Lazaro-Ibanez, J.E. Torre-Lopez, V. Granados-Soto, Participation of the nitric
oxide–cyclic GMP–ATP-sensitive Kq channel pathway in the antinociceptive action
of ketorolac, Eur. J. Pharmacol. 426 (2001) 39–44.
[54] B. Ghorbanzadeh, M.T. Mansouri, A.A. Hemmati, B. Naghizadeh, S.A. Mard,
A. Rezaie, Involvement of L-arginine/NO/cGMP/KATP channel pathway in the
peripheral antinociceptive actions of ellagic acid in the rat formalin test, Pharmacol.
Biochem. Behav. 126 (2014) 116–121.
[55] E.S. Jaios, S.A. Rahmand, S.M. Ching, A.A. Kadir, M.N.M. Desa, Z.A. Zakaria,
Possible mechanisms of antinociception of methanol extract of Melastoma malaba-
thricum leaves, Revista Brasileira de Farmacognosia 26 (2016) 586–594.
[56] W. Dal Bó, A.P. Luiza, D.F. Martins, L. Mazzardo-Martins, A.R.S. Santos, Eugenol
reduces acutepain in mice by modulating the glutamatergic and tumor necrosis
factor alpha (TNF-alfa) pathways, Fundamental and Clin. Pharmacol. 27 (5) (2013)
517–525.
[57] S. Ciura, C.W. Bourque, Transient receptor potential vanilloid 1 is required for in-
trinsic osmoreception in organum vasculosum lamina terminalis neurons and for
normal thirst responses to systemic hyperosmolality, J. Neurosci. 26 (35) (2006)
9069–9075.
[58] L.R. Bonjardim, A.P. da Silva, G.H. Gameiro, C.H. Tambeli, M.C.F.A. Veiga,
Nociceptive behavior induced by mustard oil injection into the temporomandibular
joint is blocked by a peripheral non-opioid analgesic and a central opioid analgesic,
Pharmacol. Biochem. Behav. 91 (2009) 321–326.
[59] H. Jeon, S.R. Han, M.K. Park, K. Yang, Y.C. Ba, D.K. Ahn, A novel trigeminal
neuropathic pain model: compression of the trigeminal nerve root produces pro-
longed nociception in rats, Prog. Neuro-Psychopharmacol. Biol. Psychiatry 38
(2012) 149–158.
[60] A. Viggiano, M. Monda, A. Viggiano, D. Viggiano, E. Viggiano, M. Chiefari,
C. Aurilio, B. de Luca, Trigeminal pain transmission requires reactive oxygen spe-
cies production, Brain Res. 1050 (2005) 72–78.
[61] B.B. Garrido-Suárez, G. Garrido, R. Delgado, F. Bosch, M. del C Rabí, A Mangifera
indica L. extract could be used to treat neuropathic pain and implication of man-
giferin, Molecules 15 (2010) 9035–9045.
[62] F. Ma, L. Zhang, K.N. Westlund, Reactive oxygen species mediate TNFR1 increase
after TRPV1 activation in mouse DRG neurons, Mol. Pain 5 (2009) 31–42.
[63] R. Rokyta, V. Holecek, I. Pekárkova, J. Krejcová, J. Racek, L. Trefil,
A. Yamamotová, Free radicals after painful stimulation are influenced by anti-
oxidants and analgesics, Neuro Endocrinol. Lett. 24 (2003) 304–309.
[64] A. Hacimuftuoglu, C.R. Handy, V.M. Goettl, C.G. Lin, S. Dane, R.L. Stephens Jr,
Antioxidants attenuate multiple phases of formalin-induced nociceptive response in
mice, Behav. Brain Res. 173 (2006) 211–216.
[65] R.G. Brito, A.G. Guimarães, J.S. Quintans, M.R. Santos, D.P. de Sousa, D. Badaue-
Passos Jr, W. de Lucca Jr., F.A. Brito, E.O. Barreto, A.P. Oliveira, L.J. Quintans Jr.,
Citronellol, a monoterpene alcohol, reduces nociceptive and inflammatory activities
in rodents, J. Nat. Med. 66 (2012) 637–644.
[66] D.A. Valério, S.R. Georgetti, D.A. Magro, R. Casagrande, T.M. Cunha,
F.T.M.C. Vicentini, S.M. Vieira, M.J.V. Fonseca, S.H. Ferreira, F.Q. Cunha,
W.A. Verri Jr., Quercetin reduces inflammatory pain: inhibition of oxidative stress
and cytokine production, J. Nat. Prod. 72 (2009) 1975–1979.
[67] V. Granados-Soto, C.F. Argüelles, M.I. Ortiz, The peripheral antinociceptive effect of
resveratrol is associated with activation of potassium channels, Neuropharmacology
43 (2002) 917–923.
[68] S.C. Lopes, A.V. da Silva, B.R. Arruda, T.C. Morais, J.B. Rios, M.T.S. Trevisan,
V.S. Rao, F.A. Santos, Peripheral antinociceptive action of mangiferin in mouse
models of experimental pain: role of endogenous opioids, KATP-channels and
adenosine, Pharmacol. Biochem. Behav. 110 (2013) 19–26.
F.E.A. Magalhães et al. Biomedicine & Pharmacotherapy 97 (2018) 1575–1585
1585
View publication statsView publication stats
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0195
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0200
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0200
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0205
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0205
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0205
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0210
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0210
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0215
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0215
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0215
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0215
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0220
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0220
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0220
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0225
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0225
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0225
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0225
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0230
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0230
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0230
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0230
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0230
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0235
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0235
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0240
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0240
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0245
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0245
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0245
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0250
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0250
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0250
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0255
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0255
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0260
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0260
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0260
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0265
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0265
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0265
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0270
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0270
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0270
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0270
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0275
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0275
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0275
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0280
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0280
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0280
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0280
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0285
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0285
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0285
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0285
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0290
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0290
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0290
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0290
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0295
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0295
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0295
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0295
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0300
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0300
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0300
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0305
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0305
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0305
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0310
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0310
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0315
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0315
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0315
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0320
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0320
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0320
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0325
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0325
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0325
http://refhub.elsevier.com/S0753-3322(17)34139-2/sbref0325