Artigo Científico

1. Contents lists available at ScienceDirect
Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm
Effects of fixed oil of Caryocar coriaceum Wittm. Seeds on the respiratory
system of rats in a short-term secondhand-smoke exposure model
Daniel Silveira Serraa,∗
, Allison Matias de Sousab
, Leidianne Costa da Silva Andradeb
,
Fladimir de Lima Gondimb
, João Evangelista de Ávila dos Santosc
, Mona Lisa Moura de Oliveiraa
,
Antônia Torres Ávila Pimentaa
a
Center of Technological Sciences, State University of Ceará, Av. Dr. Silas Munguba, 1700, 60714-903, Fortaleza-Ceará, Ceará, Brazil
b
Institute of Biomedical Sciences, State University of Ceará, Ceará, Brazil
c
Science Center, Federal University of Ceará, Fortaleza, CE, Brazil
A R T I C L E I N F O
Keywords:
Caryocar coriaceum
Pequi
Fatty acids
Respiratory mechanics
SHS exposure
A B S T R A C T
Ethnopharmacological relevance: Pequi fruit are obtained from the pequi tree (Caryocar coriaceum), from which
the pulp and nut are used in order to extract an oil that is commonly used in popular medicine as an antiin-
flammatory agent, particularly for the treatment of colds, bronchitis and bronchopulmonary infections. Making
use of the fixed oil of Caryocar coriaceum (FOCC), an attractive alternative for the treatment of diseases caused by
exposure to environmental tobacco smoke.
Aim of the study:: To evaluate whether oral intake FOCC provides beneficial effects in the respiratory system of
rats submitted to a short-term secondhand smoke (SHS) exposure model.
Materials and methods: The experiments were performed on Wistar rats divided into 4 groups; in the SHS + O
and SHS + T groups, the animals were pretreated orally with 0.5 mL of FOCC (SHS + O) or vehicle (Tween-80
[1%] solution) (SHS + T). Immediately after pretreatment, the animals were submitted to the SHS exposure
protocol, for a total period of 14 days. Exposures were performed 6 times per day, with a duration of 40 min per
exposure (5 cigarettes per exposure), followed by a 1-h interval between subsequent exposures. In the AA + O
and AA + T groups, animals were submitted to daily oral pretreatment with 0.5 mL of FOCC (AA + O) or
vehicle (AA + T). These animals were then subjected to the aforementioned exposure protocol, but using am-
bient air. After the exposure period, we investigated the effects of FOCC in respiratory mechanics in vivo
(Newtonian resistance -RN , tissue elastance -H, tissue resistance -G, static compliance -CST, inspiratory capacity
-IC, PV loop area) histopathology and lung parenchymal morphometry in vitro (polymorphonuclear cells –PMN,
mean alveolar diameter -Lm, bronchoconstriction index -BCI), temporal evolution of subjects’ masses, and
percent composition of the FOCC.
Results: Regarding the body mass of the animals, the results demonstrated an average body mass gain of 10.5 g
for the animals in the AA + T group, and 15.5 g for those in the AA + O group. On the other hand, the body
mass of animals in the SHS + T and SHS + O suffered an average loss of 14.4 and 4.75 g, respectively.
Regarding respiratory system analyzes, our results demonstrated significant changes in all respiratory mechanics
variables and lung parenchyma morphometry analyzed for the SHS + T group when compared to the AA + T
group (p < 0,05), confirming the establishment of pulmonary injury induced by SHS exposure. We also ob-
served that rats pretreated orally with FOCC (SHS + O) showed improvement in all variables when compared to
the SHS + T group (p < 0,05), thus demonstrating the effectiveness of FOCC in preventing lung damage
induced by short-term SHS exposure.
Conclusion: In conclusion, our results demonstrate that FOCC was able to prevent lung injury in rats submitted to
short-term SHS exposure.
https://doi.org/10.1016/j.jep.2020.112633
Received 16 September 2019; Received in revised form 21 January 2020; Accepted 23 January 2020
∗
Corresponding author.
E-mail address: daniel.silveira@uece.br (D. Silveira Serra).
Journal of Ethnopharmacology 252 (2020) 112633
Available online 27 January 2020
0378-8741/ © 2020 Elsevier B.V. All rights reserved.
T

2. 1. Introduction
Tobacco is one of the main causes of avoidable death worldwide.
Approximately 10 million deaths are estimated to occur due to tobacco-
related diseases, with 70% of these deaths projected to arise in devel-
oping nations (Pan American Health Organization, 2018). Although a
significant number of countries have implemented public health po-
licies in order to reduce smoking and secondhand smoke (SHS) ex-
posure, many others still need to take place in order to prevent cigar-
ette-related cancers (UN - United Nations, 2019). Lung cancer due to
SHS causes an estimated 21,400 deaths in non-smokers annually (Oberg
et al., 2011).
Environmental tobacco smoke is produced from the smoldering end
of cigarettes between puffs; it consists of 85% in the form of sidestream
smoke (SS) and 15% as exhaled smoke, referred to as mainstream
smoke (MS). Toxic compounds such as ammonia, volatile amines and
nitrosamines, nicotine decomposition products, and aromatic amines
are found in higher concentrations in undiluted SS, when compared to
undiluted MS (DiGiacomo et al., 2019).
A study of the Korean National Health and Nutrition Examination
Survey (KNHANES) was conducted from 2010 to 2012, with 10,532
never-smokers (8987 females and 1545 males) who were exposed daily
to SHS; the authors concluded that SHS is significantly associated with
hypertension in female never-smokers (Park et al., 2018). In a review,
the authors evaluated the existing biological evidence regarding SHS
exposure and concluded that brief, acute, transient exposures to SHS
may cause important adverse impacts on several systems of the human
body, and thus represent a significant and acute health hazard (Flouris
et al., 2009).
Moreover, exposure to SHS my induce pulmonary inflammation
(Bhat et al., 2018; Muthumalage et al., 2017), and is associated with
vascular inflammation (Adams et al., 2015), oxidative stress
(Muthumalage et al., 2017), adverse cardiovascular effects (Barnoya
and Glantz, 2005; Venn and Britton, 2007), as well as exacerbation of
upper respiratory allergies (Diaz-Sanchez et al., 2006). The knowledge
that cigarettes cause health issues not only in those who utilize them,
but also in individuals exposed to SHS, killing around 1.2 million of
people a year worldwide (World Health Organization, 2019), justifies
the need for studies to evaluate their impacts on the respiratory system,
as well as novel pharmacological alternatives that are capable of alle-
viating the effects of exposure to environmental tobacco smoke. Among
these alternatives, natural products may be an important option.
Natural products with medicinal properties are commonly used
worldwide. Popular observations regarding the use and effectiveness of
medicinal plants contribute to expressive hearsay about the purported
therapeutic qualities of plant matter. Although their chemical con-
stituents have never been elucidated, these substances are still fre-
quently prescribed due to the supposed medicinal effects they produce
(Maia-Filho et al., 2011).
A highly attractive alternative for the treatment of diseases caused
by exposure to environmental tobacco smoke may be found in fixed oil
of Caryocar coriaceum (FOCC). Pequi fruit are obtained from the pequi
tree (Caryocar coriaceum) native of dry plain areas in the northeastern
region of Brazil (Oliveira et al., 2010), from which the pulp and nut are
specifically used in order to extract an oil that is commonly used in folk
medicine for the treatment of colds and flu, rheumatism, external ul-
cers, muscle pain, and inflammation (Agra et al., 2007). Its therapeutic
properties are reportedly due to its high total phenol content, as well as
for its fatty acids, which are important contributors to its antioxidant
and antiinflammatory activity (Sena et al., 2010). The essential fatty
acids are believed to have important antiinflammatory effects on the
organism and are used as nutritional treatments for skin diseases, ar-
thritis and respiratory ailments, such as asthma (Yehuda et al., 1997;
Boissonneault, 2000; Hassig et al., 2000).
The present work studies the benefits of orally-ingested of FOCC as a
pharmacologic alternative in the treatment of respiratory disease in rats
exposed to environmental tobacco smoke. We investigated the effects of
FOCC in respiratory mechanics in vivo and in histopathology and lung
parenchymal morphometry analyses in vitro of animals submitted to a
short-term secondhand smoke (SHS) exposure model.
2. Materials and methods
2.1. Plant material
Fixed oil from the seeds of Caryocar coriaceum (FOCC) was pur-
chased commercially on July 2017, from the St. Sebastian Market, a
local farmers’ market in Fortaleza, Ceará State, Brazil. The species
Caryocar coriaceum occurs in regions comprising the states of Ceará,
Piauí, and Pernambuco (Oliveira et al., 2008).
2.2. Analysis of fixed oil Caryocar coriaceum (FOCC)
Samples of FOCC were analyzed in order to identify their compo-
nents. The fatty acid content was initially determined by adding a
0.1 mL aliquot of FOCC to a solution of hexane and methanolic po-
tassium hydroxide (1.55 g KOH in 50 mL methanol) 1:1 in a separation
funnel. The solution was mixed vigorously for 30 s and then left to rest.
The hexane fraction was then separated, dried with sodium sulphate
and subsequently analyzed using gas chromatography coupled to mass
spectrometry (GC-EM).
Analysis of the methylic esters was performed by means of GC-EM,
with a Rtx-5Ms column (30 m x0.25 mm x 0.25 μm), with helium as the
mobile phase at a flow rate of 1,0 mL/min. Column temperature started
at 40 °C and was increased to 180 °C at a rate of 4 °C/min. After this
point, temperature was increased by 20 °C/min until reaching 280 °C,
where it remained during 10 min. The injector temperature was 260 °C,
and the total time of chromatographic analysis was 50 min. Mass
spectroscopy operated in the electronic ionization mode, at 70 eV with
a temperature of 260 °C.
2.3. SHS exposure model
A rat model for short-term SHS exposure was created using an
adaptation of the protocol proposed by Ypsilantis et al. (2012). An
experimental apparatus (Fig. 1) was built containing an air pump
(Fig. 1-A) that generated a flow rate of 0.9 L/min to a SHS generation
chamber. This chamber consisted of a cylindrical acrylic recipient (ra-
dius, 8 cm; height, 27 cm) housing a lit cigarette in its interior (Fig. 1-
B); it also presented entry and exit ports. The positive pressure created
by the airflow in the interior of the cylindrical recipient kept the ci-
garette alight, thus dragging the smoke that left its tip toward the in-
terior of the exposure chamber (height, 38.7 cm; width, 39.0 cm; depth,
42.0 cm). This chamber had an internal volume of 63.4 L and contained
two exhaustors (Fig. 1-C).
The short-term SHS exposure protocol was performed during 14
days. Exposures were conducted 6 times per day, using a duration of
40 min per exposure (5 cigarettes per exposure, 30 cigarettes per day),
with a 1-h interval between exposures. The 6 daily exposures occurred
during the hours of 8:00 a.m. to 8:40 a.m., 9:40 a.m. to 10:20 a.m.,
11:20 a.m. to 12:00 a.m., 13:00 p.m. to 13:40 p.m., 14:40 p.m. to 15:20
p.m., and 16:20 p.m. to 17:00 p.m. Overall, 420 cigarettes were used
during the 14-day SHS exposure period.
Temperature, average oxygen (O2) and carbon dioxide (CO2) per-
centages, as well as the average concentrations of carbon monoxide
(CO), nitrogen oxides (NOx), sulfur dioxide (SO2), and methane (CH4)
were monitored in the interior of the SHS exposure chamber for the
duration of each individual exposure (40 min) during the 14-day ex-
perimental period, by means of a gas analyzer (Seintro-Chemist 900,
Ecil®).
D. Silveira Serra, et al. Journal of Ethnopharmacology 252 (2020) 112633
2

3. 2.4. Animals
All animals received humane care, and the experiments complied
with the following guidelines: ARRIVE; the National Institutes of Health
Guide for the Care and Use of Laboratory Animals (NIH Publications
No. 8023, revised 1978); and regulations issued by the National Council
for Controlling Animal Experimentation, Ministry of Science,
Technology and Innovation (CONCEA/MCTI), Brazil. Male Wistar rats
(7–8 weeks of age), with a body mass of 200 ± 50 g and water and
feed ad libitum, were used in this study. Rats were housed in plastic
cages under controlled environmental conditions. All animal use and
care procedures had been previously approved by the animal ethics
committee.
We used 32 animals randomly divided into four groups (n = 8). In
the SHS + O and SHS + T groups, the animals received daily oral
pretreatment with 0.5 mL of FOCC (SHS + O) or vehicle (Tween-80
[1%] solution) (SHS + T). The animals were then subsequently sub-
jected to the short-term SHS exposure protocol, as mentioned in item
2.3. In the AA + O and AA + T groups, the animals received daily oral
pretreatment with 0.5 mL of FOCC (AA + O) or vehicle (Tween-80
[1%] solution) (AA + T). The animals were then immediately exposed
to the same protocol mentioned in item 2.3, but without use of the
cigarette, thus being exposed to only ambient air.
The mean daily body mass for the animals within all groups was
monitored during the entire exposure protocol (14 days).
2.5. Respiratory system mechanics
All procedures for respiratory system mechanics analysis were
previously described (Gondim et al., 2019). In short, the animals were
anesthetized (ketamine:xylazine- 100:10 mg/kg), tracheostomized, in-
tubated (14-gauge cannula) and then connected to a computer-con-
trolled ventilator for small animals (Scireq©-flexiVent®, Montreal, QC,
Canada). The animals were ventilated at baseline settings, and paral-
yzed (pancuronium bromide - 0.5 mL/kg, i.p., Cristália, Brazil).
Immediately after standardized the mechanical history, the im-
pedance of the respiratory system (Zrs) was measured by forced oscil-
lation technique (Hantos et al., 1992), using 12 sequential 30 s-sam-
pling intervals, for a total of 6 min (12 total). Through the forced
oscillation technique we obtain data from Newtonian resistance (RN ),
and tissue elastance (H) and resistance (G). Thereafter, two quasi-static
PV curves were obtained to measure static compliance (CST), an esti-
mate of inspiratory capacity (IC), and PV loop area.
2.6. Histological study
All procedures for histological analysis were previously reported
(Gondim et al., 2019). In brief, Immediately after the determination of
respiratory system mechanics, the lungs were perfused with saline and
then removed en bloc, and was kept at functional residual capacity and
fixed in Millonig's formaldehyde (100 mL HCHO, 900 mL H2O, 18.6 g
NaH2PO4, 4.2 g NaOH). Slides containing lung sections were stained
with hematoxylin and eosin (HE) and examined by optical microscopy.
Quantitative analysis was performed using the fraction area of
collapsed alveoli and the amount of polymorphonuclear (PMN) cells
analysis, determined by the point-counting technique (Weibel, 1990).
The air-space enlargement was quantified by the mean linear intercept
length of the distal air spaces (Lm) (Knudsen et al., 2009). The
bronchoconstriction index (BCI) was determined by counting the
number of points in the airway lumen (NP) and intercepts through the
airway wall (NI), using a reticulum and applying the equation
=BCI NI NP/ (Sakae et al., 1994).
2.7. Statistical analysis
Results are presented as mean ± SD, where n represents the
number of samples. Data normal distribution and homogeneities of
variances were tested with the Kolmogorov-Smirnov (with Lilliefors's
correction) and Levene median tests, respectively. If both conditions
were satisfied, the Student's t-test was used. If any condition was re-
futed, a Mann-Whitney non-parametric test was used instead. A dif-
ference was considered significant if p < 0.05.
3. Results
Table 1 shows the percentage values of the methyl esters present in
FOCC. The most representative values were found for linoleic acid
(65.56%), palmitic acid (20.86%) and stearic acid (10.17%), which are
acids with antioxidant action.
Table 2 shows the temperatures and concentrations of some of the
gases in the interior of the test chamber during SHS exposure. Animals
Fig. 1. Experimental apparatus for exposing animals to SHS. A - Air pump; B - Container containing the cigarette; C - Animal exposure chamber.
Table 1
Percent composition of the fixed oil of Caryocar coriaceum (FOCC) obtained
by gas chromatography/mass spectrometry.
Yield (%)
Fatty acid
Palmitic acid (C16:0) 20.86
Linoleic acid (C18:2) 65.56
Stearic acid (C18:0) 10.17
Cis-11-Eicosenoic acid (C20:1) 0.55
Methyl-18-methyl-nonadecanoate (C20:0) 1.07
Behenic (C22:0) 1.17
Lignoceric (C24:0) 0.33
D. Silveira Serra, et al. Journal of Ethnopharmacology 252 (2020) 112633
3

4. exposed to ambient air were subjected to an average temperature of
24.22 ± 0.23 °C, an average percentage of 21.00 ± 0.01 O2, and to
an environment free of CO, NOx, SO2 e CH4 gases.
The mean daily body mass gain for the groups before the start of
exposure protocols (day 0), as well as during the 14 days of exposure to
either ambient air (AA + T and AA + O groups) or SHS (SHS + T and
SHS + O groups), was also monitored (Fig. 2). The results of the 14-day
protocol period demonstrated an average body mass gain of 10.5 g for
the animals in the AA + T group, and 15.5 g for those in the AA + O
group. On the other hand, the body mass of animals in the SHS + T and
SHS + O suffered an average loss of 14.4 and 4.75 g, respectively.
Fig. 3 shows the respiratory system mechanical data of AA + T
(RN =0.103 ± 0.013, G = 0.89 ± 0.13, H = 2.64 ± 0.29,
CST = 0.99 ± 0.11, CI = 10.18 ± 1.38, PV Loop Area = 41.91 ±
5.86), in which the animals had been pretreated with 0.1% Tween 80
and exposed to ambient air for 14 consecutive days. Values are also
shown for the AA + O group (RN =0.083 ± 0.018, G = 0.87 ± 0.16,
H = 2.54 ± 0.31, CST = 1.01 ± 0.12, CI = 9.79 ± 0.98, PV Loop
Area = 40.58 ± 4.92), where the animals had been pretreated with
FOCC and exposed to ambient air for 14 consecutive days. Values were
as follows for SHS + T: RN = 0.151 ± 0.023, G = 1.44 ± 0.27,
H = 3.89 ± 0.65, CST = 0.73 ± 0.10, CI = 7.52 ± 1.30, PV Loop
Area = 59.58 ± 8.32 (with animals pretreated with 0.1% Tween 80
and then exposed to SHS for 14 consecutive days). The SHS + O group
exhibited the following values: RN = 0.083 ± 0.012,
G = 0.91 ± 0.19, H = 2.75 ± 0.37, CST = 0.98 ± 0.12,
CI = 9.32 ± 1.32, PV Loop Area = 43.96 ± 5.61 (animals pretreated
with FOCC and then exposed to SHS for 14 consecutive days).
Our results demonstrated significant changes in all respiratory me-
chanics variables analyzed for the SHS + T group, when compared to
the AA + T group, confirming the establishment of respiratory lesions
induced by SHS exposure. Additionally, when comparing the AA + T
group to the AA + O, no changes were observed, proving that daily
treatment with 0.5 mL of FOCC was not toxic to the respiratory system.
We also observed that rats exposed to SHS and treated with 0.5 mL
of oral FOCC (SHS + O) showed improvement in all respiratory me-
chanics variables, when compared to the group submitted to SHS ex-
posure and treated with 0.1% Tween 80 (SHS + T), thus demonstrating
the effectiveness of FOCC in preventing lung injury induced by short-
term SHS exposure.
Fig. 4 depicts representative lung histological images for the
AA + T, AA + O, SHS + T and SHS + O groups. Alveolar collapse,
thickened septa and cellular infiltrates were observed in the photo-
micrographs of the pulmonary parenchyma of the SHS + T group.
Table 3 displays the alveolar collapse, amount of polymorpho-
nuclear cells, mean alveolar diameters and bronchoconstriction indices.
We observed an increase in all these parameters, in comparison to those
of the AA + T group. Altogether, these findings suggest pulmonary
inflammation and bronchoconstriction.
4. Discussion
Products of plant origin, such as FOCC, are commonly used in
popular medicine due to their anti-inflammatory action in wound
healing, as well as to treat various ailments of the respiratory system,
such as cough, bronchial inflammation and asthma (Matos, 2007).
Several studies have reported on the beneficial effects of FOCC, such as
anticonvulsant (Oliveira et al., 2017), anti-inflammatory (Oliveira
et al., 2010; Saraiva et al., 2011), antibacterial activity and antibiotic
modifying action (Pereira et al., 2019), potential cardioprotective
(Kerntopf et al., 2015) and antioxidant activity (Pereira, 2016).
Thus, due to reports of the antiinflammatory and antioxidant effects
of FOCC, and considering the scarcity of reports on the effects of this
compound on lung function, particularly in lung lesions caused by ex-
posure to cigarette smoke, this study sought to expand the knowledge
about the therapeutic use of oral FOCC in animals submitted to short-
term SHS exposure. For this purpose, we evaluatedrespiratory me-
chanics in vivo, as well as lung histopathology and lung parenchymal
morphometry in vitro.
The chemical analysis of the compounds present in FOCC are shown
in Table 1. The most representative values are for linoleic acid
(65.56%), palmitic acid (20.86%) and stearic acid (10.17%), which are
substances that have known antioxidant activity (Henry et al., 2002).
In the present study, we identified linoleic acid (C18:2) in FOCC as
the most highly present constituent of the oil (Table 1). This compound
Table 2
Data on temperature and pollutant concentrations while in the SHS exposure chamber.
Gas Concentration and Temperature
Time (min) O2 (%) CO2 (%) CO (ppm) NOx (ppm) SO2 (ppm) CH4 (ppm) Temperature (°C)
5 20.74 0.07 171 9 12 95 27.1
10 20.74 0.08 194 11 14 100 27.0
15 20.77 0.07 96 7 10 64 27.1
20 20.74 0.07 169 9 12 96 27.1
25 20.77 0.06 146 9 11 88 27.3
30 20.75 0.07 175 10 11 98 27.3
35 20.74 0.07 153 10 11 96 27.4
40 20.74 0.08 168 10 12 99 27.6
Mean 20.75 0.07 159.00 9.38 11.63 92.00 27.24
SD 0.01 0.01 29.23 1.19 1.19 11.89 0.20
Fig. 2. Temporal evolution of subjects' masses. Data obtained from the daily
measurements of the animals of groups AA + T, AA + O, SHS + T and
SHS + O. The masses of all animals were measured on day 0 (day before the
start of exposure protocols) and during the next 14 days of exposure to ambient
air, after receiving a daily pretreatment with vehicle (Tween-80 [1%] solution)
(AA + T Group) or fixed oil of Caryocar coriaceum (FOCC) (AA + O Group); or
exposure to SHS for 14 days, after a daily pretreatment with vehicle (Tween-80
[1%] solution) (SHS + T group) or FOCC (SHS + O group). 8 animals per
group. Values are mean ± SD.
D. Silveira Serra, et al. Journal of Ethnopharmacology 252 (2020) 112633
4

5. is known for its antiinflammatory activity (Dipasquale et al., 2018) and
antioxidant properties (Ni et al., 2015). Linoleic acid is not naturally
synthesized by mammals; thus, it is known as one of the essential fatty
acids that must be acquired through the diet (Moreira et al., 2002).
The environments in which the animals were exposed to ambient air
(AA + T and AA + O groups) or SHS (SHS + T and SHS + O groups)
were monitored throughout the experimental study (14 days).
Temperature, concentrations of O2, CO, NOx, SO2, CH4 and the per-
centage of CO2 present in the exposure chamber are all shown in
Table 2. The harmful impacts on health caused by exposure to these and
other pollutants present in SHS have been extensively documented in
the literature, such as those associated with sleep disturbances
(Morioka et al., 2018), with higher odds of asthma exacerbations and
having poorly-controlled asthma with a need for increasing dose–r-
esponse pharmacology, even at low levels of exposure (Neophytou
et al., 2018). Oxygen concentrations in the exposure chamber did not
change significantly.
The average temperature to which animals from the AA + T and
AA + O groups were exposed (24.22 ± 0.23 °C) was lower than that
faced by the animals from the SHS + T and SHS + O groups
(27.24 ± 0.20 °C). When a cigarette is lit, the tobacco is subjected to
combustion (burning), generating smoke which contains thousands of
chemical substances. Once initiated, combustion is a self-sustaining
process that lasts as long as there is enough available tobacco (fuel) and
oxygen present. While this combustion occurs, the temperature in the
tip of the cigarette may reach values exceeding 900 °C (Baker, 1974),
which would explain the higher temperature in the interior of the
chamber during SHS exposure.
Animal body mass was verified continuously during the 14-day
period. Our results demonstrate increased mass among the animals of
Fig. 3. Pulmonary mechanics. Data obtained by performing the forced oscillation technique (RN , G and H) and PV curve (CST, IC and PV loop area) in animals
exposed to ambient air for 14 days after daily pretreatment with vehicle (Tween-80 [1%] solution) (AA + T Group) or fixed oil of Caryocar coriaceum (FOCC)
(AA + O Group); and in animals exposed to SHS for 14 days after daily pretreatment with vehicle (Tween-80 [1%] solution) (SHS + T group) or FOCC (SHS + O
group). 8 animals per group. Values are mean ± SD. One-way ANOVA followed by Student–Newman–Keuls test was performed. a Difference from AA + T group
(p < 0.05). b Different from SHS + T group (p < 0.05).
D. Silveira Serra, et al. Journal of Ethnopharmacology 252 (2020) 112633
5

6. the AA + T and AA + O groups, a characteristic not observed in the
SHS + T and SHS + O groups. It has been reported that exposure to
cigarette smoke inhibits appetite and is detrimental to body develop-
ment (Castardeli et al., 2016), which may explain the body mass loss
experienced by the animals in the SHS + T and SHS + O groups.
This phenomenon can also be explained through analysis of the
temperature to which the subjects of the different groups were exposed,
as the animals of the AA + T and AA + O groups were submitted to a
mean temperature of 24.22 ± 0.23 °C, while those of the SHS + T and
SHS + O groups were exposed to an average temperature of
27.24 ± 0.20 °C. Studies report that ambient temperature influences
animal metabolic processes (Damy et al., 2010), and that mice are
particularly susceptible to changes in environmental conditions
(Chorilli et al., 2007). Small fluctuations in temperature (2 °C–3 °C)
may cause changes in physiology (Jeyaseelan et al., 2005).
Additionally, we perceived a larger weight gain among the animals
in the AA + O group, when compared to the AA + T group, as well as a
lower body mass loss among the animals of the SHS + O group, when
compared to the SHS + T group. This finding may be due to the nu-
tritional importance of pequi as a source of energy, especially its al-
mond with high percentages of protein, zinc, manganese, copper and
phosphorus (Oliveira, 2009).
The results of lung function were obtained by forced oscillation
technique (constant phase model) and quasi-static PV curve (Fig. 3). In
the constant phase model, we evaluate the variables of Newtonian re-
sistance (RN ), tissue resistance (G) and elastance (H) (Bates et al.,
2009), and in the quasi-static PV curve, the variables of static com-
pliance (CST), estimate of inspiratory capacity (IC), the PV loop area.
We can assume that the significantly higher values of RN in the
group pretreated with Tween-80 and submitted to SHS (SHS + T),
when compared to the group pretreated with Tween-80 and exposed to
ambient air (AA + T) (Fig. 3), indicate narrowing of the airway lumen
caused by inflammatory process and/or increased stiffness of the
airway smooth muscle. This hypothesis was supported by the mor-
phometric data (Table 3, BCI – PMN cells).
In addition, this can be explained by the fact that inhalation of ir-
ritants through SHS exposure can induce enhanced bronchial con-
tractile responses mediated by 5-hydroxytryptamine 2A (5-HT2A) re-
ceptors, as well as by endothelin type B (ETB) and type A (ETA)
receptors in rat bronchial smooth muscle cells; these receptors mediate
contractility and airway hyperreactivity (AHR) (Cao et al., 2012). Ad-
ditionally, SHS exposure has been seen to enhance the expression of
CXC chemokine ligand 5 (CXCL5) in the airways and lung parenchyma
(Balamayooran et al., 2012). CXCL5 is produced by alveolar epithelial
Fig. 4. Photomicrographs of pulmonary parenchyma in animals exposed to animals exposed to ambient air for 14 days after daily pretreatment with vehicle (Tween-
80 [1%] solution) (AA + T Group) or fixed oil of Caryocar coriaceum (FOCC) (AA + O Group); and in animals exposed to SHS for 14 days after daily pretreatment
with vehicle (Tween-80 [1%] solution) (SHS + T group) or FOCC (SHS + O group). Photomicrographs of lung parenchyma stained with hematoxylin–eosin. Gray
arrow = alveolar septal thickening; black arrow = cellular infiltrate; Circle = areas of atelectasis.
Table 3
Morphometric parameters. Values are mean ± SD of AA + T, AA + O, SHS + T and SHS + O groups. The data were collected in ten matched fields per rat.
a Difference from AA + T group (p < 0.05). b Different from SHS + T group (p < 0.05). By one-way ANOVA followed by the multiple comparisons corrected with
the Bonferroni's test. PMN, polymorphonuclear; BCI, bronchoconstriction index.
Groups Alveolar Collapse (%) PMN Cells (x10−3
/μm2
) Mean alveolar diameter (μm) BCI
AA + T 5.78 ± 1.26 16.54 ± 3.94 44.40 ± 3.66 2.12 ± 0.13
AA + O 4.13 ± 1.09 16.30 ± 3.16 43.23 ± 3.88 2.05 ± 0.17
SHS + T 30.70 ± 3,08 a
30.47 ± 6.37 a
36.88 ± 5.22 a
2.80 ± 0.18 a
SHS + O 6.53 ± 4.03b
17.44 ± 6.31 b
43.12 ± 4.66 b
2,06 ± 0,20 b
D. Silveira Serra, et al. Journal of Ethnopharmacology 252 (2020) 112633
6

7. Type II (AEII) cells (Jeyaseelan et al., 2005), and these findings suggest
that CXCL5 can play an important role in the pathogenesis of SHS-in-
duced airway inflammation (Balamayooran et al., 2012).
We also observed that the SHS + O group did not present increased
resistance of the airways, thus showing avoidance of installation of
airway smooth muscle lesions. This result may be related to the anti-
inflammatory (Dipasquale et al., 2018) and antioxidant (Ni et al., 2015)
properties of the pequi oil compounds, thus preventing injury to airway
smooth muscle.
Tissue resistance (G) and elastance (H) are influenced by the in-
trinsic properties of the tissue. We observed increased G and H (Fig. 3)
in the pulmonary mechanics of the SHS + T group, when compared to
those of the AA + T group. These findings can be explained by tissue
changes such alveolar septa thickening and collapse, as well as cellular
infiltrates in the pulmonary parenchyma of animals from the SHS + T
group (Fig. 4). Added to that, the increased in percentage of collapsed
alveoli, number of polymorphonuclear cells (PMN cells) and the de-
crease in mean alveolar diameter (Table 3) of animals from the
SHS + T group, may indicate the release of inflammatory cytokines,
lipid mediators and enzymes capable of promoting edema and tissue
injury (Holz et al., 2008).
Regarding the analysis of the variables obtained from the use of PV
curve, the reduction of CST and IC, corroborates the stiffening of lung
tissue indicated by the increased G and H, in the SHS + T groups, in
comparison to the AA + T group (Fig. 3). We also observed a statisti-
cally significant increase in the value of this variable in the SHST + T
group when compared to the AA + T group (Fig. 3). This findings
corroborates with can also be attributed to tissue changes, such alveolar
collapse, edema and greater presence of PMN cells, as well a me-
chanism associated with alveolar surfactant (Muller et al., 1998,
Wagers et al., 2002).
The statistically significant differences found for all respiratory
mechanics variables (R G H C IC, , , ,N ST and PV loop area), as well as for
the pulmonary parenchyma morphometry (percentage of collapsed al-
veoli, PMN cells, mean alveolar diameter and BCI), of animals from the
SHS + O group, when compared to those from the SHS + T group,
demonstrate the potential of FOCC in preventing the establishment of
pulmonary lesions induced by SHS exposure. Furthermore, the lack of
alterations seen for these variables among the animals from the AA + O
group, when compared to the AA + T group, may indicate that oral
ingestion of 0.5 mL of FOCC during 14 days did not present toxic pul-
monary effects.
5. Conclusion
In conclusion, our results demonstrated that FOCC was able to
prevent acute lung injury in rats submitted to short-term SHS exposure;
further studies are necessary to confirm wich main mechanism of ac-
tion. However, the present study adds important information regarding
the effect of this oil on respiratory system mechanics, as an alternative
therapy in the treatment of lung diseases arising from exposure to ci-
garette smoke. Our results suggest that consumption of pequi-based
products (eg FOCC) used in the pre-treatment of the short-term SHS
exposure, has the potential to provide health benefits.
Author contributions
Serra, D.S., Oliveira, M.L.M. and Pimenta, A.T.A. conceived and
designed the experiments. Sousa, A.M., Andrade, L.C.S. and Gondim,
F.L, performed the animal experiments and Serra, D.S. analyzed the
data. Pimenta, A.T.A. and Santos, J.E.A. performed the chemical ana-
lyzes. Oliveira, M.L.M. performed the pollutant analyzes. Serra, D.S.,
Oliveira, M.L.M. and Pimenta, A.T.A. helped acquired data and statis-
tical analysis. All authors wrote and corrected the paper.
Declaration of competing interest
The authors declare that there is no conflict of interests regarding
the publication of this manuscript.
Acknowledgement
This study was financed in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) -
Finance Code 001.
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