Effects of fixed oil of Caryocar coriaceum Wittm. Seeds on the respiratory system of rats in a short-term secondhand-smoke exposure model

Journal Pre-proof 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 Serra, Allison Matias de Sousa, Leidianne Costa da Silva Andrade, Fladimir de Lima Gondim, João Evangelista de Ávila dos Santos, Mona Lisa Moura de Oliveira, Antônia Torres Ávila Pimenta PII:

S0378-8741(19)33680-3

DOI:

https://doi.org/10.1016/j.jep.2020.112633

Reference:

JEP 112633

To appear in:

Journal of Ethnopharmacology

Received Date: 16 September 2019 Revised Date:

21 January 2020

Accepted Date: 23 January 2020

Please cite this article as: Serra, D.S., Matias de Sousa, A., Costa da Silva Andrade, L., de Lima Gondim, F., Evangelista de Ávila dos Santos, Joã., Moura de Oliveira, M.L., Ávila Pimenta, Antô.Torres., Effects of fixed oil of Caryocar coriaceum Wittm. Seeds on the respiratory system of rats in a short-term secondhand-smoke exposure model, Journal of Ethnopharmacology (2020), doi: https://doi.org/10.1016/ j.jep.2020.112633. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

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 Serra* (Serra, D.S.), Center of Technological Sciences, State University of Ceará, Ceará, Brazil; Av. Dr. Silas Munguba, 1700, zip: 60714-903, Fortaleza-Ceará, Brazil. Corresponding Author: [email protected]; Allison Matias de Sousa (Sousa, A.M.), Institute of Biomedical Sciences, State University of Ceará, Ceará, Brazil; Leidianne Costa da Silva Andrade (Andrade, L.C.S.), Institute of Biomedical Sciences, State University of Ceará, Ceará, Brazil; Fladimir de Lima Gondim (Gondim, F.L.), Institute of Biomedical Sciences, State University of Ceará, Ceará, Brazil; João Evangelista de Ávila dos Santos (Santos, J.E.A.), Science Center, Federal University of Ceará, Fortaleza, CE, Brazil; Mona Lisa Moura de Oliveira (Oliveira, M.L.M.), Center of Technological Sciences, State University of Ceará, Ceará, Brazil. Antônia Torres Ávila Pimenta (Pimenta, A.T.A.), Science Center, Federal University of Ceará, Fortaleza, CE, Brazil;

Abstract

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 antiinflammatory 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 minutes per exposure (5 cigarettes per exposure), followed by a 1-hour 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 ambient air. After the exposure period, we investigated the effects of FOCC in respiratory mechanics in vivo (Newtonian resistance - , tissue elastance - , tissue resistance - , static compliance - , inspiratory capacity - , PV loop area) and in histopathology and lung parenchymal morphometry in vitro (polymorphonuclear cells – PMN, mean alveolar diameter - , bronchoconstriction index ), 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 observed 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.

Key words: Caryocar coriaceum; pequi; Fatty acids; Respiratory mechanics; SHS exposure.

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 developing nations (Pan American Health Organization, 2018). Although a significant number of countries have implemented public health policies in order to reduce smoking and secondhand smoke (SHS) exposure, many others still need to take place in order to prevent cigarette-related cancers (UN, 2019). Lung cancer due to SHS causes an estimated 21,400 deaths in non-smokers annually (Oberg et al., 2012). 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 alleviating 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 constituents have never been elucidated, these substances are still frequently prescribed due to the supposed medicinal effects they produce (MaiaFilho 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 ulcers, 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, arthritis 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 purchased 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 components. The fatty acid content was initially determined by adding a 0.1 mL aliquot of FOCC to a solution of hexane and methanolic potassium hydroxide (1.55 g KOH in 50 mL methanol) 1:1 in a separation funnel. The solution was mixed vigorously for 30 seconds 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 (GCEM) Analysis of the methylic esters was performed by means of GC-EM, with a Rtx5Ms 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 minutes. The injector temperature was 260 °C, and the total time of chromatographic analysis was 50 minutes.

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 (Figure 1) was built containing an air pump (Figure 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 (radius, 8 cm; height, 27 cm) housing a lit cigarette in its interior (Figure 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 cigarette alight, thus dragging the smoke that left its tip toward the interior 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 (Figure 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 minutes per exposure (5

cigarettes per exposure, 30 cigarettes per day), with a 1-hour 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) percentages, 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 minutes) during the 14-day experimental period, by means of a gas analyzer (Seintro-Chemist 900, Ecil®). 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 subjected 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, intubated (14-gauge cannula) and then connected to a computer-controlled ventilator for small animals (Scireq©flexiVent®, Montreal, QC, Canada). The animals were ventilated at baseline settings, and paralyzed (pancuronium bromide - 0.5 mL/kg, i.p., Cristália, Brazil). Immediately after standardized the mechanical history, the impedance of the respiratory system (Zrs) was measured by forced oscillation technique (Hantos et al., 1992), using 12 sequential 30 s-sampling intervals, for a total of 6 minutes (12 total). Through the forced oscillation technique we obtain data from Newtonian resistance (

), and tissue elastance ( ) and resistance ( ). Thereafter, two quasi-static PV curves

were obtained to measure static compliance (

), an estimate of inspiratory capacity

( ), 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 pointcounting technique (Weibel et al., 1990). The air-space enlargement was quantified by the mean linear intercept length of the distal air spaces ( bronchoconstriction index ( the airway lumen (

) (Knudsen et al., 2010). The

) was determined by counting the number of points in

) and intercepts through the airway wall (

and applying the equation

=

⁄√

), using a reticulum

(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 refuted, a Mann-Whitney non-parametric test was used instead. A difference 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 exposed to ambient air were subjected to an average temperature of 24.22±0.23ºC, 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 (Figure 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. Figure (

3

shows

=0.103±0.013,

the

respiratory

=0.89±0.13,

system

=2.64±0.29,

mechanical =0.99±0.11,

data

of

AA+T

=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

(

=0.083±0.018,

=0.87±0.16,

=2.54±0.31,

=1.01±0.12,

=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:

=0.151±0.023,

=1.44±0.27,

=3.89±0.65,

=0.73±0.10,

=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:

=0.083±0.012,

=0.91±0.19,

=2.75±0.37,

=0.98±0.12,

=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 mechanics 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 mechanics variables, when compared to the group submitted to SHS exposure and treated with 0.1% Tween 80 (SHS+T), thus demonstrating the effectiveness of FOCC in preventing lung injury induced by shortterm SHS exposure. Figure 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 photomicrographs of the pulmonary parenchyma of the SHS+T group. Table 3 displays the alveolar collapse, amount of polymorphonuclear 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 exposure 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 mechanics 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 is known for its

antiinflammatory activity (Dispasquale 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 percentage 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 (Mirioka et al., 2018), with higher odds of asthma exacerbations and having poorly-controlled asthma with a need for increasing dose–response 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±0.23ºC) 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 900ºC (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 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 development (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±0.23ºC, 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 to 3°C) may cause changes in physiology (Johnson-Delaney 1996). 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 nutritional importance of pequi as a source of energy, especially its almond 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 (Figure 3). In the constant phase model, we evaluate the variables of Newtonian resistance (

), tissue resistance ( ) and

elastance ( ) (Bates 2009), and in the quasi-static PV curve, the variables of static compliance (

), estimate of inspiratory capacity ( ), the PV loop area.

We can assume that the significantly higher values of

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) (Figure 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 morphometric data (Table 3,

– PMN cells). In addition, this can be explained by the fact that inhalation of irritants through

SHS exposure can induce enhanced bronchial contractile responses mediated by 5hydroxytryptamine 2A (5-HT2A) receptors, 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). Additionally, SHS exposure has been seen to enhance the expression of CXC chemokine ligand 5 (CXCL5) in the airways and lung parenchyma (Balamayooran et al., 2018). CXCL5 is produced by alveolar epithelial Type II (AEII) cells (Jeyaseelan et al., 2005), and these findings suggest that CXCL5 can play an important role in the pathogenesis of SHS-induced airway inflammation (Balamayooran et al., 2018).

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 antiinflammatory (Dispasquale 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 intrinsic properties of the tissue. We observed increased

and

(Figure 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 (Figure 4). Added to that, the increased in percentage of collapsed alveoli, number of polymorphonuclear cells (PMN cells) and the decrease 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 increased

and

, corroborates the stiffening of lung tissue indicated by the

and , in the SHS+T groups, in comparison to the AA+T group (Figure 3).

We also observed a statistically significant increase in the value of this variable in the SHST+T group when compared to the AA+T group (Figure 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 mechanism associated with alveolar surfactant (Muller et al., 1998, Wagers et al., 2001). The statistically significant differences found for all respiratory mechanics variables (

, , ,

,

and PV loop area), as well as for the pulmonary parenchyma

morphometry (percentage of collapsed alveoli, PMN cells, mean alveolar diameter and ), 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 pulmonary 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 action. 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 cigarette 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. Conflicts of 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. 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 analyzes. Oliveira, M.L.M. performed the pollutant analyzes. Serra, D.S., Oliveira, M.L.M. and Pimenta, A.T.A. helped acquired data and statistical analysis. All authors wrote and corrected the paper. 6.

References

Agra, M.D.F., Freitas, P.F.D., Barbosa-Filho, J.M., 2007. Synopsis of the plants known as

medicinal and poisonous in Northeast of Brazil. Braz. J. Pharmacogn. 17(1), 114140, http://dx.doi.org/10.1590/S0102-695X2007000100021 Adams, T., Wan, E., Wei, Y., Wahab, R., Castagna, F., Wang, G., Memet, E., Russo, C., Homma, S., Jemtel, T.H., Jelic, S., 2015. Secondhand smoking is associated with vascular inflammation. Chest. 148 (1), 112-119, https://doi.org/10.4049/jimmunol.1701417 Baker, R.R., 1974. Temperature Distribution Inside a Burning Cigarette. Nature. 247, 405–406 Balamayooran, G., Batra, S., Cai, S., Mei, J., Worthen, G.S., Penn, A.L., Jeyaseelan, S., 2012. Role of CXCL5 in leukocyte recruitment to the lungs during secondhand

smoke exposure. American journal of respiratory cell and molecular biology. 47 (1), 104-111, https://doi.org/10.1378/chest.10-2298 Barnoya, J., Glantz, S.A., 2005. Cardiovascular effects of secondhand smoke: nearly as large as smoking. Circulation. 111 (20), 2684-2698, https://doi.org/10.1161/circulationaha Bates, J.H., Rincon, M., Irvin, C.G., 2009. Animal Models of Athma. Am J Physiol Lung Cell and Mol Physiol. 297 (3), 401–410. https://doi.org/10.1152/ajplung.00027.2009 Bhat, T.A., Kalathil, S.G., Bogner, P.N., Miller, A., Lehmann, P.V., Thatcher, T.H., Phipps, R.P., Sime, P.J., Thanavala, Y., 2018. Secondhand smoke induces inflammation and impairs immunity to respiratory infections. The Journal of Immunology. 200 (8), 2927-2940, https://doi.org/10.4049/jimmunol.1701417 Boissonneault, G.A., 2000. Dietary fat, immunity, and inflammatory disease. Food Sci. Technol. 6, 777-807. Cao, L., Zhang, Y., Cao, Y.X., Edvinsson, L., Xu, C.B., 2012. Secondhand smoke exposure causes bronchial hyperreactivity via transcriptionally upregulated endothelin and 5-hydroxytryptamine 2A receptors. PloS one. 7 (8), 2-9, https://doi.org/10.1371/journal.pone.0044170 Castardeli, C., Ramos, E.A., Sant'Ana, P.G., Leopoldo, A.S., Lima, L.A.P., Castardeli, E., 2016. Influence of exposure to cigarette smoke and physical training in appetite and body development in rats. Mundo da saúde. 40 (2), 169-179. http://doi.org/10.15343/0104-7809.20164002169179 Chorilli, M., Michelin, D.C., Salgado, H.R.N., 2007. Laboratory animals: the mouse. Rev. Ciênc. Farm. Básica Apl. 28 (1), 11-23.

Damy, S. B., Camargo, R. S., Chammas, R., Figueiredo, L. F. P. D., 2010. Fundamental aspects on animal research as applied to experimental surgery. Revista da Associação Médica Brasileira, 56 (1), 103-111, http://dx.doi.org/10.1590/S010442302010000100024 Diaz-Sanchez, D., Rumold, R., Gong, Jr.H., 2006. Challenge with environmental tobacco smoke exacerbates allergic airway disease in human beings. Journal of allergy and clinical immunology. 118 (2), 441-446, https://doi.org/10.1016/j.jaci.2006.04.047 DiGiacomo, S.I., Jazayeri, M.A., Barua, R.S., Ambrose, J.A., 2019. Environmental Tobacco Smoke and Cardiovascular Disease. Int. J. Environ. Res. Public Health. 16 (1), 96, https://doi.org/10.3390/ijerph16010096 Dipasquale, D., Basirico, L., Morera, P., Primi, R., Tröscher, A., Bernabucci, U., 2018. Antiinflammatory effects of conjugated linoleic acid isomers and essential fatty acids in bovine mammary epithelial cells. Animal. 12 (10), 2108-2114, https://doi.org/10.1017/S1751731117003676

Flouris, A.D., Vardavas, C.I., Metsios, G.S., Tsatsakis, A.M., Koutedakis, Y., 2009. Biological evidence for the acute health effects of secondhand smoke exposure. American Journal of Physiology-Lung Cellular and Molecular Physiology. 298 (1), 3-12, https://doi.org/10.1152/ajplung.00215.2009 Gondim, F.L., Serra, D.S., Cavalcante, F.S.A., 2019. Effects of Eucalyptol in respiratory system mechanics on acute lung injury after exposure to short-term cigarette smoke. Respiratory physiology & neurobiology. 266 (1), 33-38. https://doi.org/10.1016/j.resp.2019.04.007 Hantos, Z., Daroczy, B., Suki, B., Nagy, S., Fredberg, J.J., 1992. Input impedance and peripheral inhomogeneity of dog lungs, J Appl Physiol. 72 (1), 168–178, https://doi.org/10.1152/jappl.1992.72.1.168 Hassig, A., Liang, W.X., Stampfil, K., 2000. Bronchial asthma: information on phytotherapy with essential fatty acids: interactions between essential fatty acids and steroid hormones. Med. Hypotheses. 54, 72-74, https://doi.org/10.1054/mehy.1998.0829 Holz, O., Seiler, T., Karmeier, A., Fraedrich, J., Leiner, H., Magnussen, H. Jorres, R.A., Welker, L., 2008. Assessing airway inflammation in clinical practice–experience with spontaneous sputum analysis. BMC pulmonary medicine, 8 (1), 5. Jeyaseelan, S., Manzer, R., Young, S.K., Yamamoto, M., Akira, S., Mason, R.J., Worthen, G.S., 2005. Induction of CXCL5 during inflammation in the rodent lung involves activation of alveolar epithelium. American journal of respiratory cell and molecular biology. 32 (6), 531-539, https://doi.org/10.1165/rcmb.20050063OC Kerntopf, M.R., de Figueiredo, P.R.L., Felipe, C.F.B., de Oliveira A.W., de Menezes, I.R.A., Fernandes, G.P., Lemos, I.C.S., 2015. Pequi oil (Caryocar coriaceum W.) and potential cardioprotective activity. Essays and Science: C. Biological, Agrarian and Health, 17(4). Knudsen, L., Weibel, E.R., Gundersen, H.J.G., Weinstein, F.V., Ochs, M., 2009. Assessment of air space size characteristics by intercept (chord) measurement: an accurate and efficient stereological approach. Journal of applied physiology. 108 (2), 412-421, https://doi.org/10.1152/japplphysiol.01100.2009 Luo, P., Zhang, Z., Yi, T., Zhang, H., Liu, X., Mo, Z., 2008. Antiinflammatory activity of the extracts and fractions from Erigeron multiradiatus through bioassay-guided procedures. Journal of ethnopharmacology, 119 (2), 232-237, https://doi.org/10.1016/j.jep.2008.06.035 Maia-Filho, A. L. M., Silva, V.S., Barros, T.L., Costa, C.L.S., Maia, E.P.V.D., Araújo, K.S., Santos, I.M.S.P., Villaverde, A.G.J.B., Carvalho, F.A.S., Carvalho, R.A. (2011). Efeito do gel da babosa (Aloebarbadensis Mill.) associado ao ultrassom em processo inflamatório agudo. Revista Brasileira de Plantas Medicinais. 13 (2) 146-50.

Matos, F.J.A., 2007. Plantas medicinais: guia de seleção e emprego de plantas usadas em fitoterapia no Nordeste do Brasil. Fortaleza: Imprensa Universitária. 3, 394. Moreira, N.X., Curi, R., Mancini, Filho J., 2002. Ácidos graxos: uma revisão. Nutrire: Rev Soc Bras Aliment Nutrição. 24: 105-23. Morioka, H., Jike, M., Kanda, H., Osaki, Y., Nakagome, S., Otsuka, Y., Ohida, T., 2018. The association between sleep disturbance and second-hand smoke exposure: a large-scale, nationwide, cross-sectional study of adolescents in Japan. Sleep medicine, 50, 29-35, https://doi.org/10.1016/j.sleep.2018.04.014 Muller, B., Seifart, C., Barth, P.J., 1998. Effect of air pollutants on the pulmonary surfactant system. Eur J Clin Invest. 28 (9), 762-77. Muthumalage, T., Pritsos, K., Hunter, K., Pritsos C., 2017. Commonly used air filters fail to eliminate secondhand smoke induced oxidative stress and inflammatory responses. Toxicology mechanisms and methods. 27 (6), 458-466, https://doi.org/10.1080/15376516.2017.1320694 Neophytou, A.M., Oh, S.S., White, M.J., Mak, A.C., Hu, D., Huntsman, S., Meade, K. Davis, A., Avila, P., Thyne, S., Rodríguez-Cintrón, W., Rodríguez-Santana, J.R., Kumar, R., Brigino-Buenaventura, E., Sen, S., Lenoir, M.A., Williams, L.K., Benowitz, N.L., Balmes, J.R., Eisen, E.A., Burchard, E.G., 2018. Secondhand smoke exposure and asthma outcomes among African-American and Latino children with asthma. Thorax. 73 (11), 1041-1048, http://dx.doi.org/10.1136/thoraxjnl-2017-211383 Ni, L., Jie, X., Wang, P., Li, S., Wang, G., Li Y., Acharya, K., 2015. Effect of linoleic acid sustained-release microspheres on Microcystis aeruginosa antioxidant enzymes activity and microcystins production and release. Chemosphere. 121, 110-116, https://doi.org/10.1016/j.chemosphere.2014.11.056 Oberg, M., Jaakkola, M.S., Woodward, A., Peruga, A., Prüss-Ustün, A., 2011. Worldwide burden of disease from exposure to second-hand smoke: a retrospective analysis of data from 192 countries. The Lancet, 377(9760), 139146, https://doi.org/10.1016/S0140-6736(10)61388-8 Oliveira, M.E.B., Guerra, N.B., Barros, L.D.M., Alves, R.E., 2008. Agronomic and quality aspects of pequi. Embrapa Tropical Agro-Industry-Documents. https://www.infoteca.cnptia.embrapa.br/bitstream/doc/426706/1/Dc113.pdf. (Accessed 15 Jan, 2020). Oliveira, M.E.B., 2009. Physical, chemical and bioactive characteristics in native (Caryocar coriaceum Wittm.) Native of the Araripe-CE plateau. (Doctoral Thesis, Federal University of Pernambuco, Recife). Available in: https://repositorio.ufpe.br/bitstream/123456789/8560/1/arquivo3852_1.pdf Oliveira, M.L.M., Nunes-Pinheiro, D.C.S., Tomé, A.R., Mota, É.F., Lima-Verde, I.A., de Melo Pinheiro, F.G., Campello, C.C., De Morais, S. M., 2010. In vivo topical anti-inflammatory and wound healing activities of the fixed oil of Caryocar

coriaceum Wittm. Seeds. Journal of Ethnopharmacology, 129(2), 214-219, https://doi.org/10.1016/j.jep.2010.03.014 Oliveira, C.C., Oliveira, C.V., Grigoletto, J., Ribeiro, L.R., Funck, V.R., Meier, L., Fighera, M.R., Royes, L.F.F., Furian, A.F., Menezes, I.R.A., Oliveira, M.S., 2017. Anticonvulsant activity of Caryocar coriaceum Wittm. fixed pulp oil against pentylenetetrazol-induced seizures. Neurological research, 39(8), 667-674. https://doi.org/10.1080/01616412.2017.1324380 Pan American Health Organization, 2018. https://www.paho.org/bra/index.php?option=com_content&view=article&id=574: tabagismo&Itemid=463 (accessed 03 June 2019). Park, Y.S., Lee, C.H., Kim, Y.I., Ahn, C.M., Kim, J.O., Park, J.H., Lee, H.H., Kim, J.Y., Chun, E.M., Jung, T.H., Yoo, K.H., 2018. Association between secondhand smoke exposure and hypertension in never-smokers: a cross-sectional survey using data from Korean National Health and Nutritional Examination Survey V, 2010–2012. BMJ open. 8 (5), 1-6, http://dx.doi.org/10.1136/bmjopen-2017021217 Pereira, S.M.P.D., 2016. Study of the antioxidant potential of Caryocar coriaceum Wittm. in in vitro and in vivo experimental models (Doctoral thesis, Federal University of Santa Maria). Available in: https://repositorio.ufsm.br/bitstream/handle/1/17965/TES_PPGBT_2016_PEREIR A_SANDRA.pdf?sequence=1&isAllowed=y Pereira, F.F.G., Feitosa, M.K.S.B., Costa, M.S., Tintino, S.R., Rodrigues, F.F.G., Menezes, I.R.A., Coutinho, H.D.M., da Costa, J.G.M., de Sousa, E.O., 2019. Characterization, antibacterial activity and antibiotic modifying action of the Caryocar coriaceum Wittm. pulp and almond fixed oil, Natural Product Research, 1-5. doi: 10.1080/14786419.2018.1552955 Sakae, R.S., Leme, A.S., Dolhnikoff, M., Pereira, P.M., Do Patrocinio, M., Warth, T.N., Zin, W.A., Saldiva, P.H., Martins, M.A., 1994. Neonatal capsaicin treatment decreases airway and pulmonary tissue responsiveness to methacholine. American Journal of Physiology-Lung Cellular and Molecular Physiology. 266 (1), 23-29, https://doi.org/10.1152/ajplung.1994.266.1.L23 Saraiva, R.A., Araruna, M.K., Oliveira, R.C., Menezes, K.D., Leite, G.O., Kerntopf, M.R., Costa, J.G.M., Rocha, J.B.T., Tomé, A.R., Campos, A.R., Menezes, I.R., 2011. Topical anti-inflammatory effect of Caryocar coriaceum Wittm.(Caryocaraceae) fruit pulp fixed oil on mice ear edema induced by different irritant agents. Journal of ethnopharmacology, 136(3), 504-510. https://doi.org/10.1016/j.jep.2010.07.002 Sena, D.M., Rodrigues, F.F., Freire, P.T., de Lima., S.G., Coutinho., H.D., Carvajal., J.C., da Costa., J. G., 2010. Physicochemical and spectroscopical investigation of Pequi (Caryocar coriaceum Wittm.) pulp oil. Grasas y Aceites, 61(2), 191-196, https://doi.org/10.3989/gya.105909

UN - United Nations, 2019. https://nacoesunidas.org/agencia-especializada-ve-aumentodo-numero-de-mortes-por-cancer-no-mundo/> (accessed 03 June 2019). Venn, A., Britton, J., 2007. Exposure to secondhand smoke and biomarkers of cardiovascular disease risk in never-smoking adults. Circulation. 115 (8), 990, https://doi.org/10.1161/CIRCULATIONAHA.106.648469 Wagers, S., Lundblad, L., Moriya, H.T., Bates, J.H., Irvin, C.G., 2002. Nonlinearity of respiratory mechanics during bronchoconstriction in mice with airway inflammation. Journal of Applied Physiology. 92 (5), 1802-1807, https://doi.org/10.1152/japplphysiol.00883.2001 Weibel, E.R., 1990. Morphometry: stereological theory and practical methods. In: Models of Lung Disease: Microscopy and Structural Methods, edited by Gil J. New York: Marcel Dekker. pp 199–247. World Health Organization, 2019. WHO report on the global tobacco epidemic 2019: to quit tobacco use. offer help https://apps.who.int/iris/bitstream/handle/10665/326043/ 9789241516204-eng.pdf. (Accessed 15 Jan, 2020). Yehuda, S., Rabinovitz, S., Mostofsky, D.I., Huberman, M., Sredni B., 1997. Essential fatty acid preparation improves biochemical and cognitive functions in experimental allergic encephalomyelitis rats. Eur. J. Pharmacol. 328, 3-29, https://doi.org/10.1016/S0014-2999(97)83022-1 Ypsilantis, P., Politou, M., Anagnostopoulos, C., Kortsaris, A., Simopoulos, C., 2012. A rat model of cigarette smoke abuse liability. Comparative medicine. 62 (5), 395399.

Figure 1. Experimental apparatus for exposing animals to SHS. A - Air pump; B - Container containing the cigarette; C - Animal exposure chamber.

Figure 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

Figure 3. Pulmonary mechanics. Data obtained by performing the forced oscillation technique ( , and ) and PV curve ( , 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 peformed. a Difference from AA+T b group (p<0.05). Different from SHS+T group (p<0.05).

22

Figure 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.

23

Yield (%) Fatty acid Palmitic acid (C16:0) Linoleic acid (C18:2) Stearic acid (C18:0) Cis-11-Eicosenoic acid (C20:1) Methyl-18-methyl-nonadecanoate (C20:0) Behenic (C22:0) Lignoceric (C24:0)

20.86 65.56 10.17 0.55 1.07 1.17 0.33

Table 1. Percent composition of the fixed oil of Caryocar coriaceum (FOCC) obtained by gas chromatography/mass spectrometry.

24

Time (min) 5

O2 (%) 20.74

Gas Concentration and Temperature CO2 CO NOx SO2 CH4 (%) (ppm) (ppm) (ppm) (ppm) 0.07 171 9 12 95

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

Temperature (°C) 27.1

Table 2. Data on temperature and pollutant concentrations while in the SHS exposure chamber.

25

Groups AA+T AA+O SHS+T SHS+O

Alveolar Collapse (%) 5.78±1.26 4.13±1.09 30.70±3,08 a 6.53±4.03b

PMN Cells (x10-3/µm2) 16.54±3.94 16.30±3.16 30.47±6.37 a 17.44±6.31 b

Mean alveolar diameter (µm) 44.40±3.66 43.23±3.88 36.88±5.22 a 43.12±4.66 b

BCI 2.12±0.13 2.05±0.17 2.80±0.18 a 2,06±0,20 b

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.

26