Food Chemistry 325 (2020) 126923

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Myorelaxant action of the Dysphania ambrosioides (L.) Mosyakin & Clemants T

essential oil and its major constituent α-terpinene in isolated rat trachea
⁎
Luís Pereira-de-Moraisb,c, , Andressa de Alencar Silvac, Renata Evaristo Rodrigues da Silvaa,b,
Daniela Maria do Amaral Ferraz Navarrod, Henrique Douglas Melo Coutinhoa,b,
Irwin Rose Alencar de Menezesa,b, Marta Regina Kerntopfa,b, Francisco Assis Bezerra da Cunhaa,b,
José Henrique Leal-Cardosoc, Roseli Barbosaa,b
a
Biological Chemistry Department, Postgraduate Program in Biological Chemistry, Brazil
b
Regional University of Cariri, Pimenta Campus, 63105-010 Crato, CE, Brazil
c
State University of Ceará, Itapery Campus, 60741-000 Fortaleza, CE, Brazil
d
Federal University of Pernambuco, University City, 50670-901 Recife, PE, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: This study aimed to investigate the myorelaxant action of the Dysphania ambrosioides essential oil (EODa) and its
Trachea major constituent α-terpinene on tracheal smooth muscle isolated from rats. In tracheal smooth muscle ex vivo,
Dysphania ambrosioide in organ baths, isometric contractions recordings were done in order to evaluated the effect of EODa (1–1000 μg/
α-Terpinene mL) and α-terpinene (1–3000 μg/mL) on the following parameters: basal tone, contractions evoked by potassium
Essential oil
(KCl 60 mM), acetylcholine (ACh 10 μM) or serotonin (5-HT 10 μM). The EODa and its major constituent α-
Antispasmodic effect
terpinene, did not statistically alter basal tone; however, they induced myorelaxant effects on top of contractions
induced by KCl, ACh and 5-HT. EODa and α-terpinene also inhibited the contractions induced by barium in
presence of High [K+] (80 mM). The data suggest that the relaxation induced by these agents is caused by the
inhibition of L-type VGCC, inhibiting the inward Ca2+ current through these channels, but does not exclude the
possibility of participation of other mechanisms. Results from this study also suggest the EODa, due to their
efficacy on relaxation of the respiratory tract, posses a therapeutic potential as a antispasmodic agent for re-
spiratory tract.

1. Introduction Gurni, & Carballo, 2006).

The Dysphania ambrosioides (L.) Mosyakin & Clemants species pre-
viously known as Chenopodium ambrosioides is popularly know as
“mastruz” or “menstruço”. This species is characterized as a native herb
from South America that is cultivated in subtropical and sub-temperate
regions, mainly for the consumption of its leaves in cooking. This
widely known plant is also popular in medicine (Jabbar, Zaman, Iqbal,
Yaseen, & Shamim, 2007; Pereira et al., 2010).
The D. ambrosioides, often cited for the efficacy in the treatment of
diseases related to the digestive system and for vascular, antispasmodic,
anti-inflammatory as well as healing activities, receives a widespread
use in folk medicine for the treatment of respiratory problems (pre-
dominantly asthma and pneumonia) (De Feo & Senatore, 1993; Gadano,
Studies investigating the chemical composition and biological ac-
tivities of D. ambrosioides have also shown that in samples of essential
oils of this plant, α-terpinene, ascaridol and p-cymene were detected as
the main active compounds found in this essential oil (Cavalli, Tomi,
Bernardini, & Casanova, 2004; Hu et al., 2015).
α-terpinene is an isomeric monoterpene with the following mole-
cular formula: C10H16, with its double bonds being in positions 1 and 3
of the p-methane skeleton. α-Terpinene is a volatile component of
petroleum and a metabolite of some plants (Araujo et al., 1996). α-
Terpinene is a constituent of many essential oils, for example, plant
species from the Citrus, Eucalyptus and Juniperus genera, which possess a
lemon flavor and is used in the food industry as an aromatic agent.
Both D. ambrosioides essential oil (EODa) and α-terpinene have been

Abbreviations: 5-HT, serotonin; ACh, Acetylcholine; ANOVA, analysis of variance; Ba2+, Barium; EODa, Dysphania ambrosioides essential oil; GC–MS, gas chro-
matography coupled to mass spectrometry; SEM, standard error of the mean; VGCCs, voltage-gated calcium channels
⁎
Corresponding author at: Laboratory of Physiopharmacology of Excitable Cells, Biological Chemistry Department – Regional University of Cariri, Pimenta
Campus, Cel. Antônio Luis, 1161 – Pimenta, Crato, CE 63105-010, Brazil.
E-mail address: luispereira256@gmail.com (L. Pereira-de-Morais).

https://doi.org/10.1016/j.foodchem.2020.126923
Received 5 November 2019; Received in revised form 25 April 2020; Accepted 25 April 2020
Available online 01 May 2020
0308-8146/ © 2020 Elsevier Ltd. All rights reserved.
L. Pereira-de-Morais, et al.
suggested to be a potent inhibitor of gastrointestinal nematodes,
Trypanosoma cruzi and Leishmania amazonensis (De Almeida et al.,
2007). Infusion of Chenopodium ambrosioides or of its constituent as-
caridol promotes nematode death and do not display toxic activity over
intestinal smooth muscle (MacDonald et al., 2004). In addition, work by
Assaidi et al. (2019), attributes an endothelium derived vasodilatory
effect to Chenopodium ambrosioides. The α-terpinene, by its turn, re-
vealed a LD50 of 1680 mg via oral administration in rats, thus de-
monstrating moderate acute toxicity (Araujo et al., 1996). Some other
biological actions have been found in the literature, these being de-
scribed as: antioxidant, and antimicrobial activity (Kavoosi, Tafsiry,
Ebdam, & Rowshan, 2013), antifungal (Parveen et al., 2004) and va-
sorrelaxant (Sabino et al., 2013).
Phytotherapies that act selectively on the respiratory system are still
rare and in contrast diseases that affect the respiratory tract causing
bronchoconstriction (asthma and bronchitis) have been a growing
public health problem, especially in children. It is noteworthy, even
with the popular use of D. ambrosioides to treat respiratory tract al-
terations and even with the relevance of the biological activity α-ter-
pinene (vasorelaxant effect), reported for smooth muscle, there are no
studies of EODa and its α-terpinene constituent on tracheal muscle. The
purpose of this study is to characterize the activity of EODa and α-
terpinene on tracheal smooth muscle.
2. Materials and methods
2.1. Plant collection and identification
The Dysphania ambrosioides (L.) Mosyakin & Clemants botanical
material was collected from the Botanical Garden of the Natural
Products Research Laboratory – LPPN, Regional University of Cariri –
URCA, Crato, CE, Brazil (Coordinates: 07° 14′ 19,2″ latitude S. and 39°
24′ 52,8″ longitude W. of Greenwich). The species was identified by
Prof. Dr. Maria Arlene Pessoa da Silva as belonging to the species
Dysphania ambrosioides (L.) Mosyakin & Clemants, from the
Chenopodiaceae family. An exsicata was deposited in the Caririense
Dárdano de Andrade-Lima Herbarium at the Regional University of
Cariri – URCA, with sample # 12.208.
2.2. Essential oil extraction
Plant leaves were collected and cut into pieces of approximately
1 cm2. Subsequently, the plant material was immersed in distilled water
and extracted by hydrodistillation in a Clevenger type apparatus to
obtain the essential oil. After extraction, the oil was treated with an-
hydrous sodium sulfate in order to remove traces of water that may be
present in the sample. The oil was then filtered with cotton and
transferred using a Pasteur pipette to amber glass vials and stored at
−20 °C.
2.3. Phytochemical analysis
A 100 ppm solution of the essential oil was prepared using double
distilled hexane as the solvent. One μL of this solution was analyzed
using a gas chromatograph coupled to a mass spectrometer with a
analyzer Agilent 5975C Series (Agilent Technologies, Palo Alto, USA)
equipped with a HP-5 apolar column (Agilent J&W, 60 m × 0.25 mm
internal diameter; 0.25 μm film thickness). The samples were analyzed
under the following conditions: initial temperature of 40 °C (main-
tained for 2 min), heating ramp of 4 °C/min and final temperature of
230 °C, which was held for 5 min. Helium at a constant flow rate of
1 mL per minute was the carrier gas used and the injector temperature
was maintained at 250 °C. The compounds eluted from the chromato-
graphic column were ionized by electron impact at 70 eV. The ioniza-
tion source was maintained at 230 °C and the quadrupole at 150 °C.
Mass spectra were obtained in scan mode with a scan speed of 0.5 scan
2
Food Chemistry 325 (2020) 126923
per second, with a scanning range of 35 to 550 m/z.
An aliquot of 1 μL of each extract was injected in a 1:20 split mode.
The compounds were identified by analyzing the mass spectra of each
chromatographic peak, comparing them with authentic standards and
calculating retention indices. Linear retention rates of the essential oil
components were calculated by using the retention times of each
compound and the retention times of n-alkanes (C9 to C25) analyzed
under the same conditions. The calculated values were compared with
retention indices published in the literature (Adams, 2007).
The same solution was quantified by triplicate injection of 1 μL of
this solution in a splitless mode in a gas chromatograph (Thermo trace
GC ultra) equipped with a VB-5 apolar column (60 m × 0.25 mm in-
ternal diameter, 0.25 μm film thickness). The samples were analyzed
under the following conditions: initial temperature of 40 °C (main-
tained for 2 min), heating ramp of 4 °C/min and final temperature of
230 °C, which was held for 5 min. Nitrogen at a constant flow rate of
1 mL per minute was the carrier gas; the injector temperature was
maintained at 250 °C. The compounds eluted from the chromatographic
column were detected by flame ionization detectors (FID) at 250 °C.
After the analysis, in order to avoid variations in its chemical con-
stitution, the oil was stored in amber glass wrapped in aluminum foil to
protect it from light and kept in a freezer at a temperature of −20 °C,
from where it was briefly removed only for immediate use at the ex-
periments. α-terpinene was purchased from Sigma-Aldrich (St. Louis,
Missouri, USA) and stored according to the manufacturer's instructions,
in a freezer at a temperature of −20 °C, similarly to oil.
2.4. Animals
Male Wistar rats (Rattus novergicus), with a body weight between
200 and 300 g, 2,5–3 months old, were used. The animals were kept in
a Central Vivarium Station of the Regional University of Cariri-URCA,
under constant humidity (50–60%) and temperature conditions of
23 ± 2 °C, in a twelve-hour light/dark cycle, with access to water and
food ad libitum, treated according to the Brazilian College of Animal
Experimentation (COBEA), Brazil and using methods that minimize
animal suffering. Experimental procedures were approved by the
Committee on Ethics in the Use of Animals (CEUA)-URCA, registered
under protocol number: 24/2012.2/2012.
2.5. Solutions and drugs
Modified Tyrode's nutrient solution (MT or Tyrode) had the fol-
lowing composition in, mM: NaCl, 136.0; KCl, 5.00; MgCl2, 0.98;
NaH2PO4, 0.36; NaHCO3, 11.9; CaCl2, 2.0 and Glucose, 5.5. The cal-
cium-free or “zero calcium” (0 Ca2+) MT solution had 0.2 mM EGTA
and received no addition of CaCl2. MT solution was constantly aerated
by bubbling air at 37 °C; the pH was adjusted to 7.4 with 1 M HCl and/
or 1 M NaOH. The EODa and α-terpinene were prepared as a solution,
diluted directly in Tyrode and Tween. Nifedipine was diluted in
ethanol, and the remaining drugs and their stocks were diluted in dis-
tilled water. Potassium Chloride (60 mM KCl), acetylcholine (ACh
10 μM), serotonin (5-HT 10 μM) and Barium Chloride (0,1 – 30 mM
BaCl2), was used for induction of contraction (Aguiar et al., 2012). All
the salts and reagents used were of analytical grade and purity obtained
from the Sigma-Aldrich (St. Louis, Missouri, USA).
2.6. Tissue isolation preparation
The animals were euthanized using CO2 chambers, followed by
ventral opening of the thorax to remove the trachea. The trachea was
transferred to a Petri dish containing modified Tyrode. It was cleaned to
eliminate attached tissues and circular transverse segments of 4–5 mm
in length were sectioned.
The segments were mounted in a 10 mL organ bath containing MT
at 37 °C and were continuously aerated. The isolated tissue rings were
L. Pereira-de-Morais, et al. Food Chemistry 325 (2020) 126923

subjected to a 1 gf tension, and left to stabilize over a period of 1 h Table 1

before the addition of any substance (Pereira-Gonçalves et al., 2018). Chromatographic profile of the D. ambrosioides essential oil.
Measurements of contractile activity were recorded using a rod con- Compound RIb RIc % ST
nected to a power transducer (TRI, model 210, Panlab, Spain) and to a
differential amplifier (DATAQ, model PM-1000, USA) with input to an α-Terpinene 1014 1014 30.53 0.33
o-Cymene 1022 1021 13.68 0.13
analog to digital converter board (DATAQ DI-200) installed in a com-
Ascaridole 1234 1236 1.48 0.02
puter. The data was converted into digital traces and stored in files Piperitone epoxide cis 1250 1253 1.04 0.03
using the WINDAQ software (DATAQ Instruments, Inc. USA). All pro- Piperitone epoxide trans 1252 1256 1.43 0.09
tocols started with two subsequent contractions, induced by the hy- Ascaridol gycol trans 1266 1267 1.05 0.01
pertonic addition of KCl to bath so as to increase [K+] to 60 mM. These Ni 1285 0.46 0.46
Thymol 1289 1291 18.11 0.10
contractions were allowed to reach maximum steady-state values, a
Carvacrol 1298 1300 11.23 0.23
plateau, which was considered the maximal response (contraction) Ni 1303 17.14 0.05
obtained with the challenge with 60 mM KCl. Only experiments with Ni 1318 1.18 0.02
reproducible contractions were considered viable for the experimental Total 97.32
Unidentified 18.77
series. In the experiments that had Ba2+, as a contractile agonist, the
Identified 81.23
preparations of trachealis muscle, already in presence of Ca2+-free
solution, were depolarized by hypertonic addition of KCl so as to in- RIb Retention indices in the literature. RIc Retention indices in the experiments
crease [K+] to 80 mM (in order to verify the absence of extracellular (based in n-alkene homologous series). ST Standard deviation. % Relative
calcium). It was afterwards, while using Ba2+, maintained in Ca2+-free proportion of the essential oil constituents were expressed as percentages. Ni
and 80 mM [K+]. Not identified.
All experiments were carried out accompanied by their controls:
experiments in which rings subjected to the same procedures, except were added to MT. No statistically significant relaxant or contractile
that they received only the vehicle, Tween, diluted in MT solution. effect over the basal tone was observed (EODa P = 1.000 and α-ter-
After the two initial K+ (60 mM)-induced contractions, the contraction- pinene P = 1.000, one-way ANOVA followed by Holm-Sidak). However,
inducing agonists KCl (60 mM) or ACh (10 μM) were added to the each a small upward deviation of the EODa curve was observed with large
organ baths and followed by a crescent and cumulative addition of the concentrations (Fig. 1). Pinho-da-Silva et al. (2012), reported that trans-
EODa or α-terpinene separately, and for each new concentration of caryophyllene had no significant effect on basal tone of rat trachea,
EODa or α-terpinene enough time was allowed for the response to reach similar to our findings. Maintaining airway smooth muscle tone within
steady state, usually from 5 to 15 min. the normal range is of great importance, as some diseases (in asthma)
may be related to changes in this muscle tone. Studies have reported
2.7. Statistical analysis that some essential oils as well as their constituents had contracting
effects on basal tone; for example, terpinene-4-ol. This effect may be
Data are expressed as the mean ± S.E.M, n = 6 in all experiments. related to an interference with the storage of Ca+2 in the sarcoplasmic
Sigma Plot 11.0 software was used for statistical analysis and graphics reticulum (Câmara, Nascimento, Macêdo-Filho, Almeida, & Fonteles,
production. Effects were considered statistically significant if they had a 2003; Peixoto-Neves et al., 2010; Pinho-da-Silva et al., 2012). These
null hypothesis probability lower than 5% (p < 0.05). Student t tests results indicate that it is important to determine whether or not the
and analysis of variance (one or two-way ANOVA) were used, followed substances alter the tone of airway smooth muscle. Our results showing
by Bonferroni’s t-test and Holm-Sidak multiple comparisons method, EODa and α-terpinene do not interfere with the basal tone is of great
when appropriate. In order to calculate the IC50, the concentration of relevance for future use of this product as a therapeutic agent.
the substance which is capable of producing 50% of inhibition of its EODa and α-terpinene were evaluated on their effects on tracheal
maximum effect, a logarithmic interpolation for each experiment was smooth muscle contracted by 60 mM KCl (N = 6). Both agents relaxed
performed, and when not possible, the linear relationship between two this muscle up to 100% in a concentration-dependent manner. For the
points of the IC50 was performed.

3. Results and discussion

3.1. Phytochemical profile of the D. ambrosioide essential oil

The study began with an evaluation of the phytochemical profile of

the EODa, which was performed by the Gas Chromatography coupled to
Mass Spectrometry methodology (GC–MS). The following profile was
identified (Table 1) and showed that the major constituent was α-ter-
pinene (30.53%). Amongst secondary constituents identified it was
outstanding thymol and o-cymene, 18.11% and 13.68%, respectively.
The percentages of all the identified compounds are shown in Table 1.
Similar to our findings, Sá, Galvão, Ferreira, Soares, and Randau (2014)
demonstrated that the essential oil of Dysphania ambrosioides (also
known as Chenopodium ambrosioides), presented in its chemical com-
position the following major compounds: α-terpinene (42.14%) and α-
terpinenyl-acetate (31.57%), followed by thymol (7.90%).

3.2. Myorelaxant effect of the EODa and α-terpinene in rat tracheal rings
In order to analyze the effect of the EODa and its α-terpinene con-
stituent on the intrinsic basal tone of rat tracheal rings, increasing and
cumulative concentrations (1–1000 μg/mL) of EODa and α-terpinene
Fig. 1. Effect on the basal tone produced by the essential oil of Dysphania
ambrosioides (EODa) and α-terpinene in rat tracleal smooth muscle. Values are
expressed as mean ± S.E.M.; (EODa P = 1.000 and α-terpinene P = 1.000,
one-way ANOVA followed by Holm-Sidak) (N = 6).

3
L. Pereira-de-Morais, et al. Food Chemistry 325 (2020) 126923

Fig. 2. A: Representative experimental trace, MT Modified Tyrode; B: Relaxation produced by the essential oil of Dysphania ambrosioides (EODa) and α-terpinene in
rat tracleal preparations pre-contracted with 60 mM K+. The concentrations used were: 1–1000 μg/mL for EODa and 1–3000 μg/mL for α-terpinene. Values are
expressed as mean ± S.E.M.; *** represents the significant effects (EODa P < 0.007 and α-terpinene P < 0.001, one-way ANOVA followed by Holm-Sidak)
(N = 6).

EODa, the myorelaxant effect was statistically significant from the
concentration of 1 μg/mL, completely relaxing the tissue at 1000 μg/
mL, while α-terpinene was significant at 3 μg/mL, completely inhibiting
the contraction at the concentration of 3000 μg/mL (EODa P < 0.007
and α-terpinene P < 0.001, one-way ANOVA followed by Holm-Sidak)
(Fig. 2 B). The IC50 values obtained were the following for the EODa
and α-terpinene, respectively: 50.9 ± 1.25 and 43.7 ± 4.46 μg/mL.
It was evident in this study that the EODa and α-terpinene promoted
the relaxation of contractions induced by 60 mM KCl with maximal
pharmacological efficacy by promoting 100% relaxation of tracheal
smooth muscles. The myorelaxant effect of the EODa may be partly
attributed to its major constituent α-terpinene, which constitutes
30.53% of the mass of the oil. However, the EODa displayed a higher
potency than α-terpinene, thus indicating other constituents present in
this essential oil may be acting synergistically. Examples might be
thymol and carvacrol which constitute a considerable percentage
(18,11 and 11,23%, respectively) of EODa. These two monoterpenes
have a myorelaxant effect on tracheal smooth muscle (Boskabady &
Jandaghi, 2003; Engelbertz, Lechtenberg, Studt, Hensel, & Verspohl,
2012).
The relaxing effect of EODa and its major α-terpinene constituent on
KCl-evoked contractions (60 mM) suggests a mechanism involving,
blockade of voltage-gated Ca2+ channels (VGCCs) since the contraction
caused by elevation of K+ concentration in the extracellular medium
occurs because 60 mM [K+] causes membrane depolarization and
opening of VGCCs. Studies with essential oils of vegetable in the pre-
sence of KCl causing relaxation can be found in the literature. For ex-
ample, the studies by Lima et al. (2010) with Eucalyptus tereticornis
essential oil and by Carvalho et al. (2018), with Lippia alba essential oil
reveled that both essential oils relaxed the contractions promoted by
K+ (60 mM) in isolated rat trachea. Similarly, it has been demonstrated
that several monoterpenes have myorelaxant effects on smooth muscle
of various organs of rats, such as: peryl alcohol, carveol, citral and li-
monene on the aorta (Cardoso-Teixeira et al., 2018; da Silva et al.,
2018), citral and limonene in uterus (Pereira-de-morais et al., 2019)
and 1,8-cineol, citral and limonene in trachea (Pereira-Gonçalves et al.,
2018; Carvalho et al., 2018), among others .
According to Janbaz et al. (2014), when inducing contractions in
rabbit tracheal smooth muscle by the addition of 80 mM K+, the
Cymbopogon martinii extract was observed to possess a high relaxation
potential. In the study by Modesto et al., 2018, the Lippia origanoides
essential oil was also observed to relax isolated guinea pig trachea pre-
contracted by depolarization with 60 mM KCl in a concentration-de-
pendent manner. In sum these literature reports demonstrate that,
natural products, such as several essential oils, contain airway relaxa-
tion properties, principally reversing contractions induced by

4
L. Pereira-de-Morais, et al.
Fig. 3. A: Representative experimental trace, MT Modified Tyrode; B: Effect of essential oil of Dysphania ambrosioides (EODa) and α-terpineno on contraction induced
by acetylcholine. Effect of cumulative addition of EODa and α-terpineno in rat isolated tracleal tissue preparations pre-contracted with ACh (10 µM). The con-
centrations used were: 1–3000 μg/mL. Values are expressed as mean ± S.E.M.; *** represents the significant effects (EODa P < 0.001 and α-terpinene P < 0.002,
one-way ANOVA followed by Holm-Sidak); (N = 8).
potassium.
We also investigated the effect of EODa and α-terpinene on con-
traction induced by activation of membrane cholinergic muscarinic
receptors. The EODa was observed to concentration-dependently and
with maximal pharmacodynamics efficacy relax the sustained con-
traction of tracheal smooth muscle induced by 10 μM acetylcholine
(N = 6). By contrast, α-terpinene potentiated contraction produced by
acetylcholine (Fig. 3 A) (N = 8). The EODa-induced relaxant effect was
statistically significant from the concentration of 1000 μg/mL, com-
pletely relaxing tissues at the concentration of 3000 μg/mL, with an
IC50 of 956.3 ± 2.83 μg/mL (Fig. 3 B). On the other hand, α-terpinene
promoted a contraction which became significant from 600 μg/mL and
reached a 20% contraction increase at the concentration of 3000 μg/mL
in the presence of ACh (EODa P < 0.001 and α-terpinene P < 0.002,
one-way ANOVA, Holm-Sidak method) (Fig. 3 B).
When we administer the acetylcholine (ACh), it binds to the G-
protein coupled M3 receptor, which then binds to GTP and activates
phospholipase C (PLC). It subsequently cleaves phosphatidylinositol
bisphosphate (PIP2) in the cell membrane and produces inositol tri-
phosphate (IP3) and diacylglycerol (DAG) second messengers. IP3 then
binds to its specific receptor on the surface of the sarcoplasmic re-
ticulum (RS) and thereby increases intracellular [Ca2+]i DAG, by its
turn, activates protein kinase C (PKC) (Alotaibi, 2014; Song et al.,
2016).
In this study the EODa completely inhibited contractions induced by
10 μM Ach suggesting changes in DAG and/or IP3. The oil was less
5
Food Chemistry 325 (2020) 126923
pharmacodynamically potent in relaxing muscles contracted by ACh
than by KCl. Total relaxation in muscles contracted by ACh required
3000 μg/mL EODa while KCl contracted muscle required a concentra-
tion of 1000 μg/mL EODa. The difference in myorelaxant pharmaco-
dynamic potency of the EODa can also be shown by analyzing its IC50;
whilst for the electromechanical excitation-contraction couplig (ECC;
KCl-induced contraction) it was 50.9 ± 1.25 μg/mL, for the pharma-
comechanical ECC (contraction induced by cholinergic receptor acti-
vation) it was 956.3 ± 2.83 μg/mL. This significant difference in
pharmacodynamic potency is a suggestion that EODa acts through more
than one mechanism of action. If we are analyzing its action in the
context of a progressive increase in concentration, the effect appears
first in the electromechanical ECC, probably through of VGCCs
blocking. From this point of view it can be suggested that the blocking
effect of electromechanical ECC and, as suggested, blocking Ca2+
channels, is its most important mechanism of action.
α-Terpinene presents complete relaxation at the concentration of
3000 µM with an IC50 of 43.7 ± 4.46 μM in the electromechanical
(KCl) ECC. However, in the pharmacomechanical ECC, as mentioned
above, the opposite effect was observed, promoting a 20% potentiation
of the ACh-induced contraction (Fig. 3 B). One possible explanation is
that α-terpinene may be acting by potentiating some effect of ACh. We
hypothesize a total or partial inhibition of acetylcholinesterase (AChE)
activity. AChE is an enzyme responsible for the metabolism of ACh.
Supportive of this hypothesis, Lima et al. (2010), observed that the α
and β-pinene monoterpenes potentiated contractions promoted by ACh,
L. Pereira-de-Morais, et al.
and they suggested as possible cause, inhibition the AChE activity.
Many natural products have been shown to affect AChE. Given the
importance of cholinergic manipulation for the treatment of
Alzheimer's disease (Petronilhoa, Pintob, & Villara, 2011) studies of
natural products bear special meaning. Indeed, in previous studies, it
was observed that essential oils from some species, such as: Artemisia
dracunculus L., Inula graveolens L., Lavandula officinalis Chaix and
Ocimum sanctum L. have an inhibitory effect on the AChE enzyme (Dohi,
Terasaki, & Makino, 2009).
The AChE effect may explain the small pharmacological potency of
the relaxing effect of the essential oil on the pharmacomechanical ECC.
A possible AChE effect of α-terpinene coupled with a small pharma-
codynamic potency of its relaxant activity may also explain a myo-
contractile effect of this substance. It is also to be noticed that, despite
the oil having α-terpinene as its major constituent, it has a myorelaxant
effect, indicating that in the presence of other constituents the effect of
α-terpinene is synergistically inhibited. It is an important observation
that there is(are) in this essential oil constituent(s) with great phar-
macological potency for the contraction inhibition effect, so as to, at the
small concentration found in the essential oil, be able to overcome α-
terpinene-induced pro-contracturant activity and impose relaxation.
This fact also illustrate that an essential oil can bear pharmacological
effect very different from the major constituent, occasionally ther-
apeutically more advantageous, probably due to the mixture rather
than to a single constituent.
Our results corroborate those in the literature. Pinho-da-Silva et al.
(2012), demonstrated that the trans-caryophyllene terpene as well as α-
terpinene, decreased contractions induced by KCl in rat trachea in a
concentration dependent manner. When contractions were induced by
ACh, trans-caryophyllene also showed reduced potency in this response
which uses the cholinergic pharmacomechanic ECC.
In studies where tracheal smooth muscle contractions were evoked
by 5-HT (10 μM) another myocontracturant agonist, EODa at 1000 μg/
mL promoted a relaxation that was statistically significant reaching
98.2 ± 8.94% (P < 0.001, two-way ANOVA, Holm-sidak). α-terpi-
nene, at 3000 μg/mL, relaxed only 28.1 ± 9.47% of 5-HT-induced
contraction, which shows that, despite significance of this effect, it has
lower efficacy than EODa in this pharmacological ECC, and additionally
shows its low relaxing potency on this effect (P < 0.001, two-way
ANOVA, Holm-sidak) (Fig. 4). At the end of the experiments, tissue
contractions were reversed indicating that they remained viable even
after exposure to the oil and α-terpinene.
Fig. 4. Relaxation produced by the essential oil of Dysphania ambrosioides
(EODa) and α-terpinene in rat tracleal tissue preparations pre-contracted with
5-HT (10 µM). The concentrations used were: 1000 μg/mL for EODa and
3000 μg/mL for α-terpinene. Values are expressed as mean ± S.E.M.; ***
represents the significant effects (EODa and α-terpinene P < 0.001, two-way
ANOVA followed by Holm-Sidak) (N = 6).
6
Food Chemistry 325 (2020) 126923
Our results endorse these of Sadraei, Ghannadi, and Malekshahi
(2003) who demonstrated that Melissa officinalis essential oil, as well as
the EODa, inhibited 5-HT-induced contractions of the mouse ileum.
Jarvis, Barbosa, and Thompson (2016) demonstrated that Lippia alba
essential oil produced a concentration-dependent relaxations of con-
tractions evoked by 5-HT (10 μM) in rat tracheal smooth muscle and
guinea pig ileum. Moreover, according to Pereira-de-morais et al.
(2019), the Lippia alba essential oil (600 μg/mL) completely reversed
uterine smooth muscle contractions evoked by 5-HT (10 μM), reaching
96.2 ± 1.82% relaxation. These studies reinforce our findings for the
actions of the EODa on rat trachea.
To investigate the participation of voltage-gated L-type calcium
channels (VGCCs), experiments were performed where tracheal tissue
was kept depolarized in calcium-free medium containing 80 mM [K+].
Under these conditions the cumulative addition of Ba2+, induced con-
centration-dependent contractions. These contractions reached a max-
imum value with 30 mM BaCl2. In preparations preincubated with 300
and 600 μg/mL of EODa, it was observed that there was a complete
contraction blockade up to the 3 mM [Ba2+] and from the 10 mM
[Ba2+] upwards the preparations showed a small contractile response
of maximum 0.5 g/force. When the tracheal tissue was incubated with
1000 and 2000 µg/mL α-terpinene, it then promoted minimum
blockade of Ba-induced contraction. In presence of 1000 μg/mL of
EODa and 3000 μg/mL of α-terpinene, no Ba-induced contraction was
observed (EODa P < 0.005 and α-terpinene P < 0.003, ANOVA,
Holm-Sidak method). These similar effects were produced by nifedipine
(1 μM), a voltage-dependent selective L-type calcium channel blocker
(Fig. 5) (P < 0.05, one-way ANOVA followed by Holm-Sidak), thus
showing that EODa and α-terpinene may be acting in the same way as
nifedipine, that is, blocking L-type VGCCs. Ba2+ selectively permeates
through VGCCs (Murray & Kotlikoff, 1991) and thus EODa and α-ter-
pinene blocking effect of Ba-induced contraction strongly suggest that
this blocking activity is due to VGCCs Blockade by these agents. These
data also point out that EODa has a great pharmacological potency as L-
type calcium channel blocker, whereas α-terpinene needs a very high
concentration to effectively block these channels (has smaller phar-
macological potency as compared to EODa), showing once again that α-
terpinene participation in the action of the EODa is minimum and it is
likely that other compounds are involved in promoting such action.
Fig. 5. Effect of the essential oil of Dysphania ambrosioides (EODa) (300, 600
and 1000 μg/mL), and α-terpinene (1000, 2000 and 3000 μg/mL) on con-
tractions evoked by exogenous Ba2+. Nifedipine (1 μM) was used as a positive
control. x-axis (abscissa): varying concentrations of Ba2+ contractile agent in
mM. Significant effects in relation to control (EODa P < 0.005, α-terpinene
P < 0.003 and nifedipine P < 0.05, one-way ANOVA, Holm-Sidak method)
(N = 6).
L. Pereira-de-Morais, et al.
Nifedipine was used as a control because it is a selective L-type
calcium channel blocker (Salemme, Rebolledo, Speroni, & Milesi,
2007). In the study by Pereira-Gonçalves et al. (2018), it was shown
that 1,8-cineole monoterpene blocked barium (Ba2+)-induced con-
tractions acting on L-type calcium channels. It was also observed in the
same study that 1,8-cineol has a biphasic effect on tracheal smooth
muscle, where at certain concentrations it has a myorelaxant effect and
at other concentration it increased contractions, thus corroborating our
findings.
4. Conclusion
Both the EODa and its constituent, α-terpinene are here demon-
strated to be able to block contraction induced by several contraction-
inducing agents. EODa and α-terpinene were able to block the KCl-in-
duced contractile response, an unspecific contraction, with maximal
pharmacodynamic efficacy. This allow us to categorize EODa and α-
terpinene as antispasmodic agents. These data show that EODa, which
did not show contraction potentiation, has potential for development as
future therapeutic agents in the treatment of diseases involving tracheal
muscle contractions: these include asthma or bronchitis, since both
relax the smooth muscle of the rat isolated trachea. In the case of α-
terpinene, if our hypothesis of its anticholinesterase activity and ACh
acivity potentiation proves to be correct, it might be of potential use-
fulness in the treatment diseases in which inhibition of acet-
ylcholinesterase activity could be beneficial. Future studies should be
carried out in order to define other mechanisms involved in the effect of
EODa and its constituent α-terpinene.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
Daniel Weinreich, Ph.D-Professor Department of Pharmacology,
University of Maryland-Baltimore-USA for contributions in this article.
This research was supported by: Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior–CAPES, Conselho Nacional de
Desenvolvimento Cientifico e Tecnológico-CNPq and Fundação
Cearense de Apoio ao Desenvolvimento Científico e Tecnológico –
FUNCAP-Finance Code BPI: BP3. 00139-00072.0200/18.
Author contributions section
Andressa de Alencar Silva: data organization.
Renata Evaristo Rodrigues da Silva: Assistance in experiments.
Daniela Maria do Amaral Ferraz Navarro: Analysis of botanical
material.
Henrique Douglas de Melo Coutinho: Writing Assistance.
Irwin Rose Alencar de Menezes: Writing Assistance.
Marta Regina Kerntopf: Data analysis.
Francisco Assis Bezerra da Cunha: Essential Oil Extraction.
José Henrique Leal-Cardoso: Advisor.
Roseli Barbosa: Advisor.
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