Industrial Crops & Products 144 (2020) 112059

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Industrial Crops & Products

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Essential oil from Eugenia stipitata McVaugh leaves has antinociceptive, anti- T
inflammatory and antipyretic activities without showing toxicity in mice
Wêndeo Kennedy Costaa,*, João Ricardhis Saturnino de Oliveiraa, Alisson Macário de Oliveiraa,
Izabelly Bianca da Silva Santosa, Rebeca Xavier da Cunhaa, Anderson Felipe Soares de Freitasa,
Janderson Weydson Lopes Menezes da Silvaa, Valquíria Bruna Guimarães Silvaa,
Júlio César Ribeiro de Oliveira Farias de Aguiarc, Alexandre Gomes da Silvab,
Daniela Maria do Amaral Ferraz Navarroc, Vera Lúcia de Menezes Limaa, Márcia Vanusa da Silvaa
a
Universidade Federal De Pernambuco – Departamento De Bioquímica, Recife PE, 50670-901, Brazil
b
Universidade Federal De Pernambuco – Departamento De Antibióticos, Recife PE, 50670-901, Brazil
c
Universidade Federal De Pernambuco – Departamento De Química Fundamental, Recife PE, 50670-901, Brazil

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

Keywords: Eugenia stipitata is a Brazilian plant species used in traditional medicine for a wide range of ailments. Considering
Anti-inflammatory the biotechnological potential of herbal oils, in this study an essential oil was evaluated for toxicity, anti-
Myrtaceae nociceptive, antipyretic, and anti-inflammatory activity in mice model. The essential oil from Eugenia stipitata
Pain (EsEO) leaves was obtained by hydrodistillation and analyzed by gas chromatography-mass spectrometry.
Pyrexia
Abdominal contortion tests were performed to evaluate its antinociceptive activity. For estimating its anti-in-
Toxicological safety
Trans-Caryophyllene
flammatory activity, tests of paw edema, peritonitis, and protein denaturation were performed. The antipyretic
effect was assessed with a yeast-induced pyrexia model. Artemia salina was used for environmental biomoni-
toring and preliminary toxicity. To analyse the safety of use; acute toxicity and hemolytic activity were per-
formed. Majority of the compounds were identified by guaiol (13.77%), trans-caryophyllene (11.36%), and β-
eudesmol (8.13%) e γ-eudesmol (6.55%). Essential Oil from E. stipitata promoted mortality of A. salina nauplii,
with an LC50 of 812.45 μg/mL. No mice death(s) was noted with oral administration of the essential oil at 2000
and 5000 mg/kg, respectively; additionally, no alterations were observed with biochemical, hematological, and
histopatological analyses. Markedly reduced abdominal contortion (54.1–56.6% inhibition) at all doses as
compared with the negative control. Reduction in inflammation was also seen. In the paw edema test, a sig-
nificant decrease in edema (88.66–96.94%) was observed. Whereas, in peritonitis; a reduction in the migration
of total leukocytes (76.8–86.5%) and neutrophils (74.5–79.6%) was noted. Moreover, EsEO was capable of
inhibiting protein denaturation (74.39–82.83%) as compared with the control (45.02–100%) at the con-
centrations evaluated. It also exerted an antipyretic effect after the first 1 h of evaluation, until the end of
observation duration (4 h). The results suggested EsEO as a promising source of natural antinociceptive, anti-
inflammatory, and antipyretic constituent for application in food and pharmaceutical industries.

1. Introduction
Medicinal plants possess characteristics that aids in treating diseases
or improving people's health conditions. These plants produce sub-
stances that are capable of altering the functioning of organs and sys-
tems and therefore, becomes an excellent source for the search for new
compounds for therapeutic use (Wurtzel and Kutchan, 2016). Brazil
possesses the highest biodiversity on this planet and among the brazi-
lian phytogeographic domains, Caatinga (vegetation type with phy-
siological adaptations for constant temperature variations and water
deficit) is present. Therefore, these plants develop interesting chemical
characteristics and are used as medicinal sources (Saraiva et al., 2015).

⁎
Corresponding author.
E-mail addresses: wendeocosta@gmail.com (W.K. Costa), ricardhis@gmail.com (J.R.S.d. Oliveira), alissonmacario@hotmail.com (A.M.d. Oliveira),
izabelly.ufpe@outlook.com (I.B.d.S. Santos), rebeca_xavier@live.com (R.X.d. Cunha), afbiomedicine@gmail.com (A.F.S.d. Freitas),
jandersonweydson@gmail.com (J.W.L.M.d. Silva), valquiria.b.guimaraes@gmail.com (V.B.G. Silva), juliocrofa@gmail.com (J.C.R.d.O.F.d. Aguiar),
agsilva@live.com (A.G.d. Silva), navarrix@uol.com.br (D.M.d.A.F. Navarro), vlml@ufpe.br (V.L.d.M. Lima), marciavanusa@yahoo.com.br (M.V.d. Silva).

https://doi.org/10.1016/j.indcrop.2019.112059
Received 6 August 2019; Received in revised form 11 December 2019; Accepted 16 December 2019
Available online 21 December 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.
W.K. Costa, et al.
Genus Eugenia L., belonging to Myrtaceae family, is widely dis-
tributed in tropical and subtropical areas, found mainly in the
Americas. With more than 1000 species (Mazine et al., 2014; Villarroel
et al., 2016), plants belonging to this genus have been reported to
possess multiple biological activities such as antioxidant (Cunha et al.,
2016; Neri-Numa et al., 2013), antimicrobial (Santos et al., 2018;
Rodrigues et al., 2017), antiinflammatory (Costa et al., 2016; Lazarini
et al., 2016), antinociceptive (Basting et al., 2014), cytotoxic (Sousa
et al., 2015), gastroprotective (Basting et al., 2014), and insecticidal
activities (Gonzalez et al., 2014).
Eugenia stipitata McVaugh., is a deciduous shrub with simple leaves,
inflorescence with 2 or 5 flowers, and a globose fruit with succulent,
aromatic, and acidic pulp. (Fernández-Trujillo et al., 2011; Kumar et al.,
2016). It possesses agricultural and industrial importance due to its
traditional medicinal applications (Calvi et al., 2017). Additionally, the
exotic taste, juiciness, high acidity and unique sensory characteristics of
the fruit, makes it suitable to produce juice, nectars, jams, and jellies
(Lizcano et al., 2010).
To the best of our knowledge, there are no reports on chemical
composition of the essential oil of E. stipitata leaves from Caatinga,
however, little has been reported on its therapeutic potential.
Accordingly, this work was aimed to evaluate the acute toxicity in mice
with respect to the essential oil from E. stipitata leaves and to assess its
antinociceptive, anti-inflammatory, and antipyretic activity.
2. Materials and methods
2.1. Plant material and extraction of essential oil
Leaves of E. stipitata were collected in January 2017 (drought
period) in the municipality of Exu (07° 30′43″S 39° 43′ 27″ W),
Pernambuco, Brazil, Caatinga region. Access was recorded (A08E18B)
in the Sistema Nacional de Gestão do Patrimônio Genético e do
Conhecimento Tradicional Associado (SisGen).The botanical identifica-
tion was made by Dra. Arlene Pessoa da Silva and a voucher specimen
(13,054) was deposited in the Herbarium Dárdano de Andrade Lima of
the Universidade Regional do Cariri (URCA).
The collected leaves were washed in distilled water, dried at room
temperature and then ground in an industrial crusher to obtain a final
mass of 900 g. The processed plant material was hydrodistilled for 4 h
in a Clevenger type apparatus to obtain the essential oil which was
termed EsEO (Eugenia stipitata Essential Oil) and stored in an amber
glass jar tightly closed at -5 °C until needed to the tests.
2.2. Gas chromatography - mass spectrometry (GC/MS) analysis of
essential oil
Gas Chromatography (GC) analyses were performed in order to
determine the relative proportions of the components of the oils. GC
analyses were carried on a Thermo TraceGC Ultra (Thermo Scientific,
Milan, Italy) equipped with a flame ionization detector (FID) (Thermo
Scientific, Milan, Italy) and a VB-5 fused silica capillary column
(ValcoBond 30 m ×0.25 mm i.d.; film thickness: 0.25 mm) (Valco
Instruments Company Inc., Houston, TX, USA). Nitrogen was employed
as a carrier gas at a flow rate of 1 L/min and 30 psi inlet pressure. The
oven temperature program was initially 40 °C, was held for 2 min, was
increased to 230 °C at 4 °C/min, and was then held for 5 min. The
injector and detector temperatures were set to 250 °C and 280 °C, re-
spectively. The sample (1 μL; 2 mg/mL in n-hexane) was injected
splitless. The relative amount of each component was estimated from
the corresponding peak area and expressed as a percentage of the total
area of the chromatogram. Analyses were carried out in triplicate.
The identification of the compounds present in the oils was per-
formed by GC coupled with mass spectrometry (GC-MS). These analyses
were carried out using an Agilent 5975C Series GC/MSD (Agilent
Technologies, Palo Alto, CA, USA) quadrupole instrument equipped
2
Industrial Crops & Products 144 (2020) 112059
with an Agilent J&W non-polar DB-5 fused silica capillary column (30
m ×0.25 mm i.d.; film thickness: 0.25 μm) (Agilent Technologies, Palo
Alto, CA, USA). For each sample, 1 μL was injected in split mode (50:1)
with the injector temperature set to 250 °C. GC oven temperature was
set to 40 °C, was held for 2 min, was increased to 230 °C at 4 °C/min,
and was then held for 5 min. Helium (He) was used as a carrier gas at a
flow of 1 mL/min, maintained at a constant pressure of 7.0 psi. MS
Source and quadrupole temperatures were set to 230 °C and 150 °C,
respectively. Mass spectra were taken at 70 eV (in EI mode) with a
scanning speed of 1.0 scans from m/z 35–350. The identification of the
individual components was carried out by comparison with previously
reported values of retention indices, obtained by co-injection of oil
samples and a set of C9–C30 linear hydrocarbons, and calculated ac-
cording to the equation of Van den Dool and Kratz (1963). Subse-
quently, the MS data acquired for each component were matched with
the data available in the mass spectral library of the GC-MS system
(MassFinder 4, Dr. Hochmuth scientific consulting, Hamburg, Ger-
many); NIST08 Mass Spectral Library (ChemSW Inc. Fairfield, CA,
USA); Wiley Registry™ of Mass Spectral Data 9th Edition (Wiley, Ho-
boken, NJ, USA) and with other published mass spectral data (Adams,
2007). Analyses were carried out in triplicate.
2.3. In vivo assay
All the experimental procedures are in accordance with the
Brazilian laws for animal experimentation and were submitted to the
Animal Ethics Committee of the Universidade Federal de Pernambuco
and received a favorable opinion in accordance with protocol no.
23076.011325/2018-77. Male mice (Mus musculus) Swiss weighing
between 30 and 35 g and aged between 8 and 10 weeks, obtained from
the laboratory of the Keizo Asami Immunopathology Laboratory (LIKA)
were used under standard conditions (for example, 12 h clear / dark
cycle, 22 ± 0.1 °C and 50–55 % humidity) with appropriately fed
species and water supplied ad libitum. The animals were acclimatized
and fasted for 6−8 h and water restriction 30 min before the experi-
ments.
2.4. Hemolytic potential
Test was performed in 96-well plates using a 2% suspension of mice
erythrocytes in a 0.85% NaCl solution containing 10 mM CaCl 2 ac-
cording to a method described by Jimenez et al. (2003), in triplicate.
Essential Oil from E. stipitata was tested in concentrations (0.062–2.000
mg/mL). After incubation at room temperature for 30 min, the samples
were centrifuged and the supernatant removed for measurement of the
hemoglobin released by spectrophotometer reader at 540 nm absor-
bance.
2.5. Toxicity evaluation
2.5.1. Artemia salina lethality assay
Artemia salina cysts (San Francisco Bay Brand, Inc., USA) were in-
cubated in natural sea water at 25–30 °C, with the pH adjusted to 9.0
with sodium bicarbonate. After nauplii (first larval stage) hatching, the
assay was performed as described by Meyer et al. (1982). The Essential
Oil from E. stipitata was diluted in sea water in Falcon tubes to achieve 5
mL solutions at 1, 3, 10, 30, 100, 300, 500, 750 and 1000 μg/mL. Each
assay contained 10 nauplii. In the negative control, the nauplii were
incubated in sea water. After 24 h, the survival rates were determined
and the concentration required to kill 50% of the nauplii was calcu-
lated. Three independent experiments were performed in triplicate.
2.5.2. Acute toxicity oral
Acute toxicity of EsEO was evaluated according to the instructions
of the Organization for Economic Cooperation and Development
(2001). Female mice were separated into three groups (n = 3) that
W.K. Costa, et al.
received a single dose of the treatment by gavage (in a final volume of
0.1 mL). The control group, which received saline solution (control),
and two test groups, which received the EsEO at 2000 mg/kg and 5000
mg/kg b.w. respectively. Behavioral alterations were evaluated during
the first 60 min after treatment administration, divided into four per-
iods (0–15, 15–30, 30–45, and 45–60 min) and daily until the 14th day.
The following parameters were evaluated: piloerection, stool appear-
ance, sensitivity to sound and touch, mobility, and aggressive behavior
(Almeida et al., 1999; Malone, 1983).
Hematological analyses were carried out immediately after blood
collection using an automatic hematology analyzer (Coulter STKS,
Beckman Coulter, Miami, FL, USA) and optical microscopy. Parameters
included counting of red blood cells (RBC), hemoglobin (HB), hema-
tocrit (HCT), mean corpuscular volume (MCV), mean corpuscular he-
moglobin (MCH), mean corpuscular hemoglobin concentration (MCHC)
and white blood cells (WBC) differential leukocyte counts segmented
(SEG), lymphocytes (LYM) and monocytes (MON).
For biochemical analyses, the blood was centrifuged at 1480×g for
10 min to obtain serum that was stored at −20 °C. The following
parameters were determined: albumin (ALB), blood urea nitrogen
(BUN), creatinine (CRE), aspartate aminotransferase (AST), alanine
aminotransferase (ALT), total cholesterol (TC), triglycerides (TG), total
protein (TP), alkaline phosphatase (ALP), gamma-glutamyl transferase
(GGT) and bilirubin (BIL). The dosages of these parameters were per-
formed using specific kits (Labtest Diagnóstica, Lagoa Santa, Brazil) and
a COBAS Mira Plus analyzer (Roche Diagnostics Systems, Basel,
Switzerland).
Histological analyses of the liver, kidney, spleen, lung, and heart of
mice from the control group and the treatment groups were performed
by optical microscopy. Sections of the organs were fixed in buffered
formalin (10%, v/v), dehydrated through a graded ethanol series
(70–100%), diaphanized in xylol, and embedded in paraffin.
Histological slices (5 μm) were stained with hematoxylin-eosin and
mounted using cover slips with Entellan resin (Merck, Germany)
(Kiernan, 2008). The samples were observed using a Motic BA200 mi-
croscope coupled to a Moticam 1000 1.3 M P digital camera (Motic
Incorporation Ltd., Causeway Bay, Hong Kong).
2.6. Evaluation of antinociceptive activity (Acetic acid-induced writhing
test)
Male mice were separated into five groups (n = 6 per group) and
treated with oral saline (control), oral EsEO (40, 100 or 250 mg/kg),
intraperitoneal indomethacin (20 mg/kg). Saline solution and the EsEO
were administered 1 h before the acetic acid, indomethacin was ad-
ministered 30 min before. Each animal received an intraperitoneal in-
jection (0.1 mL/10 g) of 0.85% (v/v) acetic acid in saline, and was then
placed in a polyethylene box to record the latency period (time until the
first writhing) and the number of writhes in the interval corresponding
to 5–15 min after the injection of acetic acid (Oliveira et al., 2018;
Queiroz et al., 2010)
2.7. Antipyretic activity
Antipyretic activity was determined using the fungus-induced fever
model. After the basal rectal temperature was identified, fever was in-
duced with subcutaneous injection with yeast (Saccharomyces cerevisae,
15% w/v in saline). After 18 h, animals with temperature above 38 °C
were randomly assigned to five groups and then treated with saline
0.9% (Control), Dipirone (positive control 100 mg/kg) or EsEO (40, 100
and 250 mg/kg). The temperature was evaluated using a digital ther-
mometer every 1 h for 4 h (Winter et al., 1962).
3
Industrial Crops & Products 144 (2020) 112059
2.8. Anti-inflammatory activity
2.8.1. Carrageenan-induced paw edema
Mice were divided into five groups of six animals each. The paw
edema was induced by 2% carrageenin, injected in a volume of 15 μL/
animal in the subplantar region of the right mouse paw and 15 μL/
animal of saline 0.9% in the left paw (Winter et al., 1962). The animals
were pre-treated with different products: EsEO (40, 100 and 250 mg/
kg), Control (0.9% saline solution) or indomethacin (20 mg/kg) given
orally 1 h before subplant injections. Paw volume was measured by
pachymeter at 1, 2, 3, 4 and 24 h after, as described by Huang et al.
(2012). Inhibition of edema was calculated by (right paw - left paw).
2.8.2. Carrageenan‐induced peritonitis
Inflammation was induced by the modified method of Griswold
et al. (1987). Mice were divided into five groups of six animals each.
The oral treatments EsEO (40, 100 and 250 mg/kg), Control (0.9%
saline solution) or indomethacin (20 mg/kg) were performed 1 h prior
to the induction of peritonitis. After this time, carrageenan (0.25 mL,
0.75% w/v in saline) was injected intraperitonially. Four hours later,
the animals were euthanized by cervical dislocation, and 2 mL of he-
parinized phosphate buffered saline was injected into the peritoneal
cavity. Posteriorly, a gentle massage was made and peritoneal exudates
were removed. The total leukocyte count was performed in a Neubauer
chamber, and the differential cell determination was established. The
percentage of leukocyte inhibition was calculated using the following
formula: % of leukocyte inhibition = (1 − T/C) × 100, where T re-
presents the treated groups' leukocyte count, and C represents the
control group leukocyte count. Inhibition of neutrophil migration was
calculated by the following equation: Inhibition of neutrophil migration
= (1 − NT/NC) × 100, where NT = neutrophil counts of treated
groups, and NC = neutrophil counts of the control group.
2.8.3. Inhibition of protein denaturation (in vitro)
The in vitro anti-inflammatory activity of EsEO was performed using
the BSA (Bovine Serum Albumin) albumin denaturing technique ac-
cording to Lavanya et al. (2010) with modification. The test solution
(TS) was composed of 50 μL serial dilutions of EsEO (0.25–2.00 mg/mL)
added in 450 μl of 5% (w/v) BSA. The test control solution (TCS)
consisted of 450 μl 5% (w/v) BSA and 50 μl vehicle (0.9% saline). The
product control solution (PCS) consisted of 450 μl of distilled water and
50 μl of TS containing different concentrations of EsEO (0.25–2.00 mg/
mL). The standard solution (SS) consisted of 450 μl of 5% (w/v) BSA
and 50 μl of diclofenac sodium (0.25–2.00 mg/mL). All of the above
solutions were adjusted to pH 6.3 using 1 N HCl. Samples were in-
cubated at 37 °C for 20 min and the temperature was increased to hold
the samples at 57 °C for 3 min. After the incubation period 2.5 mL of
PBS was added to all tubes and the absorbance was measured using a
spectrophotometer at 416 nm. The percent inhibition of protein dena-
turation can be calculated: % Inhibition = [100 - (Abs TS - Abs PCS) /
(Abs TCS)] x100.
2.9. Statistical analysis
The results were expressed as mean ± standard deviation and ana-
lyzed in GraphPad Prism software, USA. Data were analysed by ANOVA
followed by Tuckey or Bonferroni Test. A p value < 0.001 was con-
sidered significant (antinoceptive, anti-inflammatory and fever) and p
value < 0.05 was considered significant (toxicity evaluation).
3. Results and discussion
3.1. Chemical composition of Eugenia stipitata essential oil (EsEO)
Hydrodistillation of E. stipitata dried leaves, produced a strong,
odorous, clear yellow oil; with a yield of 6.65 g (0.73% w/w). The EsEO
W.K. Costa, et al. Industrial Crops & Products 144 (2020) 112059

Table 1
Chemical composition of the Eugenia stipitata Essential Oil (EsEO).
a b a b
Compone RI RI % Component RI RI %

α-Thujene 924 924 0.28 γ-Muurolene 1477 1478 0.45

α-Pinene 930 932 1.76 D-Germacrene 1482 1480 2.28
Sabinene 970 969 0.82 β-Selinene 1487 1489 0.84
β-Pinene 973 974 2.39 δ-Selinene 1492 1492 0.84
β-Myrcene 988 990 0.29 Bicyclogermacrene 1497 1500 3.97
α-Terpinene 1014 1014 0.28 δ-Amorphene 1508 1511 0.33
Cymene 1020 1022 1.43 γ-Cadinene/ 1515 1514 0.26
Limonene 1024 1026 1.68 δ-Cadinene 1524 1522 0.3
Eucalyptol 1026 1028 1.45 α-Calacorene 1544 1544 0.43
γ-Terpinene 1054 1057 0.47 Elemol 1550 1548 4.39
α-Terpinolene 1086 1087 0.13 B-Germacrene 1558 1559 0.46
L-Pinocarveol 1137 1135 0.17 Palustrol 1569 1567 0.23
Terpinen-4-ol 1176 1174 4.06 Spathulenol 1579 1577 3.87
α-Terpineol 1189 1186 0.38 Caryophyllene oxide 1585 1583 3.81
Myrtenol 1195 1194 0.17 Guaiol 1600 1600 13.77
Δ-Elemene 1337 1335 0.8 5-epi-7-epi-α-Eudesmol 1607 1607 0.41
α-Copaene 1376 1374 0.71 10-epi-γ-Eudesmol 1621 1622 5.97
β-Bourbonene 1385 1387 0.65 γ-Eudesmol 1634 1630 6.55
β-Elemene 1392 1390 0.91 Bicyclo[7.2.0]undecane 1638 1639 0.12
Trans-Caryophyllene 1421 1417 11.36 β-Eudesmol 1653 1649 8.13
γ-Elemene 1434 1434 0.24 α-Eudesmol 1656 1652 5.97
Aromadendrene 1439 1439 0.35 Bulnesol 1669 1670 3.05
Guaia-6,9-diene 1444 1442 0.52 Cadalene 1677 1675 0.18
α-Humulene 1454 1452 1.66 Eudesma-4(15) 1688 1687 0.12
Aromadendrene 1462 1458 1.25 Total % 97.04

RIa = Retention rate determined; RIb = Retention index specialized literature; % = area of compost relative to EsEO.

components with their retention indices (RI) are presented in Table 1. secretion (Suijun et al., 2014).
The gas chromatography-mass spectrometry (GC-MS) analyses revealed The component γ-eudesmol has been described only for E. candol-
that the essential oil possessed a complex chemical profile, with the leana (Nakamura et al., 2010). Whereas, β-eudesmol occurrence is not
presence of a total of 49 compounds, representing 97.04% of EsEO reported in Eugenia species, however, it is present in Eucalyptus ci-
composition (based on area of the peak). Majority of the compounds triodora (Koundal et al., 2016), Blepharocalyx salicifolius (Hernández
were identified with guaiol (13.77%), trans-caryophyllene (11.36%), β- et al., 2018), and Neomitranthes obscura (Victório et al., 2018); all
eudesmol (8.13%), and γ-eudesmol (6.55%). belonging to the family Myrtaceae. Eudesmol isomers (α-, β- and γ-
The EsEO displayed similar qualitative composition with other oils eudesmol) exhibit multiple biological activities, such as anticonvulsant
from species of the same family and with species of the same genus. (Chiou et al., 1997), antiangiogenic (Tsuneki et al., 2005) and anti-
However, when compared with the study by Medeiros et al. (2003) tumor (Ma et al., 2008).
using E. stipitata leaves (collected in Azores, Portugal), a similarity
between some compounds (not among major compounds) was ob-
3.2. Toxicity evaluation
served. The main compounds obtained by Medeiros et al. (2003) were
β-cariophilene (22.7%), cariophilene oxide (15.4%), α-pinene (14.1%),
One way to assess the safety of medicinal plants is to determine
myrcene (2.9%), and limonene (2.7%).
their toxicity. Artemia salina lethality assay is a preliminary indicator of
Previously, guaiol has been reported to be present in Eugenia uni-
general plant compound(s) toxicity, as this microcrustacean is usually
flora (Ogunwande et al., 2005), E. brasiliensis, E. beaurepaireana, E.
very sensitive to toxic substances (Oliveira et al., 2018). EsEO promoted
umbeliflora (Magina et al., 2009), E. brejoensis, E. acutata, and E. can-
A. salina nauplii’s mortality with LC50 as 812.45 μg/mL. This result
dolleana (Nakamura et al., 2010) essential oils. Guaiol is known for its
suggests a toxic potential of EsEO which prompted us to evaluate its
antimicrobial (Choudhary et al., 2007), antitumor (Yang et al., 2016),
acute toxicity in mice along with assessing its possible effect(s) on he-
insecticide (Liu et al., 2013) and leishmanicidal (Garcia et al., 2018)
matological and biochemical parameters, and organ histology.
activities.
In oral toxicity assays, the mice groups (control and EsEO treated at
Trans-caryophyllene has been noted to occur in the essential oils of
2000 and 5000 mg/kg b.w.) did not exhibit alterations in behavioral
E. brejoensis (Souza et al., 2017), E. caryophyllata (Politeo et al., 2010),
signals during the 14 experiment days. Additionally, no significant
E. punicifolia (Sales et al., 2014), E. copacabanensis, E. candolleana and E.
differences in food and water consumption based on biomass gain were
acutata (Nakamura et al., 2010). Trans-caryophyllene is reported to
detected in the control, 2000, and 5000 mg/kg groups (p < 0.05)
have analgesic (Paula-Freire et al., 2014), anti-inflammatory
(Table 2). Determining such parameters is important in studying the
(Fernandes et al., 2007) activities, inhibition of atherosclerosis devel-
safety of a product with therapeutic purpose, as proper nutrient(s) and
opment (Zhang et al., 2017) and also acting in moderation. of insulin
water intake is essential for physiological status of the animals (Iversen

Tabela 2
Evaluation of food and water consumption and weight gain of animals from control and treated with the Eugenia stipitata Essential Oil (EsEO) per os.
Water consumed (mL) Food consumed (g) Weight gain (g)

Control 33.89 ± 0.12 31.6 ± 0.30 5.3 ± 0.10

EsEO 2000 mg/kg 33.82 ± 0.16 31.1 ± 0.44 5.2 ± 0.09
5000 mg/kg 33.84 ± 0.19 31.9 ± 0.39 5.8 ± 0.06

Values represent the mean ± SEM (n = 3/group). No significant differences (p > 0.05) were found in comparison with control.

4
 Table 3
Hematological parameters of mice treated with the Eugenia stipitata Essential Oil (EsEO) per os.
RBC Hct Hb MCV MCH MCHC WBC SEG LYM MON
W.K. Costa, et al.

Control 9.54 ± 0.19 46.22 ± 0.65 16.41 ± 0.55 45.23 ± 0.16 17.88 ± 0.27 37.31 ± 0.18 11.29 ± 1.02 50.34 ± 0.18 38.38 ± 0.07 11.28 ± 0.65
EsEO 2000 mg/kg 9.21 ± 0.32 45.54 ± 0.15 16.52 ± 0.22 46.01 ± 0.28 17.71 ± 0.31 37.89 ± 1.09 10.90 ± 1.17 49.34 ± 0.33 39.10 ± 0.12 11.56 ± 0.09
5000 mg/kg 9.66 ± 0.20 46.09 ± 0.17 13.39 ± 0.32* 40.86 ± 0.62* 17.33 ± 0.15 32.01 ± 0.66* 11.02 ± 1.12 50.22 ± 0.67 38.82 ± 0.21 10.96 ± 0.78

RBC: red blood cells (106/mm3); HCT: hematocrit (%); Hb: hemoglobin (g/dL); MCV: mean corpuscular volume (%); MCH: mean corpuscular hemoglobin (%); MCHC: mean corpuscular hemoglobin concentration (%);
WBC: white blood cells (103/mm3); SEG: segmented (%); LYM: lymphocytes (%); MON: monocytes (%). Values represent the mean ± SEM (n = 3/group). (*) Indicates significant difference (p < 0.05) in comparison with
control.

5
Table 4
Biochemical parameters of blood of mice treated with the Eugenia stipitata Essential Oil (EsEO) per os.
ALB ALT AST ALP GGT TP BUN CRE BIL TC TG

Control 3.89 ± 0.12 67.6 ± 0.30 110.2 ± 0.23 12.3 ± 0.22 10.21 ± 0.23 5.79 ± 0.12 38.5 ± 0.12 0.31 ± 0.06 0.22 ± 0.01 119.4 ± 2.32 135.3 ± 2.05
EsEO 2000 mg/kg 3.82 ± 0.16 67.1 ± 0.44 111.0 ± 0.51 13.5 ± 0.22 11.0 ± 0.44 5.29 ± 0.21 36.3 ± 0.16 0.34 ± 0.10 0.20 ± 0.01 118.8 ± 2.46 134.8 ± 2.09
5000 mg/kg 3.84 ± 0.19 81.9 ± 0.39* 134.7 ± 0.35* 12.3 ± 0.24 10.5 ± 0.31 5.42 ± 0.33 35.9 ± 0.18 0.29 ± 0.12 0.23 ± 0.02 119.4 ± 2.52 126.8 ± 8.26

ALB: albumin (g/dL); ALT: alanine aminotransferase (U/L); AST: aspartate aminotransferase (U/L); ALP: alkaline phosphatase (U/L); GGT: gamma-glutamyl transferase; TP: total protein (g/dL); BUN: blood urea nitrogen
(mg/dL); CRE: creatinine (mg/dL); BIL: bilirubin (mg/dL); TC: cholesterol total (mg/dL) TG: triglycerides (mg/dL). Values represent the mean ± SEM (n = 3/group). (*) indicates significant difference (p < 0.05) in
comparison with control.
Industrial Crops & Products 144 (2020) 112059
W.K. Costa, et al.
and Nicolaysen, 2003). Again, no mortality was observed, with
LD50 > 2000 mg/kg; that classified EsEO safe, as per OECD (2001)
guidelines. Therefore, EsEO could be included in Category 5 (low toxic
or atoxic) according to the Globally Harmonized System of Classifica-
tion and Labelling of Chemicals (GHS) classification.
However, hematological analysis (Table 3) demonstrated significant
(p < 0.05) hemoglobin, MCV, and MCHC reduction in EsEO (at 5,000
mg/kg) treated animals, when compared with the control. The result
indicated that EsEO components might be acting on the erythrocytes,
thereby causing hemoglobin content reduction and might be interfering
with hematopoiesis and resulting in the production of cells with lower
volume. It has been previously reported that plant secondary metabo-
lites might act directly on erythrocytes interfering with the integrity of
plasma membrane, thereby, causing shrinkage, hemoglobin content
reduction, and even cell(s) destruction (Oliveira et al., 2015; Nwankpa
et al., 2014).
Biochemical analysis (Table 4) revealed significant (p < 0.05) al-
terations in the serum levels of ALT and AST enzymes (liver function
markers) in EsEO treated animals (at 5,000 mg/kg), as compared with
the control. The group treated with a dose of 2,000 mg/kg did not
exhibit significant (p > 0.05) variations. Since, liver cells damage
usually results in a release of several enzyme types, therefore, we could
infer that EsEO did not cause a strong impairment of hepatocyte’s
membrane. Furthermore, GGT levels (a more specific marker of hepatic
damage) (Motta, 2009) were similar in all the groups, thereby corro-
borating that EsEO did not display any hepatotoxic effect.
Macroscopic analysis of the organs of EsEO treated mice did not
exhibit significant color or texture changes when compared with the
control groups. Additionally, no significant difference(s) in the relative
weights in EsEO treated mice was noted when compared with the
controls (Table 5). Fig. 1 presents photomicrographs of the liver,
kidney, lungs, and spleen of the control and treated mice groups. The
liver of EsEO treated mice revealed well delimited hepatocytes, nuclei
with visible chromatin, bile ducts, central lobular veins with preserved
characteristics, and the absence of fibrosis. No alterations were ob-
served in the kidneys, suggesting that mice treated with the highest
dose did not display a pathological process. Additionally, the par-
enchyma and interalveolar septa in the lungs of treated mice groups did
not exhibit alterations. Furthermore, the spleen of treated mice did not
display histological alterations when compared with the controls and
displayed no lymph nodes activation.
Brito (1994) and Vasconcellos et al. (2005) have pointed out the
importance of hemolytic assays resulting from the relevance and their
application to the development of scientific research with plant derived
materials, since it determines the plant’s toxic potential and the con-
tinuity of the studies. In our hemolysis test on mice erythrocytes in vitro,
the hemolysis rates ranged from 1.27% to 1.30%, at tested concentra-
tions. This result demonstrated that EsEO possessed low toxicity, lacked
hemolytic activity and did not damage the membranes (Jimenez et al.,
2003).
3.3. Antinociceptive activity
Antinociceptive activity of EsEO was evaluated by acetic acid-in-
duced writhing method. This method is a non-selective, chemical
screening model for analgesic drugs, wherein, inflammatory mediators
Table 5
Evaluation of the relative weight (g/10 g animal body weight) of mice treated with the Eugenia stipitata Essential Oil (EsEO) per os.
Brain Liver Kidney
Control 0.36 ± 0.01 2.18 ± 0.11 0.31 ± 0.01
EsEO 2000 mg/kg 0.38 ± 0.02 2.19 ± 0.19 0.29 ± 0.04
5000 mg/kg 0.39 ± 0.00 2.18 ± 0.18 0.31 ± 0.06
Values represent the mean ± SEM (n = 3/group). No significant differences (p > 0.05) were found in comparison with control.
Industrial Crops & Products 144 (2020) 112059
such as IL-1, IL-6, TNF-α, and prostaglandins (PGE2α and PGF2α) cy-
tokines are released that stimulate nociceptive neurons. It presents a
typical model of visceral inflammatory nociception, widely used in
evaluating the peripheral pathway of analgesic drugs (Ribeiro et al.,
2000).
Essential oil (EO) treatment at all doses significantly inhibited
(p < 0.01) the number of abdominal writhings induced by in-
traperitoneal administration of acetic acid as compared with that of
control mice (Fig. 2). The EsEO treated groups with 40, 100, and 250
mg/kg doses, exhibited a reduction of 54.1%, 55%, and 56.6%, re-
spectively; in the number of writhings. The reference drug in-
domethacin, also displayed antinociceptive effect with 71.8% reduc-
tion, as expected. The results revealed EsEO to be effective in reducing
local and inflammatory pain.
Similar results have been observed in other Eugenia species, as in a
previous study with E. candolleana essential oil (at 100 mg/kg con-
centration), which exhibited 89% inhibition (Guimarães et al., 2009). E.
uniflora EO displayed an effect on contortion inhibition (48%) at 200
mg/kg dose (Amorim et al., 2009). On the other hand, E. caryophyllata
EO at 33 mg/kg, significantly suppressed abdominal writhes develop-
ment in 87.7% (Taher et al., 2015). Therefore, different EO composi-
tions are noted to be involved in analgesic potential differences of the
studies presented.
Although EsEO mechanism in the present study is unclear, however,
its administration was noted to decrease abdominal writhes number
(acetic acid induced). Previous studies involving trans-caryophyllene
have demonstrated it to possess a high antinociceptive power, since, it
could act on COX-2, and TNF-α and PGE2 expression (Hernández et al.,
2007; Medeiros et al., 2007). Another mechanism of action of trans-
caryophyllene is that, it is an endocannabinoids type 2 agonist and the
compounds directed to this system could treat painful and in-
flammatory processes (Paula-Freire et al., 2014).
3.4. Anti-inflammatory activity
3.4.1. Carrageenan-induced paw edema
The experimental carrageenan-induced paw edema model presents
a biphasic response and allows the detection of active anti-in-
flammatory agents acting against acute inflammation mediators. The
first phase is mediated through histamine, serotonin, cytokines, and
kinins release; whereas, the second phase is marked by prostaglandin
and slow reaction substance’s release (Rocha et al., 2006; Salvemini
et al., 1996).
Anti-inflammatory effect of EsEO leaves on carrageenan-induced
edema in the hind paws of experimental mice is presented in Fig. 3. A
gradual increase in paw edema volume of the control group mice
(carrageenan treated group) was noted. However, in the test groups,
EsEO displayed a significant reduction in the paw edema volume. The
results revealed that EsEO at 40, 100, and 250 mg/kg; exhibited
88.66%, 91.3%, and 96.94% inhibition, respectively; while in-
domethacin exhibited 91.64%.
Similar results were observed with E. caryophyllata EO, which sup-
pressed paw edema in 50.6% at 33 mg/kg dose (Taher et al., 2015). It
could be suggested that the observed anti-edematogenic effects could
be attributed to trans-caryophyllene, which is capable of reducing PGE2
generation, the same mechanism used by different clinically used anti-
Lung Heart Spleen Intestine Ovary
0.24 ± 0.04 0.19 ± 0.01 0.29 ± 0.01 0.26 ± 0.01 0.11 ± 0.01
0.24 ± 0.2 0.20 ± 0.01 0.29 ± 0.00 0.28 ± 0.02 0.10 ± 0.02
0.24 ± 0.04 0.21 ± 0.01 0.29 ± 0.02 0.29 ± 0.03 0.07 ± 0.03
6
W.K. Costa, et al.
Fig. 2. Effect of Eugenia stipitata Essential Oil (EsEO) on abdominal contortion
induced by acetic acid. *p < 0.001 compared with Control, one-way ANOVA
followed by Tuckey’s Test.
Fig. 3. Responses of diferent concentrations of Eugenia stipitata Essential Oil
(EsEO) to paw edema induced by carragenan. *p < 0.001 compared with
Control, two-way ANOVA followed by Bonferroni’s Test.
7
Industrial Crops & Products 144 (2020) 112059
Fig. 1. Representative photomicrographs of
the liver, kidney, lung and spleen of mice from
the control group and the groups treated with
the Eugenia stipitata Essential Oil (EsEO) leaves
at the doses of 2000 and 5000 mg/kg. Liver:
the centrilobular vein (v) is seen in all images.
In control, the hepatocyte bundles (arrow
heads) are preserved and ordered. Kidney:
Renal glomeruli (*) and contorted tubules
without alterations visible in control and ex-
tract-treated groups. Lung: bronchiole (Br),
alveolar sac (As), and interalveolar septa can
be visualized, with preserved architecture in
control and treated with EsEO at 2000 and
5000 mg/kg. The alveolar duct can also be
seen in one image. No signs of degeneration,
necrosis/inflammation, or other changes.
Hematoxylin-eosin staining was used.
Magnification: 400 × . Spleen: Control group
and the groups treated with the EsEO leaves at
the doses of 2000, and 5000 mg/kg. The lymph
nodes (Nd) are well-defined in the control and
treated groups. Hematoxylin-eosin staining
was used. Magnification: 100 × .
inflammatories (Fernandes et al., 2007; Jeon et al., 2000).
3.4.2. Carrageenan-induced peritonitis
Carrageenan-induced peritonitis is related to the vascular changes
and cell migration. Additionally, there is production and release of
inflammatory mediators such as nitric oxide, PGE2, IL-1β, IL-6, and
TNF-α; being able to recruit leukocytes, such as neutrophils that par-
ticipate in the inflammatory process (Loram et al., 2007).
In the carrageenan-induced peritonitis model, the animals were pre-
treated with EsEO at the doses of 40, 100, and 250 mg/kg. A significant
decrease was noted in the migration of the total number of leukocytes
(76.9%, 81.7% and 86.5%, respectively) and neutrophils (74.5%,
79.6%, and 77.9%, respectively) whereas, the positive control reduced
leukocyte migration was lowered by 71.9% and neutrophils by 67.7%
(Table 6).
Carrageenan-induced peritonitis assay was also evaluated by the
method of Guimarães et al. (2009) with E. candolleana EO at con-
centrations of 25, 50, and 100 mg/kg; which displayed an inhibition of
58.5% and 52.6%, for highest doses. The hydroethanolic extract of E.
punicifolia, whose concentrations of 3, 30 and 300 mg/kg were used,
exhibited a decrease in the total number of leukocytes. A decrease
above 50% was observed for 30 and 300 mg/kg concentrations as
compared with dexamethasone, whereas, for E. aurata, migration in-
hibition occurred at 3, 30, and 300 mg/kg concentrations (Costa et al.,
2016). Infante et al. (2016) orally administered the ethanolic extracts of
leaves, pulp, and seeds of E. brasiliensis at a concentration of 500 mg/kg;
which demonstrated a reduction in the neutrophils influx by 47%, 41%,
and 50%, respectively; while, the positive control dexamethasone,
W.K. Costa, et al.
Table 6
Effect of treatment with the Eugenia stipitata Essential Oil (EsEO) per os on leukocyte migration and neutrophil migration in peritoneal exudation in carrageenan-
induced mice.
Dose mg/kg Leukocytes (105/ml) Leukocyte inhibition (%)
Vehicle – 8.2 ± 0.6
a a
Indometacin 20 mg/kg 2.3 ± 0.5 71.9
a b
EO 40 mg/kg 1.9 ± 0.2 76.8
a,b
100 mg/kg 1.5 ± 0.3 81.7
a,b
250 mg/kg 1.1 ± 0.3 86.5
Values represent mean ± S.E.M. (n = 6). a Statistically different from the negative control (vehicle). b Statistically different from the positive control (indomethacin)
(ANOVA followed by Bonferroni’s.
Test, p < 0.05).
decreased the influx by 44%.
In the present investigation, results of the leukocyte migration study
suggest that a mechanism associated with this activity might inhibit the
synthesis of inflammatory mediators involved in cell migration. Trans-
caryophyllene, a compound present in EsEO, possesses the potential to
reduce IL-1β levels (Paula-Freire et al., 2014) and TNF-α expression
(Fernandes et al., 2007), which are important mediators involved in
leukocyte recruitment process.
Therefore, EsEO effect(s) suggests a decrease in carrageenan-in-
duced mediators release, that corroborates with the findings described
above.
3.4.3. Inhibition of protein denaturation (in vitro)
This assay, based on EO’s ability to inhibit protein denaturation by
means of bovine serum albumin (BSA), is an in vitro method of evalu-
ating inflammatory and arthritic diseases (Bhaskar and Mohite, 2010;
Maharashtra, 2012).
All EsEO concentrations protected BSA against denaturation. At
concentration of 2 mg/mL, diclofenac sodium presented a higher in-
hibitory activity (100.00% ± 0.3%) than EsEO (82.83% ± 0.01%).
However, in other concentrations tested (1, 0.5, and 0.25 mg/mL),
EsEO displayed greater inhibitory effect on protein denaturation
(77.20% ± 0.03%, 75.79% ± 0.04%, 74.39% ± 0.01%, respectively)
as compared with the standard drug, sodium diclofenac
(67.00% ± 0.03%, 60.41% ± 0.12%, 45.02% ± 0.11%, respectively).
Although, this method is not a direct assay for evaluating the ac-
tivity of one of the enzymes of the inflammatory cascade; yet, it is
widely used to screen the anti-inflammatory potential of natural pro-
ducts. The results obtained, are indicative of EsEO’s anti-inflammatory
capacity when compared with the results obtained for the controls.
Based on this, our results reinforce the previously described tests.
3.5. Antipyretic activity (Brewer’s yeast-induced pyrexia)
Yeast-induced pyrexia is characterized as a pathogenic fever re-
sulting from the deregulation of thermoregulatory centers in the hy-
pothalamus and is directly dependent on PGE2 production. IL-1β re-
lease has been reported to stimulate prostaglandins synthesis, especially
PGE2; which is expressed mainly in response to inflammatory, in-
fectious or endotoxin agents (Salvemini et al., 1996).
Fig. 4. Antipyretic potential of Eugenia stipitata Essential Oil (EsEO) on mice
with fever induced by Saccharomyces cerevisae infection. *p < 0.001 compared
with Control, one-way ANOVA followed by Tuckey’s Test.
Industrial Crops & Products 144 (2020) 112059
Neutrophils (105/ml) Neutrophil inhibition (%)
5.9 ± 0.2
a
1.9 ± 0.5 67.7
a b
1.5 ± 0.4 74.5
b a,b b
1.2 ± 0.3 79.6
b a,b b
1.3 ± 0.2 77,9
All tested EsEO dosages revealed a reduction in temperature from
the first hour (p < 0.001). Fig. 4 displays that the temperature of EO
treated animals was below febrile values from 1 h, and continued until
the end of evaluation (4 h) and was concentration-independent when
compared with control group.
An essential oil of E. caryophyllata exhibited a proven temperature
suppression and was able to reduce the febrile condition (fungi in-
duced) from animals, 30 min after the oral administration of 33 mg/kg
oil (Taher et al., 2015). Additionally, E. uniflora EO has been noted to
reduce the rectal temperature of animals after its oral administration
(Amorim et al., 2009).
A study by Paula-Freire et al. (2014) has demonstrated trans-car-
yophyllene to be effective in reducing IL-1β levels and decreasing PGE2
production. It is suggested that EsEO has an antipyretic effect because it
possesses the potential to influence prostaglandins biosynthesis in the
hypothalamus, because it is comprised of such a component (Lopez-
Castejon and Brough, 2011).
4. Conclusion
Eugenia stipitata leaves from Caatinga, provided peculiar EO com-
position. Moreover, toxicity was observed only in high concentrations,
which indicated low toxicity and reliable use. A reduction in pain,
swallowing, and fever was noted when all the concentrations were
tested, which might be related to the capability of E. stipitata EO to
reduce cell migration and protein denaturation. These activities might
also be related to prostaglandin synthesis inhibition or the release of
other mediators involved in the inflammatory process. Overall, this
study indicated E. stipitata EO as a promising natural source of chemical
compounds with low side effects and anti-inflammatory potential,
which matches with its popularity for medicinal use.
Author’s contributions
WKC, JWLMS, JCROFA performed the extraction and analyzed of
essential oil. WKC, AMO, JRSO, VBGS performed the toxicology ana-
lyses. WKC, JRSO, AMO, IBSS, AFSF and RXC performed the anti-
nociceptive and anti-inflammatory analyses. VLML, MVS, DMAFN
contributed the reagents, materials, and analytical tools. WKC, AMO,
JRSO and AGS analyzed and interpreted the data. WKC, AMO, IBSS and
AGS wrote the paper. VLML, MVS, DMAFN performed critical revision
of the final version of the article.
Acknowledgments and funding
The authors express their gratitude to the Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) for financial support and
investigator research grants (VLML). We are also grateful to the
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES;
Finance Code 001) and the Fundação de Amparo à Ciência e Tecnologia
do Estado de Pernambuco (FACEPE) for financial support. WKC would
like to thank CNPq for the PhD scholarship. IBSS would like to thank
8
W.K. Costa, et al.
CAPES for the master scholarship. JWLMS would like to thank FACEPE
(PBPG-0822- 2.10/17) for the master scholarship. VBGS (scientific in-
itiation scholarship), RXC (master scholarship) and AMO (Process nº
380058/2019-7 for the Post-doctoral scholarship) would like to thank
CNPq.
We thank the Paus dóia community for their welcome in collecting
botanical material.
Declaration of Competing Interest
The authors declare no conflict of interest.
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