Phytomedicine 41 (2018) 82–95

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Phytomedicine
journal homepage: www.elsevier.com/locate/phymed

Original Article

Anti-inflammatory and anti-edematogenic action of the Croton campestris A. St.-Hil T

(Euphorbiaceae) essential oil and the compound β-caryophyllene in in vivo models
⁎
Cícera Datiane de Morais Oliveira-Tintinoa,b, , Renata Torres Pessoab,
Maria Neyze Martins Fernandesb, Isabel Sousa Alcântarab, Bruno Anderson Fernandes da Silvab,
Maria Rayane Correia de Oliveirab, Anita Oliveira Brito Pereira Bezerra Martinsa,
Maria do Socorro da Silvab, Saulo Relison Tintinoa, Fábio Fernandes Galvão Rodriguesb,
José Galberto Martins da Costab, Sidney Gonçalo de Limac, Marta Regina Kerntopfb,
Teresinha Gonçalves da Silvaa, Irwin Rose Alencar de Menezesb
a
Federal University of Pernambuco (UFPE), Pernambuco, Brazil
b
Regional University of Cariri (URCA), Ceará, Brazil
c
Federal University of Piauí (UFPI), Piauí, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Inflammation makes up a set of vascularized tissue reactions acting in the defense of the body
β-caryophyllene against harmful stimuli. Natural products are a lower cost alternative with better benefit, often used in popular
Croton campestris medicine in the treatment of inflammatory processes. Several species from the genus Croton have scientifically
Essential oil proven anti-inflammatory action.
Inflammation
Purpose: This study aims to analyze the chemical composition of the Croton campestris A. St.-Hil essential oil
Mice
(EOCC), derived from fresh leaves, as well as to evaluate the anti-inflammatory potential and the possible
mechanisms of action of the EOCC and its constituent β-caryophyllene.
Methods: The assays were performed in in vivo models of acute and chronic inflammation. Initially, the chemical
composition of the EOCC was determined and its oral toxicity was evaluated, followed by the evaluation of its
topical antiedematogenic effect through acute and chronic ear edema induced by Croton oil. For the systemic
verification of an anti-inflammatory action, the abdominal contortions, formalin test, paw edema induced by
carrageenan, dextran, histamine and arachidonic acid models, as well as a peritonitis test, vascular permeability
and granuloma assays were performed.
Results: The evaluation of the essential oil chemical composition revealed the presence of β-caryophyllene (15.91%),
1,8-cineol (16.98%) and germacrene-D (14.51%) as its main constituents. The EOCC had no relevant clinical toxicity on
oral administration, with an LD50 of more than 5000 mg/kg. The tested substances showed anti-inflammatory action in
the abdominal contortions, paw edema induced by carrageenan, dextran, histamine and arachidonic acid models, the
formalin test, peritonitis test and vascular permeability; however, β-caryophyllene had no significant effect on the
granuloma assay. This suggests as a hypothesis that both substances tested showed significant influence on the ara-
chidonic acid and histamine pathway reducing edema in these models.
Conclusion: The tested substances have a clinically safe profile, additionally the EOCC and β-caryophyllene
presented relevant anti-inflammatory activity. This study supports the hypothesis that β-caryophyllene, in as-
sociation with other constituents present in the EOCC such as 1,8-cineole, contributed to the anti-inflammatory
effect observed, in addition to suggesting that one of the mechanisms of action probably involves the inhibition
of cytokines with the involvement of the arachidonic acid and histamine pathways.

Abbreviations: AA, Arachidonic acid; ANOVA, Analysis of variance; CEUA, Animal Research Ethics Committee; COX, Cyclooxygenase; EOCC, Essential oil of Croton campestris; GC–MS,
Gas chromatography-mass spectrometry; i.p., Intraperitoneal injection; IL, Interleukin; LOX, Lipoxygenase; N, Number; NO, Nitric oxide; NOS, Nitric oxide synthase; NSAIDs, Non-
steroidal anti-inflammatory drugs; p.o., Per os (oral administration); PBS, Phosphate buffered saline; PG, Prostaglandin; PGI2, prostacyclin; PKC, Protein kinase C; rpm, Revolution per
min; SEM, Standard error of the mean; TNF, Tumour necrosis factor
⁎
Corresponding author at: Laboratory of Pharmotoxicological Prospecting of Bioactive Products, Department of Antibiotics, Federal University of Pernambuco (UFPE), Av. dos
Economistas, Cidade Universitária, Recife 52171-011, Brazil.
E-mail address: datianemorais@hotmail.com (C.D.d.M. Oliveira-Tintino).

https://doi.org/10.1016/j.phymed.2018.02.004
Received 25 September 2017; Received in revised form 20 December 2017; Accepted 11 February 2018
0944-7113/ © 2018 Elsevier GmbH. All rights reserved.
C.D.d.M. Oliveira-Tintino et al. Phytomedicine 41 (2018) 82–95

Introduction St. Hil essential oil (EOCC) and its main constituent β-caryophyllene
through in vivo models.
Inflammation characterizes a set of vascularized tissue reactions in
defense of the organism against endogenous or exogenous noxious sti- Materials and methods
muli. It is accompanied by several vascular events, such as vasodilation,
leukocyte and interstitial fluid accumulation, sensitization of nocicep- Legal requirements for conducting research
tive terminals, as well as cellular events such as synthesis and release of
pro-inflammatory mediators such as kinins, nitric oxide and cytokines To carry out this research, a registration was made on the
such as tumor necrosis factor (TNF-α) and interleukins (ILs) such as IL- Biodiversity Information and Authorization System (SISBIO) platform
1β and IL-6 (Medzhitov, 2010; Kumar et al., 2013; Sá et al., 2014). The to obtain the license to collect botanical material, receiving the regis-
use of medicinal plants to treat inflammatory diseases is a common tration number 47584-1. To carry out the in vivo tests, the experimental
therapeutic practice in several communities in northeastern Brazil since protocols were submitted to the Animal Research Ethics Committee
most of them are easily accessible and inexpensive (Veiga-Junior et al., (CEUA) of the Regional University of Cariri, being approved under
2005). protocol number 232/2016.1.
Plant species with anti-inflammatory bioactive properties are dis-
tributed among the most diverse families, especially the Euphorbiaceae
Botanical material and extraction of the Croton campestris essential oil and
family, comprising approximately 6300 species grouped in 245 genera
obtaining β-caryophyllene
(Webster, 1994; Govaerts et al., 2000). Among these is the genus
Croton, whose species, in its majority, are characterized by the presence
Croton campestris leaves were collected in the morning in the
of latex in their stalks, being popularly known as “velames” and
Chapada do Araripe, Cariri, Ceará, under coordinates 7°21′45,9″S and
“marmeleiros”, in addition to being widely used in folk medicine
039°28′36,8″W. A sample of the exsiccate was deposited at the
(Salatino et al., 2007; Riina et al., 2009). The species Croton campestris
Caririense Dárdano de Andrade-Lima Herbarium (HCDAL), under
A. St.-Hil, popularly known as “velame-do-campo”, “velame-branco”,
number 12.065. The fresh leaves were washed, crushed and immersed
“velame-verdadeiro”, “capimcigui”, “curraleira” and “canela-de-urubu”
in distilled water in a round-bottomed flask and the oil extracted by the
is a shrub originating in Brazil, occurring mainly in the Southeast and
steam distillation technique using a Clevenger system (Santos et al.,
Northeast regions (Corrêa, 1975; Dantas, 2007).
2004), obtaining a yield of 0.021%. The mixture of β-caryophyllene
Ethnopharmacological studies point to the use of this species in folk
isomers (code: 22,075 and purity > 80%) was purchased from the
medicine for general pains, influenza, constipation, bronchitis, pneu-
Sigma-Aldrich Corporation.
monia, asthma, stroke, eye problems and mouth sores (Costa-Neto and
Oliveira, 2000; Franco and Barros, 2006; Borges and Bautista, 2010;
Analysis of the chemical composition of the Croton campestris A. St. Hil
Oliveira-Júnior and Conceição, 2010; Chaves and Barros, 2012; Silva
essential oil (EOCC) by GC–MS
et al., 2012b); nasal congestion, bronchitis, throat afflictions, intestinal
problems, rheumatism, hemorrhoids, arthritis, venereal diseases and as
Analysis of the oil was performed on a Shimadzu GC-17A/MS
a depurative (Albuquerque et al., 2007; Oliveira et al., 2007); hema-
QP5050A (GC/MS system): DB-5HT capillary column
tologic disorders, inflammation, dermatoses, wounds, allergies, fever,
(30 m × 0.251 mm, 0.1 µm film thickness); carrier gas: helium 1.0 ml/
infection, sinusitis, kidney disorders, headache, gastritis, ocular
min; column inlet pressure 56.7 kPa; column flow = 1.0 ml/min; linear
cleaning, purgative, syphilis treatment and snake bites (Brito-Júnior
velocity = 36.5 cm/s; total flow 32 ml/min; injector temperature
et al., 2015; Brandão et al., 2012).
260 °C; detector temperature 270 °C; column temperature 60
Several Croton campestris A. St.-Hil biological activities have already
(2 min)–180 °C (4 min) at 4 °C/min, then 180–260 °C at 10 °C/min
been proven, with emphasis on its antimicrobial actions (Matias et al.,
(10 min). Mass spectrometer operating conditions of 70 eV ionization
2010a, 2010b; El Babili, 2009, 2012), antibiotic modulating action
energy. Identification of Individual components was based on their
(Matias et al., 2011; Brito-Júnior et al., 2011; Coutinho et al., 2011;
mass spectral fragmentation using two computer library MS searches
Almeida et al., 2013; Lavor et al., 2014), antiulcerogenic (Brito-
(Wiley 229), retention indices and comparison with literature data
Júnior et al., 2013, 2014) and molluscicidal activity (El Babili et al.,
(Alencar et al., 1990; Adams, 2007).
2006).
Studies have shown that the chemical composition of the Croton
campestris essential oil presents mainly β-caryophyllene (17%) Drugs
(Almeida et al., 2013). Several biological activities were attributed to
this sesquiterpene, among them the anti-inflammatory activity (Cho For the positive controls, the drugs indomethacin, dexamethasone,
et al., 2007; Fernandes et al., 2007), antiviral (Dunkić et al., 2011), promethazine, morphine and diazepam were used. Croton oil, acetic
antibacterial (Goren et al., 2011), anticarcinogenic (Loizzo et al., 2008; acid, formalin, carrageenan, dextran, histamine and arachidonic acid
Legault et al., 2013), antimutagenic (Di Sotto et al., 2010), local an- were used as phlogistic agents. For the negative control 0.9% saline was
esthetic (Ghelardini et al., 2001), and antarthritic activities used and as vehicles, distilled water and acetone were used. All drugs
(Vijayalaxmi et al., 2015). In addition, β-caryophyllene has been shown and reagents were purchased from the Sigma Aldrich Corporation. Pre-
to be a potent inhibitor of nitric oxide synthesis (Tung et al., 2008), the treatment administrations were performed according to the animal's
expression of IL-1β, IL-6, IL-8 and TNF-α (Gertsch et al., 2008) and of body mass (0.1 ml/10 g).
the levels of TNF-α, PGE2, INOs and COX-2 (Fernandes et al., 2007;
Dahham et al., 2015). Animals
The use of anti-inflammatory drugs are well tolerated by some pa-
tients, however these can cause severe adverse effects in others, mainly Mus musculus, albino, Swiss strain mice, weighing between 20–30 g
due to changes in coagulation and gastric problems that these drugs can were used. All mice were kept in polypropylene boxes, specific for the
cause. In this sense, essential oils and their isolated constituents can be vivarium, at a temperature of 24 ± 1 °C, with ration (Labina, Purina®)
used as an alternative to solve inflammatory conditions (Sá et al., and water ad libitum and maintained in a light/dark cycle of 12 h. All
2014). Thus, this study evaluated the potential of the anti-inflammatory the tests were performed with a number of 6 mice per group, accli-
action and the possible mechanisms of action of the Croton campestris A. matized at the experiment site for 24 h prior to the experiment.

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C.D.d.M. Oliveira-Tintino et al.
Acute oral toxicity test in mice
Acute toxicity studies were performed with Swiss male mice, as
described by OECD 425 (OECD, 2005), with slight modifications. The
animals were randomly divided into two groups (n = 3) and fasted
overnight with free access to water. The mice were orally treated with
Croton campestris essential oil at a dose of 5000 mg/kg. The first 30, 60,
120, 240 and 360 min intervals were observed and then every 24 h for
14 days after the treatment, observing the number of deaths and the
occurrence of parameters present in the Malone table (Malone and
Robichaud, 1962). This study served as a guide for the selection of
treatment doses, with values lower than 5% of the LD50 value estab-
lished. The dosage of the β-caryophyllene compound was determined at
an equivalent dose taking into account the content of this constituent as
identified by GC–MS.
Pharmacological triage of the anti-inflammatory action
Ear edema induced by single and multiple Croton oil application
The mice were topically pre-treated with 20 µl in the right ear ac-
cording to the groups 0.9% saline, 40 mg/kg dexamethasone and EOCC
25, 50, 100 and 200 mg/kg, with the vehicle being administered in the
left ear. In the acute oedema assay, 1 h after treatment, oedema was
induced with 20 µl of 5% Croton oil in the right ear, and the vehicle
acetone was administered to the left ear. After 4 h, the animals were
euthanized, 6 mm disks were removed from the ears with a metallic
punch and their mass was verified in an analytical balance (De Young
et al., 1989; Lapa, 2007). In the chronic oedema asssay the induction of
oedema with 20 µl of Croton oil was performed on alternate days with a
duration of nine days. The ears had daily measurements using a digital
calliper. The treatment occurred on alternate days from the fifth day of
the experiment, extending to the ninth day when the animals were
euthanized and the ear discs removed (Stanley et al., 1991).
Acetic acid-induced abdominal contortions
The animals were pre-treated (p.o.) with 0.9% saline, indomethacin
10 mg/kg, EOCC 25, 50, 100 and 200 mg/kg and 4.97, 9.94, 19.88,
39.76 mg/kg of the β-caryophyllene compound, and 0.6% (i.p.) acetic acid
was given 1 h after treatment. Immediately after the induction, the number
of abdominal contortions, characterized by abdominal rotation and total
stretching of the hind paws (Lapa, 2007), were counted for 30 min.
Formalin test
The animals were pre-treated with 0.9% saline, indomethacin
10 mg/kg, morphine 5 mg/kg (i.p.), EOCC 25, 50, 100 and 200 mg/kg
and β-caryophyllene 4.97, 9.94, 19, 88.37 mg/kg. After 30 min (for
morphine) or 1 h (for the other groups) 2.5% formalin was injected into
the right hind paw. Then, the time spent paw licking was timed during
the period 0–5 min after formalin administration, which corresponds to
Phase 1 or the neurogenic phase, as well as during the period
25–40 min, corresponding to Phase 2, or the inflammatory phase
(Woolfe and MacDonald, 1944; Fasmer et al., 1985; Hunskaar and Hole,
1987; Spindola et al., 2012).
Paw edema induced by intraplantar injection of carrageenan or dextran
The animals had a baseline measurement of their hind paws volume
measured by plethysmometry. Subsequently, the animals were pre-
treated orally with 0.9% saline, 10 mg/kg indomethacin (positive
control in carrageenan-induced edema), 6 mg/kg promethazine (posi-
tive control of dextran-induced edema) and 25, 50, 100 and 200 mg/kg
of the EOCC, and the doses of 4.97, 9.94, 19.88, 39.76 mg/kg of β-
caryophyllene. After 1 h, the animals received 1% carrageenan or 1%
dextran (20 µl/paw) in the right hind paw and vehicle in the left hind
paw. The volume of the hind paws of each animal were recorded after
1, 2, 3 and 4 h of the phlogistic agent injection (Lapa, 2007).
84
Phytomedicine 41 (2018) 82–95
Mechanisms of anti-inflammatory activity
Paw edema induced by intraplantar injection of arachidonic acid or
histamine
The basal volume of the hind paws of each mouse were measured by
plethysmometry. Subsequently, the animals were pre-treated (p.o.)
with 0.9% saline, 10 mg/kg indomethacin (positive control for arachi-
donic acid), or 6 mg/kg promethazine (histamine positive control),
EOCC at 100 mg/kg and β-caryophyllene at 19.88 mg/kg. After 1 h, the
animals received 1% arachidonic acid or 1% histamine in the right hind
paw (20 µl/paw) and vehicle in the left paw. The hind paw volume was
measured at 15, 30, 45 and 60 min after 1% arachidonic acid injection
(Dimartino et al., 1987). In the histamine assay, the hind paw volume of
each animal was recorded after 30, 60, 90 and 120 min of the phlogistic
agent injection (Maling et al., 1974).
Peritonitis
The mice were treated (p.o.) with 0.9% saline solution, 5 mg/kg
dexamethasone, EOCC at 100 mg/kg, β-caryophyllene at 19.88 mg/kg
and the naive group, which received no treatment or induction. After
1 h of the treatment, the animals received an intraperitoneal injection
of 1% carrageenan. After 4 h, the animals were euthanized, and 3 ml of
heparinized PBS were injected into the peritoneal cavity. The peritoneal
lavage sample was read in the ABX Micros 60 where the number of
leukocytes were counted and expressed as a percentage (Lapa, 2007).
Vascular permeability induced by Evans Blue extravasation
The mice received the same treatment cited in item 2.7.5. After 1 h
of treatment, the animals received an intraperitoneal injection of 1%
carrageenan. Concomitantly with carrageenan, 200 µl of Evans Blue
was administered to the retro-orbital plexus. After 4 h, the animals were
euthanized and 3 ml of PBS were injected into the peritoneal cavity. The
samples were centrifuged for 2 min at 6000 rpm, and the supernatant
was read by 520 nm filter spectroscopy (Lapa, 2007).
Granuloma assay
Four cotton pellets weighing 10 mg (0.01 g) each were placed on the
dorsum of mice anesthetized with 80 mg/kg ketamine and 20 mg/kg
xylazine. The animals were treated (p.o.) with 0.9% saline, 5 mg/kg
dexamethasone, 100 mg/kg EOCC and 19.88 mg/kg β-caryophyllene.
This assay lasted ten days where the treatment was performed daily. On
the tenth day, the animals were euthanized and the cotton pellets were
removed from the dorsal region, and dried for 24 h at 37 °C in an in-
cubator and the pellets were then weighed. Then, a homogenate from
the pellets was made for total protein dosage where a biuret containing
a reagent that reacts with the proteins present in the sample develops a
purple coloration proportional to the protein concentration was applied
to the homogenate. A standard solution of 40 mg/ml of Albumin was
used as a positive control for the reaction. After 10 min, the sample was
read by 550 nm filter spectroscopy (Swingle and Shideman, 1972).
Statistical analysis
All data were subjected to analysis using the GraphPad Prism 5.0
software. The comparison procedure used was a One-way ANOVA or
Two-way ANOVA, with Tukey's test being applied as a post hoc test for
both cases.
Results and discussion
Chemical composition of the Croton campestris A. St.-hil essential oil
The analysis of the oil's chemical composition revealed the presence
of 20 constituents, predominantly the oxygenated monoterpene 1,8-
cineol (16.98%) and sesquiterpenes β-caryophyllene (15.91%), ger-
macrene-D (14.51%) and bicyclogermacrene (10.41%) (Table 1).
C.D.d.M. Oliveira-Tintino et al. Phytomedicine 41 (2018) 82–95

Other studies with the Croton campestris essential oil performed by from the 6th day of the test there was a significant decrease in edema
Almeida et al. (2013) demonstrated β-caryophyllene as the major with an inhibition percentage of 43%, 22%, 26.7%, 32% and 37%,
constituent, a result similar to that of this study where this compound is respectively. This reduction can also be observed on the other days of
present as one of the major constituents. the procedure, where on the 7th day a reduction in edema of 51.9%,
Analysis of the composition of essential oils extracted from several 55.5%, 29.6%, 36.7% and 41.9% was observed in the dexamethasone,
species of the genus Croton revealed the presence of β-caryophyllene in EOCC 200, 100, 50 and 25 mg/kg groups, respectively. On the 8th day,
its constitution. Among these species are: Croton cajucara (Silva et al., a reduction of 80.7%, 45%, 55.5%, 57% and 65.5% was observed in the
2012a), Croton isabelli (Vunda et al., 2012), Croton bonplandianus same groups and on the 9th day there was a reduction of 77.6%, 43.1%,
(Joshi, 2014) and Croton zehntneri (Donati et al., 2015). In some studies, 47.4%, 51% and 65.5%, respectively (Fig. 2A). However, when evalu-
the composition analysis of other Croton species revealed the presence ating the edema at the end of the experiment by the mass of the ears on
of β-caryophyllene associated with other constituents, such as 1,8-ci- the ninth day of the experiment, a significant statistical difference was
neol, as is the case of Croton nepetaefolius (Lima-Accioly et al., 2006), observed only for the 25 mg/kg EOCC and dexamethasone groups,
Croton zehntneri (Coelho-de-Souza et al., 2013), Croton adamantinus compared to the saline group (Fig. 2B).
(Ximenes et al., 2013) and Croton kimosorum (Rabehaja et al., 2014). Croton oil is an irritant compound which, when applied to the
The presence of germacrene-D was observed in the essential oils of the epidermis, triggers an inflammatory response (Pawlaczyk et al., 2013).
Croton adenocalyx, Croton pullei, Croton urucurana, (Craveiro et al., This is due to the main irritants present in the composition of the
1990; Rocha et al., 2008; Simionatto et al., 2009) and Croton flavens Croton oil, this being 12-O-tetradecanoylphorbol-13-acetate (TPA),
(Sylvestre et al., 2006) species. In contrast, Croton sonderianus presents whose inflammation occurs by induction of the phospholipase A2 en-
β-caryophyllene, germacrene-D and 1,8-cineol in its composition zymatic action by protein kinase C (PKC) activation leading to the
(Dourado and Silveira, 2005). formation of arachidonic acid metabolites such as leukotrienes and
prostaglandins (Garg et al., 2008). Its application triggers effects such
Acute oral toxicity as the infiltration of neutrophils and polymorphonuclear leukocytes,
the release of histamine and serotonin, and an increase in vascular
The administration of the Croton campestris A. St.-Hil essential oil permeability with edema formation (Patel et al., 2012; Passos et al.,
did not present central toxic effects when evaluating the behavioral 2013; Trivellatograssi et al., 2013). Cyclooxygenase and 5-lipoxygenase
parameters of the Malone and Robichaud (1962) table; moreover, no inhibitors are potent inhibitors of the inflammatory process initiated by
mortality was observed at the 5000 mg/kg dose. Both the EOCC and β- TPA (Saraiva et al., 2011). Therefore, anti-edematogenic agents sup-
caryophyllene had no significant influence on locomotor, anxiolytic, press ear edema by inhibiting phospholipase A2 and, therefore, the
anxious or depressant activity of the Central Nervous System (CNS). No cyclooxygenase and lipoxygenase enzymes, such as is the case of dex-
significant macroscopic change was observed in the organs. This is the amethasone (Zhang et al., 2007).
first report on the acute toxicity of this oil. In this sense, to perform the The EOCC demonstrated a topical anti-inflammatory action at the
pharmacological screening tests, the doses were determined respecting concentration of 25 mg/kg in both the acute and chronic phases. In
the maximum limit of 10% of the LD50. Therefore, the determined doses chronic edema, a reduction in edema thickness was observed from the
were 25, 50, 100 and 200 mg/kg of the EOCC. The doses of the β-car- first day of treatment, a result that corroborates with acute edema,
yophyllene compound used in the pharmacological screening were however when evaluating the ear mass at the end of the experiment, no
4.97, 9.94, 19.88 and 39.76 mg/kg, which correspond to the same edema reduction action by the oil was observed at higher doses. This
concentrations of this sesquiterpene present in the EOCC, representing result indicates that components of the oil, in chronic use and at high
15.91%. concentrations, may be responsible for edematogenic effects. A similar
Although there is no literature data on the lethality of this essential result has already been observed in the literature by Veras et al. (2013),
oil, data from the literature have shown that other species from the which demonstrated that the essential oil from Lippia sidoides Cham and
genus Croton such as C. sonderianus and C. argyrophylloides did not thymol present dermal irritation effects when applied chronically. The
present toxic effects (LD50 > 6000 mg/kg), whereas the oils from C. data presented by Oliveira et al. (2014), also indicated that the Lippia
nepetaefolius and C. zehntneri presented moderately toxic effects (LD50 of sidoides Cham essential oil at high concentrations had irritating effects
3840 and 3464 mg/kg, respectively) (Lima et al., 2013). In the study
performed by Hiruma-Lima et al. (1999), oral administration of the C.
Table 1
cajucara essential oil gave an LD50 value of 9260 mg/kg. These results Chemical composition of Croton campestris A. St.-Hil essential oil.
corroborate with the data presented in this study, which verified that
some essential oils are known as non-toxic, being metabolized rapidly RT (min) Compound % KI Lit.
by the liver (Bakkali et al., 2008). These data are relevant considering
5.21 α-pinene 3.48 939
the importance of low toxicity in the planning and development of new 6.20 Sabinense 2.38 976
drugs (Korolkovas, 1988; Wermuth, 2003; Ferreira, 2003). 6.79 Myrcene 5.53 991
7.65 p-cymene 5.40 1026
Pharmacological screening of anti-inflammatory action 7.84 Eucalyptol 16.98 1033
8.71 ϒ-terpinene 5.45 1062
10.10 Linalool 1.08 1098
Acute and chronic ear edema induced by Croton oil 15.98 bornyl acetate 3.50 1285
In the evaluation of the topical anti-inflammatory activity of the 18.05 α-terpinyl acetate 1.68 1346
essential oil on acute edema, a reduction in edema was observed in the 18.74 α-copaene 1.74 1376
40 mg/kg dexamethasone groups and 25 mg/kg EOCC of 81.73% and 20.07 β-caryophyllene 15.91 1418
21.02 α-humulene 2.10 1452
30%, respectively, compared to the edema presented by the saline 21.93 germacrene D 14.51 1480
group. Meanwhile, the 50, 100 and 200 mg/kg EOCC groups did not 22.38 Bicyclogermacrene 10.41 1494
present edema reduction, obtaining statistically similar values (Fig. 1). 23.19 δ-cadinene 1.10 1524
In the evaluation of the chronic topical anti-edematogenic activity 24.65 Spathulenol 3.68 1576
24.74 caryophyllene oxide 1.73 1581
of the essential oil, a significant reduction in edema was observed on
25.31 Viridiflorol 1.22 1590
days 6, 7, 8 and 9 of the trial, corresponding to the days of treatment in 26.47 α-cadinol 2.10 1653
the dexamethasone and EOCC (at concentrations of 200, 100, 50 and
25 mg/kg) groups. In these same groups, it was possible to observe that RT – Retention Time; KI Lit. – Kovats Index.

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C.D.d.M. Oliveira-Tintino et al. Phytomedicine 41 (2018) 82–95

Fig. 1. Topical effect of the Croton campestris essential oil on the croton oil-induced ear
acute edema.
The values represent the arithmetic mean ± S.E.M. (standard error of the mean). One-way
ANOVA followed by the Tukey test. a4: p < 0.0001 vs saline; a3: p < 0.001 vs saline; ns:
not significant.

on the skin of mice, potentializing the inflammatory effects, demon-

strated by increased skin thickness, erythema formation, loss of hy-
dration and skin elasticity.
The essential oils do not seem to have specific cellular targets. Due
to their lipophilic characteristic, they pass through the cytoplasmic
membrane, causing perturbations in the structure of the poly-
saccharide, fatty acid and phospholipid layers affecting the perme-
ability. This phenomenon appears to be responsible for membrane da-
mage, leading to macromolecule leakage and lysis (Raut and
Karuppayil, 2014). Studies have shown that essential oils can be safe at
low concentrations, however some exhibit toxicity at high concentra-
tions, represented as lethal dosages (Sinha et al., 2014).

Acetic acid-induced contortions
In the evaluation of the number of abdominal contortions induced
by acetic acid, the group treated with indomethacin presented a 75.4%
reduction in the number of contortions observed, and the oil treated
groups showed a reduction of 90.3% at the concentration of 200 mg/kg;
74.6% at the concentration of 100 mg/kg; 60.4% in the group treated at
the concentration of 50 mg/kg and 59.7% at the concentration of
25 mg/kg. The indomethacin, 200 and 100 mg/kg EOCC groups showed
no statistical difference between them (Fig. 3A). In the treatment with
β-caryophyllene at 39.76, 19.88, 9.94 and 4.97 mg/kg doses, an edema
reduction of 88.5%, 90.4%, 75.4% and 87.3%, respectively, was ob-
served, all of which were similar to the indomethacin group (Fig. 3B).
The acetic acid-induced abdominal contortions test is suitable both
for assessing the action of centrally acting pain reducing drugs, such as
opioids, and peripherally acting anti-inflammatory drugs, such as
NSAIDs (Barrot, 2012; Pavão-de-Souza et al., 2012).
The application of acetic acid in the intraperitoneal cavity stimu-
lates an increase in cyclooxygenase and lipoxygenase products, the
release of inflammatory mediators, such as substance P, bradykinin,
histamine, serotonin, proinflammatory cytokines such as TNF-α, IL-1β,
and IL- 8 and degranulation of mast cells in the peritoneal region. In
addition, there is an increase in prostaglandin levels, especially PGE2,
PGF2 PGI2, thereafter there is increased vascular permeability (Radu
and Chernoff, 2013; Bahamonde et al., 2013). These released en-
dogenous substances will stimulate nociceptive nerve endings inducing
pain of an inflammatory genesis (Ikeda et al., 2001; Taesotikul et al.,
2003; Afsar et al., 2015). In this sense, the use of natural substances has
Fig. 2. Topical effect of the Croton campestris essential oil on the croton oil-induced ear
chronic edema.
The values represent the arithmetic mean ± S.E.M. (standard error of the mean). (A)
Thickness of ears evaluated for nine days. Two-way ANOVA, followed by the Tukey test.
(B) Mass of the ear on the ninth day. One-way ANOVA, followed by the Tukey test. a4:
p < 0.0001 vs saline; ns: not significant.
a relevant anti-inflammatory potential and there is growing interest in
understanding the mechanisms underlying its anti-inflammatory ac-
tivity.
The Croton cajucara essential oil at a dose of 1000 mg/kg reduced
abdominal contortions induced by acetic acid (Bighetti et al., 1999).
The essential oil from Croton sonderianus at 50, 100 and 200 mg/kg
doses reduced the number of contortions induced by acetic acid in re-
lation to the control, in addition to presenting β-caryophyllene and 1,8-
cineol as its components (Santos et al., 2005). Croton adamantinus in
addition to presenting β-caryophyllene and 1,8-cineol in its constitu-
tion, was also effective in this assay at 50 and 100 mg/kg doses
(Ximenes et al., 2013). Studies with 1,8-cineol showed effective action
in the reduction of abdominal contortions at 100, 200 and 400 mg/kg
doses (Santos and Rao, 2000).
These studies reinforce that essential oils constituents can influence
the observed activity through a joint action. Among several mechan-
isms known to be involved in the preventive activity of ROS in-
flammation, the efficacy of free radical scavenging is considered to be
important; however, other effects are present, such as lipoxygenase
and cyclooxygenase enzyme inhibition, leukotriene and prostanoid
synthesis prevention, proinflammatory cytokine inhibition such as

86
C.D.d.M. Oliveira-Tintino et al. Phytomedicine 41 (2018) 82–95

Fig. 3. Effect of the Croton campestris essential oil and β-caryophyllene on the acetic acid-induced abdominal contortions.
The values represent the arithmetic mean ± S.E.M. (standard error of the mean). (A) OECC. (B) β-caryophyllene. One-way ANOVA, followed by the Tukey test. a4: p < 0.0001 vs saline;
ns: not significant.

interleukin-1 (IL-1) and tumor necrosis factor (TNF-α), as well as the
repression of proinflammatory genes (Raut and Karuppayil, 2014).
Therefore, the substances tested in the present study may possibly have
shown their effectiveness by inhibiting the action of cyclooxygenase or
by their direct action on their metabolites such as prostaglandin, as is
the case with NSAIDs.
Formalin test
In phase 1 of the formalin test, i.e. the neurogenic phase, a 100%
reduction in paw licking time was observed in the morphine treated
animals compared to the saline control, while in the indomethacin
treated animals, a 57.9% reduction in licking time was observed. The
animals treated with the essential oil at the concentrations of 200, 100,

Fig. 4. Effect of the Croton campestris essential oil and β-caryophyllene on the formalin test.
The values represent the arithmetic mean ± S.E.M. (standard error of the mean). (A and B) OECC. (C and D) β-caryophyllene. One-way ANOVA, followed by the Tukey test. a4:
p < 0.0001 vs saline; a3: p < 0.001 vs saline; a2: p < 0.01 vs saline; ns: not significant.

87
C.D.d.M. Oliveira-Tintino et al.
50 and 25 mg/kg showed a reduction of 50.8%, 52.4%, 53.6% and
34.1%, respectively. It is observed that the results presented by in-
domethacin are similar to those presented by the essential oil in at 200,
100 and 50 mg/kg concentrations, being statistically proven that these
groups do not present significant differences between them (Fig. 4A).
In phase 2, or the inflammatory phase, morphine retains its effect by
reducing 100% of the paw licking time in relation to the saline group.
Indomethacin showed a reduction of 76.50% in licking time, and the
EOCC showed a decrease of 73.3%, 71.6%, 55.3% and 46.6%, in the
200, 100 and 50 mg/kg concentrations. In this phase of the test, the
similarity in values presented by the EOCC at 200 and 100 mg/kg with
the indomethacin group was statistically demonstrated by the absence
of a significant difference in the values of these groups among each
other (Fig. 4B).
In the treatment with 10 mg/kg indomethacin and β-caryophyllene
at 39.76, 19.88, 9.94 and 4.97 mg/kg doses, there was a reduction in
paw licking time in phase 1 representing a reduction of 57.9%, 42.8%,
59.9%, 48.4% and 49.2%, respectively, (Fig. 4C). In phase 2, these five
groups presented respective percentage reductions of 76.5%, 70.7%,
79.8%, 81.2% and 77.4%, such that in both phases, these five groups
had similar values between them (Fig. 4D).
The formalin assay is a model that aims to explore the analgesic and
anti-inflammatory activity of compounds through a central and per-
ipheral pain mechanism (Fillingim et al., 2009; Melnikova, 2010). This
model presents two distinct phases (Soldi et al., 2008). At first, formalin
induces acute neurogenic pain caused by direct stimulation on sensory
C-fibers, in addition to being mediated by substance P, glutamate and
bradykinin. The second phase, or inflammatory phase, is characterized
by pain arising from the sensitization of nociceptors by the release of
inflammatory mediators such as histamine, prostaglandin, serotonin
and bradykinin (Hunskaar and Hole, 1987; Barrot, 2012).
Central-acting drugs, such as opioids, inhibit the two phases, while
drugs that act at the peripheral level such as NSAIDs tend to inhibit the
late phase (Almeida et al., 2001; Udobang et al., 2010). The mono-
terpenes present in essential oils present analgesic potential whose
mechanism of action involves both their action as opioid agonists to
COX enzymes modulators, or as responsible for NO synthesis
(Guimarães et al., 2013). In previous studies, the Croton zehntneri es-
sential oil at 100 and 300 mg/kg doses (Oliveira et al., 2001), Croton
sonderianus at a 100 mg/kg dose and Croton adamantinus at 50 and
100 mg/kg doses (Santos et al., 2005; Ximenes et al., 2013) demon-
strated anti-inflammatory action through the formalin test. The β-car-
yophyllene compounds (Paula-Freire et al., 2014) and 1,8-cineol
(Santos and Rao, 2000) also showed significant activity in this assay by
reducing paw licking time. This evidence suggests a further link be-
tween the EOCC action and the presence of its major constituents β-
caryophyllene and 1,8-cineol.
Carrageenan-induced paw edema
In the graph below (Fig. 5A) it can be observed that from the eva-
luation time 2, there was a reduction in the percentage of paw edema in
the indomethacin, 100, 50 and 25 mg/kg EOCC groups compared to the
saline group with 80.4%, 40.9%, 41.7%, 38.6%, respectively. At time 3,
the indomethacin, 200, 100, 50 and 25 mg/kg EOCC groups presented a
reduction percentage of 96.7%, 34.3%, 69.3%, 77.4% and 51.8% re-
spectively in comparison to the saline control. Finally, at time 4 there
was a decrease of 98%, 64.7%, 79.5%, 86.2% and 76.5% in the edema
value, in the same sequence of the aforementioned groups tested, where
they also did not present statistical difference among themselves, being
significantly similar.
In the groups treated with β-caryophyllene, there were significant
results from time 2 of evaluation where the groups with 39.76, 19.88,
9.94 and 4.97 mg/kg doses, respectively, presented a reduction of
37.6%, 61.1%, 48.6%, 32.2% in comparison to saline. At time 3 this
reduction was of 41.7%, 73.2%, 55.4%, 47.6%, respectively. At time 4
88
Phytomedicine 41 (2018) 82–95
Fig. 5. Effect of the Croton campestris essential oil and β-caryophyllene on the paw edema
induced by carrageenan. The values represent the arithmetic mean ± S.E.M. (standard
error of the mean). (A) OECC. (B) β-caryophyllene. Two-way ANOVA followed by the
Tukey test. T1: 1 h after induction; T2: 2 h after induction; T3: 3 h after induction; T4: 4 h
after induction. a4: p < 0.0001 vs saline.
all sesquiterpene treated groups were statistically similar to the in-
domethacin group (Fig. 5B).
Carrageenan is a sulphated polysaccharide widely used as a phlo-
gistic agent promoting acute inflammation inducing a pro-in-
flammatory response (Sadeghi et al., 2011; Necas and Bartosikova,
2013; Solanki et al., 2015). The response to its induction is character-
ized by a biphasic inflammatory process, where in the first phase lasting
up to 2 h, rapid production of mediators such as histamine, serotonin
and bradykinin occurs (Boughton-Smith et al., 1993; Bhukya et al.,
2009). In the second phase or late phase that lasts between 3 and 6 h,
prostaglandins release mainly prostaglandin E2, contributing to an in-
crease in vascular permeability, producing edema and the mobilization
of leukocytes (Campo et al., 2009; Busnardo et al., 2010). Proin-
flammatory cytokines such as TNF-α and IL-1β are also released, as well
as nitric oxide (NO) (Halici et al., 2007; Feldmann and Maini, 2008;
Codarri et al., 2010). The latter is a potent vasodilator and is also in-
volved in the formation of edema (Cheng et al., 2010).
Both the EOCC and β-caryophyllene, at all doses tested, significantly
reduced edema. Essential oils from other species of the genus Croton
showed anti-inflammatory activity against the carrageenan-induced
paw edema model, such as Croton argyrophyllus at 10, 30 and 100 mg/
kg doses (Ramos et al., 2013) as well as in Croton cajucara
(Bighetti et al., 1999). In these studies, it is possible to observe the
presence of β-caryophyllene as a component of their compositions
(Silva et al., 2012a; Ramos et al., 2013).
Dahham et al. (2015) showed that β-caryophyllene at the 50, 100
and 200 mg/kg doses reduced edema and showed a dose-dependent
effect on carrageenan-induced inflammation. In other studies, 1,8-ci-
neol was potent in its anti-inflammatory action by reducing carra-
geenan paw edema at 100, 200 and 400 mg/kg doses (Santos and
Rao, 2000). These studies are in agreement with the data revealed in
the present study, where, due to the presence of these substances in the
EOCC, effective anti-inflammatory action was demonstrated in paw
edema induced by carrageenan. Thus, the two major constituents may
have a direct influence on the effect demonstrated by the essential oil
possibly acting to inhibit the production of cytokines or inhibit the
cyclooxygenase cascade.
C.D.d.M. Oliveira-Tintino et al.
Dextran-induced paw edema
At time 1, or the first h of evaluation, the 6 mg/kg promethazine and
200 and 100 mg/kg EOCC groups presented a reduction in the per-
centage of edema of 76.7%, 30.5% and 40.5%, respectively. At eva-
luation time 2, these same groups showed a significant reduction in
edema in relation to the saline group, with a reduction of 71.2%, 44.1%
and 99.5%, respectively. At time 3 or 3 h of evaluation, all groups were
statistically different in relation to saline control, where the 6 mg/kg
promethazine, EOCC 200, 100, 50 and 25 mg/kg groups presented
68.7%, 58.8%, 62.1%, 40.6%, and 25% reduction in edema, respec-
tively. At the same time of the evaluation, the groups treated with the
essential oil in the concentration of 200 and 100 mg/kg, presented no
statistical difference in relation to the group treated with the com-
mercialized drug promethazine. At time 4, there was a generalized re-
duction in paw volume in all groups, with a significant difference be-
tween the saline group and the other groups (Fig. 6A).
In the evaluation of the β-caryophyllene action, it was observed that
in time 2, where the peak of dextran action occurred, the groups treated
with 6 mg/kg promethazine and the sesquiterpene at 39.76 and
19.88 mg/kg doses, respectively, presented a reduction of 99.7%,
43.1% and 58.3% in paw edema, compared to saline. In the third and
fourth h of evaluation, all groups reduced edema in relation to the
control, showing statistically similar results (Fig. 6B).
Dextran is a polysaccharide that causes mastocyte degranulation
with the release of vasoactive amines, histamine and serotonin, which
contributes to increased vascular permeability and fluid extravasation
responsible for edema formation presenting peak action in the second h
(Sartori et al., 2003; Silva et al., 2014). Unlike carrageenan-induced
edema, dextran edema shows peak action in the second h and the
formed exudate contains few proteins and neutrophil infiltrate, while
carrageenan induces edema with a high concentration of both (El-
Shenawy et al., 2002).
Both essential oils and their isolated constituents can reduce this
inflammatory condition. In the study by Silva et al. (2003) with es-
sential oils from Eucalyptus citriodora, Eucalyptus tereticornis and Eu-
calyptus globulus, a greater anti-edematogenic activity against dextran
induced edema was clearly demonstrated, concluding that part of this
Fig. 6. Effect of the Croton campestris essential oil and β-caryophyllene on the paw edema
induced by dextran.
The values represent the arithmetic mean ± S.E.M. (standard error of the mean). (A)
OECC; (B) β-caryophyllene. Two-way ANOVA followed by the Tukey test. T1: 1 h after
induction; T2: 2 h after induction; T3: 3 h after induction; T4: 4 h after induction. a4:
p < 0.0001 vs saline.
89
Phytomedicine 41 (2018) 82–95
action was due to the presence of its major constituent 1,8-cineol.
Cho et al. (2007) have shown that β-caryophyllene at 30 and 300 mg/
kg doses have an anti-inflammatory effect against the dextran-induced
colitis model, as well as reducing IL-6 mRNA expression, with the
300 mg/kg dose also directly reducing the concentration of IL-6. In the
present study, both the EOCC and its major constituent showed a sig-
nificant reduction in edema at the peak time of dextran inflammation,
indicating a possible influence on the reduction of the expression of
such inflammatory cytokines and the inhibition of the release of va-
soactive amines.
Mechanisms of anti-inflammatory activity
For the following mechanism assays, the effective dose of the EOCC
was standardized, representing the lowest dose that evoked the best
effect in the screening tests, this being the 100 mg/kg EOCC dose fol-
lowed by the equivalent 19.88 mg/kg β-caryophyllene dose.
Arachidonic acid-induced paw edema
In this experimental trial, the indomethacin, 100 mg/kg EOCC and
19.88 mg/kg β-caryophyllene groups showed similar results, as they
showed no statistically significant differences from the evaluations
times between 15 min and 60 min. In addition, indomethacin, 100 mg/
kg EOCC and 19.88 mg/kg β-caryophyllene showed a significant re-
duction in edema percentage in relation to the control at the evaluation
times from 15 to 45 min, presenting, respectively, with 92%, 86% and
53.8% reduction at time 15 min, 89.6%, 90.6% and 55.7% at time
30 min and 97%, 98% and 53.4% at time 45 min (Fig. 7).
Arachidonic acid is a fatty acid present in the phospholipids of cell
membranes. Phospholipase A2 can release it, and its metabolism can be
made by the cyclooxygenase (COX) enzymes that leads to the synthesis
of prostaglandins, prostacyclin and thromboxanes; or by lipoxygenase
(LOX) enzymes that synthesize leukotrienes and lipoxins (Katzung,
2008; Kim et al., 2013). Therefore, arachidonic acid (AA) is a precursor
of PGE2 and leukotrienes, important inflammatory mediators. In this
manner, AA induces an inflammatory response causing vasodilation
and thereafter edema, as well as acting as a secondary messenger reg-
ulating the production of nitric oxide, which is also a potent vasodilator
(Kim et al., 2007; Sunita et al., 2011). Studies have shown that NSAIDs
such as aspirin and diclofenac are able to inhibit inflammation and pain
by inhibiting prostaglandin synthesis through COX enzyme inhibition
(Chen et al., 1995; Elenkov and Chrousos, 2002; Wise et al., 2008; Silva,
2010).
The anti-inflammatory effect demonstrated by the EOCC and β-
caryophyllene in this assay is possibly due to its cyclooxygenase enzyme
inhibitory properties, inhibiting, among others, the production of
prostaglandins and therefore inhibiting edema.
Fig. 7. Effect of the Croton campestris essential oil and of the β-caryophyllene on the paw
edema induced by arachidonic acid.
The values represent the arithmetic mean ± S.E.M. (standard error of the mean). Two-
way ANOVA followed by the Tukey test. T1: 15 min after induction; T2: 30 min after
induction; T3: 45 min after induction; T4: 60 min after induction. a4: p < 0.0001 vs
saline.
C.D.d.M. Oliveira-Tintino et al.
Fig. 8. Effect of the Croton campestris essential oil and of the β-caryophyllene on the paw
edema induced by histamine. The values represent the arithmetic mean ± S.E.M.
(standard error of the mean). Two-way ANOVA followed by the Tukey test. T1: 30 min
after induction; T2: 60 min after induction; T3: 90 min after induction; T4: 120 min after
induction. a4: p < 0.0001 vs saline.
Histamine-induced paw edema
The 6 mg/kg promethazine, 100 mg/kg EOCC and 19.88 mg/kg β-
caryophyllene groups showed a significant reduction in edema com-
pared to the control group during the first three h of evaluation after
induction with the phlogistic agent of 62.2%, 48.7% and 61.5% at T1,
81%, 73.1% and 84.8% at time T2 and 78.45%, 65.7%, 88.1% at time
T3 (Fig. 8), respectively.
Histamine is an important inflammatory mediator widely used as an
experimental model for assessing acute anti-inflammatory drug action
(Ghosh et al., 2015). Histamine together with serotonin are the first
vasoactive amines released during an acute inflammatory response,
causing increased vascular permeability, attracting neutrophils to the
exudate-forming inflammatory site, acting in conjunction with pros-
taglandin promoting edema (Sherwood and Toliver-Kinsky, 2004;
Vasudevan et al., 2007; Kumar et al., 2005). This mediator exerts its
function at the beginning of inflammation, this explains its rapid action
and transient duration, where the inflammatory process lasts a short
time after its induction (Gutierrez, 2015).
Fernandes et al. (2007), showed that β-caryophyllene at 50 mg/kg
was not able to reduce histamine-induced edema. However, the data
presented here disagrees with the work cited above given that β-car-
yophyllene showed activity against this model, at an even lower dose of
19.88 mg/kg, acting as further evidence of its influence in the anti-in-
flammatory action of the EOCC, where, through the direct inhibition of
histamine release, edema reduction occurs through the direct inhibition
of this agent on vessels.
This difference observed between both results may be attributed to
the different conditions to which the animals were subjected to in each
of the studies. As for example, in the work developed by
Fernandes et al. (2007), the animals were not fasted before receiving
the treatment and underwent an acclimation time corresponding to 1 h
prior to the tests, while in the present study the animals were accli-
matized in the environment for 24 h before the test, in addition to being
subjected to an 8 h fasting before the treatment.
Damy et al. (2010), points to the factors such as acclimatization
conditions and the diet under which the animals are subjected to as
some of the fundamental requirements for obtaining more accurate
results with a high degree of reproducibility, specifying the need for a
fast with a duration of at least 4 h. In relation to the acclimation time, it
was determined that at least 72 h are required for the habituation of
mice to the environment, guaranteeing the animal's well-being and its
adaptation to the experimental environment (Capdevila et al., 2007).
Peritonitis and vascular permeability
In the animals treated with dexamethasone, 100 mg/kg EOCC and
19.88 mg/kg β-caryophyllene there was a reduction in lymphocyte
percentage compared to the control by 56.4%, 42% and 28.3%, re-
spectively (Fig. 9A). These same groups caused a significant reduction
90
Phytomedicine 41 (2018) 82–95
in monocytes in relation to the control, this reduction being 64.9%,
39.9% and 65.6%, respectively, (Fig. 9B).
When comparing the saline control group to the naive group, which
did not receive any type of treatment or induction administration, it is
observed that the latter had an absorbance percentage 70.2% lower
than saline. In contrast, the groups treated with indomethacin, 100 mg/
kg EOCC and β-caryophyllene presented a respective absorbance value
of 70.3%, 59.4% and 64.3% lower than the saline group. The latter
three presented statistically similar responses, represented by the ana-
lysis, with a non-significant difference (Fig. 9C).
Carrageenan-induced peritonitis is characterized by acute in-
flammation caused by neutrophil migration in the inflammatory exu-
date (Souza et al., 1988). Intraperitoneal injection of carrageenan in-
duces the release of histamine and serotonin (Boughton-Smith et al.,
1993), the release of prostaglandin and leukocyte mobilization
(Busnardo et al., 2010), the release of TNF-α, IL-1β, IL-8 and nitric
oxide (NO) (Halici et al., 2007; Feldmann and Maini, 2008; Codarri
et al., 2010). This leads to vasodilation and increased vascular perme-
ability facilitating the migration of neutrophils through the mesenteric
venule to the peritoneal cavity (Gonçalves et al., 2011; Bitencourt et al.,
2014; Lima et al., 2014; Rzodkiewicz et al., 2014). Moreover, the
proinflammatory cytokines IL-1β and TNF-α are also involved in in-
ducing the expression of endothelial adhesion molecules, facilitating
leukocyte diapedesis (Medzhitov, 2010; Schmidt et al., 2013).
The essential oil from Croton argyrophyllus, at concentrations of 10,
30 and 100 mg/kg, significantly reduced the number of leukocytes in
relation to the control in carrageenan-induced peritonitis (Ramos et al.,
2013). In another study developed by Valer et al. (2016), the Croton
zehntneri essential oil at 125, 250 and 500 mg/kg doses were found to
reduce the intrapleural migration of leukocytes induced by carra-
geenan, and the presence of β-caryophyllene and 1,8-cineol in the es-
sential oil was evidenced in the same study.
With the increase in capillary permeability, after intraperitoneal
induction by carrageenan, the exudate will be concentrated in the in-
flamed region, containing, among others, a large concentration of acute
plasma proteins such as albumin (Silva et al., 2011; Pinheiro et al.,
2013). The Evans Blue dye, after being administered systemically, binds
strongly to albumin and its quantification by spectrophotometry allows
to indirectly evaluate changes in vascular permeability (Nagaraja et al.,
2008). It was evidenced in the literature that 1,8-cineol at 200 and
400 mg/kg doses reduced vascular permeability in this model
(Santos and Rao, 2000).
The OECC and β-caryophyllene showed positive results, as demon-
strated by the low leukocyte percentage in the intraperitoneal lavage,
where these results may be related to the inhibition of cytokine release
or inhibition of the expression of leukocyte adhesion molecules
(Sherwood and Toliver-Kinsky, 2004). Therefore, the effect observed by
the C. campestris essential oil (EOCC) and β-caryophyllene, suggests a
decrease in the release of mediators induced by carrageenan, resulting
in a decrease in permeability and protein extravasation to the affected
site, corroborating with the findings described above.
Granuloma
When the pellets were weighed on an analytical balance, the groups
treated with dexamethasone and 100 mg/kg EOCC showed a mass (g)
significantly lower than the group treated with saline, where this dif-
ference was expressed in pellets with masses 2.1 times lower than the
saline group for dexamethasone and 1.3 times lower for 100 mg/kg
EOCC (Fig. 10A).
In the absorbance analysis of the homogenate from the pellets, it
was observed that the groups treated with dexamethasone and 100 mg/
kg EOCC showed a reduction of this absorbance by 31.6% and 33.3%,
respectively, when in comparison to the saline group, demonstrating a
lower value in the number of total proteins in these groups in relation to
the control. The positive reaction control, saline and β-caryophyllene
C.D.d.M. Oliveira-Tintino et al. Phytomedicine 41 (2018) 82–95

Fig. 9. Effect of the Croton campestris essential oil and of the β-caryophyllene on the percentage of leukocytes (lymphocytes and monocytes) and on vascular permeability.
The values represent the arithmetic mean ± S.E.M. (standard error of the mean). (A) Percentage of lymphocytes. (B) Percentage of monocytes. (C) Vascular Permeability. One-way
ANOVA followed by the Tukey test. a4: p < 0.0001 vs saline; a2: p < 0.01 vs saline.

Fig. 10. Effect of the Croton campestris essential oil and of the β-caryophyllene on the granuloma.
The values represent the arithmetic mean ± S.E.M. (standard error of the mean). (A) Mass of the pellets. (B) Total proteins (absorbance of the pellets homogenate). One-way ANOVA
followed by the Tukey test. a4: p < 0.0001 vs saline; ns: not significant.

91
C.D.d.M. Oliveira-Tintino et al.
groups presented statistically similar absorbance values, demonstrating
that the latter group was inefficient in reducing such proteins
(Fig. 10B).
The in vivo granuloma assay induced by cotton pellets is widely used
to evaluate the triggering of the chronic inflammation gradual process
(Sengar et al., 2015). Granuloma formation is characterized by three
phases, the phases being transudative, exudative and proliferative
(Boonyarikpunchai et al., 2014). In the initial transudative phase that
occurs during the first three h after pellet implantation, there is an
increase in vascular permeability, where capillary fluids tend to en-
velop and fill the pellets. The exudative phase occurs from 3 h to 72 h
after implantation, where the migration of proteins around the granu-
loma occurs. Finally, the proliferative phase lasts from 3 to 6 days,
characterized by an increase in the synthesis of collagen and mucopo-
lysaccharides, penetration and proliferation of fibroblasts and finally a
vascularization of the tissue as a consequence of the release of pro-
inflammatory mediators (Hosseinzadeh et al., 2003; Pingsusaen et al.,
2015).
NSAIDs, glucocorticoids and other compounds with an anti-
proliferative effect, decrease the formation of the granulomatous tissue,
reflected in the decrease in dry weight of the granuloma due to the
attenuation of the proliferative phase of the process by these agents
(Damre et al., 2003; Verma et al., 2010). Natural substances such as the
Croton cajucara essential oil at 100 mg/kg (Bighetti et al., 1999) and
1,8-cineol at 400 mg/kg (Santos and Rao, 2000) were able to interfere
in granuloma formation, reducing thus, the mass of the pellets. Similar
data were presented in this study, where the 100 mg/kg EOCC showed a
decrease in dry pellet mass and a reduction of the total proteins found
in the pellet homogenates. This result corroborates with the observed
action on vascular permeability and leukocyte migration where there
was possibly an interference with protein migration and fibroblast ac-
tivation in the proliferative phase of the process. In contrast, β-car-
yophyllene did not present positive results in this trial, showing a
possible absence of involvement in the chronic systemic anti-in-
flammatory action of this sesquiterpene. Relating to previous studies,
this indicates that the effect of the EOCC observed in this model, is
probably largely due to the 1,8-cineol constituent.
Conclusion
Anti-inflammatory activity of essential oils and their constituents
are well documented, thus our results are consistent with the literature
and have shown that Croton campestris A. St.-Hil essential oil and its
constituent β-caryophyllene have shown relevant anti-inflammatory
action in vivo. This study supports the hypothesis that β-caryophyllene,
in association with other constituents present in the EOCC, such as 1,8-
cineol, contributed to the anti-inflammatory effect observed. The me-
chanisms of action involved in the anti-inflammatory effect of the tested
substances probably involve the inhibition of cytokines with the in-
volvement of the arachidonic acid and histamine pathways, however,
further studies are needed to elucidate the anti-inflammatory me-
chanism.
Conflict of interest
The authors declare that they have no conflict of interests.
Acknowledgments
This study was supported by grant from CNPq (134557/2015-8).
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