ARTICLE IN PRESS

G Model
JEP-6201; No. of Pages 7

Journal of Ethnopharmacology xxx (2010) xxx–xxx

Contents lists available at ScienceDirect

Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Topical anti-inflammatory effect of Caryocar coriaceum Wittm. (Caryocaraceae)

fruit pulp fixed oil on mice ear edema induced by different irritant agents
Rogério A. Saraiva a,∗ , Mariana K.A. Araruna a , Romagna C. Oliveira a , Kleber D.P. Menezes a ,
Gerlânia O. Leite a , Marta R. Kerntopf a , José G.M. Costa b , João B.T. Rocha c , Adriana R. Tomé d ,
Adriana R. Campos e , Irwin R.A. Menezes a
a
Universidade Regional do Cariri (URCA), Departamento de Química Biológica, Laboratório de Farmacologia e Química Molecular, 63105-000 Crato, CE, Brazil
b
Universidade Regional do Cariri (URCA), Departamento de Química Biológica, Laboratório de Pesquisa em Produtos Naturais, 63105-000 Crato, CE, Brazil
c
Universidade Federal de Santa Maria (UFSM), Departamento de Química, Laboratório de Bioquímica Toxicológica, 57900-000 Santa Maria, RS, Brazil
d
Universidade Estadual do Ceará (UECE). Campus do Itaperi, 60740-903 Fortaleza, CE, Brazil
e
Universidade de Fortaleza (UNIFOR). 60811-905 Fortaleza, CE, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Aim of the study: Caryocar coriaceum Wittm. fruit pulp fixed oil (CCFO) has been widely employed by com-
Received 14 January 2010 munities from Brazil Northeastern in the treatment of skin inflammation, respiratory affections, wound
Received in revised form 6 June 2010 healing and muscle pain. In this study, we evaluated the topical effect of CCFO against different irritant
Accepted 2 July 2010
agents in vivo, in order to verify its antiedematous effect as well to unravel its tentative mechanisms of
Available online xxx
action.
Materials and methods: CCFO was obtained from Caryocar coriaceum fruits using ethyl acetate as solvent.
Keywords:
Ear edema provoked by the application of Croton oil (single and multiple applications), arachidonic acid
Pequi oil
Caryocar coriaceum
(AA), capsaicin, phenol and histamine to Swiss mice was used to evaluate the topical anti-inflammatory
Skin irritants effect of CCFO. Histological analysis from mice ears sensitized with Croton oil and AA single application
Fatty acids was also performed.
Dermatitis Results: Crude CCFO (20 ␮L/ear) demonstrated significant topical antiedematous effect against Croton oil
Natural products single (inhibition of 32.0%; P < 0.05) and multiple (41.4% after 9 days, P < 0.001) applications, AA (inhibi-
tion of 49.7%; P < 0.01) and phenol (inhibition of 38.8%; P < 0.001). In contrast, CCFO did not antagonize
the edema caused by topical treatment with capsaicin and histamine when compared to control group
(P > 0.05). Histological analysis also revealed that CCFO was able to reduce the edema and the influx of
inflammatory cells in mice ears sensitized with Croton oil and AA.
Conclusions: CCFO exhibited a similar profile of topical anti-inflammatory activity to that of drugs that
classically modulate the production of arachidonic acid metabolites. The study also indicates the potential
application of CCFO as an important herbal medicine to be used against skin inflammatory diseases.
© 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction It is widely recognized that the modulation on production of

As the principal physical barrier to the external environment,
the skin provides an important bodily defense mechanism when
subjected to injury and invasion by pathogens or other exter-
nal noxious agents (Freinkel and Woodley, 2000). Normally, this
defense mechanism aims to repair the tissue damage or destroy the
microbial pathogen and do not cause serious damage. However, an
inappropriate or misdirected immune activity can implicate in the
pathogenesis of a large variety of inflammatory skin disorders, such
as psoriasis and atopic dermatitis (Kupper and Fuhlbrigge, 2004;
Maldini et al., 2009).
∗ Corresponding author. Tel.: +55 88 2101 1212.
E-mail address: rogerioaqsaraiva@hotmail.com (R.A. Saraiva).
inflammatory mediators (cytokines, neuropeptides, arachidonic
acid metabolites, etc) may be used therapeutically against skin
inflammations (Wells et al., 2004). For this propose, glucocorticoids,
antihistamines and non-steroidal anti-inflammatory drugs are gen-
erally employed (Davies et al., 2006). However, they can frequently
exhibit a set of undesirable side effects and are not effective in all
cases (Schoepe et al., 2006). As alternative, extracts and isolated
compounds from herbal medicine have been studied in order to
discover new effective and safe topical anti-inflammatory drugs
(Cordell and Colvard, 2005; Khanna et al., 2007; Lee et al., 2009).
Caryocar coriaceum Wittm. (Caryocaraceae), popularly known
in Brazil as “pequi”, is a common plant found in cerrado (savanna)
areas from Araripe plateau, Ceará State, Brazil Northeastern (Costa
et al., 2004). Pequi fruits contain excellent nutritional sources such
as antioxidant vitamins (A and E) and essential fatty acids (Sena et

0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2010.07.002

Please cite this article in press as: Saraiva, R.A., et al., Topical anti-inflammatory effect of Caryocar coriaceum Wittm. (Caryocaraceae) fruit pulp
fixed oil on mice ear edema induced by different irritant agents. J. Ethnopharmacol. (2010), doi:10.1016/j.jep.2010.07.002
 ARTICLE IN PRESS
G Model
JEP-6201; No. of Pages 7
2 R.A. Saraiva et al. / Journal of Ethnopharmacology xxx (2010) xxx–xxx
al., 2010), and are largely appreciated as food by the population of
the Cariri region, in Ceará State, and of neighboring cities of Per-
nambuco and Piauí States (De Oliveira et al., 2010). Besides being
a nutritional source, the handmade oil extracted from the pulp or
the seed is also popularly employed to treat skin inflammation,
respiratory affections, wound healing, ulcers, rheumatism, muscle
pain and contusions (Braga, 1960; Agra et al., 2007; Matos, 2007).
Previous studies from our group have confirmed that Caryocar cori-
aceum fixed oil possesses healing and gastroprotective activities in
vivo (Leite et al., 2009; Quirino et al., 2009).
Although the popular use of pequi pulp oil against inflammatory
disorders is known in Brazil Northeastern, data about the mecha-
nisms of action involved its topical anti-inflammatory activity are
not available in the literature. Thus, we were encouraged to eval-
uate the topical effect of pequi pulp oil against different irritant
agents in vivo, in order to verify its anti-inflammatory activity as
well to unravel its possible mechanisms of action.
2. Materials and methods
2.1. Plant material
Fruits of Caryocar coriaceum Wittm. were collected in a cerrado
area from Araripe plateau, Crato, Ceará State, Brazil (S 7◦ 21 53,1”;
W 39◦ 28 42,6”) in January 2009. A voucher specimen was identified
by Prof. Dr. Lígia Queiroz Matias and deposited in the Prisco Bezerra
Herbarium of the Federal University of Ceará under number 44523.
2.2. Chemicals
Croton oil, arachidonic acid, capsaicin, histamine dihydrochlo-
ride, indomethacin, and Tween 80 were purchased from Sigma
Chemical Co. (St. Louis, USA). Dexamethasone (Decadron® ) was
purchased from Aché (Brazil). Ketamine chloride and xylazine chlo-
ride were purchased from Syntec (Brazil). Acetone, ethanol and
ethyl acetate, of analytical grade, were purchased from Dinâmica
(Brazil).
2.3. Animals
Male and female Swiss mice (Mus musculus), weighting
25–35 g, were previously housed in standard polypropylene cages
under controlled conditions of temperature (22 ± 2 ◦ C) and 12-h
light/dark cycle, with free access to water and rodent chow (Labina,
Purina, Brazil). Mice were allowed to adapt to laboratory for at least
1 h before testing. All the procedures were previously approved by
Research Ethics Committee from Faculty of Medicine of Juazeiro do
Norte (Brazil), under number 2009 0218.
2.4. Extraction of fixed oil
The fixed oil from Caryocar coriaceum fruit pulp (CCFO) was
obtained through hot-extraction method. The in natura pulp from
Caryocar coriaceum fruits was removed and placed in a Soxhlet
apparatus in contact with ethyl acetate (solvent) during 3 h (60 ◦ C).
At the end of the process, a yellow solution (oil + ethyl acetate) and
an orange precipitate were obtained. The orange precipitate was
separated from the yellow solution by decantation, using a separa-
tion funnel. Ethyl acetate was removed from the yellow solution in
a rotary evaporator apparatus under reduced pressure and at a con-
trolled temperature (70 ± 2 ◦ C). The final oil extract had gold-yellow
appearance and a sui generis smell.
Please cite this article in press as: Saraiva, R.A., et al., Topical anti-inflammatory effect of Caryocar coriaceum Wittm. (Caryocaraceae) fruit pulp
fixed oil on mice ear edema induced by different irritant agents. J. Ethnopharmacol. (2010), doi:10.1016/j.jep.2010.07.002
2.5. Identification of fatty acids from CCFO
The fatty acid components were obtained as their methyl esters.
A sample of 0.2 g of the oil was subjected to reflux (30 min) with
methanolic potassium hydroxide. The oil was then converted to
methyl esters by methanolysis with acid catalyst. Isolation and
esterification of free and total fatty acids were performed according
to well-known reported procedures (Hartman and Lago, 1973). The
methyl esters of fatty acids were analyzed by gas chromatography
on a Hewlett-Packard Model 5971, equipped with capillary column
and FID detector. The operating conditions were oven temperature
program start at 35–180 ◦ C at a rate of 4 ◦ C/min, then heated at
a rate of 10 ◦ C/min to 250 ◦ C and held isothermally; injector and
detector temperature of 250 and 200 ◦ C, respectively, and H2 as a
carrier gas flowing at 0.8 ml/min. The identification was carried out
by co-injection of authentic compounds and retention times. Per-
centage area values were obtained electronically from the GC-FID
response. The oil was then characterized by a high content, 64.9%
of unsaturated fatty acids. The two major components identified
were oleic acid (55.79%) and palmitic acid (34.18%). Other con-
stituents found were palmitoleic acid (0.27%), stearic acid (1.73%),
linoleic acid (1.80%), heptadecenoic acid (5.86%) and 11-eicosenoic
acid (0.37%).
2.6. Croton oil single application-induced mouse ear edema
Groups of six Swiss mice were previously treated on the inner
and outer surfaces of the right ear with 20 ␮L of CCFO diluted in 2%
Tween 80 at concentrations of 50, 100, 200 and 400 mg/mL (1, 2, 4
and 8 mg/ear) or crude CCFO (20 ␮L = 13 mg/ear). The negative con-
trol received topically 20 ␮L of vehicle (2% Tween 80) on right ear.
Dexamethasone (0.08 mg/ear) was used as positive control. After
15 min, the edema was induced on the right ear by topical appli-
cation of 20 ␮L of Croton oil 5% (v/v) in acetone, while the left ear
received 20 ␮L of vehicle acetone. The ear edema was evaluated 6 h
after Croton oil application (Tubaro et al., 1985).
2.7. Croton oil multiple application-induced mouse ear edema
This study was conducted in 9 days (days 0–8). Croton oil 5%
(v/v) in acetone (20 ␮L/ear) was applied on the right ears and ace-
tone on the left ears of Swiss mice (n = 6/group) with a micropipette
on alternate days. The ear edema was evaluated daily by mea-
suring the ear thickness. On days 4–8, the mice were treated on
the inner and outer surfaces of the right ear with crude CCFO
(20 ␮L/ear), saline solution (NaCl 0.9%, 20 ␮L/ear) or dexametha-
sone (0.08 mg/ear) twice a day. On day 8, the mice were killed and
6-mm diameter ear punch biopsies were collected and weighted
(Stanley et al., 1991).
2.8. Arachidonic acid, capsaicin and phenol-induced mouse ear
edema
Inflammation was induced in mice (n = 6/group) by applying on
the inner and outer surfaces of the right ear 20 ␮L of the follow-
ing irritants: arachidonic acid (AA) 0.1 mg/␮L in acetone; capsaicin
0.01 mg/␮L in 90% ethanol and 10% phenol (v/v) in acetone. Fifteen
minutes before the application of each irritant agent, the right ears
were topically treated with crude CCFO (20 ␮L/ear), saline solu-
tion (negative control, 20 ␮L/ear), indomethacin (positive control
for AA, 2 mg/ear) or dexamethasone (positive control for capsaicin
and phenol, 0.08 mg/ear). The ear edema was evaluated 1 h after
AA and phenol, and 30 min after capsaicin application (Young et
al., 1984; Gábor and Razga, 1992; Gábor, 2000).
 ARTICLE IN PRESS
G Model
JEP-6201; No. of Pages 7

R.A. Saraiva et al. / Journal of Ethnopharmacology xxx (2010) xxx–xxx 3

2.9. Histamine subcutaneous application-induced mouse ear Table 1

Effect of CCFO and dexamethasone on Croton oil single application-induced ear
edema
edema.

Mice (n = 6/group) were previously anesthetized with ketamine Treatment Croton oil single application
20 mg/kg i.p. and xylazine 10 mg/kg i.p. After that, their right ears Percentage of Inhibition (%)
were treated topically with saline solution (20 ␮L/ear), dexametha- edema weight (%)
sone (0.08 mg/ear) or crude CCFO (20 ␮L/ear). Fifteen minutes later, 2% Tween 80 (Control group) 167.3 ± 9.6 –
the edema was induced on the right ear by intradermical applica- CCFO 1 mg/ear 145.3 ± 18.7 13.2
tion of 5 ␮L of histamine dihydrochloride 0.1 mg/␮L using a syringe 2 mg/ear 143.2 ± 10.1 14.4
with a 29G-hypodermical needle, while the left ear received 5 ␮L 4 mg/ear 149.8 ± 9.7 10.5
8 mg/ear 119.7 ± 9.5* 28.5*
of saline solution by the same procedure described (Sham). The ear Crude CCFO 13 mg/ear 113.8 ± 14.4* 32.0*
edema was evaluated 2 h after the histamine solution application Dexamethasone 0.08 mg/ear 24.5 ± 2.7*** 85.4***
(Brand et al., 2002).
Data expressed as mean ± s.e.m.
*
P < 0.05 and *** P < 0.001 when compared to control group (one-way ANOVA followed
by Student–Newman–Keuls test).
2.10. Ear edema measurement

Edema was expressed as percentage of the increase in ear
weight (all models) or as ear thickness variation (Croton oil multi-
ple application-induced ear edema) due to inflammatory challenge.
Ear thickness was measured with a digital caliper (Jomarca). The
digital caliper was applied near the tip of the ear just distal
to the cartilaginous ridges and the thickness was recorded in
micrometer (␮m). To evaluate the ear weight, animals were killed
by cervical dislocation, 6-mm diameter ear punch biopsies col-
lected using a metal punch, and the biopsies were individually
weighed on a MettlerToledo (AB204) balance. The extent of the
edema was expressed as percentage of increase in the ear tis-
sue weight (%), using the following formula: Percentage of edema
weight (%) = [(wRE − wLE ) × 100]/wLE , where wRE is the circle weight
obtained from the right ear (inflamed) and wLE is the circle weight
obtained from the left ear (non-inflamed). The mean of inhibition
edema percentage (%) was calculated by comparing to negative
control group.
2.11. Histology
Ear biopsies from Croton oil and arachidonic acid-induced sin-
gle application mouse ear edema were collected and fixed in 70%
ethanol for 24 h and then preserved in 10% formalin. Subsequently,
the ears were dehydrated, blocked in paraffin and then sectioned
with a microtome (5 ␮m). The cross-sections were stained with
hematoxylin and eosin for the evaluation of leukocyte accumula-
tion and edema intensity. A representative area was selected for
qualitative light microscope analysis (200× magnification).
2.12. Statistical analysis
The results are expressed as mean ± standard error of mean
(s.e.m.). The comparison between groups was assessed by one-way
analysis of variance (ANOVA) followed by Student–Newman–Keuls
test or by two-way ANOVA followed by Bonferroni test (repeated
measures) when appropriated. Values of P < 0.05 were accepted as
statistically significant.
3. Results
Topical application of Croton oil, AA, capsaicin, phenol or his-
tamine caused a significant inflammatory response in mice as
determined by the increase of ear weight, when compared to the
ears that received only vehicle of its respective irritant agent (ace-
tone, 90% ethanol or saline solution). However, it was observed that
the edematous potential of capsaicin and histamine was lower than
that of arachidonic acid, phenol and Croton oil. Further details are
discussed below.
3.1. Effect of CCFO on Croton oil single application-induced ear
edema
As reported in Table 1, CCFO 1, 2 and 4 mg/ear did not signifi-
cantly reduce the Croton oil induced ear edema when compared to
vehicle control. Only the crude CCFO (20 ␮L/ear) and CCFO 8 mg/ear
demonstrated significant reduction of edema in comparison to con-
trol group (28.5 and 32.0%, respectively, P < 0.05). Dexamethasone
(DEX) caused a significant reduction of edema (85.4%). The left ears
that received only vehicle acetone did not present visible edema.
3.2. Effect of CCFO on Croton oil multiple application-induced ear
edema
The application of crude CCFO on day 4 (in an established inflam-
matory process) caused a significant reduction in ear thickness
when compared to the saline-treated group 48 h after the first
treatment with CCFO (on day 6, P < 0.001; day 7, P < 0.01; and day
8, P < 0.001, Fig. 1A). DEX was effective in reducing significantly
the established edema 24 h after its first application (on days 5–8,
P < 0.001 for all days, Fig. 1A). The results observed on day 8 were
confirmed by the evaluation of edema weight. CCFO reduced the
edema by 49.7% (Fig. 1B).
3.3. Effect of CCFO on AA, capsaicin, phenol and
histamine-induced mice ear edema
Crude CCFO caused a significant edema reduction on sensi-
tized ears with AA (inhibition of 49.7%, P < 0.01, Fig. 2A) and
phenol (inhibition of 38.8%, P < 0.001, Fig. 2C). In contrast, CCFO did
not cause significant reduction against capsaicin and histamine-
induced edema (P > 0.05, Fig. 2B and D). Both DEX and IND were
effective when used as positive control (Fig. 2).
3.4. Histology
Histological sections of the ears submitted to a single Croton
oil application revealed a significant increase in the dermis thick-
ness, which was accompanied by loosening of connective tissue and
disorganization of the fibers from extracellular matrix. Epidermal
hyperplasia and a large number of infiltrating inflammatory cells
were also observed in the inflamed ears treated with vehicle 2%
Tween 80 (Fig. 3A), when compared to non-inflamed ear (Fig. 3D,
vehicle acetone). The ears treated with DEX and crude CCFO showed
a reduction in infiltration of inflammatory cells and lower dermis
thickness (Area 2, Fig. 3B and C) when compared to the inflamed
ear treated with 2% Tween 80 (Fig. 3A), and the ears treated with
DEX (Fig. 3C) presented a more evident effect. Histological sections
of the ears treated with CCFO 1, 2 and 4 mg/ear (data not shown)

Please cite this article in press as: Saraiva, R.A., et al., Topical anti-inflammatory effect of Caryocar coriaceum Wittm. (Caryocaraceae) fruit pulp
fixed oil on mice ear edema induced by different irritant agents. J. Ethnopharmacol. (2010), doi:10.1016/j.jep.2010.07.002
 ARTICLE IN PRESS
G Model
JEP-6201; No. of Pages 7
4 R.A. Saraiva et al. / Journal of Ethnopharmacology xxx (2010) xxx–xxx
Fig. 1. Effect of CCFO on Croton oil multiple application-induced ear edema. In (A)
is showing the time–response curve of effect from days 0 to 8. The Croton oil in
acetone was applied on alternate days. The thickness of the ear was measured daily,
using a digital caliper. On days 4–7, the ear of the animals received saline solution,
dexamethasone (DEX) or crude CCFO (arrows indicate the days when the treatment
occurred). The effect of the compounds was examined by varying the thickness of
the ear, calculated as the difference between the initial and final thickness. The
points represent the mean of 6 animals and vertical bars s.e.m (two-way ANOVA
followed by Bonferroni test). In (B) is showing the percentage of edema weight of
each group on day 8 (one-way ANOVA followed by Student–Newman–Keuls test).
*P < 0.05, **P < 0.01, and ***P < 0.001 compared to saline-treated group.
presented slight improvement when compared to control inflamed
ear.
According to the histological analysis of ears sensitized with
AA application, a significant increase in the dermis thickness and
in inflammatory cells infiltration were observed (Fig. 4A–C) when
compared to non-inflamed ear (vehicle acetone, Fig. 4D). The ears
treated with IND (Fig. 4B) and CCFO (Fig. 4C) demonstrated a
decrease in dermis thickness and a significant reduction in inflam-
matory cells infiltration when compared to the ear that received
AA and saline solution (Fig. 4A).
4. Discussion
The application of mouse models of ear edema induced by
different irritant agents (Croton oil, capsaicin, AA, phenol, his-
tamine) has been widely used to identify the probable topical
anti-inflammatory effect of the substance in study and to propose
its possible mechanism of action (Gábor, 2000).
Croton oil contains 12-o-tetracanoilphorbol-13-acetate (TPA)
and other phorbol esters as main irritant agents. TPA is able to
activate protein kinase C (PKC), which activates other enzymatic
cascades in turn, such as mitogen activated protein kinases (MAPK)
and phospholipase A2 (PLA2 ), leading to release of platelet activa-
tion factor (PAF) and AA. This cascade of events stimulates vascular
permeability, vasodilation, polymorphonuclear leukocytes migra-
tion, release of histamine and serotonin and moderate synthesis
Please cite this article in press as: Saraiva, R.A., et al., Topical anti-inflammatory effect of Caryocar coriaceum Wittm. (Caryocaraceae) fruit pulp
fixed oil on mice ear edema induced by different irritant agents. J. Ethnopharmacol. (2010), doi:10.1016/j.jep.2010.07.002
of inflammatory eicosanoids by cyclooxygenase (COX) and 5-
lipoxygenase (5-LOX) enzymes (Ferrandiz et al., 1996; Wang et
al., 2001). COX and 5-LOX inhibitors, leukotriene B4 (LTB4 ) antag-
onists and corticosteroids show topical anti-inflammatory action
in animal models of Croton oil or TPA-induced skin inflammation
(Murakawa et al., 2006). CCFO at concentrations above 8 mg/ear and
crude OFCC were effective in this model (Table 1 and Fig. 3). Due
to the best effect observed in Croton oil single application-induced
mouse ear edema, we chose the crude CCFO (20 ␮L/ear) as standard
treatment for the subsequent models, aiming to compare the effect
of the same oil concentration against different irritant agents.
Croton oil single application provides data regarding the
antiedematous activity in an acute inflammatory process, whereas
Croton oil multiple application can be used to evaluate the antiede-
matous activity in an established (chronic) inflammatory process
(Stanley et al., 1991). In fact, repeated Croton oil application is
associated with an increase in ear weight, intense neutrophilical
infiltration, macrophages and T cells (CD4+ and CD8+ ) migration
and hyperproliferative epidermis (acanthosis). Corticosteroids and
5-LOX inhibitors reduce the edematous effect in this model, while
COX inhibitors and antihistamines show little or no effect (Green
and Shuster, 1987). Therefore, this model can be used to investigate
the involvement of drugs related to release of leukotrienes (Stanley
et al., 1991). Here we observed that CCFO and dexamethasone
inhibited the edema induced by Croton oil multiple application
(Fig. 1).
The local application of AA stimulates a rapid inflammatory
response (when compared to Croton oil), contributing to an imme-
diate vasodilation and erythema in the first 5 min, followed by
development of edema after 40–60 min, which coincides with pro-
tein and leukocyte extravasion (Young et al., 1984; Crummey et
al., 1987). AA is a precursor of inflammatory eicosanoids such as
prostaglandin E2 (PGE2 ) and leukotrienes (produced via COX-1,
COX-2 and 5-LOX enzymes). COX and LOX inhibitors classically
cause significant reduction in AA-induced ear edema, while cor-
ticosteroids cause no effect or only a modest reduction in edema
when compared to non-steroidal drugs (Gábor, 2000; Carlson et
al., 1985). AA metabolites are also involved in mast cells degranu-
lation, which is accompanied by histamine release. Consequently,
antihistamines are also able to reduce AA-induced edema (Young
et al., 1984; Crummey et al., 1987; Blazsó and Gábor, 1995). We
observed that crude CCFO significantly decreased the ear edema in
this model (Fig. 2A). Histological analysis of ears sensitized with AA
and treated with CCFO also revealed little infiltration of inflamma-
tory cells, which was similar to that observed in the ears treated
with positive control IND (Fig. 4). These data indicated that CCFO
may decrease the production of inflammatory eicosanoids, which
inhibited chemotaxis of inflammatory cells and decreased vas-
cular permeability, contributing to the anti-inflammatory effect
observed (Carlson et al., 1985).
Capsaicin is an irritant alkaloid present in plants of the genus
Capsicum (peppers) and is responsible for the pungent taste of
the fruits of these species. When it contacts with epidermis, cap-
saicin activates the TRPV1 receptor, which elicits a rapid response
via the release of neuropeptides (such as calcitonin gene related
peptide, substance P and tachykinins) and monoamimes (his-
tamine and serotonin) (Gábor and Razga, 1992; Inoue et al., 1993,
1995). Inhibitors of AA metabolites do not reduce the capsaicin-
induced edema, whereas dexamethasone, antihistamines and
bradykinin receptor antagonists present significant antiedema-
tous effect (Mantione and Rodriguez, 1990; Inoue et al., 1995).
Moreover, the blockage of Ca2+ channels and ruthenium red (a
capsaicin inhibitor) also show antiedematous effect against cap-
saicin, but not against AA-induced ear edema (Inoue et al., 1993).
The topical application of CCFO did not present significant reduc-
tion on capsaicin-induced ear inflammation (P > 0.05), suggesting
 ARTICLE IN PRESS
G Model
JEP-6201; No. of Pages 7

R.A. Saraiva et al. / Journal of Ethnopharmacology xxx (2010) xxx–xxx 5

Fig. 2. Effect of crude CCFO, dexamethasone (DEX) or indomethacin (IND) on mice ear edema induced by different irritant substances. (A) Arachidonic acid (AA); (B) capsaicin;
(C) histamine; and (D) phenol. The control group received saline solution as treatment. Each point represents the mean ± s.e.m. of 6 mice. *P < 0.05, **P < 0.01 and ***P < 0.001
compared with saline-treated group (one-way ANOVA followed by Student–Newman–Keuls test).

Fig. 3. Photomicrograph of transverse sections of mice ears sensitized with topical application of Croton oil 5% (v/v) in acetone (A–C) or vehicle acetone (D, non-inflamed),
stained with hematoxylin–eosin and examined under light microscopy (magnification: 200×). Treatments: vehicle 2% Tween 80 (A), dexamethasone 0.08 mg/ear (B) and
crude CCFO 20 ␮L/ear (C). The numbers 1 and 2 indicate epidermis and dermis, respectively. Arrows indicate inflammatory cells (polymorphonuclear neutrophils) in the
dermis. The shown sections are representative of six animals per group.

Please cite this article in press as: Saraiva, R.A., et al., Topical anti-inflammatory effect of Caryocar coriaceum Wittm. (Caryocaraceae) fruit pulp
fixed oil on mice ear edema induced by different irritant agents. J. Ethnopharmacol. (2010), doi:10.1016/j.jep.2010.07.002
 ARTICLE IN PRESS
G Model
JEP-6201; No. of Pages 7

6 R.A. Saraiva et al. / Journal of Ethnopharmacology xxx (2010) xxx–xxx

Fig. 4. Photomicrograph of transverse sections of mice ears sensitized with topical application of AA 0.1 mg/␮L in acetone (A–C) or vehicle acetone (D, non-inflamed), stained
with hematoxylin–eosin and examined under light microscopy (magnification: 200×). Treatments: saline solution (A), indomethacin 2 mg/ear (B) and crude CCFO 20 ␮L/ear
(C). The numbers 1 and 2 indicate epidermis and dermis, respectively. Arrows indicate inflammatory cells (polymorphonuclear neutrophils) in the dermis. The shown sections
are representative of six animals per group.

that CCFO did not interfere in substances or receptors involved in
capsaicin-activated inflammatory pathways (Fig. 2B).
Histamine is a vasoactive amine released by mast cells activated
by complement proteins C3a and C5a and by IgE-activated lym-
phocytes, which increases vascular permeability and vasodilation.
Because of its rapid action, histamine is also involved in immediate-
type hypersensitivity reactions (Brand et al., 2002). Antihistamines
and corticosteroids (dexamethasone) reduce the edema in this
model (Fig. 2C). Crude CCFO did not modify histamine-induced
ear edema when compared to saline control (P > 0.05). This finding
suggests that CCFO did not interfere with AA-induced histamine
release, but it possibly blocked the inflammatory cascade by
decreasing the production of inflammatory eicosanoids.
When phenol comes into direct contact with the skin, ker-
atinocyte membranes are ruptured, resulting in release of cytokines
such as IL-1␣, TNF-␣ and IL-8 by a mechanism independent on PKC
activation pathway (as occur in inflammations induced by Croton
oil), which in turn results in release of other inflammatory medi-
ators such as AA metabolites and reactive oxygen species (ROS)
(Wilmer et al., 1994; Murray et al., 2007). Thus, phenol is a good
irritant agent for simulating contact dermatitis in mice (Lim et
al., 2004). Crude CCFO significantly reduced the phenol-induced
edema (Fig. 2D) when compared to the control group. This activity
may be related to the influence on the production of AA metabo-
lites and CCFO may also have some antioxidant activity mediated
by its fatty acids (Henry et al., 2002). This finding can also indicate
the potential use of CCFO in irritant contact dermatitis.
Here we have observed that CCFO exhibited a similar profile of
topical anti-inflammatory activity to that of drugs that classically
modulate the production of AA metabolites, probably via inhibition
of COX and LOX enzymes or via production of anti-inflammatory
eicosanoids (Lawrence et al., 2002). In line with this, some stud-
ies have shown that unsaturated fatty acids found in CCFO (see
Section 2.5) demonstrate anti-inflammatory activity (Calder, 2005;
Das, 2006). Henry et al. (2002) observed that oleic acid (the major
component of CCFO), at 100 ppm concentration, caused a moder-
ate in vitro inhibition of COX-1. Studies have also demonstrated
that linoleic acid (other component found in CCFO) is a precursor of
13-hydroxy-octadecaenoic acid (13-HODE) via 15-LOX, a substance
that inhibits the production of superoxide, reduces the migration
of polymorphonuclear leukocytes and modulates the actions of
leukotrienes and prostanoids (Fritsche, 2008). Linoleic acid also
inhibit COX-1 and COX-2 in vitro (Ringbom et al., 2001; Henry et al.,
2002) and decrease expression of NF-␬B and TNF-␣ genes in human
THP-1 monocytic cells, leading to decrease in IL-6, IL-1␤ and TNF-
␣ levels (Zhao et al., 2005). On the other hand, some studies have
reported that linoleic acid is also a proinflammatory substance due
to be a precursor of AA via 6-desaturase enzyme (Calder, 2005;
Fritsche, 2008), but Ziboh et al. (2000) argue that the 6-desaturase
enzyme is deficient in the epidermis, suggesting that linoleic acid
had no significant influence on production of AA via 6-desaturase
in the skin. Consequently, taken together with the results presented
here, the literature data indicate that the oleic acid and linoleic acid
can be at least in part involved in the topical anti-inflammatory
effect of CCFO.
In conclusion, our study has demonstrated the relevant topical
antiedematous effect of CCFO in mouse ear edema induced by dif-
ferent agents and indicates its potential application as an herbal
medicine to be used on skin inflammatory diseases.
Acknowledgments
The authors are thankful to Fundação Cearense de Amparo à
Pesquisa–FUNCAP, and to Conselho Nacional de Desenvolvimento
Científico e Tecnológico–MCT/CNPq (INCT for Excitotoxicity and
Neuroprotection), for providing support to this research; to Dr. Lígia

Please cite this article in press as: Saraiva, R.A., et al., Topical anti-inflammatory effect of Caryocar coriaceum Wittm. (Caryocaraceae) fruit pulp
fixed oil on mice ear edema induced by different irritant agents. J. Ethnopharmacol. (2010), doi:10.1016/j.jep.2010.07.002
 ARTICLE IN PRESS
G Model
JEP-6201; No. of Pages 7
R.A. Saraiva et al. / Journal of Ethnopharmacology xxx (2010) xxx–xxx
de Queiroz Farias (Universidade Federal do Ceará) for identifying
the voucher specimen and to Faculty of Medicine of Juazeiro do
Norte, for providing animals to this research.
References
Agra, M.F., Freitas, P.F., Barbosa Filho, J.M., 2007. Synopsis of the plants known as
medicinal and poisonous in Northeast of Brazil. Revista Brasileira de Farmacog-
nosia 17, 114–140.
Blazsó, G., Gábor, M., 1995. Effects of prostaglandin antagonist phloretin derivates
on mouse ear edema induced with different skin irritants. Prostaglandins 50,
161–168.
Braga, R., 1960. Plantas do Nordeste, especialmente do Ceará, 2a ed. Imprensa Oficial,
Fortaleza, Brazil, 540 pp.
Brand, C., Townley, S.L., Finlay-Jones, J.J., Hart, P.H., 2002. Tea tree oil reduces
histamine-induced oedema in murine ears. Inflammation Research 51, 283–289.
Calder, P.C., 2005. Polyunsaturated fatty acids and inflammation. Biochemical Soci-
ety Transactions 33, 423–427.
Carlson, R., O’Neill-Davis, L., Chang, J., Lewis, A.J., 1985. Modulation of mouse ear
edema by cycloxygenase and lipoxygenase inhibitors and other pharmacologic
agents. Agents Actions 17, 197–204.
Cordell, G.A., Colvard, M.D., 2005. Some thoughts on the future of ethnopharmacol-
ogy. Journal of Ethnopharmacology 100, 5–14.
Costa, I.R., Araujo, F.S., Lima-Verde, L.W., 2004. Flora e aspectos auto-ecológicos de
um encrave de cerrado na chapada do Araripe, Nordeste do Brasil. Acta Botanica
Brasilica 18, 759–770.
Crummey, A., Harper, G.P., Boyle, E.A., Mangan, F.R., 1987. Inhibition of arachidonic
acid-induced ear oedema as a model for assessing topical anti-inflammatory
compounds. Agents Actions 20, 69–76.
Das, U.N., 2006. Essential fatty acids – a review. Current Pharmaceutical Biotechnol-
ogy 7, 467–482.
Davies, N.M., Reynolds, J.K., Underberg, M.R., Gates, B.J., Ohgami, Y., Vega-Villa, K.R.,
2006. Minimizing risks of NSAIDs: cardiovascular, gastrointestinal and renal.
Expert Review of Neurotherapeutics 6, 1643–1655.
De Oliveira, M.E.B., Guerra, N.B., Maia, A.D.N., Alves, R.E., Matos, N.M.D., Sampaio,
F.G.M., Lopes, M.M.T., 2010. Chemical and physical–chemical characteristics in
pequi from the chapada do Araripe, Ceara, Brazil. Revista Brasileira de Fruticul-
tura 32, 114–125.
Ferrandiz, M.L., Gil, B., Sanz, M.J., Ubeda, A., Erazo, S., González, E., Negrete, R.,
Pacheco, S., Paya, M., Alcaraz, M.J., 1996. Effect of bakuchiol on leucoyte functions
and some inflammatory responses in mice. Journal of Pharmacy and Pharma-
cology 48, 975–980.
Freinkel, R.K., Woodley, D.T., 2000. The Biology of the Skin. Parthenon, New York,
pp. 15–19.
Fritsche, K. L., 2008. Too much linoleic acid promotes inflammation – doesn’t it?
Prostaglandins, Leukotrienes and Essential Fatty Acids 79, 173–175.
Gábor, M., 2000. Mouse Ear Inflammation Models and their Pharmacological Appli-
cations. Akadémiai Kiadó, Budapest.
Gábor, M., Razga, Z., 1992. Development and inhibition of mouse ear oedema induced
with capsaicin. Agents Actions 36, 83–86.
Green, C.A., Shuster, S., 1987. Lack of effect of topical indomethacin on psoriasis.
British Journal of Clinical Pharmacology 24, 381–384.
Hartman, L., Lago, R.C.A., 1973. Rapid preparation of fatty acid methyl esters from
lipids. Laboratory Practice 22, 475–477.
Henry, G.E., Momin, R.A., Nair, M.G., Dewitt, D.L., 2002. Antioxidant and cyclooxy-
genase activities of fatty acids found in food. Journal of Agricultural and Food
Chemistry 50, 2231–2234.
Inoue, H., Nagata, N., Koshihara, Y., 1993. Profile of capsaicin-induced mouse ear
oedema as neurogenic inflammatory model: comparison with arachidonic acid-
induced ear oedema. British Journal of Pharmacology 110, 1614–1620.
Inoue, H., Nagata, N., Koshihara, Y., 1995. Participation of serotonin in capsaicin-
induced mouse ear edema. Japanese Journal of Pharmacology 69, 61–68.
Khanna, D., Sethi, G., Ahn, K.S., Pandey, M.K., Kunnumakkara, A.B., Sung, B., Aggarwal,
A., Aggarwal, B.B., 2007. Natural products as a gold mine for arthritis treatment.
Current Opinion in Pharmacology 7, 344–351.
Kupper, T.S., Fuhlbrigge, R.C., 2004. Immune surveillance in the skin: mechanisms
and clinical consequences. Nature Reviews Immunology 4, 211–222.
Please cite this article in press as: Saraiva, R.A., et al., Topical anti-inflammatory effect of Caryocar coriaceum Wittm. (Caryocaraceae) fruit pulp
fixed oil on mice ear edema induced by different irritant agents. J. Ethnopharmacol. (2010), doi:10.1016/j.jep.2010.07.002
7
Lawrence, T., Willoughby, D.A., Gilroy, D.W., 2002. Anti-inflammatory lipid medi-
ators and insights into the resolution of inflammation. Nature Reviews
Immunology 2, 787–795.
Lee, D.Y., Choo, B.K., Yoon, T., Cheon, M.S., Lee, H.W., Lee, A.Y., Kim, H.K., 2009. Anti-
inflammatory effects of Asparagus cochinchinensis extract in acute and chronic
cutaneous inflammation. Journal of Ethnopharmacology 121, 28–34.
Leite, G.O., Penha, A.R.S., Quirino, G.S., Colares, A.V., Rodrigues, F.F.G., Costa, J.G.M.,
Cardoso, A.L.H., Campos, A.R., 2009. Gastroprotective effect of medicinal plants
from Chapada do Araripe, Brazil. Journal of Young Pharmacists 1, 54–56.
Lim, H., Park, H., Kim, H.P., 2004. Inhibition of contact dermatitis in animal
models and suppression of proinflammatory gene expression by topi-
cally applied flavonoid, wogonin. Archives of Pharmacological Research 27,
442–448.
Maldini, M., Sosa, S., Montoro, P., Giangaspero, A., Balick, M.J., Pizza, C., Della-Logia,
R., 2009. Screening of the topical anti-inflammatory activity of the bark of Acacia
cornigera Willdenow, Brysonima crassifolia Kunth, Sweetia panamensis Yakowlev
and the leaves of Sphagneticola trilobata Hitchcock. Journal of Ethnopharmacol-
ogy 122, 430–433.
Mantione, C.R., Rodriguez, R., 1990. A bradykinin (BK)1 receptor antagonist blocks
capsaicin-induced ear inflammation in mice. British Journal of Pharmacology
99, 516–518.
Matos, F.J.A., 2007. Plantas Medicinais: Guia de Seleção e Emprego de Plantas Usadas
em Fitoterapia no Nordeste do Brasil, 3a ed. Imprensa Universitária, Fortaleza,
Brazil, 394 pp.
Murakawa, M., Yamaoka, K., Tanaka, Y., Fukuda, Y., 2006. Involvement of necrosis
factor (TNF)-␣ in phorbol ester 12-o-tetradecaoylphorbol-13-acetate (TPA)-
induced skin edema in mice. Biochemical Pharmacology 71, 1331–1336.
Murray, A.R., Kisin, E., Castranova, V., Kommineni, C., Gunther, M.R., Shvedova, A.A.,
2007. Phenol-induced in vivo oxidative stress in skin: evidence for enhanced
free radical generation, thiol oxidation, and antioxidant depletion. Chemical
Research Toxicology 20, 1769–1777.
Quirino, G.S., Leite, G.O., Rebelo, L.M., Tomé, A.R., Costa, J.G.M., Cardoso, A.H., Campos,
A.R., 2009. Healing potential of pequi (Caryocar coriaceum Wittm.) fruit pulp oil.
Phytochemistry Letters 2, 179–183.
Ringbom, T., Huss, U., Stenholm, Å., Flock, S., Skattebøl, L., Perera, P., Bohlin, L., 2001.
COX-2 inhibitory effects of naturally occurring and modified fatty acids. Journal
of Natural Products 64, 745–749.
Sena Jr., D.M., Rodrigues, F.F.G., Freire, P.T.C., de Lima, S.G., Coutinho, H.D.M., Carvajal,
J.C.L., Costa, J.G.M., 2010. Physicochemical and spectroscopical investigation of
Pequi (Caryocar coriaceum Wittm.) pulp oil. Grasas y Aceites 61, 191–196.
Schoepe, S., Schäcke, H., May, E., Asadullah, K., 2006. Glucocorticoid therapy-induced
skin atrophy. Experimental Dermatology 15, 406–420.
Stanley, P.L., Steiner, S., Havens, M., Transposch, K.M., 1991. Mouse skin inflamma-
tion induced by multiple topical application of 12-O-tetradecanoylphorbol-13-
acetate. Journal of Pharmacological and Biophysiological Research 4, 262–271.
Tubaro, A., Dri, P., Delbello, G., Zilli, C., Della-Loggia, R., 1985. The croton oil test
revisited. Agents Actions 17, 347–349.
Wang, H.Q., Kim, M.P., Tiano, H.F., Langenbach, R., Smart, R.C., 2001. Protein kinase
C-alpha coordinately regulates cytosolic phospholipase A2 activity and the
expression of ciclooxygenase-2 trought differtent mechanism in mouse ker-
atinocytes. Molecular Pharmacology 59, 860–866.
Wells, T., Basketter, D., Schröder, K.R., 2004. In vitro skin irritation: facts and future.
State of the art review of mechanisms and models. Toxicology In Vitro 18,
231–243.
Wilmer, J.L., Burleson, F.G., Kayama, F., Kauno, J., Luster, M.I., 1994. Cytokine induc-
tion in human epidermal keratinocytes exposed to contact irritants and its
relation to chemical-induced inflammation in mouse skin. Journal of Investiga-
tive Dermatology 102, 915–922.
Young, J.M., Spires, D.A., Bedord, C.J., Wagner, B., Ballaron, S.J., De Young, L.M., 1984.
The mouse ear inflammatory response to topical arachidonic acid. The Journal
of Investigative Dermatology 82, 367–371.
Zhao, G., Etherton, T.D., Martin, K.R., Heuvel, J.P.V., Gillies, P.J., West, S.G., Kris-
Etherton, P.M., 2005. Anti-inflammatory effects of polyunsaturated fatty acids
in THP-1 cells. Biochemical and Biophysical Research Communications 336,
909–917.
Ziboh, V.A., Miller, C.C., Cho, Y., 2000. Metabolism of polyunsaturated fatty acids by
skin epidermal enzymes: generation of antiinflammatory and antiproliferative
metabolites. American Journal of Clinical Nutrition 71, 361S–366S.