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Korean J Pain 2019 April; Vol. 32, No. 2: 79-86
pISSN 2005-9159 eISSN 2093-0569
https://doi.org/10.3344/kjp.2019.32.2.79
| Original Article |
Analgesic effects of eucalyptus essential oil in mice
Ganggeun Lee
1
, Junbum Park
1
, Min Sun Kim
2
, Geun Hee Seol
3
, and Sun Seek Min
2
1Department of Anesthesiology and Pain Medicine, Eulji University Hospital, Daejeon,
2Department of Physiology and Biophysics, Eulji University School of Medicine, Daejeon,
3Department of Basic Nursing Science, Korea University School of Nursing, Seoul, Korea
Background: The use of aroma oils dates back to at least 3000 B.C., where it was applied to mummify corpses
and treat the wounds of soldiers. Since the 1920s, the term “aromatherapy” has been used for fragrance therapy
with essential oils. The purpose of this study was to determine whether the essential oil of Eucalyptus (EOE)
affects pain pathways in various pain conditions and motor coordination.
Methods: Mice were subjected to inhalation or intraperitoneal injection of EOE, and its analgesic effects were
assessed by conducting formalin, thermal plantar, and acetic acid tests; the effects of EOE on motor coord-
ination were evaluated using a rotarod test. To determine the analgesic mechanism, 5’-guanidinonaltrindole
(-opioid antagonist, 0.3 mg/kg), naltrindole (-opioid antagonist, 5 mg/kg), glibenclamide (-opioid antagonist,
2 mg/kg), and naloxone (-opioid antagonist, 4, 8, 12 mg/kg) were injected intraperitoneally.
Results: EOE showed an analgesic effect against visceral pain caused by acetic acid (EOE, 45 mg/kg); however,
no analgesic effect was observed against thermal nociceptive pain. Moreover, it was demonstrated that EOE
did not have an effect on motor coordination. In addition, an anti-inflammatory effect was observed during
the formalin test.
Conclusions: EOE, which is associated with the -opioid pain pathway, showed potential effects against somatic,
inflammatory, and visceral pain and could be a potential therapeutic agent for pain. (Korean J Pain 2019;
32: 79-86)
Key Words: Acetic acid; Analgesics; Aromatherapy; Eucalyptus; Formaldehyde; Glyburide; Intraperitoneal
injections; Mice; Naloxone; Naltrindole; Rotarod performance test; Opioid antagonists.
Received October 15, 2018. Revised January 18, 2019. Accepted January 21, 2019.
Correspondence to: Sun Seek Min
Department of Physiology and Biophysics, Eulji University School of Medicine, 77 Gyeryong-ro 771beon-gil, Jung-gu, Daejeon 34824, Korea
Te l: ＋82-42-259-1633, Fax: ＋82-42-259-1639, E-mail: ssmin@eulji.ac.kr
ORCID: https://orcid.org/0000-0001-7144-1987
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium,
provided the original work is properly cited.
ⓒThe Korean Pain Society, 2019
INTRODUCTION
Patients suffering from intense, chronic pain anticipate the
development of new medicines that have little to no side
effects; meanwhile, relying on numerous alternative medi-
cines. Inhaling the fragrance of essential oils is considered
to be an alternative medicine to achieve psychological and
physical relaxation. Fragrances are used in cosmetics,
80 Korean J Pain Vol. 32, No. 2, 2019
www.epain.org
foods, and in health-promoting agents. The utilization of
fragrances dates back to at least 3000 B.C., where essen-
tial oils were used for mummification and in beauty
products. There are even records of Hippocrates using
fragrances to cure diseases. Scientific experiments have
shown that when aromatic fragrances are inhaled, pain is
sensed differently, unlike when unpleasant odors are in-
haled [1].
Eucalyptus has been used in Brazilian traditional med-
icine as an anti-inflammatory, analgesic, and antipyretic
agent, and to treat flu, common cold, and nose bleeds
[2-4]. Modern science has also demonstrated such benefi-
cial effects of Eucalyptus. However, further studies are
necessary to confirm the precise pathways involved in the
analgesic effects of Eucalyptus.
In this study, two methods were used to observe the
analgesic effects of Eucalyptus in various pain models of
mice: 1) inhalation of the essential oil of Eucalyptus (EOE)
and 2) intraperitoneal injections for systemic adminis-
tration. Pain models included formalin-induced, acetic acid-
induced writhing, and thermal-stimulated pain. Behavioral
experiments utilizing antagonists of pain-killers have been
used to investigate which pain pathway is related to such
analgesic effects. Moreover, drowsiness and dizziness may
appear as side-effects after a medicine is injected. Cer-
tainly, side-effects need to be reduced for the medicine
to be used clinically. Therefore, in this study, the rotarod
test was used to investigate potential side effects caused
by the drug.
MATERIALS AND METHODS
1. Experimental animals
Prior to the experiment, male C57BL/6 mice aged 8 weeks
(Samtaco Inc., Osan, Korea) were maintained under a 12 h
light-dark cycle (lights on from 7 AM to 7 PM) at 21 ±
2
°
C. The animals had access to sterilized food and tap wa-
ter as they desired and were subjected to the following
tests: the formalin test, acetic acid test, rotarod test, and
plantar test. Animals were used independently for each
experiment. The guidelines of the Institutional Animal Re-
search Ethics Committee of Korea University (approval no.
KUIACUC-2011-84) were followed for the care of the ex-
perimental animals in all experimental measures.
2. Injection of EOE
Animals were injected with EOE (Aromarant Co. Ltd,
Rottingen, Germany) dissolved in almond oil at 11.25, 22.5,
45, 90, and 180 mg/kg intraperitoneally in an injection
volume of 0.1 ml/100 g body weight. Normal saline (0.9%
w/v) was administered to the control group, whereas al-
mond oil alone was injected to the vehicle group. The con-
trol, vehicle, and essential oil groups were injected 30 min
before the formalin, thermal plantar, and rotarod tests,
and 35 min before the acetic acid-induced writhing test.
3. Inhalation of EOE
The hold-board apparatus consisted of a square trans-
parent plexiglas cage (50 cm × 50 cm × 30 cm) with
4 holes in the bottom which were prepared for inhalation
of the essential oils. The EOE was first combined with the
same volume of distilled water and then absorbed in cotton
that was set on the upper side of the inhalation box to
ensure vaporization. A 1 ml aliquot of EOE was vaporized
in a petri dish (90 × 15 mm) in the EOE group, while only
distilled water was given to the control group. Before the
formalin test, mice were exposed to the translucent Plexi-
glas cage for 1, 2, 4, and 8 h, except for the group exposed
to EOE.
4. Drugs
All drugs and vehicles were injected intraperitoneally 15
min prior to the EOE injection, except in the case of the
inhalation tests, at a rate of 0.1 ml/100 g of body weight
and included morphine (4, 8 mg/kg), indomethacin (10
mg/kg), naloxone (8 mg/kg), 5
’
-guanidinonaltrindole (0.3
mg/kg), and glibenclamide (2 mg/kg).
5. Formalin test
The formalin test was similar to the one reported by
Hunskaar and Hole [5]. Twenty
l of formalin (2 % v/v) was
injected into the mice (n = 7-12) dorsal surface of the left
hind paw of the mice. The mice licking their injected paw
were instantly timed. Their response was considered,
based on the following: 1) 0-5 min after the formalin in-
jection was the nociceptive pain response and 2) 20-25
min after was the inflammatory pain response.
Lee, et al / Analgesic effects of eucalyptus essential oil in mice 81
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Fig. 1. Intraperitoneal (i.p.) injection of essential oil of
Eucalyptus (EOE) reduced licking time in the second phase
of the formalin test. Licking time in mice injected with
control (0.9% saline, i.p.), vehicle (almond oil, i.p.), essential
oil (11.25, 22.5, and 45 mg/kg, i.p.), or morphine (4 mg/kg,
i.p.) 30 min prior to the injection of 20 l of formalin (2%
v/v) into the dorsal surface of their left, hind paws.
Morphine, a positive control, reduced the licking time in th
e
first phase (0-5 min). Meanwhile, EOE (45 mg/kg) and
morphine significantly reduced licking time in the second
phase (20-25 min). Each value represents the mean and
standard error of mean. *P ＜ 0.05, **P ＜ 0.001 compared
to the vehicle group. One-way analysis of variance followed
by Tuckey’s post hoc test were performed for statistical
analyses. The control group had 12 mice while the others
had 8 mice.
6. Acetic acid-induced abdominal writhing test
In accordance to Quintans-J
ú
nior et al. [6], abdominal
writhing was assessed in mice (n = 8-10). Acetic acid was
injected intraperitoneally as a 0.5% solution (10 ml/kg).
Total writhing movements were counted during a 30-min
period starting 5 min after the acetic acid injection.
7. Thermal plantar test
To assess paw withdrawal latency (PWL) to a thermal noci-
ceptive stimulus, the Hargreaves method [7] was utilized
using intraperitoneal administrations of 0.9% saline (control
group), vehicle, or EOE (45 and 90 mg/kg) (n = 10 per
group). The plantar surface of the hind paw received the
center of a focused beam of heat. To avoid tissue damage,
the application of the beam was halted at 20 s. With at
least 1-min intervals, 4 measurements were obtained on
each hind paw. The mean PWL of each group was eval-
uated using previously determined standard values for
each hind paw.
8. Rotarod test
To measure whether the EOE could influence motor coordi-
nation, a rotarod test was applied. EOE (45, 90, and 180
mg/kg) and vehicle (almond oil) was administered
intraperitoneally. Animals were subjected to spinning at 4
rpm (n = 10 per group) using a rotating rod (Scitech Korea,
Seoul, Korea). The rotation was progressively augmented
at a rate of 1 revolutions per minute (rpm) after mouse
stabilization. Infrared sensors recorded the time that the
mice managed to remain on the rod and the speed at which
they fell off. The average of three trials was used for each
mouse.
9. Statistical analysis
Mean ± standard error was used. One-way ANOVA, fol-
lowed by Tukey
’
s post hoc test was employed to evaluate
differences among treatment groups. A P value of
＜
0.05
was considered statistically significant in all cases.
RESULTS
1. Effect of injection or inhalation of EOE on formalin test
After EOE intraperitoneal injections and pretreatment, the
first (0-5 min) and second (20-25 min) phases of licking
were recorded to investigate whether EOE has an anti-
nociceptive or anti-inflammatory effect. Licking during the
first phase was not reduced by EOE, whereas licking during
the second phase was reduced by 91% (EOE 45 mg/kg: 4.1
s vs. vehicle: 47.9 s; P
＜
0.001) (Fig. 1). First phase in-
hibition using morphine as a positive control was reduced
by 48% (morphine: 59.5 s vs. vehicle: 114.4 s; P
＜
0.01)
and by 80% during the second phase (morphine: 9.6 s vs.
vehicle: 47.9 s; P
＜
0.001). There were no substantial dif-
ferences between the EOE group (45 mg/kg) and the pos-
itive control group in the second phase.
The formalin test was used to determine whether in-
halation of EOE has any effect on licking time (Fig. 2).
82 Korean J Pain Vol. 32, No. 2, 2019
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Fig. 3. The analgesic effects of essential oil of Eucalyptus
(EOE) is via the -opioid pathway as demonstrated by the
formalin test. The relevance of antinociceptive effect of
EOE via the opioid pathway was investigated in this experi-
ment. The licking time after pretreatment with an antagonist
of the opioid receptor 15 min before the injection of the
EOE (45 mg/kg, intraperitoneal) is shown. 5’-guanidino-
naltrindole (GNTI, -opioid antagonist, 0.3 mg/kg),
naltrindole (NTD, -opioid antagonist, 5 mg/kg), and
naloxone (Nal, -opioid antagonist, 4, 8, 12 mg/kg) plus
EOE group were injected intraperitoneally. During the
second phase, there was a noteworthy variance in licking
time between the EOE group and the vehicle group. The
naloxone group (8, 12 mg/kg) and the naloxone plus EOE
group showed statistically significant differences compared
to the EOE treatment group in the second phase. Nal +
Mor: naloxone 4 mg/kg + morphine 4 mg/kg. Each
v
alue
represents the mean and standard error of mean. *P ＜
0.05, **P ＜ 0.001, compared to the EOE (45 mg/kg) onl
y
group. One-way analysis of variance followed by Tuckey’s
post hoc test were used to perform the statistical analyses.
There were 8 mice in all groups, except there were 7 in the
naloxone plus EOE group.
Fig. 2. The inhalation of essential oil of Eucalyptus (EOE)
reduced licking time in the second phase of the formalin
test. Licking time with the inhalation of control (distilled
water [DW]) or EOE mixed with the same volume of
dextrose water for 1, 2, 4, 8, and during groups before th
e
formalin test. Each value represents the mean and standard
error of mean. *P ＜ 0.05, **P ＜ 0.001, compared to the
control group. One-way analysis of variance followed by
Tu k e y ’s post hoc test was used. The number of animals in
the control group was 7 and in the during group was 8.
Licking time was reduced by inhalation of EOE in the first
phase; however, statistical significance was observed only
after 4 and 8 h of inhalation (P
＜
0.01). A significantly
shorter licking time began to appear within 1 h of in-
halation and was observed at 1, 2, 4, and 8 h compared
to the control group in the second phase (P
＜
0.01, 0.001,
0.001, and 0.001, respectively). These data suggest that
EOE exerted an antinociceptive effect on mice in the for-
malin test, especially when the results of injection and in-
halation of EOE were considered together.
2. Association between the antinociceptive effect of EOE
and opioid pathway on the formalin test
This experiment was designed to investigate the antino-
ciceptive/analgesic effects of EOE on the opioid pathway
(Fig. 3). EOE (45 mg/kg) and 5
’
-guanidinonaltrindole (0.3
mg/kg) or naltrindole (5 mg/kg)
—
which are
- and
-opioid
antagonists, respectively
—
were injected. Although there
was no statistically significant difference when compared
to that in the EOE only group, licking time was slightly in-
creased in the second phase, compared to that in the EOE
group. Naloxone decreased the licking time in the second
phase in a dose dependent manner (8 mg/kg, P
＜
0.001;
12 mg/kg, P
＜
0.001, compared to EOE only group). Also,
licking time significantly increased when naloxone and
morphine were simultaneously injected. The antinociceptive
effect of EOE acting through the
-opioid pathway in the
formalin test was demonstrated in this experiment.
3. Antinociceptive effect of EOE on acetic acid-induced
abdominal writhing test
Abdominal contraction is caused during the acetic acid-in-
duced abdominal writhing test, which is a model to test
Lee, et al / Analgesic effects of eucalyptus essential oil in mice 83
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Fig. 4. Effect of essential oil of Eucalyptus (EOE) on
writhing induced by acetic acid. Acetic acid-induced writhing
test was used to determine whether EOE has an effect on
visceral pain caused by an irritant injection. Control (0.9%
saline, i.p.), vehicle (almond oil, i.p.), essential oil (11.5,
22.5, and 45 mg/kg, i.p.), or indomethacin (10 mg/kg, i.p.)
were injected 30 min prior to administration of a 0.5% aceti
c
acid solution (10 ml/kg). Five minutes after acetic acid
injection, the total number of writhing movements were
recorded for 30 min. Writhing decreased from indo-
methacin and EOE (22.5, 45 mg/kg). Each value represents
the mean and standard error of mean. *P ＜ 0.05, **P ＜
0.001 compared to the vehicle group. #P ＜ 0.05 compared
to the control group. One-way analysis of variance followed
by Tuckey’s post hoc test was used. There were 10 mice
in the control group and 8 in the other groups. i.p.:
intraperitoneal.
Fig. 5. Essential oil of Eucalyptus (EOE) does not have
dose-dependent effects against thermal stimuli. To find out
whether EOE alters physiological pain associated with
thermal stimulus, the thermal plantar test was used.
A
pretreatment injection of EOE (45, 90, and 180 mg/kg), o
r
a vehicle were administered intraperitoneally. A focused
beam of heat was aimed at the plantar surface of the hind
paw in mice, 30 min after the pretreatment. Compared to
the vehicle group, no remarkable difference in mean
withdrawal latencies of the EOE groups (45, 90, and 180
mg/kg) was observed. Each value represents the mean and
standard error of mean. One-way analysis of variance was
used. There were 9 mice in each group.
visceral pain. Endogenous substances and other pain me-
diators (such as arachidonic acid via cyclooxygenase and
prostaglandin biosynthesis) cause such pain. This visceral
pain model varies from the formalin test, which is a model
of somatic pain. To study whether EOE has an anti-
nociceptive effect on visceral pain, this acetic acid-induced
abdominal writhing test was utilized (Fig. 4). EOE reduced
the number of writhing movements in a dose dependent
manner. The number of writhing movements were inhibited
by indomethacin and EOE (45 mg/kg) by 41.72% and
33.21%, respectively (indomethacin: 9.63, EOE 45 mg/kg:
12.1, vehicle: 29.0; P
＜
0.001, compared to the vehicle
group).
4. The effect of EOE in thermal plantar test
The thermal plantar test (Fig. 5) was used to investigate
the effect of EOE on nociceptive pain associated with ther-
mal stimulus. Compared to values of the vehicle group,
average withdrawal latencies of the EOE group presented
no significant differences (45, 90, and 180 mg/kg). These
data suggest that EOE administered at mild doses does not
affect physiological pain associated with thermal stimuli.
5. Change of latency in mice treated with EOE in rotarod
To examine the influence of EOE on motor coordination,
a rotarod test was conducted (Fig. 6). No significant differ-
ence was observed with a t-test (compared to the vehicle
group, P = 0.776), although the time EOE-treated mice
(45, 90, and 180 mg/kg) managed to remain on the rod
was slightly shorter than that of mice in the vehicle group.
Despite the high concentration of EOE used, motor coordi-
nation was not affected, and there was no significant dif-
ference between the two groups.
DISCUSSION
In Brazilian folk medicine, various Eucalyptus species from
84 Korean J Pain Vol. 32, No. 2, 2019
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Fig. 6. Essential oil of Eucalyptus (EOE) did not affect
motor coordination. A rotarod test was conducted to
investigate whether the EOE could influence motor coord-
ination. Vehicle (almond oil) or EOE (45, 90, or 180 mg/kg)
were used for pretreatment. No significant differences were
evident, although animals in the EOE group stayed on the
rod for a shorter period than animals of the vehicle group.
Each value represents the mean and standard error of
mean. One-way analysis of variance was used. There wer
e
10 mice in each group.
the Myrtaceae family are used to treat numerous medical
conditions. Traditionally, for example, hot water extracts
of the dried leaves of E. citriodora are used in cosmetics
and for culinary purposes. Moreover, the extracts are also
used to treat common cold, flu, and sinus congestion.
However, as various essential oils have different active
chemical components, they should be used appropriately
according to their physiological effects. More interestingly,
diverse analgesic effects have been shown among various
species of Eucalyptus. Therefore, individual assessments of
each species, assessing their analgesic effects, mechanism
of action, and side effects are necessary for proper aroma-
therapy. Therefore, the purpose of this study was to eluci-
date the analgesic effects, pain pathways, and motor co-
ordination effects of EOE on somatic, visceral, thermal,
and inflammatory nociception.
The formalin test was modified to evaluate inflam-
matory pain, nociceptive pain, and the central sensitization
effects of EOE. Analgesic effects on inflammatory pain
were observed through evaluation of the first and second
phases after intraperitoneal injection or inhalation of EOE
followed by hind-paw injection of 20
L of formalin (2%
v/v). An analgesic effect of EOE was observed for noci-
ceptive pain and for inflammatory pain. Moreover, com-
pared to the analgesic effect of morphine (licking times:
9.6 s), the analgesic effect of EOE was greater (licking
times: 4.4 and 4.1 s for 22.5 and 45 mg/kg, respectively).
Although there was no analgesic effect in the first phase
of the formalin test in conjunction with inhalation of EOE,
dose-dependent (1, 2, 4, and 8 h) analgesic effect was ob-
served during the second phase. In other words, a higher
analgesic effect was observed in the group that inhaled
EOE 2 h prior to the formalin test than the group that in-
haled EOE only during the formalin test. Therefore, the
longer the inhalation of EOE, the greater the analgesic
effect. In conclusion, although EOE does not have an an-
algesic effect on somatic pain, there was an analgesic ef-
fect and central analgesic effect on inflammatory pain.
In our study, we first performed an intraperitoneal in-
jection of EOE and then administered formalin 30 min lat-
er, referring to previous papers [8-11]. However, since EOE
is a mixture of various ingredients, it is unclear when the
maximum action will take place after intraperitoneal
administration. However, the main component of EOE is
1,8-cineole which is lipophilic and passes easily through
the cell membrane. Therefore, we think that the reason
for not showing the effect in the first phase of the formalin
test is that there may be no drug effect. Essential oils,
composed of many different molecules, enter the olfactory
bulb via the nose where they can exert effects on the ol-
factory cortex, which is connected to various regions of
the brain. Many studies on pain have shown that the limbic
system is connected to the amygdala and hippocampus
[11-13], which control emotion and memories.
After injecting EOE (11.5, 22.5, or 45 mg/kg each) and
indomethacin (10 mg) intraperitoneally, an anti-nociceptive
effect was observed during the writhing test. A statistically
significant analgesic effect at 45 mg/kg of EOE was ob-
served compared to that of the control group (12.1 vs. 20.3
writhing movements/30 min), and a similar effect was ob -
served for the indomethacin group (9.63 writhing move-
ments/30 min). Visceral pain involves different pain ex-
pression pathways from those of somatic pain. Sensory
innervation of the viscera not only includes bilateral spinal,
thoracic, and most abdominal organs, but also includes va-
gal afferents. Spinal afferents conduct noxious stimuli and
vagus afferents conduct chemonociception, autonomic, and
emotion responses [14,15]. Such stimulation to the viscera
is amplified and modulated by passing through voltage-
gated and ligand-gated ion channels of the spinal cord and
Lee, et al / Analgesic effects of eucalyptus essential oil in mice 85
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supraspinal area. Through these processes, the message
is diffused and poorly localized, and is specifically shown
as referred patterns. Moreover, in central levels, the an -
algesic mechanism is shown in relation to typical emotional
and autonomic responses [16]. Therefore, visceral pain is
a complex pathway in which the receptive ending, ion
channel, chemical substance, nerve transmission, and mod-
ulation are related. In this study, we can expect that, al-
though EOE did not have analgesic effects on somatic and
thermal nociception, the analgesic effect observed against
visceral pain may involve interactions in diverse parts the
body. Similarly, intraperitoneal injection (5 and 10 mg/kg)
of essential oils of C. citratus followed by the acetic
acid-induced writhing test showed inhibition (48 and 87%)
[17].
A plantar test was used to detect the thermal anti-no-
ciceptive effect of intraperitoneally injected EOE. Although
the concentrations of injected EOE (45, 90, and 180 mg/kg
each) were increased, there was no statistically significant
result, suggesting that EOE does not affect normal thermal
sensation.
A well-known analgesic effect of EOE is that it acts
as a potent inhibitor of the inflammatory response through
the arachidonic acid metabolic pathway by inhibiting the
production of leukotriene B2, prostaglandin E2, and other
arachidonic acid metabolites such as eucalyptol [4]. In this
experiment, 5
’
-guanidinonaltrindole (
-opioid antagonist,
0.3 mg/kg) and naltrindole (
-opioid antagonist, 5 mg/kg)
could not antagonize Eucalyptus, but naloxone (non-se-
lective opioid antagonist, 4, 8, 12 mg/kg) could. Naloxone
is a non-selective opioid antagonist and its binding affinity
is highest for the
-opioid receptor, then the
-opioid re-
ceptor, and lowest for the
-opioid receptor. In the present
study, the analgesic effects of Eucalyptus was not antago-
nized by the
-opioid antagonist and
-opioid antagonist.
However, the analgesic effects of Eucalyptus was antago-
nized by naloxone, a non-selective opioid antagonist. There-
fore, the
-opioid receptors are involved in the analgesic
effects of EOE.
However, naloxone failed to modify the response of
1,8-cineole (cineole), suggesting a non-participation of
-opioid receptors in the effects of cineole which is a prin-
cipal component of EOE [18]. Liapi et al. [18] used the
tail-flick and hot-plate tests, reflecting the spinal and su-
pra-spinal levels, respectively. We used the formalin test,
in which the response to formalin shows an early and a
late phase. The early phase seems to be caused predom-
inantly by C-fiber activation due to the peripheral stim -
ulus, while the late phase appears to be dependent on the
combination of an inflammatory reaction in the peripheral
tissue and functional changes in the dorsal horn of the
spinal cord. In the present study, naloxone could antago-
nize the analgesic effects of EOE in a dose dependent
manner. It is therefore likely that
-opioid receptors at the
periphery are involved in the effect of EOE, unlike the
findings of Liapi et al. [18]. Further studies are needed on
the deferent mechanism by which EOE acts in the central
and peripheral regions.
The rotarod test evaluates the effect of a drug on mo-
tor the coordination of animals and is helpful to determine
drug safety in humans. There was no statistically sig-
nificant difference in the results between the treatment
and control groups even when a high concentration of EOE
(180 mg/kg) was utilized. This shows the possibility of EOE
being developed as a new class of medicine, as it does not
affect motor coordination.
In conclusion, the present study elucidated the an-
ti-inflammatory effects of EOE. Furthermore, its inherent
medical specifications were reported in relation to species.
This study also demonstrated the analgesic effects of EOE
on nociception and inflammation, and that the anti-in-
flammatory effect of EOE likely affects the
-opioid path-
way. Moreover, EOE did not affect motor coordination.
Thus, the combined results suggest that EOE has the po-
tential to be developed into an analgesic agent to treat
various types of pain. Further studies are necessary to
improve our understanding of the pharmacology of EOE,
and to elucidate the mechanism of action of these effects
in other pain tests in addition to the formalin test using
EOE
’
s isolated active components and purified ingredients
for the treatment of pain.
ACKNOWLEDGEMENTS
This research was supported by the Bio & Medical Tech-
nology Development Program of the National Research
Foundtion (NRF) & funded by the Korean government (MSIP
& MOHW; No. 2016M3A9B6904244) and NRF (2015R1D1A1
A01061326).
86 Korean J Pain Vol. 32, No. 2, 2019
www.epain.org
CONFLICT OF INTEREST
No potential conflict of interest relevant to this article was
reported.
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