Original Papers 211

Ocimum gratissimum Essential Oil and Its

Isolated Compounds (Eugenol and Myrcene)
Reduce Neuropathic Pain in Mice

Authors Lyvia Izaura Gomes Paula-Freire 1, Graziella Rigueira Molska 2, Monica Levy Andersen 1, Elisaldo Luiz de Araújo Carlini 2

1
Affiliations Departamento de Psicobiologia, Universidade Federal de São Paulo, São Paulo, Brazil
2
Departamento de Medicina Preventiva, Universidade Federal de São Paulo, São Paulo, Brazil

Key words Abstract kg for 14 days after surgery. Pregabalin (20 mg/

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" Ocimum gratissimum
l ! kg) was used as a standard in this study. The treat-
l
" Lamiaceae
Ocimum gratissimum is used in popular medicine ment with 20 and 40 mg/kg of O. gratissimum es-
l
" eugenol
to treat painful diseases. The antihypernocicep- sential oil and at doses of 5 and 10 mg/kg of the
l
" myrcene

tive properties of O. gratissimum essential oil and active components were able to promote antihy-
l
" neuropathic pain

l
" IL‑1beta two of its active components (eugenol and myr-
cene) were tested in a model of neuropathic pain
induced by a chronic constriction injury of the sci-
atic nerve. In tests to determine chronic antinoci-
ception, adult male C57BL/6 J mice were treated
orally with corn oil (control group), O. gratissi-
mum essential oil at doses of 10, 20, or 40 mg/kg
or eugenol or myrcene at doses of 1, 5, or 10 mg/
pernociception in both mechanical (von Frey)
and thermal (hot plate) tests. The treatment with
the essential oil of the plant or eugenol was effec-
tive in reducing the levels of interleukin-1β in the
sciatic nerve. Our findings demonstrate that O.
gratissimum essential oil and its isolated active
components possess antihypernociceptive activ-
ity in neuropathic pain models.

Introduction guese) [6]. In the Amazon region, sumo leaves

received May 20, 2015
revised Sept. 23, 2015
accepted Sept. 25, 2015
Bibliography
DOI http://dx.doi.org/
10.1055/s-0035-1558165
Published online November 19,
2015
Planta Med 2016; 82: 211–216
© Georg Thieme Verlag KG
Stuttgart · New York ·
ISSN 0032‑0943
Correspondence
Lyvia Izaura Gomes de Paula
Freire
Universidade Federal de São
Paulo
Departamento de Psicobiologia
Rua Napoleão de Barros, 925
Vila Clementino
São Paulo 04024–002, SP
Brazil
Phone: + 55 (11) 21 49 01 55
Fax: + 55 (11) 21 49 01 55
lyviabiologia@gmail.com
! are used for healing, while a bath prepared with
The pain process alerts the organism about noxi-
ous stimuli that can cause tissue damage, allow-
ing for escape and defense mechanisms to be
adopted [1]. However, neuropathic pain is defined
by persistence and is often associated with altera-
tions in the pathways that transmit pain, leading
to a chronic transmission of pain signals [2].
Studies have shown that the majority of patients
treated for this pathology usually do not respond
to common analgesics, with tricyclic antidepres-
sants and anticonvulsants as the main treatments
for this type of peripheral or central pain [3, 4].
With the lack of effective treatments, certain sub-
stances have emerged as analgesic prototypes, at-
tracting the interest of researchers and the phar-
maceutical industry. Medicinal plants and their
isolated active components are examples of this,
as these natural products have contributed
greatly to the development of new therapeutic
strategies through their secondary metabolites
[5].
Ocimum gratissimum L. (Lamiaceae) is a plant
found throughout Brazil that is widely used in
cooking and as a condiment, and is commonly
known as “manjericão” or “alfavaca” (in Portu-
its leaves is considered useful against headaches,
flu, and upper airway secretions [7].
Many studies have been conducted with plants of
this genus, with most yielding positive results in
showing its remarkable biological potential. Stud-
ies with the aqueous extract of O. gratissimum
have shown acute antinociceptive and anti-in-
flammatory activities in mice [8, 9]. This same re-
sult was observed when the treatment was per-
formed with the essential oil of the same plant
via an oral route of administration [10, 11].
The essential oil of O. gratissimum contains the
chemical constituents eugenol (usually a major
component) and myrcene [12], substances with
proven activity in the central nervous system
[13–15] and acute antinociception effects [11].
The present study was designed to investigate the
ameliorative role of O. gratissimum essential oil
(OgEO) in a chronic constriction injury of sciatic
nerve-induced neuropathy and to further explore
the contribution of eugenol- and myrcene-medi-
ated beneficial effects on neuropathic pain in rats.

Paula-Freire LIG et al. Ocimum gratissimum Essential … Planta Med 2016; 82: 211–216
212 Original Papers

Results
!
Pregabalin treatment (positive control) or 20 and 40 mg/kg OgEO
treatments significantly inhibited the mechanical hypernocicep-
tion induced by the chronic constriction injury, as evaluated by
the von Frey test, compared with the control group (p < 0.001)
on the 7th and 14th days post-surgery (l " Fig. 1 A). It is interesting

to note that mice treated with the doses of 20 and 40 mg/kg for
14 days showed greater resistance to paw pressure when com-
pared with animals treated with the same doses for seven days,
indicating that the longer the treatment time, the greater the ef-
fect.
l" Fig. 1 B shows that administration of all doses of eugenol (1, 5,

and 10 mg/kg, p.o) promoted mechanical antihypernociception

in mice subjected to chronic constriction injury (CCI) when com-
pared with the animals receiving vehicle (p < 0.05) on both the
7th and 14th days of the test. The 1 mg/kg eugenol dose also re-
sulted in differences from the sham and pregabalin groups. Ad-
ministration of myrcene (5 and 10 mg/kg, p. o.; l " Fig. 1 C) also

significantly attenuated hypernociception in response to von Frey

stimulation of the injured hind paw compared with control mice

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(p < 0.05) on the 7th and 14th days post-surgery.
l" Fig. 2 A shows that the OgEO at doses of 20 and 40 mg/kg statis-

tically increased the permanence of the animals on the heated

plate on the 7th and 14th days post-surgery when compared
with the control group (p < 0.05). The 10 mg/kg dose did not pro-
mote antihypernociception. Treatment with a dose of 40 mg/kg
was the most effective, as, in addition to the intergroup differ-
ences, the intragroup analysis showed a significant increase in
the permanence of mice on the hot plate compared with baseline
values and tests conducted on the 14th day post-surgery.
Regarding the active components (l " Fig. 2 B, eugenol; l " Fig. 2 C,

myrcene), doses of 5 and 10 mg/kg were effective in reducing

the pain caused by surgery and enhanced by the thermal stimu-
lus compared with the control group (p < 0.001) on the 7th and
14th days post-surgery. The 1 mg/kg dose of either drug did not
promote antihypernociception.
The IL-1β levels of the sham-operated animals were undetect-
able. Conversely, the positive control group showed increased
levels of this cytokine. The administration of OgEO at doses of 20
and 40 mg/kg (l " Fig. 3 A) and eugenol at doses of 1, 5, and 10 mg/

kg (l" Fig. 3 B) significantly reduced (p < 0.05) the levels of IL-1β in

mice subjected to surgery for chronic constriction of the sciatic

nerve. Myrcene was not effective in reducing the levels of IL-1β
in the sciatic nerve of the animals (l " Fig. 3 C, p > 0.05).
Discussion
!
Chronic pain generated after injury of the peripheral nervous
system is a serious condition and affects much of the worldʼs
population. However, classical analgesics, such as NSAID and
opioids, are only partially effective, exhibit significant side effects
(addiction, tolerance, gastrointestinal effects, and abuse), and are
ineffective in controlling neuropathic pain [16], making the treat-
ment of painful neuropathies more difficult. In tests conducted to
alleviate neuropathic pain induced by chronic constrictive sciatic
nerve injury, mice treated with OgEO and their isolated active
components (myrcene and eugenol) showed a marked decrease
in both thermal and mechanical hypernociception.
Studies have shown that the methanol extract of the leaves of
Ocimum sanctum L. (100 and 200 mg/kg, p. o.), a plant of the same
Fig. 1 Effects of varying doses of OgEO (A), eugenol (B), and myrcene (C)
on mechanical hypernociception in mice subjected to the von Frey test.
Data are expressed as the mean ± SEM. Statistical significance is indicated
by (*) from the control group at the respective times, (#) from sham-oper-
ated animals at the respective times, and (°) as the intragroup difference
between 7 and 14 days of treatment (repeated measures ANOVA followed
by the Newman-Keuls post hoc test, p < 0.05, n = 5/group).
genus as that used in this study, was able to relieve the thermal
and mechanical hypernociception and allodynia induced by sci-
atic nerve transection in rats [17]. Recent works have shown that
O. gratissimum and O. sanctum have similar constituents in their
chemical composition, with eugenol comprising the largest com-
ponent (over 50% of the total oil composition) in both species
[18–20]. In addition to eugenol, myrcene is also found in two
plants of this genus [21]. It is believed that this antinociceptive
activity is due to a high concentration of active ingredients with
analgesic potential, mainly eugenol (major chemotype studied)

Paula-Freire LIG et al. Ocimum gratissimum Essential … Planta Med 2016; 82: 211–216

Fig. 2 The influence of OgEO (A), eugenol (B), and myrcene (C) on ther-
mal nociception after CCI of the sciatic nerve in mice. Data are expressed as
the mean ± SEM. Statistical significance is indicated by (*) from the control
group at the corresponding times, (#) from the sham group at the corre-
sponding times, and (°) as the intragroup difference at basal evaluation
(repeated measures ANOVA followed by the Newman-Keuls post hoc test,
p < 0.05, n = 5/group).
and 1,8-cineole [22, 23], or even due to synergism between these
and other substances found in smaller quantities, such as myr-
cene and trans-caryophyllene. All of these substances have dis-
tinct mechanisms of action, but all with a similar importance for
Paula-Freire LIG et al. Ocimum gratissimum Essential …
Original Papers 213
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Fig. 3 IL-1β levels in the sciatic nerve after a chronic constriction injury in
the presence or absence of OgEO (A), eugenol (B), and myrcene (C). Data
are expressed as the mean ± SEM. Statistical significance is indicated by (*)
from the control group (ANOVA followed by Tukeyʼs post hoc test, p < 0.05,
n = 5/group).
generating antinociception. Our previous study showed that the
same essential oil used in this study was able to relieve the acute
pain of animals subjected to the hot plate and the formalin paw
tests, which confirms its antinociceptive effect [11].
The results demonstrated that mice treated with the doses of 20
and 40 mg/kg for 14 days showed greater resistance to paw pres-
sure when compared with animals treated with the same doses
for seven days, indicating that the longer the treatment time,
the greater the effect. A recent study showed that treatment with
another plant that belongs to the same genus (O. sanctum) also
demonstrated a time-dependent antihypernociceptive effect
[24]. Furthermore, it is known that the CCI model generates an
Planta Med 2016; 82: 211–216
214 Original Papers
inflammatory process, with increased levels of proinflammatory
substances. These substances, such as tumor necrosis factor-α
(TNF-α), prostaglandin E2 (PGE2) and the interleukins IL-1β and
IL-6 are able to promote neurochemical alterations, leading to
hypernociception over time [25, 26]. Studies have shown that
plants from this genus promote anti-inflammatory activity, pro-
ducing the reduction of these proinflammatory cytokines [27,
28]. This effect may be responsible for decreasing the sensitiza-
tion of the nociceptive pathway during treatment, leading to the
time-dependent effect.
Eugenol is similar in chemical structure to capsaicin, a vanilloid
compound of hot chili. Capsaicin elicits pain by opening transient
receptor potential vanilloid 1 (TRPV1) channels expressed in no-
ciceptive afferent neurons [29]. However, exposure to capsaicin
produces a long-lasting desensitization of the channels, which is
the basis for the capsaicin-induced analgesia. Likewise eugenol
exhibits both irritant and analgesic actions [30]. Eugenol also in-
hibits voltage-dependent Na+ channels and N-methyl-D-aspar-
tate (NMDA) receptors; additionally, it potentiates ionotropic γ-
aminobutyric acid (GABAA) receptors. These channels are in-
volved in pain sensitivity [31, 32]. Furthermore, eugenol inhibits
compounds that are involved in action potential generation in
both A and C fibers which may explain its analgesic effects [33].
Myrcene also promoted antihypernociception in injured animals.
However, there are still no reports in the literature about the pos-
sible mechanism of action attributed to the antihypernociceptive
effect of myrcene [34] reported that in tests of acute pain, the ad-
ministration (subcutaneous and intraperitoneal) of myrcene at
the dose of 10 mg/kg provided antinociception in mice. This ef-
fect was reversed by pretreatment with naloxone (opioid antag-
onist) and yohimbine (α2 adrenergic antagonist), suggesting the
involvement of the opioid and noradrenergic systems. The results
suggest that the antinociceptive effect of myrcene is mediated by
the release of endogenous opioid-induced activation of α2 adre-
nergic receptors.
The reversal of pain hypersensitivity may be associated with the
local or plasma reduction of inflammatory mediators [35, 36].
With respect to IL-1β, treatment with OgEO and eugenol were ef-
fective in reducing the levels of this cytokine in the injured sciatic
nerve of animals. IL-1β is a cytokine belonging to the family of
proteins called interleukins and is released under conditions as-
sociated with persistent pain including inflammatory pain and
neuropathic pain [37]. Studies have shown an inadequate prod-
uction of IL-1β in a range of pathologies, including painful neu-
ropathies such as peripheral nerve injuries [38, 39]. Other studies
suggest that spinal IL-1β may be produced by glial cells and it is
know that spinal glia contributes to the development and main-
tenance of central sensitization in neurophatic pain [40]. Because
the elevation of this cytokine is important for the generation and
maintenance of neuropathic and inflammatory pain, substances
capable of reversing this increase should be investigated more
thoroughly for the development of new drugs.
Several significant proinflammatory substances are associated
with neuropathic pain process. Besides IL-1β, the levels of PGE2
and TNF-α were verified on the lesioned tissue; nonetheless, the
PGE2 and TNF-α levels for all groups were lower than the mini-
mum levels that might be detected by the kit. According to the
guidelines provided by the ELISA kit (R&D System™), the levels
of these proinflammatory mediators are lower in the tissue when
compared to the plasma levels.
The present study demonstrated that OgEO and its active compo-
nents tested in this work, eugenol and myrcene, showed marked
Paula-Freire LIG et al. Ocimum gratissimum Essential … Planta Med 2016; 82: 211–216
oral antinociceptive properties in a CCI of the sciatic nerve model
in mice, making them possible substances with therapeutic po-
tential for chronic pain. The evidence for the biological activity
of the plant justifies its popular use, which in turn encourages
further studies, particularly ones regarding its chemical constitu-
ents and their mechanisms of action.
Materials and Methods
!
Animals
Male C57BL/6 J mice (3 months old and weighing 25–30 g) from
the Centro de Desenvolvimento de Modelos Experimentais para
Medicina e Biologia (CEDEME – UNIFESP) vivarium were used.
Animals were housed in rooms with a controlled temperature
(22 ± 1 °C) and a 12-h light/dark cycle. Water and food were avail-
able ad libitum. The C57BL/6 J strain was selected because of its
higher sensitivity to behavioral tests and lower inter-animal var-
iability compared with other inbred strains [41, 42]. All studies
were conducted ethically in accordance with the Research Ethical
Committee of UNIFESP (protocol #0675/09, June 19, 2010). The
Downloaded by: University of Pittsburgh. Copyrighted material.
number of animals (n = 5) and the intensity of painful stimuli
were as small as needed to consistently demonstrate the analge-
sic effect of the drugs.
Drugs
OgEO was acquired from Centro Pluridisciplinar de Pesquisas
Químicas, Biológicas e Agrícolas (CPQBA-UNICAMP) and was
supplied with a species identification report and phytochemistry
analysis that confirmed the presence of the active compounds
(eugenol, 67.17 %; myrcene; 0.24 %). The doses of OgEO were cho-
sen based on our previous study [11]. Eugenol and myrcene were
purchased from Sigma-Aldrich. Pregabalin was acquired from
Pfizer. The purity (%) of all chemicals was over 95 %. Drugs were
freshly diluted in corn oil on the day of each experiment and ad-
ministered orally (p. o.) in a volume of 0.1 mL/10 g body weight,
always at 10 : 00 a. m.
Chronic pain tests
Induction of neuropathic pain: CCI was performed according to
the method described by [43] and modified for mice [44]. Briefly,
mice were anesthetized (with a combination of xylazine and ke-
tamine, 10 mg/kg, i. p.), and the sciatic nerve was exposed at the
level of the mid-thigh just proximal to its trifurcation. Four liga-
tures were loosely tied around the nerve at 1 mm intervals using
8–0 silk thread ligatures (Ethicon™). Sham-operated animals
(with the sciatic nerve exposed but no ligature) were used as
controls.
Mechanical hypernociception (von Frey test): Withdrawal thresh-
olds for mechanical stimuli were assessed with a von Frey elec-
tronic device. For this test, groups of five mice were treated with
corn oil (control and sham-operated), pregabalin (20 mg/kg, pos-
itive control, p. o.), OgEO (10, 20, or 40 mg/kg, p. o.), or eugenol or
myrcene (1, 5, or 10 mg/kg, p. o.) for 14 days after surgery. After
1 h of treatment, the animals were evaluated for tactile stimula-
tion by a von Frey electronic device before surgery (baseline val-
ues) and on the 7th and 14th days post-surgery. The plantar sur-
face was touched perpendicularly with the von Frey device until
the animal exhibited a characteristic “flinch” withdrawal or lick-
ing response. The intensity of the hypernociceptive response was
quantified as the change in pressure applied by subtracting the
mean of the three values obtained before the surgery from the

mean of the three values observed on days 7 and 14 post-surgery
[45].
Thermal hypernociception (hot plate test): Groups of five mice re-
ceived corn oil (control and sham-operated), pregabalin (20 mg/
kg, positive control), OgEO (10, 20, or 40 mg/kg, p. o.), or eugenol
or myrcene (1, 5, or 10 mg/kg, p. o.) for 14 days after surgery.
Thermal hypernociception was evaluated using the hot plate test.
After 1 h of treatment, the animals were placed on the hot plate
(Ugo Basile™, Biological Research Apparatus Company) at 50 °C.
The reaction time was defined as the latency for the animal to lick
its paw(s) or jump from the plate, with a maximum exposure of
60 seconds. The test occurred before surgery (baseline values)
and on the 7th and 14th postoperative days [46].
Biochemical assay
Protein extraction and enzyme-linked immunosorbent assay (ELI-
SA): After the last behavioral tests evaluating neuropathic pain,
the mice were killed by decapitation and the sciatic nerves were
dissected. Tissue was immediately frozen in dry ice and stored at
− 80 °C until analysis.
Samples for IL-1β levels were homogenized in phosphate buf-
fered saline (PBS) containing 0.4 M NaCl, 0.05 % Tween 20, 0.5 %
bovine serum albumin (BSA), 0.1 mM phenylmethyl sulfonyl flu-
oride, 0.1 mM benzethonium chloride, 10 mM EDTA, and 0.001 %
aprotinin (37.6 mg per 100 mL of PBS with EDTA). The samples
were then centrifuged at 3000 rpm for 15 min at 4 °C. The IL-1β
levels were measured using an ELISA (enzyme-linked immuno-
sorbent assay) kit, following the manufacturerʼs recommenda-
tions (R&D Systems™). The total amount of protein collected
was measured by the bicinchoninic acid method using the Micro
BCA™ Protein kit (Thermo Fisher Scientific). The absorbance was
measured using a microplate reader at 470 and 570 nm, and the
results are expressed as pg/mg total protein.
Statistical analysis
The data obtained in the IL-1β measurements were analyzed by
one-way ANOVA, followed by Tukeyʼs post hoc test when needed.
The results of the behavior tests were evaluated by repeated
measures ANOVA, followed by the Newman-Keuls post hoc test.
Data are presented as the mean ± standard error of the mean
(SEM). P < 0.05 was considered significant.
Acknowledgements
!
The authors are grateful to Fundação de Amparo a Pesquisa do Es-
tado de São Paulo (FAPESP #09/51 881–1), Centro Brasileiro de
Informações sobre Drogas Psicotrópicas (CEBRID), and Associa-
ção Fundo de Incentivo à Pesquisa (AFIP) for financial support.
Conflict of Interest
! 23 Park SH, Sim YB, Lee JK, Kim SM, Kang YJ, Jung JS, Suh HW. The analgesic
The authors have declared that there is no conflict of interest.
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