Pharmacology & Pharmacy, 2012, 3, 397-403
http://dx.doi.org/10.4236/pp.2012.34053 Published Online October 2012 (http://www.SciRP.org/journal/pp)
1
Local Peripheral Effects of β-Caryophyllene through CB2
Receptors in Neuropathic Pain in Mice
Hikari Kuwahata1,2, Soh Katsuyama2,3, Takaaki Komatsu2, Hitoshi Nakamura3,
Maria Tiziana Corasaniti4, Giacinto Bagetta5, Shinobu Sakurada6, Tsukasa Sakurada2*,
Kazuo Takahama1
1Department of Environmental and Molecular Health and Sciences, Graduated School of Pharmaceutical Sciences, Kumamoto Uni-
versity, Kumamoto, Japan; 2Department of Pharmacology, Daiichi College of Pharmaceutical Sciences, Fukuoka, Japan; 3Department
of Clinical Pharmaceutics, Tohoku Pharmaceutical University, Sendai, Japan; 4Department of Pharmacobiological Sciences, Univer-
sity of Magna Gracia of Catanzaro, Catanzaro, Italy; 5Department of Pharmacobiology, University Consortium for Adaptive Disor-
ders and Headach (UCADH), Section of Neuropharmacology of Normal and Pathological Neuronal Plasticity, University of Calabria,
Arcavacata di Rende, Italy; 6Department of Physiology and Anatomy, Tohoku Pharmaceutical University, Sendai, Japan.
Received July 13th, 2012; revised August 15th, 2012; accepted September 10th, 2012
ABSTRACT
β-Caryophyllene (BCP) is known as a common constitute of the essential oils of numerous food plants and primary
component in Cannabis. In this study, we investigated the effect of local intraplantar (i.pl.) injection of BCP on me-
chanical hypersensitivity induced by partial sciatic nerve ligation (PSNL) in mice. Relative to sham operation controls,
mice with the PSNL displayed a maximum level of hyperresponsiveness to von Frey metallic filament on post-operative
day 7. PSNL-induced allodynia was seen in the ipsilateral side of nerve ligation, but not in the contralateral side. The
i.pl. injection of BCP into the ipsilateral hindpaw to PSNL attenuated mechanical allodynia in a dose-dependent manner.
BCP injection into the contralateral hindpaw did not produce anti-allodynic effects, suggesting a local peripheral
anti-allodynic effect of BCP. Anti-allodynic effects induced by i.pl. injection of BCP were prevented by pretreatment
with the cannabinoid (CB)2 receptor antagonist AM630, but not by the CB1 receptor antagonist AM251. These data
suggest that i.pl. injection of BCP could produce anti-allodynia by activating peripheral CB2 receptors, but not CB1 re-
ceptors in a mouse model of neuropathic pain. Taken together, these results suggest that peripheral CB2 receptors may
contribute to the effectiveness of BCP in the treatment of neuropathic pain disorders.
Keywords: β-Caryophyllene (BCP); Neuropathic Pain; Partial Sciatic Nerve Ligation (PSNL); Peripheral Cannabinoid
(CB) Receptor
1. Introduction
Plants are used for various purposes including their cos-
metic, nutritive, and biomedical properties. Plant essen-
tial oils are typically composed of volatile aromatic ter-
penes and phenylpropanoids. The natural sesquiterpene
β-caryophyllene (BCP) is found in many essential oils of
different spice and food plants, such as clove, oregano,
thyme, black pepper and cinnamon [1,2], all of which
have been used as natural remedies and also as fra-
grances. This compound is also known to be anti-micro-
bial [3], anti-oxidant [3,4], and anti-carcinogenic [5] and
to possess skin penetration-enhancing properties [6].
Moreover, BCP is also a major component in the essen-
tial oil of Cannabis sativa L [7]. BCP showed marked
anti-inflammatory activity against carrageenan- and pro-
staglandin E1-induced edema in rats as well as anti-ar-
thritic activity [8-10]. Oral administration of BCP sig-
nificantly reduced the inflammation of colon [11], the
carrageenan-induced inflammatory response in wild-type
mice but not in mice lacking cannabinoid (CB)2 receptors
[12]. However, the antinociceptive efficacy of intraplan-
tar (i.pl.) BCP on partial sciatic nerve ligation (PSNL)-
induced mechanical allodynia in mice is unknown. Can-
nabinoid CB receptors, CB1 and CB2 receptors, at the
peripheral and central sites have been proposed to medi-
ate the CB-induced antinociceptive effects [13-17]. CB2
receptors are not found in the central nervous system
(CNS), but are predominantly expressed in immune cells,
their roles including the modulation of cytokine release
and immune cell migration [18,19]. CB2 receptor selec-
tive agonists produced peripheral antinociception [15,
20,21], but do not cause the effects of CNS [15,22], sug-
*Corresponding author.

Local Peripheral Effects of β-Caryophyllene through CB2 Receptors in Neuropathic Pain in Mice
398
gesting that selective activation of CB2 receptors may
achieve the goal of peripheral pain relief without CNS
effects. Therefore, in our study we used local injections
to the injured paw in order to exclude the role of central
effects, and validate the role of peripheral CB receptors
in neuropathic pain. Peripheral nerve damage can result
in long-lasting anomalous pain conditions referred to as
neuropathic pain. These abnormal pain states are often
manifested by an increased sensitivity to nociceptive
stimuli, termed hyperalgesia, as well as the perception of
typical innocuous stimuli being painful, a state referred
to as allodynia [23]. CB2 receptor selective agonists have
been also known to produce antinociception without
overt behavioral effects in neuropathic pain [20,22,24,
25]. The aims of this work was 1) to investigate whether
i.pl. injection of BCP would produce antinociception in
PSNL-induced mechanical allodynia model in mice; and
2) to determine a possible role of peripheral CB recap-
tors in BCP-induced anti-allodynic effects.
2. Materials and Methods
2.1. Animals and Neuropathic Surgery
Male mice of ddY strain weighing 22 - 24 g were pur-
chased from Kyudo Industries, Kumamoto, Japan. They
were housed in cages of 15 - 20 animals matched for
weight and placed in a colony room. Animals were al-
lowed free access to standard food (Clea Japan, Inc.,
Osaka, Japan) and tap water in an air-conditioned room
under a constant 12:12 h light/dark cycle (light on 08:00
h) at a room temperature of 22˚C - 24˚C and 50% - 60%
relative humidity. All experiments followed the Guide-
lines on Ethical Standards for Investigation of Experi-
mental Pain in Animals [26]. Additionally, the study was
approved by the Committee of Animal Experiments in
Daiichi College of Pharmaceutical Sciences.
Partial ligation of the sciatic nerve of mice was per-
formed under pentobarbital anesthesia (50 mg/kg, i.p.)
following the methods Malmberg and Basbaum [27].
Briefly, the common sciatic nerve of the right hand leg of
mice was exposed at high thigh level through a small
incision and dorsal 13 to 12 of the nerve thickness
was tightly ligated with a silk suture. The wound was
closed with a single muscle suture, and antibiotic powder
was dusted over the wound area following surgery. For
sham surgery, the sciatic nerve was exposed as described
above, but no contact was made with the nerve. To
minimize differences in technique, all operations were
done by the same person. Immediately following surgery,
the animals were kept in a soft bag cage with some food
inside so that they could feed themselves without having
difficulty in standing. The wound healed within 1 to 2
days, and the mice behaved normally. The behavior of
the mice was monitored carefully for any visual indica-
tion of motor disorders or change in weight. Testing
procedures were conducted on day 7 after PSNL, except
for the time-course experiment of PSNL-induced allody-
nia.
2.2. Mechanical Threshold
Behavioral testing was conducted from 10:00 to 16:00 in
a quiet room. Each animal received drugs only once and
was used in only one experiment. The mice were
weighed and placed individually in a Plexiglas chamber
(11.0 × 17.0 × 14.0 cm, wire mesh floor) and allowed to
acclimatize for at least 1 hour. The threshold for nocicep-
tive responsiveness to mechanical stimuli applied to the
hindpaw was assessed using an electronic version of the
von Frey test (dynamic plantar anesthesiometer, model
37400; Ugo Basile, Milan, Italy). The servo-controlled
mechanical stimulus (a pointed metallic filament) was
applied to the plantar surface, which exerted a progres-
sively increasing punctate pressure, reaching up to 3.0 g
within 5.0 s. The pressure evoking a clear voluntary hind-
paw withdrawal response (usually close to 3.0 g) was
recorded automatically and taken as the mechanical no-
ciceptive thereshold index.
2.3. Drugs
β-Caryophyllene(tra ns-(1R,9S)-8-Methylene-4,11,11-tri-
methylbicyclo [7.2.0] undec-4-ene; BCP) (Sigma, St.
Louis, MO, USA) diluted in jojoba wax (Simmondsia
chinensis) (KSA International, Co. Ltd., Kanagawa, Ja-
pan), was injected to the plantar surface of the hindpaw
in mice. N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlo-
rophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251)
and 6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-in-
dol-3-yl] (4-methoxyphenyl) methanone (AM630) (Toc-
ris Cookson, Bristol, UK), dissolved in physiological
saline was administered i.pl. or subcutaneous (s.c.) 30
min before BCP. I.pl. and s.c. injections were given in a
volume of 20 μL/site and 0.1 mL/10 g of body weight,
respectively.
2.4. Analyses of Data
All data are expressed as means ± SEM. Statistical dif-
ferences between groups were assessed with a two-way
ANOVA followed by Bonferroni’s test. The 5% (P <
0.05) level of statistical significance was set in all ex-
periments.
3. Results
3.1. The Effects of BCP on Mechanical Allodynia
The responsiveness to mechanical stimuli was deter-
mined on days 1 - 35 after PSNL. Compared with the
sham-operated mice, PSNL resulted in mechanical allo-

Local Peripheral Effects of β-Caryophyllene through CB2 Receptors in Neuropathic Pain in Mice
399
BCP, whereas AM251 (12.0 μg/paw) gave no effect
(Figure 4). AM251and AM630 used in this experiment
alone did not induce a significant effect as compared to
saline controls (data not shown).
dynia demonstrated by significantly lower threshold to
von Frey filaments from day 7 to day 28 post-PSNL
(Figure 1). The maximal decrease in withdrawal thresh-
old after PSNL was observed on day 7. On day 7 post-
PSNL, i.pl. injection of BCP (4.5, 9.0 and 18.0 μg/paw)
dose-dependently increased the mechanical response
threshold on the nerve injured side. The effect of BCP
reached a maximum at 5 min and lasted for 15 min on
nerve-ligated side. Jojoba wax control, injected into the
nerve-ligated side, did not affect the mechanical thresh-
olds. To ensure that the effects of i.pl. injections of BCP
were local and not due to systemic diffusion, BCP (18.0
μg/paw) was injected into the hindpaw contralateral.
Mechanical threshold following i.pl. injection of BCP in
the contralateral hindpaw was not statistically different
when compared with the jojoba wax control group in
PSNL mice (Figure 2).
4. Discussion
The present study demonstrates that i.pl. injection of
BCP reversed mechanical allodynia in a mouse PSNL
neuropathic pain model. The anti-allodynia induced by
BCP was inhibited by pretreatment with AM630, a selec-
tive CB2 receptor antagonist, while pretreatment with
AM251, a selective CB1 receptor antagonist, did not
change the effects of BCP. These results suggest that
BCP, injected into the plantar surface of the hindpaw,
3.2. Peripheral CB2 Receptor Mediates the
Effects of BCP
To determine if anti-allodynic effect of BCP was mediated
by peripheral CB systems, animals were pretreated sys-
temically with AM251 (3.0 mg/kg, s.c.), a selective CB1
receptor antagonist and AM630 (1.0 mg/kg, s.c.), a se-
lective CB2 receptor antagonist, 30 min before i.pl. injec-
tion of BCP (18.0 μg/paw). The selective CB2 receptor
antagonist, AM630 significantly reversed the inhibitory
effects of BCP on the PSNL-induced mechanical allo-
dynia, whereas the selective CB1 receptor antagonist,
AM251 gave no effect (Figure 3). In further experiments,
AM251 and AM630 were pretreated directly into the
same site on the hindpaw in prior to i.pl. injection of
BCP. I.pl. pretreatment with AM630 (4.0 μg/paw) could
also antagonize significantly anti-allodynic effects of
Figure 1. The time course of mechanical withdrawal thresh-
olds after partial sciatic nerve ligation in the von Frey test.
Mechanical withdrawal thresholds of the injured and sham-
operated right hindpaws are expressed as grams and data
represent the mean ± SEM of 10 mice in each group. Aster-
isks indicate significant decreases in thresholds compared to
the sham-operated mice. *P < 0.05, **P < 0.01.
Figure 2. Intraplantar injection of BCP reduced partial sciatic nerve ligation (PSNL)-induced mechanical allodynia in mice.
The withdrawal threshold was determined in PSNL and sham mice (hindpaw ipsilateral side and contralateral side) before
and 5, 10, 15, 30, 60 and 90 min after administration. Each point represents the mean ± SEM for groups of 10 mice. **P < 0.01,
*P < 0.05 when compared to jojoba wax-control.

Local Peripheral Effects of β-Caryophyllene through CB2 Receptors in Neuropathic Pain in Mice
400
Figure 3. Antagonism induced by s.c. injection of the selective CB2 receptor antagonist, AM630 and the selective CB1 receptor
antagonist, AM251 on the anti-allodynia produced by i.pl. injection of BCP in the PSNL model. Each point represents the
mean ± SEM for groups of 10 mice. **P < 0.01 when compared to saline (s.c.) + jojoba wax-control. ##P < 0.01 when compared
to s.c. saline plus BCP (18.0 μg/paw).
Figure 4. Antagonism induced by local injection of the selective CB2 receptor antagonist, AM630 and the selective CB1 recep-
tor antagonist, AM251 on the anti-allodynia produced by i.pl. injection of BCP in the PSNL model. Each point represents the
mean ± SEM for groups of 10 mice. **P < 0.01 when compared to saline (s.c.) jojoba wax-control. ##P < 0.01 when compared to
i.pl. saline + BCP (18.0 μg/paw).
produces its anti-allodynic effects by activating local pe-
ripheral CB2 receptors.
Injury to the sciatic nerve in mice as well as rats pro-
duces a prolonged mechanical allodynia and thermal hy-
peralgesia [27,28]. This experimental model has estab-
lished as a reliable and objective method to access neu-
ropathic pain. We confirmed that PSNL produced a rapid
onset and prolonged mechanical allodynia in mice. The
mechanical allodynia developed in 1 day and reached a
maximum in 7 days in mice with PSNL. PSNL-induced
mechanical allodynia was dose-dependently reversed by
local (plantar surface of the hindpaw) injection of BCP
on day 7 post-PSNL. This result suggests that i.pl. inject-
tion of BCP is an effective post-injury treatment in neu-
ropathic pain conditions.
In this study, injection of BCP into the hindpaw ipilat-
eral to the injured side could protect the mice from me-
chanical allodynia induced by PSNL. Local injection of
BCP into the contralateral hindpaw gave no effects on
mechanical allodynia, strongly supporting a local effect
of BCP on peripheral cutaneous nociceptors. It seems
that i.pl. treatment with BCP is beneficial as BCP is
anti-allodynic in neuropathic pain and have limited CNS
penetration to produce the unwanted CNS effects. It
should be noted that locally applied BCP failed to pro-
duce anti-allodynic effects in sham-operated mice at a
dose of 18.0 μg/paw. At this dose, BCP attenuated sig-
nificantly mechanical hypersensitivity in neuropathic
mice. The employment of CB receptor-selective antago-
nists allows for the involvement of CB receptor subtypes
in BCP-induced anti-allodynia. Locally applied BCP (9.0
and 18.0 μg/paw) significantly reduced the PSNL-in-

Local Peripheral Effects of β-Caryophyllene through CB2 Receptors in Neuropathic Pain in Mice 401
duced neuropathic pain in mice. The effects of BCP were
inhibited by pretreatment with the selective CB2 receptor
antagonist, AM630 (1.0 mg/kg, s.c. and 4.0 μg, i.pl.) but
not by pretreatment with the selective CB1 receptor an-
tagonist, AM251 (3.0 mg/kg, s.c. and 12.0 μg, i.pl.). The
doses AM630 (1 mg/kg) and AM251 (3 mg/kg) used in
the present experiment have been previously shown to
inhibit the effect of WIN 55,212-2, a CB receptor agonist
in vivo [18,29]. In binding assays, AM630 is CB2 selec-
tive with 165-fold lower Ki value in CB2 transfected
CHO cell membranes than in membranes of CB1 trans-
fected cells [30], and AM251 is a high degree selectivity
(306-fold) for CB1 receptors [31]. Therefore, the data
suggests that the anti-hyperalgesic effects of BCP in
neuropathic mice are mediated through activation of pe-
ripheral CB2 receptors. In line with our study, systemic
or local administration of CB2 receptor selective agonists
have been shown to produce antinociception without overt
behavioral effects in several pain models [15,20,32,33].
Besides peripherally mediated anti-allodynic effect,
peripheral administration of BCP or its containing essen-
tial oil also indicates anti-inflammatory and anti-arthritic
potential [8-11,34]. Moreover, oral administration of
BCP inhibits potent anti-inflammatory effects in wild-
type mice but not in mice lacking CB2 receptors [12].
This is consistent with our present study, suggesting the
involvement of specific CB receptor subtype, a CB2 re-
ceptor, in BCP-induced peripheral anti-allodynia. Inter-
estingly, the essential oil component (E)-BCP selectively
binds to the CB2 receptor, leading to the cellular activa-
tion as a CB2 agonist and anti-inflammatory effects [12].
Thus, the anti-allodynic effect of BCP points to the pe-
ripheral CB2 receptor as an interesting target in searching
for new peripherally active analgesics for chronic pain
therapy. The action site of BCP is of particular interest
since neuropathy observed in patients is often coupled
with not only neuropathic pain but also inflammatory
symptoms [35,36].
The peripheral mechanism by which BCP produces
anti-allodynic effect in the present findings are unclear.
However, there are various lines of evidence that selec-
tive activation of peripheral CB2 receptors is sufficient to
display antinociception in models of acute, inflammatory
and nerve injury-induced nociception [15,21,22,32,37-
39]. Indeed, CB2 receptors are expressed primarily on
mast cells and immune system such as B cells, T cells
and macrophages [19,40,41]. Activation of CB2 receptors
on mast or immune cells could inhibit the release of
molecules that sensitize the peripheral nociceptor (e.g.
histamine, serotonin, prostaglandins, interleukin-1β, tu-
mor necrosis factor-α, and nerve growth factor). A recent
study have demonstrated that the oral treatment of BCP
causes a significant reduction of prostaglandin E2 and
tumor necrosis factor-α generation in carrageenan injected
paw [34]. Therefore, it seems that activation of periph-
eral CB2 receptors might decrease the sensitivity of pri-
mary afferent neurons by inhibiting the release of sensi-
tizing substances from neighboring mast and immune
cells. Moreover, there is evidence for the presence of
CB2 receptors on peripheral nerve terminals [42,43]. In-
deed, Walczak et al. (2005) have shown that the saphe-
nous partial ligation-induced neuropathic pain model in-
creases the expression of CB2 receptors in the paw skin
[44]. Another action mechanism of BCP is that i.pl. BCP
may stimulate CB2 receptors and inhibit the responsive-
ness of primary afferent neurons by stimulating local
release of β-endorphin, an endogenous opioid peptide,
from keratinocytes, which are very abundant in skin and
have been reported to express CB2 receptors [35]. How-
ever, further studies are required to explain the specific
mechanisms underlying the observed BCP effect.
5. Conclusion
In conclusion, i.pl. injection of BCP reduced the me-
chanical allodynia by the PSNL model in mice. The anti-
allodynic effects of BCP was antagonized by s.c. and i.pl.
pretreatment with the selective CB2 receptor antagonist,
AM630, but not by the selective CB1 receptor antagonist,
AM251. The results suggest that a local effect of BCP on
cutaneous nociceptors is mediated through CB2 receptors.
BCP is predicted to be effective in treating allodynia
without the central side effects of cannabinoids-based
drugs retaining activity at the CB1 receptor.
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