Content uploaded by Jürg Gertsch
Author content
All content in this area was uploaded by Jürg Gertsch
Content may be subject to copyright.
The cannabinoid CB
2
receptor-selective
phytocannabinoid beta-caryophyllene exerts
analgesic effects in mouse models of
inflammatory and neuropathic pain
A.-L. Klauke
a,1
, I. Racz
a,
n
,1
, B. Pradier
a
, A. Markert
a
,
A.M. Zimmer
a
, J. Gertsch
b
, A. Zimmer
a
a
Institute of Molecular Psychiatry, University of Bonn, Sigmund-Freud-Straße 25, D-53127 Bonn, Germany
b
Institute of Biochemistry and Molecular Medicine, NCCR TransCure, University of Bern, CH-3012 Bern,
Switzerland
Received 23 April 2013; received in revised form 31 August 2013; accepted 12 October 2013
KEYWORDS
Inflammatory pain;
Neuropathic pain;
CB
2
;
Beta-caryophyllene;
Dietary cannabinoid
Abstract
The widespread plant volatile beta-caryophyllene (BCP) was recently identified as a natural
selective agonist of the peripherally expressed cannabinoid receptor 2 (CB
2
). It is found in
relatively high concentrations in many spices and food plants. A number of studies have shown
that CB
2
is critically involved in the modulation of inflammatory and neuropathic pain
responses. In this study, we have investigated the analgesic effects of BCP in animal models
of inflammatory and neuropathic pain. We demonstrate that orally administered BCP reduced
inflammatory (late phase) pain responses in the formalin test in a CB
2
receptor-dependent
manner, while it had no effect on acute (early phase) responses. In a neuropathic pain model
the chronic oral administration of BCP attenuated thermal hyperalgesia and mechanical
allodynia, and reduced spinal neuroinflammation. Importantly, we found no signs of tolerance
to the anti-hyperalgesic effects of BCP after prolonged treatment. Oral BCP was more effective
than the subcutaneously injected synthetic CB
2
agonist JWH-133. Thus, the natural plant
product BCP may be highly effective in the treatment of long lasting, debilitating pain states.
Our results have important implications for the role of dietary factors in the development and
modulation of chronic pain conditions.
&2013 Elsevier B.V. and ECNP. All rights reserved.
1. Introduction
Pain is an important sensory signal indicating the occurrence or
danger of tissue damage. The associated negative emotional
www.elsevier.com/locate/euroneuro
0924-977X/$ - see front matter &2013 Elsevier B.V. and ECNP. All rights reserved.
http://dx.doi.org/10.1016/j.euroneuro.2013.10.008
n
Corresponding author. Tel.: +49 228 6885 316;
fax: +49 228 6885 301.
E-mail address: iracz@uni-bonn.de (I. Racz).
1
These authors contributed equally to this work.
European Neuropsychopharmacology (]]]])],]]]–]]]
Please cite this article as: Klauke, A.-L., et al., The cannabinoid CB
2
receptor-selective phytocannabinoid beta-caryophyllene exerts
analgesic effects in mouse models of.... European Neuropsychopharmacology (2013), http://dx.doi.org/10.1016/j.euroneuro.2013.10.008
effect of pain stimulates avoidance behaviors and thus helps to
protect the organism from future dangerous situations
(Basbaum et al., 2009;Woolf and Ma, 2007). Tissue inflamma-
tion can enhance pain sensation through the sensitization of
nociceptors, peripheral neurons responding to painful stimuli,
and through a sensitization of spinal neurons leading to an
enhanced transmission of nociceptive signals. The resulting
allodynia and hyperalgesia of the inflamed tissue also contri-
butes to the recuperative process (Hauser et al., 2013). Pain
sensation typically reverts to normal levels as the inflammatory
response abates. Neuropathic pain conditions on the other hand
do not serve any useful purpose. They arise from lesion or injury
of peripheral nerves, sensory ganglia, spinal roots or central
structures, by trauma, vascular or metabolic disorders, viral
infection, neuroinflammation or autoimmune responses. These
lesions trigger inflammatory responses at the site of the nerve
injury and in the spinal projection area, characterized by an
activation of microglia and astrocytes, and an increased
production of pro-inflammatory cytokines. These responses lead
to long lasting molecular and cellular changes in the spinal cord
that account for many aspects of the chronic pain, as well as
hyperalgesia and allodynia reported by patients suffering from
neuropathic pain (Costigan et al., 2009). Neuropathic pain is
often persistent and difficult to treat with current medications.
Cannabis preparations, which have been used since thou-
sands of years for the treatment of pain (Zias et al., 1993)
have recently come again into the focus as potential
therapeutics for inflammatory and neuropathic pain condi-
tions. Currently, cannabis extracts and synthetic preparations
of the psychoactive cannabis compound Δ
9
-tetrahydrocanna-
binol (THC) have been approved in many countries for clinical
pain management at doses and formulations that show on
only minor central side effects (Ben Amar, 2006;Iskedjian
et al., 2007;Lynch and Campbell, 2011). Nevertheless,
psychoactive effects of THC-containing medications are an
important consideration for many clinicians and their
patients. These effects are caused by the activation of the
brain cannabinoid receptor 1 (CB
1
), which also mediates
many of the acute analgesic effects of THC (Hohmann and
Herkenham, 1999). Recent interest has therefore focused on
the cannabinoid receptor 2 (CB
2
), which is primarily
expressed in non-neuronal immune cells (Facci et al., 1995;
Maresz et al., 2005). CB
2
expression is induced in many
tissues and cells, including the CNS (Benito et al., 2007),
under inflammatory conditions and has therefore been
implicated as a potential therapeutic target for inflammatory
disorders including chronic pain. CB
2
-selective agonists
should have an excellent safety profile like THC, while
lacking its psychoactive effects. Indeed, CB
2
receptor ago-
nists showed good anti-nociceptive effects in several animal
models of pain (Guindon and Hohmann, 2008).
A natural selective agonist for CB
2
receptorsistheplant
volatile BCP, which represents a dietary phytocannabinoid
(Gertsch et al., 2008,2010). BCP is found in large amounts in
the essential oils of many common spices and food plants, such
as oregano (Origanum vulgare L.), cinnamon (Cinnamomum
spp.), clove (Syzygium aromaticum), rosemary (Rosmarinus
officinalis L.), thyme (Thymus serpyllum), black pepper (Piper
nigrum L.) (Bernardes et al., 2010;Hudaib et al., 2002;
Jayaprakasha et al., 2003;Mockuteetal.,2001;Orav et al.,
2004;Zheng et al., 1992). Several health effects have been
attributed to BCP or medicinal plants containing BCP (Russo,
2011), including anti-inflammatory (Gertsch et al., 2008), local
anesthetic (Ghelardini et al., 2001), anti-carcinogenic (Legault
and Pichette, 2007;Loizzo et al., 2008), anti-fibrotic (Calleja
et al., 2012)andanxiolytic-like(Galdino et al., 2012) activity.
Analgesic effects of BCP were also observed after local
intraplantar application (Katsuyama et al., 2012). There is even
some clinical evidence that aromatic essential oil massage
containing BCP has a beneficial effect on menstrual cramps
(Ou et al., 2012). We have previously shown that BCP exerts an
anti-inflammatory effect in the carrageenan-induced edema
test (Gertsch et al., 2008). More recently, it was shown that
BCP also inhibits inflammation and tissue damage in models of
colitis and nephrotoxicity in a CB
2
receptor-dependent manner
(Bento et al., 2011;Horváth et al., 2012).
In the present study, we investigated the analgesic
effects of BCP in formalin-induced inflammation model
and in a model of neuropathic pain, which involves the
partial ligation of the sciatic nerve. To assess the contribu-
tion of CB
2
receptors in the analgesic action of BCP we also
carried out experiments in mice lacking CB
2
receptors.
2. Experimental procedures
2.1. Animals
Mice with a genetic deletion of the CB
2
receptor (CB
2
/
) have been
described previously (Buckley et al., 2000). Mutant mice were
crossed for more than 10 generations to C57BL/6J animals and are
thus considered to be congenic for this background. Animals were
housed in groups of three to five mice per cage under controlled
illumination (light dark cycle 12:12 h) and environmental condi-
tions. All animals were bred in our animal facility, and had free
access to water and food. Male mice were used in this study in the
age of 3–4 months old. Experimental procedures complied with all
regulations for animal experimentation in Germany and were
approved by Landesamt für Natur, Umwelt und Verbraucherschutz
in Nordrhein-Westfalen, Germany (AZ: 9.93.210.35.07.310).
2.2. Drugs
Beta-caryophyllene (5:1 isomer mixture of (E)- and (Z)-BCP)) was
purified from a commercially available 70% pure preparation (TCI
America) by HPLC and analyzed in GC/MS. The 95% pure BCP
contained residual amounts of alpha-humulene and traces of the
BCP oxidation product BCP oxide. BCP was dissolved in olive oil
(Fluka) at a concentration of 0.02, 0.2, 1 and 2 mg ml
1
and frozen in
aliquots at 20 1C. On every treatment day aliquots were defrosted
and gavage-fed at a volume of 5 ml kg
1
with the help of a feeding‐
needle, thus resulting in doses of 0.1, 1, 5 or 10 mg kg
1
respectively.
The synthetic CB
2
agonist JWH-133 was purchased in a solution,
dissolved in TocrisolveTM 100, (Tocris Bioscience) and was further
diluted to a concentration of 0.1, 0.5, and 1 mg ml
1
,andinjected
subcutaneously in a volume of 10 ml kg
1
.TheCB
2
antagonist SR
144528 (kindly provided by NIH) was dissolved in ethanol: chremo-
phor: water (1:1:18) solution at a concentration of 0.1 mg ml
1
and
was injected in a volume of 10 ml kg
1
intraperitoneally (i.p.).
Morphine (Merck KGaA, Darmstadt, Germany) was dissolved in saline
and was injected i.p. in a doses of 5 mg kg
1
.
2.3. Inflammatory pain model: formalin test
Male wild type (CB
2
+/+
) and CB
2
/
mice were injected with 20 mlof
5% formalin into the plantar surface of the right hindpaw. The left
paw served as control. Formalin injection produces a biphasic pain
A.-L. Klauke et al.2
Please cite this article as: Klauke, A.-L., et al., The cannabinoid CB
2
receptor-selective phytocannabinoid beta-caryophyllene exerts
analgesic effects in mouse models of.... European Neuropsychopharmacology (2013), http://dx.doi.org/10.1016/j.euroneuro.2013.10.008
response. The early phase (1–5 min) is an immediate pain response
caused by the formalin, while inflammatory processes and central
sensitization contribute to late phase (15–30 min) pain. The number
of pain responses such as paw licking, shaking and lifting were
evaluated every 10 s on the ipsilateral and on contralateral sides by
an observer who was blind to experimental groups. Thirty minutes
before the formalin injection, the mice were gavage-fed with BCP,
or vehicle. Other groups of mice were injected subcutaneously with
a reference compound, JWH-133 or vehicle 30 min before the
formalin treatment. As a CB
2
antagonist SR 144528 was used and
administered i.p. 15 min before the agonists. As positive control for
analgesia, we treated CB
2
/
animals with 5 mg kg
1
morphine i.p.
2.4. Neuropathic pain model: partial sciatic nerve
ligation (PNL)
To induce neuropathic pain, a partial ligation of the right sciatic
nerve was performed as described (Racz et al., 2008). Male wild
type CB
2
+/+
and CB
2
/
mice were operated under isoflurane (2–3%
in 95% O
2
for induction and 1.5–2% in 95% O
2
for maintenance)
anesthesia. The duration of surgery was no longer than 10–15 min.
One half to one-third of the sciatic nerve at midthigh level was
tightly ligated with a polypropylene thread (9–0). In sham operated
mice the nerve was exposed without ligation.
Mechanical allodynia and thermal hyperalgesia were tested as an
indicator for the development of neuropathic pain by an observer
who was blind to the treatment strategy. Mice were habituated to
the experimental setup for 1 h on three consecutive days before the
behavioral testing started. First, baseline nociceptive responses
were determined. The partial nerve ligation was performed 1 day
later. To assess the neuropathic pain outcome the animals were
then tested on days 3, 6, 8, 10 and 14 after the partial nerve
ligation.
Mechanical allodynia was assessed with a dynamic aesthesi-
ometer (Ugo Basile Biological Research Apparatus), which consists
of an electronically controlled mobile pressure-actuator that is able
to exert a continuously increasing force. Mice were placed in a
transparent Plexiglas chamber with a metal grid floor. The tip of the
actuator (diameter: 0.5 mm) was applied to the middle of the
plantar surface of the hind paw. The forces necessary to trigger
withdrawal responses of the ipsilateral or contralateral hind paws
were recorded. Means were determined by averaging three to five
trials per paw.
Thermal hyperalgesia was evaluated using a Hargreaves appara-
tus (Ugo Basile Biological Research Apparatus, I.R. intensity: 40).
The mean withdrawal latencies were determined by averaging
three to five separate trials. Both mechanical allodynia and thermal
hyperalgesia were evaluated in the same group of animals: the tests
were separated at least by 1 h.
Mice received daily either BCP or vehicle by gavage, or the
reference compound JWH-133 or vehicle by subcutaneous injections
starting 1 day after the partial nerve ligation. On the test days,
drugs were administered directly after the experiments.
2.5. Immunohistochemistry
At the end of the behavioral experiments, 4–5 mice per group were
anesthetized using a Ketavet/Rompun (10 mg kg
1
/0.1 mg kg
1
)
mixture and intracardially perfused with ice cold phosphate buffer
(PBS) for 5 min followed by 4% PFA (paraformaldehyde) solution for
10 min. The tissue samples was quickly frozen in isopentane on dry
ice and stored at 80 1C.
The section from L4–L6 of the spinal cord was dissected,
embedded in O.C.T. compound (Tissue Tek
s
, Sakura), and sliced
in 16 μm sections on a cryostat (Leica CM 3050; Leica Microsystems).
Six sections per mice from all treatment groups were mounted on
Star frost-coated slides. The slides were permeabilized for 1 h in
0.5% Triton X-100 (Sigma). After blocking in PBS containing 3%
bovine serum albumine and 10% donkey serum, the slides were
incubated for 42 h at 4 1C with primary antibody against glial
fibrillary acidic protein (GFAP, goat polyclonal, 1:500, Santa Cruz
Biotechnology) or ionized calcium-binding adapter molecule 1
(Iba1, rabbit polyclonal, 1:1000, Wako). A red fluorescent Cy3
anti-goat secondary antibody (1:1000, Jackson ImmunoReasearch)
or green fluorescent Alexa Fluor 488 anti-rabbit secondary antibody
(1:1000, Life Technologies) were used to visualize the signals. The
slides were mounted with Mowiol 4–88 (Roth).
Pictures were acquired using a Zeiss Imager M2 fluorescent
microscope (Carl Zeiss Microimaging). To evaluate microgliosis and
astrogliosis after peripheral nerve injury, pictures of the Iba1 and
GFAP double stained sections were taken with the multichannel
mosaic function of the AxioVision software (Carl Zeiss Microimaging)
in the 20 magnification. The mosaics were converted to one
picture before quantitative analysis. To improve the overlay of the
mosaic pictures the stitching function of the software was used for
representative pictures. An observer, who was blind to experimen-
tal groups, analyzed four to five animals per treatment group using
a macro routine in the Image J 1.42q software. The percentage of
stained area in the dorsal horn compared to whole area was
analyzed. Data were expressed as the difference of the ipsilateral
staining compared to the contralateral staining (% stained area
ipsilateral side% stained area contralateral side).
2.6. Quantitative real time-PCR
To analyze the expression level of CB
2
receptor gene after PNL,
TaqMan
s
gene expression assays were used. Total RNA was isolated
according to manufacturer´s instruction and RNA concentrations
were measured using a spectrophotometer (NanoDrop). mRNA was
transcribed into double-stranded cDNA using Reverse Transcriptase
II and Oligo(dt)12-18 primer (Life Technologies). For each sample
50 ng of cDNA were mixed with gene expression master mix
(containing AmpliTaq Gold
s
DNA Polymerase, Life Technologies)
and with the CB
2
gene specific assay. Expression data were then
normalized to a housekeeping gene (β-actin) and analyzed using the
2-ΔΔCt method as described previously (Livak and Schmittgen,
2001).
2.7. Tetrad test
To exclude that BCP produces the psychoactive side effects known
from CB
1
receptor agonists, the classical tetrad test was performed
(Martin et al., 1991).
Mice were tested 45 min after an acute oral administration of
BCP (1 mg kg
1
and 10 mg kg
1
) for motor activity in the open-
field, immobility (catalepsy) on a ring, anti-nociception in the tail
flick test and the rectal temperature was measured. The control
group of mice was injected intraperitoneally (i.p.) with a dose of
10 mg kg
1
THC (1:1:18 ethanol, cremophor, saline) and tested
20 min after the injection. An observer, unaware of the treatments,
recorded the values.
2.7.1.Open-field test
To assess motor activity mice were placed in the center of an open-
field apparatus (44 40 30 cm
3
) in a dimly illuminated (20 lux at
the ground) room and their activity was tracked by an automatic
monitoring system during 10 min (TSE Systems). Horizontal motor
activity was evaluated by calculating the total distance (m) traveled
by the animals.
2.7.2.Catalepsy
Mice were placed on a vertical tube (diameter: 5.5 cm, heights: 16 cm).
The immobility time was recorded for 5 min. Animals that fell down or
3Analgesic effect of beta-caryophyllene
Please cite this article as: Klauke, A.-L., et al., The cannabinoid CB
2
receptor-selective phytocannabinoid beta-caryophyllene exerts
analgesic effects in mouse models of.... European Neuropsychopharmacology (2013), http://dx.doi.org/10.1016/j.euroneuro.2013.10.008
jumped off the ring were allowed five more trials. One animal was
excluded from the experiments after five jumps off the ring.
2.7.3.Tail flick
Tail flick latencies were determined using a tail flick apparatus
(Columbus Instruments, OH, USA). Mice were manually restraint and
the tail was placed over a radiant heat source. We measured the
tail withdrawal latency setting the cut-off time at 12 s.
2.7.4.Rectal body temperature
We assessed the body temperature using a rectal thermometer
(BAT-12, Harvard Instruments, Millis, Massachusetts). Temperatures
were recorded before and 1 h after the drug administration. In case
of THC treatment the second measurement was performed 40 min
after the injection. Data are expressed as the difference between
the two body temperatures (ΔT=Tafter treatmentTbefore
treatment).
The results of the cannabinoid tetrad were analyzed by one-way
ANOVA or repeated measurement ANOVA (body temperature)
2.8. Statistical analysis
For all experimental results mean values and the standard error of
the means (SEM) were calculated. The statistical analysis was
carried out using Statistika 7.1 (StatSoft Inc.). In case of the
neuropathic pain experiments repeated measurements ANOVA was
used to analyze the treatment effects (categorical factors: nerve
injury and treatment. In the formalin test, repeated measurements
ANOVA was used to analyze the time course of pain reactions. For
the analysis of the BCP effect and the JWH-133 treatment, an area
under curve (AUC) was calculated and compared using factorial
ANOVA. The results of the cannabinoid tetrad were analyzed by
one-way ANOVA or repeated measurement ANOVA (body tempera-
ture). In the histological analysis of the astrocyte density the Mann-
Whitney U test was performed. In all tests the level of significance
was set at pr0.05.
3. Results
3.1. BCP reduced inflammatory pain responses in
the late phase of the formalin test
To assess the effect of oral BCP on formalin-induced pain
responses, we administered BCP by gavage 30 min before
the formalin injection. In wild type mice, BCP significantly
altered formalin-induced pain reactions when administered
at a dose of 5 mg kg
1
(Figure 1A, F
1,34
=7.06, p=0.012). By
Figure 1 BCP significantly decreased the pain responses in the late phase of the formalin test in CB
2
+/+
, but not CB
2
/
mice.
A Pain responses of CB
2
+/+
mice after 1 and 5 mg kg
1
BCP treatment compared to vehicle treated mice in the formalin test (vehicle
group: n=12; BCP groups: n=12). Analysis of the area under the curve (AUC) showed that late phase responses (15–30 min after
formalin injection) were significantly reduced after oral administration of 5 mg kg
1
BCP (po0.00001). (B) CB
2
/
mice did not show
any significant changes in pain responses after 5 mg kg
1
BCP treatment compared to the vehicle group (vehicle group: n=10;
5mgkg
1
BCP group: n=10). ***, treatment effect po0.0001. Means7SEM are indicated.
A.-L. Klauke et al.4
Please cite this article as: Klauke, A.-L., et al., The cannabinoid CB
2
receptor-selective phytocannabinoid beta-caryophyllene exerts
analgesic effects in mouse models of.... European Neuropsychopharmacology (2013), http://dx.doi.org/10.1016/j.euroneuro.2013.10.008
analyzing the area under the curve (AUC), we found no
significant treatment effect for the early phase (0–5 min), but
asignificant attenuation of pain responses during the late
phase (15–30 min; F
1,34
=24.41, po0.0001). BCP at a dose of
1mgkg
1
was ineffective (Figure 1 A). The synthetic CB
2
agonist JWH-133 was also effective (F
1,34
=6.66, p=0.014) at
a relatively high dose of 10 mg kg
1
(Figure 2A). After
calculating the early and late phase AUC, JWH-133 similarly
showed a significant analgesic effect only in the late phase
(F
1,35
=4.74, p=0.037). Both substances were completely
ineffective in CB
2
/
animals (BCP treatment: F
1,23
=0.87
p=n.s. JWH-133 treatment: F
1,14
=0.109 p=n.s.), thus indi-
cating that the antinociceptive effects are indeed mediated
by CB
2
.
To further substantiate the involvement of CB
2
receptors,
we also blocked CB
2
receptor signaling with the selective
antagonist SR 144528. The injection of SR 144528 15 min
before JWH-133 at a dose of 1 mg kg
1
(Figure 3A) blocked
JWH-133 analgesia during the late phase (F
1,28
=2.67 p=n.
s.). Interestingly, a dose of 3 mg kg
1
SR 144528 was
necessary to antagonize the analgesic effect of BCP
(Figure 3B, F
1
,
28
=3.53, p=n.s.). A dose of 1 mg kg
1
was
not effective (data not shown). The late phase AUC also did
not differ significantly in the BCP-SR 144528 treated group
compared to the vehicle treated mice (F
1,28
=2.67, p=n.s.).
Additionally, we treated CB
2
/
mice with morphine (5 mg
kg
1
) as a reference compound, which exerts its effect
through the non-cannabinoid mopioid receptors. Morphine
significantly reduced the animal’s nociceptive response
(Figure 4,F
1,21
=4.96, p=0.037). AUC analysis showed that
morphine exerted a highly significant analgesic effect in the
early phase (F
1,21
=17.67, po0.001), but not in the late
phase (F
1,21
=3.39, p=n.s.).
3.2. Neuropathic pain symptoms are diminished
in BCP treated mice
We next measured mechanical allodynia (von Frey test) and
thermal hyperalgesia (Hargreaves test) in BCP treated
versus vehicle treated wild type mice after partial sciatic
nerve ligation, an animal model of neuropathic pain. Base-
line responses were similar in all groups for both tests.
Figure 2 JWH-133 significantly decreased the pain responses in the formalin test in CB
2
+/+
but not CB
2
/
mice. A JWH-133
treatment (10 mg kg
1
) slightly but not significantly decreased the pain responses in CB
2
+/+
mice compared to vehicle treated mice
in the formalin test (vehicle group: n=12; JWH-133 group: n=10). Analysis of the area under the curve (AUC) showed that late phase
responses (15–30 min after formalin injection) differed significantly between the JWH-133 and vehicle treated groups (p=0.037).
(B) CB
2
/
mice did not show any significant changes in pain responses after 10 mg kg
1
JWH-133 treatment (vehicle group: n=10;
JWH-133 treated group: n=10). Calculation of area under the curve revealed the same results. Means7SEM are indicated.
5Analgesic effect of beta-caryophyllene
Please cite this article as: Klauke, A.-L., et al., The cannabinoid CB
2
receptor-selective phytocannabinoid beta-caryophyllene exerts
analgesic effects in mouse models of.... European Neuropsychopharmacology (2013), http://dx.doi.org/10.1016/j.euroneuro.2013.10.008
The partial ligation of the right sciatic nerve induced mechan-
ical allodynia (Figure 5 A, F
1,18
=387.34, po0.001) and
thermal hyperalgesia (Figure 5B, F
1,18
=20.40, po0.001) on
the ipsilateral, but not the contralateral hind paw. Sham
operated animals showed no alterations in thermal or mechan-
ical pain responses (Supplementary Figure 1).
Figure 3 Pretreatment with a selective CB
2
receptor antagonist, SR 144528 blocked the analgesic effects of both JWH-133 and BCP
in the formalin test. (A) After 1 mg kg
1
SR 144528 treatment the synthetic CB
2
receptor agonist, JWH-133 did not show any
analgesic effect in the late phase of formalin test. (B) Interestingly, the analgesic effect of BCP could be antagonized only with a
higher dose, 3 mg kg
1
of SR 144528. (JWH 133+1mgkg
1
SR 144528 treated group n=10, BCP+3mgkg
1
SR 144528 treated group
n=10). Means7SEM are indicated.
Figure 4 Morphine significantly decreased the pain responses in the formalin test in CB
2
/
mice. 5 mg kg
1
morphine significantly
reduced the pain reaction in the formalin test (p=0.037). AUC analysis showed that early phase responses were significantly reduced
after morphine treatment (po0.001). (vehicle group: n=10; morphine group: n=10) ***, treatment effect po0.0001. Means7SEM
are indicated.
A.-L. Klauke et al.6
Please cite this article as: Klauke, A.-L., et al., The cannabinoid CB
2
receptor-selective phytocannabinoid beta-caryophyllene exerts
analgesic effects in mouse models of.... European Neuropsychopharmacology (2013), http://dx.doi.org/10.1016/j.euroneuro.2013.10.008
The animals received daily doses of BCP (0.1, 1, 5 or
10 mg kg
1
) by gavage, starting 1 day after the surgery. The
doses of 1 mg kg
1
(F
1,18
=20.66, po0.001) and 5 mg kg
1
(F
1,19
=20.47, po0.0001) BCP increased mechanical with-
drawal thresholds of ipsilateral hind paws (Figure 5A).
Treatment with 10 mg kg
1
BCP directly exhibited a robust
effect on day 3, which then persisted over the testing
period (F
1,18
=33, po0.001). In the Hargreaves test only
1mgkg
1
BCP, but not other doses, reduced thermal
hyperalgesia gradually over the 2-week testing period even
up to the basal level (F
1,18
=9.12, p=0.007; time treat-
ment: F
4,72
=2.93, p=0.026). Administration of the syn-
thetic CB
2
agonist, JWH-133 at the doses of 1 and 5 mg kg
1
significantly increased mechanical withdrawal thresholds
(Figure 5A; 1 mg kg
1
:F
1,16
=9.59, p=0.0069; 5 mg kg
1
:
F
1,18
=30,71, po0.001), but failed to diminish thermal
hyperalgesia in the Hargreaves test (Figure 5B). These
results show that CB
2
agonists are effective in reducing
mechanical allodynia, but not thermal hyperalgesia, after
sciatic nerve injury.
3.3. BCP inhibits neuropathic pain through CB
2
receptor activation
To determine if BCP exerts its effects on the development of
neuropathic pain via CB
2
receptor activation, the experi-
ments were repeated in CB
2
/
mice (Figure 6). We have
previously shown that baseline responses of CB
2
/
mice
were similar to CB
2
+/+
animals (Racz et al., 2008). Further-
more, the sham surgery did not influence the pain responses
to mechanical and thermal stimuli (Figure 6C and D). After
the partial nerve ligation vehicle treated CB
2
/
mice
developed mechanical allodynia (F
1,14
=25.97, po0.001)
and thermal hyperalgesia in the ipsilateral side (F
1,19
=
4.97, p=0.038) and a mirror image of mechanical hyper-
algesia in the contralateral side (F
1,14
=8.39, p=0.012) as
reported previously (Racz et al., 2008). In these mice
1mgkg
1
BCP failed to show any effects on mechanical
allodynia or thermal hyperalgesia (Figure 6A and B). Also,
BCP treatment did not influence the responses of sham-
operated CB
2
/
mice (Figure 6C and D).
Figure 5 Effect of different doses (0.1, 1, 5 and 10 mg kg
1
) of BCP and JWH-133 (1 and 5 mg kg
1
) treatment on mechanical
allodynia and thermal withdrawal latencies in CB
2
+/+
mice. A Chronic treatment with 1, 5 and 10 mg kg
1
BCP and both doses of
JWH-133 significantly rescued PNL induced mechanical allodynia in the ipsilateral side. (B) Chronic treatment with 1 mg kg
1
BCP
significantly rescued PNL induced thermal hyperalgesia in the ipsilateral side, while 1 and 5 mg kg
1
JWH-133 treatment showed no
effect. The mice were tested at day 3, 6, 8, 10 and 14 after surgery. Vehicle group: n=10; 0.1 mg kg
1
BCP group: n=8; 1 mg kg
1
BCP group: n=10; 5 mg kg
1
BCP group: n=11; 10 mg kg
1
BCP group: n=10; 1 mg kg
1
JWH-133 group: n=8; 5 mg kg
1
JWH-133
group: n=10. **, treatment effect po0.01; ***, treatment effect po0.001. Means7SEM are indicated.
7Analgesic effect of beta-caryophyllene
Please cite this article as: Klauke, A.-L., et al., The cannabinoid CB
2
receptor-selective phytocannabinoid beta-caryophyllene exerts
analgesic effects in mouse models of.... European Neuropsychopharmacology (2013), http://dx.doi.org/10.1016/j.euroneuro.2013.10.008
3.4. BCP treatment reduces the density of spinal
cord glia cell-markers after peripheral nerve injury
Development of neuropathic pain is accompanied by the
activation and proliferation of glia cells in the spinal cord
(Scholz and Woolf, 2007). Therefore, we performed histo-
logical stainings with the microglia marker ionized calcium-
binding adapter molecule 1 (Iba1) and astrocyte marker
glial fibrillary acidic protein (GFAP).
The partial nerve ligation strongly increased microglia
density in the ipsilateral dorsal horns of lumbar spinal cord
sections (Figure 7A and B). Thus, the stained area in
vehicle-treated mice was significantly larger compared to
sham operated animals (surgery effect: U=25, po0.001).
We also observed a microgliosis after BCP treatment (sur-
gery effect: U=23, po0.001), but at a significantly lower
level when compared to vehicle controls (treatment effect:
U=164, p=0.006). The partial nerve ligation induced a
significant increase in density of astrocytes in the ipsilateral
dorsal horn of the lumbar spinal sections compared to sham
operation (Figure 7C and D; surgery effect: U=164; p=0.003)
in vehicle controls, but not in BCP-treated animals (U=188,
p=n.s). Together, these results show that BCP treatment
prevented astrocytosis and reduced microgliosis.
3.5. PNL induced the CB
2
gene expression in the
spinal cord
To analyze the expression pattern of CB
2
receptors after PNL
and BCP treatment, we performed quantitative real-time
PCR. The partial ligation of the sciatic nerve induced an
increase in CB
2
receptor mRNA in the ipsilateral side, but
not in the contralateral side, as revealed by a significant
interaction of surgery and side in the factorial ANOVA
(F
1,29
=4.63, p=0.03). The following Fisher-LSD post-hoc
test revealed that the expression of CB
2
in the ipsilateral
Figure 6 Chronic BCP treatment did not attenuate PNL induced mechanical allodynia (A) and thermal hyperalgesia (B) in CB
2
/
mice. Additionally to the ipsilateral side, CB
2
/
mice developed a mirror image of pain at the contralateral side after surgery (Racz
et al., 2008). Ipsilateral and contralateral hindpaws of mice were tested in the von Frey test and Hargreaves test to evaluate
mechanical allodynia (vehicle group: n=8; 1 mg kg
1
BCP group: n=9) and thermal hyperalgesia (vehicle group: n=10; 1 mg kg
1
BCP group: n=12). (C) Mechanical withdrawal thresholds of sham operated animals (vehicle group: n=8; 1 mg kg
1
BCP group:
n=11). (D) Thermal withdrawal latencies of sham operated animals (vehicle group: n=11; 1 mg kg
1
BCP group: n=14). CB
2
/
mice were tested at day 3, 6, 8, 10 and 14 after surgery. Means7SEM are indicated. B=basal responses.
A.-L. Klauke et al.8
Please cite this article as: Klauke, A.-L., et al., The cannabinoid CB
2
receptor-selective phytocannabinoid beta-caryophyllene exerts
analgesic effects in mouse models of.... European Neuropsychopharmacology (2013), http://dx.doi.org/10.1016/j.euroneuro.2013.10.008
side was significantly increased compared to ipsilateral
sham values (Figure 8;p=0.0096). BCP treatment did not
influenced the CB
2
receptor expression (treatment: F
1,29
=
1.51; p=n.s).
3.6. Absence of psychoactive effects after BCP
treatment
To determine if BCP exerts acute psychomimetic effects
similar to CB
1
receptor agonists, we tested BCP-treated mice
in the classical cannabinoid tetrad of tests (Figure 9). Oral
administration of 1 and 10 mg kg
1
BCP did not produce any
significant changes in the open-field activity (F
2,20
=0.59,
p=n.s.) or body temperature (F
2,20
=1.3, p=n.s). It also
produced no ring catalepsy (F
2,20
=1.14, p=n.s.) and had no
analgesic effect in the tail flick test (F
2,20
=0.33, p=n.s.). In
contrast, our reference compound Δ
9
-THC elicited the typical
cannabinoid-induced hypomotility (F
1,11
=29.03, po0.001),
catalepsy (F
1,11
=24.13, po0.001) and hypothermia (F
1,10
=
54.7, p40.0001), although tail flick analgesia just failed to
reach significance (F
1,11
=4.51, p=0.057; data not shown).
4. Discussion
Here we demonstrate that the phytocannabinoid BCP, admi-
nistered orally, exerts analgesic effects in animal models of
inflammatory and neuropathic pain. The effects were absent
in CB
2
/
mice and blocked by the CB
2
antagonist SR 144528.
They were thus dependent on CB
2
receptor signaling. To our
knowledge, this is the first demonstration that a common food
ingredient is highly effective in animal models of chronic pain
at physiologically relevant doses.
The plant volatile and food additive BCP is a selective
natural CB
2
receptor agonist with no significant affinity for CB
1
receptors (Gertsch et al., 2008,2010)AcuteBCPtreatment
significantly reduced carrageenan-induced edema formation,
Figure 7 Chronic treatment with 1 mg kg
1
BCP reduced the density of microglia and astrocytes in the dorsal horn of spinal cord.
Representative pictures of the Iba1 (A) and GFAP (C) immunostaining in the ipsilateral dorsal horns of the lumbal spinal cord sections
recorded with the 20 magnification objective. Quantitative analysis revealed a reduction of microglia (B) and astrocyte (D) signals
in the ipsilateral dorsal horn after 2 weeks of 1 mg kg
1
BCP treatment (n=4–5 per group). **, treatment effect po0.01; ***, surgery
effect po0.001. Means7SEM are indicated.
9Analgesic effect of beta-caryophyllene
Please cite this article as: Klauke, A.-L., et al., The cannabinoid CB
2
receptor-selective phytocannabinoid beta-caryophyllene exerts
analgesic effects in mouse models of.... European Neuropsychopharmacology (2013), http://dx.doi.org/10.1016/j.euroneuro.2013.10.008
demonstrating that BCP is able to suppress inflammation via
activation of CB
2
receptors. Our present results show that BCP
is also efficacious in another inflammatory pain model, the
formalin test, which is considered as a model of central pain
sensitization (Beltramo et al., 2006). BCP had an analgesic
effect in the late phase of formalin test after acute treat-
ment. Anti-inflammatory and analgesic effects were similarly
reported for other synthetic CB
2
receptor agonists like
GW405833 (Claytonetal.,2002) and AM1241(Beltramo
et al., 2006) AM1241 also produced analgesia in different
chemical-induced (capsaicin, formalin) pain models and sub-
stance P-induced extravasation, when treated locally or
systemically (Hohmann et al., 2004;Ibrahim et al., 2003).
The CB
2
-selective agonist O-3223 reduced inflammatory and
neuropathic pain without any CB
1
-dependent psychomimetic
side effect (Kinsey et al., 2011). Our reference compound
JWH-133 is a highly selective synthetic CB
2
agonist with full
efficacy on rodent CB
2
receptor (Beltramo, 2009). However,
there are several discrepancies in the literature considering its
anti-inflammatory effect, which may depend on its poor
bioavailability and overall pharmacokinetic properties. In this
study, JWH-133, injected subcutaneously at a relatively high
dose presented a significant analgesic effect in the formalin
test during the 30 min observation period. This effect was
restricted to the late phase and could be reversed by
treatment of CB
2
receptor antagonist, SR 144528. Similar
effects were observed with BCP, which in our hands showed
a very pronounced analgesic effect and interestingly, we also
needed higher SR 144528 concentration to antagonize the BCP
effect than in case of our reference compound, JWH-133. A
possible explanation could be that a natural agonist has a
higher affinity to the CB
2
receptorsandmightimprovethe
efficiency of CB
2
signaling (Shoemaker et al., 2005). We
suppose that reduction of inflammatory processes by BCP
and other CB
2
agonists directly contribute to their anti-
nociceptive effects in different inflammatory pain models.
Neuropathic pain produces similar symptoms of hyper-
algesia, allodynia, and spontaneous pain as peripheral tissue
inflammation, but the underlying pathological processes are
induced by nerve injury (Devor, 2006). Pharmacotherapies
for neuropathic pain using morphine, THC, or a combination
Figure 9 Treatment with BCP did not elicit psychoactive side effects in CB
2
+/+
mice. Mice were tested 45 min after an acute oral
administration of 1 and 10 mg kg
1
BCP or vehicle (olive oil) in the classical cannabinoid tetrad for motor activity in the open-field,
immobility on a ring (catalepsy), anti-nociception in the tail flick test and hypothermia (n=7–8 per group). Means +SEM are
indicated.
Figure 8 PNL induced a significant up-regulation of the CB
2
mRNA level. Expression of the CB
2
receptor gene was deter-
mined by quantitative RT-PCR in the lumbar spinal cord dorsal
horn. CB
2
expression increased in the ipsilateral side after PNL,
but was not affected by BCP treatment. **, PNL effect
p=0.0096, n=4–5 per group, means+SEM are indicated.
A.-L. Klauke et al.10
Please cite this article as: Klauke, A.-L., et al., The cannabinoid CB
2
receptor-selective phytocannabinoid beta-caryophyllene exerts
analgesic effects in mouse models of.... European Neuropsychopharmacology (2013), http://dx.doi.org/10.1016/j.euroneuro.2013.10.008
of THC and cannabidiol (Sativex) produce inadequate pain
relief and at higher doses they have unwanted psychoactive
side effects (Berman et al., 2004;Naef et al., 2003). It is
now well-documented that peripherally expressed CB
2
receptors play an important role in the development of
neuropathic pain and can be targeted to reduce chronic
pain associated with nerve injury (Beltramo, 2009;Guindon
and Hohmann, 2008). Our data demonstrate that chronic
BCP treatment reduces mechanical allodynia and thermal
hyperalgesia in a model of neuropathic pain. Importantly,
we found no signs of tolerance during the 2-week adminis-
tration period. On the contrary, the BCP effect became
stronger during the treatment period. As reported for other
CB
2
agonists (Kinsey et al., 2011;Rahn et al., 2011), BCP did
not induce any of the CB
1
receptor-specific psychomimetic
responses, like hypomotility, catalepsy, hypothermia, or tail
flick analgesia.
The synthetic CB
2
selective agonists NESS400, AM1241 and
GW405833 dose-dependently attenuated tactile and thermal
hypersensitivity produced by spinal nerve ligation and a
spared nerve injury model (Beltramo et al., 2006;Ibrahim
et al., 2003). Similar to our results, NESS400 diminished the
nerve injury induced inflammatory processes in the spinal
projection area, like microglia and astroglia activation,
further increased expression of anti-inflammatory interleukin
(IL)-10 and reduced expression of IL-1ß and interferon (IFN)-γ
(Luongo et al., 2010). Intrathecal administration of JWH-133
reversed partial sciatic nerve ligation-induced mechanical
allodynia and this effect was completely abolished in CB
2
/
animals (Yamamoto et al., 2008). Together these findings
clearly demonstrated that CB
2
receptor activation alone is
sufficient for attenuation the neuropathic pain symptoms and
suggest a role of spinal CB
2
receptors for amelioration the
neuropathic pain symptoms.
Microglia are thought to initiate the neuropathic pain
processes by the release of proinflammatory chemokines
and cytokines that in turn activates astrocytes and leads to
further microglia activation (Tanga et al., 2004). In an
earlier study we could show that CB
2
activation decreases
spinal microglia and astrocyte activation in the spinal cord
after nerve injury (Racz et al., 2008). Latest studies have
shown that in the neuropathic pain processes, microglia
activation decreases to baseline after three weeks, though
astrocytes activation and hypersensitivity remains (Hald
et al., 2009;Tanga et al., 2004). In contrast, CB
2
/
mice
showed an exaggerated glial response that spread to the
contralateral dorsal horn (Racz et al., 2008). BCP treatment
now reduced expression of microglia and astrocytes markers
in the dorsal horn of lumbar spinal cord, which is in line with
the reduced hypersensitivity detected in the behavioral
tests. These findings support our hypothesis that BCP exerts
its anti-inflammatory properties through attenuation of the
spinal glia activation. It was previously shown that BCP
inhibits TH1 cytokines in LPS stimulated whole blood
18
.
For thermal hyperalgesia, a low dose of BCP, 1 mg kg
1
was
more efficacious than higher doses, thus probably reflecting a
bell shaped dose-response curve, which is commonly observed
with cannabinoid receptor agonists (Calabrese, 2008;Malfait
et al., 2000;Sulcova et al., 1998). The mechanisms underlying
this phenomenon are still not clarified.Itispossiblethatdose-
dependent changes in receptor occupation result in a differ-
ential activation of intracellular signaling cascades and thus in
distinct physiological outcomes (Beltramo et al., 2006;Sulcova
et al., 1998). Indeed, there is evidence that 2-AG induced ERK-
MAPK phosphorylation with low ED
50
, stimulated Ca
2+
transi-
ents with a higher ED
50
, and inhibited adenylate cyclase with
highest ED
50
(Shoemaker et al., 2005).Mechanical hyperalgesia,
which is clinically more relevant, did not show such a pro-
nounced dose dependency. It is thus conceivable that thermal
hyperalgesia and mechanical allodynia, which are mediated by
different nociceptive neurons, are differently affected by CB
2
agonism.
BCP is the first natural CB
2
receptor agonist, which could
orally reduce inflammatory responses in different animal
models of pain. Recently it was shown that BCP inhibits
colon inflammation and protects from cisplatin-induced
nephrotoxicity via CB
2
receptors (Bento et al., 2011). An
important question to be considered now is whether or not
this dose is relevant to humans? It has been suggested to
calculate the human dose equivalent based on normal-
ization of the body surface areas (Reagan-Shaw et al.,
2008), which means that an effective dose in mice has to
be divided by 12.3 in order to estimate the human
equivalent dose of a 60 kg adult. Thus, a human daily dose
of 0.08–0.41 mg/kg BCP would correspond to the optimal
dose in mice. We have previously estimated an average
daily BCP intake in the range of 10–200 mg, which corre-
sponds to a dose of 0.16–3.3 mg/kg for a 60 kg human. This
dose would certainly be sufficient for significant CB
2
canna-
binoid receptor activation. Thus, it is likely that BCP belongs
to a group of common plant natural products with major
potential impact on human health. The oral intake of this
dietary cannabinoid with vegetable food could be advanta-
geous in the daily routine clinical practice over synthetic
cannabinoid agonists.
Role of funding source
This work was supported by Grants from the Bundesministerium für
Forschung und Lehre (NGFN2 FKZ 01GS0853) and the Deutsche
Forschungsgemeinschaft (FOR926). The sponsors had no further role
in study design; in the collection, analysis and interpretation of
data; in the writing of the report; and in the decision to submit the
paper for publication.
Contributors
IR and AZ designed the experiments. AK, IR and AZ drafted the
manuscript. AK and IR performed the experiments and the data
analysis. AM and BP contributed to the experiments. AK and AMZ
performed tetrad test. JG provided the BCP and read critically the
manuscript. All authors contributed to and have approved the final
manuscript.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
We thank Eva Drews for critically reading the manuscript.
11Analgesic effect of beta-caryophyllene
Please cite this article as: Klauke, A.-L., et al., The cannabinoid CB
2
receptor-selective phytocannabinoid beta-caryophyllene exerts
analgesic effects in mouse models of.... European Neuropsychopharmacology (2013), http://dx.doi.org/10.1016/j.euroneuro.2013.10.008
Appendix A. Supplementary material
Supplementary data associated with this article can be
found in the online version at http://dx.doi.org/10.1016/
j.euroneuro.2013.10.008.
References
Basbaum, A.I., Bautista, D.M., Scherrer, G., Julius, D., 2009.
Cellular and molecular mechanisms of pain. Cell 139, 267–284.
Beltramo, M., 2009. Cannabinoid type 2 receptor as a target for
chronic-pain. Mini Rev. Med. Chem. 9, 11–25.
Beltramo, M., Bernardini, N., Bertorelli, R., Campanella, M.,
Nicolussi, E., Fredduzzi, S., Reggiani, A., 2006. CB
2
receptor-
mediated antihyperalgesia: possible direct involvement of
neural mechanisms. Eur. J. Neurosci. 23, 1530–1538.
Ben Amar, M., 2006. Cannabinoids in medicine: a review of their
therapeutic potential. J. Ethnopharmacol. 105, 1–25.
Benito, C., Romero, J.P., Tolón, R.M., Clemente, D., Docagne, F.,
Hillard, C.J., Guaza, C., Romero, J., 2007. Cannabinoid CB
1
and
CB
2
receptors and fatty acid amide hydrolase are specific
markers of plaque cell subtypes in human multiple sclerosis. J.
Neurosci. 27, 2396–2402.
Bento, A., Marcon, R., Dutra, R., Claudino, R., Cola, M., Leite, D.,
Calixto, J., 2011. Caryophyllene inhibits dextran sulfate sodium-
induced colitis in mice through CB
2
receptor activation and
PPARγpathway. Am. J. Pathol. 178, 1153–1166.
Berman, J., Symonds, C., Birch, R., 2004. Efficacy of two cannabis
based medicinal extracts for relief of central neuropathic pain
from brachial plexus avulsion: results of a randomised controlled
trial. Pain 112, 299–306.
Bernardes, W., Lucarini, R., Tozatti, M., Flauzino, L., Souza, M.,
Turatti, I., Andrade e Silva, M., Martins, C., da Silva Filho, A.,
Cunha, W., 2010. Antibacterial activity of the essential oil from
Rosmarinus officinalis and its major components against oral
pathogens. Z. Naturforsch. C: J. Biosci. 65, 588–593.
Buckley, N.E., McCoy, K.L., Mezey, E., Bonner, T., Zimmer, A.,
Felder, C.C., Glass, M., Zimmer, A., 2000. Immunomodulation
by cannabinoids is absent in mice deficient for the cannabinoid
CB(2) receptor. Eur. J. Pharmacol. 396, 141–149.
Calabrese, E.J., 2008. Drug therapies for stroke and traumatic brain
injury often display U-shaped dose responses: occurrence,
mechanisms, and clinical implications. Crit. Rev. Toxicol. 38,
557–577.
Calleja, M.A., Vieites, J.M., Montero-Meterdez, T., Torres, M.I.,
Faus, M.J., Gil, A., Suárez, A., 2012. The antioxidant effect of
β-caryophyllene protects rat liver from carbon tetrachloride-
induced fibrosis by inhibiting hepatic stellate cell activation. Br.
J. Nutr. 1, 1–8.
Clayton, N., Marshall, F.H., Bountra, C., O’Shaughnessy, C.T., 2002.
CB
1
and CB
2
cannabinoid receptors are implicated in inflamma-
tory pain. Pain 96, 253–260.
Costigan, M., Scholz, J., Woolf, C.J., 2009. Neuropathic pain: a
maladaptive response of the nervous system to damage. Annu.
Rev. Neurosci. 32, 1–32.
Devor, M., 2006. Response to nerve injury in relation to neuropathic
pain.In:McMahon,S.,Koltzenburg,M.(Eds.),WallandMelzack's
Textbook of Pain. Elsevier Churchill Livingstone, London,
pp. 905–927.
Facci,L.,DalToso,R.,Romanello,S.,Buriani,A.,Skaper,S.,Leon,A.,
1995. Mast cells express a peripheral cannabinoid receptor with
differential sensitivity to anandamide and palmitoylethanolamide.
Proc. Natl. Acad. Sci. USA 92, 3376–3380.
Galdino, P.M., Nascimento, M.V.M., Florentino, I.F., Lino, R.C.,
Fajemiroye, J.O., Chaibub, B.A., de Paula, J.R., de Lima, T.C.M.,
Costa, E.A., 2012. The anxiolytic-like effect of an essential oil
derived from Spiranthera odoratissima A.St.Hil.leavesandits
major component, β-caryophyllene, in male mice. Prog. Neurop-
sychopharmacol. Biol. Psychiatry 38, 276–284.
Gertsch, J., Leonti, M., Raduner, S., Racz, I., Chen, J.Z., Xie, X.Q.,
Altmann, K.H., Karsak, M., Zimmer, A., 2008. Beta-caryophyllene is
a dietary cannabinoid. Proc. Natl. Acad. Sci. USA 105, 9099–9104.
Gertsch, J., Pertwee, R.G., Di Marzo, V., 2010. Phytocannabinoids
beyond the Cannabis plant—do they exist? Br. J. Pharmacol. 160,
523–529.
Ghelardini, C., Galeotti, N., Di Cesare Mannelli, L., Mazzanti, G.,
Bartolini, A., 2001. Local anaesthetic activity of beta-caryophyllene.
Farmaco 56, 387–389.
Guindon, J., Hohmann, A.G., 2008. Cannabinoid CB
2
receptors: a
therapeutic target for the treatment of inflammatory and
neuropathic pain. Br. J. Pharmacol. 153, 319–334.
Hald, A., Nedergaard, S., Hansen, R.R., Ding, M., Heegaard, A.-M.,
2009. Differential activation of spinal cord glial cells in murine
models of neuropathic and cancer pain. Eur. J. Pain 13, 138–145.
Hauser, J., Hsu, B., Nader, N.D., 2013. Inflammatory processes
in complex regional pain syndromes. Immunol. Invest. 42,
263–272.
Hohmann, A., Herkenham, M., 1999. Localization of central canna-
binoid CB
1
receptor messenger RNA in neuronal subpopulations
of rat dorsal root ganglia: a double-label in situ hybridization
study. Neuroscience 90, 923–931.
Hohmann, A.G., Farthing, J.N., Zvonok, A.M., Makriyannis, A.,
2004. Selective activation of cannabinoid CB 2 receptors sup-
presses hyperalgesia evoked by intradermal capsaicin. J. Phar-
macol. Exp. Ther. 308, 446–453.
Horváth, B., Mukhopadhyay, P., Kechrid, M., Patel, V., Tanchian, G.,
Wink, D.A., Gertsch, J., Pacher, P., 2012. β-Caryophyllene
ameliorates cisplatin-induced nephrotoxicity in a cannabinoid
2 receptor-dependent manner. Free Radic. Biol. Med. 52,
1325–1333.
Hudaib, M., Speroni, E., Di Pietra, A.M., Cavrini, V., 2002. GC/MS
evaluation of thyme (Thymus vulgaris L.) oil composition and
variations during the vegetative cycle. J. Pharm. Biomed. Anal.
29, 691–700.
Ibrahim, M.M., Deng, H., Zvonok, A., Cockayne, D.A., Kwan, J.,
Mata, H.P., Vanderah, T.W., Lai, J., Porreca, F., Makriyannis, A.,
Malan, T.P., 2003. Activation of CB
2
cannabinoid receptors by
AM1241 inhibits experimental neuropathic pain: pain inhibition
by receptors not present in the CNS. Proc. Natl. Acad. Sci. USA
100, 10529–10533.
Iskedjian, M., Bereza, B., Gordon, A., Piwko, C., Einarson, T., 2007.
Meta-analysis of cannabis based treatments for neuropathic and
multiple sclerosis-related pain. Curr. Med. Res. Opinions 23, 17–24.
Jayaprakasha, G.K., Jagan Mohan Rao, L., Sakariah, K.K., 2003.
Volatile constituents from Cinnamomum zeylanicum fruit stalks
and their antioxidant activities. J. Agric. Food Chem. 51,
4344–4348.
Katsuyama,S.,Mizoguchi,H.,Kuwahata,H.,Komatsu,T.,Nagaoka,K.,
Nakamura, H., Bagetta, G., Sakurada, T., Sakurada, S., 2012.
Involvement of peripheral cannabinoid and opioid receptors in
beta-caryophyllene-induced antinociception. Eur. J. Pain 17,
664–675.
Kinsey, S.G., Mahadevan, A., Zhao, B., Sun, H., Naidu, P.S.,
Razdan, R.K., Selley, D.E., Imad Damaj, M., Lichtman, A.H., 2011.
The CB
2
cannabinoid receptor-selective agonist O-3223 reduces pain
and inflammation without apparent cannabinoid behavioral effects.
Neuropharmacology 60, 244–251.
Legault, J., Pichette, A., 2007. Potentiating effect of β-
caryophyllene on anticancer activity of α-humulene, isocaryo-
phyllene and paclitaxel. J. Pharm. Pharmacol. 59, 1643–1647.
Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene
expression data using real-time quantitative PCR and the 2
(-Delta Delta C(T)) Methods 25, 402–408.
A.-L. Klauke et al.12
Please cite this article as: Klauke, A.-L., et al., The cannabinoid CB
2
receptor-selective phytocannabinoid beta-caryophyllene exerts
analgesic effects in mouse models of.... European Neuropsychopharmacology (2013), http://dx.doi.org/10.1016/j.euroneuro.2013.10.008
Loizzo, M.R., Tundis, R., Menichini, F., Saab, A.M., Statti, G.A.,
Menichini, F., 2008. Antiproliferative effects of essential oils and
their major constituents in human renal adenocarcinoma and
amelanotic melanoma cells. Cell Proliferation 41, 1002–1012.
Luongo, L., Palazzo, E., Tambaro, S., Giordano, C., Gatta, L.,
Scafuro, M.A., Rossi, F.S., Lazzari, P., Pani, L., De Novellis, V.,
Malcangio, M., Maione, S., 2010. 1-(2',4'-dichlorophenyl)-6-
methyl-N-cyclohexylamine-1,4-dihydroindeno[1,2-c]pyrazole-3-
carboxamide, a novel CB
2
agonist, alleviates neuropathic pain
through functional microglial changes in mice. Neurobiol. Dis.
37, 177–185.
Lynch, M.E., Campbell, F., 2011. Cannabinoids for treatment of
chronic non-cancer pain; a systematic review of randomized
trials. Br. J. Clin. Pharmacol. 72, 735–744.
Malfait, A.M., Gallily, R., Sumariwalla, P.F., Malik, A.S., Andreakos, E.,
Mechoulam, R., Feldmann, M., 2000. The nonpsychoactive canna-
bis constituent cannabidiol is an oral anti-arthritic therapeutic in
murine collagen-induced arthritis. Proc. Natl. Acad Sci. USA 97,
9561–9566.
Maresz, K., Carrier, E.J., Ponomarev, E.D., Hillard, C.J., Dittel, B.N.,
2005. Modulation of the cannabinoid CB 2 receptor in microglial
cells in response to inflammatory stimuli. J. Neurochem. 95,
437–445.
Martin, B.R., Compton, D.R., Thomas, B.F., Prescott, W.R., Little, P.J.,
Razdan, R.K., Johnson, M.R., Melvin, L.S., Mechoulam, R.,
Ward, S.J., 1991. Behavioral, biochemical, and molecular modeling
evaluations of cannabinoid analogs. Pharmacol. Biochem. Behav.
40, 471–478.
Mockute, D., Bernotiene, G., Judzentiene, A., 2001. The essential
oil of Origanum vulgare L. ssp. vulgare growing wild in Vilnius
district (Lithuania). Phytochemistry 57, 65–69.
Naef, M., Curatolo, M., Petersen-Felix, S., Arendt-Nielsen, L.,
Zbinden, A., Brenneisen, R., 2003. The analgesic effect of oral
delta-9-tetrahydrocannabinol (THC), morphine, and a THC-
morphine combination in healthy subjects under experimental
pain conditions. Pain 105, 79–88.
Orav, A., Stulova, I., Kailas, T., Müürisepp, M., 2004. Effect of
storage on the essential oil composition of Piper nigrum L. fruits
of different ripening states. J. Agric. Food Chem. 52, 2582–2586.
Ou, M.C., Hsu, T.F., Lai, A.C., Lin, Y.T., Lin, C.C., 2012. Pain relief
assessment by aromatic essential oil massage on outpatients
with primary dysmenorrhea: a randomized, double-blind clinical
trial. J. Obstet. Gynaecol. Res. 38, 817–822.
Racz, I., Nadal, X., Alferink, J., Banos, J.E., Rehnelt, J., Martin, M.,
Pintado, B., Gutierrez-Adan, A., Sanguino, E., Manzanares, J.,
Zimmer, A., Maldonado, R., 2008. Crucial role of CB
2
cannabi-
noid receptor in the regulation of central immune responses
during neuropathic pain. J. Neurosci. 28, 12125–12135.
Rahn, E.J., Thakur, G.A., Anne, J., Wood, T., Zvonok, A.M.,
Makriyannis, A., Hohmann, A.G., 2011. Pharmacological char-
acterization of AM1710, a putative cannabinoid CB 2 agonist
from the cannabilactone class: antinociception without central
nervous system side-effects. Pharmacol. Biochem. Behav. 98,
493–502.
Reagan-Shaw, S., Nihal, M., Ahmad, N., 2008. Dose translation from
animal to human studies revisited. FASEB J. 22, 659–661.
Russo, E.B., 2011. Taming THC: potential cannabis synergy and
phytocannabinoid-terpenoid entourage effects. Br. J. Pharmacol.
163, 1344–1364.
Scholz, J., Woolf, C.J., 2007. The neuropathic pain triad: neurons,
immune cells and glia. Nat. Neurosci. 10, 1361–1368.
Shoemaker, J.L., Ruckle, M.B., Mayeux, P.R., Prather, P.L., 2005.
Agonist-directed trafficking of response by endocannabinoids
acting at CB
2
receptors. J. Pharmacol. Exp. Ther. 315, 828–838.
Sulcova, E., Mechoulam, R., Fride, E., 1998. Biphasic effects of
anandamide. Pharmacol. Biochem. Behav. 59, 347–352.
Tanga, F.Y., Raghavendra, V., DeLeo, J.A., 2004. Quantitative real-
time RT-PCR assessment of spinal microglial and astrocytic
activation markers in a rat model of neuropathic pain. Neuro-
chem. Int. 45, 397–407.
Woolf, C.J., Ma, Q., 2007. Nociceptors—noxious stimulus detectors.
Neuron 55, 353–364.
Yamamoto, W., Mikami, T., Iwamura, H., 2008. Involvement of
central cannabinoid CB
2
receptor in reducing mechanical allo-
dynia in a mouse model of neuropathic pain. Eur. J. Pharmacol.
583, 56–61.
Zheng, G., Kenney, P., Lam, L., 1992. Sesquiterpenes from clove
(Eugenia caryophyllata) as potential anticarcinogenic agents. J.
Nat. Prod. 55, 999–1003.
Zias, J., Stark, H., Sellgman, J., Levy, R., Werker, E., Breuer, A.,
Mechoulam, R., 1993. Early medical use of cannabis. Nature
363, 215.
13Analgesic effect of beta-caryophyllene
Please cite this article as: Klauke, A.-L., et al., The cannabinoid CB
2
receptor-selective phytocannabinoid beta-caryophyllene exerts
analgesic effects in mouse models of.... European Neuropsychopharmacology (2013), http://dx.doi.org/10.1016/j.euroneuro.2013.10.008
