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Immunopharmacology and inflammation
Evaluation of the anti-inflammatory, anti-catabolic and pro-anabolic
effects of E-caryophyllene, myrcene and limonene in a cell model
of osteoarthritis
Ana Teresa Rufino
a,b
, Madalena Ribeiro
a,b
, Cátia Sousa
a,b
, Fernando Judas
c,d
,
Lígia Salgueiro
a,e
, Carlos Cavaleiro
a,e
, Alexandrina Ferreira Mendes
a,b,
n
a
Centre for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal
b
Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal
c
Orthopedics Department and Bone Bank, University and Hospital Center of Coimbra, Coimbra, Portugal
d
Faculty of Medicine, University of Coimbra, Coimbra, Portugal
e
Centro de Estudos Farmacêuticos, Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal
article info
Article history:
Received 23 October 2014
Received in revised form
14 January 2015
Accepted 16 January 2015
Available online 23 January 2015
Keywords:
Osteoarthritis
Chronic inflammation
NF-
κ
B
MAPK
Natural products
abstract
Osteoarthritis is a progressive joint disease and a major cause of disability for which no curative
therapies are yet available. To identify compounds with potential anti-osteoarthritic properties, in this
study, we screened one sesquiterpene, E-caryophyllene, and two monoterpenes, myrcene and limonene,
hydrocarbon compounds for anti-inflammatory, anti-catabolic and pro-anabolic activities in human
chondrocytes. At non-cytotoxic concentrations, myrcene and limonene inhibited IL-1
β
-induced nitric
oxide production (IC
50
¼37.3
μ
g/ml and 85.3 mg/ml, respectively), but E-caryophyllene was inactive.
Myrcene, and limonene to a lesser extent, also decreased IL-1
β
-induced NF-
κ
B, JNK and p38 activation
and the expression of inflammatory (iNOS) and catabolic (MMP-1 and MMP-13) genes, while increasing
the expression of anti-catabolic genes (TIMP-1 and -3 by myrcene and TIMP-1 by limonene). Limonene
increased ERK1/2 activation by 30%, while myrcene decreased it by 26%, relative to IL-1
β
-treated cells.
None of the compounds tested was able to increase the expression of cartilage matrix-specific genes
(collagen II and aggrecan), but both compounds prevented the increased expression of the non-cartilage
specific, collagen I, induced by IL-1
β
. These data show that myrcene has significant anti-inflammatory
and anti-catabolic effects in human chondrocytes and, thus, its ability to halt or, at least, slow down
cartilage destruction and osteoarthritis progression warrants further investigation.
&2015 Elsevier B.V. All rights reserved.
1. Introduction
Osteoarthritis (OA) is a multifactorial degenerative joint disease
characterized by inflammation and progressive loss of the articular
cartilage, associated with changes in the subchondral bone and other
jointtissues.Itaffects10–15% of the world population and is a major
cause of disability, not only in the elderly, as well as in the workforce
population (Zhang and Jordan, 2010). Existing therapeutic approaches
are mainly symptomatic, thus novel drugs with disease-modifying and
chondroprotective properties, the so-called disease-modifying osteoar-
thritis drugs, are required to halt disease progression and decrease its
huge socio-economic impact (Goldring and Goldring, 2007; Kaplan
et al., 2013).
Plant-derived compounds show important biological properties that
can be explored in the context of OA for identification of compounds
with potential anti-osteoarthritic activity (Calixto et al., 2004; Khalife
and Zafarullah, 2011). Among compounds of plant origin, those found
in essential oils present favorable pharmacokinetic properties, namely
lipophilicity and low molecular weight (Miguel, 2010). Our previous
studies have been focused in identifying essential oils with anti-
inflammatory and anti-catabolic properties in human chondrocytes to
be used as sources of compounds with potential anti-osteoarthritic
activity (Neves et al., 2009; Rufino et al., 2014a). In this context, we
recently identified the essential oils of Eryngium duriaei subsp. juresia-
num and Lavandula luisieri as possessing anti-inflammatory properties
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/ejphar
European Journal of Pharmacology
http://dx.doi.org/10.1016/j.ejphar.2015.01.018
0014-2999/&2015 Elsevier B.V. All rights reserved.
n
Corresponding author at: Center for Neuroscience and Cell Biology, University
of Coimbra, Edifício da Faculdade de Medicina, Polo I, 11piso, Rua Larga, 3004-504
Coimbra, Portugal. Tel.: þ351 239 480209; fax: þ351 239 488503.
E-mail addresses: ana.t.rufino@gmail.com (A.T. Rufino),
madalena_ribeiro8@hotmail.com (M. Ribeiro),
catota_coura@hotmail.com (C. Sousa), fernandojudas@gmail.com (F. Judas),
ligia@ff.uc.pt (L. Salgueiro), Cavaleir@ff.uc.pt (C. Cavaleiro),
afmendes@ff.uc.pt (A.F. Mendes).
European Journal of Pharmacology 750 (2015) 141–150
in human chondrocytes (Rufino et al., 2014a). The current study
aims at identifying active compounds of these essential oils and
further characterizing their pharmacological properties in human chon-
drocytes.
For this, mechanisms relevant as pharmacological targets for
the development of anti-osteoarthritic drugs need to be addressed.
Although OA etiology is not yet completely understood, pro-
inflammatory cytokines, like interleukin-1
β
(IL-1
β
), play a central
role in disease development and progression by inducing the
expression of cartilage matrix-degrading enzymes and impairing
anabolic and anti-catabolic responses in chondrocytes (Goldring
et al., 2008). The consequent upregulated degradative process,
together with impaired reparative responses, results in progressive
cartilage loss, the hallmark of OA.
Matrix metalloproteases (MMPs) and aggrecanases are the main
enzymes responsible for hydrolyzing the major articular cartilage-
specific matrix components, collagen II and aggrecan. This is accom-
panied by impaired reparative responses involving downregulation of
thenaturalMMPinhibitors,thetissueinhibitorofmetalloproteases
(TIMP) family, decreased synthesis of collagen II and aggrecan and
increased expression of non-articular cartilage matrix components, like
collagen I (Troeberg and Nagase, 2012). Moreover, increased production
of pro-inflammatory and pro-catabolic mediators, like nitric oxide
(NO), amplifies and perpetuates cartilage destruction (Boileau et al.,
2002; Rosa et al., 2008; Sasaki et al., 1998). The transcription factor,
Nuclear Factor-
κ
B(NF-
κ
B), and the family of mitogen-activated protein
kinases (MAPK) play a central role in modulating the expression of
those catabolic and inflammatory mediators (Goldring and Otero, 2011)
and, thus, agents that suppress their activity have the potential to
effectively decrease cartilage destruction and, therefore, OA progression
(Berenbaum, 2004). Furthermore, compounds that can also restore
anabolic and anti-catabolic gene expression have the potential to pro-
mote cartilage repair.
Therefore, we used primary human chondrocyte cultures
stimulated with IL-1
β
as an in vitro cartilage degradation model
that emulates the damage seen in OA. Using this model, we
evaluated the inhibition of IL-1
β
-induced NO production as a
simple screening assay. The essential oils of E. duriaei subsp.
juresianum and L. luisieri were separated into several fractions
that were then screened using the assay mentioned above. The
chemical composition of the fractions tested was elucidated and
compounds present in the active fractions and commercially
available were selected for screening using the same assay. The
compounds selected were obtained with a high degree of purity
from commercial sources and those found to be capable of
inhibiting IL-1
β
-induced NO production were selected for further
assessment of anti-osteoarthritic potential. For this, we evaluated
their ability to modulate IL-1
β
-induced signaling pathways invol-
ved in the expression of inflammatory and catabolic genes, namely
activation of NF-
κ
B and the MAPK family members, Jun terminal
Kinase (JNK), p38 and Extracellular signal-Regulated Kinase 1/2
(ERK1/2). Then, we evaluated the ability of those compounds to
counteract the effects of IL-1
β
on the expression of inflammatory
(iNOS), catabolic (MMP-1 and -13), anti-catabolic (TIMP-1 and -3)
and extracellular matrix (collagen I, collagen II and aggrecan)
genes in human articular chondrocytes.
2. Material and methods
2.1. Essential oil fractionation and chemical analysis
The essential oils of E. duriaei subsp. juresianum and L. luisieri
were fractionated by flash chromatography on silica gel 63–
200
μ
m (Merck) using a 2 cm 40 cm Omnifit (Sigma-Aldrich)
glass columns. Elution was made in step gradients from 100% of
n-pentane, n-pentane /ethyl ether mixtures, till a final concentra-
tion of 100% ethyl ether. Collected fractions were analyzed by
gas chromatography–mass spectroscopy (GC/MS) and combined
if having similar composition. Analysis was performed using a
Hewlett-Packard 6890 gas chromatograph fitted with a HP1 fused
silica column (polydimethylsiloxane 30 m 0.25 mm i.d., film
thickness 0.25
μ
m), interfaced with an Hewlett-Packard mass
selective detector 5973 (Agilent Technologies) operated by HP
Enhanced ChemStation software, version A.03.00. GC–MS para-
meters: oven temperature program: 70–220 1C(31C min
1
),
220 1C (15 min); injector temperature: 250 1C; carrier gas: helium,
adjusted to a linear velocity of 30 cm s
1
; splitting ratio 1:40;
interface temperature: 250 1C; MS source temperature: 230 1C; MS
quadrupole temperature: 150 1C; ionization energy: 70 eV; ioniza-
tion current: 60
μ
A; scan range: 35–350 units; scans s
1
: 4.51.
Compounds were identified through their GC retention and mass
spectra. Retention indices, calculated by linear interpolation rela-
tive to retention times of C
8
–C
23
of n-alkanes, were compared
with those of authentic products included in laboratory data-
base (CEF/Faculty of Pharmacy, University of Coimbra) and/or the
literature data. Acquired mass spectra were compared with refer-
ence spectra from the laboratory database, Wiley/NIST library
(McLafferty, 2009) and literature data (Adams, 1995; Cavaleiro
et al., 2011; Joulain, 1998; Videira et al., 2013). Relative amounts
of individual components were calculated based on the total ion
chromatogram peak areas.
2.2. Cartilage samples and chondrocyte isolation
Human chondrocytes were isolated by enzymatic digestion
(Rosa et al., 2008) of knee cartilage from the distal femoral
condyles of multi-organ donors (20–70 years old, mean¼52.9,
n¼31) and patients (58–73 years old, mean¼65, n¼5) undergoing
total knee arthroplasty at the Orthopaedic Department and Bone
Bank of the University and Hospital Center of Coimbra (CHUC).
The cartilage samples presented variable degrees of degradation,
ranging from intact to severely damaged. All procedures were
approved by the Ethics Committee of CHUC (protocol approval
numbers 8654/DC and HUC-13-05).
2.3. Cell cultures and treatments
Primary non-proliferating chondrocyte cultures were estab-
lished from non-pooled cartilage samples. The human chondrocy-
tic cell line, C28/I2, kindly provided by Prof. Mary Goldring
(currently at the Hospital for Special Surgery, New York) and
Harvard University, was used to evaluate NF-
κ
B–DNA binding
activity. The cell cultures were serum-starved for at least 8 h and
maintained thereafter in serum-free culture medium. The test
compounds, diluted in DMSO (Sigma-Aldrich, St Louis, MO, USA)
to achieve the concentrations indicated in figures and their
legends, were added to the chondrocyte cultures 30 min before
the pro-inflammatory stimulus (IL-1
β
, 10 ng/ml) and maintained
for the experimental period indicated in figure legends. The final
concentration of DMSO did not exceed 0.1% (v/v). E-caryophyllene
(purity 498.5%) and myrcene (purity Z95.0%) were from Sigma-
Aldrich (St Louis, MO, USA) and limonene (purity 90%) was
from Fluka.
2.4. Nitric oxide production
Nitric oxide production was evaluated as the amount of nitrite
accumulated in primary chondrocyte culture supernatants after
24 h treatment with IL-1
β
, following pre-treatment with non-
cytotoxic concentrations of the fractions of the essential oils or the
test compounds. Nitrite concentration was measured using the
A.T. Rufino et al. / European Journal of Pharmacology 750 (2015) 141–15 0142
spectrophotometric method based on the Griess reaction (Green
et al., 1982).
2.5. Western blot analysis
Total and cytoplasmic extracts were prepared as described
before (Goldring and Otero, 2011). Proteins were separated by
SDS-PAGE under reducing conditions and electrotransferred onto
PVDF membranes. These were probed overnight with the follow-
ing primary antibodies: mouse monoclonal anti-human iNOS
(R&D systems, Minneapolis, MN), mouse polyclonal anti-human
phospho-I
κ
B-
α
or rabbit polyclonal anti-human I
κ
B-
α
, anti-
human phospho-JNK, anti-human phospho-p38 or anti-human
phospho-ERK1/2 (Cell Signaling Technology, Inc., Danvers, MA)
and then with anti-rabbit or anti-mouse alkaline phosphatase-
conjugated secondary antibodies (GE Healthcare, UK). Mouse anti-
human
β
-Tubulin monoclonal antibody was used to detect
β
-Tubulin as a loading control. Enhanced ChemiFluorescence
reagent (GE Healthcare) was used to detect immune complexes.
Image analysis was performed with ImageQuant TL software (GE
Healthcare). The results presented are the normalized ratio
between the intensities of the bands corresponding to the protein
of interest and to the protein used as loading control.
2.6. NF-
κ
B transcription factor assay
A colorimetric ELISA-based assay (NoShift Transcription Factor
Assay Kit, Novagen, La Jolla, CA) was used to evaluate the presence
of active NF-
κ
B dimers, capable of binding to the cognate con-
sensus oligonucleotide sequence. For this purpose, nuclear extracts
from C-28/I2 cells were incubated with a biotinylated consensus
NF-
κ
B oligonucleotide (NoShift NF-
κ
B Reagents; Novagen) and the
assay performed according to the manufacturer's instructions. The
absorbance intensity in each sample is directly proportional to the
amount of NF-
κ
B–DNA complexes formed and, thus, to the
amount of active NF-
κ
B dimers present in each sample. In parallel,
the specificity of the reaction was confirmed in competition assays
where addition of a 10-fold molar excess of non-biotinylated wild-
type or mutant oligonucleotides (NoShift NF-
κ
B Reagents; Nova-
gen) to binding reactions containing nuclear extracts from IL-1
β
treated cells abrogated or did not affect, respectively, the forma-
tion of NF-
κ
B–DNA complexes.
2.7. Total RNA extraction and quantitative real-time RT-PCR (qRT-
PCR)
Total RNA was extracted from human condrocytes using TRI-
zol
s
Reagent (Invitrogen, Life Technologies, Co; Paisley, UK) and
quantified using a NanoDrop ND-1000 spectrophotometer at
260 nm. Purity and integrity of RNA were assessed as the 240/
260 and 280/260 ratios. The cDNA was reverse-transcribed using
the iScript Select cDNA Synthesis Kit (Bio-Rad), beginning with
1mg of RNA. Specific sets of primers for iNOS, MMP-1, MMP-13,
TIMP-1, TIMP-3, collagen II, collagen I, aggrecan and HPRT-1
(Table 1) were designed using Beacon Designer software (Premier
Biosoft International, Palo Alto, CA). PCR reactions were performed
using 25
μ
g/ml of transcribed cDNA in a final volume of 20 mL.
The efficiency of the amplification reaction for each gene was
calculated using a standard curve of a series of diluted cDNA
samples, and the specificity of the amplification products was
assessed by analyzing the melting curve generated in the process.
The results for each gene of interest were normalized against
HPRT-1, the housekeeping gene found to be the most stable under
the experimental conditions used. Gene expression changes were
analyzed using the built-in iQ5 Optical system software v2, which
enables the analysis of the results with the Pfafflmethod, a
variation of the
ΔΔ
CT method corrected for gene-specificeffi-
ciencies (Nolan et al., 2006)
2.8. Statistical analysis
Results are presented as mean7S. E. M. Each subject contrib-
uted only once to the statistical analysis which was performed
using GraphPad Prism (version 5.00). The Kolmogorov–Smirnov
test was used to assess normality for the observations themselves
or for the observed differences. As this test showed that in all
experiments the results were normally distributed, the statistical
analysis was performed using the paired t-test for comparison of
each condition with its respective control and one-way ANOVA for
comparison of all conditions. Results were considered statistically
significant at Po0.05.
3. Results
3.1. Selection of inhibitors of IL-1-induced iNOS expression and NO
production
Various fractions of the essential oils of E. duriaei subsp.
juresianum and L. luisieri were separated in amounts sufficient
for pharmacological screening. The composition of these fractions
was fully elucidated and is reported in Table 2.
The fractions obtained were then tested at non-cytotoxic
concentrations ranging from 10 to 50
μ
g/ml (Supplementary
data). The results (Fig. 1 panel A) show that the hydrocarbon-
containing fractions (F
1
and F
A
) of both essential oils decreased IL-
1
β
-induced NO production in a concentration-dependent manner,
the highest concentrations achieving an inhibition of 55% and 75%,
respectively, relative to cells treated with IL-1
β
alone. The other
three fractions of the essential oil of L. luisieri also showed some
inhibitory activity which, nonetheless, did not exceed 35% relative
to IL-1
β
-treated cells. Fractions F
2.4
and F
2.5
of the essential oil of E.
duriaei subsp. juresianum were only tested at a concentration of
10 mg/ml, as higher concentrations were found to be cytotoxic
(Supplementary data). These fractions, as well as fraction F
2.7
at
Table 1
Oligonucleotide primer pairs used for qRT-PCR.
Gene name Genbank accession number Forward sequence Reverse sequence
iNOS NM_000625.4 5
0
-AATCCAGATAA GTGACATAAG-3
0
5
0
-CTCCACATTGT TGTTGAT-3
0
MMP-1 NM_001145938.1 5
0
-GAGTCTCCCAT TCTACTG-3
0
5
0
-TTATAGCATCA AAGGTTAGC-3
0
MMP-13 NM_002422.3 5
0
-GTTTCCTATCTA CACCTACAC-3
0
5
0
-CTCGGAGACTGG TAATGG-3
0
TIMP-1 NM_003254.2 5
0
-TGTTGCTGTGGC TGATAG-3
0
5
0
-CTGGTATAAGGT GGTCTGG-3
0
TIMP-3 NM_000362.4 5
0
-CCATACACTATCCAC -3
0
5
0
-TAACAGCATTGAACA -3
0
Collagen II NM_001844.4 5
0
-GGCAGAGGTA TAATGATAAGG-3
0
5
0
-ATTATGTCGTC GCAGAGG-3
0
Collagen I NM_000088.3 5
0
GGAGGAGAGTCAGGA-3
0
5
0
-GCAACACAGTTACAC-3
0
Aggrecan NM_001135 5
0
-CCTGGTGTGGCT GCTGTC-3
0
5
0
-CTGGCTCGGT GGTGAACTC-3
0
HPRT-1 NM_000194.2 5
0
-TGACACTGGCA AAACAAT-3
0
5
0
-GGCTTATATCC AACACTTCG-3
0
A.T. Rufino et al. / European Journal of Pharmacology 750 (2015) 141–150 143
a concentration of 50 mg/ml, also significantly inhibited IL-1
β
-ind-
uced NO production, although to a lesser extent than found with
F
1
. No significant effects were obtained with F
2.3
at any of the
concentrations tested. Therefore, the hydrocarbon-containing frac-
tions (F
1
and F
A
) of both essential oils were considered the most
promising for selection of compounds for further studies.
As shown in Table 2, these fractions (F
A
and F
1
) are composed of
monoterpene and sesquiterpene hydrocarbons, mainly
α
-pinene
and 3,5-dimethylene-1,4,4-trimethylcyclopentene, and E-caryo-
phyllene,
α
-neocallitropsene, germacrene D,
β
-selinene and bicy-
clogermacrene, respectively. Of these compounds, only
α
-pinene
and E-caryophyllene are readily available from commercial sources
Table 2
Composition of the fractions of the essential oils of E. duriaei subsp juresianum and Lavandula luisieri.
Eryngium duriaei subsp juresianum %Lavandula luisieri %
F
1
E-caryophyllene 29.5 F
A
3.5-dimethylene-1.4.4-trimethylcyclopentene 10.4
α-neocallitropsene 50.2 α-pinene 26.9
Germacrene D 2.7 β-pinene 3.5
β-selinene 2.7 Δ-3-carene 5.2
Bicyclogermacrene 6.3 Limonene 3.0
Limonene 0.1 E-β-ocimene 1.6
Myrcene tCyclosativene 2.0
F
2.3
Octanal 9.3 α-copaene 2.2
Caryophyllene oxide 31.6 E-caryophyllene 3.9
Isocaryophyllene-14-al 44.4 Alloaromadendrene 1.2
F
2.4
Unknown 1 22.8 β-selinene 2.9
Spathulenol 9.8 α-selinene 3.5
14-hydroxy-β-caryophyllene 38.0 δ-cadinene 6.7
Unknown 2 6.6 Selina-3.7(11)-diene 4.5
F
2.5
Unknown 1 8.8 F
B
trans-α-necrodyl acetate 30.5
Spathulenol 21.4 Lavandulyl acetate 8.2
14-hydroxy-β-caryophyllene 45.2 cis-α-necrodyl acetate 3.8
Unknown 2 2.9 1.8-cineole 32.4
Unknown 3 3.7 Lyratyl acetate 2.4
F
2.7
Caprylic acid 8.1 F
C
1.10-di-epi-cubenol 4.8
Buthyhidroxytoluene (solvent contaminant) 8.6 2.3.4.4-tetramethyl-5-methylene-cyclopent-2-enone 8.6
Tetradecanoic acid 40.5 Linalool 11.9
Hexadecanoic acid 8.5 epi-cubenol 1.3
trans-α-necrodol 20.1
Lavandulol 2.2
Viridiflorol 8.2
T-cadinol 2.4
T-muurolol 4.4
cis-linalool oxide (THP) 3.2
Unkown (C
10
H
16
O) 2.3
1.1.2.3-tetramethyl-4-hidroxymethyl-2-ciclopentene 7.2
14-norcadin-5-ene-4-one (Isomer) 4.3
F
D
trans-linalool oxide (THF) 12.6
α-terpineol 7.1
Verbenone 2.5
α-muurolol 1.3
trans-verbenol 3.1
Globulol 2.5
α-cadinol 48.7
Fig. 1. Effects of the fractions of the essential oils of Eryngium duriaei subsp juresianum and Lavandula luisieri (panel A) and of the test compounds, E-caryophyllene, myrcene
and limonene, (panel B) on IL-1β-induced NO production. Human chondrocytes were left untreated (Control, Ctrl) or treated with IL-1β, 10 ng/ml, for 24 h, following pre-
treatment for 30 min with the indicated concentrations of each fraction or pure compound. Each column represents the mean7S. E. M. of, at least, 4 independent
experiments.
n
Po0.05;
nn
Po0.01,
nnn
Po0.001 relative to IL-1βtreated cells.
§
Po0.05;
§§
Po0.01,
§§§
Po0.001 relative to control cells and
#
Po0.05;
##
Po0.01,
###
Po0.001
between the conditions indicated.
A.T. Rufino et al. / European Journal of Pharmacology 750 (2015) 141–15 0144
and we have recently reported the differential activity of
α
-pinene
enantiomers as inhibitors of pro-inflammatory and catabolic path-
ways in human chondrocytes (Rufino et al., 2014b). Nonetheless,
both essential oils have other minor components in common,
namely the monoterpene hydrocarbons, myrcene and limonene,
which were thus selected for pharmacological evaluation. Thus,
high purity E-caryophyllene, myrcene and limonene (structural
formulas are depicted in Fig. 2), obtained from commercial sources
indicated in Section 2, were screened for their ability to inhibit
IL-1
β
-induced NO production.
The obtained results (Fig. 1 panel B) show that, at non-cytotoxic
concentrations (Supplementary data), myrcene and limonene
effectively inhibited IL-1
β
-induced NO production, while E-caryo-
phyllene had no significant effect at any of the concentrations
tested.
Since myrcene and limonene showed inhibitory activity towards
IL-1
β
-induced NO production, various non-cytotoxic concentrations
were then tested to determine the respective concentration required
to inhibit NO production by 50% (IC
50
) and thus, to compare their
relative potencies. The IC
50
values obtained are 37.371.1
μ
g/ml for
myrcene and 85.3 71.2 mg/ml for limonene.
To determine whether the observed inhibition of NO produc-
tion by myrcene and limonene is due to inhibition of iNOS
expression, its mRNA (Fig. 3 panel B) and protein (Fig. 3 panel A)
levels were evaluated. Treatment with myrcene, 50 mg/ml, signifi-
cantly diminished IL-1
β
-induced iNOS mRNA and protein levels by
78% and 69%, respectively, while inhibition by limonene, even at
a concentration four fold higher, did not exceed 39% and 60%,
respectively.
3.2. Inhibition of IL-1
β
-induced NF-
κ
B activation by myrcene and
limonene
To further elucidate the mechanisms by which the two com-
pounds, myrcene and limonene, inhibit iNOS expression and to
further evaluate their potential as anti-osteoarthritic drugs, their
ability to inhibit IL-1
β
-induced NF-
κ
B activation was determined.
NF-
κ
B activation requires the phosphorylation, ubiquitination and
proteasomal degradation of its natural inhibitor, NF-
κ
B Inhibitor-
α
(I
κ
B-
α
), which, in unstimulated cells, retains NF-
κ
B dimers in
the cytoplasm. Once I
κ
B-
α
is degraded, the freed NF-
κ
B dimers
translocate to the nucleus and bind to specific sequences in the
promoter region of target genes promoting their transcription
(Hayden and Ghosh, 2008). Thus, we evaluated the protein levels
of phosphorylated and total I
κ
B-
α
by western blot and the binding
of the freed NF-
κ
B dimers to a specific DNA sequence by ELISA.
Since human cartilage samples are scarce and a large number
of cells is required, the ability of the test compounds to inhibit
Fig. 2. Structural formulas of the pure compounds tested.
Fig. 3. Effects of myrcene and limonene on IL-1β-induced iNOS protein (panel A) and mRNA (panel B) levels in human chondrocytes left untreated (Control, Ctrl) or treated
with IL-1-β, 10 ng/ml, for 24 h (panel A) or 6 h (panel B), following pre-treatment for 30 min with the indicated concentrations of the test compounds. Each column
represents the mean7S. E. M. of, at least, 4 independent experiments. A representative image is shown.
n
Po0.05,
nnn
Po0.001 relative to IL-1β-treated cells;
§
Po0.05,
§§
Po0.01 relative to control cells.
A.T. Rufino et al. / European Journal of Pharmacology 750 (2015) 141–150 145
IL-1
β
-induced NF-
κ
B-DNA binding was evaluated in the human
chondrocytic cell line, C-28/I2, while the levels of phosphorylated
and total I
κ
B-
α
were evaluated in primary human chondrocytes.
The results show that treatment with IL-1
β
dramatically increased
I
κ
B-
α
phosphorylation (Fig. 4 panel A) and decreased total I
κ
B-
α
levels, reflecting its almost complete degradation (Fig. 4 panel B),
followed by increased NF-
κ
B-DNA binding (Fig. 4 panel C). Treat-
ment with Bay 11-7082 (Bay), a specific NF-
κ
B inhibitor that
selectively prevents I
κ
B-
α
phosphorylation, or with the test
compounds markedly reduced IL-1
β
-induced I
κ
B-
α
phosphoryla-
tion (Fig. 4 panel A) and degradation (Fig. 4 panel B), as well as NF-
κ
B–DNA binding (Fig. 4 panel C). Interestingly, although the
degree of inhibition of I
κ
B-
α
phosphorylation and of NF-
κ
B-DNA
binding achieved with Bay was significantly higher than that
obtained with myrcene and limonene, I
κ
B-
α
degradation was
similarly decreased by the three compounds.
3.3. Effects of myrcene and limonene on IL-1
β
-induced MAPK
activation
Together with NF-
κ
B activation, signaling pathways involving
activation of members of the MAPK family also play an important
role in the proteolytic cartilage degradation process, namely in the
expression of MMPs (Mengshol et al., 2000). Thus, the ability of
myrcene and limonene to inhibit IL-1
β
-induced MAPK activation
was assessed by evaluating their phosphorylation levels.
Myrcene and limonene showed quite distinct effects on IL-1
β
-
induced JNK (Fig. 5 panel A), p38 (Fig. 5 panel B) and ERK1/2 (Fig. 5
panel C) phosphorylation. Myrcene significantly reduced IL-1
β
-
induced phosphorylation of the three MAPKs, while limonene was
effective in reducing p38 phosphorylation by near 39%, but
increased phosphorylated ERK1/2 by 30% and had no significant
effect on JNK.
3.4. Modulation of inflammatory, catabolic, anti-catabolic and
extracellular matrix gene expression by myrcene and limonene
Then, we evaluated the ability of the compounds tested to
counteract the effects of IL-1
β
on the expression of catabolic, anti-
catabolic and extracellular matrix genes, which, at least in part, are
mediated by NF-
κ
B and the MAPKs (Goldring and Otero, 2011). As
expected, treatment of human chondrocytes with IL-1
β
(10 ng/ml)
increased MMP-1 and -13 mRNA levels by nearly 9- and 5-fold,
respectively (Fig. 6 panel A), while decreasing TIMP-1 and -3
expressions (Fig. 6 panel B). Myrcene decreased the mRNA levels
of both MMPs by nearly 60%, relative to IL-1
β
, while limonene
reduced MMP-1 and -13 levels by 51% and 39%, respectively.
Nevertheless, the reduction of MMP-13 levels elicited by limonene
did not reach statistical significance.
On the other hand, limonene did not significantly change the
inhibitory effect of IL-1
β
on TIMP-1 and -3 mRNA levels, even
though it showed a tendency to increase TIMP-1 levels that did not
Fig. 4. Effects of myrcene and limonene on IL-1β-induced NF-κB activation, evaluated as the levels of phosphorylated (panel A) and total (panel B) IκB-αand NF-κB–DNA
complexes (panel C). C28/I2 cells were left untreated (Control, Ctrl) or treated with IL-1β, 10 ng/ml, for 5 min (panel A) or 30 min (panels B and C) following pretreatment
with or without the test compounds or the specific NF-κB inhibitor, Bay 11-7082 (Bay, 5 mM). Each column represents the mean7S. E. M. of 3 to 5 independent experiments.
n
Po0.05,
nn
Po0.01
nnn
Po0.001 relative to IL-1β-treated cells;
§
Po0.05,
§§§
Po0.001 relative to control cells.
A.T. Rufino et al. / European Journal of Pharmacology 750 (2015) 141–15 0146
Fig. 5. Effects of myrcene and limonene on IL-1β- induced activation of JNK, p38 and ERK1/2 in human chondrocytes. Phosphorylated levels of JNK (panel A), p38 (panel B)
and ERK (1/2) (panel C) were analyzed in total cell extracts of human chondrocytes left untreated (Control, Ctrl) or treated for 5 min with IL-1β, 10 ng/ml, following a pre-
treatment for 30 min with the indicated concentrations of myrcene, limonene or a specific inhibitor of the activation of each MAPK. Each column represents the mean7S. E.
M. of, at least, 4 independent experiments. Representative images are shown.
nn
Po0.01,
nnn
Po0.001 relative to IL-1β-treated cells and
§§§
Po0.001 relative to control cells.
iJNK: JNK inhibitor, SP600125 (20 mM); ip38: p38 inhibitor, SB203580 (20 mM); iERK: ERK1/2 inhibitor, U0126 (10 mM).
Fig. 6. Effects of myrcene and limonene on IL-1β-induced changes in the expression of catabolic and anti-catabolic genes. mRNA levels of MMP-1 and MMP-13 (panel A) and
TIMP-1 and TIMP-3 (panel B) were evaluatedby qRT-PCR. Each bar represents the mean7S. E. M. of, at least, 4 independent experiments in which human chondrocytes were
left untreated (Control, Ctrl) or treated for 12 h (panel A) or 24 h (panel B) with IL-1β, 10 ng/ml, in the presence or absence of the indicated compounds added to the cell
cultures 30 min before IL-1β.
n
Po0.05,
nn
Po0.01,
nnn
Po0.001 relative to IL-1β-treated cells and
§
Po0.05,
§§
Po0.01,
§§§
Po0.001 relative to control cells.
A.T. Rufino et al. / European Journal of Pharmacology 750 (2015) 141–150 147
reach statistical significance. On the contrary, myrcene not only
completely reversed the inhibitory effect exerted by IL-1
β
,asit
effectively increased TIMP-1 and -3 levels approximately 2- and
1.3-fold above those in untreated control cells, respectively, which
correspond to even larger increases if compared to TIMP-1 and
-3 mRNA levels in cells treated with IL-1
β
alone.
To assess the potential ability of the test compounds to inhibit the
deleteriouseffectsofIL-1
β
in anabolic responses that are essential for
repair of damaged articular cartilage, the expression of collagen II and
aggrecan was evaluated. Furthermore, the ability of the test com-
pounds to decrease the expression of the non-cartilage specific,
collagen I gene, induced by IL-1
β
, was also evaluated. Chondrocyte
treatment with 10 ng/ml IL-1
β
,for24h,significantly increased
collagen I mRNA levels, while decreasing those of collagen II and
aggrecan, relative to untreated control cells (Fig. 7). Treatment of
human chondrocytes with myrcene or limonene caused no significant
changesoncollagenII(Fig. 7 panel A) and aggrecan (Fig. 7 panel B)
mRNA levels compared to those in cells treated with IL-1
β
alone.
Nonetheless, both treatments were able to completely abolish or even
reverse the increase in collagen I mRNA levels induced by IL-1
β
.
4. Discussion
The results obtained in this study identify two monoterpene
hydrocarbons, myrcene and limonene, as capable of inhibiting IL-1
β
-
induced NO production in human chondrocytes. The specific activities
(activity/
μ
g) relative to inhibition of IL-1-induced NO production, of
myrcene and limonene are 1.33%/
μ
gand0.57%/
μ
g, respectively, while
for the active fractions (F
1
and F
A
) of the essential oils of E. duriaei
subsp. juresianum and L. luisieri they are 1%/
μ
g and 3.0%/
μ
g, respec-
tively. Since myrcene and limonene are only minor components of
those fractions, it is likely that other constituents are also active and
contribute to the effects observed. Moreover, since both fractions
contain several distinct compounds, none of which is present in
sufficiently high amounts to justify the effects observed, either the
active compound in those fractions is significantly more potent than
myrcene and limonene or various active compounds, including these
two, act synergistically, or at least, additively, to achieve a similar or
even higher degree of inhibition of IL-1-induced NO production.
Unfortunately, as mentioned in Section 3.1, the major components of
those fractions are either not readily available from commercial
sources or have been previously studied, as is the case for (þ)-
α
-
pinene that we showed to have anti-inflammatory and anti-catabolic
activities in human chondrocytes (Rufino et al., 2014b). Thus, identi-
fication of the other active compounds in those fractions is, at present,
impracticable.
On the other hand, the sesquiterpene hydrocarbon, E-caryo-
phyllene, which is a major component of the active fraction of the
essential oil of E. duriaei subsp. juresianum, is completely inactive.
This finding is somewhat unexpected as E-caryophyllene has been
reported to exert anti-inflammatory effects by activating cannabi-
noid CB2 receptors (Bento et al., 2011; Medeiros et al., 2007) and
endogenous and synthetic cannabinoids have been reported to
decrease inflammation in animal models of arthritis (Sumariwalla
et al., 2004) and to inhibit IL-1-induced NO production in bovine
chondrocytes (Mbvundula et al., 2005).
The two active compounds, myrcene and limonene, show clear
qualitative and quantitative differences in terms of ability to
inhibit IL-1
β
-induced responses. Myrcene was the most potent
in inhibiting NO production, as indicated by an IC
50
value less than
half of that found for limonene. Myrcene was also more effective
than limonene in preventing other inflammatory and catabolic
responses in human chondrocytes, namely expression of iNOS,
MMP-1 and MMP-13 induced by IL-1
β
, likely reflecting, at least in
part, the stronger inhibition of NF-
κ
B and the ability to inhibit all
three MAPKs. These findings are in agreement with another study
that reported anti-inflammatory properties of myrcene in a mouse
model of pleurisy induced by zymosan and bacterial lipopolysac-
charide where it inhibited the production of NO and inflammatory
cytokines (Souza et al., 2003). Furthermore, myrcene, but not
limonene, caused a net increase in the expression of the anti-
catabolic genes, TIMP-1 and -3, which in combination with the
decrease in MMP-1 and -13 expression can cause a significant
reduction of the catabolic milieu characteristic of OA.
On the other hand, myrcene also completely prevented the
increase in collagen I induced by IL-1
β
. Collagen I is not normally
found in articular cartilage and its expression increases in OA and in
association with chondrocyte dedifferentiation, a process that involves
several alterations of chondrocyte gene expression and morphology
and leads to the formation of fibrocartilage (Martin et al., 2001).
Therefore, even though it did not increase the specific anabolic
responses of human chondrocytes, myrcene may be effective in
preventing chondrocyte dedifferentiation associated with increased
collagen I expression, while decreasing inflammatory and catabolic
processes directly involved in cartilage destruction.
Reports on pharmacological properties of limonene are scarce, but
it has been shown to have antimicrobial properties (Bevilacqua et al.,
Fig. 7. Effects of myrcene and limonene on IL-1β-induced changes in the expression of extracellular matrix genes. mRNA levels of collagens I and II (panel A) and aggrecan
(panel B) were evaluated by qRT-PCR. Each bar represents the mean7S. E. M. of, at least, 4 independent experiments in which human chondrocytes were left untreated
(Control, Ctrl) or treated for 24 h with IL-1β, 10 ng/ml, in the presence or absence of the indicated compounds added to the cell cultures 30 min before IL-1β.
n
Po0.05,
nnn
Po0.001 relative to IL-1β-treated cells and
§§
Po0.01,
§§§
Po0.001 relative to control cells.
A.T. Rufino et al. / European Journal of Pharmacology 750 (2015) 141–15 0148
2010)andanti-inflammatory effects in a mouse model of LPS-induced
acute lung injury by suppressing MAPK and NF-
κ
Bpathways(Chi
et al., 2012). The results presented here only partially agree with this
study, since limonene inhibited NF-
κ
B and p38 activation, but did not
affect IL-1
β
-induced JNK and actually potentiated ERK1/2 activation,
suggesting that this compound has cell- and/or stimulus-specific
effects. On the other hand, ERK1/2 is required for a number of cellular
processes, including activation of c-fos expression which, among other
functions, is involved in cell survival (Karin et al., 1997; Shaulian and
Karin, 2002). Whether increased activation of ERK1/2 by limonene
contributes to enhance chondrocyte survival was not addressed in this
study, but is an interesting possibility to study further, as increased
chondrocyte death is a relevant feature of OA (Johnson et al., 2008).
Nonetheless, since ERK1/2 has also been shown to inhibit proteoglycan
synthesis and to promote inflammatory and catabolic responses in
chondrocytes (Scherle et al., 1997), the net effect resulting from its
induction by limonene is likely undesirable, compromising its poten-
tial utility as a therapeutic agent in OA.
Form another point of view, it is intriguing that limonene
induced ERK1/2 activation while decreasing p38 and not affecting
JNK activation. This is even more puzzling as myrcene was able to
inhibit the activation of all three MAPKs. Even though we cannot
provide an explanation for the differential effects of both com-
pounds, it seems reasonable to admit that they may act on distinct
molecular targets. Although the exact signaling pathways that link
IL-1
β
binding to its receptor to downstream events are still
incompletely understood, the immediate upstream activators of
each MAPK have been identified. While some of those, namely the
mitogen-activated protein kinase kinase 4 (MKK4 or MEK4), can
activate both JNK and p38, others specifically activate each of these
MAPK. MKK3 and MKK6 are p38 activators while MKK7 activates
JNK and MKK1 activates ERK1/2 (Weber et al., 2010). Thus,
limonene may inhibit MKK3 or MKK6 without affecting MKK4 or
MKK7 or any other upstream intermediate, while inducing MKK1
activation, either direct or indirectly. On the other hand, myrcene
may act on another intermediate common to the three MAPK and
also to NF-
κ
B or instead it may independently inhibit the MAPK
and NF-
κ
B signaling pathways. Clearly, further studies are required
to identify the specific molecular targets of myrcene and limonene.
Nonetheless, this may be a difficult task given the complexity of
each of these pathways and the extensive cross-talk between them
(Virtue et al., 2012; Weber et al., 2010).
In comparison with (þ)-
α
-pinenethatwepreviouslyreportedto
have anti-inflammatory and anti-catabolic properties in human
chondrocytes (Rufino et al., 2014b), myrcene shows potential advan-
tages since, besides inhibiting iNOS expression and activity and NF-
κ
B
and JNK activation to a similar extent but at lower concentrations, it
further decreased ERK1/2 and p38 activation and increased anti-
catabolic responses, namely TIMP-1 and -3 expression. Moreover,
myrcene can also promote the maintenance of the differentiated
chondrocyte phenotype, as it also decreased collagen I expression.
Nonetheless, none of the compounds tested, nor (þ)-
α
-pinene, seem
effective in promoting the expression of articular cartilage matrix-
specific genes and, thus, are unlike to promote the repair of areas
already damaged. Moreover, the potency of myrcene is relatively low
which may also hinder its therapeutic efficacy.
5. Conclusions
Myrcene has significant anti-inflammatory and anti-catabolic
properties in vitro suggesting that it may be useful to halt or, at
least, slow down cartilage destruction and, thus, OA progression.
Future studies in in vivo models of OA are thus warranted to
evaluate its potential as a disease-modifying osteoarthritis drug.
Acknowledgment
This work was co-funded by FEDER (QREN), through Programa Mais
Centro under project CENTRO-07-ST24-FEDER-002006, and through
Programa Operacional Factores de Competitividade –COMPETE and
national funds via FCT –Fundação Portuguesa para a Ciência e a
Tecnologia under projects PestC/SAU/LA0001/20132014, Pest-OE/SAU/
UI0177/2011, PTDC/EME-TME/113039/20 09, and the PhD fellowships,
SFRH/BD/47470/2008, SFRH/BD/78188/2011 and SFRH/BD/79600/2011.
Appendix A. Supporting information
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.ejphar.2015.01.018.
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