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Evaluation of the anti-inflammatory, anti-catabolic and pro-anabolic effects of E-caryophyllene, myrcene and limonene in a cell model of osteoarthritis


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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 monoterpene, 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-1β-induced nitric oxide production (IC50=37.3 μg/ml and 85.3 µg/ml, respectively), but E-caryophyllene was inactive. Myrcene, and limonene to a lesser extent, also decreased IL-1β-induced NF-κB, 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-1β-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-1β. 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.
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Immunopharmacology and inammation
Evaluation of the anti-inammatory, anti-catabolic and pro-anabolic
effects of E-caryophyllene, myrcene and limonene in a cell model
of osteoarthritis
Ana Teresa Runo
, Madalena Ribeiro
, Cátia Sousa
, Fernando Judas
Lígia Salgueiro
, Carlos Cavaleiro
, Alexandrina Ferreira Mendes
Centre for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal
Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal
Orthopedics Department and Bone Bank, University and Hospital Center of Coimbra, Coimbra, Portugal
Faculty of Medicine, University of Coimbra, Coimbra, Portugal
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
Chronic inammation
Natural products
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-inammatory, anti-catabolic and pro-anabolic activities in human
chondrocytes. At non-cytotoxic concentrations, myrcene and limonene inhibited IL-1
-induced nitric
oxide production (IC
g/ml and 85.3 mg/ml, respectively), but E-caryophyllene was inactive.
Myrcene, and limonene to a lesser extent, also decreased IL-1
-induced NF-
B, JNK and p38 activation
and the expression of inammatory (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
-treated cells.
None of the compounds tested was able to increase the expression of cartilage matrix-specic genes
(collagen II and aggrecan), but both compounds prevented the increased expression of the non-cartilage
specic, collagen I, induced by IL-1
. These data show that myrcene has signicant anti-inammatory
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 inammation and progressive loss of the articular
cartilage, associated with changes in the subchondral bone and other
jointtissues.Itaffects1015% 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 identication 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-
inammatory and anti-catabolic properties in human chondrocytes to
be used as sources of compounds with potential anti-osteoarthritic
activity (Neves et al., 2009; Runo et al., 2014a). In this context, we
recently identied the essential oils of Eryngium duriaei subsp. juresia-
num and Lavandula luisieri as possessing anti-inammatory properties
Contents lists available at ScienceDirect
journal homepage:
European Journal of Pharmacology
0014-2999/&2015 Elsevier B.V. All rights reserved.
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: (A.T. Runo), (M. Ribeiro), (C. Sousa), (F. Judas), (L. Salgueiro), (C. Cavaleiro), (A.F. Mendes).
European Journal of Pharmacology 750 (2015) 141150
in human chondrocytes (Runo et al., 2014a). The current study
aims at identifying active compounds of these essential oils and
further characterizing their pharmacological properties in human chon-
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-
inammatory cytokines, like interleukin-1
), 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-
specic matrix components, collagen II and aggrecan. This is accom-
panied by impaired reparative responses involving downregulation of
(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-inammatory and pro-catabolic mediators, like nitric oxide
(NO), amplies and perpetuates cartilage destruction (Boileau et al.,
2002; Rosa et al., 2008; Sasaki et al., 1998). The transcription factor,
Nuclear Factor-
B), and the family of mitogen-activated protein
kinases (MAPK) play a central role in modulating the expression of
those catabolic and inammatory 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
as an in vitro cartilage degradation model
that emulates the damage seen in OA. Using this model, we
evaluated the inhibition of IL-1
-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
-induced NO production were selected for further
assessment of anti-osteoarthritic potential. For this, we evaluated
their ability to modulate IL-1
-induced signaling pathways invol-
ved in the expression of inammatory and catabolic genes, namely
activation of NF-
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
on the expression of inammatory
(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 ash chromatography on silica gel 63
m (Merck) using a 2 cm 40 cm Omnit (Sigma-Aldrich)
glass columns. Elution was made in step gradients from 100% of
n-pentane, n-pentane /ethyl ether mixtures, till a nal concentra-
tion of 100% ethyl ether. Collected fractions were analyzed by
gas chromatographymass spectroscopy (GC/MS) and combined
if having similar composition. Analysis was performed using a
Hewlett-Packard 6890 gas chromatograph tted with a HP1 fused
silica column (polydimethylsiloxane 30 m 0.25 mm i.d., lm
thickness 0.25
m), interfaced with an Hewlett-Packard mass
selective detector 5973 (Agilent Technologies) operated by HP
Enhanced ChemStation software, version A.03.00. GCMS para-
meters: oven temperature program: 70220 1C(31C min
220 1C (15 min); injector temperature: 250 1C; carrier gas: helium,
adjusted to a linear velocity of 30 cm s
; 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
A; scan range: 35350 units; scans s
: 4.51.
Compounds were identied through their GC retention and mass
spectra. Retention indices, calculated by linear interpolation rela-
tive to retention times of C
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 (2070 years old, mean¼52.9,
n¼31) and patients (5873 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-
BDNA 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 gures and their
legends, were added to the chondrocyte cultures 30 min before
the pro-inammatory stimulus (IL-1
, 10 ng/ml) and maintained
for the experimental period indicated in gure legends. The nal
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
, 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. Runo et al. / European Journal of Pharmacology 750 (2015) 14115 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
or rabbit polyclonal anti-human I
, 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-
-Tubulin monoclonal antibody was used to detect
-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-
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-
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
B oligonucleotide (NoShift NF-
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-
BDNA complexes formed and, thus, to the
amount of active NF-
B dimers present in each sample. In parallel,
the specicity of the reaction was conrmed in competition assays
where addition of a 10-fold molar excess of non-biotinylated wild-
type or mutant oligonucleotides (NoShift NF-
B Reagents; Nova-
gen) to binding reactions containing nuclear extracts from IL-1
treated cells abrogated or did not affect, respectively, the forma-
tion of NF-
BDNA complexes.
2.7. Total RNA extraction and quantitative real-time RT-PCR (qRT-
Total RNA was extracted from human condrocytes using TRI-
Reagent (Invitrogen, Life Technologies, Co; Paisley, UK) and
quantied 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. Specic 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
g/ml of transcribed cDNA in a nal volume of 20 mL.
The efciency of the amplication reaction for each gene was
calculated using a standard curve of a series of diluted cDNA
samples, and the specicity of the amplication 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 Pfafmethod, a
variation of the
CT method corrected for gene-specicef-
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 KolmogorovSmirnov
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
signicant at Po0.05.
3. Results
3.1. Selection of inhibitors of IL-1-induced iNOS expression and NO
Various fractions of the essential oils of E. duriaei subsp.
juresianum and L. luisieri were separated in amounts sufcient
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
g/ml (Supplementary
data). The results (Fig. 1 panel A) show that the hydrocarbon-
containing fractions (F
and F
) of both essential oils decreased IL-
-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
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
-treated cells. Fractions F
and F
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
Table 1
Oligonucleotide primer pairs used for qRT-PCR.
Gene name Genbank accession number Forward sequence Reverse sequence
iNOS NM_000625.4 5
MMP-1 NM_001145938.1 5
MMP-13 NM_002422.3 5
TIMP-1 NM_003254.2 5
TIMP-3 NM_000362.4 5
Collagen II NM_001844.4 5
Collagen I NM_000088.3 5
Aggrecan NM_001135 5
HPRT-1 NM_000194.2 5
A.T. Runo et al. / European Journal of Pharmacology 750 (2015) 141150 143
a concentration of 50 mg/ml, also signicantly inhibited IL-1
uced NO production, although to a lesser extent than found with
. No signicant effects were obtained with F
at any of the
concentrations tested. Therefore, the hydrocarbon-containing frac-
tions (F
and F
) of both essential oils were considered the most
promising for selection of compounds for further studies.
As shown in Table 2, these fractions (F
and F
) are composed of
monoterpene and sesquiterpene hydrocarbons, mainly
and 3,5-dimethylene-1,4,4-trimethylcyclopentene, and E-caryo-
-neocallitropsene, germacrene D,
-selinene and bicy-
clogermacrene, respectively. Of these compounds, only
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 %
E-caryophyllene 29.5 F
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
Octanal 9.3 α-copaene 2.2
Caryophyllene oxide 31.6 E-caryophyllene 3.9
Isocaryophyllene-14-al 44.4 Alloaromadendrene 1.2
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
Unknown 1 8.8 F
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
Caprylic acid 8.1 F
1.10-di-epi-cubenol 4.8
Buthyhidroxytoluene (solvent contaminant) 8.6 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
Viridiorol 8.2
T-cadinol 2.4
T-muurolol 4.4
cis-linalool oxide (THP) 3.2
Unkown (C
O) 2.3 7.2
14-norcadin-5-ene-4-one (Isomer) 4.3
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
Po0.001 relative to IL-1βtreated cells.
Po0.001 relative to control cells and
between the conditions indicated.
A.T. Runo et al. / European Journal of Pharmacology 750 (2015) 14115 0144
and we have recently reported the differential activity of
enantiomers as inhibitors of pro-inammatory and catabolic path-
ways in human chondrocytes (Runo 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
-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
-induced NO production, while E-caryo-
phyllene had no signicant effect at any of the concentrations
Since myrcene and limonene showed inhibitory activity towards
-induced NO production, various non-cytotoxic concentrations
were then tested to determine the respective concentration required
to inhibit NO production by 50% (IC
) and thus, to compare their
relative potencies. The IC
values obtained are 37.371.1
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, signi-
cantly diminished IL-1
-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%,
3.2. Inhibition of IL-1
-induced NF-
B activation by myrcene and
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
-induced NF-
B activation was determined.
B activation requires the phosphorylation, ubiquitination and
proteasomal degradation of its natural inhibitor, NF-
B Inhibitor-
), which, in unstimulated cells, retains NF-
B dimers in
the cytoplasm. Once I
is degraded, the freed NF-
B dimers
translocate to the nucleus and bind to specic 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
by western blot and the binding
of the freed NF-
B dimers to a specic 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.
Po0.001 relative to IL-1β-treated cells;
Po0.01 relative to control cells.
A.T. Runo et al. / European Journal of Pharmacology 750 (2015) 141150 145
-induced NF-
B-DNA binding was evaluated in the human
chondrocytic cell line, C-28/I2, while the levels of phosphorylated
and total I
were evaluated in primary human chondrocytes.
The results show that treatment with IL-1
dramatically increased
phosphorylation (Fig. 4 panel A) and decreased total I
levels, reecting its almost complete degradation (Fig. 4 panel B),
followed by increased NF-
B-DNA binding (Fig. 4 panel C). Treat-
ment with Bay 11-7082 (Bay), a specic NF-
B inhibitor that
selectively prevents I
phosphorylation, or with the test
compounds markedly reduced IL-1
-induced I
tion (Fig. 4 panel A) and degradation (Fig. 4 panel B), as well as NF-
BDNA binding (Fig. 4 panel C). Interestingly, although the
degree of inhibition of I
phosphorylation and of NF-
binding achieved with Bay was signicantly higher than that
obtained with myrcene and limonene, I
degradation was
similarly decreased by the three compounds.
3.3. Effects of myrcene and limonene on IL-1
-induced MAPK
Together with NF-
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
-induced MAPK activation
was assessed by evaluating their phosphorylation levels.
Myrcene and limonene showed quite distinct effects on IL-1
induced JNK (Fig. 5 panel A), p38 (Fig. 5 panel B) and ERK1/2 (Fig. 5
panel C) phosphorylation. Myrcene signicantly reduced IL-1
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 signicant
effect on JNK.
3.4. Modulation of inammatory, 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
on the expression of catabolic, anti-
catabolic and extracellular matrix genes, which, at least in part, are
mediated by NF-
B and the MAPKs (Goldring and Otero, 2011). As
expected, treatment of human chondrocytes with IL-1
(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
, 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 signicance.
On the other hand, limonene did not signicantly change the
inhibitory effect of IL-1
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-κBDNA
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 specic NF-κB inhibitor, Bay 11-7082 (Bay, 5 mM). Each column represents the mean7S. E. M. of 3 to 5 independent experiments.
Po0.001 relative to IL-1β-treated cells;
Po0.001 relative to control cells.
A.T. Runo et al. / European Journal of Pharmacology 750 (2015) 14115 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 specic inhibitor of the activation of each MAPK. Each column represents the mean7S. E.
M. of, at least, 4 independent experiments. Representative images are shown.
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.
Po0.001 relative to IL-1β-treated cells and
Po0.001 relative to control cells.
A.T. Runo et al. / European Journal of Pharmacology 750 (2015) 141150 147
reach statistical signicance. On the contrary, myrcene not only
completely reversed the inhibitory effect exerted by IL-1
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
To assess the potential ability of the test compounds to inhibit the
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 specic,
collagen I gene, induced by IL-1
, was also evaluated. Chondrocyte
treatment with 10 ng/ml IL-1
,for24h,signicantly 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 signicant
changesoncollagenII(Fig. 7 panel A) and aggrecan (Fig. 7 panel B)
mRNA levels compared to those in cells treated with IL-1
Nonetheless, both treatments were able to completely abolish or even
reverse the increase in collagen I mRNA levels induced by IL-1
4. Discussion
The results obtained in this study identify two monoterpene
hydrocarbons, myrcene and limonene, as capable of inhibiting IL-1
induced NO production in human chondrocytes. The specic activities
g) relative to inhibition of IL-1-induced NO production, of
myrcene and limonene are 1.33%/
g, respectively, while
for the active fractions (F
and F
) of the essential oils of E. duriaei
subsp. juresianum and L. luisieri they are 1%/
g and 3.0%/
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
sufciently high amounts to justify the effects observed, either the
active compound in those fractions is signicantly 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)-
pinene that we showed to have anti-inammatory and anti-catabolic
activities in human chondrocytes (Runo et al., 2014b). Thus, identi-
cation of the other active compounds in those fractions is, at present,
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 nding is somewhat unexpected as E-caryophyllene has been
reported to exert anti-inammatory 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 inammation 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
-induced responses. Myrcene was the most potent
in inhibiting NO production, as indicated by an IC
value less than
half of that found for limonene. Myrcene was also more effective
than limonene in preventing other inammatory and catabolic
responses in human chondrocytes, namely expression of iNOS,
MMP-1 and MMP-13 induced by IL-1
, likely reecting, at least in
part, the stronger inhibition of NF-
B and the ability to inhibit all
three MAPKs. These ndings are in agreement with another study
that reported anti-inammatory properties of myrcene in a mouse
model of pleurisy induced by zymosan and bacterial lipopolysac-
charide where it inhibited the production of NO and inammatory
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 signicant
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
. 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 brocartilage (Martin et al., 2001).
Therefore, even though it did not increase the specic anabolic
responses of human chondrocytes, myrcene may be effective in
preventing chondrocyte dedifferentiation associated with increased
collagen I expression, while decreasing inammatory 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.
Po0.001 relative to IL-1β-treated cells and
Po0.001 relative to control cells.
A.T. Runo et al. / European Journal of Pharmacology 750 (2015) 14115 0148
2010)andanti-inammatory effects in a mouse model of LPS-induced
acute lung injury by suppressing MAPK and NF-
et al., 2012). The results presented here only partially agree with this
study, since limonene inhibited NF-
B and p38 activation, but did not
affect IL-1
-induced JNK and actually potentiated ERK1/2 activation,
suggesting that this compound has cell- and/or stimulus-specic
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 inammatory 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
binding to its receptor to downstream events are still
incompletely understood, the immediate upstream activators of
each MAPK have been identied. While some of those, namely the
mitogen-activated protein kinase kinase 4 (MKK4 or MEK4), can
activate both JNK and p38, others specically 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-
B or instead it may independently inhibit the MAPK
and NF-
B signaling pathways. Clearly, further studies are required
to identify the specic molecular targets of myrcene and limonene.
Nonetheless, this may be a difcult 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)-
have anti-inammatory and anti-catabolic properties in human
chondrocytes (Runo et al., 2014b), myrcene shows potential advan-
tages since, besides inhibiting iNOS expression and activity and NF-
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)-
-pinene, seem
effective in promoting the expression of articular cartilage matrix-
specic 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 efcacy.
5. Conclusions
Myrcene has signicant anti-inammatory 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.
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.
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...  1-Tetradecanol, conocido también como alcohol miristílico en la elaboración de productos cosméticos es utilizado como un emoliente (Avci et al., 2014), y ayuda en la solubilidad de los fluidos al trabajar con metales (Geier et al., 2006).  (E)-Cariofileno, en estudios previos del aceite esencial de Melinis minutiflora, ha sido detectado como constituyente mayoritario (Kimani et al., 2000;Tolosa et al., 2019), en cuanto a su actividad biológica ha sido determinado como un antiinflamatorio, anticatabólico, pro anabólico (Rufino et al., 2015), actividad disuasoria contra la ovoposición de Aedes aegypti (trasmisor del dengue) (Santos et al., 2015) y como bioinsecticida para el control de la hormiga Dorymyrmex thoracicus (Oliveira et al., 2019).  Germacreno D, Kimani et al. (2000), reporta la presencia de Germacreno D, en un 5,0%. ...
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El presente estudio determinó los componentes químicos volátiles de los aceites esenciales (AEs) de las especies Melinis minutiflora y Lantana camara, mediante cromatografía de gases acoplado a espectrómetro de masas, las especies se recolectaron en los cantones de Quilanga y Loja, la extracción se hizo mediante destilación por arrastre de vapor. En el aceite esencial (AE) de M. minutiflora se identificaron 20 compuestos, representan el 93,21%, los compuestos en mayor concentración: 1-tetradecanol (16,30%), (E)-cariofileno (12,44 %), germacreno D (10,99%), (E)-nerolidol (8,28 %), δ-cadineno (5,61 %), α-humuleno (5,36 %), viridiflorol (4,78 %) y (Z)-β-farneseno (4,76 %). En el AE de L. camara se identificaron 68 compuestos, representan el 96,54%, los compuestos en mayor concentración (E)-cariofileno (15,46%), germacreno D (12,21%), α-humuleno (9,92%), bicyclogermacreno (7,06%), γ-terpineno (5,97%) y germacreno B (4,66%); las especies M. minutiflora y L. cámara, presentan propiedades repelentes, acaricidas en larvas, adultas de Amblyomma cajennense y Rhipicephalus (Boophylus) microplus.
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Butter extracted from bacuriseeds (Platonia insignis) is widely used as a raw material in the formulation of cosmetic products. Furthermore, with the advances in research, other biological properties beneficial to health have been investigated. Therefore, tests to assess the safety for consumption are essential. This study aimed to evaluate in vitroand in vivotoxicity parameters of industrialized butter obtained from bacuriseeds (MIB). Cytotoxic activity was performed using the MTT assay in murine macrophages and in sheep blood erythrocytes and the acute oral toxicological evaluation in vivowas performed using the Fixed Dose Test. In the in vitroassays, a concentration-dependent reduction in cell viability was observed by evaluating the mitochondrial activity, with a mean cytotoxic concentration value (CC50) equal to 124.036 μg/mL and low hemolytic activity, since MIB promoted less than 10% hemolysis at the highest concentration tested (800 μg/mL), and it is not possible to determine the average hemolytic concentration. In the in vivotest, no deaths or alterations were observed in the evaluated clinical and behavioral parameters, as well as in the serum biochemical parameters, in the relative weight and in the macroscopic evaluation of the organs. All these results demonstrate an adequate safety profile, enabling the performance of subchronic toxicity tests, in order to allow the safe exploration of the therapeutic potential of this product.
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Efficient functionality of the immune system is needed to fight against the development of infectious diseases, including, among others, serious recurrent chronic infections. Research has shown that many modern common diseases, such as inflammatory bowel diseases and cardiovascular diseases, e.g., thromboembolism, cancer, obesity, or depression, are connected with inflammatory processes. Therefore, new, good stimulators of the immune system’s response are sought. They include synthetic compounds as well as biological preparations such as lipopolysaccharides, enzymes, bacterial metabolites, and secondary metabolites of plants, demonstrating a multidirectional effect. Essential oils are characterized by many invaluable activities, including antimicrobial, antioxidant, anti-inflammatory, and immunostimulating. Essential oils may stimulate the immune system via the utilization of their constituents, such as antibodies, cytokines, and dendritic cells. Some essential oils may stimulate the proliferation of immune-competent cells, including polymorphonuclear leukocytes, macrophages, dendritic cells, natural killer cells, and B and T lymphocytes. This review is focused on the ability of essential oils to affect the immune system. It is also possible that essential oil components positively interact with recommended anti-inflammatory and antimicrobial drugs. Thus, there is a need to explore possible synergies between essential oils and their active ingredients for medical use.
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Cervical cancer is the fourth most common malignancy among women, which also turns out to be the most common cause of death in women worldwide. Medicinal plants have traditionally been used to treat various diseases and disorders. The current study utilizes the molecular docking technique to investigate the anticancer potential of Juglans regia phytoconstituents against cervical cancer target proteins. This study includes the microarray dataset analysis of GSE63678 from the NCBI Gene Expression Omnibus database to identify differentially expressed genes. Furthermore, network biology approaches were employed to construct protein-protein interaction of differentially expressed genes. Next, the computation of topological parameters utilizing Cytohubba renders the top five hub genes (IGF1, FGF2, ESR1, MYL9, and MYH11). In addition, Juglans regia phytocompounds mined from the IMPPAT database were subjected to molecular docking analysis against identified hub genes. The application of molecular dynamics simulation validated the stability of prioritized docked complexes with minimum binding energy.
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Diabetes is a metabolic disorder of multiple etiologies, which is mainly characterized by persistently high levels of glucose in the blood. Estimates from the World Health Organization (WHO) indicate that approximately 422 million people have diabetes and 1.6 million deaths are directly attributed to the disease each year. Faced with such relevant numbers, natural therapies are sought that are effective, efficient and low-cost for the referred pathology. Several natural products are already being used as adjuvants in the treatment of diabetes because they have hypoglycemic action, such as monoterpenes. However, there is a strong intention to expand the portfolio of natural products with hypoglycemic compounds. Thus, the objective of this study was to list information from the literature on diabetes and the applicability of natural products, emphasizing the antidiabetic action of the monoterpenes R-(+)-Limonene and (-)-Carveol. A narrative review was performed. The selected articles were directed to the pathophysiological parameters of diabetes and its complications, aspects of the use of natural products, monoterpenes and their therapeutic effects as antidiabetic agents. The research was carried out in the following databases: PubMed, Scielo, Portal Periódicos Capes, Scopus, Web of Science and Science Direct. Studies show that diabetes is a complex disease that causes a series of complications and can be treated with oral hypoglycemic agents, insulin, diet and physical activity. Researches related to R-(+)-Limonene and (-)-Carveol, in rats, showed positive results with antidiabetic action, acting in the reduction of the plasmatic levels of glucose, triglycerides and cholesterol and of the expression of inflammatory markers, as well as in the increased effect of antioxidant enzymes. However, studies involving these monoterpenes are still incipient, making this research timely. Thus, R-(+)-Limonene and (-)-Carveol are promising candidates for further research to elucidate their therapeutic effects alone or in combination with other antidiabetic monoterpenes.
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Adams, R. P. 2007. Identification of essential oil components by gas chromatography/ mass spectrometry, 4th Edition. Allured Publ., Carol Stream, IL Is out of print, but you can obtain a free pdf of it at
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Essential oils are complex mixtures isolated from aromatic plants which may possess antioxidant and anti-inflammatory activities of interest in thye food and cosmetic industries as well as in the human health field. In this work, a review was done on the most recent publications concerning their antioxidant and anti-inflammatory activities. At the same time a survey of the methods generally used for the evaluation of antioxidant activity and some of the mechanisms involved in the anti-inflammatory activities of essential oils are also reported.
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The discovery of miRNAs has revolutionized the way we examine the genome, RNA products, and the regulation of transcription and translation. Their ability to modulate protein expression through mRNA degradation and translation repression resulted in avid scientific interest in miRNAs over the past decade. This research has led to findings that indicate miRNAs can regulate an array of cellular functions such as cellular apoptosis, proliferation, differentiation, and metabolism. Specifically, the capability of miRNAs to finely-tune gene expression naturally lends itself to immune system regulation which requires precise control for proper activity. In fact, abnormal miRNAs expression is often seen with inflammatory disorders like rheumatoid arthritis, systemic lupus erthematosus, experimental autoimmune encephalomyelitis, and inflammatory cancers. As a result, research investigating miRNAs modulation of immune cell proliferation, differentiation, and cellular signaling has yielded fruitful results. Specifically, in this review, we will examine the impact of miRNAs on tolllike receptor (TLRs) and interleukin-1beta (IL-1beta) signaling, which are integral in the proper functioning of the innate immune system. These signaling pathways share several key downstream signaling adaptors and therefore produce similar downstream effects such as the production of pro-inflammatory cytokines, chemokines, and interferons. This review will examine in depth the specific interactions of miRNAs with receptors, adaptor molecules, and regulator molecules within these cellular pathways. In addition, we will discuss the modulation of miRNAs' expression by TLR and IL-1R signaling through positive and negative feedback loops.
Context: Effective drugs to treat osteoarthritis (OA) and inflammatory bowel disease (IBD) are needed. Objective: To identify essential oils (EOs) with anti-inflammatory activity in cell models of OA and IBD. Materials and methods: EOs from Eryngium duriaei subsp. juresianum (M. Laínz) M. Laínz (Apiaceae), Laserpitium eliasii subsp. thalictrifolium Sennen & Pau (Apiaceae), Lavandula luisieri (Rozeira) Rivas-Martínez (Lamiaceae), Othantus maritimus (L.) Hoff. & Link (Asteraceae), and Thapsia villosa L. (Apiaceae) were analyzed by GC and GC/MS. The anti-inflammatory activity of EOs (5-200 μg/mL) was evaluated by measuring inducible nitric oxide synthase (iNOS) and nuclear factor-κB (NF-κB) activation (total and phosphorylated IκB-α), in primary human chondrocytes and the intestinal cell line, C2BBe1, stimulated with interleukin-1β (IL-1β) or interferon-γ (IFN-γ), IL-1β and tumor necrosis factor-α (TNF-α), respectively. Results: The EO of L. luisieri significantly reduced iNOS (by 54.9 and 81.0%, respectively) and phosphorylated IκB-α (by 87.4% and 62.3%, respectively) in both cell models. The EO of E. duriaei subsp. juresianum caused similar effects in human chondrocytes, but was inactive in intestinal cells, even at higher concentrations. The EOs of L. eliasii subsp. thalictrifolium and O. maritimus decreased iNOS expression by 45.2 ± 8.7% and 45.2 ± 6.2%, respectively, in C2BBe1 cells and were inactive in chondrocytes. The EO of T. villosa was inactive in both cell types. Discussion and conclusion: This is the first study showing anti-inflammatory effects of the EOs of L. luisieri and E. duriaei subsp. juresianum. These effects are specific of the cell type and may be valuable to develop new therapies or as sources of active compounds with improved efficacy and selectivity towards OA and IBD.
Previous studies have suggested that α-pinene, a common volatile plant metabolite, may have anti-inflammatory effects in human chondrocytes, thus exhibiting potential antiosteoarthritic activity. The objective of this study was to further characterize the potential antiosteoarthritic activity of selected pinene derivatives by evaluating their ability to modulate inflammation and extracellular matrix remodeling in human chondrocytes and to correlate the biological and chemical properties by determining whether the effects are isomer- and/or enantiomer-selective. To further elucidate chemicopharmacological interactions, the activities of other naturally occurring monoterpenes with the pinane nucleus were also investigated. At noncytotoxic concentrations, (+)-α-pinene (1) elicited the most potent inhibition of the IL-1β-induced inflammatory and catabolic pathways, namely, NF-κB and JNK activation and the expression of the inflammatory (iNOS) and catabolic (MMP-1 and -13) genes. (-)-α-Pinene (2) was less active than the (+)-enantiomer (1), and β-pinene (3) was inactive. E-Pinane (4) and oxygenated pinane-derived compounds, pinocarveol (5), myrtenal (6), (E)-myrtanol (7), myrtenol (8), and (Z)-verbenol (9), were less effective or even completely inactive and more cytotoxic than the pinenes tested (1-3). The data obtained show isomer- and enantiomer-selective anti-inflammatory and anticatabolic effects of α-pinene in human chondrocytes, (+)-α-pinene (1) being the most promising for further studies to determine its potential value as an antiosteoarthritic drug.
One of the most important features in Alzheimer's disease is the generation and deposition of neurotoxic β-amyloid peptide (Aβ). The inhibition of BACE-1, a key enzyme in Aβ formation, is considered a promising therapeutic alternative for this disease. Natural products from plants have been extensively used as memory enhancers and in dementia therapy, mostly as acetylcholinesterase inhibitors. The low molecular weight and high hydrophobicity of terpenoids are properties that provide a good chance for them to cross cellular membranes and the blood–brain barrier, an essential attribute for BACE-1 inhibition in vivo. In this study, several essential oils were screened for their inhibitory activity on BACE-1. One of them, the essential oil from Lavandula luisieri, was signalized as inhibiting the enzyme. Moreover, this oil was tested on the endogenous BACE-1 in cultured cells, being responsible for a reduction in Aβ production, with no significant toxicity. The essential oil was characterized by a combination of gas chromatography and gas chromatography–mass spectrometry data, showing high contents of oxygen-containing monoterpenes, mainly necrodane derivatives, which are absent from any other oil. We also describe the bio-guided fractionation of the oil, as well as tests of the constituents on recombinant BACE-1 and on a cell line. The main inhibitory activity was assigned to the monoterpenic ketone 2,3,4,4-tetramethyl-5-methylene-cyclopent-2-enone, one of the distinctive components of L. luisieri essential oil. Taken together, these results showed that this essential oil and its components inhibited BACE-1 activity, both in enzymatic and cellular assays, thus presenting the ability to permeate cell membranes, a novel finding in Alzheimer's drug research. Copyright © 2013 John Wiley & Sons, Ltd.
Evolving definitions of osteoarthritis and improvements in risk factor measurement that use advanced imaging, systemic and local biomarkers, and improved methods for measuring symptoms and their impact can help elucidate mechanisms and identify potential areas for intervention or prevention.
The present study aimed to investigate the protective role of limonene in lipopolysaccharide (LPS)-induced acute lung injury (ALI). ALI was induced in mice by intratracheal instillation of LPS (0.5 mg/kg), and limonene (25, 50, and 75 mg/kg) was injected intraperitoneally 1 h prior to LPS administration. After 12 h, bronchoalveolar lavage fluid (BALF) and lung tissue were collected. Limonene pretreatment at doses of 25, 50, and 75 mg/kg decreased LPS-induced evident lung histopathological changes, lung wet-to-dry weight ratio, and lung myeloperoxidase activity. In addition, pretreatment with limonene inhibited inflammatory cells and proinflammatory cytokines including tumor necrosis factor-α, interleukin-1β, and interleukin-6 in BALF. Furthermore, we demonstrated that limonene blocked the phosphorylation of IκBα, nuclear factor-κB (NF-κB) p65, p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase, and extracellular signal-regulated kinase in LPS-induced ALI. The results presented here suggest that the protective mechanism of limonene may be attributed partly to decreased production of proinflammatory cytokines through the inhibition of NF-κB and MAPK activation.