Ethyl rosmarinate inhibits lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in alveolar macrophages

Article abstract of DOI:10.1016/j.ejphar.2018.01.042

In this study, a series of rosmarinic acid and analogs were investigated for their anti-inflammatory potential against LPS-induced alveolar macrophages (MH-S). Our results showed that, among the test compounds, ethyl rosmarinate (3) exhibited the most pot

Full text of DOI:10.1016/j.ejphar.2018.01.042

EuropeanJournalofPharmacology824(2018)17–23
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
Ethyl rosmarinate inhibits lipopolysaccharide-induced nitric oxide and
prostaglandin E2 production in alveolar macrophages
Hathairat Thammasona, Pichit Khetkamb, Wachirachai Pabuprapapb, Apichart Suksamrarnb,⁎
,
⁎⁎
Duangkamol Kunthalerta,c,
a
Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand
Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand
b
c
Centre of Excellence in Medical Biotechnology, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand
A R T I C L E I N F O
A B S T R A C T
Keywords:
Ethyl rosmarinate
Nitric oxide
Prostaglandin E2
Alveolar macrophages
Chronic obstructive pulmonary disease
In this study, a series of rosmarinic acid and analogs were investigated for their anti-inflammatory potential
against LPS-induced alveolar macrophages (MH-S). Our results showed that, among the test compounds, ethyl
rosmarinate (3) exhibited the most potent inhibitory effect on NO production in LPS-induced MH-S cells, with
low cytotoxicity. Compound 3 exhibited remarkable inhibition of the production of PGE2 in LPS-induced MH-S
cells. The inhibitory potency of compound 3 against LPS-induced NO and PGE2 release was approximately two-
fold higher than that of dexamethasone. Compound 3 significantly decreased the mRNA and protein expression
of iNOS and COX-2 and suppressed p65 expression in the nucleus in LPS-induced MH-S cells. These results
suggested that compound 3 inhibited NO and PGE2 production, at least in part, through the down-regulation of
NF-κB activation. Analysis of structure-activity relationship revealed that the free carboxylic group did not
contribute to inhibitory activity and that the alkyl group of the corresponding alkyl ester analogs produced a
strong inhibitory effect. We concluded that compound 3, a structurally modified rosmarinic acid, possessed
potent inhibitory activity against lung inflammation, which strongly supported the development of this com-
pound as a novel therapeutic agent for the treatment of macrophage-mediated lung inflammatory diseases, such
as COPD.
1. Introduction
was evident in patients with COPD (Keatings et al., 1996; Pesci et al.,
1998). Furthermore, the number of parenchymal alveolar macrophages,
Lung inflammation is fundamental to the etiology and persistence of
respiratory disease conditions, including asthma and chronic ob-
structive pulmonary disease (COPD). In particular, COPD is a major
health problem that results in morbidity and mortality worldwide
(Barnes, 2015). A recent systemic review and meta-analysis suggested a
high and growing prevalence of COPD, both globally and regionally
(Adeloye et al., 2015). Among the various cell types, alveolar macro-
phages and neutrophils are thought to be responsible for COPD-related
inflammation (Barnes, 2004; Meijer et al., 2013). However, there is
increasing evidence to show that alveolar macrophages play a key role
in the pathogenesis of COPD (Barnes, 2004). COPD is a disease of the
small airways and lung parenchyma, of which inflammation and tissue
destruction are the pathological hallmarks (Di Stefano et al., 2004).
Alveolar macrophages are the predominant cell type in the lung
(Shapiro, 1999; Pons et al., 2005), and a marked increase (5–10 fold) in
the numbers of macrophages both in airways and in lung parenchyma
but not neutrophils, was found to be directly proportional to the se-
verity of the disease (Finkelstein et al., 1995; Di Stefano et al., 1998).
Moreover, the pathological role of alveolar macrophages has been de-
monstrated, as the depletion of lung macrophages conferred protection
against the development of emphysema in an experimental model of
COPD (Beckett et al., 2013). Upon activation by harmful stimuli (pre-
dominantly cigarette smoke and bacterial infections) alveolar macro-
phages release inflammatory mediators including nitric oxide (NO),
prostaglandin E2 (PGE2) and pro-inflammatory cytokines such as tumor
necrosis factor-α (TNF-α) and interleukin-6 (IL-6) (Barnes, 2016). The
uncontrolled production or excessive accumulation of these mediators
can destroy lung tissue, which leads to respiratory failure and dys-
function. As a vital organ for gaseous exchange, chronic or excessive
inflammation of the lung can be life threatening. Currently, broncho-
dilators and inhaled corticosteroids are prescribed as the first-line
therapies for COPD. However, owing to the limited improvements in
⁎
Corresponding authors.
⁎⁎
Corresponding author at: Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand.
E-mail addresses: asuksamrarn@yahoo.com, s_apichart@ru.ac.th (A. Suksamrarn), kunthalertd@yahoo.com, duangkamolk@nu.ac.th (D. Kunthalert).
https://doi.org/10.1016/j.ejphar.2018.01.042
Received 6 January 2018; Received in revised form 21 January 2018; Accepted 25 January 2018
Availableonline31January2018
0014-2999/©2018ElsevierB.V.Allrightsreserved.

H. Thammason et al.
EuropeanJournalofPharmacology824(2018)17–23
symptom control and survival rate (Callahan et al., 1991; Jiang and
Zhu, 2016), this approach has not provided a satisfactory solution.
More importantly, a certain proportion of patients with COPD are re-
ported to be resistant to corticosteroids and experience a deterioration
of their symptoms (Marwick et al., 2007; Malhotra et al., 2011; Jiang
and Zhu, 2016). When used at high doses or for a prolonged time,
corticosteroids can lead to problematic side effects, such as osteo-
porosis, aseptic joint necrosis, adrenal insufficiency, gastrointestinal,
hepatic, and ophthalmologic effects, hyperlipidemia, growth suppres-
sion, and possible congenital malformations (Buchman, 2001). A sig-
nificant increase in the risk of pneumonia with the long-term use of
inhaled corticosteroids has also been reported in patients with COPD
(Sonal and Loke, 2010). Therefore, the discovery and development of
new agents for the effective treatment of COPD are an urgent necessity.
Rosmarinic acid (1), an ester of caffeic acid and 3,4-dihydrox-
yphenyllactic acid, is commonly found in several medicinal plant spe-
cies, including Rosmarinus officialis (Kim et al., 2015) and Hyptis sua-
veolens (Prawatsri et al., 2013). Interest in this compound has increased
considerably in the last decade owing to its diverse pharmacological
activities, including anti-oxidant (Kelm et al., 2000; Al-Musayeib et al.,
2011), anti-cancer (Moore et al., 2016), anti-angiogenic (Cao et al.,
2016), anti-viral (Arabzadeh et al., 2013), and anti-microbial properties
(Abedini et al., 2013). Rosmarinic acid also exerts anti-inflammatory
effects, as demonstrated by its ability to inhibit NO and PGE2 release in
macrophage-mediated inflammation by using RAW 264.7 murine
macrophages of peritoneal origin as a model (Huang et al., 2009). In
addition, the anti-allergic activity of rosmarinic acid was also been
shown in an experimental model of allergic asthma (Costa et al., 2012;
Liang et al., 2016). These results indicated the immunomodulatory
properties of rosmarinic acid and suggested the possible use of this
compound for the treatment of airway inflammation. Nevertheless,
marked differences between the inflammatory cells involved in the
pathology of asthma and COPD have been described (Ichinose, 2009;
Moldoveanu et al., 2009). Moreover, there are functional and pheno-
typic differences among macrophages from different tissue sites (Guth
et al., 2009; Karagianni et al., 2013; Hussell and Bell, 2014). As such, an
appropriate therapeutic target for certain airway inflammatory diseases
would be of significant concern. As alveolar macrophages and the re-
lease of inflammatory mediators play a pivotal role in the pathogenesis
of COPD, and owing to the absence of scientific evidence addressing the
effects of rosmarinic acid on alveolar macrophages, this study therefore
investigated the anti-inflammatory potential of rosmarinic acid in LPS-
induced mouse alveolar macrophage MH-S cell lines. We also designed
and prepared analogs of this compound and examined their inhibitory
activities. The underlying mechanism responsible for the inhibitory
effect was also explored.
Fig. 1. Chemical structures of the test rosmarinic acid and analogs.
with a Finnigan LC-Q mass spectrometer) (see Supplementary data).
The presence of the alkoxyl groups from the starting alcohols was
evident from the NMR spectra of the esters. The purity of the com-
pounds was approximately 95%. These compounds were dissolved with
dimethyl sulfoxide (DMSO; ≥ 99.5%, Sigma, France) and further di-
luted in culture medium before bioassay.
2.2. Cell culture and conditions
Murine alveolar macrophage cell line MH-S was obtained from
American Type Culture Collection (ATCC, Manassas, VA, USA). Cells
were cultured in Roswell Park Memorial Institute medium-1640 (RPMI-
1640; HyClone, Utah, USA) supplemented with 10% fetal bovine serum
(Gibco,
South
America),
10 mM
4-(2-hydroxyethyl)-1-piper-
azineethanesulfonic acid (HEPES; HyClone, Utah, USA), 2 mM L-gluta-
mine (Gibco, Brazil) and 100 U/ml and penicillin and 100 μg/ml
streptomycin (Gibco, USA) in a 5% CO2 atmosphere at 37 °C.
2.3. Cell viability assay
Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay as previously described
(Mosmann, 1983). MH-S cells (1 × 105 cells/well) were plated in a 96-
well plate (Nunc™, Roskilde, Denmark) and incubated for 1 h at 37 °C in
a humidified atmosphere containing 5% CO2. The cells were then ex-
posed to test compounds (final concentration 25 μM) for 24 h. Cells
without the test compounds and cells treated with dimethyl sulfoxide
(DMSO) served as untreated and vehicle controls, respectively. After
the incubation, culture supernatant was discarded and 20 μl MTT so-
lution (5 mg/ml; Sigma, St. Louis, MO, USA) was added. Cells were
incubated for another 3 h and the supernatant was removed, subse-
quently 100 μl of DMSO was added to solubilize the formazan. Absor-
bance of solubilized formazan solution was measured at 540 nm using a
microplate reader (Rayto RT-2100C microplate reader, China). Per-
centage of viable viability was calculated according to the equation:
(absorbance of treated cells/ absorbance of untreated cells) × 100.
2. Materials and methods
2.1. Rosmarinic acid (1) and analogs 2–11
Rosmarinic acid (1) and methyl rosmarinate (2) (Fig. 1) are natural
compounds isolated from the weeds Hyptis sauveolens by our group
(Prawatsri et al., 2013). Structural modifications of rosmarinic acid to
the corresponding ester analogs, compounds 3–11, (Fig. 1) were
achieved by conventional esterification of rosmarinic acid to the cor-
responding esters using appropriate alcohols, with concentrated sul-
furic acid as a catalyst at room temperature, except for the benzyl ester
10 that was synthesized by coupling reaction between rosmarinic acid
(1) and benzyl alcohol using EDCI [1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide] and DMAP (4-dimethylaminopyridine) in triethy-
lamine and dichloromethane. The products were purified by column
chromatography (Merck silica gel 60, particle size < 0.063 mm) and
the structures of the synthesized analogs were confirmed by NMR
spectroscopic data (recorded on a Bruker AVANCE 400 FT NMR spec-
trometer, operating at 400 MHz), and ESI mass spectral data (measured
2.4. Nitrite and PGE2 measurements
MH-S cells (1 × 105 cells/well) were plated in a 96-well plate
(Nunc™) and incubated for 1 h at 37 °C in an 5% CO2 atmosphere.
18

H. Thammason et al.
EuropeanJournalofPharmacology824(2018)17–23
Subsequently, the cells were treated with 1 μg/ml lipopolysaccharide
(LPS; Sigma, St. Louis, MO, USA) in the presence or absence of the test
compounds and incubated for 24 h (for NO) and 48 h (for PGE2). The
culture supernatant was collected and the nitrite (NO2–), a stable pro-
duct of NO was determined using the Griess reagent system (Promega
Corporation, USA). Briefly, 50 μl of culture supernatant was reacted
with 50 μl of 1% sulfanilamide in 5% phosphoric acid for 10 min at
room temperature in the dark. Then, 50 μl of 0.1% naphthylethylene-
diamine dihydrochloride was added. After 10 min incubation at room
temperature, the optical density was measured at 540 nm using a mi-
croplate reader (BioTek Synergy HT, USA) and nitrite concentration
calculated against a sodium nitrite standard curve. The levels of PGE2 in
culture medium were quantified by enzyme-immunoassay (R&D
Systems, Minneapolis, MN, USA) according to the manufacturer's in-
structions.
Santa Cruz Biotechnology) and β-actin (ab-170325, Abcam). The
membranes were washed with TTBS twice and subsequently incubated
with peroxidase-conjugated AffiniPure goat anti-mouse IgG (115-035-
003, Jackson ImmunoResearch Laboratories, USA) or horseradish per-
oxidase-conjugated donkey anti-goat IgG (sc-2020, Santa Cruz
Biotechnology), as appropriate. After 1 h incubation at room tempera-
ture, the immunoreactive bands were detected by Clarity™ Western ECL
Blotting Substrates (Bio-Rad, USA) according to the manufacturer's in-
structions. Chemiluminescent signals were visualized using
ImageQuant LAS 4000 biomolecular imager (GE Healthcare Life
Sciences, UK). The relative intensities of the protein bands were mea-
sured by ImageJ software and then normalized with β-actin.
2.7. Statistical analysis
Experimental values were presented as mean S.E.M. of at least
two independent experiments. Statistical analyses were carried out
Student's t-test using SPSS version 23.0 statistical program (Chicago, IL,
USA). The differences were considered statistically significant at
P < 0.05.
2.5. Reverse transcription polymerase chain reaction (RT-PCR)
MH-S cells (2 × 106 cells/well) were plated in 6-well plates
(Nunc™) and incubated at 37 °C in 5% CO2 for 1 h. Cells were stimu-
lated with LPS (1 μg/ml; Sigma) in the presence or absence of com-
pound 3 (2.5, 5, 10 and 25 μM). After 24 h-incubation, total cellular
RNA was extracted using TRIzol® reagent (Invitrogen, USA) according
to the manufacturer's instructions. The concentrations of extracted RNA
were determined by Nanodrop spectrophotometer (NanoDrop
Technologies, USA). Total RNA (2 µg) was reverse-transcribed to cDNA
using RevertAid First Strand cDNA Synthesis kit (Thermo Scientific,
USA) following the manufacturer's instructions. The resulting cDNA
was used as template for PCR analysis. The primer sequences of target
genes were: iNOS forward 5′-CTC AGC CCA ACA ATA CAA G-3′, reverse
5′-CTA CAG TTC CGA GCG TCA-3′, COX-2 forward 5′-AAG CCT TCT
CCA ACC TCT-3′, reverse 5′-ACA CTC TGT TGT GCT CCC- 3′ and β-actin
forward 5′-TGT TAC CAA CTG GGA CGA CA-3′, reverse 5′-AAG GAA
GGC TGG AAA AGA GC-3′ (Gao et al., 2014). Amplification conditions
were 25 cycles at 95 °C for 10 s, 61.7 °C for 40 s and 72 °C for 10 s (for
iNOS and β-actin) and 95 °C for 3 s, 56.4 °C for 40 s and 72 °C for 10 s
(for COX-2). The PCR products were separated by electrophoresis on
1% agarose gel for 35 min. Gels were then stained with 1 mg/ml ethi-
dium bromide and visualized by UV illumination. The intensity of each
band was determined by ImageJ software and the relative expression
level of iNOS or COX-2 gene was calculated using β-actin gene as a
reference housekeeping gene.
3. Results
3.1. Effect of rosmarinic acid (1) and analogs 2–11 on cell viability
The effect of rosmarinic acid (1) and the analogs 2–11 on MH-S
macrophage cell viability was examined by using an MTT assay. As
shown in Fig. 2, the viabilities of cells exposed to the test compounds at
25 μM showed individual variability. A clear decrease in viability was
observed after the cells were treated with compounds 4, 5, 6, 7, 8, 9
and 10, compared with the untreated control. However, no changes in
viability were seen after exposure to compounds 1, 2, 3 and 11, which
suggested that these compounds were not cytotoxic to MH-S macro-
phage cells. Furthermore, no differences in cell viability were found
between the vehicle and untreated control.
3.2. Effect of rosmarinic acid (1) and analogs 2–11 on LPS-induced NO
production in MH-S macrophages
Initially, 25 μM rosmarinic acid (1) and analogs 2–11 were in-
vestigated for their inhibitory activities against LPS-induced NO pro-
duction in MH-S macrophages and the results are shown in Fig. 3A. The
treatment with compounds 2, 3, 4, 5, 6, 7, 8, 9 and 10 markedly re-
duced the LPS-stimulated production of NO compared with the pro-
duction in the LPS-treated group. Compound 11 exhibited a mild in-
hibitory effect, whereas compound 1 had no inhibitory effect. Through
the combination of inhibitory activity and cytotoxicity, compounds 2
and 3 were selected for further assessment of their dose-dependent
2.6. Western blotting analysis
MH-S cells (2 × 106 cells/well) were plated in 6-well plates
(Nunc™) and incubated at 37 °C in 5% CO2 for 1 h. Cells were stimu-
lated with LPS (1 μg/ml; Sigma) in the presence or absence of com-
pound 3 (2.5, 5, 10 and 25 μM) for 30 min (for NFκB p65), 24 h (for
iNOS) and 48 h (for COX-2). Cells were scraped into 1.5 ml-micro-
centrifuge tubes, washed with cold phosphate-buffered saline (PBS) and
pellets. The cell pellets were then lysed using RIPA lysis buffer
(Amresco, OH, USA) containing Halt™ protease and phosphatase in-
hibitor cocktails (Pierce Biotechnology, IL, USA). Nuclear and cytosolic
proteins were extracted using NE-PER™ Nuclear and Cytoplasmic
Extraction Kit (Pierce Biotechnology) and a Halt™ protease and phos-
phatase inhibitor cocktails (Pierce Biotechnology). Protein concentra-
tion was determined using a Bradford Protein assay kit (Bio-Rad). The
extracted proteins (25 μg) subjected to electrophoresis using 10%
Novex™ NuPAGE™ Bis-Tris protein gels (Invitrogen, USA). Separated
proteins were transferred to nitrocellulose membranes (Bio-Rad,
Germany) and non-specific bindings were blocked with 5% skim milk in
TTBS (20 mM Tris, 150 mM NaCl, 0.1% Tween20) for 1 h at room
temperature. The membranes were then incubated overnight with pri-
mary antibodies specific to iNOS (sc-7271, Santa Cruz Biotechnology,
USA), COX-2 (sc-1745, Santa Cruz Biotechnology), NF-κB p65 (sc-8008,
Fig. 2. Effect of rosmarinic acid (1) and analogs 2–11 on viability of MH-S macro-
phages. Cells were treated with the test compounds at 25 µM for 24 h, and the viability
was examined by an MTT assay. Data are given as mean S.E.M. of independent ex-
periments.
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H. Thammason et al.
EuropeanJournalofPharmacology824(2018)17–23
Fig. 4. Effect of ethyl rosmarinate (3) on LPS-induced PGE2 production in MH-S
macrophages. Cells were stimulated with LPS (1 µg/ml) in the presence or absence of
compound
Concentrations of PGE2 in culture supernatant were determined by enzyme-im-
munoassay. Data are presented as mean S.E.M. of independent experiments.
3 (2.5, 5, 10 and 25 μM) or dexamethasone (Dex; 10 µM) for 48 h.
#
P < 0.05 compared with untreated group; * P < 0.05 compared with LPS-treated group.
3.4. Effects of ethyl rosmarinate (3) on iNOS and COX-2 mRNA and
protein expression
In order to determine whether the inhibitory effects of compound 3
on NO and PGE2 were related to the regulation of iNOS and COX-2
enzymes, the expression levels of these two enzymes were examined by
RT-PCR and Western blot analysis. As presented in Fig. 5A, the mRNA
level of iNOS was markedly increased after LPS treatment; this increase
was significantly (P < 0.01) inhibited by treatment with compound 3.
Western blotting analysis also demonstrated that expression of iNOS
protein was upregulated by exposure to LPS; similarly this increase was
significantly inhibited as increasing concentrations of compound 3 were
applied (Fig. 5B). At 25 μM, compound 3 reduced the expression of
iNOS mRNA and protein to similar levels of unstimulated cells
(P > 0.05). Furthermore, treatment with compound 3 inhibited the
LPS-induced expression of COX-2 mRNA and protein in a dose-depen-
dent manner (Fig. 5C and D). Unexpectedly, although the effect on
COX-2 protein expression was not observed at 24 h (see Supplementary
data), the reduction in LPS-stimulated COX-2 protein expression
mediated by compound 3 was evident at 48 h, which suggested that the
effect may be modulated by translational and/or post-translational
regulation effects in the late stage.
Fig. 3. Inhibition of rosmarinic acid (1) and analogs 2–11 on LPS-induced NO
production in MH-S macrophages. (A) Initial screening. Cells were stimulated with LPS
(1 µg/ml) in the presence or absence of the test compounds (25 μM) for 24 h. The con-
centration of nitrite in culture supernatant was determined by Griess assay. (B) NO in-
hibitory activity of compounds 2 and 3. MH-S cells were stimulated with LPS (1 µg/ml) in
the presence or absence of the test compounds at 2.5, 5, 10 and 25 μM or dexamethasone
(Dex; 10 μM) for 24 h and nitrite concentration measured by Griess assay. Data are pre-
sented as mean S.E.M. of independent experiments. ### P < 0.001 compared with
untreated group; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with LPS-treated
group.
inhibitory effects against LPS-induced NO release, and their IC50 values
were determined. As shown in Fig. 3B, dose-dependent inhibition of
LPS-induced NO release was caused by these two compounds, with the
IC50 values of 12.42 and 15.09 μM for compounds 3 and 2, respectively.
At 10 μM, compound 3 inhibited NO production by 42.23% 3.86%;
at the same concentration, dexamethasone only resulted in
19.89% 4.37% inhibition. Given the strong inhibitory activity of
ethyl rosmarinate (3), this compound was therefore selected for sub-
sequent experiments.
3.5. Effect of ethyl rosmarinate (3) on NF-κB activation
To further investigate whether the inhibition of NO by compound 3
was mediated by the NF-κB activation pathway, we examined the ex-
pression level of p65, part of the NF-κB transcription factor, in the
presence of compound 3 by using Western blotting. The results in Fig. 6
demonstrated that the expression level of p65 in the nuclear fraction
increased dramatically after stimulation with LPS for 30 min compared
with the untreated control. However, although the difference was not
statistically significant, treatment with compound 3 decreased the ex-
pression level of p65 in the nucleus to a value close to the baseline in
unstimulated MH-S macrophage cells (P > 0.05).
3.3. Effect of ethyl rosmarinate (3) on LPS-induced PGE2 production in
MH-S macrophages
As shown in Fig. 4, compound 3 strongly inhibited the production of
PGE2 in LPS-induced MH-S macrophages; the effect occurred in a dose-
dependent manner. It was noted that the complete inhibition of PGE2
production in LPS-treated MH-S cells was occurred after treatment at
25 μM. When expressed as a percentage inhibition, 10 μM compound 3
inhibited PGE2 production by 90.31% 9.67%, whereas 10 μM dex-
amethasone caused 49.38% 14.97% inhibition.
4. Discussion
Alveolar macrophages, which play a critical role in the pathogenesis
of COPD, NO and PGE2 are considered to be important biomarkers in
this lung inflammatory disorder (Barnes, 2016). Elevated PGE2 levels
and significantly increased iNOS expression and activity in the lungs of
patients with COPD have been well described, and this has been sug-
gested to be associated with the development of the severe impairments
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H. Thammason et al.
EuropeanJournalofPharmacology824(2018)17–23
Fig. 5. Effects of ethyl rosmarinate (3) on iNOS and COX-2 mRNA and protein expression in LPS-induced MH-S macrophages. Cells were stimulated with LPS (1 µg/ml) in the
presence or absence of compound 3 (2.5, 5, 10 and 25 μM) as indicated in Materials and Methods. Expressions of iNOS and COX-2 mRNA and protein were determined by RT-PCR and
Western blotting analysis, respectively. Data are presented as mean S.E.M. of independent experiments. # P < 0.05, ## P < 0.01 compared with untreated group; * P < 0.05, **
P < 0.01 compared with LPS-treated group.
in COPD (Maestrelli et al., 2003; Brindicci et al., 2010). Owing to their
influence on lung inflammation, alveolar macrophages are a good
therapeutic target for airway inflammatory diseases, particularly COPD
(Costa et al., 2015). In the present study, the anti-inflammatory po-
tentials of rosmarinic acid (1) and its analogs 2–11 were investigated in
an LPS-induced inflammatory model of alveolar macrophages. LPS was
used to induce alveolar macrophage inflammation as this component of
gram-negative bacterial cell wall is responsible for inducing patholo-
gical features of COPD (Ghorani et al., 2017). LPS also plays a sig-
nificant role in the bacterial infection-induced exacerbation of COPD,
which contributes to the development of the disease (Patel et al., 2002;
Wright et al., 2008; Pera et al., 2011). Although cigarette smoke is the
most important risk factor for COPD (Li et al., 2012), a large body of
evidence has revealed that LPS is one of the major active components in
cigarette smoke (Larsson et al., 2008). Recently, a methodological
review determined that LPS was among the three most commonly used
agents for the induction of COPD in an animal model that mimics the
important features of COPD (Fehrenbach, 2006; Wright et al., 2008).
Both in vitro and in vivo LPS-induced inflammation models have been
well-accepted as valuable tools for the investigation of COPD (Chen
et al., 2007; Gao et al., 2014; Ghorani et al., 2017). The results from this
study clearly showed that, among the test compounds, ethyl rosmar-
inate (3) exhibited the most potent inhibitory action on NO production
in LPS-induced MH-S cells and exhibited low cytotoxicity. Compound 3
also showed strong inhibitory activity against the production of PGE2 in
LPS-induced MH-S cells. In particular, the inhibitory potency of com-
pound 3 against LPS-induced NO and PGE2 release was approximately
two-fold higher than that of the reference drug, dexamethasone
(Ardestani et al., 2017). As compound 3 significantly inhibited both
inflammatory mediators, it would therefore provide the most potent
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H. Thammason et al.
EuropeanJournalofPharmacology824(2018)17–23
rosmarinic acid (1) after the structural modification to ethyl rosmar-
inate (3) has also been reported (Wicha et al., 2015). Although higher
alkyl ester analogs 4–10 displayed strong inhibitory activities, such
modifications led to cellular cytotoxicity, which would limit the future
therapeutic applications of these compounds. To investigate whether
the introduction of polar functionality to the ethyl ester group would
give analog with higher inhibitory activity, the corresponding hydroxyl
analog, compound 11, was synthesized. Unexpectedly, a sharp decrease
in inhibitory activity was observed, which suggested that an additional
hydroxyl group abolished the activity. Collectively, these findings in-
dicated the importance of hydrophobic ester group and the ethyl
(CH2CH3) group seems to exert the most potent inhibitory activity
against LPS-induced alveolar macrophage inflammation and its pre-
sence produced low toxicity.
5. Conclusion
This study demonstrated for the first time that ethyl rosmarinate
(3), a structurally modified rosmarinic acid (1), exerted potent in-
hibitory activity against lung inflammation. This compound strongly
inhibited NO and PGE2 production in LPS-induced alveolar macro-
phages, without the induction of cellular toxicity. The mechanism re-
sponsible for the inhibitory effect on NO and PGE2 production was the
suppression of iNOS and COX-2 expression, which occurred at least in
part through the NF-κB activation pathway. As ethyl rosmarinate in-
hibited key inflammatory mediators, our findings provided scientific
evidence to support this compound as a promising novel therapeutic
agent for the treatment of macrophage-mediated lung inflammatory
diseases. Nevertheless, future investigations in an in vivo COPD model
are required to support the future therapeutic applications of this
compound.
Fig. 6. Effect of ethyl rosmarinate (3) on NF-κB p65 activation in LPS- stimulated
MH-S macrophages. Cells were treated with compound 3 at 2.5, 5, 10 and 25 μM and
LPS 1 μg/ml. After 30 min incubation, the expression of NF-κB p65 in the nuclear fraction
was determined by Western blotting. Results are presented as mean S.E.M. of in-
dependent experiments. # P < 0.05 compared with untreated group.
anti-inflammatory effects. Our results strongly indicated the potential
of compound 3 as a novel anti-inflammatory agent for the treatment of
macrophage-mediated inflammatory lung diseases, such as COPD.
NO and PGE2 are synthesized by inducible NO synthase (iNOS) and
cyclooxygenase-2 (COX-2), respectively (Kanwar et al., 2009; Gao et al.,
2014). To explore the anti-inflammatory mechanisms, the effects of
compound 3 on the LPS-induced iNOS and COX-2 mRNA and protein
expression were investigated. Our results demonstrated that compound
3 significantly decreased both the LPS-induced mRNA and protein up-
regulation of iNOS and COX-2 in MH-S macrophages (Fig. 5), and such
decreases were correlated with the accumulation of NO (Fig. 3) and
PGE2 (Fig. 4). As NF-κB p65 is critical for the induction of iNOS and
COX-2 expression in LPS-induced alveolar macrophages (Li et al.,
2002), the effect of compound 3 on the LPS-induced activation of NF-κB
p65 was assessed further. Given that NF-κB pathway is central to the
pathogenesis of COPD, this pathway offers an excellent therapeutic
target for the treatment of inflammatory diseases, including COPD
(Edwards et al., 2009). We found that the decrease in the p65 expres-
sion level in the nucleus after compound 3 treatment (Fig. 6) was no-
ticeably reduced to a level comparable with that of unstimulated al-
veolar macrophages. Although other pathways (e.g., AP-1 and IRF-3)
may be involved and cannot be excluded, all our results suggested that
compound 3 inhibited NO and PGE2 production by decreasing the ex-
pression of iNOS and COX-2 mRNA and protein, at least partially
through the downregulation of the NF-κB p65 activation pathway.
Through the investigation of anti-inflammatory activities by using a
series of rosmarinic acid (1) and analogs 2–11 with different chemical
structures, a relationship between the structure and the inhibitory ac-
tivity could be drawn from this study. We found that although a natural
rosmarinic acid (1) exerted no inhibitory activity, its methyl ester
analog (compound 2), which is also a natural compound, exhibited
strong activity. The sharp increase in the inhibitory activity of NO
production in going from the free carboxylic acid function to the methyl
ester function has prompted us to investigate whether larger alkyl
groups would give rise to analog(s) with higher inhibitory activity and a
number of alkyl ester analogs, compounds 3–11, were therefore syn-
thesized. Ethyl rosmarinate (3) is the first synthetic analog of the series.
This compound showed markedly stronger inhibitory activity than the
parent compound 1. An improvement of vasorelaxant activity of natural
Acknowledgements
This work was supported by the Naresuan University Research
Fund. Supports from The Thailand Research Fund (Grant no. DBG
5980003) and Center of Excellence for Innovation in Chemistry, Office
of the Higher Education Commission are gratefully acknowledged.
Conflict of interest
The authors declare no conflict of interest.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the
online version at http://dx.doi.org/10.1016/j.ejphar.2018.01.042.
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