Basic & Clinical Pharmacology & Toxicology, 2015, 116, 398–413 Doi: 10.1111/bcpt.

12335

Anti-inflammatory Effect of Rosmarinic Acid and an Extract of

Rosmarinus officinalis in Rat Models of Local and Systemic
Inflammation
Joao Rocha1, Maria Eduardo-Figueira1, Andreia Barateiro1, Adelaide Fernandes1, Dora Brites1, Rosario Bronze2, Catarina MM Duarte2,
Ana Teresa Serra2, Rui Pinto1, Marisa Freitas3, Eduarda Fernandes3, Beatriz Silva-Lima1, Helder Mota-Filipe1 and Bruno Sepodes1
1
iMed.ULisboa, Faculty of Pharmacy - University of Lisbon, Lisboa, Portugal, 2ITQB/IBET, Quinta do Marqu^es Estacß~ao Agronomica Nacional,
Oeiras, Portugal and 3REQUIMTE, Laboratory of Applied Chemistry, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto,
Porto, Portugal
(Received 14 July 2014; Accepted 25 September 2014)

Abstract: Rosmarinic acid is a polyphenolic compound and main constituent of Rosmarinus officinalis and has been shown to
possess antioxidant and anti-inflammatory properties. We aimed to evaluate the anti-inflammatory properties of rosmarinic acid
and of an extract of R. officinalis in local inflammation (carrageenin-induced paw oedema model in the rat), and further evaluate
the protective effect of rosmarinic acid in rat models of systemic inflammation: liver ischaemia–reperfusion (I/R) and thermal
injury models. In the local inflammation model, rosmarinic acid was administered at 10, 25 and 50 mg/kg (p.o.), and the extract
was administered at 10 and 25 mg/kg (equivalent doses to rosmarinic acid groups) to male Wistar rats. Administration of ros-
marinic acid and extract at the dose of 25 mg/kg reduced paw oedema at 6 hr by over 60%, exhibiting a dose–response effect,
suggesting that rosmarinic was the main contributor to the anti-inflammatory effect. In the liver I/R model, rosmarinic acid was
administered at 25 mg/kg (i.v.) 30 min. prior to the induction of ischaemia and led to the significant reduction in the serum con-
centration of transaminases (AST and ALT) and LDH. In the thermal injury model, rosmarinic acid was administered at 25 mg/
kg (i.v.) 5 min. prior to the induction of injury and significantly reduced multi-organ dysfunction markers (liver, kidney, lung)
by modulating NF-jB and metalloproteinase-9. For the first time, the anti-inflammatory potential of rosmarinic acid has been
identified, as it causes a substantial reduction in inflammation, and we speculate that it might be useful in the pharmacological
modulation of injuries associated to inflammation.

Rosmarinus officinalis L., popularly named rosemary, has
been used in folk medicine with several pharmacological
effects being associated to its consumption, including its anti-
inflammatory effects [1,2], and rosmarinic acid (RA) is one of
its main phenolic compounds [3].
Two studies have evaluated the kinetics of rosmarinic
acid when administered orally to rats [4,5]. These studies
showed that rosmarinic acid was readily absorbed in the
gastrointestinal tract (according to Konishi and Kobayashi
[6], it crosses intestinal epithelium by passive diffusion) and
reaches the peak plasma concentration at 0.5 hr post-admin-
istration. Metabolites formed are a result of glucuronidation,
sulphation and methylation of rosmarinic acid and are then
eliminated in the urine. The effect of R. officinalis and ros-
marinic acid on metabolizing enzymes was also studied in
Wistar rats [7]. This study demonstrated that the extract of
R. officinalis was able to induce the enzymes CYP1A1,
CYP2B1/2, CYP2E1, glutathione S-transferase and UDP-glu-
curonosyl transferase, but this effect was not observed
when rosmarinic acid was administered alone. The authors
have attributed this effect to the presence of flavones and
monoterpenes.
Author for correspondence: Bruno Sepodes, Faculty of Pharmacy,
University of Lisbon, Avenida Prof. Gama Pinto, 1649-003 Lisbon,
Portugal (fax +351 217946470, e-mail bsepodes@ff.ul.pt).
It has been widely recognized for many years that certain
types of inflammatory tissue injury are mediated by reactive
oxygen metabolites and that in addition to promoting direct
toxicity, they may also initiate and/or amplify inflammation
via the up-regulation of several genes involved in the inflam-
matory response [8].
A vast amount of circumstantial evidence implicates oxy-
gen-derived free radicals and high-energy oxidants as media-
tors of inflammation, shock and ischaemia/reperfusion injury
[9]. Antioxidant effects of extracts of R. officinalis have been
reported in in vitro studies, exhibiting scavenging of superox-
ide anion, peroxynitrite and nitric oxide [10–13].
Also, reactive oxygen species play a major role in the organ
injury of many inflammatory-mediated events, including sys-
temic inflammation and ischaemia–reperfusion [9], particularly
of the liver [14]. Hepatic ischaemia–reperfusion is a major
component of injury in vascular occlusion both during liver
surgery and during liver transplantation. The pathophysiology
of hepatic ischaemia–reperfusion includes several mechanisms
that contribute to various degrees of the overall organ damage,
and formation of reactive oxygen species (ROS) and oxidant
stress are the most invoked disease mechanisms in ischaemia–
reperfusion injury [15].
Generation of reactive oxygen species during the reperfu-
sion process can be prevented or attenuated, and that has been
shown to reduce liver injury in experimental models of ischae-

© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
 EFFECT OF ROSMARINIC ACID IN INFLAMMATION
mia–reperfusion [9,16]. Therefore, given the potential antioxi-
dant and anti-inflammatory effects of R. officinalis and ros-
marinic acid, they might have protective effects in the injury
sustained by the liver subjected to ischaemia–reperfusion.
Lungs represent the first frontier between oxygen entry into
the organism and delivery to the mitochondria and are there-
fore exposed to higher concentrations of oxygen and reactive
oxygen species [17], leading to the amplification of inflamma-
tory processes [18]. A disruption of oxidative balance was
found to be important in the pathogenesis of lung inflamma-
tory diseases, such as acute lung injury (ALI) and acute respi-
ratory distress syndrome (ARDS) [19], pneumonia, burn,
chronic obstructive pulmonary disease and lung ischaemia–
reperfusion [20–22].
Our aim was to determine the in vitro antioxidant activity
of a R. officinalis extract and rosmarinic acid and to evaluate
and compare their in vivo anti-inflammatory effect on a local
model of inflammation – the carrageenin-induced paw oedema
in the rat. We further evaluated the protective effect of ros-
marinic acid administration in a hepatic ischaemia–reperfusion
model and in a thermal injury model in the rat, taking into
account the previously described role of oxidative stress in
these systemic inflammatory injuries.
Materials and Methods
Chemicals. Rosmarinic acid was obtained from Sigma-Aldrich
Quımica SA (Sintra, Portugal). Dried leaves of R. officinalis were
obtained from Ervital – Plantas Aromaticas e Medicinais, Lda. (Mezio,
Portugal). Apyrogenic saline (0.9% NaCl) was obtained from B. Braun
Medical Lda. (Queluz, Portugal). Unless otherwise stated, all other
compounds were obtained from Sigma-Aldrich Quımica SA (fig. 1).
Production of the R. officinalis extract. A methanolic extract was
prepared with the dried leaves of R. officinalis and performed with a
continuous Soxhlet extractor equipment (Soxtherm Automatic;
Gerhardt Bonn, Germany). The extraction was performed boiling 7 g
of R. officinalis dried leaves, with 50 mL of methanol resulting in an
extract of 20 mL.
Characterization of the chemical profile of the R. officinalis extract.
High-performance liquid chromatography (HPLC-UV). Acetonitrile
and methanol were acquired from Lab-Scan (Dublin, Ireland), and
phosphoric acid was acquired from Riedel-deHaen (Seelze,
Germany). Deionized water (conductivity of 0.050) was obtained by
a Milli-Q system (Millipore, Molsheim, France). A Surveyor
equipment from Thermo Finnigan with a diode array detector
(Thermo Finnigan Surveyor, San Jose, CA, USA), a fluorescence
Fig. 1. Chemical structure of rosmarinic acid.
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
399
detector (Shimadzu, RF-535 Kyoto, Japan) and an electrochemical
detector (Dionex, ED40 Bannockburn IL, USA) with a vitreous
carbon electrode were used. Separations were performed at 35°C
with a LiChrospher C18 (5 lm, 250 mm 9 4 mm i.d.) column from
Merck Darmstadt, Germany with a guard column of the same type.
The samples were injected using a 20-lL loop. The separations
were carried out with a flow rate of 700 lL/min., and the mobile
phase consisted of a gradient mixture of eluent A (phosphoric acid
0.1%) and eluent B (phosphoric acid–acetonitrile–water 5:400:595,
v/v/v). The following gradient of eluents was used: 0–15 min. from
0 until 20% eluent B; 10 min. with 20% eluent B; 25–70 min. from
20 until 70% eluent B; 70–75 min. with 70% eluent B; 75–85 min.
from 70 until 100% eluent B; 85–90 min. with 100% eluent B.
Diode array detection (DAD) was performed between 200 and
800 nm. Electrochemical detection was programmed for a linear
variation of 1.0 V at 1.0 V in 1.00 sec. (detection by integrated
voltammetry using a cyclic variation of the potential). The data
acquisition systems were ChromQuest version 4.0 (Thermo Finnigan
Surveyor) for the diode array detector and software 4880 (Unicam
Linda-a-Velha, Portugal) for the electrochemical and fluorescence
detectors. A standard solution of rosmarinic acid was prepared
(180 p.p.m.), and the peaks of both the standard solution and
sample solution (diluted 1:40) were compared.
Liquid chromatography with mass spectrometry (LC-MS). A Waters
HPLC equipment (Waters 2695) with a DAD detector (Waters 2996
Milford, USA) and a Micromass MS equipment (Micromass,
Quattro micro API) were used. Separations were performed with a
Waters dC18 column (5 lm, 2.1 9 150 mm). The samples were
injected using a loop of 20 lL. The separations were carried out
with a flow rate of 700 lL/min., and the mobile phase consisted of
a gradient mixture of eluent A (formic acid 0.5%) and eluent B
(acetonitrile). The following gradient of eluents was used: 0–
60 min. from 5 until 20% eluent B; 60–80 min. from 20 to 28%
eluent B; 80–82 min. from 28 until 30% eluent B; 82–87 min. with
30% eluent B; 87–95 min. from 30 until 5% eluent B; 95–
100 min. with 0% eluent B. Diode detection was performed
between 210 and 600 nm.
Characterization of the antioxidant capacity.
Total phenolic content. The total concentrations of phenolic
compounds present in the natural extracts were determined according
to the Folin–Ciocalteu colorimetric method [23]. Briefly, the
appropriate diluted solutions of extracts were oxidized with a Folin–
Ciocalteu reagent (Panreac, Barcelona Spain), and the reaction was
neutralized with sodium carbonate. The absorbance of the samples
was measured at 765 nm on a spectrophotometer (GenesysTM 10UV
ThermoScientific, Waltham MA, USA) after 30 min. at 40°C. Gallic
acid (Fluka, Seelze, Germany) was used as standard, and the result
was expressed as means of three replicates (mg of gallic acid equiv/L
of extract – mg GAE/L) [20].
Oxygen radical absorbance capacity (ORAC). ORAC assay was used
to evaluate the antioxidant capacity of the samples towards peroxyl
radicals. The assay was carried out using the method described by
Serra et al. [24], which measures the ability of the antioxidant species
present in the sample to inhibit the oxidation of FL catalysed by
AAPH-generated peroxyl radicals (ROO˙). The composition of the
reaction mixture was 6.3 9 10 8 M FL, 1.28 9 10 2 M AAPH
(prepared in 75 mM PBS, pH 7.4) and the appropriate diluted sample,
making up a total volume of 1.8 mL. The reaction was started by the
addition of AAPH to the mixture placed in a 10-mm-wide
fluorescence cuvette at 37°C. Fluorescence emitted by the reduced
form of FL was measured and recorded every 1 min. at the emission
wavelength of 515 nm and excitation wavelength of 493 nm
400 JOAO ROCHA ET AL.
(fluorescence spectrophotometer with thermostatic bath, model Cary
Eclipse; Varian Santa Clara CA, USA) for a period of 30 min.
Phosphate buffer (PBS) was used as a blank, and 1, 5, 12.5, 25 and
50 lM Trolox solutions were used as control standards. All samples,
including the blank and the controls, were analysed in triplicate. Final
ORAC values were calculated using a regression equation between the
Trolox concentration and the net area under the FL decay curve. Data
are expressed as millimolar of Trolox equivalent antioxidant capacity
(TEAC), and the experiment was performed in triplicate [24].
Hydroxyl radical absorbance capacity (HORAC). The HORAC assay
was based on a previously reported method [24,25], modified for the
FL800 microplate fluorescence reader (Bio-Tek Instruments, Isaza,
Portugal). The FL800 microplate fluorescence reader was used with
fluorescence filters for an excitation wavelength of 485  20 nm and
an emission wavelength of 530  25 nm, and the plate reader was
controlled by software Gen5 (Biotek, Winnoski VT, USA. Caffeic
acid was used as the standard as it provides a wider linear range as
compared to gallic acid. Data were expressed as mM of caffeic acid
equivalents (CAE). All samples were analysed as triplicates.
Neutrophil oxidative burst. Isolation of human neutrophils.
Following informed consent, venous blood was collected from healthy
volunteers by antecubital venipuncture, into K3EDTA vacuum tubes.
The isolation of human neutrophils was performed by the gradient
density, as previously reported [26,27].
Evaluation of neutrophils’ oxidative burst. The chemiluminescent
probe luminol has been thoroughly studied and used for monitoring the
production of reactive species by neutrophils, namely the superoxide
anion radical (O2˙ ), hydrogen peroxide (H2O2), hydroxyl radical
(HO˙), hypochlorous acid (HOCl), nitric oxide (˙NO) and peroxynitrite
anion (ONOO ) [28]. The measurement of neutrophils’ oxidative burst
was undertaken by chemiluminescence, by monitoring ROS-induced
oxidation of luminol, according to a previously described procedure
[29]. The reaction mixtures contained neutrophils (1 9 106 cells/mL)
and the following reagents at the indicated final concentrations (in a
final volume of 250 lL): tested compounds at various concentrations,
luminol (500 lM) and phorbol myristate acetate (PMA) (160 nM).
Cells were pre-incubated with luminol and the tested compounds for
5 min. before the addition of PMA, and the measurements were carried
out at 37°C, under continuous soft shaking. Kinetic readings were
initiated immediately after cell stimulation. Measurements were taken at
the peak of the curve. This peak was observed at around 10 min. Effects
are expressed as the per cent inhibition of luminol oxidation. Each study
corresponds to, at least, four individual experiments, performed in
triplicate in each experiment.
In vivo anti-inflammatory activity.
Experimental methods. Experiments were conducted according to the
Home Office Guidance in the Operation of Animals (Scientific
Procedures) Act 1986, published by Her Majesty’s Stationary Office,
London, UK, and the Institutional Animal Research Committee Guide
for the Care and Use of Laboratory Animals published by the US
National Institutes of Health (NIH Publication No. 85-23, revised
1996), as well as to the currently adopted EC regulations. Finally, the
studies are in compliance with the ARRIVE Guidelines for Reporting
Animal Research summarized at www.nc3rs.org.uk. Paw oedema
studies were carried out using male Wistar rats weighing 100–150 g
(Harlan Iberica, Barcelona, Spain). Hepatic ischaemia–reperfusion
studies were carried out using male Wistar rats weighing 250–300 g
(Harlan Iberica). Thermal injury studies were carried out using male
Wistar rats weighing 320–380 g. All animals received a standard diet
and water ad libitum.
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
Carrageenin-induced paw oedema. The carrageenin-induced paw
oedema of the rat hind paw is a suitable model to study acute local
inflammation and widely considered to be one of the most useful
models in the evaluation of anti-inflammatory activity of
investigational compounds [30,31].
Paw oedema was induced by a single subplantar injection into the
rat left hind paw of 0.1 mL of a 1% k-carrageenin sterile saline solu-
tion. Paw volume was measured by means of a volume displacement
method using a plethysmometer (Digital Plethysmometer LE7500; Le-
tica Scientific Instruments (Reagente 5, Porto, Portugal)). Paw volume
was measured immediately after the injection of carrageenin (V0 or
basal volume) and 6 hr later (V6 hr).
Paw oedema was expressed as percentage of increase in paw vol-
ume 6 hr after carrageenin injection relative to the basal values
according to the equation: % = (V6 hr V0)/V0 9 100.
The rats were randomly allocated into eight experimental groups as
described:
1 Control Group: animals were subjected to subplantar injection into
the rat left hind paw of 0.1 mL sterile saline and administered
with vehicle dimethyl sulfoxide – DMSO 10% (1 mL/kg, p.o.)
(n = 6);
2 Carrageenin Group: animals subjected to paw oedema induction
by injection into the rat left hind paw of 0.1 mL of k-carrageenin
(1%) and administered with vehicle (1 mL/kg, p.o.) (n = 10);
3 RA10 Group: animals subjected to paw oedema induction and
pre-treated with rosmarinic acid (10 mg/kg, p.o.) 30 min. before
k-carrageenin injection (n = 8);
4 RA25 Group: animals subjected to paw oedema induction and
pre-treated with rosmarinic acid (25 mg/kg, p.o.) 30 min. before
k-carrageenin injection (n = 8);
5 RA50 Group: animals subjected to paw oedema induction and
pre-treated with rosmarinic acid (50 mg/kg, p.o.) 30 min. before
k-carrageenin injection (n = 8);
6 E10 Group: animals subjected to paw oedema induction and pre-
treated with R. officinalis extract (10 mg/kg, p.o.) 30 min. before
k-carrageenin injection (n = 8);
7 E50 Group: animals subjected to paw oedema induction and pre-
treated with R. officinalis extract (50 mg/kg, p.o.) 30 min. before
k-carrageenin injection (n = 8);
8 Indomethacin Group: animals subjected to paw oedema induction
and pre-treated with indomethacin (10 mg/kg, p.o.) 30 min. before
k-carrageenin injection (n = 8).
Hepatic ischaemia–reperfusion. The rats were randomly allocated into
five experimental groups as described:
1 Control Group: the data were obtained from non-manipulated ani-
mals, that is rats that were not subjected to any surgical procedure
(n = 6).
2 Sham Group: rats that were subjected to the surgical procedures
described below except for liver I/R. Rats were administered vehi-
cle (saline with 10% DMSO, 1 mL/kg i.v.) 30 min. prior to liver
I/R (n = 4);
3 I/R Group: rats that were subjected to the surgical procedures
described below and underwent liver ischaemia for 45 min. fol-
lowed by reperfusion for 2 hr (n = 8);
4 RA + I/R Group: rats that were administered with RA (25 mg/kg
i.v.) 30 min. prior to liver I/R (n = 8);
5 RA Sham Groups: rats that were administered with (25 mg/kg
i.v.) and that were subjected to the surgical procedures described
below except for liver I/R (n = 4).
Hepatic ischaemia–reperfusion model was performed as previously
described [16,32]. Briefly, anaesthetized rats were placed onto a ther-
mostatically controlled heating mat (Harvard Apparatus Ltd, Kent,
 EFFECT OF ROSMARINIC ACID IN INFLAMMATION
UK), and body temperature maintained at 37  0.5°C by means of a
rectal probe attached to a homoeothermic blanket. A tracheotomy was
performed to maintain airway patency and to facilitate spontaneous
respiration. The jugular vein was cannulated (PP25, internal diameter
0.40 mm; Portex, Hythe, Kent, UK) for the administration of saline or
anaesthesia as required. A midline laparotomy was performed to care-
fully expose the liver. For the liver I/R surgical procedures, ligament
attachments connecting the liver, diaphragm, abdominal wall and
neighbouring organs were divided. The liver hilus was exposed to find
the common hepatic artery and the portal vein. A vascular microclamp
was used to interrupt the portal venous and arterial hepatic blood sup-
ply to the three cephalic lobes of the liver during 45 min. The three
caudal lobes retained an intact portal inflow and venous out-flow, pre-
venting the intestinal venous congestion and possible leakage of bacte-
ria or bacterial products to the circulation. Reperfusion commenced
once the vascular clip was removed, and was allowed to proceed for
2 hr. Occlusion was verified visually by the change in the colour of
the liver to a paler shade, and reperfusion by a blush. Other rats were
subjected to sham operation (sham operated), which underwent surgi-
cal procedures similar to the I/R group rats but did not undergo I/R of
the liver clamping and were maintained under anaesthesia for the dur-
ing the experiment. For these experiments, all rats were anaesthetized
with sodium pentobarbital (60 mg/kg i.p., Eutasil; Sanofi Veterinaria
Porto Salvo, Portugal), and anaesthesia was maintained by supplemen-
tary i.p. boli. At the end of all experiments, rats were killed by an
overdose of anaesthetic.
At the end of the reperfusion period for the liver I/R experiments
and whenever appropriate regarding other experiments, blood was col-
lected from the catheter placed in the right carotid artery into a serum
SST gel and clot activator tube (Becton, Dickinson and Company
Franklin Lakes NJ, USA) and was centrifuged (2000 g for 10 min. at
4°C) to separate the serum. The serum was analysed within 24 hr (Co-
bas c111 benchtop analyzer; Roche Sistemas de Diagnosticos, Lda.
Amadora, Portugal), and liver injury was assessed by measuring the
rise in the serum levels of alanine aminotransferase (ALT, a specific
marker for hepatic parenchymal injury), aspartate aminotransferase
(AST, a non-specific marker for hepatic injury) and lactate dehydroge-
nase (LDH, a marker of non-specific cellular injury).
Thermal injury. The rats were randomly allocated into 4 experimental
groups as described:
1 Sham Group: rats that were subjected to the surgical procedures
described below except for thermal injury. Rats were administered
vehicle (saline with 10% DMSO, 1 mL/kg i.v.) 5 min. prior to
thermal injury (n = 9);
2 Thermal Injury Group: rats that were subjected to the surgical pro-
cedures described below and underwent thermal injury (n = 14);
3 RA+Thermal Injury Group: rats that were administered with RA
(25 mg/kg i.v.) 5 min. prior to thermal injury (n = 14);
4 RA Sham Group: rats that were administered with (25 mg/kg i.v.)
and that were subjected to the surgical procedures described below
except for thermal injury (n = 6).
Briefly, anaesthetized rats were placed onto a thermostatically con-
trolled heating mat (Harvard Apparatus Ltd), and body temperature
maintained at 37  0.5°C by means of a rectal probe attached to a ho-
moeothermic blanket. A tracheotomy was performed to maintain airway
patency, facilitate spontaneous respiration and removal of secretions.
The jugular vein was cannulated for the administration of saline or anaes-
thesia as required, and the carotid artery was cannulated (PP50, I.D.
0.58 mm; Portex Kent, UK) for haemodynamic monitoring by a pressure
transducer (MLT0380 BP Transducer; AD Instruments Oxford, UK) and
blood collection at the end of the experiment. A 30% third-degree skin
burn was induced by immersing dorsal-shaved skin into 99°C water for
10 sec. using a synthetic foam template after surgical procedure. The
sham control groups were submitted to identical procedures as the other
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
401
groups except that room temperature water was used instead of heated
water. After-burn animals were dried and placed over the heating mat for
6 hr. For these experiments, all rats were anaesthetized with sodium pen-
tobarbital (60 mg/kg i.p., Eutasil; Sanofi Veterinaria), and anaesthesia
was maintained by supplementary boli. At the end of all experiments,
rats were killed by an overdose of anaesthetic.
At the of the experiment, blood was collected from the catheter
placed in the right carotid artery into a serum SST gel and clot activa-
tor tube and was centrifuged (3000 r.p.m. for 10 min. at 4°C) to sepa-
rate the serum. The serum was analysed within 24 hr, and liver injury
was assessed by measurement of the rise in the serum levels of alanine
aminotransferase (ALT, a specific marker for hepatic parenchymal
injury), aspartate aminotransferase (AST, a non-specific marker for
hepatic injury) and lactate dehydrogenase (LDH, a marker of non-spe-
cific cellular injury), and kidney injury was assessed by measurement
of the rise in the serum levels of creatinine and urea. Measurements of
the serum levels of pro-inflammatory cytokines (TNF-a, IL-1b, IL-6)
were also taken by ELISAs (Quantikineâ Rat TNF-a/TNFSF1A
Immunoassay, Quantikineâ Rat IL-1b/IL-1F2 Immunoassay,
Quantikineâ IL-6 Immunoassay from R&D Systems Inc., Citomed,
Lda, Lisboa, Portugal).
Assessment of lung injury. At the end of the experiments,
bronchoalveolar lavage fluid was collected into 15-mL Falcon tubes.
Collection was performed by instillation of 10 mL of saline at 4°C
through the trachea cannula. The samples were centrifuged (3000
r.p.m., 10 min., 4°C), the supernatant was divided into aliquots to
determine albumin and cytokine levels (TNF-a, IL-1b, IL-6), and the
sediment was reconstituted in saline for the polymorphonuclear cell
count. Albumin levels were measured in a Cobas c111 benchtop
analyzer (Roche Sistemas de Diagnosticos, Lda.). Polymorphonuclear
cell count was performed on an Automated Hematology System
(ADVIA 2120; Siemens Munich, Germany) following May–
Grunwald–Giemsa coloration.
Lung tissue samples were obtained at the end of the 6-hr period
after thermal injury and were fixed in 4% paraformaldehyde in PBS
for 72 hr at room temperature, dehydrated through a graded ethanol
series and embedded in paraffin (n = 3 per group). Haematoxylin and
eosin (H&E) staining was performed as previously described [33], and
images were acquired using a brightfield Axioskop microscope (Zeiss,
G€ottingen, Germany).
For Western blot analysis of Akt and glycogen synthase kinase 3-
beta (GSK-3b) expression, frozen tissue sample cells were lysed in
RIPA buffer containing Tris 50 mM (pH 8.0), 5 mM EDTA (pH
8.0), 150 mM NaCl, 1% NP-40, 10% glycerol and 0.1% SDS, and
sonicated for 20 sec. The lysate was centrifuged at 14,000 9 g for
10 min. at 4°C, and the supernatants were collected and stored at
80°C. Protein concentrations were determined using Nanodrop ND-
1000 (ThermoScientific, Wilmington DE, USA. Cell extracts contain-
ing equal amounts of protein (100–150 lg) were separated on
sodium dodecyl sulphate–polyacrylamide gel electrophoresis and
transferred to a nitrocellulose membrane. The membranes were
blocked with 5% non-fat milk, incubated with the primary antibody
overnight at 4°C rabbit anti-p-Akt [1:1000 (#12178; Cell Signalling,
Beverly, MA, USA)], rabbit anti-Akt [(1:1000) (#4691; Cell Signal-
ling)], goat anti-p-GSK-3b [(1:200) (#sc-11757; Santa Cruz, CA,
USA)], mouse anti-GSK-3b [(1:1000) (#9832; Cell Signalling)] and
then with a horseradish peroxidase-labelled secondary antibody for
1 hr at room temperature. After extensive washes, immunoreactive
bands were detected by LumiGLOâ (Cell Signalling) and visualized
by autoradiography with Hyperfilm ECL. Phosphorylation levels of
Akt and GSK-3b were analysed by the ratio of the phosphorylated
form to total enzyme levels and expressed as fold change.
Evaluation of factor nuclear kappa B (NF-kB) activity. Nuclear
extracts were prepared according to the rapid Dignam method [34].
402 JOAO ROCHA ET AL.
Briefly, at the end of the incubation, cells were resuspended in
400 lL ice-cold buffer [10 mM HEPES (pH 7.9), 10 mM KCl,
0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT) and
0.5 mM phenylmethylsulphonyl fluoride (PMSF)] for 20 min. on ice
and vortexed vigorously for 5 sec. after the addition of 25 lL Nonidet
P40 (10%). Cell lysates were centrifuged at 10,000 9 g for 30 sec. at
4°C, and the pelleted nuclei were lysed with 50 lL ice-cold buffer
[10 mM HEPES (pH 7.9), 0.4 M NaCl, 10 mM EDTA, 10 mM
EGTA, 1 mM DTT, 1 mM PMSF] for 20 min. at 4°C. Nuclear
lysates were centrifuged at 14,000 9 g for 10 min. at 4°C, and the
nuclear proteins recovered in the supernatants were stored at 80°C.
After protein determination using the protein assay kit (Bio-Rad,
Hercules, CA, USA), the p65 NF-jB subunit expression in each
compartment was analysed by Western blot as previously described
[35]. Extracts containing equal amounts of protein (50 lg) were
separated on sodium dodecyl sulphate–polyacrylamide gel
electrophoresis and transferred to a nitrocellulose membrane. The
membranes were blocked with 5% non-fat milk, incubated with the
primary antibody overnight at 4°C [rabbit anti-p65 NF-jB subunit
(1:1000) (SC-372; SantaCruz Biotechology)] and then with a
horseradish peroxidase-labelled secondary antibody for 1 hr at room
temperature. After extensive washes, immunoreactive bands were
detected by LumiGLOâ (Cell Signalling) and visualized by
autoradiography with Hyperfilm ECL. Results were normalized to
total protein lane content measured following Amido Black staining.
Evaluation of cell death. Cell death was evaluated by caspase-3
activation as previously described [36]. The activity of caspase-3 was
determined in tissue homogenates by enzymatic cleavage of
chromophore p-nitroanilide (pNA) from the substrate Ac-DEVD-pNA
for caspase-3, according to manufacturer’s instructions. The proteolytic
reaction was carried out in protease assay buffer [50 mM HEPES (pH
7.4); 100 mM NaCl; 0.1% (w/v) CHAPS; 10 mM DTT; 0.1 mM
EDTA; 10% (v/v) glycerol], containing 2 mM specific substrate. After
the incubation of the reaction mixtures for 1–2 hr at 37°C, the formation
of pNA was measured in a microplate reader (PR 2100; BioRad
Laboratories, Inc.) at k=405 nm with a reference filter at 620 nm.
Readings were normalized to total protein content determined using a
protein assay kit (Bio-Rad) according to the manufacturer’s
specification, and expressed as fold change of respective control.
Gelatin zymography (Metalloproteinases assay). Activity of matrix
metalloproteinase (MMP-9) was assessed by gelatin zymography as
described before [37]. Aliquots of tissue homogenates were analysed
by SDS-PAGE zymography in 0.1% gelatine–10% acrylamide gels
Fig. 2. Chromatogram profile (1:10) of the Rosmarinus officinalis extract on wavelength 280 nm.
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
under non-reducing conditions. After electrophoresis, gels were
washed for 1 hr with 2.5% Triton X-100 (in 50 mM Tris pH 7.4;
5 mM CaCl2; 1 lM ZnCl2) to remove SDS and renature the matrix
metalloproteinases (MMP) species in the gel. Then, the gels were
incubated in the developing buffer (50 mM Tris pH 7.4; 5 mM
CaCl2; 1 lM ZnCl2) overnight to induce gelatine lysis. For enzyme
activity analysis, the gels were stained with 0.5% Coomassie
Brilliant Blue R-250 (Sigma-Aldrich Quimica SA, Sintra Portugal)
and destained in 30% ethanol/10% acetic acid/H2O. Gelatinase
activity, detected as a white band on a blue background, was
quantified by computerized image analysis and normalized with total
cellular protein. MMP-9 was detected as the band with 92 kDa.
Statistical analysis. The results were expressed as the mean  S.E.M.
and were compared using a one-factorial ANOVA test, followed by a
Bonferroni’s post hoc test performed with a GraphPad Prism
Statistical Package (version 5.0 GraphPad Software, La Jolla CA,
USA)). A p-value less than 0.05 was considered to be statistically
significant.
Results
Characterization of the chemical profile of the R. officinalis
extract.
Chemical characterization of the extract by HPLC-UV and
HPLC-MS revealed a high concentration of rosmarinic acid
(4684 p.p.m., corresponding to 1.34 g of rosmarinic acid per
100 g dried leaves) as demonstrated by the chromatographic
profile of a 280-nm HPL-UV (fig. 2), indicating that the
extraction procedure had optimal conditions for the preferen-
tial extraction of rosmarinic acid. Chemical profiling of the
extract also revealed the presence of other phenolic com-
pounds, although in minor concentrations (caffeic acid, p-
coumaric acid, chlorogenic acid, ellagic acid, ferulic acid,
rosmanol, rutin, luteolin, methoxyluteolin, nepitin).
Characterization of the antioxidant capacity.
The antioxidant capacity assays demonstrated that both ros-
marinic acid and R. officinalis extract have high antioxidant
capacities (table 1), although R. officinalis extract presented a
much higher activity.
Rosmarinic acid
 EFFECT OF ROSMARINIC ACID IN INFLAMMATION 403

Neutrophil oxidative burst. Hepatic ischaemia–reperfusion.

Pre-incubation of the neutrophils with rosmarinic acid resulted
in an inhibition of luminol oxidation, induced by neutrophil
activation by phorbol myristate acetate (PMA) (fig. 3). The
reduction was statistically significant compared with control
neutrophils (incubation with PMA only) and exhibits a con-
centration–response relation. The inhibitory concentration 50%
(IC50) was 1.2  0.3 lM.
Carrageenin-induced paw oedema.
As expected, intraplantar injection of carrageenin in rats led to
an increase in paw volume after 6 hr (114  11.2%) when
compared with the control group. This increase was signifi-
cantly reduced in a dose-dependent manner by pre-treatment
with rosmarinic acid and R. officinalis extract, with the most
significant effect observed at the doses 25 and 50 mg/kg
(increase in paw volume of 45.3  12.8% and 44.4  13%,
respectively) (fig. 4).
A comparison of the effect of a single administration of ros-
marinic acid (25 mg/kg) with the effect of lycopene (50 mg/
kg), tempol (30 mg/kg), Trolox (30 mg/kg) and indomethacin
(10 mg/kg), known antioxidant and anti-inflammatory sub-
stances, showed that rosmarinic acid administration reduced
oedema formation at the same magnitude as those substances
(fig. 5).
Table 1.
Characterization of the antioxidant capacity of rosmarinic acid and
Rosmarinus officinalis extract.
Total phenolic
content ORAC
Rosmarinic – 112 mM TEAC
acid
R. officinalis 62.5 mM GAE 1384 mM TEAC
extract
GAE, gallic acid equivalents; CAE, caffeic acid equivalents; TEAC,
Trolox equivalent antioxidant capacity.
In sham-operated rats, the surgical procedure did not produce
a significant change in serum levels of AST, ALT or LDH
when compared with the control group. When compared with
sham-operated rats, I/R of the liver resulted in significant rises
in the serum levels of the marker enzymes AST, ALT and
LDH, demonstrating the development of hepatocellular injury.
In rats subjected to I/R pre-treated with rosmarinic acid, the
serum rise in AST, ALT and LDH was significantly reduced
by 35%, 35% and 45%, respectively. Rosmarinic acid by
itself, when administered to rats not subjected to I/R injury,
had no significant effect on AST, ALT and LDH serum levels
(fig. 6).
Thermal injury model.
Effect of rosmarinic acid on liver and kidney injury. When
compared with sham-operated rats, thermal injury resulted in
significant rises in the serum levels of the marker enzymes AST
and ALT, demonstrating the development of hepatocellular
injury. No significant rise was detected in rats subjected to
rosmarinic acid treatment in comparison with sham-operated
rats, indicating that rosmarinic acid administration prevented the
development of hepatocellular injury. Termal injury also led to
a significant rise in creatinine and urea serum levels indicating
the development of kidney injury or dysfunction. In rats
subjected to treatment with rosmarinic acid, the rise in
creatinine serum level was significantly attenuated, although
urea levels were not statistically different from rats subjected
only to thermal injury. Rats subjected to thermal injury also
exhibited a rise in serum levels of LDH, a non-specific marker
HORAC of cell injury. No significant rise in LDH levels was detected in
rats subjected to rosmarinic acid treatment in comparison with
66 mM CAE
sham-operated rats (fig. 7).
1516 mM CAE
Effect of rosmarinic acid on cytokine serum levels. When
compared with sham-operated rats, thermal injury resulted in
significant rises in the serum levels of pro-inflammatory

100
Luminol oxidation by activated
neutrophils (% inhibition)

***
75 ***
***

50 **

25

0
0.2 0.5 0.9 1.9 3.8 7.5
μM

Fig. 3. Inhibitory effect of rosmarinic acid (0.2–7.5 lM) on human neutrophils’ oxidative burst stimulated with PMA. **p < 0.01 and
***p < 0.001 compared with the control assay (PMA alone). The values are given as the mean  S.E.M. (n ≥ 4).

© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
404 JOAO ROCHA ET AL.

Fig. 4. Effect of Rosmarinus officinalis extract and rosmarinic acid administration on the rat paw oedema development elicited by carrageenin 6 hr
after oedema induction. Effect of a single administration of R. officinalis extract (E10 mg/kg, n = 8; and E25 mg/kg, n = 8, p.o.) in comparison
with the effect of a single administration of rosmarinic acid (RA10 mg/kg, n = 8; RA25 mg/kg, n = 8; RA50 mg/kg, n = 8, p.o.) and indometha-
cin (10 mg/kg, p.o., n = 8). The data are presented as means with their standard errors. *p < 0.001 versus control group; ‡p < 0.01 versus control
group; #p < 0.01 versus carrageenin group.

130 *
120
110
100
% paw volume increase

90
80
70
# #
60 # #
#
50
40
30
20
10
0
–10 Control Carrageenan RA 25 Lycopene Tempol Trolox Indomethacin

Fig. 5. Comparison of the anti-inflammatory effect of rosmarinic acid with known antioxidant and anti-inflammatory substances. Effect of a single
administration of rosmarinic acid (RA25 mg/kg, n = 8; p.o.), lycopene (50 mg/kg, n = 8; p.o.), tempol (30 mg/kg, n = 8; p.o.), Trolox (30 mg/kg,
n = 8; p.o.) and indomethacin (10 mg/kg, p.o., n = 8). The data are presented as means with their standard errors. *p < 0.001 versus control group;
#
p < 0.01 versus carrageenin group (Bignotto et al., 2009).

cytokines (TNF-a, Il-1b and IL-6). No significant rise was
detected in rats subjected to rosmarinic acid treatment in
comparison with sham-operated rats (fig. 8).
Effect of rosmarinic acid on cytokine bronchoalveolar lavage
fluid (BALF) levels. When compared with sham-operated
rats, thermal injury resulted in significant rises in the BALF
levels of pro-inflammatory cytokines (TNF-a, IL-1b and IL-
6). No significant rise in the BALF levels of TNF-a and
IL-6 was detected in rats subjected to rosmarinic acid
treatment in comparison with sham-operated rats, and the
rise in the BALF levels of IL-1b was significantly reduced
(fig. 9).
Effect of rosmarinic acid on albumin concentration on
bronchoalveolar lavage fluid (BALF). When compared with
sham-operated rats, thermal injury resulted in significant rises
in the BALF levels of albumin demonstrating the disruption
of the alveolo-capillary membrane. No significant rise was
detected in rats subjected to rosmarinic acid treatment in
comparison with sham-operated rats (fig. 10).
Effect of rosmarinic acid on the polymorphonuclear (PMN)
cell presence on bronchoalveolar lavage fluid (BALF). When
compared with sham-operated rats, thermal injury resulted in
significant rises in the leucocyte and neutrophil number in
BALF and also an increase in neutrophil percentage. No
significant rise was detected in these parameters in rats
subjected to rosmarinic acid treatment in comparison with
sham-operated rats (fig. 11).
Effect of rosmarinic acid on lung histology. When compared
with sham-operated rats, thermal injury resulted in structural
changes that indicate lung tissue injury, namely neutrophil
accumulation on the peri-alveolar zone, hyaline membrane
formation with thickening of the alveolar wall and intra-
alveolar (clots) and tissue haemorrhage (patchy formation). In
rats subjected to rosmarinic acid treatment, a marked reduction

© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
 EFFECT OF ROSMARINIC ACID IN INFLAMMATION 405

2500
2500
*
2000 *
2000
* #
AST (U/L)

1500

ALT (U/L)
* # 1500

1000
1000

500 500

0 0
Control Sham I/R RA + I/R RA Control Sham I/R RA + I/R RA

9000
*

7500

6000 * #
LDH (U/L)

4500

3000

1500

0
Control Sham I/R RA + I/R RA

Fig. 6. Effect of rosmarinic acid administration (25 mg/kg, i.v.) on the serum levels of aspartate aminotransferase (AST), alanine aminotransferase
(ALT) and lactate dehydrogenase (LDH), in the rat model of hepatic ischaemia–reperfusion. Control group, n = 6; sham group, n = 4; I/R group,
n = 8; RA+I/R group, n = 8; RA group, n = 4. *p < 0.001 versus sham group; #p < 0.01 versus I/R group.

of these changes was observed. Haemorrhage areas in patches
are still observable but occupy a lesser area and are more rare.
Neutrophil accumulation on the peri-alveolar zone was also
rare, and no alveoli were found with thickening of the
alveolar wall (fig. 12).
Effect of rosmarinic acid on the activation of Akt. When
compared with sham-operated rats, thermal injury resulted in a
significant rise in the activation of Akt (expressed as increased
ratio of p-Akt/Akt). In rats subjected to treatment with
rosmarinic acid, the rise in Akt activation was significantly
attenuated (fig. 13).
Effect of rosmarinic acid on the activation of GSK-3b. When
compared with sham-operated rats, thermal injury resulted in a
significant decrease in the activation of GSK-3b (expressed as
increased ratio of p-GSK-3b/GSK-3b). In rats subjected to
treatment with rosmarinic acid, there was no significant
change in the activation of GSK-3b (fig. 14).
Effect of rosmarinic acid on the activation of NF-jB. When
compared with sham-operated rats, thermal injury resulted in a
significant increase in the expression and activation of NF-jB
(expressed as increased nuclear translocation of the p65
NF-jB subunit). In rats subjected to treatment with rosmarinic
acid, there was a significant increase in the expression and
nuclear translocation of NF-jB (fig. 15).
Effect of rosmarinic acid on the activation of caspase-
3. When compared with sham-operated rats, thermal injury
resulted in a significant increase in the activation of caspase-3.
In rats subjected to treatment with rosmarinic acid, although a
trend appears to exist, there was no significant decrease in the
activation of caspase-3 (fig. 16).
Effect of rosmarinic acid on the activation of
metalloproteinase-9 (MMP-9). When compared with sham-
operated rats, thermal injury resulted in significant rises in the
activation of MMP-9. No significant rise was detected in rats
subjected to rosmarinic acid treatment in comparison with
sham-operated rats (fig. 17).
Discussion
Inflammatory response develops along several stages,
apparently mediated through different mechanisms. The
carrageenin-induced paw oedema model induces a biphasic
oedema consisting of an early-phase (up to 2 hr) followed by
a more sustained late-phase (2–6 hr). The late-phase is related
to neutrophil infiltration [38] and production of reactive

© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
406 JOAO ROCHA ET AL.

2500
2500
*
2000 *
2000
* #
1500
AST (U/L)

1500

ALT (U/L)
* #

1000
1000

500 500

0 0
Control Sham I/R RA + I/R RA Control Sham I/R RA + I/R RA

9000
*

7500

6000 * #
LDH (U/L)

4500

3000

1500

0
Control Sham I/R RA + I/R RA

Fig. 7. Effect of rosmarinic acid administration (25 mg/kg, i.v.) on the serum levels of aspartate aminotransferase (AST), alanine aminotransferase
(ALT), lactate dehydrogenase (LDH), creatinine and urea, in the rat model of thermal injury. Sham group, n = 9; thermal injury (TI) group,
n = 14; RA + TI group, n = 14; RA group, n = 6. *p < 0.05 versus sham group; #p < 0.05 versus TI group.

250 175 *
*
150
200
125
TNF-α (pg/mL)
IL-6 (pg/mL)

150
100 #
#

100 75

50
50
25

0 0
Sham TI RA + TI Sham TI RA + TI

500 *

400
IL-1β (pg/mL)

300 #

200

100

0
Sham TI RA + TI

Fig. 8. Effect of rosmarinic acid administration (25 mg/kg, i.v.) on the serum levels of TNF-a, IL-1b and IL-6, in the rat model of thermal injury. Sham
group, n = 9; thermal injury (TI) group, n = 14; RA + TI group, n = 14; RA group, n = 6. *p < 0.05 versus sham group; #p < 0.05 versus TI group.

© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
 EFFECT OF ROSMARINIC ACID IN INFLAMMATION 407

250 * 25 *

200 20
# #

TNF-α (pg/mL)
IL-6 (pg/mL)

150 15

100 10

50 5

0 0
Sham Q AcR + Q Sham Q AcR + Q

450 *
400

350

300
IL-1β (pg/mL)

250 #

200

150

100

50

0
Sham Q AcR + Q

Fig. 9. Effect of rosmarinic acid administration (25 mg/kg, i.v.) on the bronchoalveolar lavage fluid levels of TNF-a, IL-1b and IL-6, in the rat
model of thermal injury. Sham group, n = 9; thermal injury (TI) group, n = 14; RA + TI group, n = 14; RA group, n = 6. *p < 0.05 versus sham
group; #p < 0.05 versus TI group.

species such as hydrogen peroxide, superoxide radical, perox- the inflammatory process [41,42] and contribute greatly to tis-
ynitrite [39] and pro-inflammatory prostanoids [40]. This sue injury caused by inflammation [43]. In fact, reactive oxy-
model has been used greatly in research and has become the gen species are already considered as a valid therapeutic target
mainstay in the elucidation of pharmacodynamic properties in in the development of new drugs for the treatment of diseases
the support of non-clinical development of most of the non- with a strong inflammatory component [44,45], and several
steroidal anti-inflammatory drugs [30]. plant-derived products are already in a late stage for drug
Several in vitro and in vivo studies have shown that reactive development aiming for therapeutic indications where oxida-
oxygen species play a central role in the physiopathology of tive stress and inflammation might play a significant role [46].
The high concentration of phenolic acids in extracts of R. offi-
cinalis has been considered to be related to its antioxidant
4
capacity [47,48], anti-inflammatory [12] and antibacterial
Albumin concentration on BALF (mg/dL)

activity [47]. Given the central role of oxidation and reactive

*
oxygen species in inflammation and the known antioxidant
3
effects of rosmarinic acid and R. officinalis, we aimed to eval-
#
uate their effect in this model of acute local inflammation.
#
2
Our results suggest that pre-treatment with rosmarinic acid or
R. officinalis extract significantly reduces the increase in paw
volume by inhibiting the inflammatory processes associated
1
with oedema formation.
Administration of an extract of R. officinalis and rosmarinic
acid led to an anti-inflammatory effect as demonstrated by a
0
60% reduction in paw oedema volume in comparison with
Sham TI RA + TI RA control animals. Administration of the extract, in a dose equiv-
alent to 25 mg/kg of rosmarinic acid, exhibited the same mag-
Fig. 10. Effect of rosmarinic acid administration (25 mg/kg, i.v.) on
nitude of effect, suggesting that it was RA that mainly
the bronchoalveolar lavage fluid levels of albumin, in the rat model of
thermal injury. Sham group, n = 9; thermal injury (TI) group, n = 14; contributed to the anti-inflammatory effect observed. Given
RA + TI group, n = 14; RA group, n = 6. *p < 0.05 versus sham the naturally higher antioxidant capacity of the extract in rela-
group; #p < 0.05 versus TI group. tion to rosmarinic acid alone, it is possible to assume that the

© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
408
Fig. 11. Effect of rosmarinic acid administration (25 mg/kg, i.v.) on the bronchoalveolar lavage fluid number and percentage of polymorphonuclear
cells, in the rat model of thermal injury. Sham group, n = 9; thermal injury (TI) group, n = 14; RA + TI group, n = 14; RA group, n = 6.
*p < 0.05 versus sham group; #p < 0.05 versus TI group.
anti-inflammatory effect exhibited in this model of acute
inflammation might not be solely related to the antioxidant
capacity, but also to other mechanisms that may need further
investigation. The same magnitude of effect exhibited by ros-
marinic acid compared with other known and extensively stud-
ied antioxidant and anti-inflammatory substances (tempol,
Trolox, lycopene and indomethacin) suggests that rosmarinic
acid may have the potential to reduce inflammatory-induced
tissue injury in other inflammation models and through differ-
ent mechanisms of action.
Most interestingly, rosmarinic acid was also able to inhi-
bit the inflammatory process associated with hepatic ischae-
mia–reperfusion, thereby reducing liver injury sustained after
reperfusion. Liver injury caused by ischaemia–reperfusion
consists of an interruption of blood supply to the liver fol-
lowed by reperfusion. When the blood supply is restored,
the organs are usually subjected to a further insult, aggra-
vating the injury created within the ischaemic period [32].
This reperfusion insult is a direct consequence of a complex
interplay between different mechanisms. The initial phase
(until 2 hr after reperfusion) is characterized by oxidative
stress. The destructive effects of ischaemia–reperfusion result
from the acute generation of ROS subsequent to reoxygen-
ation. These ROS inflict direct tissue damage and initiate a
cascade of deleterious cellular responses leading to inflam-
mation, cell death and organ failure [49]. A previous work
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
JOAO ROCHA ET AL.
has also demonstrated the protective effect of rosmarinic
acid on liver injury. Administration of rosmarinic acid
(135 mg/kg, p.o.) reduced lipopolysaccharide-induced liver
injury in D-galactosamine-sensitized mice, and the liver pro-
tection exhibited by RA was related to the scavenging or
reducing activities of superoxide and peroxynitrite [50].
Osakabe et al. also observed that the magnitude of rosmari-
nic effect was similar to that of administration of an extract
of Perilla frutescens with an equivalent dose of RA, attrib-
uting the protective effect of the extract to its main constit-
uent, rosmarinic acid.
Thermal injury has a very severe clinical prognosis and lim-
ited therapeutic options. Severe thermal injury leads frequently
to systemic inflammatory response syndrome with subsequent
multi-organ dysfunction syndrome and a potentially fatal out-
come, being the main cause of death in burned patients [51].
Acute lung injury (ALI) and acute respiratory distress
syndrome (ARDS) are at the top of the list of early complica-
tions in burned patients and associated with a high mortality
[52–54].
Several physiopathological events are considered the main
contributors in the development of acute lung injury, includ-
ing systemic release of inflammatory mediators (cytokines),
chemotaxis and activation of neutrophils (oxidative burst)
and activation of cellular pathways involved in cell death
[55].
 EFFECT OF ROSMARINIC ACID IN INFLAMMATION 409

A B

C D

E F

Fig. 12. Effect of rosmarinic acid administration (25 mg/kg, i.v.) on the lung tissue histological features, in the rat model of thermal injury. (A)
Sham group (1009); (B) sham group (4009); (C) thermal injury group (1009); (D) thermal injury group (4009); (E) rosmarinic acid + thermal
injury group (1009); and (F) rosmarinic acid + thermal injury group (4009). Images are representative of at least 4 experiments performed on dif-
ferent days.

The scalding burn model used in this model is widely cited
in the literature [56–58], and we were able to prove that it
leads to the internationally acknowledged main features of an
experimental model of ALI/ARDS [59].
We demonstrated that administration of rosmarinic acid was
able to reduce systemic release of pro-inflammatory cytokines
and also attenuated the multi-organ injury (liver, kidney and
lung) induced by scald. Specifically on the lung, the histologi-
cal analysis of the lung showed that there was a marked
reduction in the histological signs of lung tissue injury with
an appearance closer to sham-operated rats, not subjected to
thermal injury.
Along with the formation of ROS, the infiltration and accu-
mulation of polymorphonuclear leucocytes within the tissues
has been considered a hallmark of the acute inflammatory
response, including local inflammation [34] hepatic ischae-
mia–reperfusion [14,60], acute lung injury [61,62] and sys-
temic inflammation [63,64]. A prominent feature of acute
inflammation is enhanced vascular permeability resulting in
oedema formation. Such changes in vascular permeability have
been known to be dependent upon polymorphonuclear leuco-
cyte interactions with the vascular endothelium [63]. Our
results revealed inhibition of the neutrophils’ oxidative burst
by rosmarinic acid in a dose-dependent manner suggesting that
its anti-inflammatory effect might be related, at least in part,
by inhibition of neutrophil activation.
Mechanistic studies performed on the lung tissue in the
thermal injury model revealed that the beneficial effect
exhibited by rosmarinic acid might not be related to modu-
lation of the PI3K-Akt-GSK-3b pathway but rather through
activation of the NF-jB pathway and inhibition of MMP-9
activation. MMP-9 has been shown to regulate the activa-
tion of pro-apoptotic factors such as the Fas/FasL complex
[65], and direct inhibition of MMP-9 or its gene transcrip-
tion has been shown to be beneficial in several lung injury
experimental models [66–68]. Given the importance of the
NF-jB pathway on cell survival [69] and its role in the
crosstalk with several other pathways related to apoptosis,
cell fate and inflammation [70,71], we might speculate that
rosmarinic acid might exhibit pleiotropic mechanisms and
act through the modulation of NF-jB closely related path-
ways.
Given that the inflammatory process is a multi-factorial
network of different mediators and that oxidative stress and

© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
410 JOAO ROCHA ET AL.

Sham TI RA + TI Sham TI RA + TI
C N C N C N
P-AKT - - 60 kDa
NF-κB- - 65 kDa

AKT total - - 60 kDa

#
20 Cytoplasm
2.5 Nucleus
*

NF-κB protein expression

15
#
pAKT/AKT protein expression

2.0

(fold change)
10
*
(fold change)

1.5

5
1.0

0
0.5 Sham TI RA + TI

Fig. 15. Effect of rosmarinic acid administration (25 mg/kg, i.v.) on

0.0 the lung tissue NF-jB activation, in the rat model of thermal injury.
Sham group, n = 9; thermal injury (TI) group, n = 14; RA + TI
am

TI

TI
+

group, n = 14; RA group, n = 6. *p < 0.05 versus sham group;

Sh

A
R

#
p < 0.05 versus TI group.
Fig. 13. Effect of rosmarinic acid administration (25 mg/kg, i.v.) on
the lung tissue Akt activation, in the rat model of thermal injury.
Sham group, n = 9; thermal injury (TI) group, n = 14; RA + TI
group, n = 14; RA group, n = 6. *p < 0.05 versus sham group;
#
p < 0.05 versus TI group.

Sham TI RA + TI

P-GSK3β - - 46 kDa

GSK3β total - - 46 kDa

Fig. 16. Effect of rosmarinic acid administration (25 mg/kg, i.v.) on
the lung tissue caspase-3 activation, in the rat model of thermal injury.
Sham group, n = 9; thermal injury (TI) group, n = 14; RA + TI
2.5 group, n = 14; RA group, n = 6. *p < 0.05 versus sham group;
#
p < 0.05 versus TI group.
pGSK/GSK protein expression

2.0 #
(fold change)

1.5
neutrophil oxidative burst play an important role in both
models of inflammation used in this work, it is possible to
1.0 speculate that rosmarinic acid might be exhibiting an
anti-inflammatory activity by a net effect, which includes its
0.5 antioxidant properties, inhibition of neutrophil activity, inhi-
bition of MMP-9 activity and modulation of the NF-jB
0.0 pathway.
Therefore, rosmarinic acid administration might be useful in
TI
am

TI

+
Sh

the management of inflammatory processes, and further

A
R

research on anti-inflammatory effects of rosmarinic acid and

Fig. 14. Effect of rosmarinic acid administration (25 mg/kg, i.v.) on
R. officinalis might lead to the discovery of new pharmacolog-
the lung tissue GSK-3b activation, in the rat model of thermal injury.
Sham group, n = 9; thermal injury (TI) group, n = 14; RA + TI ical tools in the treatment of inflammatory diseases. Given the
group, n = 14; RA group, n = 6. *p < 0.05 versus sham group; recognized role of inflammation and oxidative stress in neuro-
#
p < 0.05 versus TI group. degenerative diseases [72], cardiovascular diseases [73],

© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
 EFFECT OF ROSMARINIC ACID IN INFLAMMATION
Fig. 17. Effect of rosmarinic acid administration (25 mg/kg, i.v.) on
the lung tissue metalloproteinase-9 (MMP-9) activation, in the rat
model of thermal injury. Sham group, n = 9; thermal injury (TI)
group, n = 14; RA + TI group, n = 14; RA group, n = 6. *p < 0.05
versus sham group; #p < 0.05 versus TI group.
respiratory diseases [74] and cancer [75], these promising
results might unveil a potential research field for rosmarinic
acid and its clinical applications.
Acknowledgements
João Rocha was supported by FCT under a Doctoral Grant
(SFRH/BD/64180/2009). Marisa Freitas acknowledges the
financial support by FCT for the Pos-doc grant (SFRH/BPD/
76909/2011), in the scope of ‘QREN e POPH e Tipologia 4.1
e Formaç ão Avanç ada’, cosponsored by FSE and by National
Funds of MCTES.
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