Bioorganic & Medicinal Chemistry 21 (2013) 7570–7577

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Antioxidant action of propolis on mouse lungs exposed to short-term

cigarette smoke
Alan Aguiar Lopes a, Thiago Santos Ferreira b, Renata Tiscoski Nesi b, Manuella Lanzetti a,
Karla Maria Pereira Pires a, Ari Miranda Silva c, Ricardo Moreira Borges c, Antonio Jorge Ribeiro Silva c,
Samuel Santos Valença b, Luís Cristóvão Porto a,⇑
a
Programa de Pós-graduação em Biologia Humana e Experimental, Instituto de Biologia Roberto Alcântara Gomes, Universidade do Estado do Rio de Janeiro,
Laboratório de Reparo
Tecidual, Av Marechal Rondon 381, Rio de Janeiro, R.J. CEP 20.950-003, Brazil
b
Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
c
Núcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Propolis is a natural product with antioxidant properties. In this study, we tested the efficacy of propolis
Received 10 August 2013 against acute lung inflammation (ALI) caused by cigarette smoke (CS). C57BL6 male mice were exposed to
Revised 19 October 2013 CS and treated with propolis (200 mg/kg orally, CS+P) or only with propolis (P). A Control group treated
Accepted 28 October 2013
with propolis was sham-smoked (Control+P). We collected the lungs for histological and biochemical
Available online 7 November 2013
analyses. We observed an increase in alveolar macrophages and neutrophils in the CS group compared
with the Control+P. These counts reduced in the CS+P group compared to the CS group. The treatment
Keywords:
with propolis normalized all biochemical parameters in the CS+P group compared with the CS group,
Propolis
Oxidative stress
including nitrite, myeloperoxidase level, antioxidant enzyme activities (superoxide dismutase, catalase
Inflammation and glutathione peroxidase), reduced glutathione/oxidized glutathione ratio and malondialdehyde. Addi-
Lung tionally, TNF-a expression reduced in the CS+P group when compared with the CS group. These data
Mice imply a potential antioxidant and anti-inflammatory role for propolis with regard to ALI caused by CS
in mice.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Cigarette smoke (CS) is composed of 4000 substances. CS is the

Propolis (bee glue) is a resinous material produced by Apis that
protects the hive against intruders. Some studies have reported
that propolis contains phenolic-like compounds and esters, differ-
ent types of flavonoids, fatty acids, terpenes, steroids, amino acids,
polysaccharides, hydrocarbons and alcohols.1–4 Propolis also has
immunomodulatory,5,6 anti-bacterial,7 anti-viral,8,9 anti-tumoral,10
anti-ulcer,1,11 and radioprotective properties.12,13 Other studies
have investigated the antioxidant properties of propolis and have
reported that they were able to not only decrease lipid peroxida-
tion and DNA damage14,15 but also act as a free radical scavenger
and reduce interferon-gamma (INF-c) production.5,16 Clinically,
propolis has shown antioxidative actions without altering human
blood parameters17 or increasing respiratory parameters in asth-
matic patients.18 However, more studies are necessary to under-
stand the mechanisms that are involved in the beneficial
activities of propolis.
primary risk factor for developing Chronic Obstructive Pulmonary
Diseases (COPD).19 Although acute lung inflammation (ALI) is
caused by CS, but does not represent a good model of all the char-
acteristics of COPD pathogenesis,20 two processes act as hallmarks:
accumulation of inflammatory cells21 and lung oxidative stress.22
CS contains oxidants (1014 radicals/cigarette puff)23 that stimulate
phagocytes to produce reactive oxygen species (ROS), such as
hydrogen peroxide and radical hydroxyl, which generate lipid
peroxidation in cells and promote NF-jB signaling24 and TNF-a
production.25 Therefore, increasing our knowledge regarding the
antioxidant properties of propolis is pivotal for improving treat-
ment strategies for diseases related to tobacco dependence. The
aim of the present study was to evaluate the anti-inflammatory
and antioxidant actions of propolis against ALI caused by CS.
2. Materials and methods
2.1. Reagents

⇑ Corresponding author. Tel./fax: +55 21 23342426. Bovine serum albumin (BSA), bromophenol blue, 5,50 -dithi-
E-mail address: lcporto@uerj.br (L.C. Porto). obis-(2-nitrobenzoic acid) (DTNB), eosin, glycerol, hematoxylin,

0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.10.044
 A. A. Lopes et al. / Bioorg. Med. Chem. 21 (2013) 7570–7577
hexadecyltrimethylammonium bromide (HTAB), 2-mercaptoethanol,
naphthylenediamide dihydrochloride, nicotinamide adenine dinu-
cleotide phosphate (NADPH), oxidized glutathione, phosphoric
acid, reduced glutathione, sodium acetate, sodium dodecyl sulfate
(SDS), sodium nitrite, sulfanilamide, 3,30 ,5,50 -tetramethylbenzidine
(TMB), triethanolamine, Tris–HCl, Tween 20, and 2-vinylpyridine
were purchased from Sigma Chemical (St. Louis, MO, USA).
Bradford reagent was purchased from Bio-Rad (Hercules, CA,
USA). Ethanol, formalin, hydrogen peroxide, potassium phosphate,
and sodium chloride (NaCl) were purchased from Vetec (Duque de
Caxias, Brazil). A commercial ethanolic extract of propolis (30% in
ethanol) was purchased from Coapi (São Gonçalo, Brazil).
2.2. Chemical analysis of the ethanolic extract of propolis
We performed full-scan ESI-MS analyses using a MicrOTOF II
mass spectrometer (Bruker Daltonics, Inc., Boston, MA, USA). We
infused samples directly into the source at a flow rate of
0.12 mL/h. The source temperature was set at 180 °C, the drying
gas (nitrogen) flow rate was 4.0 L/min and the nebulizer gas (nitro-
gen) pressure was 0.4 bar. In negative mode, the capillary voltage
was 3.8 kV, the capillary exit voltage was 150 V, the skimmer 1
and 2 voltages were 50 V and 15 V, respectively, the hexapole 1
voltage was 23 V, the hexapole RF voltage was 300 Vpp, the lens
1 transfer was 88 ls and the lens 1 pre plus stage was 15 ls. Data
were acquired in negative mode in the range of 50–2000 m/z. Mass
calibration was achieved by infusing ammonium formate in an iso-
propanol–water mixture (1:1, v/v) as an external standard. All data
were analyzed using Bruker Daltonics ESI Compass Data Analysis
Version 4.0 SP 1 (Bruker Daltonics Inc., MA, USA). Mass error (the
difference between measured and theoretical mass) and sigma (a
parameter calculated by the software that accounts for the differ-
ence between theoretical and measured isotopic pattern; smaller
values of sigma indicate better matching; data not shown) calcu-
lated for each datum.
2.3. Animals
Eight-week-old, male, C57BL/6 mice (18–22 g) were purchased
from the Instituto de Veterinária—Universidade Federal Flumin-
ense (Niterói, Brazil). The mice were fed Purina chow and allowed
unrestricted access to water in a controlled environment main-
tained at 21 °C ± 2 °C, 54–56% relative humidity, and a 12-h
light/dark cycle. The mice were allowed to acclimate for two weeks
prior to the experimental procedures. The Ethics Committee from
Instituto de Biologia Roberto Alcantara Gomes/Universidade do
Estado do Rio de Janeiro approved the use of animals for this exper-
iment (CEA). The CEA follows guidelines from the Intramural
Animal Care and Use (ACU) program of the National Institutes of
Health (NIH).
2.4. CS exposure and procedures
Thirty C57BL6 mice were divided into three groups (10 mice/
group): a control group that was sham-smoked and treated with
200 mg/kg of propolis (100 lL),26,27 a group that was exposed to
CS and treated with vehicle (100 lL of ethanol) and a CS group
treated with propolis (100 lL). All treatments were administered
orally by gavage after last cigarette smoke exposure from each
day. The ethanolic extract of propolis (200 mg/kg) was prepared
daily and this dose was choosing after in vitro experiment of
dose–response by using RAW 264.7 cells (data not shown). CS
exposure was performed with 12 commercial full-flavored Marl-
boro cigarettes (10 mg tar, 0.9 mg nicotine, and 10 mg carbon
monoxide) per day for 5 days with the use of a smoking chamber,
7571
as described previously.28,29 Briefly, the animals were placed in the
inhalation chamber (40 cm long, 30 cm wide and 25 cm high) in-
side an exhaust hood with the exhaust fan off. A cigarette was cou-
pled to a plastic 60-mL syringe, and puffs of smoke were drawn
into the syringe and then expelled into the inhalation chamber.
One liter of smoke from each cigarette was aspirated with this syr-
inge (20 puffs of 50 mL each), and each puff was immediately in-
jected into the chamber. The animals were maintained in this
smoke-filled environment (±3%) for 6 min. Then, the cover of the
inhalation chamber was removed, and the exhaust fan of the hood
was turned on to evacuate the smoke. The smoke was evacuated
within 1 min. This exposure to CS was repeated four times (4
 6 min) with an exhaust interval of 1 min after each exposure.
We repeated this procedure three times per day (morning, noon
and afternoon) which resulted in exposure to the smoke of twelve
cigarettes over 72 min. Each cigarette produced 300 mg/m3 of total
particulate matter in the chamber (measured by weighing the
material collected on Pallflex filters). Carboxyhemoglobin (COHb)
levels were measured to confirm that the treatment was not toxic,
as described previously.30
2.5. Broncoalveolar lavage and lung homogenates
Twenty-four hours after CS exposure and propolis treatment,
the mice were sacrificed by cervical dislocation. A broncoalveolar
lavage (BAL) was performed in the left lung of each animal. Briefly,
the right lung was clamped, and a cannula was inserted into the
trachea. The airspaces were washed with buffered saline solution
(500 lL) three times and the flow-through (final volume 1.2–
1.5 mL) was maintained on ice. Next, the BAL fluid was centrifuged,
and the supernatant was collected and stored on ice for subsequent
analyses of nitrite and MPO content. Then, the left lungs of each
group were removed and immediately homogenized (Homoge-
nizer NovaTécnica mod NT 136, Piracicaba, Brazil) in 1.0 mL potas-
sium phosphate buffer (pH 7.5). The homogenates were
centrifuged at 7000g (Centrifuge FANEM mod 243 M, São Paulo,
Brazil) for 10 min, and the supernatants were stored at 20 °C
for analyses of SOD, CAT, GPx, GSH/GSSG ratio, malondialdehyde
and TNF-a expression. The total protein in the samples (tissues
and BAL) was determined by the Bradford method.31
2.6. Tissue processing and morphometry
The right ventricles of all mice were perfused with saline to re-
move the blood. Next, the right lungs of all animals were inflated
with 4% phosphate buffered formalin (pH 7.2) at 25 cm H2O pres-
sure for 2 min and then ligated, removed and weighed. The inflated
lungs were fixed for 48 h before embedding in paraffin. Sagittal,
4-lm serial sections of the right lungs were stained with hematox-
ylin and eosin (H&E) for histological analyses. Alveolar macrophage
and neutrophil numbers were estimated by counting ten different
random fields (5 random fields from 2 different sections) per lung.
Morphometry was performed by using a microscope (20 objec-
tive lens) and total area analyzed was 2 mm2/lung. Two investi-
gators performed morphometry by counting coded sections.
2.7. Nitrite content
The nitrite contents in the BAL fluid were determined using a
method based on the Griess reaction.32 A quantity of 100 lL of
sample was mixed with 100 lL of Griess reagent (1% sulfanilamide
in 5% phosphoric acid and 0.1% naphthylenediamide dihydrochlo-
ride in water) and was incubated at room temperature for
10 min. The absorbance was measured with a plate reader at
550 nm (Bio-Rad Microplate Reader model 680, Hercules, CA,
USA). Nitrite concentrations in the samples were determined from
7572 A. A. Lopes et al. / Bioorg. Med. Chem. 21 (2013) 7570–7577
a standard curve that was generated with different concentrations
of sodium nitrite.
2.8. Myeloperoxidase activity
The MPO activity in the BAL fluid was measured with HTAB,
TMB, and hydrogen peroxide.33 Initially, 100 lL of sample was cen-
trifuged with 900 lL of HTAB at 14,000g for 15 min. Then, 75 lL of
the supernatant was incubated with 5 lL of TMB for 5 min at 37 °C.
Next, 50 lL of hydrogen peroxide was added, and the mixture was
incubated for 10 min at 37 °C. Finally, 125 lL of sodium acetate
buffer was added. The reaction was measured with a microplate
reader at 630 nm.
2.9. Superoxide dismutase, catalase, and glutathione peroxidase
activities
SOD, CAT, and GPx activities were determined in lung homoge-
nates. SOD activity was assayed by monitoring the inhibition of
adrenaline auto-oxidation at 480 nm.34 CAT activity was measured
by the rate of decrease of hydrogen peroxide concentrations mon-
itored at 240 nm.35 GPx activity was measured by monitoring the
oxidation of NADPH at 340 nm in the presence of hydrogen
peroxide.36
2.10. Reduced glutathione/oxidized glutathione ratio
The GSH/GSSG ratio was estimated in lung homogenates by first
reacting GSH and GSSG with DTNB and then measuring the
2-nitro-5-thiobenzoate (TNB) chromophore produced in the reac-
tion.37 To determine GSSG, the samples were treated with 2-vinyl-
pyridine, which covalently reacts with GSH (but not GSSG). The
excess 2-vinylpyridine was neutralized with triethanolamine.
Then, the rate of formation of TNB, which was measured at
412 nm, was found to be proportional to the concentration of GSSG
in the sample. The GSH or GSSG concentrations in the samples
were determined from a standard curve, which was generated with
different concentrations of purified GSH or GSSG.
2.11. Oxidative damage
As an index of oxidative damage induced by lipid peroxidation,
we used the thiobarbituric acid reactive substances (TBARS) meth-
od to analyze malondialdehyde (MDA) products during an acid-
heating reaction, as previously described by Draper et al.38 Briefly,
samples from lung homogenates were mixed with 1 mL of 10% tri-
chloroacetic acid and 1 mL of 0.67% thiobarbituric acid; the sam-
ples were then heated in a boiling water bath for 30 min. TBARS
levels were determined by absorbance at 532 nm and expressed
as MDA equivalents.
2.12. Western blotting
Samples from lung homogenates were denatured in sample
buffer (50 mM Tris–HCl pH 6.8, 1% SDS, 5% 2-mercaptoethanol,
10% glycerol, 0.001% bromophenol blue) and heated in boiling
water for 3 min. The samples (50 lg of total protein) were
dissolved by 15% SDS–polyacrylamide gel electrophoresis, and
the separated proteins were transferred to nitrocellulose
membranes (Amersham Pharmacia Biotech GE Healthcare Bio-
Sciences Corp., Piscataway, NJ, USA). Rainbow markers (Amersham
Pharmacia Biotech GE Healthcare Bio-Sciences Corp.) were run in
parallel to estimate molecular weights. The membranes were
blocked with Tween-TBS (20 mM Tris–HCl, pH 7.5, 500 mM NaCl,
0.5% Tween-20) containing 2% BSA, and incubated with goat
anti-mouse primary TNF-a antibodies (Santa Cruz Biotechnology,
Santa Cruz, CA, USA). After extensive washing in Tween-TBS, the
membranes were incubated with biotin-conjugated donkey
anti-goat immunoglobulin G (Santa Cruz Biotechnology) for 1 h.
Then, the blots were developed with the Electrochemiluminescent
Western Detection Reagent (Amersham Pharmacia Biotech GE
Healthcare Bio-Sciences Corp.), according to the manufacturer’s
instructions. The membranes were incubated with ponceau stain
as an internal control.
2.13. Statistical analyses
The values for all the measurements are expressed as the means
± SEM. Analyses of cell counts, nitrite, MPO, SOD, CAT and GPx
were performed with a one-way ANOVA, followed by a Tukey
post-hoc test (p <0.05 was considered significant). Analysis of
GSH/GSSG ratio was performed with the Kruskal–Wallis test, fol-
lowed by the Dunn’s post-hoc test (p <0.05 was considered signif-
icant). GraphPad Prism software was used to perform the statistical
analyses (GraphPad Prism version 5.0, San Diego, CA, USA).
3. Results
3.1. ESI-MS analysis of propolis
Data acquired with ESI-MS analysis enable us to recognize the
major constituents found in Brazilian green propolis (Fig. 1). Arte-
pillin C, drupanin and baccharin, flavonoids derivatives, caffeoyl-
quinic acid (this particular compound might be the bioactive
chlorogenic acid) and dicaffeoylquinic acid were identified
(Table 1). Those constituents are typical bioactive secondary
metabolites with a wide range of physiological effects.
3.2. Effects of propolis on cell infiltration
Lung sections were examined by light microscopy to determine
whether propolis was protective against the effects of CS. No
changes were observed in lung histoarchitecture in the CS group
(Fig. 2b) compared with the control (Fig. 2a) or CS+P groups
(Fig. 2c), but an increased cellularity in the alveolar septa in the
CS group was observed. However, alveolar macrophages (AMs)
and neutrophils (PMNs) were estimated in lung sections stained
with H&E (Fig. 3). The number of AMs and PMNs in the CS group
were significantly higher than in the Control+P group (p <0.01
and p <0.001, respectively).
3.3. Effects of propolis on nitrite content
The nitrite contents in BAL fluids increased 116% in the CS group
(Table 2) compared to the Control+P group (p <0.001). However,
the nitrite content in the CS+P group (p <0.001) were reduced
53% in comparison to those observed in the CS group.
3.4. Propolis reduced myeloperoxidase activity
The MPO activity in the BAL fluids increased fourfold in the CS
group (Table 2) compared to the Control+P group (p <0.001). The
MPO activity in the CS+P group was reduced approximately 1.5-
fold in comparison to the CS group (p <0.05).
3.5. Propolis enhanced antioxidant enzyme activities
The SOD, CAT, and GPx activities (Table 2) in lung homogenates
increased 113%, 148% and 102%, respectively, in the CS group com-
pared to the Control+P group (p <0.001 for all). The SOD, CAT, and
 A. A. Lopes et al. / Bioorg. Med. Chem. 21 (2013) 7570–7577 7573

Intens. -MS, 0.1-1.0min #(7-57)

x105
1.50
299.16473

1.25

1.00

0.75 231.10211

0.50 275.09155 329.17235

257.15392 315.15802
0.25 515.11895
187.11312 401.30339
353.08852 373.27265
429.26267 447.24307 489.15876
0.00
200 250 300 350 400 450 500 m/z
Intens. -MS, 0.1-1.0min #(7-57)
x105
1.50
299.16473

1.25

1.00

0.75 231.10211

0.50 275.09155 329.17235

257.15392 315.15802
0.25 284.03253
247.09810
353.08852 363.16243
0.00
240 260 280 300 320 340 360 m/z
Intens. -MS, 0.1-1.0min #(7-57)
x105
1.50
299.16473
299.05632
1.25

1.00

0.75
301.07075
0.50
300.16725
0.25

0.00
299.00 299.25 299.50 299.75 300.00 300.25 300.50 300.75 301.00 301.25 m/z

Figure 1. Data acquired with ESI-MS analysis with regard to major constituents found in Brazilian green propolis. Artepillin C, drupanin and baccharin, flavonoids derivatives,
caffeoylquinic acid, and dicaffeoylquinic acid can be identified.

Table 1
Full-scan ESI-MS analyses of propolis

Proposed identification Formula Exact mass [MH] Experimental mass Error (ppm)
Artepillin C C19H24O3 300.17254 299.16472 299.1647 0.00
Drupanin C14H16O3 232.10994 231.10212 231.1021 0.00
Baccharin C23H24O4 364.16746 363.15963 363.1625 7.70
Caffeoylquinic acida C16H18O9 354.09508 353.08726 353.08852 3.58
Dicaffeoylquinic acid C25H24H12 516.12678 515.11895 515.1190 0.00
Trihydroxymethoxyflavan C16H12O6 300.06339 299.05556 299.0562 2.53
n.i. C17H22O2 258.16198 257.15415 257.1539 0.91
n.i. C15H16O5 276.09977 275.09195 275.0915 1.45
Trihydroxymethoxyflavanone C16H14O6 302.07904 301.07121 301.0707 1.54
Artepillin C+OH C19H24O4 316.16746 315.15963 315.1579 5.44
Artepillin C+OCH3 C20H26O4 330.18311 329.17528 329.1722 8.91
n.i. C26H42O3 402.31339 401.30557 401.3034 5.43
n.i. C21H30O13 490.16864 489.16082 489.1588 4.20

n.i.: not identified.

a
Chlorogenic acid.

GPx activities in the CS+P group were reduced 47%, 64%, and 58%, ever, the MDA in the CS+P group was significantly lower (52%, p
respectively, when compared with the CS group (p <0.001 for all). <0.01) than that observed in the CS group.

3.6. Propolis improved the GSH/GSSG ratio

3.8. Propolis reduced TNF-a expression
The GSH/GSSG ratio was reduced 67% in the CS group compared
to the Control+P group (p <0.05) (Table 2). However, the GSH/GSSG TNF-a expression increased in the CS group compared to
ratio in the CS+P group increased 171% when compared with that the Control+P group (Fig. 4). The CS+P group exhibited reduced
of the CS group (p <0.05). TNF-a expression compared to the CS group.

3.7. Propolis reduced oxidative damage 4. Discussion

The oxidative damage assessed by the TBARS method and ex- Propolis is a natural product that has been shown to have both
pressed as MDA equivalents was increased in the CS group anti-inflammatory and antioxidant properties.1,2,4,17,18,39 In this
(261%, p <0.001) compared to the Control+P group (Table 2). How- study, we evaluated the effects of propolis on CS-induced ALI in
7574 A. A. Lopes et al. / Bioorg. Med. Chem. 21 (2013) 7570–7577

Figure 2. This is a histological analysis of lung tissue obtained from mice exposed to ambient air or cigarette smoke (CS). Propolis treatment was administered to the
Control+P group and to one CS+P group. Although, the CS group had no lung parenchyma destruction, inflammatory cell influxes were evident. The CS+P group was similar to
the control group. The groups are Control+P (a), CS (b), and CS+P (c). The photomicrographs are representative of each group studied. Lung histology was performed on all
mice (n = 10 for all groups), and lungs were stained with hematoxylin & eosin with magnification of 200.

Table 2
Biochemical analyses of nitrite, MPO, antioxidant enzyme activities (SOD, CAT and
GPx), GSH/GSSG ratio and MDA

Groups Control+P CS CS+P

Nitrite (lM/mg 50.01 ± 4.19 108.70 ± 7.73*** 58.84 ± 3.42###
ptn)–
MPO (mU/mg 134.00 ± 12.99 526.50 ± 34.72*** 385.10 ± 27.64#
ptn)–
SOD (U/mg ptn)–– 234.50 ± 19.78 523.50 ± 29.60*** 246.80 ± 15.69###
CAT (U/mg ptn)–– 37.38 ± 3.39 92.68 ± 6.24*** 59.84 ± 4.55###
GPx (U/mg ptn)–– 2.23 ± 0.17 4.51 ± 0.31*** 2.64 ± 0.19###
GSH:GSSG–– 1.08 ± 0.17 0.73 ± 0.07* 1.25 ± 0.10#
MDA (nM/mg 0.26 ± 0.05 0.94 ± 0.07*** 0.49 ± 0.06##
ptn)––

Values for all measurements are expressed as the mean ± SEM. All measurements
were analyzed with a one-way ANOVA, followed by a Tukey post-hoc test (p <0.05
was considered significant), except for the GSH:GSSG ratio, which was analyzed
with the Kruskal–Wallis test, followed by the Dunn’s post-hoc test (p <0.05 was
Figure 3. Morphometry of mice exposed to ambient air or cigarette smoke (CS). considered significant). (n = 10 for all groups). MPO - myeloperoxidase, SOD -
Alveolar macrophages and neutrophils increased in the CS group compared to the superoxide dismutase, CAT - catalase, GPx - glutathione peroxidase, GSH - reduced
Control+P group. Propolis treatment reduced inflammatory cell counts. The values glutathione, GSSG - glutathione disulfide, MDA - malondialdehyde equivalents.
–
are the mean ± SEM. Analysis was performed with ANOVA, followed by the Tukey Analysis made from BAL samples.
––
post-hoc test (p <0.05 was considered significant). ⁄⁄p <0.01 and ⁄⁄⁄p <0.001 when Analysis made from tissue samples.
*
compared with Control+P; ##p <0.01 and ###p <0.001 when compared with CS. p <0.05 compared with the control group.
***
(n = 10 for all groups.) p <0.001 compared with the control group.
#
p <0.05 compared with the CS group.
##
p <0.01 compared with the CS group.
###
p <0.001 compared with the CS group.
mouse lungs. This is the first study of the efficacy of propolis treat-
ment against CS-induced ALI. CS is known to be a primary factor for
the development of COPD through a progressive and constant state
of inflammation, which is characterized by parenchyma structure
changes, inflammatory cell influxes, a redox imbalance, and nitric
oxide and cytokine overproduction.40 The compounds identified
in our propolis are regularly found in other Brazilian propolis,41,41–
45
but the phytochemical compounds and their properties are Figure 4. Western blotting for TNF-a in mice exposed to ambient air or cigarette
responsible for anti-inflammatory and antioxidant propolis prop- smoke (CS). TNF-a expression is evident in the CS group. Propolis treatment
erties is a question which we need to investigate yet. reduced TNF-a expression. (n = 2 for each group).
 A. A. Lopes et al. / Bioorg. Med. Chem. 21 (2013) 7570–7577
In this work, we evaluated the effects of propolis on acute
inflammation caused by CS exposed in mice during five days, as de-
scribed previously.28–30 We observed its anti-inflammatory and
antioxidant actions and investigated the main biochemical param-
eters involved in inflammation and oxidative stress. We adminis-
tered 200 mg/kg/day of propolis because this dose has previously
been shown to have antioxidant properties with regard to bone
fracture healing46 and anti-inflammatory actions on cytokine pro-
duction in spleen and blood studies.26,27 Furthermore, higher doses
(300 mg/kg) have been shown to produce pro-oxidant activity,
increasing SOD and GPx activities and lipid peroxidation in lysate
of erythrocytes and plasma, respectively.47
We first observed that propolis administration did not alter
lung histoarchitecture in both the CS+P and Control+P groups. This
result was different from that of the CS-exposed group, for which
we observed increased cellularity in the alveolar septa. Bhadauria
et al.48 have reported that propolis promoted a histological protec-
tion effect against CCL4-induced liver injury by inhibitory actions
against lipid peroxidation and by its free radical scavenging ability.
Besides, El-Kenawy et al.49 have reported that propolis showed an
antagonistic and antioxidant effects against aluminum chloride
(AlCl3)-induced histopathological kidneys tissues, avoiding mor-
phological changes in proximal and distal convoluted tubules and
appearance of glomerular capsular space. In addition, propolis
compounds, such as artepillin C, coumaric acid and drupanin, have
antioxidant effects against environmental oxidant agents, such as
UV rays, in skin tissue.50 These antioxidant propolis properties
are against superoxide radicals, alkoxyl radicals and in the oxida-
tion reaction of luminol on hydrogen peroxide. Therefore, we
hypothesized that these compounds could protect against oxidants
delivered by CS and act to decrease tissue oxidative damage and,
therefore, avoid the initiation of the inflammatory process in the
lung parenchyma.
In vitro and in vivo studies have shown that influxes of inflam-
matory cells like macrophages are highly dependent on certain
cytokines, such as TNF-a.24,51 In our study, propolis administration
resulted in decreased inflammatory cell counts in the alveoli. These
figures are associated with decreasing TNF-a expression in the CS+P
group, despite its unclear results. These results suggest that propo-
lis prevented inflammatory cell influxes by reducing the level of
this pro-inflammatory cytokine. That scenario was associated to
other studies as reduction both TNF-a production and inflamma-
tory cell influxes in lung tissue in OVA-sensitized mice52 and
TNF-a modulation may be caused by a down-regulation of the tran-
scription factor kappa B (NF-jB) in different kind of cells just like
Treg cells, 3T3 adipocytes cells and LNCaP prostate cancer cells. In
the last work, the result obtained was caused by the artepillin C
present in propolis.53,54 Besides that, Szliszka and colleagues inves-
tigated propolis effects in several cytokines expression, in TNF-a,
over LPS+, IFN-c, and phorbol 12-myristate 13-acetate stimulated
RAW 264.7 macrophages cells and they observed decrease of cyto-
kines expressions through artepillin C anti-inflammatory action.55
These studies strongly give a suggestion which artepillin C com-
pound is involved in propolis action in TNF-a expression, but more
studies are necessary to understand it and clarify how artepillin C
acts in pro-inflammatory cytokine mechanisms of action in ciga-
rette smoke-stimulated inflammatory process.
MPO is an enzyme produced mainly by neutrophils and serves
as an indicator of neutrophilia and inflammation.56 Therefore,
understanding the action of propolis on neutrophil and MPO
metabolism with regard to CS exposure is pivotal. In the present
study, propolis treatment decreased the MPO levels in BAL samples
induced by CS exposure. Despite a few studies of propolis effects
on MPO activity, there are studies, which investigated propolis
effects on neutrophils. Krol and colleagues investigated propolis
effects on neutrophils which were stimulated by phorbol myristate
7575
acetate and they found out phenolic compounds like caffeic-
acid-phenylethyl-ester, galangin, kaempferol and kaempferid are
responsible for decreasing ROS and RNS levels by their antioxidant
effects.57 Other study of Simoes-Ambrosio et al.58 suggested
aromadendrin-40 -methyl ether, artepillin C, and baccharin have
antioxidant effects against ROS produced by neutrophils. Artepillin
C, baccharin and flavone derivates are compounds found in propo-
lis extract used in this work, these informations suggest that the
decrease in the number of neutrophils in the lung parenchyma
was caused by the antioxidant effects of propolis over ROS pro-
duced by neutrophils and probably over MPO’s oxidative product,
hypochlorous acid.56
Antioxidant enzymes activities were analyzed to understand
the redox balance in mouse lungs after CS exposure. In the present
study, we observed an increase in SOD, CAT, and GPx activity levels
in the CS group, which is in agreement with the results of previous
reports of our group28–30,59, but in CS+P and Control+P groups, en-
zyme activities were lower. Propolis administration resulted in a
significant decrease in the above-mentioned enzymes activity lev-
els in lung homogenates. Few studies have reported the effects of
propolis on SOD and GPx activity in lungs. Nagai et al. have de-
scribed propolis as having an SOD-like property.60 This description
was based on the ability of propolis to scavenge superoxide anions
in a way similar to artepillin C and drupanin,60 both of which have
antioxidant properties over superoxide anion.61 Probably, in this
study, the SOD-like action of propolis resulted of antioxidant prop-
erties of artepillin C and drupanin over superoxide anion, causing a
decrease in the superoxide anion levels in the CS+P group, this
probably influenced in superoxide dismutase activity decreased
as observed in this study. We also suggest that the SOD-like effects
of propolis indirectly influence hydrogen peroxide levels and may
have decreased CAT activity in the CS+P group. Because propolis
and its compounds have antioxidant properties over hydrogen per-
oxide,45,61 we suggest that these properties influence GPx activity
in propolis-treated groups. This suggestion can be confirmed by a
high GSH:GSSG ratio. This parameter is important not only because
of the intrinsic redox relationship between GSH and GPx levels but
also its importance as an oxidative stress indicator.37 In addition,
Capucho et al. study showed that propolis increased sulfidryl
groups, probably through its phytochemical compounds as artepil-
lin C, caffeolyquinic acid and drupanin which are able to capture
free radicals,62 maintaining GSH levels high in epididymis and
sperm study. In this study, we observed an increase in the
GSH:GSSG ratio in the group exposed to CS and treated with prop-
olis. This increase might be attributable to the action of propolis in
preventing GSH depletion, just like was observed in propolis-
treated liver injuries.63
Following an interaction with superoxide anions, nitric oxide
forms peroxynitrite during CS exposure.64 In our study, the propo-
lis-treated group decreased nitrite contents differently from the CS
group. Song et al. have described reduced nitric oxide synthase
gene expression following propolis administration in RAW 264.7
cells.65 Additionally, other studies have demonstrated that artepil-
lin C66 and hydroxycinnamic acid67 regulate NO production in mice
in inflammation process and in protein synthesis of inducible nitric
oxide synthase, respectively. Therefore, we suggest a possible
action for propolis in nitric oxide metabolism that resulted in de-
creased nitric oxide levels in the CS+P group, possibly through
mechanisms as described by other studies before. This mechanism
could be associated with the decrease in SOD activity levels
described previously, causing a reduction of peroxynitrite
production.
Malondialdehyde is a product of lipid peroxidation and serves
as an oxidative damage marker in several tissue types.38 The pres-
ent data corroborate our previous findings. In addition, propolis
administration decreased MDA levels in lung homogenates. Simi-
7576 A. A. Lopes et al. / Bioorg. Med. Chem. 21 (2013) 7570–7577
larly, previous works have shown that artepillin C41 and coumaric
acid68 prevent lipid peroxidation by scavenging activity for oxida-
tive radicals in mice lungs and eye cells. These data suggest that
propolis has an antioxidant effect against CS exposure in lungs
due to its scavenger action against lipid radicals, but the study
how propolis acts against the lipid peroxidation process, investi-
gate levels of other markers of lipid peroxidation are necessary like
4-hydroxynoneal and hydroperoxydes.
This present work is the first study, which the anti-inflamma-
tory and antioxidant actions of propolis over cigarette smoke, stim-
ulated inflammation and oxidative stress are described. Despite
our results and literature background, which suggest evidences
about propolis properties, more studies are necessary to investi-
gate the anti-inflammatory and antioxidant mechanisms in propo-
lis effects over this type of inflammation process, especially to
compare to sham group. A limitation of our study was about a lack
in control group with vehicle, which could be important to
compare over control group with propolis, although our previous
studies with cigarette smoke with similar exposure time did not
show any modification in controls.29,69,70 In conclusion, the present
study has shown for propolis treatment exerts anti-inflammatory,
through cell influx and TNF-alpha expression decreasing, and
antioxidant, through antioxidant enzymes activity and oxidative
damage increase avoided. These propolis effects with respect to
ALI caused by CS exposure in mouse lungs, probably happened
by phytochemical compounds, especially artepillin C, for its anti-
inflammatory and antioxidant effects described previously.
Acknowledgments
S.S.V. and L.C.P. are grateful to Brazilian Research agencies for
providing Grants (CNPq and FAPERJ). A.A.L., M.L., K.M.P.P. and
R.T.N. have bursaries from CAPES. This study is part of A.A.L.’s
Masters in Science Thesis (BHEx-UERJ).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.10.044.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
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