Content uploaded by Rodrigo J S Dalmolin
Author content
All content in this area was uploaded by Rodrigo J S Dalmolin on Oct 01, 2018
Content may be subject to copyright.
FULL COMMUNICATION
Turnera subulata Anti-Inflammatory Properties
in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages
Nata
´lia Cabral Souza,
1
Juliana Medeiros de Oliveira,
1
Maurı
´lio da Silva Morrone,
2
Ricardo D’Oliveira Albanus,
3
Maria do Socorro Medeiros Amarante,
4
Christina da Silva Camillo,
4
Silvana Maria Zucolotto Langassner,
4
Daniel Pens Gelain,
2
Jose
´Cla
´udio Fonseca Moreira,
2
Rodrigo Juliani Siqueira Dalmolin,
1
and Matheus Augusto de Bittencourt Pasquali
1,5
1
Institute of Tropical Medicine of Rio Grande do Norte, Federal University of Rio Grande do Norte, Natal, RN, Brazil.
2
Department of Biochemistry, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil.
3
Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, USA.
4
Department of Morphology, Federal University of Rio Grande do Norte, Natal, RN, Brazil.
5
Department of Food Engineering, Federal University of Campina Grande, Campina Grande, PB, Brazil.
ABSTRACT In South America, particularly in the Northeastern regions of Brazil, Turnera subulata leaf extract is used
as an alternative traditional medicine approach for several types of chronic diseases, such as diabetes, hypertension,
chronic pain, and general inflammation. Despite its widespread use, little is known about the medicinal properties of the
plants of this genus. In this study, we evaluate the antioxidant and anti-inflammatory of T. subulata leaf extract in an in
vitro model of inflammation, using lipopolysaccharide-stimulated RAW-264.7 macrophage cell line. We observed that
cotreatment with T. subulata leaf extract was able to reduce the oxidative stress in cells due to inflammatory response.
More importantly, we observed that the leaf extract was able to directly modulate inflammatory response by altering
activity of members of the mitogen-activated protein kinase pathways. Our results demonstrateforthefirsttimethat
T. subulata have antioxidant and anti-inflammatory properties, which warrant further investigation of the medicinal
potential of this species.
KEYWORDS: inflammation oxidative stress RAGE TLR-4 Turnera subulata
INTRODUCTION
The health benefits of plant compounds acquired
through the ingestion of tea, juice, fruits, and derivatives
are an area of active research and have been approached by
many different studies. In fact, the biological composition of
these beverages and foods has been regarded as an important
factor to reduce risk of chronic diseases.
1–3
Moreover, with the
development of new technologies to produce industrialized
foods, the interest inconsuming natural compounds that prove
to be beneficial to human health has increased in last few years.
Of particular note are the compounds with anticancer, anti-
inflammatory, and antioxidants properties, which have been
extensively investigated in plant extracts and derivatives.
Turnera subulata is a tropical plant that occurs mainly in
Northeastern Brazil.
4
It is used in traditional medicine for
treatment of different diseases, such as diabetes, hyperten-
sion, chronic pain, and inflammation.
5
Some authors have
also reported aphrodisiacs and anxiolytic properties in
plants of the genus Turnera. Characteristically, phenolic
compounds, flavonoids, alkaloids, and tannins have been
related as the main biological molecules of genus Turnera,
which can be responsible for mediating biological activities
in organisms (Table 1).
6
Some reports have demonstrated
the antioxidant effects of extracts obtained from Turnera
species.
7–9
However, the biological properties and effects of
Turnera subulata leaf extract remain unknown.
Recently, studies have associated positive effects attrib-
uted to genus Turnera with higher concentrations of arbutin,
a biological compound found in different parts of the
plant.
10–12
The decrease of cytokine secretion, such as
TNF-a, IL-1, and IL-6, observed in treatments with Turnera
diffusa or Turnera ulmifolia in different models of in vivo
inflammation was reported as positive effects of those
plants.
5,7,13,14
Moreover, the antioxidant properties of those
extracts were demonstrated through the decreased lipoper-
oxidation and modulation of antioxidant enzymes such as
glutathione peroxidase, superoxide dismutase (SOD), and
catalase (CAT) in the liver of CCl4-treated rats.
Manuscript received 28 March 2016. Revision accepted 27 July 2016.
Address correspondence to: Matheus Augusto de Bittencourt Pasquali, MSc, PhD,
Unidade de Engenharia de Alimentos, Universidade Federal de Campina Grande,
Campina Grande, Paraı
´ba 58109-900, Brazil, E-mail: matheuspasquali@gmail.com
JOURNAL OF MEDICINAL FOOD
J Med Food 00 (0) 2016, 1–9
#Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2016.0047
1
The role of reactive oxygen species (ROS) and reactive
nitrogen species (RNS) in mechanisms that lead to patho-
logical diseases are largely studied. It is known that ROS/
RNS is involved in normal signaling pathways and meta-
bolic processes. ROS/RNS products, such as superoxide
radical anion (O
2
-
$), hydrogen peroxide (H
2
O
2
), hydroxyl
radical (OH$), and nitric oxide, can trigger cells to prolif-
erate and differentiate, and cell apoptosis and death.
15–19
However, an imbalance in the organism between ROS/RNS
production and antioxidant defenses can lead to a condition
known as oxidative stress.
20–22
Cancer, neurodegenerative
diseases, diabetes, and cardiovascular diseases are patho-
logical conditions that present oxidative stress in its eti-
ology. Moreover, ROS/RNS also plays central involvement
in activation and progression of inflammatory pathways.
23,24
Due to the complex biological composition, the consump-
tion of plants extracts has been largely recommended to
prevent risk of diseases. In part, this is suggested as a result
of antioxidant properties that extracts exhibit.
In this study, we investigated the antioxidant and anti-
inflammatory effects of leaf extract from Turnera subulata in
an in vitro model of inflammation. We observed that leaf
extracts exhibit antioxidant activities. Moreover, the extract
was able to inhibit phosphorylation in mitogen-activated
protein kinases (MAPKs) p38, ERK ½, and JNK, which was
mediated by lipopolysaccharide (LPS)-stimulated macro-
phages. Markers of macrophage response for inflammation
such as toll-like receptor 4 (TLR4), receptor for advanced end
glycation products (RAGE), and CD40 were also investi-
gated. Secretion of cytokines TNF-aand IL-1bin the medium
was measured. Leaf extract of Turnera subulata inhibited
the effect mediated by LPS in our model of inflammation.
Our data demonstrate for the first time the potential anti-
inflammatory and antioxidant effects of Turnera subulata.
MATERIALS AND METHODS
Chemicals
Culture analytical grade reagents were purchased from
Sigma Chemical Co. (St.Louis, MO, USA). Antibodies
against total and phospho-SAPK-JNK, total and phospho-
p38 MAPK, total and phospho-ERK, TLR4, and b-actin
were all purchased from Cell Signaling (Beverly, MA,
USA). Anti-RAGE and CD40 antibodies were purchased
from AbCam (Cambridge, United Kingdom).
Cell culture
The mouse macrophage cell line RAW 264.7 was grown
in RPMI-1640 and 10% FBS, and maintained at 37Cinan
atmosphere containing 5% CO
2
. The media were supple-
mented with 1% penicillin/streptomycin.
Extract
The leaves of Turnera subulata were collected in Parna-
mirim (555021.000 S-3511050.500 W)–Rio Grande do Norte
-Brazil. The plant material was identified by the Pharmacy
Faculty Center of Federal University of Rio Grande do Norte–
UFRN–Brazil. Specimen from Turnera subulata was depos-
ited at the Herbario do Departamento de Bota
ˆnica, Ecologia
e Zoologia of Federal University of Rio Grande do Norte–
UFRN–Brazil. To prepare extract, the leaves of Turnera
subulata were air-dried at 40C, powdered, and extracted by
infusion with boiling water (95C, plant solvent 1:10, g/mL)
for 10 min. The aqueous extract was filtered, lyophilized, and
stored at -80C until tested.
Treatments
The extract was dissolved in a medium. Concentrated
stocks were prepared immediately before experiments by
dissolving extract into the medium and solution was kept
protected from light and high temperatures during all pro-
cedures. Cells were treated with different concentrations of
extract (0.5, 5, 500 lg/mL). All treatments were initiated by
adding concentrated solutions to reach final concentrations in
the well.
Thiobarbituric acid-reactive species
The cells were plated onto flasks of 25 cm
2
.After 24 h of
treatment, the cells were collected and homogenized. As an
index of lipid peroxidation, we measured the formation of
thiobarbituric acid-reactive species (TBARS) during an
acid-heating reaction, which is widely adopted for mea-
surement of lipid redox state, as previously described.
25
Measurement of protein carbonyls
Cells were plated onto flasks of 25 cm
2
. After 24 h of
treatment, the cells were collected and homogenized. The
oxidative damage to proteins was measured by the quanti-
fication of carbonyl groups based on the reaction with di-
nitrophenylhydrazine as previously described.
26
Measurement of protein thiol content
The cells were plated onto flasks of 25 cm
2
. After 24 h of
treatment, the cells were collected and homogenized. Pro-
tein thiol content in samples was analyzed to estimate oxi-
dative alterations in proteins.
27
Estimation of antioxidant enzyme activities
The cells were plated onto flasks of 25 cm
2
. After 24 h of
treatment, the cells were collected and homogenized. The
Table 1. Chemical Constituents Found in Extracts
of Genus Turnera
Chemical constituent
Phenolic Arbutin
Flavonoid Luteolin
Flavonoid Quercetin
Flavonoid Apigenin
Flavonoid Pinocembrin
Flavonoid Syringetin
Principal chemical compounds found in different species of genus Turnera.
2SOUZA ET AL.
catalase (EC 1.11.1.6) (CAT) activity was assayed by
measuring the rate of decrease in H
2
O
2
absorbance in a
spectrophotometer at 240 nm, and the results are expressed
as units of CAT/mg of protein.
28
The SOD (EC 1.15.1.1)
activity was assessed by quantifying the inhibition of
superoxide-dependent adrenaline autoxidation in a spectro-
photometer at 480 nm, as previously described, and the re-
sults are expressed a units of SOD/mg of protein.
29
Determination of intracellular RS production
(real-time dichlorofluorescein oxidation assay)
Intracellular reactive species production was determined
by the DCFH-DA-based real-time assay using intact living
cells. Briefly, RAW 264.7 cells were plated onto 96-well
plates and incubated for 1 h with DCFH-DA 100 lM (stock
solution in DMSO, 10 mM) in 1% FBS culture medium at
5% CO
2
and 37C.
30
Cells were then washed and treatments
were carried out. During treatment, changes in the fluores-
cence by the oxidation of DCFH into the fluorogen DCF
were monitored in a microplate fluorescence reader (F2000,
Hitachi Ltd., Tokyo, Japan) for 1 h at 37C.
MTT assay
The cells were plated onto 96-well plates. When the
culture reached 60% confluence, the culture medium of the
RAW 264.7 cells was removed and the treatments were
added. After 24 h of leaf extract treatment, RAW 264.7 cell
viability was assessed by the MTT assay.
31
H
2
O
2
300 lM
was used as positive control for cell death.
Sulforhodamine B assay
This colorimetric assay was performed to assess growth.
It estimates cell numbers indirectly by staining total cellular
protein with sulforhodamine B (SRB).
32
Immunoblot
To perform immunoblot experiments, RAW 264.7 cells
were lysed in Laemmli sample buffer (62.5 mM Tris–HCl,
pH 6.8, 1% [w/v] SDS, 10% [v/v] glycerol) and equal
amounts of cell protein (30 lg/well) were fractionated by
SDS-PAGE and electroblotted onto nitrocellulose mem-
branes. Protein loading and electroblotting efficiency were
verified through Ponceau S staining, and the membrane was
blocked in Tween–Tris-buffered saline (TTBS: 100 mM
Tris-HCl, pH 7.5, containing 0.9% NaCl and 0.1% Tween
20) containing 5% albumin. Membranes were incubated
overnight at 4C with antibodies in the presence of 5% skim
milk and then washed with TTBS. Anti-rabbit IgG
peroxidase-linked secondary antibody was incubated with
the membranes for an additional 1 h (1:5000 dilution range),
washed again, and the immunoreactivity was detected by
enhanced chemiluminescence using the ECL Plus kit.
Densitometric analysis of the films was performed with
Image J software. Blots were developed to be linear in the
range used for densitometry.
Indirect ELISA
TNF-awas quantified by indirect ELISA. A 96-well
ELISA plate was coated with 200 lL of culture medium and
purified recombinant TNF-aprotein (Abcam—Cambridge,
United Kingdom) diluted in 50 mM carbonate buffer, pH 9.0,
for standard curve calculation. After 24 h of incubation,
plates were washed thrice with TTBS (100 mM Tris–HCl,
pH 7.5, containing 0.9% NaCl, and 0.1% Tween-20). Sub-
sequently, 200 lLofanti-TNFa(1:8000 dilution range)
was added and incubation was carried for 24 h at 4C. The
plate was washed thrice with TTBS and incubated with
200 lL of a rabbit IgG peroxidase-linked secondary anti-
body (1:7000 dilution range) for 2 h. After washing the plate
thrice with TTBS, 200 lL of substrate solution (TMB spec-
trophotometric ELISA detection kit) was added to each
well and incubated for 15 min. The reaction was terminated
with 50 lL/well of 12 M sulfuric acid stopping reagent and
the plate read at 450 nm. IL-1bwas detected by Abcam
IL-1 beta Mouse ELISA (Enzyme-Linked Immunosorbent
Assay) kit Immunoassay Kit following the manufacturer’s
instructions.
Statistical analysis
Results are expressed as mean value –standard error
of the mean; P-values were considered significant when
P<.05. Differences in experimental groups were determined
by one-way ANOVA followed by the post-hoc Tukey’s test
whenever necessary.
RESULTS
We first analyzed the viability of cells treated with leaf
extracts at the concentration of 0.5, 5, and 500 lg/mL
(Fig. 1). We observed that all treatments did not alter cell
viability, as indicated by MTT and SRB-based assays. We
also analyzed the effects of leaf extract when used in co-
treatment during inflammatory response mediated in mac-
rophage cells through LPS treatment at 1 lg/mL. At all
concentrations of 0.5, 5, and 500 lg/mL, cotreatment with
leaf extract blocked the effect mediated by LPS in viability
of macrophages (Fig. 1).
The antioxidant properties were evaluated by measuring
parameters of oxidative stress in macrophage cells treated
with different concentrations of leaf extract for 24 h. The
extract, when used alone in macrophage cells, did not alter
the levels of lipid peroxidation. However, in LPS-induced
macrophage cells, we observed decreased lipid peroxidation
levels in macrophage cells that received leaf extract co-
treatment (Fig. 1). Moreover, these effects observed were
dose dependent. The protein carbonylation levels also were
evaluated. The treatment with extract did not alter the levels
of protein carbonylation in macrophage cells. Similar to ef-
fects observed in lipid peroxidation, the cotreatment with
extract in LPS-induced macrophage cells blocked the in-
crease in protein carbonylation levels (Fig. 1). Interestingly,
protein thiol content was not altered by leaf extract treatment.
However, cotreatments in LPS-induced macrophage cells
TURNERA SUBULATA ANTIOXIDANT EFFECTS 3
blocked the effect mediated by LPS in protein thiol content
(Fig. 1).
The activity of antioxidant enzymes was altered by leaf
extract treatment. We observed a decrease in CAT and SOD
activities in cells treated with leaf extract (Fig. 1) alone. In
LPS-induced macrophage cells, the cotreatment with leaf
extract blocked effects in CAT and SOD activities (Fig. 1).
In addition, the effects observed in antioxidant enzymes
activities were dose dependent (Fig. 1). Together, these data
indicate that leaf extract of Turnera subulata presents an-
tioxidant properties that result in the modulation of redox
parameters induced by LPS.
To confirm antioxidant properties of leaf extract, we
evaluated the production of ROS/RNS in macrophage cells
treated with leaf extract using real-time DCFH oxidation
assay. We observed that leaf extract at all concentrations
inhibited cellular ROS/RNS production (Fig. 1). To confirm
these antioxidant properties of the leaf extract, we cotreated
LPS-induced macrophage cells and the rate of intracellular
reactive species production was evaluated by DCF fluores-
cence. Once more, leaf extract treatment exhibits antioxi-
dant properties (Fig. 2H), which were responsible for
decreased ROS/RNS detection in the DCFH assay. To-
gether, these results demonstrate that leaf extract was effi-
ciently able to reduce cellular ROS/RNS production and
prevent increase in ROS/RNS levels when the cells are
exposed to oxidative stress conditions.
ROS/RNS is involved in the genesis of inflammatory
response. Due to the cellular signaling pathways that could
be affected by the oxidative stress during inflammatory re-
sponse, we decided to investigate the effects of leaf extract
in MAPKs, which are a family of proteins involved in in-
flammatory response signaling. We analyzed the effects of
Turnera subulata leaf extract in inflammatory response,
mediated in macrophage cells through LPS treatment, on the
activation state of MAPK ERK ½, SAPK/JNK, and p38.
MAPK phosphorylation is generally triggered within few
minutes after cell stimulation; so we preincubated macro-
phage cells with leaf extract at different doses for 60 min and
then LPS at 1 lg/mL was added to cells. Subsequently, we
performed immunoblots to detect the phosphorylated (i.e.,
active) forms of these protein kinases. It is known that LPS
treatment stimulates ERK ½, JNK, and p38 phosphoryla-
tion within few minutes. ERK ½ phosphorylation steadily
increased with time and peaked at 60 min, while p38 and
JNK activation peaked at 30 min of incubation. Turnera
subulata leaf extract cotreatment at the concentration
500 lg/mL inhibited the LPS-induced phosphorylation of
ERK ½ (Fig. 2). In p38 (Fig. 2), no changes were observed
in the activation states in macrophage cells that were co-
treated with leaf extracts. Similar behavior was observed in
SAPK/JNK activation (Fig. 2). These results indicate that
Turnera subulata leaf extract was able to block effects in
LPS-induced cells only through ERK ½ activation states.
The other members of MAPK proteins evaluated in this
study were not modulated by Turnera subulata treatments.
During cellular response induced by LPS treatment, there
occurs secretion of cytokines, such as TNF-aand IL-1b.
These cytokines regulate critical cellular processes, such as
apoptosis, inflammation, and proliferation. In this study, we
observed that cotreatment at 500 lg/mL of Turnera subulata
leaf extract reduced the secretion of both TNF-aand IL-1b
(Fig. 3) in LPS-induced macrophages cells. The other con-
centration used in cotreatment did not block TNF-aand IL-
1bsecretion mediated by LPS. In macrophages, TLR4 can
be modulated by LPS treatment. In addition, it is also known
that RAGE and CD40 have their expression modulated
during an inflammatory process induced by LPS. Therefore,
we decided to investigate the immunocontent of these re-
ceptors in cells treated with LPS and cotreated with Turnera
subulata leaf extract. We found that cotreatment with leaf
extract did not inhibit the increase of TLR4 mediated by
LPS treatment (Fig. 3). However, the effects mediated by
LPS in RAGE and CD40 immunocontent were inhibited
through cotreatment with Turnera subulata leaf extract at
500 lg/mL of concentration. Taken together, these data in-
dicate that the Turnera subulata leaf extract used in our
study have anti-inflammatory properties. In addition, the
results found in this study suggests that the action of the leaf
extract could be triggered by ERK ½ inactivation.
DISCUSSION
The role of plants extract exerts in traditional medicine
has been largely studied. The anticancer, anti-inflammatory,
and antioxidant properties of diverse extracts have been
associated to innumerous secondary metabolites found in
different parts of the plants. The beneficial effects described
for these molecules have stimulated the use of natural plant
products in nutritional supplements. In addition, innumerous
authors have suggested the positive relationship in con-
sumption of natural products and decrease of risk of chronic
diseases.
33–35
In these lines, the use of plant extracts has
represented along the years, a promising tool to prevent and
decrease the onset of chronic diseases.
In this study, our work demonstrates antioxidant and anti-
inflammatory effects that leaf extract of Turnera subulata
exhibited when used in a model of in vitro inflammation.
Leaf extract decreased lipid peroxidation and protein car-
bonylation, which was demonstrated through dose-dependent
effects during LPS-induced treatment. In addition, activities
of CAT and SOD were also modulated by leaf extract of
Turnera subulata, principally in LPS-induced model. Au-
thors recently described that the genus Turnera is a source of
different secondary compounds, such as phenolic, alkaloids,
cyanogenic glycosides, steroids, saponins, and flavonoids
that are likely associated with the positive effects of plant
products.
6,36,37
Some of these compounds can exhibit higher
antioxidant activities, which in almost all studies are sug-
gested to be used to reduce ROS/RNS production in bio-
logical systems.
The antioxidant properties of secondary metabolites of
plants can be exerted by different mechanisms. Authors have
shown that compounds found in plants can act as reducing
agents, scavengers of free radicals, metal ion chelators, co-
factors of enzymes catalyzing oxidative reactions, inhibitors
4SOUZA ET AL.
FIG. 1. Parameters of cell viability and oxidative stress in RAW 264.7 cells treated with Turnera subulata leaf extract for 24 h and in RAW
264.7 cells lipopolysaccharide(LPS) stimulated (1 lg/mL) and cotreated with Turnera subulata leaf extract for 24 h. RAW 264.7 cells were treated
with leaf extract at 0.5, 5, and 500 lg/mL. Different assays were performed to evaluate cell viability after incubation; LPS (1 lg/mL) was used as a
positive control for loss of viability. (A) MTT reduction assay and (B) SRB–incorporation assay. Parameters of oxidative stress: (C) carbonyl
levels were quantified to evaluate cell protein oxidative damage; (D) thiobarbituric acid reactive species (TBARS) levels were assessed as an
index for cellular lipid peroxidation; and (E) thiol levels were assessed to verify protein redox modification. The activities of the antioxidant
enzymes (F), catalase (CAT), and (G) superoxide dismutase (SOD) were also evaluated. Intracellular reactive species production by RAW 264.7
cells subjected to leaf extract treatment was evaluated. (H) Cells were treated with different concentrations of leaf extract for 1 h and the total
production of reactive species by living cells was evaluated by the real-time DCFH oxidation assay; LPS (1 lg/mL) was used as a positive control
for reactive species production and fluorescence intensity was calculated relative to control cells. Control group is represented in all graphs by
‘‘Control.’’ Data represent mean –SEM from three independent experiments (n=6 per group). One-way ANOVA followed by the post hoc
Tukey’s test, *P<.05 versus the control group. SRB, sulforhodamine B. SEM, standard error of the mean. Color images available online at
www.liebertpub.com/jmf
TURNERA SUBULATA ANTIOXIDANT EFFECTS 5
of oxidases, terminators of radical chain reactions, and sta-
bilizers of free radicals.
38–41
To reinforce these findings, it is
known that phenolic compounds are associated with a de-
crease in O
2
-
$production.
42–44
Furthermore, they are able
to inhibit OH$formation in a system containing iron and
hydrogen peroxide (H
2
O
2
) through the Fenton reaction.
45,46
In this study, we demonstrated that leaf extract of Turnera
subulata was able to reduce the lipid peroxidation and protein
carbonylation in LPS-induced macrophages. LPS treatment
is known for upregulating both O
2
-
$and OH$production in
macrophage cells. Therefore, our findings suggest that leaf
extract exerted an antioxidant effect and these effects could
be associated to secondary compounds present in leaf extract.
Besides, their actions may be mediated through the syner-
gistic action of these biological compounds.
The capacity of genus Turnera to modulate enzymes
through inhibition/activation has been reported in different
studies. It is known that Turnera diffusa extract had prop-
erties to inhibit aromatase enzyme. Most of this effect was
associated with higher flavonoid content in the extract.
12
However, the effects of genus Turnera in modulating anti-
oxidant enzymes responsible for the decrease in ROS/RNS
are unclear. Interestingly, we found that leaf extract of
Turnera subulata was able to modulate antioxidant enzyme
activities, such as CAT and SOD activities, in macrophages
cells. These effects were more clear in LPS-induced mac-
rophage cells. It is well described that LPS treatment in
macrophage cells leads to an impairment of the electron
transfer system, thus increasing the rate of O
2
-
$produc-
tion.
47–49
In this study, our results confirmed the increased
SOD activity and O
2
-
$production during LPS-induced
treatment in macrophage cells. Moreover, we observed a
decrease in the CAT activity. In these terms, it is well de-
scribed that O
2
-
$is a potent inhibitor of CAT.
50,51
The co-
treatment with Turnera subulata inhibited the decrease in
CAT and SOD activities of LPS-induced macrophage cells.
These effects corroborate with data found in lipid perox-
idation and protein carbonylation, where leaf extract showed
antioxidant properties during cotreatment. It is known that a
large portion of biological properties and functions involv-
ing protein structure, enzyme catalysis, and redox signaling
pathways depends on the redox state of the cells. The latter
can trigger the cells during proliferation, differentiation,
and/or inflammation.
52,53
Moreover, innumerous reports
have demonstrated that the increase in protein carbonylation
may be involved in the formation of protein aggregates,
which are very likely to culminate in widespread cellular
dysfunction.
54
The increased damage to proteins might re-
sult in increased free iron, because of its release from
damaged ferritin and other iron-containing proteins, favor-
ing the maintenance of the pro-oxidative state. The corre-
lation between protein damage and inflammatory response
is related with the increase of ROS/RNS production that
occurs during inflammatory response. The decrease in ROS/
RNS production is associated with the anti-inflammatory
response, and biological molecules that present activities to
inhibit the ROS/RNS production are target of recent stud-
ies.
55–57
In this study, we found that the leaf extract of
Turnera subulata was able to reduce damage in proteins and
lipids in macrophage cells subjected to LPS treatment. The
inhibition of damage in biological molecules might con-
tribute to maintenance of structure and function of pro-
teins and lipids, which in turn leads to the anti-inflammatory
response in the organism.
58–61
During proinflammatory response occurs characteristi-
cally the release of cytokines, such as IL-1band TNF-a.
FIG. 2. Effect of Turnera subulata leaf extract preincubation (0.5, and 5 lg/mL) on the phosphorylation of (A) EKR ½ (60 min), (B) SAPK/JNK
(30 min), and (C) p38 (30 min), in RAW 264.7 cells and RAW 264.7 LPS stimulated. Representative images (western blots) reveal detection of
phosphorylated isoforms of EKR ½, SAPK/JNK, and p38. Graphs exhibit the relative quantification of phosphorylated isoforms of EKR ½, SAPK/
JNK, and p38 in relationship to their total immunocontent. Data represent mean–SEM from three independent experiments (n=3 per group). One-
way ANOVA followed by the post hoc Tukey’s test, *P<.05 versus the control group. Color images available online at www.liebertpub.com/jmf
6SOUZA ET AL.
IL-1bis an important mediator involved in the inflammatory
response of cells.
62,63
Here, our study demonstrated that
cotreatment with leaf extract of Turnera subulata induces
inhibition of IL-1bsecretion by LPS-induced macrophages.
Interestingly, cotreatment with leaf extract did not inhibit
the increase in TLR4 immunocontent. It is known that LPS
treatment induces the expression of TLR4 and expression of
this receptor is associated with inflammatory response. The
class of TLR receptors is involved in activation of different
cellular pathways that regulate the expression of cytokines,
including IL-1b.
64
TNF-asecretion was also inhibited by the
leaf extract cotreatment. TNF-ais known for regulating
proinflammatory responses in different cells, such as endo-
thelial cells, and contributes to an increase of ROS/RNS
production in immune system cells involved in inflamma-
tory response.
65
The involvement of CD40 in inflammation
is well-known, as it is responsible for regulating different
pathways involved in the expression of cytokines such as
TNF-aand IL-1b.
66
In this study, our results showed that
leaf extract cotreatment was able to inhibit the expression of
CD40 and RAGE in LPS-induced macrophage cells. Taken
together, our results demonstrate that leaf extract of Turnera
subulata have anti-inflammatory properties.
The leaf extract cotreatment also inhibited the phosphory-
lation of ERK½. The association of MAPK signaling path-
ways during inflammatory response has been demonstrated in
numerous reports; moreover, transient MAPK activation is
associated with cell proliferation, whereas prolonged MAPK
activation may be involved in promoting cell death. Previous
works have shown the involvement of MAPK activation in
regulatory mechanisms of RAGE, CD40, TNF-a, IL-1b,and
TLR4 expression in LPS-induced models.
67
In part, LPS
treatment effects in MAPKs can be mediated through the in-
volvement of ROS/RNS. MAPK activation can stimulate
different transcript factors such as nuclear factor kappa B (NF-
jB), nuclear E2-related factor 2 (Nrf2), and p53, which are
classically known for their ubiquitous roles in inflammatory,
immune, and stress-related responses, and regulation of cell
survival in all tissues.
68
Inhibition of MAPK phosphorylation
mediated by LPS has been suggested as an anti-inflammatory
FIG. 3. Effect of Turnera subulata leaf extract (0.5, and 5 lg/mL) on the inflammatory biomarkers and cytokine release. (A) TLR-4, (B) CD40,
and (C) RAGE immunocontent in RAW 264.7 cells and RAW 264.7 LPS stimulated after 24 h. Representative images (western blots) reveal
detection of immunocontent of proteins in total cell homogenates. Graphs exhibit the relative quantification of immunocontent of protein in
relationship to their b-actin total immunocontent. (D) TNF-aand (E) IL-1bcontent in medium of incubation. Release of cytokines was performed
by ELISA. Data represent mean –SEM from three independent experiments (n=3 per group for Western blotting, and n=6 per group to ELISA
assays). One-way ANOVA followed by the post hoc Tukey’s test, *P<.05 versus the control group. RAGE, receptor for advanced end glycation
products. Color images available online at www.liebertpub.com/jmf
TURNERA SUBULATA ANTIOXIDANT EFFECTS 7
mechanism for different biological compounds in numerous
studies. These effects, at least in part, are due to MAPK sig-
naling blocking, which in turn leads to inhibition of tran-
scription factor activation such as NF-jB and decrease in the
cytokine expression in macrophages and other cells of im-
mune system.
69
Therefore, our results strongly indicate that
leaf extract of Turnera subulata has the capacity of modulat-
ing MAPK signaling pathways. We speculatethat inhibition of
ERK½ phosphorylation mediated by leaf extract was able to
block the inflammatory response in macrophage cells. The
same dose of leaf extract that inhibited the ERK½ phosphor-
ylation in LPS-induced macrophages, also blocked the in-
crease in RAGE and CD40 immunocontent and reduced the
secretion of TNF-aand IL-1b.
In conclusion, the results presented in this study demon-
strate for the first time that the leaf extract of Turnera
subulata presents antioxidant and anti-inflammatory prop-
erties. Moreover, the data reinforce the importance of po-
tential health benefits that the consumption of plants of
genus Turnera may promote. Our findings may also be
useful for better comprehension of the properties and
mechanism of action mediated by Turnera compounds.
ACKNOWLEDGMENTS
This work was funded by National Counsel of Technolo-
gical and Scientific Development (CNPq) (grants: 400805/
2014-6, 444856/2014-5, 443514/2014-3, and 467393/2014-1).
Matheus Augusto de Bittencourt Pasquali received fellowship
from CNPq.
AUTHOR DISCLOSURE STATEMENT
No competing financial interests exist.
REFERENCES
1. Dauchet L, Amouyel P, Hercberg S, Dallongeville J: Fruit and
vegetable consumption and risk of coronary heart disease: A
meta-analysis of cohort studies. J Nutr 2006;136:2588–2593.
2. Tohill BC, Seymour J, Serdula M, Kettel-Khan L, Rolls BJ:
What epidemiologic studies tell us about the relationship be-
tween fruit and vegetable consumption and body weight. Nutr
Rev 2004;62:365–374.
3. Cooke LJ, et al.: Demographic, familial and trait predictors of
fruit and vegetable consumption by pre-school children. Public
Health Nutr 2004;7:295–302.
4. de Carvalho Nilo Bitu V. et al.: Ethnopharmacological study of
plants sold for therapeutic purposes in public markets in North-
east Brazil. J Ethnopharmacol 2015;172:265–272.
5. Brito NJN, et al.: Antioxidant activity and protective effect of
Turnera ulmifolia Linn. var. elegans against carbon tetrachloride-
induced oxidative damage in rats. Food Chem Toxicol 2012;50:
4340–4347.
6. Kumar S, Taneja R, Sharma A: The genus Turnera: A review
update. Pharm Biol 2005;43:383–391.
7. Estrada-Reyes R, Carro-Jua
´rez M, Martı
´nez-Mota L: Pro-sexual
effects of Turnera diffusa Wild (Turneraceae) in male rats
involves the nitric oxide pathway. J Ethnopharmacol 2013;146:
164–172.
8. Soriano-Melgar LDAA, et al.: Antioxidant and trace element
content of damiana (Turnera diffusa Willd) under wild and cul-
tivated conditions in semi-arid zones. Ind Crops Prod 2012;37:
321–327.
9. Garza-Jua
´rez A, Salazar-Cavazos MDLL, Salazar-Aranda R,
Pe
´rez-Meseguer J, de Torres NW: Correlation between chro-
matographic fingerprint and antioxidant activity of Turnera dif-
fusa (Damiana). Planta Med 2011;77:958–963.
10. Takebayashi J, et al.: Reassessment of antioxidant activity of
arbutin: Multifaceted evaluation using five antioxidant assay
systems. Free Radic Res 2010;44:473–478.
11. Avelino-Flores MDC, Cruz-Lo
´pez M. del C, Jime
´nez-Montejo
FE, Reyes-Leyva J: Cytotoxic activity of the methanolic extract
of Turnera diffusa Willd on breast cancer cells. J Med Food
2015;18:299–305.
12. Zhao J, Dasmahapatra AK, Khan SI, Khan IA: Anti-aromatase
activity of the constituents from damiana (Turnera diffusa). J
Ethnopharmacol 2008;120:387–393.
13. Galvez J, et al.: Intestinal antiinflammatory activity of a lyoph-
ilized infusion of Turnera ulmifolia in TNBS rat colitis. Fito-
terapia 2006;77:515–520.
14. Szewczyk K, Zidorn C: Ethnobotany, phytochemistry, and bio-
activity of the genus Turnera (Passifloraceae) with a focus on
damiana—Turnera diffusa. J Ethnopharmacol 2014;152:424–443.
15. Huang J, Lam GY, Brumell J: Autophagy signaling through
reactive oxygen species. Antioxid Redox Signal 2011;14:2215–
2231.
16. Liu Z, Lenardo MJ: Reactive oxygen species regulate autophagy
through redox-sensitive proteases. Dev Cell 2007;12:484–485.
17. Vernon PJ, Tang D: Eat-Me: Autophagy, phagocytosis, and re-
active oxygen species signaling. Antioxid Redox Signal 2012;18:
120918063726003.
18. Azad MB, Chen Y, Gibson SB: Regulation of autophagy by re-
active oxygen species (ROS): Implications for cancer progression
and treatment. Antioxid Redox Signal 2009;11:777–790.
19. Chen Y, Azad MB, Gibson SB: Superoxide is the major reactive
oxygen species regulating autophagy. Cell Death Differ 2009;
16:1040–1052.
20. Gutteridge JM, Halliwell B: Free radicals and antioxidants in the
year 2000. A historical look to the future. Ann N Y Acad Sci
2000;899:136–147.
21. Fang YZ, Yang S, Wu G: Free radicals, antioxidants, and nu-
trition. Nutrition 2002;18:872–879.
22. Halliwell B: Free radicals, antioxidants, and human disease:
Curiosity, cause, or consequence? Lancet 1994;344:721–724.
23. Mittal M, Siddiqui MR, Tran K, Reddy SP, Malik AB: Reactive
oxygen species in inflammation and tissue injury. Antioxid Redox
Signal 2014;20:1126–1167.
24. Khodr B, Khalil Z: Modulation of inflammation by reactive
oxygen species: Implications for aging and tissue repair. Free
Radic Biol Med 2001;30:1–8.
25. Draper HH, et al.: A comparative evaluation of thiobarbituric
acid methods for the determination of malondialdehyde in bio-
logical materials. Free Radic Biol Med 1993;15:353–363.
26. Levine RL, et al.: Determination of carbonyl content in oxida-
tively modified proteins. Methods Enzymol 1990;186:464–478.
27. Ellman GL: Tissue sulfhydryl groups. Arch Biochem Biophys
1959;82:70–77.
8SOUZA ET AL.
28. Aebi H: Catalase in vitro. Methods Enzymol 1984;105:121–126.
29. Misra Hara P, Fridovich I: The role of superoxide anion in the
autoxidation of epinephrine and a simple assay for superoxide
dismutase. J Biol Chem 1972;247:3170–3175.
30. Wang H, Joseph JA: Quantifying cellular oxidative stress by
dichlorofluorescein assay using microplate reader. Free Radic
Biol Med 1999;27:612–616.
31. De Bittencourt Pasquali MA, et al.: Vitamin A (retinol) down-
regulates the receptor for advanced glycation endproducts
(RAGE) by oxidant-dependent activation of p38 MAPK and
NF-kB in human lung cancer A549 cells. Cell Signal 2013;25:
939–954.
32. Skehan P, et al.: New colorimetric cytotoxicity assay for
anticancer-drug screening. JNatlCancerInst1990;82:1107–1112.
33. Mukhtar M, et al.: Antiviral potentials of medicinal plants. Virus
Res 2008;131:111–120.
34. Halliwell B: Oxidative stress, nutrition and health. Experimental
strategies for optimization of nutritional antioxidant intake in
humans. Free Radic Res 1996;25:57–74.
35. Lo
´pez-Alarco
´n C, Denicola A: Evaluating the antioxidant ca-
pacity of natural products: A review on chemical and cellular-
based assays. Anal Chim Acta 2013;763:1–10.
36. Speranza PR, Seijo JG, Grela IA, Sol Neffa VG: Chloroplast DNA
variation in the Turnera sidoides L. complex (Turneraceae): Bio-
geographical implications. J Biogeogr 2007;34:427–436.
37. Szewczyk K, Zidorn C: Ethnobotany, phytochemistry, and bio-
activity of the genus Turnera (Passifloraceae) with a focus on
damiana—Turnera diffusa. J Ethnopharmacol 2014;152:424–
443.
38. Rice-Evans Ca, Miller NJ, Paganga G: Antioxidant properties of
phenolic compounds. Trends Plant Sci 1997;2:152–159.
39. Rice-Evans C, Miller N, Paganga G: Antioxidant properties of
phenolic compounds. Trends Plant Sci 1997;2:152–159.
40. Miller NJ, Rice-Evans CA: Factors influencing the antioxidant
activity determined by the ABTS.+radical cation assay. Free
Radic Res 1997;26:195–199.
41. Ruiz-Larrea MB, et al.: Antioxidant activity of phytoestrogenic
isoflavones. Free Radic Res 1997;26:63–70.
42. Roleira FMF, et al.: Plant derived and dietary phenolic antioxi-
dants: Anticancer properties. Food Chem 2015;183:235–258.
43. Ferna
´ndez-Moriano C, Go
´mez-Serranillos MP, Crespo A: Anti-
oxidant potential of lichen species and their secondary metabo-
lites. A systematic review. Pharm Biol 2015;54:1–17.
44. Costa G, Francisco V, Lopes MC, Cruz MT, Batista MT:
Intracellular signaling pathways modulated by phenolic com-
pounds: Application for new anti-inflammatory drugs discovery.
Curr Med Chem 2012;19:2876–2900.
45. Wang G, et al.: The JAK2/STAT3 and mitochondrial pathways
are essential for quercetin nanoliposome-induced C6 glioma cell
death. Cell Death Dis 2013;4:e746.
46. Moretti E, et al.: Effect of quercetin, rutin, naringenin and epi-
catechin on lipid peroxidation induced in human sperm. Reprod
Toxicol 2012;34:651–657.
47. Awad N. et al.: N-acetyl-cysteine (NAC) attenuates LPS-induced
maternal and amniotic fluid oxidative stress and inflammatory
responses in the preterm gestation. Am J Obstet Gynecol 2011;
204:450.e15–e20.
48. Wang H, et al.: N-Acetylcysteine attenuates lipopolysaccharide-
induced apoptotic liver damage in D-galactosamine-sensitized
mice. Acta Pharmacol Sin 2007;28:1803–1809.
49. Zhou R, Yazdi AS, Menu P, Tschopp J: A role for mito-
chondria in NLRP3 inflammasome activation. Nature 2011;469:
221–225.
50. Pasquali MAB, et al.: Retinol and retinoic acid modulate cata-
lase activity in Sertoli cells by distinct and gene expression-
independent mechanisms. Toxicol In Vitro 2008;22:1177–1183.
51. Gelain DP, et al.: Retinol increases catalase activity and protein
content by a reactive species-dependent mechanism in Sertoli
cells. Chem Biol Interact 2008;174:38–43.
52. Bindoli A, Fukuto JM, Forman HJ: Thiol chemistry in peroxidase
catalysis and redox signaling. Antiox Redox Signal 2008;10:
1549–1564.
53. Winterbourn CC, Hampton MB: Thiol chemistry and specificity
in redox signaling. Free Radic Biol Med 2008;45:549–561.
54. Bourdon E, Blache D: The importance of proteins in defense
against oxidation. Antioxid Redox Signal 2001;3:293–311.
55. Cheng X, Ku CH, Siow RCM: Regulation of the Nrf2 antioxidant
pathway by microRNAs: New players in micromanaging redox
homeostasis. Free Radic Biol Med 2013;64:4–11.
56. Chapple SJ, Siow RCM, Mann GE: Crosstalk between Nrf2 and
the proteasome: Therapeutic potential of Nrf2 inducers in vascular
disease and aging. Int J Biochem Cell Biol 2012;44:1315–1320.
57. Veit F, Pak O, Brandes RP, Weissmann N: Hypoxia-dependent
reactive oxygen species signaling in the pulmonary circulation:
Focus on ion channels. Antioxid Redox Signal 2015;22:537–552.
58. Ho
¨hn TJA, Grune T: The proteasome and the degradation of
oxidized proteins: Part II—protein oxidation and proteasomal
degradation. Redox Biol 2013;2:99–104.
59. Ho
¨hn TJA, Grune T: The proteasome and the degradation of
oxidized proteins: Part III-redox regulation of the proteasomal
system. Redox Biol 2014;2:388–394.
60. Grune T, Merker K, Sandig G, Davies KJA: Selective degrada-
tion of oxidatively modified protein substrates by the proteasome.
Biochem Biophys Res Commun 2003;305:709–718.
61. Davies KJA: Degradation of oxidized proteins by the 20S pro-
teasome. Biochimie 2001;83:301–310.
62. Gelain DP, et al.: Serum heat shock protein 70 levels, oxidant
status, and mortality in sepsis. Shock 2011;35:466–470.
63. Andrades ME
´,et al.: Plasma glycation levels are associated with
severity in sepsis. Eur J Clin Invest 2012;42:1055–1060.
64. Shon W-J, Lee Y-K, Shin JH, Choi EY, Shin D-M: Severity of
DSS-induced colitis is reduced in Ido1-deficient mice with down-
regulation of TLR-MyD88-NF-kB transcriptional networks. Sci
Rep 2015;5:17305.
65. Tilstra JS, et al.: Pharmacologic IKK/NF-jB inhibition causes
antigen presenting cells to undergo TNFadependent ROS-
mediated programmed cell death. Sci Rep 2014;4:3631.
66. Georgopoulos NT, et al.: A novel mechanism of CD40-induced
apoptosis of carcinoma cells involving TRAF3 and JNK/AP-1
activation. Cell Death Differ 2006;13:1789–1801.
67. Freund A, Orjalo AV, Desprez P-Y, Campisi J: Inflammatory
networks during cellular senescence: Causes and consequences.
Trends Mol Med 2010;16:238–246.
68. Ryter SW, Xi S, Hartsfield CL, Choi AMK: Mitogen activated
protein kinase (MAPK) pathway regulates heme oxygenase-1
gene expression by hypoxia in vascular cells. Antioxid Redox
Signal 2002;4:587–592.
69. Gaestel M, Kotlyarov A, Kracht M: Targeting innate immunity
protein kinase signalling in inflammation. Nat Rev Drug Discov
2009;8:480–499.
TURNERA SUBULATA ANTIOXIDANT EFFECTS 9
