Inflamm. Res.
DOI 10.1007/s00011-014-0728-2
ORIGINAL RESEARCH PAPER
Sulfated polysaccharides isolated from the green seaweed
Caulerpa racemosa plays antinociceptive and anti-inflammatory
activities in a way dependent on HO-1 pathway activation
Natássia Albuquerque Ribeiro • Ticiana Monteiro Abreu • Hellı́ada Vasconcelos Chaves
Mirna Marques Bezerra • Helena Serra Azul Monteiro • Roberta Jeane Bezerra Jorge •
Norma Maria Barros Benevides
Received: 20 September 2013 / Revised: 20 February 2014 / Accepted: 24 February 2014
Ó Springer Basel 2014
Abstract
Objective Marine algae are abundant sources of sulfated
polysaccharides with various biological activities. Conse-
quently, their biomolecules are of great of commercial
interest. In this study, we investigated the potential antin-
ociceptive activity of a sulfated polysaccharide obtained
from the green seaweed Caulerpa racemosa (CrII) and the
involvement of the hemoxigenase-1 (HO-1) pathway in its
anti-inflammatory effect.
Methods We used a systemic evaluation to verify possi-
ble toxic effects of Crll after consecutive treatments. Swiss
mice and Wistar rats were used for all experiments.
Results In Swiss mice, CrII (0.01, 0.1 and 1.0 mg/kg)
significantly reduced the number of abdominal contortions
and the duration of paw licking in the second phase after
treatment with acetic acid and formalin, respectively.
Responsible Editor: Mauro Teixeira.
Electronic supplementary material The online version of this
article (doi:10.1007/s00011-014-0728-2) contains supplementary
material, which is available to authorized users.
N. A. Ribeiro  T. M. Abreu  N. M. B. Benevides (&)
Department of Biochemistry and Molecular Biology, Federal
University of Ceará, Fortaleza, CE 60455-760, Brazil
e-mail: nmbb@ufc.br
H. V. Chaves
Faculty of Dentistry, Federal University of Ceará, Sobral,
CE, Brazil
M. M. Bezerra
Faculty of Medicine, Federal University of Ceará, Sobral,
CE, Brazil
H. S. A. Monteiro  R. J. B. Jorge
Department of Physiology and Pharmacology, Federal
University of Ceará, Fortaleza, CE, Brazil
Inflammation Research
•
However, CrII was unable to prolong the reaction time of
thermally stimulated animals. The anti-inflammatory effect
of CrII (0.01, 0.1 and 1.0 mg/kg) was evidenced by a
decreased number of leukocytes in the peritoneal cavities
of the rats. CrII (0.01, 0.1 and 1.0 mg/kg) also reduced the
amount of paw edema induced by carrageenan (Cg) and
dextran. The anti-inflammatory effect of CrII was con-
firmed by reduced levels of myeloperoxidase in the paw
tissue of the Cg groups. After inhibition with ZnPP IX, a
specific HO-1 phenotype inhibitor, the anti-inflammatory
effect of CrII was no longer observed in Cg-induced paw
edema tests. Consecutive Crll (1.0 mg/kg) for 14 days did
not change any biochemical or histopathological parame-
ters, or cause mortality of mice.
Conclusions CrII did not produce any signs of toxicity
and effectively decreased nociception and inflammation.
Also, the anti-inflammatory effect of Crll is at least in part
dependent on the integrity of the HO-1 pathway.
Keywords Marine alga  Sulfated polysaccharide 
Inflammation  Nociception  Caulerpa racemosa
Introduction
Pain transmission occurs through a mechanism that
involves a very complex interaction between the peripheral
and central structures of the skin surface and the central
cerebral cortex [1]. This transmission occurs through
nociceptors that can detect a wide variety of stimuli,
including those of physical and chemical natures, and
convert such stimuli into electrochemical signals [2].
Inadequate treatment of acute pain can lead to persistent
and chronic pain, indicating the importance of rapid and
effective pain management [3]. The treatment of chronic
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pain is a serious problem due to the adverse effects of the
available medications, which vary according to the class of
agent used and include dependence, tolerance, abuse and
gastrointestinal effects [4].
The inflammatory process is an important bodily
defense mechanism that is composed of complex responses
or a complex response of vascularized tissue. The inflam-
matory response aims to destroy, immobilize or dilute the
harmful agent, isolate the lesion, inactivate toxins and
prepare the tissue or organ for healing and repair [5].
However, excessive or inappropriate inflammation is the
cause of many diseases [6].
One of the mechanisms involved in the resolution of
inflammation is the expression of the enzyme hemoxi-
genase-1 (HO-1). In inflammatory conditions, HO-1
becomes the limiting enzyme in the catabolism of free
heme groups, which produces equimolar amounts of
carbon monoxide (CO), iron (Fe) and biliverdin (BV).
These products reduce inflammation and prevent the
development of inflammatory diseases [7]. Substances
called metalloporphyrins can inhibit HO-1 activity. These
compounds were identified as competitive inhibitors of
the hemoxygenase pathway [8]. Zinc protoporphyrin
(ZnPP) is a metalloporphyrin that competes with heme
(natural substrate) by binding to the active site of HO-1
and inactivating the enzyme [9].
In the quest to discover new anti-inflammatory and
analgesic effects, scientists continue to use bioproducts as
anti-inflammatory agents in the various stages of inflam-
mation [10]. According to Cardozo et al. [11] important
new anti-inflammatory agents have been isolated from
various classes of algae. For example, sulfated polysac-
charides have diverse biological characteristics, such as
anti-inflammatory properties, and are generally nontoxic
[12].
In previous studies, the polysaccharides of Caulerpa
racemosa were shown to be selective inhibitors of the
reference strains TK—aciclovirus, and resistant strains of
herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2)
in Vero cells [13]. Polysaccharide fractions obtained by
ion exchange chromatography with DEAE-52 cellulose
displayed strong antitumor activity in vitro and in vivo,
and inhibited the proliferation of K562 cells in vitro
[14].
The objective of this study was to investigate the
unexplored antinociceptive and anti-inflammatory activ-
ities of a sulfated polysaccharide fraction isolated from
the marine green alga C. racemosa (CrII) and perform a
systemic evaluation to verify possible toxic effects of
Crll after consecutive treatments. We also investigated
whether the anti-inflammatory effect of this polysac-
charide was related to the integrity of the HO-1
pathway.
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N. A. Ribeiro et al.
Materials and methods
Animals
Male and female Swiss mice (20–25 g) and Wistar rats
(males 180–250 g) from the Animal Care Unit of the
Federal University of Ceará (Fortaleza, Brazil) were used
for all experiments. The animals were housed in a tem-
perature-controlled room with a 12/12-h light/dark cycle
and free access to water and food. For each experiment,
groups of six animals were segregated and handled sepa-
rately. All procedures and animal treatments were
performed at a controlled ambient temperature (20–22 °C),
and special care was taken to avoid environmental distur-
bances that might influence the animals’ responses. This
study was conducted in accordance with the Committee for
Research and Ethical Issues of the International Associa-
tion for the Study of Pain (IASPÒ), Institutional Animal
Care, and with the approval of the Ethics Committee of the
Federal University of Ceará, Fortaleza, Brazil (CEPA n°.
80/10).
Drugs and reagents
The following drugs and reagents were purchased from
Sigma Aldrich (St. Louis, MO, USA): dextran sulfate, k-
carrageenan, cetylpyridinium chloride (CPC), 1-9 dime-
thyl-methylene blue (DMB), DEAE-cellulose, o-
dianisidine dihydrochloride, N-acetyl-N,N,N-trimethylam-
monium bromide, hexadecyltrimethylammonium bromide
(HTAB), zinc protoporphyrin IX (ZnPP IX), 37 % form-
aldehyde, cystein, papain and bovine serum albumin
(BSA). Gelatin was purchased from Oxoid Ltd. (Basing-
stoke, UK). Glacial acetic acid (Reagen; Rio de Janeiro,
RJ, Brazil), chlorohydrate of morphine (Dimorf, Cristália;
Itapira, SP, Brazil), dexamethasone (Decadron, Aché;
Campinas, SP, Brazil) and indomethacin (Indocid, Merck
Sharp and Dohme; Campinas, SP, Brazil) were obtained
from the indicated companies. Ethylenediaminetetraacetic
acid (EDTA) and chloral hydrate were purchased from
Vetec Quı́mica Fina, Ltda (SP, Brazil). The enzymatic kits
used to evaluate the systemic toxicity of Crll were pur-
chased from LABTEST (Diagnostic Tests, Brazil). All
chemicals were analytical grade.
Isolation of sulfated polysaccharides
Caulerpa racemosa specimens were collected on the beach
at Pedra Rachada in São Gonçalo-Ce, Brazil. The collected
algae were taken to the Carbohydrates and Lectins Labo-
ratory (CarboLec; Department of Biochemistry and
Molecular Biology, Federal University of Ceará), cleaned
to remove sand and epiphytes, dried at room temperature
Sulfated polysaccharides isolated from the green seaweed Caulerpa racemosa
(25 °C), macerated with liquid nitrogen and stored in glass
jars at room temperature. A voucher specimen (no. 52418)
was deposited in the Herbarium Prisco Bezerra in the
Department of Biological Sciences (Federal University of
Ceará, Brazil).
A 5-g sample of dry tissue was rehydrated with 250 ml
of 0.05 M sodium acetate buffer (pH 6.0) containing
5 mM EDTA and 5 mM cystein, and then incubated for
6 h with papain (1,020 mg) at 60 °C before centrifugation
(5,000g; 20 min; 4 °C), as previously described by Farias
et al. [15]. The total sulfated polysaccharide (Cr-PS)
obtained from C. racemosa (100 mg) was dissolved in
50 ml of 50 mM sodium acetate buffer (pH 6.0) and
applied to a DEAE-cellulose column (30 cm 9 2 cm) that
was equilibrated with the same buffer. The analytes were
separated using a step-wise gradient from 0 to 1.5 M
NaCl at 0.25 M intervals in the same buffer. The flow
rate through the column was 3 ml/min. Fractions (3 ml)
were collected and analyzed for sulfated polysaccharides
using the metachromatic assay (A525 nm) with DMB, as
previously described [16]. The obtained Cr-PS and frac-
tions were analyzed by 0.5 % agarose gel electrophoresis
[17]. The biological protocols were performed with the
fraction that showed the highest yield, which was termed
CrII.
Chemical composition
The sulfate content was estimated after acid hydrolysis of
the soluble polysaccharides in 1 M HCl at 110 °C for 5 h,
in accordance with the previously described gelatin–bar-
ium method, using Na2SO4 as the standard [18]. The
protein content was measured using Coomassie Brilliant
Blue G-250, using BSA as a standard [19] to assess protein
contamination.
Antinociceptive activity
Writhing test
A chemical model of nociception was used to assess
analgesic activity [20]. First, the male mice received CrII
(0.01, 0.1 or 1.0 mg/kg; i.v.) or sterile saline (0.9 %, w/v,
NaCl). After 30 min, abdominal constrictions were
induced by intraperitoneal (i.p.) injection of 0.8 % acetic
acid (v/v; 0.1 ml/10 g body weight). Shortly after the
acetic acid was administered, the number of abdominal
constrictions was counted for consecutive 30 min. The
percentage of nociception inhibition was calculated by
comparing the mean number of constrictions obtained
from the control group (injected with only acetic acid)
with that of the experimental group (pre-treated with
CrII).
Formalin test
The formalin test was performed following the method
described by Dubuisson and Dennis [21], as modified by
Hunskaar et al. [22]. This method is characterized by a
local tissue injury to the paw, which induces pain and
localized inflammation. Male mice were injected with
either CrII (0.01, 0.1 or 1.0 mg/kg; i.v.) or sterile saline
(0.9 %, w/v, NaCl). After 30 min, 20 ll of 2 % formalin
solution was injected into the right paw of male mice, and
the duration of licking was recorded during the first 5 min
(first phase, which corresponds to the direct application of a
chemical stimulus to the nociceptors—the initial phase)
and for an additional 5 min after a 20 min time period
(second phase, which involves inflammation—late phase).
Hot-plate test
Central analgesic activity was evaluated using the hot plate
test, according to the method described by Eddy and
Leimbach [23]. This test involves recording the time(s) that
male mice require to manifest a response when in contact
with a heated metal plate (51 ± 1 °C), which corresponds
to the act of removing or licking the hind paw and/or
jumping. This specific test is used to verify central noci-
ception. The animals were first acquainted with the hot
plate to observe the control reaction time. Animals with a
reaction time exceeding 10 s were discarded from the test.
Immediately after the trial (control reaction time), the mice
were divided into groups of six. The mice then received an
injection of sterile saline (0.9 %, w/v), CrII (0.01, 0.1 or
1.0 mg/kg; i.v.), morphine (5 mg/kg; s.c.) or indomethacin
(5 mg/kg; s.c.), and the reaction times were measured at 0
30, 60 and 90 min after drug administration. A cutoff time
of 40 s was used to avoid paw lesions.
Anti-inflammatory activity
Peritonitis model
The rats were injected with carrageenan (Cg) (700 lg/cav;
i.p.) or sterile saline (1 ml). Then, the animals were treated
with CrII (0.01, 0.1 or 1.0 mg/kg; i.v.) 30 min before being
exposed to stimuli. Dexamethasone (1 mg/kg; s.c.), a
synthetic glucocorticoid with potent anti-inflammatory and
immunosuppressant properties, was administered 1 h
before Cg as a reference compound [24]. Four hours later,
the animals were euthanized, and each peritoneal cavity
was washed with 10 ml of saline, containing 5 IU/ml of
heparin. The peritoneal fluid was recovered, and the total
and differential leukocytes were counted. The results are
expressed as the mean ± standard error (SEM) of the
number of cells 103/ml of peritoneal fluid [25].
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Carrageenan-induced rat paw edema
Carrageenan-induced paw edema was achieved according
to the method described by Winter et al. [26]. Cg (700 lg/
paw) was administered subcutaneously (s.c.). Rats were
pre-treated with CrII at doses of 0.01, 0.1 or 1.0 mg/kg
(0.1 ml/100 g body weight) 30 min before the Cg injec-
tion. As a reference, dexamethasone (1 mg/kg; s.c.) was
administered 1 h before Cg. An additional group received
only sterile saline (0.15 M NaCl; s.c.). The volumes of the
right hind paw of each animal were measured using a
plethysmometer before injection of the inflammatory
stimulus (time zero), and at intervals of 1, 2, 3, 4 and 5 h
after administration of the inflammatory stimulus. Swelling
was calculated as the difference between the volumes of
fluid displaced by the paw at time zero and at each time
point after the stimulus.
Dextran-induced rat paw edema
The dextran-induced paw edema model was generated
according to the method described by Maity et al. [27].
Dextran (300 lg/paw) was administered subcutaneously.
The rats were pre-treated with CrII at doses of 0.01, 0.1
or 1.0 mg/kg (0.1 ml/100 g body weight; s.c.) 1 h prior
to receiving the stimuli. Control animals received the
same volume of sterile saline (0.9 % w/v, NaCl). The
volumes of the right hind paw of each animal were
measured using a plethysmometer prior to injection of
the inflammatory stimulus (time zero) and at intervals of
30 min, 1, 2, 3 and 4 h after administration of the
inflammatory stimulus. Swelling was calculated as the
difference between the volumes of fluid displaced by the
paw at time zero and at each time point after the
stimulus.
Determination of myeloperoxidase (MPO) activity
Myeloperoxidase is an enzyme found primarily in azuro-
philic granules within neutrophils, and it has been used
extensively as a biochemical marker of granulocyte infil-
tration in various tissues. Neutrophil accumulation in rats
paw tissues was measured using an MPO activity assay, as
previously described [28]. Briefly, 50 mg of paw tissue was
homogenized in 1 ml of 50 mM potassium phosphate (pH
6.0) containing 0.5 % HTAB using a Polytron homoge-
nizer (two cycles of 10 s). After centrifugation at
4,0009g for 12 min at 4 °C, samples of the supernatant
(7 ml) were added to phosphate buffer (200 ml) containing
dihydrochloride (1 mM), o-dianisidine and 0.0005 %
hydrogen peroxide in a 96-well microplate. The absorbance
was measured at 450 nm, and two readings were taken at
60 s intervals. MPO activity was determined by measuring
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N. A. Ribeiro et al.
the change in absorbance at 450 nm using o-dianisidine
dihydrochloride and 1 % hydrogen peroxide. One unit of
MPO activity was defined as the activity required to con-
vert 1 mol of hydrogen peroxide to water in 1 min at
22 °C. The results are reported as MPO units/mg of tissue.
Analysis of HO-1 pathway involvement in the anti-
inflammatory effect of CrII
To analyze the involvement of HO-1 in the anti-
inflammatory activity of CrII, rats were pre-treated (s.c.)
with ZnPP IX (3 mg/kg) and then injected with Crll
(0.1 mg/kg; i.v.) 60 min later. After 30 min, Cg (500 lg/
paw) was injected s.c. [29], and the paw volume was
measured immediately before (0 h) the stimulus and at
selected time intervals (1, 2, 3, 4 and 5 h) using a ple-
thysmometer. The results are expressed as the variation
in paw volume (ml), which was calculated relative to the
basal volume (0 h).
Evaluation of repeated dose toxicity
Body mass loss, organ weight alteration and blood levels of
the biochemical parameters [alanine amino transferase
(AST), aspartate amino transferase (ALT), alkaline phos-
phatase (ALP) and urea] were evaluated after once-daily
treatment with CrII (1 mg/kg; i.v.) or sterile saline (0.9 %
w/v, NaCl) for seven consecutive days. After treatment, the
male and female mice were weighed, and peripheral blood
was collected for biochemical analysis (determined by
enzymatic and colorimetric tests—LABTEST). After the
animal was euthanized, the liver, kidney, spleen, thymus,
lymph nodes and heart were removed and weighed. Pos-
sible ulcerative lesions or hemorrhaging were quantified
and macroscopically measured.
Histological analysis
After euthanasia, the liver, kidney, spleen, thymus, lymph
nodes and heart were fixed with formalin. The material was
then dehydrated using ethanol and processed for embed-
ding in paraffin. The resulting blocks were sliced into
5-lm-thick sections, stained with hematoxylin-eosin (HE)
and observed under a light microscope.
Statistical analysis
The data are presented as the mean ± SEM of six animals
per group. Analysis of variance (ANOVA) was performed
using Bonferroni’s test and Student’s t test for unpaired
values. Values of p \ 0.05 were considered to be statisti-
cally significant.
Sulfated polysaccharides isolated from the green seaweed Caulerpa racemosa

Fig. 1 a Separation of CrII by

DEAE-cellulose. Fractions were
collected and checked by
metachromasia using 1,9-
dimethylmethylene blue
(diamond). The fractions were
eluted ‘‘step wise’’ with NaCl at
concentrations of 0.5, 0.75 and 1
(line). b Agarose gel
electrophoresis of isolated CrII.
CrPS and their fractions, F I
(0.5 M), F II (0.75 M) and F III
(1.0 M), were stained with
0.1 % toluidine blue

Results Antinociceptive activity

Isolation of sulfated polysaccharides
Total sulfated polysaccharides yielded low levels of poly-
saccharide (2.2 %) and free sulfate (15.17 %) and trace
amounts of protein. Cr-PS produced three different fractions
(FI, FII and FIII, eluted with 0.5, 0.75 and 1.0 M of NaCl,
respectively) when separated in a DEAE-cellulose column
(Fig. 1a). These fractions had yields of 10.5, 20.5, and 17.6 %
and sulfate contents of 5.91, 20.84 and 28.39 %, respectively,
with no protein contamination. The Cr-PS and the three
obtained fractions exhibited different charge densities when
analyzed using 0.5 % agarose gel electrophoresis. The FII
fraction was more concentrated than the FIII fraction. The FI
fraction was not detected in the gel, most likely because of the
low number of sulfated groups in the chemical structure of this
polysaccharide fraction (Fig. 1b).
Pretreatment with CrII (0.01, 0.1 or 1.0 mg/kg; i.v.) sig-
nificantly reduced the number of abdominal constrictions
induced by acetic acid in mice in a dose-dependent manner
(32.60, 49.26 and 60.75 % for 0.01, 0.1 and 1.0 mg/kg,
respectively). For this experiment, animals that were pre-
treated with either morphine or indomethacin (5 mg/kg;
s.c.) were used as positive controls. Treatment with mor-
phine and indomethacin inhibited 96 and 54 % of the
abdominal constrictions, respectively (Fig. 2a). To evalu-
ate whether CrII exhibited antinociceptive effects in
another model of peripheral analgesia, we tested its effi-
cacy in the formalin model. Intraplantar injection of a 2 %
formalin solution in mice induced a nociceptive response
that was characterized by an increased licking duration. No
reduction in the licking duration was observed during the
first phase for any of the tested Crll doses (Fig. 2b).

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Fig. 2 Effect of CrII in nociceptive models. Thirty minutes prior to
the application of stimuli, the rats were treated with sterile saline
(0.15 M NaCl, i.v.), morphine (5 mg/kg, s.c.), indomethacin (5 mg/
kg, s.c.) or CrII (0.01, 0.1 and 1.0 mg/kg, i.v.). Data are expressed as
the mean ± SEM of six animals for each group (ANOVA; Bonfer-
roni’s test). a Effect of CrII on the writhing response induced by
acetic acid in mice. Asterisk significantly different from the sterile
saline group (p \ 0.05). b Effect of CrII on the formalin test in mice.
However, CrII (0.01, 0.1 or 1.0 mg/kg; i.v.) injected
30 min prior to the administration of formalin inhibited the
formalin response during the second phase by 97.37, 97.37
and 98.68 %, respectively. Similarly, morphine and indo-
methacin inhibited the second phase by 99 and 92.26 %,
respectively (Fig. 2b).
In the hot plate test, saline (0.15 M, NaCl; i.v.), CrII
(0.01, 0.1 or 1.0 mg/kg; i.v.) or indomethacin (5 mg/kg;
s.c.) did not increase the average reaction time to thermal
stimulus compared to the control (morphine), which sig-
nificantly increased the reaction time to thermal stimulation
at 30, 60 and 90 min (Fig. 2c).
Anti-inflammatory activity
When CrII (0.01, 0.1 or 1.0 mg/kg; i.v.) was administered
30 min before Cg administration in the rats’ peritoneal
cavity, 46.41, 56.69 and 33.96 % reductions in the neu-
trophil counts were observed, respectively. Likewise,
pretreatment with dexamethasone reduced the neutrophil
counts by 59.94 % (Fig. 3). In the paw edema model in
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N. A. Ribeiro et al.
The duration of licking was determined during the first 5 min (first
phase) and during the period from 20 to 25 min (second phase) after
the injection of 2 % formalin. Asterisk significantly different from the
saline group (p \ 0.05), and Hash significantly different from the
morphine group (p \ 0.05). c Effect of CrII on the reaction times
after thermal stimuli in mice. Asterisk significantly different from the
saline group (p \ 0.05)
rats, CrII (0.01, 0.1 or 1.0 mg/kg; i.v.) was also able to
diminish the temporal course of edema over the 5 h testing
period after Cg administration (700 lg/paw; s.c.) (Fig. 4a).
Particularly during the third hour, CrII (0.01, 0.1 or 1.0 mg/
kg; i.v.) significantly reduced the occurrence of edema by
60.3, 73.0 and 79.3 %, respectively, in response to the
stimulus. Dexamethosone pretreatment inhibited edema by
82.5 %. These data were confirmed using MPO activity
assays, which revealed that CrII (0.01, 0.1 or 1.0 mg/kg;
i.v.) inhibited neutrophil accumulation in the paw by 44.92,
71.56 and 53.78 %, respectively. Dexamethasone also
inhibited MPO activity (79.67 %) (Fig. 4b). Additionally,
dextran (300 lg/paw; s.c.) induced a significant increase in
vascular permeability; the maximum effect was observed
between 30 main and 1 h after administration, and it
decreased over time. Treatment (i.v.) with different doses
of CrII (0.01; 0.1 or 1.0 mg/kg) inhibited the paw edema
induced by dextran at 1 h by 47.36, 59.64 and 47.36 %,
respectively (Fig. 5).
To investigate the role of HO-1 activity in the anti-
inflammatory effect of CrII, rats were pretreated (s.c.) with
Sulfated polysaccharides isolated from the green seaweed Caulerpa racemosa
Fig. 3 Effect of CrII on neutrophil migration in rats. Before
receiving an injection of carrageenan (700 lg/cav; i.p.), the rats
received sterile saline or CrII (0.01, 0.1 and 1.0 mg/kg, i.v.). Then,
dexamethasone (1 mg/kg; s.c.) was injected. Another group received
only saline (s.c.), without Cg. Data are expressed as the mean ± SEM
of six animals for each group. Asterisk indicates a significant
difference (p \ 0.05) from the carrageenan group (ANOVA; Bon-
ferroni’s test)
ZnPP IX (3 mg/kg), a specific HO-1 inhibitor. The anti-
inflammatory effect of CrII (0.1 mg/kg; i.v.) on the Cg-
induced rat paw edema was abolished in the presence of
ZnPP-IX (Fig. 6).
Repeated dose toxicity
After seven consecutive days of CrII (1.0 mg/kg, i.v.)
administration, no mice died and no physical, behavioral or
body mass changes were observed (p [ 0.05). Following
euthanasia and organ removal, there were no significant
changes in the wet weights of the liver, kidney, thymus,
lymph nodes and heart compared to control animals. The
serum levels of AST, ALT, ALP (a measure of hepatic
function) and urea (an indicator of renal function) did not
differ between CrII treated mice and the saline control
group (Table 1).
Additionally, histological analyses of the organs were
conducted to verify the presence or absence of abnormal
cellular or tissue morphology. No significant changes were
observed in the kidney, heart, thymus, spleen and lymph
nodes in any of the groups, with the exception of the liver,
which presented discrete cytoplasmic vacuolation (degen-
eration, swelling or edema). This change in the liver was
characterized by the accumulation of water in the cyto-
plasm, which resulted in a thick and pale nucleus that was
normally positioned. In all groups, these changes appeared
to be reversible, including the untreated (Fig. 7). There-
fore, there were no consistent signs of systemic damage in
response to Crll treatment.
Fig. 4 Effect of CrII using the carrageenan (Cg)-induced paw edema
model and myeloperoxidase (MPO) activity in the supernatant of paw
section homogenates injected with carrageenan. a Rats were treated
for 30 min before the injection of carrageenan (700 lg/paw, i.pl.),
with sterile saline (0.15 M NaCl, i.v.), dexamethasone (1 mg/kg, s.c.)
or CrII (0.01, 0.1 and 1.0 mg/kg, i.v.). Paw edema was measured 1, 2,
3, 4 and 5 h after the injection of Cg, and data are expressed as the
increase in paw volume (ml). b CrII (0.01, 0.1 and 1.0 mg/kg, i.v.),
dexamethasone (Dexa, 1 mg/kg, s.c.) or sterile saline (NaCl 0.15, s.c.)
were administered at 30 min, 1 and 1 h, respectively, before the s.c.
injection of carrageenan (Cg) (700 lg/paw). The results are expressed
in activity units of MPO/mg of tissue. Data are expressed as the
mean ± SEM of six animals for each group (ANOVA; Bonferroni’s
test). Asterisk indicates statistically significant difference (p \ 0.05)
compared to carrageenan control
Discussion
Since antiquity, substances derived from natural products
have been used for various purposes, including the
treatment of pain and inflammation [4, 12]. The results
reported here demonstrate that a polysaccharide fraction
(CrII) obtained from the alga C. racemosa by ion
exchange chromatography on DEAE-cellulose (eluted
with 0.75 M NaCl) has antinociceptive and anti-inflam-
matory effects in models of nociception (writhing,
formalin and hot-plate tests) and inflammation (peritonitis
and paw edema tests).
A writhing test induced by acetic acid is a widely used
model of inflammatory pain used to search for new agents
with analgesic, peripheral and anti-inflammatory properties
[30]. CrII (1.0 mg/kg, i.v.) displayed enhanced behavior in
writhing tests compared to sulfated polysaccharides
(3.0 mg/kg; i.v.) obtained from the green seaweed Caul-
erpa cupressoides, which belongs to the same genus of
algae [31]. However, the acetic acid-induced abdominal
123

Fig. 5 Effect of CrII in the dextran-induced paw edema model. The
animals were treated with sterile saline (0.15 M NaCl, i.v.) or CrII
(0.01, 0.1 and 1.0 mg/kg, i.v.) 30 min prior to the injection of dextran
(300 lg/paw, i.pl.). Paw edema was measured 1, 2, 3, 4 and 5 h after
dextran injection and is expressed as the increase in paw volume (ml).
Data are expressed as the mean ± SEM of six animals for each group
(ANOVA; Bonferroni’s test). Asterisk indicates a statistically signif-
icant difference (p \ 0.05) compared to the dextran control
Fig. 6 The HO-1 activity in paw edema induced by carrageenan in
the rats. Before receiving an injection of carrageenan (700 lg/paw,
i.v.), the groups of animals received CRII (0.1 mg/kg) with or without
ZnPP IX (3 mg/kg, s.c.). Another group received only saline without
Cg. Data are expressed as mean ± SEM of six animals for each group
(ANOVA; Bonferroni’s test). Asterisk indicates statistically signifi-
cant difference (p \ 0.05) when compared to control carrageenan.
Hash indicates statistically significant difference (p \ 0.05) when
compared to Cg ? CrII (0.1 mg/kg) ? ZnPP IX group
contortion test is also known to be nonspecific [32]. Thus,
to determine the best analgesic effect, CrII was assessed
using the formalin (2 %) test.
123
N. A. Ribeiro et al.
Formalin-induced nociception is widely used as model
of persistent pain [33]. This test is specific and is charac-
terized by a distinct, biphasic response; the first response is
termed neurogenic, and the second is inflammatory. Drugs
that act primarily on the central nervous system have been
reported to inhibit both phases, while others act peripher-
ally, such as anti-inflammatory and nonsteroidal agents and
corticosteroids that predominantly inhibit the second phase
[22, 34]. Therefore, because CrII is only effective in the
second test phase, this compound likely possesses proper-
ties similar to those of anti-inflammatory and nonsteroidal
agents and corticosteroids, which exert peripheral effects
(inflammatory pain) on nociceptors. These results corrob-
orate previously published reports that sulfated
polysaccharide fractions from the red seaweed Solieria fi-
liform (9 mg/kg; i.v.) only affected the duration of paw
licking in the second phase of the formalin test. Impor-
tantly, the seaweed used in this study produced a similar
result at a dose that was 900 times less than the doses used
in the work of Araújo et al. [35].
The hot plate test is widely used as an experimental
method to study nociception in rats and mice [36], and it is
considered to be a gold standard test for the evaluation of
analgesics that elicit a central effect, such as opioids, which
exert their analgesic effects via supra spinal receptors [37,
38]. Therefore, the results obtained using CrII in this model
suggest that its antinociceptive activity is not related to
central opioid receptors and that it likely acts through a
peripheral mechanism. These results are similar to those
obtained for the peripheral antinociceptive action of sul-
fated polysaccharide fractions from the alga S. filiform
[35], but differ from those of Rodrigues et al. [31], who
reported that the sulfated polysaccharide fraction of the
green alga C. cupressoides was able to significantly delay
the response to thermal stimuli.
Because of the well-established link between the
development of pain and inflammatory processes, we
investigated the anti-inflammatory activity of CrII using
the peritonitis and paw edema tests. The inflammatory
effect of Cg involves the action of a series of mediators,
characterized by the initial release of histamine, serotonin
and bradykinin, followed by increased levels of prosta-
glandins, which coincide with leukocyte migration to
sufficiently amplify the inflammatory response and trigger
the production of other mediators [39]. Peritonitis has been
well characterized as an experimental model of acute
inflammation that allows for the quantification and corre-
lation of cell migration in sets with several inflammatory
mediators [40]. In this model, all doses of CrII significantly
decreased the leukocyte count in the peritoneal cavities.
The anti-inflammatory effect elicited by CrII was similar to
the reported effects of the sulfated galactan from the red
alga Pophyridium sp., which also showed anti-
Sulfated polysaccharides isolated from the green seaweed Caulerpa racemosa

Fig. 7 Four-millimeter-thick
photomicrographs of the organs
taken from Swiss mice after
receiving no treatment, saline or
CrII (7 days of CrII, 1.0 mg/kg,
i.v.): a kidney, b heart, c timo,
d spleen, e lymph node and
f liver; note the discrete
cytoplasmic vacuolization
(white arrows). 4009
magnification

123
 N. A. Ribeiro et al.

Table 1 Systemic effects of CrII (1.0 mg/kg) in mice

Parameters Treatment (1 mg/kg, i.v.)
Males Females
Saline C. racemosa Saline C. racemosa

Body mass (g) before treatment 27.75 ± 0.60 30.29 ± 0.28 27.86 ± 0.37 24.83 ± 0.30
Body mass (g) after treatment 29.83 ± 0.89 33.51 ± 0.52 30.83 ± 0.57 26.51 ± 0.30
Heart (g)/body mass 0.65 ± 0.03 0.69 ± 0.03 0.75 ± 0.01 0.76 ± 0.02
Liver (g)/body mass 5.94 ± 0.11 5.83 ± 0.19 5.70 ± 0.16 5.21 ± 0.11
Spleen (g)/body mass 0.39 ± 0.01 0.46 ± 0.03 0.47 ± 0.03 0.54 ± 0.02
Kidney (g)/body mass 0.96 ± 0.03 0.96 ± 0.02 0.82 ± 0.03 0.83 ± 0.01
Thymus (g)/body mass 0.30 ± 0.01 0.29 ± 0.04 0.39 ± 0.05 0.42 ± 0.02
Lymph nodes (g)/body mass 0.27 ± 0.02 0.28 ± 0.02 0.27 ± 0.02 0.24 ± 0.02
Urea (mg/dl) 38.00 ± 2.59 45.37 ± 3.23 40.71 ± 2.37 35.07 ± 3.17
AST (UI/l) 73.34 ± 17.97 83.81 ± 12.70 75.43 ± 8.28 93.72 ± 11.47
ALT (UI/l) 57.11 ± 10.49 107.1 ± 26.59 54.37 ± 8.40 65.92 ± 10.50
ALP (UI/l) 104.60 ± 4.44 99.71 ± 5.77 80.31 ± 6.60 104.5 ± 12.93
Animals were weighed and injected once daily with CrII over seven consecutive days. After seven days of treatment, animals were weighed, the
blood samples were collected for biochemical dosage (AST, ALT, ALP and urea), mice were sacrificed, and the wet weight of organs
determined. Values are reported as mean ± SEM. Bonferroni t test for unpaired values

inflammatory activity [41]. Interestingly, the genus Caul-
erpa is known to possess anti-inflammatory activity.
According to Rodrigues et al. [31], a sulfated polysaccha-
ride fraction (FII) from the green alga C. cupressoides
significantly inhibited neutrophil migration in a dose-
dependent manner compared to Cg. In another study,
alcoholic and metanóicos extracts from the green seaweed
Caulepa Mexican were used in an inflammatory model of
peritonitis induced by Zymosan, and these extracts were
able to suppress cell migration to the peritoneal cavity [42].
Due to the anti-inflammatory effect of CrII on the Cg-
induced peritonitis, this compound was tested in Cg-
induced and dextran-induced models of rat paw edema.
Paw edema induced by dextran increases vascular perme-
ability by releasing vasoactive amines, such as histamine
and serotonin, which cause osmotic edema with low levels
of protein and neutrophils [43]. At all of the doses tested,
CrII inhibited both Cg-induced and dextran-induced rat
paw edema and neutrophil migration, as well as the activity
of MPO. Oral administration of total sulfated polysaccha-
rides (2.5, 5, 10 or 20 mg/kg) from the brown seaweed
Turbinaria ornate also inhibited the Cg-mediated induction
of paw edema in rats [12]. Again, CrII had a more satis-
factory effect at a 1,000-fold lower dose compared to the
polysaccharides from the brown seaweed T. ornate. This is
the first report of a sulfated polysaccharide that is capable
of inhibiting dextran-induced paw edema. Therefore, the
anti-edematogenic effect of CrII suggests that the inhibi-
tion of Cg-induced and dextran-induced paw edema is
related to inflammatory events that involve the inhibition of
multiple mediators, including histamine, serotonin, brady-
kinin, prostaglandins and vasoactive amines.
Heme oxygenase is an enzyme involved in the degra-
dation of heme groups, and its involvement in
inflammatory contexts is well established in the literature.
HO-1 production is elevated in inflammatory cells during
the resolution phase of inflammation [44]. HO-1 induction
significantly suppresses inflammation; however, inhibition
of this enzyme potentiates the inflammatory response in
different inflammation models [45–50]. Additionally, the
anti-inflammatory effects of HO-1 have been reported in
Cg-induced rat models [44]. Considering these data, we
explored the involvement of HO/BVD/CO in the anti-
inflammatory effect of CrII using ZnPP IX, a specific HO-1
inhibitor. After the pretreatment with ZnPP IX, the anti-
inflammatory efficacy of CrII in Cg-induced paw edema in
rats was not observed, suggesting that HO-1 activity is
involved in the inhibitory effects of CrII, corroborating
other data showing that the inhibition of HO-1 pathway is
associated with worsening of inflammatory response [51–
53]. In this regard, we have recently demonstrated that
ZnPP IX treatment potentiated the effect of acetic acid by
increasing the number of writhes and reducing bilirubin
levels [54]. Further, we also showed that some sulfated
polysaccharides from seaweeds may exert anti-inflamma-
tory activity through the HO-1 pathway [55], corroborating
the results observed in this study.
Although the reports regarding the safety of seaweed
compounds in animals are still limited, several studies have
reported on their possible effects. Araujo et al. [35]

123
Sulfated polysaccharides isolated from the green seaweed Caulerpa racemosa
evaluated the potential toxic effects of sulfated polysac-
charides from the seaweed S. filiform in a 14-day subchronic
toxicity test. Their results showed that intraperitoneal
administration of sulfated polysaccharides produced no
signs of toxicity in mice. The sulfated polysaccharides from
the seaweed, Gracilaria cornea, were also administered
intraperitoneally in rats for 14 consecutive days, and they
also produced no significant signs of toxicity [56]. To
evaluate the safety of CrII administration, we assessed the
integrity of the liver, kidney, thymus, lymph nodes and
heart in mice injected with CrII. Biochemical analyses
revealed no changes in the enzymatic activities of trans-
aminases and urea in the serum of treated mice. In addition,
histological analysis of tissues taken from the organs of
Crll-treated animals revealed no damage.
Conclusion
In conclusion, we demonstrated the antinociceptive and
anti-inflammatory efficacy of sulfated polysaccharides
from the green seaweed C. racemosa (Crll). Additional, our
results strongly suggest that the anti-inflammatory CrII
efficacy at least in part depends on the integrity of the HO-
1 pathway. Further, we verified that CrII is safe in the
effective dose. Taking into account the well-demonstrated
antinociceptive and anti-inflammatory efficacy of CrII, the
designing of alternative compounds to classical anti-
inflammatory and analgesic agents is very much encour-
aged, to define new pharmacological targets for the
treatment of inflammatory pain.
Acknowledgments This work was supported by Conselho Nacional
de Desenvolvimento Cientı́fico e Tecnológico (CNPq) and Coorde-
nacão de Aperfeiçoamento de Pessoal de Nı́vel Superior (CAPES).
H. S. A. Monteiro and N. M. B. Benevides are senior investigators of
CNPq/Brazil.
Conflict of interest No conflict of interest is declared.
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