Basic & Clinical Pharmacology & Toxicology, 2014, 115, 335–342
Doi: 10.1111/bcpt.12234
Peripheral Antinociception and Anti-Inflammatory Effects of
Sulphated Polysaccharides from the Alga Caulerpa mexicana
Jose Gerardo Carneiro1,2, Jose Arievilo Gurgel Rodrigues1, Edfranck de Sousa Oliveira Vanderlei1, Ricardo Basto Souza1, Ana Luıza
Gomes Quindere1, Chistiane Oliveira Coura1, Ianna Wivianne Fernandes de Ara
ujo1, Hellıada Vasconcelos Chaves3, Mirna Marques
Bezerra4 and Norma Maria Barros Benevides1
1
Department of Biochemistry and Molecular Biology, Federal University of Ceara, Fortaleza, Brazil, 2Federal Institute of Education, Science and
Technology of Ceara, Acarau, Brazil, 3Faculty of Dentistry, Federal University of Ceara, Sobral, Brazil and 4Faculty of Medicine, Federal
University of Ceara, Sobral, Ceara, Brazil
(Received 14 November 2013; Accepted 7 March 2014)
Abstract: Sulphated polysaccharides from marine algae are widely used in biotechnological and pharmaceutical areas. In this
study, we evaluated the effects of sulphated polysaccharides from the green marine alga Caulerpa mexicana (Cm-SPs) in nociceptive and inflammatory models in rodents. Cm-SPs (10 or 20 mg/kg), administered i.v. in Swiss mice, significantly reduced
nociceptive responses, as measured by the number of writhes in response to acetic acid. Cm-SPs (10 or 20 mg/kg) also reduced
second-phase responses in the formalin test, but did not exhibit a significant antinociceptive effect in the hot plate test, suggesting that its antinociceptive action occurs through a peripheral mechanism. Cm-SPs (5, 10 or 20 mg/kg), administered s.c. in wistar rats 1 hr before carrageenan, dextran, histamine or serotonin, were tested in paw oedema models. Cm-SPs (10 or 20 mg/kg)
reduced carrageenan-induced paw oedema and myeloperoxidase activity in the paw. In addition, Cm-SPs (20 mg/kg) inhibited
dextran- or histamine-induced paw oedema, but not serotonin-induced oedema, suggesting that histamine is the major target of
Cm-SPs anti-oedematogenic activity. Finally, Cm-SPs (20 mg/kg) administered in mice did not show significant signs of toxicity.
In conclusion, Cm-SPs appear to be promising natural modulatory agents for pain and inflammatory conditions.
Inflammation is a protective response that serves to eliminate
pathogens and other offending agents which have potential to
cause cell injury, as well as malignant and necrotic cells; it
involves a co-ordinated series of events that can be divided
into two parts, called the ‘acute’ and ‘chronic’ phases [1,2].
The acute phase is generally initiated by the activation of
tissue-resident sentinel cells as a result of recognition of
danger-associated molecules; these cells rapidly release soluble
effector molecules that work together to increase vasodilation
and vascular permeability and to recruit neutrophils and platelets to the site of inflammation. Acute inflammatory responses
can provide a powerful boost that helps to rapidly clear the
infection and resolve the inflammation. However, if it is not
properly controlled, a persistent inflammatory state (chronic
phase) may be installed, characterized by tissue damage mediated by leucocytes and which can lead to the development of
inflammatory diseases [1,2].
Nociception is the process by which intense thermal, mechanical or chemical stimuli are detected by a subpopulation of
peripheral nerve fibres called nociceptors [3]. Peripheral sensitization results from inflammation-associated changes in the
chemical environment of the nerve fibre. Furthermore, accumulation of endogenous factors released from activated nociceptors
or non-neural cells often results in tissue damage. Collectively,
these factors represent a wide array of signalling molecules,
including neurotransmitters, peptides (substance P, CGRP,
bradykinin), eicosanoids and related lipids (prostaglandins,
Author for correspondence: Norma Maria Barros Benevides, Department of Biochemistry and Molecular Biology, Federal University of
Ceara, s/n Humberto Monte Avenue, Pici Campus 60455-760, Fortaleza, Brazil (e-mail [email protected]).
thromboxanes, leukotrienes, endocannabinoids), neurotrophins,
cytokines and chemokines, as well as extracellular proteases and
protons [4].
The development of novel drugs to treat inflammatory diseases is constantly under debate and research [5]. Non-steroidal anti-inflammatories, glucocorticoids and opioids are drugs
that undoubtedly provide significant health benefits in the
treatment of pain and inflammation. However, their prolonged
use is followed by complications, including gastric perforations, stomach ulcers, bleeding, euphoria, respiratory depression, beyond physical and psychological dependence,
respectively [6,7]. Therefore, the discovery of new bioactive
compounds presenting anti-inflammatory and analgesic activities with minimal adverse effects is of great importance.
Marine algae are the most important source of non-animal
sulphated polysaccharides, and the chemical structure of these
polymers varies according to the algal species [8]. Sulphated
polysaccharides comprise a group of heterogeneous macromolecules presenting various biological properties, such as antiviral [9], immunomodulating [10], antitumoral [11],
anticoagulant [12], antioxidant [13], anti-inflammatory [14],
antinociceptive and pro-inflammatory effects [15].
Caulerpa mexicana Sonder ex K€
utzing is a marine green
alga of the Cauleparceae family, which can be found in the
temperate seas, but especially in tropical areas, and is widely
encountered along the coast of Brazil [16]. Aqueous and methanolic extracts obtained from this alga species have presented
antinociceptive and anti-inflammatory effects in models of ear
oedema and cell migration [17]. However, no study has
described the effects of sulphated polysaccharides from
C. mexicana in nociception and inflammation.
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
GERARDO CARNEIRO ET AL.
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336
In the present study, we investigated the effects of sulphated
polysaccharides from the green seaweed C. mexicana on acute
inflammation and nociception using several well-established
experimental animal models in rodents.
Materials and Methods
Drugs and reagents. The following drugs and reagents were used:
dextran sulphate, k-carrageenan, cetylpyridinium chloride (CPC), 1,9
dimethylmethylene blue, o-dianisidine dihydrochloride, N-acetyl-N,N,
N trimethylammonium bromide, potassium phosphate monobasic,
potassium phosphate dibasic, hexadecyltrimethylammonium bromide
(HTAB), cysteine, papain and bovine serum albumin (BSA), which
were purchased from Sigma Aldrich (St. Louis, MO, USA).
Dexamethasone was purchased from Ache (Guarulhos, SP, Brazil).
Gelatin was purchased from Oxoid Ltd (Basingstoke, Hampshire,
UK). Ethylenediaminetetraacetic acid (EDTA), formaldehyde, glacial
acetic acid and hydrate of chloral were purchased from VETEC
Quımica Farm, LTDA (SP, Brazil). The enzymatic kits used for the
evaluation of the systemic toxicity were of the LABTEST (Diagnostic
Tests, Lagoa Santa, Minas Gerais, Brazil). All other chemicals were of
analytical grade.
Animals. Male and female Swiss mice (20–25 g) and male Wistar rats
(180–240 g) from the Animal Care Unit of the Federal University of
Ceara, Fortaleza, Brazil, were used in the experiments. For each
experiment, groups of six animals were segregated and handled
separately. This study was conducted in accordance with the
guidelines set forth by the U.S. Department of Health and Human
Services and with the approval of the Ethics Committee of the Federal
University of Ceara, Fortaleza, Brazil (CEPA no. 71⁄12).
Marine alga. Specimens of Caulerpa mexicana (Caulerpaceae,
Bryopsidales) were collected at Flecheiras Beach, Brazil, and
transported to the Carbohydrates and Lectins Laboratory (Ceara State,
Brazil), Department of Biochemistry and Molecular Biology, Federal
University of Ceara. The material was cleaned of epiphytes, washed
with distilled water and stored at 20°C until use. A voucher
specimen (no. 47304) was deposited in the Herbarium Prisco Bezerra
(EAC) in the Department of Biology, Federal University of Ceara.
[19]. The density of charge of Cm-SPs was also checked by 0.5%
agarose gel electrophoresis according to Dietrich and Dietrich (1976)
[20].
Infrared (IR) spectroscopy. Cm-SPs were also characterized by IR
spectroscopy. The Fourier transform IR spectra were recorded with a
Shimadzu IR spectrophotometer (model 8300) between 400 and
4000 cm 1. The samples were analysed as a potassium bromide (KBr)
pellet.
Antinociceptive activity.
Writhing test. This test was performed to examine the antinociceptive
effect [21]. For this assay, mice received i.v. Cm-SPs (5, 10 or
20 mg/kg; 0.1 ml/10 g body-weight) or sterile saline (control group,
0.9%, w/v). After 30 min., 1% (v/v) of acetic acid was injected
intraperitoneally (0.1 ml/10 g body-weight). The number of writhes,
consisting of abdominal muscle contractions and hind paw extensions,
occurring between 0 and 30 min. after the chemical stimuli, was
recorded. Morphine (5 mg/kg; s.c.), a non-selective opioid agonist, or
indomethacin (5 mg/kg; s.c.), a non-specific inhibitor of
cyclooxygenase, which is the enzyme responsible for prostaglandin
synthesis [22], were used as controls.
Formalin test. The formalin test in mice is a valid and reliable model
of nociception and is sensitive for various classes of analgesic drugs
[23]. Mice received i.v. Cm-SPs (5, 10 or 20 mg/kg) or sterile saline
(0.9%, w/v) 30 min. before formalin injection. Then, 1% formalin
(20 ll) was injected into the right hind paw of the mice. The animals
responded to the formalin injection by running around, shaking the
paw and squeaking, and thereafter started to lick the paw. The licking
response was recorded from 0 to 5 min. (phase 1) and from 20 to
30 min. (phase 2). Morphine and indomethacin (both 5 mg/kg, s.c.)
were used as controls.
Hot plate test. This test also measures analgesic activity [24,25].
Groups of six mice each received sterile saline (0.9%, w/v, i.v.), CmSPs (5, 10 or 20 mg⁄kg, i.v.), morphine (5 mg/kg, s.c.) or indomethacin
(5 mg/kg, s.c.). Animals were individually tested and were not
habituated to the apparatus prior to testing. To register the reaction
times (licking the paw or jumping), each mouse was placed on the
heated plate (51 1°C) four times, separated by 30-min. intervals.
Therefore, reaction times were measured at time zero (0 min.) and 30,
60 and 90 min. after the administration of compounds with a cut-off
time of 30 sec. to avoid paw lesions. Each animal was tested only once.
Extraction of sulphated polysaccharides. Sulphated polysaccharides
(SPs) were obtained according to Farias et al. [12]. Essentially, 5 g of
the dehydrated algal tissue at 25°C was macerated with liquid nitrogen
and subjected to digestion with crude papain solution (30 mg/ml) in
100 mM sodium acetate buffer (pH 5.0) containing 5 mM cysteine and
5 mM EDTA at 60°C for 6 hr. The material was then centrifuged
(2295 9 g, 30 min., 10°C), and the SPs in solution were precipitated
with 16 ml of 10% cetylpyridinium chloride (CPC) solution. After
24 hr at room temperature, the mixture was centrifuged at 2560 9 g
for 20 min. at 5°C. The SPs in the pellet were washed with 200 ml of
0.05% CPC solution, dissolved with 100 ml of a 2 M NaCl–ethanol
(100:15, v/v) mixture and precipitated with 100 ml of absolute ethanol.
After 24 hr at 4°C, the precipitate was collected by centrifugation
(2560 9 g for 20 min. at 5°C), washed twice with 100 ml of 80%
ethanol and washed once with 100 ml of absolute ethanol. The final
precipitate was dialysed and freeze-dried. After these procedures, the
total SPs from C. mexicana (Cm-SPs) were obtained.
Carrageenan-induced rat paw oedema. Carrageenan (500 lg/paw;
100 ll) was injected i.pl. into hind right paws of the rats. Animals
were treated s.c. with Cm-SPs at doses of 5, 10 or 20 mg/kg (0.1 ml/
100 g body-weight) 1 hr before the stimuli. Dexamethasone (1 mg/kg,
s.c.), a synthetic glucocorticoid with potent anti-inflammatory and
immunosuppressant properties [15], was administered 1 hr before
carrageenan, as a reference drug. Paw volumes were measured
immediately before (zero time) the stimulus and at selected time
intervals (1, 2, 3 and 4 hr) using a plethysmometer (Panlab, Spain).
The results were expressed as the variation in paw volume (ml),
calculated as the difference from the basal volume [26].
Chemical composition. After acid hydrolysis of the soluble
polysaccharides (1 M HCl, 110°C, 5 hr), sulphate content was
measured by gelatin-barium method, using sodium sulphate (Na2SO4)
as a standard [18]. The protein content was measured by binding of
Coomassie Brilliant Blue G-250 to protein, using BSA as a standard
Myeloperoxidase (MPO) activity assay. The extent of neutrophil
accumulation in the paw tissue was measured by an MPO activity
assay as previously described [27]. Briefly, 50–70 mg of paw tissue
was homogenized in potassium phosphate buffer containing 0.5%
HTAB (1 ml buffer per 50 mg of tissue). The homogenate was then
Anti-inflammatory activity.
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
EFFECTS OF SULPHATED POLYSACCHARIDES FROM THE ALGA CAULERPA MEXICANA
Dextran-induced rat paw oedema. Dextran (400 lg/paw; 100 ll), a
common inductor osmotic oedema, was injected i.pl. into hind right
paws of the rats [28]. Animals were treated s.c. with Cm-SPs at doses
of 5, 10 or 20 mg/kg (0.1 ml/100 g body-weight) 1 hr before the
stimuli. Paw volumes were measured immediately before (zero time)
the stimulus and at selected time intervals after the stimulus (0.5, 1, 2,
3 and 4 hr) using a plethysmometer (Panlab Spain). The results were
expressed as the variation in paw volume (ml), calculated as the
difference from the basal volume (0).
Histamine- and serotonin-induced rat paw oedemas. Histamine
(100 lg/paw; 100 ll) or serotonin (20 lg/paw; 100 ll) was injected
i.pl. into hind right paws of the rats. Animals were treated s.c. with
either Cm-SPs (20 mg/kg) or sterile saline (0.9%, w/v) 1 hr before the
stimuli. Paw volumes were measured immediately before (zero time)
the stimulus and at selected time intervals after the stimulus (0.5, 1, 2,
3 and 4 hr) using a plethysmometer (Panlab Spain). The results were
expressed as the variation in paw volume (ml), calculated as the
difference from the basal volume (0).
Acute toxicity assay. The acute toxicity model is widely used in
assays to assess the toxicity of sulphated polysaccharides from algae
[29–32]. Cm-SPs (20 mg/kg; 0.1 ml/10 g body-weight) or saline was
injected i.v. into groups of male and female mice. Animals were
maintained with free access to water and food. Survival rate and
abnormal pattern of behaviour (piloerection, tachycardia, cyanosis,
tachypnea, pruritus, convulsions, sedation and death) were observed
for 48 hr. After this period, mice were then killed and blood was
collected for plasma biochemistry analyses. Levels of alanine
aminotransferase (ALT), aspartate aminotransferase (AST), creatinine
and alkaline phosphate (ALP) were determined using enzymatic and
colorimetric tests (LABTEST). Body mass, organ weight alteration,
morphological and histopathological analyses of organs (liver, heart,
kidney, thymus, lymph nodes and spleen) were also performed.
After killing, liver, heart and right kidney were fixed with formalin.
The material was then dehydrated using ethanol and embedded in paraffin. The resulting blocks were sliced into 5-lm-thick sections,
stained with haematoxylin–eosin (HE) and observed under a light
microscope.
Statistical analyses. All data were analysed using the program
GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA).
Student’s t-test for unpaired values and one-way analysis of variance
(ANOVA) followed by Bonferroni’s test were performed for the analysis
of data with Gaussian distribution. All data shown are mean S.E.M.
Values of p < 0.05 were considered to be statistically significant.
Results
purity of Cm-SPs was verified by agarose gel electrophoresis
procedure, a polydisperse band was obtained (data not
shown).
IR spectroscopy.
The IR spectrum of Cm-SPs (Fig. 1) presented a characteristic
band around 1251 cm 1, which corresponds to ester sulphate
groups (S=O). The signal corresponding to the stretching
vibration at 815 cm 1 was related to the presence of galactose-6-sulphate (C–O–S sulphate axial), indicating sulphate
groups located at C-6 residues of D-galactose. Typical absorptions provided information on the presence of uronic acid (at
1642–1406 cm 1, COO ) and galactan (at 1051 cm 1) in the
analysed polymer. The spectrum also displays absorbance
bands at 3444 and 2934 cm 1, which are associated with the
occurrence of OH and CH contents, respectively (Fig. 1).
Antinociceptive effect.
Acetic acid-induced writhing test. Cm-SPs at the higher doses
(10 or 20 mg/kg) reduced in a dose-dependent manner the
number of abdominal constrictions by 74.5% (18.40 4.7)
and 88.9% (6.66 3.64), respectively, when compared with
the control group (p < 0.05). No significant reduction in the
number of abdominal constrictions was observed at a dose of
5 mg/kg. Morphine (95.7%, 2.60 1.38) and indomethacin
(50.5%,
29.75 4.09)
also
exhibited
(p < 0.05)
antinociceptive effects (Fig. 2A).
Formalin test. A significant reduction in the licking time was
demonstrated in the second phase (inflammatory) at higher
doses tested i.v. of Cm-SPs (10 mg/kg – 88.6%
(12.06 7.78 ml), 20 mg/kg – 98.5% (6.37 5.51 ml), in a
dose-dependent manner from the control group (p < 0.05). No
inhibitory effect in the first phase (neurogenic) was observed
(p > 0.05). As expected, morphine (5 mg/kg, s.c.) reduced the
licking time by 94 and 97% in the first and second phases,
respectively (p < 0.05). Indomethacin (5 mg/kg, s.c.)
exhibited analgesic action of 55.6% only in the second phase
of the experiment (p < 0.05) (Fig. 2B).
0.65
3444
0.60
1051
0.55
Absorbance
centrifuged at 40,000 9 g for 7 min. at 4°C. MPO activity was
determined by measuring the change in absorbance at 450 nm using
o-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. One
unit of MPO activity was defined as the activity required to convert 1
lmol of hydrogen peroxide to water in 1 min. at 22°C. Results were
reported as MPO units/mg of tissue.
337
1251
1642
0.50
0.45
1374
1406
2934
0.40
815
Yield and chemical composition analysis and physicochemical
characterization.
The yield of the lyophilized crude extract containing sulphated
polysaccharides obtained by papain digestion from the green
marine alga C. mexicana was 1.7% from the dehydrated algal
tissue. Quantitative determination revealed a sulphate content
of 21.5%. No protein contamination was found. When the
0.35
0.30
0.25
4000
3500
3000
2500
2000
1500
cm–1
Fig. 1. IR spectral analysis of Cm-SPs at 400–4000 cm 1.
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
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GERARDO CARNEIRO ET AL.
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B
Fig. 2. Effect of Cm-SPs in nociceptive tests.
(A) Effect of Cm-SPs on the writhing
response induced by acetic acid in mice. (B)
Effect of Cm-SPs on the formalin test in
mice. The licking time was determined during
the first 5 min. (1st phase) and during the
period of 20–25 min. (2nd phase) after 1%
formalin injection in mice. (C) Effect of
Cm-SPs on reaction times to thermal stimuli
in mice. Mice received i.v. sterile saline or
Cm-SPs (5, 10 or 20 mg/kg). Morphine or
indomethacin (both 5 mg/kg) was given s.c.
30 min. before stimuli. Data are expressed as
the mean S.E.M. of six animals for each
group (one-way ANOVA; Bonferroni’s test).
*Significant difference from the saline group
(p < 0.05).
C
Hot plate test. Cm-SPs (5, 10 or 20 mg/kg, i.v.) did not
modify the reaction time during the 90 min. of observation
compared to the control group (Fig. 2C). However, morphine
(5 mg/kg, s.c.), which was used as control positive, increased
the latency time. Sterile saline (0.9%, w/v, i.v.) or
indomethacin (5 mg/kg, s.c.) did not increase the reaction time
at all intervals analysed.
Anti-inflammatory effect.
Carrageenan-induced rat paw oedema and MPO activity
assay. Carrageenan (500 lg/paw) elicited intense paw
oedema, which reached a maximum level at 3 hr
(0.86 0.06 ml) after injection and then decreased over the
subsequent hour (0.72 0.06 ml). Cm-SPs (5, 10 or 20 mg/
kg, s.c.) reduced the oedema formation (p < 0.05) at 3 hr by
21% (0.66 0.01 ml), 51% (0.41 0.03 ml) and 43%
(0.49 0.04 ml), respectively. Cm-SPs (20 mg/kg) presented
a similar inhibitory profile compared to dexamethasone (1 mg/
kg, s.c.; at 3 hr – 72% (0.23 0.05 ml) (Fig. 3A). Cm-SPs
(10 and 20 mg/kg) reduced neutrophilic infiltration in the paw
(43 and 53%, respectively), as demonstrated by the MPO
activity assay (p < 0.05) (Fig. 3B).
Dextran-, histamine- and serotonin-induced rat paw
oedemas. Dextran (400 lg/paw) elicited a significant
inflammatory response by an increase in vascular
permeability, reaching the maximal level at 30 min.
(0.61 0.08 ml) and then diminishing its action over
the subsequent hours. Administration of Cm-SPs (10 or
20 mg/kg, s.c.), 1 hr prior to the injection of dextran,
inhibited the oedema (p < 0.05) by 47% (0.32 0.01 ml)
and 44% (0.34 0.03 ml), respectively, at 30-min. interval.
Cm-SPs (5 mg/kg, s.c.) did not present anti-oedematogenic
activity (0.59 0.01 ml) (Fig. 4A).
In addition, Cm-SPs treatment (20 mg/kg, s.c.), 1 hr prior
to injection of histamine (100 lg/paw, i.pl.), reduced
(p < 0.001) the paw oedema by 36% (0.26 0.02 ml, at
30 min.) and 37% (0.25 0.01 ml, at 1 hr), respectively,
compared with control group at 30 min. (0.41 0.03 ml) and
at 1 hr (0.40 0.01 ml), respectively (Fig. 4B). However,
Cm-SPs (20 mg/kg, s.c.) was ineffective on serotonin-induced
rat paw oedema (20 lg/paw, i.pl.) (Fig. 4C).
Acute toxicity assay. Systemically administered Cm-SPs in a
single dose (20 mg/kg, i.v.) over 48 hr did not show lethality.
In addition, mice body-weight, organ wet weight or general
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
EFFECTS OF SULPHATED POLYSACCHARIDES FROM THE ALGA CAULERPA MEXICANA
A
A
B
B
339
C
Fig. 3. (A) Effect of Cm-SPs on carrageenan-induced rat paw oedema.
Animals received s.c. sterile saline, Cm-SPs or dexamethasone (Dexa)
1 hr before receiving an injection of carrageenan (500 lg/paw;
100 ll). Another group received only sterile saline (i.pl.) in the paw.
(B) Myeloperoxidase (MPO) activity in the supernatant of homogenates of the paw tissue from rats submitted to carrageenan-induced paw
oedema. Cm-SPs (5, 10 or 20 mg/kg), dexamethasone (1 mg/kg) or
saline was administered s.c. before the carrageenan injection. MPO
activity was expressed as units per mg of tissue. Data are expressed as
the means S.E.M. of six rats for each group. *p < 0.05 indicates a
significant difference from the carrageenan group (one-way ANOVA,
Bonferroni’s test).
aspects were not altered compared to the saline-treated group,
except the approximately twofold increase in the spleen
weight (p < 0.05). With respect to the biochemical analyses,
the ALT, AST, ALP and creatinine values did not differ from
the controls (Table 1). Furthermore, morphological and
histopathological analyses did not present any signs of
toxicity. The increased spleen size did not present signs of
inflammatory infiltration or cell degeneration based on
histological structures (data not shown).
Discussion
There is a great deal of current interest in identifying new
natural compounds for a wide variety of pharmaceutical applications. In our study, the yield and the sulphate content of
Cm-SPs are in accordance with data obtained in studies with
other algae species of the same genus [14,33,34]. The absence
of protein contamination in the samples of Cm-SPs could be
the result of proteolytic enzyme used during sulphated polysaccharides extraction. Cm-SPs presented a polydisperse band
on agarose gel electrophoresis, which is typical of sulphated
polysaccharides from marine algae [30–34].
The IR technique provides useful information on sulphate
and monosaccharide content in algae polysaccharides [30,35].
The IR spectrum (Fig. 1) showed several bands suggestive of
Fig. 4. (A) Effect of Cm-SPs on dextran-induced rat paw oedema.
Animals received s.c. sterile saline or Cm-SPs (5, 10 or 20 mg/kg)
1 hr before receiving an injection of dextran (400 lg/paw; 100 ll).
Another group received only sterile saline (i.pl.) in the paw. (B) Effect
of Cm-SPs on histamine-induced rat paw oedema. Animals received
s.c. sterile saline or Cm-SPs (20 mg/kg) 1 hr before receiving an
injection of histamine (100 lg/paw; 100 ll). Another group received
only sterile saline (i.pl.) in the paw. (C) Effect of Cm-SPs on serotonin-induced rat paw oedema. Animals received s.c. sterile saline or
Cm-SPs (20 mg/kg) 1 hr before receiving an injection of serotonin
(20 lg/paw; 100 ll). Another group received only sterile saline (i.pl.)
in the paw. Data are expressed as the means S.E.M. of six rats for
each group. *p < 0.05 indicates a significant difference from the carrageenan group (one-way ANOVA, Bonferroni’s test).
a sulphated galactan. The bands at 1374 cm 1 common to all
the sulphate esters [36] and at 1251 cm 1 corresponding to
the stretching vibration of SO sulphate and at 815 cm 1 bending vibrations derived from the COS sulphate in axial indicate
that sulphate groups are located at C-6 residues of D-galactose
[33,36,37]. Moreover, the bands at 1642 and 1406 cm 1 are
due to the asymmetric stretch vibration of uronic acids
(COO ), and the band at 1051 cm 1 is a typical galactan
band [36,37].
In recent years, the medical potential of sulphated polysaccharides derived from marine algae has significantly attracted
the attention of researchers due to their safety and pharmacological and biomedical potentials [29–32]. In the present
study, Cm-SPs were initially used on the acetic acid-induced
writhing test, an animal model of nociception commonly
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
GERARDO CARNEIRO ET AL.
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Table 1.
Systemic effects of Cm-SPs in mice. Animals were weighed and a single dose of Cm-SPs (20 mg/kg, i.v.) was injected. After 48 hr, animals were
weighed and anaesthetized, and the blood samples were collected for biochemical analyses (AST, ALT, ALP and creatinine). Mice were then killed
and the wet weights of organs were taken.
Treatment (i.v.)
Female
Parameters
Body mass (g) before
Body mass (g) after
Liver (g)/body mass
Kidney (g)/body mass
Heart (g)/body mass
Spleen (g)/body mass
Thymus (g)/body mass
Lymph nodes (g)/body mass
ALP (UI/l)
AST (UI/l)
ALT (UI/l)
Creatinine (UI/l)
Saline
25.93
26.29
0.559
0.887
0.657
0.431
0.413
0.299
48.3
61.7
50.32
0.52
0.51
0.54
0.014
0.025
0.027
0.027
0.068
0.021
5.53
10.55
7.49
9.54
Male
Cm-SPs (20 mg/kg)
27.08
27.39
0.559
0.879
0.663
0.833
0.343
0.249
39.8
67.43
45.78
0.48
0.42
0.43
0.008
0.027
0.029
0.115*
0.032
0.020
4.47
7.62
4.57
10.3
Saline
30.78
31.21
0.607
1.009
0.704
0.394
0.350
0.287
50.5
80.5
42.30
0.98
0.44
0.47
0.021
0.030
0.019
0.021
0.028
0.035
5.49
5.5
5.56
10.0
Cm-SPs (20 mg/kg)
29.30
29.60
0.648
1.010
0.637
0.719
0.328
0.206
45.3
77.48
39.82
0.68
0.49
0.50
0.033
0.031
0.033
0.062*
0.060
0.023
5.6
4.45
5.37
13.0
Data are expressed as the mean S.E.M. (n = 6).
*p < 0.05 indicates a significant difference from the saline group. Student’s t-test for unpaired values.
employed as a screening tool for evaluating analgesic properties. Weak analgesics present antinociceptive effects when
evaluated by the acetic acid method, which is an advantage of
this method. On the other hand, it lacks specificity [38].
The inflammatory pain accompanying the writhing caused
by intraperitoneal injections of acetic acid is associated with
the release of inflammatory mediators, such as bradykinin,
substance P, prostaglandins and several cytokines, including
IL-1b, TNF-a and IL-8 [39]. For this assay, mice that received
Cm-SPs exhibited antinociceptive response at the doses of 10
or 20 mg/kg, suggesting that Cm-SPs could inhibit the inflammatory mediators released in response to the chemical inductor
[29–32]. This antinociceptive profile of Cm-SPs was in accordance with a sulphated polysaccharide fraction isolated from
C. cupressoides [14].
The formalin-induced nociception test was also used in
order to characterize the effects of Cm-SPs on behavioural
nociception. Local formalin injection induces acute tissue
injury in the paw, leading to a licking response. The first
phase starts immediately after formalin injection, probably due
to direct chemical stimulation of nociceptors [23,40], and lasts
for 3–5 min. Experimental data indicate that formalin predominantly evokes activity in C fibres and Ad afferents [41–43],
and even that presumably non-nociceptive Ab-fibres are activated during first phase [41,42]. Experimental results have
indicated that substance P and bradykinin participate in
the early-phase. The second phase (starts approximately
15–20 min. after formalin injection) is elicited by a combination of stimuli from inflammation of the peripheral tissues to a
mechanism of central sensitization, including the participation
of several chemical mediators (e.g. serotonin, histamine,
prostaglandins, bradykinin) in response to formalin [23,44].
The antinociceptive response of Cm-SPs occurred only in
the second phase, suggesting that the action of these molecules
was related to inflammatory pain, similar to the modulating
action of non-steroidal anti-inflammatory agents and corticosteroids [6] and of other natural products derived from algae
previously studied in mice [30,31]. The hot plate test often
reflects central drug actions mediated via supraspinal and
spinal receptors [24].
Heat is more selective in the way it stimulates cutaneous
receptors. Consequently, specific categories of peripheral axons,
including thermosensitive and nociceptive fibres, can be excited.
Tests like hot plate observe reaction times for which a synchronous excitation of fibres is required, allowing an appropriate
study of neural phenomena into sensory systems. However, the
major limitation is the activity of non-opioid analgesics that
cannot be revealed by the hot plate test. In addition, this model
is not very sensitive to the analgesic effects of non-steroidal
anti-inflammatory agents [38].
In the present study, morphine caused a significant increase
in reaction time, whereas Cm-SPs produced no significant
antinociceptive effects. These results suggest that the antinociceptive action of Cm-SPs occurs via a peripheral mechanism
similar to the sulphated polysaccharide fraction from the marine algae Solieria filiformis [30] and Spatoglossum shroederii
[45]. In contrast, Coura et al. [32] and Rodrigues et al. [14]
reported that the sulphated polysaccharides from Gracilaria
cornea and C. cupressoides, respectively, inhibited both
phases of the formalin test and increased the reaction times in
the hot plate test in mice, suggesting that these polymers could
act on nociception as drugs that interact with the opioid system, like morphine.
Based on the hypothesis that there is a connection between
the antinociceptive action and the inflammatory pain, Cm-SPs
effects were also evaluated in a model of acute inflammation
(rat paw oedema) induced by different inflammatory agents
(carrageenan, dextran or vasoactive amines). Cm-SPs were initially investigated in the carrageenan-induced rat paw oedema,
which is a classic test to evaluate the effectiveness of anti-
© 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
EFFECTS OF SULPHATED POLYSACCHARIDES FROM THE ALGA CAULERPA MEXICANA
inflammatory agents [26], and it is modulated by several
mediators that lead to intense neutrophilic infiltrate [1,26,46].
Cm-SPs significantly inhibited the oedema at all intervals,
especially at the third hour, which involves higher level of exudates and neutrophil accumulation, and the participation of
diverse mediators, mainly prostaglandins [26,46]. Cm-SPs also
decreased neutrophilic migration as demonstrated by reduced
MPO activity. This enzyme is extensively used as a biochemical marker of neutrophilic infiltration into various tissues
[27]. Thus, the important role of Cm-SPs in the acute inflammatory process was revealed.
In order to better understand Cm-SPs anti-inflammatory
potency, their effect was also examined on dextran-induced rat
paw oedema. Dextran-induced inflammation is characterized
by the release of vasoactive amines (e.g. histamine and serotonin) and increased vascular permeability, producing an osmotic event with reduced levels of protein and neutrophils in the
exudate. In this experiment, Cm-SPs inhibited dextran-induced
rat paw oedema, suggesting that their anti-oedematogenic
action involves vasoactive amines [29,30,32]. To confirm this
hypothesis, histamine- and serotonin-induced paw oedemas
were performed. The highest dose of Cm-SPs (20 mg/kg) was
chosen due to its greater anti-inflammatory potency in the previous experiments. Our results demonstrated that Cm-SPs,
administered 1 hr prior to serotonin, did not reduce the
oedema. Interestingly, Cm-SPs exhibited its most potent inhibitory effect on the histamine-induced paw oedema and in the
second phase of the formalin, which is characterized by the
involvement of histamine H1 receptors [47]. Similarly, Sousa
et al. [48], investigating a sulphated polysaccharide fraction
from the red marine alga Gelidium crinale, reported an antioedematogenic response by the inhibition of histamine-induced
rat paw oedema, but no inhibition of the serotonin-induced rat
paw oedema was observed. In contrast, Quindere et al. [29]
reported that a sulphated polysaccharide fraction from the red
marine alga Acanthophora muscoides reduced both histamineand serotonin-induced rat paw oedemas.
Finally, the safety of Cm-SPs administration was evaluated.
After 48 hr, we did not observe any signs of toxicity.
Biochemical, morphological and histological analyses of mice
performed after the experimental period revealed no hepatic or
renal alteration, or apparent systemic damage in organs. Acute
toxicity studies are appropriate to assess potential lethality or
toxicity by administering a single dose and subsequent hippocratic screening evaluation [49,50] and are widely used in
assays to assess the toxicity of sulphated polysaccharides from
algae [29–32]. Several studies have shown safety of these
compounds in mice toxicity models [14,15,29–32].
Conclusion
Sulphated polysaccharides from the green marine alga Caulerpa mexicana exhibited antinociceptive and anti-inflammatory
effects reducing osmotic oedema, mainly caused by histamine,
and inhibiting neutrophils migration. Such actions appear to
reflect peripheral effects based on the reduction of histamine
release. In addition, important systemic damage in vivo was
341
not observed, indicating their safety in toxicological analysis.
Further studies aiming to elucidate the molecular mechanisms
of action of the antinociceptive and anti-inflammatory effects
of sulphated polysaccharides from marine green algae need to
be performed.
Acknowledgements
This research was supported by Conselho Nacional de Desenvolvimento Cientıfico e Tecnol
ogico (CNPq), Coordenacß~ao
de Aperfeicßoamento de Pessoal de Nıvel Superior (CAPES),
Fundacß~ao Cearense de Apoio ao Desenvolvimento Cientıfico e
Tecnol
ogico (FUNCAP), Programa Rede Nordeste de Biotecnologia (RENORBIO), N
ucleo de Biotecnologia de Sobral
(NUBIS) and Instituto Federal do Ceara (IFCE). We thank Dr.
Dalgimar Beserra de Menezes from the Department of Pathology and Forensic Medicine at Federal University of Ceara for
his helpful assistance in the histological analyses and
Dr. Regina Celia Monteiro de Paula from the Department of
Organic and Inorganic Chemistry for the use of the Shimadzu
IR spectrophotometer (model 8300). N.M.B. Benevides is a
senior investigator of CNPq/Brazil.
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