Antinociceptive activity and acute toxicological study of a novel sulfated polysaccharide from Caulerpa cupressoides var . lycopodium ( Chlorophyta ) in Swiss mice

Caulerpa cupressoides var. lycopodium (Chlorophyta) contains three sulfated polysaccharides (SPs) fractions (SP1, SP2 and SP3); but, their pharmacological properties have been limited. We investigated the antinociceptive activity of non-anticoagulant fraction (SP1) and then its acute toxicological study in male Swiss mice was performed. Animals (19-25 g) received i.v. SP1 30 min. prior to injection 0.8% acetic acid (10 mL kg, i.p.); 1% formalin (20 μL, i.pl.) or were subjected to thermal stimuli. Open-field test was also performed. Mice were treated i.p. with SP1 or 0.9% saline (0.1 mL 10 g ) for 72h. On the 4 day, the animals were anesthetized and sacrificed in order to collect blood and organs. SP1 (3, 9 or 27 mg kg) reduced (p < 0.05) the number of writhes induced by acetic acid by 44.21, 47.72 and 90.87%, respectively. SP1 inhibited (p < 0.05) the second phase of the formalin test, without antinociceptive effect in the hot-plate test, suggesting that its analgesic action occurs through of peripheral mechanisms. SP1 did not modify the locomotor activity. SP1 (27 mg kg ) did not cause hepatic or renal dysfunctions, but affected the spleen of animals (p < 0.05). Therefore, SP1 has analgesic action with high tolerance by the animals, presenting its potential applicability in pain conditions.

The genus Caulerpa Lamouroux (1809) of green seaweed of the family Caulerpaceae encompasses about one hundred species, found in tropical and subtropical waters.Algae from this genus have highly invasive capacity in the marine environment, being important contributors to the algal biomass of coral reefs and lagoons (TRI, 2009).Some metabolites with chemotaxonomic and medicinal significance have been isolated (MAO et al., 2011;RODRIGUES et al., 2012).In Brazil, some species of this genus have already been registered (RODRIGUES et al., 2012;VANDERLEI et al., 2010).Anticoagulant, antithrombotic, antitumor, antiviral and prothrombotic activities of the Caulerpa SPs have been described in the literature (GHOSH et al., 2004;HAYAKAWA et al., 2000;JI et al., 2008;RODRIGUES et al., 2011b).However, there are few studies on the biological potential of Caulerpa SPs.In addition, report in animal models of nociception and locomotion of seaweeds SPs, to the best of our knowledge, has not been associated to data.
Pain promotes an uncomfortable sensation to patients, and may be caused by tissue lesions or by an independent manner, being thus a complex interaction between peripheral and central structure from skin to central cerebral cortex.Tissue damage (due to inflammation or injury) may result in chronic neuropathic pain caused by increased sensitivity to painful stimuli (MOALEM; TRACEY, 2006).Animal models of nociception have been widely used as important tools for discovering new natural analgesic compounds (ARAÚJO et al., 2011;ASSREUY et al., 2008;COURA et al., 2012;FARIAS et al., 2011;VANDERLEI et al., 2010;VIANA et al., 2002).
The non-steroidal anti-inflammatory drugs are commonly used in the suppression of the inflammatory reaction in medical clinic, having the property to inhibit initial or latter manifestations.However, the prolonged administration of these therapeutic agents is usually followed by complications (gastric perforations, stomach ulcers and bleeding) (IWALEWA et al., 2007).
Issues about the safety of SPs are still rarely reported (CAMPO et al., 2009;LI et al., 2005;RODRIGUES et al., 2011c).Almeida-Lima et al. (2011) examined the subchronic toxicity in vivo of a heterofucan (Fucan A) from Spatoglossum schröderi (Phaeophyta) and no toxicologically significant changes were observed in the biochemical or hematological parameters in treated Wistar rats.In the present study, it was investigated the effects of a novel antinociceptive non-anticoagulant SP from C. cupressoides var.lycopodium (Chlorophyta) using experimental animal models of nociception and locomotion.The safety of this compound was also evaluated using an acute toxicity model in Swiss mice.

Experimental design
The crude SP was extracted from C. cupressoides and the antinociceptive activity of a nonanticoagulant fraction (SP 1 ) was tested in vivo with experimental animal models of nociception and locomotion using male mice.The effects on some biochemical parameters were also evaluated in mice treated for 72h using an acute toxicity model.

Animals
Male Swiss mice (19-25 g) were randomly selected from the Animal House of the Federal University of Ceará, maintained on a 12h light/dark cycle, at temperature-controlled rooms and received water and food ad libitum.For the experiments, a total of 246 animals were used.All procedures and animal treatments were conducted in accordance with the guidelines for Institutional Animal Care and Use of the Federal University of Ceará, Ceará State, Brazil, previously approved by 80/10 protocol.In addition, the tested doses and administration route of SP 1 used in the experimental models were determined as previously reported (COURA et al., 2012).

Hot-plate test
This test is also used to measure the antinociceptive activity (EDDY; LEIMBACH, 1953).Groups of six animals were used.Each mouse was dropped twice on the heated plate (51 ± 1°C), separated by a 30 min.interval.The first trial familiarized the animal with the test procedure and the second served as the control reaction time (licking the paw or jumping).Animals showing a reaction time longer than 10 s were discarded.Immediately after the second trial (control reaction time), groups of six mice each received intravenously (i.v.) 0.9% saline in a volume of 10 mL kg -1 , SP 1 (3, 9 or 27 mg kg -1 body weight), morphine (Dimorf ® , Cristália; Itapira, São Paulo State, Brazil) or indomethacin (Indocid ® , Merck Sharp and Dohme; Campinas, São Paulo State, Brazil), both 5 mg kg -1 (s.c.).Reaction times were measured at time zero (0 time) and 30, 60 and 90 min.after administration, with a cut-off time of 40 s to avoid paw lesions.

Open-field test
This test was performed according to Archer (1973).The open-field area was made of acrylic transparent walls and black floor (30 × 30 × 15 cm), divided into nine squares of equal area, to evaluate the locomotor activity of the animal for 5 min.The following parameters were observed: number of squares crossed and number of grooming and rearings.The animals were divided into five groups of 8 animals each.The groups were treated with: control saline (0.9%, i.v.), SP 1 (3, 9 or 27 mg kg -1 , i.v.) and Diazepam (2 mg kg -1 , i.p.) (União Química; Campinas, São Paulo State, Brazil).

Acute toxicity model
This assay was based on Monji et al. (2011).Body mass loss, organ weight change, and blood biochemical parameters (alanine aminotransferase (AST), aspartate aminotransferase (ALT) and urea) were evaluated after acute treatment with single doses of SP 1 (27 mg kg -1 , i.p.), with an important antinociceptive effect, or 0.9% saline (i.p.) for 72h.For the in vivo assay, the male mice (6 animals per group) were weighed before the SP 1 (dissolved in 0.9% saline) administrations.During the assay, all animals had free access to water and food.Also, the animals were daily observed considering the following aspects: body mass variation, survival rate, mucosa, eyes, hair erection, scratching or licking paws, freezing reactions, general behavior, among others.After treatment, mice were again weighed and peripheral blood was collected for biochemical dosage (determined by enzymatic and colorimetric tests-Labtest, São Paulo, São Paulo State, Brazil).After sacrificing the animal, liver, kidney, heart, spleen, thymus and lymph nodes were removed and weighed.

Statistical analyses
All results are presented as mean ± standard error of the mean (S.E.M.).Data from the animals submitted to nociception or open-field tests were analyzed by ANOVA followed by Bonferron's or Student-Newman-Keuls post hoc test.p < 0.05 or p < 0.001 was considered as significant.Student ttest was also used, considering p < 0.05 as significant, when the group of animals that received SP 1 for 72 h on acute assay was evaluated.For these analyses, the software GraphPad Prism (5.0 version) was used.

Results and discussion
The C. cupressoides SPs have been extracted and studied.This species contains three different SPs fractions (SP 1 , SP 2 , and SP 3 ), when eluted at different NaCl concentrations (0.5, 0.75 and 1 M, respectively) on DEAE-cellulose column (RODRIGUES et al., 2011b(RODRIGUES et al., , 2012)).The Table 1 shows the chemical composition of SPs fractions obtained by ion-exchange chromatography (DEAEcellulose) of the C. cupressoides crude polysaccharide extract.According to the quantitative chemical analysis, the S and TSs contents were high in all SP fractions, being the lowest data found for SP 1 , with about 16.93% S and 13.67% TSs, respectively.As also reported, CPs were not detected in the crude extract and SP fractions from this species (RODRIGUES et al., 2011b).In previous studies, it was demonstrated that only the SP 2 fraction from C. cupressoides showed an anticoagulant activity (in vitro) by the activated partial thromboplastin time test.Fractions SP 1 and SP 3 were not capable of prolonging the coagulation time in vitro (RODRIGUES et al., 2011b(RODRIGUES et al., , 2012)).Fraction SP 2 also presented antithrombotic and prothrombotic activities (in vivo) (RODRIGUES et al., 2011b).Li et al. (2005) reported that the use of anticoagulant SPs in animal studies significantly altered the clotting time, and it could be considered a adverse effect for treating patients subjected to chronic renal diseases, when administrated at high doses (900 and 2500 mg kg -1 , oral administration).Recently, a non-anticoagulant SP, but with in vivo anti-inflammatory effect from five sequential extractions, was obtained from the marine brown alga Lobophora variegata (SIQUEIRA et al., 2011).Based on these hypotheses, we chose the SP 1 (that had higher yield compared with SP 3 ) to be further investigated in nociception and locomotion animal models.
On the other hand, it has been reported the low specificity of writhing assays (COLLIER et 1968), requiring caution when interpreting the results until the performance of other tests.In order to support this hypothesis, two more complementary tests (formalin and hot-plate) were performed to prove the antinociceptive action of SP 1 .
SP 1 presented a greater antinociceptive effect in the second phase, suggesting that its action is also related to inflammatory pain (Figure 2) (ARAÚJO et al., 2011;COURA et al., 2012;VANDERLEI et al., 2010).Moreover, the late phase seems to be an inflammatory response to inflammatory pain that can be modulated by anti-inflammatory drugs, which inhibit inflammatory mediators, such as serotonin, bradykinin and prostaglandin (HUNSKAAR et al., 1985).In recent studies, an anticoagulant SP from Champia feldmannii (Rhodophyta) produced a pro-inflammatory effect (ASSREUY et al., 2008), whereas a nonanticoagulant SP from the brown alga L. variegata was an anti-inflammatory compound (SIQUEIRA et al., 2011).Araújo et al. ( 2012) reported a crude extract containing ι-carrageenans from Solieria filiformis (Rhodophyta) without anti-clotting effect, but able to inhibit the cellular infiltrate in the peritoneal cavity of rats.There is a lack of structurefunction relationship data concerning the antiinflammatory action of these molecules (POMIN, 2012).
In this study, the SP 1 (27 mg kg -1 ) also inhibited the first phase (Figure 2, panel A) compared with saline group, suggesting a possible interaction of this molecule with opioid systems.A similar result was suggested for a SP from S. schroederi (Phaeophyta) (FARIAS et al., 2011) and SPs extracted from G. cornea (Rhodophyta) (COURA et al., 2012).
Literature describes that the inflammatory hypernociception occurs at least in part as a phenomenon of sensitization of primary afferent nociceptors (MOALEM; TRACEY, 2006).In the inflammatory process, the neutrophils migration occurs in response to mechanical hypernociception, where the release of mediators is observed, such as prostaglandin (CUNHA et al., 2008).Formalininduced nociception is attributed to an increase in peritoneal fluid levels of several mediators, such as histamine, serotonin, cytokine, and eicosanoid (HUNSKAAR et al., 1985).The greater antinociceptive effect of SP 1 in the second phase (Figure 2, panel B) could be the result of drugs related to inflammatory pain (ARAÚJO et al., 2011;VANDERLEI et al., 2010), inhibiting the pain in terms of synaptic transmission at the spinal level in response to formalin-action (HUNSKAAR et al., 1985).
Based on these findings, it was suggested that the SP 1 from green seaweed C. cupressoides exhibits antinociceptive effects predominantly through a peripheral mechanism similar to the lectin (protein) isolated from this same species by Vanderlei et al. (2010).These antinociceptive actions of SP 1 seemed not to be associated with central neurotransmission (FARIAS et al., 2011).

Effect of pretreatment with SP 1 on the open-field test
Once the SP 1 (C.cupressoides) showed antinociceptive effects, it was also explored this molecule on the open-field test, a classic animal model that evaluates autonomic effects and general activity (ARCHER, 1973).Our data showed that the SP 1 did not induce changes (p > 0.05) in the locomotor activity for number of squares, grooming, and rearing of mice.Diazepam (2 mg kg -1 ) influenced (p < 0.05) the locomotor behavior of the animals (Table 2).

Toxicological evaluation
In order to investigate the safety for 'short term' use of C. cupressoides SP 1 (27 mg kg -1 , i.p., dose selected from antinociceptive assay data obtained in Figure 2), a toxicological assay was performed with male Swiss mice for 72h (Table 3).The administration with a single injection of SP 1 did not cause animal mortality as well as variation in body mass or wet weight of organs (heart, kidney, liver, thymus, and lymph nodes) compared with saline group.
During the experimental period, some physical and behavior parameters were also observed in animals, such as mucosa or eyes, hair erection, scratching or licking paws, freezing reactions and general behavior.All these aspects were considered normal (ALMEIDA-LIMA et al., 2011;ARAÚJO et al., 2011;ASSREUY et al., 2008;COURA et al., 2012;LI et al., 2005;LINS et al., 2009;MONJI et al., 2011;RODRIGUES et al., 2011c;SIQUEIRA et al., 2011).Except for the spleen that presented an increase more than 2.5-fold compared with control (p < 0.05) (Table 2), suggesting that SP 1 stimulated the immune function of mice (COURA et al., 2012).Probably, SP 1 induced a proliferation of T lymphocytes and production of specific antibodies (LINS et al., 2009;ZHANG et al., 2003).Furthermore, the histological structure of the spleen of animals treated with SP 1 was essentially preserved (data not shown), based on Coura et al. (2012).Investigating the effects of a SP fraction isolated from Porphyra haitanesis (Rhodophyta), Zhang et al. (2003) observed an in vivo antioxidant activity (reducing the risks of lipid peroxidation) and increased size of spleen and thymus in mice.Spleen is an organ where T and B cells differentiate, and mature.Lins et al. ( 2009) examined a SP from the red alga C. feldmannii as antitumor agent.The authors noted that the compound increased the relative spleen weight, induced a hyperplasia of lymphoid follicles with nest of megakaryocytes in spleens, and reversed leucopenia, supporting the hypothesis of an immunostimulant agent.In this way, our data suggest that the exact mechanism of SP 1 (C.cupressoides) related to immune function needs further investigation.
In respect to biochemical analyses, the SP 1 administrations did not cause any hepatic or renal dysfunctions (data not shown) based on Araújo et al. (2011) and Rodrigues et al. (2011c).Seaweeds have various phytochemicals (ANDRADE et al., 2010;MAO et al., 2011;MARINHO-SORIANO et al., 2006), including toxic secondary metabolites (MORALES et al., 2006).In this study, the lack of systemic toxicity in vivo of SP 1 over the considered biochemical parameters could be of pharmacological value (ALMEIDA-LIMA et al., 2011).Enzymatic extraction performed with papain digestion could also have eliminated any toxic compound in the SP 1 , being consistent with the hypothesis of Heo et al. (2005).
Seaweeds SPs have also been extensively studied as anticoagulant and antithrombotic agents.Anticoagulant activity of Caulerpa SPs is positively correlated with the sulfate content and charge density (RODRIGUES et al., 2011b(RODRIGUES et al., , 2012)); but, the position of sulfate radicals and/or the occurrence of dissulfated units in the chemical structure of these molecules have also been suggested as requisite for anticoagulant action (PEREIRA et al., 2005).However, the toxicity in vivo of these compounds has been little investigated.Li et al. (2005) extracted fucoidan from L. japonica (Phaeophyta) and evaluated the toxicological effects in Wistar rats.The results indicated no toxicity when 300 mg kg -1 body weight per day fucoidan was orally given, but with 900 and 2500 mg kg -1 body weight per day, the clotting time was significantly altered.Fucoidan with strong anticoagulant activity in vivo may have clinical importance.However, it could be considered as limited therapeutic option for the treatment of chronic renal failure.An anticoagulant SP fraction from red alga C. feldmannii was evaluated in mice by i.v administration up to 30 mg kg -1 , and no adverse effect was observed on acute toxicity after 48h (ASSREUY et al., 2008).
Although SP 1 does not have in vitro anticoagulant effect (RODRIGUES et al., 2011b(RODRIGUES et al., , 2012)), algae SPs may also exhibit in vivo actions (LI et al., 2005).Almeida-Lima et al. (2011) investigated the acute and subchronic toxicity in vivo of a heterofucan (Fucan A), and observed that it practically devoid of anticoagulant activity (in vitro) and hemorrhagic effect (in vivo), but with antithrombotic effect (in vivo) when endovenously injected in the vena cava of Wistar rats.This compound had a pharmacological potential given the absence of toxicity in vivo.
In a previous study, it was demonstrated that an anticoagulant SP fraction (SP 2 , Table 1) from C. cupressoides inhibited the thrombin activity by antithrombin (in vitro) (RODRIGUES et al., 2011b) and this interaction mechanism was different from other studied Caulerpa SPs (HAYAKAWA et al., 2000).In addition, this fraction also inhibited the venous thrombosis and produced prothrombotic activity and no hemorrhagic effect in Wistar rats (RODRIGUES et al., 2011b).The result of the present study is very important, considering that the heparin, a commercial anticoagulant SP widely used in medical clinic and recognized by its side effects, may induce discrete alterations in the enzymatic activity of aminotransferases in plasma (MAJERUS;TOLLEFSEN, 2005).
Overall, C. cupressoides features a nonanticoagulant SP fraction (SP 1 ) with antinociceptive effect which seems to be tolerable in mice systematically treated, and representing thus a novel potential source of analgesic compound to be explored in biomedical research.In addition, its analgesic action was observed to be completely dissociated from the absence of anti-clotting action (POMIN, 2012;RODRIGUES et al., 2012).New applications of C. cupressoides SPs in 'long term' doses could be of interest from oral administration of future dosage forms, registering the safety profile in repeated dose studies (MONJI et al., 2011).Additional studies are in progress by our group.

Conclusion
A non-anticoagulant sulfated polysaccharide from the green seaweed Caulerpa cupressoides var.lycopodium has interesting peripheral antinociception without important systemic alterations.
a -SPs eluted at different NaCl concentrations (DEAE-cellulose); b, c and d -Dosages of total sugars [TSs], sulfate [S] and contaminant proteins [CPs], respectively.Data are the mean value of three determinations; -not detected.

Figure 1 .
Figure 1.Effect of SP 1 on writhing response induced by acetic acid in mice.Data are expressed as mean ± S.E.M. (n = 6).*p < 0.001 compared with saline group; ∆ p < 0.001 compared with the dose of 27 mg kg -1 (ANOVA, Bonferroni test).

Table 1 .
Chemical composition of crude extract and SP fractions from Caulerpa cupressoides var.lycopodium.

Table 2 .
Open-field test with mice treated with SP 1 from Caulerpa cupressoides var.lycopodium and diazepam.

Table 3 .
Body mass and weight of organs (w w -1 ) of mice treated with SP 1 from Caulerpa cupressoides var.lycopodium after 72h.