Pharmacological Reports Copyright © 2012 2012, 64, 282292 by Institute of Pharmacology ISSN 1734-1140 Polish Academy of Sciences Antinociceptiveandanti-inflammatoryactivities ofasulfatedpolysaccharideisolatedfrom thegreenseaweed Caulerpacupressoides JoséA.G.Rodrigues1,EdfranckdeS.O.Vanderlei2,LuanaM.C.M.Silva2, IannaW.F.deAraújo1,IsmaelN.L.deQueiroz2,GabrielaA.dePaula1, TicianaM.Abreu 2,NatássiaA.Ribeiro2,MirnaM.Bezerra3,HellíadaV. Chaves3,VilmaLima4,RobertaJ.B.Jorge4,HelenaS.A.Monteiro4, EddaL.Leite5,NormaM.B.Benevides1,2 1 NortheastBiotechnologyNetwork,FederalUniversityofCeará,CampusdoPici,Fortaleza,Ceará,Av.MisterHull, CEP:60455-760,Brazil 2 DepartmentofBiochemistryandMolecularBiology,Bloco907,FederalUniversityofCeará,CampusdoPici, Fortaleza,Ceará,Av.MisterHull,CEP:60455-760,Brazil 3 FacultyofMedicine,FederalUniversityofCeará,Av.ComteMaurocélioRochaPontes,no.100,Sobral,Ceará, CEP:62042-280,Brazil 4 DepartmentofDentistClinical,FacultofPharmacy,NursingandDentistry,FederalUniversityofCeará,Rua:Cel. NunesdeMelo,no.1127,Fortaleza,Ceará,CEP:60431-970,Brazil 5 DepartmentofBiochemistry,FederalUniversityofRioGrandedoNorte,Av.SenadorSalgadoFilho,LagoaNova, Natal,RioGrandedoNorte,CEP:59072-970,Brazil Correspondence: NormaM.B.Benevides,e-mail: [email protected] Abstract: Background: Red and brown algae sulfated polysaccharides (SPs) have been widely investigated as antinociceptive and/or anti- inflammatory agents; however, no description of these biological properties concerning green algae SPs have been reported. Caul- erpa curpressoides (Chlorophyta) presents three SPs fractions (Cc-SP1, Cc-SP2, and Cc-SP3). Anticoagulant (in vitro) and anti- and pro-thrombotic (in vivo) effects of Cc-SP2 had been recently reported. We evaluated the effects of Cc-SP2 using models of nocicep- tionandacuteinflammation invivo. Methods: Male Swiss mice received Cc-SP2 (iv) 30 min prior to receiving 0.6% acetic acid (10 ml/kg, ip), 1% formalin (20 µl, sc) or were subjected to thermal stimuli (51 ± 1°C). Cc-SP2 was injected sc to male Wistar rats in a peritonitis model or a paw edema model using carrageenan (ip or ipl, 500 µg). To analyze the systemic effects, Cc-SP2 (27 mg/kg, sc) was administrated to both genders mice beforewaitingfor14days. Results: Cc-SP2 (3, 9 or 27 mg/kg) reduced (p < 0.05) the number of writhes induced by acetic acid by 57, 89.9 and 90.6%, respec- tively, the licking time in the first (9 or 27 mg/kg with 42.47 and 52.1%, respectively) and the second (3, 9 or 27 mg/kg with 68.95, 82.34 and 84.61%, respectively) phases. In the hot-plate test, the antinociceptive effect of Cc-SP2 (9 mg/kg) was primarily observed at 60 min (26.7 ± 1.2 s), with its effect reversed by naloxone (8.6 ± 1.3 s), suggesting the involvement of the opioid system. Cc-SP2 (3, 9 or 27 mg/kg, sc, p < 0.05) showed anti-inflammatory effects by decreasing neutrophils migration by 64, 69 and 73%, respec- tively, and potently reduced the paw edema, especially at the second (0.16 ± 0.02, 0.16 ± 0.03 and 0.12 ± 0.05 ml) and third (0.16 ± 0.03, 0.18 ± 0.02 and 0.14 ± 0.04 ml) hours, respectively. Cc-SP2 did not cause hepatic or renal alterations or affect body mass or the macroscopy of the organs examined (p > 0.05). Histopathological analyses of the liver and kidney showed that both organs were affectedbyCc-SP2treatment,buttheseeffectswereconsideredreversible. Conclusion: The results indicate that the analgesic and anti-inflammatory effects of Cc-SP2 could be of biomedical applicability as anew,naturaltoolinpainandacuteinflammatoryconditions. Keywords: marinealga, Caulerpacupressoides,sulfatedpolysaccharide,nociception,inflammation 282 Pharmacological Reports, 2012, 64, 282292 PharmacologicaleffectsfromCaulerpaceaesulfatedpolysaccharide José A.G. Rodrigues et al. Introduction and, thus, representing inhibitors of the classical path- way of complement activation. It was demonstrated by Ananthi et al. [2] that the crude polysaccharide from Pain is one of the most important symptoms of Turbinaria ornata (Phaeophyta) could be considered as inflammatory disease because it directly affects peo- a potential antioxidant and anti-inflammatory agent. ple’s daily lives and is the primary reason why Cardoso et al. [6] isolated a fucoidan from the brown patients pursue specialized treatment [38, 41]. Im- algae Fucus vesiculosus that was capable of reducing mune response intervention, with the use of anti- the cellular influx and nitric oxide levels in the articu- inflammatory drugs, is required to modulate the exag- lar cavity caused by zymosan-induced arthritis. Re- gerated and uncontrolled inflammatory responses that cently, a novel antinociceptive SP (k-carrageenan) can evocate the development of chronic and acute in- from the red seaweed Solieria filiformis was isolated flammatory diseases [21, 38]. Therefore, the discov- by Araújo et al. [3]. However, no description in the ery of new, natural, bioactive compounds with poten- models of nociception and inflammation of green sea- tial therapeutic action and minimal side effects have weeds SPs, to the best of our knowledge, has yet been beenexamined[9,17,34,35,39,43]. reported. Sulfated polysaccharides (SPs) comprise a group of Caulerpa cupressoides (Vahl) C. Agardh (Cc) is heterogeneous and complex macromolecules of non- a green alga belonging to the family Caulerpaceae and mammalian origin that are found at high concentra- is widely encountered along the coast of Brazil. From tions in the extracellular matrix and are involved in this species, Vanderlei et al. [39] isolated the lectin the ionic, mechanical and osmotic functions in marine (a protein) possessing antinociceptive and anti- algae [18, 27]. Recently, several studies have focused inflammatory activities. In addition, an anti-thrombin- on the polymers that have been isolated from different dependent SP possessing anti-thrombotic and pro- algae species due to their large biomedical applicabil- thrombotic effects has been isolated by our group ityandrelativelylowtoxicity[1,3–5,31,35]. [31]. In this study, we investigated the effects of this Although SPs have been commonly studied as anti- SP in nociception and acute inflammation using ex- coagulant and antithrombotic agents [4, 13, 23, 27], perimental animal models. A systemic evaluation of they are also capable of modulating many other bio- thisSP wasalsoperformed. logical activities. For example, for the SPs extracted from the marine red algae Bryothamnion seaforthii, Viana et al. [40] reported antinociceptive activity in mice. Qi et al. [29], investigating a highly sulfated heteropolysaccharide isolated from the marine green MaterialsandMethods algae Ulva pertusa, observed an in vitro antioxidant activity. SPs from the marine brown algae Lobophora variegata exerted anti-inflammatory effects in rats Animals [23]. In another study, Siqueira et al. [35] suggested that the anti-edematogenic action of an SP from this Male and female Swiss mice (25–30 g) and male Wis- species occurred via inhibition of nitric oxide syn- tar rats (180–220 g) were obtained from the Animal thase(NOS-2)andcyclooxygenase(COX)activities. House of the Federal University of Ceará. These ani- According to Leiro et al. [20], acid polysaccharides mals were maintained on a 12 h light/dark cycle in from the marine green algae Ulva rigida (Chloro- temperature-controlled rooms and received water and phyta) have been shown to stimulate the macrophage food ad libitum. The Institutional Animal Care and (in vitro) to secrete prostaglandins (PGE2) and induce Use Committee of the Federal University of Ceará, an increase in COX-2 and NOS-2 expression. More Fortaleza-CE, Brazil, approved all procedures and recently, Assreuy et al. [4] reported that an SP isolated animal treatments used in this study (protocol number from red algae Champia feldmannii had antinocicep- 125/07)inaccordancewithinternationalguidelines. tive effects. The red algae Delesseria sanguinea was described to have SPs with the possibility of cosmetic Drugsandreagents applications [14]. In addition, Grünewald et al. [15] discovered that SPs from D. sanguinea have anti- Glacial acetic acid (Reagen; Rio de Janeiro, RJ, Bra- complement effects, reducing the extent of hemolysis zil) and 37% formaldehyde (Sigma; St Louis, MO, Pharmacological Reports, 2012, 64, 282292 283 USA) were both diluted in saline (0.9% NaCl). Car- 9 or 27 mg/kg, iv) or sterile saline (control group, 0.9%, rageenan lambda type IV (Sigma; St Louis, MO, w/v) 30 min before acetic acid injection. Morphine or USA), chlorohydrate of morphine (Dimorf, Cristália; indomethacin (both 5 mg/kg, sc) was administered 30 min Itapira, SP, Brazil) and dexamethasone (Decadron, before acetic acid as a reference compound. Aché; Campinas, SP, Brazil) were diluted in saline so- lution. Naxolone (Sigma, St. Louis, MO, USA) and Formalintest indomethacin (Indocid, Merck Sharp & Dohme; Campinas, SP, Brazil) were dissolved in saline with This test, which produces a local tissue injury to the 5% sodium bicarbonate. When necessary, chloral hy- paw, has been used as model for tonic pain and local- drate (Vetec; Rio de Janeiro, RJ, Brazil) was used to ized inflammation pain [16]. Twenty microliters of anesthetize the animals. The enzymatic kits used for 1% formalin was administered ipl into the right hind evaluation of sulfated polysaccharide systemic toxic- paw of mice. The licking time was then recorded from ity were from LABTEST (Diagnostic Tests, 0.9%; 0 to 5 min (phase 1, neurogenic) and from 20 to 25 min SãoPaulo,SP,Brazil). (phase 2, inflammatory) after