Pharmacological Reports Copyright © 2012 2012, 64, 282292 by Institute of Pharmacology ISSN 1734-1140 Polish Academy of Sciences
Antinociceptive and anti-inflammatory activities of a sulfated polysaccharide isolated from the green seaweed Caulerpa cupressoides
José A.G. Rodrigues1, Edfranck de S.O. Vanderlei2, Luana M.C.M. Silva2, Ianna W.F. de Araújo1, Ismael N.L. de Queiroz2, Gabriela A. de Paula1, Ticiana M. Abreu 2, Natássia A. Ribeiro2, Mirna M. Bezerra3, Hellíada V. Chaves3, Vilma Lima4, Roberta J.B. Jorge4, Helena S.A. Monteiro4, Edda L. Leite5, Norma M. B. Benevides1,2
1 Northeast Biotechnology Network, Federal University of Ceará, Campus do Pici, Fortaleza, Ceará, Av. Mister Hull, CEP: 60455-760, Brazil 2 Department of Biochemistry and Molecular Biology, Bloco 907, Federal University of Ceará, Campus do Pici, Fortaleza, Ceará, Av. Mister Hull, CEP: 60455-760, Brazil 3 Faculty of Medicine, Federal University of Ceará, Av. Comte Maurocélio Rocha Pontes, no. 100, Sobral, Ceará, CEP: 62042-280, Brazil 4 Department of Dentist Clinical, Facult of Pharmacy, Nursing and Dentistry, Federal University of Ceará, Rua: Cel. Nunes de Melo, no. 1127, Fortaleza, Ceará, CEP: 60431-970, Brazil 5 Department of Biochemistry, Federal University of Rio Grande do Norte, Av. Senador Salgado Filho, Lagoa Nova, Natal, Rio Grande do Norte, CEP:59072-970, Brazil
Correspondence: Norma M. 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- tion and acute inflammation in vivo. 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 before waiting for 14 days. 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 affected by Cc-SP2 treatment, but these effects were considered reversible. Conclusion: The results indicate that the analgesic and anti-inflammatory effects of Cc-SP2 could be of biomedical applicability as a new, natural tool in pain and acute inflammatory conditions.
Key words: marine alga, Caulerpa cupressoides, sulfated polysaccharide, nociception, inflammation
282 Pharmacological Reports, 2012, 64, 282292 Pharmacological effects from Caulerpaceae sulfated polysaccharide 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 been examined [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 ity and relatively low toxicity [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- this SP was also performed. 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 Materials and Methods 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) and cyclooxygenase (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) in accordance with international guidelines. tive effects. The red algae Delesseria sanguinea was described to have SPs with the possibility of cosmetic Drugs and reagents 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 Formalin test 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 São Paulo, SP, Brazil). (phase 2, inflammatory) after formalin administration [10, 16]. The mice (n = 6 per group) were then treated SPs (iv) with Cc-SP2 (3, 9 or 27 mg/kg) or sterile saline (0.9%) 30 min before formalin injection. Morphine or C. cupressoides (Vahl) C. Agardh was classified by Ana indomethacin (both 5 mg/kg, sc) was also adminis- Cecília Fortes Xavier at the Prisco Bezerra Herbarium tered 30 min before the formalin injection and used as (Department of Biology of Federal University of Ceará, a reference compound. Brazil) as number 4977. The total sulfated polysaccha- ride (TSP) was extracted by protease digestion (60°C, Hot plate test 6 h) in 100 mM sodium acetate buffer (pH 5.0) contain- ing EDTA and cysteine (both 5 mM). TSP (30 mg) was The hot plate test also measures analgesic activity [11]. dissolved in 15 ml of 50 mM sodium acetate buffer Each mouse was placed twice onto a heated plate (pH 5.0) and applied to a DEAE-cellulose column (12 × (51 ± 1°C) separated by a 30 min interval. The first trial 1.5 cm) equilibrated with the same solution. The column familiarized the animal with the test procedure, and the was developed using a stepwise gradient of 0 to 1.5 M second trial served as the control for the reaction time NaCl at 0.25 M intervals in the same solution. The flow (licking the paw or jumping). Animals showing a reaction rate of the column was 1.0 ml/min. Fractions of 3 ml time greater than 10 s were excluded. After the second were collected and analyzed for SPs using the metachro- trial (control reaction time), groups of animals (n = 6) re- matic assay (A525 nm) containing dimethylmethylene ceived sterile saline (0.9%, 10 ml/kg, iv), Cc-SP2 (3, 9 or blue [12]. TSP was separated into three SP fractions 27 mg/kg, iv), morphine or indomethacin (both 5 mg/kg, (Cc-SP1, Cc-SP2 and Cc-SP3 with 0.5, 0.75 and 1 M of sc). To detect the involvement of the opioid system, mice NaCl, respectively), as previously described [31]. The were pretreated with naloxone (2 mg/kg, sc) 15 min be- biological protocols were performed with the fraction fore treatment with Cc-SP2 or morphine. The reaction that showed a higher yield (Cc-SP2). The charge density times were measured at time zero (0 time) and 30, 60 and and purity of the obtained fractions were also verified by 90 min after the compounds were administered, with agarose gel electrophoresis. a cut-off time of 40 s to avoid paw lesions.
Antinociceptive activity of Cc-SP2 Anti-inflammatory activity of Cc-SP2
Acetic acid-induced writhing test Peritonitis model in rats
The acetic-acid writhing test was used to evaluate an- The rats (n = 6 per group) received Cc-SP2 (3, 9 or algesic activity [7]. The mice (n = 6 per group) were 27 mg/kg, sc), dexamethasone (1 mg/kg, sc) or sterile injected (ip) with 0.6% acetic acid (10 ml/kg body weight), saline (0.9%, w/v, sc) 1 h before carrageenan (500 µg/ and the intensity of nociception was quantified by counting cav). After 4 h, the animals were sacrificed, and the the total number of writhes that occurred between 0 and peritoneal cavity was washed with 10 ml of saline 30 min after the injection. The animals received Cc-SP2 (3, containing heparin (5 IU/ml). The peritoneal fluid
284 Pharmacological Reports, 2012, 64, 282292 Pharmacological effects from Caulerpaceae sulfated polysaccharide José A.G. Rodrigues et al.
was collected, and the total leukocyte count was per- Histological and morphological analyses formed using the Neubauer chamber. To differentiate the leukocyte count, samples were stained by hema- Livers, hearts and right kidneys were fixed with for- toxylin-eosin (HE) method, and the cells were maldehyde. Next, they were dehydrated with increas- counted by microscopy. The data were expressed as ing concentrations of 70% ethanol and processed in the means ± SEM of the number of cells × 103 ml of paraffin. The resulting blocks were sliced in 5 µm peritoneal fluid [37]. thick sections, stained with HE and observed under a light microscope (400×).
Carrageenan-induced rat paw edema Statistical analysis The rats (n = 6 per group) were treated with Cc-SP2 The data are presented as the mean ± SEM of at least (3, 9 or 27 mg/kg, sc) or sterile saline (0.9%, w/v, sc) six animals per group. Variance analysis (ANOVA) 1 h before carrageenan injection. Carrageenan (500 µg, followed by a Bonferroni’s test were used to com- 0.1 ml, ipl) was injected into the right hind paw [42]. pared the means. Values of p < 0.05 were considered Dexamethasone (1 mg/kg, sc) was also administered to be statistically significant. 1 h before carrageenan as a reference compound. Paw volume was measured immediately before (zero time) the carrageenan injection and at selected time inter- vals (1, 2, 3 and 4 h) with a hydroplethysmometer (Ugo Basile, Italy). The results were expressed as Results a variation in paw volume (ml), which was calculated as the difference between the basal volume and the Acetic acid-induced writhing test volume at the selected time interval [19]. Cc-SP2 significantly inhibited the number of writhes Evaluation of systematic and biochemical in mice induced by acetic acid in a dose-dependent effects after subchronic treatment of mice with manner, with a maximal effect at the highest dose Cc-SP2 (90.6%). Furthermore, Cc-SP2 (3 or 9 mg/kg) showed significant inhibition of the nociceptive response by This subchronic toxicity model was based on Araújo 57 and 89.90%, respectively, as compared to the con- et al. [3] and Rodrigues et al. [30]. The mice (n = 6 trol group (p < 0.05). The groups treated with the ref- per group, both genders) were weighed and injected erence drugs, morphine or indomethacin (both 5 mg/ sc daily with a single dose of C. cupressoides SP kg, sc), also significantly diminished the number of (27 mg/kg) or sterile saline (0.9%) for 14 consecutive writhes (97.9 and 53.83%, respectively) (Fig. 1). days. Body mass, survival rate, mucosa, eyes, hair, erection, scratch or licking paws, freezing reactions, Formalin test and general behavior were evaluated. On the fifteenth day, the animals were reweighed A significant reduction in the licking time was ob- and anesthetized with 10% chloral hydrate (400 mg/ served during the first phase following formalin ad- kg, ip), and their peripheral blood was drawn for bio- ministration (neurogenic) by 42.47 and 52.1% with 9 chemical analysis (urea, alanine aminotransferase and 27 mg/kg of Cc-SP2, respectively (Fig. 2A). In (ALT) and aspartate aminotransferase (AST) levels). addition, the Cc-SP2 (3, 9 or 27 mg/kg) that was in- The animals were then sacrificed by cervical disloca- jected 30 min prior to the formalin administration tion, and their livers, kidneys and hearts were re- (20 µl, ipl) elicited a significant inhibition (p < 0.05) moved and weighed. The wet weight of each organ of the formalin response during the second phase (in- was expressed as 100/g of body mass and compared flammatory) (Fig. 2B) by 68.95, 82.34 and 84.61%, to the saline-injected group (control). Any ulcerative respectively. As expected, morphine (5 mg/kg, sc) lesions or hemorrhages were quantified and macro- significantly reduced the formalin response in both scopically measured [33]. The blood chemistry pa- phases. The morphine inhibitory effect varied from rameters were determined using enzymatic and color- a 93% reduction in licking time in the first phase to imetric assays. 98% in the second phase.
Pharmacological Reports, 2012, 64, 282292 285 as a positive control and also induced antinociception (29.9 ± 1.2 s), and its effect was blocked by naloxone at 30 (8.2 ± 0.7 s), 60 (9.1 ± 1.2 s) and 90 (6.9 ± 0.9 s) min (data not shown). Similarly, the antinociceptive effect of Cc-SP2 (at an effective dose of 9 mg/kg) was also blocked by naloxone at 30 (8.1 ± 1.2 s), 60 (8.6 ± 1.3 s) and 90 (10.1 ± 1.3 s) min, respectively (data not shown). Sterile saline (0.9%, iv) or indomethacin (5 mg/kg, sc) did not increase the reaction time.
Peritonitis model
Carrageenan (500 µg/cav, ip) induced significant leu-
Fig. 1. Effect of systemic administration of Cc-SP2 on the writhing re- kocyte and neutrophil migrations when injected into sponse induced by acetic acid. Thirty minutes before acetic acid in- the peritoneal cavity of rats. Cc-SP2 produced an jection, mice received saline or Cc-SP2 (3, 9 or 27 mg/kg, iv) or mor- phine or indomethacin (both 5 mg/kg, sc). Data are expressed as the anti-inflammatory effect elicited by a decrease in leu- mean ± SEM of six mice for each group. * p < 0.05 indicates a signifi- cant difference from the saline group (ANOVA, Bonferroni’s test) kocyte migration into the peritoneal cavity of these animals. Cc-SP2 (3, 9 or 27 mg/kg, sc) showed a sig- nificant inhibition of leukocyte migration into the peritoneal cavity of the animals by 46, 49 and 53%, Hot-plate test respectively (Fig. 4A). As the control, dexamethasone The treatment of mice with Cc-SP2 (3, 9 or 27 mg/kg, (1 mg/kg, sc) decreased the carrageenan-induced neu- iv) after the second hot-plate trial produced significant trophil migration by 70%. Similarly, at all doses tested, antinociceptive effects, as measured by the reaction Cc-SP2 also inhibited (p < 0.05) neutrophil migration time, during the 90 min of observation (Fig. 3). Cc-SP2 induced by the inflammatory stimuli (Fig. 4B). Further, (9 mg/kg) showed a maximal effect at 60 min (26.7 ± dexamethasone (1 mg/kg, sc) decreased the car- 1.2 s). As expected, morphine (5 mg/kg, sc) was used rageenan-induced neutrophil migration by 79%.
st Fig. 2. Effect of Cc-SP2 on the formalin test in mice. The licking time was determined during the first 5 min (1 phase, panel A) and during nd 20–25 min (2 phase, panel B) after a 1% formalin injection in mice. Cc-SP2 (3, 9 or 27 mg/kg) or saline were given iv 30 min before the forma- lin. Morphine or indomethacin (both 5 mg/kg) was administrated sc. Data are expressed as the mean ± SEM of six mice for each group. * p < & 0.05 indicates significant difference from the saline group. * Absence of significant difference among groups (ANOVA, Bonferroni’s test)
286 Pharmacological Reports, 2012, 64, 282292 Pharmacological effects from Caulerpaceae sulfated polysaccharide José A.G. Rodrigues et al.
Fig. 3. Effects of Cc-SP2, morphine or indomethacin on the reaction Fig. 5. Effect of Cc-SP2 on paw edema induced by carrageenan in times to thermal stimuli (hot-plate) induced in mice. Mice received rats. Before receiving an injection of carrageenan (Cg, 500 µg/cav, morphine or indomethacin (both 5 mg/kg, sc). Saline or Cc-SP2 (3, 9 ipl), rats received saline or Cc-SP2 (3, 9 or 27 mg/kg, sc). Dex- or 27 mg/kg) was injected iv. Data are expressed as the mean ± SEM amethasone (1 mg/kg) was injected sc. Another group received only of six mice for each group. * p < 0.05 indicates significant difference saline (sc) without Cg. Data are expressed as the mean ± SEM of six from the saline group (ANOVA, Bonferroni’s test) rats for each group. * p < 0.05 indicates significant difference from the saline group (ANOVA; Bonferroni’s test)
Carrageenan-induced rat paw edema
Carrageenan (500 µg, ipl) produced intense paw edema and reached a maximum level at 3 h (0.56 ± 0.03 ml) after administration, and then the edema decreased over the subsequent hour. Cc-SP2 (3, 9 or 27 mg/kg, sc) significantly inhibited the carrageenan- induced paw edema when it was administered 1 h be- fore the local injection of carrageenan at all time intervals, especially at the second (0.16 ± 0.02, 0.16 ± 0.03 and 0.12 ± 0.05 ml with 3, 9 or 27 mg/kg, respectively) and third (0.16 ± 0.03, 0.18 ± 0.02 and 0.14 ± 0.04 ml with 3, 9 or 27 mg/kg, respectively) hour (Fig. 5). Cc-SP2 also demonstrated a similar level of inhibition compared to dexamethasone (1 mg/kg, sc).
Evaluation of systemic effects of Cc-SP2
A single-dose of Cc-SP2 (27 mg/kg, sc) over 14 con- secutive days did not show any sign of toxicity in male and female mice. Cc-SP2 treatment did not af- fect the animals’ body mass or the wet weight of the livers, kidneys or hearts compared to the controls in- jected with sterile saline; however, the female heart was significantly altered (p < 0.05). All organs evalu- Fig. 4. Effect of Cc-SP2 on leukocyte (A) and neutrophil (B) migra- ated appeared normal with an absence of edema after tions induced in rats. Before receiving an injection of carrageenan Cc-SP2 treatment. The values obtained for urea and (Cg, 500 ìg/cav, ip), rats received saline or Cc-SP2 (3, 9 or 27 mg/kg, sc). Dexamethasone (1 mg/kg) was injected sc. Another group ALT and AST did not differ from the controls, except received only saline (sc) without Cg. Data are expressed as the mean ± SEM of six rats for each group. * p < 0.05 indicates significant dif- for urea (Tab. 1); however, the alterations were not ference from the Cg group (ANOVA, Bonferroni’s test) considered to be toxicologically significant.
Pharmacological Reports, 2012, 64, 282292 287 Tab. 1. Systemic effects of Cc-SP2 in mice. Animals were weighed and injected daily with single doses of Cc-SP2 during 14 consecutive days. After 14 days of treatment, animals were weighed, blood samples collected for biochemical analysis (AST, ALT and urea), sacrificed, and the wet weight of organs taken. Values are expressed as the mean ± SEM Student t-test for unpaired values (* p < 0.05)
Treatment (27 mg/kg, sc)
Parameters Female Male
Saline Cc-SP2 Saline Cc-SP2 Body mass (g) before 22.16 ± 0.4573 22.83 ± 0.8201 24.32 ± 0.8262 25.23 ± 0.0572 Body mass (g) after 25.69 ± 0.4741 24.59 ± 1.3147 32.12 ± 0.9747 31.88 ± 0.7321 Liver (g)/body mass 1.350 ± 0.0543 1.643 ± 0.0885 1.745 ± 0.0500 1.961 ± 0.0439 Kidney (g)/body mass 0.163 ± 0.0095 0.1801 ± 0.0050 0.2399 ± 0.0132 0.2721 ± 0.0133 Heart (g)/body mass 0.1711 ± 0.0072 0.1357 ± 0.0084* 0.1729 ± 0.0137 0.1723 ± 0.0109 Urea (mg/dL) 45.43 ± 0.0512 33.67 ± 1.8123* 62.18 ± 0.1786 47.24 ± 9.5156* AST (U/L) 43.25 ± 13.9000 44.55 ± 6.9820 48.45 ± 12.5401 40.45 ± 6.3282 ALT (U/L) 20.33 ± 1.4356 21.25 ± 2.8249 16.10 ± 1.0600 19.27 ± 2.9640
Histopathology and morphological analyses of treated mice with Cc-SP2
The histological analyses of the analyzed tissues are shown in Figure 6. Our results suggest that the cardiac tissue did not change because its muscle fibers were preserved. In the liver, subcapsular hepatocytes de- generation appeared cloudy compared to saline group. To the kidney, light tubular subcapsular degeneration was noted, when compared to saline group. All of these conditions were considered reversible.
Discussion
Because the use of commercially available analgesic and anti-inflammatory drugs (opioids and non- steroidal anti-inflammatory drugs) exerts different side effects [38], there is currently a strong interest in developing new therapeutic agents from natural prod- ucts [3, 9, 17, 21, 34, 39]. Agents that inhibit different mediators involved in the evolution of inflammatory processes are of great interest [8, 38, 43]. Researchers have suggested investigating this class of macromole- Fig. 6. Histopathological evaluation of the organs from mice after cules in animal models [2, 4, 6, 13, 23, 28, 35, 40] and subchronic treatment with Cc-SP2 (27 mg/kg, sc) for 14 days. Saline group: (A) heart, (B and C) liver and (D and E) kidney. Cc-Sp2 group: evaluating their potential as biotechnological tools in (F) heart, (G and H) liver and (I and J) kidney. Organs were recovered the development of new pharmacological drugs. and fixed with paraformaldehyde and stained with hematoxylin and eosin. Subcapsular hepatocytes with degeneration cloudy in the liver In this study, we explored a polysaccharide fraction and light tubular subcapsular degeneration in the kidney were noted from C. cupressoides (Cc-SP2) in in vivo models of (green sets). These processes are reversible. The tissue sections were observed under a microscope at 400´. Scale bar 10 µm nociception and acute inflammation. It was demon-
288 Pharmacological Reports, 2012, 64, 282292 Pharmacological effects from Caulerpaceae sulfated polysaccharide José A.G. Rodrigues et al.
strated that Cc-SP2 from the green seaweed C. cu- of the formalin response in both phases of the test pressoides produced antinociceptive and anti-inflam- (Fig. 2). In the formalin test, the early phase effect is matory effects in models of nociception in mice (ace- a direct effect on nociceptors and prostaglandins do tic acid-induced abdominal writhing, formalin test not play an important role during this phase. In con- and hot plate test) and acute inflammation in rats trast, the late phase has been shown to be an inflam- (peritonitis and paw edema tests). matory response that could be inhibited by nonsteroi- The acetic acid-induced writhing reaction has been dal anti-inflammatory drugs and corticosteroids [10, commonly used as a screening tool for the assessment 16]. Therefore, in the formalin test, our results indi- of analgesic or anti-inflammatory properties. Accord- cate that the antinociceptive effect of Cc-SP2 (3 mg/kg) ing to Collier et al. [7], acetic acid indirectly induced was related to inflammatory pain (similar to nonster- the release of endogenous mediators sensitive to non- oidal drugs) and was similar to other studies using steroidal anti-inflammatory drugs and opioids. This natural products from algae [3, 39]. Because Cc-SP2 substance also promoted an increase in peritoneal (9 and 27 mg/kg) inhibited both phases of the forma- fluid levels of prostaglandins (mediators of inflamma- lin test, the results could also be related to acute neu- tion), bradykinin, substance P and cytokines (IL-1b, rogenic pain sensitive to drugs that interact with the TNF-a, and IL-8) [25]. In this experiment, animals opioid system [32]. that were pretreated with Cc-SP2 modified the no- To distinguish between central and peripheral ciceptive response induced by acetic acid in a dose- antinociceptive actions, we examined the effects of dependent manner, suggesting that the antinociceptive Cc-SP2 using the hot plate test. This test evaluates action of Cc-SP2 could also be inhibiting the release a possible specific central action, in which the opioid of mediators in response to acetic acid. agents exert their analgesic effects via supraspinal and There have been previous studies involving algae spinal receptors [25]. SPs as analgesic agents. Of these, Assreuy et al. [4] In this study, Cc-SP2 produced significant antino- demonstrated that an SP isolated from the red sea- ciceptive effects (Fig. 3). Interestingly, Cc-SP2 (9 mg/ weed C. feldmannii exhibited antinociceptive effects. kg) showed a similar increase in the latency for jump- This effect was similar when compared to SPs iso- ing or licking when compared to morphine, but the lated from the red marine algae Bryothamnion seafor- maximal latency effect for the mice was obtained at thii and B. triquetrum reported by Viana et al. [40]. 60 min. A natural SP (k-carrageenan) (1 mg/kg) from Recently, Araújo et al. [3] isolated an SP (k-carra- S. filiformis (Rhodophyta) increased the reaction time geenan) from the red seaweed S. filiformis with (at 60 min) compared to the other doses tested (3 and antinociceptive effects. Its antinociceptive action sug- 9 mg/kg) [3]. In contrast, at 27 mg/kg, Cc-SP2 did not gests a ligand to mediator and/or direct action on the have the same antinociceptive profile. It is possible nerve terminals. that an intermediate dose (9 mg/kg) would be “more Formalin-induced nociception was another test util- effective” for its action. At the higher dose, Cc-SP2 ized in this study. It is considered the most predictive could be important for nociceptors and could result in test of acute pain because formalin causes local tissue a lack of a reaction response of the animals. These re- injury to the paw and has been previously used as a test sults suggest a role in the opioid system even though for tonic and localized inflammatory pain [10]. This the antinociceptive action of Cc-SP2 (9 mg/ kg) was test exhibits two distinct phases in the licking response. reversed with naloxone, which is an opioid antagonist The first phase is characterized by neurogenic pain [32]. Therefore, the results indicate that the antino- caused by a direct chemical stimulation of nociceptors, ciceptive action of Cc-SP2 could also occur via a cen- and the second phase is characterized by inflammatory tral-acting mechanism, similar to SPs isolated from pain triggered by a combination of stimuli, including the red marine algae B. seaforthii and B. triquetrum inflammation of the peripheral tissues and mechanisms [40]. of central sensitization. In the latter phase, different It is believed that there is an association between chemical mediators have been shown to be involved, the antinociceptive action and inflammatory pain. Silva such as excitatory amino acids, neuropeptides, prosta- et al. [34] and Vanderlei et al. [39] reported a relation- glandins, nitric oxide, and kinins [16]. ship between antinociceptive and anti-inflammatory In the present study, when injected 30 min prior to effects of lectins from seaweeds Pterocladiella capil- formalin, Cc-SP2 produced different inhibitory forms laceae and C. cupressoides, respectively. These lect-
Pharmacological Reports, 2012, 64, 282292 289 ins functioned on inflammatory pain (formalin test) Based on our findings, Cc-SP2 significantly inhib- and inhibited the inflammatory process (peritonitis ited the paw edema at all time intervals after the ad- and paw edema models). ministration of carrageenan (Fig. 5). As a result of this In order to evaluate this correlation, the anti- study, the anti-edematogenic response of Cc-SP2, in- inflammatory activity of Cc-SP2 (sc) was initially in- hibiting the entire time-course induced by carrageenan, vestigated using a peritonitis model in rats (Fig. 4). In could confirm the hypothesis that its anti-inflammatory this model, carrageenan induced neutrophil migration activity involves neutrophil migration (Fig. 4), which into the rat peritoneal cavity by indirectly activating suggests the relevant role for this polysaccharide in in- macrophages [36]. Cc-SP2 elicited a decrease in leu- terfering in the acute inflammatory response [23, 28]. kocyte migration into the peritoneal cavity of the ani- The oral administration of the crude polysaccharide mals (Fig. 4A), and it also inhibited neutrophil migra- from the brown algae T. ornata significantly reduced tion induced by the inflammatory stimuli (Fig. 4B). the paw edema in a dose-dependent manner compared These events could be related to the ability of Cc-SP2 to carrageenan-induced rats [2]. In contrast, acidic to interact closely with cell-receptors, such as L-se- SPs from the green seaweed U. rigida induced lectins. The selectin-adhesion molecules have been a greater than a two-fold increase in the expression of shown to be responsible for mediating leukocyte roll- several chemokines, stimulated macrophage secretion ing along the vascular endothelium at sites of inflam- (in vitro) of PGE2 and induced an increase in COX-2 mation, and the neutrophils participate in the cascade and NOS-2 expression [20]. A sulfated galactan iso- of events leading to mechanical hypernociception [8]. lated from the red seaweed C. feldmannii has been The possibly interaction between Cc-SP2 and endo- shown to demonstrate potent edematogenic activity thelial cells could be reveling an important role of this associated with an increased vascular-permeability molecule in the inflammatory process in vivo. Wunzhi and stimulation of neutrophil migration. These data et al. [43] discovered that the puerarin, an isoflavone show that the C. feldmannii polysaccharide activated extracted from Radix puerariae (a leguminous plant a vascular event in the inflammatory process [4]. native found at Eastern Asia), was capable of inhibit- It has also been reported to be involved in nitric oxide ing in vitro the expression of C reactive protein, iNOS synthase and in the cyclooxygenase activities of the and COX-2 mRNA via suppression of NF-kB activa- L. variegata SP anti-inflammatory effect [35]. These tion in LPS-induced RAW264.7 macrophage cells. reports demonstrate that each polysaccharide could Previous studies examining the role of SPs models exhibit different biological actions in vivo. of inflammation have focused on red and brown algae In addition, the safety of Cc-SP2 (27 mg/kg, sc) [2, 4, 35]. Preobrazhenskaya et al. [28], investigating was evaluated after 14 days in treated male and fe- a fucoidan from brown seaweed, demonstrated that it male mice. No animal mortality or toxicity was ob- inhibited leukocyte recruitment in a model of perito- served (Tab. 1). Cc-SP2 did not affect the animals’ neal inflammation in the rat and blocked the interac- body mass but caused a decrease in the females’ heart tion of P-selectin with its carbohydrate ligand. In ad- weight compared to the controls injected with sterile dition, SPs from the brown algae, Lobophora varie- saline, indicating less cardiac stress [30]. Further, gata and Fucus vesiculosos, decreased leukocyte Cc-SP2 did not cause hepatic or renal dysfunction in migration into the peritoneal cavity (55 and 70%, re- mice. No alteration in the enzymatic activity of the spectively) and ear swelling caused by croton oil [23]. transaminases in the peripheral blood from the treated An SP isolated from the brown algae Fucus vesiculosus mice was observed (Tab. 1). Previous studies demon- exerted its anti-inflammatory action in a zymosan- strated that SPs and lectins from other marine algae induced model of acute inflammation in rats [6]. species were tolerated in experimental animals [3, 4, To confirm the anti-inflammatory data in this study, 22, 30, 34, 35, 39]. Cc-SP2 was also tested in the paw edema model. The The histological analyses (Fig. 6) of the Cc-SP2- edema induced by carrageenan (500 µg, ipl) was tem- treated mice indicated cloudy degeneration in the poral and multi-mediated, involved in the participa- liver. Based on our findings, no damage to the cardiac tion of a diverse set of mediators, such as histamine, tissue was observed. However, the morphological serotonin, bradykinin, nitric oxide, and prostaglan- changes were reversible because the interstitial tissue dins, and is characterized by an intense neutrophil in- was preserved. These results were consistent with filtrate [24]. data indicating that there was no alteration in the en-
290 Pharmacological Reports, 2012, 64, 282292 Pharmacological effects from Caulerpaceae sulfated polysaccharide José A.G. Rodrigues et al.
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