Mar. Drugs 2013, 11, 2722-2740; doi:10.3390/md11082722 OPEN ACCESS marine drugs ISSN 1660-3397 www.mdpi.com/journal/marinedrugs Article Evaluation of Anti-Nociceptive and Anti-Inflammatory Activities of a Heterofucan from Dictyota menstrualis Ivan Rui Lopes Albuquerque 1,2, Sara Lima Cordeiro 1, Dayanne Lopes Gomes 1, Juliana Luporini Dreyfuss 3, Luciana Guimarães Alves Filgueira 1, Edda Lisboa Leite 1, Helena Bonciani Nader 3 and Hugo Alexandre Oliveira Rocha 1,2,* 1 Laboratory of Biotechnology of Natural Polymers (BIOPOL), Department of Biochemistry, Federal University of Rio Grande do Norte (UFRN), Natal-RN 59078-970, Brazil; E-Mails: [email protected] (I.R.L.A.); [email protected] (S.L.C.); [email protected] (D.L.G.); [email protected] (L.G.A.F.); [email protected] (E.L.L.) 2 Graduate Program in Health Sciences, Federal University of Rio Grande do Norte (UFRN), Natal-RN 59078-970, Brazil 3 Department of Biochemistry, Federal University of São Paulo (UNIFESP), São Paulo-SP 04044-020, Brazil; E-Mails: [email protected] (J.L.D.); [email protected] (H.B.N.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +55-84-32153416 (ext. 207); Fax: +55-84-32119208. Received: 3 May 2013; in revised form: 4 June 2013 / Accepted: 17 June 2013 / Published: 2 August 2013 Abstract: Fucan is a term that defines a family of homo- and hetero-polysaccharides containing sulfated L-fucose in its structure. In this work, a heterofucan (F2.0v) from the seaweed, Dictyota menstrualis, was evaluated as an antinociceptive and anti-inflammatory agent. F2.0v (20.0 mg/kg) inhibits 100% of leukocyte migration into the peritoneal cavity after chemical stimulation. However, F2.0v does not alter the expression of interleukin-1 beta (IL-1β) and interleukin-6 (IL-6), as well as tumor necrosis factor alpha (TNF-α). F2.0v (20.0 mg/kg) has peripheral antinociceptive activity with potency similar to dipyrone. On the other hand, it had no effect on pain response on the hot plate test. Confocal microscopy analysis and flow cytometry showed that F2.0v binds to the surface of leucocytes, which leads us to suggest that the mechanism of action of anti-inflammatory and antinociceptive F2.0v is related to its ability to inhibit the migration of leukocytes to the site of tissue injury. In summary, the data show that F2.0v compound has great potential as an antinociceptive Mar. Drugs 2013, 11 2723 and anti-inflammatory, and future studies will be performed to further characterize the mechanism of action of F2.0v. Keywords: fucan; brown seaweed; pain; analgesic; dictyotales 1. Introduction The main function of the sensory system is to keep the brain informed of external conditions, the environment and internal conditions of the body. Among the sensations of the body, pain can be classified as one of the most important for the maintenance of homeostasis. The feeling of pain serves as an alarm, which leads, in most cases, to an affected individual having an avoidance behavior, especially when it is sharp and intense, which reduces the damage to the attacked area of the body [1]. The term, nociception, being related to the recognition of pain signals through the nervous system, is complexity and is a result of evolutionary pressure, experienced by species, to prevent damage to organisms. The process of nociception and pain involves diverse mechanisms, and it is obvious that a single drug will not be able to relieve the pain completely. An effective plan includes different classes of analgesic drugs acting at different points of the pathophysiological mechanisms that involve pain. Although the pain has its protective effect, the persistent soreness can lead to negative behaviors, such as depression and irritability, causing social and economic problems [1]. Although there are several drugs currently being used as modulators of the nociceptive system, there is always a search for new more potent and safe drugs, which may be used in specific situations. Furthermore, in this search, a tool that has performed with great value for the identification of compounds is anti-nociceptive animal models [1]. Natural products are sources of diverse bioactive molecules. In this context, one can mention the seaweeds. Furthermore, among the bioactive compounds synthesized in greater amounts by seaweeds, the sulfated polysaccharides stand out. They are located in the mucilaginous matrix of seaweeds, and their biological function is related to the solar protection against dehydration during periods of low tide; they provide greater flexibility to the seaweeds, like its growth in the aquatic environment and sufficient rigidity to remains extended and, thus, capturing light and nutrients more effectively [2]. Among the sulfated polysaccharides from seaweed, those that stand out, because they have pharmacological activity, are fucans. They are a family of homo- and hetero-polysaccharides having in its constitution monomer sulfated L-fucose [3]. Recent studies have demonstrated that fucans presented various pharmacological activities, such as: anticoagulant [4], antioxidant [5], immune modulator [6], anti-viral [7,8], anti-inflammatory [9] and anti-tumoral [10]. Spatoglossum schröederi is a seaweed from the Dictyotaceae order and synthesizes three different types of sulfated fucans, which were named fucans A, B and C, according to their electrophoretic mobility in agarose gel in 1,3-diaminopropane acetate buffer [11]. These fucans exhibit antiproliferative [12], anti-adhesive [13] and antithrombotic activities [14]. Recently, Farias and colleagues [15] demonstrated that one of the fucans from S. schröederi presents anti-nociceptive activity. At best, we know this is a unique study that analyzed the anti-nociceptive activity of fucans. Dictyota menstrualis is a different seaweed from the Dictyotaceae order; it is found in almost all of the Mar. Drugs 2013, 11 2724 Brazilian coast (about 8000 km), as well as in the Caribbean and Mexico. This, added to its constant presence and biomass production, makes this organism an excellent choice for prospecting bioactive compounds. Studies with fucans-rich extract from this seaweed showed that it has antioxidant activity in vitro and cytotoxicity against tumor cells (HeLa) [16]. In a previous work, we extracted and purified heterofucans from D. menstrualis [17]. Those that were obtained in larger quantities were named fucans F1.0v, F1.5v and F2.0v. The fucans, F1.0v and F1.5v, have presented anticoagulant activity. However, the fucan known as F2.0v showed no anticoagulant activity, which allows the use of this fucan for other new applications, since the anticoagulant effect could be an unwanted side effect. Thus, this study was aimed at evaluating the potential anti-inflammatory and antinociceptive activities of fucan F2.0v from the seaweed, Dictyota menstrualis. 2. Results and Discussion 2.1. Obtaining Fucan F2.0v from D. menstrualis The polysaccharides of D. menstrualis were extracted by proteolysis at 60 °C followed by acetone fractionation and ion exchange chromatography. The fucan, F2.0v, was precipitated with two volumes of acetone, as described in the Methods. Thereafter, it was subjected to ion exchange chromatography on diethylaminoethanol cellulose (DEAE-cellulose) and eluted with increasing concentrations of NaCl (0.3–4.0 M). The elution profile was monitored by measurement of total sugars [18] and fucose [19]. Only using 3.0 M of NaCl was a peak of sugar observed. This material was dialyzed, lyophilized and subjected to gel filtration chromatography on a Sephadex G-75. The elution profile can be seen in Figure 1A; again, only one peak was identified. This fraction was dialyzed, dried, suspended in distilled water and subjected to agarose gel electrophoresis (see Methods). As can be seen in Figure 1B, fucan 2.0v is shown as a single band with electrophoretic mobility similar to the seaweed fucan C (Fuc C), from Spatoglossum schröederi, which confirms the homogeneity of F2.0v and its identity as fucan C. The amount of F2.0v obtained after purification steps correspond to 0.1% of seaweed dry weight. Chemical analysis showed that F2.0v is composed by fucose:xylose:galactose:sulfate in the ratio of 1:0.4:1.5:1.3, respectively; traces of glucuronic acid have also been identified. The presence of protein in the sample was observed. This composition is very similar to Fuc C from S. schröederi [20], and in both cases, the two fucans are sulfated galactofucans. Such polymers are not as common; however, there is a description of galactofucans in seaweeds from the Laminariales order (Undaria pinnatifida [7], Saccharina longicruris [21]), the Ectocarpales order (Adenocystis utricularis [8]), the Dictyotales order [13,20] and the Fucales order (Sargassum horneri [22]). Furthermore, antithrombotic [18] and anti-viral [7] activities have been described for these heterofucans. Mar. Drugs 2013, 11 2725 Figure 1. (A) Elution profile of F2.0v in gel filtration chromatography (A). Gel filtration chromatography fractions of a 1 mL/tube were collected and checked by the method of phenol/sulfuric [18], as well as by the presence of fucose [19]; (B) F2.0v representative agarose gel electrophoresis in 1,3-diaminopropane acetate buffer, stained with toluidine blue. (A) (B) 2.2. Infrared Analysis of F2.0v The FTIR analysis of F2.0v is shown in Figure 2. Characteristic sulfate absorptions were identified in the FTIR spectra of compounds: Bands around 1256 cm−1 for asymmetric S=O stretching vibration −1 and bands around 1075 cm for symmetric C–O vibration associated with a C–O–SO3 group. The peaks at 810–850 were caused by the bending vibration of C–O–S [23]. At 3000–3400 cm−1, Fuc C showed bands from the stretching vibration of O–H and C–H, respectively [24], at 2932 cm−1 and Fuc C showed stretching vibrations of CH2 [25].