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Print version ISSN 0102-695X
Rev. bras. farmacogn. vol.22 no.4 Curitiba July/Aug. 2012 Epub Apr 17, 2012
Thaisa Francielle Souza DomingosI,II; Fredy A. Ortiz-RamírezI; Roberto C. VillaçaI; Diana N. CavalcantiI; Eladio F. SanchezIII; Valéria L. TeixeiraI; André Lopes FulyII,*
IPrograma de Pós-graduação em Biologia Marinha, Instituto de Biologia, Universidade Federal Fluminense, Brazil
IIDepartamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Brazil
IIIFundação Ezequiel Dias, Centro de Pesquisa e Desenvolvimento, Minas Gerais, Brazil
The ability of crude extracts of the brown seaweed Spatoglossum schröederi to counteract some of the biological activities of Lachesis muta snake venom was evaluated. In vitro assays showed that only the extract of S. schröederi prepared in ethyl acetate was able to inhibit the clotting of fibrinogen induced by L. muta venom. On the other hand, all extracts were able to inhibit partially the hemolysis caused by venom and those prepared in dichloromethane or ethyl acetate fully neutralized the proteolysis and hemorrhage produced by the venom. Moreover, the dichloromethane or ethyl acetate extracts inhibited the hemolysis induced by an isolated phospholipase A2 from L. muta venom, called LM-PLA2-I. In contrast, the hexane extract failed to protect mice from hemorrhage or to inhibit proteolysis and clotting. These results show that the polarity of the solvent used to prepare the extracts of S. schröederi algae influenced the potency of the inhibitory effect of the biological activities induced by L. muta venom. Thus, the seaweed S. schröederi may be a promising source of natural inhibitors of the enzymes involved in biological activities of L. muta venom.
Keywords: Spatoglossum schröederi seaweed Lachesis muta snake venom bioprospecting
Marine organisms represent a valuable source of new compounds. The biodiversity of the marine environment, as well as its chemical diversity, constitute an unlimited source of substances that may lead to the development of bioactive products (Aneiros & Garateix, 2004). Through their metabolism, marine organisms produce molecules, called secondary metabolites, with ecological functions such as defense against predators, competitors and herbivores and for prevention of biofouling (Clavico et al., 2006; Teixeira, 2009). Moreover, some pharmacological properties have been reported for such metabolites, including antimicrobial (González et al., 2001), antiviral (Cirne-Santos et al., 2008), anticancer (Rocha et al., 2007), antimalarial and antituberculosis (Jongaramruong & Kongkam, 2007), antilonomic (Domingos et al., 2009) and antiophidic (Moura et al., 2010) activities. The brown algae (Phaeophyceae) are a class of almost exclusively marine organisms that have been extensively explored because of their biological potential, especially those of the family Dictyotaceae. The algae that belong to this family are the most studied and they produce a large amount of biologically-active substances, including terpenoids of different origins (De Paula et al., 2011). More than 300 diterpenes have been isolated from at least 35 species collected all over the world (Vallim et al., 2005). The genus Spatoglossum Kützing has twenty species but, despite this, only the diterpenoids spatane and secospatane were isolated from S. howellii Setchell & N.L. Gardner and S. schmittii W. R. Taylor, respectively (Gerwick & Fenical, 1983; Gerwick et al., 1983, De Paula et al., 2011).
Snakebite envenomation represents a serious health problem in tropical countries because of the high incidence and the sequels derived from accidents. Snake venoms are a complex mixture that induce a wide range of biological activities. Envenomation by the snake Lachesis muta (Linnaeus, 1766) results in systemic (hemostatic, renal, hemorrhage and neurotoxic disturbances) and local (edema, necrosis, inflammation, hemorrhage) signs and symptoms (Jorge et al., 1997; Fuly et al., 2002), which leads to high lethality and morbidity indexes (Ministério da Saúde, 2001).
The conventional treatment for snakebites is the parenteral administration of serum (antivenom), obtained from hyperimmunized equines, that is able to neutralize the toxic systemic effects. However, antivenom has several disadvantages such as: poor availability in distant regions, the potential for inducing allergic reactions and the lack of efficient neutralization of the local tissue damage induced by venoms (da Silva, 2007; Gutiérrez et al., 2009). Therefore, the search for venom inhibitors from either natural or synthetic sources that are able to act in concert with antivenom is relevant.
The present work reports the ability of extracts from the brown seaweed Spatoglossum schröederi (C. Agardh) Kützing to counteract the in vivo and in vitro activities of L. muta snake venom. Moreover, the inhibitory effect of seaweed was also evaluated against a phospholipase A2 enzyme (denoted LM-PLA2-I) previously isolated from this venom (Fuly et al., 1997).
Materials and methods
Specimens of S. schröederi (Dictyotaceae, Phaeophyceae) were collected and identified by Dr. Roberto Villaça through snorkeling at depths ranging from 0 to 5 m at Atol das Rocas reef, Rio Grande do Norte State (03°51'03''S and 33°40'29''W), Brazil, in July, 2009. Atol das Rocas reef is a marine biological reserve in northeast Brazil and is the only atoll in the South Atlantic. Seaweeds were washed with local sea water and separated from sediments, epiphytes or other associated organisms. The specimens are deposited in the herbarium of Dr. Roberto Villaça at the Department of Marine Biology, Universidade Federal Fluminense (UFF).
Preparation of crude extract
The air-dried algal material (109.3 g) was triturated and the extracts were obtained through an exhaustive and sequential extraction with solvents of increasing polarity: n-hexane, dichloromethane and ethyl acetate. For each extraction, the solvent was evaporated under reduce pressure and the chromatographic profiles of the extracts were observed on TLC plates (SiO2 60, F254) manufactured by Merck. The TLC plate was eluted with ethyl acetate: dichloromethane (6:4, v/v) as the solvent system. The eluted plate was revealed by spraying with ceric sulfate, followed by heating at 120 °C.
Snake venom and animals
Lyophilized L. muta snake venom was provided by the Fundação Ezequiel Dias, Belo Horizonte-MG, Brazil, and LM-PLA2-I was isolated according to Fuly and collaborators (1997; 2002). Balb/c mice (18-20 g) were obtained from the Núcleo de Animais de Laboratório of the UFF. They were housed under controlled conditions of temperature (24±1 oC) and light and all of the experiments performed were approved by the UFF Institutional Committee for Ethics in Animal Experimentation (CEPA: 200/10) and were in accordance with the guidelines of the Brazilian Committee for Animal Experimentation.
The degree of hemolysis induced by L. muta venom or LM-PLA2-I was determined by the indirect hemolytic test using human erythrocytes and hen's egg yolk emulsion as substrate (Fuly et al., 2002). The amount of L. muta venom (µg/mL) that produced 80% hemolysis was denoted as the Minimum Indirect Hemolytic Dose (MIHD). Inhibitory experiments were performed by incubating S. schröederi extracts with one MIHD for 30 min at room temperature and then the hemolytic activity was evaluated. Control experiments were performed by incubating venom with DMSO or saline instead of S. schröederi extracts.
The clotting activity of L. muta venom was determined using an Amelung Model KC4A coagulometer (Labcon, Germany). Different concentrations of L. muta venom were mixed with bovine fibrinogen solution (2 mg/mL) or with normal citrated human plasma (donated by the blood bank of Hospital Universitário Antônio Pedro, UFF). The amount of venom that clotted fibrinogen or plasma in 60 s was denoted as the Minimum Coagulant Dose (MCD). To evaluate the inhibitory effect, S. schröederi extracts were incubated for 30 min at room temperature with one MCD of venom. The mixture was then added to fibrinogen or plasma and the clotting time recorded. Control experiments were performed by incubating venom with DMSO or saline instead of S. schröederi extracts.
Hydrolytic activity upon chromogenic substrate
The hydrolysis of the chromogenic synthetic substrate S-2238 (used for monitoring thrombin-like enzymes) by L. muta venom was measured in a Thermo-max Microplate reader (Molecular Devices, Menlo Park, CA, USA). L. muta venom was incubated for 30 min at room temperature with increasing concentrations of S. schröederi extracts. Then, reaction was triggered by adding S-2238 (0.1 mM, final concentration) and the reaction was monitored at 405 nm at 37 °C. The reading obtained after 20 min of reaction in the absence of extract was taken as 100% activity and compared with the values in the presence of the extracts. Control experiments were performed by incubating L. muta venom with DMSO (1% v/v, ﬁnal concentration) or saline.
Hemorrhagic lesions produced by L. muta venom were quantified by using a procedure described by Kondo et al. (1960) with minor modifications (Moura et al., 2010). Briefly, samples were injected intradermally (i.d.) into the abdominal skin of mice. Two hours later, the animals were euthanized and the abdominal skin removed, stretched and inspected for visual changes in the internal aspect in order to localize hemorrhagic spots. Hemorrhage was quantified as the Minimum Hemorrhagic Dose (MHD), defined as the amount of venom (µg/g) able to produce a hemorrhagic halo of 10 mm (Nikai et al., 1984). The inhibitory effect of S. schröederi extracts was investigated by incubating extracts with one MHD for 30 min at room temperature and the mixture was then injected into mice and hemorrhage measured. Hemorrhagic activity was expressed as the mean diameter (in millimeters) of the hemorrhagic halo induced by L. muta venom in the presence or in the absence of S. schröederi extracts. Negative control experiments were performed by injecting DMSO or saline.
The proteolytic activity of L. muta venom was determined using azocasein as substrate (0.2% w/v, in 20 mM Tris-HCl, 8 mM CaCl2, pH 8.8), with minor modification (Garcia et al., 1978; Moura et al., 2010). An effective concentration (EC) was defined as the amount of L. muta venom (µg/mL) able to produce a variation of about 0.2 OD units at 420 nm. S. schröederi extract or DMSO alone were incubated with two EC of venom for 30 min at room temperature and then proteolysis was measured.
Results are expressed as the mean±SEM obtained with the indicated number of animals or experiments performed. The statistical significance of differences among experimental groups was evaluated using the Student's t test and p values of ≤0.05 were considered statistically significant.
Results and Discussion
Bioprospection studies with marine organisms for the identification of active molecules are growing and different types of substances with high biotechnological and pharmacological potential have been isolated from them (Pereira et al., 2005; Mayer et al., 2011; Cirne-Santos et al., 2008; Abrantes et al., 2010; Bianco et al., 2009a, b). Previous studies showed the ability of dolastane and secodolastane diterpenes isolated from C. cervicornis marine brown alga to inhibit hemolytic, proteolytic, hemorrhagic and clotting activities of L. muta venom (Moura et al., 2010; Domingos et al., 2011). We have now evaluated the ability of S. schröederi algal extracts prepared with solvents of increasing polarities (n-Hexane, dichloromethane, ethyl acetate) to neutralize the hemolytic, proteolytic, hemorrhagic and clotting activities of crude L. muta venom.
The S. schröederi extracts (90-900 µg/mL) inhibited the hemolysis induced by L. muta venom (3.6 µg/mL) or LM-PLA2-I (9.0 µg/mL) in a concentration-dependent manner, but with potencies that varied according to the extraction solvent (Table 1). The extracts of alga prepared in dichloromethane at a 1:25 venom:alga ratio (w/w) inhibited 8.5% and 100% of the hemolysis induced by L. muta venom or LM-PLA2-I, respectively (Table 1). A 100% inhibition of hemolysis was also observed for extracts of alga prepared in ethyl acetate, but not for the hexane ones. When compared to LM-PLA2-I, extracts of alga inhibited the hemolysis induced by L. muta venom less well (Table 1). Phospholipase A2 (PLA2) enzymes are considered to be the most active pharmacological component in snake venoms (De Paula et al., 2009). PLA2 enzymes are involved in prey digestion and also produce a wide range of pharmacological and toxic effects such as hemolysis, neurotoxicity, cardiotoxicity, effects on platelet aggregation, myotoxicity, and edema, that often contribute to the envenomation symptoms (Gutiérrez & Ownby, 2003; Otero et al., 2000). L. muta venom contains PLA2 enzyme isoforms (Damico et al., 2008), one of which, denoted LM-PLA2-I, has been isolated (Fuly et al., 2002). As shown in Table 1, at a 1:25 venom:alga ratio, the ethyl acetate extract inhibited 30% and 60% of the hemolysis induced by L. muta venom or LM-PLA2-I, respectively. Curiously, at higher ratios (1:50 or 1:100), no inhibitory effect was achieved for L. muta venom. In contrast, at such ratios, the hemolysis induced by LM-PLA2-I was totally inhibited (Table 1). At higher extract concentrations, less inhibition was observed for L. muta venom. At any concentration, S. schröederi extracts or DMSO did not induce hemolysis and DMSO did not interfere with the degree of hemolysis of L. muta venom or LM-PLA2-I (data not shown).
As shown in Figure 1A, proteolysis caused by L. muta venom (0.75 µg/mL) was inhibited by different concentrations of S. schröederi extracts (18.75, 37.5 and 75 µg/mL). The dichloromethane and ethyl acetate extracts presented the highest inhibitory effect, from 90 to 100% at the three venom:alga ratios (1:25, 1:50 and 1:100). The n-hexane extract inhibited proteolysis from 10 to 20% (Figure 1A, group 1). In vivo assays showed that intradermal injection of one MHD of L. muta venom (1.2 µg/g) produced a hemorrhage halo of about 10 mm in mice (data not shown). Figure 1B shows that only the extract prepared in ethyl acetate fully protected mice from hemorrhage caused by L. muta venom, while the n-hexane and dichloromethane extracts inhibited 4% and 40%, respectively (Figure 1B). The animals that received saline or S. schröederi extracts showed no hemorrhagic halo (data not shown). We suggest that the inhibitory mechanism of action of S. schröederi extracts on hemorrhagic activity could occur through an interaction between compounds present in the alga and the catalytic sites of the metalloproteases of venom or, alternatively, such algal compounds might chelate metal ions (Zn2+) that are essential for the enzymatic activity of the metalloproteases. Envenomation by L. muta venom usually produces hemorrhage due to the degradation of blood vessels or the consumption of fibrinogen or other blood clotting factors, thus preventing clot formation (Markland, 1998). Moreover, L. muta venom is a procoagulant. Hemorrhage or clotting effects are associated with specific protease groups, the metalloprotease and serine protease. A large number of bioactive molecules with anticoagulant activity have been described in marine organisms (Jurd et al., 1995; Lee et al., 1998; Mayer et al., 2011). These molecules are regularly produced via their primary or secondary metabolism, leading to the formation of polysaccharides (Camara et al., 2011) or diterpenes (Moura et al., 2010, 2011), respectively. Rocha et al. (2005) isolated polysaccharides from S. schröederi with potent in vivo antithrombotic effect upon venous thrombosis.
As observed in Figure 2A, even at the highest concentration (62 µg/mL) the three extracts of S. schröederi did not inhibit plasma clotting induced by L. muta venom (0.62 µg/mL), but at the same concentration the ethyl acetate extract inhibited clotting of commercial fibrinogen (Figure 2B). Hence, we infer that S. schröederi compounds somehow interfere with the serine protease enzymes of venom. DMSO did not affect the clotting induced by L. muta venom (Figure 2A, B). Thrombin, a serine protease enzyme, is a pivotal enzyme in the human clotting system. It is responsible for generating thrombus through the cleavage of fibrinogen, leading to formation of a fibrin net. Thrombin also induces platelet aggregation and may active other blood clotting factors. These effects are mediated by two distinct sites, a catalytic and a pharmacological one. To test the catalytic activity of thrombin or thrombin-like enzymes, a chromogenic substrate, S-2238, is often used. We therefore evaluated the ability of S. schröederi extracts to inhibit the hydrolysis of S-2238. Figure 3 shows that all extracts prevented hydrolysis by L. muta venom, suggesting that the anticlotting effect of S. schröederi extracts upon fibrinogen might be associated with thrombin-like enzymes present in L. muta venom. Again, the ethyl acetate extract proved to be the most powerful, since at 1:50 or 1:100 venom:alga ratios a 100%-inhibition of the hydrolysis of S-2238 (Figure 3) or fibrinogen clotting (Figure 2) was seen.
Based on our results, the polarity of the solvents (n-hexane, dichloromethane and ethyl acetate) influenced on the inhibition profile for the biological activities evaluated in this work. The greater the polarity of the extraction solvent, the higher was the inhibitory percentage. This could be clearly observed since the dichloromethane and ethyl acetate extracts neutralized more efficiently all of the biological activities tested. As shown in Figure 4, extracts of S. schröederi subjected to TLC revealed heterogeneity in each extract composition. The lanes corresponding to the dichloromethane and ethyl acetate extracts showed the presence of more polar compounds than that of the hexane extract. Such differences in extract composition could explain the different inhibitory profiles of the extracts; and suggest that polar molecules are responsible for the observed inhibitory effects on L. muta biological activities.
In conclusion, S. schröederi extracts may be a promising source of molecules to improve the treatment against L. muta snakebites and may be useful for the development of new antiophidian molecules.
This work was supported by grants from the International Foundation for Science (IFS grant F/4571-1) and from Brazilian funding agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro Carlos Chagas Filho, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior and Universidade Federal Fluminense/Pró-reitoria de Pesquisa e Pós-graduação e Inovação.
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André Lopes Fuly
Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, 24020-150 Niterói, RJ, Brazil
Tel. +55 21 2629 2294
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