Toxicon 46 (2005) 318327 www.elsevier.

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Rosmarinic acid, a new snake venom phospholipase A2 inhibitor from Cordia verbenacea (Boraginaceae): antiserum action potentiation and molecular interaction
Fabio K. Ticlia, Lorane I.S. Hagea, Rafael S. Cambraiab, Paulo S. Pereirab, ngelo J. Magroc, Marcos R.M. Fontesc, Rodrigo G. Stabelid, Jose R. Giglioe, A b a, Suzelei C. Franca , Andreimar M. Soares *, Suely V. Sampaioa
Departamento de Analises Clnicas, Toxicologicas e Bromatologicas, FCFRP, Universidade de Sao Paulo, USP, Ribeirao Preto-SP, Brazil b Unidade de Biotecnologia, Universidade de Ribeirao Preto, UNAERP, Ribeirao Preto-SP, Brazil c Departamento de Fsica e Biofsica, IB, Universidade Estadual Paulista, UNESP, Botucatu-SP, Brazil d Laboratorio de Bioqumica do Instituto de Pesquisas em Patologias Tropicais (IPEPATRO), FioCruz, UNIR, Porto Velho-RO, Brazil e Departamento de Bioqumica e Imunologia, FMRP, Universidade de Sao Paulo, USP, Ribeirao Preto-SP, Brazil Received 3 February 2005; revised 27 April 2005; accepted 28 April 2005 Available online 29 June 2005
a

Abstract Many plants are used in traditional medicine as active agents against various effects induced by snakebite. The methanolic extract from Cordia verbenacea (Cv) signicantly inhibited paw edema induced by Bothrops jararacussu snake venom and by its main basic phospholipase A2 homologs, namely bothropstoxins I and II (BthTXs). The active component was isolated by chromatography on Sephadex LH-20 and by RP-HPLC on a C18 column and identied as rosmarinic acid (Cv-RA). Rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid [2-O-cafeoil-3(3,4-di-hydroxy-phenyl)-R-lactic acid]. This is the rst report of RA in the species C. verbenacea (baleeira, whaler) and of its anti-inammatory and antimyotoxic properties against snake venoms and isolated toxins. RA inhibited the edema and myotoxic activity induced by the basic PLA2s BthTX-I and BthTX-II. It was, however, less efcient to inhibit the PLA2 activity of BthTX-II and, still less, the PLA2 and edema-inducing activities of the acidic isoform BthA-I-PLA2 from the same venom, showing therefore a higher inhibitory activity upon basic PLA2s. RA also inhibited most of the myotoxic and partially the edema-inducing effects of both basic PLA2s, thus reinforcing the idea of dissociation between the catalytic and pharmacological domains. The pure compound potentiated the ability of the commercial equine polyvalent antivenom in neutralizing lethal and myotoxic effects of the crude venom and of isolated PLA2s in

Abbreviations Cv-ME, Cordia verbenacea methanolic extract; Cv-RA, rosmarinic acid from Cordia verbenacea; PLA2, phospholipase A2; PLIs, phospholipase A2 inhibitors; BthTX-I, B. jararacussu bothropstoxin-I; BthTX-II, B. jararacussu bothropstoxin-II; BthA-I-PLA2, B. jararacussu acidic phospholipase A2; COSY, COrrelation SpectroscopY; HMQC, heteronuclear multiple quantum coherence; HMBC, heteronuclear multiple bond coherence; CD, circular dichroism. * Corresponding author. Tel.: C55 16 602 4714; fax: C55 16 633 1936. E-mail addresses: andreims@fcfrp.usp.br (A.M. Soares), suvilela@fcfrp.usp.br (S.V. Sampaio). 0041-0101/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2005.04.023

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experimental models. CD data presented here suggest that, after binding, no signicant conformation changes occur either in the Cv-RA or in the target PLA2. A possible model for the interaction of rosmarinic acid with Lys49-PLA2 BthTX-I is proposed. q 2005 Elsevier Ltd. All rights reserved.
Keywords: Cordia verbenacea; Rosmarinic acid; Anti-inammatory; Antimyotoxic; Antiophidian; Phospholipase A2 inhibitor; Bothrops jararacussu; Snake venom

1. Introduction Plants have often been used by humans, sometimes successfully, against numerous diseases caused by different pathological agents. Pharmacological studies have demonstrated that the extracts and fractions from some of these plants used in traditional medicine possess anti-inammatory, antiviral and antiophidian properties (Phillipson and Anderson, 1989; Martz, 1992; Mors et al., 2000). The antiophidian activity of several plant species in general use in some Brazilian communities has been investigated scientically (Mors et al., 2000; Batina et al., 2000; Borges et al., 2000, 2001; Biondo et al., 2003, 2004; Januario et al., 2004; Veronese et al., 2005; Esmeraldino and Sampaio, in press; da Silva et al., in press; Oliveira et al., 2005). Snake venoms are complex mixtures of proteins including phospholipases A2, myotoxins, hemorrhagic metalloproteases and other proteolytic enzymes, cytotoxins, cardiotoxins and others. The pathophysiology of snake envenomation involves a complex series of events that depend on the combined action of these venom components (Gutierrez, 2002). Phospholipases A2 (PLA2; EC 3.1.1.4) are abundant in snake venoms. Besides playing a digestive role in phospholipid hydrolysis, they may also exert a wide variety of pharmacological activities such as neurotoxicity, myotoxicity, edema-inducing activity and others (Gutierrez and Lomonte, 1995; Soares et al., 2004b). Local edema, a typical manifestation of Bothrops envenomation, usually in addition to pain, is due to the action of the venom upon mastocytes, kininogens and phospholipids, culminating with release of endogenous mediators (Teixeira et al., 2003). The hydroalcoholic extract from Cordia verbenacea (baleeira, whaler) has been used by Brazilian folk as cicatrizant and anti-inammatory (Sertie et al., 1988). We report now, for the rst time, the anti-inammatory and antimyotoxic activity of the extract from C. verbenacea and its active principle, rosmarinic acid, against these effects induced by Bothrops jararacussu snake venom and by its main isolated phospholipases A2. A possible model for the interaction of rosmarinic acid with Lys49-PLA2 BthTX-I is proposed. 2. Material and methods 2.1. Materials The leaves from C. verbenacea were collected during the blooming period in the Campus of the University of

Ribeirao Preto (UNAERP). A voucher specimen (No. 259) identied by specialist Prof. Dr Lin Chau Ming (Departa mento de Botanica, UNESP, Botucatu, SP, Brazil) has been preserved in the Unidade de Biotecnologia Herbarium, UNAERP. B. jararacussu venom was purchased from Sandrin Bioagents serpentarium, Batatais, SP. B. jararacussu PLA2s were isolated on Sephadex G-75 followed by cation-exchange chromatography as previously described (Andriao-Escarso et al., 2000, 2002). PLA2 homogeneity was assessed by native and SDS-PAGE and reverse-phase HPLC. 2.2. Preparation of plant extract After identication, the leaves were dried in a stove with circulating air at 40 8C. They were then grounded (375 g) and macerated with chloroform three times during three days, then with methanol, followed by ltration and evaporation of the methanol in a rotary evaporator wherefrom the dried methanolic extract (Cv-ME) was obtained. 2.3. Purication and identication of rosmarinic acid A preliminary Sephadex LH-20 column was used for the rst fractionation of Cv-ME, using 300 mL of methanol as mobile phase for elution. The resulting fractions, after drying, were analyzed by thin-layer chromatography and revealed with vanillin sulfuric acid reagent. Fraction 3 was then applied on a HPLC semipreparative Supelcosil C18 column, using a concentration gradient of methanol:water at a ow rate of 2 mL/min. Seven new fractions were so obtained, which were assayed for edema inhibition, from which rosmarinic acid (Cv-RA) was fraction 6, as identied by NMR analysis. NMR spectra were recorded with a Brucker DPX-300 spectrophotometer, operating at 300 mHz for 1H and 75 mHz for 13C. For that, 15 mg samples were used, dissolved in dimethyl-d6-sulfoxide (Aldrich). 2.4. Edema-inducing activity Edema was induced by i.d. injection, in the right foot pad of male Swiss mice (1822 g), of B. jararacussu venom (25 mg) and its puried PLA2s (50 mg). Inhibition studies were performed by incubating venom or PLA2 with Cv-ME/Cv-RA. Control groups were injected with 50 mL of phosphate-buffered saline (PBS, pH 7.2) alone, or Cv-ME/Cv-RA alone. The progression of edema was evaluated with a low pressure pachymeter (Mitutoyo,

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Japan) at various time intervals after injection (Soares et al., 2000). 2.5. Enzymatic activities PLA2 activity was determined in a gel plate containing egg yolk (6 egg yolks/L), CaCl2 (0.56 g/L) and agar (20 g/L) , according to Gutierrez et al. (1988). Crude venom (1 mg), BthTX-II (1 mg) and BthA-I-PLA2 (1 mg) were inoculated and the diameter (mm) of the resulting halos were measured after 2 h. Anticoagulant activity (Alvarado and Gutierrez, 1988) was evaluated using human plasma (200 mL), CaCl2 (0.25 mM) and BthTX-II (0.5 mg). The plasma was primarily equilibrated at 37 8C in a water bath with or without Cv-RA/Cv-ME. BthTX-II was then added and, after 10 min, the CaCl2 solution (25 mL). Plasma which did not

clot after 45 min was considered incoagulable. The control tube received PBS replacing BthTX-II, where the plasma should clot within 36 min. 2.6. Myotoxic activity Swiss male mice (1822 g) were injected intramuscularly in the right gastrocnemius muscle with solutions containing doses of 25 mg/50 mL of Bothrops venoms or toxins. The mixtures of venom or toxin/Cv-RA (1:1 and 1:10, w/w) were then evaluated. Controls received PBS or inhibitor alone. Mice were bled from the tail 3 h after injections and blood was collected into heparinized capillary tubes. Plasma creatine kinase activity was determined using the Kit 47-UV (Sigma Chemical Co.) (Soares et al., 2000). Activity was expressed in units/L, one unit corresponding to the production of one micromole of NADH per min at 30 8C.

Fig. 1. Purication of Cv-RA from Cordia verbenacea methanolic extract. (A) Fractionation on Supelcosil C18, by HPLC, of fraction F3 from the Sephadex LH-20. (B) TLC of fraction F3-CL6 (rosmarinic acid). Mobile fase:ethyl acetate:formic acid:acetic acid:water (100:11:11:26, v/v). Staining: NP/PEG, under UV. (C) Assay for purity of rosmarinic acid by HPLC-C18.

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Fig. 2. Rosmarinic acid from Cordia verbenacea methanolic extract. (A) Molecular structure of Cv-RA from resonance studies (NMR 1H and NMR 13C). (B) 3D-molecular structure of Cv-RA.

Fig. 3. Inhibition of the edema-inducing activity by rosmarinic acid. (A) Effect of Cv-ME (1:10, w/w) on the edema induced by B. jararacussu crude venom, Lys49-BthTX-I and Asp49-BthTX-II. (B) Effect of Cv-RA (1:3.5, w/w) on the edema induced by the crude venom, BthTX-I and BthTX-II. Results are expressed by the meanGSD (nZ6). Means are statistically signicantly different from the control means.

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2.7. Potentiation of anti-bothropic serum action Cv-RA was added to polyvalent antivenom (Instituto Butantan-SP, Brasil) at an ED50 (effective dose to neutralize 50% of myotoxicity as dened by levels of creatine kinase in plasma after 3 h post-injection), incubated with B. jararacussu venom or isolated PLA2s in a nal volume of 50 mL for 30 min at 37 8C and injected intramuscularly in mice as previously described (Lizano et al., 2003). 2.8. Circular dichroism of Cv-RA Far UV circular dichroism spectra (190250 nm) were measured with a JASCO 810 (JASCO, Inc., Tokyo, Japan) using 1 mm path length cuvettes and protein concentrations of 150 mg/mL for both the target myotoxic PLA2s and 450 mg/mL for Cv-RA. In the case of mixtures, the total

protein concentration was 150 mg/mL. In all cases, a total of 10 spectra were collected, averaged and corrected by subtraction of a buffer blank. 2.9. Molecular modeling The molecular model of the monomeric BthTX-I (da Silva-Giotto et al., 1998) complexed with rosmarinic acid was elaborated using the program O (Jones et al., 1990). The complex was rened and its energy was minimized using the program CNS (Brunger et al., 1998). 2.10. Statistical analysis Results are presented as the mean valueGSD obtained with the indicated number of tested animals. The statistical signicance of differences between groups was evaluated

Fig. 4. Inhibition of the PLA2 activity by rosmarinic acid. Effect of Cv-RA on the PLA2 activity induced by B. jararacussu crude venom (A), Asp49-PLA2 BthTX-II (B) and Asp49-PLA2 BthA-I-PLA2 (C) at rations 1:5, 1:10 and 1:50 (venom:inhibitor, w/w). Results are expressed by the meanGSD (nZ6). Means are statistically signicantly different (*) from the control means.

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using Students unpaired t-test. A P-value !0.05 was considered to indicate signicance.

3. Results and discussion In many countries, plant extracts have been traditionally used in the treatment of snakebite envenomations (Martz, 1992; Mors et al., 2000; Soares et al., 2004a), although only in a few cases there has been a scientic validation of such claims. Snake venoms are complex mixtures of proteins and, among these, are phospholipases A2, hemorrhagins, proteases and myotoxins that act by different mechanisms (Gutierrez, 2002). A number of PLA2s has been characterized from Bothrops venoms, some which are devoid of catalytic activity upon articial substrates due to the substitution of Lys at position 49 for Asp (Soares et al., 2004b). Fig. 1 shows the purication of Cv-RA from the C. verbenacea methanolic extract. Cv-ME represented

2.25% (8.5 g) of the dried leaves. From its fractionation on the Sephadex LH-20 column, three fractions were obtained from which fraction 3 was less heterogeneous and corresponded to 0.19% (0.7 g) of the dried leaves. Among the seven subfractions resulting from the HPLC rechromatography of fraction 3, subfraction 6 (CL-6) showed to be highly puried (HPLC-C18) and represented 0.03% (0.112 g) of the dried leaves. Spectroscopic analysis of subfraction 6 identied it as rosmarinic acid. Its chemical and tridimensional structure is shown in Fig. 2A and B, respectively Rosmarinic acid was rst isolated from Rosmarinus ofcinalis, but recently its synthetic preparation was described. RA is often described as anti-inammatory. It is a polyphenolic compound, isolated from several plants of Boraginaceae and Laminaceae families (Petersen and Simmonds, 2003). This is the rst report of rosmarinic acid in the species C. verbenacea and explains the efciency of this plant regarding anti-inammatory and antimyotoxic properties against snake venoms and isolated toxins.

Fig. 5. Inhibition of the myotoxic activity by rosmarinic acid. Effect of Cv-RA on the myotoxic activity induced by B. jararacussu crude venom (A), Lys49-PLA2 BthTX-I (B) and Asp49-PLA2 BthTX-II (C) at rations 1:1 and 1:10 (venom:inhibitor, w/w). Results are expressed by the meanGSD (nZ6). Means are statistically signicantly different (*) from the control means.

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Fig. 6. Analysis of B. jararacussu venom and isolated PLA2s, BthTX-I and BthTX-II by SDS-PAGE 12%. Before incubation with Cv-ME or Cv-RA: Lanes: 1, BthTX-II; 2, BthTX-I; 3, B. jararacussu venom; 4, Cv-ME or Cv-RA. After incubation with the Cv-ME or Cv-RA: Lanes: 1, Cv-RACBthTX-II; 2, Cv-RAC BthTX-I; and 3, Cv-RACB. jararacussu.

Edema-inducing activity is a multifactorial pharmacological activity, depending on the combined action of various toxins, suggesting that enzymatic activity is not strictly required to induce this effect. Cv-ME inhibited near 20, 60 and 10% the edema induced by B. jararacussu crude venom, BthTX-I and BthTX-II, respectively (Fig. 3A), while Cv-RA inhibited 5, 60 and 10% the edema induced by

these same samples (Fig. 3B). Cv-ME was more efcient in neutralizing the edema induced by the crude venom than Cv-RA, thus suggesting that other active principles are presented in Cv-ME other than Cv-RA. An active avonoid component, artemetin, has previously been isolated from this plant and shown to have anti-inammatory effects (Sertie et al., 1990). RA showed to be more efcient in neutralizing the PLA2 activity induced by the basic Asp49 BthTX-II (Fig. 4B) than that induced by the crude venom (Fig. 4A) and by the acidic isoform Asp49 BthA-I-PLA2 (Fig. 4C). These data suggest a more specic binding with basic PLA2s, intermediated by a probable electrostatic interaction. Biondo et al. (2003) also showed that the aqueous extract from Mandevilla velutina showed a wide inhibition spectrum of toxic, enzymatic and pharmacological activities of snake venoms and isolated toxins. However, this extract was more specic for Crotalus venom and the neurotoxic basic PLA2 when compared with Bothrops acidic PLA2. A partial dissociation between the catalytic and edemainducing domains is also likely to exist in these PLA2s, since a 60% inhibition of the edema induced by the basic Lys49 BthTX-I, enzymatically inactive, was observed, against only 10 and 50% inhibition of the edema and PLA2 activity, respectively, induced by the basic Asp49 BthTX-II. These data agree with several authors who suggest distinct domains or partial overlapping between the catalytic and other pharmacological sites (Soares and Giglio, 2003). Muscle tissue damage, myonecrosis, is a common consequence of envenomation by crotaline snakes of the genus Bothrops (Gutierrez, 2002). Muscle damaging activity of Bothrops venoms is partially caused by a group

Fig. 7. Enhancement of the antimyotoxic properties of polyvalent anti-bothropic immunoglobulin antivenom by supplementation with Cordia verbenacea rosmarinic acid (Cv-RA). Cv-RA was added to commercial polyvalent (Crotalinae) antivenom (Instituto Butantan, Brazil) at an ED50 incubated with B. jararacussu venom or isolated PLA2s for 30 min at 37 8C, and injected intramuscularly in mice. Results are expressed by the meanGSD (nZ6). Means are statistically signicantly different (*) from the control means.

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Fig. 8. Analysis of circular dichroism spectra for RA in association with BthTX-I. Spectra for the BthTX-I (open squares) alone or BthTX-I and RA (closed squares), a mixture at a 1:3 molar ratio are shown. The spectra shown are unsmoothed and corrected only by subtraction of buffer blanks as described in Section 2.

of highly basic proteins with PLA2 structure. Rosmarinic acid inhibits the myotoxic activity of both Asp49 BthTX-II and Lys49 BthTX-I phospholipases A2 from B. jararacussu (Fig. 5). Rosmarinic acid did not inhibit the myotoxic activity of the crude venom as effectively as that of the puried PLA2s. This could very well be a result of the contribution to myonecrosis made by the strong hemorrhagic toxins in the crude venom which are lacking in the puried PLA2 preparations. A series of PLA2s inhibitors

(PLIs) has been isolated from natural sources, such as marine organisms, snakes and plants (Lizano et al., 2003). Wedelolactone and 12-methoxy-4-methylvoachalotine (MMV), compounds isolated from Eclipta prostata and Tabernamontana catharinensis, respectively, effectively inhibits the myotoxic activity of the venoms of Crotalus durissus terricus, B. jararacussu, B. jararaca and Lachesis muta, as well as various isolated myotoxic PLA2s (Mors et al., 2000; Soares et al., 2004a). Although the mechanism of action of C. verbenacea methanolic extract (Cv-ME) and/or rosmarinic acid (Cv-RA) is still unknown, the nding that no visible change was detected in the electrophoretic pattern of B. jararacussu venom, BthTX-I and BthTX-II, after incubation with Cv/RA (Fig. 6), excludes proteolytic degradation as a potential mechanism. Preliminary studies on supplementation of conventional antivenom against B. jararacussu or isolated myotoxic PLA2s with the Cv-RA from C. verbenacea show that the inhibitor enhances the neutralization potential of the antivenom in mice (Fig. 7), which is often only partially effective in neutralizing myotoxicity in vivo. As far as toxicity is concerned, at least mice inoculated with B. jararacussu venom and subsequently treated by intravenous injection of the inhibitor or antivenom immunoglobulins supplemented with Cv-RA show no detectable signs of toxicity or adverse effects to the addition of inhibitor. Similarly, adjuvant effects and antiserum action

Fig. 9. Molecular model of rosmarinic acid and monomeric BthTX-I complex. Drawn with the program RIBBONS (Carson, 1997). The residues interacting with the rosmarinic acid and the BthTX-I are shown in ball-stick representation.

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potentiation by a compound Hemidesmus indicus 2-hidroxy4-methoxy-benzoic acid against Vipera russelli venom were described (Alam and Gomes, 1998). Possible secondary structural changes either in the Cv-RA or the target PLA2 following binding of the inhibitor were evaluated by circular dichroism (CD) spectroscopy (Fig. 8). The result for mixtures of the Cv-RA with Lys49-BthTX-I was described, which reveal that the CD spectra for the mixture of the two components is equal the sum of the two individual spectra of the Cv-RA and the BthTX-I alone. The experiments in which Asp49-BthTX-II substituted for the BthTX-I yielded similar results (data not shown), which suggests that no signicant secondary structure changes occurred on association of the Cv-RA with the PLA2s tested. In order to study the possible mechanism of Lys49-PLA2 BthTX-I inhibition by rosmarinic acid, a molecular model of the complex was made. The rosmarinic acid was modeled into the hydrophobic channel leading to the active site. After energy minimization, the rosmarinic acid remained in the hydrophobic channel with a hydroxyl group of one of the aromatic rings bound to His48 and the carboxyl group bound to the Lys69 (Fig. 9). This is a possible way for the rosmarinic acid to interact with a phospholipase A2 leading to its inhibition. His48 belongs to the catalytic network for class II PLA2s being a strictly conserved residue to this class of proteins. The majority of PLA2-inhibitor complexes have these molecules bound to His48 (Watanabe et al., 2005), which is seen to be essential for the inhibition process. Lys69 is conserved residue in the most part of class II PLA2s and is sited in a loop between a-helix 2 and b-wing known as pancreatic loop. While, for Asp49-PLA2s, this residue is associated with anticoagulant activities (Carredano et al., 1998), no activity is associated with this residue for Lys49-PLA2 until now. The presence of PLA2 inhibitory proteins and other compounds in plants opens the possibility to search for natural inhibitors of snake venom myotoxic effects in plants for therapeutic purposes. It is likely that other plants may also serve as sources for PLIs that could be used in the future as potent antivenom compounds.

Supplementation of antiophydian serum with natural anti-toxins such as anti-hemorrhagins and anti-PLA2s could increase the ability of serum to neutralize snake toxins. It is interesting to speculate that Cv-RA, or a derivative, may prove useful in the treatment of snakebite victims, or more importantly in the treatment of the many human diseases in which PLA2 enzymes have been implicated. In particular, the use of cell impermeable PLA2 inhibitors could be a favorable therapeutic approach in the treatment of inammatory processes.

Acknowledgements The authors gratefully acknowledge the nancial support ` by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientco e Tecnologico (CNPq). Thanks are also due to Joao J. Franco (FCFRP-USP), Adelia C.O. Cintra (FCFRPUSP), Eliandra G. Silva (TT-FAPESP) and Vanessa C. Fernandes (TT-FAPESP) for their helpful technical collaboration.

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4. Conclusions Cv-ME and Cv-RA inhibit the edema and myotoxicity induced by B. jararacussu crude venom and its main phospholipases A2 homologs, thus showing that this plant is a good tool with potential antiophidian activity. Cv-RA was much more efcient to inhibit the edema induced by the Lys49 PLA2 BthTX-I than its isoform Asp49 BthTX-II. However, it neutralized equally the myotoxicity induced by both toxins. This fact suggests the presence of distinct domains for these activities. Co-crystallization studies of this inhibitor with Lys49 PLA2s are in progress for a better insight into the mechanism of action of these enzyme and/or inhibitor.

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