Hyperalgesic and edematogenic effects of peptides isolated from the venoms of honeybee (Apis mellifera) and neotropical social wasps (Polybia paulista and Protonectarina sylveirae)

Stings by bees and wasps, including Brazilian species, are a severe public health problem. The local reactions observed after the envenoming includes typical inflammatory response and pain. Several studies have been performed to identify the substances, including peptides that are responsible for such phenomena. The aim of the present study is to characterize the possible nociceptive (hyperalgesic) and edematogenic effects of some peptides isolated from the venoms of the honeybee (Apis mellifera) and the social wasps Polybia paulista and Protonectarina sylveirae, in addition to characterize some of the mechanisms involved in these phenomena. For this purpose, different doses of the peptides mellitin (Apis mellifera), Polybia-MP-I, N-2-Polybia-MP-I (Polybia paulista), Protonectarina-MP-NH2 and Protonectarina-MP-OH (Protonectarina sylveirae) were injected into the hind paw of mice. Hyperalgesia and edema were determined after peptide application, by using an electronic von Frey apparatus and a paquimeter. Carrageenin and saline were used as controls. Results showed that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH₂ and Protonectarina-MP-OH peptides produced a dose- and time-related hyperalgesic and edematogenic responses. Both phenomena are detected 2 h after melittin, Polybia-MP-I, N-2-Polybia-MP-I injection; their effects lasted until 8 h. In order to evaluate the role of prostanoids and the involvement of lipidic mediators in hyperalgesia induced by the peptides, indomethacin and zileuton were used. Results showed that zileuton blocked peptide-induced hyperalgesia and induced a decrease of the edematogenic response. On the other hand, indomethacin did not interfere with these phenomena. These results indicate that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH₂, and Protonectarina-MP-OH peptides could contribute to inflammation and pain induced by insect venoms.     

Characterization of two peptides isolated from the venom of social wasp Chartergellus communis (Hymenoptera: Vespidae): Influence of multiple alanine residues and C-terminal amidation on biological effects

Chatergellus communis is a wasp species endemic to the neotropical region and its venom constituents have never been described. In this study, two peptides from C. communis venom, denominated Communis and Communis-AAAA, were chemically and biologically characterized. In respect to the chemical characterization, the following amino acid sequences and molecular masses were identified:Communis: Ile-Asn-Trp-Lys-Ala-Ile-Leu-Gly-Lys-Ile-Gly-Lys-COOH (1340.9 Da)Communis-AAAA: Ile-Asn-Trp-Lys-Ala-Ile-Leu-Gly-Lys-Ile-Gly-Lys-Ala-Ala-Ala-Ala-Val-Xle-NH₂ (1836.3 Da).Furthermore, their biological effects were compared, accounting for the differences in structural characteristics between the two peptides. To this end, three biological assays were performed in order to evaluate the hyperalgesic, edematogenic and hemolytic effects of these molecules. Communis-AAAA, unlike Communis, showed a potent hemolytic activity with EC₅₀ = 142.6 μM. Moreover, the highest dose of Communis-AAAA (2nmol/animal) induced hyperalgesia in mice. On the other hand, Communis (10nmol/animal) was able to induce edema but did not present hemolytic or hyperalgesic activity. Although both peptides have similarities in linear structures, we demonstrated the distinct biological effects of Communis and Communis-AAAA. This is the first study with Chartegellus communis venom, and both Communis and Communis-AAAA are unpublished peptides.     

Nocistatin: a novel neuropeptide encoded by the gene for the nociceptin/orphanin FQ precursor

We identified a novel neuropeptide and named it "nocistatin." Its presence was expected by analysis of the precursor for the neuropeptide nociceptin or orphanin FQ (Noc/OFQ), previously identified as an endogenous ligand for the orphan opioid receptor-like receptor. The precursor prepronociceptin/orphanin FQ (ppNoc/OFQ) comprises at least two bioactive peptides, nocistatin and Noc/OFQ. Noc/OFQ is involved in a broad range of pharmacological actions in various tissues from the central nervous system to the periphery. In pain transmission, Noc/OFQ is reported to have different effects including nociception, no effect, and analgesia, depending on the animal species tested, doses, route of administration, and so on. We found that intrathecal administration of Noc/OFQ induced pain responses including allodynia and hyperalgesia. Simultaneous administration of nocistatin blocked the allodynia and hyperalgesia induced by Noc/OFQ, whereas anti-nocistatin antibody decreased the threshold for the Noc/OFQ-induced allodynia. The endogenous heptadecapeptide nocistatin was isolated from bovine brains and recently identified in mouse, rat, and human brain and in human cerebrospinal fluid. Although human, rat and mouse ppNoc/OFQ produced larger respective counterparts with 30, 35, and 41 amino acid residues, all peptides showed the antinociceptive activity. This activity was ascribed to the carboxyl-terminal hexapeptide of nocistatin, Glu-Gln-Lys-Gln-Leu-Gln, which is conserved beyond species. Nocistatin also attenuated the allodynia and hyperalgesia evoked by prostaglandin E(2) and the inflammatory hyperalgesia induced by formalin or carrageenan/kaolin, and reversed the Noc/OFQ-induced inhibition of morphine analgesia at picogram doses. Furthermore, nocistatin counteracted the impairment of learning and memory induced by Noc/OFQ or scopolamine. Nocistatin is widely present in the spinal cord and brain. Although nocistatin did not bind to the Noc/OFQ receptor, it bound to the membrane of mouse brain and spinal cord with a high affinity. Nocistatin is a novel bioactive peptide produced from the same precursor as Noc/OFQ, and it plays important roles in the regulation of pain transmission and learning and memory processes in the central nervous system.     

Sea Anemone Peptide with Uncommon β-Hairpin Structure Inhibits Acid-sensing Ion Channel 3 (ASIC3) and Reveals Analgesic Activity

Three novel peptides were isolated from the venom of the sea anemone Urticina grebelnyi. All of them are 29 amino acid peptides cross-linked by two disulfide bridges, with a primary structure similar to other sea anemone peptides belonging to structural group 9a. The structure of the gene encoding the shared precursor protein of the identified peptides was determined. One peptide, π-AnmTX Ugr 9a-1 (short name Ugr 9-1), produced a reversible inhibition effect on both the transient and the sustained current of human ASIC3 channels expressed in Xenopus laevis oocytes. It completely blocked the transient component (IC₅₀ 10 ± 0.6 μm) and partially (48 ± 2%) inhibited the amplitude of the sustained component (IC₅₀ 1.44 ± 0.19 μm). Using in vivo tests in mice, Ugr 9-1 significantly reversed inflammatory and acid-induced pain. The other two novel peptides, AnmTX Ugr 9a-2 (Ugr 9-2) and AnmTX Ugr 9a-3 (Ugr 9-3), did not inhibit the ASIC3 current. NMR spectroscopy revealed that Ugr 9-1 has an uncommon spatial structure, stabilized by two S-S bridges, with three classical β-turns and twisted β-hairpin without interstrand disulfide bonds. This is a novel peptide spatial structure that we propose to name boundless β-hairpin.     

Crotalphine, a novel potent analgesic peptide from the venom of the South American rattlesnake Crotalus durissus terrificus

We have shown that the venom of the South American rattlesnake Crotalus durissus terrificus induces a long-lasting antinociceptive effect mediated by activation of kappa- and delta-opioid receptors. Despite being mediated by opioid receptors, prolonged treatment with the crotalid venom does not cause the development of peripheral tolerance or abstinence symptoms upon withdrawal. In the present study, we have isolated and chemically characterized a novel and potent antinociceptive peptide responsible for the oral opioid activity of this crotalid venom. The amino acid sequence of this peptide, designated crotalphine, was determined by mass spectrometry and corroborated by solid-phase synthesis to be 相似文献   

C-fos antisense oligodeoxynucleotide decreases subcutaneous bee venom injection-induced nociceptive behavior and fos expression in the rat

Oligodeoxynucleotide complementary to c-fos mRNA was applied to characterize its effect on the spinal cord Fos expression and relevant nociceptive behaviors challenged by subcutaneous injection of bee venom to the rat hind paw. Nociceptive behavioral responses (spontaneous pain and hyperalgesia) following bee venom (0.2 mg/50 microl) injection were assessed in adult male Sprague-Dawley rats receiving intrathecal administration of c-fos antisense oligodeoxynucleotide (ASO, 50 microg/10 microl), sense oligodeoxynucleotide (SO, 50 microg/10 microl) and saline (10 microl) 4 h prior to bee venom injection. The lumbar spinal cord expression of Fos protein 2 h after bee venom injection in the ASO-, SO- and saline-treated animals was observed by immunohistochemistry. The results showed that pretreatment of c-fos ASO markedly reduced the flinching response and primary thermal hyperalgesia, but without significant effects on mechanical hyperalgesia and secondary thermal hyperalgesia. At the same time, ASO treatment also significantly decreased the expression of Fos protein within the lumbar region of the spinal cord ipsilateral to the injection. The results provide further evidence that Fos protein contributes to the activation of the spinal dorsal horn neurons and the generation and/or maintenance of spontaneous pain and primary thermal hyperalgesia induced by subcutaneous injection of bee venom.     

Structure–function relationships of the peptide Paulistine: A novel toxin from the venom of the social wasp Polybia paulista

Background

The peptide Paulistine was isolated from the venom of wasp Polybia paulista. This peptide exists under a natural equilibrium between the forms: oxidised — with an intra-molecular disulphide bridge; and reduced — in which the thiol groups of the cysteine residues do not form the disulphide bridge. The biological activities of both forms of the peptide are unknown up to now.

Methods

Both forms of Paulistine were synthesised and the thiol groups of the reduced form were protected with the acetamidemethyl group [Acm-Paulistine] to prevent re-oxidation. The structure/activity relationships of the two forms were investigated, taking into account the importance of the disulphide bridge.

Results

Paulistine has a more compact structure, while Acm-Paulistine has a more expanded conformation. Bioassays reported that Paulistine caused hyperalgesia by interacting with the receptors of lipid mediators involved in the cyclooxygenase type II pathway, while Acm-Paullistine also caused hyperalgesia, but mediated by receptors involved in the participation of prostanoids in the cyclooxygenase type II pathway.

Conclusion

The acetamidemethylation of the thiol groups of cysteine residues caused small structural changes, which in turn may have affected some physicochemical properties of the Paulistine. Thus, the dissociation of the hyperalgesy from the edematogenic effect when the actions of Paulistine and Acm-Paulistine are compared to each other may be resulting from the influence of the introduction of Acm-group in the structure of Paulistine.

General significance

The peptides Paulistine and Acm-Paulistine may be used as interesting tools to investigate the mechanisms of pain and inflammation in future studies.     

Intrathecal CART (55-102) attenuates hyperlagesia and allodynia in a mouse model of neuropathic but not inflammatory pain

CART peptides are found in brain and spinal cord areas involved in pain transmission. In the present study, we investigated the role of rat CART (55-102) in the modulation of chronic pain using models of chronic neuropathic (nerve injury model) and inflammatory (carrageenan test) pain models in the mouse after intrathecal administration. The results show that CART (55-102) was highly effective in reversing the hyperalgesia and allodynia signs of chronic neuropathic pain in a dose-related manner at doses (0.05-2 microg/mouse) that did not affect motor coordination of the animals. These effects lasted for at least 3 h after injection and were not blocked by naloxone, an opiate antagonist. Although CART (55-102) attenuated carrageenan-induced hyperalgesia, it failed to reduce the inflammation associated with this model. These results suggest the involvement of the CART peptides in the development of hyperalgesia and allodynia associated with neuropathic pain.     

The natriuretic peptide/helokinestatin precursor from Mexican beaded lizard (Heloderma horridum) venom: Amino acid sequence deduced from cloned cDNA and identification of two novel encoded helokinestatins

Natriuretic peptides are common components of reptile venoms and molecular cloning of their biosynthetic precursors has revealed that in snakes, they co-encode bradykinin-potentiating peptides and in venomous lizards, some co-encode bradykinin inhibitory peptides such as the helokinestatins. The common natriuretic peptide/helokinestatin precursor of the Gila Monster, Heloderma suspectum, encodes five helokinestatins of differing primary structures. Here we report the molecular cloning of a natriuretic peptide/helokinestatin precursor cDNA from a venom-derived cDNA library of the Mexican beaded lizard (Heloderma horridum). Deduction of the primary structure of the encoded precursor protein from this cloned cDNA template revealed that it consisted of 196 amino acid residues encoding a single natriuretic peptide and five helokinestatins. While the natriuretic peptide was of identical primary structure to its Gila Monster (H. suspectum) homolog, the encoded helokinestatins were not, with this region of the common precursor displaying some significant differences to its H. suspectum homolog. The helokinestatin-encoding region contained a single copy of helokinestatin-1, 2 copies of helokinestatin-3 and single copies of 2 novel peptides, (Phe)⁵-helokinestatin-2 (VPPAFVPLVPR) and helokinestatin-6 (GPPFNPPPFVDYEPR). All predicted peptides were found in reverse phase HPLC fractions of the same venom. Synthetic replicates of both novel helokinestatins were found to antagonize the relaxing effect of bradykinin on rat tail artery smooth muscle. Thus lizard venom continues to provide a source of novel biologically active peptides.     

SjAPI,the First Functionally Characterized Ascaris-Type Protease Inhibitor from Animal Venoms

Background

Serine protease inhibitors act as modulators of serine proteases, playing important roles in protecting animal toxin peptides from degradation. However, all known serine protease inhibitors discovered thus far from animal venom belong to the Kunitz-type subfamily, and whether there are other novel types of protease inhibitors in animal venom remains unclear.

Principal Findings

Here, by screening scorpion venom gland cDNA libraries, we identified the first Ascaris-type animal toxin family, which contains four members: Scorpiops jendeki Ascaris-type protease inhibitor (SjAPI), Scorpiops jendeki Ascaris-type protease inhibitor 2 (SjAPI-2), Chaerilus tricostatus Ascaris-type protease inhibitor (CtAPI), and Buthus martensii Ascaris-type protease inhibitor (BmAPI). The detailed characterization of Ascaris-type peptide SjAPI from the venom gland of scorpion Scorpiops jendeki was carried out. The mature peptide of SjAPI contains 64 residues and possesses a classical Ascaris-type cysteine framework reticulated by five disulfide bridges, different from all known protease inhibitors from venomous animals. Enzyme and inhibitor reaction kinetics experiments showed that recombinant SjAPI was a dual function peptide with α-chymotrypsin- and elastase-inhibiting properties. Recombinant SjAPI inhibited α-chymotrypsin with a Ki of 97.1 nM and elastase with a Ki of 3.7 μM, respectively. Bioinformatics analyses and chimera experiments indicated that SjAPI contained the unique short side chain functional residues “AAV” and might be a useful template to produce new serine protease inhibitors.

Conclusions/Significance

To our knowledge, SjAPI is the first functionally characterized animal toxin peptide with an Ascaris-type fold. The structural and functional diversity of animal toxins with protease-inhibiting properties suggested that bioactive peptides from animal venom glands might be a new source of protease inhibitors, which will accelerate the development of diagnostic and therapeutic agents for human diseases that target diverse proteases.     

Isolation and Characterization of a Natriuretic Peptide from Crotalus oreganus abyssus (Grand Canyon Rattlesnake) and its Effects on Systemic Blood Pressure and Nitrite Levels

Studies on the therapeutic potential of venom peptides have significantly advanced the development of new peptide drugs. A good example is captopril, a synthetic peptide drug, which acts as an anti-hypertensive and potentiating bradykinin, inhibiting the angiotensin-converting enzyme, whose precursor was isolated from the venom of Bothrops jararacussu. The natriuretic peptide (NPs) family comprises three members, ANP (atrial natriuretic peptide), BNP (B-type natriuretic peptide) and CNP (C-type natriuretic peptide), and has an important role in blood pressure regulation and electrolyte homeostasis. In this study, we describe, for the first time, the isolation and characterization of a novel natriuretic-like peptide (Coa_NP), isolated from Crotalus Oreganus abyssus venom. The peptide has 32 amino acids and its complete sequence is SKRLSNGCFGLKLDRIGAMSGLGCWRLINESK. The Coa_NP has an average molecular mass of 3510.98 Da and its amino acid sequence presents the loop region that is characteristic of natriuretic peptides (17 amino acids, NP domain consensus; CFGXXXDRIXXXSGLGC). Coa_NP is a natriuretic peptide of the ANP/BNP-like family, since the carboxy terminal region of CNP has its own NP domain. The functional experiments showed that Coa_NP produced biological effects similar to those of the other natriuretic peptides: (1) a dose-dependent decrease in mean arterial pressure; (2) significant increases in plasma nitrite levels, and (3) vasorelaxation in thoracic aortic rings that were pre-contracted with phenylephrine. The structural and biological aspects confirm Coa_NP as a natriuretic peptide isolated from snake venom, thus expanding the diversification of venom components.     

Spinal processing of bee venom-induced pain and hyperalgesia

Subcutaneous injection of bee venom causes long-term neural activation and hypersensitization in the dorsal horn of the spinal cord, which contributes to the development and maintenance of various pain-related behaviors. The unique behavioral 'pheno-types' of nociception and hypersensitivity identified in the rodent bee venom test are believed to reflect a complex pathological state of inflammatory pain and might be appropriate to the study of phenotype-based mechanisms of pain and hyperalgesia. In this review, the spinal processing of the bee venom-induced different 'phenotypes' of pain and hyperalgesia will be described. The accumulative electrophysiological, pharmacological, and behavioral data strongly suggest that different 'phenotypes' of pain and hyperalgesia are mediated by different spinal signaling pathways. Unraveling the phenotype-based mechanisms of pain might be useful in development of novel therapeutic drugs against complex clinic pathological pain.     

Analgesic Effect of Photobiomodulation on Bothrops Moojeni Venom-Induced Hyperalgesia: A Mechanism Dependent on Neuronal Inhibition,Cytokines and Kinin Receptors Modulation

BackgroundEnvenoming induced by Bothrops snakebites is characterized by drastic local tissue damage that involves an intense inflammatory reaction and local hyperalgesia which are not neutralized by conventional antivenom treatment. Herein, the effectiveness of photobiomodulation to reduce inflammatory hyperalgesia induced by Bothrops moojeni venom (Bmv), as well as the mechanisms involved was investigated.Conclusion/SignificanceThese data demonstrate that LLLT interferes with mechanisms involved in nociception and hyperalgesia and modulates Bmv-induced nociceptive signal. The use of photobiomodulation in reducing local pain induced by Bothropic venoms should be considered as a novel therapeutic tool for the treatment of local symptoms induced after bothropic snakebites.     

Effect of the C-terminus of murine S100A9 protein on experimental nociception

Calcium-binding protein S100A9 induces antinociception in mice evaluated by the writhing test. Similarly, a peptide identical to the C-terminus of murine S100A9 (mS100A9p) inhibits the hyperalgesia induced by jararhagin, a metalloprotease. Thus, we investigated the effect of mS100A9p on different models used to evaluate nociception. mS100A9p induced a dose-dependent inhibitory effect on the writhing test, and on mechanical hyperalgesia induced by carrageenan. mS100A9p inhibited thermal hyperalgesia induced by carrageenan. mS100A9p did not modify the nociceptive response in hot plate or tail-flick tests. These data demonstrate that the C-terminus of S100A9 protein interferes with control mechanisms of inflammatory pain.     

Discovery of an MIT-like atracotoxin family: spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins

This project identified a novel family of six 66–68 residue peptides from the venom of two Australian funnel-web spiders, Hadronyche sp. 20 and H. infensa: Orchid Beach (Hexathelidae: Atracinae), that appear to undergo N- and/or C-terminal post-translational modifications and conform to an ancestral protein fold. These peptides all show significant amino acid sequence homology to atracotoxin-Hvf17 (ACTX–Hvf17), a non-toxic peptide isolated from the venom of H. versuta, and a variety of AVIT family proteins including mamba intestinal toxin 1 (MIT1) and its mammalian and piscine orthologs prokineticin 1 (PK1) and prokineticin 2 (PK2). These AVIT family proteins target prokineticin receptors involved in the sensitization of nociceptors and gastrointestinal smooth muscle activation. Given their sequence homology to MIT1, we have named these spider venom peptides the MIT-like atracotoxin (ACTX) family. Using isolated rat stomach fundus or guinea-pig ileum organ bath preparations we have shown that the prototypical ACTX–Hvf17, at concentrations up to 1 μM, did not stimulate smooth muscle contractility, nor did it inhibit contractions induced by human PK1 (hPK1). The peptide also lacked activity on other isolated smooth muscle preparations including rat aorta. Furthermore, a FLIPR Ca²⁺ flux assay using HEK293 cells expressing prokineticin receptors showed that ACTX–Hvf17 fails to activate or block hPK1 or hPK2 receptors. Therefore, while the MIT-like ACTX family appears to adopt the ancestral disulfide-directed β-hairpin protein fold of MIT1, a motif believed to be shared by other AVIT family peptides, variations in the amino acid sequence and surface charge result in a loss of activity on prokineticin receptors.     

Thromboxane-induced pulmonary vasoconstriction: involvement of calcium

Infusion of tert-butyl hydroperoxide (t-bu-OOH) or arachidonic acid into rabbit pulmonary arteries stimulated thromboxane B2 (TxB2) production and caused pulmonary vasoconstriction. Both phenomena were blocked by cyclooxygenase inhibitors or a thromboxane synthase inhibitor. The increase in pulmonary arterial pressure caused by either t-bu-OOH or arachidonic acid infusion correlated with the concentration of TxB2 in the effluent perfusate. The concentration of TxB2 in the effluent perfusate, however, was always 10-fold greater after arachidonic acid infusion. In the rabbit pulmonary vascular bed lipoxygenase products did not appear involved in the vasoactive response to t-bu-OOH or exogenous arachidonic acid infusion. Calcium entry blockers or a calcium-free perfusate prevented the thromboxane-induced pulmonary vasoconstriction. Calmodulin inhibitors also blocked the pulmonary vasoconstriction induced by t-bu-OOH without affecting the production of TxB2 or prostacyclin. These results suggest that thromboxane causes pulmonary vasoconstriction by increasing cytosol calcium concentration.     

Peripheral neuronal nitric oxide synthase activity mediates the antinociceptive effect of Crotalus durissus terrificus snake venom, a delta- and kappa-opioid receptor agonist

Previous work has shown that nitric oxide (NO) mediates the antinociceptive effect of Crotalus durissus terrificus venom on carrageenin-induced hyperalgesia. In the present study the role of constitutive neuronal or of inducible form of nitric oxide synthase on venom effect was determined. The rat paw prostaglandin E(2) (PGE(2))-induced mechanical hyperalgesia model was used for nociceptive evaluation. The venom (200 microg/kg) administered per oz immediately before prostaglandin induced antinociception that persisted for 120 h. The characterisation of the antinociceptive effect of the venom in this model of hyperalgesia showed that kappa and delta-opioid receptors are involved in this effect. 7-nitroindazole (7-NI), a neuronal nitric oxide synthase (NOS) inhibitor, but not L-N(6)-(1-iminoethyl)lysine (L-NIL), an inhibitor of the inducible form of NOS, injected by intraplantar (i.pl.) route, antagonized the antinociceptive effect of the venom. The i.pl. administration of 1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a selective guanylate cyclase inhibitor, blocked antinociception, whereas Rp-cGMP triethylamine, a cGMP-dependent protein kinase inhibitor, partially reversed this effect. These data indicate that peripheral kappa- and delta-opioid receptors are involved in the antinociceptive effect of Crotalus durissus terrificus on prostaglandin E(2)-induced hyperalgesia. Peripheral nitric oxide, generated by neuronal NO synthase, and cGMP/PKc are responsible, at least partially, for the molecular mechanisms of venom effect.     

Inhibitory effect of the red seaweed Plocamium brasiliense against the toxic effects of Lachesis muta snake venom

The ability of extracts and fractions of the red seaweed Plocamium brasiliense to inhibit hemorrhagic, edematogenic, hemolytic, clotting and proteolytic activities of Lachesis muta snake venom was analyzed. In Brazil, snakebites by L. muta are low (2 %) when compared to Bothrops genus (90 %); however, their lethality indexes are three times higher than Bothrops. Envenomation by L. muta venom results in hemorrhage, pain, necrosis, hemolysis, myotoxicity, and death. Since antivenom does not efficiently neutralize local effects, a large number of researchers have attempted to identify molecule(s) from natural sources to inhibit such activities to use them as an alternative treatment for snakebite. We tested four extracts of seaweed P. brasiliense obtained with solvents of increasing polarities: n-hexane (HEX), dichloromethane (DCM), ethyl acetate (ACE), and hydroalcohol (HYD). Extracts of alga or fractions were incubated with L. muta venom, and then, biological assays were performed. The extracts, except the HYD, inhibited all the assays but with different potencies. The DCM extract fully inhibited all activities. Moreover, DCM and HEX extracts inhibited hemolysis induced by a phospholipase A₂ isolated from L. muta venom (LM-PLA₂-I). A fraction from HEX enriched in cholesterol isolated from HEX extract inhibited proteolysis by L. muta venom and hemolysis by LM-PLA₂-I; in contrast, monoterpenes isolated from DCM extract did not inhibit both activities. Seaweeds may be a promising source of natural inhibitors of the toxic effects caused by snakebite by L. muta venom, and they may be used to develop new strategies for antivenom treatment.