Effect of Seaweed-Derived Fucoidans from Undaria pinnatifida and Fucus vesiculosus on Coagulant, Proteolytic, and Phospholipase A2 Activities of Snake Bothrops jararaca, B. jararacussu, and B. neuwiedi Venom.

BACKGROUND: Snakebite envenomation (SBE) causes diverse toxic effects in humans, including disability and death. Current antivenom therapies effectively prevent death but fail to block local tissue damage, leading to an increase in the severity of envenomation; thus, seeking alternative treatments is crucial. METHODS: This study analyzed the potential of two fucoidan sulfated polysaccharides extracted from brown seaweeds Fucus vesiculosus (FVF) and Undaria pinnatifida (UPF) against the fibrinogen or plasma coagulation, proteolytic, and phospholipase A2 (PLA2) activities of Bothrops jararaca, B. jararacussu, and B. neuwiedi venom. The toxicity of FVF and UPF was assessed by the hemocompatibility test. RESULTS: FVF and UPF did not lyse human red blood cells. FVF and UPF inhibited the proteolytic activity of Bothrops jararaca, B. jararacussu, and B. neuwiedi venom by approximately 25%, 50%, and 75%, respectively, while all venoms led to a 20% inhibition of PLA2 activity. UPF and FVF delayed plasma coagulation caused by the venoms of B. jararaca and B. neuwiedi but did not affect the activity of B. jararacussu venom. FVF and UPF blocked the coagulation of fibrinogen induced by all these Bothropic venoms. CONCLUSION: FVF and UPF may be of importance as adjuvants for SBE caused by species of Bothrops, which are the most medically relevant snakebite incidents in South America, especially Brazil.

Pictolysin-III, a Hemorrhagic Type-III Metalloproteinase Isolated from Bothrops pictus (Serpentes: Viperidae) Venom, Reduces Mitochondrial Respiration and Induces Cytokine Secretion in Epithelial and Stromal Cell Lines.

From the venom of the Bothrops pictus snake, an endemic species from Peru, we recently have described toxins that inhibited platelet aggregation and cancer cell migration. In this work, we characterize a novel P-III class snake venom metalloproteinase, called pictolysin-III (Pic-III). It is a 62 kDa proteinase that hydrolyzes dimethyl casein, azocasein, gelatin, fibrinogen, and fibrin. The cations Mg2+ and Ca2+ enhanced its enzymatic activity, whereas Zn2+ inhibited it. In addition, EDTA and marimastat were also effective inhibitors. The amino acid sequence deduced from cDNA shows a multidomain structure that includes a proprotein, metalloproteinase, disintegrin-like, and cysteine-rich domains. Additionally, Pic-III reduces the convulxin- and thrombin-stimulated platelet aggregation and in vivo, it has hemorrhagic activity (DHM = 0.3 µg). In epithelial cell lines (MDA-MB-231 and Caco-2) and RMF-621 fibroblast, it triggers morphological changes that are accompanied by a decrease in mitochondrial respiration, glycolysis, and ATP levels, and an increase in NAD(P)H, mitochondrial ROS, and cytokine secretion. Moreover, Pic-III sensitizes to the cytotoxic BH3 mimetic drug ABT-199 (Venetoclax) in MDA-MB-231 cells. To our knowledge, Pic-III is the first SVMP reported with action on mitochondrial bioenergetics and may offer novel opportunities for promising lead compounds that inhibit platelet aggregation or ECM-cancer-cell interactions.

An Emergent Role for Mitochondrial Bioenergetics in the Action of Snake Venom Toxins on Cancer Cells.

Beyond the role of mitochondria in apoptosis initiation/execution, some mitochondrial adaptations support the metastasis and chemoresistance of cancer cells. This highlights mitochondria as a promising target for new anticancer strategies. Emergent evidence suggests that some snake venom toxins, both proteins with enzymatic and non-enzymatic activities, act on the mitochondrial metabolism of cancer cells, exhibiting unique and novel mechanisms that are not yet fully understood. Currently, six toxin classes (L-amino acid oxidases, thrombin-like enzymes, secreted phospholipases A2, three-finger toxins, cysteine-rich secreted proteins, and snake C-type lectin) that alter the mitochondrial bioenergetics have been described. These toxins act through Complex IV activity inhibition, OXPHOS uncoupling, ROS-mediated permeabilization of inner mitochondrial membrane (IMM), IMM reorganization by cardiolipin interaction, and mitochondrial fragmentation with selective migrastatic and cytotoxic effects on cancer cells. Notably, selective internalization and direct action of snake venom toxins on tumor mitochondria can be mediated by cell surface proteins overexpressed in cancer cells (e.g. nucleolin and heparan sulfate proteoglycans) or facilitated by the elevated Δψm of cancer cells compared to that non-tumor cells. In this latter case, selective mitochondrial accumulation, in a Δψm-dependent manner, of compounds linked to cationic snake peptides may be explored as a new anti-cancer drug delivery system. This review analyzes the effect of snake venom toxins on mitochondrial bioenergetics of cancer cells, whose mechanisms of action may offer the opportunity to develop new anticancer drugs based on toxin scaffolds.

Study on the synthesis and structure-activity relationship of 1,2,3-triazoles against toxic activities of Bothrops jararaca venom.

Snakebite envenoming is a health concern and has been a neglected tropical disease since 2017, according to the World Health Organization. In this study, we evaluated the ability of ten 1,2,3-triazole derivatives AM001 to AM010 to inhibit pertinent in vitro (coagulant, hemolytic, and proteolytic) and in vivo (hemorrhagic, edematogenic, and lethal) activities of Bothrops jararaca venom. The derivatives were synthesized, and had their molecular structures fully characterized by CHN element analysis, Fourier-transform infrared spectroscopy and Nuclear magnetic resonance. The derivatives were incubated with the B. jararaca venom (incubation protocol) or administered before (prevention protocol) or after (treatment protocol) the injection of B. jararaca venom into the animals. Briefly, the derivatives were able to inhibit the main toxic effects triggered by B. jararaca venom, though with varying efficacies, and they were devoid of toxicity through in vivo, in silico or in vitro analyses. However, it seemed that the derivatives AM006 or AM010 inhibited more efficiently hemorrhage or lethality, respectively. The derivatives were nontoxic. Therefore, the 1,2,3-triazole derivatives may be useful as an adjuvant to more efficiently treat the local toxic effects caused by B. jararaca envenoming.

Synthesis, characterization and utilization of a new series of 1,2,3-triazole derivatives to neutralize some toxic activities of Bothrops jararaca snake venom

Abstract Snake envenomation is a public health problem, and while serum therapy prevents death, the local effects of venoms can lead to amputations or morbidities. Thus, alternative treatments deserve attention. In this study, we tested eight derivatives of 1,2,3-triazole against some toxic activities of Bothrops jararaca venom. The derivatives were synthesized, and their structures analyzed by infrared and nuclear magnetic resonance. After that, the ability of compounds to inhibit hemolysis, coagulation, proteolysis, hemorrhaging, edema, and lethal activities of B. jararaca venom was investigated. The derivatives were incubated with B. jararaca venom (incubation protocol), administered before (prevention protocol) or after (treatment protocol) injecting venom into the mice. Then, hemorrhaging assay occurred. As a result, most of the derivatives inhibited the activities, even if they were incubated, injected before or after B. jararaca venom. However, the derivatives TRI 07 and TRI 18 seemed to be the most efficient in impairing hemorrhaging. The derivatives showed a low drug score of toxicity based on an in silico technique. Therefore, the derivatives fulfilled physicochemical and biological requirements to become drugs, and they may be a brand new initiative for designing antivenom molecules to complement antivenom therapy to efficiently block tissue necrosis or any other local effects.

The plant Stryphnodendron adstringens (Mart.) Coville as a neutralizing source against some toxic activities of Bothrops jararacussu snake venom.

Snakebites produce several toxic effects in victims, such as hemorrhage, tissue necrosis, hemostatic, renal, or cardiotoxic alterations, inflammation, and death. To counteract these symptoms, antivenom is the official treatment. Although such therapy prevents death, it does not efficiently neutralize necrosis or other local effects, leading to amputation or morbidities of the affected limb. Therefore, the search for better and more efficient therapies deserves attention; further, plants have been used to ameliorate a number of diseases and medical conditions, including snakebites, for many years. Thus, the aim of this work was to evaluate the antivenom effect of the crude extract, fractions (aqueous and diethyl acetate), and subfractions derived from the aqueous fraction (P1, P2, P3, and P4) of the plant Stryphnodendron adstringens against in vitro (coagulation and proteolytic) and in vivo (edema, hemorrhage, and myotoxic) activities caused by Bothrops jararacussu venom. Overall, all extracts inhibited the toxic effect of B. jararacussu venom, but with different potencies, regardless of whether plant samples were incubated together with venom or injected before or after venom injection into animals; the crude extract and aqueous fraction were found to be the most effective. Indeed, phytochemical and mass spectrometry analysis of S. adstringens samples revealed the presence of flavonols, tannins, and saponins. In conclusion, the plant S. adstringens may represent a promising natural source of molecules to treat the toxic effects associated with envenomation by B. jararacussu snakebites.

Utilization of the Plant Clusia Fluminensis Planch & Triana Against Some Toxic Activities of the Venom of Bothrops jararaca and B. jararacussu Snake Venom Toxic Activities.

BACKGROUND: In Brazil, the Bothrops genus accounts for 87% of registered snakebites, which are characterized by hemorrhage, tissue necrosis, hemostatic disturbances, and death. The treatment recommended by governments is the administration of specific antivenoms. Although antivenom efficiently prevents venom-induced lethality, it has limited efficacy in terms of preventing local tissue damage. Thus, researchers are seeking alternative therapies able to inhibit the main toxic effects of venoms, without compromising safety. OBJECTIVE: The study aimed to test the ability of aqueous extracts of leaves, stems, and fruits of the plant Clusia fluminensis to neutralize some toxic effects induced by the venoms of Bothrops jararaca and Bothrops jararacussu. METHODS: The plant extracts were incubated with venoms for 30 min. at 25 °C, and then in vitro (coagulant and proteolytic) and in vivo (hemorrhagic, myotoxic, and edematogenic) activities were evaluated. In addition, the extracts were administered to animals (by oral, intravenous or subcutaneous routes) before or after the injection of venom samples, and then hemorrhage and edema assays were performed. In addition, a gel solution of the fruit extract was produced and tested in terms of reducing hemorrhage effects. A chemical prospection was performed to identify the main classes of compounds present in the extracts. RESULTS: All the extracts inhibited the activities of the two venoms, regardless of the experimental protocol or route of administration of the extracts. Moreover, the gel of the fruit extract inhibited the venom-induced-hemorrhage. The extracts comprised of tannins, flavonoids, saponins, steroids, and terpenoids. CONCLUSION: Antivenom properties of C. fluminensis extracts deserve further investigation in order to gain detailed knowledge regarding the neutralization profile of these extracts.

L-amino acid oxidase from Bothrops atrox snake venom triggers autophagy, apoptosis and necrosis in normal human keratinocytes.

Snake venom L-amino acid oxidases (LAAOs) are flavoproteins, which perform diverse biological activities in the victim such as edema, myotoxicity and cytotoxicity, contributing to the development of clinical symptoms of envenomation. LAAO cytotoxicity has been described, but the temporal cascade of events leading to cell death has not been explored so far. This study evaluates the involvement of LAAO in dermonecrosis in mice and its cytotoxic effects in normal human keratinocytes, the major cell type in the epidermis, a tissue that undergoes extensive necrosis at the snakebite site. Pharmacological inhibition by the antioxidant NAC (N-acetyl cysteine) prevented B. atrox venom-induced necrosis. Consistent with the potential role of oxidative stress in wounding, treatment with purified LAAO decreased keratinocyte viability with an Effective Concentration (EC50) of 5.1 µg/mL. Cytotoxicity caused by LAAO was mediated by H2O2 and treated cells underwent autophagy, followed by apoptosis and necrosis. LAAO induced morphological alterations that precede cell death. Our results show the chronological events leading to cell death and the temporal resolution from autophagy, apoptosis and necrosis as distinct mechanisms triggered by LAAO. Fluorescently-labelled LAAO was efficiently and rapidly internalized by keratinocytes, suggesting that catalysis of intracellular substrates may contribute to LAAO toxicity. A better understanding of LAAO cytotoxicity and its mechanism of action will help to identify potential therapeutic strategies to ameliorate localized snake envenomation symptoms.

Potential Utilization of a Polysaccharide from the Marine Algae Gayralia oxysperma, as an Antivenom for Viperidae Snakebites.

Worldwide, snakebites have serious implications for human health. The administration of antivenom is the official treatment used to reverse the toxic activities of envenomation. However, this therapy is not efficient to treat the local effects, leading to the amputation or deformity of affected limbs. As such, alternative treatments are needed. Here, we analyze the ability of a polysaccharide from the green marine alga Gayralia oxysperma (Go3) to inhibit the effects of venom from Bothrops jararaca and Lachesis muta. B. jararaca or L. muta venoms were incubated together with sulfated heterorhamnans from Go3, and the in vitro (coagulation, proteolytic, and hemolytic) and in vivo (hemorrhagic, myotoxic, edematogenic, and lethal) activities of venoms were assessed. Additionally, Go3 was injected before and after the injection of venoms, and the toxic activities were further tested. When incubated with the venoms, Go3 inhibited all activities, though results varied with different potencies. Moreover, Go3 neutralized hemorrhagic, myotoxic, and edematogenic activities when injected before or after injection with B. jararaca and L. muta venom. Go3 also blocked the coagulation of plasma in mice caused by the venoms in an ex vivo test. Therefore, Go3 has the potential to be used as antivenom for B. jararaca and L. muta bites, notably exhibiting higher efficacy on L. muta venom.

Proteomic profile, biological activities and antigenic analysis of the venom from Bothriopsis bilineata smaragdina ("loro machaco"), a pitviper snake from Peru.

In order to determine Bothriopsis bilineata smaragdina venom (BbsV) composition, proteomic approaches were performed. Venom components were analyzed by RP-HPLC, SDS- PAGE and nano LC on line with LTQ Orbitrap XL. Results showed a total of 189 identified proteins, grouped into 11 different subgroups, which include snake venom metalloproteinases (SVMPs, 54.67%), snake C-type lectins (Snaclecs, 15.78%), snake venom serine proteinases (SVSPs, 14.69%), cystein-rich secretory proteins (CRISP, 2.61%), phospholipases A2 (PLA2, 1.14%), phosphodiesterase (PDE, 1.17%), venom endothelial growth factor (VEGF, 1.06%) 5'nucleotidases (0.33%), L-amino acid oxidases (LAAOs, 0.28%) and other proteins. In vitro enzymatic activities (SVMP, SVSP, LAAO, Hyal and PLA2) of BbsV were also analyzed. BbsV showed high SVSP activity but low PLA2 activity, when compared to other Bothrops venoms. In vivo, BbsV induced hemorrhage and edema in mice and showed intraperitoneal median lethal dose (LD50) of 92.74 (± 0.15) µg/20 g of mice. Furthermore, BbsV reduced cell viability when incubated with VERO cells. Peruvian and Brazilian bothropic antivenoms recognize BbsV proteins, as detected by ELISA and Western Blotting. Both antivenoms were able to neutralize in vivo edema and hemorrhage. SIGNIFICANCE: In Peru, snakebite is a public health problem, especially in the rain forest, as a result of progressive colonization of this geographical area. This country is the second in Latin America, after Brazil, to exhibit the largest variety of venomous snakes. B. atrox and B. b. smaragdina snakes are sympatric species in Peruvian Amazon region and are responsible for approximately 95% of the envenomings reported in this region. B. b. smaragdina may cause a smaller share (3 to 38%) of those accidents, due to its arboreal habits, that make human encounters with these snakes less likely to happen. Despite B. b. smaragdina recognized medical importance, its venom composition and biological activities have been poorly studied. Furthermore, BbsV is not a component of the antigenic pool used to produce the corresponding Peruvian bothropic antivenom (P-BAV). Our results not only provide new insights on BbsV composition and biological activity, but also demonstrate that both P-BAV and B-BAV polyvalent antivenoms have a considerable recognition of proteins from BbsV and, more importantly, neutralized hemorrhage and edema, the main local effects of bothropic envenomation.

Protective Effect of the Sulfated Agaran Isolated from the Red Seaweed Laurencia aldingensis Against Toxic Effects of the Venom of the Snake, Lachesis muta.

Snakebite is a serious occupational hazard affecting mainly rural populations of tropical and subtropical developing countries. Lachesis muta (Bushmaster) bites are extremely serious but are rarely reported in the literature. Bushmaster envenomings are characterized by intense local pain, edema, neurotoxicity, hypotension, local hemorrhage, and dramatic systemic alterations. Antivenom treatment has regularly been used for more than a century; however, it fails to neutralize local tissue damage and hemorrhage, leading to morbidity or disabilities in victims. Thus, the production and clinical use of antivenom must be improved. The present work characterizes, for the first time, a sulfated polysaccharide from the red seaweed, Laurencia aldingensis, including its neutralizing effect on some toxic activities of L. muta venom. Chemical and spectroscopic analyses showed that L. aldingensis produces sulfated agarans with the A-units partially C-2 sulfated or 6-O-methoxylated presetting the B-units in the cyclized (3,6-anhydro-α-L-galactose) or in the non-cyclized form (α-L-galactose). The latter is significantly substituted by sulfate groups on C-6. In vitro and in vivo assays showed that this sulfated agaran inhibited hemolysis, coagulation, proteolysis, edema, and hemorrhage of L. muta venom. Neutralization of hemorrhagic activity was also observed when the agaran was administered by different routes and after or before the venom injection. Furthermore, the agaran blocked the edema caused by a phospholipase A2 isolated from the L. muta venom. Experimental evidence therefore indicates that the sulfated agaran of L. aldingensis has potential to aid antivenom therapy of accidents caused by L. muta venom and may help to develop more effective antivenom treatments of snake bites in general.

Data for a direct fibrinolytic metalloproteinase, barnettlysin-I from Bothrops barnetti (barnett(,)s pitviper) snake venom with anti-thrombotic effect.

Initial association of platelets after vascular injury is mediated by glycoprotein (GP)Ib-IX-V binding to von Willebrand factor (vWf) immobilized on exposed collagens and eventually leads to thrombus formation. This article provides data about a new P-I class snake venom metalloproteinase (SVMP), barnettlysin-I (Bar-I), purified from the venom of Bothrops barnetti. This Data in Brief manuscript complements the main research article by providing additional data of the biochemical characterization of Bar-I 10.1016/j.bbagen.2015.12.021[1].

Sulfated Galactan from Palisada flagellifera Inhibits Toxic Effects of Lachesis muta Snake Venom.

In Brazil, snakebites are a public health problem and accidents caused by Lachesis muta have the highest mortality index. Envenomation by L. muta is characterized by systemic (hypotension, bleeding and renal failure) and local effects (necrosis, pain and edema). The treatment to reverse the evolution of all the toxic effects is performed by injection of antivenom. However, such therapy does not effectively neutralize tissue damage or any other local effect, since in most cases victims delay seeking appropriate medical care. In this way, alternative therapies are in demand, and molecules from natural sources have been exhaustively tested. In this paper, we analyzed the inhibitory effect of a sulfated galactan obtained from the red seaweed Palisada flagellifera against some toxic activities of L. muta venom. Incubation of sulfated galactan with venom resulted in inhibition of hemolysis, coagulation, proteolysis, edema and hemorrhage. Neutralization of hemorrhage was also observed when the galactan was administered after or before the venom injection; thus mimicking a real in vivo situation. Moreover, the galactan blocked the edema caused by a phospholipase A2 isolated from the same venom. Therefore, the galactan from P. flagellifera may represent a promising tool to treat envenomation by L. muta as a coadjuvant for the conventional antivenom.

The red seaweed Plocamium brasiliense shows anti-snake venom toxic effects

Background Snakebite is considered a neglected tropical disease by the World Health Organization. In Brazil, about 70% of the envenomation cases are caused by Bothrops snakes. Its venom may provoke hemorrhage, pain, necrosis, hemolysis, renal or cardiac failure and even death in victims. Since commercial antivenom does not efficiently neutralize the local toxic effects of venoms, natural products have been tested in order to provide alternative or complementary treatment to serum therapy. Therefore, the present study aimed to evaluate the ability of the seaweed Plocamium brasiliense and its active derivatives to neutralize hemorrhagic, edematogenic, hemolytic, coagulant and proteolytic activities of B. jararaca venom. Methods Specimens of P. brasiliense were collected in Rio de Janeiro state, Brazil, dried and submitted to oil extraction using four solvents of increasing polarities, n-hexane (HEX), dichloromethane (DCM), ethyl acetate (ETA) and hydroalcoholic solution (HYD). The solvents were evaporated, yielding HEX, DCM, ETA and HYD extracts. Further, all extracts were dissolved in dimethylsulfoxide. In addition, two monoterpenes (8-bromo-3,4,7-trichloro-3,7-dimethyl-1E, 5E-octadiene and 1,8-dibromo-3,4,7-trichloro-3,7-dimethyl-1E, 5E-octadiene) and a cholesterol fraction were isolated from the extract of P. brasiliense prepared in hexane. Algal samples were incubated for 30 minutes with B. jararaca venom, and then tested for lethality; hemorrhagic, edematogenic, hemolytic, coagulant and proteolytic effects. Results Most of the algal extracts inhibited the toxic effects with different potencies. The DCM extract was the most effective, since it inhibited all types of toxic activity. On the other hand, the HYD extract failed to inhibit any effect. Moreover, the isolated products inhibited proteolysis and protected mice from hemorrhage in 30% of the cases, whereas 8-bromo-3,4,7-trichloro-3,7-dimethyl-1E, 5E-octadiene inhibited 100% and 20% of the hemorrhagic and proteolytic activities, respectively. None of the algal products were toxic to mice. Conclusion Seaweeds may be a promising source of inhibitors against toxic effects caused by B. jararacaenvenomation, which may contribute to antivenom treatment.(AU)

The red seaweed Plocamium brasiliense shows anti-snake venom toxic effects.

BACKGROUND: Snakebite is considered a neglected tropical disease by the World Health Organization. In Brazil, about 70% of the envenomation cases are caused by Bothrops snakes. Its venom may provoke hemorrhage, pain, necrosis, hemolysis, renal or cardiac failure and even death in victims. Since commercial antivenom does not efficiently neutralize the local toxic effects of venoms, natural products have been tested in order to provide alternative or complementary treatment to serum therapy. Therefore, the present study aimed to evaluate the ability of the seaweed Plocamium brasiliense and its active derivatives to neutralize hemorrhagic, edematogenic, hemolytic, coagulant and proteolytic activities of B. jararaca venom. METHODS: Specimens of P. brasiliense were collected in Rio de Janeiro state, Brazil, dried and submitted to oil extraction using four solvents of increasing polarities, n-hexane (HEX), dichloromethane (DCM), ethyl acetate (ETA) and hydroalcoholic solution (HYD). The solvents were evaporated, yielding HEX, DCM, ETA and HYD extracts. Further, all extracts were dissolved in dimethylsulfoxide. In addition, two monoterpenes (8-bromo-3,4,7-trichloro-3,7-dimethyl-1E, 5E-octadiene and 1,8-dibromo-3,4,7-trichloro-3,7-dimethyl-1E, 5E-octadiene) and a cholesterol fraction were isolated from the extract of P. brasiliense prepared in hexane. Algal samples were incubated for 30 minutes with B. jararaca venom, and then tested for lethality; hemorrhagic, edematogenic, hemolytic, coagulant and proteolytic effects. RESULTS: Most of the algal extracts inhibited the toxic effects with different potencies. The DCM extract was the most effective, since it inhibited all types of toxic activity. On the other hand, the HYD extract failed to inhibit any effect. Moreover, the isolated products inhibited proteolysis and protected mice from hemorrhage in 30% of the cases, whereas 8-bromo-3,4,7-trichloro-3,7-dimethyl-1E, 5E-octadiene inhibited 100% and 20% of the hemorrhagic and proteolytic activities, respectively. None of the algal products were toxic to mice. CONCLUSION: Seaweeds may be a promising source of inhibitors against toxic effects caused by B. jararaca envenomation, which may contribute to antivenom treatment.

Effect of diterpenes isolated of the marine alga Canistrocarpus cervicornis against some toxic effects of the venom of the bothrops jararaca snake.

Snake venoms are composed of a complex mixture of active proteins and peptides which induce a wide range of toxic effects. Envenomation by Bothrops jararaca venom results in hemorrhage, edema, pain, tissue necrosis and hemolysis. In this work, the effect of a mixture of two secodolastane diterpenes (linearol/isolinearol), previously isolated from the Brazilian marine brown alga, Canistrocarpus cervicornis, was evaluated against some of the toxic effects induced by B. jararaca venom. The mixture of diterpenes was dissolved in dimethylsulfoxide and incubated with venom for 30 min at room temperature, and then several in vivo (hemorrhage, edema and lethality) and in vitro (hemolysis, plasma clotting and proteolysis) assays were performed. The diterpenes inhibited hemolysis, proteolysis and hemorrhage, but failed to inhibit clotting and edema induced by B. jararaca venom. Moreover, diterpenes partially protected mice from lethality caused by B. jararaca venom. The search for natural inhibitors of B. jararaca venom in C. cervicornis algae is a relevant subject, since seaweeds are a rich and powerful source of active molecules which are as yet but poorly explored. Our results suggest that these diterpenes have the potential to be used against Bothropic envenomation accidents or to improve traditional treatments for snake bites.

The red seaweed Plocamium brasiliense shows anti-snake venom toxic effects

Background Snakebite is considered a neglected tropical disease by the World Health Organization. In Brazil, about 70% of the envenomation cases are caused by Bothrops snakes. Its venom may provoke hemorrhage, pain, necrosis, hemolysis, renal or cardiac failure and even death in victims. Since commercial antivenom does not efficiently neutralize the local toxic effects of venoms, natural products have been tested in order to provide alternative or complementary treatment to serum therapy. Therefore, the present study aimed to evaluate the ability of the seaweed Plocamium brasiliense and its active derivatives to neutralize hemorrhagic, edematogenic, hemolytic, coagulant and proteolytic activities of B. jararaca venom. Methods Specimens of P. brasiliense were collected in Rio de Janeiro state, Brazil, dried and submitted to oil extraction using four solvents of increasing polarities, n-hexane (HEX), dichloromethane (DCM), ethyl acetate (ETA) and hydroalcoholic solution (HYD). The solvents were evaporated, yielding HEX, DCM, ETA and HYD extracts. Further, all extracts were dissolved in dimethylsulfoxide. In addition, two monoterpenes (8-bromo-3,4,7-trichloro-3,7-dimethyl-1E, 5E-octadiene and 1,8-dibromo-3,4,7-trichloro-3,7-dimethyl-1E, 5E-octadiene) and a cholesterol fraction were isolated from the extract of P. brasiliense prepared in hexane. Algal samples were incubated for 30 minutes with B. jararaca venom, and then tested for lethality; hemorrhagic, edematogenic, hemolytic, coagulant and proteolytic effects. Results Most of the algal extracts inhibited the toxic effects with different potencies. The DCM extract was the most effective, since it inhibited all types of toxic activity. On the other hand, the HYD extract failed to inhibit any effect. Moreover, the isolated products inhibited proteolysis and protected mice from hemorrhage in 30% of the cases, whereas 8-bromo-3,4,7-trichloro-3,7-dimethyl-1E, 5E-octadiene inhibited 100% and ...

Use of a synthetic biosensor for neutralizing activity-biased selection of monoclonal antibodies against atroxlysin-I, an hemorrhagic metalloproteinase from Bothrops atrox snake venom.

BACKGROUND: The snake Bothrops atrox is responsible for the majority of envenomings in the northern region of South America. Severe local effects, including hemorrhage, which are mainly caused by snake venom metalloproteinases (SVMPs), are not fully neutralized by conventional serum therapy. Little is known about the immunochemistry of the P-I SVMPs since few monoclonal antibodies (mAbs) against these molecules have been obtained. In addition, producing toxin-neutralizing mAbs remains very challenging. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report on the set-up of a functional screening based on a synthetic peptide used as a biosensor to select neutralizing mAbs against SVMPs and the successful production of neutralizing mAbs against Atroxlysin-I (Atr-I), a P-I SVMP from B. atrox. Hybridomas producing supernatants with inhibitory effect against the proteolytic activity of Atr-I towards the FRET peptide Abz-LVEALYQ-EDDnp were selected. Six IgG1 Mabs were obtained (named mAbatr1 to mAbatr6) and also two IgM. mAbatrs1, 2, 3 and 6 were purified. All showed a high specific reactivity, recognizing only Atr-I and B. atrox venom in ELISA and a high affinity, showing equilibrium constants in the nM range for Atr-I. These mAbatrs were not able to bind to Atr-I overlapping peptides, suggesting that they recognize conformational epitopes. CONCLUSIONS/SIGNIFICANCE: For the first time a functional screening based on a synthetic biosensor was successfully used for the selection of neutralizing mAbs against SVMPs.

Inhibitory effect of plant Manilkara subsericea against biological activities of Lachesis muta snake venom.

Snake venom is composed of a mixture of substances that caused in victims a variety of pathophysiological effects. Besides antivenom, literature has described plants able to inhibit injuries and lethal activities induced by snake venoms. This work describes the inhibitory potential of ethanol, hexane, ethyl acetate, or dichloromethane extracts and fractions from stem and leaves of Manilkara subsericea against in vivo (hemorrhagic and edema) and in vitro (clotting, hemolysis, and proteolysis) activities caused by Lachesis muta venom. All the tested activities were totally or at least partially reduced by M. subsericea. However, when L. muta venom was injected into mice 15 min first or after the materials, hemorrhage and edema were not inhibited. Thus, M. subsericea could be used as antivenom in snakebites of L. muta. And, this work also highlights Brazilian flora as a rich source of molecules with antivenom properties.