Neutralization of toxic activities of Bothrops asper snake venom by ethnomedicinal plants used in Central America, with emphasis in Guatemala: a review / Neutralización de actividades tóxicas del veneno de la serpiente Bothrops asper por plantas etnomedicinales utilizadas en Centroamérica, con énfasis en Guatemala: Revisión

There are few scientific studies that explore the use of medicinal plants for snakebite envenoming in Central America, although plant-based therapies have been traditionally used in the region. This work reviews the studies conducted in Central America to assess the ability of extracts obtained from plants of local ethnomedical use to inhibit toxic activities of the venom of Bothrops asper, the snake responsible for approximately half of the snakebite envenomings in these countries. The search prioritized the description of the plants used in Guatemala, since most of the studies described in this work were conducted in that country, although references to other countries are included. Information concerning secondary metabolites and other pharmacological activities of these plant species, relevant to the treatment of snakebites, was also described. The literature search was conducted in the Google Scholar, PubMed and Scopus databases and completed with locally available literature. It was found that extracts of 12 plant species inhibited the hemorrhagic effect of the venom and three neutralized the edema-forming activity, while inhibition of proteolytic and phospholipase A2 (PLA2) activities was achieved by three and one plant species, respectively. Only Brownea rosa-de-monte was able to effectively counteract the in vitro coagulant effect of the venom. Some plant extracts screened in Guatemala demonstrated procoagulant or anti-thrombin intrinsic effects that might aggravate the coagulopathy induced by the venom. These findings underscore the need of carrying out scientific studies aimed to validate the inhibitory potential of Central American plant extracts and their metabolites against B. asper venom.

Pocos estudios científicos han explorado el uso de plantas medicinales para el tratamiento del envenenamiento ofídico en Centroamérica, a pesar de que las terapias basadas en plantas son de uso tradicional en la región. Este trabajo recopiló información sobre los estudios realizados en Centroamérica para evaluar la capacidad de extractos de plantas de uso etno-médico para inhibir las actividades tóxicas del veneno de Bothrops asper, la serpiente responsable de aproximadamente la mitad de los envenenamientos ofídicos en Centroamérica. La búsqueda priorizó la descripción de plantas utilizadas en Gua-temala, ya que la mayoría de los estudios aquí descritos fueron realizados en ese país. También se incluyó la descripción de los metabolitos secundarios y otras actividades farmacológicas de las especies evaluadas, que podrían explicar su uso como antiofídicos. La búsqueda de literatura se realizó en las bases de datos de Google Scholar, PubMed, Scopus, y se completó con literatura disponible localmente. Se determinó que 12 extractos de plantas inhibieron el efecto hemorrágico del veneno y tres el efecto edematígeno; la actividad proteolítica fue inhibida por extractos de tres especies y la fosfolipasa A2 (PLA2) por una especie. Solamente Brownea rosa-de-monte demostró inhibir efectivamente el efecto coagulante del veneno in vitro. Algunos extractos de las plantas tamizadas en Guatemala demostraron efectos procoagulantes o anti-trombina intrínsecos, que podrían agravar las alteraciones inducidas por el veneno en la coagulación. Estos hallazgos enfatizan la necesidad de validar el potencial de extractos de plantas centroamericanas y sus metabolitos secundarios para neutralizar el veneno de B. asper.

Inhibición de las actividades proteolítica y fosfolipasa A2 del veneno de Bothrops asper por el extracto etanólico de Neurolaena lobata (L) Cass / Inhibition of proteolytic and phospholipase A2 activities of Bothrops asper venom by the ethanolic extract of Neurolaena lobata (L) Cass

Neurolaena lobata es utilizada tradicionalmente en Centroamérica para tratar la mordedura de serpiente, pero su efectividad para contrarrestar el envenenamiento producido por Bothrops asper ha sido poco estudiada. Se evaluó la capacidad del extracto etanólico de sus hojas para inhibir las actividades proteolítica, fosfolipasa A2 (PLA2; evaluada como hemólisis indirecta) y coagulante del veneno in vitro. El material vegetal fue colectado en Izabal, Guatemala, secado, se hicieron extracciones con etanol y se evaluó la presencia de actividades proteolítica, PLA2 y coagulante in-trínsecas en ensayos de concentración-actividad. Los efectos inhibitorios de la actividad proteolítica y PLA2 del veneno se evaluaron después de pre-incubar concentraciones variables del extracto con concentraciones fijas de veneno. La inhibición de la actividad coagulante del veneno no fue evaluada porque el extracto presentó actividad anticoagulante intrínseca dependiente de la concentración. El extracto inhibió completamente las actividades proteolítica (CE50 = 15.7 µg/µl) y PLA2 (CE50 = 32.5 µg/µl) del veneno. El análisis fitoquímico utilizando ensayos macro y semimicrométricos de cromatografía en capa fina, demostró la presencia de flavonoides, cumarinas, saponinas, taninos, sesquiterpenlactonas y aceites esenciales en el extracto. Su efecto sobre las proteínas del veneno se evaluó por electroforesis SDS-PAGE, mostrando cambios en el patrón electroforético atribuidos a la formación de complejos moleculares con los metabo-litos del extracto. Los resultados indican que el extracto podría inhibir los efectos tóxicos del veneno inducidos por las metaloproteinasas dependientes de zinc (SVMPs) y PLA2s, pero podría afectar las alteraciones en la coagulación, coadyuvando en la desfibrinogenación inducida por el veneno.

Neurolaena lobata has been used by traditional healers in Central America to treat snakebite, but its ability to neutralize Bothrops asper envenomations needs to be proved. This study evaluated the inhibitory potential of the ethanolic extract of the leaves of N. lobata against proteolytic, phospholipase A2 (PLA2) and coagulant activities of the venom in vitro. Leaves were collected in Izabal, Guatemala, dried, extracted with ethanol and concentration-response assays were conducted to detect intrinsic proteolytic, PLA2 (evaluated as indirect hemolysis) and coagulant activities. Assays for anti-proteolytic and anti-PLA2 activities were performed after pre-incubation of several amounts of extract with a fixed concentration of venom. Inhibition assay for the coagulant effect of the venom was not tested because pre-incubation of thrombin with the extract prolonged the clotting time of plasma in a concentration-dependent manner. Proteolytic (EC50 = 15.7 µg/µl) and PLA2 (EC50 = 32.5 µg/µl) activities of the venom resulted completely inhibited by the extract. Phytochemical profiles, determined by micrometric assays and semi microanalysis by thin layer chro-matography, showed the presence of flavonoids, coumarins, saponins, tannins, sesquiterpene lactones and essential oils in the extract. SDS-PAGE was used to assess the action of the extract on the venom proteins. Results showed changes in the electrophoretic profile, probably due to the formation of insoluble complexes with plant specialized metabolites. These findings demonstrated that the extract could be able to inhibit toxic effects triggered by zinc-dependent snake venom metalloproteinases (SVMPs) y PLA2s but might aggravate the alterations induced by the venom in coagulation.

Neutralización de la actividad letal del veneno de serpiente Bothrops atrox por suero hiperinmune de llama (Lama glama) / Neutralization of the lethal activity from Bothrops atrox venom by hyperimune llama serum (Lama glama)

RESUMEN Objetivos: Evaluar la capacidad del suero hiperinmune de llama (Lama glama) para neutralizar la letalidad del veneno de la serpiente Bothrops atrox en ratones de laboratorio. Materiales y métodos: Se calculó la dosis letal media (DL50) de un pool de venenos de serpientes de Bothrops atrox de Perú, y se midieron los títulos de anticuerpos por ensayo ELISA; así como la potencia de neutralización del suero inmune por el cálculo de la dosis efectiva media (DE50) durante el periodo de inmunización. Resultados: La DL50 del veneno fue de 3,96 µg/g, similar a otros trabajos realizados en Bothrops atrox en Perú. Los títulos de anticuerpos contra el veneno se incrementan rápidamente en la llama mostrando una rápida respuesta inmune; sin embargo, la capacidad de neutralización se incrementa más lentamente y requiere de varias dosis y refuerzos de las inmunizaciones alcanzado una DE50 de 3,30 µL/g ratón y una potencia de neutralización 3,6 mg/mL después de 15 inmunizaciones. Conclusiones: El suero hiperinmune de llama es capaz de neutralizar la letalidad del veneno de la serpiente Bothrops atrox de Perú en ratones de laboratorio.

ABSTRACT Objectives: To evaluate the capacity of the hyperimmune llama serum (Lama glama) to neutralize the lethal activity of Bothrops atrox venom in laboratory mice. Materials and methods: Mean lethal dose (LD50) was calculated from a Bothrops atrox venom sample pool from Peru. The antibody titers were measured by ELISA assay; and the immune serum neutralization potency was measured by calculating the mean effective dose (ED50) during the immunization period. Results: The venom's LD50 was 3.96 μg/g; similar to what was found in other studies about Bothrops atrox carried out in Peru. The titers of antibodies against the venom increased rapidly in the llama, demonstrating a fast immune response; however, the neutralization capacity increased slowly and required several doses and immunization reinforcements, obtaining a ED50 of 3.30 μL/g mouse and a neutralization potency of 3.6 mg/mL after 15 immunizations. Conclusions: The hyperimmune llama serum is able to neutralize the lethality of the Bothrops atrox venom from Peru in laboratory mice.

Anti-ophidian activity of Bredemeyera floribunda Willd. (Polygalaceae) root extract on the local effects induced by Bothrops jararacussu venom

Bredemeyera floribunda roots are popularly used to treat snakebites in the semiarid region of Northeast Brazil, and previous studies indicate the anti-ophidian actions of triterpenoid saponins found in its roots. To assess B. floribunda root extract (BFRE) activity against the effects of Bothrops jararacussu venom (BjuV), antiphospholipasic, antiproteolytic, antihemorrhagic, antinecrotic, and anti-edematogenic activities were investigated in mice. Phytochemical analysis revealed the presence of saponins, flavonoids, and sugars, with rutin and saccharose being the major constituents of BFRE. Acute toxicity was determined and BFRE was nontoxic to mice. Phospholipase A2 and proteolytic activities induced by BjuV were inhibited in vitro by BFRE at all concentrations tested herein. BFRE (150 mg/kg) inhibited paw edema induced by BjuV (50 µg/animal), reducing total edema calculated by area under the curve, but carrageenan-induced paw edema was unchanged. Hemorrhagic and necrotizing actions of BjuV (50 µg/animal) were considerably decreased by BFRE treatment. Thus, BFRE blocked the toxic actions of B. jararacussu venom despite having no anti-inflammatory activity, which points to a direct inhibition of venom's toxins, as demonstrated in the in vitro assays. The larger amounts of rutin found in BFRE may play a role in this inhibition, since 3′,4′-OH flavonoids are known inhibitors of phospholipases A2.

Preclinical efficacy against toxic activities of medically relevant Bothrops sp. (Serpentes: Viperidae) snake venoms by a polyspecific antivenom produced in Mexico / Eficacia de un antiveneno poliespecífico producido en México para neutralizar, a nivel preclínico, las actividades tóxicas de venenos de Bothrops sp (Serpentes: Viperidae) de importancia médica

Abstract:The assessment of the preclinical neutralizing ability of antivenoms in Latin America is necessary to determine their scope of efficacy. This study was aimed at analyzing the neutralizing efficacy of a polyspecific bothropic-crotalic antivenom manufactured by BIRMEX in Mexico against lethal, hemorrhagic, defibrinogenating and in vitro coagulant activities of the venoms of Bothrops jararaca (Brazil), B. atrox (Perú and Colombia), B. diporus (Argentina), B. mattogrossensis (Bolivia), and B. asper (Costa Rica). Standard laboratory tests to determine these activities were used. In agreement with previous studies with bothropic antivenoms in Latin America, a pattern of cross-neutralization of heterologous venoms was observed. However, the antivenom had low neutralizing potency against defibrinogenating effect of the venoms of B. atrox (Colombia) and B. asper (Costa Rica), and failed to neutralize the in vitro coagulant activity of the venom of B. asper (Costa Rica) at the highest antivenom/venom ratio tested. It is concluded that, with the exception of coagulant and defibrinogenating activities of B. asper (Costa Rica) venom, this antivenom neutralizes toxic effects of various Bothrops sp venoms. Future studies are necessary to assess the efficacy of this antivenom against other viperid venoms. Rev. Biol. Trop. 65 (1): 345-350. Epub 2017 March 01.

ResumenEs necesario estudiar a nivel preclínico la capacidad neutralizante de los antivenenos producidos en América Latina, para conocer su espectro de cobertura. En este estudio se analizó la eficacia preclínica de un antiveneno poliespecífico botrópico-crotálico producido por BIRMEX, en México, para neutralizar los efectos letal, hemorrágico, desfibrinogenante y coagulante in vitro de los venenos de Bothrops jararaca (Brasil), B. atrox (Perú y Colombia), B. diporus (Argentina), B. mattogrossensis (Bolivia) y B. asper (Costa Rica). Se emplearon metodologías de laboratorio estándar en los análisis. En consonancia con estudios anteriores con diversos antivenenos botrópicos en América Latina, se observó un amplio patrón de neutralización de estos venenos heterólogos en la mayoría de los efectos estudiados. Sin embargo, el antiveneno mostró una baja capacidad neutralizante contra el efecto desfibrinogenante de los venenos de B. atrox (Colombia) y B. asper (Costa Rica) y no neutralizó la actividad coagulante in vitro del veneno de B. asper (Costa Rica) a la máxima razón antiveneno/ veneno empleada.

Validación del uso tradicional de especies de asclepias contra el veneno de bothrops diporus (yarará chica) en el nordeste de Argentina / Validation of the traditional use of asclepias species against bothrops diporus (yarará chica) venom in northeast of Argentina

La familia Asclepiadaceae posee tradición en etnomedicina. En el nordeste argentino, A.mellodora y A. curassavica se utilizan como cataplasmas en accidentes de ofidios. En este trabajo, los extractos acuosos, etanólicos y hexánicos de A. mellodora y A. curassavica se evaluaron por SDS-PAGE para determinar su actividad alexitérica. El estudio in vitro de la capacidad inhibitoria de las actividades proteolítica, hemolítica indirecta y coagulante permitieron determinar que ambas especies manifiestan actividad, siendo A. mellodora más activa. Estadísticamente los extractos de A. mellodora fueron igualmente activos contra el veneno de Bothrops diporus y no mostraron diferencias significativas respecto del órgano utilizado en la inhibición de la actividad coagulante. Este resultado está en consonancia con la forma tradicional de su uso como cataplasma. Sobre el extracto etanólico de las raíces de A. mellodora se realizó un fraccionamiento bioguiado que permitió identificar fracciones de compuestos responsables de la actividad.

The Aclepiadaceae family has been reported by its use in ethnomedicine. In the northeast of Argentina, A. mellodora and A. curassavica are traditionally used in ofidic accidents as poultices. In this work, aqueous, alcoholic and hexanoic extracts were analyzed by SDS-PAGE to determine their anti-snake activity. The in vitro study of the inhibitory ability of the following activities: proteolytic, indirect hemolytic activity and inhibition of the coagulant activity, allowed demonstrating that both species were active against venom, being A. mellodora the most active. Statistically, all extracts of A. mellodora were active against venom in the inhibition of the coagulant activity, without significant differences with respect to the organ used; which is consistent with the traditional use as external poultice. The alcoholic extract of A. mellodora roots was subjected to a bio-guided separation. The fractions obtained were enriched in compounds which could probably be responsible for the activity against venom.

Caracterización tóxica del veneno de Bothrops (Rhinocerophis) alternatus de diferentes regiones de la provincia de Córdoba (Argentina) / [The toxicity of venom of Bothrops (Rhinocerophris) alternatus in different areas of Cordoba State in Argentina].

Snake venoms can show biochemical and toxicological variability even in specimens from the same specie. The geographical localization of the snakes is one of the factors that can influence those variations. By these reasons the venom from specimens of Bothrops (Rhinocerophis) alternatus ("crucera", "yararágrande"), one of the snakes of highest medical importance in Argentina, from three different regions of Córdoba was studied. Lehtal potency, hemorrhagic, coagulant on plasma and thrombin like activities as well as the electrophoretic patterns of venom from snakes of Calamuchita, Traslasierras and the East of the province were determined. The venom from the snakes of the three regions showed the characteristic activities of the venom of the majority of Bothrops, causing hemorrhage, hemostatic disturbances acting on plasma or directly on fibrinogen with a "thrombin like activity". The different samples were very similar regarding their biochemical characteristics and toxic potencies at difference of previous observations on venoms from the same specie in different regions of other provinces fro Argentina. Bivalent antivenom, the one used by the Provincial Ministry of Health to treat the bothropic accidents, neutralized in all the cases the toxic activities of the venom in very similar range of neutralizing potency.

Pulsed ultrasound therapy accelerates the recovery of skeletal muscle damage induced by Bothrops jararacussu venom

We studied the effect of pulsed ultrasound therapy (UST) and antibothropic polyvalent antivenom (PAV) on the regeneration of mouse extensor digitorum longus muscle following damage by Bothrops jararacussu venom. Animals (Swiss male and female mice weighing 25.0 ± 5.0 g; 5 animals per group) received a perimuscular injection of venom (1 mg/kg) and treatment with UST was started 1 h later (1 min/day, 3 MHz, 0.3 W/cm², pulsed mode). Three and 28 days after injection, muscles were dissected and processed for light microscopy. The venom caused complete degeneration of muscle fibers. UST alone and combined with PAV (1.0 mL/kg) partially protected these fibers, whereas muscles receiving no treatment showed disorganized fascicules and fibers with reduced diameter. Treatment with UST and PAV decreased the effects of the venom on creatine kinase content and motor activity (approximately 75 and 48%, respectively). Sonication of the venom solution immediately before application decreased the in vivo and ex vivo myotoxic activities (approximately 60 and 50%, respectively). The present data show that UST counteracts some effects of B. jararacussu venom, causing structural and functional improvement of the regenerated muscle after venom injury.

SDS-PAGE: una herramienta útil en la evaluación preliminar de la actividad alexítera de extractos vegetales / SDS PAGE: a useful tool for preliminary screening of antisnake activity of plant extracts

Some results obtained during our research work in the search of anti-snake compounds from plant origin, allow us to propose sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a valuable method for a fast and reliable screening in order to evaluate plant extracts activity on snake proteins from Bothrops diporus (yarará chica). Such approach will allow to process a larger number of plant extracts and to select the active ones. Venoms used in this study came from B. diporus which was previously vacuum dried. Extracts (aqueous, alcoholic and hexanic) were from native plants: Aristolochia elegans, Aristolochia gibertii, Asclepia curassavica, Cissampelos pareira, Dorstenia brasiliensis, Eclipta prostrata, Iresine diffusa, Mikania micrantha, M. periplocifolia, M. coridifolia, Nectandra angustifolia, N. megapotamica, Sapium haematospermum and Trixis divaricata. The results obtained by SDS-PAGE were compared with those obtained from in vitro assays (coagulation and hemolysis inhibition). The correlation between results obtained from electroforetic and in vitro assays allowed to suggest SDS-PAGE as a suitable technique to assist in preliminary plant screenings for anti-snake activity by snake venom protein interaction with plant compounds.

El desarrollo de nuestro trabajo de investigación en la búsqueda de compuestos alexíteros de origen vegetal nos permite proponer la electroforesis en geles de poliacrilamida en condiciones desnaturalizantes, como método de screening rápido y confiable, para evaluar la actividad de extractos vegetales sobre proteínas del veneno de yarará, de manera de procesar mayor número de muestras vegetales y seleccionar aquellas que son activas. Para el desarrollo de la metodología, se utilizó un pool de veneno de Bothrops diporus desecado al vacío y extractos acuosos, alcohólicos y hexánicos de plantas autóctonas Aristolochia elegans, A. gibertii, Asclepia curassavica, Cissampelos pareira, Dorstenia brasiliensis, Eclipta prostrata, Iresine diffusa, Mikania micrantha, M. periplocifolia, M. coridifolia, Nectandra angustifolia, N. megapotamica, Sapium haematospermum y Trixis divaricata. Se realizaron pruebas in vitro (inhibición de la coagulación y hemólisis) para contrastar con los resultados obtenidos por SDS-PAGE. La correlación de los resultados obtenidos con técnicas in vitro validadas, permite sugerir el empleo de la técnica de SDS-PAGE como una herramienta útil en la evaluación preliminar de la actividad alexítera de extractos vegetales, propiedad evidenciada por la modificación en el perfil de bandas proteicas cuando se compara el veneno puro con el producto de la interacción extracto vegetal-veneno.

Anti-venom potential of aqueous extract of stem bark of Mangifera indica L. against Daboia russellii (Russell’s viper) venom.

Several plant extracts rich in pharmacologically active compounds have shown to antagonize venom of several species. Mangifera indica has been used against snakebite by the traditional healers, However, there is paucity of scientific data in support. In this study, we evaluated the antivenom potential of aqueous extract of stem bark of M. indica against D. russellii venom-induced pharmacological effects such as life myotoxicity, edema, LD50 etc. The extract inhibited the phospholipase, protease, hyaluronidase, 5`nucleotidase, ATPase and alkaline phosphomonoesterase activities with varying IC50 values. It significantly inhibited both metalloproteases and serine proteases activities. Further, the extract significantly reduced the myotoxicity of the venom, as evident by the reduction of serum creatin kinase and lactate dehydrogenase activities. Though the extract completely inhibited in vitro PLA2 activity, it was unable to completely inhibit in situ hemolytic and in vivo edema-inducing activities, usually brought about by PLA2s. In lethality studies, co-injection of the venom preincubated with the extract showed higher protection than the independent injection of venom, followed by the extract in the mice. However, in both the cases the extract -a cocktail of inhibitors significantly increased the survival time, when compared to that of mice injected (i.p) with the venom alone. These results encourage further studies on the potential use of cocktail of inhibitors in improving the treatment of snake envenomation. Further, this study substantiates the use of M. indica as an antidote against snakebite by the traditional healers.

In vivo pharmacological study on the effectiveness of available polyclonal antivenom against Hemiscorpius lepturus venom

The available Razi Institute antivenom is still, empirically, used by intramuscular (IM) administration for the treatment of scorpion stings in humans by six medically dangerous species including Hemiscorpius lepturus (H. lepturus). The aim of this study was to assess the neutralizing ability and effectiveness of the antivenom in inhibiting hemoglobinuria, biochemical changes, increased microalbuminuria and urinary lactate dehydrogenase (LDH) following H. lepturus sting. Simultaneous intramuscular administration of 10 μL and 100 μL of antivenom, after 24 hours, had no significant preventive effect on the extent and degree of hemoglobinuria or proteinuria produced in venom-treated rats. After IM administration of antivenom, no significant changes in decreased red blood cell (RBC) count and hemoglobin were observed. Immediate intramuscular administration of 10 μL of antivenom had no significant effects on both LDH and microalbuminuria. The present findings did not present correlation with clinical signs. Therefore, to fully assess the efficacy of the available antivenom and make appropriate recommendations, more in vivo or in vitro investigations including antigen-antibody interaction, enzymatic analysis and route-dependent administration are required.(AU)

Características biológicas e inmunológicas del veneno de Bothrops cotiara (Serpentes: Viperidae) / Biological and immunological characteristics of the poison of Bothrops cotiara (Serpentes: Viperidae)

Bothrops cotiara is a venomous snake sporadically found in the province of Misiones in Argentina, South of Brazil and Paraguay. Data on the clinics of the envenomation produced by its bite and on its venom are scarce. There is no information on the neutralizing capacity of the antivenoms available. In this study, the lethal potency, hemorrhagic, necrotizing, coagulant and thrombin-like, defibrinogenating, indirect hemolytic and fibrinolytic activities of the venom of B. cotiara specimens from the province of Misiones were determined. The toxic activities were within the range of those described for the other Bothrops species from Argentina, and the electrophoretic and chromatographic studies showed similarities with those described for the other bothropic venoms. The immunochemical reactivity of six South American anti Viper antivenoms (ELISA) have a strong reactivity with all the antivenoms studied. The neutralizing capacity of three of these therapeutic antivenoms against the lethal potency and hemorrhagic, necrotizing, coagulant, thrombin-like and hemolytic activities showed a very close neutralizing capacity. Our data strongly suggest that the antivenoms for therapeutic use available in this area of South America are useful to neutralize the toxic and enzymatic activities of the venom of this uncommon specie of Bothrops.

Bothrops cotiara es una serpiente que se encuentra en la provincia de Misiones (Argentina), el Sur de Brasil y Paraguay. La información sobre las características clínicas de los accidentes por esta serpiente es muy escasa y existen pocos datos sobre su veneno y la capacidad neutralizante de las actividades tóxicas del mismo por antivenenos terapéuticos. En este trabajo se estudiaron características bioquímicas, actividades tóxicas y la reactividad inmunoquímica del veneno de B. cotiara. Seis antivenenos anti Viperinos Sudamericanos fueron estudiados frente a este veneno por el método ELISA y se probó la capacidad neutralizante de tres de estos frente a las actividades hemorrágica, necrotizante, procoagulante, trombina-símil, hemolítica indirecta y la potencia letal de veneno de ejemplares de B. cotiara de la provincia de Misiones. Los patrones cromatográficos y electroforéticos mostraron características similares a los de otros venenos de Bothrops. Las actividades tóxicas estuvieron dentro de los ámbitos descritos para los venenos botrópicos. Los seis antivenenos mostraron gran reactividad inmunoquímica por ELISA y las potencias neutralizantes de los tres estudiados fueron muy próximas para las actividades letal, hemorrágica, necrotizante, hemolítica indirecta, coagulante y trombina-símil. Los resultados de los estudios de neutralización indicarían que ante la mordedura de esta poco común especie de Bothrops, pueden usarse los diferentes tipos de antivenenos botrópicos o botrópico-crotálicos para uso terapéutico disponibles en esa región.

Rabbit IgG antibodies against Phospholipase A2 from Crotalus durissus terrificus neutralize the lethal activity of the venom

Crotalus durissus terrificus (C.d.t.) (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2) and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75). Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 µg) with Freund adjuvant. Groups of six mice (20 + 2 g) were inoculated with 0.5 ml i.p. of C. d. t. venom (4 µg) or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms

El veneno de Crotalus durissus terrificus (C.d.t.) (Cascabel de Sud América) posee actividad miotóxica y neurotóxica, actividades que también exhibe el complejo crotoxina, principal componente tóxico de este veneno. El complejo crotoxina está constituido por una fosfolipasa A2 básica (PLA2) y una proteína acídica no tóxica, el crotapotín. En este trabajo se estudió la capacidad neutralizante de anticuerpos IgG anti-PLA2 sobre la letalidad inducida por el veneno entero. El antígeno PLA2, fue aislado por cromatografía de filtración en gel (Sephadex G-75). Se inocularon conejos machos por vía subcutánea e intramuscular, con 700 µg de PLA2 y adyuvante para la obtención de anticuerpos específicos. La capacidad neutralizante del antisuero se analizó en ratones por inoculación con diluciones de veneno entero preincubado con un volumen adecuado de anticuerpos IgG anti-PLA2. Se inocularon ratones controles con 0.5 ml i.p. de veneno (4 µg.ml-1). El número de muertes fue contabilizado a las 24 y 48 h posteriores a la inoculación, demostrándose que la capacidad neutralizante de los anticuerpos IgG anti-PLA2 fue superior a la obtenida con el antiveneno crotálico. Los resultados obtenidos demuestran la potencial aplicación de antivenenos constituidos por anticuerpos específicos contra PLA2, y/o la inclusión de estos anticuerpos como suplementos en antivenenos polivalentes

Severe systemic effects of Merrem's hump-nosed viper bite.

OBJECTIVE: To report unpredictable severe systemic effects of hump-nosed viper envenomation. SETTING: Medical unit, General hospital, Anuradhapura. METHODS: The clinical outcome of seven patients bitten by Merrem's hump-nosed viper were monitored until recovery or death. Limited autopsies were performed on the latter. Offending snakes were positively identified by medical officers and in one instance by a herpetologist. CONCLUSION: Merrem's hump-nosed viper bites, caused an array of potentially fatal systemic manifestations. One patient developed neurological effects, severe Raynaud's syndrome leading to ascending gangrene of distal limbs and adult repiratory distress syndrome (ARDS), in addition to the known complications of severe renal cortical necrosis and haemostatic dysfunction. Two patients who developed acute renal failure and prolonged coagulopathy recovered completely. The combination of extensive renal cortical necrosis, disseminated intravascular coagulation, and ARDS proved fatal in three.

The immunochemical reactivity and neutralizing capacity of polyvalent Vipera (European) antivenom on enzymatic and toxic activities in the venoms of Crotalids from Argentina

The immunochemical reactivity and neutralizing capacity of polyvalent Vipera antivenom (Vipera ammodytes, Vipera aspis, Vipera berus, Vipera lebetina, and Vipera xanthina) were tested on the enzymatic and biological activities of Crotalus durissus terrificus and the following Bothrops venoms from Argentina (Bothrops alternatus, Bothrops ammodytoides, Bothrops neuwiedii, Bothrops jararaca, Bothrops jararacussu, and Bothrops moojeni). The Vipera antivenom reacted weakly when tested by double immunoprecipitation (DIP) and reacted with all the venoms when tested by ELISA. Several components in all the venoms studied were recognized in Western blots. Vipera antivenom deactivated to different degrees in vitro procoagulant, (indirect) hemolytic, and proteolytic activities in all the venoms studied. Preincubation of Bothrops alternatus venom with Vipera antivenom neutralized a lethal potency of 4.5 LD50 in mice with an ED50 of 1.25 ñ 0.25 µl per µg of venom, and with 1.0 µl/µg inhibited 54 per cent of the hemorragic activity and 48 per cent of necrotic activity. Vipera antivenom (2.0 µl per µg toxin) inhibited the phospholipase A2 activity of purified crotoxin and decreased its lethal potency by 60 per cent, while the neutralizing capacity on the lethal potency of crude Crotalus durissus terrificus venom was poor even at a level of 5.0 µl/µg of venom.

Farmacología de venenos y antivenenos de serpiente / Pharmacology on poisons and antipoisonss of snake

Contiene: 1. Estudio farmacológico del veneno de Lachesis muta muta "SHUSHUPE"; 2. Antivenenos

Methodological care in the evaluation of the LD50 and of the neutralization of the lethal effect of Crotalus terrificus venom by the plant Peschiera fuchsiaefolia (Apocynaceae)

In the present study, we demonstrate that the volumes in which a given protein mass of Crotalus durissus terrificus venom or of a lyophilized stabilized aqueous extract (LSAE) of Peschiera fuchsiaefolia, an antivenom agent injected intramuscularly, have a decisive influence on the results. The LD50 of C. d. terrificus venom injected i.m. in a final volume of 200µg (2µl/g) (saline solution, 0.9 per cent NaCl) was 180µg/100g rat body weight (p<0.05, 161 to 202µ/100g body weight) and the LD50 of the venom injected i.m. in a volume of 50µl (0.5µl/g) was 120µg/100g body weight (p<0.05, 107 to 134µg/100g body weight). The reduction of the final volume injected i.m. also required a reduced mass of LSAE necessary to neutralize the lethal effect of C. d. terrificus venom. The dose of 60mg LSAE/100g rat body weight in a final volume of 200µl administered i.m., 20 seconds after venom injection, and that of 40mg LSAE/100g body weight/200µl mixed and incubated with the venom for 1h at 25º before i.m. injection were able to neutralize the lethal activity of 2LD50. However, the LSAE doses that neutralized the 2LD50 were reduced to 20mg LSAE/100g body weight in a final volume of 50µl when administered i.m. 20 seconds after venom injection and to 2.5mg LSAE/100g body weight/50µl when mixed and incubated for 1h at 25º with the venom i.m. injection. The LD50 of C. d. terrificus venom and the doses of P. fuchsiaefolia LSAE that neutralized the venom lethal activity were, therefore, significantly lower when the final volume injected i.m. was reduced.

Estudo experimental da cinética do veneno e do antiveneno do escorpiäo Tityus serrulatus no soro e tecidos de camundongos / Experimental study of the kinetics of Tityus serrulatus scorpion venom in serum and tissues of mices

O presente trabalho descreve a farmacocinética (do envenenamento experimental) da peçonha do escorpiäo Tityus serrulatus, a farmacocinética do antiveneno escorpiônico e os efeitos da sua administraçäo sobre as concentraçöes do veneno no soro e em vários tecidos de camundongos da raça CF1, determinados através da técnica de ELISA descrita por Chávez-Olórtegui et al. (1994). Descreve também a distribuiçäo tecidual da peçonha do escorpiäo Tityus serrulatus determinada através de imunohistoquímica...

Capacidad neutralizante de los sueros antiofidicos frente al veneno de Bothrops moojeni (Caisaca, Lanzadera) / Neutralizing ability of antiophidic serum against the venom of Bothrops moojeni (lance-headed viper)

La serpiente Bothrops moojeni ("caisaca", "lanzadera", "lance-headed viper") es un crotalíneo de frecuencia de aparción escasa en la Argentina. Solamente se la ha encontrado y clasificado correctamente en la provincia de Misiones. Se realizó el estudio del veneno por electroforesis en condiciones no desnaturalizantes y en SDS-PAGE y se determinó la Dosis Letal 50. La reactividad inmunoquímica frente a los antivenenos botrópicos Bivalente y Tetravalente y por el Botrópico-Crotálico producidos por el Instituto Nacional de producción de Biológicos A.N.L.I.S Dr. Carlos G. Malbrán se determinó por doble inmunoprecipitación, inmunoelectroforesis, ELISA y Western-blot. Se estudió la capacidad neutralizante de estos sueros frente a las actividades letal, hemorrágica, necrótica, proteolítica, hemolítica indirecta (fosfolipásica) y procoagulante del veneno. Estos estudios dieron como resultado un muy alto grado de reactividad inmunoquímica y de neutralización heteróloga de las diferentes actividades del veneno por los antivenenos probados. Se puede concluir que los sueros antiofídicos antibotrópicos o botrópico-crotálico, distribuídos gratuitamente a las Delegaciones Sanitarias del país por el Ministerio de Salud y Acción Social, podrían ser utilizados ante los accidentes ofídicos por Bothrops moojeni debido a que neutralizan eficientemente las actividades tóxicas del veneno de este ofidio.

A comparative study of severe scorpion envenomation in children caused by Tityus bahiensis and Tityus serrulatus

No periodo de janeiro de 1984 a maio de 1994, de 239 criancas com ate 15 anos de idade, picadas por escorpioes pertencentes as especies T. bahiensis (84,9 por cento) e T. serrulatus (15,1 por cento), 17 apresentaram envenenamento grave...