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.

Optimización en la recuperación de inmunoglobulinas, como molécula entera y fragmento F(ab´)2, en la producción de antivenenos / Optimization in the immunoglobulins recovery, as whole molecule and F(ab´)2 fragment, in the antivenim production

En la actualidad se utilizan, principalmente, dos métodos de purificación de anticuerpos a partir de plasmas equinos hiperinmunes para la producción de antivenenos a nivel industrial, obteniéndose preparaciones enriquecidas en moléculas de inmunoglobulinas G ó fragmentos F(ab´)2. Con ambos métodos, luego de la precipitación, se observa una importante pérdida de capacidad neutralizante en comparación con la capacidad neutralizante de los plasmas de partida. En este trabajo, se realizó el fraccionamiento de plasmas equinos hiperinmunes utilizando ácido caprílico con y sin digestión enzimática con pepsina. El objetivo del trabajo fue dar a conocer la proporción de recuperación de la capacidad neutralizante luego del fraccionamiento; resultando ésta menor cuando el plasma se trató enzimáticamente. Adicionalmente, se propuso establecer cuál sería la etapa responsable de la diferencia en la recuperación de anticuerpos entre una metodología y otra. Cuando se purificaron las inmunoglobulinas enteras, se recuperó aproximadamente un 53% de la capacidad neutralizante mientras que cuando la muestra se purificó luego de ser tratada enzimáticamente, se obtuvo alrededor del 30% de esa actividad. Una relación de similar magnitud se verifica en la recuperación de la masa de proteínas solubles luego de remover los contaminantes, entre una metodología y otra. La insolubilización del fragmento Fc generado durante la digestión sería el responsable de esa pérdida adicional de proteína y capacidad neutralizante.

Today two methods are mainly used for the purification of antibodies from hyperimmune equine plasma at industrial level obtaining enriched preparations of immunoglobulin G (IgG) molecules or F(ab´)2 fragments. In both methods, after the precipitation, an important loss in the neutralizing capability was observed compared to the one of the original plasma. In this work, we performed the fractionation of hyperimmune equine plasma using caprylic acid, with and without enzymatic digestion with pepsin. The aim was to explain the percentage of recovery of the neutralizing capability after the fractionation; which resulted minor when the plasma was enzymatically treated. Additionally, we intended to establish which stage, in the purification process, was the responsible for the difference in the antibody recovery between one methodology and the other. When entire immunoglobulins were purified, approximately 53% of the neutralizing capacity was recovered, but when the sample was purified after the enzymatic treatment, around the 30% of the activity was obtained. A ratio of similar magnitude is verified on the recovery of the soluble protein mass after the removal of contaminants, between the two methods. The insolubilization of the fragment Fc generated during digestion would be responsible for the additional loss of protein and neutralizing capacity.

CORAL SNAKE ANTIVENOM PRODUCED IN CHICKENS (Gallus domesticus) / Antiveneno de serpiente coral producido en gallinas (Gallus domesticus)

The production of anti-snake venom from large mammal's blood has been found to be low-yielding and arduous, consequently, antivenom immunoglobulins for treatment are achieved regularly as polyvalent serum. We have standardized an undemanding technique for making purified immunoglobulin IgY antivenom consisting of polyclonal antibodies against coral snake venom in the egg yolk of immunized hens. We have adapted a reported process of antibody purification from egg yolks, and achieved 90% antibody purity. The customized technique consisted of the removal of lipids from distilled water-diluted egg yolks by a freeze–thaw sequence. The specific immunoglobulins were present in the egg yolk for up to 180 days postimmunization. Therefore, by means of small venom quantities, a significant amount of immunoglobulins were found in an adequately purified state (The obtained material contained about 90% pure IgY). The antigen binding of the immunoglobulins was detected by a double immunodiffusion test. Titers of antibodies in the yolk were estimated with a serum protection assay (Median effective dose = ED50) (ED50= 477 mg/kg). Given that breeding hens is economically feasible, egg gathering is noninvasive and the purification of IgY antibodies is quick and easy, chicken immunization is an excellent alternative for the production of polyclonal antibodies. To the best of our knowledge, this is the first coral snake antivenom prepared in birds.

La producción de antiveneno de serpiente usando sangre de grandes mamíferos se ha encontrado que es de bajo rendimiento y de trabajo arduo, en consecuencia, las inmunoglobulinas antiveneno para el tratamiento se obtienen generalmente, como suero polivalente. Hemos estandarizado una técnica poco exigente para la fabricación de inmunoglobulina purificada IgY, que consistió en generar anticuerpos policlonales contra el veneno de la serpiente coral en huevos de gallinas inmunizadas. La técnica consistió en la eliminación de lípidos de las yemas del huevo, diluidas en agua y en una secuencia de congelación-descongelación. Las inmunoglobulinas específicas estuvieron presentes en la yema de huevo hasta 180 días después de la inmunización. La unión del antígeno a las inmunoglobulinas se detectó mediante un ensayo de inmunodifusión doble. Los títulos de anticuerpos en la yema fueron estimados con un ensayo de protección (dosis efectiva media = ED50). Dado que las gallinas reproductoras son económicamente viables, la recolección de huevos es no invasiva y la purificación de anticuerpos IgY es rápida y fácil, la inmunización de la gallina es una excelente alternativa para la producción de anticuerpos policlonales. A nuestro entender, esta es el primer anti-veneno contra serpiente de coral preparado en aves.

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.

Antisnake venom activity of ethanolic seed extract of Strychnos nux vomica Linn.

The whole seed extract of S. nux vomica (in low doses) effectively neutralized Daboia russelii venom induced lethal, haemorrhage, defibrinogenating, PLA2 enzyme activity and Naja kaouthia venom induced lethal, cardiotoxic, neurotoxic, PLA2 enzyme activity. The seed extract potentiated polyvalent snake venom antiserum action in experimental animals. An active compound (SNVNF) was isolated and purified by thin layer chromatography and silica gel column chromatography, which effectively antagonised D. russelii venom induced lethal, haemorrhagic, defibrinogenating, oedema, PLA2 enzyme activity and N. kaouthia induced lethal, cardiotoxic, neurotoxic, PLA, enzyme activity. Polyvalent snake venom antiserum action was significantly potentiated by the active compound. Spectral studies revealed it to be a small, straight chain compound containing methyl and amide radicals. Detailed structure elucidation of the compound (SNVNF) is warranted before its clinical trials as a snake venom antagonist.

New findings on the purification and characterization of an antibothropic factor from Didelphis marsupialis (opossum) serum

We have used DEAE-Sephacel and Sephacryl S-200 to separate protein fractions from Didelphis marsupialis serum capable of protecting mice from the lethal effect of Bothrops jararaca venom. the fractions separated were homogeneous by conventional electrophoresis using cellulose acetate and polyacrylamide; however, they were heterogeneous on PAGE-SDS, showing similar electrophoretic patterns with or without mercaptoethanol. The protein bands obtained were glycoproteins with a molecular weight of 42,000 to 58,000 Daltons

Production of monovalent anti-Bothrops asper antivenom: development of immune response in horses and neutralizing ability

A monovalent antivenom was produced by immunozing two horses with venom of the pit viper Bothrops asper (Ophidia: Viperidae). Although development of the immune response against four toxic and enzymatic activitiesof the venom was similar in both horses during the first two thirds of the immunization schedule, antibody response in one of the horses reached much higher levels in the last part of the immunization. Immunoelectrophoretic analysis indicates that there were precipitating antibodies in the sera of these horses during all the stages of immunization. However, immunoprecipitation did not correlated with the ability of sera to neutralize toxic activities of B. asper venom. Monovalent antivenom was more effective than the commercialy available polyvalent antivenom in the neutralization of Bothrops asper venom. On the other hand, despite the fact that it neutralizes lethal and hemorrhagic activities of the venoms of Lachesia muta and Crotalus durissus, it was less effective than polyvalent antivenom in these neutralizations. Moreover, it does not neutalize defibrinating activity induced by these two venoms, whereas it neutralizes this effect in the case of B. asper venom. It is proposed that monovalent antivenom my be highly effective in the case of envenomations induced by Bothrops asper venom; its use in treating accidentsby L. muta and C. durissus would be indicated only of polyvalent antivenom is not available. Results also demonstrate that it is important to monitor antibody response individually in horses being immunized for antivenom production, due to the conspicuous variability in the response of different animals.