The antisnake venom activities of Parkia biglobosa (Mimosaceae) stem bark extract.

Snake bites in rural Nigeria are commonly treated with plant extracts. We have studied the ability of one such traditionally used plant (Parkia biglobosa; [Jacq.] Benth., Mimosaceae) to reduce the effects of two snake venoms (Naja nigricollis, and Echis ocellatus) in several experimental models. A water-methanol extract of P. biglobosa stem bark significantly (p<0.001) protected the chick biventer cervicis (cbc) muscle preparation from N. nigricollis venom-induced inhibition of neurally evoked twitches when it was added to the bath 3-5 min before or after the venom. The extract also reduced the loss of responses to acetylcholine (Ach), carbachol and KCl, which are normally blocked by N. nigricollis venom, and significantly reduced the contractures of the preparation induced by venom. P. biglobosa extract (75, 150 and 300 microg/ml) significantly (p<0.05) protected C2C12 murine muscle cells in culture against the cytotoxic effects of N. nigricollis and E. ocellatus venoms. The extract protected egg embryos exposed to lethal concentrations of E. ocellatus venom for more than 12 h and completely blocked the haemorrhagic activity of the venom at concentrations of 5 and 10 microg/1.5 microl. P. biglobosa extract (400 mg/kg) did not protect mice injected i.p. with 5 and 2.5 mg/kg of E. ocellatus and N. nigricollis venoms, respectively. It, however, protected 40% of the mice from death caused by E. ocellatus venom after the extract and venom were pre-incubated for 30 min before injecting the mixture.     

Study of the efficacy of the black stone on envenomation by snake bite in the murine model.

The black stone (BS) has been used since antiquity to treat envenomations. Since no actual clinical trial has ever been performed we used an experimental approach to evaluate its efficacy against the venoms of Bitis arietans, Echis ocellatus and Naja nigricollis. Local application of BS after intramuscular venom injection had no demonstrable effect on the outcome of envenomationa and it did not change the LD(50) of B. arietans venom. Our results show that, contrary to widespread belief, no efficacy to treat envenomation may be expected of the BS.     

The neutralizing capacity of Androctonus crassicauda antivenom against Mesobuthus eupeus scorpion venom

Scorpion envenomations are considerable health problem in tropical and subtropical regions. There are approximately 1500 species of scorpions worldwide. The number of dangerous species in the Buthidae family is significantly higher than in other families of scorpions. Mesobuthus eupeus is a member of Mesobuthus genus, Buthidae family. In this study, the potency and para-specific activities of Androctonus crassicauda antivenom were investigated against M. eupeus scorpion venom. The median lethal dose of M. eupeus and A. crassicauda scorpion venoms were found to be 0.18mg/kg, 15.45mug/kg by i.c.v injection route. The antivenom showed neutralization effect against the venoms of M. eupeus. One milliliter of A. crassicauda antivenom neutralizes 464LD(50) of M. eupeus and 940LD(50)A. crassicauda venom on mice. Western blotting demonstrated immunologic reaction with the venoms. The monovalent antivenom has immunoactivity and neutralizing capacity to the scorpion venoms. This study indicates that the antivenom produced by Refik Saydam Hygiene Center could be used for the treatment of M. eupeus stings in Turkey.     

Cloning of a prothrombin activator-like metalloproteinase from the West African saw-scaled viper, Echis ocellatus.

Systemic envenoming by the saw-scaled viper, Echis ocellatus, is responsible for more deaths than any other snake in West Africa. Despite its medical importance, there have been few investigations into the toxin composition of the venom of this viper. Here we describe the isolation of E. ocellatus venom gland cDNAs encoding a protein of 514 amino acids that showed 91% sequence similarity to Ecarin, a prothrombin-activating metalloproteinase from the venom of the East African viper, E. pyramidum leakeyi, that induces severe consumption coagulopathy. Structural similarities between the E. ocellatus metalloproteinase and analogues in venoms of related vipers suggest that antibodies raised to phylogenetically conserved E. ocellatus metalloproteinase domains may have potential for cross-specific and cross-generic neutralisation of analogous venom toxins.     

Biological and biochemical properties of Scatophagus argus venom.

Scatophagus argus of the family Scatophagidae inflicts painful wounds in fishermen during handling. The clinical picture is characterized by excruciating and persistent local pain disproportionate to the size of injury, redness, swelling and a throbbing sensation that extends to the limbs, followed by dizziness. The biological properties of the S. argus venom were studied to assess its risk and lethal factors with regard to human welfare. In contrast to other fish venoms, S. argus showed relatively low LD50 (9.8 mg/kg via i.p.). Haemolytic activity in human erythrocytes was recorded. Platelet lysis expressed as LDH activity of lysed cells was dose dependent. S. argus venom failed to induce any clot in human plasma. No PLA(2) activity was found in S. argus venom. Mild proteolytic activity was observed. The injection of venom in mice produced lesions and nociception, which were not inhibited by antihistamine pheniramine maleate, suggesting that histamine was not involved in the inflammatory process. The increase in serum creatine kinase activity indicated myotoxicity. Cytotoxicity on HeLa cells was observed. The spectrum of activity in experimental animals of S. argus crude venom resembles those of other fish venoms previously studied and well correlated to the systemic manifestations that are described for S. argus envenomation.     

Hemostatic properties of Venezuelan Bothrops snake venoms with special reference to Bothrops isabelae venom

In Venezuela, Bothrops snakes are responsible for more than 80% of all recorded snakebites. This study focuses on the biological and hemostatic characteristics of Bothrops isabelae venom along with its comparative characteristics with two other closely related Bothrops venoms, Bothrops atrox and Bothrops colombiensis. Electrophoretic profiles of crude B. isabelae venom showed protein bands between 14 and 100 kDa with the majority in the range of 14-31 kDa. The molecular exclusion chromatographic profile of this venom contains five fractions (F1-F5). Amidolytic activity evaluation evidenced strong thrombin-like followed by kallikrein-like activities in crude venom and in fractions F1 and F2. The fibrinogenolytic activity of B. isabelae venom at a ratio of 100:1 (fibrinogen/venom) induced a degradation of Aα and Bβ chains at 15 min and 2 h, respectively. At a ratio of 100:10, a total degradation of Aα and Bβ chains at 5 min and of γ chains at 24 h was apparent. This current study evidences one of rarely reported for Bothrops venoms, which resembles the physiologic effect of plasmin. B. isabelae venom as well as F2 and F3 fractions, contain fibrinolytic activity on fibrin plate of 36, 23.5 and 9.45 mm²/μg, respectively using 25 μg of protein. Crude venom and F1 fraction showed gelatinolytic activity. Comparative analysis amongst Venezuelan bothropoid venoms, evidenced that the LD₅₀ of B. isabelae (5.9 mg/kg) was similar to B. atrox-Puerto Ayacucho 1 (6.1 mg/kg) and B. colombiensis-Caucagua (5.8 mg/kg). B. isabelae venom showed minor hemorrhagic activity, whereas B. atrox-Parguasa (Bolivar state) was the most hemorrhagic. In this study, a relative high thrombin-like activity was observed in B. colombiensis venoms (502-568 mUA/min/mg), and a relative high factor Xa-like activity was found in B. atrox venoms (126-294 mUA/min/mg). Fibrinolytic activity evaluated with 10 μg protein, showed that B. isabelae venom contained higher specific activity (50 mm²/μg) than B. colombiensis and B. atrox venoms, which should encourage the isolation of these fibrinolytic molecules to improve the quality of immunotherapy.     

Anti-Echis carinatus venom antibodies from chicken egg yolk: isolation, purification and neutralization efficacy.

High titer antibodies (IgY) were raised in egg yolk of white leghorn chicken (Gallus gallus domesticus) by immunizing with the venom of Echis carinatus (Saw scaled viper or carpet viper), an Indian venomous snake belonging to the family Viperidae. The anti-snake venom antibodies (antivenom) were isolated from egg yolk by the water dilution method, enriched by 19% sodium sulfate precipitation and purified by immunoaffinity chromatography. A single, electrophoretically pure IgY band of 180-200 kDa was obtained on SDS-PAGE. Immunoblot analysis revealed not only the specific binding of the antivenom but also dose-dependent blocking of antivenom by venom proteins. In neutralization studies, a preincubated mixture of both affinity-purified (50 mg/kg body weight) as well as partially purified (210 mg/kg body weight) anti-E. carinatus IgY with 2 LD(50) dose of E. carinatus venom (2 x 6.65 mg/kg body weight) gave 100% protection in mice when administered subcutaneously.     

An in vivo examination of the stability of venom from the Australian box jellyfish Chironex fleckeri.

We have previously characterised the pharmacological activity of a number of jellyfish venoms with a particular emphasis on the profound cardiovascular effects. It has been suggested that jellyfish venoms are difficult to work with and are sensitive to pH, temperature and chemical changes. The current study aimed to examine the working parameters of the venom of the Australian box jellyfish Chironex fleckeri to enable fractionation and isolation of the toxins with cardiovascular activity. C. fleckeri venom was made up fresh each day and subjected to a number of different environments (i.e. a pH range of 5-9 and a temperature range of 4-30 degrees C). In addition, the effect of freeze drying and reconstituting the venom was investigated. Venom (50 microg/kg, i.v.) produced a transient hypertensive response followed by cardiovascular collapse in anaesthetised rats. This biphasic response was not significantly effected by preparation of the venom at a pH of 5, 7 or 9. Similarly, venom (50 microg/kg, i.v.) did not display a loss of activity when exposed to temperatures of 4, 20 or 30 degrees C for 1.5h. However, the cardiovascular activity was abolished by boiling the venom. Freeze drying, and then reconstituting, the venom did not significantly affect its cardiovascular activity. However, repeated freeze drying and reconstituting of extracted venom resulted in a significantly loss of activity. This study provides a more detailed knowledge of the parameters in which C. fleckeri venom can be used and, while supporting some previous studies, contradicts some of the perceived problems of working with the venom.     

Biochemical and biological characterization of the venoms of Bothriopsis bilineata and Bothriopsis taeniata (Serpentes: Viperidae).

Snake venom is a complex mixture containing diverse protein components with different structures and functions that are used for prey immobilization and death. Snake venoms from the family Viperidae cause pronounced local and systemic effects, such as pain, edema, hemorrhage and necrosis. Here, we investigated the enzymatic and biological activities of venoms from two Amazonian snakes, Bothriopsis bilineata and Bothriopsis taeniata. Both venoms presented high enzymatic activities for proteases kallikrein, thrombin and plasmin, low levels of trypsin, cathepsin C and leucine aminopeptidase activities, while lacked acetylcholinesterase activity. B. taeniata and B. bilineata crude venoms caused inflammation inducing neutrophil recruitment into peritoneal cavity of mice 4h after injection. Neutrophil recruitment induced by B. taeniata venom was accompanied by hemorrhage. EDTA treatment profoundly impaired neutrophil recruitment, suggesting the involvement of a metalloproteinase on venoms-induced neutrophil recruitment. Pretreatment with dexamethasone and zileuton, a 5-lipoxygenase inhibitor, significantly reduced neutrophil migration, but indomethacin and montelukast, a cysteinyl leukotriene receptor antagonist, had no effect, suggesting the involvement of lipoxygenase-derived metabolites, probably LTB(4). Together, these results show that B. bilineata and B. taeniata venoms induce a marked inflammatory reaction, with leukocyte recruitment, and hemorrhage, which parallels to a high proteolytic activity found in these venoms.     

Comparison of the effect of Crotalus simus and Crotalus durissus ruruima venoms on the equine antibody response towards Bothrops asper venom: Implications for the production of polyspecific snake antivenoms

Antivenoms are preparations of immunoglobulins purified from the plasma of animals immunized with snake venoms. Depending on the number of venoms used during the immunization, antivenoms can be monospecific (if venom from a single species is used) or polyspecific (if venoms from several species are used). In turn, polyspecific antivenoms can be prepared by purifying antibodies from the plasma of animals immunized with a mixture of venoms, or by mixing antibodies purified from the plasma of animals immunized separately with single venom. The suitability of these strategies to produce polyspecific antibothropic-crotalic antivenoms was assessed using as models the venoms of Bothrops asper, Crotalus simus and Crotalus durissus ruruima. It was demonstrated that, when used as co-immunogen, C. simus and C. durissus ruruima venoms exert a deleterious effect on the antibody response towards different components of B. asper venom and in the neutralization of hemorrhagic and coagulant effect of this venom when compared with a monospecific B. asper antivenom. Polyspecific antivenoms produced by purifying immunoglobulins from the plasma of animals immunized with venom mixtures showed higher antibody titers and neutralizing capacity than those produced by mixing antibodies purified from the plasma of animals immunized separately with single venom. Thus, despite the deleterious effect of Crotalus sp venoms on the immune response against B. asper venom, the use of venom mixtures is more effective than the immunization with separate venoms for the preparation of polyspecific bothropic-crotalic antivenoms.     

General biochemical and immunological characteristics of the venom from Peruvian scorpion Hadruroides lunatus

This communication describes the general biochemical properties and some immunological characteristics of the venom from the Peruvian scorpion Hadruroides lunatus, which is the most medically relevant species in Peru. The soluble venom of this scorpion is toxic to mice, the LD₅₀ determined was 0.1 mg/kg and 21.55 mg/kg when the venom was injected intracranial or intraperitoneally, respectively. The soluble venom displayed proteolytic, hyaluronidasic, phospholipasic and cardiotoxic activities. High performance liquid chromatography of the soluble venom resulted in the separation of 20 fractions. Two peptides with phospholipasic activity were isolated to homogeneity and their molecular masses determined by mass spectrometry (MALDI TOF). Anti-H. lunatus venom sera were produced in rabbits. Western blotting analysis showed that most of the protein content of this venom is immunogenic. H. lunatus anti-venom displayed consistent cross-reactivity with venom antigens from the new World-scorpions Tityus serrulatus and Centruroides sculpturatus venoms; however, a weaker reactivity was observed against the venom antigens from the old World-scorpion Androctonus australis Hector.     

Observations on white and yellow venoms from an individual southern Pacific rattlesnake (Crotalus viridis helleri)

Biochemical differences in white and yellow venoms produced in the separate venom glands of an individual southern Pacific rattlesnake (Crotalus viridis helleri) were investigated. Compared to the yellow venom, the white venom contained fewer low molecular weight components and was considerably less toxic. Although the exact LD50 was not determined, the white venom did not produce toxic effects in mice when injected i.v. at concentrations up to 10 mg/kg. The i.v. LD50 of the yellow venom was approximately 1.6 mg/kg. Both white and yellow venoms had hemorrhagic activity, but the white venom caused less intradermal hemorrhage in mice. No L-amino acid oxidase activity was measured in the white venom and protease and phospholipase A2 activities of the white venom were much less than in the yellow venom. The white and yellow venoms both produced myonecrosis at 1, 3 and 24 hr after i.m. injection into mice, however, there were some qualitative differences in the myonecrosis produced. When the venom samples were reacted against Wyeth's polyvalent (Crotalidae) antivenom using immunodiffusion, three precipitin bands formed against the yellow venom, whereas only one formed against the white venom. When reacted against an antiserum to myotoxin alpha from C. viridis viridis venom, both the white and yellow venoms produced one precipitin band each.     

Studies on egyptian Cerastes cerastes antivenin

A monovalent C. cerastes antivenin was prepared in horses. At the end of the immunization course 1 ml of serum neutralized 92 ₅₀ of its homologous venom. Cross neutralization was evident against Cerastes vipera venom (53 ₅₀'s per ml serum), but was low against the venoms of Echis carinatus, Bitis gabonica, B. arietans. Dendroaspis polylepis, D. angusticeps, Naja haje, N. nigricollis, N. nivea, N. naja and W. aegyptia (8–20 ₅₀ per ml) and there was no protection against T. flavoviridis venom. Precipitation bands obtained by Ouchterlony immunodiffusion studies, gave comparable results showing stronger reaction against the venoms of C. cerastes and C. vipera and weaker reactions against the other venoms.     

Protease activity and lethal toxicity of venoms from some little known rattlesnakes

Information on yield, lethality, and protease activity is given for venoms of Crotalus exul, C. p. pricei, C. pusillus, C. w. willardi and Sistrurus ravus. Lethal toxicity of C. tigris venom (LD50 i.v. 0.056 mg/kg; s.c. 0.21 mg/kg) is the highest known for any rattlesnake venom. The lethal potency of C. pricei venom is high by i.v. but not by s.c. injection. Both these venoms lack protease activity. C. pusillus venom is lowest in lethality.     

Immunological studies on Egyptian polyvalent antivenins

Polyvalent antivenin was prepared against the Egyptian snake venoms Naja haje, N. nigricollis, Cerastes cerastes and C. vipera, and tested by neutralization and immunodiffusion tests. The antivenin was of high potency against the Egyptian snake venoms, especially C. cerastes and C. vipera, followed by Echis carinatus, E. coloratus, N. haje, N. nigricollis and Walterinnesia aegyptia. The titre against the African and Indian cobra venoms N. nivea, N. haje (Ethiopian) and N. naja was lower, while poor or no effect was shown against Bitis arietans, B. gabonica and Trimeresurus flavoviridis venoms. The polyvalent serum prepared in horses not previously immunized, was comparable to sera obtained by using horses, which were previously immunized against a single venom. However, the latter sera, still maintained their higher titre against the original sensitizing venom. Comparison of polyvalent and monovalent N. nigricollis antivenins, revealed that while the polyvalent serum was of better effectiveness against the Egyptian viper venoms especially cerastes venoms, more protection was provided by the monovalent serum against the naja venoms.     

Role of IgG(T) and IgGa isotypes obtained from arachnidic antivenom to neutralize toxic activities of Loxosceles gaucho, Phoneutria nigriventer and Tityus serrulatus venoms.

The ability of IgG(T) and IgGa subclasses--isolated by liquid chromatography from equine arachnidic antivenom (AAV)-to neutralize toxic activities of Loxosceles gaucho, Phoneutria nigriventer and Tityus serrulatus venoms as well as to remove venom toxins from circulation was investigated. These subclasses showed similar antibody titers against L. gaucho, P. nigriventer and T. serrulatus venoms, and by immunoblotting few differences were observed in the recognition pattern of venom antigens. IgG(T) and IgGa neutralized 100% lethality induced by L. gaucho and 50% of P. nigriventer venom, but IgGa failed to neutralize T. serrulatus venom, in contrast to IgG(T). Both subclasses neutralized local reactions and dermonecrosis induced by L. gaucho venom in rabbits. In mice, IgG(T) and IgGa partially neutralized the edematogenic activity induced by P. nigriventer and T. serrulatus venoms, but only IgG(T) neutralized (ca. 81%) the nociceptive activity induced by T. serrulatus venom. Both subclasses failed to neutralize nociceptive activity induced by P. nigriventer venom. IgG(T) reduced the serum venom levels of animals injected with L. gaucho, P. nigriventer or T. serrulatus venoms, while IgGa solely reduced L. gaucho and P. nigriventer venoms levels. Our results demostrate that IgG(T) and IgGa subclasses neutralize toxic activities induced by P. nigriventer, T. serrulatus and L. gaucho venoms with different efficacies, as well as depurate these venoms from circulation.     

Extracts of Renealmia alpinia (Rottb.) MAAS Protect against Lethality and Systemic Hemorrhage Induced by Bothrops asper Venom: Insights from a Model with Extract Administration before Venom Injection

Renealmia alpinia (Rottb.) MAAS, obtained by micropropagation (in vitro) and wild forms have previously been shown to inhibit some toxic activities of Bothrops asper snake venom if preincubated before injection. In this study, assays were performed in a murine model in which extracts were administered for three days before venom injection. R. alpinia extracts inhibited lethal activity of B. asper venom injected by intraperitoneal route. Median Effective Dose (ED₅₀) values were 36.6 ± 3.2 mg/kg and 31.7 ± 5.4 mg/kg (p > 0.05) for R. alpinia wild and in vitro extracts, respectively. At a dose of 75 mg/kg, both extracts totally inhibited the lethal activity of the venom. Moreover, this dose prolonged survival time of mice receiving a lethal dose of venom by the intravenous route. At 75 mg/kg, both extracts of R. alpinia reduced the extent of venom-induced pulmonary hemorrhage by 48.0% (in vitro extract) and 34.7% (wild extract), in agreement with histological observations of lung tissue. R. alpinia extracts also inhibited hemorrhage in heart and kidneys, as evidenced by a decrease in mg of hemoglobin/g of organ. These results suggest the possibility of using R. alpinia as a prophylactic agent in snakebite, a hypothesis that needs to be further explored.     

Identification and characterization of venom proteins of two solitary wasps, Eumenes pomiformis and Orancistrocerus drewseni

Secretory proteins were identified in the venoms of two solitary hunting wasps, Eumenes pomiformis and Orancistrocerus drewseni, by SDS-PAGE in conjunction with mass analysis. More than 30 protein bands (2-300 kDa) were detected from the crude venom of each wasp. With the aid of the previously constructed venom gland/sac-specific EST libraries, a total of 31 and 20 proteins were identified from 18 to 20 distinctive protein bands of E. pomiformis and O. drewseni venoms, respectively. Arginine kinase was the most predominant protein in both wasp venoms. Along with the full-length arginine kinase, a truncated form, which was known to have paralytic activity on a spider, was a common predominant protein in the two wasp venoms. Insulin/insulin-like peptide-binding protein was abundantly found only in E. pomiformis venom, which might be due to its unique behaviors of oviposition and provision. The presence of various immune response-related proteins and antioxidants suggested that wasps might use their venom to maintain prey fresh while feeding wasp larvae by protecting the prey from microbial invasion and physiological stresses. It seemed that some venom proteins are secreted into venom fluid from venom gland cells via exosomes, not by signal sequence-mediated transport processes.     

Role of cyclooxygenases in oedema-forming activity of bothropic venoms.

The venoms of Bothrops asper (BaV) and Bothrops jararaca (BjV), two of the most medically important poisonous snakes of Latin America, cause pronounced oedema in the victims through poorly understood mechanisms. In the present study, we examined the possible role of cyclooxygenases (COX) in the genesis of mouse paw oedema caused by BaV and BjV injections. BaV at 2.5 microg/paw and BjV at 0.75 microg/paw induced significant oedema that persisted for up to 6h following subplantar injection. Treatment with indomethacin (2 mg/kg), rofecoxib, (10 mg/kg), or dexamethasone (2 mg/kg) significantly reduced the BaV- and BjV-induced oedema formation. Treatment with SC-560 (30 mg/kg) significantly reduced the oedema formation induced by BjV but had no effect on that induced by BaV. Both venoms induced significant increases in the levels of prostaglandin E(2) (PGE(2)) and the expression of COX-1 and COX-2 in paw tissue. The peak of oedema formation and PGE(2) release correlated with marked expression of COX-2 in the paw tissue. These results demonstrate that injection of BaV and BjV results in a rapid increase in oedema formation that is, at least partially, mediated by arachidonic acid metabolites formed by COX-2. In the case of BjV, COX-1-derived prostanoids also appear to contribute significantly to the inflammatory changes.