Inhibitory Effects of Varespladib, CP471474, and Their Potential Synergistic Activity on Bothrops asper and Crotalus durissus cumanensis Venoms.

Snakebite is a neglected tropical disease that causes extensive mortality and morbidity in rural communities. Antivenim sera are the currently approved therapy for snake bites; however, they have some therapeutic limitations that have been extensively documented. Recently, small molecule toxin inhibitors have received significant attention as potential alternatives or co-adjuvant to immunoglobulin-based snakebite therapies. Thus, in this study, we evaluated the inhibitory effects of the phospholipase A2 inhibitor varespladib and the metalloproteinase inhibitor CP471474 and their synergistic effects on the lethal, edema-forming, hemorrhagic, and myotoxic activities of Bothrops asper and Crotalus durissus cumanensis venoms from Colombia. Except for the preincubation assay of the lethal activity with B. asper venom, the mixture showed the best inhibitory activity. Nevertheless, the mix did not display statistically significant differences to varespladib and CP471474 used separately in all assays. In preincubation assays, varespladib showed the best inhibitory activity against the lethal effect induced by B. asper venom. However, in independent injection assays, the mix of the compounds partially inhibited the lethal activity of both venoms (50%). In addition, in the assays to test the inhibition of edema-forming activity, the mixture exhibited the best inhibitory activity, followed by Varespladib, but without statistically significant differences (p > 0.05). The combination also decreased the myotoxic activity of evaluated venoms. In these assays, the mix showed statistical differences regarding CP471474 (p < 0.05). The mixture also abolished the hemorrhagic activity of B. asper venom in preincubation assays, with no statistical differences to CP471474. Finally, the mixture showed inhibition in studies with independent administration in a time-dependent manner. To propose a mode of action of varespladib and CP471474, molecular docking was performed. PLA2s and SVMPs from tested venoms were used as targets. In all cases, our molecular modeling results suggested that inhibitors may occupy the substrate-binding cleft of the enzymes, which was supported by specific interaction with amino acids from the active site, such as His48 for PLA2s and Glu143 for the metalloproteinase. In addition, varespladib and CP471474 also showed interaction with residues from the hydrophobic channel in PLA2s and substrate binding subsites in the SVMP. Our results suggest a synergistic action of the mixed inhibitors and show the potential of varespladib, CP471474, and their mixture to generate new treatments for snakebite envenoming with application in the field or as antivenom co-adjuvants.

Inhibitory effect of pinostrobin from Renealmia alpinia, on the enzymatic and biological activities of a PLA2.

Pinostrobin is a flavanone isolated from Renealmia alpinia, a plant used in folk medicine to treat snakebites. We tested the inhibitory ability of pinostrobin on the enzymatic, anticoagulant, myotoxic and edema-inducing activities of a PLA2 isolated from Crotalus durissus cumanensis venom. The compound displayed IC50 values of 1.76mM and 1.85mM (95% Confidence intervals: 1.34-2.18 and 1.21-2.45) on the PLA2 enzymatic activity, when either aggregated or monodispersed substrates were used, respectively. When mice were injected with PLA2 preincubated with 0.4, 2.0 and 4.0mM of pinostrobin, myotoxic activity induced by the PLA2 was inhibited up to 87%. Nevertheless, these values decreased up to 56% when the pinostrobin was injected into muscle after PLA2. Pinostrobin inhibited edema-forming and anticoagulant activities of the PLA2. In order to have insights on the mode of action of pinostrobin, intrinsic fluorescence and ultraviolet studies were performed. Results suggest that pinostrobin interacts directly with the PLA2. These findings were supported by molecular docking results, which suggested that pinostrobin forms hydrogen bonds with residues His48 and Asp49 of PLA2, besides, a π-π stacking interactions with those of residues Phe5 and Trp31, and rings C of flavanone and Tyr52 of the toxin.

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.

Inhibition of the toxic effects of Bothrops asper venom by pinostrobin, a flavanone isolated from Renealmia alpinia (Rottb.) MAAS.

ETHNOPHARMACOLOGICAL RELEVANCE: Renealmia alpinia has been traditionally used to treat snakebites by indigenous Embera-Katíos tribes belonging to the regions of Antioquia and Chocó, Colombia, and it has been shown to inhibit the enzymatic and biological activities of Bothrops venoms and their purified phospholipase A2 (PLA2) toxins. In addition to its common local usage against snakebites, Renealmia alpinia is commonly used to treat pain. To evaluate the inhibitory ability of pinostrobin, the main compound in the dichloromethane extract of Renealmia alpinia, on the toxic effects of Bothrops asper venom through in vitro and in vivo models and to evaluate its activity against pain and edema. MATERIALS AND METHODS: Pinostrobin was isolated from the dichloromethane extract of Renealmia alpinia leaves. The protective properties of the extract and of pinostrobin against the indirect hemolytic, coagulant and proteolytic effects of Bothrops asper venom were evaluated in vitro, and the anti-hemorrhagic and anti-inflammatory activity were evaluated in vivo. RESULTS: Renealmia alpinia extract significantly inhibited the proteolytic activity and indirect hemolytic activity of Bothrops asper venom at a venom:extract ratio of 1:20. Moreover, the present data demonstrate that pinostrobin may mitigate some venom-induced local tissue damage due to hemorrhagic effects, and the compound is also responsible for the analgesic and anti-inflammatory activity of the extract from Renealmia alpinia. This is the first report to describe pinostrobin in the species Renealmia alpinia and its properties in vitro against Bothrops asper venom. CONCLUSION: Our studies of the activity of Renealmia alpinia against the venom of Bothrops asper have confirmed that this species possesses inhibitory effects against Bothrops asper venom in both in vitro and in vivo models and that these effects may be due to pinostrobin, supporting the traditional usage of the plant. Additionally, pinostrobin may be responsible for the anti-hemorrhagic and analgesic activity (peripheral analgesic activity) of Renealmia alpinia.

The biflavonoid morelloflavone inhibits the enzymatic and biological activities of a snake venom phospholipase A2.

The biflavonoid morelloflavone has been reported as inhibitor of secretory PLA2s (phospholipases A2 from human synovial and bee venom sources); however, its capacity to interact and inhibit snake venom PLA2 activities has not been described. In this work we tested the inhibitory ability of morelloflavone on the enzymatic, anticoagulant, myotoxic and edema-inducing activities of a PLA2 isolated from Crotalus durissus cumanensis venom. The biflavonoid displayed IC50 values of 0.48 mM (95% Confidence intervals: 0.45-0.51) and 0.38 mM (95% Confidence intervals: 0.36-0.40) on the PLA2 enzymatic activity, when either aggregated or monodispersed substrates were used, respectively. In addition, morelloflavone inhibited in a time-dependent manner and irreversibly the PLA2 enzymatic activity. When mice were injected with PLA2 preincubated (preincubation assay) with 0.13, 0.63 and 1.26 mM of the biflavonoid, the myotoxic activity induced by the PLA2 was inhibited up to 63%. Nevertheless, these values decreased up to 38% when the morelloflavone was injected into muscle after PLA2. Moreover, morelloflavone inhibited, in a concentration-dependent manner, edema-forming activity of the PLA2 in the footpad. Morelloflavone also inhibited the anticoagulant activities of the PLA2 in concentration-dependent mode. In order to have insights on the mode of action of morelloflavone, intrinsic fluorescence studies were performed. Results of these assays suggest that morelloflavone interacts directly with the PLA2. These findings were supported by molecular docking results, which suggested that morelloflavone forms hydrogen bonds with residues Gly33, Asp49, Gly53 and Thr68 of the enzyme. In addition, our results suggested a π-π stacking interaction between rings A of morelloflavone with that of the residue Tyr52, and Van der Waals interactions with Gly32, His48 and Ala56. Our molecular modeling results suggest that morelloflavone may occupy part of substrate binding cleft of the PLA2. Morelloflavone is a candidate for the development of inhibitors to be used in snakebite envenomation.

Inhibition of venom serine proteinase and metalloproteinase activities by Renealmia alpinia (Zingiberaceae) extracts: comparison of wild and in vitro propagated plants.

ETHNOPHARMACOLOGICAL RELEVANCE: The plant Renealmia alpinia has been used in folk medicine to treat snakebites in the northwest region of Colombia. In addition, it has been shown to neutralize edema-forming, hemorrhagic, lethal, and defibrin(ogen)ating activities of Bothrops asper venom. In this work, extracts of Renealmia alpinia obtained by micropropagation (in vitro) and from specimens collected in the wild were tested and compared in their capacity to inhibit enzymatic and toxic activities of a snake venom metalloproteinase isolated from Bothrops atrox (Batx-I) venom and a serine proteinase (Cdc SII) from Crotalus durissus cumanensis venom. MATERIALS AND METHODS: We have investigated the inhibition capacity of Renealmia alpinia extracts on enzymatic and toxic actions of isolated toxins, a metalloproteinase and a serine proteinase. The protocols investigated included inhibition of proteolytic activity on azocasein, inhibition of proteolytic activity on fibrinogen, inhibition of pro-coagulant activity, inhibition of hemorrhagic activity and inhibition of edema-forming activity. RESULTS: Colorimetric assays detected the presence of terpenoids, flavonoids, tannins and coumarins in Renealmia alpinia extracts. Renealmia alpinia extracts inhibited the enzymatic, hemorrhagic and fibrinogenolytic activities of Batx-I. Extracts also inhibited coagulant, defibrin(ogen)ating and edema-forming activities of Cdc SII. Results highlight that Renealmia alpinia in vitro extract displayed comparable inhibitory capacity on venom proteinases that Renealmia alpinia wild extract. No alteration was observed in the electrophoretic pattern of venom proteinases after incubation with Renealmia alpinia extracts, thus excluding proteolytic degradation or protein denaturation/precipitation as a mechanism of inhibition. CONCLUSIONS: Our results showed that Renealmia alpinia wild and in vitro extracts contain compounds that neutralize metallo- and serine proteinases present in snake venoms. The mechanism of inhibition is not related to proteolytic degradation of the enzymes nor protein aggregation, but is likely to depend on molecular interactions of secondary metabolites in the plant with these venom proteinases.

Glycolic acid inhibits enzymatic, hemorrhagic and edema-inducing activities of BaP1, a P-I metalloproteinase from Bothrops asper snake venom: insights from docking and molecular modeling.

Glycolic acid (GA) (2-Hydroxyethanoic acid) is widely used as chemical peeling agent in Dermatology and, more recently, as a therapeutic and cosmetic compound in the field of skin care and disease treatment. In this work we tested the inhibitory ability of glycolic acid on the enzymatic, hemorrhagic and edema-inducing activities of BaP1, a P-I metalloproteinase from Bothrops asper venom, which induces a variety of toxic actions. Glycolic acid inhibited the proteolytic activity of BaP1 on azocasein, with an IC50 of 1.67 mM. The compound was also effective at inhibiting the hemorrhagic activity of BaP1 in skin and muscle in experiments involving preincubation of enzyme and inhibitor prior to injection. When BaP1 was injected i.m. and then, at the same site, different concentrations of glycolic acid were administered at either 0 or 5 min, 7 mM solutions of the inhibitor partially abrogated hemorrhagic activity when administered at 0 min. Moreover, glycolic acid inhibited, in a concentration-dependent manner, edema-forming activity of BaP1 in the footpad. In order to have insights on the mode of action of glycolic acid, UV-vis and intrinsic fluorescence studies were performed. Results of these assays suggest that glycolic acid interacts directly with BaP1 and chelates the Zn²âº ion at the active site. These findings were supported by molecular docking results, which suggested that glycolic acid forms hydrogen bonds with residues Glu143, Arg110 and Ala111 of the enzyme. Additionally, molecular modeling results suggest that the inhibitor chelates Zn²âº, with a distance of 3.58 Å, and may occupy part of substrate binding cleft of BaP1. Our results suggest that glycolic acid is a candidate for the development of inhibitors to be used in snakebite envenomation.

Biochemical and biological characterization of two serine proteinases from Colombian Crotalus durissus cumanensis snake venom.

Two clotting serine proteinases, named Cdc SI and Cdc SII, were isolated and characterized for the first time from Colombian Crotalus durissus cumanensis snake venom. The enzymes were purified using two chromatographic steps: molecular exclusion on Sephacryl S-200 and RP-HPLC on C8 Column. The molecular masses of the proteins, determined by MALDI-TOF mass spectrometry, were 28,561.4 and 28,799.2 Da for Cdc SI and Cdc SII, respectively. The aim of the present study was to evaluate enzymatic, coagulant and toxic properties of the two enzymes. The serine proteinases hydrolyzed specific chromogenic substrate (BaPNA) and exhibited a Michaelis-Menten behavior. Cdc SI had V(max) of 0.038 ± 0.003 nmol/min and K(M) of 0.034 ± 0.017 mM, while Cdc SII displayed values of V(max) of 0.267 ± 0.011 nmol/min and K(M) of 0.145 ± 0.023 mM. N-terminal sequences were VIGGDEXNIN and VIGGDICNINEHNFLVALYE for Cdc SI and Cdc SII, respectively. Molecular masses, N-terminal sequences, inhibition assays, and enzymatic profile suggest that Cdc SI and Cdc SII belong to the family of snake venom thrombin-like enzymes. These serine proteinases differed in their clotting activity on human plasma, showing a minimum coagulant dose of 25 µg and 0.571 µg for Cdc SI and Cdc SII, respectively. Enzymes also showed coagulant activity on bovine fibrinogen and degraded chain α of this protein. Toxins lack hemorrhagic and myotoxic activities, but are capable to induce defibrin(ogen)ation, moderate edema, and an increase in vascular permeability. These serine proteinases may contribute indirectly to the local hemorrhage induced by metalloproteinases, by causing blood clotting disturbances, and might also contribute to cardiovascular alterations characteristic of patients envenomed by C. d. cumanensis in Colombia.

Efecto inhibitorio de extractos de Renealmia alpinia Rottb. Maas (Zingiberaceae) sobre el veneno de Bothrops asper (mapaná) / Evaluation of the inhibitory effect of extracts from leaves of Renealmia alpinia Rottb. Maas (Zingiberaceae) on the venom of Bothrops asper (mapaná)

Introducción. La medicina tradicional es una invaluable fuente de investigación de nuevos remedios como complemento para el tratamiento del accidente ofídico, considerado como un grave problema de salud pública a nivel mundial. Objetivo. Este trabajo de investigación pretende comprobar la capacidad de neutralizar los efectos hemorrágicos, coagulantes y proteolíticos, de los extractos de hojas de Renealmia alpinia, usada tradicionalmente por los indígenas del Chocó (Colombia) contra la mordedura de la serpiente Bothrops asper, causante de la gran mayoría de los accidentes ofídicos en nuestro país. Materiales y métodos. Se llevaron a cabo ensayos de toxicidad aguda y de actividad analgésica in vivo de R. alpinia. Además, se hicieron ensayos in vitro sobre inhibición de las actividades coagulante, hemolítica y proteolítica del veneno de B. asper. Resultados. El presente estudio demuestra que R. alpinia no produce efectos tóxicos en animales de experimentación; además, presenta efectos analgésicos in vivo y antiofídicos in vitro,y protege contra los efectos letales del veneno de B. asper, in vivo. Conclusión. Renealmia alpinia puede ser una buena alternativa terapéutica como complemento al tratamiento con antiveneno en el accidente ofídico, por sus efectos analgésicos y antiofídicos.

Introduction. Traditional medicine is an invaluable source of research into new medicines as a supplement for the treatment of snakebite, considered as a serious public health problem worldwide. The extracts of the medicinal plant, Renealmia alpina, have been used traditionally by indigenous people of Chocó (Colombia) against Bothrops asper snakebite, a snake responsible for the majority of snakebite accidents in Colombia. Objective. The ability of extracts of R. alpinia leaves was tested for its ability to neutralize the hemorrhagic, coagulant and proteolytic effects of the snakebite venom of B. asper. Materials and methods. The acute toxicity tests and analgesic activity of R. alpina were evaluated in vivo. In addition, tests were undertaken in in vitro conditions to demonstrate inhibition of coagulant, haemolytic and proteolytic activity of the B. asper venom. Results. Renealmia alpinia extracts had no toxic effects in experimental animals and also provided analgesic and antiophidian effects and protection against the lethal effects of the venom of B. asper. Conclusion. Renealmia. alpinia was an effective therapeutic alternative in association with antivenom treatment in the event of a B. asper snakebite accident. It was demonstrated to protect against the lethal effects and provided analgesic properties as well.

Relationship between the structure and the enzymatic activity of crotoxin complex and its phospholipase A2 subunit: an in silico approach.

Crotoxin, one of the major toxins of South American rattlesnake Crotalus durissus subspecies, is an heterodimeric complex composed of two distinct subunits: a basic phospholipase A(2) (PLA(2), CB) and an acidic nontoxic catalytically inactive protein, crotapotin (CA). It's well known that CB has a high enzymatic activity; however the molecular aspects that determine this fact remain unknown. In this study, an in silico approach was used to predict the CA structure by homology modeling, and the crotoxin structure by means of molecular docking. CA structure was built using the software Modeller taking Crotalus atrox PLA(2) (1PP2:R) as a template. Different criteria measured by Procheck, Verify 3D and ProSA were indicative of the reliability and the proper fold for the predicted structural model of CA. Then, a combination of this model and CB crystal structure was used to build the structure of crotoxin complex through rigid-body protein-protein docking. The crotoxin-3D model suggested that by means of hydrophobic and π-π stacking interactions, CA-Y24 and CA-F119 interact with CB-F24 and CB-F119, respectively. Those interactions could prevent the interfacial adsorption of the CB onto the lipid/water interface by blocking part of the interfacial binding surface of the PLA(2). This fact could explain the differences regarding to enzymatic activity between the crotoxin complex and CB. In addition, the crotoxin-3D model showed solvent-exposed regions of CA that could bind the receptor expressed in target cells.

[Evaluation of the inhibitory effect of extracts from leaves of Renealmia alpinia Rottb. Maas (Zingiberaceae) on the venom of Bothrops asper (mapaná)]. / Efecto inhibitorio de extractos de Renealmia alpinia Rottb. Maas (Zingiberaceae) sobre el veneno de Bothrops asper (mapaná).

INTRODUCTION: Traditional medicine is an invaluable source of research into new medicines as a supplement for the treatment of snakebite, considered as a serious public health problem worldwide. The extracts of the medicinal plant, Renealmia alpina, have been used traditionally by indigenous people of Chocó (Colombia) against Bothrops asper snakebite, a snake responsible for the majority of snakebite accidents in Colombia. OBJECTIVE: The ability of extracts of R. alpinia leaves was tested for its ability to neutralize the hemorrhagic, coagulant and proteolytic effects of the snakebite venom of B. asper. MATERIALS AND METHODS: The acute toxicity tests and analgesic activity of R. alpina were evaluated in vivo. In addition, tests were undertaken in in vitro conditions to demonstrate inhibition of coagulant, haemolytic and proteolytic activity of the B. asper venom. Results. Renealmia alpinia extracts had no toxic effects in experimental animals and also provided analgesic and antiophidian effects and protection against the lethal effects of the venom of B. asper. CONCLUSION: Renealmia. alpinia was an effective therapeutic alternative in association with antivenom treatment in the event of a B. asper snakebite accident. It was demonstrated to protect against the lethal effects and provided analgesic properties as well.

Efectos inhibitorios de compuestos fenólicos de plantas sobre la actividad enzimática y citotoxica inducida por una fosfolipasa A2 de veneno de serpiente / Inhibitory effects of plant phenolic compounds on enzymatic and cytotoxic activities induced by a snake venom phospholipase A2

Los compuestos fenólicos han mostrado inhibir los efectos tóxicos inducidos por proteínas de veneno de serpiente. En éste trabajo, nosotros demostramos que el ácido gálico, el ácido ferúlico, el ácido cafeico, el propilgalato y el epigalocatequingalato inhiben la actividad enzimática de una fosfolipasa A2 (PLA2) usando yema de huevo como sustrato. Los valores de IC50 están entre 0,38 – 3,93 mM. Los compuestos mencionados también inhiben la actividad enzimática cuando un sustrato sintético es usado. Además, estos compuestos polifenólicos disminuyen el efecto citotóxico inducido por la fosfolipasa A2 miotóxica, el epigalocatequingalato exhibe la mejor capacidad inhibitoria con 90,92 ± 0,82%, mientras que el ácido ferúlico muestra la menor actividad inhibitoria con 30,96 ± 1,42%. Con el fin de determinar los posibles mecanismos de acción de los compuestos fenólicos, realizamos estudios de modelamiento molecular. Todos los polifenoles muestran puentes de hidrogeno con el sitio activo de la enzima; además el epigalocatequingalato presenta una unión más fuerte con la PLA2 que los otros compuestos. Adicionalmente, un análisis preliminar de relación estructura actividad muestra una correlación entre los valores de IC50 y el área superficial polar topológica (p = 0,0491, r = -0,8079 (-0,9878 a -0,2593)), la cual indica el área superficial requerida por cada molécula para unirse a la enzima. Además, nuestros resultados muestran al propilgalato y el epigalocatequingalato como dos nuevos productos naturales con potencial antimiotóxico. La aplicación tópica de estos polifenoles en el sitio de mordedura podría llevar a la prevención de la miotoxicidad; sin embargo, posteriores investigaciones in vivo serían necesarias para confirmar los resultados in vitro.

Inhibitory effects of bile acids on enzymatic and pharmacological activities of a snake venom phospholipase A(2) from group IIA.

Bile acids, such as cholic acid (CA) and ursodeoxycholic acid (UDCA) have shown to decrease or increase the enzymatic activity of group IB pancreatic PLA(2), depending on the concentration used. Studies suggest that the inhibition of hydrolysis rate of the substrate is due to formation in aqueous phase of a complex between bile acid and PLA(2), which is catalytically inert. For this reason, we tested the inhibition of the enzymatic activity of group IIA snake venom PLA(2) by bile acids, using an aqueous phase model. In addition, we measured the ability of bile acids to inhibit the toxic effects caused by the mentioned toxin. UDCA and CA inhibited the enzymatic activity of the PLA(2) in a competitive mode. Moreover, these compounds inhibited myotoxic, cytotoxic and edema-forming activities induced by the toxin, but UDCA was more efficient than CA. It was demonstrated that bile acids interact directly with this protein by causing slight changes in the intrinsic fluorescence spectra. Preliminary molecular docking studies suggest that bile acids interact with amino acids at the active site of the PLA(2) through different interactions, CA showed hydrogen bonds with His48, whereas, UDCA displayed with Asp49. Results obtained herein may turn UDCA and CA into promising models for the development of new molecules with anti-inflammatory and anti-snake venom PLA(2) properties.

Isolation and biological characterization of Batx-I, a weak hemorrhagic and fibrinogenolytic PI metalloproteinase from Colombian Bothrops atrox venom.

A hemorrhagic metalloproteinase, named Batx-I, was isolated from the venom of Bothrops atrox specimens (from Southeastern Colombian region) by a combination of CM-Sephadex C25 ion-exchange and Affi-gel Blue affinity chromatographies. This enzyme accounts for about 45% of venom proteins, and it has an ESI-MS isotope-averaged molecular mass of 23296.2 Da and a blocked N-terminus. Two internal fragments sequenced by mass spectrometric analysis showed similarity to other SVMPs from Bothrops venoms. To investigate the possible participation of Batx-I in the envenomation pathophysiology, proteolytic, fibrinogenolytic, hemorrhagic, and other biological activities were evaluated. The minimal hemorrhagic dose obtained was 17 microg/20 g body weight. The enzyme showed proteolytic activity on azocasein, comparable with activity of BaP1. This activity was inhibited by EDTA and 1, 10 o-phenanthroline but not by aprotinin, pepstatin A or PMSF. Fibrinogenolytic activity was analyzed by SDS-PAGE, revealing a preference for degrading the A alpha- and B beta-chains, although partial degradation of the gamma-chain was also detected. The protein lacks coagulant and defibrinating activity. The CK levels obtained, clearly reflects a myotoxic activity induced by Batx-I. The hemorrhagic and fibrinogenolytic activities exhibited by the isolated PI-SVMP may play a role in the hemorrhagic and blood-clotting disorders observed in patients bitten by B. atrox in Colombia.

Búsqueda de alternativas terapéuticas para el accidente ofídico en residuos agroindustriales de frutas tropicales / Research of therapeutic alternatives for snakebite in agroindustry residues of tropical fruits

Este estudio pretende demostrar que residuos de la agroindustria, como son las semillas de Vitis vinífera y Citrus sinensis, y la almendra de Mangifera indica, poseen capacidad neutralizante de algunos efectos enzimáticos inducidos por los venenos de Bothrops asper y Porthidium nasutum; además, actividad inhibitoria del crecimiento de algunos microorganismos de la flora normal de los colmillos y la boca de las serpientes; es así como se determina que los extractos de M. indica y V. vinífera poseen buena capacidad inhibitoria de la actividad de fosfolipasa A2 dependiente de la dosis, y una mayor potencia neutralizante hacia tal actividad del veneno de P. nasutum. Frente al efecto proteolítico inducido por el veneno de las dos víboras, V. vinífera presenta los mejores porcentajes de inhibición dependiendo de la cantidad de extracto utilizado. Sobre la actividad coagulante del veneno de B. asper, M. indica y V. vinífera logran prolongar el tiempo de coagulación hasta ~ 31 y 13 veces respectivamente, el tiempo de l correspondiente control positivo.M. indica y V. vinífera inhiben el crecimiento de Escherichia coli y Staphylococcus aureus. Por el contrario, el extracto etanólico de las semillas de C. sinensis no presenta resultados de inhibición muy prometedores.

Descripción y discusión acerca de los métodos de análisis de fibra y del valor nutricional de forrajes y alimentos para animales / Description and discussion about the methods of analysis of fiber and of the nutritional value of forages and foods for animals

Es necesario contar con un mayor volumen de forrajes de adecuado valor nutricional mejorando así la cadena agroalimentaria y disminuyendo el uso extensivo de la tierra en cultivos para ganadería. Una alternativa laconstituyen los procesos biotecnológicos con pastos mejorados por fermentaciones con hongos basidiomicetos, pero determinar el cambio en los componentes del forraje con el proceso de fermentación y su relación con el valor nutricional es complicado. Existen numerosas publicaciones sobre técnicas para evaluar componentes de un forraje; el sistema detergente es el más utilizado, aunque existen métodos más modernos. La accesibilidad y los costos de estos métodos son factores limitantes para muchos laboratorios. Algunos de ellos no están reconocidos como métodos oficiales de análisis. En este artículo se describe y discute especialmente el sistema detergente para evaluar el valor nutricional de alimentos para rumiantes, y las dificultades de interpretación. Se concluye que en el análisis de forrajes sometidos a procesos de biodegradación, los valores de lignina y celulosa por el sistema van Soest encontrados entre blancos y muestras, no corresponden al contenido total de lignina o celulosa de la muestra biodegradada. Se deben utilizar otros métodos más precisos como la espectroscopia IRTF (Infrarrojo con Transformada de Fourier) o la NIRs (Infrarrojo cercano) para caracterizar estas muestras.