In vivo and in vitro prospection of the anti-ophidic properties exercised by the extracts of Jacaranda decurrens L

The research and development of alternative treatments for snakebites (e.g., medicinal plants) is necessary due to the high costs of the existing ones. The effects of the aqueous extracts from Jacaranda decurrens leaves, roots, and xylopodium were analyzed upon the venom-induced (Bothrops spp. and Crotalus spp.) systemic and local toxicity. The extracts were able to partially inhibit the phospholipase activity of the venoms from Bothrops jararacussu and Crotalus durissus terrificus. The myotoxic, edema-inducing, coagulant, and hemorrhagic activities were also inhibited. The SDS-PAGE showed that the venom proteins were intact after their incubation with the extracts. This suggests that the possible mechanism of inhibition is not related to the degradation of the protein but rather to their binding to specific sites of the enzymes. The extracts significantly prolonged the survival time of animals in the lethality assay performed with Crotalus durissus terrificus venom and its toxin (crotoxin). The anti-ophidic activity of medicinal plants may aid in the management of snakebites in distant locations by reducing the victim’s local effects and time to heal.

In vivo and in vitro prospection of the anti-ophidic properties exercised by the extracts of Jacaranda decurrens L

The research and development of alternative treatments for snakebites (e.g., medicinal plants) is necessary due to the high costs of the existing ones. The effects of the aqueous extracts from Jacaranda decurrens leaves, roots, and xylopodium were analyzed upon the venom-induced (Bothrops spp. and Crotalus spp.) systemic and local toxicity. The extracts were able to partially inhibit the phospholipase activity of the venoms from Bothrops jararacussu and Crotalus durissus terrificus. The myotoxic, edema-inducing, coagulant, and hemorrhagic activities were also inhibited. The SDS-PAGE showed that the venom proteins were intact after their incubation with the extracts. This suggests that the possible mechanism of inhibition is not related to the degradation of the protein but rather to their binding to specific sites of the enzymes. The extracts significantly prolonged the survival time of animals in the lethality assay performed with Crotalus durissus terrificus venom and its toxin (crotoxin). The anti-ophidic activity of medicinal plants may aid in the management of snakebites in distant locations by reducing the victims local effects and time to heal.(AU)

Geographic variation of individual venom profile of Crotalus durissus snakes

South American rattlesnakes are represented in Brazil by a single species, Crotalus durissus, which has public health importance due to the severity of its envenomation and to its wide geographical distribution. The species is subdivided into several subspecies, but the current classification is controversial. In Brazil, the venoms of C. d. terrificus and C. d. collilineatus are used for hyperimmunization of horses for antivenom production, even though the distinction of these two subspecies are mostly by their geographical distribution. In this context, we described a comparative compositional and functional characterization of individual C. d. collilineatus and C. d. terrificus venoms from three Brazilian states. Methods: We compared the compositional patterns of C. d. terrificus and C. d. collilineatus individual venoms by 1-DE and RP-HPLC. For functional analyzes, the enzymatic activities of PLA2, LAAO, and coagulant activity were evaluated. Finally, the immunorecognition of venom toxins by the crotalic antivenom produced at Butantan Institute was evaluated using Western blotting. Results: The protein profile of individual venoms from C. d. collilineatus and C. d. terrificus showed a comparable overall composition, despite some intraspecific variation, especially regarding crotamine and LAAO. Interestingly, HPLC analysis showed a geographic pattern concerning PLA2. In addition, a remarkable intraspecific variation was also observed in PLA2, LAAO and coagulant activities. The immunorecognition pattern of individual venoms from C. d. collilineatus and C. d. terrificus by crotalic antivenom produced at Butantan Institute was similar. Conclusions: The results highlighted the individual variability among the venoms of C. durissus ssp. specimens. Importantly, our data point to a geographical variation of C. durissus ssp. venom profile, regardless of the subspecies, as evidenced by PLA2 isoforms complexity, which may explain the increase in venom neurotoxicity from Northeastern through Southern Brazil reported for the species.(AU)

Geographic variation of individual venom profile of Crotalus durissus snakes

South American rattlesnakes are represented in Brazil by a single species, Crotalus durissus, which has public health importance due to the severity of its envenomation and to its wide geographical distribution. The species is subdivided into several subspecies, but the current classification is controversial. In Brazil, the venoms of C. d. terrificus and C. d. collilineatus are used for hyperimmunization of horses for antivenom production, even though the distinction of these two subspecies are mostly by their geographical distribution. In this context, we described a comparative compositional and functional characterization of individual C. d. collilineatus and C. d. terrificus venoms from three Brazilian states. Methods: We compared the compositional patterns of C. d. terrificus and C. d. collilineatus individual venoms by 1-DE and RP-HPLC. For functional analyzes, the enzymatic activities of PLA2, LAAO, and coagulant activity were evaluated. Finally, the immunorecognition of venom toxins by the crotalic antivenom produced at Butantan Institute was evaluated using Western blotting. Results: The protein profile of individual venoms from C. d. collilineatus and C. d. terrificus showed a comparable overall composition, despite some intraspecific variation, especially regarding crotamine and LAAO. Interestingly, HPLC analysis showed a geographic pattern concerning PLA2. In addition, a remarkable intraspecific variation was also observed in PLA2, LAAO and coagulant activities. The immunorecognition pattern of individual venoms from C. d. collilineatus and C. d. terrificus by crotalic antivenom produced at Butantan Institute was similar. Conclusions: The results highlighted the individual variability among the venoms of C. durissus ssp. specimens. Importantly, our data point to a geographical variation of C. durissus ssp. venom profile, regardless of the subspecies, as evidenced by PLA2 isoforms complexity, which may explain the increase in venom neurotoxicity from Northeastern through Southern Brazil reported for the species.(AU)

Non-Avian Reptiles of the state of Rio de Janeiro, Brazil: status of knowledge and commented list

Abstract We assessed the current knowledge on non-avian reptile species composition in the state of Rio de Janeiro (RJ), southeastern Brazil. We used published data in indexed journals and verified voucher specimens housed in the herpetological collections of the Museu Nacional (UFRJ), and the Laboratory of Reptiles (ZUFRJ). We also confirmed vouchers from Instituto Vital Brazil (IVB) and from the Museum of Comparative Zoology (MCZ). We compiled a list containing 149 species of reptiles, distributed among Testudines (N = 15), Crocodylia (N = 1) and Squamata (N = 133; six amphisbaenians, 38 lizards and 89 snakes). Our results add 20 species to the previous list known for the state. Four species recorded are endemic to the state of Rio de Janeiro (Anolis neglectus, Glaucomastix littoralis, Leposternon scutigerum, and Liolaemus lutzae). We identified that 21 reptile species recorded in RJ state (nearly 15% of the total) are included in some threat category either in the IUCN, Brazilian (ICMBio) or state lists of endangered species. We also report that seven of the reptiles recorded are non-indigenous to the state: Cnemidophorus aff. lemniscatus (= Cnemidophorus cryptus), Anolis sagrei, Trachemys scripta, Trachemys dorbigni, Hemidactylus mabouia, Crotalus durissus terrificus, and Rhinoclemmys punctularia. The checklist presented here provides a comprehensive database for further research on the herpetofauna of the Brazilian Atlantic Forest.

Identification and characterization of the first endogenous phospholipase A2 inhibitor from a non-venomous tropical snake, Boa constrictor (Serpentes: Boidae)

Abstract Background: Endogenous phospholipase A2 inhibitors from snake blood (sbPLIs) have been isolated from several species around the world, with the primary function of self-protection against the action of toxic phospholipases A2. In American snakes, sbPLIs were solely described in pit vipers, in which the natural protection role is justified. In this study, we described a sbPLI in Boa constrictor (popularly known as jiboia), a non-venomous snake species from America. Methods: PLA2 inhibitory activity was tested in the blood plasma of B. constrictor using C. d. terrificus venom as the enzyme source. Antibodies developed against CNF, a sbγPLI from Crotalus durissus terrificus, were used to investigate the presence of homologues in the blood plasma of B. constrictor. A CNF-like molecule with a PLA2 inhibitory activity was purified by column chromatography. The encoding gene for the inhibitor was cloned from B. constrictor liver tissue. The DNA fragment was cloned, purified and sequenced. The deduced primary sequence of interest was aligned with known sbγPLIs from the literature. Results: The blood plasma of B. constrictor displayed PLA2 inhibitory activity. A CNF-like molecule (named BcNF) was identified and purified from the blood plasma of B. constrictor. Basic properties such as molecular mass, composing amino acids, and pI were comparable, but BcNF displayed reduced specific activity in PLA2 inhibition. BcNF showed highest identity scores (ISs) with sbγPLIs from pit vipers from Latin America (90-100%), followed by gamma inhibitors from Asian viperid (80-90%). ISs below 70% were obtained for BcNF and non-venomous species from Asia. Conclusion: A functional sbγPLI (BcNF) was described in the blood plasma of B. constrictor. BcNF displayed higher primary identity with sbγPLIs from Viperidae than to sbγPLIs from non-venomous species from Asia. The physiological role played by sbγPLIs in non-venomous snake species remains to be understood. Further investigation is needed.(AU)

Non-Avian Reptiles of the state of Rio de Janeiro, Brazil: status of knowledge and commented list

We assessed the current knowledge on non-avian reptile species composition in the state of Rio de Janeiro (RJ), southeastern Brazil. We used published data in indexed journals and verified voucher specimens housed in the herpetological collections of the Museu Nacional (UFRJ), and the Laboratory of Reptiles (ZUFRJ). We also confirmed vouchers from Instituto Vital Brazil (IVB) and from the Museum of Comparative Zoology (MCZ). We compiled a list containing 149 species of reptiles, distributed among Testudines (N = 15), Crocodylia (N = 1) and Squamata (N = 133; six amphisbaenians, 38 lizards and 89 snakes). Our results add 20 species to the previous list known for the state. Four species recorded are endemic to the state of Rio de Janeiro (Anolis neglectus, Glaucomastix littoralis, Leposternon scutigerum, and Liolaemus lutzae). We identified that 21 reptile species recorded in RJ state (nearly 15% of the total) are included in some threat category either in the IUCN, Brazilian (ICMBio) or state lists of endangered species. We also report that seven of the reptiles recorded are non-indigenous to the state: Cnemidophorus aff. lemniscatus (= Cnemidophorus cryptus), Anolis sagrei, Trachemys scripta, Trachemys dorbigni, Hemidactylus mabouia, Crotalus durissus terrificus, and Rhinoclemmys punctularia. The checklist presented here provides a comprehensive database for further research on the herpetofauna of the Brazilian Atlantic Forest.

Non-Avian Reptiles of the state of Rio de Janeiro, Brazil: status of knowledge and commented list

We assessed the current knowledge on non-avian reptile species composition in the state of Rio de Janeiro (RJ), southeastern Brazil. We used published data in indexed journals and verified voucher specimens housed in the herpetological collections of the Museu Nacional (UFRJ), and the Laboratory of Reptiles (ZUFRJ). We also confirmed vouchers from Instituto Vital Brazil (IVB) and from the Museum of Comparative Zoology (MCZ). We compiled a list containing 149 species of reptiles, distributed among Testudines (N = 15), Crocodylia (N = 1) and Squamata (N = 133; six amphisbaenians, 38 lizards and 89 snakes). Our results add 20 species to the previous list known for the state. Four species recorded are endemic to the state of Rio de Janeiro (Anolis neglectus, Glaucomastix littoralis, Leposternon scutigerum, and Liolaemus lutzae). We identified that 21 reptile species recorded in RJ state (nearly 15% of the total) are included in some threat category either in the IUCN, Brazilian (ICMBio) or state lists of endangered species. We also report that seven of the reptiles recorded are non-indigenous to the state: Cnemidophorus aff. lemniscatus (= Cnemidophorus cryptus), Anolis sagrei, Trachemys scripta, Trachemys dorbigni, Hemidactylus mabouia, Crotalus durissus terrificus, and Rhinoclemmys punctularia. The checklist presented here provides a comprehensive database for further research on the herpetofauna of the Brazilian Atlantic Forest.(AU)

Identification and characterization of the first endogenous phospholipase A2 inhibitor from a non-venomous tropical snake, Boa constrictor (Serpentes: Boidae)

Background:Endogenous phospholipase A2 inhibitors from snake blood (sbPLIs) have been isolated from several species around the world, with the primary function of self-protection against the action of toxic phospholipases A2. In American snakes, sbPLIs were solely described in pit vipers, in which the natural protection role is justified. In this study, we described a sbPLI in Boa constrictor (popularly known as jiboia), a non-venomous snake species from America.Methods:PLA2 inhibitory activity was tested in the blood plasma of B. constrictor using C. d. terrificus venom as the enzyme source. Antibodies developed against CNF, a sbγPLI from Crotalus durissus terrificus, were used to investigate the presence of homologues in the blood plasma of B. constrictor. A CNF-like molecule with a PLA2 inhibitory activity was purified by column chromatography. The encoding gene for the inhibitor was cloned from B. constrictor liver tissue. The DNA fragment was cloned, purified and sequenced. The deduced primary sequence of interest was aligned with known sbγPLIs from the literature.Results:The blood plasma of B. constrictor displayed PLA2 inhibitory activity. A CNF-like molecule (named BcNF) was identified and purified from the blood plasma of B. constrictor. Basic properties such as molecular mass, composing amino acids, and pI were comparable, but BcNF displayed reduced specific activity in PLA2 inhibition. BcNF showed highest identity scores (ISs) with sbγPLIs from pit vipers from Latin America (90-100%), followed by gamma inhibitors from Asian viperid (80-90%). ISs below 70% were obtained for BcNF and non-venomous species from Asia.Conclusion:A functional sbγPLI (BcNF) was described in the blood plasma of B. constrictor. BcNF displayed higher primary identity with sbγPLIs from Viperidae than...(AU)

Fractal dimension in liver histological findings of Wistar rats experimentally intoxicated with venom of Crotalus durissus terrificus

Background: Accidents caused by venom of Crotalus durissus snakes, popularly known in Brazil as rattlesnake, aresecond in relation to the occurrence and first place in deaths in humans and animals, mainly due to the great neurotoxic,myotoxic, coagulant, nephrotoxic and hepatotoxic potential of their venom. The effects observed are due to the action ofthe main poison fractions and among them we can mention crotoxin (representing 50% of the total poison), crotamine,gyroxine and conxulxin. The present study aimed to analyze the liver of experimentally poisoned Wistar rats with venomof Crotalus durissus terrificus by means of histological and fractal analysis. The hypothesis is that the venom of Crotalusdurissus terrificus is can induce hepatic damage at the dose recommended in this study, that its alterations can be quantifiedby the fractal dimension and that the antiofidic serum botropic crotalic be able to minimize the hepatic lesions inducedby the venom.Materials, Methods & Results: Ninety rats were distributed into different groups and treated with: control group (GC, n= 30) 0.9% sodium chloride solution; venom group (GV, n = 30) crotalic venom at the dose of 1 mg/kg; (GVS, n = 30)crotalic venom at the dose of 1 mg/Kg and antiofidic serum 6 h after the application of the venom at the dose recommendedby the manufacturer. Liver samples were collected at 2 h (M1), 8 h (M2) and 24 h (M3) after venom administration andsubmitted to histological analysis and fractal dimension (DF) using the ImageJ® software and box-counting method. Procedures for collecting, processing and analyzing samples were standardized. For statistical analyzes, after the normalitywas verified by the Shapiro-Wilk test and homogeneity by the Bartlett test, the data were submitted to analysis of variance(ANOVA) with Duncan test contrast with a significance level of 5%. No significant lesions were observed in GC, howevernecrosis...(AU)

Fractal dimension in liver histological findings of Wistar rats experimentally intoxicated with venom of Crotalus durissus terrificus

Background: Accidents caused by venom of Crotalus durissus snakes, popularly known in Brazil as rattlesnake, aresecond in relation to the occurrence and first place in deaths in humans and animals, mainly due to the great neurotoxic,myotoxic, coagulant, nephrotoxic and hepatotoxic potential of their venom. The effects observed are due to the action ofthe main poison fractions and among them we can mention crotoxin (representing 50% of the total poison), crotamine,gyroxine and conxulxin. The present study aimed to analyze the liver of experimentally poisoned Wistar rats with venomof Crotalus durissus terrificus by means of histological and fractal analysis. The hypothesis is that the venom of Crotalusdurissus terrificus is can induce hepatic damage at the dose recommended in this study, that its alterations can be quantifiedby the fractal dimension and that the antiofidic serum botropic crotalic be able to minimize the hepatic lesions inducedby the venom.Materials, Methods & Results: Ninety rats were distributed into different groups and treated with: control group (GC, n= 30) 0.9% sodium chloride solution; venom group (GV, n = 30) crotalic venom at the dose of 1 mg/kg; (GVS, n = 30)crotalic venom at the dose of 1 mg/Kg and antiofidic serum 6 h after the application of the venom at the dose recommendedby the manufacturer. Liver samples were collected at 2 h (M1), 8 h (M2) and 24 h (M3) after venom administration andsubmitted to histological analysis and fractal dimension (DF) using the ImageJ® software and box-counting method. Procedures for collecting, processing and analyzing samples were standardized. For statistical analyzes, after the normalitywas verified by the Shapiro-Wilk test and homogeneity by the Bartlett test, the data were submitted to analysis of variance(ANOVA) with Duncan test contrast with a significance level of 5%. No significant lesions were observed in GC, howevernecrosis...

Unique heterologous fibrin biopolymer with hemostatic, adhesive, sealant, scaffold and drug delivery properties: a systematic review

Fibrin biopolymers, previously referred as "fibrin glue" or "fibrin sealants", are natural biomaterials with diverse applications on health. They have hemostatic, adhesive, sealant, scaffold and drug delivery properties and have become widely used in medical and dental procedures. Historically, these biomaterials are produced from human fibrinogen and human or animal thrombin, and the possibility of transmission of infectious diseases by human blood is not ruled out. In the 1990s, to overcome this problem, a new heterologous biomaterial composed of a thrombin-like enzyme purified from Crotalus durissus terrificus venom and a cryoprecipitate rich in fibrinogen extracted from buffaloes Bubalus bubalis blood has been proposed. Therefore, a systematic review of studies on exclusively heterologous fibrin sealants published between 1989 and 2018 was carried out using the following databases: PubMed, SciELO and Google Scholar. The keyword used was "heterologous fibrin sealant". The search resulted in 35 scientific papers in PubMed, four in SciELO and 674 in Google Scholar. After applying the inclusion/exclusion criteria and complete reading of the articles, 30 studies were selected, which formed the basis of this systematic review. It has been observed that the only completely heterologous sealant is the one produced by CEVAP/UNESP. This heterologous biopolymer is proven effective by several studies published in refereed scientific journals. In addition, clinical trials phase I/II for the treatment of chronic venous ulcers authorized by the Brazilian Health Regulatory Agency (ANVISA) were completed. Preliminary results have indicated a safe and promising effective product. Phase III clinical trials will be proposed and required to validate these preliminary findings.(AU)

Unique heterologous fibrin biopolymer with hemostatic, adhesive, sealant, scaffold and drug delivery properties: a systematic review

Fibrin biopolymers, previously referred as “fibrin glue” or “fibrin sealants”, are natural biomaterials with diverse applications on health. They have hemostatic, adhesive, sealant, scaffold and drug delivery properties and have become widely used in medical and dental procedures. Historically, these biomaterials are produced from human fibrinogen and human or animal thrombin, and the possibility of transmission of infectious diseases by human blood is not ruled out. In the 1990s, to overcome this problem, a new heterologous biomaterial composed of a thrombin-like enzyme purified from Crotalus durissus terrificus venom and a cryoprecipitate rich in fibrinogen extracted from buffaloes Bubalus bubalis blood has been proposed. Therefore, a systematic review of studies on exclusively heterologous fibrin sealants published between 1989 and 2018 was carried out using the following databases: PubMed, SciELO and Google Scholar. The keyword used was “heterologous fibrin sealant”. The search resulted in 35 scientific papers in PubMed, four in SciELO and 674 in Google Scholar. After applying the inclusion/exclusion criteria and complete reading of the articles, 30 studies were selected, which formed the basis of this systematic review. It has been observed that the only completely heterologous sealant is the one produced by CEVAP/UNESP. This heterologous biopolymer is proven effective by several studies published in refereed scientific journals. In addition, clinical trials phase I/II for the treatment of chronic venous ulcers authorized by the Brazilian Health Regulatory Agency (ANVISA) were completed. Preliminary results have indicated a safe and promising effective product. Phase III clinical trials will be proposed and required to validate these preliminary findings.(AU)

Hormonal and ultrasonographic characterization of the seasonal reproductive cycle of male and female Crotalus durissus terrificus

Research concerning to characterize seasonal reproductive cycle in males and females of Crotalus durissus terrificus by ultrasound and hormonal measurement. Reproductive aspects (follicular and testicular cycles, and pregnancy) from 28 adult snakes (14 males and 14 females) during different months of the years were studied. Snakes housed individually in cages in an environment with controlled luminosity and humidity, and fed monthly. Females were examined by ultrasound during quiescence and active follicular phase, and pregnancy for follicular and embryo/fetal development. Males were evaluated to testicular echotexture and measurements during reproductive and non-reproductive season. The blood samples were collected from males and females for serum testosterone and progesterone determination, respectively. In 77% males the testes were identified by ultrasound and found increased size during summer, decreased serum testosterone in winter, and positive correlation between serum testosterone and testes size. There was no change in testicular echotexture in according to season. Testosterone concentration was decreased during winter and it was positively correlated with testes size. In 71% females, were observed follicular development (vitellogenesis) and gestation since winter to spring by ultrasonography. Parturition occurred mainly in summer. Pregnancy length was 123.0 ± 11.4 days, with mean 6.9 ± 1.5 newborns/female, and there was gradual increase of serum progesterone during this period. There was no variation in progesterone concentration in nongravid females. Males and females Tropical Rattlesnake show seasonal variation of reproductive cycle and was clear a biennial cycle in female. The ultrasonography can be considered an essential tool to accomplish the follicular development, pregnancy and testicular alterations in Tropical Rattlesnake.(AU)

Hormonal and ultrasonographic characterization of the seasonal reproductive cycle of male and female Crotalus durissus terrificus

Research concerning to characterize seasonal reproductive cycle in males and females of Crotalus durissus terrificus by ultrasound and hormonal measurement. Reproductive aspects (follicular and testicular cycles, and pregnancy) from 28 adult snakes (14 males and 14 females) during different months of the years were studied. Snakes housed individually in cages in an environment with controlled luminosity and humidity, and fed monthly. Females were examined by ultrasound during quiescence and active follicular phase, and pregnancy for follicular and embryo/fetal development. Males were evaluated to testicular echotexture and measurements during reproductive and non-reproductive season. The blood samples were collected from males and females for serum testosterone and progesterone determination, respectively. In 77% males the testes were identified by ultrasound and found increased size during summer, decreased serum testosterone in winter, and positive correlation between serum testosterone and testes size. There was no change in testicular echotexture in according to season. Testosterone concentration was decreased during winter and it was positively correlated with testes size. In 71% females, were observed follicular development (vitellogenesis) and gestation since winter to spring by ultrasonography. Parturition occurred mainly in summer. Pregnancy length was 123.0 ± 11.4 days, with mean 6.9 ± 1.5 newborns/female, and there was gradual increase of serum progesterone during this period. There was no variation in progesterone concentration in nongravid females. Males and females Tropical Rattlesnake show seasonal variation of reproductive cycle and was clear a biennial cycle in female. The ultrasonography can be considered an essential tool to accomplish the follicular development, pregnancy and testicular alterations in Tropical Rattlesnake.

Immunotherapeutic potential of Crotoxin: anti-inflammatory and immunosuppressive properties

For the past 80 years, Crotoxin has become one of the most investigated isolated toxins from snake venoms, partially due to its major role as the main toxic component in the venom of the South American rattlesnake Crotalus durissus terrificus. However, in the past decades, progressive studies have led researchers to shift their focus on Crotoxin, opening novel perspectives and applications as a therapeutic approach. Although this toxin acts on a wide variety of biological events, the modulation of immune responses is considered as one of its most relevant behaviors. Therefore, the present review describes the scientific investigations on the capacity of Crotoxin to modulate anti-inflammatory and immunosuppressive responses, and its application as a medicinal immunopharmacological approach. In addition, this review will also discuss its mechanisms, involving cellular and molecular pathways, capable of improving pathological alterations related to immune-associated disorders.(AU)

Evaluation of the antibacterial activity of Crotalus durissus terrificus crude venom / Avaliação da atividade antibacteriana do veneno bruto de Crotalus durissus terrificus

Snake venoms are recognized as a promising source of pharmacologically active substances and are potentially useful for the development of new antimicrobial drugs. This study aimed to investigate the antimicrobial activity of the venom from the rattlesnake Crotalus durissus terrificus against several bacteria. Antibacterial activity was determined by using the plate microdilution method and the activity on the bacterial envelope structure was screened by using the crystal violet assay. The proteins in crude venom were separated by electrophoresis and characterized regarding their proteolytic activity. C. d. terrificus venom exhibited antimicrobial action against gram-positive and gram-negative bacteria. MIC values were defined for Pseudomonas aeruginosa ATCC 27853 (62.5 µg/mL), Staphylococcus aureus ATCC 25923 (125 µg/mL), and Micrococcus luteus ATCC 9341 (≤500 µg/mL). For Salmonella enterica serovar typhimurium ATCC 14028 and Corynebacterium glutamicum ATCC 13032, the decrease in bacterial growth was not detected visually, but was statistically significant. The crystal violet assay demonstrated that the crude venom increased bacterial cell permeability and the secreted protein profile agreed with previous reports. The results suggest that the proteins with lytic activity against bacteria in C. d. terrificus venom deserve further characterization as they may offer reinforcements to the weak therapeutic arsenal used to fight microbial multidrug resistance.

Os venenos de serpentes são reconhecidos como uma fonte promissora de substâncias farmacologicamente ativas e potencialmente úteis para o desenvolvimento de novas drogas antimicrobianas. Esse trabalho teve como objetivo investigar a atividade antimicrobiana do veneno de Crotalus durissus terrificus contra várias bactérias. A determinação da atividade antibacteriana foi realizada pelo método de microdiluição em placas e a ação na estrutura do envelope bacteriano pelo ensaio violeta de cristal. As proteínas do extrato bruto foram separadas por eletroforese e caracterizadas quanto à sua atividade proteolítica. O veneno de C. d. terrificus apresentou ação antimicrobiana frente bactérias gram-positivas e gram-negativas. Os valores de MIC foram definidos para Pseudomonas aeruginosa ATCC 27853 (62.5 µg/ mL), Staphylococcus aureus ATCC 25923 (125 µg/mL) e Micrococcus luteus ATCC 9341 (≤ 500 µg/mL). Para Salmonella enterica serovar typhimurium ATCC 14028 e Corynebacterium glutamicum ATCC 13032, o decréscimo no crescimento bacteriano não foi detectado visualmente, mas foi estatisticamente significante. O teste do cristal violeta demonstrou que o veneno bruto aumentou a permeabilidade das células bacterianas e o perfil de proteína secretada está em consonância com relatos anteriores. Os resultados sugerem que as proteínas com atividade lítica contra bactérias no veneno de C. d. terrificus merecem atenção para uma melhor caracterização, uma vez que podem trazer reforços para o escasso arsenal terapêutico empregado para combater a multirresistência microbiana.

Evaluation of the antibacterial activity of Crotalus durissus terrificus crude venom / Avaliação da atividade antibacteriana do veneno bruto de Crotalus durissus terrificus

Snake venoms are recognized as a promising source of pharmacologically active substances and are potentially useful for the development of new antimicrobial drugs. This study aimed to investigate the antimicrobial activity of the venom from the rattlesnake Crotalus durissus terrificus against several bacteria. Antibacterial activity was determined by using the plate microdilution method and the activity on the bacterial envelope structure was screened by using the crystal violet assay. The proteins in crude venom were separated by electrophoresis and characterized regarding their proteolytic activity. C. d. terrificus venom exhibited antimicrobial action against gram-positive and gram-negative bacteria. MIC values were defined for Pseudomonas aeruginosa ATCC 27853 (62.5 µg/mL), Staphylococcus aureus ATCC 25923 (125 µg/mL), and Micrococcus luteus ATCC 9341 (≤500 µg/mL). For Salmonella enterica serovar typhimurium ATCC 14028 and Corynebacterium glutamicum ATCC 13032, the decrease in bacterial growth was not detected visually, but was statistically significant. The crystal violet assay demonstrated that the crude venom increased bacterial cell permeability and the secreted protein profile agreed with previous reports. The results suggest that the proteins with lytic activity against bacteria in C. d. terrificus venom deserve further characterization as they may offer reinforcements to the weak therapeutic arsenal used to fight microbial multidrug resistance.(AU)

Os venenos de serpentes são reconhecidos como uma fonte promissora de substâncias farmacologicamente ativas e potencialmente úteis para o desenvolvimento de novas drogas antimicrobianas. Esse trabalho teve como objetivo investigar a atividade antimicrobiana do veneno de Crotalus durissus terrificus contra várias bactérias. A determinação da atividade antibacteriana foi realizada pelo método de microdiluição em placas e a ação na estrutura do envelope bacteriano pelo ensaio violeta de cristal. As proteínas do extrato bruto foram separadas por eletroforese e caracterizadas quanto à sua atividade proteolítica. O veneno de C. d. terrificus apresentou ação antimicrobiana frente bactérias gram-positivas e gram-negativas. Os valores de MIC foram definidos para Pseudomonas aeruginosa ATCC 27853 (62.5 µg/ mL), Staphylococcus aureus ATCC 25923 (125 µg/mL) e Micrococcus luteus ATCC 9341 (≤ 500 µg/mL). Para Salmonella enterica serovar typhimurium ATCC 14028 e Corynebacterium glutamicum ATCC 13032, o decréscimo no crescimento bacteriano não foi detectado visualmente, mas foi estatisticamente significante. O teste do cristal violeta demonstrou que o veneno bruto aumentou a permeabilidade das células bacterianas e o perfil de proteína secretada está em consonância com relatos anteriores. Os resultados sugerem que as proteínas com atividade lítica contra bactérias no veneno de C. d. terrificus merecem atenção para uma melhor caracterização, uma vez que podem trazer reforços para o escasso arsenal terapêutico empregado para combater a multirresistência microbiana.(AU)

Immunotherapeutic potential of Crotoxin: anti-inflammatory and immunosuppressive properties

For the past 80 years, Crotoxin has become one of the most investigated isolated toxins from snake venoms, partially due to its major role as the main toxic component in the venom of the South American rattlesnake Crotalus durissus terrificus. However, in the past decades, progressive studies have led researchers to shift their focus on Crotoxin, opening novel perspectives and applications as a therapeutic approach. Although this toxin acts on a wide variety of biological events, the modulation of immune responses is considered as one of its most relevant behaviors. Therefore, the present review describes the scientific investigations on the capacity of Crotoxin to modulate anti-inflammatory and immunosuppressive responses, and its application as a medicinal immunopharmacological approach. In addition, this review will also discuss its mechanisms, involving cellular and molecular pathways, capable of improving pathological alterations related to immune-associated disorders.(AU)

Crotalus durissus terrificus crotapotin naturally displays preferred positions for amino acid substitutions

Abstract Background Classically, Crotalus durissus terrificus (Cdt) venom can be described, according to chromatographic criteria, as a simple venom, composed of four major toxins, namely: gyroxin, crotamine, crotoxin and convulxin. Crotoxin is a non-covalent heterodimeric neurotoxin constituted of two subunits: an active phospholipase A2 and a chaperone protein, termed crotapotin. This molecule is composed of three peptide chains connected by seven disulfide bridges. Naturally occurring variants/isoforms of either crotoxin or crotapotin itself have already been reported. Methods The crude Cdt venom was separated by using RP-HPLC and the toxins were identified by mass spectrometry (MS). Crotapotin was purified, reduced and alkylated in order to separate the peptide chains that were further analyzed by mass spectrometry and de novo peptide sequencing. Results The RP-HPLC profile of the isolated crotapotin chains already indicated that the chain would present isoforms, which was corroborated by the MS and tandem mass spectrometry analyses. Conclusion It was possible to observe that the Cdt crotapotin displays a preferred amino acid substitution pattern present in the chain, at positions 31 and 40. Moreover, substitutions could also be observed in and chains (one for each). The combinations of these four different peptides, with the already described chains, would produce ten different crotapotins, which is compatible to our previous observations for the Cdt venom.