- Email: [email protected]
220. Evaluation of Extracts and Partitions from Aerial Parts of Baccharis microdonta on Enzymatic Activity, Pro-Inflammatory and Myotoxic Activities Induced by Secretory Phospholipase A2 from Bothrops jararacussu
208
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
complementation by rBaltMIP. In this activity, it was observed that the snake antivenom for veterinary use was capable of neutralizing the myotoxic activity of Bothrops jararacussu venom and BthTX-I, moreover, the inhibitor rBaltMIP was more effective to neutralize the myotoxicity of these compounds compared to the antiserum tested. Therefore, when the antiserum was supplemented with rBaltMIP was possible to observe a complementation of its inhibition activity, suggesting a possible commercial application for this inhibitor. Discussion and Conclusions: It is known that myotoxins has a low immunogenicity, which means that antivenom is not fully effective for those enzymes. rBaltMIP showed high potential for complementation of snake antivenom, however, future studies should be performed. Keywords: rBaltMIP, PLI, myotoxin inhibitor, myotoxic activity 10.1016/j.toxicon.2012.04.220
220. Evaluation of Extracts and Partitions from Aerial Parts of Baccharis microdonta on Enzymatic Activity, Pro-Inflammatory and Myotoxic Activities Induced by Secretory Phospholipase A2 from Bothrops jararacussu Veronica C.G. Soares 1, Daniel Bristot 1, Camila L. Pires 1, Marcos H. Toyama 1, Paulete Romoff 2, Marcelo J. Pena 2, Oriana A. Favero 3, Daniela de O. Toyama 3 1 General Biology, UNESP - Campus Experimental do Litoral Paulista, SP-Brazil 2 Centro de Ciências e Humaniidades - Universidade Presbiteriana Mackenzie, SP-Brazil 3 Centro de Ciências Biológicas e da Saúde - Universidade Presbiteriana Mackenzie, SP-Brazil E-mail address: [email protected] (D.deO. Toyama).
Background: Several species of Baccharis have been extensively used in folk medicine for the treatment or prevention of anemia, diabetes and stomach, and other inflammatory diseases. As the inflammation involves the mobilization of arachidonic acid by phospholipase A2, this project used the sPLA2 purified from the Bothrops jararacussu as molecular target for the initial studies for characterize the anti-phospholipasic A2 of the methanol extract and its partitions in hexane, dichloromethane, ethyl acetate and Butane -1-ol. Methods: For purification of sPLA2 from Bothrops jaracussu used a combination of ion exchange and reverse phase HPLC, for obtantion methanol extraction and their respective partitions. The enzymatic activity was done using a NOB and all experiments were done on the ELISA plate reader. The Baccharis microdonta aerial parts were processed for preparation of methanolic was partitioned with hexane, butane-1-ol, dichloromethane and ethyl acetate partitions. Results: The inhibition enzymatic activity of phospholipase A2 was observed only for the partition butane-1-ol while the methanolic extract and the other partitions showed a little or absent. The paw edema test performed in mice were performed with native sPLA 2 from Bothrops jararacussu, sPLA2 previously treated with the extract and its partitions, which they have also been previously
injected into the animals 30 minutes before the administration of native sPLA2. Under these conditions, we observed that the methanol extract and dichloromethane partition abolished the edematogenic effect of sPLA2 of native Bothrops jaracussu PLA2. The partition in Ethyl acetate was injected 30 minutes before abolished the edema induced by native sPLA2. The ethyl acetate partition previously injected 30 minutes before or previously incubated with the native sPLA2 abolished the myotoxic effects of sPLA2. These results suggest that the methanol extract and dichloromethane partitions showed a promising antiinflammatory activity but did not directly inhibit phospholipase A2. The real-time PCR results showed a decrease in synthesis of COX-1, so that the partition dichloromethane and methanol extract can inhibit the inflammatory activity of sPLA 2 via inhibition of COX-1. Discussion: The previous result analysis showed that Baccharis microdonta present some bioactive compound that inhibited the sPLA2 enzymatic activity that seen only found in the butane-1-ol partition that in general allowed the purification of glycosiled flavonoids and tannins that did not extracted by other partition. Keywords: Baccharis microdonta, phospholipase A2, Bothrops jaracussu, COX 10.1016/j.toxicon.2012.04.221
221. The Interaction of the Antitoxin DM43 with a Snake Venom Metalloproteinase Analyzed by Mass Spectrometry and Surface Plasmon Resonance Guilherme D. Brand 1, Rune Salbo 2, Thomas J.D. Jørgensen 3, Carlos Bloch Jr. 1, Elisabetta B. Erba 4, Carol V. Robinson 5, Isabelle Tanjoni 6, Ana M. Moura-da-Silva 6, Peter Roepstorff 3, Gilberto B. Domont 7, 9,10, Jonas Perales 8, 9,10, Richard H. Valente 8, 9,10, Ana G.C. Neves-Ferreira 8, 9,10 1 Laboratory of Mass Spectrometry, Embrapa-Recursos Genéticos e Biotecnologia, Brazil 2 Diabetes Protein Engineering, Novo Nordisk A/S, Denmark 3 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Denmark 4 Laboratory of Organic Chemistry, ETH Zürich, Switzerland 5 Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, United Kingdom 6 Laboratory of Immunopathology, Butantan Institute, Brazil 7 Laboratory of Protein Chemistry, Chemistry Institute, Federal University of Rio de Janeiro, Brazil 8 Laboratory of Toxinology, Oswaldo Cruz Institute, Fiocruz, Brazil 9 Rio de Janeiro Proteomic Network/FAPERJ, Brazil 10 INCTTOX /CNPq, Brazil E-mail address: rhv4u@ioc.fiocruz.br (R.H. Valente).
Background: Natural inhibitors of snake toxins are promising molecules that can contribute to the rational development of new ancillary snakebite envenomation therapies. DM43 is a circulating dimeric antitoxin isolated from Didelphis aurita, a South American marsupial naturally immune to snake envenomation. This endogenous inhibitor binds non-covalently to jararhagin, the main hemorrhagic metalloproteinase from Bothrops jararaca snake venom, and efficiently neutralizes its toxicity. The aim of this study was to apply mass spectrometry and
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
complementation by rBaltMIP. In this activity, it was observed that the snake antivenom for veterinary use was capable of neutralizing the myotoxic activity of Bothrops jararacussu venom and BthTX-I, moreover, the inhibitor rBaltMIP was more effective to neutralize the myotoxicity of these compounds compared to the antiserum tested. Therefore, when the antiserum was supplemented with rBaltMIP was possible to observe a complementation of its inhibition activity, suggesting a possible commercial application for this inhibitor. Discussion and Conclusions: It is known that myotoxins has a low immunogenicity, which means that antivenom is not fully effective for those enzymes. rBaltMIP showed high potential for complementation of snake antivenom, however, future studies should be performed. Keywords: rBaltMIP, PLI, myotoxin inhibitor, myotoxic activity 10.1016/j.toxicon.2012.04.220
220. Evaluation of Extracts and Partitions from Aerial Parts of Baccharis microdonta on Enzymatic Activity, Pro-Inflammatory and Myotoxic Activities Induced by Secretory Phospholipase A2 from Bothrops jararacussu Veronica C.G. Soares 1, Daniel Bristot 1, Camila L. Pires 1, Marcos H. Toyama 1, Paulete Romoff 2, Marcelo J. Pena 2, Oriana A. Favero 3, Daniela de O. Toyama 3 1 General Biology, UNESP - Campus Experimental do Litoral Paulista, SP-Brazil 2 Centro de Ciências e Humaniidades - Universidade Presbiteriana Mackenzie, SP-Brazil 3 Centro de Ciências Biológicas e da Saúde - Universidade Presbiteriana Mackenzie, SP-Brazil E-mail address: [email protected] (D.deO. Toyama).
Background: Several species of Baccharis have been extensively used in folk medicine for the treatment or prevention of anemia, diabetes and stomach, and other inflammatory diseases. As the inflammation involves the mobilization of arachidonic acid by phospholipase A2, this project used the sPLA2 purified from the Bothrops jararacussu as molecular target for the initial studies for characterize the anti-phospholipasic A2 of the methanol extract and its partitions in hexane, dichloromethane, ethyl acetate and Butane -1-ol. Methods: For purification of sPLA2 from Bothrops jaracussu used a combination of ion exchange and reverse phase HPLC, for obtantion methanol extraction and their respective partitions. The enzymatic activity was done using a NOB and all experiments were done on the ELISA plate reader. The Baccharis microdonta aerial parts were processed for preparation of methanolic was partitioned with hexane, butane-1-ol, dichloromethane and ethyl acetate partitions. Results: The inhibition enzymatic activity of phospholipase A2 was observed only for the partition butane-1-ol while the methanolic extract and the other partitions showed a little or absent. The paw edema test performed in mice were performed with native sPLA 2 from Bothrops jararacussu, sPLA2 previously treated with the extract and its partitions, which they have also been previously
injected into the animals 30 minutes before the administration of native sPLA2. Under these conditions, we observed that the methanol extract and dichloromethane partition abolished the edematogenic effect of sPLA2 of native Bothrops jaracussu PLA2. The partition in Ethyl acetate was injected 30 minutes before abolished the edema induced by native sPLA2. The ethyl acetate partition previously injected 30 minutes before or previously incubated with the native sPLA2 abolished the myotoxic effects of sPLA2. These results suggest that the methanol extract and dichloromethane partitions showed a promising antiinflammatory activity but did not directly inhibit phospholipase A2. The real-time PCR results showed a decrease in synthesis of COX-1, so that the partition dichloromethane and methanol extract can inhibit the inflammatory activity of sPLA 2 via inhibition of COX-1. Discussion: The previous result analysis showed that Baccharis microdonta present some bioactive compound that inhibited the sPLA2 enzymatic activity that seen only found in the butane-1-ol partition that in general allowed the purification of glycosiled flavonoids and tannins that did not extracted by other partition. Keywords: Baccharis microdonta, phospholipase A2, Bothrops jaracussu, COX 10.1016/j.toxicon.2012.04.221
221. The Interaction of the Antitoxin DM43 with a Snake Venom Metalloproteinase Analyzed by Mass Spectrometry and Surface Plasmon Resonance Guilherme D. Brand 1, Rune Salbo 2, Thomas J.D. Jørgensen 3, Carlos Bloch Jr. 1, Elisabetta B. Erba 4, Carol V. Robinson 5, Isabelle Tanjoni 6, Ana M. Moura-da-Silva 6, Peter Roepstorff 3, Gilberto B. Domont 7, 9,10, Jonas Perales 8, 9,10, Richard H. Valente 8, 9,10, Ana G.C. Neves-Ferreira 8, 9,10 1 Laboratory of Mass Spectrometry, Embrapa-Recursos Genéticos e Biotecnologia, Brazil 2 Diabetes Protein Engineering, Novo Nordisk A/S, Denmark 3 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Denmark 4 Laboratory of Organic Chemistry, ETH Zürich, Switzerland 5 Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, United Kingdom 6 Laboratory of Immunopathology, Butantan Institute, Brazil 7 Laboratory of Protein Chemistry, Chemistry Institute, Federal University of Rio de Janeiro, Brazil 8 Laboratory of Toxinology, Oswaldo Cruz Institute, Fiocruz, Brazil 9 Rio de Janeiro Proteomic Network/FAPERJ, Brazil 10 INCTTOX /CNPq, Brazil E-mail address: rhv4u@ioc.fiocruz.br (R.H. Valente).
Background: Natural inhibitors of snake toxins are promising molecules that can contribute to the rational development of new ancillary snakebite envenomation therapies. DM43 is a circulating dimeric antitoxin isolated from Didelphis aurita, a South American marsupial naturally immune to snake envenomation. This endogenous inhibitor binds non-covalently to jararhagin, the main hemorrhagic metalloproteinase from Bothrops jararaca snake venom, and efficiently neutralizes its toxicity. The aim of this study was to apply mass spectrometry and