RESUMO
Calotropis procera produces a milky sap containing proteolytic enzymes. At low concentrations, they induce milk-clotting (60 µg/ml) and to dehair hides (0.05 and 0.1%). A protocol for obtaining the enzymes is reported. The latex was mixed with distilled water and the mixture was cleaned through centrifugation. It was dialyzed with distilled water and centrifuged again to recover the soluble fraction [EP]. The dialyze is a key feature of the process. EP was characterized in terms of protein profile, chemical stability, among other criteria. Wild plants belonging to ten geographic regions and grown in different ecological conditions were used as latex source. Collections were carried out, spaced at three-month, according to the seasons at the site of the study. Proteolytic activity was measured as an internal marker and for determining stability of the samples. EP was also analyzed for metal content and microbiology. EP showed similar magnitude of proteolysis, chromatographic and electrophoretic profiles of proteins. Samples stored at 25 °C exhibited reduced solubility (11%) and proteolytic capacity (11%) after six months. Enzyme autolysis was negligible. Microbiological and metal analyses revealed standard quality of all the samples tested. EP induced milk clotting and hide dehairing after storage for up to six months.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Calotropis/enzimologia , Técnicas de Química Analítica/normas , Ecossistema , Látex/química , Proteínas de Plantas/metabolismo , Estações do Ano , Pelo Animal/efeitos dos fármacos , Animais , Ácido Aspártico Endopeptidases/análise , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/farmacologia , Bovinos , Cabras , Remoção de Cabelo/métodos , Látex/isolamento & purificação , Proteínas de Plantas/análise , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Proteólise , Padrões de Referência , SolubilidadeRESUMO
BACKGROUND: Calotropis procera latex protein fraction (LP) was previously shown to protect animals from septic shock. Further investigations showed that LP modulate nitric oxide and cytokines levels. OBJECTIVES: To evaluate whether the protective effects of LP, against lethal bacterial infection, is observed in its subfractions (LPPII and LPPIII). METHODS: Subfractions (5 and 10 mg/kg) were tested by i.p. administration, 24 h before challenging with lethal injection (i.p.) of Salmonella Typhimurium. LPPIII (5 mg/kg) which showed higher survival rate was assayed to evaluate bacterial clearance, histopathology, leukocyte recruitment, plasma coagulation time, cytokines and NO levels. FINDINGS: LPPIII protected 70% of animals of death. The animals given LPPIII exhibited reduced bacterial load in blood and peritoneal fluid after 24 h compared to the control. LPPIII promoted macrophage infiltration in spleen and liver. LPPIII restored the coagulation time of infected animals, increased IL-10 and reduced NO in blood. MAIN CONCLUSIONS: LPPIII recruited macrophages to the target organs of bacterial infection. This addressed inflammatory stimulus seems to reduce bacterial colonisation in spleen and liver, down regulate bacterial spread and contribute to avoid septic shock.
Assuntos
Antibacterianos/uso terapêutico , Calotropis/química , Homeostase/efeitos dos fármacos , Inflamação/tratamento farmacológico , Látex/química , Extratos Vegetais/farmacologia , Proteínas de Plantas/uso terapêutico , Infecções por Salmonella/tratamento farmacológico , Animais , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Regulação para Baixo , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologiaRESUMO
BACKGROUND Calotropis procera latex protein fraction (LP) was previously shown to protect animals from septic shock. Further investigations showed that LP modulate nitric oxide and cytokines levels. OBJECTIVES To evaluate whether the protective effects of LP, against lethal bacterial infection, is observed in its subfractions (LPPII and LPPIII). METHODS Subfractions (5 and 10 mg/kg) were tested by i.p. administration, 24 h before challenging with lethal injection (i.p.) of Salmonella Typhimurium. LPPIII (5 mg/kg) which showed higher survival rate was assayed to evaluate bacterial clearance, histopathology, leukocyte recruitment, plasma coagulation time, cytokines and NO levels. FINDINGS LPPIII protected 70% of animals of death. The animals given LPPIII exhibited reduced bacterial load in blood and peritoneal fluid after 24 h compared to the control. LPPIII promoted macrophage infiltration in spleen and liver. LPPIII restored the coagulation time of infected animals, increased IL-10 and reduced NO in blood. MAIN CONCLUSIONS LPPIII recruited macrophages to the target organs of bacterial infection. This addressed inflammatory stimulus seems to reduce bacterial colonisation in spleen and liver, down regulate bacterial spread and contribute to avoid septic shock.