title: Protein-rich fraction of Cnidoscolus urens (L.) Arthur leaves: enzymatic characterization and procoagulant and fibrinogenolytic activities.
abstract: Proteolytic enzymes are important macromolecules in the regulation of biochemical processes in living organisms. Additionally, these versatile biomolecules have numerous applications in the industrial segment. In this study we have characterized a protein-rich fraction of Cnidoscolus urens (L.) Arthur leaves, rich in proteolytic enzymes, and evaluated its effects on the coagulation cascade. Three protein-rich fractions were obtained from the crude extract of C. urens leaves by precipitation with acetone. Fraction F1.0 showed higher proteolytic activity upon azocasein, and thus, was chosen for subsequent tests. The proteolytic activity of F1.0 on fibrinogen was dose-dependent and time-dependent. The extract demonstrated procoagulant activity on citrated plasma and reduced the APTT, not exerting effects on PT. Despite the fibrin(ogen)olytic activity, F1.0 showed no defibrinogenating activity in vivo. The fraction F1.0 did not express hemorrhagic nor hemolytic activities. The proteolytic activity was inhibited by E-64, EDTA and in the presence of metal ions, and increased when pretreated with reducing agents, suggesting that the observed activity was mostly due to cysteine proteases. Several bands with proteolytic activity were detected by zymography with gelatin, albumin and fibrinogen. The optimal enzymatic activity was observed in temperature of 60 °C and pH 5.0, demonstrating the presence of acidic proteases. In conclusion, these results could provide basis for the pharmacological application of C. urens proteases as a new source of bioactive molecules to treat bleeding and thrombotic disorders.
authors.affiliation: Laboratório de Tecnologia e Biotecnologia Farmacêutica, Departamento de Farmácia, Universidade Federal do Rio Grande do Norte, Rua Gal. Gustavo Cordeiro de Farias, s/n-Petrópolis, Natal, RN 59012-570, Brazil. yamaramenezes@yahoo.com.br.
authors.forename: Yamara A S
authors.initials: YA
authors.lastname: de Menezes
authors.affiliation: Laboratório de Tecnologia e Biotecnologia Farmacêutica, Departamento de Farmácia, Universidade Federal do Rio Grande do Norte, Rua Gal. Gustavo Cordeiro de Farias, s/n-Petrópolis, Natal, RN 59012-570, Brazil. julianafelix_rn@hotmail.com.
authors.forename: Juliana
authors.initials: J
authors.lastname: Félix-Silva
authors.affiliation: Laboratório de Tecnologia e Biotecnologia Farmacêutica, Departamento de Farmácia, Universidade Federal do Rio Grande do Norte, Rua Gal. Gustavo Cordeiro de Farias, s/n-Petrópolis, Natal, RN 59012-570, Brazil. arnobiosilva@ufrnet.br.
authors.forename: Arnóbio A
authors.initials: AA
authors.lastname: da Silva-Júnior
authors.affiliation: Laboratório de Hematologia Clínica, Departamento de Análises Clínicas e Toxicológicas, Universidade Federal do Rio Grande do Norte, Rua Gal. Gustavo Cordeiro de Farias, s/n-Petrópolis, Natal, RN 59012-570, Brazil. ivaniserebecchi@gmail.com.
authors.forename: Ivanise M M
authors.initials: IM
authors.lastname: Rebecchi
authors.affiliation: Departamento de Bioquímica, Universidade Federal do Rio Grande do Norte: Instituto de Medicina Tropical do Rio Grande do Norte, Campus Universitário, s/n-Lagoa Nova, Natal, RN 59078-970, Brazil. cisteana@yahoo.com.br.
authors.forename: Adeliana S
authors.initials: AS
authors.lastname: de Oliveira
authors.affiliation: Departamento de Biologia Celular e Genética, Universidade Federal do Rio Grande do Norte: Instituto de Medicina Tropical do Rio Grande do Norte, Campus Universitário, s/n-Lagoa Nova, Natal, RN 59078-970, Brazil. afuchoa@ufrnet.br.
authors.forename: Adriana F
authors.initials: AF
authors.lastname: Uchoa
authors.affiliation: Laboratório de Tecnologia e Biotecnologia Farmacêutica, Departamento de Farmácia, Universidade Federal do Rio Grande do Norte, Rua Gal. Gustavo Cordeiro de Farias, s/n-Petrópolis, Natal, RN 59012-570, Brazil. mpedrosa@ufrnet.br.
authors.forename: Matheus de F
authors.initials: Mde F
authors.lastname: Fernandes-Pedrosa
mesh_terms: D000818:Animals; D005591:Chemical Fractionation; D003029:Coagulants / Q000737:chemistry / Q000494:pharmacology*; D004789:Enzyme Activation; D019665:Euphorbiaceae / Q000737:chemistry* / Q000201:enzymology; D005260:Female; D005340:Fibrinogen / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008670:Metals; D051379:Mice; D010314:Partial Thromboplastin Time; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism*; D010936:Plant Extracts / Q000737:chemistry*; D018515:Plant Leaves / Q000737:chemistry* / Q000201:enzymology; D059748:Proteolysis / Q000187:drug effects; D013696:Temperature; D013918:Thrombin Time
chemical_list: D003029:Coagulants; D005343:Fibrinolytic Agents; D008670:Metals; D010936:Plant Extracts; D005340:Fibrinogen; D010447:Peptide Hydrolases