Extração, caracterização e atividades biológicas de proteínas da espécie cnidoscolus urens (L.) Arthur
The extraction, chemical and structural characterization of a wide variety of compounds derived from plants has been a major source of bioactive molecules. Several proteases have been isolated in the plant kingdom, with numerous pharmacological and biotechnological applications. Among the proteas...
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Formato: | Dissertação |
Idioma: | por |
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Universidade Federal do Rio Grande do Norte
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Endereço do item: | https://repositorio.ufrn.br/jspui/handle/123456789/13497 |
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Resumo: | The extraction, chemical and structural characterization of a wide variety of
compounds derived from plants has been a major source of bioactive molecules.
Several proteases have been isolated in the plant kingdom, with numerous
pharmacological and biotechnological applications. Among the proteases isolated
from plants, are the fibrinogenolytic, with relevant application in the treatment of
disorders in the coagulation cascade, in addition to potential use as a tool in clinical
laboratories. In this study, in addition to evaluating the effects of the protein extract of
Cnidoscolus urens (L.) Arthur (Euphorbiaceae) in the coagulation cascade also
investigates the presence of antimicrobial activity and characterizes the proteolytic
activity detected in this extract, aiming to determine their potential pharmacological
and biotechnological application. In this way, crude protein extracts obtained from the
leaves of C. urens in Tris-HCl 0.05M, NaCl 0.15M, pH 7.5, were precipitated in
different concentrations of acetone, and assessed for the presence of proteolytic
activity in azocaseína and fibrinogen. The most active fraction (F1.0) in these tests
was chosen for assessment of biological activity and biochemical characterization.
The Aα chain and Bβ of fibrinogen were completely cleaved at a concentration of
0.18 μg/μL of protein fraction in 4 minutes. Fibrinogenolytic activity presented total
inhibition in the presence of E-64 and partial in the presence of EDTA. The fraction
demonstrated coagulant activity in plasm and reduced the APTT, demonstrating
acting on the factors coagulation of the intrinsic pathway and common, not exerting
effects on the PT. Fibrinolytic activity on plasma clot was detected only in SDS-PAGE
in high concentrations of fraction, and there were no defibrinating. Although several
proteases isolated from plants and venomous animals are classically toxic, the
fraction F1.0 of C. urens not expressed hemorrhagic nor hemolytic activities. Fraction
F1.0 also showed no antimicrobial activity. In proteolytic activity on the azocasein,
the optimal pH was 5.0 and optimum temperature of 60ºC. The enzyme activity has
been shown to be sensitive to the presence of salts tested, with inhibition for all
compounds. The surfactant triton did not influence the enzyme activity, but the
tween-20 and SDS inhibited the activity. In the presence of reducing agents increase
in enzyme activity occurred, a typical feature of enzymes belonging to the class of
cysteine proteases. Several bands with proteolytic activity were detected in
zymogram, in the region of high-molecular-weight, which were inhibited by E-64. In
this study, we found that C. urens presents in its constitution cysteine proteases with
fibrinogenolytic and procoagulant activity, which may be isolated, with potential
application in treatment of bleeding disorders, thrombolytic and clinical laboratory |
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