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Previous issue date: 2013-07-04 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / 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 azocase?na and fibrinogen. The most active fraction (F1.0) in these tests
was chosen for assessment of biological activity and biochemical characterization.
The A? chain and B? of fibrinogen were completely cleaved at a concentration of
0.18 ?g/?L 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 60?C. 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 / A extra??o, caracteriza??o qu?mica e estrutural de uma grande diversidade de
compostos derivados de plantas tem sido uma fonte importante de mol?culas
bioativas. Diversas proteases t?m sido isoladas no reino vegetal, com in?meras
aplica??es farmacol?gicas e biotecnol?gicas. Dentre as proteases isoladas de
plantas, est?o as fibrinogenol?ticas, com relevante aplica??o no tratamento de
dist?rbios na cascata da coagula??o, al?m do uso em potencial como ferramenta em
laborat?rios cl?nicos. Neste trabalho, al?m de avaliar os efeitos do extrato prot?ico de
Cnidoscolus urens (L.) Arthur, pertencente ? fam?lia Euphorbiaceae, na cascata de
coagula??o, tamb?m se investigou a presen?a de atividade antimicrobiana e
caracterizou a atividade proteol?tica detectada neste extrato, tendo como objetivo
determinar sua potencial aplica??o farmacol?gica e biotecnol?gica. Desse modo,
extratos prot?icos brutos obtidos das folhas de C. urens em tamp?o Tris-HCl 0,05M,
NaCl 0,15M, pH 7,5, foram precipitados em diferentes concentra??es de acetona, e
avaliados quanto a presen?a de atividade proteol?tica em azocase?na e fibrinog?nio.
A fra??o mais ativa (F1.0) nestes testes foi escolhida para realiza??o de avalia??o
de atividade biol?gica e caracteriza??o bioqu?mica. As cadeias A? e B? do
fibrinog?nio foram completamente clivadas na concentra??o de 0.18 ?g/?L de
prote?na da fra??o em 4 minutos. A atividade fibrinogenol?tica apresentou inibi??o
total em presen?a de E-64 e parcial em presen?a de EDTA. A fra??o demonstrou
atividade coagulante sobre o plasma e reduziu o tempo de tromboplastina parcial
ativada, indicando atuar sobre os fatores da via intr?nseca e comum da coagula??o,
n?o exercendo efeitos sobre o tempo de protrombina. A atividade fibrinol?tica sobre o
co?gulo de plasma foi detectado apenas em SDS-PAGE em concentra??es
elevadas da fra??o, e apesar da atividade fibrin(ogen)ol?tica, n?o foi observada
atividade defibrinogenante in vivo. Apesar de v?rias proteases de plantas e animais
pe?onhentos serem classicamente t?xicas, a frac??o F1.0 n?o expressou atividade
hemorr?gica nem hemol?tica. A fra??o F1.0 tamb?m n?o demonstrou atividade
antimicrobiana. Na avalia??o da atividade proteol?tica sobre a azocase?na, o pH
?timo de rea??o foi 5.0, e a temperatura ?tima igual a 60?C. A atividade enzim?tica
demonstrou ser sens?vel ? presen?a dos sais testados, com inibi??o para todos os
compostos. O tensoativo triton n?o influenciou a atividade enzim?tica, por?m o
tween-20 e SDS inibiram tal atividade. Em presen?a de agentes redutores ocorreu
aumento da atividade enzim?tica, caracter?stica t?pica de enzimas pertencentes ?
classe das ciste?no proteases. Diversas bandas prot?icas com atividade proteol?tica
foram detectadas em zimograma, na regi?o de elevada massa molecular, que foram
inibidas por E-64. Neste trabalho, foi revelado que C. urens apresenta fra??o
enriquecida com ciste?no-proteases que apresentam atividade fibrinogenol?tica e
procoagulante, que podem ser isoladas, com potencial aplica??o no tratamento de
dist?rbios hemorr?gicos, como trombol?tico e em laborat?rio cl?nico / 2020-01-01
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/13497 |
| Date | 04 July 2013 |
| Creators | Menezes, Yamara Arruda Silva de |
| Contributors | CPF:96728647449, http://lattes.cnpq.br/2929963416385218, Assis, Cristiane Fernandes de, CPF:03143301600, http://lattes.cnpq.br/0034694007210837, Pereira, Wogelsanger Oliveira, CPF:02392207496, http://lattes.cnpq.br/4661963400736302, Pedrosa, Matheus de Freitas Fernandes |
| Publisher | Universidade Federal do Rio Grande do Norte, Programa de P?s-Gradua??o em Ci?ncias Farmac?uticas, UFRN, BR, Bioan?lises e Medicamentos |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Repositório Institucional da UFRN, instname:Universidade Federal do Rio Grande do Norte, instacron:UFRN |
| Rights | info:eu-repo/semantics/embargoedAccess |
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