Isolamento da vicilina do feijão mungo verde (Vigna radiata L.) e estudo de suas atividades hipocolesterolêmica e antimicrobiana
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Data
2016-09-29
Autores
Amaral, Ana Lúcia da Silva [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A homologia entre as características estruturais, sequenciais e funcionais da β- conglicinina da soja em relação a outras vicilinas de leguminosas e a ação de seus peptídeos bioativos nos estimulou a investigar a atividade da globulina vicilina (8S) do feijão mungo verde como composto funcional. Objetivo: isolar e caracterizar a globulina 8S do feijão mungo verde e verificar a ação hipocolesterolêmica e antimicrobiana de seus hidrolisados em testes in vitro. Método: a proteína majoritária 8S do feijão mungo verde foi extraída a partir do método de isolamento, em seguida foi parcialmente purificada por cromatografia de permeação molecular, determinada sua massa molecular a partir da eluição em Coluna Sephadex G-200 e suas subunidades foram caracterizadas por SDS Page. A proteína purificada foi submetida à hidrólise enzimática sequencial com pepsina-pancreatina e o hidrolisado obtido caracterizado por permeação molecular e SDS Tricina. Diferentes frações provenientes da eluição do hidrolisado em Sephadex G-25 foram testadas quanto à inibição da HMG CoAr (3 - Hidróxi - 3 - metilglutaril - CoA redutase) e de microrganismos (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 e Helicobacter pylori ATCC 43504). Resultados: A 8S do feijão mungo verde é composta por polipeptídeos de 26, 29, 48 e 61 kDa e sua massa molecular é de 158,23±10 kDa, características condizentes com as vicilinas de outras leguminosas. As frações obtidas da eluição do hidrolisado em Sepahadex G-25 (10, 12, 14, 22 e 32) mostraram significativa inibição in vitro da atividade da enzima HMG-CoAr comparativamente a drogas de reconhecida ação hipocolesterolêmica (pravastatina), sugerindo participação dessas frações peptídicas na inibição de uma importante etapa da síntese do colesterol. O hidrolisado obtido a partir da hidrólise sequencial com pepsina-pancreatina apresentou atividade antimicrobiana frente aos microrganismos testados, principalmente contra a H. pylori, sendo um promissor avanço na terapia antibacteriana. Ao contrário do ocorrido com o hidrolisado, as frações peptídicas da 8S não apresentaram ação antimicrobiana, o que sugere que este efeito esteja relacionado ao sinergismo decorrente da presença de peptídeos com diferentes massas e características estruturais. Conclusão: Os resultados mostram a eficácia nos métodos de isolamento e caracterização da fração proteica, além do efeito hipocolesterolêmico e antimicrobiano do hidrolisado da 8S do feijão mungo verde, sugerindo a utilização desta proteína como composto funcional.
The homology between the structural and functional characteristics of sequential β- conglycinin soy compared to other legume vicilin and the action of their bioactive peptides stimulated us to investigate the activity of globulin vicilin (8S) mung beans as functional compound. Objective: isolate and characterize the 8S globulin mung beans and check the hypocholesterolemic and antimicrobial activities their hydrolysates in in vitro tests. Method: The major protein 8S mung bean is extracted from the isolation method, then was partially purified by molecular permeation chromatography, its molecular weight determined from the elution Sephadex G- 200 and its subunits were characterized by SDS-PAGE. The purified protein was subjected to sequential enzymatic hydrolysis with pepsin-pancreatin and the hydrolyzate characterized by molecular permeation and Tricine SDS. Different fractions from the hydrolyzate eluted on Sephadex G-25 were tested for inhibition of HMG CoAr (3 - hydroxy - 3 - methylglutaryl - CoA reductase) and microorganisms (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Helicobacter pylori ATCC 43504). Results: 8S mung bean is comprised of polypeptides of 26, 29, 48 and 61 kDa and its molecular weight is 158.23 ± 10 kDa, consistent with the characteristics vicilins from other legumes. The fractions obtained from the elution hydrolyzate in Sepahadex G-25 (10, 12, 14, 22 and 32) demonstrated significant in vitro inhibition of HMG-CoAr enzyme activity compared with the recognized hypocholesterolemic action drug (pravastatin), suggesting involvement of these peptide fractions in inhibiting an important step in cholesterol synthesis. The hydrolyzate obtained from sequential hydrolysis with pepsin-pancreatin presented antimicrobial activity against the tested microorganisms, particularly against H. pylori, is a promising advance in antibacterial therapy. Contrary to what occurred with the hydrolyzate peptide fractions of the 8S showed no antimicrobial activity, suggesting that this effect is related to the synergism due to the presence of peptides with different masses and structural characteristics. Conclusion: The results show the effectiveness of the methods of isolation and characterization of the protein fraction, beyond the hypocholesterolemic and antimicrobial effect of the hydrolyzate 8S mung bean, suggesting the use of this protein as a functional compound.
The homology between the structural and functional characteristics of sequential β- conglycinin soy compared to other legume vicilin and the action of their bioactive peptides stimulated us to investigate the activity of globulin vicilin (8S) mung beans as functional compound. Objective: isolate and characterize the 8S globulin mung beans and check the hypocholesterolemic and antimicrobial activities their hydrolysates in in vitro tests. Method: The major protein 8S mung bean is extracted from the isolation method, then was partially purified by molecular permeation chromatography, its molecular weight determined from the elution Sephadex G- 200 and its subunits were characterized by SDS-PAGE. The purified protein was subjected to sequential enzymatic hydrolysis with pepsin-pancreatin and the hydrolyzate characterized by molecular permeation and Tricine SDS. Different fractions from the hydrolyzate eluted on Sephadex G-25 were tested for inhibition of HMG CoAr (3 - hydroxy - 3 - methylglutaryl - CoA reductase) and microorganisms (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Helicobacter pylori ATCC 43504). Results: 8S mung bean is comprised of polypeptides of 26, 29, 48 and 61 kDa and its molecular weight is 158.23 ± 10 kDa, consistent with the characteristics vicilins from other legumes. The fractions obtained from the elution hydrolyzate in Sepahadex G-25 (10, 12, 14, 22 and 32) demonstrated significant in vitro inhibition of HMG-CoAr enzyme activity compared with the recognized hypocholesterolemic action drug (pravastatin), suggesting involvement of these peptide fractions in inhibiting an important step in cholesterol synthesis. The hydrolyzate obtained from sequential hydrolysis with pepsin-pancreatin presented antimicrobial activity against the tested microorganisms, particularly against H. pylori, is a promising advance in antibacterial therapy. Contrary to what occurred with the hydrolyzate peptide fractions of the 8S showed no antimicrobial activity, suggesting that this effect is related to the synergism due to the presence of peptides with different masses and structural characteristics. Conclusion: The results show the effectiveness of the methods of isolation and characterization of the protein fraction, beyond the hypocholesterolemic and antimicrobial effect of the hydrolyzate 8S mung bean, suggesting the use of this protein as a functional compound.
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Palavras-chave
Helicobacter pylori, HMG CoAr, Composto funcional, Mungo verde, Vicilina, Functional compound, Helicobacter pylori, HMG CoAr, Mung bean, Vicilin