ABSTRACT
Streptokinase purified from Streptococcus equinus VIT_VB2 isolated from bovine milk sample was immobilized in various solid supports namely entrapment in agarose gel, calcium alginate beads and gelatin gel by cross-linking with formaldehyde. Immobilization of streptokinase in calcium alginate beads showed maximum efficiency (81.8 ± 1.06%) when compared with entrapment with agarose gel (55.6 ± 2.17%) and cross-linked gelatin formaldehyde gel (71.0 ± 1.54%). The purified SK activity was expressed maximum in calcium alginate (1%) and gelatin gel (0.25%) with 1292.68 ± 1.33 and 1121.9 ± 1.2 U mL-1, respectively. Similarly, SK entrapped in gelatin gel and calcium alginate showed maximum in vitro blood clot lysis activity with 77.67 ± 2.64% and 76.16 ± 2.72%, respectively. The immobilized SK in gelatin gel showed complete clot lysis within 15 min; hence, this application of the study could be used in the treatment of superficial thrombophlebitis, phlebitis, and venous thrombosis. These beads were used for three repeated cycles to check the conversion of substrates into their products, and we concluded that SK can be immobilized in the suitable matrices. Therefore, this helps in the drug-delivery strategies in highly efficient way, moreover, economically competent process in the pharmaceutics.
Subject(s)
Enzymes, Immobilized/metabolism , Streptococcus/enzymology , Streptokinase/metabolism , Alginates/chemistry , Animals , Blood Coagulation/drug effects , Caseins/metabolism , Cattle , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/isolation & purification , Enzymes, Immobilized/pharmacology , Glucuronic Acid/chemistry , Hexuronic Acids/chemistry , Humans , Industrial Microbiology , Milk/microbiology , Streptococcus/metabolism , Streptokinase/chemistry , Streptokinase/isolation & purification , Streptokinase/pharmacologyABSTRACT
The most practical approach to reduce morbidity and mortality of coronary heart disease (CHD) is to delay the process of thrombus by usage of clot-dissolving agents. The necessities of such safer compounds are to be critically examined for thrombolytic activity especially, from marine sources. Thrombolytic agents have been investigated as a possible treatment for thrombus. The aim of this study was to investigate the in vitro thrombolytic potential of Streptomyces sp.VITJS4 (NCIM No. 5574); (ACC No: JQ234978.1) active compounds. The fibrin degradation revealed a clear transparent zone of clearance with 500µg/mL concentration showing 24mm hydrolysis. The thrombolytic effect of Streptomyces sp.VITJS4 compounds was also demonstrated in vitro clot lysis assay where The percent of thrombolysis by the crude extract showed 90±1.7% at the concentration of 1000µg/mL, whereas percent of thrombolysis by streptokinase was found 100± 00%%. The bioactive compounds were further studied for spectrophotometric analysis. The UV-VIS profile showed different peaks ranging from 400-700 nm with different absorption respectively. The data confirmed the presence of both analogues with absorption maxima at 210 and 310 nm. A sensitive method using LC-MS technique was optimized for the separation and identification of bioactive metabolites which was indicated by the fingerprints. The results of the LC-MS analysis provided different peaks determining the presence of compounds with different therapeutic activities. The current study refers the bioactive compound as impressive thrombolytic agent for further laboratory study. Further studies should be conducted to ensure the efficacy and safety of different concentration of bioactive compounds for drug development. Hence the results reported perhaps useful for the discovery of novel thrombolytic drugs from marine origin.
A abordagem mais prática para reduzir a morbidade e a mortalidade da doença arterial coronariana (CHD, do inglês coronary heart disease) consiste em retardar o processo de trombo através da utilização de agentes de dissolução de coágulos. As necessidades de tais compostos mais seguros devem ser criticamente examinadas para a atividade trombolítica, especialmente de fontes marinhas. Agentes trombolíticos tem sido estudados como um possível tratamento para o trombo. O objetivo deste estudo foi investigar o potencial trombolítico in vitro dos compostos ativos do Streptomyces sp.VITJS4 (NCIM No. 5574); (ACC No: JQ234978.1). A degradação da fibrina revelou um clara zona livre transparente com concentração de 500µg/mL mostrando uma hidrólise de 24mm. O efeito trombolítico dos compostos de Streptomyces sp.VITJS4 também foi demonstrado no ensaio in vitro de lise dos coágulos em que a percentagem de trombólise pelo extrato bruto mostrou 90±1.7% a uma concentração de 1000µg/mL, enquanto que a percentagem de trombólise pela estreptoquinase foi de 100± 00%. Os compostos bioativos foram estudados posteriormente através da análise espectrofotométrica. O perfil ultra violeta visível (UV-VIS profile, em inglês) mostrou diferentes picos variando entre 400-700 nm com diferentes absorções respectivamente. Os dados confirmaram a presença de ambos os análogos com absorção máxima em 210 e 300 nm. Um método sensível usando a técnica LC-MS (Liquid chromatographymass spectrometry) foi otimizado para a separação e identificação metabólitos bioativos que foram indicados pelas impressões digitais (?). Os resultados da análise LC-MS forneceram diferentes picos determinando a presença de compostos com diferentes atividades terapêuticas. O estudo atual refere-se ao composto bioativo como um agente trombolítico impressionante para futuros estudos em laboratório. Estudos futuros devem ser conduzidos para assegurar a eficácia e segurança de diferentes concentrações dos compostos bioativos para o desenvolvimento de drogas. Assim, os resultados reportados talvez sejam úteis para a descoberta de novas drogas trombolíticas de origem marinha.
Subject(s)
Streptomyces , Thrombosis , In Vitro Techniques , Actinobacteria , Fibrinolytic AgentsABSTRACT
Xylarinase is a bi-functional fibrinolytic metalloprotease isolated from the culture filtrate of endophytic fungus Xylaria curta which is monomeric with a molecular mass of â¼33.76 kDa. The enzyme displayed both plasmin and tissue plasminogen activator like activity under in vitro conditions. It hydrolyses Aα and Bß chains of the fibrinogen. Optimal fibrinolytic activity of xylarinase is observed at 35 °C, pH 8. Ca(2+) stimulated the fibrinolytic activity of xylarinase while Fe(2+) and Zn(2+) inhibited suggesting it to be a metalloprotease. The Km and Vmax values of xylarinase were 240.9 µM and 1.10 U/ml for fibrinogen and 246 µM and 1.22 U/ml for fibrin, respectively. Xylarinase was found to prolong the activated partial thromboplastin time and prothrombin time. The N-terminal sequence of xylarinase (SNGPLPGGVVWAG) did not show any homology with previously known fibrinolytic enzymes. Thus xylarinase is a novel fibrinolytic metalloprotease which could be possibly used as a new clot busting enzyme.
