• 생약학회지
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• 제39권3호
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• pp.265-269
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• 2008

This study was carried out to observe the antioxidative activity, fibrinolytic activity, nitrite savenging ability and adenosine content of three medicinal mushrooms. Among water and ethanol extract of each mushroom, water extract of Inonotus obliquus (Czech) showed the highest antioxidative activity $(57.1{\mu}g/ml)$, nitrite scavenging ability (52.04%), fibrinolytic activity (86.8%) and adenosin content $(94.3{\mu}g/g)$, whereas nitrite scavenging ability of ethanol extract of Phellinus linteus showed higher than that of water extract. Apart from the above statements, water was effective than ethanol as extracting solvent in general. These results suggest that Inonotus obliquus (Czech) showed higher activities such as antioxidative activity, nitrite scavenging ability, fibrinolytic activity and adenosin content and water was effective as solvent except for nitrite scavenging ability.

• Preventive Nutrition and Food Science
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• 제9권1호
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• pp.14-20
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• 2004

Bacillus strains capable of producing fibrinolytic enzyme were isolated from traditional fermented Korean soybean paste and Japanese fermented soybean (Natto). Among the 16 strains, a selected Bacillus sp. was identified as bacillus firmus, with 80.7％ homology, by API kit analysis. Seed starter or B. firmus NA-1 was prepared with 5％ soymilk prepared from micronized soybean powder. To produce fibrinolytic enzyme by B. firmus NA-1 the liquid culture was performed with NB broth (pH 7.0) fortified with 1％ galactose, 0.1% tryptone, and 0.5% $K_2$HPO$_4$, by shaking with 180 rpm at 37$^{\circ}C$. Fibrinolytic enzyme activity reached the highest value at 7.8 unit/mL (plasmin unit) after fermentation for 72 hr. The crude fibrinolytic enzyme showed higher relative activity in the range of pH 7.0∼9.0. The activity of crude fibrinolytic enzyme was well maintained even after concentration by the vacuum evaporation at 5$0^{\circ}C$ for 1 hr.

• Natural Product Sciences
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• 제8권2호
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• pp.66-70
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• 2002

The in vitro anticoagulant and fibrinolytic activities of crude extracts from insects were evaluated in order to find effective therapeutic drugs for the treatment of myocardial and cerebral thrombosis. We prepared three types of extracts (water, methanol and ethylacetate) from 28 insects for use as raw materials for the activity assays. The fibrinolytic activity was tested using the fibrin plate method and the activated partial thromboplastin time and thrombin time were measured for blood clotting activity. With regards to the fibrinolytic system, water extracts of six kinds of insects displayed a remarkable level of activity with a plasmin-like action. The water extracts of [Catharsius molossus, Eupolyphaga sinensis, Huechys sanguinea, Mantidis $o\ddot{o}theca$, Mimela splendens, and Polistes mandarinus (Vespae Nidus)] exhibited the activity. On the other hand, the methanol extracts did not display any fibrinolytic activity. In terms of the coagulation system, an aqueous extract of silkworm Tongchunghacho (Paecilomyces japonica), Oxya japonica japonica and Buthus martensi (Scorpion) increased the clotting time significantly longer (181 times) than the control. These results suggest that crude drugs from insects are useful sources for the development of new drugs for use in treatments involving blood coagulation and fibrinolysis.

• 한국자원식물학회지
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• 제33권4호
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• pp.237-244
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• 2020

산수유 열수추출물의 혈전용해활성을 탐색하고, 유기용매로 분획화한 에틸아세테이트(ethyl acetate) 분획물에서 1.36 plasmin units/mL의 강한 혈전용해활성을 확인하였다. Amberlite IRA-400, Sephadex LH-20 column, active charcoal column과 재결정으로 정제한 혈전용해화합물을 HPLC로 분석 비교한 결과, 표준물질인 malic acid와 같은 retention time (RT)을 나타내고, LC/MS/MS 분석의 Negative mode 측정 시 m/z 133에서 같은 peak를 나타내어 정제한 화합물이 분자량 134 Da인 malic acid 임을 확인하였다. 정제한 화합물은 0.69 plasmin units/mL의 혈전용해활성을 보이고, 14.62%의 트롬빈 저해활성을 나타냈으며, 6.42%의 항산화 효과와 17.28%의 항당뇨 효과를 나타냈다. 분리한 화합물은 섬유소원의 Aα chain, Bβ chain과 반응하지 않았지만 γ chain을 분해하는 것으로 나타났다. 위 연구 결과를 바탕으로 산수유에서 분리한 혈전용해화합물은 혈관계 질환의 치료제 개발과 기능성 식품 개발을 위한 우수한 소재로 이용 가능할 것으로 예상된다.

• Journal of Microbiology and Biotechnology
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• 제12권4호
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• pp.649-656
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• 2002

Fibrinolytic microorganisms were screened from 42 samples of Korean fermented food (7 kinds of Chungook-jang, 14 kinds of commercial Doen-Jang, 5 kinds of home-made Doen-jang, and 16 kinds of Jeot-gal), 15 samples of Japanese fermented food (5 kinds of home-made soybean paste, and 10 kinds of Natto), and 19 samples of Indonesian fermented food (Tempe) as well as starters of Meju (500 microflora from Korea, and 22 from China). Initially, 11 isolates with strong fibrinolytic activity were selected for further characterization. The fibrinolytic activity of the 11 isolates ranged from 89 to 199% of standard plasmin. Four strains, M5l from Korean fermented food (Meju), I 1-1, I 1-4, and I 5-1 from Indonesian fermented food (Tempe), were chosen based on the degree of activity and reproducibility, and identified as Staphylococcus sciuri, Citrobacter or Enterobacter, Enterococcus faecalis, and Bacillus subtilis, respectively. The first two isolates are pathogenic stains while the latter two are considered as GRAS (Generally Recognized As Safe). Fibrinolytic activity of E. faecalis, characterized and designated as BRCA-5, reached a maximum, when the producer was cultivated in Ml7 broth supplemented with 1.0% glucose for 5 h at 37$^{\circ}C$ with shaking at 180 rpm. Compared to commercial fibrinolytic enzymes, the cell-free culture supernatant of 5. faecaiis BRCA-5 showed stronger activity than plasmin and streptokinase, but similar degree of specific activity as nattokinase and urokinase, aud it also demonstrated anticoagulant and antiplatelet activity ex vivo. These features of E. faecalis make it an attractive agent as a biomaterial for health-promoting foods.

• 한국미생물·생명공학회지
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• 제28권1호
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• pp.14-20
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• 2000

The transformation of Bacillus subtilis K-54 and J-10 was carried out with constructed vectors containing structure and enhancer genes of aprN and prtR, to increase their fibrinolytic enzyme activity. Bands for the aprN and prtR genes were identified from B. subtilis J-10 by PCR that was carried out with the constructed primers for the genes. In addition, the gene fragments contained promoter site based on the results of analysing their nucleotide sequence. The two gene fragments, aprN and prtR, obtained by the PCR, were, then, inserted to vector such as T-vector and E.coli/Bacillus shuttle vector. The constructed vector were designated as pAPR2 (aprN), pENC2 (prtR) and pFLA1 (aprN and prtR), respectively. The constructed vector was used for transformation of the strains of B.subtilis J-10 and B. subtilis K-54 and the fribrinolytic activity of the transformed strains was investigated. The introduction of the vector, pAPR2 and the fibrinolytic activity of the transformed strains was investigated. The introduction of the vector, pAPR2 and pFLA1, resulted in the increase of fibrinolyitic enzyme activity in B. subtilis J-10 by 27.3% and 16%, respectively. However, the introduction of pENC2 to B. subtilis J-10 did not seem to induce increase of the enzyme activity. The strain of B.subtilis K-54 transformed with pENC2 showed an increased fibrinolytic activity by 5 folds compared with that of the original strain of B. subtilis K-54.

• Journal of Microbiology and Biotechnology
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• 제31권6호
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• pp.833-839
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• 2021

Bacillus subtilis CH3-5 isolated from cheonggukjang secretes a 28 kDa protease with a strong fibrinolytic activity. Its gene, aprE3-5, was cloned and expressed in a heterologous host (Jeong et al., 2007). In this study, the promoter of aprE3-5 was replaced with other stronger promoters (P_cry3A, P₁₀, P_SG1, P_srfA) of Bacillus spp. using PCR. The constructed chimeric genes were cloned into pHY300PLK vector, and then introduced into B. subtilis WB600. The P10 promoter conferred the highest fibrinolytic activity, i.e., 1.7-fold higher than that conferred by the original promoter. Overproduction of the 28 kDa protease was confirmed using SDS-PAGE and fibrin zymography. RT-qPCR analysis showed that aprE3-5 expression was 2.0-fold higher with the P10 promoter than with the original promoter. Change of the initiation codon from GTG to ATG further increased the fibrinolytic activity. The highest aprE3-5 expression was observed when two copies of the P₁₀ promoter were placed in tandem upstream of the ATG initiation codon. The construct with P10 promoter and ATG and the construct with two copies of P10 promoter in tandem and ATG exhibited 117% and 148% higher fibrinolytic activity, respectively, than that exhibited by the construct containing P10 promoter and GTG. These results confirmed that significant overproduction of a fibrinolytic enzyme can be achieved by suitable promoter modification, and this approach may have applications in the industrial production of AprE3-5 and related fibrinolytic enzymes.

• Journal of Microbiology and Biotechnology
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• 제19권9호
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• pp.997-1004
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• 2009

Bacillus amyloliquefancies CH51 isolated from cheonggukjang, a traditional Korean fermented soy food, has strong fibrinolytic activity and produces several fibrinolytic enzymes. Among four different growth media, tryptic soy broth was the best in terms of supporting cell growth and fibrinolytic activity of this strain. A protein with fibrinolytic activity was partially purified from the culture supernatant by CM-Sephadex and Phenyl Sepharose column chromatographies. Tandem mass spectrometric analysis showed that this protein is a homolog of AprE from B. subtilis and it was accordingly named AprE51. The optimum pH and temperature for partially purified AprE51 activity were 6.0 and $45^{\circ}C$, respectively. A gene encoding AprE51, aprE51, was cloned from B. amyloliquefaciens CH51 genomic DNA. The aprE51 gene was overexpressed in heterologous B. subtilis strains deficient in fibrinolytic activity using an E. coli-Bacillus shuttle vector, pHY300PLK.

• 대한의용생체공학회:학술대회논문집
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• 대한의용생체공학회 1997년도 춘계학술대회
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• pp.51-54
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• 1997

Lumbrokinase, potent fibrinolytic enzyme purified from earthworm, was immobilized onto the total artificial heart valves using photoreaction. This valve were implanted into the lamb for three days. After experiments, thrombus was observed in the untreated valves whereas no thrombus was observed in the lumbrokinase immobilized valves. The fibrinolytic activity and proteolytic activity of the implanted valve was examined. The fibrinolytic activity of the valve was remained after the implantation. The lumbrokinase could be a suitable fibrinolytic agents in the vascular contacting devices to reduce the thrombus.

• Preventive Nutrition and Food Science
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• 제7권4호
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• pp.417-420
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• 2002

A strong fibrin-specific fibrinolytic enzyme was produced from Bacillus subtilis KH-4 isolated from Deonjang, a Korean fermented soybean paste similar to Japanese miso. The addition of glucose as a carbon source resulted in the highest levels of caseinolytic and fibrinolytic activities. Likewise, the addition of yeast extract as the nitrogen source resulted in the highest caseinolytic and fibrinolytic activities (3473.2 unit and 47.4 munit, respectively), It was observed that out of all metal ion sources only calcium (chloride) enhanced caseinolytic and fibrinolytic activities, with increases of 4949.3 unit and 58.2 unit/mg, respectively. The optimal temperature for the production of the enzyme was found to be 4$0^{\circ}C$ in the optimal medium (glucose 20 g, yeast extract 5 g, CaCl$_2$l g, and NaCl 2 g). The maximum fibrinolytic activity was observed at the late stationary phase. B. subtilis KH-4 produced a fibrinolytic enzyme at 4$0^{\circ}C$, after 30 h growth, which increased up to 54 h and then remained constant. These results suggest that Deonjang has potential as a source of physiologically active anti-thromotic enzymes.