• Journal of the Korean Society of Food Science and Nutrition
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• v.41 no.2
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• pp.168-173
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• 2012

We performed this study to develop antithrombotic agents from oriental medicine herb extracts. Polygonum cuspidatum has been traditionally used as an edible medical resources for the treatment of cancer, pyodermatitis, hepatitis, cystitis, and inflammation. However, the effects of Polygonum cuspidatum on thrombosis and platelet activation are not precisely understood. The antithrombotic and antiplatelet activities of Polygonum cuspidatum were investigated by assessing the effect of a 70% ethanol extract of Polygonum cuspidatum on blood coagulation, fibrinolysis, and platelet aggregation. Polygonum cuspidatum showed effective fibrinolytic activity at 10 mg/mL. Polygonum cuspidatum also inhibited adenosine diphosphate induced platelet aggregation. Furthermore, evaluation of anticoagulant activity showed that an extract of Polygonum cuspidatum prolonged coagulation time via activated partial thromboplastin time (APTT). Our results show that Polygonum cuspidatum can be a potential candidate for antiplatelet activity as well as a fibrinolytic agent.

• Proceedings of the Korean Society of Community Living Science Conference
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• 2009.09a
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• pp.83-83
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• 2009

암을 제외한 대부분의 성인병은 혈액순환과 관련된 혈관계 질환들로 이들에 의한 사망 비율의 합은 암의 비율과 비슷하게 나타나 혈관계 질환의 심각성을 나타내고 있다. 혈전에 의한 혈관계 질환은 혈전용해제와 트롬빈저해제를 이용하여 치료하고 예방할 수 있으며, a-glucosidase 저해제는 당뇨병 예방과 치료에 이용할 수 있다. 예로부터 민간요법에서 다양하게 이용되어 왔던 반하를 성인병 치료와 예방에 관련된 기능성식품의 자료로 사용하기 위해 반하 열수추출물을 유기 용매로 분획화하고 분획물의 혈전용해활성, 트롬빈저해활성, 혈당강하효과를 측정하였다. 혈전을 직접 용해하는 활성을 측정하기위해 100 mg/ml의 농도로 준비한 시료액을 fibrin plate 방법을 이용하여 혈전용해활성을 측정 결과 hexane 층, chloroform 층, ethyl acetate 층, butanol 층에서는 활성을 나타내지 않았지만 물 층에서 만 0.8 plasmin unit의 높은 혈전용해활성을 나타냈다. 혈전의 형성을 억제하기위해 혈전형성의 필수 효소인 트롬빈의 활성을 저해하는 트롬빈저해활성을 측정하기위해 10 배 희석한 (10 mg/ml) 시료 용액을 이용하여 트롬빈저해활성을 측정결과 hexane 층에서 75.3%의 높은 트롬빈저해활성을 나타냈으며, ethyl acetate 층과 chloroform에서도 각각 43.3%와 39.7%의 활성을 나타냈다. 또한 탄수화물의 소화를 지연시킴으로서 소장에서 포도당의 흡수를 억제하여 혈관내 당의 농도를 조절하는 혈당강하제의 개발을 위해 준비된 조효소액(10 mg/ml)을 이용하여 a-glucosidase 저해활성을 측정한 결과 ethyl acetate 층과 hexane 층에서 각각 24.9%와 23.4%의 저해활성을 확인하였다. 따라서 반하의 hexane 층과 ethyl acetate 층과 물층은 혈전관련 혈관계 질환과 당뇨병 관련 기능성 식품의 개발 소재로 이용할 수 있을 것으로 기대된다. 특히 최근 질병 치료에 효과는 크지만 부작용이 동반되는 화학약품들 대신 활성은 조금 뒤지지만 부작용이 없는 생약을 이용하려는 경향이 커지고 있다. 따라서 오랫동안 민간용법에서 사용되고 있는 반하는 안정성이 확인된 장점을 갖고 있어, 식품재료나 기능성 음료에 이용할 경우 장기간 섭취가 가능해 혈관계 질환의 치료와 예방에 큰 효과를 가져 올 수 있을 것으로 기대된다.

• Biomedical Science Letters
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• v.6 no.4
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• pp.261-268
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• 2000

Fibrinolytic enzyme (FE-2) was purified from the fruiting bodies of Tricholoma saponaceum using DEAE-Cellulose chromatography and Mono-S column chromatography, The enzyme has a molecular weight of 18.23 kDa and include Zn$^{2+}$ ion as found by ICP/MS. The N-terminal amino acid sequence of the enzyme was A-L-Y-V-G-X-S-P-X-Q-Q-S-L-L-V It has a pH optimum at pH 7.5, suggested that FE-2 was a neutral pretense. The activity of FE-2 was highly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzyme is a metalloprotease. The activity of FE-2 was increased by $Mg^{2+}$, Zn$^{2+}$, Fe$^{2+}$, and Co$^{2+}$, but the enzyme activity was totally inhibited by Hg$^{2+}$. No inhibition was found with PMSF, E-64, pepstatin and 2-mercaptoethanol. The enzyme hydrolyzed both $A\alpha$ and B$\beta$ chains of human fibrinogen. The $\gamma$ chain was resistant to hydrolysis by FE-2.

• The Korean Journal of Mycology
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• v.28 no.1
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• pp.60-65
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• 2000

Two fibrinolytic enzymes were purified from the fruiting bodies of Tricholoma saponaceum. The enzymes have a molecular weight of 18(FE-1) and 18.2(FE-2) kDa, respectively, and include $Zn^{2+}$ ion as determined by ICP/MS. The N-terminal amino acid sequence of the two enzymes were exactly the same: A-L-Y-V-G-X-S-P-X-Q-Q-S-L-L-V. The activity of FE-1 was highly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzyme is a metalloprotease. The activity of FE-1 was slightly increased by $Mg^{2+},\;Zn^{2+},\;Fe^{2+}\;and\; Co^{2+}$, however, the enzyme activity was totally inhibited by $Hg^{2+}$. Addition of $Zn^{2+}\;and\;Co^{2+}$ reversed the inhibition caused by 1,10-phenanthroline. It has a pH optimum at pH 7.5, suggested that FE-1 was a neutral protease. It shows the maximum fibrinolytic activity at $55^{\circ}C$, is completely inactivated above at $65^{\circ}C$.

• The Korean Journal of Mycology
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• v.40 no.4
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• pp.296-298
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• 2012

In this study, the biological activities of 33 kinds of wild mushroom methanol extracts were investigated. Boletellus elatus showed the greatest fibrinolytic activity (1.08 plasmin units/mL) in a fibrin plate assay, and the activity of Heterobasidion insulasis was 0.89 plasmin units. The thrombin inhibitory activities of Boletellus elatus and Heterobasidion insulasis were 93.32% and 93.69%, respectively. In a ${\alpha}$-glucosidase inhibitory activity test, Laccaria amethystina showed the greatest inhibitory activity at 81.25%. The antioxidative activities of Gomphus sp. and Geastrum lageniforme were 91.37% and 90.42%, respectively. Since Boletellus elatus and Heterobasidion insulasis have strong fibrinolytic and thrombin inhibitory and antioxidative activities, the two mushrooms can be used as material for the development of biofunctional foods for cardiovascular diseases.

• The Korean Journal of Mycology
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• v.35 no.2
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• pp.128-132
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• 2007

The aim of this study was to investigate the physiological functionalities of 60 wild mushroom methanol extracts. We determined their fibrinolytic and ${\alpha}-glucosidase$ inhibitory activities. Among them, 11 mushrooms showed fibrinolytic activity. In particular, Armillaria sp. showed the greatest enzyme activity (4.2 plasmin units/ml) in a fibrin plate assay. The fibrinolytic activities of Amanita virosa. Gymnophilus sp. and Lepista nuda were 2.3, and the activities of Amanita citrina and Cortinarius alboviolaceus were 2.1 and 2.0 plasmin units, respectively. In a ${\alpha}-glucosidase$ inhibitory activity test, Naematoloma fasciculare showed the greatest inhibitory activity at 98.5%. The ${\alpha}-glucosidase$ inhibitory activities of Suillus luteus and Gomphidius maculatus were 66.6% and 61.5%, respectively. This study suggests that both Armillaria sp. and N. fasciculare can be used as candidates for the development of new medicine effective to cardiovascular diseases and biofunctional food.

• The Korean Journal of Mycology
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• v.39 no.3
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• pp.189-193
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• 2011

To investigate the physiological activities of hot water extract and solvent fractions isolated from Pleurotus ferulea, antioxidative, fibrinolytic, thrombin inhibitory, and ${\alpha}$-glucosidase inhibitory activities were examined. Pleurotus ferulea, hot water extract was fractionated into hexane, chloroform, ethyl acetate, butanol and water fraction. Each of these was assayed individually. The antioxidative activities of ethyl acetate and butanol fractions were 86.79% and 87.82%, respectively. Using the fibrin plate method, only the ethyl acetate fraction showed a plasmin activity of 0.08 units/ml. Thrombin inhibitory activities of chloroform and ethyl acetate fractions were 74.90% and 71.08%, respectively. In the ${\alpha}$-glucosidase inhibitory activity test, butanol fraction showed the highest activity at 49.67%. From the above results, we anticipate that solvent fractions of Pleurotus ferulea can be used as a materials for the development of biofunctional foods for cardiovascular diseases.

• Proceedings of the KOSOMBE Conference
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• v.1996 no.05
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• pp.91-96
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• 1996

광반응을 이용하여 fibrinolytic 활성을 보이는 LK (lumbrokinase)를 생체재료인 PU (polyurethane) 표면에 고정화하는 방법에 관하여 연구하였다. 먼저 PU표면에 amino 작용기를 도입하기 위하여 side chain 작용기로 phenylazide 와 amino작용기를 갖는 AzPhPAL을 이용하였으며 광반응은 266nm 이상의 UV light을 약 1분간 조사시키는 방법을 이용하였다. LK를 amino작용기가 유도된 PU 표면에 공유결합시키기 위하여 amide결합 형성시 사용되는 수용성 carbodiimide인 EDC를 이용하였다. ESCA측정시, AzPhPAL으로 공유결합시킨 경우 반응하지 않은 PU표면에 비하여 N/C가 0.1717로, LK 고정시 S/C가 0.0043로 증가된것을 알 수 있었다. 이것으로부터 AzPhPAL과 LK가 PU표면에 공유결합된 것을 확인할 수 있었으며 static contact angle 측정으로 표면개질된 PU표면이 소수성에서 친수성으로 변화된 것을 확인 할 수 있었다. 또한 공유결합된 LK의 활성을 확인하기 위하여 fibrin plate test를 실시하였는데 그 결과 고정화된 후에도 LK의 활성이 존재함을 알 수 있었다.

• Journal of Microbiology and Biotechnology
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• v.31 no.2
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• pp.327-337
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• 2021

Fibrinolytic enzymes with a direct mechanism of action and safer properties are currently requested for thrombolytic therapy. This paper reports on a new enzyme capable of degrading blood clots directly without impairing blood coagulation. This enzyme is also non-cytotoxic and constitutes an alternative to other thrombolytic enzymes known to cause undesired side effects. Twenty-four Bacillus isolates were screened for production of fibrinolytic enzymes using a fibrin agar plate. Based on produced activity, isolate S127e was selected and identified as B. subtilis using the 16S rDNA gene sequence. This strain is of biotechnological interest for producing high fibrinolytic yield and consequently has potential in the industrial field. The purified fibrinolytic enzyme has a molecular mass of 27.3 kDa, a predicted pI of 6.6, and a maximal affinity for Ala-Ala-Pro-Phe. This enzyme was almost completely inhibited by chymostatin with optimal activity at 48℃ and pH 7. Specific subtilisin features were found in the gene sequence, indicating that this enzyme belongs to the BPN group of the S8 subtilisin family and was assigned as AprE127. This subtilisin increased thromboplastin time by 3.7% (37.6 to 39 s) and prothrombin time by 3.2% (12.6 to 13 s), both within normal ranges. In a whole blood euglobulin assay, this enzyme did not impair coagulation but reduced lysis time significantly. Moreover, in an in vitro assay, AprE127 completely dissolved a thrombus of about 1 cc within 50 min and, in vivo, reduced a thrombus prompted in a rat tail by 11.4% in 24 h compared to non-treated animals.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.12
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• pp.1832-1838
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• 2010

The objective of this study was to improve the flavor of Cheonggukjang prepared by the culture optimization of starter with the fibrinolytic activity. After 25 strains isolated from the commercial Cheonggukjang and Doenjang in different regions were compared, 7 Bacillus strains with proteolytic and slime-producing activities were selected. When the fibrin plate method for fibrinolytic activity was applied, CJJN-4 and 5 showed the higher activity in tripticase soy broth (TSB). All Cheonggukjang prepared with CJJN-4 and 5 also had the fibrinolytic activity, regardless of culture temperature or time. Especially, Cheonggukjang prepared at $40^{\circ}C$ showed higher activity than $45^{\circ}C$, and Cheonggukjang prepared with CJJN-4 for 48 hr at $40^{\circ}C$ showed the highest fibrinolytic activity. Although there was no significant difference in pH of Cheonggukjang prepared with CJJN-4 or 5 during 72 hr at $40^{\circ}C$, Cheonggukjang prepared with CJJN-4 at $45^{\circ}C$ had lower pH until 60 hr than $40^{\circ}C$ or CJJN-5. The total amino type nitrogen contents of Cheonggukjang were 1,139.6 (CJJN-4) and 1,027.6 mg% (CJJN-5) for 72 hr at $40^{\circ}C$, and their producing rates were also higher at $40^{\circ}C$ fermentation than $45^{\circ}C$. Meanwhile, the contents of ammonia type nitrogen induced unpleasant flavor were lower in Cheonggukjang with CJJN-4 and 5 at $40^{\circ}C$ than CJJN-5 at $45^{\circ}C$. Therefore, Cheonggukjang fermented with CJJN-4 starter at $40^{\circ}C$ had the improved flavor, such as change of amino or ammonia type nitrogen production, and higher fibrinolytic activity.