• 동의생리병리학회지
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• 제18권5호
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• pp.1494-1504
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• 2004

The present paper reports the effects of Hwaotang an atherosclerosis using a spontaneous experimental model, We have also investigated the pharmacological effect of Hwaotang on collagen- and ADP-induced blood platelet aggregation, thrombin-induced conversion of fibrinogen and fibrinolysis in in vitro experiments, and various effects on stimuli-induced superoxide generation in human neutrophils. Hwao-tang was shown to have inhibitory effect on collagen- and ADP-induced blood platelet aggregation, on thrombin-induced conversion of fibrinogen to fibrin and on the activity of plasminogen or plasmin. Hwao-tang also significantly inhibited fMLP-induced superoxide generation in a concentration-dependent manner, but not that induced by arachidonic acid. Hwao-tang inhibited neutrophil functions, including degranulation, superoxide generation, and leukotriene B4 production, without any effect on 5-lipoxygenase activity. In conclusion, the protection of extracts of Hwao-tang on the ischemic infarction induced artificially might be involved to their inhibition of thrombotic action. The results also indicate that Hwao-tang exerts the effects on superoxide generation related to the inhibition of neutrophil functions.

• BMB Reports
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• 제33권2호
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• pp.112-119
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• 2000

Three kinds of serine protease inhibitors, members of the Bowman-Birk trypsin inhibitor, were purified from Dolichos lablab seeds and named Dolichos protease inhibitor 1, 2 and 3 (DI-1, DI-2 and DI-3), respectively. Each inhibitor showed a single band with gel mobility at around 15.9, 12.1 and 14.6 kDa on 20% SDS-PAGE under reducing conditions. To characterize inhibitory specificity, the inhibition constant (Ki) for these inhibitors was measured against several known serine proteases. All three Dolichos protease inhibitors (DI-1, DI-2 and DI-3) inhibited the activity of trypsin and plasmin, but had no effect on thrombin and kallikrein (either for human plasma kallikrein or for porcine pancreas kallikrein). DI-1 inhibited chymotrypsin most effectively (Ki = $3.6{\times}10^{-9}\;M$), while DI-2 displayed inhibitory activity for porcine pancreatic elastase (Ki = $6.2{\times}10^{-8}\;M$). Pre-treatment of the 33 mg/kg of DI-mixture (active fractions from $C_{18}$ open column chromatography that included DI-1, DI-2 and DI-3) inhibited the induction of pseudomonal elastase-induced septic hypotension and prevented an increase in bradykinin generation in pseudomonal elastase-treated guinea pig plasma. Also, the increase of kallikrein activity, by injection of pseudomonal elastase, was inhibited by the pretreatment of the DI-mixture in a guinea pig. Since the DI-mixture had no inhibitory effect on kallikrein activity when Z-Phe-Arg-MCA was used as a substrate in vitro, its inhibitory activity in the pseudomonal elastase-induced septic hypotension model might not be due to a direct inhibition of plasma kallikrein in the activation cascade of the Hageman factor and prekallikrein system. These results suggest that the Dolichos DI-mixture might be used as an inhibitor in pathogenic bacterial protease-induced septic shock.

• Toxicological Research
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• 제17권
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• pp.97-104
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• 2001

Three types of proteases (MEF-1, MEF-2 and MEF-3) were purified from the egg cases of Ten-odera sinensis using ammonium sulfate fractionation, gel filtration on Bio-Gel P-60 and affinity chromatography on DEAE Affi-Gel blue gel. The proteases were assessed homogeneous by SDS-polyacrylamide gel electrophoresis and have molecular weight of 31,500, 32,900 and 35,600 Da, respectively. The N-terminal regions of the primary structure were compared and they were found to be different each other. MEFs readily digested the $A\alpha$ - and B$\beta$-chains of fibrinogen and more slowly the ${\gamma}$-chain. The action of the enzymes resulted in extensive hydrolysis of fibrinogen and fibrin, releasing a variety of fibrinopeptides. MEF-1 was inactivated by Cu$^{2+}$ and Zn$^{2+}$ and inhibited by PMSF and chymostatin. MEF-2 was inhibited by PMSF, TLCK. soybean trypsin inhibitor. MEF-3 was only inhibited by PMSF and chymostatin. Antiplasmin was not sensitive to MEF-1 but antithrombin III inhibited the enzymatic activity qf MEF-1. MEF-2 specifically bound to anti plasmin Among the chromogenic protease substrates, the most sensitive one to the hydrolysis of MEFs was benzoyl-Phe-Val-Arg-p-nitroanilide with maximal activity at pH 7.0 and 3$0^{\circ}C$. MEF-1 preferentially cleaved the oxidized B-chain of insulin between Leu15 and Tyr16. In contrast, MEF-2 specifically cleaved the peptide bond between Arg23 and Gly24. D-dimer concentrations increased on incubation of cross-linked fibrin with MEF-1, indicating the enzyme has a strong fibrinolytic activity.ity.

• Journal of Microbiology and Biotechnology
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• 제24권2호
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• pp.245-253
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• 2014

A fibrinolytic enzyme was produced by an edible mushroom of Pleurotus ostreatus using submerged culture fermentation. The enzyme was purified from the culture supernatant by applying a combination of freeze-thaw treatment, ammonium sulfate precipitation, hydrophobic interaction, and gel filtration chromatographies. The enzyme was purified by a 147-fold, with a yield of 7.54%. The molecular masses of the enzyme an determined by gel filtration and SDS-PAGE were 13.6 and 18.2 kDa, respectively. The isoelectric point of the enzyme was 8.52. It hydrolyzed fibrinogen by cleaving the ${\alpha}$ and ${\beta}$ chains of fibrinogen followed by the ${\gamma}$ chains, and also activated plasminogen into plasmin. The enzyme was optimally active at $45^{\circ}C$ and pH 7.4. The enzyme activity was completely inhibited by EDTA, whereas protease inhibitors of TPCK, SBTI, PMSF, aprotinin and pepstatin showed no inhibition on its activity. The partial amino acid sequences of the enzyme as determined by Q-TOF2 were ATFVGCSATR, GGTLIHESSHFTR, and YTTWFGTFVTSR. These sequences showed a high degree of homology with those of metallo-endopeptidases from P. ostreatus and Armillaria mellea. The purified enzyme can also be applied as a natural agent for oral fibrinolytic therapy or prevention of thrombosis.

• 한국식품과학회지
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• 제39권1호
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• pp.83-87
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• 2007

단삼 메탄올 추출물의 항혈전 및 항산화 효과를 확인하기 위하여 혈소판의 응고 억제효과, 혈장응고시간의 연장, 혈전용해 활성으로 항혈전 효과를 고찰하였으며 DPPH radical 소거능을 측정하여 항산화 효과를 검토하였다. 그 결과, 단삼의 메탄올 추출물은 항혈전과 항산화에 효과가 있는 것으로 나타났다. 이 추출물은 혈소판응고 억제, 혈액응고시간 연장 및 혈전용해 활성에 모두 효과가 좋은 것으로 나타났으며, 폴리페놀 함량이 높고 DPPH radical 소거능도 매우 좋았다. 이로서 단삼의 높은 DPPH radical 소거능이 항혈전 효과에 영향을 미치는 것으로 사료되며, 정확한 효과 및 혈소판의 기능을 조절하는 기전에 대한 연구는 이 결과들을 바탕으로 현재 진행 중에 있다.

• Archives of Pharmacal Research
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• 제20권1호
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• pp.17-23
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• 1997

A saline suspension of Lumbricus rubellus earthworm powder (EWP) was administered to rats (1 g/kg/day) orally for 15 days to evaluate an oral effectiveness for thrombotic disorders. Blood was drawn at 2-day interval after the administration. Several parameters for antithrombotic, anticoagulant and fibrinolytic activities were measured, including platelet aggregation, clotting time, plasmin activity and the levels of FDP (fibrin/fibrinogen degradation products), D-dimer, and t-PA antigen. It did not affect platelet aggregation induced by ADP and collagen but anticoagulant activity (aPTT and TT) was gradually increased to two-folds for the first 5 days of administration and back to normal. Fibrinolytic activity of euglobulin fraction was highest on the 11 th day after the administration. The level of FDP was elevated to be comparable to the positve control$ (5-10 {\mu}g/ml)$ after 9-day treatment. Oral administration of the EWP could also reduce the formation of venous thrombus induced with viper venom. Complete blood count (CBC) profiles were within normal ranges except for a slight increase in white blood cells after the oral administration for 15 days. These results suggested that the EWP may be valuable for the prevention and/or treatment of thrombotic diseases.

• 사상체질의학회지
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• 제19권1호
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• pp.160-170
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• 2007

1. Objectives This study was performed to find the activities and characteristics of purified thrombolytic enzymes from Holotrichia extracts. 2. Methods In the first time, a coarse enzyme fluid was made by using the freedried Holotrichia extracts. After manufacturing total soluble proteins and purifing enzymes, it was evauluated the activities and characteristics of this enzyme's dissolving capability to fibrin and thrombus. This study was taken using azocasein assay, fibrin-plate method, native-PAGE and fibrin zymography. 3. Results A soluble proteins were efficiently extracted form freezedried Holotrichia extracts. And, this purified enzyme had a ten times fibrinolytic capability compare with ustulation Holotrichia sample. In native PAGE and fibrin zymography, Holotrichia extracts showed the respectable fibrinolytic activity. Also, It had higher thrombolytic activities compared with general thrombolytic enzyme 'plasmin'. In experiment of various protease inhibitors of the purified enzyme from Holotrichia extracts on the azocaseinolytic activity, the enzyme was strongly inhibited by EDTA ${\cdot}$ EGTA, and weakly by APMSF ${\cdot}$ PMSF ${\cdot}$ TPCK. 4. Conclusion Holotrichia extracts has the thrombolytic activities, and it will operate directly th fibrin-clot and thrombus.

• 한국식품영양과학회지
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• 제36권4호
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• pp.395-399
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• 2007

각종 미네랄과 무기염류의 함량이 풍부한 것으로 알려진 함초를 이용하여 발효 후 그에 따른 기능성을 검토해 보고자 하였다. 유산균 접종 후 pH는 초기 4.50에서 발효 후 2.84로 변했으며, 산도는 초기 0.29에서 발효 후 0.55로 증가하였다. 한편 생균수를 측정한 결과 초기 $1.2{\times}10^{11}CFU/mL$이었던 생균수는 발효 72시간 후 $1.4{\times}10^{13}CFU/mL$를 보였다. 함초 추출물 발효액을 동결건조시킨 후 그에 따른 기능성을 검토하였으며 SOD-유사활성과 DPPH-라디칼 소거능 측정결과, 농도 의존적인 경향을 보였으며 혈전분해능 측정결과 발효 전의 함초 추출물에서는 나타나지 않았던 혈전분해능을 발효 후 동결건조분말 25%의 시료 농도에서 plasmin $3.84{\mu}g/mL$의 농도와 동일한 수준의 혈전용해능을 보였다.

• 한국지역사회생활과학회:학술대회논문집
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• 한국지역사회생활과학회 2009년도 추계학술대회
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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.

• 한국식품영양학회지
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• 제29권6호
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• pp.962-970
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• 2016

Commercialized production of onion vinegar, which has biological activities formed through alcohol and acetic acid fermentation, requires standardization. The objective of this study was to determine optimal conditions of sugar contents ($11{\sim}15^{\circ}Brix$) and agitation rate (100~300 rpm) of fermenter in the alcohol-acetic fermentation for producing onion vinegar. The alcohol and total acidity contents increased, whereas contents of total sugars decreased during alcohol fermentation. Contents of alcohol of 13 and $15^{\circ}Brix$ reactants were about 8% in 36 hr and total acidities of all samples were below 0.2% in 60 hr. During acetic fermentation, total acidity increased with highest value at 9 days (3.2% in 100 rpm), 10 days (4.1% in 200 rpm) and 8 days (4.3% in 300 rpm), respectively. From these results, sugar contents ($13^{\circ}Brix$) were measured for alcohol fermentation and agitation rate (300 rpm) for fast fermentation method of vinegar. The contents of total phenols, flavonoids and quercetin in onion vinegar were 33.3 mg/100 g, 3.0 mg/100 g and 2.0 mg/100 g, respectively. Onion vinegar showed an antimicrobial activity against Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Escherichia coli and Enterobacter aerogenes. Antioxidant effect of onion vinegar was 26.23% in DPPH radical inhibition and 58.58% in superoxide dismutase like activity, respectively. Fibrinolytic activity was 1.51 plasmin unit/mL in onion vinegar. In conclusion, onion vinegar processed by alcohol and acetic fermentation had nutritional values and potential biological activities.