• Title/Summary/Keyword: Fibrin plate assay

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Pharmacetical Characteristics of Solvent Fractions Isolated from Glycyrrhiza uralensis

  • Kim, Jun-Ho;Oh, Hae-Sook
    • Biomedical Science Letters
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    • v.16 no.3
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    • pp.161-167
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    • 2010
  • In this study, the biological activities of Glycyrrhiza uralensis were investigated, including antioxidative, fibrinolytic, thrombin inhibitory, and a-glucosidase inhibitory activity. The hot water extract of Glycyrrhiza uralensis was fractionated into hexane, $CHCl_3$, ethyl acetate, butanol, and water fractions, and each of these fractions were assayed individually. The water fraction showed the highest extraction yield of 19.45% (w/w). Using the DPPH method, the free radical scavenging activity was to be the strongest in the $CHCl_3$ fraction at 89.3%. Using the fibrin plate method, only the butanol fraction showed a substantial plasmin activity of 0.62 units/ml. In thrombin inhibitory activity tests, a 100-fold dilution of the ethyl acetate fraction showed the strongest activity of 46.9%. In the a-glucosidase inhibitory activity assay, a 100-fold dilution of the $CHCl_3$ fraction showed the strongest activity of 80.6%. In conclusion, the combined results of this study demonstrate that the extracts of Glycyrrhiza uralensis can be used as a material for the development of biofunctional foods for diabetics.

Screening Test of Wild Mushroom Methanol Extracts for Fibrinolytic and $\alpha-Glucosidase$ Inhibitory Activity

  • Kim, Jun-Ho;Yoo, Kwan-Hee;Seok, Soon-Ja
    • Biomedical Science Letters
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    • v.13 no.3
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    • pp.245-249
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    • 2007
  • We investigated the fibrinolytic and ${\alpha}-glucosidase$ inhibitory activity of 55 wild mushroom methanol extracts. Among them, 14 mushrooms showed fibrinolytic activity. In particular, Amanita virgineoides showed the greatest enzyme activity (3.9 plasmin units/ml) by a fibrin plate assay. The fibrinolytic activities of Suillus pictus and Polypolellus varius were 3.8 plasmin units, and the activity of Gomphus fujisanensis was 2.8 plasmin units. Leccinum extremiorientale and Xerocomus nigromaculatus had the same activity with 2.3 plasmin units. In a ${\alpha}-glucosidase$ inhibitory activity test, Lactarius sp. showed the greatest inhibitory activity at 97.3%. The ${\alpha}-glucosidase$ inhibitory activities of Clitocybe odora, Xerocomus nigromaculatus, Melanoleuca melaleuca, Suillus pictus, and Gyroporus castaneus were 84.3%, 77.9%, 74.6%, 68.7%, and 65.4%, respectively. According to the results, because Suillus pictus and Xerocomus nigromaculatus have strong fibrinolytic and ${\alpha}-glucosidase$ inhibitory activities, the two mushrooms will be used as materials for the development of new biofunctional food.

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Purification and Characterization of Fibrinolytic Enzyme from Armillariella mellea (뽕나무버섯으로부터 Fibrinolytic enzyme의 정제 및 특성 연구)

  • Kim, Jun-Ho;Kim, Yang-Sun
    • The Korean Journal of Mycology
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    • v.26 no.4 s.87
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    • pp.583-588
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    • 1998
  • A fibrinolytic enzyme has been isolated from the edible honey mushroom, Armillariella mellea and purified. The apparent molecular mass of purified enzyme was estimated to be 19800Da by SDS polyacryl amide gel electrophoresis and 19900Da by gel filtration, indicating that it was a monomer. The enzyme was optimal at pH 7, suggesting that the purified enzyme was a neutral proteinase. It shows the maximum fibrinolytic activity at $55^{\circ}C$, is completely inactivated above $65^{\circ}C$, and still indicates 40% of activity at $37^{\circ}C$. The fibrinolytic activity has been decreased by the addition of EDTA. Fifteen amino acid sequence was determined by protein sequencing techniques.

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Characterization of Fibrinolytic Proteases from Gloydius blomhoffii siniticus Venom

  • Choi, Suk-Ho
    • Journal of Pharmacopuncture
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    • v.14 no.3
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    • pp.71-79
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    • 2011
  • Objectives : This study was undertaken to identify fibrinolytic proteases from Gloydius blomhoffii siniticus venom and to characterize a major fibrinolytic protease purified from the venom. Methods : The venom was subjected to chromatography using columns of Q-Sepharose and Sephadex G-75. The molecular weights of fibrinolytic proteases showing fibrinolytic zone in fibrin plate assay were determined in SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) The effects of inhibitors and metal ions on fibrinolytic protease and the proteolysis patterns of fibrinogen, gelatin, and bovine serum albumin were investigated. Results : 1) The fibrinolytic fractions of the three peaks isolated from Gloydius blomhoffii siniticus venom contained two polypeptides of 46 and 59 kDa and three polypeptides of 32, 18, and 15 kDa and a major polypeptide of 54 kDa, respectively. 2) The fibrinolytic activity of the purified protease of 54 kDA was inhibited by metal chelators, such as EDTA, EGTA, and 1,10-phenanthroline, and disulfhydryl-reducing compounds, such as dithiothreitol and cysteine. 3) Calcium chloride promoted the fibrinolytic activity of the protease, but mercuric chloride and cobalt(II) chloride inhibited it. 4) The fibrinolytic protease cleaved preferentially A${\alpha}$-chain and slowly B${\beta}$-chain of fibrinogen. It also hydrolyzed gelatin but not bovine serum albumin. Conclusions : The Gloydius blomhoffii siniticus venom contained more than three fibrinolytic proteases. The major fibrinolytic protease was a metalloprotease which hydrolyzed both fibrinogen and gelatin, but not bovine serum albumin.

Antioxidant Activity, Fibrinolysis and Angiotensin I Converting Enzyme Inhibitory Activity of Pine Mushroom Juice (Tricholoma matsutake Sing) (송이즙의 항산화 활성, 혈전용해활성 및 Angiotensin I Converting Enzyme의 저해활성 검색)

  • Kim, Young-Eon;Kwon, Eun-Kyung;Han, Dae-Seok;Kim, In-Ho;Ku, Kyung-Hyung
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.37 no.5
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    • pp.535-541
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    • 2008
  • Pine mushroom (Tricholoma matsutake Sing.) is an expensive and highly prized delicacy in Korean and Japanese cuisines with its unique flavor and functional properties. The biological activities of pine mushroom juice (soluble solid contents $4.3^{\circ}$Brix) were evaluated using different tests; DPPH radical scavenging assay for its antioxidant activity, fibrin plate method for fibrinolysis and angiotensin I converting enzyme (ACE) inhibitory activity for anti-hypertensive effect. Free radical scavenging activity of the pine mushroom juice was $48.3{\pm}2.2%$ at the concentration of 1.0 mg/mL. The fibrinolytic activity of pine mushroom was about 2 times greater than that of plasmin used as positive control and the activity increased dose-dependently. The pine mushroom juice inhibited ACE activities dose-dependently and $IC_{50}$ value of ACE activity was $1.03^{\circ}$Brix. These results suggest that pine mushroom is a healthy delicacy.

Purification and Characterization of a Thrombolytic Enzyme Produced by a New Strain of Bacillus subtilis

  • Frias, Jorge;Toubarro, Duarte;Fraga, Alexandra;Botelho, Claudia;Teixeira, Jose;Pedrosa, Jorge;Simoes, Nelson
    • 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.

Characterization of a Fibrinolytic Metalloenzyme from a Wild Mushroom, Tricholoma sejunctum (쓴송이버섯으로부터 분리한 혈전용해 금속효소의 특성 연구)

  • Kim, Jun-Ho;Cho, Seung-Koo
    • The Korean Journal of Mycology
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    • v.32 no.2
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    • pp.119-124
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    • 2004
  • Metalloenzyme was purified from the fruiting bodies of Tricholoma sejunctum. MALDI-TOF and ICP/MS analyses revealed that the enzyme had a molecular weight of 18788.25 and includes $Zn^{2+}$ ion. The N-terminal amino acid sequence of the enzyme was Ala-Thr-Tyr-Lys-Ile-X-Ser-Ala-Thr-His-Gln-X-X-Leu-Val. The activity of the enzyme was inhibited by EDTA and 1,10-phenanthroline, indicating that the enzyme was a metalloprotease. No inhibition was found with E-64 and pepstatin. It has broad substrate specificity for synthetic peptides. The enzyme was stable up to $40^{\circ}C$. The activity of the enzyme was increased by $Zn^{2+}$ and $Co^{2+}$, while it was totally inhibited by $Hg^{2+}$. The enzyme hydrolyzes $A{\alpha}$ subunit of human fibrinogen but did not show any reactivity for $B{\beta}$ and ${\gamma}$ form of human fibrinogen.

Purification and Characterization of Fibrinolytic Enzyme from Tricholoma saponaceum (II) (할미송이버섯으로부터 혈전용해효소의 정제 및 특성 연구 (II))

  • 김준호
    • 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.

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Purification and Characterization of Fibrinolytic Enzymes from Tricholoma saponaceum (할미송이버섯으로부터 혈전용해효소의 정제 및 특성 연구)

  • Kim, Jun-Ho
    • 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 $65^{\circ}C$.

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Detection of Biological Activities of Wild Mushroom Methanol Extracts (야생버섯 메탄올추출물의 생리활성 검색)

  • Kim, Jun-Ho;Yoo, Kwan-Hee;Kim, Yang-Sup;Seok, Soon-Ja
    • 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.