• BMB Reports
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• 제45권7호
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• pp.390-395
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• 2012

Oleanolic acid (OA), a triterpenoid known for its anti-inflammatory and anti-cancer properties, is commonly present in several medicinal plants but its anticoagulant activities have not been studied. Here, the anticoagulant properties of OA were determined by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrin polymerization as well as cell-based thrombin and activated factor X (FXa) generation activities. Data showed OA prolonged aPTT and PT significantly and inhibited thrombin catalyzed fibrin polymerization. In addition, OA inhibited the activities of thrombin and FXa and inhibited the generation of thrombin or FXa in human endothelial cells. OA also inhibited TNF-${\alpha}$-induced tissue factor expression on human endothelial cells. In accordance with these anticoagulant activities, OA showed an anticoagulant effect in vivo. These results indicate that OA possesses antithrombotic activities and suggest that daily consumption of a herb containing OA may be preventing thrombosis in pathological states.

• 한국균학회지
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• 제40권3호
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• pp.159-163
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• 2012

노루궁뎅이버섯을 이용하여 성인병 치료와 예방을 위한 기능성식품 개발을 위한 기초자료를 얻기 위해 노루궁뎅이버섯 물 추출물과 유기용매 분획물을 이용하여 성인병관련 생리활성을 확인하였다. 노루궁뎅이버섯 물 추출물은 87.78%의 항산화활성을 나타냈으며, 부탄올 분획물과물 분획물은 87.91%와 92.27%의 높은 활성을 나타냈다. 에틸 아세테이트 분획물은 2.96 plasmin unit의 높은 혈전용해 활성을 보였다. 핵산 분획물로부터 77.67%와 90.53%의 높은 트롬빈 저해활성과 ${\alpha}$-glucosidase 저해활성을 확인하였다. 실험 결과로부터 노루궁뎅이버섯 에틸아세테이트 분획물의 높은 혈전용해 활성과 핵산 분획물의 높은 트롬빈 저해활성과 ${\alpha}$-glucosidase 저해활성은 혈관계질환 치료나 예방을 위한 기능성식품 개발에 이용 가능할 것으로 기대된다.

• Archives of Pharmacal Research
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• 제22권2호
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• pp.119-123
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• 1999

Parathyroid hormone (PTH) treatment of bone and kidney-derived cells not only activates adenyly cyclase buy also increases intracellular free calcium, and translocates protein kinase C (PKC) from cytosol to plasma membranes. We have found that acute phorbol ester pretreatment significantly decreases PTH-induced calcium transients and the effect of phorbol ester was antagonized by staurosporine (ST). Although the major effect of ST in that study was the reversal of the action of phorbol ester, it appeared that ST may also have promoted the effect of PTH directly. To further investigate the observation, we examined the effect of ST on the intracellular calcium transients induced by PTH and $\alpha$-thrombin ($\alpha$-TH). For calcium transient experiments, UMR-106 cells were loaded with 2 mM fluo-acetoxymethylester for 30 min at room temperature. The cells were then washed and suspended in buffer containing 1 mM calcium. Fluorescence was detected at 530 nm, with excitation at 505 nm. ST alone did not cause calcium transients, but enhanced the transients elicited by PTH response. added 5 min before the hormone. Another protein kinase inhibitor H-7 likewise enhanced the calcium responses elicited by PTH, while genistein did not affect PTH response. Calcium transients elicited by $\alpha$-TH were also enhanced by ST. The results suggest that there might be tonically activated endogenous protein kinase(s) which inhibit calcium signaling of some calcemic agents.

• 대한의생명과학회지
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• 제18권1호
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• pp.35-41
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• 2012

Physiological activities of hot water extract and solvent fractions isolated from Astragalus membranaceus were examined and the antioxidative, fibrinolytic, thrombin inhibitory and a-glucosidase inhibitory activity were measured. The hot water extract of Astragalus membranaceus was fractionated into hexane, chloroform, ethyl acetate, butanol and water fractions, and each of these fractions was individually assayed. The antioxidative activities of ethyl acetate and chloroform fractions were 89.96% and 87.36%, respectively. Using the fibrin plate method, only the hot water extract showed a plasmin activity of 0.41 units/ml. The thrombin inhibitory activity of the ethyl acetate fraction was the highest with a value of 82.73%. The hot water extract displayed a-glucosidase inhibitory activity of 64.91%. In conclusion, the hot water extract and the ethyl acetate fraction can be used as materials for the development of biofunctional foods to prevent cardiovascular diseases.

• Journal of Microbiology and Biotechnology
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• 제1권4호
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• pp.266-273
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• 1991

Hirudin, a 65-amino acid protein isolated from the salivary gland of the bloodsucking leech, Hirudo medicinalis, is a potent thrombin-specific inhibitor and blocks the thrombin-mediated conversion of fibrinogen to fibrin in clot formation. We have studied the gene expression and secretion of hirudin in yeast. Saccharomyces cerevisiae. A gene coding for hirudin was synthesized based on the amino acid sequence and cloned into a yeast expression vector $YEG{\alpha}-1$ containing the ${\alpha}-mating$ factor pre-pro leader sequence and galactose-inducible promoter, GALl0. Recombinant S. cerevisiae was found to secrete biologically active hirudin into the extracellular medium. The secreted recombinant hirudin was recovered from the culture medium and purified with ultrafiltration and reverse phase high performance liquid chromatography. Approximately 1 mg of hirudin per liter was produced under suboptimal culture conditions and brought to about 90% purity in two steps of purification.

• BMB Reports
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• 제32권6호
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• pp.567-572
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• 1999

We have previously shown that a DNA fragment is responsible for the anticoagulatory effect of an earthworm, Lumbricus rubellus. The anticoagluant increased the activated partial thromboplastin time (APTT) and also inhibited the thrombin activity observed with either N-${\alpha}$-p-tosyl-L-arginine methyl ester (TAME) or H-D-phenyl-alanyl-L-pipecoil-L-arginine-p-nitroanilide (S-2238). Since trypsin digestion of the anticoagulant further increased the APTT, the possible presence of a regulatory protein for the anticoagulatory DNA was investigated by digesting the anticoagulant with trypsin and isolating the DNA fragment with C4-reversed phase HPLC. The DNA fragment lacking a regulatory protein was eluted in the flow-through fraction, and analyzed with thrombin and activated factor X. Activated factor X activity was more strongly inhibited than thrombin activity. For DNA digestion, we treated the anticoagulant with DNase and purified the DNA-binding protein with a FPLC Resource-S cation exchange column. The regulatory protein, with an $M_r$ of 55.0 kDa, reduced the anticoagulatory effect of the DNA fragment.

• 한국균학회지
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• 제39권3호
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• pp.189-193
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• 2011

아위버섯으로부터 성인병 치료와 예방 관련된 생리활성물질을 확인하고 이 물질의 추출을 위한 최적 조건을 확립하기위해 아위버섯 열수추출물과 유기용매 분획물을 사용하여 성인병 관련 생리활성을 측정하였다. 아위버섯 열수추출액에서는 작은 항산화효과. 트롬빈저해효과, 혈당강하효과가 확인되었지만 혈전용해효과는 나타나지 않았다. 그러나 에틸아세테이트 분획물과 부탄올 분획물에서 86.79%와 87.82%의 높은 항산화효과가 확인 되었으며, 에틸아세테이트 분획물에서 0.08 plasmin unit의 작은 혈전용해활성을 확인할 수 있었다. 클로로포름 분획물과 에틸아세테이트 분획물에서 74.90%와 71.88%의 높은 트롬빈저해효과와 함께 부탄올 분획물에서 49.67%의 혈당강하효과를 확인하였다. 따라서 아위버섯의 에틸아세테이트 분획물과 부탄올 분획물은 혈관계 질환을 위한 기능성 식품의 개발에 이용할 수 있을 것으로 판단된다.

• Journal of Ginseng Research
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• 제40권4호
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• pp.359-365
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• 2016

Background: Glycoprotein IIb/IIIa (${\alpha}aIIb/{\beta}_3$) is involved in platelet adhesion, and triggers a series of intracellular signaling cascades, leading to platelet shape change, granule secretion, and clot retraction. In this study, we evaluated the effect of ginsenoside Ro (G-Ro) on the binding of fibrinogen to ${\alpha}aIIb/{\beta}_3$. Methods: We investigated the effect of G-Ro on regulation of signaling molecules affecting the binding of fibrinogen to ${\alpha}aIIb/{\beta}_3$, and its final reaction, clot retraction. Results: We found that G-Ro dose-dependently inhibited thrombin-induced platelet aggregation and attenuated the binding of fibrinogen to ${\alpha}aIIb/{\beta}_3$ by phosphorylating cyclic adenosine monophosphate (cAMP)-dependently vasodilator-stimulated phosphoprotein (VASP; $Ser^{157}$). In addition, G-Ro strongly abrogated the clot retraction reflecting the intensification of thrombus. Conclusion: We demonstrate that G-Ro is a beneficial novel compound inhibiting ${\alpha}aIIb/{\beta}_3$-mediated fibrinogen binding, and may prevent platelet aggregation-mediated thrombotic disease.

• 한국균학회지
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• 제40권4호
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• pp.296-298
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• 2012

본 연구는 야생버섯으로부터 혈관계질환 관련 생리활성을 확인하여 혈관계질환을 위한 기능성식품 개발에 재료로 사용하기 위해 야생버섯 33종의 야생버섯 메탄올 추출물을 제조하여 심혈관계 질환 생리활성을 확인하였다. 긴대밤그물버섯(Boletellus elatus)과 벽돌빛뿌리버섯(Heterobasidion insulasis)이 높은 혈전용해 활성과 트롬빈저해효과, 항산화활성을 나타내 혈관계질환 치료나 예방을 위한 기능성식품 개발에 이용 가능할 것으로 기대된다.

• BMB Reports
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• 제29권6호
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• pp.500-506
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• 1996

We have isolated a water-extracted novel regulator for blood coagulation from an earthworm, Lumbricus rubellus. As a folk remedy, the earthworm has been known to facilitate blood circulation. After complete heat inactivation of endogenous proteases in the earthworm, an anticoagulant(s) was purified through ammonium sulfate fractionation and three consecutive gel permeation chromatography of Sephacryl S-300, Sephadex G-75, and G-150 by measuring activated partial thromboplastin time (APTT) The anticoagulant was further purified to 2,800 fold with a C4 reversed-phase HPLC This activity was stable under heat ($100^{\circ}C$ for 30 min) and acidic conditions (0.4 N HCl). The effects of this partially purified anticoagulant on thrombin were observed with various substrates such as N${\alpha}$-benzoyl-DL-arginine-p-nitroanilide (BApNA), H-D-phenylalanyl-L-pipecoyl-L-arginine-p-nitroanilide (S-2238), N${\alpha}$-p-tosyl-L-arginine methyl ester (TAME), and fibrinogen as a natural substrate. Only TAME hydrolysis, due to an esterase activity of the enzyme, was inhibited among the chromogenic substrates. In addition, the anticoagulant not only inhibited the conversion of fibrinogen to fibrin but also prolonged the fibrin clot formation monitored with the in vitro coagulation test. Based on these observations, we suggest the significance of measuring the ability of antithrombotic drugs to inhibit the esterase activity of thrombin. In this report, it was also shown that the earthworm indeed contained a water-extractable, heat- and acid-stable anticoagulant which could be used as a novel antithrombotic agent.