• 한국식품영양과학회지
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• 제27권6호
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• pp.1204-1210
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• 1998

This study was focused on the purification, characterization and promotion mode of an anticoagulant polysaccharide from Hizikia fusiforme. The anticoagulant crude polysaccharide(HF 0) was obtained by using hot water extraction at 100oC for 3 hrs after homogenizing desalted Hizikia fusiforme. The anticoagulant polysaccharide(HF 2 3 1a) was purified from the crude extract(HF 0) through stepwise gradient ethanol precipitation(HF 2), DEAE Toyopearl 650C(HF 2 3), Sephadex G 75(HF 2 3 1), Sepharose CL 6B(HF 2 3 1a) chromatography and HPLC to homogeneity. HF 2 3 1a was estimated at 5.3$\times$105 Da molecular weight and composed of fucose(51.92%), galactose(19.34%), mannose(13.92%), xylose (7.14%), arabinose(3.95%) and rhamnose(3.78%), and comprimised 29.7 % sulfate residue. The sulfated anticoagulant polysaccharide from HF 2 3 1a was proposed to inhibit via the intrinsic pathway and common pathway in the blood coagulation. The HF 2 3 1a exhibited the anticoagulant activity by activating an antithrombin III and the activity depended on the concentration of HF 2 3 1a. Acute toxicity of HF 2 in mice was not detected. Only 14 of 33 control mice(11.4%) that had taken saline survived for 30 min after injecting thrombin(100 NIH unit/ml).

• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.503-511
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• 2008

An edible marine red alga, Grateloupia filicina, collected from Jeju Island of Korea was hydrolyzed by cheap food-grade carbohydrases (Viscozyme, Celuclast, AMC, Termamyl, and Ultraflo) to investigate its anticoagulant activity. Among the tested enzymatic extracts of G. filicina, a Termamyl extract showed the highest anticoagulant activity. Anion-exchange chromatography on DEAE-cellulose and gel-permeation chromatography on Sepharose-4B were used to purify the active polysaccharide from the crude polysaccharide fraction of G. filicina. The purified sulfated polysaccharide (0.42 sulfate/total sugar) showed ${\sim}1,357kDa$ molecular mass and was comprised mainly of galactose(98%) and 1-2% of glucose. The sample showed potential anticoagulant activity on activated partial thromboplastin time (APTT) thrombin time (TT) assays. The purified G. filicina anticoagulant (GFA) inhibited the coagulation factor X (92%), factor II (82%), and factor VII (68%) of the coagulation cascade, and the molecular interaction (protein-polysaccharide) was highly enhanced in the presence of ATIII (antithrombin III). The dissociation constant of polysaccharide towards serine proteins decreased in the order of FXa (58.9 nM) >FIIa (74.6 nM) >FVII (109.3 nM). The low/less cytotoxicity of the polysaccharide benefits its use in the pharmaceutical industry; however, further studies that would help us to elucidate the mechanism of its activity are needed.

• 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.

• Archives of Pharmacal Research
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• 제29권5호
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• pp.418-423
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• 2006

Tabanus anticoagulant protein (TAP) was isolated from the whole body of the tabanus, Tabanus bivittatus, using three purification steps (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and ion exchange chromatography on DEAE Sephadex gel). The purified TAP, with a molecular weight of 65 kDa, was assessed to be homogeneous by SDS-polyacrylamide gel electrophoresis, and an isoelectric point of 7.9 was determined by isoelectric focusing. The internal amino acid sequence of the purified protein was composed of Ser-Leu-Asn-Asn-Gln-Phe-Ala-Ser-Phe-lle-Asp-Lys-Val-Arg. The protein was activated by $Cu^{2+}\;and\;Zn^{2+}$, and the optimal conditions were found to be at pH $3\sim6\;and\;40\sim70^{\circ}C$. Standard coagulation screen assays were used to determine thrombin time and activated partial thromboplastin time. Chromogenic substrate assays were performed for thrombin and factor Xa activity. TAP considerably prolonged human plasma clotting time, especially activated partial thromboplastin time in a dose-dependent manner; it showed potent and specific antithrombin activity in the chromogenic substrate assay. Specific anti-factor Xa activity in TAP was not detected. Overall, this result suggested that TAP has significant anticoagulant activity on blood coagulation system.

• Archives of Pharmacal Research
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• 제28권7호
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• pp.816-822
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• 2005

Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gin Asp Phe Val Glu Glu lie Leu. CPM-2 was inactivated by $Cu^{2+}\;and\;Zn^{2+}$ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and ${\alpha}_1$-antitrypsin. However, EDTA, EGTA, cysteine, $\beta$-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved $A{\alpha}-$ and $B{\beta}$-chains of fibrinogen and fibrin, and y-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.

• 대한한방내과학회지
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• 제17권1호
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• pp.34-50
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• 1996

The present experiment was desinged to investigate the effects of Tongdosan water extracts on the Cardiovascular System in the Experimental Animals. Thus, the changes of blood pressure and heart rate were measured after oral admini- stration. Measurment of Mortality rate was observed for measuring the effect of Tongdosan water extract. Tongdosan water extract against pulmonary thrombo- embolism induced by collagen the mixture(0.1ml/10g, 2mg/kg B.W) plus serotonin(5mg/kg B.W) in mouse. The effect of Tongdosan water extract was examined by observing the change of collagen-induced platelet aggregation, coagulation activity, ex vivo and in vitro fibrinolytic activity of euglobulin fraction in rats. The results were summarized as followings. 1. Tongdosan dropped the blood pressure in spontaneous hypertensive rat. 2. The drug increased the auricular blood flow in rabbit. 3. The drug relaxed the artery contraction by pretreated norepinephrine in rat. 4. The drug inhibited the death rate of mouse which was led to thromboembo- lism by serotonin and collagen. 5. The drug inhibited the platelet aggregation in rat. 6. The drug prolonged the prothrombin time and activated partial thromboplastin time on the test of plasma coagulation factor activity in rat, but was not valuable. 7. The drug reduced the fibrinogen lyses time of rat ex vivo assay and lyses area was increased. 8. Tongdosan reduced fibrinogen lyses time of rat in vitro assay. According to the above mentioned results, Tongdosan increased the blood flow and dropped the blood pressure by the dilation of blood vessel. And the drug presented the antithrombin acivity, inhibited the platelet aggregation.

• 대한한방내과학회지
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• 제20권1호
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• pp.57-72
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• 1999

This study was designed to elucidate the anti-inflammatory, cardiovascular, anti-thrombotic, and analgesic effect of Whalrakdan. The anti-inflammatory effects was measured by the method of carrageenin induced edema, protein leakage test using CMC-pouch, and the effect of Whalrakdan on the cardiovascular system was observed by the change of flow rate of Ringer solution in the vascular system in the ear of rabbit. and the contraction and dilatation of rat tail artery. Death rate, platelet aggregation, plasma coagulation activity, antithrombin activity was observed for the measurement of the anti-thrombotic effect of Whalrakdan, and the analgesic effect was measured by the acetic acid method and hot plate method. The result was as follows: 1. After 2 or 3hour of Whalrakdan administration, carrageenin induced edema and CMC-pouch protein leakage was significantly decreased. 2. The slight anagesic effect of Whalrakdan extract was confirmed by the observation of writhing syndrome, paw licking time, and escape time. 3. The droplet of Ringer solution increased according to the increase of concentration of Whalrakdan extract, and the vasoconstriction decreased dependantly to the concentration of Whalrakdan extract. 4. The anti-thrombotic effect of Whalrakdan was observed by the decrease of death rate, the inhibition of platelet aggregation, and the increase of anti-thrombin activity.

• BMB Reports
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• 제30권1호
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• pp.37-40
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• 1997

Earthworm extracts are known for anti-inflammatory, analgesic. antipyretic, and anticancer effects but can also influence blood circulation. It was previously shown that an earthworm, Lumbricus rubelius. contained a water-extractable anticoagulant which was a heat- and acid-stable molecule with hydrophilic property. In order to uncover the biochemical nature of this molecule, the anticoagulant was processed with various hydrolases such as trypsin, DNase, RNase. and lysozome. When the digested samples were analyzed with an in vitro coagulation test measuring activated partial thromboplastin time (APTT) and agarose gel electrophoresis, the anticoagulant proved to be a relatively homogeneous DNA fragment with relative molecular size around 72 base pairs. Interestingly, the activity was further stimulated with a trypsin digestion. RNA. on the other hand, did not prolong the APTT. It was also demonstrated that the DNA accelerated the antithrombin III (AT-III) inhibition of thrombin from $IC_{50}$ of 0.34 to 0.16 unit determined with S-2238 as a substrate, whereas heparin, a popular anticoagulant. shifted the value to 0.05. Therefore, it is suggested that the DNA could be considered as an alternative antithrombotic agent to heparin, which would exhibits bleeding side effects.

• 약학회지
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• 제36권4호
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• pp.350-356
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• 1992

Anticoagulant activities were tested for the fifteen kinds of medicinal plants by measuring activated partial thromboplastin time (aPTT). Of them five kinds or species (Artemisia princeps, Sanguisorba officinalis, Artemisia apiacea, Eclipa alba, Schizonepeta tenuifolia) were selected and fractionated for the preparation of acidic polysaccharides. They were extracted with water by refluxing and the extracts were precipitated with ethanol. The precipitates were separated based on charge using a DEAE-Sephadex. The low salt and high salt fractions were sulfated with anhydrous pyridine and chlorosulfonic acid complex. In vitro anticoagulant activities of sulfated polysaccharides were tested by measuring aPTT, prothrombin time (PT), and factor Xa clotting time using normal human plasma. No relationship was found between the amount of uronic acids and anticoagulant activities, but the sulfated ones show the increase of activities. In vivo anticoagulant properties of the sulfated polysaccharide from Artemisia apiacea were also tested by the intraveneous administration of three different doses (3,5 and 10 mg/kg) to rats. APTT and PT were increased significantly and the action of factor Xa and thrombin mediated through antithrombin III were inhibited slightly.

• 생명과학회지
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• 제17권4호
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• pp.535-539
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• 2007

식용 및 약용으로 광범위하게 이용되고 있는 산사자의 유용 생리활성 검토를 목적으로 산사자 추출물 및 분획물을 조제하여, 혈액내의 혈전 생성에 직접 관여하는 인간 트롬빈의 thrombin time(TT) 및 activated partial thromboplastin time(aPTT) 증가 활성을 측정하여 항혈전 활성을 평가하였다. 산사자의 항혈전 물질은 물 추출보다는 메탄을 추출이 적합하며(추출효율 40.4%), butanol 분획에서 가장 강력한 활성을 나타내었다. 산사자의 butanol 분획물은 1.25 mg/ml의 농도에서 835%의 TT 증가, 315%의 aPTT 증가를 보여 아스피린보다 2.9배 이상의 강력한 항혈전 활성을 나타내었다. 이러한 항혈전 활성은 기존의 보고된 quercetin, kaempferol, myricetin 및 rutin과는 무관하였다. 또한 산사자의 활성물질은 0.5 N HCl 처리시 120분 이상 안정하였으나, $100^{\circ}C$, 30분 열처리에는 85% 이상의 활성소실이 나타나 열에 매우 민감한 물질로 확인되었다. 본 연구결과는, 비열 처리와 적합한 가공 공정이 가능하다면 산사자 추출물이 혈액순환 장해 및 혈관계 질환 예방 및 치료제로 개발 가능함을 제시하고 있다.