• Applied Biological Chemistry
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• v.39 no.2
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• pp.159-164
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• 1996

Screening of anticoagulant activity was conducted for the hot water extracts of 73 kinds of medicinal herbs, 41 kinds of Korean edible plants, and 5 kinds of sea weeds using plasma recalcification test(Tr). In the first screening several extracts of the plants, Alisma calndiculatum, Corydalis ternata Panax notoginseng, Allium sativum, Ganderma luidum, Codium fragile, showed high activities. When the plants were reextracted with various solvent conditions, acidic water extracts of Codium fragile showed the highest activity in APTT. A crude polysaccharide fraction(CF-1) was prepared by methanol reflux, ethanol precipitation, dialysis and Iyophilization of the acid extracts. CF-1 comprised 80.8% total sugar consisting of arabinose, galactose and glucose as the main monomers, 8.7% protein, and 13.3% sulfate. The anticoagulant activity of CF-1 was not changed by pronase digestion, but decreased by periodate oxidation, and this indicated that the anticoagulant activity was attributed to the polysaccharide portion.

• Applied Biological Chemistry
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• v.42 no.2
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• pp.140-146
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• 1999

We have isolated two anticoagulant polysaccharides from an acidic extract of Codium fragile. The purification was conducted using three consecutive chromatographies of DEAE-Toyopearl 650C, Sephadex G-100 (G-75), and Sepharose CL-6B by measuring activated partial thromboplastin time (APTT). The two purified anticoagulant polysaccharides, CF-1-VIa-1 and CF-1-VIIa-1, were found to be nearly homogenous on HPLC using a gel permeation column and appeared to have molecular weights of about 80,000 Da and 40,000 Da, respectively. The polysaccharides consisted mainly of arabinose and galactose in a molar ratio of about 2 : 1, and also comprised 12-13% of sulfates at their constituent sugars. CF-1-VIa-1 and CF-1-VIIa-1 inhibited blood coagulation via both the intrinsic and the extrinsic pathways. The polysaccharides unlike heparin showed an inhibitory activity on thrombin when a pure fibrinogen without antithrombin III was used as a substrate. Structural modifications using sulfation and desulfation affected the anticoagulant activities directly, suggesting that the content of sulfate plays an important role in the blood coagulation cascade. The polysaccharides may inhibit some proteases involved in the blood coagulation cascade, judging from the independence of calcium concentrations in their anticoagulant activity.

• Journal of Life Science
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• v.9 no.4
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• pp.423-429
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• 1999

Carrageenan oligosaccharides prepared from -carrageenan by carrageenase from Pseudomonas alcaligenes. The oligosaccharides showed three spots on TLC and the degree of Polymerization of the C1, C2 and C3 spot were each 9.0$\pm$1.0, 6.0$\pm$1.5 and 2.5$\pm$1.5, respectively. Each hydrolysates and spots-C1, C2, C3-were tested the several functionalities such as antimicrobial activity, anticavity activity and anticoagulant activity. The antimicrobial and anticavity activity of carrageenan hydrolysates and oligosaccharide fractions were very low, but the anticoagulant activity was identified in all samples.

• BMB Reports
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• v.32 no.6
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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.

• BMB Reports
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• v.30 no.1
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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.

• YAKHAK HOEJI
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• v.36 no.4
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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.

• Journal of Microbiology and Biotechnology
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• v.28 no.2
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• pp.275-283
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• 2018

Ischemic stroke can result from blockage of blood vessels, forming fibrin clots in the body and causing irreparable brain damage. Remedial thrombolytic agents or anticoagulants have been studied; however, because the FDA-approved tissue plasminogen activator has low efficacy and side effects, it is necessary to develop safer and more effective treatment candidates. This study aimed at assessing the fibrinolytic and anticoagulation features of a novel serine protease extracted and purified from Diopatra sugokai, a polychaeta that inhabits tidal flats. The purified serine protease was obtained through ammonium sulfate precipitation, affinity chromatography, and ion-exchange chromatography. Its molecular size was identified via SDS-PAGE. To characterize its enzymatic activities, the protease activity at various pH and temperatures, and in the presence of various inhibitors, was measured via azocasein assay. Its fibrinolytic activity and anticoagulant effect were assessed by fibrin zymography, fibrin plate assay, and fibrinogenolytic activity assays. The novel 38 kDa serine protease had strong indirect thrombolytic activity rather than direct activity over broad pH (4-10) and temperature ($37^{\circ}C-70^{\circ}C$) ranges. In addition, the novel serine protease exhibited anticoagulant activity by degrading the ${\alpha}$-, ${\beta}$-, and ${\gamma}$-chains of fibrinogen. In addition, it did not produce cytotoxicity in endothelial cells. Therefore, this newly isolated serine protease is worthy of further investigation as a novel alkaline serine protease for thrombolytic therapy against brain ischemia.

• ALGAE
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• v.22 no.3
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• pp.247-252
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• 2007

Crude fucoidan was extracted from the sporophyll of Korean Undaria pinnatifida collected at a coastal area ofWando, Korea, mainly by dilute acid extraction, ethanol precipitation, CaCU Precipitation, with an yield of approxi-mately 3.9% in mass. It was further purified by DEAE-cellulose column chromatography and its chemical composi-don and in vitro anticoagulant activity was determined. The average molecular mass of the purified fucoidan wasestimated about 2.1 x 103 kDa by size-fractionation HPLC and it consisted of neutral sugar (52.34% in mass), uronicacid (26.2%), and sulfate esters (7.4%). From the HPAEC-PAD analysis, the monosaccharide composition of thepurified fucoidan was shown to be fucose, galactose, xylose, and mannose, with a molar ratio of 1, 0.2, 0.02, 0.15,respectively, demonstrating that major monosacd-iande was fucose (72.3% in mol percentage) and other sugars,xylose (1.5%), galactose (14.6%), and mannose (10.9%) were present as minor component. The results suggested thatthis fucoidan is a sulfated, U-type fucoidan. The activated partial thrombloplastin time (APTT) assay of the purifiedfucoidan showed that the purified fucoidan elicited anticoagulant activity in a dose-dependent manner. Five jUg ofsporophyll fucoidan delayed the blood clotting time up to 5 times than untreated control and also up to 1.5 timesthan the same amount of the commercial fucoidan, respectively. Although it is preliminary, these results suggestthat the fucoidan of Korean Undaria vinnatifida sporophyll would be promising candidates for the development ofan anticoaeulant.

• Archives of Pharmacal Research
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• v.20 no.1
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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.

• Journal of Applied Biological Chemistry
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• v.63 no.4
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• pp.439-442
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• 2020

In vitro antioxidant and anticoagulant activities of 50% ethanolic extract from Ganghwa medicinal mugwort (Artemisia princeps Pampanini) were investigated. The polyphenol and flavonoid contents of the mugwort extract were 106.9±3.3 and 34.1±0.4 mg/g-extract, respectively. The mugwort extract possessed a potent scavenging activity against radicals and nitrite, reducing power, and lipid peroxidation inhibition activity in a concentration-dependent manner. Also the mugwort extract delayed the plasma coagulation time through the inhibition of common coagulation pathway in a dose-dependent manner.