• Archives of Pharmacal Research
• /
• 제28권9호
• /
• pp.1037-1041
• /
• 2005

Geranium (Pelargonium inquinans Ait) leaves were extracted with $80\%$ MeOH, and partitioned into n-hexane, ethyl acetate, BuOH and $H_2O$ to isolate the anticoagulant principles. The EtOAc fraction was found to be the most active, and was further purified using silica and octadecylisilane column chromatography employing a bioassay-guided fractionation method. The active compound was isolated and identified as $1,2,3,4,6-pentagalloyl-\beta-D-glucopyranose$(PGG) (compound I). The isolated anticoagulant significantly prolonged the activated partial thrombin time (APTT) and thrombin time (TT) using normal human plasma. One microgram of $1,2,3,4,6-pentagalloyl-\beta-D-glucopyranose$ showed 0.063 heparin units in the APTT and 2.73 heparin units in the TT for anti-thrombosis. This is the first report of the isolation of PGG from geranium plants.

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

• Archives of Pharmacal Research
• /
• 제28권6호
• /
• pp.667-674
• /
• 2005

The leaves of Persimmon (Diospyros kaki L.) has long been used for tea in Korea since it was thought to be effective against hypertension. An anticoagulant fraction was purified through gel filtration G-100, hydrophobic, gel filtration G-150, and FPLC, Phenyl superpose column chromatographies. The purified fraction was homogenous and its Mr was estimated 10,000 Da by gel filtration and SDS-PAGE. The purified fraction was sensitive to treatment of subtilisin B, but not to heat and its activity was not changed after periodate oxidation, indicating that the activity was not due to carbohydrates. It delayed thrombin time (TT), activated partial thromboplastin time (APTT), and prothrombin time (PT) using human plasma. TT was more sensitive than APTT and PT, suggesting that the anticoagulant activity may be caused by a degradation or a defect of fibrin or thrombin. It did not cause the hydrolysis of fibrin after incubation. However, it inhibited thrombin-catalyzed fibrin formation with a competitive inhibition pattern. These results indicate that it may be an antithrombotic agent and that it is bound to fibrinogen binding sites of thrombin.

• Archives of Pharmacal Research
• /
• 제24권2호
• /
• pp.109-113
• /
• 2001

Curdlan is a natural $\beta$-1,3-glucan produced by Agrobacterium biovar 1. In this study, the anticoagulant activity of sulfoalkyl derivatives of curdlan was investigated by carrying out activated partial thromboplastin time (APTT) assay and compared with that of o-sulfonated curdlan. Approximately 100-fold higher concentration of o-sulfonated curdlan than heparin was required to obtain the same level of the clotting time. Anticoagulant activity of curdlan derivatives was dependent on the degree of sulfation in prolonging the clotting time. However, the chain length of the substituent did not play a role in prolonging the clotting time. The curdlan derivatives enhanced thrombin inhibition by mediating through antithrombin III. The inhibition of thrombin by o-sulfonated curdlan was found to be approximately 10-fold weaker than that by heparin.

• BMB Reports
• /
• 제29권3호
• /
• pp.225-229
• /
• 1996

Effects on thrombin by an amphipathic cation, dequalinium, which has been recognized as an anticarcinoma agent were investigated with small chromogenic substrates such as Na-benzoyl-DL-argininep-nitroanilide (BApNA), H-D-phenylalanyl-L-pipecoyl-L-arginine-p-nitroanilide (S-2238), and Na-p-tosyl-L-arginine methyl ester (TAME). Among them, only TAME hydrolysis due to an esterase activity of the enzyme was significantly activated to 81% at 20 ${\mu}M$ dequalinium in the absence of NaCl. This stimulation became even higher in the presence of 0.2 M NaCl to 3.5-fold at 60 ${\mu}M$ dequalinium. This specific activation of thrombin was well correlated with the results of in vitro coagulation tests measuring the activated partial thromboplastin time (APTT) and the prothrombin time (PT) It is pertinent. therefore, to suggest that the esterase activity should be examined in addition to the effects on 5-2238 hydrolysis when especially any regulators not directed to an active site of thrombin need to be studied. We also expect that dequalinium could be a useful tool for studying structure-function relationship of thrombin and blood coagulation.

• 한국식품영양과학회지
• /
• 제33권8호
• /
• pp.1262-1267
• /
• 2004

한약재의 재료로 사용되는 옥발에서 항응고물질(anticoagulant)을 추출하여 정제하였다. 정제된 항응고물질의 분자량은 135 kDa으로 측정되었고 옥발로부터 정제된 항응고물질의 회수율은 11%이고, 24배 농축되었다. 옥발의 항응고물질은 열에 안정하고 비특이적 단백질 가수분해 효소 처리 후에도 활성을 유지하였으나, periodate 산화 처리시 현저하게 활성이 소실되어 항응고물질의 성분은 당류인 것으로 추정된다. 옥발의 항응고물질을 thin layer chromatography(TLC)와 Bio-LC로 구성당을 분석한 결과 galactose, glucose, mannose, glucosamine, galactosamine, fucose로 구성되어있고, IR을 통하여 확인된 OH, N-H bond가 확인되어 항응고물질이 중성당과 아미노당으로 구성된 것을 뒷받침해주고 있다. 옥발의 항응고물질은 thrombin time(TT)에 가장 민감하고, TT에서 항응고 활성을 2배, 3배 지연시키는 농도는 60 nM와 88 nM이었다. Prothrombin time(PT)에도 민감하였지만, snake venom activity와 activated partial thromboplastin time(APTT)의 clotting activity에는 상대적으로 별 영향을 끼치지 않았다. 옥발의 항응고물질을 정제된 fibrinogen을 이용하여 thrombin과의 반응 시 fibrinogen의 농도가 증가함에 따라 응고 저해 정도가 감소하여 thrombin에 대해fibrinogen과 결합자리를 경쟁하고, 이 때 Ki 값은 23 nM이다.

• 한국식품영양과학회지
• /
• 제29권2호
• /
• pp.335-341
• /
• 2000

허브를 비롯한 식용되는 채소류로부터 항응고 활성을 검색하기 위하여 93종을 대상으로 냉수(Fr. I), 메탄올(Fr. ll), 열수(Fr. lll)추출물을 각각 조제하였다. 내인성 경로에 작용하는 항응고 활성은 수용성 획분인 Fr. I과 Fr.lll를 대상으로 activated partial thromboplastin time(APTT)으로 검색한 결과 양파, 마늘, 정향, 쑥, 울금 등이 높은 활성을 보였으며, 외인성 경로와 공통경로에 작용하는 항응고 활성은 저분자임을 감안하여 Fr. I과 Fr. II를 대상으로 각각 prothrombin time(PT) 과 thrombin time(TT)으로 검색한 결과 민트, 이탈리안 시즈닝, 로즈메리, 타라곤, 울금, 백리향, 와사비는 공통경로에 항응고 활성을 나타내였으나 외인성 경로에 단독으로 항응고 활성을 나타내는 시료는 없었다. 1, 2차 검색 결과 가장 높은 항응고 활성을 보인 정향(Eugenia caryophyllate, clove)을 대상으로 산지를 달리하여 항응고 활성을 검색한 결과 홀랜드산 정향의 활성이 가장 높았다.

• 한국응용약물학회:학술대회논문집
• /
• 한국응용약물학회 1998년도 Proceedings of UNESCO-internetwork Cooperative Regional Seminar and Workshop on Bioassay Guided Isolation of Bioactive Substances from Natural Products and Microbial Products
• /
• pp.177-177
• /
• 1998

In the previous report, tetrandrine (TET) and fangchinoline (FAN) showed antithrombotic and antiplatelet aggregation activities. The present study was undertaken to investigate the effects of tetrandrine and fangchinoline on human platelet aggregation, formation of thromboxane B$_2$ and coagulation of platelet poor plasma. TET and FAN inhibited platelet activating factor (PAF) induced human platelet aggregation, but didn't inhibit the specific binding of PAF to its receptor. Meanwhile, TET and FAN also inhibited PAF, thrombin and arachidonic acid induced thromboxane B$_2$ formation in human washed platelets. In addition, neither TET nor FAN showed any anticoagulation activities in the measurement of the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using human platelet poor plasma. These results suggest that antithrombotic effects of TET and FAN in mice may be mainly related to the antiplatelet aggregation activities, and the antiplatelet aggregation effects may be related to the intracellular messenger system such as TXA$_2$ formation etc., but not to the binding of PAF to PAF-receptor on the platelet membrane directly.

• 한국양식학회지
• /
• 제19권1호
• /
• pp.33-39
• /
• 2006

Anticoagulant activities of a fermented edible brown alga, Laminaria ochotensis was investigated. L. ochotensis was fermented with 15% sugar (w/v) at $25^{\circ}C$ for 10 weeks. Anticoagulant activity was measured from the supernatant of algal mixture at biweekly intervals up to $10^{th}$ week by activated partial thromboplastin (APTT), prothrombin time (PT) and thrombin time (TT) assay using citrated human plasma. Sample having high APTT activity $(6^{th}\;week)$ was filtered, ethanol precipitated and freeze-dried. The polysaccharide compound having anticoagulant activity was purified by DEAE ion exchange chromatography followed by Sepharose-4B gel filtration chromatography. Anticoagulant activity, polysaccharide concentration, and heparin like activity were determined for the collected fractions by APTT, $phenol-H_2SO_4$, and glycosaminoglycan assay, respectively. The anticoagulant activity assay showed that the activity was increased up to $6^{th}$ week, and decreased thereafter. The concentration of our purified compound was $31.0{\mu}g/ml$ and showed higher APTT activity than commercial heparin. At the same concentration of $31.0{\mu}g/ml$, the heparin showed 186.5 sec activity while our purified compound showed an activity of 386 sec. Single spot on agarose gel electrophoresis showed that the compound was purified and polyacrylamide gel electrophoresis (PAGE) results revealed that the molecular mass of the purified polysaccharide compound was between 60 and 500 kDa. Therapeutic interest of the algal polysaccharide as an anticoagulant has recently been in highlighted. This purified anticoagulant compound from fermented L. ochotensis can be used as a model for anticoagulant agent or could be developed as an anticoagulant agent. This study can be extended to identify the structure and chemical composition of the purified polysaccharide, and to establish a relationship between structure and the function of the identified anticoagulant compounds.

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