纳豆激酶抗凝血作用的研究
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摘要
纳豆激酶是一种新型抗血栓药物，具有安全性好，作用迅速且持续时间长，口服易吸收，可由纳豆菌发酵生产而造价低廉等特点。纳豆激酶的溶血栓作用目前已有报道，但其抗凝血作用的研究还未见有报导，本文就其抗凝血作用进行了研究和探讨，以便发现其新的作用，以期为纳豆激酶的合理应用，提供实验依据。本实验采用纤维蛋白平板法测定了纳豆激酶的活性，采用试管法测定了家兔全血凝固时间、血浆复钙时间、凝血酶时间、大鼠凝血活酶时间、凝血酶引起的纤维蛋白凝固时间；采用Quick氏法测定了大鼠凝血酶原时间；分别采用静脉血栓结扎法和Chandler法观察了纳豆激酶对大鼠静脉血栓和体外血栓形成的影响；采用ELISA法测定了家兔血浆中TXB_2的含量；用荧光分光光度法测定了大鼠血浆中NO的含量。结果表明，固体粉末NK的活性为565510U／g，是UK的3.134倍；液体NK的活性为15233.86U／ml；纳豆激酶能显著延长家兔全血凝固时间、血浆复钙时间、凝血酶时间、大鼠凝血活酶时间、凝血酶引起的纤维蛋白凝固时间、大鼠凝血酶原时间(P＜0.01)；能显著抑制大鼠静脉血栓和动脉血栓的形成(P＜0.01)；能显著降低家兔血浆中TXB_2的含量(P＜0.01)和升高大鼠血浆中NO的含量(P＜0.01)，从而说明纳豆激酶具有抗凝血作用而抑制大鼠体内外血栓形成。
Nattokinase(NK) is a new anti-thrombus drug produced by Bacillus natto, Which has good safety and absorption. Thrombolytic action of NK has been already studied, but its anticoagulative action has not been researched. In order to find its new mechanism, Anticoagulative action of NK was investigated in the paper. Activity of NK was tested by fibrin plate method. Coagulation time(CT), recalcification time(RT), partial thrombopla-stin time(PTT), fibrin coagulation time(FCT) and Thrombin time(TT) was tested using test tube method. Prothrombin time(PT) was determined by Quick method. Effects of venous thrombosis in vivo and arterial thrombosis in vitro was observed after i.v NK,using venous thrombus ligating and Chandler method respectively in rat.Content of thrombo-xaneB2(TXB2) and nitric oxide(NO) was tested by ELISA and fluoro- spectrophotometry separately. The results showed that: activty of NK pound and NK liquid was 565510U/g and 15233.86U/ml respectively. NK could significantly lengthen Coagulation time, r
ecalcification tune, partial thromboplastin time, fibrin coagulation time, thrombin time and prothrombin time and inhibit venous and arteryial thrombosis. NK also could obviously reduce content of TXB2 and increase content of NO. The results indicated that NK had notable anticoagulation in rat both in vivo and in vitro.
Nattokinase(NK) is a new anti-thrombus drug produced by Bacillus natto, Which has good safety and absorption. Thrombolytic action of NK has been already studied, but its anticoagulative action has not been researched. In order to find its new mechanism, Anticoagulative action of NK was investigated in the paper. Activity of NK was tested by fibrin plate method. Coagulation time(CT), recalcification time(RT), partial thrombopla-stin time(PTT), fibrin coagulation time(FCT) and Thrombin time(TT) was tested using test tube method. Prothrombin time(PT) was determined by Quick method. Effects of venous thrombosis in vivo and arterial thrombosis in vitro was observed after i.v NK,using venous thrombus ligating and Chandler method respectively in rat.Content of thrombo-xaneB2(TXB2) and nitric oxide(NO) was tested by ELISA and fluoro- spectrophotometry separately. The results showed that: activty of NK pound and NK liquid was 565510U/g and 15233.86U/ml respectively. NK could significantly lengthen Coagulation time, r
ecalcification tune, partial thromboplastin time, fibrin coagulation time, thrombin time and prothrombin time and inhibit venous and arteryial thrombosis. NK also could obviously reduce content of TXB2 and increase content of NO. The results indicated that NK had notable anticoagulation in rat both in vivo and in vitro.
引文
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2 徐仲，赵晓东等．纳豆激酶的纯化及活力测定．生物技术，1997，7(4)：16-18
3 付利，杨志兴．纳豆激酶的研究与应用[J]．生物工程进展，1995，15(5)：46-49
4 董明盛，江晓，江汉湖等．溶栓纳豆菌的筛选鉴定及其发酵特性研究．南京农业大学学报，2001，24 (1)：95-98
5 须见洋行．纳豆纤溶系．化学生物，1991，29(2)：119-123
6 Fujita M. Purifiction and characterization of a strong fibrinolytic enzyme (Nattonkinase) in the vegetable cheese natton, a popular soybean fermented food in Japan. Biochem Biophy Res commun, 1993, 197 (3): 1340-1347
7 Fujita M. Characterization of Nattonkinase-degraded produces from human fibrinogen or cross-linked fibrin. Fibrinolysis, 1995, 9 (3): 157-164
8 徐仲，赵晓东等．纳豆菌纤溶活性分析．东北农业大学学报，1998，29(4)：380-383
9 Sumi H, Hamada H, Tsushima H, et al. Anovel fibrinolytic enzeme (Nattokinase) in th vegetable cheese natto, a typical and popular soybean food in the Japanes diet. Experientia, 1987, 43: 1110
10 刘北域，宋后燕．纳豆激酶的研究进展．药物生物技术，1998，5(4)：248-251
11 黄志立，罗立新等．纳豆激酶．生命的化学，2000，20(2)：82-83
12 康明官．中外著名发酵食品生产工艺手册．北京：化学工业出版社，1997，157-158
13 Ogawa Y, Yamaguchi F, Yuasa K. Efficient production of gama-polyglutamic acid by Bacillus subtillis in jar fermanter. Bioscience Biotechmology and Biochemisty, 1997, 61 (10): 1684-1687
14 Sawa S, Tsushima H. Isolation and purification of polyglutamic acid produced by Bacillus subtilis[J].Agric Chemisty Society of Japan, 1973, 47 (3): 159-165
15 Eiji Ichishima, Yukihiro Takada, kijiro Tijiro Taira. Specificities of extracellular and ribosomal serine proteinases from Bacillus Natto, a food microorganism. Biochimica Biophysica Acta, 1986, 869: 178-184
16 H. Sumi, H Hanmada, Anovel fibrinolytic enzyme(Nattokinase) in the Vegetable cheese Natto, atypical and popular soybean food in the Japanese diet. Experientia, 1987,43: 1110
17 江晓，董明盛．纳豆．纳豆激酶与人体健康．中国酿造，2001，4：1-3
18 须见洋行，健康食品[纳豆]．科技资料，1996，2(39)：85-91
19 谢秋玲，郭勇等．产纳豆激酶之菌株的分离与培养．中南林学院学报，1999，19(4)：50-52
20 周秀琴．超级健康食品纳豆．中国食品，1991，12：
21 Fukutake M, Takahashi M, Ishida K. Quantification of genistein and genistein in soybean and soybean products in Japan. Food and Chemical Toxicolygy, 1996,34(5): 457461
22 Miyagi Y, Shinjo S, Nishida P, Miyagi C. Trypsin inhibtitor activity in commercial soybean products in Japan. Journal of Nutrition Science and vitaminology, 1997, 43(5): 575-580
23 胡景柱，夏寿华等．纳豆冻干粉的安全性研究．安徽农业科学，2000，28(3)：380-381
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