跳到主要內容

臺灣博碩士論文加值系統

(3.236.84.188) 您好!臺灣時間:2021/08/01 19:53
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:陳錦紋
研究生(外文):Chin-Wen Chen
論文名稱:納豆激酶之體內活性分析之研究
論文名稱(外文):In vivo Characterization of Nattokinase
指導教授:蕭世裕
指導教授(外文):Shyh-Yu Shaw
學位類別:碩士
校院名稱:國立成功大學
系所名稱:化學系碩博士班
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:70
中文關鍵詞:納豆激酶口服溶血栓活性抗凝血作用
外文關鍵詞:NattokinaseOral fibrinolytic activityAnti-thrombosis
相關次數:
  • 被引用被引用:0
  • 點閱點閱:689
  • 評分評分:
  • 下載下載:269
  • 收藏至我的研究室書目清單書目收藏:1
納豆激酶(Nattokinase)來自於納豆菌(Bacillus subtilis natto)的泌外蛋白質中,是很好的溶血栓酵素,能預防缺血性心臟病與中風。近年來保健食品中常添加納豆菌或納豆萃取液,可見納豆激酶的吸引性,然而沒有人知道納豆激酶是如何可以不受消化道系統的影響,而最終能夠到血液中溶解血栓。本實驗是從納豆醱酵初萃液中純化出納豆激酶,了解其相關活性能力的探討,並以納豆醱酵初萃液、納豆膠囊來進行大鼠動物的口服實驗。實驗結果顯示,本實驗能夠用簡易純化方法得到納豆激酶,也了解此酵素的溶血纖維蛋白活性,於動物實驗方面,以口服方式餵食七週大的大鼠,每日兩次(每日劑量3.6 kFU/kg大鼠),五天之後測試大鼠血液中納豆激酶活性,結果兩隻大鼠可以測得納豆激酶活性,但八隻大鼠無法測得其活性。同樣七週大鼠以高劑量(28 kFU/kg大鼠)長期餵食測試,四隻有兩隻觀察到NK存在血液活性,但出現的天數並不一致,故只能知道NK確實有通過小腸存有活性。所以接下來實驗將測試以一蛋白質藥物包覆的方式來探討NK是如何通過小腸進入到血液循環中。
Nattokinase is a secreted protein from the Bacillus subtilis natto, and is a strong fibrinolytic enzyme. The Bacillus subtilis natto is a very popular health food in Taiwan. It is believed that Bacillus subtilis natto can reduce the risk for heart attacks or strokes. Even nattokinase is a strong fibrinolytic enzyme but no one has proved it can pass through the gastrointestinal tract and go into circulation system. In this study, we used both the extract from Bacillus subtilis natto and purified nattokinase to feed Spraque-Dawley rat, and examined the fibrinolytic activity of nattokinase in the blood. In conclusion, The Spraque-Dawley rats were fed with nattokinase (3.6 kFU/kg rat per day by oral) for five days, and the blood was drawn for test. The results showed that two out of ten rats was detcected nattokinase activity in the blood, while the other eights was not. Then, we performed a long-term feeding experiment at a doses of nattokinase (28 kFU/kg rat) to elucidate the possible pathway of nattokinase passing through the gastrointestinal tract. There are two out of four rats was detcected nattokinase activity in the blood, but nattokinase appear in the different day between the rats. The result confirm nattokinase could pass through the gastrointestinal tract, so we will elucidate the possible pathway using other way by drug delivery
摘要 I
Abstract II
目錄 III
表目錄 VII
圖目錄 VIII


壹.緒論 1
一.納豆與納豆激酶 1
1.納豆起源 1
2.納豆食品 2
3.納豆激酶Nattokinase 3
4.其它醱酵菌中酵素 4
二.心血管疾病 5
1.形成血栓栓塞之原因 5
1.1高血脂與缺血性心臟病,中風 5
1.2高血壓與腦栓塞,頸動脈硬化 7
1.3高血糖 8
2.治療藥物種類 8
2.1抗凝血劑 8
2.2血栓溶解劑 10
三.文獻探討 12
1.溶血栓 12
2.胞漿素原的活化抑制酶使之去活性 12
3.降血壓,降低了血液黏稠度 13
4.降解類澱粉蛋白纖維,抗氧化壓力 14
四.研究動機 15
貳.材料與方法 16
一.材料 16
二.儀器 17
三.實驗方法 18
1.純化納豆激酶及水解纖維蛋白活性測試 18
1.1.1鹽析(Salting out) 18
1.1.2快速蛋白質純化液相層析儀(FPLC) 18
1.2一維電泳 19
1.3血纖維蛋白塊測試-Fibrin clot test 19
1.4血纖維蛋白平板測試-Fibrin plate test 20
2.納豆激酶測試 21
2.1測試血漿中的蛋白質與納豆激酶關係 21
2.2蛋白酵素電泳法-Zymographic Gel 22
2.3抗凝結測試-Anticoagulation test 23
2.4西方墨點法-Western blot 24
3.動物口服實驗 25
3.1材料準備 25
3.2動物實驗設計 26
3.3以SDS-PAGE 和 Western blot血液分析 28
參.結果與討論 29
1.純化納豆激酶及水解纖維蛋白活性測試 29
2.納豆激酶測試 30
2.1 SDS-PAGE 30
2.2抑制與活化蛋白質鑑定-Zymographic Gel 32
2.3西方墨點法-Western blot 32
2.4抗凝結測試-Anticoagulation test 32
3.動物口服實驗 33
肆.結論 36
伍.參考文獻 39
1.蘇遠志, 專題報導-納豆菌代謝產物的開發與應用, 生物產業,vol 11, No 2, p117, 2003.
2.陳信琮, 納豆激酶之純化與活性分析之研究, 國立成功大學化學研究所碩士論文, 成功大學, 2008
3.須洋見行, 納豆菌+青蔬汁遠離心肌梗塞、腦中風、癡呆、糖尿病!!, 鼎樂科技藝術, 臺北市, 2004
4.砂山聰, 圖解循環系統的疾病與機制, 世茂出版, 臺北縣, 2005
5.楊榮森等15位臺大醫師與營養師合著, 慢性病的飲食處方, 原水, 臺北市,2005
6.陳明豐, 心臟血管疾病知多少, 健康文化, 台北市, 2007
7.William F. Ganong, Review of Medical Physiology醫學生理學, 合記圖書, 台北市, 2008
8.Stuart Ira Fox, Human physiology 人體生理學, 新文京, 臺北市, 2006
9.楊雅涵, 製備磁性奈米粒子作為藥物載體, 國立成功大學生物科技碩士論文, 成功大學, 2007
10.Mine Y, Wong AHK, and Jiang B, Fibrinolytic enzymes in Asian traditional fermented foods. Food Research International, 2005. 38(3): p. 243-250
11.Sumi H, Hamada H, Tsushima H, Mihara H, Muraki H, A novel fibrinolytic enzyme (Nattokinase) in the vegetable cheese natto: a typical and popular soybean food in the Japanese diet. Experientia, 1987. 43(10): p. 1110-1111.
12.Fujita M, Nomura K, Hong K, Ito Y, Asada A, Nishimuro S, Purification and characterization of a strong fibrinolytic enzyme (Nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan.. Biochemical and Biophysical Research Communications, 1993. 197(3): p. 1340-1347.
13.Zheng ZL, Zuo ZY, Liu ZG, Tsai KC, Liu AF, Zou GL, Construction of a 3D model of nattokinase, a novel fibrinolytic enzyme from Bacillus natto - A novel nucleophilic catalytic mechanism for nattokinase. Journal of Molecular Graphics & Modelling, 2005. 23(4): p. 373-380.
14.Zheng ZL, Ye MQ, Zuo ZY, Liu ZG, Tai KC, Zou GL, Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation, Biochemical Journal, 2006. 395: p. 509-515.
15.Wu S, Feng C, Zhong J, Huan L., Roles of S3 site residues of nattokinase on its activity and substrate specificity. Journal of Biochemistry, 2007. 142(3): p. 357-364.
16.Kim W, Choi K, Kim Y, Park H, Choi J, Lee Y, Oh H, Kwon I, Lee S, Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp strain CK 11-4 screened from Chungkook-Jang. Applied and Environmental Microbiology, 1996. 62(7): p. 2482-2488.
17.Kim DC, Chae HJ, and M.J. In, Existence of stable fibrin-clotting inhibitor in salt-fermented anchovy sauce. Journal of Food Composition and Analysis, 2004. 17(1): p. 113-118.
18.Kim SB, Lee DW, Cheigh CI, Choe EA, Lee SJ, Hong YH, Choi HJ, Pyun YR, Purification and characterization of a fibrinolytic subtilisin-like protease of Bacillus subtilis TP-6 from an Indonesian fermented soybean, Tempeh. Journal of Industrial Microbiology & Biotechnology, 2006. 33(6): p. 436-444.
19.Chang CT, Fan MH, Kuo FC, Sung HY, Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1. Journal of Agricultural and Food Chemistry, 2000. 48(8): p. 3210-3216.
20.Ko JH, Yan JP, Zhu L, Qi YP, Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02. Comparative Biochemistry and Physiology C-Toxicology & Pharmacology, 2004. 137(1): p. 65-74.
21.Wong AHK. and Mine Y, Novel fibrinolytic enzyme in fermented shrimp paste, a traditional Asian fermented seasoning. Journal of Agricultural and Food Chemistry, 2004. 52(4): p. 980-986.
22.Wang CT, Ji BP, Li B, Ji H, Enzymatic properties and identification of a fibrinolytic serine protease purified from Bacillus subtilis DC33. World Journal of Microbiology & Biotechnology, 2006. 22(12): p. 1365-1371.
23.Sherry, S., Recombinant tissue plasminogen-activator (rt-PA): is it the thrombolytic agent of choice for an evolving acute myocardial-infraction. American Journal of Cardiology, 1987. 59(9): p. 984-989.
24.Sumi H, Hamada H, Nakanishi K, Hiratani H, Enhancement of the fibrinolytic-activity in plasma by oral-administration of Nattokinase, Acta Haematologica, 1990. 84(3): p. 139-143.
25.Sumi H, Yanagisawa Y, Yatagai C, Saito J, Natto bacillus as an oral fibrinolytic agent: Nattokinase activity and the ingestion effect of Bacillus subtilis natto. Food Science and Technology Research, 2004. 10(1): p. 17-20.
26.Fujita M, Hong K, Ito Y, Fujii R, Kariya K, Nishimuro S, Thrombolytic effect of Nattokinase on a chemically-induced thrombosis model in rat., Biological & Pharmaceutical Bulletin, 1995. 18(10): p. 1387-1391
27.Fujita M, Hong K, Ito Y, Misawa S, Takeuchi N, Kariya K, Nishimuro S, Transport of Nattokinase across the rat intestinal-tract, Biological & Pharmaceutical Bulletin, 1995. 18(9): p. 1194-1196.
28.Yamashita T, Oda E, Giddings JC, Yamamoto J, The effect of dietary bacillus natto productive protein on in vivo endogenous thrombolysis. Pathophysiology of Haemostasis and Thrombosis, 2003. 33(3): p. 138-143.
29.Suzuki Y, Kondo K, Ichise H, Tsukamoto Y, Urano T, Umemura K, Dietary supplementation with fermented soybeans suppresses intimal thickening. Nutrition, 2003. 19(3): p. 261-264.
30.Suzuki Y, Kondo K, Matsumoto Y, Zhao BQ, Otsuguro K, Maeda T, Tsukamoto Y, Urano T, Umemura K, Dietary supplementation of fermented soybean, natto, suppresses intimal thickening and modulates the lysis of mural thrombi after endothelial injury in rat femoral artery. Life Sciences, 2003. 73(10): p. 1289-1298.
31.Omura K, Hitosugi M, Kaketani K, Zhu X, Maeda H, Tokudome S, Fibrinolytic and anti-thrombotic effect of NKCP, the protein layer from Bacillus subtilis (natto). Biofactors, 2004. 22(1-4): p. 185-187.
32.Urano T, Sumiyoshi K, Nakamura M, Mori T, Takada Y, Takada A, Fluctuation of tPA and PAI-1 antigen levels in plasma-difference of their fluctuation paterns between male and female, Thrombosis Research, 1990. 60(2): p. 133-139.
33.Urano T, Ihara H, Umemura K, Suzuki Y, Oike M, Akita S, Tsukamoto Y, Suzuki I, Takada A, The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis cleaves and inactivates plasminogen activator inhibitor type 1. Journal of Biological Chemistry, 2001. 276(27): p. 24690-24696.
34.Kim JY, Gum SN, Paik JK, Lim HH, Kim KC, Ogasawara K, Inoue K, Park S, Jang Y, Lee JH, Effects of Nattokinase on Blood Pressure: A Randomized, Controlled Trial. Hypertension Research, 2008. 31(8): p. 1583-1588.
35.Pais E, Alexy T, Holsworth RE Jr, Meiselman HJ, Effects of nattokinase, a pro-fibrinolytic enzyme, on red blood cell aggregation and whole blood viscosity. Clinical Hemorheology and Microcirculation, 2006. 35(1-2): p. 139-142.
36.Hsu RL, Lee KT, Wang JH, Lee LY, Chen RP, Amyloid-Degrading Ability of Nattokinase from Bacillus subtilis Natto. Journal of Agricultural and Food Chemistry, 2009. 57(2): p. 503-508.
37.Ko J, Yan J, Zhu L, Qi Y, Subtilisin QK, a fibrinolytic enzyme, inhibits the exogenous nitrite and hydrogen peroxide induced protein nitration, in vitro and in vivo. Journal of Biochemistry and Molecular Biology, 2005. 38(5): p. 577-583
38.Hames BD, Rickwood D, Gel Electrophoresis of Proteins: A Practical Approach, Oxford university press, New York, 1990
39.NKA, Japan NattoKinase Association, http://j-nattokinase.org/index.html
40.Kim SH and Choi NS, Electrophoretic analysis of protease inhibitors in fibrin zymography. Analytical Biochemistry, 1999. 270(1): p. 179-181.
41.Rye DB, Saper CB, Wainer BH, Stabilization of the tetramethylbenzidine (TMB) reaction product application for retrograde and anterograde tracing, and combination with immunohistochemistry, Journal of Histochemistry & Cytochemistry, 1984. 32(11): p. 1145-1153.
42.Mr. Bright 柏氏健康生活網
43.康達生命科學有限公司 Contek Life Science Co., Ltd.
連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top