:::

| | | | | | 第 | | 筆

詳目顯示區塊

第 14 筆
| (以作者名查詢海洋大學圖書館館藏系統)
| (以作者名查詢全國圖書書目資訊網)
| (以作者名查詢全國博碩士系統)
論文名稱: | 蜆抗癌有效成分之分離及化學結構鑑定
| Antitumor compound of freshwater clam
研究生: | 梁惠如 Hui-Ru Liang
指導教授: | 廖若川 Yeul Chuen Liu
| 孫寶年 Bonnie Sun Pan
學位類別: | 碩士
學校名稱: | 國立臺灣海洋大學
系所名稱: | 食品科學系
學號: | M92320023
畢業學年度: | 93
語文別: | 中文
關鍵字: | 蜆 freshwater clam
| 分離純化 isolation
| 細胞毒性 cytotoxicity
全文點閱次數: | 20

全文影像連結:

論文全文(pdf)

論文頁數: | 89
摘要: | 摘要
|
| 由最近的研究發現，水產貝類具有抗腫瘤等生理活性，如文蛤、牡蠣及鮑魚等具有抗肝癌、胃癌、抗病毒及提高免疫力的作用；另外蜆被認為有保肝作用，民間常以熬煮蜆服食作為肝病治療，目前已有研究證實蜆肉水萃取物具有抑制人類肝癌細胞增生及誘導發生凋亡的效果。雖然研究已證實貝類具有抗腫瘤等生理活性，但其抗癌物質卻鮮少被發現，因此本研究目地為：1. 蜆萃取物抗癌的有效成分之分離及純化、2. 有效物質的化學結構鑑定。
| 初步研究發現台灣蜆以MeOH 萃取後再以EtOAc/H2O 進行分層萃取之FC-Me-EA，可造成人肝癌細胞(Hep-J2)凋亡。因此研究將甲醇萃取後EtOAc 分層的FC-Me-EA，以管柱層析(Al2O3, SiO2)、薄層層析以及高效液相層析等，進行分離純化，所得的各層，以人類肝癌細胞(6KK、HepG2)以及血癌細胞(HL60)培養，依所得抗癌效果bioactive-guide，進行分離純化，得抑制 HepH2 生長效果佳(4μg/ml)之112-5 fraction 由於所分離之化合物其產率太低，再進行大量製備後，由 NMR、MS 等進行化學結構評估，目前分離純化所得之 LWR-200-7為易分解之物質，初步鑑定為具 sterol 骨架的化合物。
|
| Abstract
|
| Recently, the marine shellfish have shown to inhibit cancer proliferation and other bioactivities. Hard calm, oyster and the abalone have been reported that could suppress the growth of liver carcinoma and gastric carcinoma, as well as antiviral and immunomodulated activities.  In addition, local people believe that the soup made with freshwater clam have hepatoprotective activity. Although the freshwater calm aqueous extract has demonstrated to inhibit the proliferation of human liver carcinoma and induce the apoptosis of carcinoma and possess significant hepatoprotective activity, the study of bioactive compounds was not extensive. The goal of this study was to search the bioactive compound based on the anti-cancer activity-guided approach.
| In the preliminary study, the ethyl acetate fraction after partition with water of freshwater calm methanolic extract induced apoptosis of Hep-J2 cell line. FC-Me-EA was further purified with column chromatography including Al2O3, SiO2 column chromatography and reverse phase high performance liquid chromatography based on bioactivity-guided approach. Different cell-lines including 6KK, HepG2 (human liver hepatoma) and HL-60 (human premyelocytic leukemia) were employed to study the cytotoxicities of fresh-clam extract and used to guide to isolate active fraction. This study demonstrated that the lipophilic fraction of fresh-clam possessed anti-cancer activities. One of fraction 112-5 was demonstrated to have significant anti-hepatoma activity (4 μg/ml). After further purification, a pure compound 200-7 consisting sterol skeleton was obtained.
|
論文目次: | 目錄
| 中文摘要………………………………………………………………Ⅰ
| 英文摘要………………………………………………………………Ⅱ
| 壹、緒論……………………………………………………………….1
| 研究目的…………………………………………………………..2
| 貳、    文獻整理………………………………………………………..3
| ㄧ、貝類之藥用. …………………………………………………3
| 1. 文蛤藥用價值…………………………………………..4
| 2. 牡蠣藥用價值…………………………………………..5
| 3. 蜆的藥用價值…………………………………………..6
| 二、貝類具抗癌的有效物質…………………………………….7
| 1. 水產貝類之活性物質………………………………….7
| 2. Spisulosine的抗癌細胞生長能力………………………8
| 3. 作用機制……………………………………………….10
| 三、保肝作用：…………………………………………………12
| 1. 肝臟生理功能………………………………………….13
| 2. 四氯化碳引起肝損傷………………………………….14
| 3. 肝功能指數： ALT 及 AST…………………………14
| 4. 抗氧化測定 TBARs…………………………………15
| 5. Silymarin  肝炎之治療藥物…………………………15
| 四、Sterol 之細胞毒性………………………………………..16
| 参、實驗方法與材料……………………………………………….22
| ㄧ、材料………………………………………………..……..22
| （一）    實驗材料：………………………………………..22
| （二）    化學藥品…………………………………………..22
| （三）    儀器設備…………………………………………..23
| 二、實驗方法…………………………………………..…….25
| （一）蜆之萃取及分離………………………………….25
| （二）產率與純度之測定……………………………….26
| （三）有效成分之純化分離及分析……………………26
| （四）TLC 之呈色反應………………………………..30
| （五）化合物之化學結構鑑定…………………………32
| （六）活性評估：………………………………………33
| 1. 細胞培養…………………………………………34
| 2. 細胞存活率………………………………………34
| 3. 蜆萃取物之肝癌細胞活性評估…………………34
| 肆、結果與討論…………………………………………….35
| （一）蜆之萃取…………………………………………35
| （二）蜆萃取粗分離物抑制癌細胞生長之活性………35
| （三）有效成分之分離、分析及其抗癌活性…………36
| （四）蜆分離物之化學結構之評估……………………38
| （五）結論
| 伍、參考文獻………………………………………………69
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
| 圖目錄
| 表一、已知化合物 10、11、7 與研究分離所得 200-7 於13C-NMR 上位移訊號……………………………………………………………….42
| 圖一、實驗流程………………………………………………………..45
| 圖二、蜆萃取物分離後各層抑制人類血癌細胞 HL60 生長活性….46
| 圖三、蜆萃取物分離後各層抑制人類肝癌細胞 6KK 生長活性…..47
| 圖四、LWR-1-104-9 之 HPLC 圖譜…………………………….......48
| 圖五、93-3 管柱分離各層之 TLC 分析…………………………….49
| 圖六、 104-9 之分離…………………………………………………50
| 圖七、LWR-1-200-5 之 HPLC 圖譜………………………………..51
| 圖八、LWR-1-200-7 之 HPLC 圖譜………………………………..52
| 圖九、104-9 RP-HPLC 分離各層抑制人類肝癌細胞 HepG2 生長活      性………………………………………………………………53
| 圖十、200-7 分解後之 HPLC 分析………………………………..54
| 圖十一、LWR-1-212 管柱分離……………………………………..55
| 圖十二、LWR-1-212-3 分解後分析………………………………..56
| 圖十三、083-7分離各層抑制人類血癌細胞 HL60 生長活性…...57
| 圖十四、83-5~83-6 分離物抑制人類肝癌細胞 6KK 生長活性…60
| 圖十五、83-5~83-6 分離物抑制人類血癌細胞 HL60 生長活性..61
| 圖十六、LWR-1-96-3 之 HPLC 分析圖譜……………………….62
| 圖十七、096-3、-4分離物抑制肝癌細胞 HepG2 生長活性……63
| 圖十八、112-5  之 HPLC 分析圖譜…………………………….64
| 圖十九、112-5 之分離…………………………………………….65
| 圖二十、208-6之 HPLC 分析圖譜………………………………66
| 圖二十一、208-9之 HPLC 分析圖譜……………………………67
| 圖二十二、096-6之 HPLC 分析圖譜……………………………68
|
參考文獻: | 伍、文獻
| 丁雲源；何雲達；http://www.tfrin.gov.tw/service/handbook/ts-1.html水產養殖手冊，水產試驗所。 2004/08/30
| 王穎；馬安；張惠珍；趙振軍；傅方浩；周光炎；牡蠣提取物抗腫瘤作用的實驗研究。中國海洋藥物，1997，1，18-22。
| 尹淑敏；李風謙；牡蠣的藥用。中國藥學雜誌，1994，29，751-752。
| 行政院衛生署食品衛生處。http://www.doh.gov.tw/NewVersion/index.asp 健康食品安全及功效評估， 2004/08/30
| 李時珍；本草綱目。文化圖書公司，台北，1995，46，1407-1411。
| 林志生；陳琦裴；蜆萃物供飲對四氯化碳誘發大鼠亞急性肝炎之影響。台灣農業化學與食品科學，2003，41，159-166。
| 林志生；蜆萃取物對酒精性肝損傷之影響。中華民國營養學雜誌，2003，28，26-33。
| 李俊逸等編譯；圖解生理學（B. R. Mackenna, R.Callander 原著）。台北，1999。
| 許實波；海洋生物製藥。化學工業出版社，北京；2002。
| 佟麗；陳江華；陳育展；牡蠣提取物對脾虛模型小鼠免疫功能的影響及抗應激反應的實驗研究。中國實驗臨床免疫研究，1998，10，50-53，。
| 祝雯；林志堅；吳祖建；林奇英；謝聯輝；河蜆醣蛋白對人肝癌細胞凋亡的影響。中國公共衛生，2004，20，674-675。
| 孫謚；向葆卿；王繞軍；馬英杰；易仁元；扇貝內臟提取勿對藥物性肝損傷的保護作用。海洋水產研究， 1996，17，20-28。
| 陳長堅；膳食蜆粉或蜆精對正常及四氯化碳誘發之肝障害大白鼠脂質過氧化的影響。國立台灣海洋大學食品科學系碩士論文，基隆。2002。
| 張善玉；朴惠順；金英順；金在久；申英愛；夏枯草等3種中藥對四氯化碳性肝損傷的影響。時珍國醫國藥，2003，14，658-660。
| 張廣欽；禹志領；趙厚長；文蛤肉水解降血脂作用的實驗研究。中國海洋藥物，1997，2，1-24。
| 楊奕蕾，綠島海域脈指型軟珊瑚 Sinularia gibberosa 及 Isis hippuris 所含天然物之研究。國立中山大學海洋資訊所碩士論文，台灣，2003。
| 馮貢國；台灣養殖蜆的熱水萃取物和蜆肉酵素水解物的生物活性評估。國立台灣海洋大學食品科學系碩士論文，基隆。1999。
| 賴呈委；文蛤抗腫瘤成分誘導肝癌細胞凋亡之探討。國立台灣海洋大學食品科學系碩士論文，基隆。1999。
| 劉清；李瑞聲；鮑化學成分的研究發展。中國海洋藥物雜誌，1995，1，40-43。
| 魏永泉；藥膳調治肝炎。家庭醫學，1994，14，45。
| 竇昌貴；黃芳；黃夢生；李佩珍；肖文東；文蛤多醣免疫藥理作用的研究。中國海洋藥物，1999，2，15-19。
| 駱抗先；乙型肝炎基礎和臨床。人民衛生出版社，北京，1996。
| Ahn, E. H.; Schroeder, J. J. Sphingoid bases and ceramide induce apoptosis in HT-29 and HCT-116 human colon cancer cells. Exp. Biol. Med. (Maywood). 2002, 227, 345-53.
| Aiello, A.; Fattorusso, E.; Menna, M.; Carnoccio, R.; Iuvone, T. New cytotoxic steroids from the marine sponge Dysidea fragilis coming from the lagoon of Venice. Steroids. 1995, 60, 666-673.
| Alfons, L.; Apoptosis - an introduction. Bio. Essays 2003, 25, 888-896.
| Bird, R. P.; Draper, H. H. Comparative studies on difference methods of malonaldehyde determination. Meth. in enzymology. 1984, 105, 299-303.
| Blitterswijk, W.; vanderLuit, A.; Veldman, R.; Verheij, M.; Borst, J. Ceramide: second messenger or modulator of membrane structure and dynamics? Biochem J. 2003, 369, 199-211.
| Bok, J. W.; Leonard, L.; Chilton, J.; Hans, G.; Klingeman, G. H. Antitumor sterols from the mycelia of Cordyceps sinensis. Phytochem. 1999, 51, 891-898.
| Bonini, C.; Cooper, B. C. Minor and trace sterols in marine invertebrates; 41    structure and stereochemistry of naturally occurring 9,11-seco sterols. J. Org. Chem., 1983, 48, 2108-2111.
| Claude, S.; Bertrand, R.; Josiane, C.; Takashi, O.; Lester, P. Selective inhibition of NF-κB activation by the flavonoid hepatoprotector silymarin in HepG2. FEBS Letters 1998, 440, 8-12.
| Glannin, C.; Zampella, A.; Debitus, C.; Menou, J.; Roussakis, C.; Valeria, M. Isolation and structural elucidation of Crellastatins I-M: cytotoxic biis-steroid derivatives from the Vanuatu marine sponge Crella sp. Tetrahedron 1999, 55, 13749-13756.
| Connor, CE.; Burrows J.; Hearps AC.; Woods, GM.; Lowenthal, RM.; Ragg ,SJ. Cell cycle arrest of hematopoietic cell lines after treatment with ceramide is commonly associated with retinoblastoma activation. Cytometry. 2001, 43, 164-149.
| Cuadros, R.; Montejo de Garcini, E.; Wandosell, F.; Faircloth, G.; Fernández-Sousa, J.M. The marine compound spisulosine, an inhibitor of cell proliferation, promotes the disassembly of actin stress fibers.  Cancer Letters. 2000, 152, 23-29.
| Kazlauskas, R; Murphy, P. T. Spermidine derivatives and 9,11-seco steroids from a soft coral (Simalaria sp.) Aust. J. Chem., 1982, 35, 69-75.
| Kenneth, F.; Martin H.; Hugo, R.; Kent, B. Milk thistle (Silymarin marianum) for the therapy of liver disease. The Am. J. Gastroent. 1998, 93, 139-143.
| Kiso, Y.; Tohkin, M.; Hikino, H. Assay method for antihepatotoxic activity using carbon tetrachloride induced cytotoxicity in primary cultured hepatocytes. Plant Med. 1983, 49, 222-225.
| Koljia, R.; Lopp, A.; Pehk, T.; Varvas, K.; Muurisepp, A.; Jarving, I.; Samel, N. New cytotoxic sterols from the soft coral Gersemia fruticosa. Tetrahedron 1998, 54, 179-186.
| Kolesnick, R. The therapeutic potential of modulating the ceramide/sphingomyelin pathway. J. Clin. Invest. 2002, 110, 3-8.
| Kong, Z. L.; Chiang, L. C.; Pan, P. Programmed Cell Death of Cultured Human Hepatoma Induced by Extracts of Hard Clam, Meretrix lamarcki , Animal Cell Technology: Basic and Applied Aspects. 1997, 8, 415-419.
| Lawen, A.; Apoptosis-an introduction. Bioessays. 2003, 25, 88-96.
| Leah, J.; Richard, N.; Marco, C. Ceramide Channels Increase the Permeability of the Mitochondrial Outer Membrane to Small Proteins. J. Biol. Chem. 2002, 227, 26796-26803.
| Leah, J. S.; Richard N. K.; Marco, C.; Ceramide Channels Increase the Permeability of the Mitochondrial Outer Membrane to Small Proteins. J. Bio. Chem. 2002, 277, 26796–26803.
| LI, H. J.; Lin, Y. C.; VRIJMOED, L. L. P.; JONES, E. B. G. A New Cytotoxic Sterol Produced by an Endophytic Fungus from Castaniopsis fissa from the South China Sea. Chin. Chem. Lett., 2004, 15, 419-422.
| Mackay, D.J.; Hall, A.; Rho GTPase. J. Biol. Chem. 1998, 273, 20685-20688.
| Maraschiello, C.; Miranda, E.; Millan, E.; Floriano, P.; Vilageliu, J.; Phenylisothiocyanate and dansyl chloride precolumn derivatizations
| for the high-performance liquid chromatography analysis of the
| antitumoral agent ES-285 in dog plasma. J. Chromatog. B. 2003, 791, 1-11.
| Makoto, T.; Toshiyuki, T.; Ryusuke, N.; Tatsuo, W.; Kenzo, S. Molecular    process in acute liver injury and regeneration induced by carbon tetrachloride. Life Sci.  2004 , 75, 1539–1549
| Merck, E. Dyeing reagents for thin layer and paper chromatography. 1976, Darmstadt, Germany.
| Monique, W. J.;, Bastiaan, N.; Elena, M.; Pablo, F.; Simon, M.; Ignacio, M.; Jos, H. B.; Development and validation of a liquid chromatography-ultraviolet absorbance detection assay using derivatisation for the novel marine anticancer agent ES-285•HCl [(2S,3R)-2-amino-3-octadecanol hydrochloride] and its pharmaceutical dosage form. J. Chromatogr. A. 2003, 1020, 251-258.
| Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods. 1983, 65: 55-63.
| Ohkawa, H.; Ohishi, N.; Yagi, K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 1979, 95, 351-358.
| Pedram, G.; Sabine, D. K.; Olivier, S.; Ursula, S.; Martin, P.; Sonia, R.; Christoph, R. Ceramide Induces Cytochrome c Releasefrom Isolated Mitochondria. J. Biol. Chem. 1999, 274, 6080–6084.
| Peter Guengerich, F. Common and Uncommon Cytochrome P450 Reactions Related to Metabolism and Chemical Toxicity. Chem. Res. Toxicol. 2001, 14, 611-650.
| Prescott, B.; Li, C.; Caldes, G.; Martino, C. Chemical studies of paolin Ⅱ, an antiviral substance from oyster. P.S.E.B.M. 1966, 123, 460-464.
| Raymond, F.; James, M.; Kuldeep, P. Relationship of oxygen and glutathione in protection against carbon tetrachloride-induced hepatic microsomal lipid peroxidation and covalent binding in the rat. J. Clin. Invest. 1984, 74, 1996-2001.
| Samsel, L.; Zaidel, G.; Drumgoole, H.; Jelovac, D.; Drachenberg, C.; Rhee, J.; Brodie, A.; Bielawska, A.; Smyth, M. The ceramide analog, B13, induces apoptosis in prostate cancer cell lines and inhibits tumor growth in prostate cancer xenografts. Prostate. 2004, 58, 382-393.
| Santafe, G.; Paz, V.; Rodrı´guez, J.; Jime´nez, C. Novel Cytotoxic Oxygenated C29 Sterols from the Colombian Marine Sponge Polymastia tenax. J. Nat. Prod. 2002, 65, 1161-1164
| Simon, G. and Alexander I. G. Isolation by Planar Chromatography. 1998, 234-237. Nature Products Isolation. Cannell, R. J. P., Ed. Humana Press Inc., New Jersey.
| Tatyana, I.; Kou-Yi, T.; Charles, H. Direct Inhibition of Mitochondrial Respiratory Chain Complex III by Cell-permeable Ceramide. J. Biol. Chem. 1997, 272, 24154–24158.
| Tepper, A.; Ruurs, P.; Wiedmer, T.; Sims, P.; Borst, J.; Blitterswijk, W. Sphingomyelin hydrolysis to ceramide during the execution phase of apoptosis results from phospholipid scrambling and alters cell-surface morphology. J Cell Biol. 2001, 150, 155-64.
| Vesper, H.; Schmelz, E.; Nikolova-Karakashian, M.; Dillehay, D.; Lynch, D.; Merrill, A. Sphingolipids in food and the emerging importance of sphingolipids to nutrition. J Nutr. 1999, 129, 1239-50.
| Wang, G. S.; Li, F. Y.; Zeng, L. M.; Ma, L. B.; Tu, G. Z. Structure determination of Nephalsterol A and B with 19-hydroxy Group from Soft Coral Nephthea. Chem. J. Chin. Univ. 1992, 13, 623–627.
| Watanabeb, K. Molecular process in acute liver injury and regeneration induced by carbon tetrachloride. Life sciences 2004, 75, 1539–1549.
| Nobuhiro, F.; Drug from the sea. Karger, Tokyo, 2000.
| PharmaMar:http://www.biospace.com/news_story.cfm?StoryID=13031220&full=1 ES-285, 2004/08/30 Reproductive & Cardiovascular Disease research group, Department of Biochemistry and Immunology at St. George's Hospital Medical School, London.http://www.sghms.ac.uk/depts/immunology/~dash/apoptosis/intro.html, 2004/08/30
|

|

預覽及輸出檢索結果

記錄

欄位

| 本頁 1 筆
| 記錄 14 至 26
| 選擇之記錄

| 標題
| 全部欄位
| selected fields new window
| APA Style
| Chicago Style
| MLA Style
| CNS-13611 Style
| EndNote Style

e-mail 地址 | : |
e-mail 標題 | : |

| | |

系統規劃：海洋大學圖書館
系統製作：飛資得資訊有限公司

2009 年 11 月 22 日 23:37