臺灣博碩士論文加值系統

台灣蜆 (Corbicula fluminea) 在民間被視為有治肝病的療效，具有很高的經濟價值，為台灣最重要的淡水養殖貝類，文獻指出台灣蜆富有保健功能，具有保肝、抗氧化、抗癌及降血壓等生理活性，但是其活性成分物質的研究並不廣泛，因此本研究目的為分離純化鑑定出台灣蜆中具有細胞毒性及保肝生理活性之有效成分。
整個實驗主要分為兩大部分，第一部分為純化分離出具有細胞毒性之化合物，主要以MTT assay為評估手段，台灣蜆經由管柱層析、薄層層析及逆向高速液相層析分離純化出純物FE-FEe-2-p-1，FE-FEe-2-p-1具有顯著之細胞毒性，其抑制肝癌細胞Hep G2及Hep 3B細胞之IC50分別為7 ug/mL及6 ug/mL，經由NMR結構鑑定後，證明為cholestenone類似物，為3,5,9-trihydroxy-cholest-7-en-6-one，而此一化合物為新發現之固醇類化合物。
第二部分為純化分離出具有保肝生理活性之化合物，主要以S9為基質四氯化碳誘導的肝損傷模式追蹤篩選出具有保肝功效之成份，追蹤純化發現FE-FJd-2具有顯著之保肝效果，同時探討FE-FJd-2對於CYP2E1酵素活性之影響，結果顯示FE-FJd-2可能是透過抑制CYP2E1酵素活性而達到保肝功效；而由FE-FJd-2進一步分離純化出之純物FE-FJd-2-1發現具有細胞毒性，其抑制肝癌細胞Hep G2之IC50為10 ug/mL，經由NMR結構鑑定後，證明為3,7,9-trihydroxy-cholest- 5-ene，此一化合物為一新發現之固醇類化合物。

Corbicula fluminea is the highest cultivated freshwater clam in Taiwan. It is consumed as soup in the folk medicine of Taiwan and commonly behued to have hepatoprotective activitice. Recently, some reports have shown that the Corbicula fluminea possessed a broad range of biological activities including hepatoprotective, antioxidant, anticancer and antihypertension. However, the active constituents of Corbicula fluminea is not studied extensively. The main objective of this study was to isolate and identification of cytototic and hepatoprotective compounds from Corbicula fluminea.
The whole experiment is divided into two major parts mainly, first part have isolate and purification cytototic compounds from Corbicula fluminea. The anti-cancer activity of Corbicula fluminea was evaluated by MTT assays, the FE-FEe-2-p-1 and FE-FEe-2-p-2 of Corbicula fluminea were purified with column chromatography, thin layer chromatography and reverse phase high performance liquid chromatography. FE-FEe-2-p-1 has significantly inhibitory cell activity against cell viability of Hep G2 and Hep 3B cells, it IC50 were 7 ug/mL and 6 ug/mL, respectively. The pure compound FE-FEe-2-p-1 was characterized with NMR, it is a new sterol, 3,5,9-trihydroxy-cholest-7- en-6-one .
Second part have isolate and purification hepatoprotective compounds from Corbicula fluminea. The hepatoprotective activity of Corbicula fluminea was evaluated by TBARS induced by CCl4, the FE-FJd-2 was purified base on hepatoprotective bioactivity-oriented approach. The effects of FE-FJd-2 on CYP2E1 were also investigated. The results showed that protective effects of FE-FJd-2 against the CCl4-induced hepatotoxicity may be due to its ability to inhibition the expression and activity of CYP2E1. The pure compound FE-FJd-2-1 was isolated from FE-FJd-2. It has significantly inhibitory cell activity against cell viability of Hep G2, with IC50 10 ug/mL. The pure compound was characterized with NMR, it is a new sterol, 3,7,9-trihydroxy-cholest- 5-ene.
This studies have demonstrated that the lipophilic fraction of Corbicula fluminea possessed anti-cancer and inhibited CCl4-induced lipid peroxidation in rat liver S9 in vitro.

壹、 緒論..............................1

貳、 文獻整理..........................3
一、保肝中草藥..............................3
(一) 水飛薊…………………………………………3
1、水飛薊簡介………………………………………3
2、Silymarin化學成分介紹………………………3
3、Silymarin的相關研究應用………………………4
(二) 葉下珠..................................7
1、Phyllanthus niruri抗氧化及保肝功能評估...8
2、Phyllanthus niruri對抗人類B肝病毒有效成分...9
二、海洋生物之固醇類化合物........................................10
(一) 海綿...................................10
(二) 柳珊瑚.................................13
(三) 軟珊瑚..................................18
(四) 藻類....................................20
(五) 蜆......................................22
1、簡介....................................22
2、蜆的生理活性及其相關研究................23

参、實驗材料與方法..............................26
第一部份、蜆之細胞毒性成分純化鑑定及活性分析...26
一、 材料...................................26
二、 化學藥品…………………………………………26
三、 儀器設備…………………………………………28
四、 實驗方法…………………………………………28
五、 實驗步驟與流程…………………………………29
(一) 蜆之前處理..............................29
(二) 蜆之細胞毒性有效成分純化分離............29
(三) TLC之呈色反應......................33
(四) 活性評估..............................33
(五) 化學結構鑑定............................36
第二部分、蜆之保肝有效成分純化鑑定及活性分析....37
一、 材料.................................37
二、 化學藥品..........................37
三、 儀器設備..............................38
四、 實驗方法..............................39
(一) 實驗原理...............................39
五、 實驗步驟與流程.........................41
(一) 蜆之保肝有效成分純化分離...............41
(二) TLC之呈色反應..........................44
(三) 活性評估..............................44
(四) 化學結構鑑定...........................46

肆、實驗結果..................................47
第一部份、蜆之細胞毒性有效成分純化鑑定及活性分析....47
一、 蜆之分離純化fractions之生理活性分析........47
二、 蜆各層有效fractions之HPLC鑑定分析........49
三、 NMR結構鑑定.............................50
第二部份、蜆之保肝有效成分純化鑑定及活性分析.....53
一、 蜆之分離純化fractions 之保肝生理活性分析..53
二、 蜆有效fractions之HPLC鑑定分析.............54
三、 NMR結構鑑定.............................54

伍、討論...........................................57
第一部份、蜆之細胞毒性有效成分純化鑑定及活性分析...57
一、 蜆細胞毒性fractions之生理活性分析.........57
二、 NMR結構鑑定........................59
第二部份、蜆之保肝有效成分純化鑑定及活性分析.........61
一、 蜆保肝fractions之活性分析...............61
1、 抗氧化..............................61
2、 CYP2E1酵素活性影響…………………………61
3、 細胞毒性............................62
二、 NMR結構鑑定........................64

陸、結論.........................................66

柒、參考文獻.....................................67

行政院衛生署。健康食品護肝功能評估。2000年7月31日公告。
行政院衛生署。中華民國九十四年死因統計結果摘要。 http://www.doh.gov.tw/statistic/data/死因摘要/94年/94.htm 2006/8/15。
江孟燦；陳長堅；姚賢宗。2005。蜆精對四氯化碳誘發大白鼠急性肝臟損傷之影響。台灣農業化學與食品科學。43，402-409。
巫文隆。1979。台灣蜆形態測定學的研究。貝類學報。6，1-12。
吳書瑜。2006。探討文蛤之保肝功能研究。國立臺灣海洋大學食品科學系碩士學位論文，基隆。
林志生。2003。飲用蜆萃出物對酒精性肝臟損傷之影響。中華民國營養學會雜誌。28，26-33。
林國泰。2005。蜆抽出物之抗氧化活性與其季節及產地之變動。國立臺灣海洋大學食品科學系碩士學位論文，基隆。
邱思魁；蕭泉源；藍惠玲。1997。養殖台灣蜆化學成分之季節變化。食品科學。24，469-478。
邱建中；張隆仁。2003。保健植物-水飛薊 ( Silybum marianum ) 。台中區農業專訊。 43，20-21。
祝雯；林志鏗；吳祖建；林奇英；謝聯輝。2004a。河蜆糖蛋白對人肝癌細胞凋亡的影響。中國公共衛生。20，674-675。
祝雯；林志鏗；吳祖建；林奇英；謝聯輝。2004b。河蜆中活性蛋白CFp-a的分離純化及其活性。中國水產科學。11，349-353。
陳其潮。2002。河川湖泊中的保健食品。食品資訊。7，48-54。
陳琦斐；林志生。2003。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。41。159-166。
粱惠如。2005。蜆抗癌有效成分之分離及化學結構鑑定。國立臺灣海洋大學食品科學系碩士學位論文，基隆。
Ahmed, A. F., Dai, C-F., Kuo, Y-H. and Sheu, J-H. 2003. 1α,3β,5β- Trihydroxy-24-methylenecholestan-6-one: a novel steroid from a soft coral Sinularia gibberosa. Steroids. 68, 377-381.
Bultel-Poncé, V., Etahiri, S. and Guyot, M. 2002. New ketosteroids from the red alga Hypnea musciformis. Bioorganic and Medicinal Chemistry Letters. 12, 1715-1718.
Dhiman, R. K. and Chawla, Y. K. 2005. Herbal medicines for liver diseases. Digestive Diseases and Sciences. 50, 1807-1812.
Elbarbry, F., Wilby, K. and Alcorn, J. 2006. Validation of a HPLC method for the determination of p-nitrophenol hydroxylase activity in rat hepatic microsomes. Journal of Chromatography B. 834, 199-203.
Ferenci, P., Dragosics, B. and Dittruch, H. 1989. Randomized controlled trial of silymarin treatment in patients with cirrhosis of the liver. Joumal of Hepatology. 9, 105-113.
Fernández, J., Pérez-Álvarez, JA. and Fernández-López, JA. 1997. Thiobarbituric acid test for monitoring lipid oxidation in meat. Food Chemistry. 59, 345-353.
Flora, K., Hahn, M. and Rosen, H. 1998. Milk thistle ( Silybum marianum ) for the therapy of liver disease. The American Journal of Gastroenterology. 93, 139-143.
Fujita, M., Nakao, Y., Matsunaga, S., Seiki, M., Itoh, Y., Soest, R W. M. V., Heubes, M., Faulkner, D. J. And Fusetani, N. 2001. Isolation and structure elucidation of two phosphorylated sterol sulfates, MT1-MMP inhibitors from a marine sponge Cribrochalina sp.: revision of the structures of haplosamates A and B. Tetrahedron. 57, 3885-3890.
Garrido, L., Zubía, E., Ortega, M. J. and Salvá, J. 2000. Isolation and structure elucidatuion of new cytotoxic steroids from the gorgonian Leptogorgia sarmentosa. Steroids. 65, 85-88.
González, N., Barral. M. A., Rodríguez. J. and Jiménez. C. 2001. New cytotoxic steroids from the gorgonian Isis hippuris. Structure-activity studies. Tetrahedron. 57, 3487-3497.
Halliwell, B. and Gutteride, J. M. C. 1990. The measurement and mechanism of lipid peroxidation in biological system. Trends in Biochemical Sciences. 15, 129-135.
Harish, R. and Shivanandappa, T. 2006. Antioxidant activity and hepatoprotective potential of Phyllanthus niruri. Food Chemistry. 95, 180-185.
Huang, Y. T., Huang, Y. H., Hour, T. C., Pan, B. S., Liu, Y. C. and Pan, M. H. 2006. Apoptosis-inducing active components from Corbicula fluminea through activation of caspase-2 and production of reactive oxygen species in human leukemia HL-60 cells. Food and Chemical Toxicology. 44, 1261-1272.
Huang, R. L., Huang, Y. L., Ou, J. C., Chen, C. C., Hsu, F. L. and Chang, C. 2003. Screening of 25 compounds isolated from Phyllanthus species for anti-human hepatitis B virus in vitro. Phytotherapy Research. 17, 449-453.
Jaime, L., Mendiola, J., Herrero, M., Soler-Rivas, C., Santoyo, S., Señorans, F. J., Cifuentes, A. and Ibáñez, E. 2005. Separation and characterization of antioxidants from Spirulina platensis microalga combining pressurized liquid extraction, TLC, and HPLC-DAD. Journal of Separation Science. 28, 2111–2119.
Jeong, H. G., YOU, H. J., Park, S. J., Moon, A. R., Chung, Y. C., Kang, S. K. and Chun, H. K. 2002. Hepatoprotective effects of 18β-glycyrrhetinc acid on carbon tetrachloride-induced liver injury: inhibition of cytochrome P450 2E1 expression. Pharmacological Research. 46, 221-227.
Koljak, R., Lopp, A., Pehk, T., Varvas, K. and Müürisepp, A-M. 1998. New cytotoxic sterols from the soft coral Gersemia fruticosa. Tetrahedron. 54, 179-186.
Khanns, A.K., Rizvi, F. and Chander , R. 2002. Lipid lowering activity of Phyllanthus niruri in hyperlipemic rat. Journal of Ethnopharmacology.82, 19-22.
Khatoon, S., Rai, V., Rawat , A. and Mehrotra, S. 2005. Comparative pharmacognostic studies of three Phyllanthus species. Journal of Ethnopharmacology. 104, 79-86.
Kvasnička. F., Biba. B., Sevcik. R. and Voldrich. M. 2003. Analysis of the active components of silymarin. Journal of Chromatography A. 990, 239-245.
Letteron, P., Labbe, G., Degott, C., Berson, A., Fromenty, B., Delaforge, M., Larrey, D. and Pessayre, D. 1990. Mechanism for the protective effects of silymarin against carbon tetrachloride-induced lipid peroxidation and hepatotoxicity in mice. Biochemcial Pharmacology.. 39, 2027-2034.
Luper, S. 1998. A review of plants used in the treatment of liver disease: part I. Alternative Medicine Review. 3, 410-421.
Merck, E. 1976. Dying reagents for thin layer and paper chromatography.
Morazzoni, P. and Bombardelli, E. 1995. Silybum marianum ( Carduus marianus ) . Fitoterapia. LXVI, 3-42.
Miyamoto, T., Kodama, K., Aramaki, Y., Higuchi, R. and Soest. R W. M. V. 2001. Orostanal, a novel abeo-sterol inducing apoptosis in leukemia cell from a marine sponge, Stelletta hiwasaensis. Tetrahedron. 42, 6349-6351.
Morazzoni, P. and Bombardelli, E. 1995. Siliybum marianum (Carduus marianus). Fitoterapia. LXVI:3-42.
Pares, A., Planas, R., Torre, M. 1998. Effects of silymarin in alcoholic patients with cirrhosis of the liver: results of a controlled, double-blind, randomized and multicenter trial. Joumal of Hepatology. 28, 615-621.
Pepping, J. 1999. Milk thistle: Silybum marianum. American Journal of Health-System Pharmacy. 56, 1195-1197.
Plumb, J. A., Milroy, G. C. and Kaye, S. B. 1989. Effect of the pH dependence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay. Cancer Recerch. 49, 4435-4440.
Quaglia, M. G., Bossu. E., Donati. E., Mazzanti. G. and Brandt, A. 1999. Determination of silymarin in the extract form the dried Silybum marianum fruits by high performance liquid chromatography and capillary electrophoresis. Pharmaceutical and Biomedical Analysis. 19, 435-442.
Riccardis, F. D., Minale, L., Iorizzi, M., Debitus, C. And Lévi, C. 1993. Marine sterols. Side-chain-oxygenaed sterols, possibly of abiotic origin, from the new caledonian sponge Stelodoryx Chlorophylla. Journal of Natural Products. 56, 282-287.
Santafé, G., Paz, V., Rodríguez, J. And Jiménez, C. 2002. Novel cytotoxic oxygenated C29 sterols from the colombian marine sponge Polymastia tenax. Journal of Natural Products. 65, 1161-1164.
Salmi, H. A. and Sarna, S. 1981. Effect of silymarin on chemical, functional, and morphological alterations of the liver. Scand J Gastroenterol. 17, 517-521.
Schuppan, D., Dong, J., Brinkhaus, B. and Hahn, E. G. 1999. Herbal products for liver diseases： a therapeutic challenge for the new millennium. Hepatology. 30, 1099-1104.
Shamasundar, K. V., Singh, B. Thakur, R. S., Hussain, A., Kiso, Y. and Hikino, H. 1985. Antihepatoprotective principles of Phyllanthus niruri herbs. Journal of Ethnopharmacology. 14, 41–44.
Sheu, J-H., Chang, K-C. and Duh, C-Y. 2000. A Cytotoxic 5α,8β- Epidioxysterol from a Soft Coral Sinularia Species. Journal of Natural Products. 63, 149-151.
Sheu, J-H., Huang, S-Y. and Duh, C-Y. 1996. Cytotoxic oxygenated desmosterols of the red alga Galaxaura marginata. Journal of Natural Products. 59, 23-26.
Sheu, J-H., Liaw, C-C. and Duh, C-Y. 1995. Oxygenated clerosterols isolated from the marine alga Codium arabicum. Journal of Natural Products. 58, 1521-1526.
Simon, G. and Alexander, I. G. 1998. Isolation by Planar Chromatography. Nature Products Isolation. 234-237. Cannell, R. J. P., Ed. Humana Press Lnc., New Jersey.
Suja, S. R., Latha. P. G., Pushpangadan. P. and Rajasekharan. S. 2004. Evaluation of hepatoprotective effects of Helminthostachys zeylanica (L.) Hook against carbon tetrachloride-induced liver damage in Wistar rats. Journal of Ethnopharmacol. 92, 61–66.
Tanaka, J., Trianto, A., Musman, M., Issa, H. H., Ohtani, I. I., Ichiba, T., Higa, T., Yoshida, W. Y. and Scheuer, P. J. 2002. New polyoxygenated steroids exhibiting reversal of multidrug resistance from the gorgonian Isis hippuris. Tetrahedron. 58, 6259-6266.
Tsai, J. S., Lin, T. C., Chen, J. L. and Pan, B. S. 2006. The inhibitory effects of freshwater clam (Corbicula fluminea, Muller) muscle protein hydrolysates on angiotensin I converting enzyme. Process Biochemistry. 41, 2276-2281.
Valenzuela, A. and Garrido, A. 1994. Biochemical bases of the pharmacological action of the flavonoid silymarin and of its structural isomer silibinin. Biological Research. 27, 105-112.
Yaoita, Y., Amemiya, K., Ohnuma, H., Furumura, K., Masaki, A., Matsuki, T. and Kikuchi, M. 1998. Sterol constituents from five edible mushrooms. Chemical and pharmaceutical bulletin. 46, 944-950.