臺灣博碩士論文加值系統

摘要
大豆屬一條根 (Glycine tomentella Hayata) 具有抑制不飽和脂肪酸自氧化、清除自由基、螯合鐵離子之能力及延緩低密度脂蛋白氧化。於吳郭魚低溫緊迫試驗中，可降低吳郭魚血液流動之起始應力、黏稠度及血小板 5-, 12-, 15- 脂氧合?活性。在一條根 95% 乙醇萃取物 (GTE) 中，確定含有 daidzein、daidzin、malonyldaidzin、genistein 及 glycitin，但尚有較強於 異黃酮類之抗氧化功效成分未被鑑定。本研究為找出 GTE 抗氧化力最高之成分，以抑制 15-脂氧合?之活性能力為篩選依據，並檢測其對環氧合?之抑制功效。
GTE 經乙酸乙酯萃取、silica gel 管柱層析後可得 5 個區分，以第三區分 (GTE-EA-III) 抑制 15-脂氧合?之能力最高，於濃度 2 μg/mL 之抑制率約為 92.76%。GTE-EA-III 經 silica gel 管柱層析得 GTE-EA-III-A~J 共十區分物，其中 GTE-EA-III-C於濃度 2 μg/mL 對 15-LOX 抑制率約為 98.70%，高於 genistein 且與脂氧合?抑制劑 (nordihydroguaiaretic acid, NDGA) 於濃度為0.2 μg/mL 之抑制率相似。GTE-EA-III-C 經 silica gel 管柱層析得 GTE-EA-III-C-a~m，共十三區分，經檢測抑制 15-脂氧合?活性，得知以 GTE-EA-III-C-f & g 區分抗氧化力較強，而其中以 GTE-EA-III-C-g 區分回收量較 f 區分高，因此進一步以半製備型 RP-HPLC分離 GTE-EA-III-C-g ，共分 GTE-EA-III-C-g-1~4 四區分物，GTE-EA-III-C-g-3 抑制 15-脂氧合?活性之 IC50 為 0.3 μg/mL，劑量為 NDGA 的 7.9倍。
GTE-EA-III-C-g-3的紫外光光譜中於 245 nm 與 345 nm 有吸收波峰，在 306 nm 有小肩峰；再以 LC/MS 分析，發現陽離子質量為 464 Da，以核磁共振光譜分析，初步推測為 prenylate alkaloids。

目錄
摘要 I
Abstract III
目錄 IV
圖目錄 VIII
表目錄 X
壹、前言 1
貳、文獻回顧 4
一、豆科植物之異黃酮 (isoflavone) 成分 4
二、一條根生理活性 5
1. 一條根之來源 5
2. 一條根的藥理作用 5
3. 一條根之成份及藥理活性 6
4. 闊葉大豆之毒性試驗 7
三、黃酮類與異黃酮化合物之結構 8
四、黃酮類對心血管之影響 10
1. 預防紅血球溶血 11
2. 預防心血管疾病 13
五、異黃酮類之生理活性 14
1. 減緩更年期症候群 15
2. 預防骨質疏鬆症 16
3. 預防癌症 16
4. 預防心血管疾病 17
六、黃酮類之抗氧化機制 18
1. 清除活性氧自由基 18
2. 螯合金屬離子 19
3. 抑制脂質氧化 20
七、發炎及過敏的發生機制 20
八、Lipoxygenase (LOX) 生成物及其生理活性 22
1. 5-Lipoxygenase (5-LOX) 之氧化產物及發炎反應 23
2. 12-Lipoxygenase (12-LOX) 氧化作用及相關疾病(Ref) 23
3. 15-Lipoxygenase (15-LOX) 氧化作用及對血管的影響 24
九、Cyclooxygenase (COX) 區分及氧化產物 25
十、黃酮類抑制 LOX 及 COX 之作用 26
?、 實驗流程 27
一、 一條根 28
二、 方法 28
1. 一條根萃取物 (GTE) 之製備 28
2. 一條根萃取物之分離 29
2.1 乙酸乙酯極性區分 29
2.2 silica gel 管柱層析 29
2.3 薄層層析分析 30
2.4 分離物之 HPLC 分析 31
2.5 半製備型HPLC分析 32
3. 抗氧化性分析測定脂氧合?總活性測定 32
4. LC/MS/MS測定分析 33
5. 核磁共振光譜 33
6. 統計分析 33
伍、結果與討論 34
一、一條根抗氧化成分之分離 34
1. 一條根經 95% 乙醇萃取 34
2. GTE經乙酸乙酯極性劃分 35
3. Silica gel 60 管柱層析 37
3.1 GTE-EA 之區分產率及對 15-LOX 抑制活性 37
3.2 GTE-EA-III 之區分產率及對 15-LOX 抑制活性 40
3.3 GTE-EA-III-C 之區分產率及對 15-LOX 抑制活性 43
4. GTE-EA-III-C-g半製備型 HPLC 之區分及對 15-LOX 抑制活性 46
二、 一條根區分物之鑑定 49
1. 異黃酮之 HPLC 圖譜檢測 49
1.1 GTE 及 GTE-EA 之RP-HPLC 圖譜比較 49
1.2 GTE-EA-I~V 區分之 RP –HPLC 圖譜 51
1.3 GTE-EA-III-A~J 區分之 RP-HPLC 圖譜 53
1.4 genistein之 LC/MS 圖譜與定量 59
1.5 GTE-EA-III-C-a~m 區分之 RP-HPLC 圖譜 65
2. 類黃酮分類之鑑定 70
陸、結論 82
柒、參考文獻 83

柒、參考文獻
吳禮宇、吳岳文、施純青、柯裕仁、林文川。2001。台灣金線連和闊葉大豆（金門產一條根）之初步基因毒性試驗。中國醫藥學院雜誌。12:173-178。
林文川、柯裕仁、吳岳文。2000。闊葉大豆（金門產一條根）水粗萃物對大鼠28天餵食毒性作用。中國醫藥學院雜誌。11:2050215。
柯裕仁、謝文全、邱年永、陳忠川。1999。是售一條根類藥材之藥用植物考察。中國醫藥學院雜誌。8:33-46。
柯裕仁。2003。ㄧ條根類藥材之鑑別及藥理活性研究。中國醫藥學院中國藥學研究所博士論文。台中。
許桂森、林明忠及蕭哲志。2001。抗發炎藥物。簡明圖解藥理學。藝軒出版社。479-500。
郭瑩玉。2003。大豆屬與千觔拔屬一條根生理活性之比較及其酚類化合物之分離。國立臺灣海洋大學食品科學研究所碩士論文。基隆。
陳冠丞。2002。闊葉大豆（金門一條根）之抗氧化特性及其有效多酚化合物之分離。國立臺灣海洋大學食品科學研究所碩士論文。基隆。
陳冠良。2005。大豆屬一條根異黃酮類化合物含量與抗氧化性之鑑定。國立臺灣海洋大學食品科學研究所碩士論文。基隆。
游家芷。2006。大豆屬一條根抑制脂質氧化成分之分離與鑑定。國立臺灣海洋大學食品科學研究所碩士論文。基隆。
Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M. and Fukami, Y. 1987. Genistein, a specific inhibitor of tyrosine-specific protein kinases. The Journal of Biological Chemistry. 262:5592-5596.
Alekel, D.L., Germain, A.S., Peterson, C.T., Hanson, K.B., Stewart, J.W. and Toda, T. 2000. Isoflavone-rich soy protein isolate attenuates bone loss in the lumbar spine of perimenpausal women. American Journal of Clinical Nutrition. 72:844-852.
Arora, A., Nair, M.G. and Strasburg, G.M. 1998. Structure-activity relationships for antioxidant activities of a series of flavonoids in a liposomal system. Free Radical Biology and Medicine. 24:1355-1363.
Bonnet, C., Bertin, P., Cook-Moreau, J., Chable-Rabinovitch, H., Treves, R., and Rigaud, M. 1995. Lipoxygenase products and expression of 5-lipoxygenase and 5-lipoxygenase-activating protein in human cultured synovial cells. Prostaglandins. 50:127-135
Brash, A.R. 1999. Lipoxygenases: occurrence, functions, catalysis, and acquisition of substrate. Journal Biol. Chemistry. 274:23679-23682.
Cassidy, A., Bingham, S. and Setchell, K.D.R. 1994. Biological effects of a diet of soy protein rich in isoflavones on the menstrual cycle of premenopausal women. American Journal of Clinical Nutrition. 60:333-340.
Cesquini, M., Torsoni, M.A., Stoppa, G.R. and Ogo, S.H. 2003. t-BOOH-induced oxidative damage in sickle red blood cells and the role of flavonoids. Biomedicine and Pharmacotherapy. 57:124-129.
Chen, H.Y. and Yen, G.C. 1998. Free radicals, antioxidant defenses and human health. Nuritional Sciences Journal. 23: 105-121.
Cook, N.C. and Samman, S. 1996. Flavonoids-chemistry, metabolism, cardioprotective effects, and dietary sources. Nutritional Biochemistry. 7:66-76.
Cuong, N.M., Hung, T.Q., Sung, T.V. and Taylor, W.C. 2004. A New Dimeric Carbazole Alkaloid from Glycosmis stenocarpa Roots. Chemical & Pharmaceutical Bulletin. 52(10):1175—1178.
Dai, F., Miao, Q., Zhou, B., Yang, L. and Liu, Z.L. 2006. Protective effects of flavonols and their glycosides against free radical-induced oxidative hemolysis of red blood cells. Life Sciences. 78 :2488-2493.
Dannhardt, G. and Kiefer, W. 2001. Cyclooxygenase inhibitors-current status and future prospects. Eur. J. Med. 36:109-126.
Drazen, J. M. 1999. Asthma therapy with agents preventing leukotriene synthesis or action. Proceedings of the Association of American Physicians. 111:547–559
Eldrige, A.C. and Kwolek, W.F. 1983. Soybean isoflavones: effect of environment and variety on composition. Journal of Agricultural and Food Chemistry. 31:394-396.
Gaudette, D.C. and Holub, B.J. 1990. Effect of genistein, a tyrosinekinase inhibitor on U46619-induced phosphoinositide phosphorylation in human platelets. Biochemical and Biophysical Research Communications. 170:238-242.
George, J., Afek, A., Shaish, A., Levkovitz, H., Bloom, N., Cyrus, T., Zhao, L., Funk, C. D., Sigal, E. and Harats, D. 2001. 12/15-lipoxygenase gene disruption attenuates atherogenesis in LDL receptor-deficient mice. Circulation. 104:1646-1650.
Ghost, J. and Myers, C. E. 1997. Arachidonic acid stimulates prostate cancer cell growth: critical role of 5-lipoxygenase. Biochem. Biophys. Res. Commun. 235:418-423.
Goodwin, D.C., Landino, L.M. and Marnett, L.J. 1999. Effects of nitric oxide and nitric oxide-derived species on prostaglandin endoperoxide synthase and prostaglandin biosynthesis. FASBEB. J. 13:1121-1136.
Greedale, G.A., Lee, N.P. and Arriola, E.R. 1999. The menopause. The Lancet. 353:571-580.
Gugliucci, A. and Menini, T. 2002. Three different Pathways for human LDL oxidation are inhibited in vitro by water extracts of the medicinal herb Achyrocline satureoides. Life Sci. 71:693-705.
Han, K.K., Soares, J.M., Haidar, M.A., Lima, G.R.de and Baracat, E.C. 2002. Benefits of soy isoflavone therapeutic regimen on menopausal symptoms. The American College of Obstetricians and Gynecologists. 99:389-394.
Herderson, W. R. 1994. The role of leukotrienes in flammation. Ann. Intern. Med. 121:684-697.
Hertrampf, T., Schmidt, S., Laudenbach-Leschowsky, U., Seibel, J. and Diel, P. 2005. Tissue-specific modulation of cyclooxygenase-2 (Cox-2) expression in the uterus and the v. cava by estrogens and phytoestrogens. Molecular and Cellular Endocrinology. 243:57-53.
Ingram, D., Sanders, K., Kolybaba, M. and Lopez, D. 1997. Case-control study of phyto-oestrogens and breast cancer. Lancet. 350:990-994.
Izumi, T., Pikula, M.K., Osawa, S., Obata, A., Tobe, K., Saito, M., Kataoka, S., Kubota, Y. and Kikuchi, M. 2000. Soy isoflavone aglycones are absorbed faster and in higher amount than their glucosides in humans. The Journal of Nutrition. 130:1695-1699.
Jenkins, D.JA, Kendall, C.WC, Jackson, C.J.C., Connelly, P.W., Parker, T., Faulkner, D., Vidgen, E., Cunnane, St.C., Leiter, L.A. and Josse, R. G. 2002. Effects of high- and low-isoflavone soyfoods on blood lipids, oxidized LDL, homocysteine, and blood pressure in hyperlipidemic men and women. American Society for Clinical Nutrition. 76:365-372.
Kim, J.A., Hong, S.B., Jung, W.S., Yu, C.Y., Ma, K.H., Gwag, J.G. and Chung, I. M. 2006. Comparison of isoflavones composition in seed, embryo, cotyledon and seed coat of cooked-with-rice and vegetable soybean (Glycine max L.) varieties. Food Chemistry.
Kondo, Y., Kawai,Y., Miyazawa, T., Matsui, H. and Mizutani, J. 1994. An assay for lipoxygenase activity by chemiluminescence. Biosci. Biotech. Biochem. 58:421-422.
Kuhn, H. and Borchert, A. 2002. Regulation of enzymatic lipid peroxidation: the reduction of expression of VEGF receptors. Life Sci. 73:1299-1313.
Kuhn, H. and Thiele, B.J. 1999. The diversity of the lipoxygenase family. FEBS Lett. 499:7-11.
Lagiou, P., Samoli, E., Lagiou, A., Tzonou, A., Kalandidi, A., Peterson, J., Dwyer, J. and Trichopoulos, D. 2004. Intake of specific flavonoid classes and coronary heart disease-a case-control sturdy in Greece. European Journal of Clinical Nutrition. 58:1643-1648.
Lamartiniere, C.A. 2000. Protection against breast cancer with genistein: a component of soy. American Journal of Clinical Nutrition. 71:1705-1707.
Lapointe, A., Couillard, C. and Lemieux, S. 2006. Effects of dietary factors on oxidation of low-density lipoprotein particles. Journal of Nutritional Biochemistry. 17:645-658.
Lee, C.S., Kwon, S.J., Na, S.Y., Lim, S.P. and Lee, J.H. Genistein supplementation inhibits atherosclerosis with stabilization of the lesions in hypercholesterolemic rabbits. The Korean Academy of Medical Sciences. 19: 656-61.
Lichtenstein, A.H. 1998. Soy protein, isoflavones and cardiovascular disease risk. The Journal of Nutrition. 128:1589-1592.
Limer, J.L. and Speirs, Valerie. 2004. Phyto-oestrogens and breast cancer chemoprevention. Breast Cancer Research. 6:119-127.
Lin, S.J., Lay, H.L., Wu, S.T. and Thseng, F.S. 2005. Content of certain isoflavones in Glycine dolichocarpa, Glycine tabacina and Glycine tomentella collected in Taiwan. Journal of Food and Drug Analysis. 13:260-266.
Middleton, Jr E., Kandawami, C. and Theoharides, T.C. 2000. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease and cancer. Pharmacological Reviews. 52:673-751.
Mira, L., Fernandez, M.T., Santos, M., Rocha, R., Florencio, M.H. and Jennings, K.R. 2002. Interactions of flavonoids with iron and copper ions: a mechanism for their antioxidant activity. Free radical Research. 36:1199-1208.
Munro, I.C., Harwood, M., Hlywka, J.J., Stephen,,A.M., Doull, J., Flamm, W.G. and Adlercreutz, H. 2003. Soy isoflavones: a safety review. Nutrition Reviews. 61:1-33.
Murkies, A.L., Lombard, C., Strauss, B.J.G., Wilcox, G., Burger, H.G. and Morton, M.S. 1995. Dietary flour supplementation decreases postmenopausal hot flushes: effect of soy and wheat. Maturitas. 21:189-195.
Murphy, P.A., Song, T., Buseman, G. and Barua, K. 1997. Isoflavones in soy-based infant formulas. Journal of Agricultural and Food Chemistry. 45:4635-4638.
Newton, K.M., LaCroix, A.Z., Levy, L., Li, S.S., Qu, P., Potter, J.D. and Lampe, J.W. 2006. Soy protein and bone mineral density in older men and women: a randomized trial. Maturitas. the European menopause journal. 55: 270–277.
Raffaella, F., Piovan, A., Innocenti, G., Caniato, R. and Cappelletti, E.M. 1998. Production of coumarin compounds by haplophyllum patavinum in vivo and in vitro. Phytochemistry. 49:2337-2340.
Pan, B.S., Kao, Y.Y., Chen, T.Y. and Liu, Y.C. 2005. Anti-oxidative and anti-inflammatory activities of two different 2 species of a Chinese herb I-Tiao-Gung. Life Science. 77:2830-2839.
Parthasarathy, S., Weiland, E. and Steinberg, D. 1989. A role for endothelial cell lipoxygenase in the oxidative modification of low density lipoprotein. Proc. Natl. Acad. Sci. U. S. A. 86:1046-1050.
Pascal, C., Caroline, B., Claude, C. and Isabelle, V. 2002. 15-Lipoxygenase: A janus enzyme? Am. J. Respir. Cell Mol. Biol. 27: 655–658.
Peterson, J. and Dwyer, J. 1998. Flavonoids: dietary occurrence and biochemical activity. Nutrition Research. 18:1995-2018.
Pietta, P.G. 2000. Flavonoids as antioxidants. Journal of Natural Products. 63:1035-1042.
Potter, S.N., Baum, J.A., Teng, H., Stillman, R.J., Shay, N.F. and Erdman, J.W. 1998. Soy protein and isoflavones : their effects on blood lipids and bone density in postmenopausal women. American Journal of Clinical Nutrition. 68:1375-1379.
Rijke, E., Out, P. Niessen, W.M.A., Ariese, F., Gooijer, C. and Brinkman. U.A.Th. 2006. Analytical separation and detection methods for flavonoids. Journal of Chromatography A. 1112 :31–63.
Reilly, K.B., Srinivasan, S., Hatley, M.E., Patricia, M.K., Lannigan, J., Bolick, D.T., Vandenhoff, G., Pei, H., Natarajan, R., Nadler, J.L. and Hedrick, C.C. 2004. 12/15-lipoxygenase activity mediates inflammatory monocyte/endothelial interactions and atherosclerosis in vivo. J. Biol. Chem. 279:9440-9450.
Rocca, B. and Gerald, G.A.F. 2002. Cyclooxygenases and prostaglandins: shaping up the immune response. Intern Immunopharmcol. 2:603-630.
R?fer, C.E. and Kulling, S.E. 2006. Antioxidant Activity of Isoflavones and Their Major Metabolites Using Different in Vitro Assays. J. Agric Food. Chem. 54:2926-2931.
Schleicher, R.L., Lamartiniereb, C.A., Zhenga, M. and Zhang, M. 1999. The inhibitory effect of genistein on the growth and metastasis of a transplantable rat accessory sex gland carcinoma. Cancer Letters. 136:195:201.
Schoene, N.W. and Guidry, C.A. 1999. Dietary soy isoflavones inhibit activation of rat platelets. Journal of Nutritional Biochemistry. 10:421-426.
Silva, E.L., Abdalla, D.S.P. and Terao, J. 2000. Inhibitory effect of flavonoids low-density lipoprotein peroxidation catalyzed by mammalian 15-lipoxygenase. IUBMB Life. 49:289-295.
Tedesco, I., Russo, M., Russo, P. and Iacomino, G. 2000. Antioxidant effect of red wine polyphenols on red blood cells. The Journal of Nutrition Biochemistry. 11:114-119.
Teede, H.J., Dalais, F.S., Kotsopoulos, D., Liang, Y.L., Davis, S. AND Mcgrath, B.P. 2001. Dietary Soy Has Both Beneficial and Potentially Adverse Cardiovascular Effects: A Placebo-Controlled Study in Men and Postmenopausal Women. The Journal of Clinical Endocrinology & Metabolism. 86(7):3053-3060.
Wang, H. and Murphy, P.A. 1994. Isoflavone content in commercial soybean foods. Journal of Agricultural and Food Chemistry. 42:1666-1673.
Wei, H., Zhang, X., Wang, Y. and Lebwohl, M. 2002. Inhibition of ultraviolet light-induced oxidative events in the skin and internal organs of hairless mice by isoflavone genistein. Cancer Letters. 185:21-29.
Wiseman, H. 1997. Dietary phytoestrogens: disease prevention versis potential hazards. Nutrition and Food Science. 1:32-38.
Yamamoto, S. 1992. Mammalian lipoxygenase: molecular structures and functions. Biochem. Biophys. Acta. 1128:117-131.
Yamashita, H., Nakamura, A., Noguchi, N., Niki, E. and Kuhn, H. 1999. Oxidation of low density lipoprotein and plasma by 15-lipoxygenase and free radicals. FEBS Lett. 445:287-290.
Yao, L.H., Jiang, Y.M., Shi, J., Tomas-Barberan, F.A., Datta, N. Singanusing, R. and Chen, S.S. 2004. Flavonoids in food and their health benefits. Plant Foods for Human Nutrition. 59: 113–122.
Ye, F., Wu, J., Dunn, T., Yi, J., Tong, X. and Zhang, D. 2004. Inhibition of cyclooxygenase-2 activity in head and neck cancer cells by genistein. Cancer Letters. 211:39-46.