臺灣博碩士論文加值系統

花色素係為重要之植物色素，廣泛存於植物體當中，為各種植物顏色（紅、紫、紫紅）之主要來源，因對人體無害，所以具有良好的開發潛力。但因結構複雜，安定性容易受到pH、高溫、氧氣、光和維生素丙之影響。因此，針對紫蕪所存在的花色素進行結構鑑定。
利用高效率液相層析(HPLC)分離純化，分離得到三種花色素，將主要三帶花色素經光譜吸收分析、質譜分析、濾紙層析及降解產物分析結果，推論其結構為：
化合物一： sinapyl cyanidin 3-sophoroside 5-glucoside
化合物二： feruloyl cyanidin 3-sophoroside 5-glucoside
化合物三： feruloyl, sinapyl cyanidin 3-sophoroside 5-glucoside
此三化合物結構亦存在於紅高麗菜中，藉此可鑑別兩種作物間的種源關係。

Anthocyanins are water-soluble and among the most important group of plant pigment. Because of its safety for human consumption they are highly desirable substitute for synthetic red colorants which have been banned by regulatory action. The study was conducted to identify the anthocyanins in Purple vienna (Brassica Oleracea) .
three anthocyanins from Purple vienna were separated by HPLC. Based on absorption spectral analysis,mass spectrometry analysis , paper chromatography and instrumental analysis of the degradated products analysis , the structure of antocyanins from Purple vienna were tentatively concluded to be:
compoundⅠ: sinapyl cyanidin 3-sophoroside 5-glucoside.
compoundⅡ: feruloyl cyanidin 3-sophoroside 5-glucoside.
compoundⅢ: feruloyl ,sinapyl cyanidin 3-sophoroside 5-glucoside.
These three band also found in red cabbage. The results has been compared with those anthocyanins from red cabbage published to find the relationship between them.

目 錄
壹、中文摘要………………………………………………………… 1
貳、英文摘要…………………………………………………………. 3
參、研究動機…………………………………………………………. 5
肆、文獻整理…………………………………………………………. 7
一、紫蕪簡介…………………………………………………….. 8
二、花色素簡介……………………………………………… 10
三、花色素之結構鑑定……………………………………… 15
四、影響花色素穩定之因素………………………………… 17
五、花色素作為食品著色劑………………………………… 28
六、天然色素發展概況………………………………………… 30
七、花色素之生理療效……………………………………… 31
八、花色素之抗氧化性………………………………………… 33
伍、實驗材料與方法…………………………………………………. 37
實驗材料………………………………………………………. 38
實驗方法………………………………………………………. 39
一、花色素之萃取及分離…………………………………… . 39
二、花色素之分離及純化…………………………………… . 39
三、花色素之光譜分析……………………………………… . 41
四、花色素之濾紙層析性質…………………………………. ..42
五、花色素的測定…………………………………………….…..42
六、醯化合物之測定……………………………………………. . 44
七、糖之測定………………………………………………………45
八、花色素分子量測定………………………………………. . 49
陸、結果與討論……………………………………………………….. 50
一、花色素之分離與純化…………………………………… . 51
二、花色素之光譜分析……………………………………… . 53
三、花色素化學結構鑑定………………………………… …. 61
1、花色素之TLC、HPLC分析…………………………. …..61
2、醯化物之分析………………………………………… ……63
3、糖組成份之分析……………………………………… ……67
四、花色素分子量之測定………………………………… ……..70
柒、結論………………………………………………………………. ..72
捌、附錄……………………………………………………………… . 74
玖、參考文獻…………………………………………………………. .84
圖 目 錄
圖一、紫蕪外觀………………………………………………………. 9
圖二、flavylium的基本構造………………………………………… 12
圖三、類黃酮之生合成路徑………………………………………….. 16
圖四、在酸性中花色素之四種結構……………………………….. 17
圖五、加熱100℃下對色素吸光值之影響…………………………… 20
圖六、25℃下光照對色素吸光值之影響…………………………….. 21
圖七、花色素在不同pH值下轉化反應………………………….. 24
圖八、維生素C之裂解機制…………………………………………. 27圖九、添加Cy-3-g與否對老鼠肝臟受損之酵素濃度指標………… 35
圖十、添加Cy-3-g與否對老鼠肝臟、血清中TBARs濃度之影響…36
圖十一、毛細管電泳檢測裝置圖…………………………………….. 46
圖十二、紫蕪花色素之HPLC分析圖……………………………. 52
圖十三、紫蕪去醯花色素之HPLC圖…………………………… . 57
圖十四、紫蕪花色素在不同pH之紫外-可見光吸收光譜圖……. 60
圖十五(A)、四種酸標準品之HPLC圖……………………………… 65
圖十五(B)、四種酸標準品之毛細管電泳圖…………………………..65
圖十六、紫蕪各帶醯基毛細管電泳圖……………………………….. 66
表 目 錄
表一、自然界存在之花色素配基之結構…………………………. 13
表二、作為食品著色劑花色素來源………………………………. 29
表三、花色素之生理功能………………………………………….. 32
表四、紫蕪花色素之HPLC分析條件…………………………… 40
表五、用於各種層析之溶劑系統……………………………………. 43
表六、紫蕪醯基之毛細管電泳分析條件……………………………. 47
表七、分析糖基所用GC條件………………………………………. 48
表八、紫蕪花色素與去醯花色素之光譜及濾紙性質………….. 58
表九、紫蕪花色素在不同pH值之L.a.b值……………………… 59
表十、紫蕪花色素之花色素HPLC及TLC分析表……………… 62
表十一、紫蕪花色素以GC分析糖分之酯化物滯留時間表………68
表十二、紫蕪花色素之各組成份……………………………………69
表十三、紫蕪花色素之FAB-mass訊號表………………………. .71
附 圖 目 錄
附圖一、紫蕪花色素之吸收光譜圖………………………………. 75
附圖二、紫蕪去醯花色素之吸收光譜圖…………………………. 76
附圖三、紫蕪花色素在不同pH值下之色澤變化………………. 77
附圖四、紫蕪花色素之花色素HPLC圖………………………… 78
附圖五、紫蕪各帶花色素之HPLC圖…………………………… 79
附圖六、紫蕪花色素糖基之GLC分析圖………………………… 80
附圖七(A)、紫蕪第一帶花色素之FAB-mass圖…………………. 81
附圖七(B)、紫蕪第二帶花色素之FAB-mass圖…………………. 82
附圖七(C)、紫蕪第三帶花色素之FAB-mass圖…………………. 83

薛聰賢 (1997) 書香果樂。台灣普綠有限公司。
劉心綺 (1999) 天然色素資源簡介。 食品資訊。
葉碧櫻 (1993) 甘藷花色素之鑑定和三種天然花色素萃取物安
定性之比較研究。東海大學碩士論文。
楊正憲 (1995) 桑葚、紅皮甘藷花色素之研究與花色素之氧化和抗
氧化之探討。東海大學碩士論文。
蔡正宗、王昭君、謝淑惠 (1998) 反轉電滲流毛細管電泳高效率液相
層析分離四種Cinnamic acid 衍生物之比較。食品科學
25（6）: 758-764
蔡正宗、陳淑凰 (1996) 黑糯米中兩種主要花色素之結構鑑定。
食品科學 23(3) : 444-452。
蔡正宗、陳中文、楊正憲 (1995) 紅鳳菜( Gynura bicolor) 所含兩種
主要花色素之研究。
Alessandro, B ., Annalisa, R ., Nadia M. and Bruno, C. (1995) HPLC/MS application to anthocyanins of Vitis vinfera L. J. Argic. Food Chem. 43: 2104-2109.
Bridle, P., Garcia, C. (1996) Analysis of anthocyanins by capillary zone electrophpresis. J. Liq. Chrom. & Rel.Technol. 19 (4) : 537-545.
Bridle, P., Tomberlake, C. F. (1997) Anthocyanins as natural food colours —selected aspects. Food Chemistry. 58: 103-109.
Christine, E.L., John, R.L. and Jane, E. L. (1995) Effect of polysaccharides on the colur of anthocyanins. Food Chemistry. 54: 315-319.
Crisina Garcia-Viguera, Peter Bridle. (1999) Influence of structure on color stability of anthocyanins and flavylium salts with ascorbic acid. Coccinea. Phytochem. 49: 875-880.
Daka, E., Yamakita, I., Ogawa, H., Kondo, T. T., Yamamoto, M., and Goto T. (1987) Structure of three diacylated anthocyanins isolated from red cabbage Brassica oleracea. Chemistry Letters . 1213-1216.
Davies, A.J., and Mazza, G. (1993) Copigment of simple and acylated anthocyanins with colorless phenolic compounds. J. Agric. Food Chem. 41: 716-720.
Eiichi, I., Kaneyoshi, S., Hiroshi, Y., Toshihiko, O., Tadao, k. and Toshio G.(1987) Structure of monoacylated anthocyanins isolated from red cabbage, Brassica Oleracea .Chemistry Letters. 145-148.
Englyst, H., Wiggins, H. S., and Cummings, J.H. (1982) Determination of the non-starch polysaccharides in plant foods by Gas-liquid chromatography of constituent sugars as alditol acetates. Analyst. 107: 307-318.
Esterbauer, H., Gebicki, and Jiirgens ,G., (1992) The role of lipid peroxidation and anthocyanins in oxidative modification of LOL. Free Rad. Bio Med. 13: 341-390 .
Fast atombombardment mass spectrometry of the anthocyanins violanin and platyconin. Phytochem. 22: 1002.
Fiorini, M. (1995) Preparative high-performance liquid chromatography for the purification of natural anthocyanins. Journal of Chromatography. 213-215.
Francis, F. J.(1986) Handbook of Food Colorant Patents 1969-1985
Gao, L., and Mazza, G. (1994) Rapid method for complete chemica characterization of simple and acylated anthocyanins by HPLC and capillary LC. J. Agric. Food Chem. 42: P 118-125.
Goto, T., Kondo, T., Imagawa, H., Takase, S., Atobe, M., and Miura, I. (1981) Structure confirmation of tris- deacyl Heavenly blue anthocyanins, An alkaline hydrolysis product of heavenly blue anthocyanin obtained from of Morning Glory “Heavenly blue”. Chemistry Letters. 883-886 .
Harborne, J. B. (1958) Spectral methods of characterizing anthocyanins.
J. Biochemistry. 70: 22.
Harborne, J. B. (1967) Comparative Biochemistry of the flavonoids. Academic Press. London and New York.
Harborne, T.B. (1964) Plant Polyphenols-XI the structure of acylated anthocyanins. Phytochem. 3:151.
Hebrero, E., Gercia-Rodriguez, C., Santos-Buelga, C. and Rives-Gonzaio, J. C. (1989) Analysis of anthocyanins by High Performance Liquid Chromatography-Diode Arry spectroscopy in a Hybrid grape variety cvitis uinifera and vitis berlandieri 41B. Am.J. Enol. Vitic. 40: (4).
Helen, M. I., Franck L.,and Lydia, M. (1999) Analysis of polysaccharides and monosacchardides in the root mucilage of maize (Zwa. Maysl.) by gas chromatograpy. Journal of Chromatography. 831:267-276.
Hideo Yamasaki., Hirotaka Vefuji., and Yasuko Sakihama. (1996) Blenching the red anthocyanun induced by superoxide redical. Archives of Biochemistry and Biophysics. 332: (1) 183-186.
Hosokawa, K., Chihaur, F., and Tamamura, S. (1995) Three acylated cyaniding glucosides in pink flowers of gentiana. Phytochemistry. 40: 941-947.
Hrazdina, G., Iredale, H., and Mattick, I. R. (1977) Anthocyanin composition of Brassica oleracea cv.red Danish. Phytochemistry. 16: 297-299.
Igarashi, K., Takanashi, K., Makino, M., and Yasui, Y., (1989) Antioxidative activity of major anthocyanin isolated from wild Grapes(Vitis oignetiae) Nippon Shokuhin Kogyo Gakaishi. 36: 8525-8562.
Ikeda, K., Kihuzaki, H., Nakatani,N. (1987) Structure of two acylated anthocyanins from red cabbage(Brassica oleracea). Chem.Express 2: 563-566.
Imbert, M.P., and Seaforth, C. (1968) Anthocyanins in Diosorea alata. L. Experientia. 24: 417.
John, I. and Bertford, H. (1958) Spectral methods of characterizing anthocyanins. Journal Biochemistry. 70: 23-27.
Jose, A. F., Luis, A., Jose, A. M., Venancio, H., and Juan, C., (1998) Dependance between color and individual anthocyanin content in ripening grapes. Food Research International. 31: (9) 667-672.
Kunijiro Yoshitama and Kenzo. (1996) Chromatographic and spectral characterizated of 3’-glycosylation in anthocyanins. Phytochemistry.
Liu-Y. M., and S-J. Sheu (1994) Separation of aromatic acids by reversed electroosmotic flow capillary . Journal Chromatogr 663: 239。
Luis, C., Torgils, F. and Yvind M., (2000) Color and stability of the six common anthocyanidin 3-glucosides in aqueous solutions. Food Chemistry. 68: 101-107.
Markakis, P. (1982) Stability of anthocyanins in food. In Anthocyanins as food colors. Markakis. P. Academic Press. N.Y.NY.
Mazza, G. and Miniati, M.(1993) Anthocyanins in fruits, vegetables,and Grains. P.1 CRC Press, Inc., Florida.
Murai, K., and Wilkins, D. (1990) Natural red color derived from red cabbage. Food Technology. June 131.
Nakatani,N., Ikeda, K., Nakamura, M. and Kikuzaki, H. (1987) Structure of diacylated anthocyanins from red cabbage. Chem.Express.2: 555-562.
Nobji,N.,Kayo, N., Mikio, N. and Hiroe K. (1987) Structure of diacylated anthocyanins from red cabbage, Brassica Oleracea . Chemistry Express. 2: (9) 555-588.
Petter Bride & Cristrina Garcia-viguera. (1997) Analysis of anthocyanins in strawberries and elederberries. A comparision of capillary zone electrophoresis and HPLC. Food Chemistry. 59: 299-304.
Regina, D., Barbara Pliszka., and Teresa Olszewska. (1999) Fluorescence properties of plant anthocyanin pigments. I. Fluorescence of anthocyanins in Brassica Oleracea L.extracts. Journal of Photochemistry. 50: P 53-58.
Saito, N., Tatsuzawa, F., Kasahara, K., Iida S. and Honda, T. (1998) Acylated cyanidin 3-sophorosides in the brownish-red flowers of Ipomoea purpurea. Phytochem 49: P 875-880.
Sapers, G.M., Taffer, I., Ross, L.R. (1981) Functional properties of a Food colorant prepared from red cabbage. J. Food. Sci 46(1): 105-109.
Saskia A.B.E., Vanacker., Michel, N.T.L. Tromp., Guido, R.M.M. Haenen., Wim, J.F van der vijgh and Aalt Bast. (1995) Flavonoids as scavengers of niric oxide radical. Biochemical and Biophysical Reseach Communication. 214: (3).212-215
Shewfelt, R. L., Ahmed, E. M.(1977) Anthocyanin extracted from red cabbage shows promise as coloring for dry beverage mixed.Food Chemistry. 45:265-277.
Takanori, T., Fumihika, H., Tunzoh, K. and Toshihiko, O. (1999) Protetive effects of dietary cyaniding 3-O-β-D-glucoside on liver ischemia —reperfusion injury in rates. Avchives of Biochemistry and Biophysics. 368: (4) 361-366.
Tanchev, S.S. and Timberlake C.F. (1969) The anthocyanins of red cabbage (Brassica Oleracea) . Phytochemistry. 8: 1825-1827.
Tatsuzawa, F., Saito, N., Yokio , M., Shigihara, A. and Honda T. (1998)
Torgil Fossen., Luis Cabrita., and Oyvind M. Andersen. Colour and stability of pure anthocyanins influenced by pH including the alkaline region. Food Chemistry. 63: (4) 435-440.
Toshiro, Watanabe and Shigeru Terabe. (2000) Analysis of natural food pigments by capillary electrophoresis. Journal of chromatography. 311-322.
Totsuka-ku,and Yokohama.(1993) Anthocyanin intramolecular copigment effect. Phytochemistry. 34(1):119-124.
Tsuda,T.,Ohshima, K., Kawakishi, S. and Osawa, T. (1994) Antioxidative pigments isolate from the seeds of Pheseoleus vulgaris . L. J . Agric. Food Chem. 42: 248.
Victor, H., Ronald, E. and Wrolstad. (1990) Use of HPLC separation /photodiode array detection for characterization of anthocyanins.
J. Argic. Food Chem. 38: 708-715.
Watanabe, S., Sakamura, S. and Obata, Y. (1966) The structures of acylated anthocyanins in eggplant and perilla and the position of acylation. Agriculature Chem. 30: 420-422.
Wesche-Ebeling, P., Argaiz-Jamet, A., Hemandez-Porras, L.G. and Lopez-Malo, A. (1996) Preservation factors and processing effects on anthocyanin pigments in plums. Food Chemistry. 57:(3) 399-403.
Wilhelmina, Lalt., Charles, F., Forney., Antonio, M. and Ronald, L. P. (1999) Antioxidant capacity , Vitamin C , Phenolics , and Anthocyanins after fresh storage of small fruits. J. Agric. Food Chem. 47: 1638-4644.
Yoshida, K., Kondo, T., Kameda, K., Kawakishi, S., Lubag, A.M., Mendoza, A. M. T. and Goto, T. (1991) Structure of alatanin A,B and C isolated from edible purple yam Dioscorea alata.Tetrahedron Letters 32: 5575-5578.
Yoshida, K. Kondo, T. and Goto, T. (1991) Usually stable monoacylated anthocyanin from purple yam dioscorea alata. Tatrahcdron Letters. 32: (40) 5579-5580.
Yoshitama, K. and Abe, K. (1977) Chromatographic and spectral characterization of 3''-glucosylation in anthocyanins.Phytochem 16: 591.
Yukihide, Abe. and Kczo, H. (1995) Further studies on paper chromatography of anthocyanins, Involving an examination of glycoside types by partial hydrolysis. Bot. Mag. Tokyo. 69: 579-585.