臺灣博碩士論文加值系統

綠茶已被證實富含茶多酚，尤其是兒茶素類，三種主要的兒茶素成分為epicatechin (EC)、epigallocatechin gallate (EGCG)以及epicatechin gallate (ECG)，其中以EGCG為含量最多且活性最高的成分。本研究探討含環糊精(β-CD)矽膠吸附劑的最適化製備技術，並將其應用於綠茶萃取物中層析分離與純化 EGCG。研究目的在透過自行製備的β-CD矽膠吸附劑，提高對兒茶素中主要成分EGCG的吸附選擇率，使能更有效分離純化綠茶萃取物中的咖啡因及各類兒茶素。
在吸附劑的製備，包含底下二個階段：(1)將環氧基矽氧烷(GOPS)修飾在矽膠表面，得GOPS_silica顆粒，(2)將β-環糊精(β-CD)與氫化鈉(NaH)反應後獲得Na-β-CD，再與GOPS_silica進行接枝反應，得β-CD_silica吸附劑。以熱重分析(TGA)與元素分析(EA)鑑定的結果如下：矽膠的GOPS修飾量約為0.32~0.39 mmol GOPS/g Silica，而β-CD接枝量約為0.06~0.15 mmol β-CD/g Silica，平均2.5~4.0 mmol GOPS可接枝1 mmol β-CD。
首先以液-液萃取法將粗綠茶萃取物濃縮製備為綠茶濃縮物，將其做為批次吸附與管柱層析操作的原料。液-液萃取的條件為乙酸乙酯溶劑、單次萃取、進料濃度30 mg/mL、水油比例=1：1、萃取溫度=30℃、萃取時間=2小時及保持原綠茶溶液的pH值。三種兒茶素的總純度由約9%濃縮至約45%左右，濃縮比約5.4倍；EGCG的純度由約5.6%濃縮至約30.4%，濃縮比約5.4倍。將此綠茶濃縮物做為批次吸附與管柱層析操作的原料。
在批次吸附與脫附操作方面，首先，由吸附動力實驗得知，120min為最佳吸附平衡時間；吸附平衡實驗得知，平衡濃度Ce約在1.4 mg/ml時達吸附飽和，飽和吸附量qe約為153.94 mg/g。接著，本研究進行批次吸附與脫附實驗，結果顯示，於水溶液時，吸附能力最好（吸附率>95%），但幾乎無法以甲醇脫附。改使用氰甲烷溶液時，吸附率降至54.85%，但依舊無法有效脫附大部分的兒茶素。另外，在未接枝β-CD之前，活化Silica與GOPS_silica對咖啡因與三種兒茶素並沒有很好的吸附效果(吸附率<10%)，但在接枝β-CD之後，對於兒茶素中EGCG的吸附能力有非常明顯的提升(EGCG吸附率>95%)，所以可以確定本研究確實成功合成出β-CD_silica吸附劑。
在管柱沖提層析操作方面，管柱沖提層析有良好的分離效果，且注入物幾乎能被有效脫附與收集。8.7公分管柱有較好的分離效果，尤其以氰甲烷-甲醇系統的分離效果較為良好，但僅能分離三種兒茶素與咖啡因；而水-甲醇系統在2ml的注入量下，雖然分離效果不及氰甲烷-甲醇系統，但可得到較高純度的EGCG和ECG。

Green tea is recognized for its high content of tea polyphenols, in particular tea catechins. The four represented catechins in green tea are, epicatechin (EC), epigallocatechin gallate (EGCG), and epicatechin gallate (ECG). Among these four major compounds, EGCG is the most abundant and biologically active compound in green tea. In this study, the optimal techniques for preparation of silica adsorbents containing β-cyclodextrin (β-CD) and their use in column chromatography were investigated. The objective is using the self-prepared silica adsorbents containing β-CD to enhance the adsorptivity and selectivity of EGCG and then to effectively separate and purify caffeine (CA) and each of the four catechins from green tea extracts.
The preparation of silica adsorbents containing β-CD includes the following two steps: (1) modification of 3-glycidoxypropyl-trimethoxysilane (GOPS) on silica particles to produce epoxy terminated GOPS_silica particles, (2) chemical grafting of Na-β-CD, obtained from the reaction of β-CD with sodium hydride, on GOPS_silica particles to produce β-CD_silica adsorbents. The amount of GOPS modified on GOPS_silica and the amount of β-CD grafted on β-CD_silica, analyzed by elemental analysis (EA) and thermo-gravimetric analysis (TGA), were about 0.32~0.39 mmol GOPS/g Silica and about 0.06~0.15 mmol β-CD/g Silica respectively. That means about 2.5~4.0 mmol GOPS grafted 1 mmol β-CD.
First, the crude green tea extracts were concentrated by liquid-liquid extraction using ethyl acetate as the extractant and under the following conditions: concentration of 30 mg/mL, water/oil ratio of 1:1, extraction temperature of 30℃, extraction time of 2 hr, and without pH adjustment. The purity of the three catechins was raised from 9.0% to about 45.0% (a concentration ratio of about 4) and the purity of EGCG was raised from 5.6% to about 30.4% (a concentration ratio of about 5.4). The green tea concentrates were then used as the feed materials in the following batch adsorption and column chromatographic experiments.
In the batch adsorption and desorption experiments, first, the adsorption kinetic studies showed that 120min is the best equilibrium time for adsorption, and the adsorption equilibrium experiments showed that the adsorption got saturation at equilibrium concentration Ce of about 1.4 mg/ml, at which the equilibrium adsorption capacity qe reached 153.94 mg/g. Then, the batch adsorption and desorption experiments showed that a very high adsorption ratio (>95%) obtained when water was used as the solvent, but there was nearly no desorption by methanol. When the solvent was changed to acetonitrile, the adsorption ratio reduced to 54.85%, however, the desorption ratio was still very low for the major catechins. Also, when the bare silica and the GOPS_silica were used as the adsorbents, the adsorption ratios for caffeine and the four catechins were found to be very low (<10%). But, when the β-CD_silica were used as the adsorbents, the adsorption behavior of the four catechins was significantly improved and a very high adsorption ratio for EGCG (>95%) was obtained. The great improvement in adsorption capacity proved that the β-CD_slica adsorbents were successfully prepared in this study.
In the column chromatographic experiments operated in the step-gradient elution mode, it had better separation selectivity and the injected samples could be effectively eluted and almost collected. The column of 8.7 cm had better separation than the column of 3.7 cm, especially in the acetonitrile-methanol system, but it only could separate three catechins and caffeine. In contrast, the 8.7 cm column operated in water-methanol system could get higher purity of EGCG and ECG than that in the acetonitrile-methanol system for the 2 ml injection.

目錄

中文摘要---------------------------------------------- i
英文摘要------------------------------------------------------ iii
誌謝------------------------------------------------------------- vi
目錄---------------------------------------------------------------- vii
表目錄----------------------------------------------------------------- ix
圖目錄---------------------------------------------------------------- x
一、緒論-------------------------------------------------------------- 1
1.1 前言----------------------------------------------------------------1
1.2 研究動機---------------------------------------------------------- 2
1.3 研究方法---------------------------------------------------------- 3
二、文獻回顧與理論部分------------------------------------------------- 5
2.1 兒茶素及咖啡因的介紹----------------------------------------------- 5
2.1.1 兒茶素的組成----------------------------------------------------- 5
2.1.2 兒茶素的保健功效與應用------------------------------------------- 5
2.1.3 咖啡因的功效與應用----------------------------------------------- 9
2.2 兒茶素與咖啡因的分析方法------------------------------------------- 9
2.3 濃縮純化兒茶素與咖啡因之相關研究------------------------------------13
2.4 含環糊精吸附劑之製備及其應用的研究----------------------------------17
三、實驗部分----------------------------------------------------------- 23
3.1 實驗材料與儀器----------------------------------------------------- 23
3.1.1 材料與標準品----------------------------------------------------- 23
3.1.2 溶劑------------------------------------------------------------- 23
3.1.3 實驗儀器與設備-------------------------------------------------- 24
3.2 實驗方法----------------------------------------------------------- 28
3.2.1 矽膠吸附劑的前處理---------------------------------------------- 28
3.2.2 β-CD _silica吸附劑的製備--------------------------------------- 28
3.2.2.1 GOPS_silica吸附劑的製備--------------------------------------- 30
3.2.2.2 β-CD _silica的製備-------------------------------------------- 30
3.2.2.3 GOPS_silica與β-CD_GOPS_silica的定量與定性分析---------------- 33
3.2.3 三種兒茶素及咖啡因的HPLC定量分析-------------------------------- 36
3.2.3.1 逆相HPLC定性分析---------------------------------------------- 36
3.2.3.2 標準品檢量線的製作-------------------------------------------- 36
3.2.4 以液-液萃取法提取濃縮兒茶素------------------------------------- 36
3.2.5 批次吸脫附實驗--------------------------------------------------- 40
3.2.5.1 批次吸附實驗--------------------------------------------------- 40
3.2.5.2 批次脫附實驗----------------------------------------------------40
3.2.6 以管柱層析法分離純化綠茶萃取物中的咖啡因與兒茶素---------------- 42
3.2.6.1 管柱充填------------------------------------------------------- 42
3.2.6.2 製備級沖提層析裝置與操作系統---------------------------------- 42
四、結果與討論-------------------------------------------------------- 45
4.1 含β-CD矽膠吸附劑的製備與鑑定------------------------------------- 45
4.1.1 熱重分析(TGA)定量GOPS與β-CD的修飾量---------------------------- 45
4.1.2 元素分析(EA)定量GOPS與β-CD的修飾量----------------------------- 45
4.1.3 β-CD_silica兩種定量分析結果的比較------------------------------ 51
4.2 綠茶粉原料的定量分析及濃縮其中的兒茶素與咖啡因-------------------- 51
4.2.1 綠茶粉中三種兒茶素及咖啡因的HPLC定量分析------------------------ 52
4.2.2 以液-液萃取法提取濃縮綠茶粉中的兒茶素與咖啡因-------------------- 52
4.3 以批次法分離純化咖啡因與兒茶素------------------------------------- 56
4.3.1 時間效應--------------------------------------------------------- 56
4.3.2 EGCG吸附平衡等溫關係--------------------------------------------- 56
4.3.3 吸附與脫附性能--------------------------------------------------- 62
4.3.4 與其他吸附劑吸附性能的比較(接枝鑑定)----------------------------- 62
4.4 以β-CD_silica管柱沖提層析分離與純化各類兒茶素--------------------- 68
4.4.1 製備級沖提層析系統(3.7公分管柱)---------------------------------- 68
4.4.2 製備級沖提層析系統(8.7公分管柱)---------------------------------- 79
4.4.3 沖提層析系統總結----------------------------------------------- 83
4.5 管柱沖提層析與批次吸脫附操作之比較----------------------------------88
五、結論-------------------------------------------------------------- 89

陳俊安，茶葉的科學研究，徐氏基金會科學圖書編譯委員會，258-260，1983。
印壽根、李晨曦、宋正紀、黃文強、何炳林，樹脂對茶多酚與咖啡因的吸附分
離，離子交換與吸附，第14卷，第1期，73-77，1998。
廖慶樑，茶葉中兒茶素的用途與萃取製程，食品資訊，174期，22-25，2000。
翁家瑞，各種常見飲品的成份功效，食品資訊，180期，44-47，2000。
程玲，茶兒茶素類之機能及其應用，食品資訊，180期，34-43，2000。
陳清泉，茶葉之兒茶素的機能與應用，食品市場資訊，8期，16-23，2001。
王重、史作清、施榮富、範雲鴿，酚醛型吸附樹脂對咖啡因吸附效能的研究，
高分子學報，第2卷， 211-215，2003。
蕭名傑，陳怡雯，賴世明，應用製備級液相層析術於天然藥物的濃縮與純化，
化工,第50卷，第5期，2003。
張志玲，喝茶防癌及減重的科學依據，科學發展，379期，30-35，2004。
吳亮宜，孫璐西，茶與健康，科學發展，384期，18-23，2004。
胡家碩，蕭千祐，茶與咖啡-喝出健康好氣色，大樹林出版社，2004。
駱少君，呂毅，就是這樣喝茶才健康，紅螞蟻圖書，2004。
黃詠勤，應答曲面實驗設計法蒐尋螺旋膜組分離綠茶多酚最適條件，國立中興
大學化學工程研究所碩士論文，2005。
張愉敏，由綠茶粉製備級提取與分離兒茶素，國立雲林科技大學化學工程研究
所碩士論文，2006。
李文龍，基材的表面改質及其在感測與分離上的應用，國立雲林科技大學化學
工程與材料工程所博士論文，2008。
李文龍、黃炳豪、賴世明， Preparation of silica adsorbents containing cyclodextrins
and their use in the separation and purification of catechins and caffeine
from green tea powders, 化工年會，台中中興大學，2009。
健健康康網站，認識多酚，http://www.jjkkusa.com/ modules/news/ php?storyid
article.=415，2010
黃炳豪，含環糊精矽膠吸附劑及其在由綠茶粉分離純化兒茶素上的應用，國立雲
林科技大學化學工程與材料工程所碩士論文，2010。
Adachi T., S. Ando and J. Watanabe, Characterization of synthetic adsorbents with fine particle sizes for preparative-scale chromatographic separation, Journal of Chromatography A, 944, 41-59, 2002.
Amarowicz, R. and F. Shahidi, A rapid chromatographic method for separation of individual catechins from green tea, Food Research International, 29, 1, 71-76, 1996.

Aoki, N., M. Nishikawa, and K. Hattori, Synthesis of chitosan derivatives bearing cyclodextrin and adsorption of p-nonylphenol and bisphenol A, Carbohydrate Polymers, 52, 219-223, 2003.
Armstrong, D. W., W. Demond, A. Alak, W. L. Hinze, T. E. Riehl, and K. H. Bui, Liquidchromatographic separation of diastereomers and structural isomers on cyclodextrin-bonded phases, Anal. Chem., 57, 234-237, 1985.
Bailey, D. T., R. L. Yuhasz and B. Z. Wayne, Method for isolation of caffeine-free catechins from green tea, US Patent, 6,210,679, 2001.
Bonrath W., D. C. Burdick, P. Schirg and A. Thum, Procss for concentrating epigallocatechin gallate, US Patent, 6,383,392, 2002.
Chiu, S. H., T. W. Chung, R. Giridhar, and W. T. Wu, Immobilization of β-cyclodextrin in chitosan beads for separation of cholesterol from egg yolk, Food Research International, 37, 217-223, 2004.
Copeland, E. L., M. N. Clifford and C. M. Williams, Preparation of (–)-epigallocatechin gallate from commercial green tea by caffeine precipitation and solvent partition, Food Chemistry, 61, 1/2, 81-87, 1998.
Crini, G. and M. Morcellet, Synthesis and applications of adsorbents containing cyclodextrins, J.of Separation Science, 25, 789-813, 2002.
Crini, G., Recent developments in polysaccharide-based materials used as adsorbents in wastewatertreatment, Progress in Polymer Science, 30, 38-70, 2005.
Dalluge, J. J. and B. C. Nelson, Determination of tea catechins, Journal of Chromatography A, 881, 411-424, 2000.
Ding, M., H. Yang and S. Xiao, Rapid, direct determination of polyphenols in tea by reverse-phase column liquid chromatography, Journal of Chromatography A., 849, 637-640, 1999.
Ekanayake, K., J. R. Bunger, and M. J. Mohlenkamp Jr., Green tea extract subjected to cation exchange treatment and nanofiltration to improve clarity and color, US Patent, 5,879,733, 1999.
Fan, Y., Y. Q. Feng, and S. L. Da, On-line selective solid-phase extraction of 4-nitrophenol with β-cyclodextrin bonded silica, Analytica Chimica Acta, 484, 145-153, 2003.
Faraji, H., β-Cyclodextrin-bonded silica particles as the solid-phase extraction medium for the determination of phenol compounds in water samples followed by gas chromatography with flame ionization and mass spectrometry detection, J. of Chromatography A, 1087, 283–288, 2005.
Feng, Y. Q., M. J. Xie, and S. L. Da, Preparation and characterization of an L-tyrosine-derivatzed β-cyclodextrin-bonded silica sationary phase for liquid chromatography, Analytica Chimica Acta, 403, 187-195, 2000.
Fujimura, K., T. Ueda, and T. Ando, Retention behavior of some Aromatic compounds on chemically bonded cyclodextrin sillic stationary phase in liquid chromatography, Anal. Chem., 55, 446–450, 1983.
Fujimura, K., S. Suzuki, K. Hayashi, and S. Masuda. Retention behavior and chiral recognitionmechanism of several cyclodextrin-bonded stationary phases for dansyl amino acids, Anal. Chem.,62, 2198–2205, 1990.
Furusaki, E., Y. Ueno, N. Sakairi, N. Nishi, and S. Tokura, Facile preparation and inclusion ability of a chitosan derivative bearing carboxymethyl- β-cyclodextrin, Carbohydrate polymers, 29, 29-34, 1996.
Goto, T., Y. Yoshia, M. Kiso and H. Nagashima, Simultaneous analysis of individual catechins and caffeine in green tea, Journal of Chromatography A., 749, 295-299, 1996.
He, P., N. Hu, Electrocatalytic properties of heme proteins in layer-bylayer films assembled with SiO2 nanoparticles., Electroanalysis, 16, 1122–1131, 2004.
Hu, Y., Y. Zheng, and G. Li, Solid-phase microextraction of phenol compounds using a fused-silica fiber coated with β-cyclodextrin-bonded silica particles, Analytical Sciences, 20, 667-671, 2004.
Joseph, J. D., B. C. Nelson, J. B. Thomas and L. C. Sander, Selection of column and gradient elution system for the separation of catechins in green tea using high-performance liquid chromatography, Journal of Chromatography A., 793, 265-274, 1998.
Kawaguchi, Y., M. Tanaka, M. Nakae, K. Funazo, and T. Shono, Chemically bonded cyclodextrin stationary phases for liquid chromatographic separation of aromatic compounds, Anal. Chem., 55, 1852-1857, 1983.
Khokhar, S., D. Venema, P. C. H. Hollman, M. Dekker and Wim Jongen, A RP-HPLC method for the determination of tea catechins, Cancer Letters, 114, 171-172, 1997.
Kim, J. I., S. B. Hong, K. H. Row, Effect of particle size in preparative reversed-phase high-performance liquid chromatography on the isolation of epigallocatechin gallate from Korean green tea, Journal of Chromatography A, 949, 275-280, 2002.
Li, P., Y. Wang, R. Ma, and X. Zhang, Separation of tea polyphenol from green tea leaves by a combined CATUFM-adsorption resin process, Journal of Food Engineering, 67, 253-260, 2005.
Lunder, T. L., Process for obtaining catechin complexes, US Patent, 5,107,000, 1992.
Martel, B., M. Weltrowski, D. Ruffin, and M. Morcellet, Polycarboxylic acids as crosslinking agents for grafting cyclodextrins onto cotton and wool fabrics: Study of the process parameters, J. Appl. Poly. Sci., 83, 1449-1456, 2002.
Nah, T, H., E, H, Cho., M, D, Jang,.Y, K., Lee,.J, H, Park,.Binding forcescontributing to reversed-phase liquidchromatographic retention on a β-cyclodextrin bonded phase, Journal of Chromatography A, 722, 41-46, 1996.
Niwuha, V., Novel studies on membrane extraction of bioactive components of green tea in organic solvents: part I, Journal of Food Engineering, 44, 233-238, 2000.
Pelillo, M., M. Bonoli, B. Biguzzi, A. Bendini, T. Gallina Toschi and G. Lercker, An investigation in the use of HPLC with UV and MS-electrospray detection for the quantification of tea catechins, Food Chemistry, 87, 465-470, 2004.
Phan, T. N. T., M. Bacquet, and M. Morecellet, The removal of organic pollutants from water using new silica-supported β-cyclodextrin derivatives, Reactive and Functional Polymers, 52, 117-125, 2002.
Ponchel, A., S. Abramson, J. Quartararo, D. Bormann, Y. Barbaux, and E. Monflier, Cyclodextrin silica-based materials: advanced characterizations and study of their complexing behavior by diffuse reflectance UV–Vis spectroscopy, Microporous and Mesoporous Materials, 75, 261–272, 2004.
Sakanaka S., A Novel Convenient Process To Obtain a Raw Decaffeinated Tea Polyphenol Fraction Using a Lignocellulose Column, Journal Agric. Food Chemistry, 51, 3140-3143, 2003.
Su, Y. L., L. L. Kwok, Y. Huang, Z. Y. Chen, 2003, Stability of tea theaflavins and catechins, Food Chemistry, 83, 189-195.
Tojima, T., H. Katsura, M. Nishiki, N. Nishi, S. Tokura, and N. Sakairi, Chitosan beads with pendant α-Cyclodextrin: preparation and inclusion property to nitrophenolates, Carbohydrate Polymers, 40, 17-22, 1999.
Tsuchiya, H., M. Sato, H. Kato, T. Okubo, L. R. Juneja and M. Kim, Simultaneous determination of catechins in human saliva by high-performance liquid chromatography, Journal of Chromatography B., 703, 253-258, 1997.
Wang, H., K. Helliwell, Epimerisation of catechins in green tea infusions, Food Chemistry, 70, 337-344, 2000.
Wang, H., K. Helliwell and X. You, Isocratic elution for the determination of catechins, caffeine and gallic acid in green tea using HPLC, Food Chemistry, 68, 115-121, 2000.
Wang, H., G. J. Provan and K. Helliwell, HPLC determination of catechins in tea extract using relative response factors, Food Chemistry, 81, 307-312, 2003.
Xu J., G. Zhang, T. Tan and J. C. Janson, One-step purification of epigallocatechin gallate from crude green tea extracts by isocratic hydrogen bond adsorption chromatography on β-cyclodextrin substituted agarose gel media, Journal of Chromatography B, 824, 323-326, 2005.
Xu, J., T. Tan, J. C. Janson, L. K. and C. Sandstrom, NMR Studies on the interaction between(−)-epigallocatechin gallate and cyclodextrins, free and bonded to silica gels, Carbohydrate Research, 342, 843-850, 2007.
Yoshida, Y., M. Kiso and T. Goto, Efficiency of the extraction of catechins from green tea, Food Chemistry, 67, 429-433, 1999.
Yu, E. K. C., Novel decaffeination process using cyclodextrins, Applied Microbiology Biotechnology, 28, 546-552, 1988.
Yukihiko H., Process for the production of tea catechins, US Patent, 4,613,672, 1986.
Zhao, X. B., and B. L. He, Synthesis and characterization of polymer-immobilized β-cyclodextrin with an inclusion recognition functionality, Reactive Polymers, 24, 9-16 (1994).
Zuo, Y., H. Chen and Y. Deng, Simultaneous determination of catechins, caffeine and gallic acids in green, Oolong, black and puerh teas using HPLC with a photodiode array detector, Talanta, 57, 307-316, 2002.