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研究生： 王思惠
Wang, Sz-Huei
論文名稱： 應用分子模版從不同大豆溶液中選擇性萃取異黃酮素
The application of molecularly imprinted polymers to the selective extraction of isoflavones from various soybean solutions
指導教授： 周澤川
Chou, Tse-Chuan
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程學系
Department of Chemical Engineering
論文出版年： 2007
畢業學年度： 95
語文別： 中文
論文頁數： 147
中文關鍵詞： 分子模版 、分離 、大豆 、異黃酮素
外文關鍵詞： soybean, isoflavones, separation, molecularly imprinted polymer
相關次數： 點閱：149  下載：1 
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    • 本研究目的在於利用分子模版技術，代替傳統的分離純化程序，自大豆中提煉大豆異黃酮素(Soy isoflavones)。一般而言，異黃酮素於每克大豆內的總含量為1.2～3.4毫克，且其含量會因大豆的種類、生長年數以及生長的區域的不同而變化。由於大豆異黃酮素具有抗氧化、預防骨質疏鬆、降低心血管疾病的罹患率及抗癌等作用，使得大豆異黃酮素為近幾年許多研究人員積極研究的物質。
    若能利用低成本、提煉步驟簡易的分子模版技術成功地將大豆內之異黃酮素提煉出來，則將能解決傳統分離純化程序所遭遇到的耗能、又費時的問題。
    本研究以金雀異黃酮為目標分子，進行分子模版的製備。利用批式再吸附測試，以及微熱卡計的恆溫滴定方法，對分子模版的組成進行最適化設計。比較批式再吸附以及恆溫滴定的結果，乙二醇二甲基丙烯酸酯(EGDMA)以及四甲基吡啶(4VP)分別是於被測試的單體中，最適交聯劑與功能性單體。隨功能性單體對目標分子的莫耳比(FM/T)由2增加至8，微熱卡計所記錄的功能性單體與目標分子溶液間作用熱由30 mJ減少成14 mJ，但隨更多的功能性單體滴定入系統中，微熱卡計所顯示的訊號為一定值，不再改變。於本研究中，以乙腈為溶劑，功能性單體對目標分子的莫耳比為8的組成比例，使用沈澱聚合方法製備分子模版，所得模版之α值約為2。
    利用Scatchard plot探討模版吸附的動力機制，可知道分子模版對金雀異黃酮的吸附，是由特異性吸附行為，以及非特異性吸附行為共同貢獻，其中兩吸附行為的親和性的差異相差了約17倍。而分子模版不管於單一成份系統中或於多成份複雜系統中，皆可藉由分子的大小、形狀與化學結構的不同，對分子進行辨識，然而當模版於多成份之複雜系統之下，對於目標分子的辨識效果會因其他分子的競爭而降低。
    實際應用模版於大豆萃取液對異黃酮素分離純化的結果顯示，於模版再脫附的溶液組成中，異黃酮素的組成比例有所提高，由原本於大豆萃取液中佔42%提高至65%；而其他非異黃酮素類的物質於溶液內的組成相對減少，代表分子模版於真實樣品溶液內，對異黃酮素分子具有吸附能力以及選擇性。但由於模版對異黃酮素的選擇性以及吸附能力未達到理想，因此未能得到預期的突破曲線，於模版設計方面必需再以加強，才能真正簡化分離純化之程序。

    The purpose of this research was to replace the traditional separation process by molecular imprinting technique to separate and purify soy isoflavones from soybean. In general, soybeans contain 1.2~4.2 mg of total isoflavones/g of sample, and the content will vary with the variety, crop year and growth location of the soybean samples.
    Due to the antioxidant, anti-osteoporotic, anti-atherogenic, and anti-carcinogenic activities, isoflavones were the compounds that researchers are very interested in recent years.
    The problems of power-consuming and tedious procedure of traditional separation process could be overcome with cost-down and simplified process by using molecularly imprinted polymer.
    In this study, genistein was chosen as template to prepare the molecularly imprinted polymer. The batch re-binding study and isothermal titration method were introduced to investigate the optimized formulation of genistein-imprinted polymer. Comparing the results of batch re-binding study and isothermal titration, EGDMA and 4VP were the optimized cross-linker and functional monomer among the monomers tested in this study. The interaction between 4VP and genistein solution recorded by micro-calorimeter decrease from 30 mJ to 14 mJ with increasing the molar ratio of functional monomer to template (FM/T) from 2 to 8, and the interaction became a constant with further injecting. The imprinting factor of genistein-imprinted polymer preparing with acetonitrile as solvent, FM/T ratio 8 is about 2. The result of scatchard plot shows that both specific binding and non-specific binding sites contribute the adsorption of genistein on the polymer. And the affinity of specific binding sites are 17 times then the non-specific ones. The genistein-imprinted polymer could recognize the target molecules by the size, shape and the structure of the compounds in both single-component and multi-component systems. However, the selectivity of the imprinted polymer for genistein decreased in multi-component system due to competition of other structural related compounds.
    The composition of isoflavones in the after-desorbing solution was higher than the composition in the soybean extract while the composition of the other components decreases. This revealed that the imprinted polymer exhibit selectivity and affinity for isoflavones. However, in this study, the break-through curve couldn’t be obtained due to the performance of the imprinted polymer wasn’t better enough. The genistein-imprinted polymer could be applied to simplify the separation process by preparing imprinted polymer with the optimized formulation.

    中文摘要………………………………………………………I 英文摘要………………………………………………………III 誌謝……………………………………………………………V 目錄……………………………………………………………VII 表目錄.……………………………………………………….XI 圖目錄…………………………………………………………XIV 專有名詞縮寫對照表………………………………………..XIX 符號說明……………………………………………………..XX 第一章　緒論………………………………………………..1 1-1 大豆之簡介………………………………………………1 1-2 黃酮素與異黃酮素之簡介………………………………2 1-2-1 黃酮素…………………………………………………2 1-2-2 異黃酮素…………………………………………....2 1-3 異黃酮素的療效…………………………………………5 1-4 異黃酮素之萃取、純化方法……………………………7 1-5 研究動機…………………………………………………9 第二章 原理………………………………………………….10 2-1 分子模版…………………………………………………11 2-1-1 起源與發展……………………………………………11 2-1-2 分子模版之原理………………………………………14 2-1-3 影響分子模版辨識性的因素…………………………15 2-1-3.1 模版分子與功能性單體間的作用力種類…………15 2-1-3.2 交聯劑的選擇………………………………………17 2-1-3.3功能性單體的選擇及使用劑量…………………….17 2-1-4 聚合反應………………………………………………24 2-1-5 分子模版的效能測試…………………………………26 2-1-5.1 批次再吸附方法……………………………………27 2-1-5.2 層析方法……………………………………………29 2-1-6 分子模版的之應用……………………………………29 2-2 微熱卡計…………………………………………………31 第三章 實驗設備與方法…………………………………….36 3-1 藥品與儀器………………………………………………36 3-1-1 藥品……………………………………………………36 3-1-2 儀器……………………………………………………38 3-2 實驗的流程與方法………………………………………39 3-2-1 等溫微熱卡計之滴定…………………………………39 3-2-2 高效能液相層析儀之操作……………………………42 3-2-3 校正曲線之製作………………………………………43 3-2-4 高分子粉體製備………………………………………44 3-2-4.1 沈澱聚合法…………………………………………44 3-2-4.2 總體聚合法…………………………………………47 3-2-5 模版之再吸附測試……………………………………49 3-2-5.1 飽和吸附曲線………………………………………49 3-2-5.2 單一成份系統測試…………………………………49 3-2-5.3 多成份系統測試……………………………………50 3-2-5.4 Scatchard plot之製作……………………………50 3-2-6 自大豆粉萃取異黃酮素………………………………50 3-2-7 實際利用異黃酮素分子模版從大豆萃取液提煉異黃 酮素之效能評估…………………………………51 第四章 實驗結果與討論…………………………………….52 4-1 待測物之校正曲線製作…………………………………53 4-2 異黃酮素分子模版的設計………………………………57 4-2-1 交聯劑之選擇…………………………………………57 4-2-2 功能性單體之選擇……………………………………62 4-2-2.1 以微熱卡計量測功能性單體對於金雀異黃酮的作 用熱……………………………………………62 4-2-2.2 不同功能性單體製備的高分子粉體對金雀異黃酮 之親和性………………………………………68 4-2-3 功能性單體與模版分子之鍵結比例測定……………71 4-2-4 功能性單體與交聯劑間組成比例設定………………75 4-3 各變因對異黃酮素分子模版影響之探討………………77 4-3-1 聚合時所用之溶劑對模版效能之影響………………79 4-3-2 聚合方法對模版效能之影響…………………………83 4-3-3 再吸附溶劑環境對模版效能之影響…………………88 4-4 最佳化分子模版對於常見異黃酮素的吸附測試………90 4-4-1 飽和吸附曲線…………………………………………90 4-4-2 Scatchard Plot………………………………………99 4-4-3 異黃酮素分子模版對干擾物之吸附…………………107 4-4-3.1 獨立成份系統下異黃酮素分子模版對干擾物之吸 附………………………………………………108 4-4-3.2 多成份系統下異黃酮素分子模版對干擾物之吸附112 4-5 異黃酮素分子模版實際應用於萃取大豆異黃酮素……114 4-5-1 大豆萃取液內異黃酮素組成份析……………………114 4-5-2 分子模版於真實樣品溶液下之效能評估……………115 第五章 綜合討論…………………………………………….122 第六章 結論………………………………………………….130 參考文獻………………………………………………………133 附錄A 各高分子粉體的型態…………………………………137 附錄B 各產品內金雀異黃酮之含量…………………………140 附錄C 模版於乙醇溶液中之選擇性…………………………143 附錄D 萃取方法比較…………………………………………144

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