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研究生:蕭名傑
研究生(外文):Ming-jie Siao
論文名稱:應用製備級液相層析術純化大豆萃取物中的異黃酮素
論文名稱(外文):Purification of Isoflavones from Soybean Extracts by Preparative Liquid Chromatography
指導教授:賴世明賴世明引用關係
指導教授(外文):Shih-ming Lai
學位類別:碩士
校院名稱:國立雲林科技大學
系所名稱:工業化學與災害防治研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:124
中文關鍵詞:大豆萃取物製備級液相層析術純化異黃酮素
外文關鍵詞:purificationpreparative liquid chromatographysoybean extractsisoflavones
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異黃酮素為大豆中的重要成份,具有預防癌症與骨質疏鬆症等功效,且有助於膽固醇之降低,因此可作為健康食品及醫療藥品。目前市售純的葡萄糖配醣體(glucosides)異黃酮素Daidzin(D)、Genistin(G)、Glycitin(Gly),因價格昂貴而不易取得。本研究藉由市售的大豆萃取物所含葡萄糖配醣體(glucosides)異黃酮素(約占14 %),製備生產出高純度(>90%)、高回收率(>80%)的異黃酮素Daidzin、Genistin、Glycitin之標準品,以供定量色層分析異黃酮素之使用。
研究結果顯示,在大豆萃取物中異黃酮素的進一步濃縮方面,以大豆萃取物稱約150 mg轉溶於30 ml甲醇中,在純度方面,可達到大於7%以上的D、大於4%以上的Gly以及大於9%以上的G,在回收率方面,均可達到90%以上的回收率。第一階段製備級純化G的系統,以逆相操作的水(1%醋酸)/甲醇系統為最佳的條件,在操作量方面,可達到5 mg/ml的注入濃度以及10 ml的注入體積,在純度方面,可達到大於96%以上的G,在回收率方面,可達到93%以上的回收率,在產量方面,經過五次注入濃縮大豆萃取物(250 mg),可得到G約23 mg的產率。第二階段製備級純化D與Gly的系統,以正相操作的正己烷(0.1%醋酸)/乙酸乙酯系統為較佳的條件,在操作量方面,可達到0.2 mg/ml的注入濃度以及10 ml的注入體積,在純度方面,可達到大於95%純度的D與大於90%純度的Gly,在產量方面,經過十次注入第一階段所收集的D與Gly混合產物(20 mg),可得到D約12 mg的產率與Gly約2 mg的產率,在回收率方面,D的回收率較佳,而Gly回收率甚差(約30%)。第二階段有必要再尋找更佳的層析系統來改善產率以及回收率。
Isoflavones are considered to be important fractions in soybean. They possess the functions of preventing cancer and osteoporosis and reducing the cholesterol levels in the human body, so they can be used as nutrition foods and medicines. The pure Daidzin(D), Genistin(G), and Glycitin(Gly) of isoflavone glucosides commercially available are very expensive and not easy to get. This study preparatively produced the pure isoflavone glucosides D, G, and Gly with high purity (>90%) and high recovery (>80%) from commercial soybean extracts containing about 14% isoflavone glucosides, which can be used as the standards in quantitatively chromatographic analysis of isoflavones.
The results show that, for the further concentration of isoflavones in soybean extracts, dissolving 150 mg soybean extracts in 30 ml methanol, the purities of D, Gly, and G of 7+%, 4+%, and 9+%, respectively, and the recovery of 90+% for each component were reached. For the first stage of preparative purification of G, the optimal chromatographic separation system was found to be the water (1% acetric acid) / methanol system of reversed phase. Injecting the sample with a concentration of 5 mg/ml and a sample size of 10 ml, the purity of G of 96+% and the recovery of G of 93+% were reached. After five times of the above injection (250 mg soybean extracts), the yield of G was about 23 mg. For the second stage of preparative purification of D and Gly, the appropriate chromatographic separation system was found to be the n-hexane(0.1% acetric acid) / ethyl acetate system of normal phase. Injecting the sample with a concentration of 0.2 mg/ml and a sample size of 10 ml, the purities of D and Gly of 95+% and 90+%, respectively, and the recoveries of D and Gly of 99+% and about 30%, respectively, were reached. After ten times of the above injection (20 mg of the mixture of D and Gly), the yields of D and Gly were about 12 mg and 2 mg, respectively. The chromatographic separation system in the second stage needs to be further improved in order to enhance the recovery and the yield.
中文摘要----------------------------------------------------------------- i
英文摘要----------------------------------------------------------------- ii
目錄--------------------------------------------------------------------- iv
表目錄------------------------------------------------------------------- vi
圖目錄------------------------------------------------------------------- vii
符號說明----------------------------------------------------------------- ix
一、緒論----------------------------------------------------------------- 1
1.1 前言----------------------------------------------------------- 1
1.2研究動機-------------------------------------------------------- 3
1.3研究方向-------------------------------------------------------- 3
二、文獻回顧與理論部分--------------------------------------------------- 5
2.1大豆異黃酮的概述------------------------------------------------ 5
2.1.1 大豆異黃酮的組成----------------------------------------------- 5
2.1.2 大豆異黃酮的藥理功效------------------------------------------- 8
2.2 分析與分離純化------------------------------------------------- 9
2.2.1 分析方法------------------------------------------------------- 9
2.2.2 分離純化方法--------------------------------------------------- 9
2.3 製備級液相層析術----------------------------------------------- 13
2.3.1 液相層析的分類------------------------------------------------- 14
2.3.2 製備級液相層析的分離機制--------------------------------------- 14
2.3.3 製備級液相層析的操作模式--------------------------------------- 23
2.4 沖提層析法量測分離解析度--------------------------------------- 29
2.5 濃縮純化性能--------------------------------------------------- 30
三、實驗部分------------------------------------------------------------ 32
3.1 材料與藥品----------------------------------------------------- 32
3.2 大豆異黃酮的分析----------------------------------------------- 34
3.2.1 設備與儀器----------------------------------------------------- 34
3.2.2 實驗方法與步驟------------------------------------------------- 34
3.2.2.1 五種異黃酮素標準品檢量線的製作--------------------------------- 34
3.2.2.2 大豆萃取物之HPLC定性與定量分析--------------------------------- 36
3.3 階段純化異黃酮素的策略----------------------------------------- 38
3.4 大豆萃取物中異黃酮素的進一步濃縮------------------------------- 40
3.4.1 設備與儀器----------------------------------------------------- 40
3.4.2 實驗方法與步驟------------------------------------------------- 40
3.5 製備級液相層析術純化異黃酮素----------------------------------- 41
3.5.1 設備與儀器----------------------------------------------------- 41
3.5.2 實驗方法與步驟------------------------------------------------- 41
3.5.2.1 第一階段純化--------------------------------------------------- 41
3.5.2.2 第二階段純化--------------------------------------------------- 44
3.6 其他設備與儀器------------------------------------------------- 46
四、結果與討論---------------------------------------------------------- 47
4.1 大豆萃取物之HPLC定性與定量分析(未經水解與經水解步驟所得分析結果
的比較)-------------------------------------------------------- 47
4.2 大豆萃取物中異黃酮素的進一步濃縮------------------------------- 47
4.3 第一階段純化--------------------------------------------------- 50
4.3.1 最佳層析系統的選取--------------------------------------------- 54
4.3.2 Genistin的製備級純化------------------------------------------- 54
4.3.3 第一階段純化效果總結------------------------------------------- 70
4.4 第二階段純化--------------------------------------------------- 71
4.4.1 最佳層析系統的選取--------------------------------------------- 72
4.4.2 Daidzin、Glycitin的製備級純化---------------------------------- 72
4.4.3 第二階段純化效果總結------------------------------------------- 82
五、結論----------------------------------------------------------------- 86
六、參考文獻------------------------------------------------------------ 90
附錄A-------------------------------------------------------------------- 94
附錄B-------------------------------------------------------------------- 99
附錄C-------------------------------------------------------------------- 104
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