大豆異黃酮定量方法之改良及加工方式對大豆異黃酮種類轉換之研究

Abstract

大豆及大豆製品為大豆異黃酮的主要攝取來源，大豆異黃酮為植物雌激素的一種，目前認為對於人體健康方面有保健效果。已有關於大豆異黃酮定量方法已有許多報告，一般係使用乙腈水溶液萃取大豆異黃酮。然後在30℃下真空濃縮後再覆溶於80%甲醇水溶液中，定容後以HPLC進行定量分析，此種樣品製備方法複雜、耗時、容易造成含量被低估、標準偏差大的缺點。在本篇研究係探討一種較為迅速且較為簡易的萃取方法以用於大豆食品中的異黃酮定量分析。在本研究中係利用65%甲醇水溶液作為萃取溶劑，選用benzoic acid或fluorescein化學安定性高的化合物作為定量所需的內標準品，萃取後直接取部份萃取液離心取上清液部分以高效率層析儀分析定量；並可同時選用兩種內標準品雙重確認（double check）。本方法有過程較簡便，且再現性高的優點。65%甲醇水溶液為萃取大豆異黃酮的最佳溶劑，萃取1小時便可達最佳的萃取率。 目前大部份的研究報告中所建立的定量方法，主要都是針對大豆中全部12種異黃酮，針對12種個別異黃酮進行分析定量相當耗時且不經濟，在本研究並建立一種異黃酮總量的快速定量方法。此方法之優點為不需準備全部12種大豆異黃酮的標準品且可以縮短分析所需的時間。根據本研究，此方法係使用1.0 M sodium methylate在60℃下與樣品中之acetylglycoside、malonylglycoside反應20分鐘，將其轉化為7-O-glycoside，反應完成後以2.5N鹽酸水溶液中和，調整溶液為80%甲醇溶液並在60℃下繼續萃取30分鐘然後進行HPLC分析；由於僅需要分析兩種形式的大豆異黃酮化合物（aglycones及7-O-glycosides），因此可以有效地縮短分析時間至30分鐘以內。本研究中所提出之總異黃酮含量的定量方法為目前相關研究中最迅速的定量方式。 由於malonylglycoside、acetylglycoside無法被人體有效吸收，且不能被微生物代謝轉化，因此研究之另一目的為探討將這類型的大豆異黃酮化合物轉化為生物可利用性較高之7-O-glycosides、aglycones的方法。結果發現在加熱的情況下會造成12種大豆異黃酮的組成改變，於40℃作為加工條件時，alycone為組成份中主要的形式，約佔大豆異黃酮總含量的67%；於90℃的加熱時，則以glycoside為主要的形式，佔總量的69%。

Soybean and soy products are the main sources of isoflavones, a well-studied group of phytoestrogens with numerous biological effects. Several methods have bean published to quantify soy isoflavones in food samples. The aqueous acetonitrile solutions are the most frequently used solvent for isoflavone extraction. However, for some reasons, the extraction should be concentrated to almost dryness by vacuum evaporation at a temperature not higher than 30℃, and then re-dissolved in a different solvent rather than the primary extraction solvent. No doubt, the complicated extraction process is time-consuming, and will cause underestimation as well as a quite large standard deviation. In this study, we developed a rapid and simple method for the quantification of isoflavones in soybean foods. The method includes the extraction of isoflavones from soybean foods by using 65% aqueous methanol containing a chromatographically suitable and chemically stable internal standard (benzoic acid and/or fluorescein) as the extraction solvent, and clarifying an aliquot of extract by microcentrifugation , followed by injection of the supernatant for HPLC analysis. The solvent has a high efficiency for isoflavone extraction, and it reached the maximum extraction yield of isoflavones in one hour. The standard deviation is very small due to the simplicity of extraction process before HPLC analysis. Several methods have been published to quantify all 12 kinds of isoflavone compounds in soybean or soy foods. However, it is time-consuming, and needs many kinds of authentic isoflavone compounds who do not need to collect information about the conjugate pattern in soy samples. Besides building up the extraction method for all 12 forms of isoflavones, in this study, I developed a new quantitative method for determination of total amount of isoflavones. According to our method, we can quantify the total amount of isoflavone more rapid and conventiently without preparing all 12 kinds of standards.In this study, acetyl- and malonyl- glycosides were converted to 7-O-glycosides by reacting with 1M methanolic sodium methylate at 60℃ for 20 min. Then, the solution was neutralized with 2.5N HCL and extracted for further 30 min at 60℃. Aglycones and 7-O-glycosides were then analysed by HPLC. The total analytical time of this method is shorter than any other published quantitative methods. Because the malonyl- and acetyl- glycoside could not bean absorbed by human or metabolized by microorganism. We decided to convert those kinds of isoflavone compounds to more bioavailble forms like 7-O-glycosides or aglycones. I treated the processing temperature on the conversion of isoflavones. In this sudy, I found that the composition of 12 forms of isoflavones was changed during heating in water bath. When processing at 40℃, the isoflavones were mainly composed by aglycones (67%). Besides it, the glycosidic forms are the main composition (56%) when processing at 80℃.