山苦瓜萃取暨其酵素水解最佳化與三萜類成分之探討

Optimization of bitter melon processing including enzyme hydrolysis and investigation of triterpenoids in Momordica charantia

林德岳(Te-Yueh Lin)

指導教授 ： 黃青真

國立臺灣大學/生命科學院/生化科技學系/碩士(2013年)

https://doi.org/10.6342/NTU.2013.00793

摘要

糖尿病是目前影響人類健康重大的公共醫藥衛生問題，此種代謝性疾病常導致許多嚴重併發症。文獻指出苦瓜具有改善血糖調節之潛力，其中所富含之三萜類，為近年研究所聚焦之活性化合物，尤其是苷元形式的三萜類化合物。唯此類化合物多數以帶一個或多個醣基之型式存在於苦瓜中。本研究目的首在探討山苦瓜中去醣基水解酶之酵素活性與特性，並建立其蛋白質純化方法；其次在探討山苦瓜經過不同條件酵素處理所得樣品中三萜類化合物之譜型。
在固定條件下，山苦瓜蛋白質粗抽物其 β-glucosidase 活性表現優於cellu- lase及a-glucosidase。選定β-glucosidase 經由硫酸銨沉澱、離子交換與膠體過濾等步驟，進行蛋白質純化。取電泳分離之蛋白質色帶進行LC-MS/MS分析，預測片段之序列。其次，利用 UPLC-MS 分析預測，山苦瓜萃取物於外加酵素水解所得樣品中各種三萜類化合物之譜型。分析山苦瓜以冷水 (CWE) 於室溫萃取之樣品，發現能夠產生許多帶醣與不帶醣之三萜類化合物，顯示萃取過程中山苦瓜內源性酵素有發生作用。接著探討三種市售去糖基酵素 amyloglucosidase (ag), β-glucosidase (bg) 與 cellulase (cel) 分別 (ag, bg, cel) 或組合cel+bg (cb) 對於山苦瓜萃取物之作用，將這些酵素分別添加於苦瓜之甲醇或正丁醇萃取物進行水解反應，並取其乙酸乙酯區分物進行分析。結果顯示，甲醇萃物以 amyloglucosidase (ag) 的酵素水解，在質譜分析結果顯示能夠產生較多的三萜類化合物。基於未加酵素之對照組似亦有較多的三萜類化合物，乃依對照組反應條件進行山苦瓜粉萃取，即分成：中溫 (MWE, 37℃) 或高溫 (HWE, 60℃)，又各分成是否調整 pH 值以及外加酵素與否等處理，嘗試找出最簡便經濟且能促進苦瓜內源性酵素活性，提高山苦瓜水萃取物中苷元形式的三萜類化合物含量之處理條件。結果以 HWEa 與 MWE 能夠產生較多的三萜類化合物，外加酵素於這些水萃取物中，並未能夠產生更多的三萜類化合物。
綜之，本研究是第一個確認苦瓜具有β-glucosidase, cellulase , α-glucosidase 等酵素活性，並初步建立了β-glucosidase 純化方法。也發現天然之花蓮四號山苦瓜可能含有 3β-hydroxycucurbita-5(10),6,22(E),24-tetraen-19-al, (23E)-cucurbita- 5, 23, 25-triene-3,7-dione, 5β,19-epoxy-3β,25-dihydroxycucurbita-6,23(E)-diene, Goyagly- cosides (151), (23E)-5β,19-epoxycucurbita-6,23-diene-3β,25-diol, Kara- vilagenin E, oleanolic acid, 25-oxo-27-normomordicoside L, momordicosed F2 及 momordico- side I 等三萜類化合物。以水進行山苦瓜萃取時，在水萃取物中外加酵素的去醣基效果並不顯著，可能是需要外加更多的酵素，才能有顯著的效果；其中以 HWEa (高溫加酸水萃取處理) 與 MWE (中溫水萃取處理) 兩種處理方法能夠更簡便經濟地產生較多苷元形式的三萜類化合物。也可能這種處理能夠藉由促進山苦瓜內源性酵素的活性，產生更多的三萜類化合物，進一步提升山苦瓜降血糖的活性。

關鍵字

山苦瓜 ； 降血糖 ； 三萜類 ； 酵素水解 ； β-glucosidase

並列摘要

Type 2 Diabetes mellitus has been an increasing public health concern. This meta- bolic disorder diagnosed by hyperglycemia could lead to serious complications. Bitter melon (Momordica Charantia, BM) has been shown to improve glucose dys-regula- tion and recent reports focused on its triterpenoids, especially aglycone forms, as ac- tive compounds. This study aims to examine endogeneous deglycosidation enzyme activity of BM, to isolate and characterize β-glucosidase enzyme from BM, and to ex- amine the triterpenoids profiles in BM extracts subjected to deglycosidation enzyme treatments.
Using specific substrates and a fixed assay condition, BM extract showed acti- vity of β-glucosidase, cellulase and a-glucosidase. The β-glucosidase enzyme of BM was purified by ammonium sulfate precipitation, ion exchange and gel filtration chromatography. The isolated protein band from electrophoresis was analyzed by LC-MS/MS and partial sequence predicted. Furthermore, the triterpenoids profiles of BG extracts subjected to deglycosilation enzyme treatments were examined using UPLC and QTOF-MS and predicted by the software based on a self-constructed BM triterpenoid database. When analyzing the BG water extract prepared by cold water (CWE), we found out that much triterpenoids (including aglycons and glycosides) were found in CWE, suggesting the BM endogenous enzyme might be effective. Next, three commercial enzymes, amyloglu- sidase (ag), β-glucosidase (bg), and cellulase (cel) were used to treat methanol or butanol extracts of BM, individually (ag, bg, cel) or cel + bg successively (cb), and the ethyl acetate (EA) partition of extracts analyzed. Among the enzyme treated samples, the methanol extracts treated with amyloglucosidase (ag) showed highest amount of triterpenoids. As some of the control samples (no enzyme treatment in incubations) also had high triterpenoid profile, water extracts were further prepared at medium (MWE, 37℃) or high temperature (HWE, 60℃) with or without the adjust- ment of pH (pH = 5), and with or without exogeneous enzymes (ag, cb). Analysis of the EA extracts of these samples revealed that MWE and HWE had higher triterpe- noids. Exogeneous enzymes did not increase triterpenoids.
This is the first study that demonstrated glycoside hydrolase activities in BM, and the β-glucosidase was partially purified. Also the following triterpenoids were predicted in the Hualien No.4 wild bitter melon used in this study. 3β-hydroxycucur- bita-5(10),6,22(E),24-tetraen-19-al, (23E)-cucurbita-5,23,25-triene-3,7-dione, 5β,19- epoxy-3β,25-dihydroxycucurbita-6,23(E)-diene, Goyaglycosides (151), (23E)-5β,19- epoxycucurbita-6,23-diene-3β,25-diol, Karavilagenin E, oleanolic acid, 25-oxo-27- normomordicoside L, momordicosed F2 and momordicoside I. Extraction method of HWEa and MWE might be a good choice to obtain more aglycone triterpenoids.

並列關鍵字

Momordica charantia ； bitter melon ； bitter gourd ； hypoglycemic ； triterpenoids ； enzyme hydrolysis ； β-glucosidase

參考文獻

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