愛玉子瘦果渣水萃物中果膠酯酶抑制物之研究

Abstract

本研究從愛玉子瘦果渣分離其中之果膠酯酶抑制物，並探討其理化性質，同時建立一快速檢測抑制果膠酯酶活性之測定方法，用以輔助追蹤愛玉子中果膠酯酶抑制物之化學組成。 以甲基紅及亞甲基藍混合作為指示劑，利用其於鹼性下呈現綠色，酸性則為紫紅色之特性，發現此指示劑於果膠溶液中之OD 527 nm變化量與反應液中pH之變化量呈現良好之線性關係，故可做為檢測果膠酯酶活性抑制程度之指示劑。 將愛玉子瘦果渣以水均質萃取，加入等體積3%之NaCl後，破壞乳化相去除部分雜質，再經由90%丙酮沉澱去除不可溶之蛋白質，將丙酮上清液減壓濃縮去除丙酮後，通過HP-20管柱進行疏水性層析區分，結果顯示甲醇流洗出的區分具有最顯著的抑制活性，可有效分離愛玉子中果膠酯酶抑制物。 愛玉子中果膠酯酶抑制物即使經過90%丙酮處理後，仍在上清液顯示具有抑制活性，且具有高耐熱性，推測此抑制物之成份並非多肽；此外，此抑制物可被PVPP所吸附，並經酸水解後會失去抑制活性且酸水解後之游離胺基酸之含量並無增加，推測此抑制物可能為酚類化合物；依其與蛋白質水溶液混合後會產生沉澱之特性，與HPLC圖譜hump-like之現象，推論其可能為單寧類化合物。以HPLC分析此抑制物之酸水解產物，發現水解物中出現有鞣花酸﹙ellagic acid﹚單元，推測此果膠酯酶抑制物可能屬於一種可水解之鞣花酸單寧。 此外，90%丙酮可溶之PEI水溶液﹙PEI-AS﹚經HP-20管柱流洗所得之甲醇區分物可抑制枯草桿菌微生物之a-amylase及牛胰蛋白脢﹙trypsin﹚之活性，但是對豬脂解酶﹙lipase﹚活性則有促進之效果。

In this study, we isolated pectinesterase inhibitors (PEI) from aqueous extract of jelly fig (Ficus awkeotsang Makino) residue, which is the by-product of jelly curd making and studied on the physico-chemical characteristics of the pectinesterase inhibitors. In addition, we developed a simple and effective procedure to monitor the the change of pH value in the reaction solutions by means of using a devised indicator of methylene blue and methyl red for tracing the fractions of PEI during the isolation processes. The process for isolating PEI from jelly fig residue was firstly conducted by extraction with 12 fold of distilled water (w/v). Then, the same volume of 3% NaCl was added into the extracted solutions. After centrifugation, a clear crude extract solution of PEI was obtained. The further fraction of PEI from crude extract solution was preceded by adding volumes of acetone into the extract for removing the protein of materials. Following removing acetone under vacuum, the 90 % acetone-soluble aqueous solution was subjected into a Diaion HP-20 column. The most level of PEI fraction was obtained from methanol elution of the hydrophobic chromatography. Acroding to the physico-chemical characteristics of PEI tested in this study, the PEI of jelly fig achenes was supposed as belonging to a polyphenolic component. Furthermore, the results from HPLC pattern profile, protein-binding capability and analysis of hydrolysate were revealed that the PEI of jelly fig achenes was considered as being a novel tannin. Besides, we found out the PEI also inhibited the activities of a-amylase (bacillus spp.) and trypsin (bovine pancreas), but had a enhanced effect toward lipase (porcine pancreas).