特定位置乙醯化葡萄甘露聚醣的製備及其對腸道菌生成短鏈脂肪酸之影響

Abstract

石斛為常用之蘭科中藥材，分布於亞洲熱帶和亞熱帶至大洋洲地區，曾被報導具有抗氧化、抗糖尿病、調控免疫及新陳代謝等活性。研究發現活性來自其葡萄甘露多醣。為了探討石斛多醣之作用機理，本研究透過餵食小鼠兩種石斛水萃並透過化學結構修飾葡萄甘露寡醣以及結構解析來了解石斛多醣如何影響生理。 為分析餵食金童石斛及金皇石斛水萃物之小鼠糞便中三種主要短鏈脂肪酸，實驗使用氣相層析，但因DB-5管柱之作用力過弱而使用了作用力較佳的DB-FFAP管柱，在低初始溫度下，解析度皆高於1.5。分析餵食金童石斛水萃物的小鼠糞便中丁酸的濃度與控制組相比下降約30%，乙酸則上升了約5%；而在金皇組中，糞便中的丁酸約上升30%，乙酸約下降25%。 在糞便腸道菌群分析中，金童組中的Allobaculum sp.及Muribaculaceae sp.的數量上升約70%，但其假長雙歧桿菌的數量卻約下降60%。由於假雙歧桿菌能夠產生大量的乳酸並進一步被代謝為丁酸，可能為金童組之丁酸大幅下降，而乙酸只有略為增加的原因。在金皇組中則發現大量的糞擬桿菌，有助於丁酸的產生，可能為金皇組丁酸含量較高之原因。 在乙醯基對活性之影響方面，本研究嘗試使用內切纖維素酶與Lipozyme TL IM和碳酸鉀來降解與乙醯化蒟蒻葡萄甘露聚醣。內切纖維素酶可將順利將其降解，而Lipozyme和碳酸鉀皆可將其乙醯化，但兩種乙醯化方法都無專一性。又由於石斛的多醣片段曾被報導與其生物活性有關而使用了史密斯降解來獲得含有乙醯基的寡醣片段。在反應完成並利用粒徑篩析層析管柱以及質譜來分析其降解完的片段之分子量後發現降解完的寡醣中有單乙醯化及雙乙醯化之質譜訊號。 本研究顯示金童石斛以及金皇石斛之水萃物會改變腸道菌群和糞便中短鏈脂肪酸比例。同時也製備了乙醯化葡萄甘露寡醣以及金童石斛水萃物之史密斯降解產物以做為往後活性測試之材料。

Dendrobium plants belong to Orchidaceae family and are distributed from tropical and sub-tropical Asia to Oceania. Dendrobium plants are commonly used in traditional Chinese medicine and were reported to possess the activity of anti-oxidant, anti-diabetes, and regulation of immune system and metabolism. Research had shown that the bioactivities are from their glucomannan. In order to discover the bioactivity mechanism of Dendrobium polysaccahrides (DPs) , mice were fed with two different Dendrobium water extracts and glucomannan were chemically modified to understand how DPs effect the physiology. To analyze the three main short chain fatty acids (SCFAs) in the feces from the mice fed with Dendrobium Cassiope water extract (DC-PS) and Dendrobium Taiseed Tosnobile water extract (DTT-PS). Gas spectrometry were used as the analytical instrument and DB-FFAP as the GC column instead of DB-5 beacuse of the strong interaction in FFAP. With the lower intial temperature, all resolutions are greater than 1.5. After analyzing the feces from the mice fed with DC-PS, a decrease of 30% and an increase of 5% in butyrate and acetate concentration were found. And in DTT-PS group, there was an increase of 30% in butyric acid, and a decrease of 25% in acetic acid. The gut microbiota analysis showed that the abundance of Allobaculum sp. and Muribaculaceae sp. in DC-PS group all increased 70% while Bifidobacterium pseudolongum decreased 60%. As B. pseudolongum produces considerable amount of lactic acid, and can be further transformed into butyric acids, this could be the reason for the large decreasing and minor increasing in butyric acid and acetic acid. DTT-PS group on the other hand, large amount of Bacteroides caccae were observed. B. caccae could be the reason for the increasing of butyric acid because of its assistance for the production in butyric acid. And considering the relationship between acetylation and bioactivity, endo-glucanase and Lipozyme TL IM or K2CO3 were used to degrade and acetylate konjac glucomannan (KGM). The degradation and acetylation were all successful. However, both acetylation methods had no selectivity. Fragments of Dendrobium polysaccharides were reported to be related the bioactivity, Smith degradation was used to obtain the acetylated oligosaccharides. After the reaction, the fragments were analyzed by SEC column and MS. The result showed there are mono- and di-acetylation on the oligosaccharides. In this research, the water extracts of DC and DTT were shown to changed the ratio of gut microbes and SCFAs. Acetylated oligo-glucomannan and the Smith degradation product of DC-PS were prepared for further biological test.