金童石斛多醣與乙醯化衍生物對高脂飲食小鼠生理數值與腸內菌相之影響

Effect of the polysaccharides from Dendrobium Cassiope and their acetylated derivatives on the physiological indicators and gut microbiota of high-fat diet-fed mice

陳玄燁(Hsuan-Yeh Chen)

指導教授 ： 張嘉銓

國立臺灣大學/醫學院/藥學研究所/碩士(2022年)

https://doi.org/10.6342/NTU202203932

摘要

金童石斛為金釵石斛與銅皮石斛的雜交種，其特徵為生長快速以及富含水溶多醣。過去本實驗室成員已鑑定出金童石斛的水溶性多醣結構為部分乙醯化之葡萄甘露多醣，並且在正常飲食小鼠模型中顯出具有降血糖功效；此外，有研究表示蒟蒻的葡萄甘露多醣經乙醯化後，促進細胞素分泌的活性顯著上升，暗示著多醣活性與其乙醯基團間可能有所關聯。
為了探討多醣乙醯基、腸內菌及降血糖活性間的關係，本研究將高脂飲食 (HFD) 添加金童石斛粗多醣 (DC-PS) 或其乙醯化衍生物 (DC-acPS) 後餵予實驗小鼠。DC-PS是從金童石斛水萃物以酒精沉澱所得，再透過與乙酸酐以及吡啶反應48小時得到DC-acPS。藉由1H NMR與HSQC解析，確認DC-acPS在甘露醣基的2、3與6號位發生乙醯基取代。
24隻C57BL/6J小鼠被分為四組：HFD、HFD+DC-PS、HFD+DC-acPS及控制組，餵養期間紀錄生理數值，並在9週後犧牲並收集其肝臟、脾臟、血漿、白色脂肪組織、大腸與糞便進行分析。相較於控制組，多醣餵食組小鼠之大腸顯著增長；此外，餵食DC-acPS的小鼠有最低與穩定的空腹血糖。在糞便短鏈脂肪酸方面，餵食4週與9週的DC-PS變化不大，而餵食DC-acPS的小鼠其主要短鏈脂肪酸在這5週間增加1.3~3.5倍，且第9週時有組間最高的丁酸濃度。以次世代定序分析小鼠糞便菌叢的16S rRNA基因V3-V4區段，DC-acPS組的小鼠相較HFD組小鼠，毛螺菌科 (Lachnospiraceae) 與瘤胃菌科 (Ruminococcaceae) 之相對豐度增加1.8與3.2倍，而丹毒絲菌科 (Erysipelotrichaceae) 減少3.1倍。然而，各試驗組在G-CSF表現量上沒有差異，且多醣餵食組的小鼠在葡萄糖耐受性，以及肝臟損傷狀況相較於HFD組的小鼠更差。
本研究透過化學修飾增加金童石斛多醣之乙醯化度，發現能對高脂飲食小鼠的生理狀況與腸內菌相產生不同程度的影響，顯示出乙醯基與其活性的關聯。

關鍵字

金童石斛 ； 葡萄甘露多醣 ； 乙醯化 ； 高脂飲食

並列摘要

Dendrobium Cassiope, the hybrid of D. nobile and D. moniliforme, is characterized by the fast growth rate and high polysaccharide production. In the previous study, we found that the bioactive polysaccharide extracted from D. Cassiope (DC-PS) was composed of 1,4-β-glucomannan with partial acetylation and showed hypoglycemic activity on normal diet mice. Moreover, some researches showed that the konjac glucomannans with high degree of acetylation enhanced higher cytokines expression than that of normal konjac glucomannans, and revealed possible relationships between the bioactivity and the acetyl groups of polysaccharides.
To investigate the relationship among O-acetyl group of polysaccharide, gut microbiota and anti-hyperglycemic effect. The mice were fed with high fat diet (HFD) contained DC-PS or its acetylated derivatives (DC-acPS). The DC-PS was isolated from the water extract of D. Cassiope stem by ethanol precipitation, then reacted with acetic anhydride and pyridine for 48 hour to produce DC-acPS. The results of NMR show that DC-acPS was acetylated at the 2, 3 and 6 positions of mannosyl residues.
Twenty-four male C57BL/6J mice were separated into 4 groups: normal diet, HFD only, HFD with DC-PS, and DC-acPS. Physiological data were recorded during the feeding period. After 9 weeks, liver, spleen, serum, pancreas, white adipose tissue, colon and feces were collected after sacrifice. Compared with the control group, the polysaccharide-treated groups were with significantly longer values in colon length. Furthermore, the DC-acPS group showed the most stable and lowest fasting blood sugar level. The results of fecal short-chain fatty acids (SCFAs) analysis show that there were little difference after fed with DC-PS for 4 and 9 weeks; in contrast, the mice fed with DC-acPS showed a 1.3 to 3.5-fold elevation of SCFAs between 5 weeks, especially butyric acid. In the sequencing analysis of fecal bacterial 16S rRNA gene V3-V4 region, the DC-acPS group had 1.8 and 3.2 times the relative abundance of Lachnospiraceae and Ruminococcaceae, and 0.3 times the Erysipelotrichacea compared to HFD group. However, the expression of G-CSF showed less difference between each HFD-treated group, and polysaccharide-treated group had the worse glucose tolerance and liver damage statue than HFD group.
In this research, polysaccharide from D. Cassiope was acetylated by chemical modification, and showed a different influence on physiology statue and gut microbiota of HFD mice. These results indicate the relationship between acetyl group and bioactivity.

並列關鍵字

Dendrobium Cassiope ； glucomannan ； acetylation ； high-fat diet

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