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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張嘉銓(Chia-Chuan Chang) | |
| dc.contributor.author | Hsuan-Yeh Chen | en |
| dc.contributor.author | 陳玄燁 | zh_TW |
| dc.date.accessioned | 2023-03-19T22:09:45Z | - |
| dc.date.copyright | 2022-10-13 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-09-26 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/84375 | - |
| dc.description.abstract | 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。 | zh_TW |
| dc.description.abstract | 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. | en |
| dc.description.provenance | Made available in DSpace on 2023-03-19T22:09:45Z (GMT). No. of bitstreams: 1 U0001-2309202216324600.pdf: 6041674 bytes, checksum: 1ef7eaa6c32954f4b9d9dd36eade5d66 (MD5) Previous issue date: 2022 | en |
| dc.description.tableofcontents | 總目錄 (List of contents) 口試委員審定書 I 致謝 (Acknowledgement) II 中文摘要 III Abstract IV 總目錄 (List of contents) VI 流程圖目錄 (List of schemes) IX 表目錄 (List of tables) IX 圖目錄 (List of figures) IX 附圖目錄 (List of supporting figures) XI 詞彙縮寫 XII 1. 緒論 1 1.1 前言與研究目的 1 1.2 植物簡介 2 1.3 O-乙醯化多醣 (O-acetylated polysaccharide) 4 1.3.1 生合成途徑 4 1.3.2 乙醯基對多醣活性之影響 6 1.4 腸內菌簡介 8 1.4.1 腸內菌與飲食 10 1.4.1.1 膳食纖維 10 1.4.1.2 蛋白質 15 1.4.1.3 脂肪 17 2. 實驗部分 20 2.1 儀器與材料 20 2.1.1 理化性質測定儀器 20 2.1.2 分離及分析用之儀器與材料 20 2.1.3 生化性質分析儀器 20 2.1.4 試劑與溶媒 21 2.1.5 生物試驗試劑 22 2.1.6 PCR引子 23 2.2 金童石斛多醣樣品之製備與定性 24 2.2.1 植物來源 24 2.2.2 金童石斛多醣之萃取 24 2.2.3 金童石斛多醣之乙醯化 24 2.2.4 核磁共振 (NMR) 圖譜測定 25 2.2.5 羥胺-氯化鐵法測定乙醯化度 26 2.2.6 皂化法測定乙醯化度 27 2.2.7 動物飼料之製備 29 2.3 動物實驗模式 30 2.3.1 實驗動物 30 2.3.2 動物實驗設計之時間軸 30 2.3.3 空腹血糖與血糖耐受性測定 31 2.3.4 GOT與GPT測定 31 2.3.5 組織切片 32 2.3.6 糞便菌叢分析 32 2.3.6.1 糞便中細菌genomic DNA (gDNA) 萃取 32 2.3.6.2 聚合酶連鎖反應 (PCR) 33 2.3.7 西方墨點法 34 2.3.7.1 緩衝溶液配製 34 2.3.7.2 蛋白質萃取 34 2.3.7.3 蛋白質定量 35 2.3.7.4 SDS-PAGE、轉印與抗體反應 35 2.3.8 短鏈脂肪酸含量測定 36 2.3.9 統計方式 37 3. 實驗結果與討論 38 3.1 DC-acPS結構 38 3.1.1 一維及二維核磁共振圖譜解析 38 3.1.2 乙醯化度測定 43 3.1.2.1 羥胺-氯化鐵法 43 3.1.2.2 皂化法 45 3.2 DC-PS與DC-acPS對高脂飲食小鼠生理之影響 48 3.2.1 生理數值變化與比較 48 3.2.2 解剖構造 50 3.2.3 組織切片 51 3.3 以西方墨點法偵測G-CSF表現量 53 3.4 短鏈脂肪酸含量測定 54 3.5 DC-PS與DC-acPS對高脂飲食小鼠腸道菌相之影響 58 3.5.1 糞便中細菌gDNA萃取 58 3.5.2 α多樣性分析 60 3.5.2.1 Observed species分析 60 3.5.2.2 Chao1指數分析 60 3.5.2.3 Shannon指數分析 61 3.5.2.4 Phylogenetic diversity分析 62 3.5.3 β多樣性分析 63 3.5.3.1 Bray-Curtis相異度 63 3.5.4 腸內菌比例差異 65 3.6 討論 72 參考文獻 77 附錄 90 流程圖目錄 (List of schemes) Scheme 1. Extraction of DC-PS and preparation of DC-acPS. 25 Scheme 2. Schematic representation of treatment groups and timeline for mice admini-stration. 31 Scheme 3. Proposed relationship between each phenomenon observed in the experiment. 73 表目錄 (List of tables) Table 1. Traits of selected medicinal Dendrobium species. 3 Table 2. Fiber substrates, their dietary source and SCFA-producing bacteria. 14 Table 3. PCR reagents. 33 Table 4. PCR program setting. 34 Table 5. DS values of DC-PS-deae and DC-PS obtained at different reaction time. 44 Table 6. The degree of acetylation of DC-acPS from different batches measured by saponification assay. 47 Table 7. Concentration and purity of microbial gDNA after extraction and PCR. 58 Table 8. Comparison of results between DC-PS and DC-acPS. 74 圖目錄 (List of figures) Figure 1. Appearance of Dendrobium Cassiope ’Taiseed Golden Boy No.1’. 2 Figure 2. Proposed structure of DC-PS1. 4 Figure 3. Model of the wall polysaccharides O-acetylation system in various organisms. 5 Figure 4. Effects of PSG and its derivatives on pinocytic activity of macrophages. 6 Figure 5. Effect of desulfated, deacetylated and desulfated–deacetylated CAF on NO production in RAW 264.7 cells. 7 Figure 6. Microbiota in the different sections of human lower gastrointestinal tract. 8 Figure 7. Temporal dietary modulation of the gut microbiota. 9 Figure 8. Simplified pathway of bacterial SCFAs formation. 11 Figure 9. Metabolism of SCFAs from dietary fiber to systemic circulation. 11 Figure 10. SCFA cellular signaling pathway. 13 Figure 11. Signaling cascade of microbial metabolites of aromatic amino acids. 16 Figure 12. Metabolic pathway of PUFA by L. plantarum AKU 1009a. 17 Figure 13. Main associations between dietary fat, gut microbiota, and metabolic markers. 19 Figure 14. Reaction of hydroxamic acid with the esters and ferric chloride in sequence. 26 Figure 15. Linear calibration curve for the saponification assay. 28 Figure 16. The calorie distribution of two different chows. 29 Figure 17. Design of Fuji Dri-Chem Slide 31 Figure 18. Linear calibration curve for protein quantification. 35 Figure 19. 1H NMR spectra of four batches of DC-acPS and DC-PS. 38 Figure 20. HSQC spectra of DC-PS-deae and DC-PS. 40 Figure 21. M2 and M3 effect in partially acetylated 1,4-β-mannotriose. 41 Figure 22. HSQC spectra of DC-acPS overlapped with DC-PS and per-O-acetylated konjac glucomannan from literature 42 Figure 23. Linear calibration curve of different reaction time for hydroxylamine-ferric trichloride assay and relationship between amounts of sugar unit and relative change of OD500 from reaction for 20 minutes to 1440 minutes. 44 Figure 24. 1H NMR spectra of deacetylated DC-PS-deae and DC-acPS. 45 Figure 25. ATR-FTIR spectra of DC-PS-deae and DC-acPS before and after alkali treatment.. 46 Figure 26. Food intake and body weight gain of each groups during the period.. 48 Figure 27. Effects of DC-PS and DC-acPS administration on blood glucose parameters in HFD-fed mice. 49 Figure 28. GPT and GOT of each group at the 12th week. 50 Figure 29. Colonic length and adipose weight were measured after sacrifice and the macroscopic liver images. 51 Figure 30. H&E stained liver tissue from mice fed with pure high fat diet or accompanied with DC-acPS or DC-PS, and fed with normal diet.. 52 Figure 31. G-CSF expression in the spleen of HFD (with/without DC-acPS or DC-PS) and normal diet mice measured by western blot. 53 Figure 32. The SCFAs concentration in feces of mice fed with HFD (with/without DC-PS or DC-acPS) and normal diet for 4 and 9 weeks 55 Figure 33. Concentration of major SCFAs in feces and ceca and ratio of major SCFAs in ceca of mice fed with HFD 57 Figure 34. Result of gel electrophoresis. 59 Figure 35. Total number of bacterial species in the feces of mice fed with HFD (with/without DC-PS or DC-acPS) and normal diet. 60 Figure 36. The Chao1 indices of gut microbiota of mice fed with HFD (with/without DC-PS or DC-acPS) and normal diet. 61 Figure 37. The Shannon indices of gut microbiota of mice fed with HFD (with/without DC-PS or DC-acPS) and normal diet. 62 Figure 38. The phylogenetic diversity indices of gut microbiota of mice fed with HFD (with/without DC-PS or DC-acPS) and normal diet. 63 Figure 39. The PCoA scatter plot of Bray-Curtis dissimilarity. 64 Figure 40. Firmicutes/Bacteroidetes ratio and relative abundance of fecal microbiota at phylum level. 65 Figure 41. Relative abundance of fecal microbiota at genus level. 66 Figure 42. Relative abundance of Lachnospiraceae at family and genus level 67 Figure 43. Relative abundance of Ruminococcaceae and Muribaculaceae 68 Figure 44. Relative abundance of Faecalibaculum rodentium and Bacteroides acidifaci-ens JCM 10556. 69 Figure 45. Relative abundance of several probiotics at genus and species level 71 Figure 46. Relative change in gut microbiota average relative abundance of DC-acPS or DC-PS administration mice compared with HFD group.. 76 附圖目錄 (List of supporting figures) Figure S1. 1H NMR spectrum of DC-PS-deae. (DMSO-d6, 500 MHz) 91 Figure S2. HSQC spectrum of DC-PS-deae. (DMSO-d6, 500 MHz) 92 Figure S3. 1H NMR spectrum of DC-PS. (DMSO-d6, 500 MHz) 93 Figure S4. HSQC spectrum of DC-PS. (DMSO-d6, 500 MHz) 94 Figure S5. 1H NMR spectrum of DC-acPS. (DMSO-d6, 800 MHz) 95 Figure S6. HSQC spectrum of DC-acPS. (DMSO-d6, 800 MHz) 96 Figure S7. Full GC chromatogram of alkali treated-DC-acPS (4). 97 Figure S8. Full GC chromatogram of SCFA standards. 98 Figure S9. Full GC chromatogram of fecal SCFAs (9th week, cage no.4). 99 Figure S10. Full GC chromatogram of cecal SCFAs (mouse no.714). 100 Figure S11. Relative abundance at genus level of fecal microbiota in each mouse. 101 | |
| dc.language.iso | zh-TW | |
| dc.subject | 乙醯化 | zh_TW |
| dc.subject | 金童石斛 | zh_TW |
| dc.subject | 葡萄甘露多醣 | zh_TW |
| dc.subject | 高脂飲食 | zh_TW |
| dc.subject | Dendrobium Cassiope | en |
| dc.subject | glucomannan | en |
| dc.subject | acetylation | en |
| dc.subject | high-fat diet | en |
| dc.title | 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 | zh_TW |
| dc.title | Effect of the polysaccharides from Dendrobium Cassiope and their acetylated derivatives on the physiological indicators and gut microbiota of high-fat diet-fed mice | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 110-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李水盛(Shoei-Sheng Lee),盧美光(Mei-Kuang Lu),劉慧康(Hui-Kang Liu) | |
| dc.subject.keyword | 金童石斛,葡萄甘露多醣,乙醯化,高脂飲食, | zh_TW |
| dc.subject.keyword | Dendrobium Cassiope,glucomannan,acetylation,high-fat diet, | en |
| dc.relation.page | 101 | |
| dc.identifier.doi | 10.6342/NTU202203932 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2022-09-27 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 藥學研究所 | zh_TW |
| dc.date.embargo-lift | 2022-10-13 | - |
| 顯示於系所單位： | 藥學系 | |
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