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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張嘉銓(Chia-Chuan Chang) | |
| dc.contributor.author | Wei-Yeh Chen | en |
| dc.contributor.author | 陳韋曄 | zh_TW |
| dc.date.accessioned | 2021-07-10T21:37:21Z | - |
| dc.date.available | 2021-07-10T21:37:21Z | - |
| dc.date.copyright | 2020-09-10 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-17 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76803 | - |
| dc.description.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。 | zh_TW |
| dc.description.abstract | 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. | en |
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| dc.description.tableofcontents | 總目錄 口試委員審定書.............................................................................................................I 中文摘要.......................................................................................................................II Abstract ........................................................................................................................III 目錄..............................................................................................................................IV 表目錄 (List of tables)..............................................................................................VIII 圖目錄 (List of figures)….......................................................................................VIIII 辭彙...........................................................................................................................XIII 目錄 1.前言及簡介 1.1緒論及研究目的..............................................................................................1 1.2 石斛簡介.........................................................................................................2 1.2.1 金童石斛..............................................................................................2 1.2.2 金皇石斛..............................................................................................6 1.3 腸內菌簡介.....................................................................................................9 1.3.1 腸內菌於腸道的分佈........................................................................10 1.3.2 腸內菌在腸道的分佈比例................................................................14 1.3.3腸內菌與人體的健康關係.................................................................15 1.4 腸內菌與多醣的交互作用...........................................................................24 1.4.1 腸內菌降解多醣為雙醣或單醣........................................................24 1.4.2 單醣經由糖解作用降解為短鏈脂肪酸............................................28 1.4.3 腸內菌產生之短鏈脂肪酸在腸內菌群的交互影響........................38 1.4.4. 腸內菌與腸內菌之間的交互影響...................................................41 1.4.5 腸內菌代謝各種多醣所產生之短鏈脂肪酸對於人體的影響........42 1.5石斛多醣活性之相關文獻............................................................................46 1.6 與本研究相關之腸內菌介紹.......................................................................53 1.6.1 Allobaculum spp. .................................................................................53 1.6.2 Muribaculaceae spp. (S24-7 spp.) .......................................................53 1.6.3 Clostridiales spp. (梭菌目) .................................................................51 1.6.4 Bifidobacterium pseudolongum (假長雙歧桿菌) ...............................54 1.6.5 Bacteroides caccae (糞擬桿菌) ..........................................................54 1.7短鏈脂肪酸之分析方法回顧........................................................................54 2. 實驗部分.................................................................................................................57 2.1 儀器與材料...................................................................................................57 2.1.1理化性質測定儀器.............................................................................57 2.1.2 成分分離之儀器與材料....................................................................57 2.1.3 試劑與溶媒........................................................................................58 2.2植物來源........................................................................................................59 2.3金童石斛以及金皇石斛的水萃物萃取........................................................59 2.4餵食小鼠石斛水萃物之動物實驗................................................................59 2.5短鏈脂肪酸GC分析......................................................................................60 2.6 菌種分析方法...............................................................................................60 2.7蒟蒻多醣之水解............................................................................................61 2.8蒟蒻寡醣之乙醯化........................................................................................61 2.9金童石斛水萃物之史密斯降解....................................................................62 2.10以普魯蘭標準品製作多醣分子量較正曲線..............................................62 3. 實驗結果與討論.....................................................................................................63 3.1 使用GC-FID分析糞便中SCFAs的方法開發...........................................63 3.1.1 以DB-5作為短鏈脂肪酸之分析管柱.............................................63 3.1.2 以DB-FFAP作為短鏈脂肪酸分析之管柱......................................67 3.2 餵食金童與金皇石斛水萃物之老鼠糞便的短鏈脂肪酸含量...................71 3.3 短鏈脂肪酸含量與腸內菌OTU比例之關聯.............................................73 3.4 蒟蒻葡萄甘露聚醣之水解與區域選擇性乙醯化之結果...........................77 3.5 金童石斛水萃物結構以及其史密斯降解產物(Smith degradation)分析...83 3.6 結論...............................................................................................................89 參考文獻......................................................................................................................90 附圖............................................................................................................................106 表目錄 Table 1. Monosaccharide composition of (A) DNP-W1 (B) JCS1, two main polysaccharides isolated from the stems of D. nobile......................................................4 Table 2. Monosaccharide composition of DMP isolated from the stems of D. moniliforme....................................................................................................................5 Table 3. Monosaccharide composition of DTTPS-N polysaccharide extracted from the stem of D. Taiseed Tosinobile.........................................................................................7 Table 4. Monosaccharide composition of DTP-N polysaccharide extracted from the stem of D. tosaense.........................................................................................................8 Table 5. The pH value and major gut microbiota group in different part of digestion tract...............................................................................................................................12 Table 6. Amount of GH and PL genes in different gut microbial phylum.....................27 Table 7. Selected genera present in human gut microbiome and their metabolism.....33 Table 8. Plant polysaccharides and their chemical composition, covalent linkage, source, abundance and degree of digestion..................................................................42 Table 9. Summary of findings about the biological efficacies of polysaccharides against various metabolic diseases............................................................................................43 Table 10. Selected Dendrobium polysaccharides and its bioactivity.............................48 圖目錄 Figure 1. Picture of Dendrobium Cassiope...................................................................3 Figure 2. Proposed structure of DC-PS1 extracted from D. Cassiope..........................3 Figure 3. Proposed structure of polysaccharide JCS1 extracted by boiling water from D. nobile.........................................................................................................................4 Figure 4. Picture of Dendrobium Taiseed Tosnobile.....................................................6 Figure 5. The treatment of DTTPS increases the population of splenic lymphocytes in BALB/c mice..................................................................................................................7 Figure 6. (A) Splenic NK cytotoxicity against YAC-1 cells and (B) splenocytes phagocytic activity of D. tosaense-polysaccharide treated or contol BALB/c mice................................................................................................................................8 Figure 7. Temporal aspects of microbiota establishment and maintenance and factors influencing microbial composition.................................................................................9 Figure 8. A global overview of the relative abundance of key phyla of the human microbiota composition in different stages of life.........................................................10 Figure 9. The nutrient concentration, pH, oxygen, and bacterial load in different part and cross section of digestion tract................................................................................11 Figure 10. Bacterial growth of 14 different gut microbiota from Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, and Verrucomicrobia on different polysaccharides............................................................................................................13 Figure 11. The ratio of gut microbiome genera in mice, rats, non-human primates, and human...........................................................................................................................14 Figure 12. Leaky gut can cause lipopolysaccharide (LPS) to enter the vascular system which induces low-grade inflammation that leads to metabolic syndromes..................16 Figure 13. The vicious cycle of leaky gut caused by the inflammation from LPS......17 Figure 14. The concentration of GLP-2 is promoted by the production of SCFAs by gut microbes, which then tighten the junction integrity of gut cells....................................19 Figure 15. Gene expression of PYY and GCG (GLP-1 producing gene) after giving 2mM of acetate, propionate, and butyrate.....................................................................20 Figure 16. PYY peptide secretion increases both in 2 mM butyrate and 500 nM HDAC inhibitor Trichostatin A, while no significant increase in GLP-1 secretion...................20 Figure 17. Schematic overview of the actions of TGR5 in different cells and tissue.....21 Figure 18. EC50 of TGR5 activated by primary and secondary bile acids...................22 Figure 19. Bile acids metabolized by gut microbiota have higher activity to FXR receptor, which has the effect of anti-inflammation and maintaining epithelial barrier integrity.........................................................................................................................23 Figure 20. Degradation of polysaccharides by Sus-like system....................................25 Figure 21. Degradation of polysaccharides by ATP-binding cassette transport system...........................................................................................................................26 Figure 22. Degradation of polysaccharides by cellulosome-like scaffolded enzyme system...........................................................................................................................26 Figure 23. The percentage of CAZymes in CAZy database........................................27 Figure 24. The metabolic pathway of glycolysis through the EMP pathway, PP pathway, and ED pathway............................................................................................................28 Figure 25. Reaction pathway of acetate formation.......................................................30 Figure 26. Reaction pathways of butyrate formation....................................................30 Figure 27. Reaction pathways of propionate formation................................................31 Figure 28. A total scheme of all fermentation pathways to produce acetic acid, propioinic acid, butyric acid, and other end products....................................................31 Figure 29. Bifid shunt pathway....................................................................................32 Figure 30. The arabinoxylo-oligosaccharide (AXOS) fermentation product concentration of acetate (▲), butyrate (○), ethanol (●), formate (■), and lactate (◆). (A) Only Bifidobacterium longum NCC2705 (B) Only Eubacterium rectale ATCC 33656 (C) Both..............................................................................................................................39 Figure 31. A simplified scheme of the co-fermentation of Bifidobacterium longum NCC2705 and Eubacterium rectale ATCC 33656.......................................................40 Figure 32. Bifidobacteria produces lactic acid, which can be further transformed to butyric acid by butyrate-producing bacteria.................................................................40 Figure 33. Bdellovibrio bacteriovorus decrease the amount of Salmonella.................41 Figure 34. IL-12 and IL-13 secretion in duodenum, jejunum, ileum, and colon from mice fed with distilled water (control), 200mg/kg/day GXG hydrolysate (hydrolysate), 50mg/kg/day GXG (GXGlow), and 200mg/kg/day (GXGhigh).......................................47 Figure 35. Fatty acids concentration in feces of mice that were fed with chow diet and D. officinale polysaccharide DOW-5b..........................................................................47 Figure 36. GC analysis of SCFAs by split injection (ratio 12:1; dissolved in methanol) ......................................................................................................................................63 Figure 37. GC analysis of SCFAs by split injection (ratio 5:1; dissolved in water).......64 Figure 38. GC analysis of SCFAs by split injection (ratio 100:1; dissolved in acidified water)............................................................................................................................64 Figure 39. GC analysis of SCFAs with different split ratio. (Dissolved in acidified water) (A) 120:1 (B) 150:1 (C) 220:1.......................................................................................66 Figure 40. Structure of coating in DB-FFAP GC column and its interaction to fatty acids..............................................................................................................................67 Figure 41. GC analysis of three SCFAs using DB-FFAP column by the reported condition.......................................................................................................................68 Figure 42. GC analysis of water-extracted fatty acids from mouse feces using (A) reported method and (B) modified method...................................................................69 Figure 43. The resolution of close peaks before and after adjustment..........................69 Figure 44. (A) GC chromatogram of AA, PPA, BA, and 2-EB; Linear regression of (B) acetic acid, (C) propionic acid, and (D) butyric acid from the concentration and area ratio between the analytes and internal standard...........................................................70 Figure 45. The SCFAs concentration in DC-PS-fed BALB/c mouse feces................71 Figure 46. The SCFAs concentration in C57BL/6J mice feces from DC, DTT, and control group................................................................................................................72 Figure 47. The OTU counts of Allobaculum sp., Clostridiales sp., Muribaculaceae sp., and B. pseudolongum in control group, DC group, an DTT group..............................73 Figure 48. The OTU reads of B. caccae in DC group, DTT group, and control group.............................................................................................................................74 Figure 49. The interaction between (A) Dendrobium Cassiope water extract, (B) Dendrobium Taiseed Tosnobile water extract and mice microbiota............................76 Figure 50. The SEC chromatogram of the hydrolyzed konjac glucomannan (hKGM) analyzed by TSKgel G3000 PWXL column...................................................................77 Figure 51. HSQC spectrum of (A) per-acetylated hKGM and (B) hKGM....................79 Figure 52. HSQC spectrum of K2CO3 catalyzed acetylated hKGM.............................80 Figure 53. HSQC spectrum of Lipozyme TL IM catalyzed acetylated hKGM...........81 Figure 54. The molecular weight of D. cassiope water extract, analyzed by TSKgel G3000 PWXL SEC column....................................................................................................................82 Figure 55. HSQC spectrum of DC water extract....................................................................84 Figure 56. (A) The mechanism of Smith degradation. (B) The tentative structure of DC-PS1.........................................................................................................................85 Figure 57. (A)The products of Smith degradation products of DC water extract analyzed by TSKgel G-Oligo-PW column. (B) The tentative structure of DC-PS1...............................................................................................................................86 Figure 58. Analysis of Smith degradation products by TSKgel-G3000PWXL SEC column..........................................................................................................................87 Figure 59. The MS spectrum of degraded DC-PS separated by G-Oligo-PW SEC column..........................................................................................................................88 Figure 60. The structure of degraded fragments from DC-PS....................................89辭彙 (A)XOS (Arabino)xylo-oligosaccharide 2-EB 2-Ethylbutyric acid AA Acetic acid ABC transport system ATP-binding cassette transport system ADP Adenosine diphosphate AGEs Advanced glycation end products AH Anhui Ara Arabinose ATP Adenosine triphosphate BA Butyric acid BAT Brown adipose tissue CA Cholic acid CAZyme Carbohydrate-active enzyme CDCA Chenodeoxycholic acid ConA Concanavalin A D2 Thyroid hormone-activating enzyme type 2 iodothyronine deiodinase DC Dendrobium Cassiope DCA Deoxycholic acid DCs Dendritic cells DDP D. denneanum polysaccharide DHAP Dihydroxyacetone phosphate DMP Dendrobium moniliforme polysaccharide DMSO Dimethyl sulfoxide DNP Dendrobium nobile polysaccharide DOP Dendrobium officinale polysaccharide DOPA-1 L-3,4-Dihydroxyphenylalanine DOW D. officinale polysaccharide DPs Dendrobium polysaccharides DTP Dendrobium tosaense polysaccharide DTT Dendrobium Taiseed Tosnobile DTTPS Dendrobium Taiseed Tosnobile polysaccharide DvP D. devonianum polysaccahride ED pathway Entner–Doudoroff pathway ELSD Evaporative light scattering detectors EMP pathway Embden–Meyerhof–Parnas pathway F/B ratio Firmicutes and Bateroidetes ratio FFAR Free fatty acid receptor FID Flame ionization detector FOS Fructo-oligosaccharide FXR Farnesoid X receptor Gal Galactose GAPDH Glyceraldehyde-3-phosphate dehydrogenase GC Gas chromatography GCS-F Granulocyte colony-stimulating factor GH Glycoside hydrolase Glc Glucose GLP Glucagon-like peptide GM-CSF Granulocyte-macrophage colony-stimulating factor GXG Dendorbium huoshanense polysaccharide HDAC Histone deacetylase HD-Bedllo Bdellovibrio bacteriovorus HD100 hKGM Hydrolyzed konjac glucomannan HMBC Heteronuclear multiple bond correlation HMO Human-milk oligosaccharide HPLC High performance liquid chromatography HSCoA Coenzyme A HSQC Heteronuclear single quantum coherence spectroscopy IBA Isobutyic acid IFN-γ Interferon gamma IL Interleukin IM Inner membrane IVA Isovaleric acid KGM Konjac Glucomannan LBP Lipopolysaccharide binding protein LCA Lithocholic acid LDA Linear discriminant analysis LPS Lipopolysaccharide Man Mannose MS Mass spectrometry Mw Molecular weight NAD(P)H Nicotinamide adenine dinucleotide (phosphate) NAFLD Non-alcoholic fatty liver disease NGS Next generation sequence NK Nature killer cell NOS Nitric oxide synthase OAc O-Acetyl OM Outer membrane OTU Operational taxonomic unit PCR Polymerase chain reaction PFK Phosphofructokinase PL Polysaccharide lyase PP pathway Pentose phosphate pathway PPA Propionic acid PPRs Pathogen recognition receptors PS Polysacchairde PYY Peptide tyrosine tyrosine SCFAs Short chain fatty acids SEC Size exclusion chromatography sp. specie SPE Solid phase microextraction spp. plural species Sus-like Starch utilizing system-like TBDT TonB-dependent transporter TGR5 Takeda G protein-coupled receptor-5 TLR Toll-like receptor TNF-α Tumor Necrosis Factor Alpha UV Ultraviolet VA Valeric acid Xyl Xylose YN Yunnan ZJ Zhejiang | |
| dc.language.iso | zh-TW | |
| dc.subject | 史密斯降解 | zh_TW |
| dc.subject | 金童石斛 | zh_TW |
| dc.subject | 金皇石斛 | zh_TW |
| dc.subject | 短鏈脂肪酸 | zh_TW |
| dc.subject | 葡萄甘露聚醣 | zh_TW |
| dc.subject | 乙醯化 | zh_TW |
| dc.subject | Dendrobium Taiseed Tosnobile | en |
| dc.subject | acetylation | en |
| dc.subject | glucomannan | en |
| dc.subject | short chain fatty acids | en |
| dc.subject | Dendrobium Cassiope | en |
| dc.subject | Smith degradation | en |
| dc.title | 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 | zh_TW |
| dc.title | Regio-selective acetylation of glucomannans and their effects on the short-chain fatty acid formation by gut microbiota | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李水盛(Shoes-Sheng Lee),盧美光(Mei-Kuang Lu),林雲蓮(Yun-Lian Lin) | |
| dc.subject.keyword | 金童石斛,金皇石斛,短鏈脂肪酸,葡萄甘露聚醣,乙醯化,史密斯降解, | zh_TW |
| dc.subject.keyword | Dendrobium Cassiope,Dendrobium Taiseed Tosnobile,short chain fatty acids,glucomannan,acetylation,Smith degradation, | en |
| dc.relation.page | 112 | |
| dc.identifier.doi | 10.6342/NTU202003836 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-08-17 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 藥學研究所 | zh_TW |
| 顯示於系所單位： | 藥學系 | |
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