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研究生:蔣健興
研究生(外文):Chiang Chien-Hsing
論文名稱:金線連水溶性多醣區分之組成與免疫活性分析
論文名稱(外文):Physiochemical characteristics and potential immuo-modulating activities of the water-soluble polysaccharide fractions from Anoectochilus formosanus Hayata
指導教授:呂廷璋
指導教授(外文):Lu Ting-Jang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:食品科技研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:117
中文關鍵詞:金線連多醣免疫調節活性高效能陰離子交換層析法
外文關鍵詞:Anoectochilus formosanuspolysaccharideimmuno-modulating activityhigh-performance anion-exchange chromatography
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由金線連(Anoectochilus formosanus Hayata)中分離出具免疫調節作用潛力的水溶性多醣,並對其物理化學性質及免疫活性進行分析。金線連含2.5 ± 0.3水溶性膳食纖維。由水溶性膳食纖維中分離出水溶性多醣(AFP),含量為水溶性膳食纖維之70%,以陰離子交換樹脂層析出中性(AFPN)及酸性(AFPA)多醣區分,而中性(AFPN)與酸性(AFPA)多醣區分的比例約為1 : 1。利用Methanolysis 與trifluoroacetic acid水解,再以高效能陰離子交換層析法配合脈衝式電流檢測,測定單糖組成,結果顯示中性區分的多醣含有甘露糖、半乳糖、及葡萄糖,其相對的莫耳比例為28.4 : 10.6 : 1.0。酸性區分的多醣含有半乳糖、甘露糖、阿拉伯糖、及葡萄糖,並含有兩種醛醣酸:半乳糖醛酸及葡萄糖醛酸,其相對的莫耳比例為 5.2 : 2.5 : 1.9 : 1.6 : 4.3: 1.0。酸性區分的多醣含有1.06 %的2-keto-3-deoxy-D-manno-octulosonic acid (KDO),並對β-D-glucosyl-Yariv antigen有專一性的反應活性,因此推測酸性區分屬於果膠質多醣類的區塊。
AFP、AFPN及AFPA於200 μg/ml濃度下皆可顯著的刺激人類單核球細胞(PBMC) 產生TNF-α及IL-6 (P﹤0.01),對於經PHA (phytohemagglutinin)活化的人類單核球細胞,AFP、AFPN及AFPA皆可顯著的增加IL-6含量,但不影響TNF-α含量。AFP、AFPN及AFPA對於PBMC細胞,無論是否經由PHA刺激,皆不具誘導細胞產生IL-2的活性。而AFPA經endo-polygalacturonase水解後仍保有大部分的大分子量多醣結構, 但其誘導細胞產生TNF-α及IL-6的活性略有下降。
Abstract
Physiochemical characteristics and potential immuo-modulating activities of the water-soluble polysaccharide fractions of Anoectochilus formosanus Hayata were studied. Anoectochilus formosanus Hayata contained 2.5 % of soluble dietary fiber (SDF). The water-soluble polysaccharides (AFP) fraction was composed of 70 % of SDF. The water soluble polysaccharide fraction was composed of neutral (AFPN) and acidic (AFPA) polysaccharide with a approximate equally amout in weight and were further separated using DEAE-anion exchange chromatography.
The sugar composition was determined using methanolysis, trifluoroacetic acid hydrolysis and high-performance anion-exchange chromatography couple with pulsed amperometric detection. The neutral fraction was composed of mannose, galactose, and glucose with molar ratios of 28.4 : 10.6 : 1.0. Acidic fraction was composed of galactose, mannose, arabinose and glucose and two uronic acids, galacturonic and glucuronic acids with molar ratios of 5.2 : 2.5 : 1.9 : 1.6 and 4.3: 1.0 . The acidic fraction was considered as branched domains of pectic substance for its containing 1.06 % of 2-keto-3-deoxy-D-manno- octulosonic acid (KDO) and specific reaction to β-D-glucosyl-Yariv antigen.
AFP, AFPN and AFPA at 200μg/ml concentration level shows a significant stimulating effect on the human peripheral blood mononuclear cell (PBMC) to produce TNF-αand IL-6 (P﹤0.01). AFP, AFPN and AFPA also can induce IL-6 production but not TNF-α on PHA (phytohemagglutinin) -activated PBMC. AFP, AFPN and AFPA did not stimulate PBMC to produce IL-2 whether it has been activated by PHA or not. Endo- polygalacturonase resistant portion of AFPA reduced its activity to stimulate PBMC to produce TNF-αand IL-6.
目錄
摘要………………………………………………………………….......Ⅰ
Abstract…………………………………………………………………. Ⅱ
目錄……………………………………………………………………...Ⅲ
圖目錄…………………………………………………………………...Ⅵ
表目錄…………………………………………………………………...Ⅷ
壹、前言…………………………………………………………………..1
貳、文獻回顧……………………………………………………………..3
一、多醣作為生物反應修飾物(BRM)………………………………...3
(一) (1→3;1→6)-β-D-glucan………………………………………4
1.(1→3)-β-D-glucan主鏈鍵結結構對生理活性表現的影響..6
2.分支度對(1→3)-β-D-glucan生理活性表現的影響……….6
3.分子量區分對(1→3)-β-D-glucan生理活性表現的影響….7
4.化學修飾對(1→3)-β-D-glucan生理活性表現的影響…….7
5.三級結構對(1→3)-β-D-glucan生理活性表現的影響…….8
(二)果膠酸多醣Pectic polysaccharide……………………………..10
1.Homogalacturonan(HG)…………………………………….12
2.RhamnogalacturonanⅠ(RGⅠ)………………….…...……..14
3.RhamnogalacturonanⅡ(RGⅡ)……………………………..16
(三)α-D-glucans……………………………… ………………..….22
(四) Mannan聚甘露醣與聚甘露醣主幹之多醣…………………...27
二、多醣結構分析之單糖組成份分析法…………………………....31
1.酸水解……………………………………………………….31
2.Methanolysis………………………………………………...32
3.Acetolysis……………………………………………............33
三、台灣金線連簡介…………………………………………....…....35
(一)形態與分佈…………………………………………………….35
(二)栽培環境因子………………………………………………….36
(三)金線連品種品系……………………………………………….36
(四)金線連生理活性測試………………………………………….37
參、實驗材料與方法…………………………………………………....40
一、實驗材料………………………………………………………....40
二、膳食纖維含量測定……………………………………………....40
三、樣品製備方式………………………………………………….... 41
(一)製備水溶性膳食纖維……..……………………………….…. 41
(二) 中性與酸性多醣之區分……………………………………...41
(三)endo-polygalacturonase酵素水解………………..……………41
四、樣品分析方法……………………………………………………44
(一)糖含量測定…………………………………………………….44
(二)醛糖酸含量測定……………………………………………….44
(三)KDO(2-keto-3-deoxy-D-manno-octulosonic acid)含量測定.…44
(四)還原糖含量測定……………………………………………….44
(五)蛋白質含量測定……………………………………………….44
(六)單糖組成測定………………………………………………….45
(七) Acetolysis……………………………………………………...47
(八)β-glucosyl-Yariv antigen test……………………………….......47
(九)分子量測定.................................................................................47
五、免疫分析試驗…………………………………………….….......47
(一)製備分離人類週邊單核球細胞…………………………....….47
(二)多醣樣品對於PBMC cell 產生細胞激素活性分析……........48
六、統計分析方法……………………………………………………48
肆、結果與討論………………………………………………………….49
一、膳食纖維含量……………………………………………………49
二、金線連多醣之分離………………………………………………50
三、金線連多醣區分之化學性質…………………………………....52
(一)金線連多醣區分組成份與單糖組成………………………...…52
(二)金線連中性多醣AFPN之性質………………………………...66
1. Acetolysis……………………………………………………..66
2. 分子量測定…………………………………………………...70
(三)金線連酸性多醣AFPA之性質…………………………………71
1.金線連酸性多醣(AFPA)對endo-polygalacturonase的水解
抗性............................................................................................71
2. β-D-glucosyl-Yariv reagent test………….……………………76
四、免疫分析試驗..................................................................................79
(一)多醣樣品誘導PBMC 細胞產生腫瘤壞死因子(tumor necrosis factor alpha, TNF-α)的檢測........................................................79
(二)多醣樣品誘導PBMC細胞產生間白-6 (interleukin-6, IL-6)......84
(三)多醣樣品誘導PBMC細胞產生間白素-2(interleukin-2)的檢測89
(四) endo-polygalacturonase酵素水解AFPA對於免疫調節作用的
影響..............................................................................................92
伍、結論....................................................................................................93
陸、參考文獻............................................................................................95
圖目錄
圖一、具抗腫瘤活性的(1→6)-β-D-glucosyl branched (1→3)-β-D- glucopyran.化學組成鍵結結構………….................................…4
圖二、3種果膠質多醣homogalacturonan(HG), rhamnogalacturonan I(RG I),及rhamnogalacturonan II (RG II)彼此間以共價鍵結型式
存在....................................................................................... ..... 11
圖三、Homogalacturonan(HG)分子一級結構………………………….13
圖四、rhamnogalacturonan I (RG I)基本結構示意圖……………….….15
圖五、rhamnogalacturonan II (RG II) 一級結構圖………………....….19
圖六、RG II分子與borate 形成dimer型式的兩種立體異構物……..20
圖七、Cnidium officinale(川芎)多醣可能的鍵結結構…..……………..24
圖八、赤芝孢子粉中多醣可能的鍵結結構……………………………25
圖九、金線連多醣樣品製備流程………………………………………43
圖十、利用陰離子交換層析分離水溶性金線連多醣中的中性與酸性多醣的層析圖…………………………………………………… 51
圖十一、不同單糖樣品濃度與酚硫酸呈色吸光值標準曲線……...…..54
圖十二、九種標準單糖樣品於HPAEC-PAD層析圖譜………………57
圖十三、金線連水溶性多醣methanolysis/TFA水解液之HPAEC-PAD圖譜…………………………………………………………….58
圖十四、金線連中性多醣TFA水解液之HPAEC-PAD圖譜………...59
圖十五、金線連酸性多醣methanolysis/TFA 水解液之HPAEC-PAD
圖譜………………………………………………………..…....60
圖十六、酸性標準單糖樣品於HPAEC-PAD層析圖譜……………….61
圖十七、金線連水溶性多醣methanolysis/TFA 水解液 HPAEC-PAD
圖譜……………………………………………………………62
圖十八、金線連酸性多醣methanolysis/TFA 水解液 HPAEC-PAD
圖譜…………………………………………………………….63
圖十九、金線連中性多醣acetolysis前(A)後(B)分子篩HW55(F)區分圖………………………………………………………………..67
圖二十、guar gum acetolysis前(A)後(B)分子篩HW55(F)區分圖…68
圖二十一、金線連中性區分多醣高效能分子篩層析圖譜……………70
圖二十二、endopolygalacturonase酵素水解金線連酸性多醣其水解液以HW-50 gel分離區分層析圖…………….. ……………………73
圖二十三:endo-polygalacturonase酵素水解金線連酸性多醣AFPA其水解液以HW-55 (F) gel 分離區分層析圖,金線連酸性多醣(A);金線連endopolygalacturonase resistant酸性多醣(B) ..…74
圖二十四、金線連酸性多醣(A);金線連endopolygalacturonase resistant酸性多醣 (B) 高效能分子篩層析圖譜…..……………….…..75
圖二十五、不同樣品對於β-D-glucosyl-Yariv antigen 膠體擴散圖.....78
圖二十六、金線連水溶性多醣區分對於PBMC細胞產生TNF-α的個體差異…………………………………………………………..82
圖二十七、金線連水溶性多醣對PBMC細胞產生TNF-α的濃度
效應……………………………………………………………..83
圖二十八、金線連水溶性多醣區分對於PBMC細胞產生IL-6的個體
差異……………………………………………………………..87
圖二十九、金線連水溶性多醣對PBMC細胞產生IL-6 的濃度效應..88
圖三十、金線連水溶性多醣區分對於PBMC細胞產生IL-2的個體差異………………………………………………………………..91
表目錄
表一、Grifolan, Lentinan,及Schizophyllan基本物化性質……………..5
表二、具有免疫調節生理活性的α-D-glucan…………………….……26
表三、蘆薈多醣結構………………………………………………...….29
表四、蘆薈多醣Acemannan(CarrisynTM)生物活性反應…………….. 30
表五、雙糖乙醯基化降解(acetolysis)反應速率………….……………34
表六、金線連生理活性一覽表……………………………………….....39
表七、金線連膳食纖維含量……………………………………………49
表八、金線連多醣區分之化學組成份…………………………………54
表九、不同單糖酸水解前後對於脈衝式電流檢測器反應相對因子…64
表十、標準單糖於脈衝式電流檢測器中濃度對反應曲線公式………64
表十一、金線連多醣區分之單醣組成…………………………………65
表十二、金線連中性多醣區分及guar gum經acetolysis水解後理化性質分析…………………………………………………………..69
表十三、金線連多醣區分對於PBMC細胞產生TNF-α的影響………81
表十四、金線連多醣區分對於PBMC細胞產生IL-6的影響………..86
表十五、金線連多醣區分對於PBMC細胞產生IL-2的影響………..90
表十六、AFPA及其經endo-polygalacturonase水解處理產物AFPA-PR對於PBMC細胞產生TNF-α及IL-6的影響………………......92
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