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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/37792完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 吳世雄(Shih-Hsiung Wu) | |
| dc.contributor.author | Ya-Mei Chien | en |
| dc.contributor.author | 簡雅梅 | zh_TW |
| dc.date.accessioned | 2021-06-13T15:44:04Z | - |
| dc.date.available | 2013-07-14 | |
| dc.date.copyright | 2008-07-14 | |
| dc.date.issued | 2008 | |
| dc.date.submitted | 2008-07-03 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/37792 | - |
| dc.description.abstract | 長久以來，黑大豆一直廣泛地使用於傳統醫藥中。黑大豆多醣具有免疫調節活性、抑制人類血癌細胞U937細胞生長，並誘導血癌細胞分化成正常細胞及具有活化造血系統的功能。
為了進一步的了解黑大豆多醣的免疫調節功能，本研究首先先將黑大豆磨成粉狀、再利用二氯甲烷及乙醇去除脂溶性及小分子物質，最後使用70 ℃的二次水來萃取黑大豆多醣。經過二次水的萃取、冷凍乾燥、Sephadex LH 20管柱層析及TSK HW-55F管柱層析後，再利用proteinase K將蛋白質水解，即可得到黑大豆多醣。醣的檢測部分利用酚-硫酸法，最後利用小鼠J774A.1巨噬細胞株檢測多醣的免疫調節活性。 本實驗利用氣相層析質譜儀來分析黑大豆多醣的單糖組成分，G1、G1P1及G2P1的主要單糖成分是甘露糖與半乳糖、只有含少量的葡萄糖及其他單糖，但是G2P2則以葡萄糖、半乳糖為主要單糖成分，只含少量的甘露糖。免疫調節活性方面顯示，甘露糖含量越多的多醣則免疫調節活性越佳。 經由1H-核磁共振分析G2P2之結構發現，G2P2就是水蘇醣，水蘇醣是由一個果糖、半乳糖及葡萄糖組成，經由α-1,6的鍵結將半乳糖及葡萄糖鍵結在一起，在氣相層析質譜儀也有相同的結果。比較水蘇醣及G2P2的免疫調節活性發現，G2P2有些微的免疫調節活性，但是在水蘇醣中並沒有免疫調節活性。探究其原因可能是因為G2P2含有一些可以刺激巨噬細胞分泌細胞激素的甘露糖殘留。 | zh_TW |
| dc.description.abstract | Black-soybean [Glycine max (L.) Merr.] has been used extensively as a healthy food and a traditional Chinese medicine for promoting good health. The related therapeutically qualities including immunomodulative activity, inhibition of human leukemic U937 cell proliferation, inducement of cell differentiation and promotion of hematopoiesis were reported from it’s polysaccharide.
To further investigate the immunomodulatory property of black-soybean polysaccharides, black-soybean powder was first dissolved in dichloromethane and ethanol in order to remove the lipid-soluble and low-molecule weight components, and then extracted by water at 70 oC. The water soluble crude extracts were collected, lyophilized, further fractionated by Sephadex LH 20, TSK HW-55F chromatographies and deproteinized by proteinase K. The fractions containing polysaccharides were detected by phenol-sulfuric acid and the immuno-stimulatory activities were conducted in vitro by mouse J774A. 1 macrophage cell line. The carbohydrate composition analysis of crude black soybean extract was analyzed by Gas Chromatography-Mass Spectroscopy (GC-MS). Our result suggests that the monosaccharide composition of G1, G1P1, and G2P1 all pinpointed mannose and galactose as the major components together with smaller components of other sugars; G2P2 are composed of glucose and galactose as major and minor mannose. With regard to imuno-modulative activities, the more mannoses the better the activity. Through NMR, the structure of G2P2 is determinated as stachyose that contains one fructose, one glucose and two of galactose. The linkage of G2P2 was terminal- fructose, terminal-hexose and two of α-1,6-hexose that determinated by Gas Chromatohraphy-Mass Spectroscopy (GC-MS), and the data was similar to NMR. Comparing G2P2 and stachyose immuno-modulative activity we found that G2P2 had immuno-modulative activities depending on dose-dependent, but stachyose were bio-inactive. The reason for immuno-modulative activity was that G2P2 was contaminated by mannose residue that can interact with macrophage and induce the cytokines secreted. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T15:44:04Z (GMT). No. of bitstreams: 1 ntu-97-R95B46032-1.pdf: 1726011 bytes, checksum: 6dce92a368abb0867c752e803dd72889 (MD5) Previous issue date: 2008 | en |
| dc.description.tableofcontents | 目錄
口試委員審定書........................................i 誌謝..................................................ii 中文摘要..............................................iii 英文摘要..............................................iv 內文目錄..............................................vii 圖目錄................................................viii 表目錄................................................x 附錄目錄..............................................xi 內文目錄 第一章 前言........................................... 1 第一節 黑大豆......................................... 1 第二節 免疫系統....................................... 4 第三節 免疫細胞....................................... 5 第四節 多醣與結構..................................... 9 第五節 研究動機與目的................................. 13 第二章 材料與方法..................................... 14 第一節 實驗材料....................................... 14 第二節 實驗方法....................................... 18 第三章 結果........................................... 30 第一節 Sephadex LH 20管柱層析......................... 30 第二節 TSK HW-55F管柱層析—G1......................... 32 第三節 TSK HW-55F管柱層析—G2......................... 33 第四章 討論........................................... 35 第一節 黑大豆活性成分與結構之探討..................... 35 第二節 多醣結構與生物活性............................. 37 第三節 甘露糖接受器(mannose receptor)與免疫活性....... 39 參考文獻. ............................................. 41 圖目錄 圖1 萃取黑大豆多醣之流程示意圖....................... 51 圖2 黑大豆多醣純化流程及產率之示意圖................. 52 圖3 利用Sephadex LH 20管柱進行Gel filtraction純化黑豆多醣，並利用phenol-sulfuric acid分析各fraction中醣類分佈.... 53 圖4 利用Bradford方法測試蛋白質含量................... 54 圖5 水解G1蛋白質的proteinask K使用量測定............. 55 圖6 LPS contamination測試............................ 56 圖7 Sephadex LH20 column各fraction刺激J774A.1巨噬細胞分泌細胞激素(TNF-α)之含量.................................. 57 圖8 G1經proteinase K切除蛋白質後，刺激J774A.1巨噬細胞分泌細胞激素(TNF-α)之含量.................................. 58 圖9 G1、G2經100 ℃加熱使蛋白質性後，刺激J774A. 1細胞分泌細胞激(TNF-α)之含量 ....................................59 圖10 黑大豆萃取物細胞毒性測試........................ 60 圖11 利用TSK HW-55F管柱進行Gel filtration純化黑豆多醣(G1)，並利用phenol-sulfuric acid分析各fraction中醣類分佈.... 61 圖12 TSK HW-55F管柱純化黑大豆(G1)各fraction，刺激小鼠J774A.1巨噬細胞分泌細胞激素(TNF-α)之含量............. 62 圖13 水解G1P1蛋白質的proteinask K使用量測定.......... 63 圖14 G1經proteinase K切除蛋白質後，刺激J774A.1巨噬細胞分泌細胞激素(TNF-α)之含量................................ 64 圖15 利用TSK HW-55F管柱進行Gel filtration純化黑豆多醣(G2)phenol-sulfuric acid分析各fraction中醣類分佈.......... 65 圖16 TSK HW-55F管柱純化黑大豆(G2)各fraction，刺激小鼠J774A.1巨噬細胞分泌細胞激素(TNF-α)之含量............. 66 圖17 stachyose與G2P2之生物活性比較................... 67 表目錄 表1 黑大豆單糖組成................................... 68 表2 G2P2之GC-MS單糖鍵結分析.......................... 68 表3 G2P2 1H-與13C-NMR資料............................ 69 表4 Antitumor polysaccharide-protein complexes from mushrooms ..............................................70 附錄 附錄 1 免疫反應示意圖................................ 71 附錄 2 細胞激素列表.................................. 72 附錄 3 具有免疫調節生理活性的α-D-glucan............. 73 附錄 4 單糖組成分析之單糖衍生化流程圖................ 74 附錄 5 醣類鍵結分析之PMAA流程圖...................... 74 附錄 6 各單糖標準品之GC-MS分析....................... 75 附錄 7 GC-MS分析G1PK之單糖組成....................... 75 附錄 8 GC-MS分析G1P1PK之單糖組成..................... 76 附錄 9 GC-MS分析G2P1之單糖組成....................... 76 附錄 10 GC-MS分析G2P2之單糖組成...................... 77 附錄 11 GC-MS分析G2P2單糖鍵結方式.................... 77 附錄 12 G2P2之GC-MS PMAA分析圖 (14.6min)............. 78 附錄 13 G2P2之GC-MS PMAA分析圖 (15.9min)............. 78 附錄 14 G2P2之GC-MS PMAA分析圖 (17.2min)............. 79 附錄 15 G2P2之GC-MS PMAA分析圖 (17.58min)............ 79 附錄 16 G2P2之1H-NMR分析圖........................... 80 附錄 17 G2P2之13C-NMR分析圖.......................... 80 附錄 18 G2P2之2D COSY分析圖.......................... 81 附錄 19 G2P2之HSQC分析圖............................. 81 附錄 20 G2P2之HSQC-TOCSY分析圖....................... 82 附錄 21 G2P2之HMBC分析圖............................ 82 附錄 22 G2P2 (stachyose)之結構圖..................... 83 附錄 23 G2P2與stachyose 1H-NMR分析圖................. 83 | |
| dc.language.iso | zh-TW | |
| dc.subject | 細胞激素 | zh_TW |
| dc.subject | 黑豆 | zh_TW |
| dc.subject | 多醣 | zh_TW |
| dc.subject | 免疫調節 | zh_TW |
| dc.subject | 多醣-蛋白質複合體 | zh_TW |
| dc.subject | immuno-modulation | en |
| dc.subject | cytokines | en |
| dc.subject | polysaccharide-protein complex | en |
| dc.subject | black soybean | en |
| dc.subject | polysaccharide | en |
| dc.title | 具有免疫調節活性的黑豆物質之研究---萃取、純化及結構分析 | zh_TW |
| dc.title | Isolation, purification and structural analysis of substance with immuno-modulative activity from black soybean [Glycin max(L.) Merr.] | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 96-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳水田(Shui-Tein Chen),趙國評(Louis-Kuoping Chao),游宜屏(Yi-Ping Yu) | |
| dc.subject.keyword | 黑豆,多醣,免疫調節,多醣-蛋白質複合體,細胞激素, | zh_TW |
| dc.subject.keyword | black soybean,polysaccharide,immuno-modulation,polysaccharide-protein complex,cytokines, | en |
| dc.relation.page | 83 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2008-07-03 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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