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研究生:方銘志
研究生(外文):Ming-Chih Fang
論文名稱:山藥多醣體之免疫調節機能探討
論文名稱(外文):Immune modulating functions of yam polysaccharide from Dioscorea pseudojaponica
指導教授:龔瑞林龔瑞林引用關係
指導教授(外文):Z. L. Kong
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:食品科學系
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:2002
畢業學年度:90
語文別:中文
論文頁數:112
中文關鍵詞:山藥免疫調節多醣體發酵乳
外文關鍵詞:yamdioscireaimmune modulatingpolysaccharideyogurt
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山藥自古就做為中國人養生滋補的草藥,本研究針對免疫調節機能方面的探討,對基隆山藥不同方式的萃取物,評估其免疫生理調節活性。
以冷水萃取,經硫酸銨劃分後的山藥醣蛋白,在蛋白質1.7 mg/ml濃度時對細胞的增生沒有影響。山藥的鹽酸、硫酸冷水萃取物,及鹽酸水解萃取物亦不能促進細胞增生,推測其原因可能在低pH值下山藥中的成分產生化學反應,破壞其中成分。
以熱水萃取山藥多醣,經去除脂質、蛋白質、澱粉後,可得約1%乾重的山藥粗多醣,其醣濃度約100μg/ml時可促進HL-60、THP-1、UMΦ、mice PEC、小鼠脾臟細胞增生,並活化細胞,促進巨噬細胞分泌NO,及促進THP-1細胞產生細胞內超氧陰離子,但不能刺激非免疫相關細胞株6KK、Hela、B16、MCF-7增生,推測山藥多醣可能能直接刺激免疫細胞,而造成細胞增生與活化現象。進而以膠過濾管柱HW-65F層析山藥粗多醣,得到分子量200 kDa左右的山藥多醣YPS M,在醣濃度25μg/ml時即對細胞有增生與活化之現象,在濃度200μg/ml以下並能促進自然殺手細胞及吞噬細胞活性。
以體內動物實驗評估口服山藥多醣粗萃取液對小鼠免疫力的影響,結果證實口服山藥多醣0.5 g/kg/day,經七天的短期刺激下,能增強小鼠血清中抗體IgG生成量,促進脾臟細胞增生,及增強自然殺手細胞活性,具有調節小鼠免疫力之功效。
結合山藥與發酵乳的山藥發酵乳,在以健康食品為訴求的發展上,評估山藥發酵乳之免疫調節活性,山藥發酵乳體外試驗能促進細胞增生,在將其添加於飼料中餵食小鼠四週的體內動物實驗中,餵食7%山藥發酵乳能增進脾臟細胞增生,並促進血清抗體IgG分泌。結果顯示,山藥發酵乳可以提昇小鼠的免疫能力。
In traditional Chinese medicine, the tuber of Dioscorea spp. was a good material for health care. We discuss with the immune modulation function of yam different extracts in vitro and in vivo. Yam (Dioscorea japonica Thumb. Var pseudojaponica (Hatata) Yamamoto) bought from Gungliau Shiang, Taipei. Yam glycoprotein was prepared by ammonium sulfate. In vivo test, yam glycoprotein was no effect on cells proliferation. Yam acid extracts from HCl and H2SO4, and hydrochloric acid hydrolysis extracts were not promoted cell viability in cell culture mode, also.
To extracted yam polysaccharide, the tube was to cut into slices, and powdered after dry, then remove lipid and sterol by ethanol. The residue was extracted by hot water, and remove proteins by TCA. The yam crude polysaccharide YCPS was got from ethanol precipitate. YCPS 100μg/ml induced UMΦ, THP-1, HL-60 immuno-cell lines proliferation, but not 6KK, MCF-7, B16, Hela tumor cell lines. Cells incubated containing 5% FCS. In an 18hr culture, macrophage from mice peritoneal produced significantly greater amounts of NO upon activation with YCPS, detected by Griess method. The phagocyte respiratory burst as assessed by NBT reduction assay also displayed a 140% enhancement in YCPS administered. We suggest YPS M may direct to stimulate on cells but the mechanism is not clear. Purification of YCPS by gel filtration, crude polysaccharide divided into three fractions according to molecular weight, YPS L is M.W.>1000kDa, YPS M is M.W.=200kDa, YPS S is M.W.=50kDa, YPS M treated cells exhibit best proliferation and activation by MTT, Griess assay, and NBT reduction. In addition, YPS M can promoted NK cells activity and phagocytosis. We discussion yam polysaccharide can mudulation immune function.
For in vivo assay, BALB/C mice were divided into four groups. Group A is control, Group B, C and D was fed with 0.25, 0.5 and 1.0 g/day/kg bodyweight dosage of YCPS. After 7 days, eight mice from each group were sacrificed. In cellular response, the results revealed that splenic lymphocyte proliferation index in group B, C and D were higher than control group both stimulated with Con A (p<0.05) or LPS. In splenic nature killer cell activity and serum IgG were higher than control. We suggested that YCPS were effective in enhancing mice immunity.
In healthy food development, yam to manufacture into yam yogurt, yam yogurt’s immuno-function had be studied in mice mode. Spleens from mice receiving the supplemented diet had higher Con A and LPS induced cell proliferation than those from control mice. In addition, mice serum IgG produced higher than control mice, also. We conclude that yam yogurt may modulate the immunity in mice.
目錄
中文摘要 I
英文摘要 II
壹、 前言 1
貳、 文獻整理 2
一、 國內保健食品概況 2
二、 多醣類的免疫生理活性物質研究 2
1. 幾丁聚醣的免疫生理活性 3
2. 海藻多醣的免疫生理活性 3
3. 香菇多醣的免疫生理活性 4
4. 核酸多醣的免疫生理活性 6
三、 乳酸桿菌屬在免疫調節上的功效 6
四、 台灣山藥 7
1. 台灣山藥種類之鑑別 7
2. 台灣山藥之栽培 8
3. 山藥的生理功能 9
(1) 概況 9
(2) 歷代醫療價值記載 9
(3) 降膽固醇、降血脂 9
(4) 降血糖 10
(5) 促進細胞分化、抗氧化、抗神經退化 11
(6) 抗細胞增生 11
(7) 增強免疫力 12
五、 免疫活性物質之評估方法 12
1. 免疫細胞的活化 13
(1) T細胞的活化 13
(2) 自然殺手細胞的活化 13
(3) 吞噬細胞的活化 14
2. 巨噬細胞細胞膜上甘露醣接受器之功能 14
3. 免疫活性因子的分析方法 15
(1) 細胞增生(MTT還原分析法) 15
(2) 吞噬球呼吸傷 (respiratory burst) NBT還原分析法 15
(3) 毒胞活性NO產生分析法 16
(4) 自然殺手細胞活性分析法 16
參、 材料與方法 18
一、 實驗大綱 18
二、 實驗材料與藥品 20
1. 細胞株 20
2. 實驗動物 20
3. 動物飼料 20
4. 山藥來源 21
5. 細胞培養之藥品 21
6. 山藥蛋白製備之藥品 21
7. 山藥多醣製備之藥品 22
8. 山藥發酵乳製備之藥品 22
9. 蛋白質定量實驗之藥品 22
10. 總醣量分析實驗之藥品 22
11. 細胞存活率測定之藥品 22
12. 淋巴細胞活化實驗之藥品 23
13. 毒胞性及自然殺手細胞活性實驗之藥品 23
14. 吞噬作用之藥品 23
15. 聚丙烯胺膠電泳分析之藥品 23
16. 醣蛋白鑑別實驗之藥品 23
17. 小鼠血清抗體測定實驗之藥品 24
18. 小鼠脾臟細胞分離之藥品 24
19. 小鼠脾臟細胞增生倍率試驗之藥品 24
20. 小鼠脾臟NK細胞活性測定之藥品 25
21. 山藥粗多醣管柱層析之材料與藥品 25
22. 儀器設備 25
三、 實驗方法 26
1. 動物細胞培養 26
(1) 懸浮性細胞培養 26
(2) 附著性細胞之培養 27
2. 實驗動物之飼養 27
3. 山藥蛋白之製備 28
4. 山藥多醣體之製備 28
(1) 酸萃取法 28
(2) 熱水萃取法 28
5. 山藥粗多醣管柱層析 29
6. 蛋白質定量 29
7. 總含糖量分析 30
8. 膠電泳分析 (SDS-PAGE及Native-PAGE) 30
9. 醣蛋白鑑別 32
10. 細胞存活率 33
11. 小鼠腹腔細胞之取得 33
12. 小鼠脾臟細胞之取得 34
13. 淋巴細胞活化試驗 34
(1) NO產生量 34
(2) NBT 還原反應 34
14. 自然殺手細胞活性評估 35
15. 脾臟細胞毒胞性試驗 36
16. 吞噬作用 36
17. 餵食山藥粗多醣對小鼠免疫作用之影響 37
18. 山藥發酵乳之製備 37
19. 山藥發酵乳飼料之製備 38
20. 餵食山藥發酵乳對小鼠免疫作用之影響 38
21. 小鼠脾臟細胞增生倍率試驗 38
22. 小鼠血清抗體免疫球蛋白量評估 39
23. 統計分析 39
肆、 結果與討論 40
一、 山藥蛋白的分離與免疫生理活性 40
二、 山藥多醣之萃取與免疫生理反應 41
1. 酸萃取法 41
2. 熱水萃取法 42
三、 山藥粗多醣對細胞的影響 42
1. 促進免疫細胞增生 42
2. 促進一氧化氮分泌 43
3. 促進超氧陰離子產生 44
四、 山藥粗多醣之純化 44
五、 純化多醣分化對細胞的影響 45
六、 山藥粗多醣對小鼠免疫的影響 47
七、 山藥發酵乳之製備與體外試驗 49
八、 山藥發酵乳對小鼠免疫的影響 50
伍、 結論 52
陸、 參考文獻 54
柒、 圖表 67
圖表目錄
圖 1. 糖含量分析檢量線。使用葡萄糖及甘露糖做為標準品,以酚硫酸法測量總糖含量。 67
圖 2. 蛋白含量分析測定之檢量線。以牛血清白蛋白 (BSA)做為標準品。 68
圖 3. 山藥醣蛋白經硫酸銨劃分後之蛋白質電泳圖,分別以蛋白質染色及醣染色。 69
圖 4. 山藥醣蛋白經硫酸銨40%-60%劃分後對UMΦ、HL-60、 HB4C5生長比較圖。不同濃度的山藥醣蛋白與細胞培養48小時後,進行MTT分析其細胞存活率。 70
圖 5. 山藥醣蛋白經硫酸銨60%-80%劃分後對UMΦ、HL-60、 HB4C5生長比較圖。不同濃度的山藥醣蛋白與細胞培養48小時後,進行MTT分析其細胞存活率。 71
圖 6. 山藥粉末0.1 N H2SO4萃取物對細胞HB4C5、J774.1、K562成長的影響。 72
圖 7. 山藥粉末HCl萃取物對細胞HB4C5、J774.1、K562成長的影響。 73
圖 8. 新鮮山藥以HCl 121℃熱萃取物對細胞HL-60、HB4C5、UMΦ、mice PEC、J774.1成長的影響。 74
圖 9. 新鮮山藥以HCl 121℃熱萃取物對細胞HL-60、HB4C5、UMΦ、mice PEC、J774.1成長的影響。 75
圖 10. 不同濃度之山藥粗多醣對人類淋巴癌細胞株HL-60、及人類急性前骨髓淋巴癌細胞株THP-1及巨噬細胞株UMΦ生長百分率圖。不同濃度之山藥粗多醣,在無血清狀況下與細胞培養48小時後,進行MTT分析其細胞存活率。 76
圖 11. 不同濃度之山藥粗多醣對人類淋巴癌細胞株HL-60、及人類急性前骨髓淋巴癌細胞株THP-1及巨噬細胞株UMΦ生長百分率圖。不同濃度之山藥粗多醣,在5%血清狀況下與細胞培養48小時後,進行MTT分析其細胞存活率。 77
圖 12. 山藥粗多醣對小鼠脾臟細胞及小鼠腹腔細胞成長的影響。 78
圖 13. 不同濃度之山藥粗多醣對人類肝癌細胞株6KK、及人類子宮頸癌細胞株Hela、老鼠黑色腫瘤細胞株B16及人類乳癌細胞株MCF-7生長百分率圖。不同濃度之山藥粗多醣,在5%血清狀況下與細胞培養48小時後,進行MTT分析其細胞存活率。 79
圖 14. 山藥粗多醣對小鼠腹腔細胞NO產生量的影響。*P<0.05。 80
圖 15. 添加不同劑量TPA對細胞株THP-1、UMΦ、U937 NBT還原量的影響。 81
圖16. THP-1細胞添加山藥粗多醣對細胞NBT還原量的影響。細胞添加不同濃度山藥粗多醣培養18小時後,在含10 ng/ml TPA下測NBT還原量。*P<0.05。 82
圖 17. 以Toyopearl HW-65F管柱分離山藥粗多醣之層析圖。 83
圖 18. 山藥多醣YPS L對細胞株HL-60、THP-1、UMΦ成長的影響。 84
圖 19. 山藥多醣YPS M對細胞株HL-60、THP-1、UMΦ成長的影響。 85
圖 20. 山藥多醣YPS S對細胞株HL-60、THP-1、UMΦ成長的影響。 86
圖 21. 山藥粗多醣經膠過濾管柱HW-65F層析後,三個劃分區YPS L、YPS M、YPS S對小鼠脾臟細胞成長的影響。 87
圖 22. 山藥粗多醣經膠過濾管柱HW-65F層析後,三個劃分區YPS L、YPS M、YPS S對小鼠腹腔細胞成長的影響。 88
圖 23. 山藥多醣對小鼠腹腔細胞NO產生量的影響。 89
圖 24. THP-1細胞添加山藥粗多醣對細胞NBT還原量的影響。細胞添加山藥粗多醣YPS L、YPS M、YPS S培養18小時後,在含10 ng/ml TPA下測NBT還原量。*P<0.05。 90
圖 25. 山藥多醣刺激小鼠脾臟細胞18小時後,對毒殺U937細胞的影響 (ET ratio = 10:1)。 91
圖 26. 山藥多醣刺激小鼠脾臟細胞18小時後,對毒殺U937細胞的影響 (ET ratio = 20:1)。 92
圖 27. 經山藥多醣YPS M刺激後之小鼠脾臟細胞,對癌細胞株U937的毒胞性 (10:1, 20:1 effecter/target ratio)。 93
圖 28. 山藥多醣刺激小鼠脾臟細胞18小時後,對毒殺YAC-1細胞的影響 (ET ratio = 20:1)。 94
圖 29. 小鼠脾臟細胞在含200 ng/ml Con A下,添加山藥多醣培養18小時後,對毒殺YAC-1細胞的影響 (ET ratio = 20:1)。 95
圖 30. 山藥多醣YPS M對小鼠脾臟細胞自然殺手細胞活性之影響。 96
圖 31. 山藥多醣YPS M對THP-1吞噬作用之影響。 97
圖 32. THP-1細胞吞噬FITC螢光染色之酵乳菌顯微鏡照相圖。 98
圖 33. 山藥多醣YPS M對THP-1吞噬作用之影響。 99
表1. 以不同劑量山藥粗多醣管胃小鼠一週後,對小鼠其器官與體重比之影響。 100
圖 34. 比較不同劑量山藥粗多糖對小鼠血清免疫球蛋白γ的影響分別以ELISA方式,測小鼠餵食前 (day 0)與犧牲前 (day 7)之免疫球蛋白γ之量。 101
圖 35. 管灌不同濃度山藥粗多醣對小鼠脾臟細胞增生率的影響。小鼠脾臟細胞在不同濃度刺激原(Con A)下,以MTT測其細胞存活率,除以未加刺激原的值後,其增生倍率受餵食山藥粗多醣的影響。 102
圖 36. 管灌不同濃度山藥粗多醣對小鼠脾臟細胞增生率的影響。小鼠脾臟細胞在不同濃度刺激原(LPS)下,以MTT測其細胞存活率,除以未加刺激原的值後,其增生倍率受餵食山藥粗多醣的影響。 103
圖 37. 餵食不同劑量山藥粗多糖對小鼠脾臟自然殺手細胞活性的影響。 104
圖38. 不同濃度之山藥發酵乳萃取物對人類淋巴癌細胞株HL-60、人類急性前骨髓淋巴癌細胞株THP-1及巨噬細胞株UMΦ生長百分率圖。不同濃度之山藥發酵乳萃取物,與細胞培養48小時後,進行MTT分析其細胞存活率。 105
圖 39. 餵食不同劑量山藥發酵乳對小鼠體重與攝食量之影響。分別探討餵食山藥發酵乳4%、7%與未添加山藥發酵乳control對體重的影響。以及餵食山藥發酵乳對攝食率的影響。 106
表 2. 以不同劑量山藥發酵乳餵食小鼠四周後,對小鼠其器官與體重比之影響。 107
圖40. 比較添加不同劑量山藥發酵乳飼料control、飼料添加4%山藥發酵乳與飼料添加7%山藥發酵乳,對小鼠血清免疫球蛋白γ的影響。分別以ELISA方式,測小鼠餵食前(7 week)、餵食二星期 (9 week)與犧牲前 (11 week)之免疫球蛋白γ之量。 108
圖 41. 餵食不同濃度山藥發酵乳,對小鼠脾臟細胞增生率的影響。小鼠脾臟細胞在不同濃度刺激原concanavalin A下,以MTT測其細胞存活率,除以未加刺激原的值後,其增生倍率受餵食山藥發酵乳的影響。 109
圖 42. 餵食不同濃度山藥發酵乳,對小鼠脾臟細胞增生率的影響。小鼠脾臟細胞在不同濃度刺激原(LPS)下,以MTT測其細胞存活率,除以未加刺激原的值後,其增生倍率受餵食山藥粗多醣的影響。 110
表 3. 小鼠攝食劑量與其他種生物換算表。 111
【附錄一】台灣山藥之品種 112
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