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研究生:鄒裕民
研究生(外文):Yu-Min Tsou
論文名稱:幾丁聚醣-麩胱甘肽-龍鬚菜多醣萃取物複合奈米粒之製備及抗氧化特性探討
論文名稱(外文):Preparation and antioxidative properties of chitosan-glutathione-Gracilaria polysaccharide extract nanoparticles
指導教授:張克亮
指導教授(外文):Ke-Liang Chang Bruce
學位類別:碩士
校院名稱:國立臺灣海洋大學
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:2006
畢業學年度:94
語文別:中文
論文頁數:111
中文關鍵詞:幾丁聚醣龍鬚菜多醣麩胱甘肽奈米科技抗氧化藻類
外文關鍵詞:chitosanGracilaria polysaccharidesglutathionenanoparticlesantioxidantagar
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摘要
龍鬚菜為臺灣常見之大型藻類。本研究以幾丁聚醣與龍鬚菜多醣水解液為原料,製備出具抗氧化等機能性成分之幾丁聚醣-麩胱甘肽-龍鬚菜多醣萃取物複合奈米粒,並對其進行特性分析。
將龍鬚菜多醣以cellulase水解後,還原醣含量會隨著水解時間的延長而逐漸升高,且水解24小時後之水解液可有80.5 %清除DPPH自由基、73.5 %螯合亞鐵離子及93.6 %清除超氧陰離子能力。經光散射分析儀與包覆率分析發現,以乳化沉澱法在幾丁聚醣、麩胱甘肽與龍鬚菜多醣水解液濃度0.28 %、0.28 %與0.82 %所製備出之奈米粒為最佳條件,平均粒徑約306 nm,包覆率有78.2 %,而以SEM觀察奈米粒近於圓形,粒徑介於100 ~ 150 nm之間。
在清除DPPH自由基上,麩胱甘肽與龍鬚菜多醣水解液皆有不錯之抗氧化能力,在濃度1 %時,清除率可達80 %左右。於螯合亞鐵離子方面,龍鬚菜多醣水解液可有約90 %之螯合率。至於清除超氧陰離子部分,龍鬚菜多醣萃取與水解液亦有70 %左右之清除能力。而奈米粒之抗氧化效果,有隨溶解時間增加而持續上升的趨勢,在溶解時間180分鐘時,清除DPPH自由基、螯合亞鐵離子及清除超氧陰離子能力方面,依序為43.8、40.1及32.1%。
以溶離試驗機進行奈米粒之溶離測試,發現多酚與麩胱甘肽皆於模擬胃液中有較高之釋放速率,於溶離時間120分鐘時,分別溶出60.18與0.80 μg / ml。
將龍鬚菜多醣水解液與奈米粒添加至細胞中進行細胞存活試驗,兩者對正常細胞(CCD-966sk)影響不大,存活率於最高濃度(1.667 mg / ml)下高於七成,而癌細胞方面,水解液似乎可抑制人類腸道上皮癌與乳癌細胞生長,於最高濃度(1.667 mg / ml)時可使細胞存活率降至67.06及52.13 %。
至於奈米粒於不同酸鹼值之熱安定性與儲存安定性部分,顯示隨著加熱及存放時間的延長,奈米粒之抗氧化能力會逐漸降低,但在鹼性環境中,DPPH自由基之清除能力較能保持,可有40 %的清除率,可是在清除超氧陰離子方面,卻反而是低pH值時表現較佳,可有近50 %之清除能力。
Abstract
The Gracilaria tenuistipita is a common macroalgae in Taiwan. This research investigated the preparation and characterization of antioxidant capacity of chitosan-glutathione-Gracilaria polysaccharide extract nanoparticles using chitosan and Gracilaria polysaccharides hydrolysates as ingredients.
After the hydrolysis of Gracilaria polysaccharides by cellulase, the reducing sugar increased gradually with increasing hydrolysis time. After hydrolysis for 24 hours, 80.5 % of the DPPH radical scavenging effect and 73.5 % of ferrous ion chelating effect and 93.6 % of the superoxide anion radical scavenging effect can be reached by the hydrolysate. Based on the light scattering measurement and entrapment efficiency analysis, the nanoparticles made by emulsification-droplet coalescence method using 0.28 % chitosan and 0.28 % glutathione and 0.82 % Gracilaria ploysaccharides hydrolysates has the best combination of size and encapsulation properties. The average particle diameter was 306 nm from light scattering measurement and entrapment efficiency was 78.2 %. However, SEM pictures show that they are 84 nm.
In the scavenging of DPPH radical, both the glutathione and Gracilaria polysaccharide hydrolysates have good antioxidant capacity. At a concentration of 1 %, the scavenging effect of DPPH radical can reach about 80 % by polysaccharide extract from Gracilaria; About 90 % of ferrous ion can be chelated by Gracilaria polysaccharide hydrolysates. For Gracilaria polysaccharide extract and hydrolysates, the radical scavenging effect of the superoxide anion was about 70 %. The antioxidant capacity of nanoparticles increases continuously with increasing dissolution time. After a dissolution time of 180 minutes, 43.8 % DPPH radical scavenging effect and 40.1 % ferrous ion chelating effect and 32.1 % superoxide anion radical scavenging effect can be reached.
The dissolution of the nanoparticles in a dissolution testing system is performed. The glutathione and polyphenol have higher release rates in simulated gastric fluid. After dissolution for 120 minutes, the total amount of glutathione and polyphenol released is 60.18 and 0.80 μg / ml respectively.
The cell viability after the addition of Gracilaria polysaccharide hydrolysates and nanoparticles is examined. They have little effect on the growth of normal cell (CCD-966sk) and the survival rate is higher than 70%. It seems that the Gracilaria polysaccilaride hydrolysates can suppress the growth of HT-29 and MCF-7 cells. Their cell survival rates drop to 67.06 % and 52.13 % after the addition of 1.667 g / ml hydrolysates.
The thermal and storage stability of nanoparticles vary in different pH value. With longer heating and storage time, the antioxidant capacity of the nanoparticles reduces gradually. In high pH value environment, the scavenging of DPPH radical remains relatively unchanged at 40 %. But for the scavenging of superoxide anion radical, the scavenging effect is close to 50 % at low pH value.
目錄
目錄..…………………………………………………………….…..........I
圖表目錄.………………………………………………………………VII
附圖表目錄…………………………………………………………..….X
中文摘要………………………………………………………………..XI
英文摘要……………………………………………………………...XIII
壹、研究背景與目的……………………………………………..………1
貳、文獻整理………………………………………………………..……5
一、抗氧化物質……………………………………………………..…5
1. 自由基與活性氧………………………………………………….5
2. 抗氧化物質種類………………………………………………….6
2.1 天然抗氧化物………………………………………………….6
2.2 人體自行製造的抗氧化酶…………………………………….7
二、麩胱甘肽(Glutathione)……………………………………….…..8
1. 麩胱甘肽之合成及代謝………………………………………….8
2. 麩胱甘肽之抗氧化功能………………………………………….8
3. 麩胱甘肽與老化之關係………………………………………….9
三、海藻多醣…………………………………………………………..9
1. 海藻多醣定義及特性…………………………………………….9
2. 影響海藻多醣萃取因子………………………………………...11
2.1 浸漬處理……………………………………………………...11
2.2 萃取溫度……………………………………………………...11
2.3 萃取時間……………………………………………………...12
2.4 酸鹼處理……………………………………………………...12
2.5 酵素處理……………………………………………………...13
3. 海藻多醣生理活性……………………………………………...13
4. 龍鬚菜多醣……………………………………………………...14
4.1 龍鬚菜簡介…………………………………………………...15
4.2 龍鬚菜多醣生理活性………………………………………...16
4.3 龍鬚菜寡醣生理活性………………………………………...16
四、幾丁質類物質……………………………………………………17
1. 幾丁質與幾丁聚醣之簡介……………………………………...17
2. 幾丁質與幾丁聚醣之溶解性質………………………………...18
3. 幾丁聚醣包覆核心物質方法…………………………………...18
4. 幾丁聚醣於生醫材料上之應用………………………………...19
五、奈米技術於藥物輸遞之應用………………………..…………20
1. 奈米級藥物載體……………………………………………….20
2. 奈米級藥物載體的製造方法………………………………….21
2.1 溶劑揮發法………………………………………………….21
2.2 自發性乳化 / 溶劑擴散法………………………………...21
2.3 乳化沉澱法………………………………………………….22
2.4 聚合法……………………………………………………….22
2.5 應用親水性聚合物製備奈米級顆粒………………………23
叁、實驗材料及方法……………………………………………………24
一、實驗藥品…………………………………………………………24
二、實驗儀器…………………………………………………………25
三、實驗流程圖………………………………………………………28
四、實驗步驟…………………………………………………………29
1. 龍鬚菜多醣物質萃取…………………………………………...29
1.1 龍鬚菜前處理………………………………………………...29
1.2 龍鬚菜多醣體製備…………………………………………...29
1.3 龍鬚菜多醣酵素水解………………………………………...29
a. Agarase水解…………………………………………………...30
b. Cellulase水解………………………………………………….30
2. 龍鬚菜萃取物、水解液成分分析……………………………….31
2.1 粗成分分析…………………………………………………...31
a. 水分…………………………………………………………...31
b. 粗蛋白………………………………………………………...31
c. 粗纖維………………………………………………………...32
d. 粗脂肪………………………………………………………...32
e. 灰分…………………………………………………………...32
f. 碳水化合物……………………………………………………33
2.2 元素分析……………………………………………………...33
2.3 化學成分分析………………………………………………...33
a. 全醣量………………………………………………………...33
b. 蛋白質定量…………………………………………………...33
c. 3,6-anhydro-galactose含量測定………………………………34
d. 還原醣測定…………………………………………………...34
e. 硫酸酯含量測定……………………………………………...35
2.4 抗氧化成分分析……………………………….......................36
a. 總酚類化合物(polyphenol)含量測定……………………….37
b. 類黃酮(flavonoid)含量測定………………………………....37
3. 幾丁聚醣-麩胱甘肽-龍鬚菜多醣萃取物複合奈米粒製備…….37
3.1 離子聚合法…………………………………………………...37
3.2 乳化沉澱法…………………………………………………...38
4. 複合奈米粒之特性分析………………………………………...39
4.1 粒徑分析……………………………………………………...39
a. 光散射分析…………………………………………………...39
b. 掃描式電子顯微鏡(SEM)…………………………………...39
4.2 包覆率測定…………………………………………………...39
4.3 抗氧化能力試驗……………………………………………...40
a. 清除DPPH(1,1-diphenyl-2-picrylhydrazyl)自由基能力……40
b. 螯合亞鐵離子能力…………………………………………...41
c. 清除超氧陰離子能力………………………………………...42
4.4 溶離釋放試驗………………………………………………...43
4.5 細胞存活試驗………………………………………………...45
a. 類人類巨噬細胞(UM)……………………………………….45
b. 人類纖維母細胞(CCD-966sk)………………………………45
c. 人類腸道上皮癌細胞(HT-29)……………………………….45
d. 人類乳癌細胞(MCF-7)......………………………………….45
e. 人類黑色素瘤細胞(A375)…………………………………...45
f. 人類肝癌細胞(sk-Hep1)……………………………………..45
4.6 安定性試驗…………………………………………………...45
a. 不同酸鹼條件下之熱安定性……………………………….45
b. 儲存安定性之試驗………………………………………….46
肆、結果與討論…………………………………………………………47
一、龍鬚菜多醣萃取、水解液之特性分析…………………………..47
1. 成分分析………………………………………………………...47
2. 抗氧化性測定…………………………………………………...48
二、不同製備方法及樣品濃度對複合奈米顆粒之粒徑與包覆率影 響…………………………………………………………….....49
三、幾丁聚醣-麩胱甘肽-龍鬚菜多醣萃取物複合奈米粒之特性探討……………………………………………………………….51
1. 粒徑分析………………………………………………………...51
2. 奈米粒抗氧化性測定…………………………………………...52
3. 溶離釋放試驗…………………………………………………...54
4. 細胞存活試驗…………………………………………………...55
5. 安定性試驗……………………………………………………...56
伍、結論…………………………………………………………………58
陸、參考文獻……………………………………………………………60
圖………………………………………………………………………..76
表………………………………………………………………………..97
附圖……………………………………………………………………106
附表…………………………………………………………………..110
圖表目錄
圖一、龍鬚菜外觀………………………………………………………76
圖二、全醣含量測定之檢量線,分別以葡萄糖及鼠李醣為標準品…..77
圖三、蛋白與3,6-anhydro-galactose含量測定之檢量線,分別以牛血清蛋白及果糖為標準品………..………………………………78
圖四、還原醣與硫酸酯含量測定之檢量線,分別以葡萄糖及Na2SO4為標準品………………………………...……………………...79
圖五、總酚類與類黃酮含量測定之檢量線,分別以沒食子酸及榭固酮 為標準品…………………..…………………………...……….80
圖六、幾丁聚醣、麩胱甘肽、龍鬚菜萃取與水解液之清除DPPH自由基能力…………………….……………………………...……..81
圖七、幾丁聚醣、麩胱甘肽、龍鬚菜萃取與水解液之螯合亞鐵離子能力…………………………………...…………………………...82
圖八、幾丁聚醣、麩胱甘肽、龍鬚菜萃取與水解液之清除超氧陰離子能力……………………...……………………………………...83
圖九、麩胱甘肽含量測定之檢量線一,以甲醛溶液及5,5-dithio-bis (2-nitrobenzoic acid) 試劑進行檢測……………………..…….84
圖十、以掃描式電子顯微鏡觀察幾丁聚醣-麩胱甘肽-龍鬚菜多醣萃取物奈米粒………………………………………………………..85
圖十一、幾丁聚醣-麩胱甘肽-龍鬚菜多醣萃取物奈米粒中多酚於胃液(pH 1.24)120分鐘及腸液(pH 7.48)180分鐘之釋放情形……………………………………………………………...86
圖十二、麩胱甘肽含量測定之檢量線二,以5,5-dithio-bis (2-nitrobenzoic acid) 試劑進行檢測……………..………….87
圖十三、幾丁聚醣-麩胱甘肽-龍鬚菜多醣萃取物奈米粒中麩胱甘肽於胃液(pH 1.24)120分鐘及腸液(pH 7.48)180分鐘之釋放情形…………………………………………………………...88
圖十四、龍鬚菜多醣水解液與幾丁聚醣-麩胱甘肽-龍鬚菜多醣萃取物奈米粒對(a)人類纖維母細胞;(b)類人類巨噬細胞存活率之影響。………………………………………………..…...89
圖十五、龍鬚菜多醣水解液與幾丁聚醣-麩胱甘肽-龍鬚菜多醣萃取物奈米粒對人類癌細胞存活率之影響………………………...90
圖十六、幾丁聚醣-麩胱甘肽-龍鬚菜多醣萃取物奈米粒於不同pH值之清除DPPH自由基熱安定性…………………..………......91
圖十七、幾丁聚醣-麩胱甘肽-龍鬚菜多醣萃取物奈米粒於不同pH值之螯合亞鐵離子熱安定性………………………….………..92
圖十八、幾丁聚醣-麩胱甘肽-龍鬚菜多醣萃取物奈米粒於不同pH值之清除超氧陰離子熱安定性…………………………….…..93
圖十九、儲藏不同時間之幾丁聚醣-麩胱甘肽-龍鬚菜多醣萃取物奈米粒清除DPPH自由基之能力………………………..………..94
圖二十、儲藏不同時間之幾丁聚醣-麩胱甘肽-龍鬚菜多醣萃取物奈米粒螯合亞鐵離子之能力……………………….………..........95
圖二十一、儲藏不同時間之幾丁聚醣-麩胱甘肽-龍鬚菜多醣萃取物奈米粒清除超氧陰離子之能力……………………….……..96
表一、龍鬚菜藻體及多醣萃取物粉末之粗成分分析…………………97
表二、龍鬚菜藻體及多醣萃取物粉末之元素分析……………………98
表三、龍鬚菜多醣萃取物經cellulase及agarase水解後之還原醣含量與抗氧化能力……………..………..…………………………....99
表四、龍鬚菜多醣萃取液及水解液之化學組成分析………………..100
表五、以不同濃度幾丁聚醣、龍鬚菜水解液及麩胱甘肽製備奈米粒之中心組合實驗設計條件………………………………………..101
表六、不同濃度之幾丁聚醣、龍鬚菜水解液及麩胱甘肽以離子聚合法製備奈米粒之粒徑與包覆率………………..…………………102
表七、不同濃度之幾丁聚醣、龍鬚菜水解液及麩胱甘肽以乳化沉澱法製備奈米粒之粒徑與包覆率…………………..………………103
表八、幾丁聚醣-麩胱甘肽-龍鬚菜多醣萃取物奈米粒之抗氧化能…104
表九、不同奈米粒溶解三小時後之抗氧化能力……………….....….105
附圖表目錄
附圖一、麩胱甘肽之化學結構………………………………………..106
附圖二、麩胱甘肽在細胞內合成過程………………………………..107
附圖三、洋菜多醣之結構特徵………………………………………..108
附圖四、纖維素、幾丁質及幾丁聚醣之化學結構……………………109
附表一、自然抗氧化物質成份來源及效用…………………………..110
附表二、現代藥物輸遞技術的主要用途……………………………111
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5. 趙強,1997。對抗疾病與老化的新發現-自由基與抗氧化物質。美食天下。64:116-117。
6. 蔡震壽、邱宗甫,1996。酵素處理海菜水解物與蛋白質結合物之乳化性質。食品科學。23(1):35-44。
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