臺灣博碩士論文加值系統

摘要
本研究以幾丁聚醣、矽酸鈉與硫酸鋅、超氧歧化酶為原料，以乳化沉澱法製備出具抗氧化能力之含有硫酸鋅、硫酸鋅+超氧歧化酶或超氧歧化酶之幾丁聚糖-二氧化矽複合奈米粒，並分析其特性。含有超氧歧化酶之幾丁聚醣-二氧化矽奈米粒，於幾丁聚醣及矽酸鈉濃度分別為0.55% 及 0.55% 時為最佳製備條件，平均粒徑約36 nm，超氧歧化酶包覆率為89% ；在90℃加熱2小時後，清除超氧陰離子能力仍可維持在70% ；儲存於pH=3之檸檬酸buffer中340小時後，清除超氧陰離子能力仍可維持在50%。含有超氧歧化酶及硫酸鋅之幾丁聚醣-二氧化矽奈米粒，於幾丁聚醣及矽酸鈉濃度皆為0.87% 時為最佳製備條件，SEM觀察奈米粒近圓形，平均粒徑約27 nm，硫酸鋅包覆率為82 %，超氧歧化酶包覆率則為94%；在90℃加熱2小時後，清除超氧陰離子能力仍可維持在90%；儲存於pH=3之檸檬酸buffer中340小時後，清除超氧陰離子能力仍有約60%。含硫酸鋅之幾丁聚醣-二氧化矽奈米粒，於幾丁聚醣及矽酸鈉濃度分別為0.87 % 及 0.87% 時為最佳製備條件，平均粒徑約28 nm，硫酸鋅包覆率有93%；清除過氧化氫能力為51%。三種奈米粒均可抑制對A549肺癌細胞及SK-Hep-1肝癌細胞生長，又以含超氧歧化酶之幾丁聚醣-二氧化矽複合奈米粒效果最為顯著，於高濃度 ( 167 μg / ml ) 時可使A549肺癌細胞及SK-Hep-1肝癌細胞之細胞存活率降至38% 及 50% 。
雖然隨儲存時間的延長，奈米粒之抗氧化效果會逐漸降低，以奈米粒固定超氧歧化酶確實可以增進其抗氧化能力及熱與酸環境下之安定性。

Abstract
This research explores the method of using chitosan and silica to immobilize superoxide dismutase and zinc sulphate on nanoparticles by emulsion-droplet coalescence.
The optimal combination of particle size and entrapment efficiency of superoxide dismutase (SOD) was obtained by using 0.55% chitosan and 0.55% silicate . The average particle diameter was 36 nm and the entrapment efficiency of SOD was 89%. The superoxide anion radical scavenging effect reached 70% after heating for 2 hrs at 90℃. After stored for 340 hrs in citric acid buffer (pH=3), the superoxide anion radical scavenging effect was 50%.
The optimal combination of particle size and entrapment efficiency of both SOD and zinc sulphate was obtained by using 0.87% chitosan and 0.87% silicate. The average particle diameter was 27 nm and the entrapment efficiency of SOD was 94%. The superoxide anion radical scavenging effect was maintained at 90% after heating for 2 hrs at 90℃. After stored for 340 hrs in citric acid buffer (pH=3), the superoxide anion radical scavenging effect was 60%.
The optimal combination of particle size and entrapment efficiency of zinc sulphate was obtained by using 0.87% chitosan and 0.87% silicate. The average particle diameter was 28 nm and the entrapment efficiency of Zn was 93%. The hydroden peroxide scavenging effect was maintained at 48% after stored for 340 hrs in citric acid buffer (pH=3).
All three nanoparticles could inhibit the growth of both A549 and SK-HEP-1 cancer cell lines. The cell proliferation decreased to 38% and 50% at the concentration of 167 μg/ml of chitosan-silica–SOD nanoparticles. Apparently chitosan-silica containing SOD, zinc, or both SOD and zinc can inhibit the growth of several tumor cells in vitro.
The antioxidant effect of nanoparticles decreased with inereasing storage time. However, the encapusation/immobilization of SOD by chitosan-silica nanoparticles help retain the antioxidative ability and increase its thermal stability.

目錄
目錄…………………………………………………………………….Ⅰ
圖目錄…………………………………………………………..………V
表目錄…………………………………………………………….........VII
中文摘要………………………………………………………………. X
英文摘要…………………………………………………………...….XII
壹、研究背景與目的.................................................................................1
貳、文獻整理…………………………………………………………….3
1.1自由基………………………………………………………………..3
1.2自由基的產生………………………………………………………..3
1.3自由基之種類…………………………………................................4
1.4 自由基對生物體之傷害…………………………………………….5
2 生物之抗氧化機制………………………………................................6
2.1 超氧歧化酶……………………………………………………..…...7
2.2 超氧歧化酶之分類…………………………………………...……7
2.3 超氧歧化酶之生理功能性……………………………………..…..9
3. 鋅的功能性及代謝………………………………….......................12
4. 酵素固定化…………………………………..................................14
4.1 固定化技術…………………………………................................14
5 奈米載體………………………………….......................................16
5.1 奈米載體的製造方法………………………………….…….........17
6. 幾丁類物質…………………………………..................................18
6.1 幾丁質 ( Chitin ) ………………………………….......................18
6.2 幾丁聚醣…………………………………....................................19
参、實驗材料與方法
一、實驗藥品………………………………………………………..21
二、實驗儀器………………………………………………………..23
三、實驗流程圖……………………………………………………..24
四、實驗步驟.…………………………………………………….…25
1. 幾丁聚醣-二氧化矽-超氧歧化酶、超氧歧化酶+硫酸鋅、硫酸鋅複合奈米粒製備……………………………………………………25
1.1 乳化沉澱法………………………………..…………………..….25
2. 複合奈米粒之特性分析………………………………………….25
2.1 粒徑分析…………………………………………..…………….26
a. 光散射分析………………………………………………………...26
b. 掃描式電子顯微鏡( SEM )………..………………………………26
2.2 包覆率測定…………………………………………………….…26
2.3 抗氧化能力試驗……….………………………………………....27
a. 清除超氧陰離子能力……………………………………………...27
b. 清除過氧化氫能力………………………………………...…..…..29
2.4 細胞存活試驗…………………………………………..………...30
a. 肺癌細胞 ( A 549 )………………………………………………….30
b. 肝癌細胞 ( SK-Hep-1 )………………………………….………….30
c. 胃癌細胞 ( MKN-28 )………………………………………………30
d. 大腸癌細胞( HT-29 )………………………………………………..30
2.4 安定性試驗……………………………………………………..….30
a. 超氧歧化酶之最適pH值…………………………………………...31
b. 奈米粒固定化酵素之不同酸鹼條件下室溫儲存性試驗………….31
c. 奈米粒固定化酵素之熱穩定性實驗……………………………...31
肆、結果與討論…………………………………………………............33
1、乳化沉澱法製備複合奈米粒之粒徑及包覆率之探討…....…..….33
2、幾丁聚醣-二氧化矽-超氧歧化酶、超氧歧化酶+硫酸鋅、硫酸鋅
複合奈米粒之特性探討………………………………………....34
2.1粒徑分析…………………………………………………………….34
2.2細胞存活試驗………………………………….………..…….....…35
2.3奈米粒之抗氧化性測定…………………………………………….36
2.4奈米粒固定化酵素之安定性試……………………………….....…37
伍、結論…………………………………………………………….…...42
陸、參考文獻……………………………………………………..……..44
圖…………………………………………………………………….….52
表..………………………………………………………………………66
附圖……………………………………………………………………..76
附表……………………………………………………………………..79














圖目錄
圖一、超氧歧化酶活性測定之標準曲線圖…..………………………..52
圖二、鋅濃度測定之檢量線……………………………………..…….53
圖三、以掃描式電子顯微鏡觀察含超氧歧化酶及硫酸鋅之幾丁聚醣-二氧化矽奈米粒…………………………………………….….54
圖四、以掃描式電子顯微鏡觀察含超氧歧化酶之幾丁聚醣-二氧化矽奈米粒……………………………………………………..……55
圖五、以掃描式電子顯微鏡觀察含硫酸鋅之幾丁聚醣-二氧化矽奈米粒…………………………………………………………..…..56
圖六 、含硫酸鋅、超氧歧化酶或硫酸鋅+超氧歧化酶之幾丁聚醣-二氧化矽奈米粒對(a) 肺癌細胞 (b) 肝癌細胞 (c) 胃癌細胞 (d) 大腸癌細胞存活率之影響………………………….…..57
圖七、超氧歧化酶之最適pH值……………………………………...58
圖八、超氧歧化酶與含超氧歧化酶、硫酸鋅或超氧歧化酶+硫酸鋅之幾丁聚醣-二氧化矽奈米粒清除超氧陰離子能力於不同溫度之熱安定性…………………………..……………………………59
圖九、超氧歧化酶與含有超氧歧化酶、硫酸鋅或超氧歧化酶+硫酸鋅之幾丁聚醣-二氧化矽奈米粒清除超氧陰離子能力於 90℃之熱安定性……………………………………………………..…60
圖十、超氧歧化酶於不同pH值溶液中儲存不同時間後之清除超氧陰離子能力………………………………………………………..61
圖十一、含超氧歧化酶之幾丁聚醣-二氧化矽奈米粒於不同pH值溶液中之清除超氧陰離子自由基室溫儲存安定性..……………62
圖十二、含超氧歧化酶及硫酸鋅之幾丁聚醣-二氧化矽奈米粒於不同
pH值溶液中之清除超氧陰離子自由基室溫儲存安定性...63
圖十三 、含硫酸鋅之幾丁聚醣-二氧化矽奈米粒於不同pH值之溶液中清除超氧陰離子自由基室溫儲存安定性………………..64
圖十四 、含硫酸鋅或超氧歧化酶+硫酸鋅之幾丁聚醣-二氧化矽奈米粒於不同pH值之溶液中清除過氧化氫能力於室溫中之儲存安定性…………………………………………………....65

表目錄
表一、以不同濃度幾丁聚醣及矽酸鈉製備奈米粒之中心組合實驗設計條件……………………………………………………………66
表二、由不同濃度幾丁聚醣與矽酸鈉以乳化沉澱法製備含 500 unit / ml 超氧歧化酶奈米粒之粒徑與包覆率……………….…...…67
表三、由不同濃度幾丁聚醣與矽酸鈉以乳化沉澱法製備含 500 unit / ml 超氧歧化酶及5 % 硫酸鋅奈米粒之粒徑與包覆率…..…68
表四、由不同濃度幾丁聚醣與矽酸鈉以乳化沉澱法製備含 5 % 硫酸鋅奈米粒之粒徑與包覆率……………………..………………69
表五、含超氧歧化酶、超氧歧化酶+硫酸鋅或硫酸鋅之幾丁聚醣-二氧化矽奈米粒之清除超氧陰離子能力……………………..……70
表六、含超氧歧化酶、超氧歧化酶+硫酸鋅或硫酸鋅之幾丁聚醣-二氧化矽奈米粒之清除過氧化氫能力…………………………..…71
表七、不同奈米粒加入溶液中三小時後之抗氧化能力………..……72
表八、含超氧歧化酶之幾丁聚醣-二氧化矽奈米粒於不同pH值緩衝溶液中之清除超氧陰離子能力…………………………….….…73
表九、含超氧歧化酶 + 硫酸鋅之幾丁聚醣-二氧化矽-奈米粒於不同pH值緩衝溶液中之清除超氧陰離子能力………………..…74
表十、含硫酸鋅之幾丁聚醣-二氧化矽-奈米粒於不同pH值緩衝溶液中之清除超氧陰離子能力……………..……………………..75









附件目錄
附圖一、微脂粒對超氧歧化酶之保護作用………………..…………76
附圖二、活性氧來源及超氧歧化酶抗氧化機制………..…..………..77
附圖三 、丙二醛形成途徑……………………………………..……..78
附表一 、人體自行製造的抗氧化酵素………………………………79
附表二 、六類含鋅酵素……………………………..…………………80

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