臺灣博碩士論文加值系統

幾丁聚醣是一種具生物相容性，可分解性且無毒性之天然高分子化合物，在生物醫藥，食品及化學工業具有廣泛之用途。然幾丁聚醣在生物應用上仍有難溶於水及高黏度之缺點，顯然水溶性幾丁聚醣之製備及其生物活性之探討甚為重要，因此本研究製備三種水溶性低分子量幾丁聚醣衍生物，並探討及其生物活性，包括抗氧化能力及α-葡萄糖苷酶抑制活性。
三種不同幾丁聚醣衍生物之製備係將市售幾丁聚醣 (分子量1000 kDa，去乙醯度84%)以化學修飾劑 2-chloroethanol，2-chloroacetate及2-chloroethylamino hydrochloride於第6 C上衍生不同基團，其接上幾丁聚醣之分支鏈分別為ethylene glycol (EG)，carboxymerthyl (CM) 和aminoethyl (AE)等基團。將製備之水溶性幾丁聚醣衍生物進一步以愛玉子乳汁幾丁聚醣酶降解，產生三種低分子量幾丁聚醣 (LMWC) 衍生物分別為EG-LMWC、CM-LMWC及AE-LMWC。以Superose 12 HR管柱膠體過濾層析測得三種低分子量幾丁聚醣之分子量分別為11.2，11.2及8.89 kDa。
低分子量幾丁聚醣衍生物之抗氧化活性以ABTS、超氧陰離子及氧自由基清除效應模式評估之。比較ABTS自由基之trolox當量抗氧化能力 (TEAC)，CM-LMWC具有最高清除活性，其次為AE-LMWC和EG-LMWC，CM-LMWC之TEAC值為45.97 ± 0.1 mg trolox/ g sample。比較超氧陰離子自由基之trolox當量，AE-LMWC優於CM-LMWC及EG-LMWC，AE-LMWC之TEAC值為1853 ± 52 mg trolox/ g sample。比較氧自由基吸收能力 (ORAC)，AE-LMWC及CM-LMWC優於EG-LMWC，其ORAC值分別為2715 ± 113 及1894 ± 347 μmol trolox/ g sample。
低分子量幾丁聚醣衍生物之降血糖活性係以EG-LMWC和CM-LMWC抑制α-葡萄糖苷酶活性評估之。結果顯示兩種低分子量幾丁聚醣對α-葡萄糖苷酶水解p-nitrophenyl α-glucoside基質均具有顯著抑制活性，EG-LMWC和CM-LMWC之半抑制濃度 (IC50)分別為2.96 ± 0.05及 2.65 ± 0.03 mg/mL。以Lineweaver-Burk 做圖進行抑制動力學分析，顯示EG-LMWC屬非競爭性抑制物，而CM-LMWC則屬混合型抑制物。

Chitosan is a non-toxic, biocompatible and biodegradable natural macromolecule, and shown to be a valuable biopolymer because of its widespread applications in biomedical, food and chemical industries. However, chitosan has several drawbacks to be utilized in biological applications, including poor solubility under physiological conditions and high viscosity. Apparently it is important to prepare water-soluble chitosans and explore their biological activities. Therefor, in this study, three kinds of water-soluble low-molecular-weight chitosan (LMWC) derivatives were prepared and their biological activities including antioxidant capacity and α-glucosidase inhibitory activity were investigated.
To synthesize three different chitosan derivatives, commercial chitosan with molecular mass of 1000 kDa and 84% of deacetylation were chemically modified by grafting 2-chloroethanol, 2-chloroacetate and 2-chloroethylamine hydrochloride at C-6 position. The attached substituting branched-chain on chitosan were ethylene glycol (EG), carboxymethy (CM) and aminoethyl (AE) groups, respectively. After digested by the jelly fig latex chitosanase, the synthesized water-soluble chitosan derivatives were depolymerized and three kinds of water-soluble LMWC derivatives, namely EG-LMWC, CM-LMWC and AE-LMWC, were generated. The molecular masses of EG-LMWC, CM-LMWC and AE-LMWC were 11.2, 11.2, and 8.89 kDa, respectively, as estimated by gel filtration on Superose 12 HR column.
Antioxidant activities of the prepared LMWC derivatives were evaluated with models of scavenging effects on ABTS, superoxide and oxygen radicals. By comparison of the trolox equivalent antioxidant capacities (TEAC) toward ABTS radicals, CM-LMWC showed the highest scavenging activity, followed by AE-LMWC and EG-LMWC. The TEAC value of CM-LMWC toward ABTS radicals was 45.97 ±0.1 mg trolox/ g sample. By comparison of trolox equivalent (TE) toward superoxide radicals, AE-LMWC was superior to CM-LMWC and EG-LMWC. The TE values for AE-LMWC toward superoxide radicals was 1853 ± 52 mg trolox/ g sample. By comparison of oxygen radical absorption capacities (ORAC), AE-LMWC and CM-LMWC were superior to EG-LMWC. The ORAC values of AE-LMWC and CM-LMWC were 2715 ± 113 and 1894 ± 347 μmol trolox/ g sample, respectively.
To evaluate the potential of LMWC derivatives being an anti-diabetic agent, inhibitory activity of EG-LMWC and CM-LMWC on the α-glucosidase was assessed. It was found that EG-LMWC and CM-LMWC had potent inhibitory effects on the hydrolysis of p-nitrophenyl- α-glucoside by α-glucosidase. The IC50 values for EG-LMWC and CM-LMWC were 2.96 ± 0.05 and 2.65 ± 0.03 mg/mL, respectively. The inhibition kinetics were analyzed by Lineweaver-Burk plots, which revealved that EG-LMWC was a noncompetitive inhibitor whereas CM-LMWC was a mixed type inhibitor.

目錄 I
表目錄 IV
圖目錄 V
縮寫表 VII
中文摘要 VIII
英文摘要 X
第一章 前言 1
第一節 研究背景及相關文獻 1
第二節 研究目的 3
第二章 文獻回顧 4
第一節 幾丁質 (chitin)、幾丁聚醣 (chitosan)之結構、性質與應用 4
壹、 幾丁質與幾丁聚醣之結構 4
貳、 幾丁聚醣的製備方法 5
參、 幾丁質與幾丁聚醣之物化特性 6
肆、 幾丁質與幾丁聚醣在食品工業上之應用 6
第二節 幾丁聚醣及寡醣之生物活性 8
壹、 抗菌作用 8
貳、 降血脂作用 9
參、 降尿酸功能 11
肆、 增強免疫、抗腫瘤和抗癌功能 11
伍、 抗氧化活性 12
陸、 抗發炎和修復關節組織 12
柒、 降血壓功能 13
捌、 抗病毒活性 13
玖、 幾丁聚醣之安全性評估 13
第三節 低分子量幾丁聚醣之製備及其生物活性 14
壹、 低分子量幾丁聚醣製備 14
貳、 低分子量幾丁聚醣之生物活性 14
第四節 自由基與天然抗氧化物質 21
壹、 自由基 (free radicals) 和活性氧 (reactive oxygen species, ROSs) 之生成與特性 21
貳、 氧化壓力與疾病 23
參、 抗氧化之物質 27
第五節 糖尿病與α-葡萄糖苷酶抑制劑 32
壹、 糖尿病簡介 32
貳、 糖尿病的診斷及分類 32
參、 糖尿病治療藥物 35
肆、 α-葡萄糖苷酶抑制物 37
第三章 材料及方法 40
第一節 實驗材料 40
壹、 幾丁聚醣(chitosan) 40
貳、 愛玉子果實乳汁 40
參、 化學藥品 40
肆、 重要器材及儀器 41
第二節 實驗方法 42
壹、 水溶性幾丁聚醣衍生物及其水解產物製備 42
貳、 水溶性低分子量幾丁聚醣衍生物之抗氧化活性測定 50
參、 水溶性低分子量幾丁聚醣衍生物之酵素抑制活性 58
肆、 統計分析 64
第四章 結果與討論 65
第一節 水溶性低分子量幾丁聚醣之製備 65
壹、 水溶性幾丁聚醣製備 65
貳、 水溶性幾丁聚醣之酵素水解產物 66
參、 水溶性低分子量幾丁聚醣產物之分子量 66
第二節 水溶性低分子量幾丁聚醣產物之抗氧化活性 69
壹、 ABTS自由基清除活性 69
貳、 超氧陰離子清除能力測定 73
參、 氧自由基吸收能力 77
第三節 水溶性低分子量幾丁聚醣之酵素抑制活性 89
壹、 α-葡萄糖苷酶抑制活性 89
貳、 α-葡萄糖苷酶抑制型式及抑制常數測定 93
第五章 結論 100
第六章 未來展望 101
第七章 參考文獻 102
附錄一 120

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