臺灣博碩士論文加值系統

褐藻多醣 (Fucoidan) 是從褐藻中萃取出的硫酸多醣，具有多種的生理活性，目前許多的研究都集中在褐藻多醣聚合物的抗癌活性上，文獻指出其能有效抑制結腸癌等多種癌症，為極具潛力之抗癌成份。褐藻多醣可根據其分子量以不同的方式調節細胞生長，然而其為親水性大分子，通過腸胃道後常不易被吸收，而導致生物利用率明顯降低。本研究探討來自 Laminaria japonica 的褐藻多醣以過氧化氫降解後再進行超過濾，分離出三個不同分子量褐藻多醣的抗癌活性，並製備 pH 敏感型幾丁聚醣/褐藻多醣奈米粒 (Chitosan/Fucoidan nanoparticles, CS/F NPs)，探討其經口服傳輸系統對抗結腸癌的效果。本實驗成功製備 436 kDa、23-128 kDa 及 8 kDa 三種不同分子量的高、中、低分子量褐藻多醣 (HF、MF 及 LF)，與幾丁聚醣經由電荷交互作用形成 CS/F NPs，粒徑約 149-228 nm；當幾丁聚醣及褐藻多醣的重量比為 3:1 時，奈米粒具 pH 值敏感性，CS/F NPs 能於仿腸道 pH 環境 (pH=7.4) 中崩解，使褐藻多醣自奈米粒中釋放。細胞實驗結果顯示 LF 抑制 HT-29 結腸癌細胞生長效果較佳，其 IC50 為 245.53 μg/mL；經製備為奈米粒後 IC50 為 203.29 μg/mL，表示褐藻多醣經包覆後可提高其抗癌效果。於細胞跨膜電位實驗顯示，此奈米粒可促使 Caco-2 腸道上皮細胞開啟，使釋放的褐藻多醣經由細胞間隙之路徑被吸收。綜合上述，褐藻多醣能藉由 CS/F NPs 提高其生物利用率，且 LF 較有抗腸癌之潛力，透過製備為 CS/F NPs 可提升褐藻多醣之抗癌效果及腸道吸收效果。

Fucoidan is a complex sulfated polysaccharide that is found in brown algae. Recently, the diverse biological activities of fucoidan have been studied intensively. In particular, the antitumor activity has recently attracted considerable attention. Fucoidan has also been shown to induce a substantial reduction in viable cell numbers and apoptosis of human colon cancer HT-29 cells. Fucoidan polymers can modulate cell growth in different manners depending on their molecular weight.　However, in the stomach and small intestine may lead hydrophilic macromolecules of fucoidan cannot diffuse across the cells through the lipid-bilayer cell membranes. In this study, fucoidan was extracted from the Laminaria japonica, hydrolyzed by hydrogen peroxide and subsequently fractionated using an ultrafiltration system, which produced three fucoidan fractions with different molecular weights. To overcome the poor oral bioavailability, a pH sensitive Chitosan/Fucoidan nanoparticle (CS/F NP) was prepared to against colon cancer. The objectives of this study were to determine the physicochemical properties of these fucoidan fractions and to investigate the relationship between their molecular characteristics and promote in vitro anticancer activity with CS/F NPs. We successfully prepared three fucoidan fractions with 436 kDa, 23-128 kDa and 8 kDa, named HF, MF and LF, respectively. The results suggest that the nanoparticles composed of chitosan and fucoidan were successfully prepared based on their electrostatic interaction. The size of CS/F NPs was around 149-228 nm. The CS/F NPs reveals significant pH sensitive property and high loading efficiency as the weight ratio of chitosan to fucoidan was 3:1. According to control release studies, the encapsulated fucoidan was released from NPs under simulated intestinal pH environment (pH=7.4). In vitro studies suggested that LF inhibited the growth of HT-29 cell better with the IC50 was 245.53 μg/mL. After preparation for CS/LF NPs, the IC50 was 203.29 μg/mL. Indicating that the preparation of fucoidan nanoparticles will not affect its anticancer activity. The results of transepithelial electric resistance (TEER) of Caco-2 show that NPs effectively enhanced opening of cell tight junctions. Briefly, all the results mentioned above suggest that LF should be explored as a potential anticancer agent, and the CS/F NPs developed in this study are a promising carrier for oral delivery of fucoidan against colon cancer and improve its bioavailability.

摘要 i
Abstract ii
目錄 iii
圖目錄 v
表目錄 vii
第一章、前言 1
第二章、文獻回顧 2
2.1 褐藻多醣 2
2.1-1 褐藻多醣的抗癌活性 2
2.2 褐藻多醣降解 4
2.3 褐藻多醣奈米粒 4
2.4 幾丁聚醣/褐藻多醣奈米粒 5
2.4-1 幾丁聚醣/褐藻多醣奈米粒之腸道吸收效果 6
第三章、實驗目的 7
第四章、實驗架構 8
第五章、實驗流程 9
第六章、實驗材料與方法 10
6.1 實驗材料 10
6.2 實驗藥品 10
6.3 實驗儀器 12
6.4 實驗溶液配製 14
6.5實驗方法 16
6.5-1 幾丁聚醣的製備 16
6.5-2 褐藻多醣的製備 16
6.5-3 製備幾丁聚醣/褐藻多醣奈米粒 18
6.5-4 幾丁聚醣/褐藻多醣奈米粒之物理化學特性探討 18
6.5-5褐藻多醣之控制釋放分析 20
6.5-6 奈米粒的抗癌效果 20
6.5-7 奈米粒的腸道吸收效果 21
6.5-8 統計分析 23
第七章、結果討論 24
7.1 幾丁聚醣的製備 24
7.2 褐藻多醣之物理化學特性 24
7.2-1 化學成分分析 24
7.2-2 傅立葉紅外線光譜 (FT-IR) 定性分析 25
7.2-3 X光繞射晶體 (XRD) 分析 25
7.3 幾丁聚醣/褐藻多醣奈米粒之物理化學特性 25
7.3-1 粒徑大小、Zeta 電位及多分散性分布指數 25
7.3-2 傅立葉紅外線光譜 (FT-IR) 定性分析 26
7.3-3 X光繞射晶體 (XRD) 分析 26
7.3-4 pH 敏感特性分析 26
7.3-5穩定度測試 27
7.4 奈米粒構型觀察 27
7.5 不同 pH值下控制釋放 28
7.6 細胞實驗 28
7.6-1 褐藻多醣的抗癌效果 28
7.6-2 幾丁聚醣/褐藻多醣奈米粒的抗癌效果 29
7.6-3 幾丁聚醣/褐藻多醣奈米粒的腸道吸收效果 30
第八章、結論 33
第九章、參考文獻 34
第十章、圖表 43

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