臺灣博碩士論文加值系統

胰島素阻抗性 (Insulin resistance, IR) 是第二型糖尿病的特徵之一，本研究目的為評估石蓴正己烷萃取物 (Ulva fasciata hexane extract, UF) 或石蓴寡醣 (Ulva fasciata oligosaccharides, UO) 於人體腸道及肝臟細胞之抗糖尿病效果並探討作用機制。首先，脂肪酸成分分析顯示UF主成分為α-次亞麻油酸 (17.51 mg/g)、亞麻油酸 (9.68 mg/g)、油酸 (1.98 mg/g) 及棕櫚油酸 (1.08 mg/g)；其總類胡蘿蔔素、葉黃素a及葉黃素b分別為0.30 g/g、0.09 mg/g及0.10 mg/g。UO總醣含量約15%，分子量小於1 kDa (曾, 2018)。體外試驗顯示20 μg/mL UF及1000 μg/mL UO皆具有抗氧化活性，其清除DPPH自由基能力分別相當於2.0 μg/mL維生素E及0.3 μg/mL維生素C、螯合亞鐵離子能力相當於3.0 μg/mL 及4.0μg/mL EDTA、清除超氧陰離子能力相當於54.6 μg/mL及95.8 μg/mL没食子酸。以UF (2.5 ~ 20 μg/mL) 或UO (7.8 ~ 125 μg/mL) 分別處理人類腸道Caco-2細胞，發現抑制蔗糖酶活性最高可達控制組58.8%或39.1%，其中 7.8 ~ 125 μg/mL UO抑制Caco-2細胞蔗糖酶活性與抗糖尿病藥物Acarbose (125 μg/mL) 相當。人類肝臟HepG2細胞模式發現，UF (2.5 ~ 20 μg/mL) 顯著增加細胞膜表面之葡萄糖運輸蛋白GLUT4表現 (達控制組114.5 ~ 173.8%)、促進葡萄糖攝入量 (102.7 ~ 176.2%)、提升細胞內肝醣 (較控制組增加 0.07 μg/μL) 及ATP含量 (達132.3 ~ 159.6%)、並抑制細胞外乳酸產生 (較控制組降低26.4 ~ 61.6 pmol/μL)，因此UF有助於IR肝臟細胞利用葡萄糖產能及合成肝醣；Western blot結果顯示此現象與UF可抑制IRS-1 (Ser307) 磷酸化及促進PI3K、AMPK、AKT磷酸化有關。此外，UF (10 μg/mL) 及UO (500 ~ 1000 μg/mL) 皆可清除HepG2細胞內活性氧分子 (分別為控制組56.0%及44.0 ~ 60.8%)，並降低IL-8分泌 (分別較控制組降低115.0 pg/mL及136.1 ~ 186.9 pg/mL)。綜上，UF能抑制腸細胞之蔗糖酶活性並活化肝細胞PI3K訊息、提升GLUT4 translocation、增加葡萄糖利用、減少ROS及IL-8，因此UF具有改善IR之潛力。

Insulin resistance (IR) is one of the characteristics of type 2 diabetes. This study was aimed to investigate the anti-diabetics effects and mechanisms of Ulva fasciata hexane extract (UF) or Ulva fasciata oligosaccharides (UO) in human intestinal and hepatic cells. Firstly, the fatty acid analysis revealed that the most abundant unsaturated fatty acids in UF were α-linolenic acid (C18:3 n3; 17.51 mg/g), linoleic (C18:2 n6c; 9.68 mg/g), oleic (C18:1 n9c; 1.98 mg/g) and palmitoleic (C16:1; 1.08 mg/g). The contents of carotenoids, chlorophyll a and chlorophyll b were 0.30 g/g, 0.09 mg/g and 0.10 mg/g, respectively. The total carbohydrate content of UO was about 15%, molecular weight was less than 1 kDa. (曾，2018). UF and UO demonstrated antioxidative activity. The DPPH radical-scavenging assay revealed that the potential of 20 μg/mL UF and 1000 μg/mL UO were equivalent to 2.0 μg/mL of vitamin E and 0.3 μg/mL of vitamin C. Iron chelating asaay demonstrated that the potential of 20 μg/mL UF and 1000 μg/mL UO were equivalent to 3.0 μg/mL and 4.0 μg/mL of EDTA. Superoxide radical-scavenging assay found that the potential of 20 μg/mL UF and 1000 μg/mL UO were equivalent to 54.6 μg/mL and 95.8 μg/mL of gallic acid. Besides, UF (2.5 ~ 20 μg/mL) and UO (7.8 ~ 125 μg/mL) significantly inhibited sucrase activity in human intestinal Caco-2 cells to 58.8% and 39.1% of control group, respectively. The sucrase inhibitory effect of UO (7.8 ~ 125 μg/mL) was equivalent to anti-diabetic drug Acarbose (125 μg/mL) in Caco-2 cells. In human hepatic HepG2 cells, UF (2.5 ~ 20 μg/mL) significantly improved translocation of glucose tranporter GLUT4 to cell membrane (114.5 ~ 173.8% of control), enhanced glucose uptake (102.7 ~ 176.2%), and increased intracellular glycogen content (0.07 μg/μL more than control), as well as accelated ATP content (132.3 ~ 159.6%) and declined lactate production (26.4 ~ 61.6 pmol/μL less than control). Notable, the regulatory effects of UF were correlated with decreased IRS-1 (Ser307) phosphorylation and activated PI3K/AMPK/AKT signaling. Additionally, treatment with UF (10 μg/mL) or UO (500 ~ 1000 μg/mL) decreased ROS production (56.0% or 44.0 ~ 60.8% of control) and suppressed interleukin (IL)-8 secretion (115.0 pg/mL or 136.1 ~ 186.9 pg/mL less than control). In conclusion, the anti-diabetic effects of UF may through decreasing intestinal sucrase activity, activation hepatic PI3K signaling, and increasing translocation of GLUT4, as well as increasing glucose utilization, and ameliorate intracellular ROS and IL-8 production. Therefore, UF is a potent agent to attenuate IR.

摘要 I
Abstract II
縮寫表 IV
目次 VI
圖目次 IX
表目次 X
第一章 前言 1
第二章 文獻回顧 2
第一節 石蓴 (Ulva fasciata) 2
1. 石蓴 2
2. 石蓴生理活性 2
3. 石蓴萃取物於T2DM動物之調節機制 3
第二節 高血糖誘發葡萄糖代謝之影響 4
第三節 Caco-2細胞模式 4
第四節 HepG2細胞模式 4
第五節 血糖恆定 (glucose homeostasis) 5
1. 葡萄糖攝入肝細胞之作用 5
2. 糖新生作用 6
第六節 胰島素 (insulin) 6
1. 胰島素訊息傳遞路徑 6
2. PI3K/AKT 7
第七節 腺苷單磷酸活化蛋白激酶 (AMP-activated protein kinase, AMPK) 7
1. AMPK之結構 (structure of AMPK) 7
2. AMPK之調控 (regulation of AMPK) 8
第八節 延緩高血糖之症狀 8
第三章 研究設計 10
第一節 實驗架構 ： 石蓴之成分及抗氧化活性分析 10
第二節 實驗架構 ： 腸道細胞雙醣酶活性 11
第三節 實驗架構 ： 肝細胞胰島素抗性模式 12
第四章 研究材料及方法 13
第一節 研究材料 13
1. 細胞株 13
2. 細胞培養用試劑及耗材 13
3. 材料及藥品 13
4. 儀器設備 15
第二節 研究方法 16
1. 石蓴之一般成分分析 16
2. 萃取石蓴之脂溶性成分、脂肪酸及總類胡蘿蔔素測定 18
3. 萃取石蓴寡醣及分子量篩選 19
4. 抗氧化試驗 19
5. 細胞實驗 21
第五章、結果 29
第一節 石蓴之成分分析及抗氧化活性測定 29
第二節 UF及UO顯著抑制Caco-2細胞之蔗糖酶活性 29
第三節 高濃度葡萄糖細胞培養液誘導HepG2細胞 30
第四節 UF增加HepG2細胞膜上GLUT4表現 30
第五節 UF增加HepG2細胞之葡萄糖攝取量 31
第六節 UF增加HepG2細胞之肝醣含量 31
第七節 UF促進HepG2細胞之 ATP 生成 32
第八節 UF抑制HepG2細胞之乳酸生成 32
第九節 UF抑制IRS-1 (Ser307) 之磷酸化 32
第十節 UF活化HepG2 細胞之PI3K、AMPK、AKT訊息 33
第十一節 UO抑制HepG2 細胞ROS產生及IL-8分泌 33
第六章、討論 35
第一節 石蓴萃取物之成分分析及抗氧化活性比較 35
第二節 石蓴萃取物抑制 Caco-2 細胞之雙醣酶活性 36
第三節 高濃度葡萄糖細胞培養液誘導HepG2細胞 36
第四節 石蓴萃取物對葡萄糖攝取之影響 37
第五節 石蓴萃取物對肝臟葡萄糖之新陳代謝影響 38
第六節 石蓴萃取物於胰島素訊息傳遞之調控 38
第七節 石蓴萃取物抑制IR-HepG2 細胞之氧化壓力 39
第七章、結論 40
第八章、參考文獻 41

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