臺灣博碩士論文加值系統

肥胖是人類許多重大疾病發展之危險因子，包括心血管疾病、糖尿病及癌症因此肥胖是大眾健康與預防醫學上的重要議題。脂質吸收與胰脂解酶活性具有密切的關係，前脂肪細胞分化在脂肪生合成之機制上扮演著相當重要的角色。目前關於荷葉甲醇萃取物及其區分物對胰脂解酶活性及3T3-L1 前脂肪細胞分化之研究甚少。本實驗目的是探討荷葉甲醇萃取物對胰脂解酶活性及 3T3-L1 前脂肪細胞分化之影響。以胰脂解酶活性抑制率與降低前脂肪細胞脂肪合成作用兩項指標篩選荷葉甲醇萃取物，期望能找到具有降低肥胖趨勢之有效區分層、次區分層及活性成分。由荷葉甲醇萃取物中主要分離到 6 種純化合物，包括 quercetin、quercetin-3-O-glucoside、quercetin-3-O-glucuronide、quercetin-3-O-galactoside、catechin 和 kaempferol-3-O-glucoside。在抑制胰脂解酶活性試驗中，quercetin在濃度50 μM 下，與對照組相較下，能顯著地抑制胰脂解酶活性75%（p＜0.05）。在 3T3-L1 前脂肪細胞分化實驗中，各溶劑萃取物皆有降低3T3-L1脂肪細胞三酸甘油酯累積之趨勢；次區分層中以乙酸乙酯層之第 1、3及4 次區分層 (LLME~1、3及4) 在濃度為 50 μg/mL 時，能顯著地抑制3T3-L1脂肪細胞 25% 之三酸甘油酯累積，此外，quercetin 及 quercetin-3-O-galactoside在濃度為 50 μM 下，能抑制3T3-L1 脂肪細胞 15% 及 18% 三酸甘油酯之累積。LLME~3 能降低 PPARγ 和C/EBPα mRNA的表達，尤其是抑制 C/EBPα 的表現較 PPARγ 更具有顯著性 。綜合結果，荷葉甲醇萃取物能降低胰脂解酶之活性及抑制 3T3-L1 前脂肪細胞分化，減少脂肪細胞內三酸甘油酯之累積。

Obesity is the most common nutritional disorder and it is considered to be a risk factor associated with the development of the major human diseases, including cardiovascular disease, diabetes, and cancer. It is an important topic in the world of public health and preventive medicine. It has close relations with lipid absorption and pancreatic lipase activity. The differentiation of 3T3-L1 preadipocytes plays an important role on the mechanism of adipogenesis. In the research about lotus (Nelumbo nucifera Gaertn.) leaf methanolic extract (LLM) on pancreatic lipase activity and differentiation of 3T3-L1 preadipocytes is rare. The aim of the present study was to assess the effects of LLM on pancreatic lipases activity and differentiation of 3T3-L1 preadipocytes. LLM were screened for inhibitory effects on pancreatic lipase (PL) activities as well as on triglyceride (TG) accumulation in 3T3-L1 preadipocytes to find the effective fractions, subfractions and active compounds that having the anti-obesity trend. Six flavonoids were separated from LLM, including quercetin, quercetin-3-O-glucoside, quercetin-3-O-glucuronide, quercetin-3-O-galactoside, catechin and kaempferol-3-O-glucoside. In pancreatic lipase activity-inhibiting assay, quercetin exhibited significant inhibitory effects at the concentration of 50 μM with 75% pancreatic lipase activity v.s control (p<0.05). In 3T3-L1 preadipocyte experiment, subfractions 1, 3 and 4 of LLME (LLM ethyl acetate partition) markly inhibited about
v
25% of lipid accumulation in 3T3-L1 cells at the concentration of 50 μg/mL. Moreover, quercetin and quercetin-3-O-galactoside were found to inhibit TG accumulation in 3T3-L1 cells at the concentration of 50 μΜ with 15% and 18%. Subfraction 3 of LLME could reduce expression of PPARγ and C/EBPα mRNA, especially mRNA expression-inhibiting of C/EBPα is more apparent than PPARγ. Consequently, LLM could reduce pancreatic lipase activity, inhibit the differentiation of 3T3-L1 preadipocytes and decrease TG accumulation in 3T3-L1 cells.

目 錄
頁數
口試委員會審定書-------------------------------------------i
誌謝------------------------------------------------------ii
中文摘要-------------------------------------------------iii
英文摘要--------------------------------------------------iv
目錄------------------------------------------------------vi
圖目錄----------------------------------------------------ix
表目錄-----------------------------------------------------x
英文縮寫對照表--------------------------------------------xi
第一章、總論-----------------------------------------------1
1.1 前言---------------------------------------------------1
1.2 文獻整理-----------------------------------------------1
1.2.1 肥胖症的介紹-----------------------------------------1
1.2.2 脂肪組織與脂肪細胞的介紹-----------------------------5
1.2.2.1 脂肪組織功能---------------------------------------5
1.2.2.2 脂肪細胞的研究-------------------------------------8
1.2.2.3 脂肪細胞分化及調節代謝之相關轉錄因子--------------20
1.2.3 胰脂解酶抑制劑--------------------------------------23
1.2.4 荷葉------------------------------------------------24
1.2.4.1 荷葉介紹------------------------------------------24
1.2.4.2 荷葉之成分分析------------------------------------25
1.2.4.3 荷葉之藥理研究------------------------------------26
1.2.4.4 荷葉中分離出純化合物之介紹------------------------29
1.3 研究動機與目的----------------------------------------35
1.4實驗設計與架構-----------------------------------------36

第二章、荷葉甲醇萃取物對胰脂解酶活性的探討----------------37
2.1 前言--------------------------------------------------37
2.2 材料與方法--------------------------------------------37
2.2.1 荷葉來源與處理--------------------------------------37
2.2.2 藥品------------------------------------------------38
2.2.3 酵素活性測試----------------------------------------38
2.3 統計分析----------------------------------------------39
2.4 結果--------------------------------------------------39
2.5 討論--------------------------------------------------41

第三章、荷葉甲醇萃取物對3T3-L1前脂肪細胞分化的影響--------42
3.1 前言--------------------------------------------------42
3.2 材料與方法--------------------------------------------42
3.2.1 樣品來源與處理--------------------------------------42
3.2.2 細胞株----------------------------------------------42
3.3.3 藥品------------------------------------------------42
3.2.4 細胞培養與分化--------------------------------------44
3.2.4.1細胞解凍-------------------------------------------44
3.2.4.2細胞繼代培養---------------------------------------44
3.2.4.3細胞冷凍保存---------------------------------------45
3.2.4.4細胞分化-------------------------------------------45
3.2.4.5樣品添加-------------------------------------------45
3.2.6 樣品收集與分析--------------------------------------46
3.2.6.1 細胞存活率分析 (MTT assay)------------------------46
3.2.6.2 胞內三酸甘油酯含量分析----------------------------46
3.2.6.3 油紅染色 (Oil-Red O staining)---------------------49
3.2.6.4 總RNA萃取-----------------------------------------49
3.2.6.5 反轉錄反轉譯cDNA合成------------------------------50
3.2.6.6 聚合酶連鎖反應 (PCR)------------------------------50
3.2.6.7 DNA電泳與膠片定量分析-----------------------------51
3.2.4 統計分析--------------------------------------------51
3.3 結果--------------------------------------------------52
3.3.1荷葉甲醇萃取物及其不同區分層對3T3-L1脂肪細胞存活率之影響--------------------------------------------------------52
1.荷葉甲醇萃取物及其不同溶劑區分層------------------------52
2.荷葉正丁醇層 (LLMB) 次區分層----------------------------52
3.荷葉乙酸乙酯層 (LLME) 次區分層--------------------------52
4.荷葉中分離純化物----------------------------------------53
3.3.2. 荷葉甲醇萃取物及其不同區分層對3T3-L細胞脂質累積之影響--------------------------------------------------------53
1.荷葉甲醇萃取物及其不同溶劑區分層------------------------53
2.荷葉正丁醇層 (LLMB) 次區分層----------------------------54
3.荷葉乙酸乙酯層 (LLME) 次區分層--------------------------54
4.荷葉中分離純化物對3T3-L1細胞脂質累積之影響--------------54
3.3.3 荷葉有效次區分層對3T3-L1細胞分化相關因子之影響------55
3.4 討論--------------------------------------------------56
參考文獻--------------------------------------------------78
附錄------------------------------------------------------94

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