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研究生:何瑋倫
研究生(外文):Wei-Lun, He
論文名稱:紫菜油相萃取物區分對3T3-L1前脂肪細胞分化的影響
論文名稱(外文):Effects of Extracted Oil Fractions from Porphyra crispata on 3T3-L1 Preadipocyte Differentiation
指導教授:龔瑞林龔瑞林引用關係
指導教授(外文):Kong, Zwe-Ling
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:147
中文關鍵詞:紫菜前脂肪細胞抗氧化不飽和脂肪酸
外文關鍵詞:Porphyra crispata3T3-L1 preadipocyteantioxidantEPA
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肥胖會造成高血脂和胰島素抗性等代謝症候群現象,而導致糖尿病、高血壓或引起心血管疾病。因此能預防肥胖及改善代謝症候群的天然物廣泛的被研究,不飽和脂肪酸及為其中之一。紫菜中即含有豐富的多元不飽和脂肪酸及並有許多的抗氧化成分,因此本實驗即是利用前脂肪細胞模式來探討紫菜的油相萃取物(PCO)對肥胖的影響。將紫菜以甲醇/氯仿萃取法得到PCO,以GC分析脂肪酸發現其EPA佔總脂肪酸的30.4%,以TLC分析發現其含有固醇類及少量的多酚類,進一步以GC/MS分析,其含膽固醇(cholesterol)及Methyl(25RS)-3beta-hydroxy-5-cholesten -26-one 等兩種固醇類。在抗氧化相關分析方面,發現PCO及其區分(frac含有0.56~1.86%的總酚類,0.22~21.67 �慊/mL的葉綠素a(chlorophyll a),0.37~13.37�慊/mL的葉綠素b(chlorophyll b)和1.11~130.06�慊/mL 類胡蘿蔔素(carotenoid)。另外,含有相當於92.70~776.43 �慊 Trolox的總抗氧化力及10.62~41.38% 的DPPH自由基清除能力。另用乙醇萃取物(Et-PCO)、紫菜甲基化區分(PCF)和紫菜非皂化區分(PCS)進行細胞實驗。Et-PCO對3T3-L1的分化和三酸甘油酯(TG)的累積沒有明顯的影響,PCS和PCF則會促進3T3-L1的分化及TG的累積,且在第零天加入樣品時影響最明顯,由油紅染色則可以發現PCF會促進油脂的累積在細胞中形成大油滴。在胞內膽固醇分析方面則以PCS的影響較大。而PCF對PGC-1的基因表現有顯著的影響。在抑制細胞活性氧(ROS)的部分也是以PCS和PCF的效果較佳。
Obesity is a major risk factor for diabetes, hyperlipidemia, hypertension, cardiovascular diseases, and metabolic syndrome. Omega-3 polyunsaturated fatty acid (PUFA) of marine origin predicted to regulated lipid metabolism and antioxidants could reduced the oxidative stress which induced by obesity. We extracts the oil from Porphyra crispate, a kind of Rhodophyta, by methanol/chloroform, there is 30.4% eicosapentaenoic acid (EPA) of the fatty acids. We also determine the content of antioxidants in otal extract (PCO) and fractions. There were 0.56~1.82% total phenolic content, 0.22~21.67 �慊/mL chlorophyll a,0.37~13.37�慊/mL chlorophyll b and 1.11~130.06�慊/mL carotenoid in PCO and fractions. PCO and fractions also had total antioxidant activity 89.95~771.08 (�慊 trolox equivalents/g extract) and 10.62~41.38% DPPH radical scavenging activity. In this report, we investigated the effects of ethanol extract of PCO ( Et- PCO), methyl esterification (PCF) and nonsaponifiable (PCS) on the 3T3-L1 differentiation. Et-PCO was no significantly effect on cell differentiation. PCS and PCF could promoted cell differentiation and triglyceride accumulation in dose dependent, especially treated at Day 0. On the other hand, PCS was increasing the content of cholesterol in 3T3-L1. PCF could induced PGC-1 mRNA expression. Furthermore, PCS and PCF could reduce the ROS production. Our results suggest that antioxidant of PCO may prevent the obesity-associated metabolic syndrome and PCF and PCS may decrease hyperlipidemia by promoting triglyceride accumulation.
目錄 i
圖目錄 iv
表目錄 vi
附錄 vii
摘要 viii
Abstract ix
名詞縮寫(Abbreviation) x
壹、前言 0
貳、文獻整理 2
一 、肥胖(obesity) 2
1. 肥胖的成因 2
1.1 遺傳因子 3
1.2 環境因素 4
2. 肥胖與慢性發炎 5
3. 肥胖與氧化壓力的關係 6
二、代謝症候群(metabolic syndromes) 7
1. 代謝症候群定義: 7
2. 活性氧(ROS)與代謝症候群: 7
三、氧化壓力 8
1. 氧化壓力的形成 8
2. 氧化壓力對細胞所造成的傷害 9
3. 體內抗氧化機制 10
3.1 酵素系統: 11
3.2 非酵素系統: 12
四、脂肪細胞之介紹 13
1. 脂肪細胞之特性與種類 13
2. 脂肪細胞之分化 14
3. 3T3-L1脂肪細胞 17
4. 分化轉錄因子之介紹 18
五、不飽和脂肪酸 23
1. 不飽和脂肪酸的種類及來源 23
2. 不飽和脂肪酸的生理功能 23
六、紫菜的特性 24
1. 紫菜之組成 25
2. 紫菜膳食纖維之功能 25
3. 抗腫瘤之活性 26
4. 免疫活性與抗致突變性 27
5. 紫菜之抗氧化性 28
參、實驗設計 30
肆、實驗材料與方法 31
一、實驗材料 31
二、實驗方法 36
1. 紫菜油溶性物質萃取方式 36
2. 紫菜油之成分分離及分析 37
3. 總多酚含量分析 39
4. 葉綠素及類胡蘿蔔素含量分析 40
5. 總抗氧化力測定 40
6. 清除DPPH自由基之能力測定 40
7. 前脂肪細胞培養 41
8. 脂肪細胞之分化培養 41
9. 細胞存活率 (MTT assay) 42
10. 油紅 O 染色 (Oil red O stain) 42
11. 細胞內三酸甘油酯 (Triglyceride) 含量之分析 42
12. 細胞內膽固醇含量之分析 43
13. 抽取 3T3-L1 細胞 total RNA 44
14. 反轉錄反應 (RT) 44
15. 聚合?連鎖反應 (PCR) 45
16. DNA電泳與膠片定量分析 45
17. 吞噬球呼吸爆 (Respiratory burst) NBT還原分析 46
18. 蛋白質定量 46
19. 統計分析 46
伍、結果與討論 47
一、PCO 之組成分析。 47
1. 油溶性萃取物產率之比較 47
2. 油溶性萃取物之定性分析 47
二、PCO及其區分之抗氧化成分分析及抗氧化能力測試 48
1. 總酚類含量分析 48
2. 葉綠素a,b和類胡蘿蔔素含量分析 48
3. 總抗氧化力測試 49
4. DPPH自由基清除能力分析 49
三、紫菜油乙醇萃取物(ET-PCO)、甲基化脂肪酸(PCF)和非皂化物固
醇類區分(PCS)對3T3-L1前脂肪細胞存活率影響 49
四、紫菜之油溶性萃取物及其區分對前脂肪細胞的分化的影響。 50
1. 脂肪細胞的培養與分化 50
2. 紫菜之油溶性萃取物及其區分對前脂肪細胞分化的影響 50
五、紫菜之油相萃取物及其區分對前脂肪細胞三酸甘油酯及膽固醇累
積的影響。 51
1. 不同濃度的Et-PCO, PCS, PCF對前脂肪細胞三酸甘油酯累積的影響。 51
2. 不同時間點添加Et-PCO, PCS, PCF對前脂肪細胞三酸甘油酯累積的影響。 51
3. 不同濃度的Et-PCO、 PCS、 PCF對前脂肪細胞膽固醇累積的影響。 52
4. 不同時間點添加Et-PCO, PCS, PCF對前脂肪細胞膽固醇累積的影響。 53
六、紫菜之油相萃取物對前脂肪細胞的氧化壓力影響。 53
1. 分化過程中ROS的生成。 53
2. Et-PCO、PCS、PCF對脂肪細胞ROS生成的影響。 54
陸、結論 55
柒、參考文獻 56
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