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研究生:劉力綱
研究生(外文):Li-Kaung
論文名稱:桑椹萃取物降低高油脂高膽固醇飲食引起之高血脂與體脂肪形成之作用與分子機制
論文名稱(外文):Effects of Mulberry (Morus alba L.) Extracts on Hypolipidemic and Prevention of Fat Accumulation in High Fat /Cholesterol Diets fed Hamsters
指導教授:王朝鐘王朝鐘引用關係
指導教授(外文):Chau-Jong Wang
學位類別:博士
校院名稱:中山醫學大學
系所名稱:生化暨生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:100
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本研究欲驗證一天然植物桑椹之水萃取物(mulberry water extracts ; MWEs)是否具有降血脂與不易形成體脂之作用,並探討其作用機制。以六週齡體重約為90-100公克之雄性倉鼠(Syrian Golden hamster)進行動物實驗,動物單獨餵食含組成比例0.2% cholesterol與10% coconut oil之高油脂高膽固醇飲食(high fat/cholesterol diets ; HFCD)或於HFCD中混入0.5%、1%與2%的MWEs餵食十週,誘導動物產生高血脂症與脂肪沉積之現象。結果發現添加MWEs的組別(MWEs組)與單獨餵食HFCD組別(誘導組)相較,其動物血清中三酸甘油酯(TG)、總膽固醇(TC)、脂肪酸(FA)、低密度脂蛋白(LDL)的濃度均降低(p<0.05),罹患心血管疾病的風險值(risk ratio)也下降(p<0.05);而動物肝臟內之TG與TC濃度也分別較誘導組減少(p<0.05),顯示MWEs具有降低由HFCD誘導的高血脂作用。在動物體重變化方面,誘導組的體重較正常飲食餵食之組別(正常組)增加約15%的重量,但MWEs組則較誘導組減少約10%的體重(p<0.05);此外MWEs組的動物腎臟周邊與睪丸周邊組織的大小,也較誘導組小(p<0.05),顯示MWEs可減少HFCD誘導的脂肪沉積作用。
肝臟是動物體中脂質合成代謝主要之器官,接續探討動物肝臟中調控脂質合成代謝之相關蛋白的表現與變化。結果顯示在MWEs組中,動物肝臟蛋白中膽固醇、三酸甘油酯與脂肪酸的合成效率限制的關鍵酵素:HMG-CoA reductase、glycerol-3-phosphate acyltransferase(GPAT)與fatty acid synthease(FAS)的表達均降低(p<0.05),顯示肝臟細胞內脂質生合成作用減少。另外,促進脂肪酸代謝的β-氧化作用之效率限制關鍵酵素:CPTΙ(camitine palmitoyltransferase Ι)的表現量則增加(p<0.05),顯示MWEs組中肝臟對脂肪酸之代謝作用增加,可避免造成脂質沉積。為避免動物肝臟中脂質合成代謝相關蛋白之變化是因HFCD引起之回饋抑制作用,故培養Hep G2 細胞株並加入不同濃度之MWEs (1.0、3.0、6.0 mg/mL)於serum free的培養液共同培養反應後,萃取細胞蛋白與mRNA分別進行蛋白與基因表現分析,結果顯示Hep G2細胞內HMG-CoA reductase、GPAT 與FAS的蛋白與基因表現均有降低(p<0.05)。此外也發現MWEs會增加Hep G2 細胞LDLR的表達,與其uptake LDL之能力(p<0.05)。另也發現MWEs也可調控粒線體β-氧化作用相關蛋白表現,增加Hep G2 細胞內脂肪酸代謝。
由動物實驗結果顯示MWEs具有降血脂與減緩體脂形成之作用,而其作用機制輔以細胞實驗驗證,可能是透過調降肝臟細胞內脂質合成代謝相關蛋白表現來減少脂質生合成;另一方面,也增加肝臟LDLR表達與uptake 血中膽固醇能力,並促使脂肪酸進行粒線體β-氧化作用,藉由減少脂質合成與增加脂質代謝雙方面的作用,達到降血脂與減少脂質沉積的效果。


The objective of this study was to investigate the lipid and fat accumulation-lowering effects of mulberry water extracts (MWEs). To evaluate these effects of MWEs, hamsters were fed with either high fat/cholesterol diets (HFCD) or HFCD supplemented with 0.5, 1 and 2% MWEs for 10 weeks. Plasma total cholesterol (TC) and triglyceride (TG) levels of hamsters fed HFCD with MWEs were significantly reduced by about 44-60% and 14-45%, respectively, as compared to those without MWEs. Similar results were also measured in hepatic TC and TG of hamsters fed HFCD with MWEs. LDLR gene expression and the uptake ability of LDL in HepG2 cells were also upregulated by additions of MWEs. MWEs also decreased the gene expressions of enzymes involved in the TG and TC biosyntheses. Results suggest that hypolipidemic effects of MWEs are via an enhancement of LDLR gene expression and the clearance ability of LDL and a decrease in the lipid biosynthesis.
In addition, HFCD-fed groups with MWEs had less perirenal and epididymal fat pads, moreover the weight gain were significantly decreased compared with HFCD-fed groups. These effects probably resulted from MWEs increased the expressions of proteins involved in lipolysis. We further examined the effects of MWEs on protein expressions of the key enzyme of β-oxidation via the HepG2 cell model. Data revealed that the addition in protein expression of PPARα and CPTI in HepG2 cells were detected treatment of 3 and 6 mg/ml of MWEs, respectively, as compared to those without MWEs treatment.
The present study reveals for the MWEs could improve hyperinsulinemia and prevent body fat accumulation induced by HFCD. Therefore, MWEs could be used as a natural agent against hyperlipidemia and obesity.


中文摘要……………………………………………………………………………Ⅰ
英文摘要……………………………………………………………………………Ⅳ
縮寫表………………………………………………………………………………Ⅷ
圖表目錄……………………………………………………………………………Ⅸ
研究動機……………………………………………………………………………...1
第一章、緒論
1.桑椹(mulberry)……………………………………………………………..3
1.1.桑椹之型態特徵與傳統醫藥之應用…………………………………..3
1.2.桑椹果實之生理保健功能……………………………………………..5
1.3.桑椹花青素……………………………………………………………..6
1.3.1.花青素之結構與生理活性……………………………………...6
1.3.2.桑椹花青素……………………………………………………...9
2. 人體脂質代謝 (lipid metabolism)……………………………………….11
2.1. 人體內脂質與膽固醇之來源與恆定……………………………......12
2.2. 高血脂症(hyperlipidemia)………………………………………...12
2.3. 脂肪沉積(fat accumulation)與肥胖(obesity)……………….....14
3.高血脂症與肥胖之預防與治療方法………………………………………...15
3.1. 增加細胞對外源性脂質、膽固醇的代謝作用……………………...15
3.1.1. 肝臟LDL receptor 表達之調控機制………………………16
3.2.增加細胞內源性脂質的降解作用……………………………………17
3.2.1. 脂肪酸β-氧化作用…………………………………………...17
3.3.減少細胞內源性脂質的合成作用:相關蛋白與機制………………18
3.3.1. HMG CoA reductase………………………………………..18
3.3.2. 固醇調節結合蛋白(SREBPs)……………………………...20
3.3.3. 脂肪酸生成酶(fatty acid synthease)………………………..22
3.3.4. 過氧化體增殖活化受體(PPARs)…………………………...22
第二章、實驗模式與架構
1.動物實驗模式………………………………………………………………...25
2.細胞實驗模式………………………………………………………………...28
第三章、實驗方法
1.桑椹水萃取物(MWEs)製備………………………………………………30
2.桑椹水萃取物(MWEs)成份分析…………………………………………30
3.動物實驗分析………………………………………………………………...33
4.細胞實驗分析………………………………………………………………...37
5.統計分析方法………………………………………………………………...41
第四章、實驗結果
1.桑椹水萃取物(MWEs)成份………………………………………………42
2.各組動物血清中TG、TC與FFA濃度之影響變化…………………………42
3.各組動物血清中LDL與HDL濃度之影響變化……………………………43
4.各組動物血漿脂質過氧化程度之影響變化………………………………44
5.各組動物肝臟TG與TC濃度之影響變化…………………………………45
6.各組動物肝臟功能指數:AST、ALT之影響變化…………………………45
7.各組動物腎臟功能指數:BUN、CRE 之影響變化………………………46
8.各組動物正常生理功能指標:血中酮體、電解質濃度之影響變化………46
9.各組動物飼養期間體重變化………………………………………………47
10.各組動物副睪脂體(epididymal fat pad)變化…………………………48
11.各組動物副腎脂體(perirenal fat pad)變化……………………49
12.各組動物肝臟物理型態重量變化………………………………49
13.各組動物肝臟中脂質生合成相關蛋白的表達變化………………………50
14.各組動物肝臟中粒線體β-氧化作用相關蛋白的表達變化……………51
15. MWEs對HepG2細胞中脂質生合成代謝相關基因表達的作用………51
16. MWEs對HepG2細胞中脂質生合成相關蛋白表達的作用………51
17. MWEs對HepG2細胞中β-氧化作用相關蛋白的作用……………52
18. MWEs對HepG2細胞中LDL uptake ability的作用………………52
第五章、實驗討論
1. MWEs降血脂與減少脂質沉積之生理活性成分…………………………54
2. MWEs於HFCD餵食動物中降低血脂與減少脂質沉積之作用………55
3. MWEs在脂質合成代謝相關蛋白之調控…………………………………58
4. MWEs降血脂與減少脂肪沉積之作用機制………………………………60
實驗數據圖表………………………………………………………………………63
參考文獻……………………………………………………………………………84



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