Development of Monascus Functional Food for the Prevention of Fat Accumulation

肥胖是導致心血管疾病與糖尿病之危險因子，本研究利用體外細胞實驗及活體動物實驗評估紅麴米及紅麴山藥對體脂肪生成之影響。體外模式以 3T3-L1 前脂肪細胞株作為試驗材料，探討紅麴米及紅麴山藥萃取物對前脂肪細胞增生、分化之影響，並分析成熟脂肪細胞脂解作用 (lipolysis) 及脂蛋白脂解酶 (lipoprotein lipase, LPL) 活性之變化。體內評估部分以雄性 Wistar 大白鼠作為試驗動物，研究中同時餵食高油脂飼料與紅麴發酵產物，飼養 6 週後，利用各項評估指標判定其減少體脂肪生成之效果。細胞實驗結果顯示，紅麴米水萃取物、乙醇萃取物與紅麴山藥乙醇萃取物皆具有抑制前脂肪細胞增生、分化之效果；紅麴山藥水萃取物僅具抑制分化作用。紅麴米及紅麴山藥水萃取物皆可提高 lipolysis 效率，其中以紅麴米之效果較佳。紅麴山藥乙醇萃取物能抑制脂肪細胞肝素釋放脂蛋白脂解酶 (heparin-releasable LPL, HR-LPL) 活性。動物實驗結果顯示，於高油脂飲食中添加 0.4%、2% (w/w) 紅麴米能顯著抑制腎臟及副睪周圍脂肪細胞增大，改善脂肪組織堆積情形，並且可減緩體重上升，其可能因素包括提高腎臟及副睪周圍脂肪組織 lipolysis 效率及減少攝食量。紅麴山藥僅在高劑量組 (2%, w/w) 有明顯抑制脂肪細胞增大及體重上升之效果，這個效果可能與提高副睪周圍脂肪組織 lipolysis 效率、抑制腎臟周圍脂肪組織 HR-LPL 活性及減少攝食量有關。雖然在本研究之動物模式中，紅麴發酵產物之功效主要來自於抑制脂肪細胞增大，但其仍具有抑制前脂肪細胞增生及分化之影響，可避免過多的脂肪細胞生成。Monacolin K (lovastatin) 能降低大鼠攝食量，抑制副睪周圍脂肪細胞增大，並且有抑制前脂肪細胞增生之效果，可能是紅麴發酵產物的功能性成分之ㄧ。另外，餵食紅麴發酵產物可降低高油脂飲食大鼠血中總膽固醇、低密度脂蛋白膽固醇 (low density lipoprotein cholesterol, LDL-C) 及胰島素濃度，對於肝臟脂質、血糖及血液生化值無顯著影響。本研究首次提出紅麴發酵產物能夠改善高油脂飲食引起之體脂肪堆積、血脂紊亂及高血胰島素症狀，有潛力發展為不易形成體脂肪之保健食品。Obesity is associated with high risk for developing cardiovascular disease and diabetes. In this study, we used 3T3-L1 cell line as in vitro model to examin the effects of extracts of red mold rice (RMR) and red mold dioscorea (RMD) on proliferation and differentiation in preadipocyte as well as lipolysis and heparin-releasable lipoprotein lipase activity in mature adipocyte. Furthermore, we also investigated the in vivo antiobesity effects of Monascus fermented products by animal experiment. Male Wistar rats were fed high-fat diet (HF) and Monascus fermented products at the same time. After six weeks, rats were sacrificed for evaluation. As results in cellular experiment, ethanol extracts of RMR and RMD and water extract of RMD had inhibitive effects on 3T3-L1 preadipocyte proliferation and differentiation. However, water extract of RMD was not able to affect preadipocyte proliferation but reduce the triglyceride content in cell during adipogenesis. The water extracts but not ethanol extracts of RMR or RMD could induce lipolysis in mature adipocyte, and RMR water extract had stronger effect. 3T3-L1 adipocyte treated with the ethanol extract of RMD had lower heparin-releasable LPL (HR-LPL) activity. In animal experiment, rats fed HF diet supplemented with 0.4% and 2% (w/w) RMR had less perirenal and epididymal fat pads, moreover the weight gain were significantly decreased compared with HF control. These effects probably resulted from reducing calorie intake as well as increasing lipolysis activity in perirenal and epididymal adipose tissues. Rats fed with high dose RMD (2%, w/w) also had less adipose tissue mass and weight gain. Stimulating lipolysis in epididymal fat pads and lowering HR-LPL activity in perierenal fat pads as well as decreasing calorie intake might illustrate the influence of high dose RMD. In our animal model, the major effect of Monascus fermented products was inhibiting hypertrophy in adipocyte. However, the Monascus fermented products had inhibitive effects on proliferation and differentiation in preadipocyte, which could avoid fat cell formation. Monacoin K (lovastatin) had inhibitive effects on food intake and differentiation in 3T3-L1 preadipocyte, which may be one of the functional ingredients in Monascus fermented products. On the other hand, rat fed Monascus fermented products had lower serum total cholesterol, low density lipoprotein cholesterol (LDL-C) and insulin while hepatic lipids and serum parameters without any significant difference. This study reveals for the first time that Monascus fermented products could prevent body fat accumulation and improve dyslipidemia and hyperinsulinemia induced by high-fat diet, which has potential to be an antiobesity functional food.縮寫表 I 中文摘要 III 英文摘要 IV 第一章 前言 1 第二章 文獻回顧 3 一、體外脂肪細胞分化 3 (一) 前脂肪細胞株 3 (二) 脂肪細胞分化過程 3 (三) 調節分化之主要轉錄因子 7 二、成熟脂肪細胞儲存與分解脂質之調控 8 (一) 脂蛋白脂解酶 (lipoprotein lipase, LPL) 8 (二) 脂解作用 (lipolysis) 10 三、肥胖與健康 16 (一) 脂肪組織增加直接造成之疾病 16 (二) 脂肪組織增加引起之代謝失調疾病 16 四、紅麴 18 (一) 紅麴之菌種特性 18 (二) 紅麴生產之功能性二次代謝物 18 (三) 抑制脂肪細胞分化之相關研究 25 五、研究大綱與目的 27 第三章 材料與方法 29 一、實驗材料 29 二、實驗方法 32 (一) 體外細胞實驗 32 (二) 體內動物實驗 39 第四章 結果與討論 46 一、 體外細胞實驗 46 (一) Monacolin K 含量之測定 (HPLC 分析) 46 (二) 前脂肪細胞增生率之測定 (MTT 試驗) 46 (三) 細胞毒性分析 (trypan blue dye exclusion 試驗) 50 (四) 前脂肪細胞分化率之測定 53 (五) 成熟脂肪細胞 lipolysis 作用之測定 (甘油釋放濃度) 61 (六) 成熟脂肪細胞 heparin-releasable LPL 活性之測定 (pNPB 酵素分析法) 66 (七) 小結 70 二、體內動物實驗 73 (一) 功能性指標之評估 73 (二) 安全性指標之評估 85 (三) 小結 90 第五章 綜合討論 94 第六章 參考文獻 9