- 學位論文
預防高血脂與阿茲海默症之多功效紅麴保健產品開發
Development of Monascus Multifunctional Product for The Prevention of Hyperlipidemia and Alzheimer’s Disease
摘要
近年來紅麴菌 (Monascus species) 之相關研究日益增加，紅麴保健功效除了降膽固醇與降血壓之外，抗氧化、防癌、抗疲勞與抑制脂肪細胞肥大等保健功效皆陸續被發現。於未來之發展上紅麴將可被發展成為一複合式多功效之保健食品。台灣目前已邁入老年化社會，其中老年癡呆症中之阿茲海默症則是老年化伴隨而來的主要疾病之一。引起阿茲海默症的主要因素是類澱粉樣蛋白 (amyloid β-protein, Aβ) 於腦部大量沈積所造成，病情會隨著膽固醇的增加、神經傳導物質的缺乏與氧化發炎反應而逐漸加重，近來研究指出 statin 類的藥物具有治療阿茲海默症的效果，紅麴中的 monacolin K 亦為 statin 化合物的一種，且紅麴中的γ-胺基丁酸 (γ-aminobutyric acid，GABA) 與抗氧化物質可能與預防阿茲海默症有關，利用紅麴預防與改善阿茲海默症之可行性將大幅提高。因此，本研究主要目的為開發具預防阿茲海默症功效的紅麴保健食品。本研究之第一階段主要目的為建立最適紅麴菌株並評估是否具發展為健康食品之潛力。並於第二階段探討對阿茲海默症危險因子的降低與記憶學習能力的改善效果。 首先建立紅麴米中monacolin K 與 citrinin 之分析方法，以鑑定與篩選較佳的紅麴發酵產物。本研究利用一種萃取方法與同一支 C18 逆向層析管柱進行分離，並串聯螢光偵測器與紫外光偵測器以同步定量 citrinin (CT)、monacolin K lactone form (MKL) 與 monacolin K acid form (MKA)。在萃取方法部分，1 g 的紅麴粉末以 10 mL 95% ethanol 於 60oC 靜置萃取 30 min 具有最好的萃出效果。HPLC 分析條件，以 acetonitrile-water-trifluoroacetate (550:450:0.5) 為等位流洗的移動相，可有效的分離 CT、MKL 與 MKA 之波峰。在菌種開發上，自紅麴米中篩選得高 monacolin K 生產力之紅麴菌株 Monascus purpureus HM 105，利用傳統變異法篩選得到 M. purpureus NTU 568，為一可生成高量 monacolin K (9,500 mg/kg) 之紅麴菌株。如此高 monacolin K 生成量之紅麴菌株至今於文獻中皆尚未被提出。經本研究進一步探討後發現此紅麴變異菌株具有良好的穩定性，具有較佳的抗氧化能力。此外最適菌株發酵之紅麴米於降血脂功效評估結果顯示，紅麴米確實具有降低血液中總膽固醇、三酸甘油酯與低密度脂蛋白膽固醇的效果。並對紅麴產品的安全性進行分析。研究結果顯示，M. purpureus NTU 568 之米發酵產物並不會造成肝臟的危害。 紅麴米進一步評估降低阿茲海默症危險因子與改善記憶學習能力的效果。在抑制 Aβ40 之神經細胞毒性方面，M. purpureus NTU 568 發酵之紅麴米乙醇萃取物 (ethanol extract of red mold rice fermented by M. purpureus NTU 568，RE 568) 中的 monacolin K 可藉由抑制下游產物 geranylgeranyl pyrophosphate (GGpp) 的生成，使 small G-protein 不被活化而抑制 Aβ40 所誘發的發炎反應，RE 568 亦提供有效的抗氧化能力以防止 Aβ40 所引發的氧化壓力。此外，RE 568 所呈現的效果會更勝於單純的 lovastatin 藥物處理，這可能是紅麴米中的 monacolin K、抗氧化物質與抗發炎物質共同呈現的協同效果。在體內動物評估方面，以 Aβ40 連續 28 天輸注於大鼠腦部，使 Aβ40 直接於腦部大量沉積而引發阿茲海默症，Aβ40 會提升腦部乙醯膽鹼酶活性、活性氧原子 (reactive oxygen species，ROS) 生成量與脂質過氧化的程度，並降低總抗氧化力與超氧歧化酶 (superoxide dismutase，SOD) 活性。而紅麴米的餵食可顯著抑制這些因腦部輸注 Aβ40 所造成的損傷，並且其效果較 lovastatin 藥物治療組佳，此外，藉由抑制氧化發炎反應而使輸注之 Aβ40 無法於海馬迴組織中大量沈積。第二部分抑制Aβ 等相關危險因子生成方面，體外試驗以可表現內源性類澱粉樣前驅蛋白 (amyloid precursor protein，APP) 之IMR-32 與 PC-12 細胞株做為細胞模式。結果顯示，外添加 mevalonate 與膽固醇會提高可溶性類澱粉樣前驅蛋白β-secretase 剪切片段 (soluble amyloid precursor protein β-secretase cleaved fragment，sAPPβ) 與 Aβ 的生成量，而 RE 568 則可有效降低受 mevalonate 或膽固醇誘發的 Aβ 與 sAPPβ生成量，並且提高可溶性的 sAPPα 生成量，紅麴主要是藉由抑制細胞膽固醇的生成而降低β-secretase 活性，以達到抑制 Aβ 的生成。體內試驗之結果顯示紅麴米顯著改善水迷宮與被動迴避試驗的記憶學習能力，同時腦部輸注 Aβ40 與高熱量飲食會造成腦部危害，包含提高乙醯膽鹼酶活性與加劇氧化發炎反應。而這些危害可因紅麴米的餵食而被顯著抑制。紅麴米亦抑制高熱量飲食提高腦部膽固醇濃度，且降低危險因子 ApoE 表現量與β-secretase 的活性，最後達到降低 Aβ40 於海馬迴組織的累積量，並提高神經細胞保護因子 sAPPα 的生成量。紅麴米首次被證實具降低阿茲海默症危險因子與改善記憶學習能力的效果。且其效果會較 lovastatin 藥物更具顯著效果，期望本研究可有助於紅麴在預防阿茲海默症之保健食品的開發。
並列摘要
Recently, the Monascus research was gradually popular. The function of Monascus was possessed of antioxidant activity, cancer prevention and antifatigue in addition to anti-hypercholesterolemic and hypotensive effect. Monascus fermented product will be developed as a multi-functional food in the future. Taiwan has been gradually turned into a geriatric society. Therefore, the geriatric disease was respected by people, and senile dementia (also called Alzheimer’s disease (AD)) was major one of the diseases. Amyloid β-protein (Aβ) is the principal proteinaceous compound of amyloid plaques in brain of AD patients. Deposition of Aβ is expressed with increasing cholesterol concentration, decreasing neurotransmitter and serious oxidative stress and inflammatory response. Recently, one epidemiological study suggests that statin therapy may provide protection against AD. According as monacolin K is one of the statin compounds, and Monascus fermented products γ-aminobutyric acid (GABA) and antioxidant are possible to prevent AD development. Therefore, we considered that AD prevented and improved by Monascus was a feasibility study. The goal of this study is to develop a novel Monascus functional food with prophylactic effect on AD pathogenesis. The purpose of first section is to search and establish the optimal Monascus strain, and evaluate the potential for developing as a functional food. The evaluation of lowering AD risk factors and ameliorating impairment of memory and learning ability in AD rat model would be further carried out in the second section. First, the analysis method of monacolin K and citrinin is set up in order to screen and examine the optimal red mold rice (RMR). In the result, citrinin (CT), monacolin K lactone form (MKL) and monacolin K acid form (MKA) are extracted using the same extraction method, and are then separated in a reverse phase C18 column. The elution from the C18 column is then passed through a UV detector and introduced directly into the fluorescence detector. As shown in the result, higher recovery rate of CT, MKL, and MLK are yielded from RMR powder by extracting with 95% ethanol (10 mL) at 60°C for 30 minutes. The peaks of CT, MKL, and MKA can be clearly separated from any noise peaks by isocratic elution with optimum mobile phase, acetonitrile-water-trifluoroacetate (55:45:0.05). In the development of Monascus strain, Monascus purpureus NTU 568 was selected from the mutants of M. purpureus HM 105 because it could produce high monacolin K production at 9,500 mg/kg. Such a Monascus species with high monacolin K productivity has never been published in the reference according to our view. M. purpureus NTU 568 was proven that it could perform stable production of secondary metabolite and well antioxidative ability. Regarding the hypolipidemia effect of M. purpureus NTU 568 fermented RMR, oral administration of RMR significantly lowers serum total cholesterol (TC), triglyceride (TG), and low density lipoprotein cholesterol (LDL-C), and does not result in liver damage. In the results of RMR on repressing Aβ-induced neurotoxicity, monacolin K of RE fermented by M. purpureus NTU 568 (RE 568) represses Aβ40 toxicity via repressing small G-protein-mediated inflammation in PC12 cell. RE 568 also exhibits more robust anti-oxidative ability on the protection against Aβ-induced oxidative stress. Importantly, stronger effects on repressing the Aβ40-induced cell death, inflammation, and oxidative stress are performed by RE 568 than that by the equal levels of lovastatin, which results from a synergism mechanism made up of monacolin K, antioxidants and anti-inflammatory agents. The effects of dietary administration of RMR on memory and learning abilities are confirmed in an animal model of AD rats infused with Aβ40 into cerebral ventricle. The in vivo results indicate that RMR administration potently reverses the memory deficit in the memory task. Aβ40 infusion increases acetylcholinesterase (AChE) activity, reactive oxygen species, and lipid peroxidation, and decreases total antioxidant status and superoxide dismutase activity in brain, but these damages were potently reversed by RMR administration and the protection were more significant than that of lovastatin administration. In addition, these neuroprotection provided by RMR would lead to the prevention of Aβ40 accumulation in hippocampus. Regarding the section of RMR on repressing the formation of Aβ and related AD risk factors, PC-12 cell and IMR-32 cell are used as an in vitro cell model because of the expression of amyloid precursor protein (APP). Mevalonate and cholesterol supplementation would significantly enhance Aβ and soluble amyloid precursor protein α-secretase cleaved fragment (sAPPα) secretion in PC-12 cell and IMR-32 cell. Importantly, RE 568 can exhibit stronger effect on repressing cholesterol-mediated Aβ and sAPPβ secretion than lovastatin. At the same time, neuroprotective factor— sAPPα can be increase by RE 568 supplementation. In addition, RE 568 would repress the activity of β-secretase and indirectly inhibit Aβ secretion. In the in vivo results, orally administered RMR potently reversed the memory deficit in the water maze and passive avoidance tasks. Although Aβ40 infusion and cholesterol feeding caused great damage in brain involved in the increase of AChE activity, oxidative stress, and inflammation, the damage was potently reversed by RMR administration. Regarding the cholesterol diet-raised risk factor, RMR administration reduces brain cholesterol levels, apolipoprotein E (ApoE) expression, and β-secretase activity in cerebral cortex and hippocampus, and finally represses Aβ40 accumulation and stimulates sAPPα formation in hippocampus. Daily food supplement—RMR is the first to be applied to repress the formation of AD risk factors and ameliorate impairment of memory and learning ability. Importantly, RMR have an advantage over lovastatin for the prophylaxis of AD pathogenesis. We hope this study will be a great benefit to the development of Monascus functional food on the prophylaxis of AD.