奈米化紅麴菌發酵產物之安全性試驗與保健成份之血脂調節評估

Abstract

紅麴菌是中國已使用數千年之食品發酵菌種，被視為傳統的自然食品呈色劑，而紅麴菌的次級代謝產物—monacolin K、γ-胺基丁酸 (γ-amino butyric acid，GABA) 及 dimerumic acid 經證實具有降低膽固醇、降低血壓及抗氧化的功效，因具有多種生理功效，使得紅麴成為目前保健食品的主要研究課題之一。 本研究之目的在探討如何將紅麴米粉末，利用濕式介質研磨的方式研磨成奈米微粒，並進行生物安全性試驗與功效評估試驗。研磨方法為將紅麴米粉末與水進行共同研磨以製得奈米微粒懸浮液，分別利用掃描式電子顯微鏡、穿透式電子顯微鏡，雷射光散射法 (dynamic laser scattering)、高效液相層析儀來了解奈米化紅麴的物化性質。在安全性評估方面：進行基因毒性試驗、細胞毒性試驗與二十八天餵食毒性試驗；在功效性評估方面：則以誘發性高膽固醇倉鼠為動物模式，採取管餵餵食的方式投予奈米紅麴或未經研磨的紅麴米粉末，於實驗四週與八週後犧牲以觀察調節血脂之功效。實驗結果顯示，紅麴米粉末在濕式研磨後所形成之水相奈米微粒懸浮液，其平均粒徑由 20.2 μm 降至 259 nm。雷射光散射法分析發現：本研究之奈米化方法可達到研磨粒徑均ㄧ與分散性良好的效果。利用 HPLC 來分析二次代謝物中降膽固醇成分 monacolin K 與黴菌毒素 citrinin 之差異，結果顯示，二次代謝物 monacolin K 降低為原來的 75.2%，citrinin 降低為原來之 74.6%。在冷藏儲存兩個月時，奈米化顆粒有部分凝聚的結果。細胞毒性實驗中，相當於 4.22 mg/mL 奈米紅麴粉末之酒精萃取物會對肝癌細胞 Hep G2 造成 50% 之細胞毒殺效果；基因毒性試驗中，紅麴與奈米紅麴萃取物 (1 mg/plate) 對 Salmonella typhimurium TA 98 、TA 100 與 TA 102 無毒性且無致突變現象；二十八天餵食毒性試驗，無毒性作用劑量 (NOAEL) 為雄性大鼠為 500 mg/kg/day；雌性大鼠為 1000 mg/kg/day。功效性評估方面：八週時，餵食奈米紅麴在劑量 20 mg/110 g 倉鼠體重/每天時，在降低 LDL/HDL 比値上比紅麴米組有降低之趨勢，且與高膽固醇組有顯著差異 (p<0.05)。而在肝臟脂質分析中，此劑量之奈米化紅麴在 TC 與 TG 有顯著降低的效果。 利用濕式介質研磨技術可將紅麴米製備成一種穩定且具生物活性的奈米微粒懸浮液，體內與體外安全性試驗也證實奈米化紅麴發酵產物不具致突變性與毒性。

Monascus, one of the Chinese traditional fermentation fungi, has been used for thousands of years. It has been recorded in ancient Chinese books for its special functions and food applications. For instance, in China, monascus have been widely used as a natural food coloring agent for many kinds of foods. The metabolite of Monascus species, specifically, monacolin K, γ-amino butyric acid (GABA) and dimerumic acid, have been proven to have cholesterol-lowering effects, blood-pressure-lowering effects, and antioxidant effects. Nowadays, the public has recognized the importance of monascus products for its many health benefits; therefore, it is not surprising that monascus has become the focus of many studies as functional foods. The aim of this study is to determine the feasibility of using wet milling technology to formulate crude red mold rice (RMR) as stable nanoparticulate dispersion and evaluate its safety and cholesterol lowering effect. Red mold rice nanoparticulate formulation (NRMR) was reproducibly obtained after milling RMR in the presence of distilled water. The physical and chemical properties of these particles were studied using electron microscopy, laser light scattering and high performance liquid chromatography (HPLC). To verify safety and/or functionality evaluations, Ames test, cytotoxicity, in vivo 28-day feeding toxicity in Wistar rats and in vivo cholesterol-lowering effect in hypercholesterolemic hamsters were processed using RMR and NRMR. RMR (mean size = 20.2 μm) was processed using wet milling technology to form an aqueous-based nanoparticle dispersion with a mean particle size of 259 nm. The formulation was homogeneous and exhibited a unimodal particle size distribution profile using dynamic laser scattering techniques. In addition, HPLC analyses, performed on the second metabolite, demonstrated that monacolin K was reduced to 75.2% of its base level, and citrinin was reduced to 74.6% of its base level. Partial agglomeration has been observed in RMR dispersion when stored in refrigerator after 2 months. Cytotoxicity test demonstrates that 4.22 mg/mL of RMR nanoparticulate, extracted with alcohol, has resulted in 50% lethality in Hep G2 cells. Ames test shows the absence of genotoxicity in Salmonella typhimurium TA 98, TA 100 and TA 102 when treated with less than 1 mg of extract per plate. In feeding toxicity, the no observed adverse effect level (NOAEL) of NRMR, administered to Wistar rats via oral gavages for 28 days, was 500 mg/kg/day for male rats and 1000 mg/kg/day for female rats. In vivo cholesterol-lowering effect in hypercholesterolemic hamsters, plasma LDL/HDL ratio (LDL: low density lipoprotein; HDL high density lipoprotein), liver TC (total cholesterol) and TG (triglyceride) levels in hamster fed with NRMR dispersion at dosage (20 mg/110 g bw/day) for 8 weeks were significantly lower than that in hyperlipidemia hamster. In conclusion, red mold rice can be formulated as a stable and bioactive nanoparticulate dispersion using wet milling technology. And in vitro and in vivo safety evaluations indicate that no mutagenic or toxic responses were observed in this study.