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  • 學位論文

Monascus purpureus NTU 568 生成黃色素與橘色素之抗發炎活性探討

Anti-inflammatory properties of yellow and orange pigments from Monascus purpureus NTU 568

指導教授 : 潘子明
共同指導教授 : 郭曜豪

摘要


紅麴菌 (Monascus) 為中國傳統發酵真菌,運用在食品上已有數千年的歷史。本研究室利用傳統變異法篩選得到紅麴菌株Monascus purpureus NTU 568,經研究證實具有降低膽固醇、抗發炎、抗腫瘤與抑制腫瘤轉移等功效。本研究利用山藥與米做為基質,以 M. purpureus NTU 568 進行發酵,分離出 20 種黃色素與橘色素,並以 lipopolysaccharide (LPS) 誘發老鼠巨噬細胞 (RAW 264.7) 發炎,進行抗發炎功效評估,發現黃色素與橘色素可抑制 nitric oxide 生成量。因此選擇具安全性且為主量的色素,包含:黃色素 ankaflavin (IC50 = 21.8 uM)、monascin (IC50 = 29.1 uM) 以及結構相近之 monaphilone A (IC50 = 19.3 uM)、monaphilone B (IC50 = 22.6 uM),橘色素 monascorubrin (IC50 > 40 uM)、rubropunctatin (IC50 = 21.2 uM) 與另外四個新化合物 monaphilol A ~ D (IC50 = 1.0 ~ 3.8 uM),進行大量分離純化,以利於後續細胞與動物實驗,探討其抗發炎機轉與功效。於細胞實驗中,以 Western blot 及 ELISA kits 分析發炎相關酵素與促發炎細胞激素,發現黃色素與橘色素能夠明顯降低 inducible nitric oxide synthase 之蛋白表現,對 cyclooxygenase-2 之蛋白表現則具輕微抑制效果。除此之外,亦可減少 tumor necrosis factor-a (TNF-a)、interleukin-1b (IL-1b) 與 interleukin-6 (IL-6) 之分泌量。除細胞實驗外,本研究進一步以兩種動物模式,驗證黃色素與橘色素之抗發炎功效。在 TPA 誘發小鼠耳朵水腫模式中,預先處理黃色素與橘色素可減緩水腫達 60%;於LPS腹腔注射模式,預先投予黃色素與橘色素,可降低 LPS 誘發之血液 NO 與促發炎細胞激素 TNF-a、IL-1b 與 IL-6 之含量。綜合以上結果,本論文探討之紅麴黃色素與橘色素抗發炎活性良好,其中又以橘色素 monaphilol A ~ D 效果最佳,具高度潛力發展成為具有預防保健功效之抗發炎機能性食品或藥物。

並列摘要


The Monascus species is a Chinese traditional fermentation fungus used on food for over thousands of years in China. In our previous reports, the red mold rice (RMR) fermented by Monascus purpureus NTU 568, mutated by UV irradiation, have been proved for the suppression cholesterol production, anti-inflammation, anti-tumor and suppression tumor metastasis. In this study, twenty yellow and orange pigments were isolated from the fermented rice and dioscorea of M. purpureus NTU 568. We treated murine macrophages cell RAW 264.7 with these pigments and screened for their anti-inflammatory activity to suppress the lipopolysaccharide (LPS)-induced secretion of nitric oxide. For anti-inflammatory studies, we performed large-scale preparation for ten pigments, including the two main yellow pigments ankaflavin (IC50 = 21.8 uM) and monascin (IC50 = 29.1 uM), their structural analogues monaphilone A (IC50 = 19.3 uM), monaphilone B (IC50 = 22.6 uM), two main orange pigments, monascorubrin (IC50 > 40 uM) and rubropunctatin (IC50 = 21.2 uM), and four new orange pigments monaphilol A ~ D (IC50 = 1.0 ~ 3.8 uM). Using Western blot and ELISA kits, we investigated the inflammatory enzymes and pro-inflammatory cytokines, and demonstrated that the protein expression of iNOS was severely decreased with the treatment of yellow and orange pigments, whereas the expression of COX-2 was just slightly decreased with the same treatment. Moreover, yellow and orange pigments suppressed the production of tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), and interleukin-6 (IL-6). In order to investigate the anti-inflammatory potential of yellow and orange pigments, we used two in vivo assays. In 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema model, the applications of yellow and orange pigments before TPA administered can inhibit the ear edema approximately 60%. In LPS-injection mice model, we also proved that yellow and orange pigments inhibited the plasma NO, TNF-a, IL-1b and IL-6 level. In a conclusion, these yellow and orange pigments from M. purpureus NTU 568 had great anti-inflammatory activities, especially monaphilol A ~ D, possessed high potential to develop as a kind of chemopreventive food or drugs for inflammatory-associated disease.

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