紅麴Monascus purpureus NTU 568發酵產物中防癌及其相關活性成分之研究

Abstract

本實驗室利用傳統變異法篩選得到紅麴菌株Monascus purpureus NTU 568，經由動物實驗證實本菌株的發酵產物除了有降血脂功效之外，對減少體脂肪生成和改善阿茲海默症也有效果，在抗腫瘤的研究也發現M. purpureus NTU 568發酵的紅麴米之酒精萃取物可以延緩老鼠肺癌的成長，對於DMBA致癌劑誘發的老鼠口腔癌也有減輕效果。紅麴的主要色素係由黃、橘和紅色素組成，其屬於azaphilone化學結構，已被證實具有抗發炎和抑制癌症生長之效果。本論文研究為探討M. purpureus NTU 568發酵產物中的癌症化學預防成分，其中包含抗癌和抗發炎的azaphilone衍生物以及抗氧化的成分。 利用各種管柱層析及高效液相層析的分離方法，分別從紅麴米甲醇萃取物中分離到14個azaphilone化合物 (1-14) 、紅麴山藥丙酮萃取物中分離到8個azaphione化合物 (1、2、15-20)、紅麴米50%酒精萃取物分離到2個化合物 (33、34)。此外，以兩個黃色素ankaflavin (1) 和monascin (2) 經化學反應，製備6個azaphilone衍生物 (21-26)，從兩個橘色素monascorubrin (15) 和 rubropunctatin (16) 則製備4個azaphione衍生物 (27-30) 以及兩個已知的紅色素monascorubramine (31) 和rubropunctamine (32)。上述化合物1-34的結構判斷係經由NMR、MASS、IR、UV等光譜資料的解析；其中11個化合物被確定為首次分離的新化合物，分別為monaphilone A-C (3-5)、monapurpyridine A-B (9-10)、monapurfluore A-B (13-14)、monaphilol A-D (17-20)，而化合物21-30則係經由半合成反應所得的新衍生物。 將所獲致的azaphilone類化合物 (1-32) 分析其對人類喉癌 (HEp-2)、人類大腸癌 (WiDr) 和人類乳癌 (MCF-7) 等癌細胞株的抑制生長情形，大多數的化合物具中等程度的抑制效果 (IC50值在20-80 μM之間)，化合物 monaphilol A-D (17-20) 的抑制效果最好 (IC50值可達8.6-15.6 μM)；且部分化合物對癌細胞具有選擇性抑制作用， 其中monascin (2) 只對HEp-2有抑制作用，FK-17-P2b1 (6)、monasflure A (13) 和monasflure B (12) 則針對MCF-7具抑制作用。進一步探討橘色素 (15-16) 及其衍生物 (17-20、27-32) 的化學結構與生物活性的相關性，發現橘色素8號位置的酮基還原成氫氧基時 (17-20)，對HEp-2和WiDr細胞的抑制活性可以提升4-7倍，但如果同時還原14號位置的酮基時 (27-30)，原本的抑制效果則會降低，顯示有無8和14號位置的酮基會影響橘色素的對癌細胞生長的抑制活性。 以LPS誘發巨噬細胞 (RAW 264.7) 發炎成生NO的模式分析azaphilone衍生物的抗發炎效果，發現化合物 1-14中只有monasphilone C (5) 和FK-17-P2b1 (6) 沒有抑制發炎效果，其他化合物的抗發炎效果IC50值均在20-30 μM之間，其中只有monapurpyridine B (10) 對RAW 264.7細胞有毒性。化合物 monaphilol A-D (17-20) 在5 μg/mL可以抑制100% 的NO生成，IC50值在1.0-1.7 μM之間，抑制發炎效果較正控制組檞黃素 (quercetin, IC50 = 13.2 μM) 佳。以抑制DPPH自由基生成能力做為抗氧化活性評估指標，由紅麴米50% 酒精萃取物，共分離得到2個抗氧化化合物dimerumic acid (33) 和 deferricoprogen (34)，其抑制DPPH自由基生成活性之IC50值分別為16和15 μg/mL，而deferricoprogen (34) 為首次由紅麴中分離到的抗氧化成分。進一步利用HPLC分析紅麴米50% 酒精萃取物的主要成分含量，發現dimerumic acid (33) 和 deferricoprogen (34) 是紅麴米50%酒精萃取物的主要成分，其含量分別為0.85 mg/g和3.00 mg/g。 本實驗從紅麴M. purpureus NTU 568的發酵產物中分離得到21個具抗癌和抗發炎的azaphilone衍生物以及2個主要的抗氧化成分，這些成分的發現和生物活性的證實，有助於紅麴應用於癌症預防的研究，未來將利用這些活性成分探討紅麴防癌的作用機轉，並發展紅麴成為防癌的機能性食品。

In our previous reports, the red mold rice (RMR) fermented by Monascus purpureus NTU 568, mutated by UV irradiation, have been proved for hypolipidemic effects, the regulation of obesity-related factors, and the amelioration of the impairment of memory ability in vivo. Moreover, the ethanol extracts of M. purpureus NTU 568 fermented rice also could mitigate oral carcinogenesis in 7,12-dimethyl-1,2-bena[a]anthracene (DMBA)-induced oral tumor as well as reduce tumor progression of Lewis lung cancer. Monascus pigments compose of yellow, orange and red pigments which belonged to an azaphilone skeleton, and have been reported to possess anti-inflammantory and anticancer activities. Consequently, the aims of the thesis are to study the bioactive chemical constituents from M. purpureus NTU 568 fermented products and further evaluate their anti-proliferative, anti-inflammatory and anti-oxidative activities of isolates. By column chromatography and high performance liquid chromatography, fourteen (1~14), eight (1, 2, 15~20) azaphilone derivatives and two siderophore skeleton compounds (33 and 34), were isolated from methanol extracts, acetone extracts, and the 50% ethanol extracts of red mold dioscorea (RMD), respectively. Moreover, six azaphilone derivatives (21-26) were derived from yellow pigments, ankaflavin (1) and monascin (2), via chemical reaction. Four azaphione derivatives (27-30) and two known red pigments, monascorubramine (31) and rubropunctamine (32), were also acquired by a short semi-synthesis of orange pigments, monascorubrin (15) and rubropunctatin (16). Structural elucidation of isolated compounds was based on mass, infrared (IR), ultraviolet (UV), and nuclear magnetic resonance (1H-NMR, 13C-NMR, COSY, HMQC, and HMBC) spectroscropic analyses. Of the above mentioned twenty-two isolates, including monaphilone A (3), monaphilone B (4), monaphilone C (5), monapurpyridine A~B (9~10), monapurfluore A (13), monapurfluore B (14), monaphilol A (17), monaphilol B (18), monaphilol C (19), and monaphilol D (20) were verified as new compounds. Compounds 1-32 were further evaluated for their inhibitory effects on the proliferation against human tumor cell lines, HEp-2 (human laryngeal carcinoma) and WiDr (human colon adenocarcinoma), as well as MCF-7 (human breast adenocarcinoma). Bioactive data showed that most of isolates (1-32) had moderate anti-proliferative activities (IC50 : 20 to 80 μM) and monaphilol A-D (17-20) had potent anti-proliferative effect (IC50 : 8 to 15 μM). Monascin (2) exhibited a selective inhibitory effect toward HEp-2, while FK-17-P2b1 (6), monasflure A (13) and monasflure B (12) showed significant inhibitory effects agaist MCF-7. For the structure-activity relationship (SAR) studies of orange pigments (15, 16) and their derivatives (17-20 & 27-32), the anti-proliferative activities of compounds 17-20 with hydroxyl group at C-8 show 4- to 7- fold higher than that of compounds 15 and 16 with ketone group at C-8. However, the anti-proliferative activities of compounds 27-30 were less than compounds 17-20, duo to the hydroxyl groups substituted by ketone groups at C-8 and C-14. Moreover, compounds 1-14, except for monascin (2), monasphilone C (5) and FK-17-P2b1 (6), exhibited promising anti-NO production on LPS-stimulated RAW264.7 macrophages compared with the positive control, quercetin (IC50 : 13.2 μM). Monapurpyridine C (10) showed cytotoxic phenomenon for RAW264.7 cells at 10-15 μg/mL. However, monaphilol A-D (17-20) could reduce LPS-stimulated NO production completely at 5 μg/mL; whereas, they exhibited cytotoxic effects for RAW264.7 cells at 10 μg/mL. Dimerumic acid (DMA) and deferricoprogen (DFC), were isolated from the 50% ethanol extracts of M. purpureus NTU 568 fermented red mold rice (RMR) by antioxidant guided fractionation method. DMA and DFC showed potent DPPH free radical scavenging activities with IC50 value of 16 and 15 μg/mL, respectively. The concentrations of DMA and DFC in RMR were determined as 0.85 and 3.00 mg/g employing HPLC analysis. In this study, twenty-one azaphilone derivatives possessing anti-inflammatory and anti-proliferative effects, as well as two major natural anti-oxidative compounds were isolated and characterize from M. purpureus NTU 568 fermented products. The investigation of chemopreventive mechanism of these bioactive isolates remain to be established. We hope that the potential azaphilone analogues isolated from Monascus purpureus NTU 568 fermented products will be developed as a functional food of cancer chemoprevention.