- 學位論文
濱海藥用植物馬氏濱藜 (Atriplex maximowicziana) 之衍生真菌株的活性成分和天然物之策略開發
Bioactive constituents from littoral medicinal herb Atriplex maximowicziana-derived fungi and the strategic development of natural products
摘要
從台灣金門和彰化海岸採集的濱海藥用植物－馬氏濱藜 (Atriplex maximowcziana Makino) 中，分離並純化出1,534株真菌。接著以紙錠擴散法 (disk-diffusion method)，從真菌株粗萃物中篩選具有抗人體致病菌－金黃色葡萄球菌 (Staphylococcus aureus YC981)、白色念珠菌 (Candida albicans SC5314)、大腸桿菌 (Escherichia coli DH5α) 及新型隱球菌 (Cryptococcus neoformans H99) 等致病菌活性的化合物，結果發現，其中39株在1 mg/mL的濃度下具有顯著的抗菌活性，進一步挑選具開發潛力的真菌株進行後續研究，首先以DNA定序確認真菌株的分類位階，已初步完成32株真菌的鑑種，並由後續各種活性平台的評估，例如：抗病毒、抗發炎、抗血管新生、細胞毒性等，發現Curvularia intermedia Km0855 和Chaetomium globosum Km1226具有抑制第四型人類皰疹病毒活性，其抑制百分比分別為4%和17%；C. intermedia Km0855、Fusarium sp. Km1163、C. globosum Km1226、Alternaria sp. Km1934、Cochliobolus sp. Km1956、Fusarium sp. Km 2117和Fusarium sp. Km 2277具有抑制人類內皮源細胞血管新生活性，其抑制百分比分別為1%、<0%、<0%、4%、<0%、2%和4%；在小鼠神經膠質BV-2細胞上，Neocamarosporium sp. Km1910和Cochliobolus sp. Km1996並無細胞毒性，且具有顯著抑制NO產生的活性，其抑制百分比分別為35%和78%，藉由這些數據的呈現，決定以一株多化合物策略 (one strain-many compounds, OSMAC) 對其中具潛力的100株真菌株進行小量培養，再針對其發酵所含之代謝產物以液相層析串聯質譜儀 (LC-MS/MS) 進一步分析，並使用MZmine、全球天然物社群分子網絡 (GNPS) 和Cytoscape建立可視化和註釋非靶向質譜數據的分子網絡 (MN)，繪製複雜樣本的化學空間圖，以促進有潛力真菌株和分子的發現，依序將C. globosum Km1226、 C. intermedia Km0855和Stemphylium lycopersici Km1578以OSMAC策略進行大量培養，再針對其發酵液所含之代謝產物以管柱 (CC) 和高效液相層析儀 (HPLC) 進行分析、分離與純化，接著以核磁共振儀 (NMR) 作為主要的工具確認化合物的分子結構，搭配質譜 (MS)、紅外光譜 (IR)、結晶繞射 (X-ray) 和紫外光譜 (UV) 等數據佐證結構的正確性，共得到24種聚酮體 (ployketide)、4種大環內脂 (macrocyclic lactone) 和7種生物鹼 (alkaloid) ，包含3個新化合物aureonitol C (1) 和mollipilins G和 H (2 和 3)，以及32個已知化合物aureonitol (4)、mollipilins A和F (5和6)、aureonitol A (7)、chaetoglobosins A–F (8–13)、aureochaeglobosins B和C (14和15)、dihydrocoarctatin (16)、coarctatin (17)、chaetoviridin A (18)、chaetomugilin A (19)、chaetomugilin D (20)、11-epichaetomugilin A (21)、12β-hydroxychaetoviridin C (22)、(+)-(11S, 15R)-11-hydroxycurvularin (23)、(+)-(11R, 15R)-11-hydroxycurvularin (24)、(S)-dehydrocurvularin (25)、curvularin (26)、lunatin (27)、rheoemodin (28)、3,11α,12β,13β,16-pentahydroxy-11,12-dihydroperylen-6(13H)-one (29)、altersolanol A (30)、auxarthrol C (31)、infectopyrone (32)、stemphyloxin Ⅱ (33)、stemphyloxin Ⅰ (34) 和stemphyperylenol (35)，其中化合物5 在小鼠神經膠質BV-2細胞上具有顯著抑制NO產生的效果，其半抑制濃度 (IC50) 為0.7 ± 0.1 μM；化合物8在人類大腸直腸癌HCT-116細胞上具有顯著細胞毒性，其 IC50 為3.0 ± 0.1 μM；化合物11在人類內皮源細胞上具有顯著抗血管新生活性，其IC50 為0.8 ± 0.3 μM；化合物23–25在EBV上具有抑制效果，其EC50值介於1.6至3.5 μM，且化合物23的選擇指數 (SI) 為8.5；化合物29在Staphylococcus aureus NEWMAN具有顯著的抑制作用，其最低抑菌濃度測定 (MIC) 值為4.0 μg/mL。
並列摘要
1,534 fungal strains were isolated from the littoral medicinal herb Atriplex maximowicziana Makino collected from Kinmen and Changhua coast, Taiwan. Using disk-diffusion method for the screening of antimicrobial activities from the ethyl acetate extract of the bioactivities of fungal strains against Staphylococcus aureus YC981, Escherichia coli DH5α, Candida albicans SC5314 and Cryptococcus neoformans H99. The 39 fungal strains were found to exhibit significant antimicrobial activities at a concentration of 1 mg/mL, and selected the more potential fungal strains for in-depth study. Firstly, by using polymerase chain reaction to determine the fungal strains DNA sequences, 32 fungal strains already have been determined their taxonomy. In order to develop leading drug candidates, various biological activities (anti-virus, anti-inflammatory activity, anti-angiogenesis and cytotoxicity etc.) tests were practiced. Curvularia intermedia Km0855 and Chaetomium globosum Km1226 exhibited significant human herpesvirus 4 (HHV-4) inhibitory activity with the percentage of inhibition were 4% and 17%, respectively; C. intermedia Km0855, Fusarium sp. Km1163, C. globosum Km1226, Alternaria sp. Km1934, Cochliobolus sp. Km1956, Fusarium sp. Km 2117 and Fusarium sp. Km 2277 exhibited significant anti-angiogenesis activity in human endothelial progenitor cells with the percentage of inhibition were 1%、<0%、<0%、4%、<0%、2% and 4%, respectively; Neocamarosporium sp. Km1910 and Cochliobolus sp. Km1996 showed no cytotoxicity toward microglial BV-2 cells, but exhibited significant inhibitory activity of LPS-induced NO production with the percentage of inhibition were 35% and 78%, respectively. Therefore, potential 100 fungal strains were investigated intensively by applying OSMAC strategy. The secondary metabolites contained in the fermentation were further analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). The MS2 data were generated by MZmine, Global Natural Product Social Molecular Networking (GNPS) and Cytoscape to build molecular networking (MN) for visualizing and annotating non-targeted mass spectrometry data. It was used to map the chemical space of complex samples to facilitate the discovery of potential fungal strain and new molecule. The fungal strain C. globosum Km1226, C. intermedia Km0855 and Stemphylium lycopersici Km1578 were further cultured by OSMAC strategy for scale-up, resulted in the isolation of compounds 1–35. Their structures were determined by spectroscopic analysis as 24 polyketides, 4 macrocyclic lactone and 7 alkaloids, including three previously undescribed C13-polyketides, namely aureonitol C (1), mollipilins G and H (2 and 3), along with thirty-two known compounds, aureonitol (4)、mollipilins A and F (5 and 6), aureonitol A (7), chaetoglobosins A–F (8–13), aureochaeglobosins B and C (14 and 15), dihydrocoarctatin (16), coarctatin (17), chaetoviridin A (18), chaetomugilin A (19), chaetomugilin D (20), 11-epichaetomugilin A (21), 12β-hydroxychaetoviridin C (22), (+)-(11S,15R)-11-hydroxycurvularin (23), (+)-(11R,15R)-11-hydroxycurvularin (24), (S)-dehydrocurvularin (25), curvularin (26), lunatin (27), rheoemodin (28), 3,11α,12β,13β,16-pentahydroxy-11,12-dihydroperylen-6(13H)-one (29), altersolanol A (30), auxarthrol C (31), infectopyrone (32), stemphyloxin Ⅱ (33), stemphyloxin I (34), stemphyperylenol (35). Among these, 6 exhibited significant LPS-induced NO production inhibitory activity with an IC50 value of 0.7 ± 0.1 μM in microglial BV-2 cells; 8 showed potent cytotoxic activity with an IC50 value of 3.0 ± 0.1 μM in HCT-116; 11 exhibited significant anti-angiogenesis activity with an IC50 value of 0.8 ± 0.3 μM in human endothelial progenitor cells; 23–25 showed good antiviral activities against EBV with the EC50 values of 1.6 to 3.5 μM, especially selectivity index of 23 was 8.5; 29 exhibited significant antimicrobial activity with minimum inhibitory concentration value of 4.0 μg/mL against Staphylococcus aureus NEWMAN.