台灣三種海洋細菌醱酵液之生物活性成分研究

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Title:	台灣三種海洋細菌醱酵液之生物活性成分研究 Studies on the bioactive constituents of the fermented broth of three marine bacterial species isolated in TaiwanStudies on the bioactive constituents of the fermented broth of three marine bacterial species isolated in Taiwan
Authors:	李翊弘 Yi-Hung Lee
Contributors:	生藥學研究所
Keywords:	抗菌活性瓊脂擴散法綠膿桿菌紅桿菌科堅強芽孢桿菌 antibacterial activity disc agar diffusion method Pseudomonas aeruginosa Rhodobacteraceae Bacillus firmus
Date:	2008
Issue Date:	2009-08-31 11:30:41 (UTC+8)
Abstract:	本研究利用瓊脂擴散法 (disc agar diffusion method) 來篩選105株台灣海洋細菌，藉以尋找出對抗藥性金黃色葡萄球菌 (Penicillin G-resistant Staphylococcus aureus, ATCC 11631 ) 具有抑制活性的海洋細菌。所得結果根據抑菌圈的大小判斷其中2株具較明顯的抑制活性，分別為#M1B (Pseudomonas aeruginosa) 和#AW52 (Rhodobacteraceae)。此外，所有海洋菌株萃取液在血管舒張活性 (vasorelaxing activities) 上，利用Phenylephrine誘發大鼠 (Sprague-Dawley Rat) 胸主動脈血管收縮，再以細菌醱酵液萃取物進行處理，得知其中2株菌之萃取物具有抑制血管收縮的活性，分別為#M1B (P. aeruginosa) 和#MO6 (B. firmus)。隨後將上述三株海洋菌以PY培養基擴大培養，針對其代謝物進行一系列的分析、分離、純化與結構解析，計分離出15個化合物，包括: 兩個喹林生物鹼 (quinoline alkaloide)、兩個吩嗪生物鹼 (phenazine alkaloids)、五個芳香胺衍生物 (phenylamides derivatives) 、六個環狀雙胺基酸 (diketopiperazines) 和 2-胺基苯乙酯(2-aminophenyl acetate)。分別為: 2-n-heptylquinol-4-one (1)、2-aminophenyl acetate (2)、cyclo-L-Pro-L-Phe (3)、cyclo-L-Tyr-L-Pro (4)、phenazine-1-carboxamide (5)、hydroxyphenazine (6)、N-(2’-Phenylethyl)isobutyramide (7)、2-Ethyl-N-(2’-phenethyl)butyramide (8)、2-Methyl-N-(2’-phenylethyl)butyramide (9)、N-(2’-phenylethyl)isovaleramide (10)、2-heptyl-4-hydroxyquinoline-N-oxide (11)、cyclo-L-Pro-L-Ile (12)、cyclo-L-Pro-L-Val (13)、cyclo-L-Pro-L-Leu (14) 和cyclo-L-Pro-L-Met sulfoxide (15)，其中cyclo-L-Pro-L-Met sulfoxide (15) 是過去未曾報導過的新化合物。這些天然物純質更進一步進行抗菌活性試驗，結果顯示化合物1、5、6和11針對抗藥性金黃色葡萄球菌有明顯的抑制活性，其最小抑制濃度 (minimum inhibitory concentration, MIC) 分別為64、36、36和24 ?慊/ml。另外，細胞毒性檢測的結果顯示，化合物1和11具有顯著的細胞毒性，其對於 NPC-tw01、HCT-116、Jurkat和H661等四株癌細胞的半抑制濃度 (IC50) 範圍介於 7.1 ~ 14.7 ?嵱。 In this study, 105 strains of marine bacteria isolated from Taiwan were cultured for the screening of their antibacterial activities by disc agar diffusion method. Of these bacterial strains monitored, 2 strains exhibited antibacterial activities. Among them, bacterial strains M1B (Pseudomonas aeruginosa) and AW52 (Rhodobacteraceae) showed significant antibacterial activities toward Penicillin G-resistant Staphylococcus aureus. (ATCC 11631). Besides, all the marine bacterial extracts on vasorelaxing activities of SD rats induced by phenylephrine were also examined. Of these bacterial strains monitored, strains M1B (P. aeruginosa) and MO6 (B. firmus) exhibited vasorelaxing activities. Based on this findings, the 3 bacterial strains were mass cultured using PY broth. Then, the bioactive constituents were obtained by a series of chromatographic separation, and characterized by spectral analysis. Totally 15 compounds including 2 quinoline alkaloids, 2 phenazine alkaloids, 4 phenylamide derivates, 6 diketopiperazines and a 2-aminophenyl acetate were isolated and identified . Their structures were elucidated to be 2-n-heptylquinol-4-one (1), 2-aminophenyl acetate (2), cyclo-L-Pro-L-Phe (3), cyclo-L-Tyr-L-D-Pro (4), hydroxyphenazine (5), phenazine-1-carboxamide (6), N-(2’-Phenylethyl)isobutyramide (7), 2-Ethyl-N-(2’-phenethyl)butyramide (8), 2-Methyl-N-(2’-phenylethyl)butyramide (9), N-(2’-phenylethyl)isovaleramide (10), 2-heptyl-4-hydroxyquinoline-N-oxide (11), cyclo-L-Pro-L-Ile (12), cyclo-L-Pro-L-Val (13), cyclo-L-Pro-L-Leu (14), and cyclo-L-Pro-L-Met sulfoxide (15). Among them, cyclo-L-Pro-L-Met sulfoxide (15) was isolated for the first time from the natural source. The bioactivities of all the pure entities were further evaluated. The results of biological tests indicated that 1, 5, 6 and 11 exhibited strong antibacterial activity toward resistant S. aureus, with minimum inhibitory concentrations (MIC) of 64, 36, 36 and 24 ug/ml, respectively. Additionally, 1 and 11 showed significant cytotoxic activitives against NPC-tw01, HCT-116, Jurkat, and H661 cancer cell lines, with an IC50 values from 7.1 to 14.7 uM.
Data Type:	thesis
Appears in Collections:	[Graduate Institute of Pharmacognosy Science] Dissertation/Thesis

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