臺灣兩種海洋細菌之生物活性成分研究
Studies on the bioactive constituents of two marine bacterial species isolated in Taiwan
中文摘要
本研究利用瓊脂擴散法 (disc agar diffusion method) 來篩選 250 台灣株海洋細菌，藉以找 尋出具有抗菌活性的海洋細菌，所使用之五種病原菌分別為創傷弧菌 (Vibrio vulnificus, ATCC 27562)、腸炎弧菌 (V. parahaemolyticus, ATCC 17802)、魚類病原弧菌 (Listonella anguillarum, ATCC 19264)、金黃色葡萄球菌 (Staphylococcus aureus, ATCC 12600) 和 嗜水性產氣單胞菌 (Aeromonas hydrophila, ATCC 7966) 等，再根據抑菌圈的大小判斷其 中 12 株具有抗菌活性，其中較顯著的三株包括: BGN6 (Pseudoalteromonas
flavipulchra)、B0 (P. flavipulchra) 對於創傷弧菌 (V. vulnificus) 有抑制效果，而 BTN3 (V. natriegens) 則對於金黃色葡萄球菌 (S. aureus) 有抑制效果。隨後，將這三株菌以 PY 培養 基大量培養，針對其成份進行一系列的分析、分離、純化與構造解析，計分離、決定出十五個化 合物，包括：二個脂肪族生物鹼 korormicin (1) 和 norkorormicin (5)、二個嘧啶類化合物 uracil (2) 和 thymine (7)、二個酚類化合物 4-(hydroxymethyl)phenol (3) 和
p-hydroxybenzaldehyde (6)、七個雙胺基酸環化衍生物 (diketopiperazine) 包括: cyclo-L-Pro-L-Phe (4)、cyclo-L-Pro-L-Val (8) cyclo-L-Pro-L-Met (9)、cyclo-L-Pro-L-Ile (10)、cyclo-L-Pro-L-Leu (11)、cyclo-D-Pro-L-Phe (12) 和 cyclo-L-Pro-L-Tyr (13) 以 及二個吲哚類化合物 indole (14) 和 3-(3,3-diindoly)propane-1,2-diol (15)。此外，生物 活性試驗結果顯示化合物 1 和 5 對於抑制創傷弧菌 (V. vulnificus) 有最強的抑制生長活性， 其最小抑制濃度 (minimun inhibitory concentration, MIC) 為 1 mg/mL。另外，細胞毒 性測試則顯示化合物 1、4 和 12 具較為顯著的細胞毒性效果，其對於 FaDu、MT-2 和 NPC-tw01 等細胞株的半抑制濃度 (IC50) 範圍介於 2.4 ~ 25.3 mM。
英文摘要
In this study, 250 strains of marine bacteria isolated from Taiwan were cultured for the screening of their antibacterial activities by disc agar diffusion method. Five pathogenic bacteria including Vibrio vulnificus, (ATTC 27562), V. parahaemolyticus, (ATTC 17802), Listonella anguillarum, (ATTC 19264), Staphylococcus aureus, (ATTC 12600), and Aeromonas hydrophila, (ATTC 7966) were tested. Of these bacterial strains monitored, 12 strains exhibited antibacterial activities. Among them, bacterial strains BGN6 (Pseudoalteromonas flavipulchra) and B0 (P. flavipulchra) showed significant antibacterial activity toward V. vulnificus, while BTN3 (V.
natriegens) showed significant antibacterial activity specifically toward S. aureus as evidenced from the observation of significant inhibitory zones. Based on these findings, the 3 bioactive bacterial strains were mass cultured using PY medium. Then, the bioactive constituents were obtained by a series of chromatographic
separation, and characterized by spectral analysis. Totally 15 compounds including 2 aliphatic alkaloids, 2 pyrimidines, 2 phenolics, 2 indole alkaloids, and 7
diketopiperazines were isolated and identified. Their structures were elucidated to be korormicin (1), uracil (2), 4-(hydroxymethyl)phenol (3), cyclo-L-Pro-L-Phe (4), norkorormicin (5), p-hydroxybenzaldehyde (6), thymine (7), cyclo-L-Pro-L-Val (8), cyclo-L-Pro-L-Met (9), cyclo-L-Pro-L-Ile (10), cyclo-L-Pro-L-Leu (11),
cyclo-D-Pro-L-Phe (12), cyclo-L-Pro-L-Tyr (13), indole (14), and
3-(3,3-diindoly)propane-1,2-diol (15). In addition, results of biological tests indicated that 1 and 5 exhibited strong antibacterial activity toward V. vulnificus, and both their minimun inhibitory concentrations were 1 mg/mL. The cytotoxicity tests revealed that 1, 4 and 12 exerted moderate cytotoxic activities against FaDu、 MT-2 and NPC-tw01 cancer cell lines when compared with the chemotherapeutic agent, doxorubixin, with an IC50 value of 2.4 ~ 25.3 mM.
