臺灣細翼枝菜內生真菌 Trichoderma harzianum NTOU4253之成分研究

Abstract

利用抑制誘導型一氧化氮合成酶 (inducible Nitric Oxide Synthase, iNOS) 的活性分析平台來篩選真菌培養株，以篩選出具有抗發炎活性的真菌株，發現某些菌株的醱酵培養液可抑制lipopolysaccharides (LPS) -induced RAW264.7細胞之一氧化氮的產生。同時由抗菌活性評估，顯示某菌株之粗萃物可抑制革蘭氏陽性金黃色葡萄球菌 (Staphylococcus aureus) 的生長，而後選定細翼枝菜 (Pterocladiella capillacea) 內生真菌Trichoderma harzianum NTOU4253進行其化學成分分析。接著以PDY (Peptone Dextrose Yeast) 液態培養基加以擴大培養，對其醱酵液所含代謝產物進行分析、分離與純化，計獲得6個化合物，藉由各種光譜分析決定其平面結構，進一步由Mosher反應後與文獻比對，始確認其絕對立體。其中4個為新化合物，定為trichoharol A (1)、trichoharol B (4)、trihcoharol C (5) 以及 (−)-harzianolide (6)，另外2個為已知化合物，harzianumols A (2) 與harzianumols B (3)，皆屬於多聚酮類 (polyketide) 化合物。在活性試驗上，本研究發現相對於對照組，化合物2 、 4與5對於LPS-induced RAW264.7細胞產生之一氧化氮顯示有抑制效果，其半抑制濃度 (IC50) 分別為31.1、51.3及19.7 μM，且對於RAW264.7細胞不具毒殺特性。正對照組aminoguanidine及 Nω-nitro-L-arginine (L-NNA) 的半抑制濃度 (IC50) 分別為22.3 ± 0.3和146.4 ± 0.7 μM。同時，化合物5對於金黃色葡萄球菌 (S. aureus) 相較於對照組具有抑制效果，其抑菌圈大小為15.0 ± 0.3 mm。正對照組tetracycline之抑菌圈大小為25.0 ± 0.1 mm。

Bioassay-guided fractionation and separation of the active component from Trichoderma harzianum NTOU4253 resulted in the isolation of six compounds 1-6. Their structurues were determined to be linear C13 polyketides, and four new compounds were named trichoharol A (1), trichoharol B (4), trihcoharol C (5) and (−)-harzianolide (6), together with previously reported two polyketide-type C13 lipids harzianumols A (2) and harzianumols B (3). Compound 2, 4 and 5 exhibited moderate to potent inhibitory activity on NO production of RAW264.7 cells induced by lipopolysaccharide (LPS) with IC50 values of 31.1, 51.3 and 19.7 μM, respectively, without any cytotoxicity. The IC50 values of the positive controls, aminoguanidine and Nω-nitro-L-arginine (L-NNA), were 22.3 ± 0.3 and 146.4 ± 0.7 μM, respectively. Compound 5 exhibited slight antimicrobial activities against Staphylococcus aureus with 15.0 ± 0.3 mm inhibition zone compared to positive control-tetracycline with inhibition zone of 25.0 ± 0.1 mm.