食用麴菌所生產對大腸直腸癌細胞具TRAIL凋亡誘導致敏性的新型發酵產物篩選研究

Screening of novel fermented products produced by edible fungi of Aspergillus sp. that sensitizes colorectal cancer cells to TRAIL-induced apoptosis.

楊心妤(Sin-Yu Yang)

指導教授 ： 許元勳

臺北醫學大學/醫學院/醫學科學研究所/碩士(2012年)

https://doi.org/10.6831/TMU.2012.00143

摘要

本研究針對食用麴菌(edible koji)，包括製造日本清酒專用的米麴菌(Aspergillus oryzae)、泡盛麴菌(Aspergillus awamori)…等，進行與各種培養基質的發酵組合，以探討其應用在新天然抗癌藥物開發的可行性；研究標的著眼於能增強腫瘤壞死因子相關凋亡誘導配體(TNF-related apoptosis-inducing ligand, TRAIL)誘導癌細胞凋亡效果的致敏因子(senisitizers)。標的活性物質的篩選及追蹤指標包括：1.抗細菌活性試驗；2.自由基清除和抗氧化能力試驗；以及3.含硫醇基化合物N-acetylcysteine (NAC)和還原態glutathione (GSH)對生物活性的拮抗現象等觀測。根據篩選實驗結果，選定以泡盛麴菌發酵SDB 培養基的組合做為標的活性物質的生產條件。當菌種發酵上清液經乙酸乙酯(ethyl acetate)酸鹼轉溶萃取分層時，在萃取中性層(neutral fraction)可得到符合上述篩選指標的活性成分；進一步以矽膠(silica gel)管柱層析進行活性物質分離，以階段梯度沖堤，在正己烷(n-hexane)：乙酸乙酯=70：30處可得到最佳的活性表現區段，其中尤以區段No.20的活性表現最佳。此區段在室溫靜置過程中可形成無色針狀結晶，且證實此結晶物質具有降低人類大腸癌細胞HT-29存活率及增加TRAIL所誘導HT-29細胞凋亡之效果。針對此活性區段，再經矽膠薄層層析(TLC)分析後，可確定展開溶劑為苯(benzene)：乙酸乙酯：甲醇(methanol) =70：25：5，於展開阻滯值(retention factor, Rf ) 0.59處，UV254波長下可觀測到呈現明顯吸收的單一成分存在，此即為標的活性物質的純化物。以C18管柱為固定相的逆相高效能液相層析(HPLC)分析結果顯示，以UV254波長檢測，於水：甲醇=80：20沖堤條件下，可觀測到完全分離的兩個吸收峰，分別命名為 No.AWM-N01與No.AWM-N02。上述兩成分再經過製備級C18逆相HPLC的分離純化收集，可得到兼具抗細菌及NAC與GSH拮抗活性的No.AWM-N01，以及僅具有抗細菌活性的No.AWM-N02兩個活性成分，兩者的純度均接近100 %。No.AWM-N01的FT-IR圖譜顯示，在1724 cm-1 (sharp, w, C=O)、1648 cm-1 (sharp, w, C=C)可分別觀測到微弱但明顯的羰基及碳雙鍵官能基特性的吸收峰，推測No.AWM-N01為帶有α,β-共軛不飽和羰基(α,β-unsaturated conjugated carbonyl group)的化合物，此分子結構特性符合本研究初始所設定的篩選條件。本研究已證實食用麴菌在開發新型TRAIL細胞凋亡誘導致敏性天然藥物的可行性。

關鍵字

食用麴菌 ； 泡盛酒麴 ； 細胞凋亡 ； 大腸直腸癌 ； TRAIL ； 致敏因子

並列摘要

The aim of this study was to investigate the application of the secondary metabolites of edible koji, which include sake-making koji-kin (koji mold) such as Aspergillus oryzae, Aspergillus awamori... etc., in developing the fermented koji-kin/cultural medium combination products as an anticancer drug. The main focus was to seek the sensitization factors (sensitizers) that can enhance TRAIL (TNF-related of apoptosis-inducing ligand) induced apoptosis in cancer cells. The criteria in this study for screening active substances was set as follows: 1. antibacterial activity; 2. free radical scavenging and/or antioxidant activities; 3. antagonistic effect of antibacterial activity which was abolished by the thio-containing compounds, such as N-acetylcysteine (NAC) and reduced glutathione (GSH)…etc.. As a result, the combination of Aspergillus awamori and SDB medium was selected as the optimal fermentation condition for the production of target active substances. The supernatant from the cultural broth of Aspergillus awamori/SDB medium combination was extracted by ethyl acetate (EtOAc) under the process of acid/base and activity-guided fractionation in result of obtaining the neutral fraction which meet our screening criteria. The neutral fraction was then subjected to a silica gel column chromatography by stepwise elution with n-hexane/ethyl acetate to give fraction No.20 (n-hexane: ethyl acetate= 70: 30), the most potent fraction in antibacterial and antagonistic activity. The active compounds were obtained in the form of the colorless needle crystals when placed at room temperature. These crystals were confirmed to possess cytotoxic activity and TRAIL-induced apoptosis sensitization activity of human colorectal cancer cell HT-29. By using TLC (benzene: ethyl acetate: methanol= 70: 25: 5) analysis together with bio-autographic detection, the target component was confirmed to exist in the fraction No.20.The target compound was further purify by reverse phase C18 HPLC in a mode of isocratic elution with water: methanol =80:20 as the mobile phase. The result of HPLC profile revealed two completely separated components, designated as No.AWM-N01 and No.AWM-N02. These two compounds were further obtained by preparative-scale reverse phase C18 HPLC. No.AWM-N01 exhibited significant antibacterial activity which could be abolished by NAC and GSH, nevertheless No.AWM-N02 only showed antibacterial activity. The FT-IR spectrum of No.AWM-N01 indicated the characteristic spectroscopic absorbance of C=O at 1724 cm-1 (w, s) and C=C at 1648 cm-1 (w, s). This result may provide a clue for No.AWM-N01 to content an α, β-unsaturated conjugated carbonyl group in its molecule. In conclusion, we have demonstrated the feasibility of edible koji as a promising source in exploring natural novel TRAIL sensitizers for anticancer chemotherapy.

並列關鍵字

edible koji ； Aspergillus awamori ； fermentation ； colorectal cancer ； apoptosis ； TRAIL ； sensitizer

參考文獻

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