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泡盛酒麴菌Aspergillus awamori所生產新生理活性化合物之探討－抗菌、拮抗以及抗腫瘤活性
Other Title
Study on the novel physiologically active compounds produced by Aspergillus awamori - antimicrobial, antagonistic and
antitumor activities.
antitumor activities.
Type
thesis
Date Issued
2013-07-11
Author(s)
陳虹潔
Advisor
許元勳
Subjects
系所名稱：醫學科學研究所
Description
學位別：碩士
語文別：中文
指導教授：許元勳
共同指導教授：
口試委員：蘇慶華;吳宗正
中文關鍵字：泡盛酒麴菌;抗菌活性;拮抗活性;細胞毒殺活性;TRAIL
語文別：中文
指導教授：許元勳
共同指導教授：
口試委員：蘇慶華;吳宗正
中文關鍵字：泡盛酒麴菌;抗菌活性;拮抗活性;細胞毒殺活性;TRAIL
Abstract
Our laboratory has successfully isolated a series of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitizing compounds, which are effective against human colorectal cancer cell HT29, from the cultural filtrate of Aspergillus awamori (A. awamori) when fermented with modified sabouraud dextrose broth (M-SDB).After carefully analyzing the metabolites of the strain, we newly found some other bioactive ingredients existing in the ethyl acetate(EA) extractable neutral portion.
In continuing the study, we fermented the same strain by using M-SDB as the production medium. The cultivation conditions and the recovery procedure to the bioactive target compounds were re-examined and proceeded. Acid-base EA extraction, bioactivity-directed fractionation followed by 3-step chromatographic separation including silica gel column(once)and thin layer(twice) chromatography, as well as C-18 reverse phase high performance liquid chromatography(final), has led us to isolate two pure compounds, AWM-NF01 and AWM-NF02. Since AWM-NF02 is the major bioactive ingredient (>97% by content) and also showed more potent antimicrobial activity than AWM-NF01, we chose this compound for the following study. The minimum inhibitory concentration (MIC) of AWM-NF02 against Staphylococcus aureus was about 125μg/mL, nevertheless such antimicrobial activity could be abolished by glutathione (GSH) which is one of the sulfhydryl group containing compounds. The molecular weight and the molecular formula of AWM-NF02 were determined to be 308 and C20H36O2, respectively, by analyzing the elemental analytical result together with MS and NMR spectroscopic data. The cytotoxic activities of AWM-NF02 against human colorectal cancer cell HT-29 and oral squamous carcinoma cell OEC-M1 were determined to be 7.81μg/mL (60% survival rate) and 250 μg/mL (50% survival rate) by MTT assay. In addition, the sensitization function of AWM-NF02 in potentiating TRAIL-induced apoptosis to HT-29 was also determined. Our results indicated that AWM-NF02 is a previously undiscovered novel metabolite of A. awamori, exhibiting a variety of physiological activities. Moreover, we demonstrated that AWM-NF02 can indeed sensitize HT-29 to TRAIL-induced apoptosis, further studies are needed to realize the full mechanism of this new combination.
In continuing the study, we fermented the same strain by using M-SDB as the production medium. The cultivation conditions and the recovery procedure to the bioactive target compounds were re-examined and proceeded. Acid-base EA extraction, bioactivity-directed fractionation followed by 3-step chromatographic separation including silica gel column(once)and thin layer(twice) chromatography, as well as C-18 reverse phase high performance liquid chromatography(final), has led us to isolate two pure compounds, AWM-NF01 and AWM-NF02. Since AWM-NF02 is the major bioactive ingredient (>97% by content) and also showed more potent antimicrobial activity than AWM-NF01, we chose this compound for the following study. The minimum inhibitory concentration (MIC) of AWM-NF02 against Staphylococcus aureus was about 125μg/mL, nevertheless such antimicrobial activity could be abolished by glutathione (GSH) which is one of the sulfhydryl group containing compounds. The molecular weight and the molecular formula of AWM-NF02 were determined to be 308 and C20H36O2, respectively, by analyzing the elemental analytical result together with MS and NMR spectroscopic data. The cytotoxic activities of AWM-NF02 against human colorectal cancer cell HT-29 and oral squamous carcinoma cell OEC-M1 were determined to be 7.81μg/mL (60% survival rate) and 250 μg/mL (50% survival rate) by MTT assay. In addition, the sensitization function of AWM-NF02 in potentiating TRAIL-induced apoptosis to HT-29 was also determined. Our results indicated that AWM-NF02 is a previously undiscovered novel metabolite of A. awamori, exhibiting a variety of physiological activities. Moreover, we demonstrated that AWM-NF02 can indeed sensitize HT-29 to TRAIL-induced apoptosis, further studies are needed to realize the full mechanism of this new combination.