土壤分離菌Streptomyces sp. 所生產天然抗氧化成分之探索研究

Characterization of the natural antioxidants produced by the soil-born isolate of Streptomyces sp. AMBL-019C

林錦瑩(Chin-Ying Lin)

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(2001年)

摘要

現階段抗氧化劑的應用主要分為兩大類：(１) 健康食品及食品添加物，(２) 臨床治療及預防用藥。為了因應市場不同的需求和人體不同組織吸收藥物途徑的差異，我們以增加抗氧化劑的多樣性及安全性作為研發新型抗氧化劑的主要方向之一。經過各種篩選物質來源的比較評估結果認為，放線菌二次代謝物的複雜度及變化性較高適合作為生理活性物質生產源，因此在實驗設計中鎖定此一微生物素材進行抗氧化物質的篩選，希望藉此發展出能維護人體健康或者具有治療疾病功能的天然抗氧化劑。 　　實驗初期我們至全省各地採集多種土壤樣本，並從其中分離出近200株不同形態的放線菌菌株。針對這些分離菌株，進行菌種培養及二次代謝物的醱酵生產，並配合所設定的抗氧化活性檢測方法進行抗氧化物質生產菌的篩選。針對篩選目的，我們設計了以下的實驗步驟：首先進行二次代謝物回收及檢測樣本處理，亦即是將醱酵所得的培養上清液，利用乙酸乙酯 (Ethyl acetate; EtOAc) 萃取回收，並以真空減壓濃縮脫水等方式處理，以取得提供抗氧化活性篩選之檢測樣本；接著進行抗氧化活性檢測及菌種篩選：以「清除α,α-dipheny-β-picryhydrazyl (DPPH) 自由基能力之測定」、「硫丙二醯尿生成系統 【Thiobarbituric acid reactive substances (TBARS) formation system】」和「還原力(Reducing power) 測定」等檢測方法作為篩選菌株之標準。經此篩選程序，我們選獲了具有抗氧化物質高生產力的菌株，命名為 AMBL-019C ；並由菌種鑑定的結果確認 AMBL-019C屬於 Streptomyces sp.。接下來為取得足量的抗氧化物質以進行後續物質的探討，我們同時大量醱酵了 AMBL-019C 及進行其代謝物的生產回收研究；此部份我們選用YMD + 0.1% Tween80 培養基在室溫下利用通氧培養法進行菌種大量醱酵，並採乙酸乙酯萃取的方式回收其二次代謝物。關於進一步抗氧化物質的分離純化方面：實驗首先將 AMBL-019C 粗萃取物以酸鹼轉溶，再配合抗氧化分析取得中性活性層部份。後續則以矽膠薄層色層分析法 ( Thin layer chromatography; TLC) 分析所得特定活性物質 Rf 值的位置為判斷基礎，利用矽膠管柱層析法 (Column chromatography) 進行較大規模的物質分離提純。當移動相為Hexane：EtOAc＝96：4時可沖提出此特定活性物質，並將此純化物命名為AMBL-019C-TS (TS)。以高效能液相層析法 (High performance liquid chromatography; HPLC) 進一步進行 AMBL-019C-TS純化，在滯留時間7.30至7.70分鐘處可收集到活性物質，其純度亦可再由 HPLC 加以分析確認；TS 為淡黃色針狀結晶，可溶於甲醇 (Methanol) 及較甲醇極性低的有機溶劑，如乙酸乙酯、苯、正己烷等，但不溶於水。利用質譜儀 (Mass spectroscopy)、紫外光-可見光全光譜 (Ultraviolet-visible spectroscopy)、核磁共振氫譜 (1H-NMR)、核磁共振碳譜 (13C-NMR)、氫-氫二次元核磁共振圖譜 (1H-1H 2D NMR) 初步鑑定得知 TS 分子量為236，分子結構中具有苯環和羰基 (-CO)。在抗氧化活性評估方面，由於體內低密度脂蛋白 (Low density lipoprotein, LDL) 的氧化會造成粥狀動脈硬化而導致心血管疾病，故針對此我們評估了 TS 對 LDL 過氧化保護活性，以初步判定此一物質用於預防或治療心血管疾病的可能性；檢測結果顯示此物質具抑制 LDL 過氧化的作用，並與濃度成正比關係，因此我們推論 TS 具有開發成為預防或治療心血管疾病藥物的潛力，至於動物實驗的結果則有待進一步評估；此外為了檢測 TS 對於細胞受氧化傷害時的保護效果，我們亦利用 Balb-3T3 細胞進行細胞毒殺及抗細胞氧化傷害實驗，實驗結果發現TS在抑制 DPPH 所造成的細胞氧化傷害的濃度下，TS 並未被觀測出有任何細胞毒性。針對此一結果，可以初步判定 TS 不但具有抑制自由基及抗油脂氧化的活性，且對於動物細胞亦具有抗氧化保護活性和相當程度的安全性。

關鍵字

抗氧化劑 ； 放線菌 ； 二次代謝物 ； 自由基 ； 低密度脂蛋白

並列摘要

Antioxidants have at present been recognized to play two major roles in industrial applications: (1) healthy foods and food additives; (2) clinically useful supplements for the prevention of chronic diseases, such as cancers and cardiovascular diseases. Due to the social demands and the close relationship between antioxidants and the physiological function of our bodies, the objective of our research was thus aimed at new antioxidant developments in order to improve the safety and usefulness of them. Actinomycetes have been known to be a good source for the development of biologically active compounds, we therefore set these microbes as the screening targets in this experiments. Beginning to our job, we firstly collected a number of domestic soil samples from different areas, and isolated nearly 200 strains of actinomycetes. The isolates were then cultured and fermented by conventional process in order to screen antioxidant-producing strains. The samples were recovered from the microbial metabolites followed by EtOAc extraction and then evaporation to dryness in vacuo. After that, they were subjected to a series of tests in their antioxidative activity. The activity of the prepared samples screened by measurement of their DPPH radical scavenging ability, activity in the inhibition of thiobarbituric acid reactive substances (TBARS) formation, and the potency of reducing power. With above-mentioned categories, we successfully chose out a highly antioxidant producing strain, named AMBL-019C. AMBL-019C was furthermore confirmed to be a strain of Streptomyces sp. in accordance with its taxonomical characteristics. We then lavishly fermented AMBL-019C in YMD + 0.1% Tween80 medium and meanwhile, extracted its metabolites with EtOAc. In the further steps of purification, we used acid-base fractionation combined with antioxidative activity analysis to pick up the neutral phase, an active part of the fraction collected from the crude material. After that, TLC was applied to sample analysis and resulted in obtaining the Rf value of active metabolites. The active part was collected and subsequently subjected to column chromatography ( Hexane：EtOAc 96：4) for further large-scale purification and conducted to gernerate a pale yellow crystal, designed as AMBL-019C-TS (TS). In order to confirm the purity of the target compound, HPLC was further performed and TS was consequently as well as detected at retention time of 7.30~7.70 min with high purity. TS was soluble in methanol and less polar solvents but insoluble in water. The integrate results of mass spectroscopy, Ultraviolet-visible spectroscopy, 1H-NMR, 13C-NMR and 1H-1H 2D NMR to TS showed that it was a benzoic and keto group containing compound with molecule weight of 236. As for antioxidative activity, TS showed an remarkable inhibitory activity against LDL oxidation in a dose-dependent manner over a concentration ranging from 2.0 to 10.0 mM. Since oxidative modification of LDL has proved to play a crucial role in atherosclerosis and coronary artery disease (CAD), our experiments implied the potential of TS in the prevention of CAD, though supplementary animal model has not yet been established. As regards the anti-cellular oxidative activity test of Balb-3T3 cells, TS also showed protective effect to the cells, however, no cytotoxicity was observed. Therefore, further study of TS on the antioxidative activity in cellular and physiological level as well as its mechanism and applications in human will be a research topic with value in the future.

並列關鍵字

Antioxidants ； Actinomyces ； Secondary metabolites ； Free radicals ； Low density lipoproteins

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