索 及其抗氧化活性評估 土壤分離菌 Streptomyces lavandulae SY-815 所生產天然抗氧化物之探

酸敗油脂的攝取以及生物體內因氧化反應、紫外線或放射線照射等 現象所產生的氧化 自由基 (Oxidizing free radicals) 或過氧化脂質 (Lipid peroxides) ，是造成細胞損傷 、組織老化甚至生物體內各種病 變的主因之一；這樣的過氧化傷害機制，在生物體內雖可 藉由外來或內 在的各種抗氧化防禦機制予以減輕或防止，但有時並不能完全有效的對抗 多 種氧化自由基及過氧化脂質所造成的傷害，因此抗氧化物

(Antioxidants) 的適時補充或 添加有其必要性。然而目前泛用的抗氧 化物多屬人工合成，對人體的安全性堪慮，因此如 何從天然資源中找尋 更有效的安全性抗氧化物，以進一步評估其抗氧化活性及作用機制即 更 顯重要且深具開發潛力的。在過去植物起源的天然抗氧化物被報導頗多，

然深究其抗氧 化活性並不顯著，且大部份有效成份的分子結構較為特定

，因此發展範圍有限。微生物是 天然物重要的資源，從現代生物技術的 角度觀之，從微生物代謝產物中篩選具抗氧化活性 的天然物，應是生物 醫學範疇中值得探討的重要課題。 因此針對上述目的，我們首

先利用選擇性菌種分離方法 (Selective isolation method) 從所採集 的 56 個土壤試樣中分離出包括絲狀真菌 (Filamentous fungi) 和放線 菌 (Actinomycetes) 等近 500 株特定高好氣性微生物，並分別予以分類整 理及保存。在菌 種發酵培養方面，我們採用批式液態發酵方式進行往復 式振盪培養以刺激二次代謝物的生 產 ; 所得的發酵培養上清菌液再藉由 吸附性樹脂 Amberlite XAD-2 吸附、乙酸乙酯 (Ethyl acetate) 及正 丁醇 (n-Butanol) 等不同極性的有機溶劑萃取轉溶，並以甲醇

(Methanol) 振盪溶離及減壓濃縮脫水方式，以取得抗氧化活性篩選的檢 測試樣。經篩選 結果，我們從近 300 株放線菌中，篩得一具有抗氧化物 質高生產能力的菌株，命名為 SY-815 。從其生理生化特徵及形態觀測等 菌種鑑定的結果，確認該菌株為鏈黴菌中的 Streptomyces lavandulae

。接著針對該菌種代謝產物中的兩個主要抗氧化活性成份 (SY-815A 及 SY-815B) ，利用順相 (Normal phase, 矽膠 ) 及膠體過濾層析 (Gel filtration, Sephadex LH-20) 等方法取得純化物，更進一步利用元素分 析、質譜分析 (MS) 和核磁共振 (1H 及 13C-NMR) 等有機光譜的分析，

確定其構造分別為烯醇式 b －苯丙 酮酸 (Enol form b － phenylpyruvic acid) 與 2,5 －二羥苯乙酸 (2,5-Dihydroxybenzeneacetic acid ；

Homogentisic acid) 。 在抗氧化活性評估方面，我們針對幾

種不同的抗氧化機制進行了探討，即： 1. 對超 氧自由基 (Superoxide

土壤分離菌 Streptomyces lavandulae SY-815 所 生產天然抗氧化物之探 索及其抗氧化活性評估

anion radical O2- ) ﹐ ‧ 的清除作用：以 I.N.T. 系統 (2-[4- iodophenyl]-3-[4-nitrophenol]-5-phenyltetrazolium chloride

System) ； 2. 對 氫氧自由基 (Hydroxy radical OH ) ﹐ ‧ 的清除作用 : 以去氧五碳醣系統（ Deoxyribose system ） ; 以及 3. 對抑制過氧化脂質生 成的影響：包括 (1)β- 胡蘿蔔素系統 (β-Carotene system) ； (2) 紅血

球細胞膜系統 (RBC ghos t membrane system) ； (3) 老鼠肝微粒系統 (Rat liver microsome system) ； (4) 固態硫丙二醯尿系統 (Solid TBA system) 等。測 試結果證明， enol form β-phenylpyruvic aid 與

homogentisic acid 均可有效抑制脂質 過氧化反應的進行，減少脂質過 氧化所造成的傷害，尤其在紅血球細胞膜系統與固態硫丙 二醯尿系統中

，其效果均較現在常用的天然抗氧化物維生素 E (a-tocopherol) 更為顯著

； 其中 Homogentisic acid 同時亦具有類超氧化物歧化  (Superoxide dismutase mimicry ； SOD － Like) 的功能，可清除由黃嘌呤

(Xanthine) 與黃嘌呤氧化 (Xanthine oxidase) 反應後所產生的超氧自

由基，當其檢測濃度為 0.005mg/ml 時，可發揮相當於 SOD 1.731U/ml 的

活性。此外，我們亦選擇了肝癌細胞株 (Hepatoma A22T) 為模式，證實

這 些天然抗氧化 物具有對氫氧自由基 (Hydroxyl radical ， OH ) ‧ 所

造成細胞毒性的減緩 效果。

Discovery and activity evaluation of natural antioxid ants producted by the soil-born isolate of Streptom

yces lavandulae SY-815



Free radicals and lipid peroxides have been implicated as the causative factors in cell injury, aging processes and the pathogenesis of numerous diseases. These reactive oxygen species (ROS) can be generated in most biological systems by lipid rancidity, ultraviolet rays or radioirradiation.

Although such kinds of oxidative lesions can be prevented or reduced by the endogenous or exogenous defending mechanisms of oxidation, it is almost important to obtain sufficient

quantity of antioxidants through the diet in order to prevent the deleterious effects exerted by ROS. To meet this requirements, it is important to search for more potent and reliable antioxidants from environmental sources. Natural antioxidants of microbial origin have recently received considerable attention from biotechnological industry not only because of the concerns over the safety of synthetic

antioxidants, but of the low effectiveness and limited species in some plant-derived antioxidants. Microbial metabolites represent rich sources of useful biologically active

compounds. In the search for new antioxidants with commercial value, microbial products screening against specific oxidative targets is going to play an increasingly important role in the future. For the purpose mentioned above, firstly we collected 56 soil samples from different areas as the source for isolating of microorganisms. Then we used selective isolation method to isolate a total of more than 500 strains of different types of highly aerobic microorganisms, mainly filamentous fungi and bacteria of the actinomycete group, which were subjected to the preliminary classification and short time storage. The isolated strains were fermented in small scales (15-ml culture medium in a 50-ml L-type tube) and the crude extracts, which were prepared by Amberlite XAD-2

absorption, MeOH elution and EtOAc-BuOH fractionation from the fermentation broths, were used as test samples for studying antioxidant activity. According to the established screening methods,we screened nearly 300 strains of actinomycetes and

 chose out a strain of actinomycetes, designed SY-815,. which satisfied our screening criteria. From the taxonomical

characteristics of this strain, we concluded that it belongs to Streptomyces lavandulae. For the detection of its

fermentation profile, we checked the desired antioxdiants with TLC analysis in combination of bioautography. Two of the major antioxidant components (SY-815A and SY-815B) were found in the fermentation supernatant of the strain. The purification of

these two compounds was carried out mainly through a series of LC fractionation columns which included silica gel (normal phase) and Sephadex LH-20 (gel filtration) separation

techniques. Elemental analysis and MS, 1H and 13C-NMR spectral investigations confirmed the structures of SY-815A as enol

form of b-phenylpyruvic acid and SY-815B as homogentisic acid.

The antioxidant activities of these microbe-derived compounds, SY-815A and SY-815B, were then evaluated with various mechanism-based assay systems. They were: 1. Scavenge of superoxide anion radical (O2- ); 2. Inhibition of lipid ‧ peroxidation in rat liver microsome; 3. Inhibition of lipid peroxidation in erythrocyte ghost membrane; 4. Suppression of OH -induced cytotoxicity on HepA22T cells. The results showed ‧ that both SY-815A and B exhibited superior or comparable

inhibitory activity to three known antioxidants, BHA, BHT and α--tocopherol against lipid peroxidation in rat liver

microsomes as well as in erythrocyte ghost membranes at

concentrations of several mM, respectively. In the cell culture

system (HepA22T cells), two tested metabolites also showed

comparable activity to that of α--tocopherol (or trolox) on the

suppression of OH -induced cytotoxicity in vitro. ‧