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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

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.

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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

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  1. 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 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 (Gelfiltration, 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－phenylpyruvicacid) 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

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

  3. Discovery and activity evaluation of natural antioxidants producted by the soil-born isolate of Streptomyces lavandulae SY-815 • Free radicals and lipid peroxides have been implicated asthe causative factors in cell injury, aging processes and thepathogenesis of numerous diseases. These reactive oxygenspecies (ROS) can be generated in most biological systems bylipid rancidity, ultraviolet rays or radioirradiation.Although such kinds of oxidative lesions can be prevented orreduced by the endogenous or exogenous defending mechanisms ofoxidation, it is almost important to obtain sufficientquantity of antioxidants through the diet in order to preventthe deleterious effects exerted by ROS. To meet thisrequirements, it is important to search for more potent andreliable antioxidants from environmental sources. Naturalantioxidants of microbial origin have recently receivedconsiderable attention from biotechnological industry not onlybecause of the concerns over the safety of syntheticantioxidants, but of the low effectiveness and limited speciesin some plant-derived antioxidants. Microbial metabolitesrepresent rich sources of useful biologically activecompounds. In the search for new antioxidants with commercialvalue, microbial products screening against specific oxidativetargets is going to play an increasingly important role in thefuture. For the purpose mentioned above, firstly wecollected 56 soil samples from different areas as the sourcefor isolating of microorganisms. Then we used selectiveisolation method to isolate a total of more than 500 strains ofdifferent types of highly aerobic microorganisms, mainlyfilamentous fungi and bacteria of the actinomycete group, whichwere subjected to the preliminary classification and shorttime storage. The isolated strains were fermented in smallscales (15-ml culture medium in a 50-ml L-type tube) and thecrude extracts, which were prepared by Amberlite XAD-2absorption, MeOH elution and EtOAc-BuOH fractionation from thefermentation broths, were used as test samples for studyingantioxidant activity. According to the established screeningmethods,we screened nearly 300 strains of actinomycetes and

  4. chose out a strain of actinomycetes, designed SY-815,. whichsatisfied our screening criteria. From the taxonomicalcharacteristics of this strain, we concluded that it belongsto Streptomyces lavandulae. For the detection of itsfermentation profile, we checked the desired antioxdiants withTLC analysis in combination of bioautography. Two of the majorantioxidant components (SY-815A and SY-815B) were found in thefermentation supernatant of the strain. The purification ofthese two compounds was carried out mainly through a series ofLC fractionation columns which included silica gel (normalphase) and Sephadex LH-20 (gel filtration) separationtechniques. Elemental analysis and MS, 1H and 13C-NMR spectralinvestigations confirmed the structures of SY-815A as enolform 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 variousmechanism-based assay systems. They were: 1. Scavenge ofsuperoxide anion radical (O2-‧); 2. Inhibition of lipidperoxidation in rat liver microsome; 3. Inhibition of lipidperoxidation in erythrocyte ghost membrane; 4. Suppression ofOH‧-induced cytotoxicity on HepA22T cells. The results showedthat both SY-815A and B exhibited superior or comparableinhibitory activity to three known antioxidants, BHA, BHT andα--tocopherol against lipid peroxidation in rat livermicrosomes as well as in erythrocyte ghost membranes atconcentrations of several mM, respectively. In the cell culturesystem (HepA22T cells), two tested metabolites also showedcomparable activity to that of α--tocopherol (or trolox) on thesuppression of OH‧-induced cytotoxicity in vitro.

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