微生物學(xué)報(bào)Acta Microbiologica Sinica
48(8)：1035~1041; 4 August 2008
基金項(xiàng)目: 國家自然科學(xué)基金項(xiàng)目(30570189); 國家“863 計(jì)劃” (2007AA021506); 安徽高校自然科學(xué)研究重點(diǎn)項(xiàng)目(DT 200708)
*通訊作者。[email protected]; [email protected]
作者簡介: 胡豐林(1965. ), 男, 安徽銅陵人, 教授, 博士, 研究方向?yàn)橄x生真菌代謝物,。 E-mail: [email protected]
胡豐林，陸瑞利,，黃勃，李增智*
(安徽省微生物防治重點(diǎn)實(shí)驗(yàn)室, 安徽農(nóng)業(yè)大學(xué), 合肥 230036)
摘要：【目的】在一次對蟲生真菌代謝物進(jìn)行大規(guī)模的清除自由基活性物質(zhì)篩選中,，發(fā)現(xiàn)一種被毛孢（Hirsutella sp.）菌株RCEF0881 發(fā)酵液中存在有較強(qiáng)的清除自由基活性物質(zhì),。本研究目的是初步搞清這些活性成分的具體組成，并制備出一定量的純品用于進(jìn)一步的結(jié)構(gòu)鑒定,?！痉椒ā坑糜袡C(jī)溶劑法提取活性成分,；用二苯基苦基苯肼自由基（DPPH）酶標(biāo)儀法和薄層色譜法進(jìn)行活性測定；用高分辨液質(zhì)聯(lián)用方法進(jìn)行活性成分初步分析和鑒定,；用反相制備色譜法制備活性組分?！窘Y(jié)果】提取實(shí)驗(yàn)結(jié)果表明具清除自由基活性的物質(zhì)能較好地被乙酸乙酯提取出來,；液相色譜-質(zhì)譜-活性測定分析表明提取物中活性組分的可能分子式分別為C7H6O4、C8H8O3 和C12H14N2O,。結(jié)合色譜特性,、紫外光譜特征、質(zhì)譜碎片和數(shù)據(jù)庫查詢可初步推斷它們分別為二羥基苯甲酸,、羥基甲基苯甲酸和生物堿類物質(zhì),，但具體結(jié)構(gòu)還有待于進(jìn)一步確認(rèn)。從高效液相色譜和質(zhì)譜離子流的峰面積可知上述3 種活性物質(zhì)中C12H14N2O 的含量最高,。本研究成功地用反相制備色譜制備出該天然活性組分的純品,。該3 種清除自由基活性物質(zhì)都是首次發(fā)現(xiàn)存在于蟲生真菌的代謝物中。
關(guān)鍵詞：一種被毛孢（Hirsutella sp.）,；自由基清除劑,；分析；制備,；二苯基苦基苯肼自由基 （DPPH）
中圖分類號：Q939.93 文獻(xiàn)標(biāo)識碼：A 文章編號：0001-6209 (2008) 08-1035-07
Analysis and isolation of radical scavengers from metabolites of Hirsutella sp.
Fenglin Hu, Ruili Lu, Bo Huang, Zengzhi Li*
(Research Center on Entomogenous Fungi, Anhui Agricultural University, Hefei 20036, China)
Abstract: [Objective] During a screening for free radical scavengers from metabolites of entomogenous fungi, we fond a fermentation broth of the strain RCEF 0881 of Hirsutella sp. exhibited strong radical scavenging activity. To make clear of the constituents of the active compounds, and prepare some pure active compound for further structure identification we
launched this study. [Methods] We used organic solvent for active compounds extraction. DPPH-TLC and DPPH- Microplate assay were used for activity analysis. Components analysis was carried out on a HPLC-DAD-HRMS, and bioactive compound preparation on a preparative RP-HPLC. [Results] Our extraction tests showed that ethylacetate was the best solvent for the bioactive constituents extracting. HPLC-DAD-HRMS-DPPH assay revealed that the molecular formular of the radical scavengers existed in the extract were possibly C7H6O4, C8H8O3 and C12H14N2O. From the chromatographic and Uv properties, and the MS fragments, and database consulting, the compounds could be deduced as dihydroxybenzoic acid, methyl-hydroxyl benzoic acid, and an alkaloid, however, the structures are still needed to be confirmed.
The pick area of HPLC and MS showed that the compound C12H14N2O was the main component of the extract. It was isolated via activity directed fractionation. The activity of the prepared compound was confirmed with DPPH-TLC assay and its purity was confirmed with HPLC-DAD-HR-ESIMS. The occurance of the th ree active compounds in entomogenous fungi was revealed for the first time.
Keywords: Hirsutella sp.; free radical scavengers; analysis; preparation; 1,1-Diphenyl-2- picryhydrazyl hydrate (DPPH)
生物谷推薦全文下載：一種被毛孢(Hirsutella sp.)培養(yǎng)液中自由基清除劑的分析,、分離和制備
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