【作者】 吕平；

【导师】 赵学明；

【作者基本信息】 天津大学 ， 制药工程， 2009， 博士

【摘要】 大黄素（Emodin）,1,3,8-三羟基-6-甲基蒽醌,具有抗病毒、抗炎、抗肿瘤、抑菌、扩张血管、免疫抑制等多种生理功能,已经成为一种十分有前途的新型药物单体。目前大黄素的主要生产方式是从药用植物大黄、虎杖中提取,产量低,质量难以控制。而本实验室保藏的一株赭曲霉（Aspergillus ochraceus LP₀201）在自然状态下便可以产生大黄素,为通过工业发酵的方式来高效地生产大黄素提供了可能。利用制备型HPLC纯化了大黄素单体。制备HPLC的条件为:流动相,甲醇:水（60:40,v/v）;进样量,300mg;柱温,室温;流量,60mL/min;检测波长,254nm。通过ESI-MS、ESI-MS/MS、UV-VIS、FT-IR、1H-NMR、13C-NMR和DEPT确证了大黄素的结构。通过N⁺离子注入获得大黄素高产菌株Aspergillus ochraceus LP₀301,并优化了发酵条件,使其最终产率达1.453mg/L,比出发菌株提高2.96倍。优化后的培养基为:镁离子1.5 g/L,硝酸铵10g/L,蛋白胨10g/L,玉米淀粉75g/L;发酵条件为:温度30℃,转数140 r/min,初始pH为7,装液量80 mL（250 mL）,接种量10%,菌龄20h。研究了大黄素提取和精制方法。提取方式为有机溶剂回流法,提取条件为:料液比,1:12;醇浓度,100%;提取时间,1.5h。精制采用D101大孔树脂,上样流速为1.5mL/min,温度为20℃,上样液中大黄素含量为1.0mg/mL。以中性90%乙醇在50℃下,2.0mL/min的速率洗脱。短链脂肪酸、氨基酸添加和代谢抑制实验表明,大黄素由聚酮途径合成。通过RAPD分析,获得两个差异性扩增片断,一个片断与AY272043.1和AY583208.1同源,证实大黄素通过聚酮途径合成,另一个片断与XM₀02374227.1和AY585205.1同源,表明大黄素合成与氧自由基有关。通过HPLC-ESI-MS/MS对13C标记大黄素动态跟踪及13C在大黄素分子中分布,提出了大黄素合成前体物和延伸的单元均是乙酰辅酶A（或其衍生的二碳单位）,延伸共进行7轮。通过研究大黄素在细胞外对氧自由基的消除作用和测定发酵过程中SOD、CAT酶活,推测大黄素的诱发机制和生理功能可能是抑制体内氧自由基的增加。更多还原

【Abstract】 Emodin （1,3,8-trihydroxy-6-methyl-anthraquinone） which exhibited diverse biological activities including anti-virus, anti-bacteria, anti-cancer, anti-inflammatory and immunosuppressive function become an important chinese herbal monomer. The extraction yield and purity of emodin from chinese medicinal herbs is very low at present. It was found in our lab that emodin could be produced by Aspergillus ochraceus LP₀201 in natural conditions.The preparative liquid chromatography （mobile phase, 60% methanol in water; sample amounts, 300mg; flow rate, 60 mL/min; detection wavelength, 254nm） was employed to isolate and purify emodin from fermentation broth. The structure of emodin was confirmed by ESI-MS、ESI-MS/MS、UV-VIS、FT-IR、1H-NMR、13C-NMR and DEPT.The productivity of emodin was improved by N+ low-energy ion beam irradiation and optimization of medium and fermentation conditions up to 1.453mg/L. The optimized medium was composed of Mg2+ （1.5 g/L）, ammonium nitrate （10g/L）, peptone （10g/L）, corn flour （75 g/L）. The optimized fermentation conditions were temperature, 300C; initial pH, 7; rotation speed, 140 r/min; media volume in flask, 80mL（250 mL）; inoculum rate, 10%; seed age, 20h.The mixed solvent return method was used to extract emodin. The optimum extraction conditions were as follows: material-solution ratio was 1:12; ethanol concentration was 100%; extraction time was 1.5h.A method for purification of emodin using macroporous adsorptive resins D101 was investigated. The optimum absorption conditions of D101were that the absorbing velocity of flow was 1.5mg/min, the temperature of absorbing solution was 20℃and the concentration of emodin in sample was 1.0mg/mL. The optimum elution conditions of D101 were that the concentration of ethanol as elutent was 90%, the eluting velocity of flow was 2.0mL/min and the temperature of elutent was 50℃.Experiments of feeding different amino acids, short fatty acids and metabolic inhibitors were used to confirm that emodin was biosynthesized by ployketide pathways. Two polymorphic amplification segments were found by RAPD analysis. One was homologous with AY272043.1 and AY583208.1, which also indicated emodin was biosynthesized by ployketide pathways. The other was homologous with XM₀02374227.1 and AY585205.1, which indicated the biosynthesis of emodin was related to SOD and oxygen free radicals. The details that two carbon units derived from acetyl coenzyme A were not only precursors of emodin but also elongation units and there were 7 circulations in the process of emodin biosynthesis were discovered based on the dynamic change of 13C labeled emodin and 13C location in emodin molecule, which were investigated by HPLC-ESI-MS/MS.activities of SOD and CAT in the fermentation was analyzed, and the emodin function of scavenging oxygen free radical in vitro was identified. According to the results of above experiments, emodin was speculated to scavenge oxygen free radicals.更多还原