A Study Of Biosynthesis Pathway And Induced Mechanism Of Emodin By Fermentation From Aspergillus Ochraceus

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.