Optimization Of Preparation Process Of Ustiloxin A And Analyisis Of Its Biological Activity

Ustiloxins(Ust)is a cyclopeptide secondary metabolite produced by Villosiclava virens.It has the toxic activity of inhibiting cell mitosis,inhibits seed germination,and hindering grain nutrient transport and normal development at the starting stage.Ust can cause acute poisoning in chicken,rabbit and other small animals,and cause diseases in trachea,heart,liver,lung and kidney.So far,7 species of Ust have been isolated and identified,which are A,B,C,D,E,F and G,of which Ustiloxins A(Ust A)is the main component.In view of the universality and harmfulness of the pollution of Ust A,its pollution risk assessment,toxicology research and control method development have drawn extensive attention from the indUstry,which has a great demand for the pure product of Ust A.So far,there is no feasible synthetic process for Ust A,which severely limits the source of Ust A.In this study,the technique of preparing a large number of Ust A based on macroporous adsorption resin and high speed countercurrent chromatography(HSCCC)was developed by using rice curtilage from infected rice ear as raw material,and the toxicity of Ust A was preliminarily studied.The main results are as follows:(1)The extraction conditions of Ust A in rice curvilinear were optimized,and the optimal condition was 5% formic acid solution at 25?,acidic environment and material-liquid ratio of 1:20.(2)The separation methods of Ust A was optimized.In this study,the separation effects of five macroporous adsorption resins and two ion-exchange resins on Ust A were compared,and it was found that macroporous resin XAD-4 showed the best performance.Through the optimization of adsorption time curve and eluent of macroporous resin XAD-4,it is found that the performance of macroporous resin XAD-4 can reach the best when the crude extract is adsorption for 7 hours,and the concentration of methanol solution is 40% as the eluent,and the separation amount reaches 296.2 9g/g.(3)Preparation chromatography(Prep-HPLC)and HSCCC were compared to purify Ust A.The recovery and purity of Ust A purified by Prep-HPLC were 76.2% and 80%,respectively.The recovery and purity of Ust A purified by HSCCC was 88% and 95%,respectively.Moreover,Prep-HPLC,HSCCC can produce more than 200 mg of Ust A at a time.HSCCC used n-butanol /5% TFA aqueous solution(1:1)as a two-phase system,flow rate of 1 mL/min,rotation speed of 1000 r/min,column temperature of 30?.(4)The plant toxicity of Ust A was evaluated.The results showed that Ust A significantly inhibited the germination of wheat and soybean seeds.