Isolation And Structure Identification Of Secondary Metabolite From Hypoxylon Investiens NX9 And Optimization Of Fermentation Medium And Process

The bioactive ingredients and pharmacological value of the endophytic fungi Hypoxylon investiens NX9,which was isolated from Phlegmariurus phlegmaria,were explored in this paper.The experimental results are listed as follows:1.Selection of fermentation mode for Hypoxylon NX9:Hypoxylon NX9 was cultured by the two ways of liquid and solid fermentation with the volume of 2 L for 10 days.Then the fermented product was extracted with ethyl acetate and afforded two crude extracts of 90.0 mg/L and 74.0 mg/L respectively.The inhibition rate of liquid extracts(15.80%)showed higher acetylcholinesterase inhibitory activity than that of solid extracts(10.82%).Combined with the results of two kinds of crude extracts by thin layer chromatography,liquid fermentation was selected for bioactive compounds screening from Hypoxylon NX9.2.The optimization of fermentation medium of Hypoxylon NX9:Fermentation optimization was conducted by comparing acetylcholinesterase inhibitory activitiy of crude extract.The optimum fermentation medium were found to be:potato 20%;glucose 8%;mannitol 8%;ammonium sulfate 1%;potassium dihydrogen phosphate 0.05%;calcium chloride 0.05%and sodium chloride 0.05%.In addition,other desirability parameters were confirmed to 1/3 volume of medium loading and 10 days of duration.Under the condition,about 0.071g crude extract was obtained and the inhibition rate on acetylcholinesterase was improved to 34.19%.Plackett-Burman design was further introduced to optimize the three significant factors including approache.The optimium value of shaking speed,pH and temperature were found to be 193.63 rpm,7.53 and 28.96? respectively.The inhibitory rate of crude extract obtained in this condition could reach to 48.64%.3.Purification and identification of compounds from Hypoxylon investiensNX9:Hypoxylon investiens NX9 was cultured by liquid fementation in the abve optimi zed fermentation conditions with the total volume of 20L.A total of 4.448g of cr ude organic extract was obtained.Nine compounds were isolated from the crude e xtract through repeated column chromatography over RP-18,Sephadex LH-20 and silica-gel.Eight compounds ZHO1?ZHO2?ZHO3a?ZHO3b?ZHOB2?ZHOC5?ZHOA4 and ZHO10 were identified as 4,6-dihydroxy-atetralone?3,4-dihydro-3,6,8-trihydroxy-3-methyl-Naphthalenone?4,6,8-trihydroxy-3,4-dihydronaphthalen?Scytalo ne?5-Methoxy-1-tetralone?4,5-dihydroxy-3,4-dihydro-1-naphthalenone?3,4-dihydro-3,4,5-trihydroxynaphthalen and 3,4-dihydro-3,4,8-trihydroxy-l-naphthalenone respecti vely.These compounds were naphthalene derivatives.Compound 9 was sesquiterpe noid and identified as 1?,4a,5?,7?(H)-guaiane-10?,11,12-triol4.Biological activities of nine isolated compounds:5mg/mL compound ZHOB2 was found to possess the inhibitory activity on acetylcholinesterase with the inhibition rate of 52.55%and the IC50 value of 5.23mg/mL.Compound ZHOA4 showed the inhibitory activity on alpha glucosidase with the inhibition rate of 30.86%.The experimental results displayed that compound ZHO2?ZHO3?ZHOC5 and ZHOA4 possess the antioxidant activities by the method of TLC-bio-autography.In conclusion,the experimental results displayed that the main secondary met abolites of Hypoxylon investiens NX9 were naphthalene derivatives.Among the is olated compounds,ZHOB2(5-Methoxy-l-teralone)exhibits strong inhibitoryactivity on acetylcholinesterase.The study will lay a foundation for the development of pharmacological value of Hypoxylon investiens NX9.