Optimization Of The Culture Conditions Of Endophytic Fungus Aspergillus Wentii En-48 For The Anti-cancer Agents Tetranorditerpenoids And The Secondaty Metabolites Research On The Marine-derived Fungus Penicillium Simplicissimum

Due to the various structures and potent pharmaceutical activities,microbial natural products play an important role in human fighting diseases and protecting health.The ocean is the mother of life.Living in the high pressure,high salinity,low temperature,less light and oligotrophic marine environment,marine fungi have evolved new genetic and metabolic mechanism and able to produce novel secondary metabolites with potent anti-tumor,anti-microbial,antiviral and antibiotic bioactivities,which has attracted more and more attention.Recently,our group obtained several new tetranorlabdane diterpenoids with potent anti-cancer activities from a marine alga-derived endophytic fungus A.wentii EN-48.However,the yields of tetranorlabdane diterpenoids in A.wentii were relatively low under static cultures,which limited further investigations.In order to enhance the production of the anti-tumor agents tetranorlabdane diterpenoids,the fermentation conditions of A.wentii EN-48 were optimized.In this dissertation,the fungus was identified through systematic identification,and standard curves of wentilactone A,wentilactone B and asperolide A were established.Different media,medium volume,salinity,initial p H,culture time,temperature,and light conditions were tested by singal-factor experiment.And according to the results of single-factor experiments,effects of three factors(salinity,initial p H,and culture time)were chosen for further optimization employing response surface methodology(RSM)based on Box-Behnken design.Under the optimized culture condition,the yields of wentilactone A,wentilactone B and asperolide A were 13.4,6.5 and 4.5 mg/L,respectively,and 12-,13.5-and 14-fold higher than before.From the cultures of A.wentii EN-48,14 compounds were isolated and identified by repeated column chromatography on silica gel,RP-18,Sephadex LH-20,preparative thin layer chromatography and preparative high liquid chromatography as well as by recrystallization.Compounds AM1~AM5 showed different DPPH radical-scavenging activitives,but no brine shrimp lethality activity.In this dissertation,57 marine-derived fungi were obtained and screened under different medium for further investigation.The fermentation extracts were evaluated on the basis of TLC,HPLC and NMR analysis.As a result,Penicillium simplicissimum MA-332,a marine fungus derived from the rhizosphere of the mangrove Bruguiera sexangula var.Rhynchopetala,were selected for further chemical investigation.From the cultures of Penicillium simplicissimum MA-332,9 compounds were isolated and identified,including three new dihydroisocoumarin derivatives(PS1*~PS3*),one new sesquiterpene(PS4*)and a new natural products diphenyl ether(PS5).The isolated compounds were screened for antimicrobial,and DPPH radical-scavenging activity,and PS1*~PS3* exhibited broad-spectrum activities against human and aquatic pathogenic bacteria and plant pathogenic fungi.PS1 exhibited highest activities against E.coli,P.aeruginosa,V.parahaemolyticus,V.harveyi,and C.gloeosprioides,each with MIC value of 4 μg/mL.