Isolation,Synthesis And Activity Determination Of Secondary Metabolites From Aspergillus Tubingensis

ObjectiveFungal strains were screened and isolated from soil,and classified by ecological characteristics and molecular methods to identify strain types.Optimize fungal liquid and solid fermentation conditions;separate and purify secondary metabolites;identify the structure of monomeric compounds;determine the biological activity of monomeric compounds so as to lay the foundation for bio-utilization and development of new sources of green and environmentally friendly drugs;and monomer compounds are synthesized by chemical synthesis method,and natural products are combined with chemical synthesis technology to provide conditions for industrial mass production.MethodThe strain was purified by the plate scribing method at the colony level,Combining morphological methods and molecμLar biological methods to identify strain types and build system utterance.Optimize the fermentation conditions of liquid and solid fermentation;improve the traditional extraction process,obtain a large amount of extracts from the ethanol soaked fermentation product.prepare thin layer chromatography,reversed phase silica gel column chromatography,normal phase silica gel column chromatography,gel column chromatography,preparation Separation and purification of the fermentation extract of Aspergillus tubing by separation method such as silica gel scraper.The isolated monomer compounds were identified by NMRand the monomer compounds were synthesized by chemical synthesis.Antibacterial activity and exploration of the effects of growth,development and reproduction of Musca domestica.Monomeric compounds were synthesized by chemical synthesis method to further explore the antimicrobial activity of monomeric compounds and their effects on the growth,development and reproduction of housefly.Results(1)The morphological characteristics,DNA sequence alignment and phylogenetic tree construction of the isolated strains were analyzed.The DNA sequence of the strain was amplified by PCR,sequenced,and then sequenced by NCBI BLAST gene sequence.The phylogenetic tree was constructed by MEGA software,and the phylogenetic analysis was carried out.Then the morphological characteristics of the strain were consistent with the strain,and the separation was determined.The resulting strain was Aspergillus tubing.(2)Five kinds of monomers of HA,HB,DA,EB and LA were isolated by preparative thin layer chromatography,reversed phase silica gel column chromatography,normal phase silica gel column chromatography,gel column chromatography and silica gel plate scraping.Monomers of 1-(2,4,5-trimethylpiperazinyl)-2,4-hexadienone were further synthesized by chemical synthesis.(3)The activity of 1-(2,4,5-trimethylpiperazine)-2,4-hexadienone monomer compounds was determined.The inhibitory zone of the sample group was compared with that of the control group.The results showed that the LA monomers with concentrations of500mg/m L,250mg/m L and 125mg/m L had no bacteriostasis effect on Escherichia coli,Staphylococcus aureus and Bacillus subtilis.By comparing the development of normal houseflies and those fed with LA compounds of different concentrations,we can see that LA compounds of 1mg/m L,1.5mg/m L,2mg/m L,2.5mg/m L and 3mg/m L had certain inhibitory effects on the growth and development of houseflies,and could prolong the development period of houseflies.Among them,the samples with a concentration of3mg/m L had the greatest impact on the development,growth and fecundity of houseflies.No spawning was found in this group,and the life span of male and female houseflies was shortened by 3 days compared with the control group.and houseflies fed with LA compounds had higher mortality and lower spawning rates ConclusionFive monomeric compounds of HA,HB,DA,EB and LA were isolated from the fermentation metabolites of aspergillus tabin,and their structures were identified as: dimer of naphthopyranone,peroxy ergosteroid and piperazine derivatives.High yield of LA(1-(2,4,5-trimethylpiperazinyl)-2,4 hexadienone)monomers can be obtained by chemical synthesis.The biological activity experiment showed that the compound had no obvious bacteriostatic effect on Escherichia coli,Staphylococcus aureus and Bacillus subtilis at the concentrations of 500mg/m L,250mg/m L and 125mg/m L.The survival rate,life expectancy and fecundity of houseflies at a specific age were calculated by using gender-specific life table software,and the effects of LA monomer compounds on the growth and population growth of houseflies were evaluated.The results showed that the LA houseflies had problems such as growth retardation,reproductive decline,and their population growth was significantly inhibited.