Studies On Secondary Metabolites And Their Activities Of Endophytic Fungi From Neriumindicum Mill.cv Paihua

Endophytic fungi of medicinal plants are abundant in resources,which are natural resources for obtaining medicinal chemical components.Almost all the known medicinal ingredients can be obtained from endophytic fungi.Taking endophytic fungi of medicinal plants as resources,in-depth study on their active secondary metabolites is of great significance for screening new drugs and developing new medicinal resources.Studies on endogenous fungi and active secondary metabolites of Neriumindicum mill.cv Paihua have rarely been reported.Therefore,this thesis systematically studied endophytic fungi and their active secondary metabolites by bioactivity-oriented separation technology.The main contents include:isolation and identification of endophytic fungi,screening of active strains,identification of active strains and investigation of medium conditions,isolation of active metabolites,structural analysis of monomer compounds,evaluation of antibacterial,antioxidant,antitumor and hypoglycemic activities of monomer compounds.First of all,35 endophytic fungi were isolated from Neriumindicum mill.cv Paihua,which belonged to 4 orders,5 families and 9 genera respectively.Among them,8 strains were Aspergillus sp.,accounting for 22.86%.6 strains were of the Aternana sp.,accounting for 17.14%.5 strains were Trichoderma sp.,accounting for 14.29%.Five strains were Alternaria sp.?14.29%?.4 strains were Penicillium sp.,accounting for 11.43%.The two strains were Botrytis sp.?5.71%?.One strain was Penicilliopsis sp..One strain was of the Oidium sp.and Aspergillus sp.and Aternana sp.were the dominant strains.35 endophytic fungi were screened with antibacterial activity and extract yield.Three strains with strong antibacterial activity were identified as S1,R3 and R13 by rescreening the antibacterial activity of the metabolites.Secondly,the active strains S1,R3 and R13 were analyzed by ITS sequence combined with morphological identification.S1 was identified as Alternaria papavericola strain CBS116606?Genbank accession No.KC584579.1?,with 98%homology.R3 was identified as Aspergillus fumigatus Yu3-7?Genbank accession No.MG827158.1?,with 100%homologous.R13 was identified as Aspergillus strain 112822?Genbank accession No.EU583496.1?,with 99%homologous.Condition of culture medium and fermentation time was investigated,under28?,the culture condition of S1 is rice solid culture medium with 10%NaCl,pH of 7,fermentation time 20 days;The optimization medium for R3 was rice solid medium with 5%NaCl,pH of 9,fermentation time 17 days.The medium condition of R13 was rice solid medium with 10%NaCl and pH 11,fermentation time 22 days.Thirdly,The fermentation of strain S1,R3 and R13 was separated and purified by various chromatography and recrystallization,and 80 monomer compounds were obtained.The structure of 70 compounds were determined by various spectral techniques.1-hydroxy-10-methyl-sinomenine?70?is a new compound,belongs to morphine alkaloids.Compound methyl 3,5-dihydroxy-2-?2-hydroxy-6-methoxy-4-methylbenzoyl?benzoate?69?was isolated from the natural world for the first time.Fourthly,the antibacterial,antitumor,antioxidant and antidiabetic activities of monomer compounds were screened.?1?The antibacterial experiments showed that there were 26 compounds with good antibacterial effect,mainly alkaloids,organic acids,dibenzopyranones and so on.Among them,AME?Alternariol-5-methyl ether?has a strong inhibitory effect on 4 kinds of bacteria and 9 kinds of plant pathogens,especially on Cytospora sp.,Peony anthracnose,Botrytis cinerea,Pyricularia oryzae Cav.,Phytophthora capsici and Botrytis cinerea than carbendazim.AOH?Alternariol?had a strong inhibitory effect on 4 kinds of bacteria and 4 kinds of plant pathogens?Cytospora sp.,Fusarium oxysporum f.sp.niveum,Botrytis cinerea and Phytophthora capsici?.?2?MTT was used to preliminarily evaluate the antitumor activity of 8active monomer compounds in human prostate cancer cell PC3,human lung cancer cell A549 and human breast cancer cell McF-7.The results showed that Ergosterol peroxide and Fumitremorgin B had better inhibitory effects on human lung cancer cells A549,with IC₅₀ values of 14.3 mol/L and 19.8 mol/L,respectively.AOH showed antitumor activity against human prostate cancer cell PC3,human lung cancer cell A549 and human breast cancer cell McF-7,with IC₅₀0 values of 28.6 mol/L,20.3 mol/L and 47.04 mol/L,respectively.?3?Antioxidant experiment found that the sample order of DPPH radical scavenging abilities:AOH>Demethoxy-fumitremorgin C>Ergosterol peroxide>Fumitremorgin B>Alternariol-5-O-?-D-glycoside>AME>Helvolic acid>Monomethyl ochratoxin.The 8 compounds have different degrees of antioxidant activity.?4?Antidiabetic activity studies have shown that AOH and AME have good hypoglycemic activity.The inhibition of?-amylase and?-glucosidase was obvious in vitro.The experiments on STZ diabetic mice showed that AME and AOH could significantly increase the body weight and reduce the blood glucose of STZ diabetic mice.They can significantly reduce the damage of the pancreas,kidneys,testicles and liver of mice with STZ diabetes.In this dissertation,the biological activities of endophytic fungi,secondary metabolites and monomer compounds in Neriumindicum mill.cv Paihua.were systematically studied.The results laid a foundation for the study of endophytic fungi in Neriumindicum mill.cv Paihua..It has enriched the types of morphine alkaloids and diphenyl ketones,broadened the structural types of the secondary metabolites of endophytic fungi,and provided a basis for the development of new antibacterial agents and antidiabetes drugs.