Study On The Chemical Constituents And In Vitro Cytotoxic Activity Of The Fermentation Products Of Endophyte Alternaria Sp.QDFB-2 From Ficus Carica L.

Microorganisms are widely distributed in nature and constitute the largest group of organisms on Earth,accounting for approximately 60%of the total biomass.Studies have shown that microorganisms could produce secondary metabolites with rich chemical structures and significant biological activities,which are important sources of drug lead compounds.Meanwhile,microorganisms also have the characteristics of short growth cycles,strong metabolic abilities,and easily strain breeding,which can substantially increase the yield of target compounds and stimulate the production of new chemical substances by means of gene regulation and optimization of cultivation conditions.Therefore,there is enormous potential to produce novel pharmaceutical lead compounds from microorganisms.Alternaria genus fungus,belonging to the Fungus Imperfecti,is commonly found in nature,which could produce abundant pharmacologically active constituents and is an important microbial resource.Alternaria sp.QDFB-2 is an endophytic fungus isolated from the branches of Ficus carica Linn.and its chemical constituents was investigated in this dissertation.Firstly,GNPS molecular networking technology based on LC-MS/MS was used to analyse the chemical constituents in fermentation products of Alternaria sp.QDFB-2 cultured in GPY medium.The Classical Molecular Networking(MN)showed that there were significant differences in the chemical constituents of the fermentation broth and mycelium.Compared with the crude extract of the fermentation broth,the molecular clusters formed by the crude extract of the mycelium were more abundant,indicating that the mycelium contained secondary metabolites with more varied structures.The matching function of the GNPS platform was used to identify and predict the chemical constituents in the crude extract of the fermentation broth and mycelium quickly.A total of 116 compounds from 11 categories were discovered,including peptides,polyketides,fatty acids,fatty amides,nucleosides,alkaloids,tricycloalternarenes,sterols,polyols,benzoic acid derivatives,phthalate derivatives,etc.Among them,polyketides were mainly derived from the fermentation broth,while fatty acids,nucleosides and ergosterols were mainly derived from the mycelium.Further analysis for the Feature based molecular networking(FBMN)of the fermentation products revealed that the main metabolites in the fermentation broth were cyclic tetrapeptides and tricycloalternarenes,and the main metabolites in the mycelium were fatty acids and cyclic dipeptides.The results preliminarily revealed the secondary metabolite profile of the strain QDFB-2.Considering that some secondary metabolites produced by microorganisms could not secrete to the extracellular environments,resulting in chemical composition differences between mycelium and fermentation broth.Therefore,isolation and purification,and structure determination of the chemical constituents in mycelium of Alternaria sp.QDFB-2 cultured in GPY medium was carried out in this dissertation.A combination of silica gel column chromatography,Sephadex LH-20 gel column chromatography,reversed phase column chromatography and high performance liquid chromatography were used for the isolation and purification of the compounds,and modern spectroscopic techniques such as HR-ESI-MS,1D and 2D NMR spectroscopy and literature comparison were used for identification of the compounds.Eight of compounds were obtained from the crude extract of the mycelium of strain QDFB-2 and identified as alternariol(1),tentoxin(2),dihydrotentoxin(3),cyclo(Leu-N-methyl-Phe)(4),cyclo(Gly-Pro)(5),cyclo(4-hydroxy-Pro-Pro)(6)and uridine(7),respectively.Among them,compound 1 is a polyketide,compounds 2 and 3 are cyclic tetrapeptides,compounds 4-6 are cyclic dipeptides,compound 7 is a nucleoside,and the structure of compound 8 is to be further determined.The MTT method was applied to evaluate the in vitro cytotoxicity of the chemical constituents of the strain QDFB-2 in this dissertation finally.The results showed that compounds 1 and 7 exhibited potent proliferation inhibitory activities against SMMC-7721 cells,with IC50 values of 50.47 and 42.50 μg/mL(195.62 and 174.18 μM),respectively.In addition,compound 1 also showed moderate proliferation inhibitory activity against A549 cells,with an inhibition rate of 52.12%at the concentration of 100 μg/mL.The IC50 values of positive control cisplatin were 3.46 and 5.12 μg/mL(11.53 and 17.06 μM),respectively.Furthermore,it was found that the in vitro cytotoxic activity of the fermentation extracts of strain QDFB-2 varied depending on the culture medium used in experiments.The fermentation broth of rice medium and PD medium inhibited the proliferation of SMMC-7721 cells significantly,with IC50 values of 138.7 and 158.8 μg/mL,respectively.The fermentation broth of rice medium and GPY medium showed moderate proliferation inhibition activity against A549 cells,with inhibitory rates of 44.57%and 46.49%at the concentration of 200 μg/mL,respectively.However,the crude extract of other fermentation products could not inhibit the growth of the two cell lines obviously.The results indicated that the culture medium could affect the second metabolites of strains QDFB-2 obviously.Alternaria sp.QDFB-2 fermentation product contains a rich variety of chemical constituents.Polyketides,cyclics and nucleosides have been isolated from the crude extract of the mycelium,and their chemical compositions are worthy of further study.We will continue to analyse the secondary metabolite profile of QDFB-2 fermentation products by using the GNPS molecular networking technology,using the known secondary metabolites of Alternaria fungus and the compounds produced by strain QDFB-2 as the "seed"compounds.Meanwhile,based on the GNPS Molecular Network,the OSMAC strategy will be used to activate the silent biosynthetic gene clusters of QDFB-2 to explore its potential to generate novel structural molecules,providing new lead compounds for new drug development.