Study On The Secondary Metabolomics And Chemical Constituents Of Endophyte Alternaria Sp. QDFB-2 From Ficus Carica L.

With a vast territory and diverse climates,China is endowed with rich medicinal plant resources.However,the development of traditional Chinese medicine is seriously constrained by the long growth cycle of medicinal plants and especially the limited resources of endangered,rare and wild medicinal plants.Since Stierle et al isolated an endophytic fungus that could produce taxol from Taxus brevifolia in 1993,there has been an upsurge of searching for medicinal active substances from endophyte around the world.As an alternative resource,endophyte has become an important resource treasury for new drug research and development.Ficus carica Linn,a kind of medicine and food homologous plant,is rich in chemical constituents,including carotenes,peptides,organic acids,alkaloids and so on,which has rich medicinal and nutritional values.Based on the theory of endo-symbiosis,the endophyte of Ficus carica Linn is worthy of further research.Alternaria sp.QDFB-2 is an endophytic fungus isolated from the branches of Ficus carica Linn.and its fermentation condition and secondary metabolites were studied in this thesis.Firstly,considering the possible gene silencing phenomenon during fermentation,strain QDFB-2 was cultured in GPY,PD and Rice mediums respectively.Then,GNPS Molecular networking technology based on LC-MS/MS was used to construct the Classical molecular networking(MN)and Feature based molecular networking(FBMN)of the crude extracts from these three liquid cultures,for analyzing and comparing the chemical constituents in the fermentation products.The co-constructed MN and separately constructed FBMN of the three mediums fermentation products showed that secondary metabolites of strain QDFB-2 from different mediums were significantly different.GPY medium fermentation mainly produced tricycloalternarenes(TCA)and peptides,PD medium fermentation mainly produced TCAs,polyketide and peptides and Rice medium fermentation mainly produced polyketide and peptides compounds,and polyketide compounds were the main component.Furthermore,through the matching function of GNPS spectrum library,a total of 99 compounds in 8 classes were identified from the cultures of strain QDFB-2,including fatty acids,alkaloids,phthalates,polyols,polyketones,TCAs,peptides,steroids and so on.This result revealed the secondary metabolites profile of strain QDFB-2 preliminarily.It was also found that the culture medium had a significant impact on the types and production of metabolites,indicating that the medium optimization could purposefully promote the production of target compounds,improve the yield of target compounds,and discover novel structural molecules.Isolation and purification and structure identification of the chemical constituents in the fermentation broth of Alternaria sp.QDFB-2 cultured in GPY medium was further carried out in this thesis.The chemical constituents were isolated and purified by silica gel column chromatography,reversed phase column Chromatography,Sephadex LH-20 gel column chromatography and high performance liquid chromatography.Modern spectral techniques such as HR-ESI-MS,NMR spectra and literature comparison were used to determine the structure of the compounds.Ten compounds were obtained from the fermentation broth of strain QDFB-2,including five polyketides alternariol(1),altenuisol(2),stemphyperylenol(3),altertoxin I(4)and 5’-methoxy-6-methyl-biphenyl-3,4,3’-triol(10),two cyclic tetrapeptide compounds tentoxin(5)and dihydrotentoxin(6),three tricycloalternarene(TCA)compounds 6-iso-tricycloalternarene 6a(7),tricycloalternarene 6a(8)and tricycloalternarene 6b(9).Alternaria sp.QDFB-2 could produce a variety of structural types of secondary metabolites.Polyketones,tricycloaltemarene,cyclopeptides and other compounds isolated from Alternaria sp.QDFB-2 are highly potential drug lead compounds.In addition,the molecular networking analysis showed that there may be many new compounds in the fermentation broth of GPY culture medium of QDFB-2.The obtained compounds will be used as "seed" compounds to track the distribution of their derivatives in fermentation products quickly,for improving the efficiency in identifying and predicting chemical substances in fermentation products and revelling the secondary metabolites profile of QDFB-2 better.Moreover,OSMAC strategy will be continued to use to optimize the fermentation conditions of strain QDFB-2,in order to find more natural products with significant activity,novel structure and providing lead compounds for new drug research and development.