| Searching for new chemical entities with significant biological activity is the main goal of modern drug research and development.The marine environment with high salinity,high permeability,low temperature,low oxygen,low light and little nutrition has created the biodiversity and particularity of marine microorganisms,and its secondary metabolites with novel and complex structure and unique function have great medicinal value.There are a wide variety and large numbers of marine microorganisms.Marine microorganisms also have the advantages of fast growth and reproduction,and easy regulation of metabolism,so they can be cultivated on a large scale to realize industrial production.With the development of new technologies in culture and extraction of marine microorganisms,people are increasingly interested in the natural products from marine microorganisms.Mining new drugs or lead compounds from the secondary metabolites of marine microorganisms has become a promising strategy and development trend in the field of modern medicine.Marine symbiotic microorganisms are an important group of marine microorganisms,which have great potential to produce new biologically active substances.Talaromycetes sp.ML-3 is a symbiotic fungus isolated from oysters in the sea area of Qingdao,and the chemical composition of its fermentation products was studied in this thesis.Firstly,the strain ML-3 was fermented in a small amount with five kinds of media,which were rice,SWS,GPY,PDB and fungus No.1,respectively.Then the strain ML-3 was cultured in large scale with the optimal medium.The fermentation broth was extracted with ethyl acetate and the solvent was removed in vacuo to obtain the crude extract.The crude extract of fermentation broth was preliminarily separated by silica gel column chromatography to obtain various polar fraction.guided by 1H-NMR and TLC,fractions were then isolated and purified by silica gel column chromatography,Sephadex LH-20 gel column chromatography,high performance liquid chromatography and etc.The structures of the compounds were identified by modern spectroscopic techniques such as HR-ESI-MS,1D and 2D NMR spectroscopy and literature comparison.The1H-NMR spectrum and TLC showed that the fungus NO.1 medium was beneficial to product abundant secondary metabolites,which was used for large-scale fermentation of strain ML-3,the fermentation broth was extracted with ethyl acetate and the solvent was removed in vacuo to provide about 44 g of crude extract.Finally,six compounds were isolated and purified from the crude extract of culture of ML-3,and identified as emodin（1）,3-indolethanol（2）,ω-hydroxy-emodin（3）,（22E,24R）-8,14epoxyergosta-4,22-diene-3,6-dione（4）and 4-hydroxy-4-（4-methyl-5-oxocyclohex-3-en-1-yl）pentanoic acid（5）,respectively.Among them,compounds 1 and 3 are anthraquinone compounds,compound 2 is an indole alkaloid,compound 4 is an ergosterol compound,compound 5 is a new sesquiterpene compound that had never been reported yet,the structure of compound 6 needs to be further determined.There are many unexpressed biosynthetic gene clusters in microorganisms under conventional culture conditions,and changing fermentation medium is the simplest and most effective way to activate silenced genes.GNPS molecular networking technology based on LC-MS/MS was used in this thesis to construct the conventional molecular networking and feature-based molecular networking of small amount fermentation products of in five media and the large-scale liquid culture of fungus No.1 medium,respectively,for better understanding the potential of Talaromyces sp.ML-3 to produce new and/or active secondary metabolites.Analysis of the chemical constituents of strain ML-3 was carried out and the results showed that the number of nodes of small amount fermentation products of fungus No.1 medium was the largest,and the nodes are widely distributed,indicating that fungus No.1 medium was indeed beneficial to strain ML-3 to produce rich secondary metabolites,which was consistent with the previous 1H-NMR spectra.Some node clusters were unique to a certain medium,indicating that the changes of medium did have a great impact on the type of secondary metabolism of strain ML-3.The chemical constituents in fermentation products of the strain were identified by using the "seed" node and the search function of the GNPS platform,and a total of 66 chemical constituents with 10 types were identified,including anthraquinones,fatty acids,alkaloids,flavonoids,polyols,polyketones,terpenoids,cyclic peptides,steroids,and benzoic acid derivatives.These results laid a good foundation for finding out the optimal medium for the strain and revealing the secondary metabolite profile.From the chemical constituents in large-scale culture of fungus No.1 medium of strain ML-3 in this thesis and 9 anthraquinones（including 3 dianthraquinones and 6 monoanthraquinones）,3 ergosterols and many other types compounds were identified.The analysis result provided a theoretical basis for subsequent guided separation.Talaromyces sp.ML-3 can produce various structural types of secondary metabolites,among which anthraquinone and dianthraquinone compounds,cyclic peptides and steroids are all potential drug lead compounds.Due to the limited data of the GNPS spectrum database and the small number of "seed" compounds,only a small number of compounds were identified from the fermentation products of ML-3 at present.Subsequently,the identified compounds as well as the secondary metabolites found in the genus Talaromyces reported in the literature will be used as "seeds" in the further identification of chemical constituents in attempt to reveal the secondary metabolite profile of strain ML-3 more completely.GNPS molecular networking can not only be used to compare the differences of metabolites under different fermentation conditions for screening out the best fermentation conditions,but also to realize the deduplication of natural product separation.In the future,the GNPS molecular networking analysis results for the crude extract of large-scale fermentation of fungus No.1 medium of strain ML-3 will be used for directional separation of target compounds. |
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