Chemical Constituents Of The Broth Of Oyster-Associated Fungus Myceliophthora Sp. ML-1

Nowdays the medical and health system is facing more and more severe challenges,and the status and role of traditional Chinese medicine in modern treatment theory has been re-understood and acquired more higher emphasis.It plays a special function and role in modern intractable diseases with its unique compatibility and efficacy,but the long regeneration cycle of marine Chinese medicinal materials and the endangerment of some precious marine medicinal materials restrict the development of modern marine traditional Chinese medicine.The study found that symbiotic microorganisms from marine medicinal animals and plants have unique advantages.Compared with marine animals and plants,their secondary metabolites are more abundant,and some active constituents found in marine animals and plants are actually produced by the symbiotic microorganisms.Therefore,targeting the symbiotic microorganisms in marine medicinal animals and plants to carry out new drug research will solve the problem of the shortage of marine traditional Chinese medicine resources to a large extent,which has become an important field of modern medicine research and development.Oysters（Ostreae）,also known as oysters,are the most zinc-rich foods.Oysters are rich in chemical constituents such as nucleosides,polysaccharides,polypeptides and so on,which have biological activities such as anti-oxidation and anti-fatigue etc.Therefore,the secondary metabolites of its symbiotic microorganism are also worthy of attention.Myceliophthora sp.ML-1 is a symbiotic fungus isolated from oysters in the sea area of Qingdao.The chemical composition of Myceliophthora sp.ML-1 was studied in this thesis.Firstly,the strain ML-1 was fermented in small amount by using five media（PDB,GPY,SWS,rice,and fungus No.1）.The 1H-NMR spectra of the crude extract of the small amount fermentation product showed that the secondary metabolites in the culture of GPY medium are the most abundant.Therefore,GPY medium was used for large-scale fermentation of ML-1,the fermentation broth was extracted with ethyl acetate three times,and the solvent was removed in vacuo to obtain about 26.5 g of crude extract.The crude extract was separated by silica gel column chromatography to proivde 31 fractions,and then guided by 1H-NMR spectrum and TLC,isolation and purification of target compounds were performed through silica gel column chromatography,Sephadex LH-20 gel column chromatography,and high performance liquid chromatography etc.Seven compounds were isolated from the fermentation broth of Myceliophthora sp.ML-1 finally,and identified as 3-methoxy-5pentadecylphenol（1）,3-methoxy-5-heptadecylphenol（2）,3-methoxy-5nonadecylphenol（3）,3-methoxy-5-heneicosylphenol（4）and uracil（5）,respectively.Among them,compounds 1-4 are 3-alkyl-5-methoxyphenols belong to resorcinols,and this is the first time to obtain resorcinol compounds from the genus Myceliophthora.The structures of compounds 6 and 7 need to be further determined.In order to better understand the potential of Myceliophthora sp.ML-1 to produce secondary metabolites,LC-MS/MS molecular networking was used to analyze and compare the chemical constituents of small amount cultures of five medias in this thesis.The results showed that the GPY medium was more beneficial to produce more abundant chemical substances,which was consistent with the previous 1H-NMR spectra analysis.It was also found that the changes of medium significantly affected the types and quantities of secondary metabolites in the strain.Some node clusters in the molecular network were originated from one or several culture media.Myceliophthora sp.ML-1 can produce secondary metabolites of various structural types,and with the help of "seed" compounds and the search function of the GNPS spectral library,14 categories of 71 compounds（including 3 new dianthraquinone compounds）had been identified from the small amount fermentation of 5 kinds of media.They are resorcinols,emodin anthraquinones,dianthraquinones,isocoumarins,cyclic dipeptides,ergosterols,oleanane-type pentacyclic triterpenes,acyclic diterpenes,indole alkaloids,amphetamine alkaloids,benzoic acid and its derivatives,fatty acids,MO-TMS derivatives,polyalcohols and purine alkaloids.The above-mentioned results provided a theoretical basis for stimulating strains to produce new and/or active ingredients through the optimization of fermentation media.Finally,the LC-MS/MS molecular networking was used to analyze the chemical substances in large-scale fermentation broth of GPY medium.The result showed that in addition to the obtained resorcinols,the large-scale culture of GPY medium was rich in terpenoids,alkaloids and so on,which provided a new idea for the subsequent directional separation of target chemical components.Myceliophthora sp.ML-1 can produce abundant chemical substances,and its secondary metabolites were worthy of further study.The Global Natural Products Social Molecular Networking（GNPS）based on LC-MS/MS can help to identify known substances and discover new compounds quickly,and also plays an important role in microbial secondary metabolomics and compound deduplication,which would promote the research of microbial drug greatly.