Study On The Secondary Metabolites From Myrothecium Roridum IFB-E091 Based On Molecular Networking

Natural products from microorganism could provide lead compounds with novel structure and distinct biological activity,which have more druggable potential than chemical synthetic drugs.Microorganisms in special eco-environment refer to extreme environment microorganisms,marine microorganisms,microorganisms in internal environment of animals and plants,etc.Secondary metabolites produced by them have varied structure and multi-bioactivity.It is one of the important researcg fields on microbial natural medicines to excavate bioactive substances from endophyte in medicinal plants.Myrothecium roridum IFB-E091 is an endophytic fungus isolated from the root of Artemisia annua L.and the chemical constituents of its solid culture was studied in this thesis.Peptide compounds in the crude extract of solid culture were isolated and purified through silica gel column chromatography and high performance liquid chromatography firstly.Planar structures of obtained compounds were elucidated by high-resolution electrospray mass spectrometry(HR-ESI-MS/MS),1D and 2D nuclear magnetic resonance spectroscopy(NMR)and MALDI-TOF mass spectrometry then.Furthermore,Marfey’s method was used to determine the steric configuration of peptides.Four cyclic peptides MR-1～MR-4 were obtained from Fr.5-3 of the crude extract and identified as cyclo(NMe-Thr-Ile-NMe-Ala-Gly1-Phe-NMe-Ser-Leul-NMe-Phe-Gly2-Leu2)(MR-1),cyclo(NMe-Thr-Ile-NMe-Ala-Gly1-Phe-NMe-Ser-Leu1-NMe-Phe-Gly2-Leu2)(MR-2),cyclo(NMe-Thr-Val-NMe-Ala-Gly1-Leu1-NMe-Ser-Leu2-NMe-Phe-Gly2-Leu3)(MR-3)and cyclo(NMe-Thr-Val-NMe-Ala-Gly1-Leu1-NMe-Ser-Leu2-NMe-Phe-Gly 2-Leu3)(MR-4),respectively and the amino acids in MR-1 and MR-2 were all L-type(glycine has not chirality).These four cyclic decapeptides have never been reported in literature and are all new compounds.MR-2 showed significant protective activity against OGD-injured rat primary astrocytes in our research,and had great potential in the treatment of diseases related to hypoxic-ischemic injury.The pharmacological activities of other three new cyclic decapeptides need to be explored.As a new method of dereplication,LC-MS/MS molecular network technology had been attracted extensive attention of researchers and widely used in the field of natural medicine research.In order to discover the chemical constituents of the culture of M.roridum IFB-E091,LC-MS/MS molecular network based on the GNPS(Global Nature Products Social Molecular Networking)platform was used to analyze the chemical components in the culture of the strain in this thesis.The LC-MS/MS spectra of several fractions of the culture was determined,which were converted by MSconvert software and uploaded to GNPS platform to construct molecular network.At the same time,the LC-MS/MS spectrometry data were processed by MZmine2 software,and then uploaded to the GNPS network platform to construct the feature-based molecular networking.MR-1 and MR-2 were used as seed nodes to analyze the molecular network,and several analogues of cyclic peptides were found in Fr.5-3.Except for four new cyclic peptides,three cyclic peptides MR-5-MR-7 were recognized again.The presence of seven analogues nodes of Halovir C(a linear chain fatty peptide)were also observed in the fraction 4 and 5,which were named as Halovir C-I～Halovir C-VII.According to the relationship between the nodes in the molecular networking and MS2 data,the planar structures of MR-5～MR-7 and Halovir C-I～Halovir C-Ⅶ were deduced,which were all new compounds.In addition,the molecular network also showed the presence of steroids and macrolides in Fr.1,Fr.2 and Fr.4,which was consistent with the previous experimental results.GNPS is an information platform for constructing molecular networking and realizing data sharing.Analyzing the chemical composition of fermentation products by GNPS network platform could help to predict the chemical composition to a great extent,which could accelerate the identification of natural products and play a role of dereplication.Particularly,MR-1 and MR-2 were used as seed nodes to trace the distribution of other peptides,which improved the efficiency of excavating new peptides and laid the foundation for the directional isolation of new peptides.