Study On Secondary Metabolites Of Myrothecium Roridum

Endophyte widely exists in the host plants, and it symbiosises and coevolves with the host plant based on mutual benefit. The special metabolic pathway in endophyte could produce varieties of bioactive metabolites, which provide a new idea to solve some problems of pharmaceutical and ecological destruction caused by slow growth of some medicinal plant, shortage of resources and so on. The secondary metabolite of endophyte becomes one of important sources for new drugs and lead compounds.M. roridum IFB-LLY and IFB-E091were isolated by the Institute of Functional Biomolecules of Nanjing University from the stems of E. equisetina Bunge and the root of A. annua, respectively. Solid fermentation extracts of these two endophytic fungi were studied in this dissertation. The solid biomass of IFB-LLY and IFB-E091were extracted with CHCl3-CH3OH (v/v1:1) several times and then evaporated in vacuo to afford the crude extract (52g and38g, respectively). Guided by1H-NMR spectrum and TLC, the chemical constituents of the crude extract were separated and purificated through several methods such as silica gel column chromatography, Sephadex LH-20gel column chromatography, HPLC and so on. Eleven compounds were obtained from IFB-LLY (1～11) and three compounds were obtained from IFB-E091(12-14)(A new trichothecene macrolide roritoxin E、two known steroidal compounds and one known alkaloid had been obtained from IFB-E091in our previous research). Structures of the obtained compouds were determined through modern spectrum technology such as HR-ESI-MS,1D and2D NMR, UV and CD etc. They were determined as1-methoxyphenazine (1)、1-phenazinecar-boxylic acid (2)、N-methyl-1H-indole-2-carboxamaide (3)、uracil (4)、thymine (5)、 monomethylsulochrin (6)、ditryptophenaline (7)、daucosterol (8)、lumichrome (9)、1,3,9-trimethyluric acid (10)、(S)-(-)-N-[2-(3-Hydroxy-2-oxo-2,3-dihydro-1H-indol-3-yl)-eth-yl]-acetamide (12). Compound12is a new compound which has not been reported yet and the structures of11,13and14are being identified. In vitro cytotoxicity of compounds1,2,3,6,7and12were evaluated by MTT method. The result showed that compound3has obvious anti-tumor activity to hepatocarinoma SMMC-7721cells, with an IC50value of40.0μg/ml(0.230μM), while cis-platinum, the positive control, has IC50value of11.5μg/ml(0.066μM).The solid fermentation of endophytic M. roridum could produce several types of chemical compositions, such as richothecene macrolides, alkaloids, steroids etc. These natural products also have a variety of biological activities. Endophytic M. roridum could provide plenty of compound for pharmacological activity screening and its chemical constituents are worthy of further study in the future.