Studies On The Secondary Metabolites And Their Biological Activities Of Two Endophytic Fungi From Uncariar Hynchophylla (Miq.) Miq. Ex Havil.

Endophytic fungi are rich in resources.Researchers have isolated a large number of compounds with novel structures and significant activities from endophytic fungi.Natural products obtained through fungal metabolism have significant advantages in yield,economy and environmental protection.In this study,the secondary metabolites of endophytic fungi of Uncaria uncaria were systematically studied,with a view to finding novel and active drug lead compounds,and providing a basis for the development of new drugs,especially new biological pesticides.In this dissertation,the secondary metabolites of two endophytic fungi,Trichoderma sp.TMS-2011 and Hypoxylon sp.HD-2014,were systematically studied.Rice culture medium was used for mass fermentation and ethyl acetate solvent was used for ultrasonic extraction.The crude extract was separated and purified by silica gel column chromatography,Sephadex LH-20,ODS column chromatography,MCI column chromatography and semi-preparative high performance liquid chromatography.A total of 30 compounds were obtained.Their planar and three-dimensional structures were determined by means of NMR,high-resolution mass spectrometry,optical rotation,infrared spectroscopy,circular dichroism and other characterization methods.Six of them were new compounds that were not reported in the literature.At the same time,the anti-plant pathogenic fungi and anti-inflammatory activities of all the isolated compounds were evaluated.Seventeen compounds were isolated from the endophytic fungus Trichoderma sp.TMS-2011.Respectively:eight trichodermic acid derivatives（1-8）,three dioxypiperazine alkaloids（9-11）,one dipeptide（12）,one sterol compound（13）,one nucleoside compound（14）,one phenolic acid compound（15）,two fatty compound（16,17）.Compounds 1-4 are new compounds that have not been reported in the literature.Thirteen compounds were isolated from the endophytic fungus Hypoxylon sp.HD-2014.Respectively:five aliphatic compounds（18-22）,three phenolic acids（23-25）,three naphthalenones（26-28）,one benzodihydropyranone compound（29）,one coumarin（30）.Compounds 18 and 19 are new compounds that have not been reported in the literature.All compounds were tested for the minimum inhibitory concentration of plant pathogenic fungi to evaluate their anti-plant pathogenic fungi activity.The results showed that compounds 1-7 showed moderate antifungal activity in inhibiting the growth of Botrytis cinerea and Alternaria alternata（MIC:32-64μg/m L）.However,there was no significant effect on Fusarium oxysporum and Fusarium moniliforme.Compound 8 shows good antifungal activity,and its MIC to Botrytis cinerea is 4μg/m L,its MIC to Alternaria alternata is 16μg/m L.It also showed moderate antifungal activity against Fusarium oxysporum with the MIC of 32μg/m L.In order to further explore the action mechanism of compound 8 on Botrytis cinerea,the mycelium of Botrytis cinerea treated with compound 8 was observed by scanning electron microscope and transmission electron microscope.The scanning electron microscope results showed that after treatment with compound 8,the mycelial extensibility of Botrytis cinerea decreased,the mycelial branches increased significantly,and the surface folds contracted.The transmission electron microscope results showed that after treatment with compound 8,the ultrastructure of Botrytis cinerea cells changed significantly,and the cell deformation was serious,the plasma membrane was amorphous,the extracellular substances increased and distributed unevenly,the intracellular vesicles increased,enlarged and merged into a large central vacuole,and most of the intracellular organelles disappeared.Thus,the mechanism of compound 8 inhibiting the growth of Botrytis cinerea was preliminarily clarified.All compounds were tested for LPS-induced inhibition of NO production in RAW264.7 cells to evaluate their anti-inflammatory activity.The results showed that compounds 1-7 and 13 exhibited certain anti-inflammatory activity(IC₅₀=24.3-49.5μM)compared to the positive control Dexamethasone(IC₅₀=7.80μM).Other compounds did not show obvious anti-inflammatory activity(IC₅₀>60μM)。In this dissertation,the chemical composition anti-plant pathogenic fungi activity and anti-inflammatory activity of the secondary metabolites of two endophytic fungi from Uncariar hynchophylla（Miq.）Miq.ex Havil.were systematically studied.Thirty compounds were isolated and identified,some of which exhibited certain anti fungal and anti-inflammatory activities against plant pathogens,and the mechanism of compound 8 against plant pathogenic fungi was deeply explored.The research results enrich the secondary metabolites of two endophytic fungi,Trichoderma sp.TMS-2011 and Hypoxylon sp.HD-2014.It provides potential active molecules for anti-plant pathogenic fungi and anti-inflammatory,and also provides a possible research direction for the antifungal mechanism of Trichoderma sp..