Isolation Of Endophytic Fungi From Sevaral Plants,and Evaluation On Biological Activities Of Their Secondary Metabolites

Many studies have shown that endophytic fungi in medicinal plants have become a natural treasure to find novel,active substances.Therefore,five kinds of medicinal plants were selected as materials,and their endophytic fungus were isolated and purified.We studied the secondary metabolites biological activities of these endophytic fungus.The chemical constituents of some active strains were studied.The research contents and results are as follow:1.The isolation and purification of endophytic fungi: Rhododendron przewalskii,Uncaria scandens,Corydalis adunca,Ligularia hodgsonii,Thalictrum minus L.were used to isolate and purify endophytic fungus from the roots,stems and leaves of five medicinal plants.The result is as follow: 18 strains were obtained from Rhododendron przewalskii;9 strains from Uncaria scandens;1 strain from Corydalis adunca;2 strains from Ligularia hodgsonii;1 strain from Thalictrum minus L..Among them,10% of endophytic fungi were isolated from roots,19% were separated from leaves,71% were separated from stem and endophytic fungi were the most distributed in leaves.62 extracts of the bacterial and mycelial were obtained by liquid fermentation technology(PDB medium),ethyl acetate extraction and methanol reflux extraction.2.Activity detection: The MAO inhibitory of 62 extracts were evaluated by 96-well microplate method.The results showed that several endophytic fungi crude extracts have varying degrees of anti-monoamine oxidase activity.The inhibitory rate of three strains were more than 50%,which were LSDJ-J-4(51.67%),LSDJ-J-6(50.72%)and QDE-6(54.65%).The AChE inhibitory of 62 extracts were tested by modified Ellman method.Studies have shown that the inhibitory activity of the bacterial liquid and mycelial extracts against acetylcholinesterase is higher than that of monoamine oxidase inhibitory activity.Among them,the acetylcholinesterase inhibitiory rate of 16 extracts were more than 50%.The acetylcholinesterase inhibitiory rate of nine strains were more than 60%.The nine strains are LSDJ-J-1,LSDJ-J-2,LSDJ-J-6,LSDJ-J-9,LSDJ-J-10,LSDJ-G-1,QDE-6,LTTW-Y-1 and TSC-J-1.The insecticidal activity of all the endophytic fungi extracts was determined by the slide impregnation method.Three strains with high activity were obtained,which was QDE-4,QDE-5 and QDE-6.The inhibitory activity against Phytophthora infestans of all endophytic fungi was determined by the method of toxic culture.The inhibitory rate of eight strains was more than 80%,which were LSDJ-J-1,LSDJ-J-2,LSDJ-J-6,LSDJ-J-8,QDE-4,QDE-6,LTTW-Y-1 and TSC-J-1.Based on the above results,strain QDE-6(isolated from Uncaria scandens)showed high activity in a variety of activity assays,and it was worth to further study it's chemical constitutent.3.Preliminary chemical constitutent research: By TLC thin layer chromatography,macroporous resin column chromatography,silica gel column chromatography,high per formance liquid chromatography,the chemical constitutent of endophytic fungi LSDJ-J-6 and QDE-6 were studied and 11 compounds were isolated.The structures of isola ted compounds were identified by 1D-NMR,ESI-MS,EI-MS.Among 11 compounds,5 compounds were identified as Aspergiketone(5),Gliotoxin(6),Pseurotin A2(7),Br evianamide F(8),Pseurotin A1(11).4.Identification of five endophytic fungus: The strain was identified by molecular biology method and morphological analysis.Strain LSDJ-J-2 was identified as a fungi of Alternaria sp.;Strain LSDJ-J-6 was identified as a fungi of Trichodermas sp.;strain QDE-6 was identified as a fungi of Aspergillus Sp.;strain LTTW-Y-1 was identified as a fungi of Aspergillus sp.;strain TSC-J-1 was identified as a fungi of Fusarium sp..