Isolation, Identification And Their Bioactivity Of Endophytic Fungi From Five Medicinal Plants

Recent researches have shown that endophytic fungi of medicinal plants can produce multiple novel active secondary metabolites,and have become an important resource for searching novel active natural products.Advances in this field are reviewed briefly in this paper.Therefore, five medicinal plants were chosen to carry some preliminary researches on their fungi, their bioactivities and the constituents from the secondary metabolites. The main contents and results were as follows:1.46strains endophytic fungi were isolated from5medicinal plants:14strains from Nelumbo nucifera,12strains from Uncaria tomentosa,7strains from Tripterygium wilfordii,5strains from Holarrhena antidysenterica,8strains from Euphorbia nematocypha endophytes. The culture broth was filtered to separate the mycelium and the filtrate. The mycelium and the filtrate were extracted to afford2kinds of extract parts which were gained as follows:ethyl acetate extract and ethanol extract of mycelium.2. Modified Ellman’s method and spectrophotometric method in96-well microplates method were used to evaluate the anti-acetylcholinesterase and anti-monoamine oxidase avtivities of these extracts. The result showed that2endophytic extracts had remarkable AChE inhibitory activity, of which the inhibition rates were more than50%. The ethyl acetate extract of ZXM-5was the most potent extract with inhibition rate of61.81%. Anti-monoamine oxidase results showed that a total of24samples had remarkable inhibitory activities to monoamine oxidase, inhibition rate of samples were higher than50%, in which,8samples were higher than70%, The ethyl acetate extract of GT-5was the most potent extract with inhibition rate of92.53%. Samples with monoamine oxidase inhibitory activity accounted for the endophytic fungi total number of samples of each plant42.86%,58.33%,42.86%,40%and62.5, respectively.3.23compounds were isolated and purified from LGT-2, LGT-4, GT-5strains of ethyl acetate extracts and LGT-4strain mycelium alcohol extract using modern chromatographic methods, including silica gel column chromatography, preparative TLC, Sephadex LH-20,HPLC, etc.. Their structures were elucidated by extensive spectroscopic analysis (’H-NMR,13C-NMR, ESI-MS, HMBC, HSQC,’H-’HCOSY, NOESY, MS, IR), which were identified as Citrinin(1),4,6,8(14),22(23)-Tetraen-3-one-ergostane(2),2-Hydroxy-14-tricosen vicacid(3), Wortmannin(4), Wortmannolone(5),2,4-Dihydroxy-6-((R)-4-hydroxy-2-oxopentyl)-3-Methylbenzaldehyde(6),(+)-BrefeldinA(7),(22E,24R)-Ergosta-5,7,22-trien-3β-ol(8), Epoxyvirone(9), Secvironolide (10),11-Desacetoxywortmannin(11), Ergosterol-7,22-dien-3p,5a,6a-triol(cerevisterol)(12),3-Dihydrovirone(13),11-Desacotoxy wortmannin(14), Deacetylisowortmin(15), Ergosterol(16), Campesterol(17) Phenylalanine(18), Physcion(19), Chrysophanol (20), Ergosterol peroxide(22), Flufuran(23).The AChE and MAO inhibitory activity of these compounds were evaluated. The result showed that compounds4,10,23had moderate MAO inhibitory activity. At a final concentration of167μg/mL, these compounds inhibition rates were78.40%,83.55%,88.67%, of witch IC50were91.22μg/mL,40.17μg/mL,29.19μg/mL, respectively. In a final concentration of100μg/mL, the inhibitory effect angainst AChE of compound1were moderate and the IC50was147.74μg/mL.5. By using molecular biology method, including analyzing the ITS rDNA assay, contrasting with other high homology of gene in the Blast system and observing the color and the mycelia of colony morphology, strain LGT-2, GT-5and LGT-4were identified as Aspergillus sp.,Aspergillus sp. and Talaromyces sp., respectively.