| After Three Gorges Dam is constructed,the Three Gorges Reservoir（TGR）forms a new vast hydro-fluctuation belt with an elevation of 145 m in summer to 175 m in winter,a length of more than 2000 km and an area of 300 km2,which forms a unique ecological condition for species diversity and distribution of organisms in TGR area.Many field surveys have shown that a large proportion of the pre-dam riparian vegetation are gradually dying out because they are unable to adapt to reversal of the submergence time,prolonged duration of flooding,and new hydro-fluctuation belt（up to 30m in elevation）.Generally,plants have limited access to oxygen and light under flooding condition,resulting in producing excessive reactive oxygen species（ROS）,which are considered to be the key factors that hinder the growth and development of submerged plants.They are forced to undergo the oxidative pathway,and usually develop an antioxidant defense system consisting of some antioxidant enzymes and specific metabolites to convert these excessive ROS into harmless products in order to protect themselves.As symbionts,endophytic fungi can produce antioxidants that can interrupt the chain reaction of ROS to help host plants respond to various biotic and abiotic stresses.Undoubtedly,natural antioxidants should be warranted in the enhancement of human health.Additionally,the search for safer and novel drugs based on natural product from endophytes is of utmost importance because of the increasing incidence of cancer and the recent emerged and rapid evolution of superbugs due to antibiotic resistance.And,the medicinal plant Distylium chinense is a rare and evergreen shrub of Hamamelidaceae and endemic to the TGR area,which has strong flooding tolerance.Up to now,the research and development of endophytic fungi from D.chinense are still not in-depth,and there have no reports on the diversity of endophytic fungi from D.chinense at home and abroad.In this paper,we identified and screened for antioxidant,antimicrobial and anticancer activities of 154 endophtic fungi isolated from the representative plant D.chinense in the TGR.As a result,a broad-spectrum bioactive strain,Irpex lacteus DR10-1,was screened and its secondary metabolites were further isolated and identified.The mian results are discussed as follows:1.154 strains of fungal endophytes from D.chinense were identified by morphology and molecular biology.The results showed that 154 strains of endophytic fungi belonged to 2 subphyla,6 classes,9 orders,13 families and 27 taxa,which included one order,6 genus and 20 species.They are Diaporthales spp.;Phomopsis sp.,Diaporthe sp.,Paraphoma sp.,Periconia sp.,Irpex sp.,Fusarium sp.;Phoma medicaginis,Diaporthe eres,Neofusicoccum parvum,Xylaria venosula,Lasiodiplodia theobromae,Irpex lacteus,Botryosphaeria dothidea,Fusarium oxysporum,Mycorrhizalbasidiomycete,Fusariumequiseti,Penicillumochrochloron,Schizophyllum commune,Rhizoctonnia bataticola,Robillarda sessilis,Coprinellus xanthothrix,Phlebiopsis crassa,Polyporus umbellatus,Diaporthe longicolla,Flavodon flavus and Ceriporia lacerta.Among them,Ascomycota was the dominant community,with 132 endophytic fungi,accounting for 85.7%of the total strains at the phylum level;Sordariomycetes was the dominant class,with 75 endophytic fungi,accounting for 48.7%of the total strains at the class level;Botryosphaeriales was the dominant order,with 52 strains in total at the order;Phomopsis sp.and Neofusicoccum parvum were dominant taxa,with 38 and 36 endophytic fungi,accounting for 24.7%and 23.4%of the total strains,respectively.Therefore,endophytic fungi from D.chinense differed greatly in quantity,distribution and population composition,and have abundant species diversity.2.The ethyl acetate extract from fermentation broth of 154 endophytic fungi were evaluated for antioxidant activity,antimicrobial activity against 7 pathogenic microorganisms and anticancer activity against 2 cancer cell lines.As a result,153strains showed at least one biological activity,accounting for 99.4%of the total strains.Specifically,99 strains of endophytic fungi showed notable antioxidant activity,accounting for 64.3%of the total strains;153 strains of endophytic fungi exhibited antimicrobial activity against at least one tested pathogen,accounting for 99.4%of the total strains;27 strains of endophytic fungi showed anticancer activity against at least one tested cancer cell lines,accounting for 17.5%of the total strains.3.The strain Irpex lacteus DR10-1 exhibited the widest spectrum bioactivities,including notable antioxidant activity with IC50 value of 2.79±0.04μg/mL;antimicrobial activity against seven tested microorganisms and anticancer activity against two tested cancer cell lines,which deserved further study.4.The enlarged liquid fermentation of broad-spectrum active strain DR10-1was carried out to isolate,purify and identify the secondary metabolites of its ethyl acetate layer.Twenty-four compounds were obtained from acetyl acetate ex tract of DR10-1 by silica gel column chromatography,Sephadex LH-20 column chromatography,thin layer preparation chromatography.On the basis of spectral dataanalyses(1H-NMR,13C-NMR,1H-1H COSY,DEPT,HMBC,HMQC)and rel evant treferences,24 compounds were elucidated.Among them,four new comp oundswere identified as sesquiterpene irpexlacte A,furan derivatives irpexlacte B,irpexlacte C,irpexlacte D and 20 known compounds were elucidated as sesquit erpenes:irlactin E;sesquiterpene:3β-hydroxycinnamolide;sterol:stigmasta-4,6,8（14）,22-tetraen-3-one;triterpenes:20β-hydroxydammara-23-en-3-one;benzene ring derivatives:hydroperoxide,2,5-dihydro-5-methoxy-2,5-dimethyl-2-furanyl;1,2-be nzenedicarboxylic acid,1,2-dibutyl ester;1,2-benzenedicarboxylic acid,1,2-bis（2-et hylhexyl）ester;1-phenyl-（4’-hydroxy）-n-nonanone;4-hydroxybenzaldehyde;p-hydr oxyl-benzoic acid;nitrogen-containing compounds:indole-3-carboxaldehyde;indol e-3-carboxylic acid;indole-3-carboxamide;3-（3-methyl-1-oxo-2-butenyl）]1H indole;β-carboline;4-tert-butyl-2-（4-methoxyphenylamino）phenol、bis（1,2-dihydro-2,2,4-tri methylquinolin-6-yl）isophthalate;polycyclic derivatives:5-（tert-butyl）octahydro-1-b enzofuran;aliphatic long chain compounds:1,2,3-propanetriyl tris（hexadecanoate）.5.Four new compounds were evaluated for antioxidant activity and antimicrobial activity against Pseudomonas aeruginosa and anticancer activity against four typical cancer cell lines including hepatoma carcinoma（SMMC 7221）,colon adenocarcinoma（SW-480）and two breast cancer（T47D and MDA-MB-231）.They showed moderate antimicrobial activity with MIC values ranging from 23.8 to 35.4μM.Irpexlacte A and irpexlacte D showed remarkable antioxidant activity with IC50 values of 2.50 and 5.75μM respectively,which may contribute to the survival of D.chinense under waterlogging stress by eliminating the reactive oxygen species（ROS）.Four new compounds were inactivity against cancar cell lines.The results of activity test showed that the secondary metabolites of strain DR10-1 had abundant biological activity and were an effective source for the development of new active drugs.The above results showed that there were abundant endophytic fungi resources in the D.chinense plant,and the activity screening results showed that the endophytic fungi of D.chinense had abundant biological activities,which provided abundant fungi resources for the screening of new drugs.Additionally,the secondary metabolites of the broad-spectrum active strain Irpex lacteus DR10-1 showed that endophytic fungi DR10-1 had abundant metabolites,manifested in diverse structures and biological activities,which provided valuable sustainable fungal resources for natural active drugs and provided a basis for subsequent research. |
