Studies On Active Secondary Metabolites Of Three Plant Endophytic Fungi

Endophytic fungi are widely distributed in the tissues of the whole plant,and establish remarkable mutualistic symbiosis with their host plants.They can produce various types of secondary metabolites such as alkaloids,terpenoids,polyketones,quinones,esters,etc.with unique structures,and have a wide range of biological activities,such as antitumor,anti-inflammatory,antibacterial,antiviral and other pharmacological effects.In recent years,endophytic fungi have gradually become an important source for the discovery of active lead compounds.Meanwhile,it is also serve as a potential new resources to replace plant secondary metabolites.In this study,three endophytic fungi Xylaria arbuscula（Cuiluo）,Fusarium solani（MBMTL）and Bipolaris sp.（YJH-29）with potent activities and rich secondary metabolites were selected from 80 endophytic fungi as the research objects based on the results of combination of LC-MS analysis and antitumor activity test.Solid sterile rice medium was selected for large-scale fermentation,after which ethyl acetate crude extract could be gotten.Then,the crude extract was isolated and purified by normal and reversed column chromatography,Sephadex LH-20 gel column chromatography as well as high-performance liquid chromatography（HPLC）and so on.As a result,31 compounds were obtained.The structures of these compounds were identified by spectroscopy（UV,1D and 2D NMR,HR-ESI-MS）.Moreover,these compounds were tested cytotoxic activity.The results are presented as follows:1.The secondary metabolites of the endophytic fungus X.arbuscula（Cuiluo）were isolated and purified,and seventeen compounds were obtained,namely xylarialoid A（1*）,cytochalasin C（2）,cytochalasin D（3）,zygosporin G（4）,cytochalasin N（5）,cytochalasin O（6）,cytochalasin R（7）,dodecahydro-cytochalasinen D（8）,6,7-dihydro-7-oxo-cytochalasin C（9）,7-oxo-cytochalasin C（10）,7-O-acetylcytochalasin P（11）,halorosellinic acid（12）,17-dehydroxyhalorosellinic acid（13）,10,11-epoxyguaian-13-ol（14）,5-carboxylmellein（15）,rickitin A（16）,piliformic acid（17）.Among them,1 is a new compound that has not been reported in literatures;Compounds 2-11 are cytochalasins.2.The secondary metabolites of the endophytic fungus F.solani（MBMTL）were isolated and purified,and six compounds were obtained,namely fusaridioic acid E（18*）,fusaridioic acid C（19）,fusaridioic acid A（20）,L660282（21）,hymeglusin（22）,hymeglnone（23）.Among them,18 is a new compound that has not been reported in literatures.3.The secondary metabolites of the endophytic fungus B.sp.（YJH-29）were isolated and purified,and eight compounds were obtained,namely helminthosporol（24）,9-hydroxyhelminthosporol（25）,helminthosporic acid（26）,helminthosporal acid（27）,cochliobolins A（28）,prehelminthosporol（29）,fusaproliferin（30）,cochlioquinone D（31）.Compounds 24-29 were sesquiterpenoids.4.All compounds were tested for cytotoxicity against tumor cells A549 and Hep G2 by MTT method.Compared with the positive control,compounds 4 and 22 exhibited potent cytotoxic activities against A549 and Hep G2 with the IC₅₀ values ranging from 3.63 to 13.57μM;Compounds 1,3 and 10 showed good cytotoxicity against（A549 and Hep G2）cell line with the IC₅₀ values of 14.6-19.6μM;Compounds 2,6,9,18,23,25,27,28,30 and 31 showed moderate cytotoxicity against A549 and Hep G2 cell line with IC₅₀ values of 21.87-49.20μM.