Secondary Metabolites From Endophytic Fungi Harbored In The Plant Mahonia Fortunei And Their Biological Activities

Healthy plant tissues contain some fungi which caused no obvious disease symptoms and were often called endophytic fungi.With the long-term co-evolution between these endophytic fungi and their host plants,a symbiotic interspecies crosstalk has been formed and developed within plants.In this plant-endophyte relationship,endophytic fungi and their secondary metabolites display a variety of functions,such as protecting host plants from other pathogens and regulating the metabolic process of host plants.Therefore,chemical studies on plant-derived endophytic fungi could contribute to the discovery of drug leads and promote the development of innovative drugs.Mahonia fortunei is an important plant used in the Traditional Chinese Medicine（TCM）.Its roots,stems and leaves as medicine were employed to treat heat,reduce swelling,stop diarrhea,and inhibit a variety of gram-positive bacteria.Considering the long-term interaction between plants and endophytic fungi and our previous work,M.fortunei is an ideal plant host for isolation of diverse fungi.In this thesis,M.fortunei in different locations of China were firstly collected for the isolation and purification of fungal endophytes,followed by small-scale fermentation in rice media and detailed analyses of secondary metabolites and their biological activities.This pre-screening process enabled the selection of bioactive endophytic fungi for following chemical research.The targeted fungi were further subjected to large-scale fermentation which was treated with ethyl acetate to provide a crude extract.Then,column chromatography（CC）on silica gel,Sephadex LH-20 CC,and high performance liquid chromatography were used to separate and purify chemical constituents present in the above extract extract.The structures of the isolates were identified by IR,UV,MS,and NMR.Finally,their antibacterial,antifungal,quorum sensing（QS）inhibitory,and cytotoxic activities were evaluated.Five endophytic fungi（Gliocladium sp.NJ-F1,Acremonium pilosum F47,Fusarium sp.FY18,FWY40,and NJ-F5）residing within M.fortunei collected from Changsha and Nanjing,China,were selected and subjected to chemical investigation.A total of 32compounds were obtained,including twelve cyclic peptides:pullularins A,D and B（2,3,and 4）,glioperazine C（5）,cyclo-（L-Ala-L-Phe）（6）,trans-cyclo-（Pro-Phe）（7）,cyclo-（Phe-Try）（8）,cyclo-（L-Pro-L-Trp）（9）,cyclo-（L-Leu-L-Leu-D-Leu-L-Leu-L-Leu）（27）,cyclo-（L-Leu-L-Leu-D-Leu-L-Leu-L-Ile）（28）,cyclo-（L-Phe-L-Leu-L-Leu-L-Ile）（29）,and beauvericin（31）;six amides:scalusamide B（1）,phenaceturic acid（20）,N-phenylacetyl-L-cysteine（21）,N-phenethylacetamide（22）,3-（acetylaminomethyl）indole（25）and N-acetyltryptamine（30）;three fusidane-type antibiotices:fusidic acid（13）,16-desacetylfusidic acid（14）and acremonidiol B（15）;two fusarin derivatives:7-desmethyl fusarin C derivative（23）,13α-hydroxylucilactaene（24）;two polyterpenoids:glisoprenin D（10）and glisoprenin G（11）;two pyranones:acrepyrone B（12）and gibepymne A（17）;one gibberellin:GA₁₃（32）;together with four other compounds:3-pentene-1,4-diol（16）,4-methylpentanoic acid（18）,p-hydroxylbenzyl propanol（19）,4-methoxybenzeneacetic acid（26）.Among them,compounds 11,12,and 15 are new compounds.The antibacterial,antifungal,QS inhibitory and cytotoxic activities of isolated compounds were evaluated.Compound 13（0.2μg/disk）showed strong antibacterial activities against Staphylococcus aureus and Bacillus subtilis with inhibitory zones of 1.8cm and 1.5 cm,respectively.Compound 26（0.2μg/disk）possessed strong inhibitory activity against Alternaria solani.It has to be noted that compound 30（10μg/well）showed strong QS activity comparable to that of the positive control furanone C30.For cytotoxicity,compounds 2-4,10,and 11 exhibited strong efficiencies to MDA-MB-231 cells with IC₅₀values of 12.89,19.01,12.59,15.59 and 9.05μM,respectively.In summary,32 secondary metabolites,including three new compounds（11,12,and15）,were isolated and identified from five endophytic fungi harbored in the medicinal plant M.fortunei.Fusidic acid（13）is the only fusidane-type antibiotic in clinical application at present.Compound 30 showed strong QS activity comparable to that of the positive control furanone C30.What’s important is that new compound 11 had the strongest cytotoxicity among all isolated metabolites.These results suggest that fungal endophyte-derived secondary metabolites with obvious structural diversity have a wide range of biological activities and are an important source of lead compounds for the development of innovative drugs.