Isolation And Identification Of 25 Strains Of Filamentous Fungi And Their Secondary Metabolites

Plant endophytic fungi and fungi of Chaetomium sp., microbial resources with high diversity, can produce a variety of secondary metabolites with novel structures. Most of these secondary metabolites have antioxidant, antitumor, antibacterial, hypoglycemic, immunosuppressive and antiviral activities. These fungi have become an important source of novel natural products with biological activities. Therefore, plant endophytic fungi and fungi of Chaetomium sp. were selected as the objects of this study. The isolation, identification and bioactivities of secondary metabolites of these two kinds of fungi were investigated.First of all, by using traditional artificial separation method, 20 strains of endophytic fungi were selectively isolated from 29 parts of fruit, leaf and stem of 13 kinds of plants. The 28 S r DNA sequences of 20 strains of endophytic fungi were determined by molecular method. By compared with the highly homologous sequences in BLAST, analysis of phylogenies and observation of colonies, 16 strains of endophytic fungi were identified as Cladosporium sp., Paramicrothyrium chinensis, Mycosphaerella sp. H41b4, Eupenicillium sp. NF2_PD013, Colletotrichum boninense, Corynespora torulosa, Sarocladium bacillisporum, Colletotrichum sp., Pestalotiopsis sp., Guignardia mangiferae, Phoma minor, Pseudocercospora nephrolepidicola, Collembolispora barbata, Alternaria sp., Corynespora cassiicola and Glomerella acutata, the other 3 strains of fungi were identified as Fungal sp. F185(JQ747675.1), Fungal endophyte(KF435931.1) and Fungus clone Alb_O_Aug F04(GU174372.1), and the strain of ZSY-1 was a new fungus obtained in this study.The secondary metabolites of 20 strains of endophytic fungi and 5 strains of Chaetomium sp. and the effects of epigenetic reagents of nicotinamide(NA) and suberoyl bis-hydroxamic acid(SBHA) on their production were investigated. These fungi were fermented under the conditions of control, NA 50 μmol/L, NA 100 μmol/L, SBHA 100 μmol/L and SBHA 500 μmol/L. The extracts of culture media were collected and analyzed by high performance liquid chromatography(HPLC) and mass spectrometry(MS). The results showed that the secondary metabolites of endophytic fungi ZYHY and HGZJ, and Chaetomium piluliferoides were very rich. The epigenetic reagents of NA and SBHA had effects on the secondary metabolites production of most strains. NA 50 mol/L significantly affected the the secondary metabolites production of endophytic fungi HGZJ and Chaetomium piluliferoides. In addition, the preliminary exploration of lipopolysaccharide(LPS) effects on the secondary metabolites production of endophytic fungi ZYHY and HGZJ was conducted. The results showed that LPS could obviously change the secondary metabolites of these two strains of fungi.Using various separation technologies such as silica gel column chromatography, thin layer chromatography, Sephadex LH-20 gel chromatography and HPLC, the extracts of culture media of fungi ZYHY, HGZJ and Chaetomium piluliferoides were isolated and purified and 15 compounds were obtained. Their chemical structures were identified by NMR and MS spectra, and nine of them were new compounds. Four known compounds geodin, dihydrogeodin, 3’-chlorotrypacidin and monochlorosulochrin, which contain chlorine, were isolated from the secondary metabolites of fungi ZYHY. Five new compounds HGZJ-1, HGZJ-2, HGZJ-3, HGZJ-5 and HGZJ-6, and a known compound ambuic acid were obtained from the secondary metabolites of fungi HGZJ. Four new compounds CPIL-1, CPIL-3, CPIL-4 and CPIL-5, and a known compound Chaetophenol A were isolated from the secondary metabolites of Chaetomium piluliferoides. In addition, the specific optical rotation values of compounds CPIL-3 and CPIL-4 were determined as 0 and-63.5. The absolute configuration of compound CPIL-4 was determined by Mosher method and VCD method. The absolute configuration at C-2 was S and the absolute configuration at C-3 was R.The antioxidant, anticancer and anti-liver injury activities of obtained compounds were preliminarily evaluated. The results showed that geodin and CPIL-3 had significant DPPH free radical scavenging ability. Geodin, dihydrogeodin, 3’-chlorotrypacidin, monochlorosulochrin, CPIL-4 and Chaetophenol A significantly inhibited the proliferation of MCF-7 cells, while HGZJ-1, HGZJ-2, CPIL-1 and CPIL-3 exhibited moderate growth inhibitory effects on MCF-7 cells. Geodin, 3’-chlorotrypacidin and Chaetophenol A significantly inhibited the proliferation of Hep G2 cells, while dihydrogeodin, monochlorosulochrin, HGZJ-1, HGZJ-2, CPIL-1 and CPIL-3 showed moderate growth inhibitory effects on Hep G2 cells. CPIL-4 promoted the proliferation of Hep G2 cells at low concentrations, but inhibited its proliferation at high concentrations. CPIL-5 and low concentrations of dihydrogeodin and monochlorosulochrin had protective effects on CCl4-induced liver cell injury.