Secondary Metabolites Of Endophytic Fungal Mutant From Curcuma Wenyujin

Endophytic fungi are usually defined as those species that exist within the living tissues of plants, for some or all of their life cycle, without causing apparent harm to their hosts. They are an important group of microorganisms with high diversity in metabolites and physiological activities, showing the potential as sources of antimicrobial, anticancer, antioxidant, antiviral compounds and etc.The research is focused on secondary metabolites of an endophytic fungal mutant associated with the medicinal plant Curcuma wenyujin through an active tracking method. It aims at obtaining natural active substances and exploring a new way for preparing active ingredients of plants. It contains isolation and identification of endophytic fungi from C. wenyujin and selection of an active strain, molecular identification of the active strain EZG0807 and research of biological characteristics, mutation of the strain EZG0807 by a superconducting magnet and screening of mutants, optimal fermentation conditions of the mutant M7226, separation, purification and structural characterization of secondary metabolites of M7226, and bioactivities of secondary metabolites from M7226. The results are as follows:(1) Sixty-six endophytic fungi were recovered from roots, leaves and stems of healthy C. wenyujin according to the protocol of tissue surface-sterilization. They belonged to five orders, seven families including fourteen genera. The dominant genera were Penicillium, Fusarium and Aspergillus. The antibacterial assay showed that strain EZG0807 exhibited a broad antimicrobial spectrum against eight tested strains viz Escherichia coli, Proteus vulgaris, Bacillus subtilis, Staphylococcus aureus, Microzyme, Aspergillus niger, Rhizopus and Mucor. EZG0807 was identified as Gibberella moniliformis and designated as Gibberella moniliformis EZG0807 according to ITS sequence through molecular biology. Meanwhile, the ITS sequence data was deposited in GenBank (accession No. JQ003920).(2) G. moniliformis EZG0807 was mutated by a gradient superconducting magnet which could simulate the space gravity environment. The mutation mechanism was explored by means of the amplified fragment length polymorphism method. The result showed that the large gradient superconducting magnet made DNA bands of G. moniliformis EZG0807 lost and induced gene mutation.(3) The mutant M7226 with high activity and heritable stability was selected from 139 pure mutants harvested through dilution spread plate according to the bioactivities of their secondary metabolites which were determined by the disc antibacterial method and MTT. Compared with G. moniliformis EZG0807, the antimicrobial activities of metabolites from M7226 against E. coli、P. vulgaris、B. subtilis and S. aureus were improved, and the antitumor activities against tumor cells A549 (human lung adenocarcinoma epithelial cell line), H460 (lung cancer cell), MCF-7 (human breast adenocarcinoma cell line) and HepG2 (liver hepatocellular cell line) were significantly improved.(4) The fermentation condition of M7226 was optimized with Box-Behnken of response surface method. The optimal fermentation condition of M7226 was 29℃ for the temperature, 6.8 for initial pH and 10 d for fermentation time. Under the condition, five compounds were obtained from its fermentation broth through large-scale fermentation and integrated separation. They were identified as curcumin, cinnamic acid,1,4-dihydroxy anthraquinone, gibberellic acid and kaempferol according to the spectroscopic data.(5) The antibacterial activity assay of the five compounds through MIC method showed that curcumin and 1,4-dihydroxy anthraquinone could strongly inhibit E. coli, P. vulgaris, B. subtilis and S. aureus with concentration-dependent effect. The MIC of curcumin on E. coli, P. vulgaris, B. subtilis and S. aureus was 200,200,200 and 50 μg/mL, respectively; the MIC of 1,4-dihydroxy anthraquinone on E. coli, P. vulgaris, B. subtilis and S. aureus was 200,400, 400 and 200 μg/mL, respectively; gibberellic acid, cinnamic acid and kaempferol exhibited weak inhibition on the four tested bacteria with MIC greater than 400 μg/mL.(6) The cytotoxicity of the five compounds on MCF-7, A549, H460 and HepG2 detected by Alamar Blue showed that IC50 of curcumin on HepG2, H460, MCF-7 and A549 was 14.12, 24.65,20.25 and 16.42 μg/mL, respectively; IC50 of kaempferol on HepG2, H460, MCF-7 and A549 was 60.47,90.22,90.95 and 129.60μg/mL, respectively; the cell inhibition rate of HepG2 was 24.8% when it was treated with gibberellic acid at a concentration of 200 μg/mL for 24 h; the cell inhibition rate of A549 was 13.1% when it was treated with cinnamic acid at a concentration of 200 μg/mL for 24 h; and 1,4-dihydroxy anthraquinone showed weak inhibition on the four cells.(7) The morphology of HepG2 treated by curcumin was detected by the assays of Hoechst33258, AO/EB staining and cell online culture techniques. Bright blue fluorescent could be visualized in cytoplasm of HepG2 cell stained with Hoechst33258, reflecting apoptosis characteristics of nuclear chromatin condensation and nucleolus rupture. Accompanied by the increasing concentrations of curcumin, the cell of HepG2 became sparse, and a large number of orange cells with typical apoptotic morphological characteristics of chromatin condensation and nuclear fragmentation appeared, which could be seen with AO/EB under fluorescent microscope. Besides, HepG2 showed some morphological characteristics with cell shrinkage, chromatin condensation, membrane subsidence and appearance of apoptotic bodies, without membrane burst and content release under the inverted microscope, illustrating that curcumin could induce apoptosis of HepG2 cell, not cell necrosis. Based on the morphological observation, it showed that HepG2 cell treated by curcumin had obvious apoptotic characteristics.(8) The results of RT-PCR showed that Bcl-2 gene expression was down-regulated and Bax, Caspase 3 and Caspase 8 were up-regulated in HepG2 cell treated by curcumin, speculating curcumin induced apoptosis of HepG2 cell through the mitochondrial-dependent and mitochondria-independent pathways.