Endophytic Fungi From Tribulus Terrestris L.and Their Secondary Metabolites

Endophytic fungi can produce the same or similar bioactive agents as medicinal plants, or relay on the phytochemicals produced by the plants. It is urgent to isolate and study associated endophytes from the host plants. The main purpose of this dissertation was to investigate antimicrobial and anticancer compounds from the natural products of endophytes. To continue the previous work in our lab, Tribulus terrestris L., a traditional medicinal plant, was selected for further investigation. In the present study, the research was performed on several aspects including isolation and identification, activity detection, investigation on secondary metabolites of endophytic fungi from T. terrestris L.. The main contents of this dissertation are summarized as follow:1. A total of64endophytic fungi were isolated by culture-dependent method, identified by morphological and molecular methods and categorized into10genera. Among them, Phoma and Alternaria were the dominant genera, accounted for39.06%and28.12%respectively of the total strains. Among them,21strains were selected as representative strains for a further study.2. Screening strains by detection of flavonoids and saponins compounds and biological activities.(1) Flavonoids and saponins were selected as test indicators, as which were the main active ingredients of T. terrestris L. Analysis of the products from21endophytic fungi using SPE-HPLC and GC-MS showed that JL-16could produce squalene and17-(1,5-dimethylhexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-lH-cyclopenta[a]phenanthren-3-ol, both of which are precursors of saponins. However, on subculturing the fungus JL-16in axenic medium, the endophyte tended to lose their ability to produce the two secondary metabolites.(2) Antibacterial, antimycotic and cytotoxic activities of the endophytes were also evaluated. Antibacterial activity of21endophytic fungi was evaluated by Staphylococcus aureus and Escherichia coli, which are the representative of Gram-negative and Gram-positive bacteria. The strains with their inhibition zone diameter of the PDB and Czapek broth greater than10mm were accounted for19.05% (4/21) and23.81%(5/21) respectively of the total strains. Antimycotic activity of21endophytic fungi was evaluated by Magnaporthe oryzae model. The strains, of which the PDB broth and the Czapek broth with the MIC (minimum inhibitory concentration) of Magnaporthe oryzae lower than10%, were accounted for19.05%(4/21) and47.61%(10/21) respectively of the total strains. Cytotoxic activities of21endophytic fungi were evaluated by PC-3(CRL-1435) and4T1(CRL-2539) cells. The PDB fermentation broth with the inhibitory action on4T1cancer cells over50%was accounted for42.86%of the total PDB broth (9/21). And the Czapek fermentation broth with the inhibitory action on4T1cancer cells over50%was accounted for38.10%of the total Czapek broth (8/21). The PDB fermentation broth with the inhibitory action on PC-3cancer cells over50%was accounted for47.62%of the total PDB broth (10/21). And the Czapek fermentation broth with the inhibitory action on PC-3cancer cells over50%was accounted for28.57%of the total Czapek broth (6/21). JL-3, JL-7w, JL-13, JL-14, JL-16and JL-17exhibited strongly cytotoxic effect. Crude extracts were obtained by fractional extraction and evaluated in PC-3cancer cells. Trichloromethane (TCM) extracts of JL-7w had the most effective activity against PC-3cells with the IC50value of55.15μg/mL. In summary, JL-7w exhibited strongly biological activities.3. Particular identification and chemical analysis of JL-7w.(1) The test of JL-7w resulted to be Alternaria tenuissima, which was confirmed by morphological characterization, endopolygalacturonase gene analysis and UV fingerprint spectra.(2) A new secondary metabolite, named altertoxin IV, together with altertoxin II, were isolated from the fermentation broth of JL-7w using repeated silica gel column and gel column chromatography. The new structure was characterized by HR-ESI-MS, multinuclear NMR spectroscopy, and single crystal X-ray diffraction method. Altertoxin II and Altertoxin IV were tested for their anti-cancer activities in vitro on the PC-3cells. Altertoxin IV had little anti-cancer activity at the tested (highest) concentration of2.67mM; however, Altertoxin Ⅱ showed moderate anti-cancer activity with the IC50value of14.28μM.