Secondary Metabolites of Five Flamentous Fungal Strains: Structures and Bioactivities
|School||Ocean University of China|
|Keywords||Fungi Secondary metabolites Antitumor Neuroprotective|
Special ecological environment bred special biodiversity, special biodiversity often breeds a special chemical diversity and biological activity of diversity. In order to find the skeleton innovative new drug lead compounds with good therapeutic effect, the paper marine and terrestrial fungi isolated from the environment to different students as research subjects, to carry out the activity of the secondary metabolites of the structure and biological activity. Study include: Isolation and Screening of the active strains; Purification and identification of the chemical structure of the metabolites and the determination of the configuration; exploration of new compounds biosynthesis pathway; preliminary evaluation of the biological activity of the monomer compounds; active compounds role of targets and A preliminary study of the active mechanism. With sea mud collected from different latitudes (Shandong, Fujian, Guangdong, Hainan) 24 intertidal the mangrove of Thuringia mud and crater sediment samples were isolated filamentous fungi 390, the shrimp bio-lethal the law and tsFt210 cells cytotoxic activity screening model for microscopic examination, TLC and HPLC chemical screening, 20 active bacteria selected from the crater sediment sources citrinum Penicillium citrinum HGY1-5 and the mangrove root the Sea Mud sources glaucum Aspergillus Aspergillus glaucus HB1-19, as well as other sources of three fungi - deep-sea marine mud sources Penicillium Penicillium sp. F23-2 and Penicillium Penicillium sp. F1, the intertidal sea mud sources Trichoderma Trichoderma sp. f-13-as the paper's research object. The fermentation product of the five target activity, the use of extraction, thin layer chromatography, normal phase, reverse phase silica gel column chromatography, LH-20 gel column chromatography, and reverse phase high pressure liquid phase chemical separation and purification means, from Penicillium citrinum P. The isolated metabolites citrinumHGY1-5 a 47 monomer compound (1-47); from glaucum niger A. metabolites glaucusHB1-19 were isolated 34 of the monomer compound (42,43,48-79); from Penicillium Penicillium sp. F23-2 metabolites isolated 11 of the monomer compound (80-90); from Penicillium Penicillium sp. The isolated F1 metabolites 10 monomer compound (91-100); from Trichoderma of Trichoderma sp. F-13 of the metabolic product isolated 11 of the monomer compound (46,101-110); Further, Penicillium citrinum P. citrinum HGY1-5 steroidal biotransformation obtain three monomeric compounds (111-113); citrinin dimer chemical conversion of 17 to obtain an artificial product (114), a total of 114 compounds. Then combined with chemical reactions, physical and chemical properties and spectroscopic methods (IR, UV, MS, NMR, CD, X-ray) (Mosher law bromobenzoyl esterification) clarify all 114 compounds chemical structure (Fig.1), structure type classification biosynthesis sources including: the steroidal compound 21 (1-16,40,41,111-113), the terpenoids 6 (85-90), polyketone 56 compounds (17-39,42-45,48-75,114), alkaloids of 19 (76-84,91-100) 11 sorbicillin derivatives (46,101-110), brain glycoside esters of one of the compound (47). Which discovered 63 new compounds, including 13 rare bicyclo [4.4.1] A / B ring C25 steroidal the compounds (1,3-13,16), two new skeleton citrinin trimer body (19, 20), 13 novel structure citrinin the dimer (17,18,21-24,26-28,32-34,114) 2 containing novel naphtho [1,2,3 -de] CHROMENE-2 ,7-dione skeleton the glaucum neomycin class polyketone compound (48,49), the 2 contain novel Spiro [5.5] undecane skeleton of spiro ring the glaucum neomycin class polyketone compound (50,51), 14 anthracene type or naphthalene-type aromatic polyketone A compound (52-54,56,57,59-61,63-68), 2 meleagrin alkaloids (80,81, of which 80 is a novel meleagrin the composite compounds formed by the the alkaloids and conidiogenone diterpenoid link) the two type roquefortine diketopiperazine alkaloids (83, 84), 6 rare conidiogenone type diterpenoid (85-90 only two cases reported), 5 type brevicompanine diketopiperazine alkaloids (91-93,96,97), 2 sorbicillin derivatives (101,107). In the study, we for the first time using a stable isotope labeling, chemical transformation and microbial steroid transformation method to determine the C25 steroid (1-16) skeleton biosynthesis source and the side chain type compounds kinship structure; against citrus neomycin (36) artificial polymerization and degradation reaction mechanisms explore; using an X-ray single crystal diffraction methods to determine the the compound the glaucum amphotericin A new skeleton (48) of the structure, using the NMR method of isotope labeling combined identified spiro ring compounds of the new skeleton the the glaucum adriamycin C (50) of the structure, and further a combination of methods using chiral HPLC-CD and computer Quantum Chemical CD simulation, determine the nature of the the glaucum adriamycin C racemic mixture and the two a using NOESY and CD method of combining the first identified conidiogenone type the diterpene (85-90) of the absolute configuration; using a combination of methods on the NOESY and amino acid chiral HPLC analysis to determine the absolute configuration of the enantiomers; type brevicompanine diketopiperazine alkaloids (91-93,96,97) absolute configuration. In order to find the therapeutic effect of the compound, we used the anti-tumor and neuroprotective two different strategies to evaluate the biological activity of the monomeric compounds. First, using the MTT assay, the SRB method combined with flow cytometry the morphological detection method, a preliminary evaluation of the anti-tumor activity of the isolated compounds were screened out of the 20 active compounds (cytotoxic IC 50 <10μM) (17-22,24,46,48,49,61,80-82,86-88,90,102,114), further use of the active compounds molecular targets and mechanism of preliminary exploration. Including: 1. The new skeleton compound the glaucum amphotericin A (48) A-549 and HL-60 cells the IC 50 > were 0.13 and 0.28μm; compound for protein kinase inhibition at 10μM role in the inhibition rates were Src (40.5%), KDR (43.4%); the glaucum neomycin A (48) in the concentration can also significantly inhibit topoisomerase II activity; preliminary experiments that the glaucum neomycin A (48) mice S 180 sarcoma model has antitumor activity in vivo; the glaucum neomycin A (48) Pre-clinical pharmacodynamic evaluation of depth . 2. The citrinin the polymer 17-20 and 114 1-10μM concentration significantly the induction of HL-60 cells to apoptosis, and its role in a time-dependent and concentration-dependent manner; 10μM concentration effect activation withered apoptosis-related protein Caspase 3 and 8, as well as of PARP is a novel class of tumor cell apoptosis-inducing agent. 3. Alkaloids Meleagrin 82 10μM concentration can HL-60 cell cycle arrest at the G 2 / M phase, suggesting its mechanism of action may be related to 9-OCH 3 analogues oxaline play a role, by inhibiting tubulin polymerization; alkaloids and diterpenes new link type skeleton compound 10μM and 5μM concentration can significantly induced apoptosis in HL-60 cells occurred, suggesting diterpenoid fragment meleagrin B (80), introduction may make targets of change. 4. Sorbicillin derivative (46,101-110) on the cytotoxicity test of the HL-60 cells showed that the sorbyl side chain 2,3 - reduction of the double bond causes the corresponding compound cytotoxic activity decreased by 2-10 times, indicating that such compounds The side chain conjugated system is closely related to its anti-tumor activity. For 100μM concentrations showed no significant cytotoxic activity of the compound using antagonistic model of LPS-induced BV2 microglia inflammation or G-protein coupled receptor 12 (GPR12) activation model evaluation of its potential neuroprotective activity, the first times found 10 new active the compounds (1,2,8,9,12,91,92,94,96,97). Including: 1. Type Brevicompanine diketopiperazine alkaloids 92 and 97 in the concentration range of 0.1μg/ml ~ 80μg/ml LPS-induced BV2 microglia cell inflammation model has significant inhibitory activity, this is the first discovery of such dione piperazine the alkaloids of anti-inflammatory activity. 2. The C25 steroid 1,2,8,9 and 12 at 10μM concentration can significantly activate GPR12, for cAMP release. This is the first the C25 steroidal compounds of potential therapeutic activity. The papers from five fungal secondary metabolites were isolated and identified 63 new compounds discovered five novel compounds skeleton type, to determine the absolute configuration, and C25 steroidal citrinin derivatives and gray the the neomycin class polyketone new skeleton compounds biosynthesis system, enrich the content of chemical structure and biosynthesis of natural products. Activity evaluation, were screened out of the 30 active compound (including 24 novel compounds), which found a new skeleton pilot antitumor compound glaucum amphotericin A (48), and the compound has a the novel NAPHTHO [1,2, 3-de] chromene-2 ??,7-dione skeleton, in vitro cell and molecular level, effective, effective preliminary evaluation in vivo is a novel multi-target role of anti-tumor lead compounds; discovered two types of new skeleton tumor apoptosis induced agent citrinin polymer 17-20 and alkaloids diterpene link compounds meleagrin B (80), provides an important lead structure for drug research. Screened 20 anti-tumor activity of strains were isolated, and found that the three can produce a series of new skeleton glaucum anti-tumor activity of compounds fungi Aspergillus A. glaucus HB 1-1 9, Penicillium citrinum P. citrinum HGY1-5 and Penicillium Penicillium sp. F23-2, active strains of medicinal resources for the development of fungal anticancer drugs.