Studies On The Secondary Metabolites And Bioactivities Of Four Endophytic Fungus, One Actinomycete And One Higher Fungus

Based on the theories of natural products chemistry and the modern bio-fermentation technology, the chemical constituents and bioactivities of four endophytic fungi isolated from the plant Ginkgo biloba L., one marine actinomycete and one species of higher fungi were studied. Eighty five secondary metabolites were isolated from the liquid fermentation broths (mycelia and filtrate) of the microbial materials or fruiting bodies of the higher fungi through bioassay-guided fractionation by silica gel, reversed-phase (ODS), and Sephadex LH-20 column chromatography. Sixty seven compounds were elucidated by means of spectroscopic techniques including ¹D and ²D-NMR (¹H-NMR, ¹³C-NMR, ¹H-¹H COSY, HMQC, HMBC and NOESY) as well as mass spectrometry (EI-MS, ESI-MS and HR-ESI-MS) in combination with the advanced database of Germany. Among isolated compounds, five novel isolated compounds were named as chaetomugilin D , globosterol, rufoolivacin C, rufoolivacin D, rufoolivacin E, respectively; besides, five antifungal compounds (chaetoglobosin A, chaetoglobosin C, kitamycin A, antimycin A1b, and blastmycin) and eleven cytotoxic constituents (chaetoglobosin A, chaetoglobosin C, chaetomugilin D, chaetomugilin A, 4-12, vivotoxin II, vivotoxin, fructigenine A , kitamycin A, antimycin A1b, and blastmycin ) were also obtained. The results are concluded as follows:1. One hundred and sixty endophytic fungi were isolated from the plant Ginkgo biloba L., and one hundred and sixteen strains were identified by morphological taxonomy. The results of activity screening indicated that there are two strains of Chaetomium globosum with higher activity to Mucor miehei; in addition to these two C. globosum strains, we screened another two cytotoxic strains, penicillium sp. and Alternaria sp., with significant toxic effects on Artemia salina.2. Endophytic strain Chaetomium globosum showed marked antifungal and cytotoxic activities to Mucor miehei and Artemia salina. We scaled up the fermentation and isolated the chemical constituents of mycelia and filtrate, respectively, by a bioassay-guided fractionation, thirteen metabolites were isolated from mycelium extract, of which nine compounds were elucidated as ergosterol(3-1), ergosta-4, 6, 8, 22-tetraen-3-one(3-2), ergosterol peroxide(3-3), succinic acid (3-4), chaetoglobosin A (3-5), chaetoglobosin C(3-6), allantoin (3-7), uracil (3-8), and epimwsokorwnone A (3-9), respectively. Among them, chaetoglobosin A and C displayed antifungal and cytotoxic activity; eight compounds were obtained from the filtrate and seven of them were elucidated as chaetomugilin D (3-10), chaetomugilin A(3-11), p-hydroxybenzonic acid (3-12), indole-3-acetic acid (3-13), succinic acid (3-14), mannitol (3-15), and cyclo-(Phe-Gly) (3-16). Among them, both chaetomugilins D and A are cytotoxic, and chaetomugilin D is a new compound. Chaetoglobosins A, C, and chaetomugilins A and D showed significant toxicity toward brine shrimp larvae at a concentration of 10μg·mL-1 with mortality rates of 83.4%, 75.3%, 78.3%, and 75.2%, respectively. Chaetoglobosins A and C exhibited marked inhibitory effects on Mucor miehei with observed zones of inhibition of 25.4 and 15.3mm in diameter, respectively, at 10μg·disk^-1.3. Endophytic strain Chaetomium globosum ZY-22 showed marked antifungal and cytotoxic activities to Mucor miehei and Artemia salina. Under a bioassay-guided fractionation, twelve metabolites were isolated from mycelium extract, and ten compounds of them were elucidated as globosterol (4-1), uracil (4-2), cerebroside B (4-3), cerebroside C (4-4), chaetoglobosin A (4-5), ergosta-4, 6, 8, 22-tetraen-3-one (4-7), 9(11)-dehyoergosterol peroxide (4-6), allantoin (4-8), adenosine (4-9), and indole-3- carboxylic acid (4-10). Among them, chaetoglobosin A is antifungal and cytotoxic, and globosterol is a new C29-steroid with a novel side chain; five compounds were obtained from the filtrate and were elucidated as chaetomugilin D (4-11), 3-methylorsellinic acid (4-13), thymine (4-14), o-coumaric acid (4-15) and conjectural structure chaetoviridin C (4-12). Among them, both 4-11 and 4-12 are cytotoxic, and 4-12 showed toxicity toward brine shrimp larvae at a concentration of 10μg·mL^-1 with mortality rates of 59.5%.4. Endophytic strain Alternaria No.28 showed marked cytotoxic effect on Artemia salina. Ten compounds were isolated from the combined extracts of mycelium and filtrate and eight compounds of them were elucidated as alterperylenol (5-1), altertoxin I (5-2), alternariol monomethyl ether (5-3), alternariol (5-4), vivotoxin II (5-5), vivotoxin (5-6), ergosterol (5-7), and ergosta-4, 6, 8, 22-tetraen-3-one (5-8). Among them, vivotoxin II and vivotoxin are the main cytotoxic constituents with mortality rates of 73.6% and 68.9%, respectively, at a concentration of 10μg·mL-1.5. The endophytic strain Penicillium No.97 showed marked effect on fresh hatched artemia salina. Under a bioassay-guided fractionation, thirteen metabolites were isolated from the combined extracts of mycelium and filtrate and nine structures of them were elucidated as fructigenine A (6-1), anthranilamide (6-2), anthranilic acid (6-3), ferulic acid (6-4), p-hydroxybenzonic acid (6-8),β-sitosterol (6-5), indole-3-acetic acid (6-6), 3-methylorsellinic acid (6-9), orsellinic acid (6-10) and mixture (6-7). Fructigenine A is cytotoxic, with mortality rate of 66.5%, at a concentration of 10μg·mL^-1.6. The marine actinomycete Actinomycetes No.125 showed marked growth inhibition on Mucor miehei and fresh hatched artemia salina. Under a bioassay-guided fractionation, seven metabolites were isolated from the combined extracts of mycelium and filtrate and six compounds were established as hopen B (7-1),β-sitosterol (7-2), 4-hydroxy-benzeneacetic acid (7-3), kitamycin A (7-4), antimycin A1b (7-5) and blastmycin (7-6). The mortality rates of three cytotoxic constituents kitamycin A, antimycin A1b and blastmycin against artemia salina are 88.4%, 78.3% and 74.3%, respectively, at a concentration of 10μg·mL-1; they also exhibited remarkable inhibitory effects on Mucor miehei with observed zones of inhibition of 29.4, 22.3 and 26.3 mm in diameter, respectively, at 10μg·disk-1.7. The edible mushroom Cortinarius rufo-olivaceus belongs to the family Cortinariaceae. Sixteen anthraquinone metabolites were isolated from the EtOAc extract of the fruiting bodies of this fungus and thirteen compounds were elucidated as rufoolivacin (8-1), rufoolivacin C (8-2), rufoolivacin D (8-3), rufoolivacin E (8-4), physcion (8-6), (-)-(3S)-torosachrysone- 8-O-methyl ether (8-5), torachrysone-8 -O-methyl ether (8-8), 1-hydroxy-5, 7-dimethoxy- 3- methylanthraquinone (8-7), citreorosein 6, 8-dimethyl ether (8-9), sinapiquinone (8-10), (3S, 3'S, P)-anthydrophlegmacin-9,10-quinone-8'-O-methyl ether (8-11), ergosterol (8-12) and ergosterol peroxide (8-13). Rufoolivacins C, D, and E are new compounds.As a conclusion, the chemical constituents and bioactivities of four endophytic fungi isolated from the plant Ginkgo biloba L., one marine actinomycete and one higher fungus were studied in this thesis. The present results established the basis for discovery of bioactive lead compounds and provided theoretical foundation for further utilization of the microbial resources studied.