Chemical Constituents And Their Bioactivities Of Four Endophytic Fungi From Medicinal Plants Of Meliaceae

Endophytic fungi of medicinal plants can produce a large number of novel active secondary metabolites,and have become an important resource for searching novel active natural products.Thus,the chemical constituents and their bioactivities of four endophytic fungi,isolated from two Chinese medicinal plants Melia azedarach and Toona sinensis were studied.By using repeated chromatographic column on silica gel,Sephadex LH-20,revesed phase C18,PTLC and HPLC,58 compounds,including 6 new compounds and 3 new natural products were obtained from the large scale fermentation cultures of the microbial materials.The structures were elucidated on the basis of spectroscopic analysis(1H-NMR?13C-NMR?HSQC?1H-1H COSY?HMBC?NOESY?(HR-ESI)MS and X-ray).The results are concluded as follows:1.Two new metabolites,an ?-pyridone derivative,3-hydroxy-2-methoxy-5-methylpyridin-2(1H)-one(1-1)and a new ceramide derivative 3-hydroxy-N-(1-hydroxy-3-methylpentan-2-yl)-5-oxohexanamide(1-2),and a new natural product,3-hydroxy-N-(1-hydroxy-4-methylpentan-2-yl)-5-oxohexanamide(1-3),along with 15 known compounds:pycnophorin(1-4),stemphyperylenol(1-5),altenuene(1-6),chaetoglobosin C(1-7),chaetoglobosin F(1-8),altenusin(1-9),alternariol methyl ether(1-10),alternariol(1-11),5'-methoxy-6-methyl-biphenyl-3,4,3'-triol(1-12),7-hydroxy-1-isochromanone(1-13),ergosterol peroxide(1-14),?-daucosterol(1-15),5-(hydroxymethyl)-1H-pyrrole-2-carbaldehyde(1-16),5-hydroxymethylfuran-2-carbaldehyde(1-17),cerebroside C(1-18),3-O-?-D-glucopyranosyl-spinasterol(1-19),methyl octadca-8,12-dienoate(1-20),glyceryl linoleate(1-21),p-hydroxyphenylethanol(1-22),3-methylpentane-1,2-diol(1-23)and succinic acid(1-24)were isolated from the solid culture of the endophytic fungus Botryosphaeria dothidea KJ-1,collected from the stems of Melia azedarach L.These metabolites were evaluated in vitro for anti-microbial,anti-oxidant,and cytotoxicity activities.Stemphyperylenol(1-5)displayed a potent antifungal activity against the plant pathogen Alternaria solani with minimum inhibitory concentration(MIC)value of 1.57 ?M comparable to the commonly used fungicide carbendazim.Both altenusin(1-9)and alternariol methyl ether(1-10)showed markedly DPPH radical scavenging activities,their IC50 were 17.6 ± 0.23 and 18.7 ± 0.18 ?M,respectively.In addition,stemphyperylenol(1-5)and altenuene(1-6)exhibited strong cytotoxicity against HCT116 cancer cell line with median inhibitory concentration(IC50)value of 3.13 ?M in comparison with the positive control etoposide(IC50 2.13 ?M).2.Six known metabolites,dianhydro-aurasperone C(2-1),isoaurasperone A(2-2),fonsecinone A(2-3),asperpyrone A(2-4),asperazine(2-5),rubrofusarin B(2-6)and a new natural product(R)-3-Hydroxybutanonitrile(2-7)were isolated from the extracts of the Aspergillus sp.KJ-9,an endophyte isolated from the stem bark of Melia azedarach.All isolated metabolites were evaluated in vitro against phytopathogenic fungi and pathogenetic bacteria.Fonsecinone A(2-3),asperpyrone A(2-4)and asperazine(2-5)significantly inhibited the growth of phytopathogenic fungus Alternaria solani with MIC of 6.25 ?M comparable to the commonly used fungicide hymexazol.Fonsecinone A(2-3)and 3-Hydroxybutanonitrile(2-7)showed potent antifungal activity against the phytopathogenic fungus Magnaporthe grisea or Gibberella saubinetti with MIC of 12.5 ?M respectively,both of which exceed the positive control hymexazol(MIC = 25 ?M).In addition,2-3 also strongly inhibited the growth of Staphyloccocus aureus and Bacillus subtilis with equal MICs of 25?M,approximate to positive control streptomycin sulfate(MIC = 12.5 ?M).3.A new compound phomopsis acid A(3-1)and a new natural product phomopsamide A(3-2),together with 11 know compounds:eugenitol(3-3),Cytosporone C(3-4),4-Hydroxyphenethyl alcohol(3-5),1-(4-Hydroxyphenyl)ethane-1,2-diol(3-6),N-(2-hydroxy-2-phenylethyl)Acetamide(3-7),phomopene(3-8),Methyl a-D-glucopyranoside(3-9),D-Galactitol(3-10),Methyl-?-D-galactofuranoside(3-11),Methyl 4-O-methyl-?-D-glucopyranosyl-(1?4)-?-D-glucopyranoside(3-12)and uridine(3-13)were isolated from the extracts of the Phomopsis sp.KY-9,an endophyte isolated from the leaf of Melia azedarach.3-1 is a long chain trans fatty acid;3-2 is a cyclic depsipeptide.Acetylation,isopropylidene reaction and acid hydrolysis were carried out to confirm the relative and absolute configuration of 3-2.All isolated metabolites were evaluated in vitro antifungal activities against phytopathogenic fungi and toxicity against brine shrimp.3-1,3-4?3-7 and 3-8 showed different antifungal activties against four phytopathogenic fungi(Fusarium solani,Gibberella saubinetti,Botrytis cinerea and Alternaria solani).3-1 exhibited potent antifungal activities against Fusarium solani with MIC values of 25 ?M,which exceeded to the positive control hymexazol(50 ?M).In addition,3-1?3-4 and 3-6-8 showed weak toxicity against brine shrimp.4.Two new cytochalasins,cytochalasin Z21(4-1)and cytochalasin Z22(4-2),and a new phenolic glycoside derivative toonaside(4-3),together with 11 know compounds:3,5-Dimethyl-8-(?-D-glucopyranosyloxy)-isochroman-1-one(4-4),5-O-?-D-glucopyranosyl-5-hydroxymellein(4-5),terricollenes A(4-6),16-?-D-mannopyranosyloxyisopimar-7-en-19-oic acid(4-7),2,2-bis[p-(2,3-dihydroxypropoxy)phenyl]propane(4-8),2-Butyl-3-methylsuccinic acid(4-9),adenosine(4-10),uridine(4-11),2'-deoxyuridine(4-12),4-Methyl-5,6-dihydro-2H-pyran-2-one(4-13)and thymidine(4-14)were isolated from the Xylariales cf.XC-16,an endophyte isolated from Toona sinensis.Most of metabolites were evaluated in vitro antifungal activities against phytopathogenic fungi and toxicity against brine shrimp.Compounds 4-3?4-7 showed different antifungal activties against four phytopathogenic fungi(Fusarium solani,Gibberella saubinetti,Botrytis cinerea and Alternaria solani).Among them,4-4 and 4-7 showed potent antifungal activities against phytopathogenic fungus Botrytis cinerea with both MICs of 50 ?M,which were comparable with positive control hymexazol.4-7 also displayed strong antifungal activity against Fusarium solani.In addition,these metabolites showed weak toxicity against brine shrimp.