Synthesis and Biological Evaluation of 6-[(1 R)-1-Hydroxyethyl]-2,4a( R),6( S),8a( R)-tetrahydropyrano-[3,2- b]-pyran-2-one and Structural Analogues of the Putative Structure of Diplopyrone.

The phytotoxin diplopyrone is considered to be the main phytotoxin in a fungus that is responsible for cork oak decline. A carbohydrate-based synthesis of the enantiomer of the structure proposed for diplopyrone has been developed from a commercially available derivative of d-galactose. Key steps in the synthesis are a highly stereoselective pyranose chain-extension based on methyltitanium, preparation of a vinyl glycoside via Isobe C-alkynylation-rearrangement/reduction, and RCM-based pyranopyran construction. Crystallographic and NMR analysis confirms an earlier report that the structure originally proposed for diplopyrone may require revision. Structural analogues were prepared for biological evaluation, the most promising being a pyranopyran nitrile synthesized from tri- O-acetyl-d-galactal by Ferrier cyanoglycosidation, Wittig chain extension, and lactonization. Biological assays revealed potent antibacterial activity for the nitrile analogue against common bacterial pathogens Edwardsiella ictaluri and Flavobacterium columnare that cause enteric septicemia (ESC) and columnaris disease, respectively, in catfish. The IC50 value of 0.002 against E. ictaluri indicates approximately 100 times greater potency than the antibiotic florfenicol used commercially for this disease. Phytotoxic activity for all three target compounds against duckweed was also observed. The antibiotic and phytotoxic activities of the new pyranopyrans synthesized in this study demonstrate the potential of such compounds as antibiotics and herbicides.

Fungicidal Properties of Some Novel Trifluoromethylphenyl Amides.

Trifluoromethylphenyl amides (TFMPAs) were designed and synthesized as potential pesticides. Thirty-three structures were evaluated for fungicidal activity against three Colletotrichum species using direct bioautography assays. Active compounds were subsequently tested against C. fragariae, C. gloeosporioides, C. acutatum, Phomopsis obscurans, P. viticola, Botrytis cinerea and Fusarium oxysporum. The study identified 2-chloro-N-[2,6-dichloro-4-(trifluoromethyl)phenyl]acetamide (7a) as showing the strongest antifungal activity, and the broadest activity spectrum in this set against Colletotrichum acutatum (at 48 and 72 h) and Phomopsis viticola (at 144 h). The presence of triethylamine in its complex with N-[2,6-dichloro-4-(trifluoromethyl)phenyl]-2,2,3,3,3-pentafluoropropanamide (7b') played an important role in the bioactivity, and depending on the concentration or fungal species it showed higher or lower activity than the parent amide. X-Ray crystallography has shown that the complex (7b') is an ion pair, (C10 H2 Cl2 F8 NO)- (C6 H16 N)+ , where a proton is transferred from the amide nitrogen to the triethylamine nitrogen and then connected by hydrogen bonding to the acyl oxygen (N-H 0.893 Å; H⋅⋅⋅O 1.850 Å; N⋅⋅⋅O 2.711 Å; N-H⋅⋅⋅O 161.2(13)°). Although none of these compounds were better than standards, this work revealed some potential lead structures for further development of active novel compounds.

Bioassay-Guided Isolation and Structure Elucidation of Fungicidal and Herbicidal Compounds from Ambrosia salsola (Asteraceae).

The discovery of potent natural and ecofriendly pesticides is one of the focuses of the agrochemical industry, and plant species are a source of many potentially active compounds. We describe the bioassay-guided isolation of antifungal and phytotoxic compounds from the ethyl acetate extract of Ambrosia salsola twigs and leaves. With this methodology, we isolated and identified twelve compounds (four chalcones, six flavonols and two pseudoguaianolide sesquiterpene lactones). Three new chalcones were elucidated as (S)-ß-Hydroxy-2',3,4,6'-tetrahydroxy-5-methoxydihydrochalcone (salsolol A), (S)-ß-Hydroxy-2',4,4',6'-tetrahydroxy-3-methoxydihydrochalcone (salsolol B), and (R)-α, (R)-ß-Dihydroxy-2',3,4,4',6'- pentahydroxydihydrochalcone (salsolol C) together with nine known compounds: balanochalcone, six quercetin derivatives, confertin, and neoambrosin. Chemical structures were determined based on comprehensive direct analysis in real time-high resolution mass spectrometry (HR-DART-MS), as well as 1D and 2D NMR experiments: Cosy Double Quantum Filter (DQFCOSY), Heteronuclear Multiple Quantum Coherence (HMQC) and Heteronuclear Multiple Bond Coherence (HMBC), and the absolute configurations of the chalcones were confirmed by CD spectra analysis. Crystal structure of confertin was determined by X-ray diffraction. The phytotoxicity of purified compounds was evaluated, and neoambrosim was active against Agrostis stolonifera at 1 mM, while confertin was active against both, Lactuca sativa and A. stolonifera at 1 mM and 100 µM, respectively. Confertin and salsolol A and B had IC50 values of 261, 275, and 251 µM, respectively, against Lemna pausicotata (duckweed). The antifungal activity was also tested against Colletotrichum fragariae Brooks using a thin layer chromatography bioautography assay. Both confertin and neoambrosin were antifungal at 100 µM, with a higher confertin activity than that of neoambrosin at this concentration.

Antimycobacterial and antimalarial activities of endophytic fungi associated with the ancient and narrowly endemic neotropical plant Vellozia gigantea from Brazil.

BACKGROUND: Endophytic fungi, present mainly in the Ascomycota and Basidiomycota phyla, are associated with different plants and represent important producers of bioactive natural products. Brazil has a rich biodiversity of plant species, including those reported as being endemic. Among the endemic Brazilian plant species, Vellozia gigantea (Velloziaceae) is threatened by extinction and is a promising target to recover endophytic fungi. OBJECTIVE: The present study focused on bioprospecting of bioactive compounds of the endophytic fungi associated with V. gigantea, an endemic, ancient, and endangered plant species that occurs only in the rupestrian grasslands of Brazil. METHODS: The capability of 285 fungal isolates to produce antimicrobial and antimalarial activities was examined. Fungi were grown at solid-state fermentation to recover their crude extracts in dichloromethane. Bioactive extracts were analysed by chromatographic fractionation and NMR and displayed compounds with antimicrobial, antimycobacterial, and antimalarial activities. FINDINGS: Five fungi produced antimicrobial and antimalarial compounds. Extracts of Diaporthe miriciae showed antifungal, antibacterial, and antimalarial activities; Trichoderma effusum displayed selective antibacterial activity against methicillin-resistant Staphylococcus aureus and Mycobacterium intracellulare; and three Penicillium species showed antibacterial activity. D. miriciae extract contained highly functionalised secondary metabolites, yielding the compound epoxycytochalasin H with high antimalarial activity against the chloroquine-resistant strain of Plasmodium falciparum, with an IC50 approximately 3.5-fold lower than that with chloroquine. MAIN CONCLUSION: Our results indicate that V. gigantea may represent a microhabitat repository hotspot of potential fungi producers of bioactive compounds and suggest that endophytic fungal communities might be an important biological component contributing to the fitness of the plants living in the rupestrian grassland.

Fungi associated with rocks of the Atacama Desert: taxonomy, distribution, diversity, ecology and bioprospection for bioactive compounds.

This study assessed the diversity of cultivable rock-associated fungi from Atacama Desert. A total of 81 fungal isolates obtained were identified as 29 Ascomycota taxa by sequencing different regions of DNA. Cladosporium halotolerans, Penicillium chrysogenum and Penicillium cf. citrinum were the most frequent species, which occur at least in four different altitudes. The diversity and similarity indices ranged in the fungal communities across the latitudinal gradient. The Fisher-α index displayed the higher values for the fungal communities obtained from the siltstone and fine matrix of pyroclastic rocks with finer grain size, which are more degraded. A total of 23 fungal extracts displayed activity against the different targets screened. The extract of P. chrysogenum afforded the compounds α-linolenic acid and ergosterol endoperoxide, which were active against Cryptococcus neoformans and methicillin-resistance Staphylococcus aureus respectively. Our study represents the first report of a new habitat of fungi associated with rocks of the Atacama Desert and indicated the presence of interesting fungal community, including species related with saprobes, parasite/pathogen and mycotoxigenic taxa. The geological characteristics of the rocks, associated with the presence of rich resident/resilient fungal communities suggests that the rocks may provide a favourable microenvironment fungal colonization, survival and dispersal in extreme conditions.

Synthesis and Biological Evaluation of 3,5-Dimethoxystilbene Analogs.

In our continuing effort to discover natural product-based pest management agents, derivatives of 3,5-dimethoxystilbene were synthesized yielding 27 new and six known compounds. Compounds 11 and 12 showed strong Aedes aegypti larvicidal activity (LC50 45.31 and 49.93 µm, respectively). Furthermore, 11 and 12 exhibited high effectiveness against larvae of pesticide-susceptible and pyrethroid-resistant strains of Ae. aegypti; activity against the adult mosquitoes was low. Compounds 6f, 6g, and 6i at either 83.3 or 166.7 µg/ml reduced the mobility of second-stage juveniles (J2) of the root-knot nematode (Meloidogyne incognita) that hatched from eggs immersed in the test compounds for 7 days. However, there was little or no effect on J2 placed directly into these compounds, and none of the analogs suppressed M. incognita egg hatch. The compounds were tested for inhibition of some agriculturally important fungi; 6a, 7a, and 7e demonstrated strong inhibition of Colletotrichum species. Activity of the stilbenes against some human pathogens was also explored; 11, 12, and 16 showed moderate inhibitory activity against Cryptococcus neoformans, Staphylococcus aureus, methicillin-resistant S. aureus, and Mycobacterium intracellulare. Except for 11 and 12, which were active against mosquito larvae and some human pathogens, no single analog demonstrated activity in all the tests, indicating specific activities. Synthesis of the analogs and structure-activity relationships are discussed.

Molecular Phylogeny, Diversity, and Bioprospecting of Endophytic Fungi Associated with wild Ethnomedicinal North American Plant Echinacea purpurea (Asteraceae).

The endophytic fungal community associated with the ethnomedicinal plant Echinacea purpurea was investigated as well as its potential for providing antifungal compounds against plant pathogenic fungi. A total of 233 endophytic fungal isolates were obtained and classified into 42 different taxa of 16 genera, of which Alternaria alternata, Colletotrichum dematium, and Stagonosporopsis sp. 2 are the most frequent colonizers. The extracts of 29 endophytic fungi displayed activities against important phytopathogenic fungi. Eight antifungal extracts were selected for chemical analysis. Forty fatty acids were identified by gas chromatography-flame-ionization detection (GC-FID) analysis. The compounds (-)-5-methylmellein and (-)-(3R)-8-hydroxy-6-methoxy-3,5-dimethyl-3,4-dihydroisocoumarin were isolated from Biscogniauxia mediterraneaEPU38CA crude extract. (-)-5-Methylmellein showed weak activity against Phomopsis obscurans, P. viticola, and Fusarium oxysporum, and caused growth stimulation of C. fragariae, C. acutatum, C. gloeosporioides, and Botrytis cinerea. (-)-(3R)-8-Hydroxy-6-methoxy-3,5-dimethyl-3,4-dihydroisocoumarin appeared slightly more active in the microtiter environment than 5-methylmellein. Our results indicate that E. purpurea lives symbiotically with different endophytic fungi, which are able to produce bioactive fatty acids and aromatic compounds active against important phytopathogenic fungi. The detection of the different fatty acids and aromatic compounds produced by the endophytic community associated with wild E. purpurea suggests that it may have intrinsic mutualistic resistance against phytopathogen attacks in its natural environment.

Diversity and bioprospection of fungal community present in oligotrophic soil of continental Antarctica.

We surveyed the diversity and capability of producing bioactive compounds from a cultivable fungal community isolated from oligotrophic soil of continental Antarctica. A total of 115 fungal isolates were obtained and identified in 11 taxa of Aspergillus, Debaryomyces, Cladosporium, Pseudogymnoascus, Penicillium and Hypocreales. The fungal community showed low diversity and richness, and high dominance indices. The extracts of Aspergillus sydowii, Penicillium allii-sativi, Penicillium brevicompactum, Penicillium chrysogenum and Penicillium rubens possess antiviral, antibacterial, antifungal, antitumoral, herbicidal and antiprotozoal activities. Bioactive extracts were examined using (1)H NMR spectroscopy and detected the presence of secondary metabolites with chemical shifts. Our results show that the fungi present in cold-oligotrophic soil from Antarctica included few dominant species, which may have important implications for understanding eukaryotic survival in cold-arid oligotrophic soils. We hypothesize that detailed further investigations may provide a greater understanding of the evolution of Antarctic fungi and their relationships with other organisms described in that region. Additionally, different wild pristine bioactive fungal isolates found in continental Antarctic soil may represent a unique source to discover prototype molecules for use in drug and biopesticide discovery studies.

Phomalactone from a Phytopathogenic Fungus Infecting ZINNIA elegans (ASTERACEAE) Leaves.

Zinnia elegans Jacq. plants are infected by a fungus that causes dark red spots with necrosis on leaves, particularly in late spring to the middle of summer in the Mid-South of the United States. This fungal disease causes the leaves to wilt and eventually kills the plant. The fungus was isolated, cultured in potato dextrose broth, and identified as Nigrospora sphaerica by molecular techniques. Two major lactone metabolites (phomalactone and catenioblin A) were isolated from liquid culture of N. sphaerica isolated from Z. elegans. When injected into leaves of Z. elegans, phomalactone caused lesions similar to those of the fungus. The lesion sizes were proportional to the concentration of the phomalactone. Phomalactone, but not catenioblin A, was phytotoxic to Z. elegans and other plant species by inhibition of seedling growth and by causing electrolyte leakage from photosynthetic tissues of both Z. elegans leaves and cucumber cotyledons. This latter effect may be related to the wilting caused by the fungus in mature Z. elegans plants. Phomalactone was moderately fungicidal to Coletotrichum fragariae and two Phomopsis species, indicating that the compound may keep certain other fungi from encroaching into plant tissue that N. sphaerica has infected. Production of large amounts of phomalactone by N. sphaerica contributes to the pathogenic behavior of this fungus, and may have other ecological functions in the interaction of N. sphaerica with other fungi. This is the first report of isolation of catenioblin A from a plant pathogenic fungus. The function of catenioblin A is unclear, as it was neither significantly phyto- nor fungitoxic.

Chemical composition and biological effects of Artemisia maritima and Artemisia nilagirica essential oils from wild plants of western Himalaya.

Artemisia species possess pharmacological properties that are used for medical purposes worldwide. In this paper, the essential oils from the aerial parts of Artemisia nilagirica and Artemisia maritima from the western Indian Himalaya region are described. The main compounds analyzed by simultaneous GC/MS and GC/FID were camphor and 1,8-cineole from A. maritima, and camphor and artemisia ketone from A. nilagirica. Additionally, the oils were evaluated for their antibacterial, antifungal, mosquito biting deterrent, and larvicidal activities. A. nilagirica essential oil demonstrated nonselective antifungal activity against plant pathogens Colletotrichum acutatum, Colletotrichum fragariae, and Colletotrichum gloeosporioides, whereas A. maritima did not show antifungal activity. Both Artemisia spp. exhibited considerable mosquito biting deterrence, whereas only A. nilagirica showed larvicidal activity against Aedes aegypti. Antibacterial effects assessed by an agar dilution assay demonstrated greater activity of A. maritima essential oil against Staphylococcus aureus and Pseudomonas aeruginosa compared to A. nilagirica.

Biting deterrence, repellency, and larvicidal activity of Ruta chalepensis (Sapindales: Rutaceae) essential oil and its major individual constituents against mosquitoes.

The essential oil from aerial parts of Ruta chalepensis L. (Sapindales: Rutaceae) was obtained by hydrodistillation, and its chemical profile was identified using gas chromatography and gas chromatography-mass spectrometry. Compounds, 2-undecanone (43.2%), 2-nonanone (27.9%), and 2-nonyl acetate (10.6%) were the major constituents of the oil. Biting deterrent activity of R. chalepensis essential oil at 10 and 50 microg/cm2, 2-undecanone at 8.5 microg/cm2, 2-nonanone at 9 microg/cm2, and 2-nonyl acetate at 9.3 microg/cm2 was similar to DEET (N, N-diethyl-meta-toluamide) at 4.8 microg/cm2, against Aedes aegypti L. Biting deterrent activity of R. chalepensis oil at 50 microg/cm2 against Anopheles quadrimaculatus Say was statistically similar to DEET at 4.8 microg/cm2, whereas the activity was lower in the other compounds tested. In cloth patch assay, R. chalepensis essential oil was effective at 187 microg/cm2, whereas 2-undecanone was effective at 108.9 microg/cm2 against Ae. aegypti. In larval bioassays, 2-undecanone showed similar toxicity whereas toxicity of R. chalepensis essential oil and 2-nonanone was higher at 24-h posttreatment at the LD50 in An. quadrimaculatus than Ae. aegypti. This study revealed that R. chalepensis essential oil and its major compounds were active biting deterrents against Ae. aegypti at higher application rates whereas only the essential oil showed activity similar to DEET against An. quadrimaculatus. 2-undecanone was the most active compound in in vivo repellency bioassay against Ae. aegypti. Chemical composition of R. chalepensis essential oil varies because of plant production and harvest practices, and the activity level of the essential oil may depend on the source of the sample.

Insecticidal, repellent and fungicidal properties of novel trifluoromethylphenyl amides.

Twenty trifluoromethylphenyl amides were synthesized and evaluated as fungicides and as mosquito toxicants and repellents. Against Aedes aegypti larvae, N-(2,6-dichloro-4-(trifluoromethyl)phenyl)-3,5-dinitrobenzamide (1e) was the most toxic compound (24 h LC50 1940 nM), while against adults N-(2,6-dichloro-4-(trifluoromethyl)phenyl)-2,2,2-trifluoroacetamide (1c) was most active (24 h LD50 19.182 nM, 0.5 µL/insect). However, the 24 h LC50 and LD50 values of fipronil against Ae. aegypti larvae and adults were significantly lower: 13.55 nM and 0.787 × 10(-4) nM, respectively. Compound 1c was also active against Drosophila melanogaster adults with 24 h LC50 values of 5.6 and 4.9 µg/cm(2) for the Oregon-R and 1675 strains, respectively. Fipronil had LC50 values of 0.004 and 0.017 µg/cm(2) against the two strains of D. melanogaster, respectively. In repellency bioassays against female Ae. aegypti, 2,2,2-trifluoro-N-(2-(trifluoromethyl)phenyl)acetamide (4c) had the highest repellent potency with a minimum effective dosage (MED) of 0.039 µmol/cm(2) compared to DEET (MED of 0.091 µmol/cm(2)). Compound N-(2-(trifluoromethyl)phenyl)hexanamide (4a) had an MED of 0.091 µmol/cm(2) which was comparable to DEET. Compound 4c was the most potent fungicide against Phomopsis obscurans. Several trends were discerned between the structural configuration of these molecules and the effect of structural changes on toxicity and repellency. Para- or meta- trifluoromethylphenyl amides with an aromatic ring attached to the carbonyl carbon showed higher toxicity against Ae. aegypti larvae, than ortho- trifluoromethylphenyl amides. Ortho- trifluoromethylphenyl amides with trifluoromethyl or alkyl group attached to the carbonyl carbon produced higher repellent activity against female Ae. aegypti and Anopheles albimanus than meta- or para- trifluoromethylphenyl amides. The presence of 2,6-dichloro- substitution on the phenyl ring of the amide had an influence on larvicidal and repellent activity of para- trifluoromethylphenyl amides.

Characterization of volatile constituents from Origanum onites and their antifungal and antibacterial activity.

Essential oils obtained by hydrodistillation (HD) and microwave-assisted HD (MWHD) of Origanum onites aerial parts were analyzed by GC and GCIMS. Thirty-one constituents representing 98.6% of the water-distilled oil and 52 constituents representing 99.6% of the microwave-distilled oil were identified. Carvacrol (76.8% HD and 79.2% MWHD) and thymol (4.7% HD and 4.4% MWHD) were characterized as major constituents in both essential oils. Separation of carvacrol and thymol was achieved by overpressured layer chromatography. HPTLC and TLC separations were also compared. Essential oils were evaluated for antifungal activity against the strawberry anthracnose-causing fungal plant pathogens Colletotrichum acutatum, C. fragariae, and C. gloeosporioides using a direct overlay bioautography assay. Furthermore, main oil components carvacrol and thymol were then evaluated for antifungal activity; only carvacrol demonstrated nonselective antifungal activity against the three Colletotrichum species. Thymol and carvacrol were subsequently evaluated in a 96-well microdilution broth assay against Phomopsis obscurans, Fusarium oxysporum, three Colletotrichum species, and Botrytis cinerea. No activity was observed against any of the three Colletotrichum species at or below 30 pM. However, thymol demonstrated antifungal activity and produced 31.7% growth inhibition of P. obscurans at 120 h and 0.3 pM, whereas carvacrol appeared inactive. Thymol and carvacrol at 30 pM showed 51.5 and 36.9% growth inhibition of B. cinerea at 72 h. The mechanism of antibacterial activity was studied in a bioautography-based BioArena system. Thymol and carvacrol showed similar inhibition/killing effect against Bacillus subtilis soil bacteria; the action could be enhanced by the formaldehyde generator and transporter copper (II) ions and could be decreased in the presence of L-arginine, a formaldehyde capturer. Results indicated that Origanum essential oils and its major components thymol and carvacrol appear to generate antimicrobial activity through a mechanism of action where formaldehyde and its reaction products are produced.

Synthesis and biological activity of substituted urea and thiourea derivatives containing 1,2,4-triazole moieties.

A series of novel thiourea and urea derivatives containing 1,2,4-triazole moieties were synthesized and evaluated for their antifungal and larvicidal activity. Triazole derivatives 3a-e and 4a-e were synthesized by reacting thiocarbohydrazide with thiourea and urea compounds 1a-e and 2a-e, respectively, in a 130-140 °C oil bath. The proposed structures of all the synthesized compounds were confirmed using elemental analysis, UV, IR, 1H-NMR and mass spectroscopy. All compounds were evaluated for antifungal activity against plant pathogens, larvicidal and biting deterrent activity against the mosquito Aedes aegypti L. and in vitro cytotoxicity and anti-inflammatory activity against some human cell lines. Phomopis species were the most sensitive fungi to these compounds. Compounds 1b, 1c, 3a and 4e demonstrated selectively good activity against Phomopis obscurans and only 1b and 4e showed a similar level of activity against P. viticola. Compound 3d, with a LD50 value of 67.9 ppm, followed by 1c (LD50 = 118.8 ppm) and 3e (LD50 = 165.6 ppm), showed the highest toxicity against Aedes aegypti larvae. Four of these compounds showed biting deterrent activity greater than solvent control, with the highest activity being seen for 1c, with a proportion not biting (PNB) value of 0.75, followed by 1e, 2b and 1a. No cytotoxicity was observed against the tested human cancer cell lines. No anti-inflammatory activity was observed against NF-kB dependent transcription induced by phorbol myristate acetate (PMA) in human chondrosarcoma cells.

Chemical composition, antifungal and insecticidal activities of Hedychium essential oils.

The antimicrobial properties of essential oils have been documented, and their use as "biocides" is gaining popularity. The aims of this study were to analyze the chemical composition and assess the biological activities of Hedychium essential oils. Oils from 19 Hedychium species and cultivars were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) techniques. The antifungal and insecticidal activities of these oils were tested against Colletotrichum acutatum, C. fragariae, and C. gloeosporioides, and three insects, the azalea lace bug (Stephanitis pyrioides), the yellow fever mosquito (Aedes aegypti), and the red imported fire ant (Solenopsis invicta). Hedychium oils were rich in monoterpenes and sesquiterpenes, especially 1,8-cineole (0.1%-42%), linalool (<0.1%-56%), a-pinene (3%-17%), b-pinene (4%-31%), and (E)-nerolidol (0.1%-20%). Hedychium oils had no antifungal effect on C. gloeosporioides, C. fragariae, and C. acutatum, but most Hedychium oils effectively killed azalea lace bugs. The oils also show promise as an adult mosquito repellent, but they would make rather poor larvicides or adulticides for mosquito control. Hedychium oils acted either as a fire ant repellent or attractant, depending on plant genotype and oil concentration.

One-step multiple component isolation from the oil of Crinitaria tatarica (Less.) Sojak by preparative capillary gas chromatography with characterization by spectroscopic and spectrometric techniques and evaluation of biological activity.

Gas chromatographic analysis revealed that the oil of Crinitaria tatarica was rich in sabinene (32.1%), ß-pinene (8.8%), and two unknown (M+200) compounds (I) and (II) (21.4% and 3.4%). One-step multiple fractionation of the oil and separation of two unknown constituents were performed using preparative capillary gas chromatography connected to preparative fraction collector system. This combination allowed separation and recover of sufficient quantities of two unknown compounds with high purity from complex oil matrix. Separation conditions (column temperature, cooling temperature, flow rate, injection volume, cut time) were optimized to achieve the best isolation and successful collection. The target compounds were separated from the oil using a HP Innowax (Walt & Jennings Scientific, Wilmington, DE, USA) preparative capillary column in rapid one-step manner with 95.0% purity. Trapping of the isolated compounds in collector system was facilitated by cooling with liquid nitrogen. Structure determination was accomplished by spectral analysis including ultraviolet, nuclear magnetic rezonance, and high-resolution electrospray ionization mass spectrometry. Z- (I) and E-artemidin (II) were isolated for the first time from this species. Crinitaria tatarica oil and Z- (I) and E-artemidin (II) were evaluated for biological activity.

Antifungal activity of extracts from endophytic fungi associated with Smallanthus maintained in vitro as autotrophic cultures and as pot plants in the greenhouse.

The endophytic fungal assemblages associated with Smallanthus sonchifolius (Poepp.) H. Rob. and Smallanthus uvedalius (L.) Mack. ex Small growing in vitro autotrophic cultures and in the greenhouse were identified and evaluated for their ability to produce bioactive compounds. A total of 25 isolates were recovered that were genetically closely related to species of the genera Bionectria , Cladosporium , Colletotrichum , Fusarium , Gibberella , Hypocrea , Lecythophora , Nigrospora , Plectosphaerella , and Trichoderma . The endophytic assemblages of S. sonchifolius presented a greater diversity than the group isolated from S. uvedalius and demonstrated the presence of dominant generalist fungi. Extracts of all fungi were screened against the fungal plant pathogens. Ten extracts (41.6%) displayed antifungal activities; some of them had a broad antifungal activity. The phylotypes Lecythophora sp. 1, Lecythophora sp. 2, and Fusarium oxysporum were isolated from in vitro autotrophic cultures and displayed antifungal activity. The presence of bioactive endophytic fungi within S. sonchifolius and S. uvedalius suggests an ecological advantage against pathogenic attacks. This study revealed reduced numbers of endophytes in association with both Smallanthus species in controlled cultivation conditions compared with the endophytic communities of hosts collected in the wild environments. Even as reduced endophytic communities, these fungi continue to provide chemical protection for the host.

Discovery of Niphimycin C from Streptomyces yongxingensis sp. nov. as a Promising Agrochemical Fungicide for Controlling Banana Fusarium Wilt by Destroying the Mitochondrial Structure and Function.

Banana Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is the most destructive soil-borne fungal disease. Tropical race 4 (Foc TR4), one of the strains of Foc, can infect many commercial cultivars, which represents a threat to global banana production. Currently, there are hardly any effective chemical fungicides to control the disease. To search for natural product-based fungicides for controlling banana Fusarium wilt, we identified a novel strain Streptomyces yongxingensis sp. nov. (JCM 34965) from a marine soft coral, from which a bioactive compound, niphimycin C, was isolated using an activity-guided method. Niphimycin C exhibited a strong antifungal activity against Foc TR4 with a value of 1.20 µg/mL for EC50 and obviously inhibited the mycelial growth and spore germination of Foc TR4. It caused the functional loss of mitochondria and the disorder of metabolism of Foc TR4 cells. Further study showed that niphimycin C reduced key enzyme activities of the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC). It displayed broad-spectrum antifungal activities against the selected 12 phytopathogenic fungi. In pot experiments, niphimycin C reduced the disease indexes in banana plantlets and inhibited the infection of Foc TR4 in roots. Hence, niphimycin C could be a promising agrochemical fungicide for the management of fungal diseases.

Triterpenoidal saponins: bioactive secondary metabolites from Zygophyllum coccineum.

Phytochemical investigation of the aerial parts of Zygophyllum coccineum L. led to the isolation of nine ursane-type triterpene saponins (1- 9), including the new one; zygophylloside S (1), together with a known flavonoid glycoside (10) and a sterol glycoside (11). The isolated compounds were tested for antifungal activity against several important plant pathogens and for insecticidal activity against two important mosquito species. Among the isolated compounds 1, 3, 5, 6, and 9 showed 32-77 % fungal growth inhibition at a concentration of 30 µM against Phomopsis viticola. Compound 9 showed 90 % and 80 % mosquitocidal activity at 3.1 µg/0.5 µL against Aedes aegypti and Culex quinquefasciatus, respectively.

Antifungal metabolites from the roots of Diospyros virginiana by overpressure layer chromatography.

A preparative overpressure layer chromatography (OPLC) method was successfully used for the separation of two new natural compounds, 4-hydroxy-5,6-dimethoxynaphthalene-2-carbaldehyde (1) and 12,13-didehydro-20,29-dihydrobetulin (2) together with nine known compounds, including 7-methyljuglone (3), diospyrin (4), isodiospyrin (5), shinanolone (6), lupeol (7), betulin (8), betulinic acid (9), betulinaldehyde (10), and ursolic acid (11) from the acetone extract of the roots of Diospyros virginiana. Their identification was accomplished by 1D- and 2D-NMR spectroscopy and HR-ESI-MS methods. All the isolated compounds were evaluated for their antifungal activities against Colletotrichum fragariae, C. gloeosporioides, C. acutatum, Botrytis cinerea, Fusarium oxysporum, Phomopsis obscurans, and P. viticola using in vitro micro-dilution broth assay. The results indicated that compounds 3 and 5 showed high antifungal activity against P. obscurans at 30 µM with 97.0 and 81.4% growth inhibition, and moderate activity against P. viticola (54.3 and 36.6%). It appears that an optimized OPLC system offers a rapid and efficient method of exploiting bioactive natural products.