Secondary metabolite chemistry of the marine-derived fungus Massarina sp., strain CNT-016

Chemical investigation of the culture broth extracts of the marine-derived fungus Massarina sp. (strain CNT-016) has yielded two secondary metabolites, spiromassaritone (1) and massariphenone (2), as well as the previously reported fungal metabolites 6-epi-5'-hydroxy-mycosporulone (3) and enalin A (4). The structures of these compounds were established by a variety of one- and two-dimensional NMR experiments, while the relative configuration of spiromassaritone (1) was determined by X-ray crystallographic methods. The fungal strain was isolated as a sterile mycelium from an ocean mud sample and identified using ITS sequence analysis.     

Transformation of a monoterpene ketone, piperitenone, and related terpenoids using Mucor piriformis

Biotransformation of piperitenone (I), 5,5-dimethyl-2-(1-methylethylidene)-cyclohexanone (II), and 2-(1-ethyl-1-propylidene)-5-methylcyclohexanone (III) was studied using a versatile fungal strain, Mucor piriformis. The organism initiates transformation of these compounds by hydroxylation at the allylic positions or at the tertiary carbon. Transformation of piperitenone (I) by this strain yielded 5-hydroxypiperitenone (Ic), 7-hydroxypiperitenone (Id), 7-hydroxypulegone (Ie), 10-hydroxypiperitenone (If), and 4-hydroxypiperitenone (Ig) as metabolites. It was possible to block some of the metabolic activities of the organism through structural modification of piperitenone (I). This was evidenced by the fact that biotransformation of 5,5-dimethyl-2-(1-methylethylidene)-cyclohexanone (II) yielded 5,5-dimethyl-2-(1-hydroxy-1-methylethyl)-2-cyclohexen-1-one (IIb) and 5,5-dimethyl-3-hydroxy-2-(1-methylethylidene)-cyclohexanone (IIa), whereas 2-(1-ethyl-1-propylidene)-5-methylcyclohexanone (III) yielded 6-(1-ethyl-1-propylidene)-5-methyl-2-cyclohexen-1-one (IIIb) and 6-(1-ethyl-1-propylidene)-5-hydroxy-5-methylcyclohexanone (IIIa) as metabolites. Based on the identification of the metabolites, pathways for the biotransformation of I, II, and III have been proposed. The mode of biotransformation of these compounds by M. piriformis also compared to their modes of metabolism in the rat system.     

无患子内生真菌抗菌筛选与鉴定

目的筛选和鉴定具有抗菌活性的无患子内生真菌,为进一步探索其抗菌成分提供菌源。方法采用拮抗试验和体外抗菌测试方法,以大肠埃希菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)和白假丝酵母菌(Candida albicans)为指示菌,首次研究6株无患子内生真菌(HY11-1、HY11-2、P11、O、ye4、ye5)的抗菌特点。通过形态学观察和18SrDNA序列分析,对活性菌株进行菌种鉴定。结果菌株HY11-1、ye5和P11具有较强的抗菌能力,其中菌株HY11-1对金黄色葡萄球菌抑菌效果最显著,确定菌株HY11-1、ye5、P11分别为Alternaria alternate、Aspergillus ochraceus和Penicillium chrysogenum。结论 3株无患子内生真菌A.alternate HY11-1、A.ochraceus ye5和P.chrysogenum P11具有较强的抗菌效果,具有进一步研究的价值。     

Further secondary metabolites produced by the fungus Pyricularia grisea isolated from buffelgrass (Cenchrus ciliaris)

The fungal pathogen Pyricularia grisea has been studied to evaluate its production of phytotoxins for the biocontrol of the buffelgrass (Cenchrus ciliaris L.) weed. A first investigation allowed to isolate several new and known phytotoxic metabolites. However, the further investigation on the organic extract obtained from the fungus liquid culture showed the presence of other metabolites possibly contributing to its phytotoxicity. Thus, four known metabolites were isolated and identified by spectroscopic (nuclear magnetic resonance [NMR] and high-resolution electrospray ionization mass spectrometry [HRESIMS]) methods as dihydropyriculol ( 1 ), epi-dihydropyriculol ( 2 ), 3-methoxy-6,8-dihydroxy-3-methyl-3,4-dihydroisocoumarin ( 3 ), and (R)-mevalonolactone ( 4 ). The absolute configuration of 1 – 3 was determined for the first time by a computational analysis of their electronic circular dichroism (ECD) spectra. When the isolated compounds were bioassayed at a concentration of 5 × 10^–3 M in a buffelgrass coleoptile and radicle elongation test no toxicity was detected. On the contrary, compounds 1 and 3 showed a significant stimulating effect of radical elongation. Furthermore, the difference in growth stimulation between 1 and its epimer 2 highlights the tight relationship between absolute configuration and biological activity of these fungal metabolites.     

Biodegradation of tetrabromobisphenol A by oxidases in basidiomycetous fungi and estrogenic activity of the biotransformation products

Tetrabromobisphenol A (TBBPA) degradation was investigated using white rot fungi and their oxidative enzymes. Strains of the Trametes, Pleurotus, Bjerkandera and Dichomitus genera eliminated almost 1 mM TBBPA within 4 days. Laccase, whose role in TBBPA degradation was demonstrated in fungal cultures, was applied to TBBPA degradation alone and in combination with cellobiose dehydrogenase from Sclerotium rolfsii. Purified laccase from Trametes versicolor degraded approximately 2 mM TBBPA within 5 h, while the addition of cellobiose dehydrogenase increased the degradation rate to almost 2.5 mM within 3 h. Laccase was used to prepare TBBPA metabolites 2,6-dibromo-4-(2-hydroxypropane-2-yl) phenol (1), 2,6-dibromo-4-(2-methoxypropane-2-yl) phenol (2) and 1-(3,5-dibromo-4-hydroxyphen-1-yl)-2,2',6,6'-tetrabromo-4,4'-isopropylidene diphenol (3). As compounds 1 and 3 were identical to the TBBPA metabolites prepared by using rat and human liver fractions (Zalko et al., 2006), laccase can provide a simple means of preparing these metabolites for toxicity studies. Products 1 and 2 exhibited estrogenic effects, unlike TBBPA, but lower cell toxicity.     

Bioactive metabolites from the endophytic fungus Ampelomyces sp. isolated from the medicinal plant Urospermum picroides

Extracts of cultures grown in liquid or on solid rice media of the fungal endophyte Ampelomyces sp. isolated from the medicinal plant Urospermum picroides exhibited considerable cytotoxic activity when tested in vitro against L5178Y cells. Chromatographic separation yielded 14 natural products that were unequivocally identified based on their ¹H and ¹³C NMR as well as mass spectra and comparison with previously published data. Six compounds (2, 4, 5, 7, 9 and 11) were natural products. Both fungal extracts differed considerably in their secondary metabolites. The extract obtained from liquid cultures afforded a pyrone (2) and sulfated anthraquinones (7 and 9) along with the known compounds 1, 3, 6 and 8. When grown on solid rice medium the fungus yielded three compounds 4, 5 and 11 in addition to several known metabolites including 6, 8, 10, 12, 13 and 14. Compounds 4, 8 and 10 showed the strongest cytotoxic activity against L5178Y cells with EC₅₀ values ranging from 0.2–7.3 μg/ml. Furthermore, 8 and 10 displayed antimicrobial activity against the Gram-positive pathogens, Staphylococcus aureus, S. epidermidis and Enterococcus faecalis at minimal inhibitory concentrations (MIC) of 12.5 μg/ml and 12.5–25 μg/ml, respectively. Interestingly, 6 and 8 were also identified as constituents of an extract derived from a healthy plant sample of the host plant U. picroides thereby indicating that the production of bioactive natural products by the endophyte proceeds also under in situ conditions within the host plant.     

Biotransformation of (20S)-20-hydroxymethylpregna-1,4-dien-3-one by four filamentous fungi

Microbial transformation of (20S)-20-hydroxymethylpregna-1,4-dien-3-one (1) by four filamentous fungi, Cunninghamella elegans, Macrophomina phaseolina, Rhizopus stolonifer, and Gibberella fujikuroi, afforded nine new, and two known metabolites 2–12. The structures of these metabolites were characterized through detailed spectroscopic analysis. These metabolites were obtained as a result of biohydroxylation of 1 at C-6β, -7β, -11α, -14α, -15β, -16β, and -17α positions, except metabolite 2 which contain an O-acetyl group at C-22. These fungal strains demonstrated to be efficient biocatalysts for 11α-hydroxylation. Compound 1, and its metabolites were evaluated for the first time for their cytotoxicity against the HeLa cancer cell lines, and some interesting results were obtained.     

Microbial transformation of (+)-nootkatone and the antiproliferative activity of its metabolites

Six metabolites were obtained as a result of microbial transformation of (+)-nootkatone (1) by the fungal strains: Botrytis, Didymosphaeria, Aspergillus, Chaetomium and Fusarium. Their structure were established as (+)-(4R,5S,7R,9R)-9α-hydroxynootkatone (2), (+)-(4R,5S,7R)-13-hydroxynootkatone (3) and (+)-(4R,5S,7R,9R,11S)-11,12-epoxy-9α-hydroxynootkatone (4), (+)-(4R,5S,7R,11S)-11,12-epoksynootkatone (5), (+)-(4R,5S,7R)-11,12-dihydroxynootkatone (6) and (+)-(4R,5S,7R)-7,11,12-trihydroxynootkatone (7) on the basis of their spectral data. Two products: (4) and (7) were not previously reported in the literature. The antiproliferative activity of (+)-nootkatone (1) and isolated metabolites (2-7) of its biotransformation has been evaluated.     

Identification of fungal metabolites of anticonvulsant drug carbamazepine

Carbamazepine, which has been used in the treatments of epilepsy, is often found in the environment. Although metabolism of carbamazepine by humans and rats has been characterized, the environmental fate of carbamazepine has not been studied. In this study, two model fungi Cunninghamella elegans ATCC 9245 and Umbelopsis ramanniana R-56, which have previously shown diverse metabolic activities, were tested for metabolism of carbamazepine. Both fungi produced three metabolites each (C1-C3 and M1-M3). All six metabolites showed [M + H](+) at m/z 253, suggesting addition of one oxygen to the parent compound. High-performance liquid chromatography and liquid chromatography-mass spectrometric analysis detected 10, 11-dihydro-10, 11-epoxycarbamazepine as a major product (C3 (47%) and M3 (85%)) and 3-hydroxycarbamazepine (C2 (15%) and M2 (7%)) from carbamazepine through mixed mono-oxidation reactions in both fungal strains. C. elegans was confirmed to produce 2-hydroxycarbamazepine (C1 (38%)) while U. ramanniana produced a yet unidentified ring-hydroxylated metabolite (M1 (8%)). The current study suggests that carbamazepine is likely to be subjected to initially diverse mono-oxygenation reactions by fungal metabolisms, resulting in the formation of the corresponding metabolites, which were similarly found in mammalian metabolisms.     

Hepatic 15-hydroxylation of corticosteroids in the rat. Substrate specificity studied in the isolated perfused liver.

The substrate specificity of a sex-specific hepatic 15-hydroxylase active on different C21O2 and C21O3 steroids was studied in the isolated perfused liver from female rats. Liquid-chromatographic separation methods in combination with computerized gas chromatography-mass spectrometry was employed to identify the metabolites formed. The majority (between 75-90%) of 15-hydroxylated compounds isolated were present as monosulphate conjugates while smaller amounts of disulphates were also detected. Hydroxylation was found to take place exclusively at position 15 beta. A certain number of 11-deoxy-21-hydroxy, 11-oxo-21-hydroxy and 11,21-dihydroxy steroids with a 3-keto-delta4-, 3-keto-5 alpha(5 beta)-, 3alpha, 5alpha- or 3 beta, 5 beta-structure were readily converted to 15 beta-hydroxylated metabolites. Depending on the structure of the substrate, between 20 and 87% of the total metabolites formed were 15 beta-hydroxylated. 5 alpha-Reduced steroids were better substrates for the hydroxylase than the corresponding 3-keto-delta4- or 5 beta-reduced compounds. The configuration of the hydroxylgroup at C-3 did not affect the degree of 15-hydroxylation. 11 beta-Hydroxylated steroids served as better substrates than the corresponding 11-dehydro-, 11-deoxy- or 11 alpha-hydroxy compounds. 5 alpha-Dihydrocorticosterone and 3 alpha, 5 alpha-tetrahydrocorticosterone were the best substrates for the 15 beta-hydroxylase.     

Structure elucidation and antibacterial activity of new fungal metabolites of sclareol

The transformation of the antibacterial diterpene sclareol (1) by two different fungal strains was investigated (Scheme). In the presence of Rhizopus stolonifer, (3beta)-3-hydroxysclareol (2), 18-hydroxysclareol (3), (6alpha)-6,18-dihydroxysclareol (4), and (11S)-11,18-dihydroxysclareol (5) were formed. Fermentation of 1 with Fusarium lini afforded (1beta)-1-hydroxysclareol (6) and (12S)-12-hydroxysclareol (7). Compounds 4-7 were identified as new compounds, and some of them were active against Bacillus subtilis (Table 3).     

Metabolomic differential display analysis of the white-rot basidiomycete Phanerochaete chrysosporium grown under air and 100% oxygen

When the cultural atmosphere of the white-rot basidiomycete, Phanerochaete chrysosporium, was changed from air to 100% oxygen, the lyophilized mycelial weight increased and thickening of extracellular glucan layer was observed in 2-3 days. To better understand the oxygen-stress responsive mechanism of P. chrysosporium, the metabolomic differential display analysis was performed using metabolites isolated from fungal cells grown under either air or 100% O(2) atmosphere. In the GC-MS total ion chromatogram of methanol-extracts from fungal cells, at least 183 peaks were detected and 53 compounds were identified. Among them, veratryl alcohol (VA), threonate, and erythronate were identified as oxygen-stress responsive metabolites. The intracellular concentration of VA increased dramatically within 1 h after an oxygen purge, indicating that VA production is sensitive to the oxygen stress in P. chrysosporium.     

Effect of abscisic Acid on the linoleic Acid metabolism in developing maize embryos

Partially purified protein extracts from maize (Zea mays L.) embryos, whether treated or not with abscisic acid (ABA), were incubated with linoleic acid (LA) and 1-[¹⁴C]LA. The resulting LA metabolites were monitored by high performance liquid chromatography with a radioactivity detector and identified by gas chromatography-mass spectrometry. α- and γ-ketol metabolites arising from 9-lipoxygenase activity were the more abundant compounds detected in the incubates, although the corresponding metabolites produced by 13-lipoxygenase were also present in the samples. In addition, a group of stereoisomers originating from two isomeric trihydroxy acids (9, 12, 13-trihydroxy-10-octadecenoic and 9, 10, 13-trihydroxy-11-octadecenoic acids) are described. Important variations in the relative proportions of the LA metabolites were observed depending on the embryo developmental stage and on ABA treatment. Two new ABA-induced compounds have been detected. These compounds are present in embryos at all developmental stages, being more abundant in old (60 days) embryos. Furthermore, ABA induction of these compounds is maximum at very young developmental stages, decreasing as maturation progresses. A tentative structure for these compounds (10-oxo-9, 13-dihydroxy-11-octadecenoic acid and 12-oxo-9, 13-dihydroxy-10-octadecenoic acid) is also provided. This study revealed an early stage in maize embryogenesis characterized by a higher relative sensitivity to ABA. The physiological importance of ABA on LA metabolism is discussed.     

Chemical Constituents of the Culture Broth of Panus rudis

In our ongoing search for new secondary metabolites from fungal strains, one novel compound (1) and nine known compounds (2-10) were isolated from the EtOAc-soluble layer of the culture broth of Panus rudis. The culture broth of P. rudis was extracted in acetone and fractionated by solvent partition; column chromatography using silica gel, Sephadex LH-20, and Sephadex G-10; MPLC; and HPLC. The structures of isolated compounds were elucidated by one- and two-dimensional NMR and LC-ESI-mass measurements. One new compound, panepoxydiol (1), and nine known compounds, (E)-3-(3-hydroxy-3-methylbut-1-en-1-yl)-7-oxabicyclo[4.1.0]hept-3-ene-2,5-diol (2), isopanepoxydone (3), neopanepoxydone (4), panepoxydone (5), panepophenanthrin (6), 4-hydroxy-2,2-dimethyl-6-methoxychromane (7), 6-hydroxy-2,2-dimethyl-3-chromen (8), 2,2-dimethyl-6-methoxychroman-4-one (9), 3,4-dihydroxy-2,2-dimethyl-6-methoxychromane (10), were isolated from the culture broth of P. rudis. This is the first report of isolation of a new compound panepoxydiol (1) and nine other chemical constituents (2-5, 7-10) from the culture broth of P. rudis.     

Secondary metabolites released into the rhizosphere by <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> and <Emphasis Type="Italic">Fusarium</Emphasis> spp. as underestimated component of nonspecific replant disease

Background and aims

A study was performed to investigate the role of fungal metabolites released into the rhizosphere of replanted orchards as a potential biotic component of tree growth decline.

Methods

The phytotoxicity of the gamma ray-sterilized crude culture filtrates of sixteen fungal species originating from replanted apple orchards was tested in a bioassay. Low molecular weight compounds released by Fusarium spp. were analyzed.

Results

The fungal culture filtrates affected seedling growth and health with an activity that varied from growth inhibition to promotion. Three out of the six species of Fusarium tested produced species-specific mycotoxins such as equisetin and enniatin B and D (<1 μg ml^?1 and <6 μg ml^?1, respectively) associated with root-tip necrosis, whereas fusaric acid (80–230 μg ml^?1) was associated with asymptomatic plant growth inhibition. These findings were consistent with those obtained using pure compounds. Moreover, methoxyconidiol, paecilaminol, integrastatin B and other biologically active compounds, whose fungal origin and phytotoxicity have not yet been reported, were found. in all fungal filtrates.

Conclusions

Findings suggest that i) phytopathogenicity of soil borne fungi can be expressed regardless of root infection; ii) a synergistic interaction between co-occurring mycotoxins and other biologically active compounds may explain plant growth inhibition. Iii) fungal metabolites released into soil may represent an underestimated component of nonspecific replant disease.

     

Synthesis and antimicrobial activity of some new pyrazole derivatives containing a ferrocene unit

A series of new imines and amines have been synthesized by condensation of 1H-3-ferrocenyl-1-phenylpyrazole-4-carboxaldehyde with the corresponding amines, followed by reduction with sodium borohydride. The synthesized compounds have been screened for their in vitro antimicrobial activity against 11 bacteria and three fungal/yeast strains, using disc diffusion and broth microdilution susceptibility assays. They have shown a wide range of activities, from completely inactive to the highly active compounds.     

Volatile metabolites produced by six fungal species compared with other indicators of fungal growth on cereal grains.

Six fungal species, Penicillium brevicompactum, P. glabrum, P. roqueforti, Aspergillus flavus, A. versicolor, and A. candidus, were inoculated on moistened and autoclaved wheat and oat grains. They were cultivated in glass vessels provided with an inlet and outlet for air. Air was passed through the vessels to collect volatile fungal metabolites on porous polymer adsorbents attached to the outlet. Samples were collected at two fungal growth stages. Adsorbed compounds were thermally desorbed, separated by gas chromatography, and identified by mass spectrometry. Differences in the production of volatile metabolites depended more on the fungal species than on the grain type. The fungal growth stage was not an important factor determining the composition of volatiles produced. 3-Methylfuran was produced in similar amounts regardless of the fungal species and substrate (oat versus wheat). The production of volatile metabolites was compared with the production of ergosterol and CO2 and the number of CFU. The production of volatile metabolites was more strongly correlated with accumulated CO2 production than with actual CO2 production and more strongly correlated with ergosterol contents of the grain than with numbers of CFU.     

Volatile metabolites produced by six fungal species compared with other indicators of fungal growth on cereal grains.

Six fungal species, Penicillium brevicompactum, P. glabrum, P. roqueforti, Aspergillus flavus, A. versicolor, and A. candidus, were inoculated on moistened and autoclaved wheat and oat grains. They were cultivated in glass vessels provided with an inlet and outlet for air. Air was passed through the vessels to collect volatile fungal metabolites on porous polymer adsorbents attached to the outlet. Samples were collected at two fungal growth stages. Adsorbed compounds were thermally desorbed, separated by gas chromatography, and identified by mass spectrometry. Differences in the production of volatile metabolites depended more on the fungal species than on the grain type. The fungal growth stage was not an important factor determining the composition of volatiles produced. 3-Methylfuran was produced in similar amounts regardless of the fungal species and substrate (oat versus wheat). The production of volatile metabolites was compared with the production of ergosterol and CO2 and the number of CFU. The production of volatile metabolites was more strongly correlated with accumulated CO2 production than with actual CO2 production and more strongly correlated with ergosterol contents of the grain than with numbers of CFU.     

Pyrene Metabolism in Crinipellis stipitaria: Identification of trans-4,5-Dihydro-4,5-Dihydroxypyrene and 1-Pyrenylsulfate in Strain JK364

The isolation and identification of two novel metabolites in the fungal metabolism of pyrene are described. The plant-inhabiting basidiomycete Crinipellis stipitaria JK364 metabolized pyrene, a polycyclic aromatic hydrocarbon containing four rings, when grown in submerged cultures in a medium containing malt extract, glucose, and yeast extract. In experiments with [¹⁴C] pyrene, after 7 days of incubation 40% of the labeled substrate was converted into organic solvent-extractable metabolites. Metabolites isolated from cultures grown with pyrene were identified as 1-pyrenylsulfate and trans-4,5-dihydro-4,5-dihydroxypyrene. 1-Hydroxypyrene, the precursor of 1-pyrenylsulfate, was also detected. 1-Pyrenylsulfate was isolated from mycelial extracts, whereas trans-4,5-dihydro-4,5-dihydroxypyrene was recovered from the culture filtrate. Identification of the compounds was based on their UV spectra, mass spectra, and nuclear magnetic resonance spectra. This is the first report on the detoxification of a polycyclic aromatic hydrocarbon by a plant-inhabiting basidiomycete. The occurrence of 1-pyrenylsulfate and trans-4,5-dihydro-4,5-dihydroxypyrene among fungal metabolites of pyrene is also new.     

Oxyprenylated secondary metabolites: a survey of their innovative extraction methodologies

Phytochemistry Reviews - Oxyprenylated secondary metabolites of plant, fungal, and microbial origin have emerged as biologically active natural compounds with a great potential for the next future....