Bio-guided isolation of potential anti-inflammatory constituents of some endophytes isolated from the leaves of ground cherry (Physalis pruinosa L.) via ex-vivo and in-silico studies.

BACKGROUND: Due to the extensive potential of previously studied endophytes in addition to plants belonging to genus Physalis as a source of anti-inflammatory constituents, the present study aimed at isolation for the first time some endophytic fungi from the medicinal plant Physalis pruinosa. METHODS: The endophytic fungi were isolated from the fresh leaves of P. pruinosa then purified and identified by both morphological and molecular methods. Comparative evaluation of the cytotoxic and ex vivo anti-inflammatory activity in addition to gene expression of the three pro-inflammatory indicators (TNF-α, IL-1ß and INF-γ) was performed in WBCs treated with lipopolysaccharide (LPS) for the identified endophytes, isolated compounds and the standard anti-inflammatory drug (piroxicam). For prediction of the binding mode of the top-scoring constituents-targets complexes, the Schrödinger Maestro 11.8 package (LLC, New York, NY) was employed in the docking study. RESULTS: A total of 50 endophytic fungal isolates were separated from P. pruinosa leaves. Selection of six representative isolates was performed for further bioactivity screening based on their morphological characters, which were then identified as Stemphylium simmonsii MN401378, Stemphylium sp. MT084051, Alternaria infectoria MT573465, Alternaria alternata MZ066724, Alternaria alternata MN615420 and Fusarium equiseti MK968015. It could be observed that A. alternata MN615420 extract was the most potent anti-inflammatory candidate with a significant downregulation of TNF-α. Moreover, six secondary metabolites, alternariol monomethyl ether (1), 3'-hydroxyalternariol monomethyl ether (2), alternariol (3), α-acetylorcinol (4), tenuazonic acid (5) and allo-tenuazonic acid (6) were isolated from the most potent candidate (A. alternata MN615420). Among the tested isolated compounds, 3'-hydroxyalternariol monomethyl ether showed the highest anti-inflammatory potential with the most considerable reductions in the level of INF-γ and IL-1ß. Meanwhile, alternariol monomethyl ether was the most potent TNF-α inhibitor. The energy values for the protein (IL-1ß, TNF-α and INF-γ)-ligand interaction for the best conformation of the isolated compounds were estimated using molecular docking analysis. CONCLUSIONS: The results obtained suggested alternariol derivatives may serve as naturally occurring potent anti-inflammatory candidates. This study opens new avenues for the design and development of innovative anti-inflammatory drugs that specifically target INF-γ, IL-1ß and INF-γ.

Anti-Epstein-Barr Viral Agents from the Medicinal Herb-Derived Fungus Alternaria alstroemeriae Km2286.

Chromatographic separation on the liquid-state fermented products produced by the fungal strain Alternaria alstroemeriae Km2286 isolated from the littoral medicinal herb Atriplex maximowicziana Makino resulted in the isolation of compounds 1-9. Structures were determined by spectroscopic analysis as four undescribed perylenequinones, altertromins A-D (1-4), along with altertoxin IV (5), altertoxin VIII (6), stemphyperylenol (7), tenuazonic acid (8), and allo-tenuazonic acid (9). Compounds 1-6 exhibited antiviral activities against Epstein-Barr virus (EBV) with EC50 values ranging from 0.17 ± 0.07 to 3.13 ± 0.31 µM and selectivity indices higher than 10. In an anti-neuroinflammatory assay, compounds 1-4, 6, and 7 showed inhibitory activity of nitric oxide production in lipopolysaccharide-induced microglial BV-2 cells, with IC50 values ranging from 0.33 ± 0.04 to 4.08 ± 0.53 µM without significant cytotoxicity. This is the first report to describe perylenequinone-type compounds with potent anti-EBV and anti-neuroinflammatory activities.

Identification and Toxicological Characterization of Alternaria japonica Strains.

Fungi of the genus Alternaria are producers of biologically active compounds. Alternaria japonica is pathogenic to small radish and certain other crucifers, but has not been studied in sufficient detail. Discrepant data on its toxic metabolites are available in the literature, possibly because a limited set of nutritive substrates was used in culturing or species identification of the strains was incorrect. The objectives of this study were to accurately identify the Russian A. japonica strains and to assess the A. japonica toxigenic potential. Four Russian A. japonica strains were identified using a multifaceted approach, which included analyses of morphological characters (the diameter and morphology of colonies grown on the diagnostic media potato carrot agar (PCA) and yeast extract-glucose (YES) agar for one week), the conidial size, and the presence of chlamydospores), the nucleotide sequences of DNA markers (ITS and EF1α regions), and chemotaxonomic data (mycotoxin production). Biomass and extractive substance yields of A. japonica cultures were found to significantly depend on the composition of the liquid medium. Minor differences between the A. japonica strains were detected via metabolite profiling by HPLC/MS-UV. Extracts of A. japonica cultures exerted phytotoxic activity toward small radish leaves and cytotoxicity toward Paramecium caudatum to a level comparable with that of A. tenuissima extracts. Brassicicolin A, dihydrobrassicicolin A, and phomenins A and B, which are known for several species of the genus Alternaria, were identified in A. japonica extracts. Mycotoxins (alternariol, its methyl ether, tentoxin, tenuazonic acid, and altenuene), which are characteristic of the cosmopolitan species A. tenuissima, were not detected in cultures of the A. japonica strains. Extract toxicity and the yield of extractive substances were studied in the A. japonica strains, and strain MFP244011 proved promising as a producer of known and, presumably, new toxins upon culture on the M1D synthetic medium or semisynthetic liquid media (e.g., the Sabouraud medium).

Derivatives of Tenuazonic Acid as Potential New Multi-Target Anti-Alzheimer's Disease Agents.

Alzheimer's disease (AD) is generally recognized as a multifactorial neurodegenerative pathology with an increasing impact on society. Tenuazonic acid (TA) is a natural compound that was recently identified as a potential multitarget ligand with anti-cholinesterase, anti-amyloidogenic and antioxidant activities. Using its structure as a chemical scaffold, we synthesized and evaluated new derivatives (1-5), including tenuazonic-donepezil (TA-DNP) hybrids (4 and 5) due to the clinical importance of the anti-AD drug donepezil. These novel compounds all achieved activity in the micromolar range towards all selected targets and demonstrated to be potentially orally absorbed. Moreover, a selected compound (1) was further investigated as a chelating agent towards copper (II), zinc (II) and iron (III) and showed good chelating ability (pFe = 16.6, pCu = 11.6, pZn = 6.0 at pH 7.4). Therefore, the TA motif can be considered an interesting building block in the search for innovative multi-functional anti-neurodegenerative drugs, as exemplified by hybrid 5, a promising non-cytotoxic lead compound adequate for the early stages of AD, and capable of ameliorating the oxidative status of SH-SY5Y human neuroblastoma cells.

Determination of Alternaria Toxins in Sunflower Oil by Liquid Chromatography Isotope Dilution Tandem Mass Spectrometry.

Alternaria toxins have gained attention as a potential health risk and can be classified as emerging mycotoxins. As a result, they are candidates to be regulated by the European Commission. This paper describes a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for analyzing five Alternaria toxins in sunflower oil, which is a rather different type of sample to those matrices investigated in earlier published papers. An optimal sample preparation condition was achieved when samples were dissolved in n-hexane and extracted with methanol/water mixture, followed by sample pre-concentration with solvent evaporation. This study is the first focusing only on this lipophilic matrix and in using all corresponding isotopically labeled internal standards (ISTD) to compensate the matrix effect that strongly influences the LC-MS/MS analysis of toxins. Target compounds were separated on Zorbax Extend C-18 column enabling the analysis at alkaline pH of 8.8 that was necessary to obtain appropriate peak shape of tenuazonic acid and to separate the analytes at baseline. The method was validated according to the EU 2002/657/EC Decision and all the analytical performance characteristics met the requirements. The recovery was between 74% and 122% in fortified sunflower oil samples and the precision varied from 9% to 22%. The method was successfully demonstrated for sunflower seed quality check (QC) samples. Finally, 16 different sunflower oil samples were measured; and tenuazonic acid and tentoxin toxins were detected at levels close to LOQ concentrations.

Coordination of mycotoxins with lanthanides in luminescent complexes.

The ability of several chelating mycotoxins to form coordination complexes with the lanthanide metals europium and terbium was explored. The mycotoxins examined included ochratoxin A, citrinin, cyclopiazonic acid (CPA), kojic acid, and tenuazonic acid (TeA). Of these compounds, TeA and CPA resulted in the greatest luminescence. Parameters influencing luminescence of TeA were investigated further. These included the type of lanthanide and its concentration, certain environmental factors, and the effect of competing metal cations. Of the two lanthanide metals, the terbium coordination complex (TeA-Tb3+) showed greater luminescence relative to the europium complex (TeA-Eu3+). The effects of solvent type, water content, and pH on the TeA-Tb3+ system suggested that optimal conditions for luminescence were in 90% methanol with 10% aqueous buffer at pH 3. In competitive assays, the luminescence of the TeA-Tb3+ complex decreased as the concentration of competing metal cations increased. Among the cations tested, Cu2+ was the best inhibitor followed by Al3+, Au3+, Fe3+, Co2+, Mn2+, Mg2+, and Ca2+. Two cations, Na+ and K+, showed no significant inhibition. This is the first report to describe the coordination of the metal-chelating mycotoxin TeA with lanthanides and the ability of TeA to serve as an "antenna" for the efficient transfer of energy to the lanthanide with resulting luminescence. Understanding the ability of mycotoxins such as TeA to chelate metals provides insight into how they exert their toxic effects.

Alternaria toxins in South African sunflower seeds: cooperative study.

Sunflower seed samples (N = 80) from different sunflower cultivars originating from different localities in South Africa were analyzed for 15 toxins produced by fungi of the genus Alternaria by means of a simple one-step extraction dilute-and-shoot HPLC-MS/MS approach. References for valine-tenuazonic acid (Val-TeA), altenusin (ALTS), and altenuisol (ALTSOH) were isolated from fungal culture extracts and spectroscopically characterized. Additionally, valine-tenuazonic acid was tested regarding its cytotoxicity in comparison with tenuazonic acid (TeA) and showed less activity on HT-29 cells. Furthermore, alternariol monomethyl ether-3-O-ß-D-glucoside (AME-3G) was produced by fermentation of alternariol monomethyl ether (AME) with the fungus Rhizopus oryzae. The seed samples were analyzed both with and without hulls. The method covers the AAL toxins TA1 and TA2, altenuene (ALT) and iso-altenuene (iso-ALT), altenuisol, altenusin, altertoxin I (ATX-I) and altertoxin II (ATX-II), alternariol (AOH) and alternariol monomethyl ether, alternariol monomethyl ether-3-O-ß-D-glucoside, tenuazonic acid, allo-tenuazonic acid (allo-TeA) and valine-tenuazonic acid, and tentoxin (TEN). More than 80% of the samples were positive for one or more analytes above the respective limit of detection (0.2-23 µg/kg). Alternariol, its monomethyl ether, tentoxin, tenuazonic acid, altenuisol, and valine-tenuazonic acid were found in quantifiable amounts. The highest prevalences were found for tentoxin (73% positive, mean content 13.2 µg/kg, maximum level 130 ± 0.9 µg/kg) followed by tenuazonic acid (51% positive, mean content 630 µg/kg, maximum level 6300 ± 560 µg/kg). The obtained data were further analyzed statistically to identify quantitative or qualitative relationships between the levels of Alternaria toxin in the samples.

Natural occurrence of tenuazonic acid and Phoma sorghina in Brazilian sorghum grains at different maturity stages.

A survey of 100 samples of sorghum grains was carried out to determine Phoma spp. and tenuazonic acid (TA) contamination using molecular tools and LC-MS/MS. Sorghum samples were obtained at the following four grain maturity stages: milk (S1), soft dough (S2), hard dough (S3), and physiological maturity (S4). The results revealed a good correlation between Phoma and TA occurrence during grain development. The samples showed Phoma contamination with frequencies ranging from 2.4% (S1) to 87.4% (S4), and the molecular identification revealed P. sorghina as the only Phoma specie isolated. Tenuazonic acid was found in sorghum grains at all maturity stages. In S2, S3 and S4, 100% of the samples showed TA contamination with levels ranging from 20 to 1234µg/kg. Low levels of TA were detected in 36% of the samples collected at S1 stage. This is the first report of tenuazonic acid in Brazilian sorghum grains.

A Synthetic Route to ß-Hydroxytyrosine-Derived Tetramic Acids: Total Synthesis of the Fungal Metabolite F-14329.

3-Acyltetramic acids derived from ß-hydroxytyrosine are synthetically challenging. The first route to this structural motif, based upon a condensation between a Meldrum's acid conjugate bearing the acyl side chain, and a ß-hydroxytyrosinate, N-protected by an ortho-nitrobenzyl group is presented. This group enables the Dieckmann cyclization of the resulting N-(ß-ketoacyl)amino ester, after which it can be removed photolytically without compromising the delicate 3'-hydroxy group. This strategy was applied to the first total synthesis of the fungal metabolite F-14329 (1).

Biosynthetic approaches to creating bioactive fungal metabolites: Pathway engineering and activation of secondary metabolism.

The diversity of natural products is greater than that of combinatorial chemistry compounds and is similar to that of drugs. Compounds rich in sp3 carbons, such as natural products, typically exhibit high structural complexity and high specificity to molecular targets. Microorganisms can synthesize such sp3 carbon-rich compounds and can be used as excellent factories for making bioactive compounds. Here, we mainly focus on pathway engineering of two sp3 carbon-rich bioactive indole alkaloids, fumitremorgin C and terpendole E. We also demonstrate the importance of activation of secondary metabolism by focusing on tenuazonic acid, a bioactive tetramic acid compound, as an example.

Genome mining unveils widespread natural product biosynthetic capacity in human oral microbe Streptococcus mutans.

Streptococcus mutans is a major pathogen causing human dental caries. As a Gram-positive bacterium with a small genome (about 2 Mb) it is considered a poor source of natural products. Due to a recent explosion in genomic data available for S. mutans strains, we were motivated to explore the natural product production potential of this organism. Bioinformatic characterization of 169 publically available genomes of S. mutans from human dental caries revealed a surprisingly rich source of natural product biosynthetic gene clusters. Anti-SMASH analysis identified one nonribosomal peptide synthetase (NRPS) gene cluster, seven polyketide synthase (PKS) gene clusters and 136 hybrid PKS/NRPS gene clusters. In addition, 211 ribosomally synthesized and post-translationally modified peptides (RiPPs) clusters and 615 bacteriocin precursors were identified by a combined analysis using BAGEL and anti-SMASH. S. mutans harbors a rich and diverse natural product genetic capacity, which underscores the importance of probing the human microbiome and revisiting species that have traditionally been overlooked as "poor" sources of natural products.

Determination of Exposure to the Alternaria Mycotoxin Tenuazonic Acid and Its Isomer allo-Tenuazonic Acid in a German Population by Stable Isotope Dilution HPLC-MS(3).

The content of the Alternaria toxin tenuazonic acid and its isomer allo-tenuazonic acid was quantitated in urine of a German cohort (n = 48) using a newly developed and successfully validated solid phase extraction based stable isotope dilution HPLC-MS(3) method. Tenuazonic acid was detected in all of the samples and quantifiable in 97.9% of these samples in a range of 0.16-44.4 ng/mL (average = 6.58 ng/mL) or 0.07-63.8 ng/mg creatinine (average = 8.13 ng/mg creatinine). allo-Tenuazonic acid was for the first time detected in human urine (95.8% of the samples positive) and quantitated in 68.8% of the samples in a range of 0.11-5.72 ng/mL (average = 1.25 ng/mL) or 0.08-10.1 ng/mg creatinine (average = 1.52 ng/mg creatinine), representing 3.40-25.0% of the sum of both isomers (average = 12.4%). Food-frequency questionnaires were used to document food consumption of study participants to correlate mycotoxin exposure to nutritional habits. Although no statistically significant correlation between consumption of a specific food and urinary excretion of tenuazonic acid could be determined, a trend regarding elevated intake of cereal products and higher excretion of tenuazonic acid was evident. On the basis of these results, a provisional mean daily intake (PDI) for both tenuazonic acid and allo-tenuazonic acid was calculated, being 0.183 and 0.025 µg/kg body weight, respectively. A combined mean PDI for both isomers amounts to 0.208 µg/kg body weight with the highest individual PDI for one of the participants (1.582 µg/kg body weight) slightly exceeding the threshold of toxicological concern assumed for tenuazonic acid by the European Food Safety Authority of 1.500 µg/kg body weight. This is the first study to investigate the tenuazonic acid content in human urine of a larger sample cohort enabling the calculation of PDIs for tenuazonic acid and allo-tenuazonic acid.

Equisetin, reutericyclin and streptolodygin as natural product lead structures for novel antibiotic libraries.

The emergence of antimicrobial resistance has created a need for the development of novel antibacterial therapies to treat infection. Natural products that exhibit antibacterial activity offer validated starting points for library generation, and the authors report here that small molecule mimics of tetramate-containing natural products may show antibacterial activity and offer the potential for further optimization.

Gastrointestinal localization of metronidazole by a lactobacilli-inspired tetramic acid motif improves treatment outcomes in the hamster model of Clostridium difficile infection.

OBJECTIVES: Metronidazole, a mainstay treatment for Clostridium difficile infection (CDI), is often ineffective for severe CDI. Whilst this is thought to arise from suboptimal levels of metronidazole in the colon due to rapid absorption, empirical validation is lacking. In contrast, reutericyclin, an antibacterial tetramic acid from Lactobacillus reuteri, concentrates in the gastrointestinal tract. In this study, we modified metronidazole with reutericyclin's tetramic acid motif to obtain non-absorbed compounds, enabling assessment of the impact of pharmacokinetics on treatment outcomes. METHODS: A series of metronidazole-bearing tetramic acid substituents were synthesized and evaluated in terms of anti-C. difficile activities, gastric permeability, in vivo pharmacokinetics, efficacy in the hamster model of CDI and mode of action. RESULTS: Most compounds were absorbed less than metronidazole in cell-based Caco-2 permeability assays. In hamsters, lead compounds compartmentalized in the colon rather than the bloodstream with negligible levels detected in the blood, in direct contrast with metronidazole, which was rapidly absorbed into the blood and was undetectable in caecum. Accordingly, four leads were more efficacious (P < 0.05) than metronidazole in C. difficile-infected animals. Improved efficacy was not due to an alternative mode of action, as the leads retained the mode of action of metronidazole. CONCLUSIONS: This study provides the clearest empirical evidence that the high absorption of metronidazole lowers treatment outcomes for CDI and suggests a role for the tetramic acid motif for colon-specific drug delivery. This approach also has the potential to lower systemic toxicity and drug interactions of nitroheterocyclic drugs for treating gastrointestine-specific diseases.

Chemical modulation of the biological activity of reutericyclin: a membrane-active antibiotic from Lactobacillus reuteri.

Whilst the development of membrane-active antibiotics is now an attractive therapeutic concept, progress in this area is disadvantaged by poor knowledge of the structure-activity relationship (SAR) required for optimizing molecules to selectively target bacteria. This prompted us to explore the SAR of the Lactobacillus reuteri membrane-active antibiotic reutericyclin, modifying three key positions about its tetramic acid core. The SAR revealed that lipophilic analogs were generally more active against Gram-positive pathogens, but introduction of polar and charged substituents diminished their activity. This was confirmed by cytometric assays showing that inactive compounds failed to dissipate the membrane potential. Radiolabeled substrate assays indicated that dissipation of the membrane potential by active reutericyclins correlated with inhibition of macromolecular synthesis in cells. However, compounds with good antibacterial activities also showed cytotoxicity against Vero cells and hemolytic activity. Although this study highlights the challenge of optimizing membrane-active antibiotics, it shows that by increasing antibacterial potency the selectivity index could be widened, allowing use of lower non-cytotoxic doses.

New routes towards reutericyclin analogues.

A range of N-acylpyrrolo[3,4-c]isoxazoles and derived N-acyltetramides has been prepared via a nitrile oxide dipolar cycloaddition approach, as analogues of the acyltetramic acid metabolite reutericyclin, of interest for its antibiotic potential against Gram-positive bacteria including hospital-acquired infections of resistant Clostridium difficile.

Structure and tautomerism of tenuazonic acid--a synergetic computational and spectroscopic approach.

All reasonable tautomers and rotamers of tenuazonic acid, which is considered to be of the highest toxicity amongst the Alternaria mycotoxins, were investigated by DFT calculations at different levels of theory in gas phase and in solution to obtain optimized geometries for further examinations. Calculated NMR spectra of tautomeric structures are being presented and compared to experimental data to finally achieve a synergetic computational and spectroscopic approach for structure elucidation of 3-acetyltetramic acids, affording the predominant tautomer of tenuazonic acid in aqueous solution. Furthermore we were able to simulate the less hindered rotation of the exocyclic acetyl group, which occurs after dissociation of tenuazonic acid in protic solvents.

Large-scale synthesis of isotopically labeled 13C2-tenuazonic acid and development of a rapid HPLC-MS/MS method for the analysis of tenuazonic acid in tomato and pepper products.

Tenuazonic acid is a fungal secondary metabolite that is produced by a number of Alternaria species and is therefore a natural contaminant of food and feed samples. This paper describes a new strategy for the efficient and economical large-scale synthesis of the isotopically labeled internal standard (13)C(2)-tenuazonic acid via a three-step procedure. Furthermore, a new reliable and quick method based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) cleanup is presented for the determination of tenuazonic acid in food and feed samples utilizing high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) by application of the stable isotope dilution analysis. This new method has a limit of detection (LOD) of 0.86 µg/kg and a limit of quantitation (LOQ) of 2.89 µg/kg. In total 26 tomato samples and 4 bell pepper samples from the German market were analyzed. Tenuazonic acid was found in each sample with levels from 3 to 2330 µg/kg.

Minor contribution of alternariol, alternariol monomethyl ether and tenuazonic acid to the genotoxic properties of extracts from Alternaria alternata infested rice.

Alternaria spp. are known to form a spectrum of secondary metabolites with alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT) and tenuazonic acid (TA) as the major mycotoxins with respect to quantity. In the present study we investigated the contribution of these compounds for the DNA damaging properties of complex extracts of Alternaria spp. infested rice. Five different Alternaria strains were cultured on rice and analyzed for their production of AOH, AME, ALT and TA. The extracts of two strains with distinctly different toxin profiles were selected for further toxicological analysis. An extract from A. alternata DSM 1102 infested rice, found to contain predominantly TA, exhibited substantial DNA strand breaking properties in cultured human colon carcinoma cells in the comet assay, whereas TA as a single compound did not affect DNA integrity up to 200µM. An extract of A. alternata DSM 12633 infested rice, containing in comparable proportions AOH, AME and TA, exceeded by far the DNA damaging properties of the single compounds. In contrast to AOH, AME and TA, both selected extracts induced an increase of DNA modifications sensitive to the bacterial repair enzyme formamidopyrimidine DNA glycosylase (FPG) in the comet assay, indicative for oxidative DNA damage. Toxicity-guided fractionation of the DSM 12633 extract confirmed that these effects were not caused by AOH, AME or TA. Taken together, the mycotoxins AOH, AME and TA, representing the major mycotoxins with respect to quantity in A. alternata infested food, play only a subordinate role for the genotoxic properties of complex extracts and appear not to be involved in the induction of FPG sensitive sites.