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A QuEChERS (quick, easy, cheap, effective, rugged, and safe)-based multi-mycotoxin method was developed, analyzing 24 (17 free and 7 modified) Alternaria and Fusarium toxins in cereals via ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). A modified QuEChERS approach was optimized for sample preparation. Quantification was conducted using a combination of stable isotope dilution analysis (SIDA) for nine toxins and matrix-matched calibration for ten toxins. Quantification via a structurally similar internal standard was conducted for four analytes. Alternariol-9-sulfate (AOH-9-S) was measured qualitatively. Limits of detection (LODs) were between 0.004 µg/kg for enniatin A1 (ENN A1) and 3.16 µg/kg for nivalenol (NIV), while the limits of quantification were between 0.013 and 11.8 µg/kg, respectively. The method was successfully applied to analyze 136 cereals and cereal-based foods, including 28 cereal-based infant food products. The analyzed samples were frequently contaminated with Alternaria toxins, proving their ubiquitous occurrence. Interestingly, in many of those samples, some modified Alternaria toxins occurred, mainly alternariol-3-sulfate (AOH-3-S) and alternariol monomethyl ether-3-sulfate (AME-3-S), thus highlighting the importance of including modified mycotoxins in the routine analysis as they may significantly add to the total exposure of their parent toxins. Over 95% of the analyzed samples were contaminated with at least one toxin. Despite the general contamination, no maximum or indicative levels were exceeded., (© 2024. The Author(s).)

Alternaria toxins (ATs) are produced from Alternaria species that result in crop losses and harmful impacts on human health. A stable isotope dilution LC-MS/MS method was established to quantify four ATs in 15 food commodities: alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), and tenuazonic acid (TeA). Based on systematically optimization of detection conditions and pre-processing steps, the limits of detection and limits of quantification of the four ATs ranged from 0.1 to 10 μg/kg and 0.2 to 30 μg/kg, respectively. The results showed that the recoveries of the four ATs were 72.0%-119.1%. The intra-precision and inter-precision ranged from 0.7% to 11.1% and 1.1% to 13.1%, respectively. The method was successfully applied to the determination of four ATs in 35 food samples, suggesting that this method could provide meaningful occurrence data to support the assessment of emerging ATs in food commodities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

As one of the most typical pathogens in fruit postharvest diseases, Alternaria alternata (A. alternata) can produce Alternaria toxins (ATs) aggravating fruit decay and harming human health. In this study, ATs (tenuazonic acid, alternariol monomethyl ether, and alternariol) production was inhibited effectively by 200 and 8000 mg/L MF (methyl ferulate) in vitro and in vivo. 1-Octen-3-ol and 3-octanol were the potential iconic volatile organic compounds of ATs (R 2  > 0.99). MF induced oxidative stress, resulting in physiological and metabolic disorders, membrane lipid oxidation and cell damage. It decreased precursors and energy supply by disturbing amino acid metabolism, ABC transporters, citrate cycle, pentose and glucuronate interconversions to regulate ATs synthesis. MF down-regulated the genes related to ATs synthesis (PksJ, AaTAS1, and OmtI), transport (AaMFS1 and MFS), and pathogenicity to affect ATs production and virulence. This study provided a theoretical basis for the control of ATs production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Isopyrazam (IPZ) is a new chiral fungicide. For bioactivity, there was a 3.37-1578 times difference among the four stereoisomers. For Alternaria alternata and Phoma multirostrata , cis -(1 S ,4 R ,9 S ) - IPZ had the greatest activity. Using cis -IPZ might improve the efficacy and reduce the dosage of the racemate by 54.7-72.2% for A. alternata and P. multirostrata . To zebrafish, trans -IPZ showed the highest acute toxicity (LC 50 , 0.096 mg/L). The degradation half-lives of IPZ stereoisomers in the five crops ranged from 3.50 to 15.2 days. Cis -IPZ was preferentially degraded in grape, pear, and celery. The residual concentrations of IPZ in grape and celery were still higher than the maximum residue limit, and the acute and chronic dietary intake risks of IPZ in celery were unacceptable (RQ a : 146-250%, HQ: 117-200%), which were worthy of further researching. Based on the research results, it is safer and more reasonable to use IPZ in the form of a racemate with a high ratio of cis -IPZ.

Fungal secondary metabolites play a highly significant role in crop protection, which is related to their antifungal activity against agriculturally important phytopathogens. In fact, plant diseases caused by fungi including species belonging to the genera of Alternaria , Botrytis , and Fusarium have become increasingly serious affecting crop yield and quality. Hence, there is increasing awareness by the scientific community of the importance of exploiting fungal products for finding new compounds able to inhibit phytopathogens. In this study several drimane-type sesquiterpenes have been detected for the first time as products of Aspergillus xerophilus by GC-MS analysis of the organic extracts obtained from the mycelia and culture filtrates of the fungus grown on two different substrates. Seven pure drimane-type sesquiterpenes were also isolated and identified by spectroscopic methods. The inhibitory effects of the pure compounds have been investigated against three phytopathogenic fungi of agrarian crops (i.e., Botrytis cinerea , Alternaria alternata , and Fusarium oxysporum f. sp. pisi ). Among the drimane-type sesquiterpenes isolated in this study, 9,11-dihydroxy-6-oxodrim-7-ene is the most active against the three phytopathogens. Our findings also reveal the high sensitivity of A. alternata to the isolated compounds. These results pave the way for future applications in agriculture of both A. xerophilus and its metabolites.

Two endophytes from the same Ginkgo biloba host were isolated and cultured separately. Three new eremophilane sesquiterpenoids ( 1-3 ), three new furan derivates ( 6 , 8 - 9 ), one new polyketide ( 10 ), and four known compounds ( 4 , 5 , 7 , 11 ) from Paraphaeosphaeria sp. and two new 10-membered macrolides ( 12 - 13 ), a new liner polyketide ( 14 ), a new benzofuran ( 15 ), and six known compounds ( 16 - 21 ) from Nigrospora oryzae were isolated. The structures of the isolated compounds were determined by spectroscopic methods, NMR calculations, and ECD calculations. The compounds 3 - 7 , 9 - 10 , 12 , and 14 - 17 showed significant antiphytopathogenic effects against mycotoxigenic Alternaria sp. comparable to the activity of nystatin (positive control). Compounds 2 , 6 , 8 , 9 , and 18 indicated inhibitions against phytopathogen Fusarium asiaticum with MICs < 10 μg/mL. In addition, the compounds with weak antifungal activities from two endophytes were mixed to test their antifungal activity. The results showed that the metabolites from two endophytes had synergistic antifungal effects, and the beneficial interactions between natural products can induce more antifungal effects against plant pathogens than that of single compounds.

Three new lanostane triterpenoids ( 1-3 ) along with two new amides fatty compounds ( 4-5 ) were isolated from the ethyl acetate extract of a culture of the endophytic fungus Alternaria sp. gx-2. Their structures were identified by 1D and 2D NMR spectral data and HRESIMS. Compounds 1-12 were evaluated for their anti-inflammatory and tyrosinase inhibition activities. The isolated compounds did not show inhibitory activities at a concentration of 100 μM against tyrosinase, while under the concentration of 10 μM, the release of lactate dehydrogenase (LDH) inhibition rate of compound 1 was 54.45%, indicating that compound 1 had moderate anti-inflammatory activity on the activation of NLRP3 inflammasome.

Fungal endophytes are a known warehouse of bioactive compounds with multifarious applications. In the present investigation two compounds, β-Sitosterol (1) and ursolic acid (2), were isolated from Alternaria alternata , an endophytic fungus associated with Morus alba Linn for the first time. The structure of the compounds was elucidated on the basis of comprehensive spectral analysis (UV, IR, 1 H-, 13 C- and 2D-NMR, as well as HRESI-MS). In the in vitro alpha amylase inhibitory assay both compounds (1) and (2) show potent antidiabetic activity. In support, Docking studies indicate significant binding affinity of the isolated compounds. Hence from the present study, it can be concluded that endophytic fungi in Morus alba Linn can find use in antidiabetic drug development in the medicinal industry.Communicated by Ramaswamy H. Sarma.

Alterbutenolide ( 1 ), a new butenolide derivative with a long-chain aliphatic acid substitution, together with seven known phenolic compounds i.e. alternariol ( 2 ), asperigillol B ( 3 ), p -hydroxyphenylacetic acid ( 4 ), p -hydroxyphenylethyl alcohol ( 5 ), methyl p -hydroxyphenyl acetate ( 6 ), 2-(4-hydroxyphenyl)ethyl acetate ( 7 ), and 5,6-dihydro-4-methyl-2 H -pyran-2-one ( 8 ), was isolated from the cultures of a sponge-derived fungus Alternaria alternata I-YLW6-1. The structure of 1 was established on the basis of HR-MS, 1D and 2D NMR, as well as by comparison of the optical rotation data with the literature reported. Compounds 2 and 3 showed significant to moderate inhibitory activities against three harmful microalgae with IC 50 values from 3.0 to 36.2 μg/mL, whereas compound 1 only displayed moderate inhibition against Chattonella marina with IC 50 value of 34.6 μg/mL. Meanwhile, compounds 3 and 4 showed weak toxicity against brine shrimp larvae with LC 50 values >100 μg/mL.

Much more attention has been paid to the contamination of Alternaria toxins because of food contamination and the threat to human health. In this study, an ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed for the simultaneous detection of the prototypical alternariol, alternariol monomethylether, and the metabolites 4-oxhydryl alternariol, and alternariol monomethylether 3-sulfate ammonium salt of Alternaria toxins. The positive samples were used as matrix samples to optimize the different experimental conditions. 0.01% formic acid solution and acetonitrile were used as the mobile phase, and analytes were scanned in negative electron spray ionization under multiple reaction monitoring, and quantitative determination by isotope internal standard method. Application of this method to samples of human plasma and urine showed the detection of the above analytes. The results showed that the recoveries were from 80.40% to 116.4%, intra-day accuracy was between 0.6% and 8.0%, and inter-day accuracy was between 1.1% and 12.1%. The limit of detection of the four analytes ranged from 0.02 to 0.6 µg/L in urine, and 0.02 to 0.5 µg/L in plasma, respectively. Thus, the developed method was rapid and accurate for the simultaneous detection of analytes and provided a theoretical basis for the risk assessment of Alternaria toxins for human exposure., (© 2024 Wiley‐VCH GmbH.)

Four new isocoumarins, alternariethers A-C (1-3) and alternariester (4) were separated from the fermentation of the fungus Alternaria malorum FL39, purified from Myoporum bontioides. Their structures were ascertained using NMR and HR-ESI-MS spectroscopy. For compound 4, the absolute configuration was solved with the help of ECD calculation and the DP4+ method. Compared with the positive control triadimefon, compound 1 showed more potent antifungal effects on Colletotrichum musae. The antifungal effects of compounds 1, 2, and 3 on Fusarium oxysporum and Fusarium graminearum, of compound 4 on F. oxysporum, were equal to those of triadimefon. Except for compound 4 which was inactive against Escherichia coli with O78 serotype, all compounds showed moderate or weak antibacterial activity against Staphylococcus aureus ATCC 6538 and E. coli with O6 or O78 serotype., (© 2024 Wiley-VHCA AG, Zurich, Switzerland.)

Six undescribed infrequent eremophilane derivatives including diaportheremopholins A - F and its previously undescribed side chain (E)-2-methyloct-2-enoic acid, together with three known compounds (testacein, xestodecalactones B and C), were isolated from the endophytic fungus Diaporthe sp. BCC69512. The chemical structures were determined based on NMR spectroscopic information in conjunction with the evidence from NOESY spectrum, Mosher's application, and chemical reactions for corroborating the absolute configurations. The isolated compounds were evaluated for biological properties such as antimalarial, anti-TB, anti-phytopathogenic fungal, antibacterial activities and for cytotoxicity against malignant (MCF-7 and NCI-H187) and non-malignant (Vero) cells. Diaportheremopholins B (2) and E (5) possessed broad antimicrobial activity against Mycobacterium tuberculosis, Bacillus cereus, Alternaria brassicicola and Colletotrichum acutatum with MICs in a range of 25.0-50.0 μg/mL. Testacein (7) exhibited strong anti-A. brassicicola and anti-C. acutatum activities with equal MIC values of 3.13 μg/mL. Moreover, diaportheremopholin F (6) and compound 8 displayed antitubercular activity with equal MIC values of 50.0 μg/mL. All tested compounds were non-cytotoxic against MCF-7, NCI-H187, and Vero cells, except those compounds 2 and 5-7 exhibited weak cytotoxicity against both malignant and non-malignant cells with IC 50 values in a range of 15.5-115.5 μM., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The increasing oxidative stress demands potential chemical compounds derived from natural resources with good antioxidant activity to overcome adverse health issues. In this context, we investigated the antioxidant properties of four dibenzopyrone phenolic compounds obtained from the endophytic fungus Alternaria alternata : altenusin, altenusin B, alterlactone, and dehydroaltenusin using DFT calculations. Our investigation focused on understanding the structure-antioxidant property relationship. It delved into probing the activity by modelling the antioxidant mechanisms. The computed transition states and thermochemical parameters, along with the structural attributes, indicate that altenusin B has good antioxidant efficacy among the four compounds, and it follows the HAT mechanism in an aqueous phase. Remarkably, our findings indicate that altenusin B exhibits potent HOO˙ radical scavenging properties, characterized by the computed high rate constant. The molecular docking studies of these compounds with the pro-oxidant enzyme xanthine oxidase (XO) gave insights into the binding modes of the compounds in the protein environment. Furthermore, molecular dynamics (MD) simulations were employed to study the interaction and stability of the compounds inside the XO enzyme. Our exploration sheds light on the radical scavenging potential of the -OH sites and the underlying antioxidant mechanisms that underpin the compounds' effective antioxidant potential.

Alternarialone A ( 1 ), one new curvularin derivative, and two known compounds ( 2 and 3 ) were isolated from the crude extract of the mangrove-derived fungus Alternaria longipes . Their structures were elucidated by comprehensive spectroscopic analyses, including MS and NMR spectroscopic data. The absolute configuration of 1 was assigned by 13 C NMR calculations and a comparison of electronic circular dichroism (ECD) spectra. All compounds were evaluated for their antibacterial activities against Helicobacter pylori . Compounds 2 and 3 showed antibacterial activities against H. pylori G27 with MIC values of 8 and 16 µg/ml, respectively, while compound 3 also displayed antibacterial activity against H. pylori BHKS159 with the MIC value of 16 µg/ml.

A total of 181 citrus-based products, including dried fruits, canned fruits, and fruit juices, collected from China and from abroad in 2021 were analyzed for the four Alternaria toxins (ALTs): alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), and tenuazonic acid (TeA) via ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS). Although the concentrations of the four ALTs varied by product and geographically, TeA was the predominant toxin followed by AOH, AME, and TEN. Products made in China showed higher levels of ALTs than those made abroad. Maximum levels of TeA, AOH, and AME in analyzed domestic samples were 4.9-fold, 1.3-fold, and 1.2-fold, respectively, higher than those in imported products. Furthermore, 83.4% (151/181) of the analyzed citrus-based products were contaminated with at least two or more ALTs. There were significant positive correlations between AOH and AME, AME and TeA, and TeA and TEN in all analyzed samples. More importantly, the solid and the condensed liquid products had higher concentrations of ALTs than the semi-solid product samples, as well as tangerines, pummelos, and grapefruits compared to the other kinds of citrus-based products. In conclusion, co-contamination with ALTs in commercially available Chinese citrus-based products was universal. Extensive and systematic surveillance of ALTs in citrus-based products, both domestic and imported, is required to obtain more scientific data for the determination of the maximum allowable concentrations of ALTs in China.

Fungi of the genus Alternaria are ubiquitous in the environment. Their mycotoxins can leach out of contaminated plants or crop debris into the soil entering the plant via the roots. We aim to evaluate the importance of this entry pathway and its contribution to the overall content of Alternaria toxins (ATs) in wheat plants to better understand the soil-plant-phytopathogen system. A hydroponic cultivation system was established and wheat plants were cultivated for up to two weeks under optimal climate conditions. One half of the plants was treated with a nutrient solution spiked with alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TeA), whereas the other half of the plants was cultivated without mycotoxins. Plants were harvested after 1 and 2 weeks and analyzed using a QuEChERS-based extraction and an in-house validated LC-MS/MS method for quantification of the ATs in roots, crowns, and leaves separately. ATs were taken up by the roots and transported throughout the plant up to the leaves after 1 as well as 2 weeks of cultivation with the roots showing the highest ATs levels followed by the crowns and the leaves. In addition, numerous AOH and AME conjugates like glucosides, malonyl glucosides, sulfates, and di/trihexosides were detected in different plant compartments and identified by high-resolution mass spectrometry. This is the first study demonstrating the uptake of ATs in vivo using a hydroponic system and whole wheat plants examining both the distribution of ATs within the plant compartments and the modification of ATs by the wheat plants., (© 2023. The Author(s).)

Alternaria fungi are widely distributed plant pathogens that invade crop products, causing significant economic damage. In addition, toxic secondary metabolites produced by the fungi can also endanger consumers. Many of these secondary metabolites are chemically characterized as mycotoxins. In this study, Q Exactive Orbitrap mass spectrometry was used for the non-targeted analysis of the metabolome of seven Alternaria isolates cultured on Potato Carrot Agar (PCA), Potato Dextrose Agar (PDA) and Potato Sucrose Agar (PSA) medium. Due to the difficulty of detecting modified toxins, an analytical strategy with multiple visual analysis tools was also used to determine the presence of sulfate conjugated toxins, as well as to visualize the molecular network of Alternaria toxins. The results show that PSA medium exhibits more advantageous properties for the culture of Alternaria , with more toxigenic species and quantities and more obvious metabolic pathways. Based on high-resolution tandem mass spectrometry (MS/MS) data, the mycotoxins and their metabolites were mainly clustered into four groups: alternariol (AOH)/alternariol monomethyl ether (AME)/altenusin (ALU)/altenuene (ALT)/dehydroaltenusin (DHA)/Desmethyldehydroaltenusin (DMDA) families, Altertoxin-I (ATX-I) family, tentoxin (TEN) family and tenuazonic acid (TeA) family. Moreover, the PSA medium is more suitable for the accumulation of AOH, AME, ALU, ALT, DHA and DMDA, while the PDA medium is more suitable for the accumulation of ATX-I, TEN and TeA. This research may provide theoretical support for the metabolomics study of Alternaria .

Altersteroids A-D ( 1 - 4 ), four new 9,11-secosteroid-derived γ-lactones, were isolated from cultures of the ascomycete fungus Alternaria sp. Their structures were elucidated primarily by NMR experiments. The absolute configuration of 1 was established by X-ray crystallographic analysis of its di- p -nitrobenzenesulfonate 1a using Cu Kα radiation, whereas those for 2 - 4 were assigned by quantum-chemical calculations. Compounds 1 - 4 incorporate a γ-lactone moiety fused to the steroid D ring at C-13/C-14. Compound 3 showed moderate cytotoxicity toward four tumor cell lines and induced an apoptotic process in A549 cells. Notably, compound 3 showed equipotent activity against the cisplatin-sensitive MB49 and -resistant MB49 CisR cells, with an IC 50 value of 12.7 μM.

The regulation of food contaminants in the European Union (EU) is comprehensive, and there are several compounds in the register or being added to the recommendation list. Recently, European standard methods for analysis have also been issued. The quick analysis of different groups of analytes in one sample requires a number of methods and the simultaneous use of various instruments. The aim of the present study was to develop a method that could analyze several groups of food contaminants: in this case, 266 pesticides, 12 mycotoxins, 14 alkaloid toxins, and 3 Alternaria toxins. The main advantage of the herein described approach over other methods is the simultaneous analysis of tenuazonic acid (TEA) and other relevant food contaminants. The developed method unites the newly published standard methods such as EN 15662:2018, EN 17194:2019, EN 17256:2019, EN 17425:2021, EN 17521:2021, which describes the analysis of both regulated and emerging contaminants. The developed method is based on a QuEChERS sample preparation, followed by LC-MS/MS analysis under alkaline mobile phase conditions. The pH of the aqueous eluent was set to 8.3, which resulted in baseline separation among ergot alkaloids and their corresponding epimers, a symmetric chromatographic peak shape for analyzing TEA and fit-for-purpose sensitivity for MS/MS detection in both positive and negative ionization modes. Those compounds, which possess the corresponding isotopically labeled internal standards (ISTD), allowed for direct quantification by the developed method and no further confirmation was necessary. This was proven by satisfactory analyses of a number of quality control (QC), proficiency test (PT), and validation samples.

Alternariol (AOH) and alternariol monomethyl ether (AME) are two Alternaria mycotoxins with high occurrence rates in food systems. This study aimed to investigate the photodegradation of AOH and AME by ultraviolet-C (UV-C) irradiation. The effect of UV-C intensity, pH, treatment time, solvents and the exposure of food components were evaluated. After treated by UV-C irradiation at 3500 μW/cm 2 for 90 min, AOH samples in methanol, aqueous solution and solid state were degraded by 89.1%, 72.9% and 53.2%, respectively, while the degradation percentages of AME were 86.6%, 50.1% and 11.1%, respectively. Increasing irradiation intensity and prolonging irradiation time could significantly facilitate the degradation of AOH and AME. An alkaline environment (pH = 11) was more conducive to the degradation of toxins. In addition, 2.5 mg mL -1 citric acid or malic acid increased the photodegradation of AOH and AME to 94.6% and 95.3%, 93.2% and 70.5%, respectively. However, protein, polyphenols and vitamin C exerted inhibitory effects on the degradation, while 10% glucose or sucrose reduced the photodegradation of AOH and AME to 65.9% and 40.3%. UV-C treatment could effectively reduce the content of AOH and AME, with the highest efficiency achieved in methanol and alkaline environment. By contrast, UV-C irradiation is more effective in degrading toxins in some liquid foods rich in organic acids but lacking in protein. The utilization of UV-C radiation appears to be a potentially useful approach for decreasing the underlying risk of Alternaria mycotoxin contamination in foods.

Alternariol (AOH) and alternariol monomethyl ether (AME), the two Alternaria mycotoxins with the highest outbreak rates in food systems, could be effectively reduced by cold plasma. This research evaluated the impact of food components on the plasma removal of AOH and AME. The results showed that 6% whey protein or ovalbumin almost completely inhibited the reduction of AOH or AME. Polyphenols inhibited the removal of AOH and AME by up to 90.8% and 83.4%, respectively. Organic acids and Vc reduced AME removal by up to 43.4% and 31.9%, respectively, but had little effect on AOH removal. Sugars and amino acids could decrease both toxin removal by less than 10%. Proteins exhibited the most inhibitory effect on plasma removal of AOH and AME, followed by polyphenols, while the effect of other components was relatively small. AOH and AME removal by cold plasma was highly related to H 2 O 2 produced during plasma discharge., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Three new dibenzo- α -pyrone derivatives, alternolides A-C ( 1 - 3 ), and seven known congeners ( 4 - 10 ) were isolated from the marine-derived fungus of Alternaria alternata LW37 assisted by the one strain-many compounds (OSMAC) strategy. The structures of 1 - 3 were established by extensive spectroscopic analyses, and their absolute configurations were determined by modified Snatzke's method and electronic circular dichroism (ECD) calculations. Compounds 6 and 7 showed good 1,1-diphenyl-2-picrylhydrazyl (DPPH) antioxidant scavenging activities with IC 50 values of 83.94 ± 4.14 and 23.60 ± 1.23 µM, respectively. Additionally, 2 , 3 and 7 exhibited inhibitory effects against α -glucosidase with IC 50 values of 725.85 ± 4.75, 451.25 ± 6.95 and 6.27 ± 0.68 µM, respectively. The enzyme kinetics study indicated 2 and 3 were mixed-type inhibitors of α -glucosidase with K i values of 347.0 and 108.5 µM, respectively. Furthermore, the interactions of 2 , 3 and 7 with α -glucosidase were investigated by molecular docking.

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)., (© 2022. Pleiades Publishing, Ltd.)

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 EC 50 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 IC 50 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.

Alternaria mycotoxins including alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), altertoxin-I (ATX-I), tentoxin (TEN), and tenuazonic acid (TeA), are ubiquitous contaminants in agricultural products. A method for the simultaneous determination of these six toxins by ultrahigh performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) with solid phase extraction (SPE) was validated in rice, sesame, tomato, and apple juice matrices. The performance of the method was evaluated in terms of linearity ( R 2 > 0.999), the limit of detection (0.04-1.67 μg/kg), the limit of quantification (0.12-5.06 μg/kg), recovery (80.0-114.7%), and precision (<17.7%). The validated method was applied to monitor 152 marketed food samples in South Korea, as well as to investigate the co-occurrence and correlation between Alternaria toxins. The mean occurrence levels were 2.77 μg/kg for AOH, 4.36 μg/kg for AME, 0.14 μg/kg for ALT, 0.11 μg/kg for ATX-I, 0.43 μg/kg for TEN, and 104.56 μg/kg for TeA. Mean and extreme (95th percentile) daily dietary exposures of South Koreans to Alternaria toxins were estimated to be 22.93 ng/kg b.w./day and 86.07 ng/kg b.w./day, respectively.

As a filamentous and spoilage fungus, Alternaria spp. can not only infect processing tomatoes, but also produce a variety of mycotoxins which harm the health of human beings. To explore the production of Alternaria toxins in processing tomatoes during growth and storage, four main Alternaria toxins and four conjugated toxins were detected by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and ultra-performance liquid chromatography-ion mobility quadrupole time-of-flight mass spectrometry (UPLC-IMS QToF MS) in processing tomatoes on different days after being inoculated with A. alternata . The results show that the content of Alternaria toxins in an in vivo assay is higher than that under field conditions. Tenuazonic acid (TeA) is the predominant toxin detected in the field (205.86~41,389.19 μg/kg) and in vivo (7.64~526,986.37 μg/kg) experiments, and the second-most abundant toxin is alternariol (AOH). In addition, a small quantity of conjugated toxins, AOH-9-glucoside (AOH-9-Glc) and alternariol monomethyl ether-3-glucoside (AME-3-Glc), were screened in the in vivo experiment. This is the first time the potential of Alternaria toxins produced in tomatoes during the harvest period has been studied in order to provide data for the prevention and control of Alternaria toxins.

After ingestion of food commodities, the gastrointestinal tract (GIT) poses the first barrier against xenobiotics and pathogens. Therefore, it is regularly confronted with external stressors potentially affecting the inflammatory response and the epithelial barrier. Alternaria mycotoxins such as alternariol (AOH) and altertoxin II (ATX-II) are frequently occurring food and feed contaminants that are described for their immunomodulatory capacities. Hence, this study aimed at exploring the effect of AOH and ATX-II as single compounds or binary mixtures on the immune response and epithelial homeostasis in noncancerous colon epithelial cells HCEC-1CT. Both toxins suppressed mRNA levels of proinflammatory mediators interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), and secretion of IL-8, as well as mRNA levels of the matrix metallopeptidase 2 (MMP-2). Binary combinations of AOH and ATX-II reduced the response of the single toxins. Additionally, AOH and ATX-II modified immunolocalization of transmembrane proteins such as integrin β1, zona occludens 1 (ZO-1), claudin 4 (Cldn 4), and occludin (Ocln), which support colonic tissue homeostasis and intestinal barrier function. Moreover, the cellular distribution of ZO-1 was affected by ATX-II. Mechanistically, these effects could be traced back to the involvement of several transcription factors. AOH activated the nuclear translocation of the aryl hydrocarbon receptor (AhR) and the nuclear factor erythroid 2-related factor 2 (Nrf2), governing cell metabolic competence and structural integrity. This was accompanied by altered distribution of the NF-κB p65 protein, an important regulator of inflammatory response. ATX-II also induced AhR and Nrf2 translocation, albeit failing to substantiate the effect of AOH on the colonic epithelium. Hence, both toxins coherently repress the intestinal immune response on the cytokine transcriptional and protein levels. Furthermore, both mycotoxins affected the colonic epithelial integrity by altering the cell architecture.

A liquid chromatography tandem mass spectrometry (LC-MS/MS) multi-mycotoxin method was developed for the analysis of the Alternaria toxins alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), altertoxin I (ATX I), altertoxin II (ATX II), alterperylenol (ALTP), and altenuene (ALT), as well as the modified toxins AOH-3-glucoside (AOH-3-G), AOH-9-glucoside (AOH-9-G), AME-3-glucoside (AME-3-G), AOH-3-sulfate (AOH-3-S), and AME-3-sulfate (AME-3-S) in barley and malt. The toxin tenuazonic acid (TeA) was analyzed separately as it could not be included into the multi-mycotoxin method. Quantitation was conducted by using a combination of stable isotope dilution analysis (SIDA) for AOH, AME, and TeA, and matrix-matched calibration for all other toxins. Limits of detection were between 0.05 µg/kg (AME) and 2.45 µg/kg (ALT), whereas limits of quantitation ranged from 0.16 µg/kg (AME) to 8.75 µg/kg (ALT). Recoveries between 96 and 107% were obtained for the analytes when SIDA was applied, while recoveries between 84 and 112% were found for analytes quantified by matrix-matched calibration. The method was applied for the analysis of 50 barley samples and their respective malts from the harvest years 2016-2020 for their mycotoxin content, showing the overall potential of toxin formation during the malting process. The toxins ALTP and ATX I were mainly found in the malt samples, but not in barley., (© 2022. The Author(s).)

Introduction: In addition to the mycotoxin swainsonine, the locoweed endophytic fungus Alternaria oxytropis (Pleosporaceae) also produces a series of rarely reported, highly oxygenated bicyclic guaiane sesquiterpenoids. Few investigations on the electrospray tandem mass fragmentation pattern of this sesquiterpenoid have been reported., Objectives: We aimed to analyze and detect new guaiane sesquiterpenoid analogues from crude extracts of the locoweed endophytic fungus A. oxytropis by UPLC-Q-TOF-MS/MS experiments., Materials and Methods: Oxytropiols A-J (1-10) and the extract of the locoweed endophytic fungus A. oxytropis were analyzed by UPLC-Q-TOF-MS/MS in positive mode., Results: Typical neutral losses, McLafferty rearrangement, 1,2-rearrangement, and 1,3-rearrangement were considered to be the main fragmentation patterns for the [M + H] + /[M + Na] + ions of 1-10 by UPLC-Q-TOF-MS/MS experiments, and possible fragmentation pathways of 1-10 were suggested. A unique and undescribed analogue named oxytropiol K (11) was found in the extract based on UPLC-Q-TOF-MS/MS analysis. Compound 11 was isolated and elucidated by NMR spectrometry, and its UPLC-Q-TOF-MS/MS analysis was consistent with the fragmentation pathways of 1-10., Conclusion: The results further support that UPLC-Q-TOF-MS/MS is a powerful and sensitive tool for the characterization of known compounds (dereplication) and the detection of new analogues from crude extracts and imply that the locoweed endophytic fungus A. oxytropis, with few chemical investigations, is an important resource for undescribed metabolites., (© 2021 John Wiley & Sons, Ltd.)

On our ongoing searching for bioactive natural products derived from entophytes, two polyketides possessing novel skeletons, alternatones A-B ( 1 - 2 ), were identified from the culture of Alternaria alternate L-10. Their structures were established by a combination of spectroscopic and single-crystal X-ray diffraction with Cu K a radiation. Alternatone A ( 1 ) exhibited cytotoxic activity against human hepatoma carcinoma HepG-2 cell line. The putative biosynthetic pathways for compounds 1 - 2 were also proposed.

Three pairs of undescribed enantiomeric α-pyrone derivatives (1a/1b-3a/3b) and six undescribed congeners (4-9), were obtained from the fungus Alternaria brassicicola that was isolated from the fresh leaves of Siegesbeckia pubescens Makino (Compositae). The structures of these new compounds were characterized by extensive NMR spectroscopic and HRESIMS data, and their absolute configurations were further elucidated by a modified Mosher's method, chemical conversion, single-crystal X-ray diffraction analysis, and ECD calculations. This is the first report of three pairs of enantiomeric α-pyrone derivatives from the fungus A. brassicicola, and these enantiomers were successfully acquired from scalemic mixtures via chiral HPLC. Compounds 1a/1b-3a/3b and 4-9 were evaluated for the herbicidal activity against Echinochloa crusgalli, Setaria viridis, Portulaca oleracea, and Taraxacum mongolicum. At a concentration of 100 μg/mL, compounds 1a and 1b could significantly inhibit the germination of monocotyledon weed seeds (E. crusgalli and S. viridis) with inhibitory ratios ranging from 68.6 ± 6.4% to 84.2 ± 5.1%, which was equivalent to that of the positive control (glyphosate). The potential structure-herbicidal activity relationship of these compounds was also discussed. To a certain extent, the results of this study will attract great interest for the potential practical application of promising fungal metabolites, α-pyrone derivatives, as ecofriendly herbicides., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Nettle (Urtica dioica L), as a plant rich in biologically active compounds, is one of the most important plants used in herbal medicine. Studies have shown that this plant has antioxidant, antiplatelet, hypoglycemic and hypocholesterolemia effects. In this study, we characterized three Alternaria endophytic fungi isolated from their host U. dioica. We hypothesized that these endophytic fungi can produce new bioactive metabolites, which may possess the bioactive property with potential application in the medical and pharmaceutical industries. The antibacterial activity was evaluated against reference and isolated strains, including Methicillin-Resistant Staphylococcus aureus. A wide range of antimicrobial activities similar to those measured in nettle leaves was detected especially for Alternaria sorghi. Furthermore, the highest antioxidant activity detected with DPPH free radical scavenging was measured for A. sorghi and nettle leaves ethyl acetate extracts. In addition, whereas catalase activity was similar in the three isolated fungi and nettle leaves, total thiol content and superoxide dismutase activity were significantly higher in leaves. A. sorghi showed the best activities compared to other isolated fungi. The characterization and further production of bioactive compounds produced by this endophyte should be investigated to fight bacteria and especially those that develop drug multi-resistance.

Open sandwich enzyme-linked immunosorbent assay (OS-ELISA), a novel noncompetitive immunoassay format, has shown great potential in rapid detection for small molecules compared with traditional competitive format. Here, an enhanced OS-ELISA towards the mycotoxin tenuazonic acid (TeA) was developed for the first time based on heavy chain variable region (V H ) and light chain variable region (V L ) from the hybridoma cells (3F10) producing anti-TeA monoclonal antibody (mAb). The established OS-ELISA exhibited a limit of detection of 0.08 ng/mL, and was 13 times more sensitive than mAb-based indirect competitive ELISA (ic-ELISA). The proposed assay was also applied to detect TeA contents in juice, flour and tomato ketchup samples with satisfactory recoveries of 87.6%-111.3%. Finally, the great accuracy of the established OS-ELISA method was validated by the standard ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS)., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Six new polyketone metabolites, compounds (1-6) and seven known polyketone compounds (7-13) were isolated from Rhodiola tibetica endophytic fungus Alternaria sp. The structural elucidation of five new polyketone metabolites were elucidated on the basis of spectroscopic including 2D NMR and HRMS and spectrometric analysis. Inhibition rate evaluation revealed that compounds 1(EC 50  = 0.02 mM), 3(EC 50  = 0.3 mM), 6(EC 50  = 0.07 mM), 8(EC 50  = 0.1 mM) and 9(EC 50  = 0.04 mM) had inhibitory effect on the SARS-CoV-2 virus., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Alternaria toxins are considered as emerging mycotoxins, however their toxicity has not been fully evaluated in humans. Alternariol (AOH), the most prevalent Alternaria mycotoxin, was previously reported to be genotoxic and to affect hormonal balance in cells; however, its direct molecular mechanism is not known. The imbalance in androgen/estrogen ratio as well as chronic inflammation are postulated as factors in prostate diseases. The environmental agents affecting the hormonal balance might participate in prostate carcinogenesis. Thus, this study evaluated the effect of two doses of AOH on prostate epithelial cells. We observed that AOH in a dose of 10 µM induces oxidative stress, DNA damage and cell cycle arrest and that this effect is partially mediated by estrogen receptor β (ERβ) whereas the lower tested dose of AOH (0.1 µM) induces only oxidative stress in cells. The modulation of nuclear erythroid-related factor 2 (Nrf2) was observed in response to the higher dose of AOH. The use of selective estrogen receptor β (ERβ) inhibitor PHTPP revealed that AOH-induced oxidative stress in both tested doses is partially dependent on activation of ERβ, but lack of its activation did not protect cells against AOH-induced ROS production or DNA-damaging effect in case of higher dose of AOH (10 µM). Taken together, this is the first study reporting that AOH might affect basic processes in normal prostate epithelial cells associated with benign and malignant changes in prostate tissue.

The yellow peach ( Amygdalus persica ), an important fruit in China, is highly susceptible to infection by Alternaria sp., leading to potential health risks and economic losses. In the current study, firstly, yellow peaches were artificially inoculated with A lternaria alternate . Then, the fruits were stored at 4 °C and 28 °C to simulate the current storage conditions that consumers use, and the Alternaria toxins (ATs) contents from different parts of the fruits were analyzed via ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The results showed that the growth of A . alternate and the ATs production were dramatically affected by the storage temperature. At 28 °C, the fungi grew rapidly and the lesion diameter reached about 4.0 cm within 15 days of inoculation, while, at 4 °C, the fungal growth was noticeably inhibited, with no significant change in the lesion diameter. To our surprise, high contents of ATs were produced under both storage conditions even though the fungal growth was suppressed. With an increase in the incubation time, the amounts of ATs showed a steady tendency to increase in most cases. Remarkably, alternariol monomethyl ether (AME), alternariol (AOH), and tenuazonic acid (TeA) were detected in the rotten tissue and also in the surrounding tissue, while a large amount of TeA could also be found in the healthy tissue. To the best of our knowledge, this is the first report regarding the production of ATs by the infection of Alternaria sp. in yellow peach fruits via artificial inoculation under regulated conditions, and, based on the evidence herein, it is recommended that ATs be included in monitoring and control programs of yellow peach management and food safety administration.

Six new polyketides, alternaritins A-D [(±)-1-4] and isoxanalteric acid I (8), and 25 known Alternaria toxins were isolated from the culture of an endophytic fungi Alternaria sp. MG1. 3 is a rare fungal metabolite. 6 is a new natural product, and 5, 7, and 9 are known previously but their absolute configurations have not been determined. Three enantiomers [(±)-1, (±)-7, and (±)-15] were separated via chiral HPLC resolution. The structures of those polyketides (1-9) were elucidated by spectrometric analysis using MS and NMR. The absolute configurations were established using X-ray diffraction analysis and statistical comparative analysis of the experimental ECD and OR data, in conjunction with quantum mechanical calculations. All of the compounds were evaluated for their bioactivities. Known compound 27 exerted the most potent cytotoxic activities against HT-1080 and NCI-H1299 cell lines. The new compounds, 2 and 3, showed moderate inhibition on COX-2, while a pair of isomers, 8 and 9, exhibited medium activity on COX-2 and uropathogenic Escherichia coli., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Alternariol (AOH) is an emerging mycotoxin produced by Alternaria molds. It occurs as a contaminant e.g., in oilseeds, cereals, grapes, and tomatoes. Chronic exposure to AOH may cause genotoxic and endocrine disruptor effects. Our recent studies demonstrated that the fluorescence signal of AOH can be strongly affected by the environmental pH as well as by the presence of serum albumin or cyclodextrins. In the current study, we aimed to characterize the most optimal circumstances regarding the highly sensitive fluorescent detection of AOH. Therefore, the further detailed investigation of the microenvironment on the fluorescence signal of the mycotoxin has been performed, including the effects of different buffers, organic solvents, detergents, and cations. Organic solvents (acetonitrile and methanol) caused only slight increase in the emission signal of AOH, while detergents (sodium dodecyl sulfate and Triton-X100) and Ca 2+ induced considerably higher enhancement in the fluorescence of the mycotoxin. In addition, Mg 2+ proved to be a superior fluorescence enhancer of the AOH. Spectroscopic and modeling studies suggest the formation of low-affinity AOH-Mg 2+ complexes. The effect of Mg 2+ was also tested in two HPLC assays: Our results show that Mg 2+ can considerably increase the fluorescence signal of AOH even in a chromatographic system.

Five new natural compounds ( 1 - 5 ) along with four known ones, involving dibenzo- α -pyrone derivatives, a benzo- γ -pyrone derivative and an amide-type compound were obtained from Alternaria alternata , an endophyte isolated from Paeonia lactiflora. The structures of these isolates were elucidated by intensive analysis of spectroscopic data including NMR, HRMS (ESI and EI), UV and IR spectra. Compounds ( 1 - 4 ) were evaluated for their cytotoxicities against five selected human tumourtumour cell lines (A-549, MDA-MB-231, MCF-7, KB and KB-VIN), and compound 3 exhibited activities against MDA-MB-231and MCF-7 with IC 50 values of 20.1  μ M and 32.2  μ M.

Endophytic fungi are broadly dispersed residing inside plant tissues and have been demonstrated as a treasure for bioactive natural products. Unexplored harsh and heavy metal contaminant habitat of Avicennia marina may have diverse and potential fungal association. Therefore, this work aimed to isolate the culturable fungal endophytes associated with leaves of A. marina and to evaluate their medical potentialities. Seventeen isolates of endophyte fungi were isolated from healthy leaves and their antimicrobial activities were evaluated. Results showed that isolates had activity against micro-organisms in addition to their antioxidant activity produced a variety of phenolic compounds, besides exhibited a lowest cytotoxicity against ATCC-CCL-81 cell line. Consequently, selected endophytic fungal isolates were identified genetically as Chaetomium sp., Chaetomium madrasense, Chaetomium sp., Chaetomium globosum, Aspergillus hiratsukae, Aspergillus ochraceus, Alternaria tenuissima and Curvularia lunata with gene bank accession numbers MT089951, MT089952, MT089953, MT089954, MT089955, MT089956, MT089957 and MT089958 respectively. The most potent fungus extract was analysed using Gas chromatography-mass spectrometry which verified the presence of numerous bioactive compounds. These findings confirmed that new endophytic fungal strains derived from A. marina thrive in harsh ecosystem produce bioactive metabolites which can be recommended as a novel source for drug discovery., (© 2020 The Society for Applied Microbiology.)

Alternaria toxins are emerging mycotoxins whose regulation and standardization are in progress by the European Commission and the European Committee for Standardization. This paper describes a dilute and shoot approach to determine five Alternaria toxins in selected food samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The strategy involves sample extraction with acidified aqueous methanol, followed by a solvent change accomplished via sample evaporation and reconstitution. The quantification is based on isotope dilution, applying all corresponding isotopically labeled internal standards to compensate possible matrix effects of the analysis. The main advantages of the present method over other existing methods includes simple and effective sample preparation, as well as detection with high sensitivity. The five-fold sample dilution can decrease matrix effects, which were evaluated with both external and internal standard methods. The results demonstrated a limit of quantification lower than 1.0 µg/kg for all five analytes for the first time. The newly presented method showed acceptable accuracy (52.7-111%) when analyzing naturally contaminated and spiked standard samples at the described levels. The method was validated for tomato-based and flour samples (wheat, rye, and maize). The absolute recovery ranged from 66.7% to 91.6% (RSD < 10%). The developed method could be an alternative approach for those laboratories that exclude sample cleanup and pre-concentration of state-of-the-art instruments with enhanced sensitivity.

One new chromanone derivative, alterchromanone A (1), and four known curvularin-type macrolides (2-5) were isolated from the crude extract of the mangrove-derived endophytic fungus Alternaria longipes. Their structures were elucidated by MS and NMR spectroscopic analyses and by a comparison with data from the literature. The absolute configuration of 1 was assigned by combination of experimental and calculated electronic circular dichroism (ECD) spectra. Compound 1 exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity with an IC 50 value of 56.3 μg ml -1 . According to the structural features of these compounds, the plausible biosynthetic pathways of 1-5 were also proposed.

Swainsonine (SW, 1), a unique indolizine with poly-hydroxyl groups, was re-isolated from the plant endophytic fungus Alternaria oxytropis. The structure (including planar structure and relative configuration) was systematically elucidated by NMR spectra (including 1 H, 13 C, 1 H- 1 H COSY, HMQC, HMBC, and NOESY spectra in DMSO-d 6 and in CD 3 OD); 1 H NMR spectra of the modified Mosher's products were first used to determine the absolute configuration of SW. More importantly, the complex coupled features of H-7α, H-7β, and H-6α in the 1 H NMR spectrum of (1) were analyzed in details, which will provide aids for the planar and relative configuration determination of analogs., (© 2020 John Wiley & Sons, Ltd.)

Three pairs of enantiomeric dibenzo-α-pyrone derivatives ( 1 - 3 ) including two pairs of new racemates (±)-alternaone A ( 1 ) and (±)-alternaone B ( 2 ) and one new enantiomer (-)-alternatiol ( 3 ), together with five known compounds ( 4 - 8 ) were isolated from the fungus Alternaria alternata ZHJG5. Their structures were confirmed by spectroscopic data and single-crystal X-ray diffraction analysis. All enantiomers were separated via chiral high-performance liquid chromatography, with their configurations determined by electronic circular dichroism calculation. Biogenetically, a key epoxy-rearrangement step was proposed for the formation of skeletons in 1 - 3 ; (+) 1 , (-)- 1 , and 5 presented moderate antibacterial inhibition on phytopathogenic bacteria Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola . In the antifungal test, compounds 7 and 8 showed a moderate protective effect against Botrytis cinerea in vivo.

Alternaria molds produce a variety of chemically diverse secondary metabolites with potentially adverse effects on human health. However, data on occurrence in food and human exposure is inconsistent for some of these mycotoxins. Membrane filtration is a frequent step in many sample preparation procedures for LC-MS-based methods analyzing food contaminants. Yet, little is known about the possibility of adsorptive phenomena that might result in analyte losses. Thus, we treated a complex extract of Alternaria toxins with several types of syringe filters and unraveled the impact on its chemical composition by LC-MS/MS. We observed significant, and in some cases complete, losses of compounds due to filtration. Particularly, two key Alternaria toxins, alternariol (AOH) and its monomethyl ether (AME), were heavily affected. As a comparison with published food surveys indicating a correlation of the type of filtration used with lower incidence reports in food, our results point at a possible underestimation of AME in past exposure assessment. Also, perylene quinones were greatly affected by filtration, underlining the importance to take this into consideration during analytical method development. Furthermore, we applied the comet assay in HT-29 cells to elucidate the impact of filtration on the genotoxicity of the extract. We observed strong coincidences with the loss of epoxide-carrying metabolites and also an intriguing induction of oxidative DNA damage by yet toxicologically uncharacterized Alternaria toxins. In conclusion, we highlight potential issues with sample filtration and call for a critical re-evaluation of previous food occurrence data in the light of the results at hand.

Fungi of the genus Alternaria infest many agricultural crops and produce numerous mycotoxins, of which altertoxin II (ATX II) is one of the most mutagenic metabolites. ATX II carries an epoxide group but the formation of DNA adducts has not been demonstrated to date. We report now that ATX II gives rise to two covalent adducts with guanine when incubated with DNA under cell-free conditions. These adducts were demonstrated by LC-high resolution MS after enzymatic degradation of the incubated DNA to deoxynucleosides. The major adduct results from the covalent binding of ATX II, presumably through the epoxide group, to guanine, whereas the minor guanine adduct is derived from the major one by the elimination of two equivalents of water. In addition, a third adduct was detected, formed through covalent binding of ATX II to cytosine followed by the loss of two equivalents of water. The direct DNA reactivity of ATX II may explain its high mutagenicity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Molds of the genus Alternaria produce several mycotoxins, some of which may pose a threat for health due to their genotoxicity. Due to the lack of adequate toxicological and occurrence data, they are currently not regulated. Interactions between mycotoxins, gut microbiota and food constituents might occur after food ingestion, modifying the bioavailability and, therefore, overall toxicity of mycotoxins. The present work aimed to investigate the impact of in vitro short-term fecal incubation on the in vitro DNA-damaging effects exerted by 5 µg/mL of an Alternaria alternata extract, containing, among others, 15 nM alternariol, 12 nM alternariol monomethyl ether, 241 nM altertoxin II and 301 nM stemphyltoxin III, all of which are known as genotoxic. The involvement of microorganisms, undigested food constituents and soluble substances of human fecal samples in modifying the composition and the genotoxicity of the extract was investigated through the application of LC-MS/MS analysis and comet assays in HT-29 cells. Results showed that the potential of the mycotoxins to induce DNA strand breaks was almost completely quenched, even before anaerobic incubation, by contact with the different fractions of the fecal samples, while the potency to induce formamidopyrimidine DNA glycosylase (FPG)-sensitive sites was only slightly reduced. These effects were in line with a reduction of mycotoxin concentrations found in samples analyzed by LC-MS/MS. Although a direct correlation between the metabolic activity of the gut microbiota and modifications in mycotoxin contents was not clearly observed, adsorptive phenomena to bacterial cells and to undigested food constituents might explain the observed modifications.

Patchouli is a tropical medicinal and spice crop with high economic value, and the endophytic microorganism is also one of its important components and can provide new active compounds with medicinal use. In the present study, four new biphenyl compounds named 3-O-demethylaltenuisol (1), (-)-dialtenuisol (5) and (+)-dialtenuisol (6), and altertoxin VII (9), as well as six known related compounds, were isolated from the patchouli (Pogostemon cablin) endophytic fungus Alternaria sp. PfuH1. The structures of the new compounds were elucidated from spectroscopic data, ECD spectra analysis, and ECD calculations. Compounds 5 and 6 are a pair of dimeric axially chiral enantiomers. Compounds 2, 4, and 9 showed antibacterial activities against S. agalactiae with MIC values of 9.3, 85.3, and 17.3 μg/mL, respectively, and compound 4 also showed weak antibacterial activity against E. coli with MIC value of 128 μg/mL., Competing Interests: Declaration of Competing Interest The authors declared that they have no conflicts of interest to this work., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Fusicoccane-derived diterpenoids bearing a unique bridgehead double-bond-containing tricyclo[9.2.1.0 3,7 ]tetradecane (5-9-5 ring system) core skeleton represent a rarely reported class of rearranged terpenoids, which traced back to fusicoccanes with a classical dicyclopenta[a,d]cyclooctane (5-8-5 ring system) core skeleton via a crucial Wagner-Meerwein rearrangement reaction. In this research, alterbrassicenes B-D (1-3), three new rearranged fusicoccane diterpenoids bearing a rare bridgehead double-bond-containing tricyclo[9.2.1.0 3,7 ]tetradecane core skeleton, together with two known congeners, brassicicenes O and K (4 and 5), were isolated from the modified cultures of fungus Alternaria brassicicola. Their structures were elucidated by comprehensive analyses of the NMR and HRESIMS data, and the absolute configurations of 1 and 4 were further confirmed via a combination of 13 C NMR and ECD calculations and single-crystal X-ray diffraction analysis (Cu Kα). Interestingly, alterbrassicene B (1) represented the second case of bridgehead C-10-C-11 double-bond-containing natural products with a bicyclo[6.2.1]undecane core skeleton, and also featured an undescribed oxygen bridge between C-6 and C-14 to construct an unprecedentedly caged tetracyclic system. Alterbrassicenes B-D showed moderate cytotoxic activity against certain human tumor cell lines with IC 50 values in the range of 15.87-36.85 μM., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Two new meroterpenoids, tricycloalternarenes X and Y, together with one known meroterpenoid, tricycloalternarene I, were isolated from the fungus Alternaria sp. JJY-32. The structures including absolute configurations were established by the comprehensive spectroscopic data, electronic circular dichroism (ECD) spectral analyses, and biosynthesis consideration. Tricycloalternarene X showed cytotoxicity against the HL-60 and HO8910 cells with IC 50 values of 7.54 and 20.32 μm., (© 2020 Wiley-VHCA AG, Zurich, Switzerland.)