Characterization of Eurotium cristatum Fermented Thinned Young Apple and Mechanisms Underlying Its Alleviating Impacts on Experimental Colitis.

A hundred million tons of young apples are thinned and discarded in the orchard per year, aiming to increase the yield and quality of apples. We fermented thinned young apples using a potential probiotic fungus, Eurotium cristatum, which notably disrupted the microstructure of raw samples, as characterized by the scanning electron microscope. Fermentation substantially altered the metabolite profiles of samples, which are predicted to alleviate colitis via regulating inflammatory response and response to lipopolysaccharide by using network pharmacology analysis. In vivo, oral gavage of water extracts of E. cristatum fermented young apples (E.YAP) effectively alleviated DSS-induced colitis, restored the histopathology damage, reduced the levels of inflammatory cytokines, and promoted colonic expressions of tight junction proteins. Moreover, E.YAP ameliorated gut dysbacteriosis by increasing abundances of Lactobacillus,Blautia, Muribaculaceae, and Prevotellaceae_UCG-001 while inhibiting Turicibacter, Alistipes, and Desulfovibrio. Importantly, E.YAP increased colonic bile acids, such as CA, TCA, DCA, TUDCA, and LCA, thereby alleviating colitis via PXR/NF-κB signaling. Furthermore, a synbiotic combination with Limosilactobacillus reuteri WX-94, a probiotic strain isolated from feces of healthy individuals with anti-inflammatory properties, augmented anticolitis capacities of E.YAP. Our findings demonstrate that E.YAP could be a novel, potent, food-based anti-inflammatory prebiotic for relieving inflammatory injuries.

Dynamic changes on sensory property, nutritional quality and metabolic profiles of green kernel black beans during Eurotium cristatum-based solid-state fermentation.

Eurotium cristatum, a unique probiotic in Fu brick tea, is widely used in food processing to enhance added values. Here, green kernel black beans (GKBBs) were solid-fermented with E. cristatum and dynamic changes in flavour, chemical composition and metabolites during fermentation were investigated. As results, E. cristatum fermentation altered aroma profiles and sensory attributes of GKBBs, especially reduced sourness. After fermentation, total polyphenolic and flavonoid contents in GKBBs were elevated, while polysaccharides, soluble proteins and short-chain fatty acids contents were decreased. E. cristatum fermentation also induced biotransformation of glycosidic isoflavones into sapogenic isoflavones. During fermentation, dynamic changes in levels of 17 amino acids were observed, in which 3 branched-chain amino acids were increased. Non-targeted metabolomics identified 51 differential compounds and 10 related metabolic pathways involved in E. cristatum fermentation of GKBBs. This study lays foundation for the development of green kernel black bean-based functional food products with E. cristatum fermentation.

Salicylaldehyde derivatives from a marine-derived fungus Eurotium sp. SCSIO F452.

Two pairs of new salicylaldehyde derivative enantiomers, salicylaldehydiums A and B (1 and 2), along with five known analogues were isolated and identified from a marine-derived fungus Eurotium sp. SCSIO F452. Their structures and absolute configuration were determined by extensive spectroscopic analysis and electronic circular dichroism (ECD) calculations. All the new optical pure enantiomers [(+)-1, (-)-1, (+)-2, (-)-2] were evaluated for their cytotoxic and antioxidative activities. Compound (-)-1 exhibited weak cytotoxic activity.

Eurocristatine, a plant alkaloid from Eurotium cristatum, alleviates insulin resistance in db/db diabetic mice via activation of PI3K/AKT signaling pathway.

Eurocristatine (ECT) is an alkaloid isolated from Eurotium cristatum, and it has been used in multiple applications. However, its use as a treatment for type 2 diabetes mellitus (T2DM) has not yet been reported. In this study, we investigated the anti-T2DM effect of ECT and explored its potential molecular mechanism. In vivo, after treatment with ECT (20, 40 mg/kg) for 6 weeks, fasting blood glucose (FBG) was remarkably reduced in db/db mice. Moreover, glucose tolerance, insulin sensitivity and hyperinsulinemia were ameliorated treatment with ECT. The values of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) also showed that ECT could alleviate liver toxicity caused by diabetes in db/db mice. In vitro, ECT (15 and 30 µM) alleviated insulin resistance by increasing glucose consumption, glucose uptake and glycogen content in high glucose-induced HepG2 cells. The Western blotting (WB) results showed that ECT could upregulate the expression of phosphatidylinositol 3-kinase (PI3K), increase the phosphorylation of insulin receptor substrate 1 (IRS1) and protein kinase B (AKT) in vivo and in vitro. Besides, ECT improved the glycogen content by inhibiting the expression of glycogen synthase kinase3ß (GSK3ß) and promoting that of glycogen synthase (GS). Furthermore, administration of the PI3K/AKT signaling pathway inhibitor LY294002 abolished the beneficial effects of ECT. These findings are the first to verify that ECT has the potential to improve glucose metabolism and alleviate insulin resistance by activating the PI3K/AKT signaling pathway in db/db mice.

Eurotium Cristatum Fermented Okara as a Potential Food Ingredient to Combat Diabetes.

Type 2 diabetes mellitus (T2DM) is a chronic disease, and dietary modification is a crucial part of disease management. Okara is a sustainable source of fibre-rich food. Most of the valorization research on okara focused more on the physical attributes instead of the possible health attributes. The fermentation of okara using microbes originated from food source, such as tea, sake, sufu and yoghurt, were explored here. The aim of this study is to investigate fermented okara as a functional food ingredient to reduce blood glucose levels. Fermented and non-fermented okara extracts were analyzed using the metabolomic approach with UHPLC-QTof-MSE. Statistical analysis demonstrated that the anthraquinones, emodin and physcion, served as potential markers and differentiated Eurotium cristatum fermented okara (ECO) over other choices of microbes. The in-vitro α-glucosidase activity assays and in-vivo mice studies showed that ECO can reduce postprandial blood glucose levels. A 20% ECO loading crispy snack prototype revealed a good nutrition composition and could serve as a fundamental formulation for future antidiabetes recipe development, strengthening the hypothesis that ECO can be used as a novel food ingredient for diabetic management.

The Sponge-Associated Fungus Eurotium chevalieri MUT 2316 and its Bioactive Molecules: Potential Applications in the Field of Antifouling.

The need for new environmentally friendly antifouling and the observation that many marine organisms have developed strategies to keep their surface free of epibionts has stimulated the search for marine natural compounds with antifouling activities. Sponges and in particular fungi associated with them represent one of the most appropriate sources of defence molecules and could represent a promising biomass for the supply of new antifouling compounds. The objective of this work was therefore to evaluate the antifouling potency of 7 compounds isolated from the sponge derived fungus Eurotium chevalieri MUT 2316. The assessment of their activity targeted the inhibition of the adhesion and/or growth of selected marine bacteria (5) and microalgae (5), as well as the inhibition of the mussel's byssus thread formation (tyrosinase activity). The 7 compounds showed bioactivity, with various levels of selectivity for species. Cyclo-L-Trp-L-Ala was the most promising active compound, and led to the inhibition, at very low concentrations (0.001 µg ml-1 in 61.5% of cases), of adhesion and growth of all the microalgae, of selected bacteria, and towards the inhibition of tyrosinase. Promising results were also obtained for echinulin, neoechinulin A, dihydroauroglaucin and flavoglaucin, respectively, leading to inhibition of adhesion and/or growth of 9, 7, 8 and 8 microfouling species at various concentrations.

Anti-inflammatory prenylbenzaldehyde derivatives isolated from Eurotium cristatum.

Two undescribed prenylbenzaldehyde derivatives, cristaldehydes A and B, and an undescribed quinone derivative, cristaquinone A, along with seven known compounds were isolated from the fungus Eurotium cristatum. The structures of undescribed compounds were determined by spectroscopic analysis including NMR, HR-ESIMS, and single-crystal X-ray diffraction. This is the first report of identification of a dibenzannulated 6,6-spiroketal derivative, cristaldehyde B, in a natural product. Cytotoxic and anti-inflammatory activities of all compounds were evaluated. Cristaldehyde A and cristaquinone A along with five known compounds showed significant anti-inflammatory activities with IC50 values in the range from 0.37 to 14.50 µM.

Eurotiumins A⁻E, Five New Alkaloids from the Marine-Derived Fungus Eurotium sp. SCSIO F452.

Three new prenylated indole 2,5-diketopiperazine alkaloids (1⁻3) with nine known ones (5⁻13), one new indole alkaloid (4), and one new bis-benzyl pyrimidine derivative (14) were isolated and characterized from the marine-derived fungus Eurotium sp. SCSIO F452. 1 and 2, occurring as a pair of diastereomers, both presented a hexahydropyrrolo[2,3-b]indole skeleton. Their chemical structures, including absolute configurations, were elucidated by 1D and 2D NMR, HRESIMS, quantum chemical calculations of electronic circular dichroism, and single crystal X-ray diffraction experiments. Most isolated compounds were screened for antioxidative potency. Compounds 3, 5, 6, 7, 9, 10, and 12 showed significant radical scavenging activities against DPPH with IC50 values of 13, 19, 4, 3, 24, 13, and 18 µM, respectively. Five new compounds were evaluated for cytotoxic activities.

Asperflavin, an Anti-Inflammatory Compound Produced by a Marine-Derived Fungus, Eurotium amstelodami.

In the present study, 16 marine-derived fungi were isolated from four types of marine materials including float, algae, animals and drift woods along with the coast of Jeju Island, Korea and evaluated for anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated RAW 24.7 cells. The broth and mycelium extracts from the 16 fungi were prepared and the broth extract (BE) of Eurotium amstelodami (015-2) inhibited nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells without cytotoxicity. By further bioassay-guided isolation, three compounds including asperflavin, neoechinulin A and preechinulin were successfully isolated from the BE of E. amstelodami. It was revealed that asperflavin showed no cytotoxicity up to 200 µM and significantly inhibited LPS-induced NO and PGE2 production in a dose-dependent manner. In the western blot results, asperflavin suppressed only inducible NOS (iNOS), but COX-2 were slightly down-regulated. Asperflavin was also observed to inhibit the production of pro-inflammatory cytokines including TNF-α, IL-1ß, and IL-6. In conclusion, this study reports a potential use of asperflavin isolated from a marine fungus, E. amstelodami as an anti-inflammatory agent via suppression of iNOS and pro-inflammatory cytokines as well as no cytotoxicity.

Antibacterial and antibiofilm activities of the metabolites isolated from the culture of the mangrove-derived endophytic fungus Eurotium chevalieri KUFA 0006.

Five previously undescribed metabolites, including acetylquestinol, two prenylated indole 3-carbaldehyde derivatives, an anthranilic acid derivative and an isochromone derivative, were isolated, in addition to eleven known compounds: palmitic acid, ergosterol 5,8-endoperoxide, emodin, physcion, questin, questinol, (11S, 14R)-cyclo(tryptophylvalyl), preechinulin, neoechinulin E, echinulin and eurocristatine, from the culture of the endophytic fungus Eurotium chevalieri KUFA 0006. The structures of the previously undescribed compounds were established based on an extensive 1D and 2D NMR spectral analysis as well as HRMS and IR data. In case of 2-(2, 2-dimethylcyclopropyl)-1H-indole-3-carbaldehyde and 6, 8-dihydroxy-3-(2S-hydroxypropyl)-7-methylisochromone, the absolute configurations of their stereogenic carbons were established based on comparison of their experimental and calculated ECD spectra. All the compounds, except for palmitic acid and ergosterol 5, 8-endoperoxide, were evaluated for their antibacterial and antibiofilm activities against two Gram-positive and two Gram-negative bacteria, as well as multidrug-resistant isolates from the environment. Emodin not only exhibited moderate antibacterial activity against the Gram-positive bacteria but also showed strong synergistic association with oxacillin against MRSA Staphylococcus aureus.

Indolediketopiperazine Alkaloids from Eurotium cristatum EN-220, an Endophytic Fungus Isolated from the Marine Alga Sargassum thunbergii.

Four new indolediketopiperazine derivatives (1-4), along with nine known congeners (5-13), were isolated and identified from the culture extract of Eurotium cristatum EN-220, an endophytic fungus obtained from the marine alga Sargassum thunbergii. The structures of thesecompounds were elucidated on the basis of extensive spectroscopic analysis and the absolute configurations of compounds 1-4 were established by NOESY experiments and by chiral HPLC analyses of their acid hydrolysates. The absolute configuration of C-8 (a quaternary carbon substituted with a hydroxyl group) in 5 of preechinulin class was firstly determined by electronic circular dichroism (ECD) calculations. All these compounds were evaluatedfor brine shrimp (Artemia salina) lethality and nematicidal activity as well as antioxidativeand antimicrobial potency.

Two benzaldehyde derivatives and their artefacts from a gorgonian-derived Eurotium sp. fungus.

Two new benzaldehyde derivatives, named 3'-OH-tetrahydroauroglaucin (1) and(3'S*,4'R*)-6-(3',5-epoxy-4'-hydroxy-1'-heptenyl)-2-hydroxy-3-(3''-methyl-2''-butenyl)benzaldehyde (2), were isolated from a gorgonian-derived Eurotium sp. fungus. Their structures were determined by extensive spectroscopic analysis including NMR and MS spectra. Dissolved 1 in CDCl3 for several days could be detected its 2H-chromene skeleton derivatives (1a/1b), a pair of enantiomers with opposite configurations at C-3'. Compound 2 was also found to chemically convert to a pair of epimers non-enzymatically. The plausible mechanism to form the 2H-chromene artefacts with racemisation at C-3' undergoing nucleophilic substitution (SN1) was proposed.

Conditional changes enhanced production of bioactive metabolites of marine derived fungus Eurotium rubrum.

Metabolites of marine derived fungus Eurotium rubrum MPUC136 differed between cultivation on wheat medium and Czapek-Dox agar medium. Melanin synthesis inhibitory activity of crude extract of culture on wheat medium showed stronger activity than that of crude extract of culture on Czapek-Dox agar medium. A new diketopiperazine compound isoechinulin D (1) and eight reported diketopiperazines (2-9) were isolated from the crude extract of wheat medium. The structure of 1 was established using NMR, MS and IR methods. 2-5 inhibited melanogenesis using B16 melanoma cells (IC50=68, 2.4, 83, 9.1µM each). Structure-Activity-Relationships of diketopiperazines (1-10) indicated the importance of the prenyl groups at C-2, C-5 and C-7, the vinyl group at C-12 to C-25 and the sp2 carbons at C-8 and C-9. Isolated compounds (1-9) were not or slightly observed from the extracts of Czapek-Dox agar medium by HPLC analysis, suggesting that different cultivation processes could affect metabolism and enhance bioactivities.

Isolation, Stereochemical Study, and Antioxidant Activity of Benzofuranone Derivatives from a Mangrove-derived Fungus Eurotium rubrum MA-150.

Enantiomers of a 2-benzofuran-1(3H)-one derivative [(-)- and (+)-] and four known analogs () were isolated and identified from the culture extract of Eurotium rubrum MA-150, a fungus obtained from the mangrove-derived rizospheric soil. Their structures were established by detailed interpretation of nuclear magnetic resonance (NMR) data and the structure of (±)- was confirmed by single-crystal X-ray diffraction analysis. The absolute configuration of the enantiomers (-)- and (+)- was determined by means of online high-performance liquid chromatography - electronic circular dichroism (HPLC-ECD) measurements and time-dependent Density Functional Theory - electronic circular dichroism (TDDFT-ECD) calculations. Compounds (±)- as well as and exhibited potent DPPH radical scavenging activities with IC50 values of 1.23, 2.26, and 3.99 µg/mL, respectively. Chirality 28:581-584, 2016. © 2016 Wiley Periodicals, Inc.

Mycotoxigenic fungi and natural co-occurrence of mycotoxins in rainbow trout (Oncorhynchus mykiss) feeds.

Samples of rainbow trout feed were analyzed with the aim to determine the mycobiota composition and the co-occurrence of mycotoxins. A total of 28 samples of finished rainbow trout feed from hatcheries in the provinces of Río Negro and Neuquén, Argentina, were studied. Fungal counts were obtained on three culture media in the ranges of <10 to 4.2 × 104 CFU/g on Dichloran Rose Bengal Chloramphenicol Agar (DRBC), <10 to 5.1 × 104 CFU/g on Dichloran Chloramphenicol Peptone Agar (DCPA) and <10 to 3.6 × 104 CFU/g on Dichloran 18% Glycerol Agar (DG18). The most frequent mycotoxigenic fungi were Eurotium (frequency (Fr) 25.0%), followed by Penicillium (Fr 21.4%) and Aspergillus (Fr 3.6%). The most prevalent mycotoxigenic species were E. repens (Fr 21.4%) and E. rubrum (Fr 14.3%). All samples were contaminated with mycotoxins: 64% samples were contaminated with T-2 toxin (median 70.08 ppb), 50% samples with zearalenone (median 87.97 ppb) and aflatoxins (median 2.82 ppb), 25% with ochratoxin A (median 5.26 ppb) and 3.57% samples with deoxynivalenol (median 230 ppb). Eight samples had a fumonisins contamination level below the limit of detection. Co-occurrence of six mycotoxins was determined in 7% of the samples.

Rubrumazines A-C, Indolediketopiperazines of the Isoechinulin Class from Eurotium rubrum MA-150, a Fungus Obtained from Marine Mangrove-Derived Rhizospheric Soil.

Three new indolediketopiperazine alkaloids of the isoechinulin type, rubrumazines A-C (1-3), each possessing an oxygenated prenyl group either at C-7 (1 and 2) or at C-5 (3), along with 13 related analogues (4-16), were isolated and identified from a culture extract of Eurotium rubrum MA-150, a fungus obtained from mangrove-derived rhizospheric soil collected from the Andaman Sea coastline, Thailand. Their structures were established by detailed interpretation of NMR spectroscopic and mass spectrometry data analysis. The structure and absolute configuration of compound 1 were confirmed by X-ray crystallographic analysis, thus providing the first characterized crystal structure of an isoechinulin-type alkaloid. All isolated compounds were evaluated for brine shrimp lethality and antibacterial activity.

Neoechinulin B and its analogues as potential entry inhibitors of influenza viruses, targeting viral hemagglutinin.

A class of prenylated indole diketopiperazine alkaloids including 15 new compounds namely rubrumlines A-O obtained from marine-derived fungus Eurotium rubrum, were tested against influenza A/WSN/33 virus. Neoechinulin B (18) exerted potent inhibition against H1N1 virus infected in MDCK cells, and is able to inhibit a panel of influenza virus strains including amantadine- and oseltamivir-resistant clinical isolates. Mechanism of action studies indicated that neoechinulin B binds to influenza envelope hemagglutinin, disrupting its interaction with the sialic acid receptor and the attachment of viruses to host cells. In addition, neoechinulin B was still efficient in inhibiting influenza A/WSN/33 virus propagation even after a fifth passage. The high potency and broad-spectrum activities against influenza viruses with less drug resistance make neoechinulin B as a new lead for the development of potential inhibitor of influenza viruses.

Inhibitory effects of benzaldehyde derivatives from the marine fungus Eurotium sp. SF-5989 on inflammatory mediators via the induction of heme oxygenase-1 in lipopolysaccharide-stimulated RAW264.7 macrophages.

Two benzaldehyde derivatives, flavoglaucin (1) and isotetrahydro-auroglaucin (2), were isolated from the marine fungus Eurotium sp. SF-5989 through bioassay- and 1H NMR-guided investigation. In this study, we evaluated the anti-inflammatory effects of these compounds in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. We demonstrated that compounds 1 and 2 markedly inhibited LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production by suppressing inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression without affecting cell viability. We also demonstrated that the compounds reduced the secretion of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6). Furthermore, compounds 1 and 2 inhibited LPS-induced nuclear factor-κB (NF-κB) activation by suppressing phosphorylation of IkappaB (IκB). These results indicated that the anti-inflammatory effects of these benzaldehyde derivatives in LPS-stimulated RAW264.7 macrophages were due to the inactivation of the NF-κB pathway. In addition, compounds 1 and 2 induced heme oxygenase-1 (HO-1) expression through the nuclear transcription factor-E2-related factor 2 (Nrf2) translocation. The inhibitory effects of compounds 1 and 2 on the production of pro-inflammatory mediators and on NF-κB binding activity were reversed by HO-1 inhibitor tin protoporphyrin (SnPP). Thus, the anti-inflammatory effects of compounds 1 and 2 also correlated with their ability of inducing HO-1 expression.

A new prenylated indole diketopiperazine alkaloid from Eurotium cristatum.

A new prenylated indole diketopiperazine alkaloid, cristatumin F (1), and four known metabolites, echinulin (2), dehydroechinulin (3), neoechinulin A (4) and variecolorin O (5), were isolated from the crude extract of the fungus Eurotium cristatum. The structure of 1 was elucidated primarily by NMR and MS methods. The absolute configuration of 1 was assigned using Marfey's method applied to its acid hydrolyzate. Cristatumin F (1) showed modest radical scavenging activity against DPPH radicals, and exhibited marginal attenuation of 3T3L1 pre-adipocytes.

Total synthesis and structural revision of (+)-cristatumin C.

Naturally occurring (+)-cristatumin C, a bis-pyrrolidinoindoline diketopiperazine alkaloid isolated from Eurotium cristatum EN-220, is the 2R,3R,11S,15R,2'R,3'R,11'S,15'S enantiomer, as confirmed by total synthesis.