Miniaturized Cultivation Profiling (MATRIX)-Facilitated Discovery of Noonazines A-C and Noonaphilone A from an Australian Marine-Derived Fungus, Aspergillus noonimiae CMB-M0339.

Subjecting the Australian marine-derived fungus Aspergillus noonimiae CMB-M0339 to cultivation profiling using an innovative miniaturized 24-well plate format (MATRIX) enabled access to new examples of the rare class of 2,6-diketopiperazines, noonazines A-C (1-3), along with the known analogue coelomycin (4), as well as a new azaphilone, noonaphilone A (5). Structures were assigned to 1-5 on the basis of a detailed spectroscopic analysis, and in the case of 1-2, an X-ray crystallographic analysis. Plausible biosynthetic pathways are proposed for 1-4, involving oxidative Schiff base coupling/dimerization of a putative Phe precursor. Of note, 2 incorporates a rare meta-Tyr motif, typically only reported in a limited array of Streptomyces metabolites. Similarly, a plausible biosynthetic pathway is proposed for 5, highlighting a single point for stereo-divergence that allows for the biosynthesis of alternate antipodes, for example, the 7R noonaphilone A (5) versus the 7S deflectin 1a (6).

Secondary Metabolites with Agricultural Antagonistic Potential from Aspergillus sp. ITBBc1, a Coral-Associated Marine Fungus.

A marine-derived fungal strain, Aspergillus sp. ITBBc1, was isolated from coral collected from the South China Sea in Hainan province. Intensive chemical investigation of the fermentation extract of this strain afforded four new secondary metabolites (1-4), named megastigmanones A-C and prenylterphenyllin H, along with four known compounds (5-8). Their structures were elucidated by extensive spectroscopic analysis including one-and two-dimensional (1D and 2D) NMR spectroscopy and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). The modified Mosher's method was undertaken to determine the absolute configurations of new compounds. The phytotoxic activity test showed that compounds 6-8 exhibited significant antagonistic activity against the germination of Triticum aestivum L. and Oryza sativa L. seeds with a dose-dependent relationship.

New Meroterpenoids and α-Pyrone Derivatives Isolated from the Mangrove Endophytic Fungal Strain Aspergillus sp. GXNU-Y85.

Two new meroterpenoids, aspergienynes O and P (1 and 2), one new natural compound, aspergienyne Q (3), and a new α-pyrone derivative named 3-(4-methoxy-2-oxo-2H-pyran-6-yl)butanoic acid (4) were isolated from the mangrove endophytic fungal strain Aspergillus sp. GXNU-Y85, along with five known compounds (5-9). The absolute configurations of those new isolates were confirmed through extensive analysis using spectroscopic data (HRESIMS, NMR, and ECD). The pharmacological study of the anti-proliferation activity indicated that isolates 5 and 9 displayed moderate inhibitory effects against HeLa and A549 cells, with the IC50 values ranging from 16.6 to 45.4 µM.

Ergone Derivatives from the Deep-Sea-Derived Fungus Aspergillus terreus YPGA10 and 25,28-Dihydroxyergone-Induced Apoptosis in Human Colon Cancer SW620 Cells.

Ten new ergone derivatives (1-10) and five known analogues (11-15) were isolated from the deep-sea-derived fungus Aspergillus terreus YPGA10. The structures including the absolute configurations were established by detailed analysis of the NMR spectroscopic data, HRESIMS, ECD calculation, and coupling constant calculation. All the structures are characterized by a highly conjugated 25-hydroxyergosta-4,6,8(14),22-tetraen-3-one nucleus. Structurally, compound 2 bearing a 15-carbonyl group and compounds 5-7 possessing a 15ß-OH/OCH3 group are rarely encountered in ergone derivatives. Bioassay results showed that compounds 1 and 11 demonstrated cytotoxic effects on human colon cancer SW620 cells with IC50 values of 8.4 and 3.1 µM, respectively. Notably, both compounds exhibited negligible cytotoxicity on the human normal lung epithelial cell BEAS-2B. Compound 11 was selected for preliminary mechanistic study and was found to inhibit cell proliferation and induce apoptosis in human colon cancer SW620 cells. In addition, compound 1 displayed cytotoxic activity against five human leukemia cell lines with IC50 values ranging from 5.7 to 8.9 µM. Our study demonstrated that compound 11 may serve as a potential candidate for the development of anticolorectal cancer agents.

Detection of aflatoxin B1 in chili powder using attenuated total reflectance-Fourier transform infrared spectroscopy.

Aflatoxin B1, a major global food safety concern, is produced by toxigenic fungi during crop growing, drying, and storage, and shows increasing annual prevalence. This study aimed to detect aflatoxin B1 in chili samples using ATR-FTIR coupled with machine learning algorithms. We found that 83.6% of the chili powder samples were contaminated with Aspergillus and Penicillium species, with aflatoxin B1 levels ranging from 7.63 to 44.32 µg/kg. ATR-FTIR spectroscopy in the fingerprint region (1800-400 cm-1) showed peak intensity variation in the bands at 1587, 1393, and 1038 cm-1, which are mostly related to aflatoxin B1 structure. The PCA plots from samples with different trace amounts of aflatoxin B1 could not be separated. Vibrational spectroscopy combined with machine learning was applied to address this issue. The logistic regression model had the best F1 score with the highest %accuracy (73%), %sensitivity (73%), and %specificity (71%), followed by random forest and support vector machine models. Although the logistic regression model contributed significant findings, this study represents a laboratory research project. Because of the peculiarities of the ATR-FTIR spectral measurements, the spectra measured for several batches may differ, necessitating running the model on multiple spectral ranges and using increased sample sizes in subsequent applications. This proposed method has the potential to provide rapid and accurate results and may be valuable in future applications regarding toxin detection in foods when simple onsite testing is required.

Polyketides with α-glucosidase inhibitory and neuroprotective activities from Aspergillus versicolor associated with Pedicularis sylvatica.

Aspergillus versicolor, an endophytic fungus associated with the herbal medicine Pedicularis sylvatica, produced four new polyketides, aspeversins A-D (1-2 and 5-6) and four known compounds, O-methylaverufin (2), aversin (3), varilactone A (7) and spirosorbicillinol A (8). Their structures were elucidated by extensive spectroscopic data analysis, and their absolute configurations were determined by calculated electronic circular dichroism (ECD) and Mo2(AcO)4-induced CD data. Compound 5 was found to exhibit α-glucosidase inhibitory activity with an IC50 value of 25.57 µM. An enzyme kinetic study indicated that 5 was a typical uncompetitive inhibitor toward α-glucosidase, which was supported by a molecular docking study. Moreover, compounds 1-3 and 5 also improved the cell viability of PC12 cells on a 1-methyl-4-phenylpyridinium (MPP+)-induced Parkinson's disease model, indicating their neuroprotective potential as antiparkinsonian agents.

Increasing structure diversity of farnesylated chalcones by a fungal aromatic prenyltransferase.

Farnesylated chalcones were favored by researchers due to their different biological activities. However, only five naturally occurring farnesylated chalcones were described in the literature until now. Here, the farnesylation of six chalcones by the Aspergillus terreus aromatic prenyltransferase AtaPT was reported. Fourteen monofarnesylated chalcones (1F1-1F5, 2F1-2F3, 3F1, 3F2, 4F1, 4F2, 5F1, 6F1, and 6F2) and a difarnesylated product (2F3) were obtained, enriching the diversity of natural farnesylated chalcones significantly. Ten of them are C-farnesylated products, which complement O-farnesylated chalcones by chemical synthesis. Fourteen products have not been reported prior to this study. Nine of the produced compounds (1F2-1F5, 2F1-2F3, 5F1, and 6F1) exhibited inhibitory effect on α-glucosidase with IC50 values ranging from 24.08 ± 1.44 to 190.0 ± 0.28 µM. Among them, compounds 2F3 with IC50 value at 24.08 ± 1.44 µM and 1F4 with IC50 value at 30.09 ± 0.59 µM showed about 20 times stronger than the positive control acarbose with an IC50 at 536.87 ± 24.25 µM in α-glucosidase inhibitory assays.

Antimicrobial metabolites from the marine-derived fungus Aspergillus sp. ZZ1861.

Fungi from the genus Aspergillus are important resources for the discovery of bioactive agents. This investigation characterized the isolation, structural elucidation, and antimicrobial evaluation of 46 metabolites produced by the marine-derived fungus Aspergillus sp. ZZ1861 in rice solid and potato dextrose broth liquid media. The structures of these isolated compounds were determined based on their HRESIMS data, NMR spectral analyses, and data from ECD, NMR, and optical rotation calculations. Emericelactones F and G, 20R,25S-preshamixanthone, 20R,25R-preshamixanthone, phthalimidinic acid A, phthalimidinic acid B, aspergilol G, and 2-hydroxyemodic amide are eight previously undescribed compounds and (S)-2-(5-hydroxymethyl-2-formylpyrrol-1-yl) propionic acid lactone is reported from a natural resource for the first time. It is also the first report of the configurations of 25S-O-methylarugosin A, 25R-O-methylarugosin A, 5R-(+)-9-hydroxymicroperfuranone, and 5R-(+)-microperfuranone. Phthalimidinic acid A, phthalimidinic acid B, aspergilol G, and 2-hydroxyemodic amide have antifungal activity against Candida albicans with MIC values of 1.56, 3.12, 1.56, and 12.5 µg/mL, respectively, 20R,25S-preshamixanthone (MIC 25 µg/mL) shows antibacterial activity against Escherichia coli, and 20R,25R-preshamixanthone exhibits antimicrobial activity against all three tested pathogens of methicillin-resistant Staphylococcus aureus, E. coli, and C. albicans with MIC values of 50, 25, 25 µg/mL, respectively.

Discovery of an immunosuppressive functional metabolite from the insect-derived endophytic Aspergillus taichungensis SMU01.

Three p-terphenyl metabolites (1-3), three indole-diterpenoids (4-6), an herbicide sesquiterpene (7), a flavonoid (8), and five other small molecules containing nitrogen (9-13) were isolated from the medicinal insect (Periplaneta americana)-derived endophytic Aspergillus taichungensis SMU01. Their chemical structures were elucidated on the basis of spectroscopic data and quantum chemical computational methods. Biological activity of these isolates in the differentiation of mouse CD4+ T cell subsets was evaluated. Importantly, metabolites 2 targeting JAK-STAT signaling pathway could hold potential benefits in maintaining peripheral immune homeostasis and alleviating the progression of autoimmune diseases.

Characterization and antioxidant properties of three exopolysaccharides produced by the Cyclocarya paliurus endophytic fungus.

In recent years, the considerable potential of endophytic bacteria and fungi as prolific producers of exopolysaccharides (EPSs) have attracted interest. In this study, 56 endophytes were isolated from Cyclocarya paliurus, and the secondary metabolites of EPSs were extracted from Monascus purpureus, Penicillium citrinum and Aspergillus versicolor, screened, and named MPE, PCE and AVE, respectively. In this work, the physicochemical properties and antioxidant activities of three EPSs, their cell proliferation activity on IEC-6 and RAW264.7 were investigated. The three EPSs were mainly composed of neutral sugar and differ in microstructure. However, MPE had a loose structure, and PCE exhibited a dense and sheet-like structure. In addition, the three EPSs performed ordinary antioxidant activity in vitro but showed excellent cell proliferation activity on IEC-6 and RAW264.7. The cell proliferation activity of PCE was 1.4-fold that of the controls at a concentration of 800 µg/mL on IEC-6, and MPE exhibited 1.3-fold increase on RAW264.7. This study provided scientific evidence and insights into the application of endophytes as a novel plant resource possessing huge application potential.

Discovery of Antibacterial Compounds against Xanthomonas citri subsp. citri from a Marine Fungus Aspergillus terreus SCSIO 41202 and the Mode of Action.

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a severe citrus disease. Currently, copper-containing pesticides are widely used to manage this disease, posing high risks to the environment and human health. This study reports the discovery of naturally occurring anti-Xcc compounds from a deep-sea fungus, Aspergillus terreus SCSIO 41202, and the possible mode of action. The ethyl acetate extract of A. terreus was subjected to bioassay-guided isolation, resulting in the discovery of eight anti-Xcc compounds (1-8) with minimum inhibitory concentrations (MICs) ranging from 0.078 to 0.625 mg/mL. The chemical structures of these eight metabolites were determined by integrative analysis of various spectroscopic data. Among these compounds, Asperporonin A (1) and Asperporonin B (2) were identified as novel compounds with a very unusual structural skeleton. The electronic circular dichroism was used to determine the absolute configurations of 1 and 2 through quantum chemical calculation. A bioconversion pathway involving pinacol rearrangement was proposed to produce the unusual compounds (1-2). Compound 6 exhibited an excellent anti-Xcc effect with a MIC value of 0.078 mg/mL, which was significantly more potent than the positive control CuSO4 (MIC = 0.3125 mg/mL). Compound 6 inhibited cell growth by disrupting biofilm formation, destroying the cell membrane, and inducing the accumulation of reactive oxygen species. In vivo tests indicated that compound 6 is highly effective in controlling citrus canker disease. These results indicate that compounds 1-8, especially 6, have the potential as lead compounds for the development of new, environmentally friendly, and efficient anti-Xcc pesticides.

Nucleobase-Coupled Xanthones with Anti-ROS Effects from Marine-Derived Fungus Aspergillus sydowii.

A MS/MS-based molecular networking approach compared to the Global Natural Product Social Molecular Networking library, in association with genomic annotation of natural product biosynthetic gene clusters within a marine-derived fungus, Aspergillus sydowii, identified a suite of xanthone metabolites. Chromatographic techniques applied to the cultured fungus led to the isolation of 11 xanthone-based alkaloids, dubbed sydoxanthones F-M. The structures of these alkaloids were elucidated using extensive spectroscopic data, including electronic circular dichroism and single-crystal X-ray diffraction data for configurational assignments. Among these analogues, sydoxanthones F-K exhibit structure features typical of nucleobase-coupled xanthones, with sydoxanthone H being an N-bonded xanthone dimer. Notably, (±)sydoxanthones F (1a/1b), (±)sydoxanthones H (3b/3a), and (±)sydoxanthones J (5b/5a) are enantiomeric pairs, while sydoxanthones G (2), I (4), and K (6) are stereoisomers of 1, 3, and 5, respectively. Furthermore, (+)sydoxanthone H (3a) demonstrated significant rescue of cell viability in H2O2-injuried SH-SY5Y cells by inhibiting reactive oxygen species production, suggesting its potential for neuroprotection.

Ergosterols with rare peroxide, oxetane ring moiety, and a lactone ring from Aspergillus spectabilis and their immunosuppressive activities.

Ten ergostane-type steroids, including seven undescribed ones named spectasteroids A-G, were obtained from Aspergillus spectabilis. Their structures and absolute configurations were determined based on HRESIMS, NMR, ECD calculations, and single-crystal X-ray diffraction analyses. Structurally, spectasteroid A was a unique example of aromatic ergostane-type steroid that featured a rare peroxide ring moiety; spectasteroid B contained a rare oxetane ring system formed between C-9 and C-14; and spectasteroid C was an unusual 3,4-seco-ergostane steroid with an extra lactone ring between C-3 and C-9. Spectasteroids F and G specifically showed inhibitory effects against concanavalin A-induced T lymphocyte proliferation and lipopolysaccharide-induced B lymphocyte proliferation, with IC50 values ranging from 2.33 to 4.22 µM. Spectasteroid F also showed excellent antimultidrug resistance activity, which remarkable enhanced the inhibitory activity of PTX on the colony formation of SW620/Ad300 cells.

Identifying biochemical constituents involved in the mycosynthesis of zinc oxide nanoparticles.

Filamentous fungi are known to secrete biochemicals that drive the synthesis of nanoparticles (NPs) that vary in composition, size, and shape; a process deemed mycosynthesis. Following the introduction of precursor salts directly to the fungal mycelia or their exudates, mycosynthesis proceeds at ambient temperature and pressure, and near neutral pH, presenting significant energy and cost savings over traditional chemical or physical approaches. The mycosynthesis of zinc oxide (ZnO) NPs by various fungi exhibited a species dependent morphological preference for the resulting NPs, suggesting that key differences in the biochemical makeup of their individual exudates may regulate the controlled nucleation and growth of these different morphologies. Metabolomics and proteomics of the various fungal exudates suggest that metal chelators, such as hexamethylenetetramine, present in high concentrations in exudates of Aspergillus versicolor are critical for the production dense, well-formed, spheroid nanoparticles. The results also corroborate that the proteinaceous material in the production of ZnO NPs serves as a surface modifier, or protein corona, preventing excessive coagulation of the NPs. Collectively, these findings suggest that NP morphology is regulated by the small molecule metabolites, and not proteins, present in fungal exudates, establishing a deeper understanding of the factors and mechanism underlying mycosynthesis of NPs.

The Promiscuous Flavin-Dependent Monooxygenase PboD from Aspergillus ustus Increases the Structural Diversity of Hydroxylated Pyrroloindoline Diketopiperazines.

The potential of natural products as pharmaceutical and agricultural agents is based on their large structural diversity, resulting in part from modifications of the backbone structure by tailoring enzymes during biosynthesis. Flavin-dependent monooxygenases (FMOs), as one such group of enzymes, play an important role in the biosynthesis of diverse natural products, including cyclodipeptide (CDP) derivatives. The FMO PboD was shown to catalyze C-3 hydroxylation at the indole ring of cyclo-l-Trp-l-Leu in the biosynthesis of protubonines, accompanied by pyrrolidine ring formation. PboD substrate promiscuity was investigated in this study by testing its catalytic activity toward additional tryptophan-containing CDPs in vitro and biotransformation in Aspergillus nidulans transformants bearing a truncated protubonine gene cluster with pboD and two acetyltransferase genes. High acceptance of five CDPs was detected for PboD, especially of those with a second aromatic moiety. Isolation and structure elucidation of five pyrrolidine diketopiperazine products, with two new structures, proved the expected stereospecific hydroxylation and pyrrolidine ring formation. Determination of kinetic parameters revealed higher catalytic efficiency of PboD toward three CDPs consisting of aromatic amino acids than of its natural substrate cyclo-l-Trp-l-Leu. In the biotransformation experiments with the A. nidulans transformant, modest formation of hydroxylated and acetylated products was also detected.

Four sulfur-containing compounds with anti-colon cancer effect from marine-derived fungus Aspergillus terreus.

Sulfur-containing natural products possess a variety of biological functions including antitumor, antibacterial, anti-inflammatory and antiviral activities. In this study, four previously undescribed sulfur-containing compounds asperteretals L and M, terreins A and B, together with 17 known compounds were obtained from a culture of marine fungus A. terreus supplemented with inorganic sulfur source Na2SO4. Their planar structures and absolute configurations were elucidated by NMR, HRESIMS, and ECD experiments. The in vitro cytotoxicities of compounds 1-21 against HCT-116 and Caco-2 were evaluated by SRB assay. Asperteretal M (2) exhibited activity against HCT-116 with the IC50 value at 30µM. The antiproliferative effect of asperteretal M was confirmed by colony formation assay and cell death staining. Furthermore, the preliminary study on the anti-colon cancer mechanism of asperteretal M was performed by RNA-seq analysis. Western blotting validated that asperteretal M significantly decreased the expression of cell-cycle regulatory proteins CDK1, CDK4, and PCNA in a concentration-dependent manner.

Untargeted Metabolomic to Access Chemical Differences Induced by Dual Endophyte Cultures Isolated from Euphorbia Umbellata.

Endophytic fungi live asymptomatically inside vegetal tissues, and such uncommon habitat contributes to their exceptional chemical diversity. Isolating natural products from endophytic fungi could fail due to silent biosynthetic gene clusters under ordinary in vitro culture conditions, and co-culturing has been assayed to trigger their metabolism. We carried out single and dual cultures with 13 endophyte strains isolated from Euphorbia umbellata leaves. Multivariate statistics applied to untargeted metabolomics compared the chemical profiles of all endophyte cultures. PCA analysis guided the selection of the Aspergillus pseudonomiae J1 - Porogramme brasiliensis J9 dual culture for its most significant chemical differentiation: Five compounds were putatively annotated in the J1-J9 culture according to UHPLC-HRMS data, kojic acid, haliclonol and its diastereoisomer, caffeic acid, and 2-(3,4-dihydroxyphenyl)acetaldehyde. Analysis by PLS-DA using VIP score showed that kojic acid displayed the most significative importance in discriminating single and dual J1-J9 cultures.

Advancing Natural Product Discovery: A Structure-Oriented Fractions Screening Platform for Compound Annotation and Isolation.

Natural product discovery is hindered by the lack of tools that integrate untargeted nuclear magnetic resonance and mass spectrometry data on a library scale. This article describes the first application of the innovative NMR/MS-based machine learning tool, the "Structure-Oriented Fractions Screening Platform (SFSP)", enabling functional-group-guided fractionation and accelerating the discovery and characterization of undescribed natural products. The concept was applied to the extract of a marine fungus known to be a prolific producer of diverse natural products. With the assistance of SFSP, we isolated 24 flavipidin derivatives and five phenalenone analogues from Aspergillus sp. GE2-6, revealing 27 undescribed compounds. Compounds 7-22 were proposed as isomeric derivatives featuring a 5/6-ring fusion, formed by the dimerization of flavipidin E (5). Compounds 23 and 24 were envisaged as isomeric derivatives with a 6/5/6-ring fusion, generated through the degradation of two flavipidin E molecules. Furthermore, flavipidin A (1) and asperphenalenone E (28) exhibited potent anti-influenza (PR8) activities, with IC50 values of 21.9 ± 0.2 and 12.9 ± 0.1 µM, respectively. Meanwhile, asperphenalenone (26) and asperphenalenone P (27) treatments exhibited significant inhibition of HIV pseudovirus infection in 293FT cells, boasting IC50 values of 6.1 ± 0.9 and 4.6 ± 1.1 µM, respectively. Overall, SFSP streamlines natural product isolation through NMR and MS data integration, as showcased by the discovery of numerous undescribed flavipidins and phenalenones based on NMR olefinic signals and low-field hydroxy signals.

Immunosuppressive steroids quadrilisteroids A-C and derivatives from the terrestrial fungus Aspergillus quadrilineatus.

Seven undescribed compounds (1-7) along with six known compounds (8-13) were isolated from Eurotiaceae Aspergillus quadrilineatus. Their structures and absolute configurations were elucidated by NMR, HRESIMS, and ECD calculations. Quadrilisteroids A (1) and B (2) possessed an unprecedented 6/5/6/6/6/5 hexacyclic ring system in conjugation with a highly fused benzene ring, while quadrilisteroid C (3) featured a surprising 6/6/6/5/5-fused carbocyclic skeleton. Quadrilisteroid C (3) exhibited potent inhibitory activity against LPS-induced proliferation of B lymphocyte cells with an IC50 value of 1.03 µM. Compound 4, demonstrated inhibitory activity against Con A-induced proliferation of T lymphocyte cells with IC50 values of 6.42 µM.

Asperustins A-J: Austocystins with Immunosuppressive and Cytotoxic Activities from Aspergillus ustus NRRL 5856.

Ten new (1-10) and nine known (11-19) austocystins, along with four known anthraquinones (20-23), were isolated from the culture of Aspergillus ustus NRRL 5856 by bioactivity-guided fractionation. The structures of the new compounds were elucidated by spectroscopic data analysis, X-ray crystallographic study, the modified Mosher's method, [Rh2(OCOCF3)4]-induced ECD spectral analysis, and comparison of the experimental ECD spectra with those of the similar analogues. Compounds 1-8 represent the first examples of austocystins with a C-4' oxygenated substitution. The absolute configuration of 1″-hydroxy austocystin D (11) was determined by single-crystal X-ray diffraction and consideration of its biosynthetic origin. Compounds 5, 9, and 11 exhibited significant inhibitory effects against the proliferation of ConA-induced T cells with IC50 values of 1.1, 1.0, and 0.93 µM, respectively. Furthermore, these compounds suppressed the expression of IL-6 in a dose-dependent manner. Compounds 10-12 and 14 showed pronounced cytotoxicities against MCF-7 with IC50 values of 3.9, 1.3, 0.46, and 2.3 µM, respectively.