Production of kojic acid by Aspergillus flavus OL314748 using box-Behnken statistical design and its antibacterial and anticancer applications using molecular docking technique.

Kojic acid is a wonderful fungal secondary metabolite that has several applications in the food, medical, and agriculture sectors. Many human diseases become resistant to normal antibiotics and normal treatments. We need to search for alternative treatment sources and understand their mode of action. Aspergillus flavus ASU45 (OL314748) was isolated from the caraway rhizosphere as a non-aflatoxin producer and identified genetically using 18S rRNA gene sequencing. After applying the Box-Behnken statistical design to maximize KA production, the production raised from 39.96 to 81.59 g/l utilizing (g/l) glucose 150, yeast extract 5, KH2PO4 1, MgSO4.7H2O 2, and medium pH 3 with a coefficient (R2) of 98.45%. Extracted KA was characterized using FTIR, XRD, and a scanning electron microscope. Crystalized KA was an effective antibacterial agent against six human pathogenic bacteria (Bacillus cereus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Serratia marcescens, and Serratia plymuthica). KA achieves high inhibition activity against Bacillus cereus, K. pneumonia, and S. plymuthica at 100 µg/ml concentration by 2.75, 2.85, and 2.85 compared with chloramphenicol which gives inhibition zones 1, 1.1, and 1.6, respectively. Crystalized KA had anticancer activity versus three types of cancer cell lines (Mcf-7, HepG2, and Huh7) and demonstrated high cytotoxic capabilities on HepG-2 cells that propose strong antitumor potent of KA versus hepatocellular carcinoma. The antibacterial and anticancer modes of action were illustrated using the molecular docking technique. Crystalized kojic acid from a biological source represented a promising microbial metabolite that could be utilized as an alternative antibacterial and anticancer agent effectively.

Uncovering the Power of HSCCC: Separation of Diastereomeric and Regioisomeric Styrylpyrones from the Stem Bark of Goniothalamus leiocarpus.

The plant Goniothalamus leiocarpus of the Annonaceae family is used as an alternative medicine in tropical regions. Applying high-speed counter current chromatography (HSCCC), eight new bioactive styrylpyrone isomers, including 6R,7S,8R,2'S-goniolactone B (1), 6S,7S,8S,2'S-goniolactone B (2), 6R,7R,8R,2'S-goniolactone B (3), 6R,7S,8S,2'S-goniolactone C (4), 6R,7S,8R,2'S-goniolactone C (5), 6S,7R,8S,2'S-goniolactone C (6), and two positional isomers, 6R,7R,8R,2'S-goniolactone G (7) and 6S,7R,8R,2'S-goniolactone G (8), were isolated from a chloroform fraction (2.1 g) of G. leiocarpus, which had a prominent spot by TLC analysis. The structures of the new compounds were elucidated by MS, NMR, IR, and UV spectra, and their absolute configurations were determined by Mosher's method, ECD, and X-ray diffraction analysis. The isolates are characteristic components found in plants of the genus Goniothalamus and consist of two structural moieties: a styrylpyrone and a dihydroflavone unit. The isolation of the eight new compounds demonstrates the effectiveness of HSCCC in separating the isomers of natural styrylpyrone. In a bioactivity assessment, compounds 1 and 6 exhibited cytotoxic effects against the human colon carcinoma cell lines LS513 and SW620 with IC50 values ranging from 1.6 to 3.9 µM. Compounds 1, 2, 7, and 8 showed significant synergistic activity against antibiotic-resistant Staphylococcus aureus strains.

Sesquiterpenes and α-pyrones from an endophytic fungus Xylaria curta YSJ-5.

Chemical investigation of the culture extract of an endophyte Xylaria curta YSJ-5 from Alpinia zerumbet (Pers.) Burtt. et Smith resulted in the isolation of eight previously undescribed compounds including five eremophilane sesquiterpenes xylarcurenes A-E, one norsesquiterpene xylarcurene F, and two α-pyrone derivatives xylarpyrones A-B together with eight known related derivatives. Their chemical structures were extensively established based on the 1D- and 2D-NMR spectroscopic analysis, modified Mosher's method, electronic circular dichroism calculations, single-crystal X-ray diffraction experiments, and the comparison with previous literature data. All these compounds were tested for in vitro cytotoxic, anti-inflammatory, α-glucosidase inhibitory, and antibacterial activities. As a result, 6-pentyl-4-methoxy-pyran-2-one was disclosed to display significant antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus with minimal inhibitory concentration value of 6.3 µg/mL.

Biosynthesis of Epipyrone A Reveals a Highly Specific Membrane-Bound Fungal C-Glycosyltransferase for Pyrone Galactosylation.

Epipyrone A is a unique C-galactosylated 4-hydroxy-2-pyrone derivative with an antifungal potential from the fungus Epicoccum nigrum. We elucidated its biosynthesis via heterologous expression and characterized an unprecedented membrane-bound pyrone C-glycosyltransferase biochemically. Molecular docking and mutagenesis experiments suggested a possible mechanism for the heterocyclic C-glycosylation and the importance of a transmembrane helix for its catalysis. These results expand the repertoire of C-glycosyltransferases and provide new insights into the formation of C-glycosides in fungi.

Undescribed α-pyrone-containing mycotoxins and an eremophilane-type sesquiterpenoid isolated from Aspergillus aureoterreus and their cytotoxicity.

Four previously undescribed and highly oxygenated α-pyrone-containing mycotoxins designated citreoviridins (E‒H), and an unreported eremophilane-type sesquiterpenoid namely aureoterrolide N, were isolated from the culture broth of Aspergillus aureoterreus. Those isolates were inferred from extensive spectroscopic methods and theoretical computation, where their absolute configurations were unambiguously determined by coupling constants following an empirical rule for the acyclic vicinal diol, theoretical ECD calculation, and NMR computation using the GIAO method and DP4+ analysis. Among them, citreoviridins E‒H are four stereoisomers of a citreoviridin derivative, featuring a methylated α-pyrone, an oxidized polyene linker, and a tetrahydrofuran ring. Cytotoxicity assay of all isolates demonstrated that aureoterrolide N exhibited weak inhibitory effect against human cancer cell line HL-60 with an inhibition rate of 55.2% at 40.0 µM.

Antibacterial p-terphenyl and α­pyrone derivates isolated from the marine-derived actinomycete Nocardiopsis sp. HDN154086.

Assisted by OSMAC strategy, one new p-terphenyl and two new α­pyrone derivates, namely nocarterphenyl I (1) and nocardiopyrone D-E (2-3), were obtained and characterized from the marine sediment-derived actinomycete Nocardiopsis sp. HDN154086. The structures of these compounds were determined on the basis of MS, NMR spectroscopic data and single-crystal X-ray diffraction. Compound 1 with a rare 2,2'-bithiazole structure among natural products showed promising activity against five bacteria with MIC values ranging from 0.8 to 1.6 µM and 3 exhibited notable antibacterial activity against MRSA compared the positive control ciprofloxacin.

Enantiomeric α-pyrone derivatives with immunosuppressive activity from Talaromyces adpressus.

Five pairs of undescribed enantiomeric α-pyrone derivatives (±)-adprepyrones A-E (±1-±5), together with an unreported congener adprepyrone F (6), and 6-[(E)-3-Hydroxyprop-1-enyl]-4-methoxy-5-methyl-2-pyrone (7), recently reported as synthetic compound, were isolated from the fungus Talaromyces adpressus. Their structures with absolute configurations were elucidated by HRESIMS, 1D and 2D NMR, electronic circular dichroism calculations, and single-crystal X-ray diffraction analyses. (±)-Adprepyrone A (±1) possesses an unreported carbon skeleton formed by the fusion of an α-pyrone derivative with nicotinamide. Compounds (+)-2, (±)-4, (±)-5, and 7 showed moderate inhibitory activity against concanavalin A (ConA)-induced T lymphocyte proliferation with IC50 values ranging from 8.9 to 19.8 µM.

Arzanol, a natural phloroglucinol α-pyrone, protects HaCaT keratinocytes against H2O2-induced oxidative stress, counteracting cytotoxicity, reactive oxygen species generation, apoptosis, and mitochondrial depolarization.

Skin oxidative stress results in structural damage, leading to premature senescence, and pathological conditions such as inflammation and cancer. The plant-derived prenylated pyrone-phloroglucinol heterodimer arzanol, isolated from Helichrysum italicum ssp. microphyllum (Willd.) Nyman aerial parts, exhibits anti-inflammatory, anticancer, antimicrobial, and antioxidant activities. This study explored the arzanol protection against hydrogen peroxide (H2O2) induced oxidative damage in HaCaT human keratinocytes in terms of its ability to counteract cytotoxicity, reactive oxygen species (ROS) generation, apoptosis, and mitochondrial membrane depolarization. Arzanol safety on HaCaT cells was preliminarily examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and microscopic observation. The arzanol pre-incubation (5-100 µM, for 24 h) did not induce cytotoxicity and morphological alterations. The phloroglucinol, at 50 µM, significantly protected keratinocytes against cytotoxicity induced by 2 h-incubation with 2.5 and 5 mM H2O2, decreased cell ROS production induced by 1 h-exposure to all tested H2O2 concentrations (0.5-5 mM), as determined by the 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) assay, and lipid peroxidation (thiobarbituric acid reactive substances [TBARS] method). The 2-h incubation of keratinocytes with H2O2 determined a significant increase of apoptotic cells versus control cells, evaluated by NucView® 488 assay, from the dose of 2.5 mM. Moreover, an evident mitochondrial membrane potential depolarization, monitored by fluorescent mitochondrial dye MitoView™ 633, was assessed at 5 mM H2O2. Arzanol pre-treatment (50 µM) exerted a strong significant protective effect against apoptosis, preserving the mitochondrial membrane potential of HaCaT cells at the highest H2O2 concentrations. Our results validate arzanol as an antioxidant agent for the prevention/treatment of skin oxidative-related disorders, qualifying its potential use for cosmeceutical and pharmaceutical applications.

Unravelling the Secrets of α-Pyrones from Aspergillus Fungi: A Comprehensive Review of Their Natural Sources, Biosynthesis, and Biological Activities.

Aspergillus, one of the most product-rich and genetically robust genera, contains a diverse range of species with potential economic and ecological implications. Chemically, Aspergillus is one of the essential sources of polyketides, alkaloids, diphenyl ethers, diketopiperazines, and other miscellaneous compounds, displaying a variety of pharmacological activities. The α-pyrones are unsaturated six-membered lactones. Although α-pyrone has a small structure, it is responsible for the structural diversity of several natural and synthetic compounds and multiple biological activities. In this review, we have summarized approximately 178 α-pyrone containing metabolites derivatives identified/reported from terrestrial, marine, endophytic, and filamentous Aspergillus species, including their sources, biological properties, and biosynthetic pathways until mid-2023, for the first time. This review is the first to compile and analyze the available data on α-pyrone metabolites from Aspergillus, which could facilitate further research and innovation in this field. Additionally, it offers a valuable source of scaffolds for future bioactive drug development, as some of these metabolites have shown potent antimicrobial, anti-inflammatory, and anticancer effects. Therefore, this review has significant implications for the advancement of natural product chemistry, pharmacology, biotechnology, and medicine.

Neuroprotective α-pyrones from Nigrospora oryzae, an endophytic fungus residing in Taxus chinensis var. mairei.

Endophytes coevolve with plant hosts and thus are more probable to acquire the character (in favor) of producing undescribed bioactive metabolites. Consequently, the topic has been intensely investigated for over two decades, but endophytic metabolites with neuroprotective effect remain scarce. The study presents the discovery of eight undescribed (named solanapyrones U-Z and prosolanapyrones A and B) and six known pyrones (solanapyrones A-C and E-G) from the culture of Nigrospora oryzae, an endophytic fungus associated with Taxus chinensis var. mairei. The structures and absolute configurations of undescribed pyrones were elucidated by extensive spectroscopic analysis, modified Mosher's method, and induced circular dichroism (ICD) spectrum. Solanapyrones A and B and an undescribed pyrone (solanapyrone U) were demonstrated to be more neuroprotective than clenbuterol in inducing bone marrow mesenchymal stem cells (bMSCs) to secret nerve growth factor (NGF). The work updates the pyrone chemodiversity in nature and extends the biofunction repertoire of solanapyrone-related polyketides.

Structurally Diverse Metabolites from the Marine-Derived Streptomyces sp. DS-27 Based on Two Different Culture Conditions.

Nine new compounds, including streptothiomycin A-E (1-5), two cyclopentenones (6, 7), one α-pyrone (8), wailupemycin Q (20), along with sixteen known compounds were identified from a rhizosphere strain Streptomyces sp. DS-27 derived from the marine cordgrass Spartina alterniflora under two different culture conditions. All of the structures were elucidated by extensive analysis of 1D/2D NMR and HR-ESI-MS data. The absolute configurations were determined by NOESY analysis, ECD, specific rotation and GIAO NMR calculations, and DP4+ probability analysis. Bioactivity investigation showed that compounds 5 and 7 exhibited significant inhibitory effects on LPS-induced NO production in a dose-dependent manner, which indicates their anti-inflammatory potential.

Biosynthesis-Guided Discovery and Engineering of α-Pyrone Natural Products from Type I Polyketide Synthases.

Natural products containing the α-pyrone moiety are produced by polyketide synthases (PKSs) in bacteria, fungi, and plants. The conserved biosynthetic logic for the production of the α-pyrone moiety involves the cyclization of a triketide intermediate which also off-loads the polyketide from the activating thioester. In this study, we show that truncating a tetraketide natural product producing PKS assembly line allows for a thioesterase-independent off-loading of an α-pyrone polyketide natural product, one which we find to be natively present in the extracts of the bacterium that otherwise furnishes the tetraketide natural product. By engineering the truncated PKS in vitro, we demonstrate that a ketosynthase (KS) domain with relaxed substrate selectivity when coupled with in trans acylation of polyketide extender units can expand the chemical space of α-pyrone polyketide natural products. Findings from this study point toward heterologous intermolecular protein-protein interactions being detrimental to the efficiency of engineered PKS assembly lines.

In vitro DPPH Radical Scavenging Activity of α-Pyrones from Odontonema strictum (Acanthaceae).

An ethyl acetate leaf extract from Odontonema strictum has been reported to have potent antihypertensive activity by inhibiting coronary artery contractions in porcine heart. However, the phytochemistry of the active fraction was unknown. Here we report, for the first time, the isolation and characterization of four known α-pyrones from the active fraction. The antioxidant activity of umuravumbolide (IC50 = 55.7±0.027 µg/mL), deacetylumuravumbolide (IC50 = 0.24±0.0002 µg/mL), dideacetylboronolide (IC50 = 149±0 µg/mL) and deacetylboronolide (IC50 = 24±0 µg/mL) was evaluated in vitro against 2,2-diphenyl-1-picrylhydrazyl radicals. Ascorbic acid was used as a positive control (IC50 = 1.73×10-3±0.3 µg/mL). The presence of 6-substituted 5,6-dihydro-α-pyrones and phenylpropanoid glucosides in the active fraction was suggested to be responsible for the antihypertensive activity. This is the first time that the antioxidant potential of these phytochemicals has been evaluated, and the results indicate that O. strictum has potential as an herbal medicine. Thus, further chemotaxonomic studies among the genera Odontonema and Tetradenia, a known source of α-pyrones, are recommended.

Absolute configuration of cyclopropanes and the structural revision of pyrones from Marine-derived fungus Stagonospora sp. SYSU-MS7888.

Two new cyclopropane derivatives (1-2) and seven undescribed α-pyrone derivatives (3-9), along with one known congener (10) were obtained from the marine fungus Stagonospora sp. SYSU-MS7888, which was isolated from the South China Sea. Their planar structures were established through extensive spectroscopic analyses including 1D and 2D NMR and HR-ESIMS. The absolute configurations were identified on basis of the quantum chemical calculations of ECD and NMR, as well as the modified Mosher's method. It's particularly noteworthy that the tetrasubstituted furopyrans, chenopodolans A-F, possessing phytotoxicity and zootoxicity, were structural misassignments. The structures of chenopodolans featuring with furopyran skeleton were revised as common trisubstituted α-pyrones by computational chemistry, NMR spectroscopic method, and empirical rule. Compounds 1, 2, 7, and 9 showed significant anti-inflammatory activity with IC50 values ranging from 3.6 to 22.8 µM, which is better than the positive control indomethacin (IC50 = 26.5 ± 1.13 µM). This discovery holds potential for the development of new anti-inflammatory agents.

Ethylidene-Tethered Chromene-Pyrone Hybrids as Potential Plant-Growth Regulators from an Endolichenic Phaeosphaeria Species.

Phaeosphaeria sp., a lichen-associated fungus, produced six skeletally new dimeric spiciferones (1-6) and four known metabolites (7-10). The new structures were elucidated by spectroscopic analysis, and their absolute configurations were determined by electronic circular dichroism calculations. Compounds 1 and 3-6 represent the first examples of ethylidene-bridged dimers from the building blocks 4H-chromene-4,7(8H)-dione and α-pyrone, and 2 is a unique homodimer of spiciferone. Compounds 1, 2, and 5-9 significantly inhibited the growth of weed-like dicot Arabidopsis thaliana at 100.0 µM. Notably, 8 showed the strongest inhibitory activity against the fresh weight and root elongation of A. thaliana with the IC50 values of 32.04 and 26.78 µM, respectively, whereas 1, 8, and 9 stimulated the growth of A. thaliana at lower concentrations. Meanwhile, compounds 2 and 6 exhibited weak inhibitory effects on the root elongation of monocot rice, while 1 and 8 exhibited growth-promoting effects on the shoot and root elongation of rice in a roughly dose-dependent manner.

Antifungal γ-Pyrone and Isoprenylated Cyclohexanoids from the Fungus Beenakia informis.

Chemical investigation of the extract of the macroscopic fungus Beenakia informis led to the isolation of a previously unreported γ-pyrone and two new isoprenylated cyclohexanoids, together with speciocin N. Their structures were elucidated spectroscopically and the absolute configuration was determined by comparison of the experimental vs. calculated ECD curves. Three of the compounds showed very good to moderate activity against phytopathogenic fungi.

Inhibitory Effect of Styrylpyrone Extract of Phellinus linteus on Hepatic Steatosis in HepG2 Cells.

The prevalence of nonalcoholic fatty liver disease (NAFLD) is estimated to be approximately about 25.24% of the population worldwide. NAFLD is a complex syndrome and is characterized by a simple benign hepatocyte steatosis to more severe steatohepatitis in the liver pathology. Phellinus linteus (PL) is traditionally used as a hepatoprotective supplement. Styrylpyrone-enriched extract (SPEE) obtained from the PL mycelia has been shown to have potential inhibition effects on high-fat- and high-fructose-diet-induced NAFLD. In the continuous study, we aimed to explore the inhibitory effects of SPEE on free fatty acid mixture O/P [oleic acid (OA): palmitic acid (PA); 2:1, molar ratio]-induced lipid accumulation in HepG2 cells. Results showed that SPEE presented the highest free radical scavenging ability on DPPH and ABTS, and reducing power on ferric ions, better than that of partitions obtained from n-hexane, n-butanol and distilled water. In free-fatty-acid-induced lipid accumulation in HepG2 cells, SPEE showed an inhibition effect on O/P-induced lipid accumulation of 27% at a dosage of 500 µg/mL. As compared to the O/P induction group, the antioxidant activities of superoxide dismutase, glutathione peroxidase and catalase were enhanced by 73%, 67% and 35%, respectively, in the SPEE group. In addition, the inflammatory factors (TNF-α, IL-6 and IL-1ß) were significantly down-regulated by the SPEE treatment. The expressions of anti-adipogenic genes involved in hepatic lipid metabolism of 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK), sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) were enhanced in the SPEE supplemented HepG2 cells. In the protein expression study, p-AMPK, SIRT1 and PGC1-α were significantly increased to 121, 72 and 62%, respectively, after the treatment of SPEE. Conclusively, the styrylpyrone-enriched extract SPEE can ameliorate lipid accumulation and decrease inflammation and oxidative stress through the activation of SIRT1/AMPK/PGC1-α pathways.

Neuropyrones A-E, five undescribed α-pyrone derivatives with tyrosinase inhibitory activity from the endophytic fungus Neurospora dictyophora WZ-497.

Five undescribed α-pyrone derivatives, named neuropyrones A-E, were isolated from the endophytic fungus Neurospora dictyophora WZ-497 derived from the stems of Aster tataricus L. f. The structures of these α-pyrones with absolute configurations were determined by comprehensive spectroscopic analysis and computational calculations. All isolated compounds were tested for various bioactivities, including tyrosinase inhibitory activity. The results showed that neuropyrones A-C displayed potent inhibitory effects on tyrosinase with IC50 values of 0.38 ± 0.07, 0.49 ± 0.06, and 0.12 ± 0.01 mM, respectively, which were comparable to that of the positive control, kojic acid (IC50 = 0.14 ± 0.021 mM). A molecular docking study revealed the interaction between 3 and the His263, His85, Val283, Asn260, Phe264, and Val248 residues of tyrosinase.

2-(2-(Dimethylamino)vinyl)-4H-pyran-4-ones as Novel and Convenient Building-Blocks for the Synthesis of Conjugated 4-Pyrone Derivatives.

A straightforward approach for the construction of the new class of conjugated pyrans based on enamination of 2-methyl-4-pyrones with DMF-DMA was developed. 2-(2-(Dimethylamino)vinyl)-4-pyrones are highly reactive substrates that undergo 1,6-conjugate addition/elimination or 1,3-dipolar cycloaddition/elimination followed by substitution of the dimethylamino group without ring opening. This strategy includes selective transformations leading to conjugated and isoxazolyl-substituted 4-pyrone structures. The photophysical properties of the prepared 4-pyrones were determined in view of further design of novel merocyanine fluorophores. A solvatochromism was found for enamino-substituted 4-pyrones accompanied by a strong increase in fluorescence intensity in alcohols. The prepared conjugated structures demonstrated valuable photophysical properties, such as a large Stokes shift (up to 204 nm) and a good quantum yield (up to 28%).

Computational Analysis of Triazole-Based Kojic Acid Analogs as Tyrosinase Inhibitors by Molecular Dynamics and Free Energy Calculations.

Molecular docking, molecular dynamics (MD) simulations and the linear interaction energy (LIE) method were used here to predict binding modes and free energy for a set of 1,2,3-triazole-based KA analogs as potent inhibitors of Tyrosinase (TYR), a key metalloenzyme of the melanogenesis process. Initially, molecular docking calculations satisfactorily predicted the binding mode of evaluated KA analogs, where the KA part overlays the crystal conformation of the KA inhibitor into the catalytic site of TYR. The MD simulations were followed by the LIE method, which reproduced the experimental binding free energies for KA analogs with an r2 equal to 0.97, suggesting the robustness of our theoretical model. Moreover, the van der Waals contributions performed by some residues such as Phe197, Pro201, Arg209, Met215 and Val218 are responsible for the binding recognition of 1,2,3-triazole-based KA analogs in TYR catalytic site. Finally, our calculations provide suitable validation of the combination of molecular docking, MD, and LIE approaches as a powerful tool in the structure-based drug design of new and potent TYR inhibitors.