Natural Pigments from the Endophyte Aspergillus westerdijkiae and Evaluation of their Bioactivities

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Rollinger, Joanna Grzelczyk, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7546670/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Dec, 2025 Read the published version in Microbial Cell Factories → Version 1 posted 11 You are reading this latest preprint version Abstract Background The growing consumer preference for natural and sustainable products has heightened interest in biopigments across pharmaceutical, cosmetic, and food industries. In this study, we investigate endophytic fungi as a viable and eco-friendly source for the production of bioactive natural pigments. Results A promising strain, Aspergillus westerdijkiae P17, was isolated from Betula pendula and assessed for its pigment-producing potential and associated bioactivities. The cell-free culture extract was fractionated, and the resulting components were evaluated for antimicrobial, antioxidant, anticancer, neuroprotective, and peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist activities. Among the fractions, 17P2 exhibited broad-spectrum antimicrobial effects, notable antioxidant activity (83% DPPH radical scavenging at 1000 mg/mL), and cytotoxicity against MCF-7 and HepG2 cancer cell lines, with IC₅₀ values of 250 mg/mL. Isothermal titration calorimetry (ITC) demonstrated strong binding affinities of 17P2 to acetylcholinesterase (Kd = 1.63 µM) and butyrylcholinesterase (Kd = 0.03 µM), indicating potential anti-Alzheimer’s properties. Additionally, significant interactions with monoamine oxidase A and PPAR-γ suggest possible antidepressant and antidiabetic applications. Four major pigment fractions (17P1–17P4) were purified and structurally characterized using UHPLC-MS and NMR, revealing key metabolites such as aluminium and iron aspergillic acid complexes, penicillic acid, and preussin. Notably, gamma irradiation at 2000 Gy significantly enhanced pigment yield without compromising fungal viability. Conclusions Collectively, these findings position A. westerdijkiae P17 as a valuable and versatile biotechnological resource for the sustainable production of multifunctional fungal pigments with potential industrial and therapeutic applications. Natural Pigments Monoamine Oxidase Cholinesterase Inhibitors PPAR-γ Fungal Endophytes Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Colors play a fascinating part in human life profoundly influencing emotions and preferences. Anciently, a rich variety of dyes and pigments originating from natural fruit and vegetable extracts were utilized for architectural, decorative and preservative purposes [ 1 ]. Even though the previous centuries saw applications of synthetic colorants in textile, food, cosmetic and pharmaceutical industries, their toxicity and non-environment friendly manufacturing practices has started limiting its use and enhanced the search for bio-pigments from natural sources [ 2 ]. Colorants sourced from algae, insects, plants and animals, face limitations such as low yields, seasonal availability, deforestation, instability and insolubility [ 2 ]. However, microbial pigments offer significant advantages like consistent supply, greater stability, cost efficiency and higher yields [ 3 ]. Fungi are a remarkable source of natural pigments often displaying a wide spectrum of colors with improved solubility and stability. Notably, individual fungal species can biosynthesize diverse pigments with distinct properties [ 4 , 5 ]. Fungal mycelium releases these pigments as secondary metabolites, generally, as a response to environmental stress or nutrient limitation [ 6 ]. These fungal pigments also possess multifaceted biological activity such as cellular differentiation, metal transport, complex interactions with other organisms through symbiosis and competition, even providing defense against predators and insects [ 7 ], all of which generated interest in the pharmaceutical field, documenting their potential as antiviral, antifungal, antibacterial, antioxidant, anti-inflammatory, antitumor, anti-Alzheimer’s disease, anti-atherosclerotic, anti-obesity, immunosuppressive, nematocidal and cytotoxic agents [ 8 ]. Endophytic fungi comprise a ubiquitous and diverse microbial group inhabiting all plant species across a wide range of climatic and ecological zones [ 9 ]. Isolated from virtually every plant type, from trees, shrubs, herbs to ferns and marine plants, their diversity is significantly influenced by host plant genotype and physiology, environmental factors, human activities and growth season [ 10 ]. This diversity extends to differences among host plants within the same geographical area and even within different parts of a single plant [ 11 ]. Notably, tropical and subtropical regions exhibit a significantly higher diversity of fungal endophytes compared to other areas [ 12 , 13 ]. The co-evolution between fungal endophytes and their plants results in bioactive metabolites production that provide multiple benefits such as stimulating plant growth, triggering defense mechanisms against pathogens and aid in tolerating drought and salt stresses [ 14 – 16 ]. For their ability to quantitatively increase the production of secondary metabolites in host plants and their extensive distribution and diversity, these endophytic fungi are often considered superior to other fungi [ 17 ]. In this study, pigments from the endophyte Aspergillus westerdijkiae P17, isolated from the twig of Betula pendula , were separated and their bioactive properties including antimicrobial, cytotoxic, antioxidant, monoamine oxidase A, acetylcholinesterase, and butyrylcholinesterase inhibitors, and peroxisome proliferator-activated receptor gamma agonists were analysed. The impact of gamma irradiation on pigments production was evaluated. Chemical characterization of pigments fractions was also studied. Materials and Methods Isolation of endophytic fungi . Betula pendula samples were collected from Mokrzański forest, Poland. The plant was identified with the help of Dr. Katarzyna Patejuk, Department of Plant Protection, Wrocław University of Environmental and Life Sciences. Healthy plant parts were collected, stored in ice box and transported to the laboratory. The isolation of fungal endophytes was accomplished according to previous study[ 18 ]. The samples were cut into small pieces, surface-sterilized by dipping in 70% ethanol for 1 min, followed by 0.1% HgCl 2 for 1 min, and then rinsed in sterile distilled water before being dried on sterile filter paper. The sterilized plant fragments were then aseptically transferred onto potato-dextrose agar plates (supplemented with streptomycin and tetracycline) and incubated at 25°C. The inoculated plates were checked daily and the isolated cultures were sub-cultured for purity and stored at − 4°C in glycerol (15%) as a suspension of spores and mycelia. Identification of the fungal strain 17P . Among the isolated fungi, a promising strain (17P) was isolated from a twig of Betula pendula . The fungus was identified using cultural, morphological and molecular methods. Cultural characteristics were studied on Malt Extract agar (MEA), Yeast Extract Sucrose agar (YES), Czapek–Yeast Autolysate agar (CYA), and Potato-dextrose agar (PDA) at 30°C for 10 days. The identity of the fungus was confirmed by molecular characterization techniques. DNA was extracted from fungal cultures using the Genomic Mini AX Yeast kit (A&A Biotechnology, Gdańsk, Poland) following the manufacturer's protocol. The DNA concentration was then quantified using the Qubit 4.0 fluorometer. Amplification of the ITS region was performed using the ITS4 (TCCTCCGCTTATTGATATGC) and ITS5 (GGAAGTAAAAGTCGTAACAAGG) primers under the following thermal cycling conditions: an initial denaturation at 95°C for 2 min; 35 cycles of 95°C for 60 seconds, 55°C for 60 seconds, and 72°C for 90 seconds; followed by a final elongation at 72°C for 10 min. PCR products were visualized via electrophoresis on a 1% agarose gel and subsequently purified using NucleoMag magnetic beads (Macherey-Nagel). Sequencing was carried out using the Oxford Nanopore Technology (ONT) MinION platform, employing the SQK-NBD114.96 barcoding kit and a FLO-MIN114 flow cell. Library preparation followed ONT’s guidelines for barcoded samples. Raw sequence data in POD5 format were basecalled using the Dorado basecaller ( https://github.com/nanoporetech/dorado ) with the super accurate (SUP) model. FastQC ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ) was used for quality assessment, and reads were trimmed to the appropriate length with a minimum phred score of 12 using Chopper [ 19 ]. Consensus sequences were generated using Scaffold Builder [ 20 ]. Initial species identification was conducted through the MycoBank database. Sequences from the study were then incorporated into a phylogenetic analysis alongside ITS sequences of known species obtained from NCBI, including outgroup. Phylogenetic relationships were inferred using the Bayesian framework implemented in MrBayes 3.2.7a [ 21 ], with the mixed model selection. Trees were sampled every 100th generation over 10,000,000 MCMC generations, and the consensus tree was constructed from samples with an average standard deviation of split frequencies well below 0.01. Preparation and extraction of fungal biomass . Spore suspensions were obtained from 7-day-old fungal cultures. Spore concentrations were determined and standardized to 1 × 10⁶ spores·mL⁻¹ using a hemocytometer. Under aseptic conditions, 1 mL of the standardized spore suspension was inoculated into 250 mL Erlenmeyer flasks containing 50 mL of potato dextrose (PD) broth. The flasks were incubated at 30°C under static conditions for 14 days. Following incubation, cultures were filtered through Whatman No. 1 filter paper to separate the culture filtrate from the fungal biomass. The recovered mycelial biomass was homogenized using a mortar and pestle until a uniform consistency was achieved. Each homogenized sample was extracted with 25 mL of a chloroform–methanol mixture (9:1, v/v). The extraction mixtures were sonicated for 1 hour at 20 kHz and 20°C in an ultrasonic bath. Subsequently, the organic phase was separated using a separatory funnel and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure using a rotary vacuum evaporator to yield crude dry extracts. These extracts were reconstituted in a methanol–dimethyl sulfoxide mixture (2:1, v/v) for further analysis. Fractionation and chromatographic separation of the extract. The crude fungal extract was adsorbed onto silica gel (Silica Gel 60, 0.040–0.063 mm particle size; Merck, Germany) and the solvent was removed under reduced pressure using a rotary vacuum evaporator. A chromatographic column was packed to three-quarters of its volume with silica gel, after which the silica–extract mixture was added. Flash chromatography was performed using a puriFlash® XS520 Plus system (Interchim SA, France) equipped with a silica-based column (SIHP-JP, F0012). Elution was carried out using a gradient system of n-hexane and ethyl acetate. The separation commenced with 100% n-hexane, followed by stepwise increases in the ethyl acetate concentration (1%, 2%, and finally 5%, v/v), thereby increasing the polarity of the mobile phase. This process resulted in the isolation of four pigment-containing fractions, designated as 17P1, 17P2, 17P3, and 17P4. Testing bioactivities of the separated fractions Antimicrobial Activity The antimicrobial activity of the isolated pigment fractions (17P1–17P4) was assessed using the agar well diffusion method [ 22 ]. Antibacterial efficacy was evaluated against human pathogenic strains Staphylococcus aureus (ATCC 6538) and Escherichia coli (ATCC 11229), while antifungal activity was tested against Fusarium oxysporum (EUM37, a phytopathogen), Candida albicans (ATCC 10231), and Aspergillus brasiliensis (ATCC 16404). The positive control for antibacterial assays was a mixture of amoxicillin and clavulanic acid (500 µg/mL), and for antifungal assays, nystatin (100 µg/mL) was used. The solvent mixture (MeOH:DMSO, 2:1 v/v) served as the negative control. Bacterial cultures were standardized to 0.5 McFarland turbidity using spectrophotometric measurement at 600 nm (A₆₀₀ = 0.08–0.10). Fungal spore suspensions were adjusted to 1 × 10⁶ spores/mL using a hemocytometer. One hundred microliters of each microbial suspension were inoculated onto appropriate media: Mueller–Hinton Agar for bacteria, Potato Dextrose Agar for molds, and Sabouraud Dextrose Agar for Candida . Agar wells were created and filled with 50 µL of each fungal extract. Plates inoculated with E. coli , S. aureus , and C. albicans were incubated at 37°C, while those containing F. oxysporum and A. brasiliensis were incubated at 30°C for 24 h. Zones of inhibition (ZOI) were measured in millimeters. DPPH Scavenging Assay The antioxidant potential of the pigment fractions was evaluated using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay [ 23 ]. A 120 µL aliquot of 100 µM DPPH in methanol was mixed with 80 µL of each pigment extract. Ascorbic acid (10 mM) served as the positive control, while the solvent mixture (DMSO:MeOH, 1:2 v/v) served as the blank. After 15 minutes of incubation in the dark at room temperature, the absorbance was measured at 517 nm. Cytotoxic activity assay. The cytotoxic effects of pigment fractions (17P1–17P4) were evaluated against three human cancer cell lines: melanoma (A375), breast adenocarcinoma (MCF-7), and lung carcinoma (A549), along with a non-cancerous human fibroblast cell line (Hfb-4). All cell lines were procured from ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and L-glutamine (Sigma-Aldrich). All cells were tested for Mycoplasma contamination before experiments. Cytotoxicity of the pigments fractions 17P1, 17P2, 17P3 and 17P4 was evaluated against the aforementioned cell lines using the MTT assay according to the method described by Van de Loosdrecht and co-workers [ 24 ] with slight modifications. Cells were seeded at 1 × 10⁴ cells/well in 96-well plates and incubated at 37°C in a humidified 5% CO₂ atmosphere. Cells were treated with 20 µL of each pigment fraction for 24 h. Untreated control wells received 0.1% DMSO. After treatment, 10 µL of MTT (5 mg/mL) was added to each well, followed by incubation for 4 h. The resulting formazan crystals were solubilized in 100 µL of DMSO. Absorbance was read at 570 nm using a TECAN SunRise microplate reader. Cytotoxicity was calculated using: Inhibition (%) = [ 100 - (A570 of treated cells / A570 of control cells) ] × 100 AChE and BChE inhibitory potentials. Enzymatic inhibition studies were conducted using acetylcholinesterase (AChE) from Electrophorus electricus and butyrylcholinesterase (BChE) from horse serum (Sigma-Aldrich). The isothermal titration calorimetry (ITC) assay was performed using a MicroCal PEAQ-ITC200 instrument (Malvern Instruments, UK). The sample cell (0.2 mL) was loaded with a 20 µM solution of either AChE or BChE in methanol. Pigment fractions (10 mM in methanol) were injected in 2 µL increments. Binding thermodynamics were recorded at 36°C under constant stirring. Control titrations (methanol only) were subtracted from enzyme–ligand interactions. Binding parameters including dissociation constant (Kd), association constant (Ka), enthalpy change (ΔH), entropy change (ΔS), and Gibbs free energy change (ΔG) were calculated using single-site binding models via the MicroCal PEAQ-ITC software [ 25 ]. Competitive inhibition was also assessed in the presence of acetylcholine (ACh). MAO-A inhibitory potential MAO-A inhibitory potential was evaluated under similar ITC conditions with modifications. A 10 µM solution of human recombinant MAO-A was titrated with 2 mM pigment fractions and/or serotonin (5-HT, positive control). A total of 11 injections were performed over 30 minutes [ 26 ]. PPAR-γ agonist potential GST-tagged human PPAR-γ ligand-binding domain (residues 204–477; 50 µg/mL) and the natural ligand 15-deoxy-Δ¹²,¹⁴-prostaglandin J2 (≥ 95%) were obtained from Sigma-Aldrich. ITC analysis was conducted as described for BChE, with a 10 µM solution of PPAR-γ used as the target and 20 µM pigment fractions or prostaglandin J2 as ligands. The measurements were carried out at 36.6°C, with 11 injections over 30 minutes [ 27 ]. Effect of 60 Co gamma irradiation on pigments production . Spore suspensions of Aspergillus westerdijkiae P17 were subjected to γ-irradiation at doses of 250, 500, 1000, 2000, 4000, 8000, and 16000 Gy using a ⁶⁰Co gamma irradiator (MC20, Russia; dose rate 311.88 Gy/h). Post-irradiation, suspensions were incubated in the dark overnight, then 1 mL was inoculated into 50 mL of PD broth (pH 6.0) and cultured at 30°C. After incubation, biomass was harvested, extracted as previously described, and pigment concentrations were determined spectrophotometrically at 410 nm (yellow), 470 nm (orange), and 510 nm (red). Fungal biomass (g·L⁻¹) was measured by drying to constant weight at 50°C. Spore survival rates were assessed by plating 100 µL of irradiated spore suspensions on PDA, incubating at 25°C for 5 days, and counting colony-forming units. The survival rate was expressed relative to unirradiated controls (considered 100%). Chemical characterization . The whole biomass extract of the fungus was first analyzed via LC-MS dereplication using a database of over 700 fungal metabolites using procedures that have been described previously [ 28 , 29 ]. Second, UHPLC-ELSD and UHPLC-UV-MS analyses were performed on a Waters Acquity UPLC H-Class system consisting of a fraction manager, column manager, quaternary solvent manager, PDA detector, ELS detector, isocratic solvent manager and a single quadrupole mass detector (Acquity QDa) equipped with an ESI source. A BEH C 18 column (1.7 µm, 2.1 x 100 mm, Waters) was used as stationary phase; water + 0,1% formic acid (A) and acetonitrile + 0,1% formic acid (B) were used as mobile phase. The column temperature was set to 40°C, flow rate of 0.3 was used and the following gradient was applied: 5% B at 0.0 min, from 5% − 98% B in 5.0, 98% B for 10 min. Mass detection was performed in positive (cone voltage: 15 V, capillary voltage 0.8 kV) and negative (cone voltage: 30 V, capillary voltage: 0.8 kV) mode from 200–1200 Da. To increase ionization, a mixture of water:methanol (9:1) + 10 mM ammonium formate was used make-up solvent. The instrument was controlled by Empower 3. Size exclusion chromatography was conducted for the separation of 17P2, using Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) as stationary phase and MeOH as mobile phase (column dimensions: 100 cm × 2 cm). The fractions were collected in a time-dependent manner (3 min/tube) and analyzed by thin-layer chromatography (TLC) using Merck silica gel 60 PF254 plates as stationary phase and ethyl acetate as mobile phase. Detection was performed at visible light and UV366. Based on TLC fingerprints, the collected tubes were pooled into 5 fractions (17P2_I-V). 1D and 2D NMR data of 17P4 were recorded using a Bruker UltraShield 500 MHz NMR spectrometer equipped with a TCI Prodigy CryoProbe (5 mm), an AVANCE III HD console and a SampleJet. The fraction was measured at 296 K in MeOD referenced to the residual non-deuterated solvent signals (δH 3.31 ppm; δC 49.00 ppm). The resonance frequency for 1 H NMR was 500 MHz and for 13 C NMR 125 MHz. Standard 1D (1H) and gradient-enhanced 2D experiments, i.e. HSQC, and HMBC, were used as supplied by the manufacturer. The raw NMR data were processed using Topsin 4.3.0. Penicillic acid ( 5 ): identified in 17P4. 1 H NMR (MeOD, 500 MHz): 5.47 (1H, m), 5.28 (1H, s), 5.24 (1H, m), 3.89 (3H, s); 13 C NMR (MeOD, 125 MHz): 176.2 (qC), 173.5 (qC), 117.4 (CH 2 ), 91.1 (CH), 59.7 (OCH 3 ), 17.4 (CH 3 ); ESI-MS: m/z 171.13 [M + H] + . Preussin ( 6 ): identified in 17P4. 1 H NMR (MeOD, 500 MHz): 7.34 (2Hs, m, 2x H-3’), 7.32 (2Hs, m, 2x H-2’), 7.24 (1H, m, H-4’), 4.03 (1H, m, H-3), 3.12 (1H, dd, H-1a), 2.96 (1H, dd, H-1b), 3.03 (1H, m, H-2), 2.92 (1H, m, H-5), 2.67 (3Hs, s, NCH 3 ), 2.36 (1H, m, H-4a), 1.63 (1H, m, H-4b), 0.91 (3Hs, t, H-14); 13 C NMR (MeOD, 125 MHz): 139.3 (CH, C-1’), 130.3 (2x CH, C-3’), 129.7 (2x CH, C-2’), 127.7 (CH, C-4’), 75.3 (CH, C-2), 69.8 (CH, C-3), 69.3 (CH, C-5), 39.3 (CH 2 , C-4), 38.8 (NH, NCH 3 ), 32.5 (CH 2 , C-1), 14.4 (CH 3 , C-14); ESI-MS: 318.27 [M + H] + . Statistics. Isothermal titration calorimetry (ITC) data were analyzed based on the mean values obtained from three independent replicates (n = 3), with results expressed as mean ± standard deviation (SD). Statistical significance was assessed using one-way analysis of variance (ANOVA) performed in SPSS software (version 22.0; IBM Corp., NY, USA). Differences were considered statistically significant at P < 0.05. Results and discussion Identification of the 17P fungus. Figure 1 shows the colony morphology of the P17 strain maintained on the CYA (Fig. 1 a), YES (Fig. 1 b), MEA (Fig. 1 c), and PDA (Fig. 1 d). Colony characters: CYA 30°C, 7 d: Colony surface floccose; mycelial areas white to greyish yellow; sporulation pale yellow to light yellow; reverse greyish yellow. YES 30°C, 7 d: Colony surface floccose; mycelial areas pinkish and white near margin; sporulation greyish yellow; reverse dark brown. MEA 30°C, 7 d: Colony surface floccose; mycelial areas brown with white margin; sporulation light yellow; reverse dark brown. PDA 30°C, 7 d: Colony surface floccose; mycelial areas pale yellow; sporulation greyish yellow; reverse pale yellow, yellowish and white near margin. These characteristics are identical with those reviewed by previous reports [ 30 , 31 ] concerning identifications of Aspergillus westerdijkiae . For analyzed 17P sample, 1,000 high-quality reads (post-trimming) were obtained to ensure adequate sequencing depth. Consensus sequence generated in this study have been submitted to the NCBI database under accession number PV650320. Both Mycobank database-based identification and the constructed phylogenetic tree (Fig. 2 ) validated the species identifications, clustering the sample consistently within their respective species clades. In the literature, there is no information about isolation of an endophytic Aspergillus westerdijkiae from plant sources. Antimicrobial, antioxidant, and anticancer potentials . The pigment fractions 17P1, 17P2, 17P3 and 17P4 separated from the fungus Aspergillus westerdijkiae 17P were tested against prominent bacterial and fungal pathogens as depicted in Tables 1 and 2 . The results indicate Zone of Inhibition (ZOI) and Minimum Inhibitory Concentration (MIC) values of pigments against the pathogens. All the fractions showed inhibitory activity against E. coli and C. albicans , while the fraction, 17P2, showed inhibition against all tested pathogens. Notably, 17P2 displayed ZOI at 100 mg/mL against E. coli and S. aureus and 250 mg/mL against P. aeruginosa and K. pneumoniae . With fungal pathogens, 17P2 displayed ZOI at 500 mg/mL against A. brasiliensis , A. alternata and F. oxysporum and 250 mg/mL against C. albicans . Inhibitory activity of 17P4 was observed at 500 mg/mL concentration against E. coli and C. albicans and at 1000 mg/mL against F. oxysporum . 17P1 showed activity against E. coli (250 mg/mL), S. aureus (250 mg/mL) and C. albicans (500 mg/mL) while 17P3 showed activity against E. coli, S. aureus, A. alternata and C. albicans at 500 mg/mL concentration. Table 1 Antibacterial activity of the separated fractions from Aspergillus westerdijikae 17P cultures against different Gram-positive and Gram-negative human pathogenic bacterial strains. Separated fractions Bacterial pathogens E. coli S. aureus P. aeruginosa K. pneumoniae 17P1 ZOI (mm) 9.67 ± 0.88 10.43 ± 0.91 Nil Nil MIC (mg mL − 1 ) 250 250 0.00 0.00 17P2 ZOI (mm) 11.43 ± 0.87 10.78 ± 0.54 9.08 ± 0.67 11.78 ± 0.76 MIC (mg mL − 1 ) 100 100 250 250 17P3 ZOI (mm) 8.67 ± 0.56 9.35 ± 0.34 Nil Nil MIC (mg mL − 1 ) 500 500 0.00 0.00 17P4 ZOI (mm) 10.48 ± 0.98 Nil 10.39 ± 0.76 Nil MIC (mg mL − 1 ) 500 500 0.00 0.00 Amoxicillin 13.98 ± 0.77 16.79 ± 0.91 18.31 ± 0.61 11.33 ± 0.79 LSD 0.4511 0.5210 0.4388 0.3981 Amoxicillin was used as the positive control at a concentration of 500 µg mL − 1 . The calculated mean is for triplicate measurements from three independent experiments ± SD. Nil means that no ZOI was detected. LSD means Least Significant Differences. Table 2 Antifungal activity of the separated fractions from Aspergillus westerdijikae 17P cultures against different human and plant pathogenic fungi. Separated fractions Fungal pathogens A. brasiliensis A. alternata F. oxysporum C. albicans 17P1 ZOI (mm) Nil Nil Nil 14.43 ± 0.56 MIC (mg mL − 1 ) 0.00 0.00 0.00 500 17P2 ZOI (mm) 10.38 ± 0.87 9.61 ± 0.33 11.87 ± 0.31 17.39 ± 0.65 MIC (mg mL − 1 ) 500 500 500 250 17P3 ZOI (mm) Nil 10.21 ± 0.33 0.00 12.34 ± 0.57 MIC (mg mL − 1 ) 0.00 1000 0.00 1000 17P4 ZOI (mm) Nil Nil 10.31 ± 0.54 11.34 ± 0.32 MIC (mg mL − 1 ) 0.00 0.00 1000 500 Nystatin 15.34 ± 0.67 12.41 ± 0.33 14.67 ± 0.34 15.97 ± 0.61 LSD 0.3187 0.1891 0.1012 0.2198 Nystatin was used as the positive control at a concentration of 500 µg mL − 1 . The calculated mean is for triplicate measurements from three independent experiments ± SD. Nil means that no ZOI was detected. LSD means Least Significant Differences. Results of scavenging activity of the separated fractions showed that the activity increases with increasing concentration as stated in Table 3 . When compared to 17P1 and 17P3, it can be observed that the fractions 17P2 and 17P4 shows higher activity. Nearly 83% and 67% of free radicals were scavenged by fractions 17P2 and 17P4 respectively at a concentration of 1000 mg/mL, demonstrating the most promising antioxidant potential. Table 3 Antioxidant activity of the separated fractions from Aspergillus westerdijikae 17P cultures. Concentration (mg mL − 1 ) Scavenging activity (%) 17P1 17P2 17P3 17P4 0.00 0.00 0.00 0.00 0.00 0.1 0.00 27.31 ± 10.78 0.00 10.43 ± 0.77 10 8.71 ± 2.79 44.29 ± 9.39 10.89 ± 1.89 28.71 ± 2.79 100 23.54 ± 10.98 61.66 ± 10.51 29.43 ± 11.43 43.54 ± 10.98 1000 47.66 ± 12.54 83.54 ± 11.32 55.43 ± 21.78 67.66 ± 12.54 LSD 3.620 4.667 3.620 4.667 Ascorbic acid (antioxidant standard) was the control. The calculated mean is for triplicate measurements from three independent experiments ± SD, LSD = least significant differences (p ≤ 0.05). Cytotoxicity of the isolated pigments was tested against normal (Hfb-4), breast cancer (MCF-7) and liver cancer (HepG-2) cell lines (Table 4 ). Cell viability of 70–80% can be seen with fractions 17P1 and 17P3 at 500 mg/mL MIC against both cancer cell lines. At the MIC of 1000 mg/mL, 17P4 showed cell viability of 87% and 91% against breast and liver cancer cell lines. The pigment fraction 17P2 was considered more promising with a cell viability of 68% against MCF-7 and 48% against HepG-2 at a minimum inhibitory concentration of 250 mg/mL. Table 4 Cytotoxic activities of the separated fractions from Aspergillus westerdijikae 17P cultures against normal, breast cancer, and liver cancer cell lines. Separated fractions Cell line Hfb-4 (normal) MCF-7 (breast) HepG-2 (liver) 17P1 Cell viability (%) 66.52 ± 11.97 78.44 ± 14.83 70.09 ± 17.81 MIC (mg mL − 1 ) 500 500 500 17P2 Cell viability (%) 57.83 ± 10.33 67.98 ± 12.67 48.78 ± 9.76 MIC (mg mL − 1 ) 250 250 250 17P3 Cell viability (%) 84.76 ± 21.32 79.21 ± 17.43 80.32 ± 18.81 MIC (mg mL − 1 ) 500 500 500 17P4 Cell viability (%) 90.55 ± 20.51 87.32 ± 15.45 91.45 ± 18.98 MIC (mg mL − 1 ) 1000 1000 1000 Taxol 15.56 ± 1.33 17.08 ± 1.67 20.72 ± 1.87 LSD 5.839 8.728 7.451 Taxol was used as the positive control at a concentration of 50 µg mL − 1 . MTT-based assay was used for measuring the cytotoxic activities at 570 nm using MTT solution under the conditions described in Materials and Methods. Calculated mean is for triplicate measurements from three independent experiments ± SD. Nil means that no ZOI was detected. LSD means Least Significant Differences. In the literature, endophytic fungi have attracted considerable scientific interest due to their ability to produce a wide range of bioactive secondary metabolites. To date, the processes of isolation, cultivation, purification, and characterization of these fungi have led to the identification of approximately 200 structurally diverse and biologically significant compounds, including metabolites with demonstrated antimicrobial [ 32 , 33 ], antioxidant [ 34 ], and anticancer [ 35 ] activities. AChE, BChE and MAO-A inhibitory and PPAR-γ agonist potential of the separated pigment fractions. ITC analysis allows for a better understanding of the interactions between the studied enzymes and the receptors and pigments. To determine binding affinity, an analysis was performed by measuring the heat during each injection of the ligand into the protein present in the cell. This allows to monitor heat changes until all bonds are saturated. The experiment also includes a reference test in which the ligand is injected into a reagent in which the sample has been diluted. This heat is subtracted from the ligand-protein experiment [ 36 , 37 ]. The parameter n indicated one protein binding site per ligand molecule [ 38 ]. The ITC technique allows the use of low enzyme/receptor concentrations, thus demonstrating affinity independent of biological effects, avoiding non-specific binding [ 39 ]. AChE and BChE AChE was found to bind to 17P1 and 17P2, but not to bind to 17P3 and 17P4. Kd for 17P2 was 1.63 µmol/L, and for 17P1 it was 2.78 µmol/L (Table 5 ). The enthalpy, ΔS, showed a value of -31.09 J/mol*K for 17P1 and + 71.06 J/mol*K for 17P2, which means that the 17P2 compound showed a stronger interaction, confirming the high affinity between 17P2 and AChE (ΔG -13.28 kJ/mol). In the case of BChE, compounds 17P1, 17P2 and 17P3 showed strong binding to BChE. Only 17P4 showed no binding to the active site of the enzyme. The highest affinity, similarly to AChE, was shown by compound 17P2, with a value of -124.98 kJ/mol (Table 6 ), with a positive reaction enthalpy ΔH = 7.69 kJ/mol. The dissociation constant was comparable for 17P1 and 17P2, being 0.03 µmol/L, while compound 17P3 had a slightly lower value of 0.01 µmol/L. The IC50 for AChE showed the lowest ligand dose inhibiting 50% of the enzyme for 17P1, while for BChE it was the 17P1 compound. Fungi are known to produce a diverse array of bioactive metabolites capable of inhibiting acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The extent of cholinesterase inhibition by fungal extracts varies considerably; some exhibit potent AChE inhibition, while others are more selective for BChE [ 40 ]. This variability indicates the presence of multiple bioactive constituents within fungal species. Notably, out of 43 fungal strains screened, six isolates demonstrated differing levels of inhibitory activity were reported, underscoring the strain-specific nature of these effects [ 41 ]. Table 5 Thermodynamic parameters of interactions between AChE and the separated fractions from Aspergillus westerdijikae 17P cultures. Separated frctions K d (µmol L − 1 ) K a *10 3 (L mol − 1 ) ∆H (kJ mol − 1 ) ∆G (kJ mol − 1 ) ∆S (J mol*K − 1 ) Inhibitor activity [%] IC 50 (0.25 µL µM AChE i 10 µMAchE − 1 (%)) K i (µmol L − 1 ) K M ACh 35.91 17P1 2.78 ± 0.03 a 0.10 ± 0.00 a 10.00 ± 0.02 a 35.11 ± 0.45 a -71.06 ± 0.21 a 86.11 ± 0.15 a 5.28 ± 0.02 a 0.34 ± 0.01 a 17P2 1.63 ± 0.02 b 5.62 ± 0.05 b 0.18 ± 0.01 b -13.28 ± 0.45 b -31.09 ± 0.98 b 75.22 ± 0.18 b 6.04 ± 0.04 b 0.05 ± 0.00 b 17P3 Nb Nb Nb Nb Nb Nb Nb Nb 17P4 Nb Nb Nb Nb Nb Nb Nb Nb Values are expressed as mean ± SD; n = 4; different letters in one column correspond to significant differences (P < 0.05); Nb – no binding. Table 6 Thermodynamic parameters of interactions between BChE and the separated fractions from Aspergillus westerdijikae 17P cultures. Separated frctions K d (µmol L − 1 ) K a *10 3 (L mol − 1 ) ∆H (kJ mol − 1 ) ∆G (kJ mol − 1 ) ∆S (J mol*K − 1 ) Inhibitor activity [%] IC 50 (0,25 µL µM BChE i 10 µM BChE − 1 (%)) Ki (µmol L − 1 ) K M BCh 35.91 17P1 0.03 ± 0.00 a 25.40 ± 0.48 a 0.03 ± 0.00 a -25.99 ± 0.78 a -27.21 ± 0.07 a 46.86 ± 0.15 a 12.33 ± 0.03 a 0.01 ± 0.00 a 17P2 0.03 ± 0.00 a 25.13 ± 0.02 a 7.69 ± 0.02 b -124.98 ± 0.52 b -40.75 ± 0.12 b 99.37 ± 0.45 b 4.57 ± 0.02 b 0.27 ± 0.01 b 17P3 0.02 ± 0.00 b 18.30 ± 0.15 b 0.12 ± 0.01 c -18.98 ± 0.19 c -28.04 ± 0.55 a 62.23 ± 0.26 c 7.29 ± 0.03 c 0.02 ± 0.00 c 17P4 Nb Nb Nb Nb Nb Nb Nb Nb Values are expressed as mean ± SD; n = 4; different letters in one column correspond to significant differences (P < 0.05); Nb – no binding MAO-A Obtained fractions bound at the active site. The results are presented in Table 7 . The dissociation constant ranged from 0.13 to 1 µmol/L for 17P3 and 17P2, respectively. The highest interaction constant and affinity was shown by compound 17P2, which was ΔH = 100 kJ/mol and ΔG = 153 kJ/mol. On the other hand, the lowest interaction constant and the highest affinity were observed for compound 17P3, which was ΔH = 0.01 kJ/mol and ΔG= -47.23 kJ/mol. Fraction 17P3 also showed the lowest value needed to achieve half-maximal inhibitory effect, which was 4.03 µmol/µmol enzyme. Reports on metabolites with monoamine oxidase A (MAO-A) inhibitory activity from endophytic fungi remain scarce in the literature. Nevertheless, a few fungal species have been identified as sources of metabolites with MAO-A inhibitory potential. For instance, Daldinia fissa was found to produce 5-hydroxy-2-methylchroman-4-one, which exhibited dual inhibition of MAO-A and MAO-B [ 42 ]. In another study, ( R )-5-methylmellein, isolated from the mycelial fermentation of Xylaria nigripes , acted as a selective MAO-A inhibitor and contributed to the understanding of its antidepressant mechanisms [ 43 ]. Table 7 Thermodynamic parameters of interactions between MAO-A and the separated fractions from Aspergillus westerdijikae 17P cultures. Separated frctions K d (µmol L − 1 ) K a *10 3 (L mol − 1 ) ∆H (kJ mol − 1 ) ∆G (kJ mol − 1 ) ∆S (J mol*K − 1 ) Inhibitor activity [%] IC 50 (1 µmol L inhibitor − 1 : 1 µmol L − 1 MAO-A) K i (µmol L − 1 ) K M 5-HT 0.34 mmol 17P1 0.24 ± 0.01 a 0.472 ± 0.13 a 2.11 ± 0.11 a 2.64 ± 0.16 a -37.49 ± 0.33 a 77.78 ± 0.44 a 7.46 ± 0.13 a 1.13 ± 0.05 a 17P2 1.00 ± 0.00 b 0.001 ± 0.00 b 100.00 ± 1.18 b 153.40 ± 0.15 b -71.06 ± 0.85 b 81.44 ± 0.44 b 6.55 ± 0.51 b 0.84 ± 0.05 b 17P3 0.13 ± 0.01 c 118.00 ± 0.98 c 0.01 ± 0.00 c -47.23 ± 0.09 c -17.34 ± 0.14 c 97.05 ± 0.52 a 4.03 ± 0.78 c 0.88 ± 0.05 b 17P4 0.35 ± 0.01 d 5.82 ± 0.22 d 0.17 ± 0.02 d 9.94 ± 0.11 d -30.26 ± 0.44 d 94.00 ± 0.58 c 5.49 ± 0.15 b 0.94 ± 0.02 d Values are expressed as mean ± SD; n = 4; different letters in one column correspond to significant differences (P < 0.05); Nb – no binding PPAR-γ The obtained fractions bind to the active site of PPAR-γ. The results are presented in Table 8 . The dissociation constant is in the range of 7.13–8.70 µmol/L, for 17P1 and 17P3, respectively. The lowest reaction enthalpy is characterized by the 17P3 fraction, amounting to -19.65 kJ/mol, but it shows a slightly lower affinity for the receptor compared to the other fractions, amounting to -29.97 kJ/mol. The typical ligand, 15-deoxy-Δ12,14-prostaglandin J2, showed a higher affinity by about − 18 kJ/mol. Literature on peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists from endophytic fungi is extremely limited. Three studies to date have reported relevant fungal metabolites. The first described the isolation of five novel compounds from Cladosporium oxysporum [ 44 ], while the second identified four new bioactive metabolites through bioassay-guided fractionation of an unidentified fungal strain [ 45 ]. The third described six different endophytes with varying agnostic levels [ 41 ]. Table 8 Thermodynamic parameters of interactions between PPAR-γ and the separated fractions from Aspergillus westerdijikae 17P cultures. Separated fractions N K d (µmol L − 1 ) K a *10 4 (L mol − 1 ) ∆H (kJ mol − 1 ) ∆G (kJ mol − 1 ) ∆S (J mol*K − 1 ) 17P1 0.09 ± 0.01 a 7.13 ± 0.03 a 1.40 ± 0.05 a -16.72 ± 0.13 a -30.51 ± 0.15 a 44.53 ± 0.38 a 17P2 0.07 ± 0.01 b 7.80 ± 0.01 a 1.28 ± 0.01 b -11.70 ± 0.15 b -30.26 ± 0.13 a 59.92 ± 0.16 b 17P3 0.08 ± 0.00 b 8.70 ± 0.02 b 1.15 ± 0.01 c -19.65 ± 0.05 c -29.97 ± 0.28 b 33.33 ± 0.27 c 17P4 0.09 ± 0.01 a 7.82 ± 0.02 a 1.28 ± 0.02 a -10.45 ± 0.15 b -30.26 ± 0.11 a 63.97 ± 0.21 d Ligand 0.99 ± 0.02 c 0.05 ± 0.00 c 189.75 ± 2.15 d -98.81 ± 0.95 b -48.99 ± 1.35 c -0.16 ± 0.01 e Values are expressed as mean ± SD; n = 4; different letters in one column correspond to significant differences (P < 0.05); Nb – no binding Effect of gamma irradiation on pigments production. Data presented in Table 9 indicate that exposure to low doses of gamma irradiation (250 and 500 Gy) resulted in spore survival rates of 99.48% and 89.48%, respectively. However, a marked decline in spore viability was observed at 2000 Gy, with survival reduced to 79.38%. At the highest applied dose (16,000 Gy), no fungal growth was detected, indicating a complete loss of viability. These findings suggest a dose-dependent inhibitory effect of gamma irradiation on fungal viability and growth. Correspondingly, dry biomass measurements showed that higher doses of gamma irradiation (4000 and 8000 Gy) led to the most pronounced reductions in biomass accumulation, with yields declining to 3.78 g/L and 1.08 g/L, respectively. This further supports the observation of dose-dependent suppression of fungal growth. Table 9 Effect of gamma irradiation on survival rate (%), fungal growth (g L − 1 ), and yellow, orange and red pigments production by Aspergillus westerdijikae 17P. Dose (kGy) Survival (%) Dry biomass (g L − 1 ) Pigment yield (AU g − 1 fresh biomass) Red Orange Yellow 0.00 100.00 ± 0.00 10.76 ± 0.56 0.87 ± 0.02 0.54 ± 0.03 0.71 ± 0.09 0.25 99.48 ± 0.28 10.01 ± 0.87 1.01 ± 0.21 0.57 ± 0.01 0.73 ± 0.10 0.50 89.48 ± 1.88 9.43 ± 0.21 1.43 ± 0.35 0.79 ± 0.13 0.84 ± 0.15 1.00 80.78 ± 2.87 7.88 ± 1.03 1.47 ± 0.26 0.79 ± 0.21 1.06 ± 0.21 2.00 79.38 ± 6.87 5.34 ± 0.87 2.98 ± 0.81 1.19 ± 0.42 1.19 ± 0.41 4.00 39.44 ± 12.98 3.78 ± 0.98 1.81 ± 0.16 1.06 ± 0.55 0.97 ± 0.17 8.00 2.73 ± 1.02 1.08 ± 0.37 0.11 ± 0.01 0.37 ± 0.01 0.27 ± 0.11 16.00 0.00 0.00 0.00 0.00 0.00 LSD 9.1873 0.8661 0.3121 0.1198 0.2136 The concentration was measured at 410, 470, and 510 nm that corresponded to the characteristic absorbance of yellow, orange and red pigments, respectively. The calculated mean is for triplicate measurements from two independent experiments ± SD. LSD means Least Significant Differences Regarding pigment biosynthesis, exposure to 2000 Gy significantly enhanced the production of yellow, orange, and red pigments by A. westerdijkiae 17P compared to the non-irradiated control. This dose was identified as optimal for stimulating pigment biosynthesis. However, further increases in irradiation dose (4000–8000 Gy) resulted in a progressive decline in pigment production across all three pigment classes. In the literature, gamma radiation, a potent form of ionizing energy, can induce substantial biological effects, primarily by generating mutations through DNA strand breaks and errors during subsequent repair processes [ 46 , 47 ]. Mutagenesis induced by gamma rays has been widely applied in microbial biotechnology to improve strain performance [ 48 – 53 ]. This approach typically involves repeated exposure to physical or chemical mutagens to introduce random genetic variability, followed by screening for strains with enhanced biosynthetic traits [ 54 ]. Our findings demonstrate that gamma irradiation at a dose of 2000 Gy (2 kGy) can significantly enhance pigment production in A. westerdijkiae , potentially due to mutations induced within biosynthetic gene clusters responsible for secondary metabolite synthesis [ 55 ]. Chemical characterization . The whole biomass extract of the fungus was analyzed via LC-MS dereplication using a database of over 700 fungal metabolites. An [M + H] + ion peak of m/z 241.1544 was observed in the positive mode at 3.12 min, matching to the standard peak for neohydroxyaspergillic acid and with a calculated [M + H] + of m/z 241.1546 and an elution time of 2.99 min (Fig. 3 ). Further confirmation came from fragmentation analysis of the observed peak where, five out of eight of the most abundant fragments matched that of the neohydroxyaspergillic acid standard. To gain more insights into the chemical profile of the investigated fractions (17P1-17P4), UHPLC-ELSD and UHPLC-UV-MS analyses were performed (Fig. 4 , Table 10 ). The ELSD chromatograms revealed a similar chemical composition for the fraction pairs 17P1 and 17P2 as well as for 17P3 and 17P4. To determine the constituents contributing to the shiny orange color of fractions 17P1 and 17P2, size exclusion chromatography was conducted which resulted in five fractions (17P2_I-V), with 17P2_II containing the orange-colored pigments. UHPLC-UV-MS analysis of this fraction enabled the tentative annotation of two constituents ( 3, 4 ) as metal complexes, bearing either iron or aluminum as central metal ions each forming coordinate bonds with three units of aspergillic acids or its isomer, neoaspergillic acid. Such pigments have been previously described for other Aspergillus species [ 56 ]. The two main constituents ( 1 , 2 ; both m/z 499.32 in neg. mode) of 17P1 and 17P2 were found in the sub-fractions 17P2_III and 17P2_IV in mixture with other constituents (not shown). Compounds 5 and 6 present in fractions 17P3 and 17P4 were tentatively annotated as penicillic acid ( m/z 171.12 [M + H] + ) and preussin ( m/z 318.27 [M + H] + ) based on UHPLC-MS, respectively. These annotations were confirmed by 1D and 2D NMR analysis of fraction 17P4, with the detected signals aligned well with previously reported NMR data for compound 5 [ 57 ] and 6 [ 58 , 59 ]. Since compound 6 was in a mixture, its absolute configuration could not be determined. Table 10 Structural annotation of detected compounds in fractions 17P1-17P4 and 17P2_II. Peak no. RT [min] UV max [nm] m/z [pos] m/z [neg] Structure annotation 1 8.59 n. d. n. d. 499.3 unknown 2 8.71 n. d. n. d. 499.4 unknown 3 10.83 316 697.5 [M + H] + , 719.5 [M + Na] + , 735.5 [M + K] + n. d. Aspergillic acid aluminium complex 4 11.25 309 726.5 [M + H] + , 748.5 [M + Na] + , 764.5 [M + K] + n. d. Aspergillic acid iron complex 5 3.55 n. d. 371.13 [M + H] + n. d. Penicillic acid* 6 4.79 n. d. 318.27 [M + H] + n. d. Preussin* n. d., not detected; *, Identified by NMR. Conclusion Aspergillus westerdijkiae P17, isolated from Betula pendula , demonstrates strong potential as a sustainable source of multifunctional fungal pigments. Fraction 17P2 exhibited notable antimicrobial, antioxidant, anticancer, neuroprotective, and metabolic regulatory activities. Structural analysis revealed key bioactive metabolites, and pigment production was significantly enhanced by gamma irradiation without affecting fungal viability. These findings highlight the strain's promise for pharmaceutical and industrial applications of natural biopigments. Declarations Author contributions Abirami Baskaran(Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing), Andreas Wasilewicz, Judith M. Rollinger , Joanna Grzelczyk , Ilona Gałązka-Czarnecka, Grażyna Budryn, Tomasz Strzała, Tyler N. Graf, Nicholas H. Oberlies (Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – review & editing), Filip Boratyński (Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Software, Supervision, Validation, Visualization, Writing – review & editing), El-Sayed R. El-Sayed (Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing). Funding Sources This research is part of project No. 2021/43/P/NZ9/02 241 co-funded by the National Science Centre and the European Union Framework Programme for Research and Innovation Horizon 2020 under the Marie Skłodowska-Curie grant agreement no. 945339. Acknowledgments The publication was prepared within the project PROM – short-term academic exchange (project no. BPI/PRO/2024/00001/U/00001), financed by the National Agency for Academic Exchange (NAWA). Availability of data and materials All data generated or analyzed during this study are included in this published article. Ethics approval and consent to participate Not applicable. Adherence to national and international regulations Not applicable. Consent for publication Not applicable. Competing interests NHO and TNJ are members of the Scientific Advisory Board of Clue Genetics, Inc. NHO is also a member of the Scientific Advisory Boards of Mycosynthetix, Inc. and Ionic Pharmaceuticals, LLC. References Meruvu H, dos Santos JC. Colors of Life: A Review on Fungal Pigments. Crit Rev Biotechnol. 2021;41(8):1153–77. https://doi.org/10.1080/07388551.2021.1901647 . Elkhateeb W, Daba GF, Pigments. Their Diversity, Chemistry, Food and Non-Food Applications. Appl Microbiol. 2023;3(3):735–51. https://doi.org/10.3390/applmicrobiol3030051 . Toma MA, Rahman MH, Rahman MS, Arif M, Hussain N, Dufossé L. Fungal Pigments: Carotenoids, Riboflavin, and Polyketides with Diverse Applications. J Fungi. 2023;9(4):454–454. https://doi.org/10.3390/jof9040454 . Gmoser R, Ferreira JA, Lennartsson PR, Taherzadeh MJ. Filamentous Ascomycetes Fungi as a Source of Natural Pigments. Fungal Biology Biotechnol. 2017;4(1). https://doi.org/10.1186/s40694-017-0033-2 . Venil CK, Velmurugan P, Dufossé L, Renuka Devi P, Veera Ravi A. Fungal Pigments: Potential Coloring Compounds for Wide Ranging Applications in Textile Dyeing. J Fungi. 2020;6(2):68. https://doi.org/10.3390/jof6020068 . Lin L, Xu J. Production of Fungal Pigments: Molecular Processes and Their Applications. J Fungi. 2022;9(1):44. https://doi.org/10.3390/jof9010044 . Gomes DC. Fungal Pigments: Applications and Their Medicinal Potential. Fungi Bioactive Metabolites: Integration of Pharmaceutical Applications. Springer Nature Singapore; 2024. pp. 651–81. https://doi.org/10.1007/978-981-99-5696-8_21 . Lin L, Xu JF. Pigments and Their Roles Associated with Human Health. J Fungi. 2020;6(4):280. https://doi.org/10.3390/jof6040280 . Venugopalan A, Srivastava S. Endophytes as in Vitro Production Platforms of High Value Plant Secondary Metabolites. Biotechnol Adv. 2015;33(6):873–87. https://doi.org/10.1016/j.biotechadv.2015.07.004 . Gakuubi MM, Munusamy M, Liang Z-X, Ng SB. Fungal Endophytes: A Promising Frontier for Discovery of Novel Bioactive Compounds. J Fungi. 2021;7(10):786. https://doi.org/10.3390/jof7100786 . Rai N, Kumari Keshri P, Verma A, Kamble SC, Mishra P, Barik S, Kumar Singh S, Gautam V. Plant Associated Fungal Endophytes as a Source of Natural Bioactive Compounds. Mycology. 2021;12(3):139–59. https://doi.org/10.1080/21501203.2020.1870579 . Devi R, Verma R, Dhalaria R, Kumar A, Kumar D, Puri S, Thakur M, Chauhan S, Chauhan PP, Nepovimova E, Kuca K. A Systematic Review on Endophytic Fungi and Its Role in the Commercial Applications. Planta. 2023;257(4). https://doi.org/10.1007/s00425-023-04087-2 . Adeleke B, Babalola O. Pharmacological Potential of Fungal Endophytes Associated with Medicinal Plants: A Review. J Fungi. 2021;7(2):147. https://doi.org/10.3390/jof7020147 . Gupta A, Meshram V, Gupta M, Goyal S, Qureshi KA, Jaremko M, Shukla KK. Fungal Endophytes: Microfactories of Novel Bioactive Compounds with Therapeutic Interventions; a Comprehensive Review on the Biotechnological Developments in the Field of Fungal Endophytic Biology over the Last Decade. Biomolecules. 2023;13(7):1038–1038. https://doi.org/10.3390/biom13071038 . Shaban A, Zakaria Z, Abdelhamid IA, Elhakim HKA, El-Sayed E-SR. Novel Myco-Fabrication of Copper and Nickel Nanoparticles and Evaluation of Their Effects against Antibiotic Resistance Genes in Different Bacterial Strains and Anticancer Potentials. Process Biochem. 2024;149:192–203. https://doi.org/10.1016/j.procbio.2024.12.008 . Abdelkader DH, Negm WA, Elekhnawy E, Eliwa D, Aldosari BN, Almurshedi AS. Zinc Oxide Nanoparticles as Potential Delivery Carrier: Green Synthesis by Aspergillus Niger Endophytic Fungus, Characterization, and in Vitro/in Vivo Antibacterial Activity. Pharmaceuticals. 2022;15(9):1057. https://doi.org/10.3390/ph15091057 . Panwar A, Manna S, Sahini G, Kaushik V, Kumar M, Govarthanan M. The Legacy of Endophytes for the Formation of Bioactive Agents, Pigments, Biofertilizers, Nanoparticles and Bioremediation of Environment. World J Microbiol Biotechnol. 2025;41(2). https://doi.org/10.1007/s11274-025-04265-2 . El-Sayed E-SR, Zaki AG, Ahmed AS, Ismaiel AA. Production of the Anticancer Drug Taxol by the Endophytic Fungus Epicoccum Nigrum TXB502: Enhanced Production by Gamma Irradiation Mutagenesis and Immobilization Technique. Appl Microbiol Biotechnol. 2020;104(16):6991–7003. https://doi.org/10.1007/s00253-020-10712-x . Wouter DC, Rademakers R. NanoPack2: Population Scale Evaluation of Long-Read Sequencing Data. Bioinformatics. 2023;39(5). https://doi.org/10.1093/bioinformatics/btad311 . Silva GG, Dutilh BE, Matthews T, Elkins K, Schmieder R, Dinsdale EA, Edwards RA. Combining de Novo and Reference-Guided Assembly with Scaffold_builder. Source Code Biol Med. 2013;8(1):23. https://doi.org/10.1186/1751-0473-8-23 . Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. MrBayes 3.2: Efficient Bayesian Phylogenetic Inference and Model Choice across a Large Model Space. Syst Biol. 2012;61(3):539–42. https://doi.org/10.1093/sysbio/sys029 . Magaldi S, Mata-Essayag S, Hartung de Capriles C, Perez C, Colella MT, Olaizola C, Ontiveros Y. Well Diffusion for Antifungal Susceptibility Testing. Int J Infect Dis. 2004;8(1):39–45. https://doi.org/10.1016/j.ijid.2003.03.002 . Ebrahimzadeh MA, Nabavi SM, Nabavi SF, Bahramian F, Bekhradnia AR. Antioxidant and Free Radical Scavenging Activity of H. Officinalis L. Var. Angustifolius, v. Odorata, B. Hyrcana and C. Speciosum. Pak J Pharm Sci. 2010;23(1):29–34. van de Loosdrecht AA, Beelen RHJ, Ossenkoppele GJ, Broekhoven MG, Langenhuijsen MMAC. A Tetrazolium-Based Colorimetric MTT Assay to Quantitate Human Monocyte Mediated Cytotoxicity against Leukemic Cells from Cell Lines and Patients with Acute Myeloid Leukemia. J Immunol Methods. 1994;174(1–2):311–20. https://doi.org/10.1016/0022-1759(94)90034-5 . Grzelczyk J, Gałązka-Czarnecka I, Oracz J. Potentiality Assessment of the Acetylcholinesterase-Inhibitory Activity of Olive Oil with an Additive Edible Insect Powder. Molecules. 2023;28(14):5535–5535. https://doi.org/10.3390/molecules28145535 . Gach J, Grzelczyk J, Strzała T, Boratyński F, Olejniczak T. Microbial Metabolites of 3-n-Butylphthalide as Monoamine Oxidase a Inhibitors. Int J Mol Sci. 2023;24(13):10605–10605. https://doi.org/10.3390/ijms241310605 . Grzelczyk J, Budryn G, Pérez-Sánchez H. Evaluation of Affinity of Bioactive Isolates from Various Coffee Extracts through Binding with PPAR-γ with the Use of Isothermal Titration Calorimetry and Docking Simulation to Prevent Antidiabetic Effects. J Therm Anal Calorim. 2020;142(2):877–87. https://doi.org/10.1007/s10973-020-09801-w . Paguigan ND, El-Elimat T, Kao D, Raja HA, Pearce CJ, Oberlies NH. Enhanced Dereplication of Fungal Cultures via Use of Mass Defect Filtering. J Antibiot 2017, 70 (5), 553–61. https://doi.org/10.1038/ja.2016.145 El-Elimat T, Figueroa M, Ehrmann BM, Cech NB, Pearce CJ, Oberlies NH, High-Resolution MS. MS/MS, and UV Database of Fungal Secondary Metabolites as a Dereplication Protocol for Bioactive Natural Products. J Nat Prod. 2013;76(9):1709–16. https://doi.org/10.1021/np4004307 . Samson RA, Hoekstra ES, Frisvad JC. Introduction to Food and Airborne Fungi. 7th Edition, Centraalbureau voor Schimmelcultures, Utrecht. - References - Scientific Research Publishing . . https://www.scirp.org/reference/ReferencesPapers?ReferenceID=1444676 Visagie CM, Varga J, Houbraken J, Meijer M, Kocsubé S, Yilmaz N, Fotedar R, Seifert KA, Frisvad JC, Samson RA. Ochratoxin Production and Taxonomy of the Yellow Aspergilli (Aspergillus Section Circumdati). Stud Mycol. 2014;78:1–61. https://doi.org/10.1016/j.simyco.2014.07.001 . Saha P, Talukdar AD, Choudhury MD, Nath D. Bioprospecting for Fungal-Endophyte-Derived Natural Products for Drug Discovery. In: Singh B, editor. Advances in Endophytic Fungal Research. Springer International Publishing; 2019. pp. 35–49. https://doi.org/10.1007/978-3-030-03589-1_3 . El-Sayed ESR, Baskaran A, Pomarańska O, Mykhailova D, Dunal A, Dudek A, Satam S, Strzała T, Łyczko J, Olejniczak T, Boratyński F. Bioprospecting Endophytic Fungi of Forest Plants for Bioactive Metabolites with Antibacterial, Antifungal, and Antioxidant Potentials. Molecules. 2024;29(19):4746–4746. https://doi.org/10.3390/molecules29194746 . Kumar V, Prasher IB. Phytochemical Analysis and Antioxidant Activity of Endophytic Fungi Isolated from Dillenia Indica Linn. Appl Biochem Biotechnol. 2024;196:332–49. https://doi.org/10.1007/s12010-023-04498-7 . Hridoy M, Gorapi MZH, Noor S, Chowdhury NS, Rahman MM, Muscari I, Masia F, Adorisio S, Delfino DV, Mazid. Md. A. Putative Anticancer Compounds from Plant-Derived Endophytic Fungi: A Review. Molecules. 2022;27(1):296. https://doi.org/10.3390/molecules27010296 . Bastos M, Abian O, Johnson CM, Ferreira-da-Silva F, Vega S, Jimenez-Alesanco A, Ortega-Alarcon D, Velazquez-Campoy A. Isothermal Titration Calorimetry. Nat Reviews Methods Primers. 2023;3(1):1–23. https://doi.org/10.1038/s43586-023-00199-x . Wang TY, Ji H, Everton D, Le ATH, Krylova SM, Fournier R, Krylov S. N. Fundamental Determinants of the Accuracy of Equilibrium Constants for Affinity Complexes. Anal Chem. 2023;95(42):15826–32. https://doi.org/10.1021/acs.analchem.3c03557 . Wang TY, Latimer J, Jean-Luc R, Kogan I, Krylova SM, Schreiber S, Kohlmann P, Jose J, Krylov S. N. A Practical Approach to Quantitatively Assessing Equilibrium-Constant Accuracy from a Single Binding Isotherm. Precision Chem. 2024;3(2):89–104. https://doi.org/10.1021/prechem.4c00085 . Weinhäupl K, Meuret L, Desrat S, Roussi F, Morellet N, Beaupierre S, Guillou C, van Heijenoort C, Abian O, Vega S, Wolf I, Akopian T, Krandor O, Rubin E, Velazquez-Campoy A, Gauto D, Fraga H. Identification of New ClpC1-NTD Binders for Mycobacterium Tuberculosis Drug Development. Sci Rep. 2025;15(1). https://doi.org/10.1038/s41598-025-87535-1 . Singh B, Bhagat J, Chadha B, Kaur A. Cholinesterase Inhibitory Potential of Different Alternaria Spp. And Their Phylogenetic Relationships. Biologia 2014, 69 (1). https://doi.org/10.2478/s11756-013-0294-z El-Sayed ESR, Grzelczyk J, Strzała T, Gałązka-Czarnecka I, Budryn G, Boratyński F. Bioprospecting Endophytic Fungi of Forest Plants for Their Monoamine Oxidase a and Cholinesterases Inhibitors, and Peroxisome Proliferator-Activated Receptor Gamma Agonists. J Appl Microbiol. 2025;136(3). https://doi.org/10.1093/jambio/lxaf034 . Jeong G-S, Kang M-G, Han S-A, Noh J-I, Park J-E, Nam S-J, Park D, Yee S-T, Kim H. Selective Inhibition of Human Monoamine Oxidase B by 5-Hydroxy-2-Methyl-Chroman-4-One Isolated from an Endogenous Lichen Fungus Daldinia Fissa. J Fungi. 2021;7(2):84–84. https://doi.org/10.3390/jof7020084 . Huang C, Xiong J, Guan H-D, Wang C-H, Lei X, Hu J-F, Discovery. Synthesis, Biological Evaluation and Molecular Docking Study of (R)-5-Methylmellein and Its Analogs as Selective Monoamine Oxidase a Inhibitors. Bioorg Med Chem. 2019;27(10):2027–40. https://doi.org/10.1016/j.bmc.2019.03.060 . Li D, Luo X, Ying W, La Kim E, Hong J, Lee J, Jung JH. Peroxisome Proliferator Activated Receptor-γ Agonistic Compounds from the Jellyfish‐Derived Fungus Cladosporium Oxysporum . Chem Biodivers. 2023;20(9). https://doi.org/10.1002/cbdv.202300851 . Lee D, Lee JH, Cai XF, Shin JC, Lee K, Hong Y-S, Lee JJ. Fungal Metabolites, Sorbicillinoid Polyketides and Their Effects on the Activation of Peroxisome Proliferator-Activated Receptor γ. J Antibiot. 2005;58(10):615–20. https://doi.org/10.1038/ja.2005.84 . El-Sayed ESR, Mousa SA, Strzała T, Boratyński F. Enhancing Bioprocessing of Red Pigment from Immobilized Culture of Gamma Rays Mutant of the Endophytic Fungus Monascus Ruber SRZ112. J Biol Eng. 2024;18(1). https://doi.org/10.1186/s13036-024-00439-y . Parekh S, Vinci VA, Strobel RJ. Improvement of Microbial Strains and Fermentation Processes. Appl Microbiol Biotechnol. 2000;54(3):287–301. https://doi.org/10.1007/s002530000403 . Thacker J. Repair of Ionizing Radiation Damage in Mammalian Cells. Alternative Pathways and Their Fidelity. Comptes Rendus de l’Académie des Sciences - Series III - Sciences de la Vie 2000, 322 (2–3), 103–108. https://doi.org/10.1016/S0764-4469(99)80030-4 Chopra VL, Mutagenesis. Investigating the Process and Processing the Outcome for Crop Improvement. Curr Sci. 2005;89(2):353–9. https://doi.org/10.2307/24110583 . Konar A, Datta S. Strain Improvement of Microbes. In Industrial Microbiology and Biotechnology ; Verma, P., Ed.; Springer Singapore, 2022; pp. 169–193. https://doi.org/10.1007/978-981-16-5214-1_6 Ismaiel AA, Ahmed AS, El-Sayed ER. Immobilization Technique for Enhanced Production of the Immunosuppressant Mycophenolic Acid by Ultraviolet and Gamma-Irradiated Penicillium Roqueforti. J Appl Microbiol. 2015;119(1):112–26. https://doi.org/10.1111/jam.12828 . El-Sayed ESR, El-Sayyad GS, Abdel-Fatah SS, El-Batal AI, Boratyński F. Novel Nanoconjugates of Metal Oxides and Natural Red Pigment from the Endophyte Monascus Ruber Using Solid-State Fermentation. Microb Cell Fact. 2024;23(1). https://doi.org/10.1186/s12934-024-02533-8 . Zaki AG, El-Sayed E-SR. New and Potent Production Platform of the Acetylcholinesterase Inhibitor Huperzine a by Gamma-Irradiated Alternaria Brassicae under Solid-State Fermentation. Appl Microbiol Biotechnol. 2021;105(23):8869–80. https://doi.org/10.1007/s00253-021-11678-0 . Hazaa MA, Shebl MM, El-Sayed E-SR, Mahmoud SR, Khattab AA, Amer MM. Bioprospecting Endophytic Fungi for Antifeedants and Larvicides and Their Enhancement by Gamma Irradiation. AMB Express. 2022;12(1). https://doi.org/10.1186/s13568-022-01461-3 . Bleisch R, Freitag L, Ihadjadene Y, Sprenger U, Steingröwer J, Walther T, Krujatz F. Strain Development in Microalgal Biotechnology—Random Mutagenesis Techniques. Life (2075 – 1729). 2022;12(7):961–961. https://doi.org/10.3390/life12070961 . Assante G, Camarda L, Locci R, Merlini L, Nasini G, Papadopoulos E. Isolation and Structure of Red Pigments from Aspergillus Flavus and Related Species, Grown on a Differential Medium. J Agric Food Chem. 1981;29(4):785–7. https://doi.org/10.1021/jf00106a023 . Vansteelandt M, Blanchet E, Egorov M, Petit F, Toupet L, Bondon A, Monteau F, Bizec BL, Thomas OP, Pouchus YF, Bot RL, Grovel O. Ligerin, an Antiproliferative Chlorinated Sesquiterpenoid from a Marine-Derived Penicillium Strain. J Nat Prod. 2013;76(2):297–301. https://doi.org/10.1021/np3007364 . Pak CS, Lee GH. Total Synthesis of (+)-Preussin, a Novel Antifungal Agent. J Org Chem. 1991;56(3):1128–33. https://doi.org/10.1021/jo00003a040 . Gu B-B, Jiao F-R, Wu W, Jiao W, Li L, Sun F, Wang S-P, Yang F, Lin H-W. Preussins with Inhibition of IL-6 Expression from Aspergillus Flocculosus 16D-1, a Fungus Isolated from the Marine Sponge Phakellia Fusca . J Nat Prod. 2018;81(10):2275–81. https://doi.org/10.1021/acs.jnatprod.8b00662 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 31 Dec, 2025 Read the published version in Microbial Cell Factories → Version 1 posted Editorial decision: Revision requested 11 Nov, 2025 Reviews received at journal 25 Oct, 2025 Reviews received at journal 23 Oct, 2025 Reviewers agreed at journal 19 Oct, 2025 Reviewers agreed at journal 14 Oct, 2025 Reviewers agreed at journal 14 Oct, 2025 Reviewers agreed at journal 14 Oct, 2025 Reviewers invited by journal 14 Oct, 2025 Editor assigned by journal 07 Sep, 2025 Submission checks completed at journal 07 Sep, 2025 First submitted to journal 05 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7546670","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":512088330,"identity":"d9bd242e-7ad1-4608-a812-23b32d6ef9cd","order_by":0,"name":"Abirami Baskaran","email":"","orcid":"","institution":"Wrocław University of Environmental and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Abirami","middleName":"","lastName":"Baskaran","suffix":""},{"id":512088334,"identity":"64b60354-f592-4911-bcc2-fd18d841bd92","order_by":1,"name":"Andreas Wasilewicz","email":"","orcid":"","institution":"University of Vienna","correspondingAuthor":false,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Wasilewicz","suffix":""},{"id":512088335,"identity":"29a2172e-09ad-40de-b001-216fb7694858","order_by":2,"name":"Judith M. Rollinger","email":"","orcid":"","institution":"University of Vienna","correspondingAuthor":false,"prefix":"","firstName":"Judith","middleName":"M.","lastName":"Rollinger","suffix":""},{"id":512088336,"identity":"14bc8f27-a16e-4849-b4d1-edaa8f16a809","order_by":3,"name":"Joanna Grzelczyk","email":"","orcid":"","institution":"Lodz University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Joanna","middleName":"","lastName":"Grzelczyk","suffix":""},{"id":512088337,"identity":"9a3732ee-5b63-4a77-a49c-3362f1521364","order_by":4,"name":"Ilona Gałązka-Czarnecka","email":"","orcid":"","institution":"Lodz University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Ilona","middleName":"","lastName":"Gałązka-Czarnecka","suffix":""},{"id":512088338,"identity":"15f13d6b-2064-4d9c-ab38-9ab02182f722","order_by":5,"name":"Grażyna Budryn","email":"","orcid":"","institution":"Lodz University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Grażyna","middleName":"","lastName":"Budryn","suffix":""},{"id":512088339,"identity":"04c59d75-76f8-4eb3-a6e2-5511b8087db7","order_by":6,"name":"Tomasz Strzała","email":"","orcid":"","institution":"Wroclaw University of Environmental and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Tomasz","middleName":"","lastName":"Strzała","suffix":""},{"id":512088340,"identity":"02b4b86a-60f9-4572-863c-ccd966f725f2","order_by":7,"name":"Tyler N. Graf","email":"","orcid":"","institution":"University of North Carolina at Greensboro","correspondingAuthor":false,"prefix":"","firstName":"Tyler","middleName":"N.","lastName":"Graf","suffix":""},{"id":512088344,"identity":"96d8f5be-66d6-49c3-bd48-b03c7afc039a","order_by":8,"name":"Nicholas H. Oberlies","email":"","orcid":"","institution":"University of North Carolina at Greensboro","correspondingAuthor":false,"prefix":"","firstName":"Nicholas","middleName":"H.","lastName":"Oberlies","suffix":""},{"id":512088346,"identity":"c125f027-88e0-4d24-b517-78264a2cf92d","order_by":9,"name":"Filip Boratyński","email":"","orcid":"","institution":"Wrocław University of Environmental and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Filip","middleName":"","lastName":"Boratyński","suffix":""},{"id":512088348,"identity":"b6c7d0e4-b424-46ac-84a6-2000c6f78fb7","order_by":10,"name":"El-Sayed R. El-Sayed","email":"data:image/png;base64,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","orcid":"","institution":"Wrocław University of Environmental and Life Sciences","correspondingAuthor":true,"prefix":"","firstName":"El-Sayed","middleName":"R.","lastName":"El-Sayed","suffix":""}],"badges":[],"createdAt":"2025-09-05 18:53:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7546670/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7546670/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12934-025-02905-8","type":"published","date":"2025-12-31T15:58:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90886743,"identity":"7733ef51-3362-4810-af45-79c8cd97de79","added_by":"auto","created_at":"2025-09-09 10:14:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":621399,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological characteristics of the 17P strain. Colonial growth was observed on Czapek–Yeast Autolysate agar (a), yeast-sucrose agar (b), malt extract agar (c) and potato-dextrose agar (d). The plate cultures in the top shows a front view of the growth and the plate cultures in the bottom shows a reverse view of the growth after incubation for 7 days at 30°C.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7546670/v1/5a44449e9873237a2ec7188e.png"},{"id":90885310,"identity":"e8ee48d9-d204-495a-814a-0696bc217196","added_by":"auto","created_at":"2025-09-09 10:06:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":296184,"visible":true,"origin":"","legend":"\u003cp\u003eBayesian phylogenetic tree of the ITS sequence from this study and sequences possessed form NCBI (accession numbers of all used NCBI sequences are showed on the tree). Numbers along the nodes are the posterior probabilities of the nodes. \u003cem\u003eAspergillus westerdijkiae \u003c/em\u003eclade is shaded and marked with a star shape. Sample analyzed in this study is bolded.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7546670/v1/d393a787b5c7f3d92ccf4f25.png"},{"id":90885314,"identity":"1022e9ac-e3cd-4f04-82ba-578f7da681e0","added_by":"auto","created_at":"2025-09-09 10:06:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":220061,"visible":true,"origin":"","legend":"\u003cp\u003eESI chromatograms of the biomass extract of the \u003cem\u003eAspergillus westerdijkiae \u003c/em\u003eP17.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7546670/v1/81a2f94d40e35bff3cd22e7e.png"},{"id":90885312,"identity":"ff3ddb0c-50f6-43a6-aa2d-1aec98a30842","added_by":"auto","created_at":"2025-09-09 10:06:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":499673,"visible":true,"origin":"","legend":"\u003cp\u003eELSD chromatograms of the separated fraction from the biomass extract of the \u003cem\u003eAspergillus westerdijkiae \u003c/em\u003eP17.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7546670/v1/5e63a5fbdcaf9a12f5dbe67c.png"},{"id":99545321,"identity":"875fd32d-13d2-435b-85b2-8ab5fdc677bb","added_by":"auto","created_at":"2026-01-05 16:05:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4140465,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7546670/v1/835dbdca-6e99-4fd4-b7fd-85a2e3f3ed78.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Natural Pigments from the Endophyte Aspergillus westerdijkiae and Evaluation of their Bioactivities","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColors play a fascinating part in human life profoundly influencing emotions and preferences. Anciently, a rich variety of dyes and pigments originating from natural fruit and vegetable extracts were utilized for architectural, decorative and preservative purposes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Even though the previous centuries saw applications of synthetic colorants in textile, food, cosmetic and pharmaceutical industries, their toxicity and non-environment friendly manufacturing practices has started limiting its use and enhanced the search for bio-pigments from natural sources [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Colorants sourced from algae, insects, plants and animals, face limitations such as low yields, seasonal availability, deforestation, instability and insolubility [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, microbial pigments offer significant advantages like consistent supply, greater stability, cost efficiency and higher yields [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Fungi are a remarkable source of natural pigments often displaying a wide spectrum of colors with improved solubility and stability. Notably, individual fungal species can biosynthesize diverse pigments with distinct properties [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Fungal mycelium releases these pigments as secondary metabolites, generally, as a response to environmental stress or nutrient limitation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These fungal pigments also possess multifaceted biological activity such as cellular differentiation, metal transport, complex interactions with other organisms through symbiosis and competition, even providing defense against predators and insects [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], all of which generated interest in the pharmaceutical field, documenting their potential as antiviral, antifungal, antibacterial, antioxidant, anti-inflammatory, antitumor, anti-Alzheimer\u0026rsquo;s disease, anti-atherosclerotic, anti-obesity, immunosuppressive, nematocidal and cytotoxic agents [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eEndophytic fungi comprise a ubiquitous and diverse microbial group inhabiting all plant species across a wide range of climatic and ecological zones [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Isolated from virtually every plant type, from trees, shrubs, herbs to ferns and marine plants, their diversity is significantly influenced by host plant genotype and physiology, environmental factors, human activities and growth season [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This diversity extends to differences among host plants within the same geographical area and even within different parts of a single plant [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Notably, tropical and subtropical regions exhibit a significantly higher diversity of fungal endophytes compared to other areas [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The co-evolution between fungal endophytes and their plants results in bioactive metabolites production that provide multiple benefits such as stimulating plant growth, triggering defense mechanisms against pathogens and aid in tolerating drought and salt stresses [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. For their ability to quantitatively increase the production of secondary metabolites in host plants and their extensive distribution and diversity, these endophytic fungi are often considered superior to other fungi [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, pigments from the endophyte \u003cem\u003eAspergillus westerdijkiae\u003c/em\u003e P17, isolated from the twig of \u003cem\u003eBetula pendula\u003c/em\u003e, were separated and their bioactive properties including antimicrobial, cytotoxic, antioxidant, monoamine oxidase A, acetylcholinesterase, and butyrylcholinesterase inhibitors, and peroxisome proliferator-activated receptor gamma agonists were analysed. The impact of gamma irradiation on pigments production was evaluated. Chemical characterization of pigments fractions was also studied.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eIsolation of endophytic fungi\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003eBetula pendula\u003c/em\u003e samples were collected from Mokrzański forest, Poland. The plant was identified with the help of Dr. Katarzyna Patejuk, Department of Plant Protection, Wrocław University of Environmental and Life Sciences. Healthy plant parts were collected, stored in ice box and transported to the laboratory. The isolation of fungal endophytes was accomplished according to previous study[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The samples were cut into small pieces, surface-sterilized by dipping in 70% ethanol for 1 min, followed by 0.1% HgCl\u003csub\u003e2\u003c/sub\u003e for 1 min, and then rinsed in sterile distilled water before being dried on sterile filter paper. The sterilized plant fragments were then aseptically transferred onto potato-dextrose agar plates (supplemented with streptomycin and tetracycline) and incubated at 25\u0026deg;C. The inoculated plates were checked daily and the isolated cultures were sub-cultured for purity and stored at \u0026minus;\u0026thinsp;4\u0026deg;C in glycerol (15%) as a suspension of spores and mycelia.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIdentification of the fungal strain 17P\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eAmong the isolated fungi, a promising strain (17P) was isolated from a twig of \u003cem\u003eBetula pendula\u003c/em\u003e. The fungus was identified using cultural, morphological and molecular methods. Cultural characteristics were studied on Malt Extract agar (MEA), Yeast Extract Sucrose agar (YES), Czapek\u0026ndash;Yeast Autolysate agar (CYA), and Potato-dextrose agar (PDA) at 30\u0026deg;C for 10 days.\u003c/p\u003e\u003cp\u003eThe identity of the fungus was confirmed by molecular characterization techniques. DNA was extracted from fungal cultures using the Genomic Mini AX Yeast kit (A\u0026amp;A Biotechnology, Gdańsk, Poland) following the manufacturer's protocol. The DNA concentration was then quantified using the Qubit 4.0 fluorometer. Amplification of the ITS region was performed using the ITS4 (TCCTCCGCTTATTGATATGC) and ITS5 (GGAAGTAAAAGTCGTAACAAGG) primers under the following thermal cycling conditions: an initial denaturation at 95\u0026deg;C for 2 min; 35 cycles of 95\u0026deg;C for 60 seconds, 55\u0026deg;C for 60 seconds, and 72\u0026deg;C for 90 seconds; followed by a final elongation at 72\u0026deg;C for 10 min. PCR products were visualized via electrophoresis on a 1% agarose gel and subsequently purified using NucleoMag magnetic beads (Macherey-Nagel). Sequencing was carried out using the Oxford Nanopore Technology (ONT) MinION platform, employing the SQK-NBD114.96 barcoding kit and a FLO-MIN114 flow cell. Library preparation followed ONT\u0026rsquo;s guidelines for barcoded samples. Raw sequence data in POD5 format were basecalled using the Dorado basecaller (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/nanoporetech/dorado\u003c/span\u003e\u003cspan address=\"https://github.com/nanoporetech/dorado\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with the super accurate (SUP) model. FastQC (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.babraham.ac.uk/projects/fastqc/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used for quality assessment, and reads were trimmed to the appropriate length with a minimum phred score of 12 using Chopper [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Consensus sequences were generated using Scaffold Builder [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Initial species identification was conducted through the MycoBank database. Sequences from the study were then incorporated into a phylogenetic analysis alongside ITS sequences of known species obtained from NCBI, including outgroup. Phylogenetic relationships were inferred using the Bayesian framework implemented in MrBayes 3.2.7a [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], with the mixed model selection. Trees were sampled every 100th generation over 10,000,000 MCMC generations, and the consensus tree was constructed from samples with an average standard deviation of split frequencies well below 0.01.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation and extraction of fungal biomass\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eSpore suspensions were obtained from 7-day-old fungal cultures. Spore concentrations were determined and standardized to 1 \u0026times; 10⁶ spores\u0026middot;mL⁻\u0026sup1; using a hemocytometer. Under aseptic conditions, 1 mL of the standardized spore suspension was inoculated into 250 mL Erlenmeyer flasks containing 50 mL of potato dextrose (PD) broth. The flasks were incubated at 30\u0026deg;C under static conditions for 14 days.\u003c/p\u003e\u003cp\u003eFollowing incubation, cultures were filtered through Whatman No. 1 filter paper to separate the culture filtrate from the fungal biomass. The recovered mycelial biomass was homogenized using a mortar and pestle until a uniform consistency was achieved. Each homogenized sample was extracted with 25 mL of a chloroform\u0026ndash;methanol mixture (9:1, v/v). The extraction mixtures were sonicated for 1 hour at 20 kHz and 20\u0026deg;C in an ultrasonic bath.\u003c/p\u003e\u003cp\u003eSubsequently, the organic phase was separated using a separatory funnel and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure using a rotary vacuum evaporator to yield crude dry extracts. These extracts were reconstituted in a methanol\u0026ndash;dimethyl sulfoxide mixture (2:1, v/v) for further analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFractionation and chromatographic separation of the extract.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe crude fungal extract was adsorbed onto silica gel (Silica Gel 60, 0.040\u0026ndash;0.063 mm particle size; Merck, Germany) and the solvent was removed under reduced pressure using a rotary vacuum evaporator. A chromatographic column was packed to three-quarters of its volume with silica gel, after which the silica\u0026ndash;extract mixture was added.\u003c/p\u003e\u003cp\u003eFlash chromatography was performed using a puriFlash\u0026reg; XS520 Plus system (Interchim SA, France) equipped with a silica-based column (SIHP-JP, F0012). Elution was carried out using a gradient system of n-hexane and ethyl acetate. The separation commenced with 100% n-hexane, followed by stepwise increases in the ethyl acetate concentration (1%, 2%, and finally 5%, v/v), thereby increasing the polarity of the mobile phase. This process resulted in the isolation of four pigment-containing fractions, designated as 17P1, 17P2, 17P3, and 17P4.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eTesting bioactivities of the separated fractions\u003c/h2\u003e\u003cp\u003e\u003cb\u003eAntimicrobial Activity\u003c/b\u003e The antimicrobial activity of the isolated pigment fractions (17P1\u0026ndash;17P4) was assessed using the agar well diffusion method [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Antibacterial efficacy was evaluated against human pathogenic strains \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (ATCC 6538) and \u003cem\u003eEscherichia coli\u003c/em\u003e (ATCC 11229), while antifungal activity was tested against \u003cem\u003eFusarium oxysporum\u003c/em\u003e (EUM37, a phytopathogen), \u003cem\u003eCandida albicans\u003c/em\u003e (ATCC 10231), and \u003cem\u003eAspergillus brasiliensis\u003c/em\u003e (ATCC 16404). The positive control for antibacterial assays was a mixture of amoxicillin and clavulanic acid (500 \u0026micro;g/mL), and for antifungal assays, nystatin (100 \u0026micro;g/mL) was used. The solvent mixture (MeOH:DMSO, 2:1 v/v) served as the negative control.\u003c/p\u003e\u003cp\u003eBacterial cultures were standardized to 0.5 McFarland turbidity using spectrophotometric measurement at 600 nm (A₆₀₀ = 0.08\u0026ndash;0.10). Fungal spore suspensions were adjusted to 1 \u0026times; 10⁶ spores/mL using a hemocytometer. One hundred microliters of each microbial suspension were inoculated onto appropriate media: Mueller\u0026ndash;Hinton Agar for bacteria, Potato Dextrose Agar for molds, and Sabouraud Dextrose Agar for \u003cem\u003eCandida\u003c/em\u003e. Agar wells were created and filled with 50 \u0026micro;L of each fungal extract. Plates inoculated with \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, and \u003cem\u003eC. albicans\u003c/em\u003e were incubated at 37\u0026deg;C, while those containing \u003cem\u003eF. oxysporum\u003c/em\u003e and \u003cem\u003eA. brasiliensis\u003c/em\u003e were incubated at 30\u0026deg;C for 24 h. Zones of inhibition (ZOI) were measured in millimeters.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDPPH Scavenging Assay\u003c/b\u003e The antioxidant potential of the pigment fractions was evaluated using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. A 120 \u0026micro;L aliquot of 100 \u0026micro;M DPPH in methanol was mixed with 80 \u0026micro;L of each pigment extract. Ascorbic acid (10 mM) served as the positive control, while the solvent mixture (DMSO:MeOH, 1:2 v/v) served as the blank. After 15 minutes of incubation in the dark at room temperature, the absorbance was measured at 517 nm.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCytotoxic activity assay.\u003c/b\u003e The cytotoxic effects of pigment fractions (17P1\u0026ndash;17P4) were evaluated against three human cancer cell lines: melanoma (A375), breast adenocarcinoma (MCF-7), and lung carcinoma (A549), along with a non-cancerous human fibroblast cell line (Hfb-4). All cell lines were procured from ATCC and cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and L-glutamine (Sigma-Aldrich). All cells were tested for Mycoplasma contamination before experiments. Cytotoxicity of the pigments fractions 17P1, 17P2, 17P3 and 17P4 was evaluated against the aforementioned cell lines using the MTT assay according to the method described by Van de Loosdrecht and co-workers [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] with slight modifications. Cells were seeded at 1 \u0026times; 10⁴ cells/well in 96-well plates and incubated at 37\u0026deg;C in a humidified 5% CO₂ atmosphere. Cells were treated with 20 \u0026micro;L of each pigment fraction for 24 h. Untreated control wells received 0.1% DMSO. After treatment, 10 \u0026micro;L of MTT (5 mg/mL) was added to each well, followed by incubation for 4 h. The resulting formazan crystals were solubilized in 100 \u0026micro;L of DMSO. Absorbance was read at 570 nm using a TECAN SunRise microplate reader. Cytotoxicity was calculated using:\u003c/p\u003e\u003cp\u003eInhibition (%) = [ 100 - (A570 of treated cells / A570 of control cells) ] \u0026times; 100\u003c/p\u003e\u003cp\u003e\u003cb\u003eAChE and BChE inhibitory potentials.\u003c/b\u003e Enzymatic inhibition studies were conducted using acetylcholinesterase (AChE) from \u003cem\u003eElectrophorus electricus\u003c/em\u003e and butyrylcholinesterase (BChE) from horse serum (Sigma-Aldrich). The isothermal titration calorimetry (ITC) assay was performed using a MicroCal PEAQ-ITC200 instrument (Malvern Instruments, UK). The sample cell (0.2 mL) was loaded with a 20 \u0026micro;M solution of either AChE or BChE in methanol. Pigment fractions (10 mM in methanol) were injected in 2 \u0026micro;L increments. Binding thermodynamics were recorded at 36\u0026deg;C under constant stirring. Control titrations (methanol only) were subtracted from enzyme\u0026ndash;ligand interactions. Binding parameters including dissociation constant (Kd), association constant (Ka), enthalpy change (ΔH), entropy change (ΔS), and Gibbs free energy change (ΔG) were calculated using single-site binding models via the MicroCal PEAQ-ITC software [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Competitive inhibition was also assessed in the presence of acetylcholine (ACh).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMAO-A inhibitory potential\u003c/b\u003e MAO-A inhibitory potential was evaluated under similar ITC conditions with modifications. A 10 \u0026micro;M solution of human recombinant MAO-A was titrated with 2 mM pigment fractions and/or serotonin (5-HT, positive control). A total of 11 injections were performed over 30 minutes [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003ePPAR-γ agonist potential\u003c/b\u003e GST-tagged human PPAR-γ ligand-binding domain (residues 204\u0026ndash;477; 50 \u0026micro;g/mL) and the natural ligand 15-deoxy-Δ\u0026sup1;\u0026sup2;,\u0026sup1;⁴-prostaglandin J2 (\u0026ge;\u0026thinsp;95%) were obtained from Sigma-Aldrich. ITC analysis was conducted as described for BChE, with a 10 \u0026micro;M solution of PPAR-γ used as the target and 20 \u0026micro;M pigment fractions or prostaglandin J2 as ligands. The measurements were carried out at 36.6\u0026deg;C, with 11 injections over 30 minutes [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of\u003c/b\u003e \u003csup\u003e\u003cb\u003e60\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eCo gamma irradiation on pigments production\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eSpore suspensions of \u003cem\u003eAspergillus westerdijkiae\u003c/em\u003e P17 were subjected to γ-irradiation at doses of 250, 500, 1000, 2000, 4000, 8000, and 16000 Gy using a ⁶⁰Co gamma irradiator (MC20, Russia; dose rate 311.88 Gy/h). Post-irradiation, suspensions were incubated in the dark overnight, then 1 mL was inoculated into 50 mL of PD broth (pH 6.0) and cultured at 30\u0026deg;C.\u003c/p\u003e\u003cp\u003eAfter incubation, biomass was harvested, extracted as previously described, and pigment concentrations were determined spectrophotometrically at 410 nm (yellow), 470 nm (orange), and 510 nm (red). Fungal biomass (g\u0026middot;L⁻\u0026sup1;) was measured by drying to constant weight at 50\u0026deg;C.\u003c/p\u003e\u003cp\u003eSpore survival rates were assessed by plating 100 \u0026micro;L of irradiated spore suspensions on PDA, incubating at 25\u0026deg;C for 5 days, and counting colony-forming units. The survival rate was expressed relative to unirradiated controls (considered 100%).\u003c/p\u003e\u003cp\u003e\u003cb\u003eChemical characterization\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eThe whole biomass extract of the fungus was first analyzed via LC-MS dereplication using a database of over 700 fungal metabolites using procedures that have been described previously [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSecond, UHPLC-ELSD and UHPLC-UV-MS analyses were performed on a Waters Acquity UPLC H-Class system consisting of a fraction manager, column manager, quaternary solvent manager, PDA detector, ELS detector, isocratic solvent manager and a single quadrupole mass detector (Acquity QDa) equipped with an ESI source. A BEH C\u003csub\u003e18\u003c/sub\u003e column (1.7 \u0026micro;m, 2.1 x 100 mm, Waters) was used as stationary phase; water\u0026thinsp;+\u0026thinsp;0,1% formic acid (A) and acetonitrile\u0026thinsp;+\u0026thinsp;0,1% formic acid (B) were used as mobile phase. The column temperature was set to 40\u0026deg;C, flow rate of 0.3 was used and the following gradient was applied: 5% B at 0.0 min, from 5% \u0026minus;\u0026thinsp;98% B in 5.0, 98% B for 10 min. Mass detection was performed in positive (cone voltage: 15 V, capillary voltage 0.8 kV) and negative (cone voltage: 30 V, capillary voltage: 0.8 kV) mode from 200\u0026ndash;1200 Da. To increase ionization, a mixture of water:methanol (9:1)\u0026thinsp;+\u0026thinsp;10 mM ammonium formate was used make-up solvent. The instrument was controlled by Empower 3.\u003c/p\u003e\u003cp\u003eSize exclusion chromatography was conducted for the separation of 17P2, using Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) as stationary phase and MeOH as mobile phase (column dimensions: 100 cm \u0026times; 2 cm). The fractions were collected in a time-dependent manner (3 min/tube) and analyzed by thin-layer chromatography (TLC) using Merck silica gel 60 PF254 plates as stationary phase and ethyl acetate as mobile phase. Detection was performed at visible light and UV366. Based on TLC fingerprints, the collected tubes were pooled into 5 fractions (17P2_I-V).\u003c/p\u003e\u003cp\u003e1D and 2D NMR data of 17P4 were recorded using a Bruker UltraShield 500 MHz NMR spectrometer equipped with a TCI Prodigy CryoProbe (5 mm), an AVANCE III HD console and a SampleJet. The fraction was measured at 296 K in MeOD referenced to the residual non-deuterated solvent signals (δH 3.31 ppm; δC 49.00 ppm). The resonance frequency for \u003csup\u003e1\u003c/sup\u003eH NMR was 500 MHz and for \u003csup\u003e13\u003c/sup\u003eC NMR 125 MHz. Standard 1D (1H) and gradient-enhanced 2D experiments, i.e. HSQC, and HMBC, were used as supplied by the manufacturer. The raw NMR data were processed using Topsin 4.3.0.\u003c/p\u003e\u003cp\u003ePenicillic acid (\u003cb\u003e5\u003c/b\u003e): identified in 17P4. \u003csup\u003e1\u003c/sup\u003eH NMR (MeOD, 500 MHz): 5.47 (1H, m), 5.28 (1H, s), 5.24 (1H, m), 3.89 (3H, s); \u003csup\u003e13\u003c/sup\u003eC NMR (MeOD, 125 MHz): 176.2 (qC), 173.5 (qC), 117.4 (CH\u003csub\u003e2\u003c/sub\u003e), 91.1 (CH), 59.7 (OCH\u003csub\u003e3\u003c/sub\u003e), 17.4 (CH\u003csub\u003e3\u003c/sub\u003e); ESI-MS: \u003cem\u003em/z\u003c/em\u003e 171.13 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePreussin (\u003cb\u003e6\u003c/b\u003e): identified in 17P4. \u003csup\u003e1\u003c/sup\u003eH NMR (MeOD, 500 MHz): 7.34 (2Hs, m, 2x H-3\u0026rsquo;), 7.32 (2Hs, m, 2x H-2\u0026rsquo;), 7.24 (1H, m, H-4\u0026rsquo;), 4.03 (1H, m, H-3), 3.12 (1H, dd, H-1a), 2.96 (1H, dd, H-1b), 3.03 (1H, m, H-2), 2.92 (1H, m, H-5), 2.67 (3Hs, s, NCH\u003csub\u003e3\u003c/sub\u003e), 2.36 (1H, m, H-4a), 1.63 (1H, m, H-4b), 0.91 (3Hs, t, H-14); \u003csup\u003e13\u003c/sup\u003eC NMR (MeOD, 125 MHz): 139.3 (CH, C-1\u0026rsquo;), 130.3 (2x CH, C-3\u0026rsquo;), 129.7 (2x CH, C-2\u0026rsquo;), 127.7 (CH, C-4\u0026rsquo;), 75.3 (CH, C-2), 69.8 (CH, C-3), 69.3 (CH, C-5), 39.3 (CH\u003csub\u003e2\u003c/sub\u003e, C-4), 38.8 (NH, NCH\u003csub\u003e3\u003c/sub\u003e), 32.5 (CH\u003csub\u003e2\u003c/sub\u003e, C-1), 14.4 (CH\u003csub\u003e3\u003c/sub\u003e, C-14); ESI-MS: 318.27 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistics.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIsothermal titration calorimetry (ITC) data were analyzed based on the mean values obtained from three independent replicates (n\u0026thinsp;=\u0026thinsp;3), with results expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical significance was assessed using one-way analysis of variance (ANOVA) performed in SPSS software (version 22.0; IBM Corp., NY, USA). Differences were considered statistically significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cb\u003eIdentification of the 17P fungus.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the colony morphology of the P17 strain maintained on the CYA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), YES (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), MEA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), and PDA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Colony characters: CYA 30\u0026deg;C, 7 d: Colony surface floccose; mycelial areas white to greyish yellow; sporulation pale yellow to light yellow; reverse greyish yellow. YES 30\u0026deg;C, 7 d: Colony surface floccose; mycelial areas pinkish and white near margin; sporulation greyish yellow; reverse dark brown. MEA 30\u0026deg;C, 7 d: Colony surface floccose; mycelial areas brown with white margin; sporulation light yellow; reverse dark brown. PDA 30\u0026deg;C, 7 d: Colony surface floccose; mycelial areas pale yellow; sporulation greyish yellow; reverse pale yellow, yellowish and white near margin. These characteristics are identical with those reviewed by previous reports [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] concerning identifications of \u003cem\u003eAspergillus westerdijkiae\u003c/em\u003e. For analyzed 17P sample, 1,000 high-quality reads (post-trimming) were obtained to ensure adequate sequencing depth. Consensus sequence generated in this study have been submitted to the NCBI database under accession number PV650320. Both Mycobank database-based identification and the constructed phylogenetic tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) validated the species identifications, clustering the sample consistently within their respective species clades. In the literature, there is no information about isolation of an endophytic \u003cem\u003eAspergillus westerdijkiae\u003c/em\u003e from plant sources.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntimicrobial, antioxidant, and anticancer potentials\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eThe pigment fractions 17P1, 17P2, 17P3 and 17P4 separated from the fungus \u003cem\u003eAspergillus westerdijkiae\u003c/em\u003e 17P were tested against prominent bacterial and fungal pathogens as depicted in Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The results indicate Zone of Inhibition (ZOI) and Minimum Inhibitory Concentration (MIC) values of pigments against the pathogens. All the fractions showed inhibitory activity against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e, while the fraction, 17P2, showed inhibition against all tested pathogens. Notably, 17P2 displayed ZOI at 100 mg/mL against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e and 250 mg/mL against \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e. With fungal pathogens, 17P2 displayed ZOI at 500 mg/mL against \u003cem\u003eA. brasiliensis\u003c/em\u003e, \u003cem\u003eA. alternata\u003c/em\u003e and \u003cem\u003eF. oxysporum\u003c/em\u003e and 250 mg/mL against \u003cem\u003eC. albicans\u003c/em\u003e. Inhibitory activity of 17P4 was observed at 500 mg/mL concentration against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e and at 1000 mg/mL against \u003cem\u003eF. oxysporum\u003c/em\u003e. 17P1 showed activity against \u003cem\u003eE. coli\u003c/em\u003e (250 mg/mL), \u003cem\u003eS. aureus\u003c/em\u003e (250 mg/mL) and \u003cem\u003eC. albicans\u003c/em\u003e (500 mg/mL) while 17P3 showed activity against \u003cem\u003eE. coli, S. aureus, A. alternata\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e at 500 mg/mL concentration.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eAntibacterial activity of the separated fractions from\u003c/b\u003e \u003cb\u003eAspergillus westerdijikae\u003c/b\u003e \u003cb\u003e17P cultures against different Gram-positive and Gram-negative human pathogenic bacterial strains.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e\u003cp\u003eSeparated fractions\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e\u003cp\u003eBacterial pathogens\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e17P1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZOI (mm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNil\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNil\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMIC (mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e17P2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZOI (mm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e11.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMIC (mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e17P3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZOI (mm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNil\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNil\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMIC (mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e17P4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZOI (mm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNil\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNil\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMIC (mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eAmoxicillin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e18.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e11.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eLSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.4511\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.5210\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.4388\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.3981\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003eAmoxicillin was used as the positive control at a concentration of 500 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The calculated mean is for triplicate measurements from three independent experiments\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Nil means that no ZOI was detected. LSD means Least Significant Differences.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eAntifungal activity of the separated fractions from\u003c/b\u003e \u003cb\u003eAspergillus westerdijikae\u003c/b\u003e \u003cb\u003e17P cultures against different human and plant pathogenic fungi.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e\u003cp\u003eSeparated fractions\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e\u003cp\u003eFungal pathogens\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eA. brasiliensis\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eA. alternata\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eF. oxysporum\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eC. albicans\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e17P1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZOI (mm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNil\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNil\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNil\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMIC (mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e17P2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZOI (mm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e17.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMIC (mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e17P3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZOI (mm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNil\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMIC (mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e17P4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZOI (mm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNil\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNil\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e11.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMIC (mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eNystatin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e14.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eLSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.3187\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.1891\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.1012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.2198\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003eNystatin was used as the positive control at a concentration of 500 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The calculated mean is for triplicate measurements from three independent experiments\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Nil means that no ZOI was detected. LSD means Least Significant Differences.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eResults of scavenging activity of the separated fractions showed that the activity increases with increasing concentration as stated in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. When compared to 17P1 and 17P3, it can be observed that the fractions 17P2 and 17P4 shows higher activity. Nearly 83% and 67% of free radicals were scavenged by fractions 17P2 and 17P4 respectively at a concentration of 1000 mg/mL, demonstrating the most promising antioxidant potential.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eAntioxidant activity of the separated fractions from\u003c/b\u003e \u003cb\u003eAspergillus westerdijikae\u003c/b\u003e \u003cb\u003e17P cultures.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eConcentration\u003c/p\u003e\u003cp\u003e(mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eScavenging activity (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e17P1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17P2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17P3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e17P4\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.31\u0026thinsp;\u0026plusmn;\u0026thinsp;10.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.71\u0026thinsp;\u0026plusmn;\u0026thinsp;2.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e44.29\u0026thinsp;\u0026plusmn;\u0026thinsp;9.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e28.71\u0026thinsp;\u0026plusmn;\u0026thinsp;2.79\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e23.54\u0026thinsp;\u0026plusmn;\u0026thinsp;10.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e61.66\u0026thinsp;\u0026plusmn;\u0026thinsp;10.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e29.43\u0026thinsp;\u0026plusmn;\u0026thinsp;11.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e43.54\u0026thinsp;\u0026plusmn;\u0026thinsp;10.98\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e47.66\u0026thinsp;\u0026plusmn;\u0026thinsp;12.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e83.54\u0026thinsp;\u0026plusmn;\u0026thinsp;11.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e55.43\u0026thinsp;\u0026plusmn;\u0026thinsp;21.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e67.66\u0026thinsp;\u0026plusmn;\u0026thinsp;12.54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.620\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.667\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.620\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.667\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eAscorbic acid (antioxidant standard) was the control. The calculated mean is for triplicate measurements from three independent experiments\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, LSD\u0026thinsp;=\u0026thinsp;least significant differences (p\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eCytotoxicity of the isolated pigments was tested against normal (Hfb-4), breast cancer (MCF-7) and liver cancer (HepG-2) cell lines (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Cell viability of 70\u0026ndash;80% can be seen with fractions 17P1 and 17P3 at 500 mg/mL MIC against both cancer cell lines. At the MIC of 1000 mg/mL, 17P4 showed cell viability of 87% and 91% against breast and liver cancer cell lines. The pigment fraction 17P2 was considered more promising with a cell viability of 68% against MCF-7 and 48% against HepG-2 at a minimum inhibitory concentration of 250 mg/mL.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eCytotoxic activities of the separated fractions from\u003c/b\u003e \u003cb\u003eAspergillus westerdijikae\u003c/b\u003e \u003cb\u003e17P cultures against normal, breast cancer, and liver cancer cell lines.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e\u003cp\u003eSeparated fractions\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e\u003cp\u003eCell line\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHfb-4\u003c/p\u003e\u003cp\u003e(normal)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMCF-7\u003c/p\u003e\u003cp\u003e(breast)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eHepG-2\u003c/p\u003e\u003cp\u003e(liver)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e17P1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCell viability (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e66.52\u0026thinsp;\u0026plusmn;\u0026thinsp;11.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"8\" rowspan=\"9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e78.44\u0026thinsp;\u0026plusmn;\u0026thinsp;14.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"8\" rowspan=\"9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e70.09\u0026thinsp;\u0026plusmn;\u0026thinsp;17.81\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMIC (mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e17P2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCell viability (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e57.83\u0026thinsp;\u0026plusmn;\u0026thinsp;10.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e67.98\u0026thinsp;\u0026plusmn;\u0026thinsp;12.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e48.78\u0026thinsp;\u0026plusmn;\u0026thinsp;9.76\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMIC (mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e250\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e17P3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCell viability (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e84.76\u0026thinsp;\u0026plusmn;\u0026thinsp;21.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e79.21\u0026thinsp;\u0026plusmn;\u0026thinsp;17.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e80.32\u0026thinsp;\u0026plusmn;\u0026thinsp;18.81\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMIC (mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e17P4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCell viability (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e90.55\u0026thinsp;\u0026plusmn;\u0026thinsp;20.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e87.32\u0026thinsp;\u0026plusmn;\u0026thinsp;15.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e91.45\u0026thinsp;\u0026plusmn;\u0026thinsp;18.98\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMIC (mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eTaxol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e17.08\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e20.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eLSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.839\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8.728\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7.451\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eTaxol was used as the positive control at a concentration of 50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. MTT-based assay was used for measuring the cytotoxic activities at 570 nm using MTT solution under the conditions described in Materials and Methods. Calculated mean is for triplicate measurements from three independent experiments\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Nil means that no ZOI was detected. LSD means Least Significant Differences.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn the literature, endophytic fungi have attracted considerable scientific interest due to their ability to produce a wide range of bioactive secondary metabolites. To date, the processes of isolation, cultivation, purification, and characterization of these fungi have led to the identification of approximately 200 structurally diverse and biologically significant compounds, including metabolites with demonstrated antimicrobial [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], antioxidant [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], and anticancer [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] activities.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAChE, BChE and MAO-A inhibitory and PPAR-γ agonist potential of the separated pigment fractions.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eITC analysis allows for a better understanding of the interactions between the studied enzymes and the receptors and pigments. To determine binding affinity, an analysis was performed by measuring the heat during each injection of the ligand into the protein present in the cell. This allows to monitor heat changes until all bonds are saturated. The experiment also includes a reference test in which the ligand is injected into a reagent in which the sample has been diluted. This heat is subtracted from the ligand-protein experiment [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The parameter n indicated one protein binding site per ligand molecule [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The ITC technique allows the use of low enzyme/receptor concentrations, thus demonstrating affinity independent of biological effects, avoiding non-specific binding [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eAChE and BChE\u003c/b\u003e AChE was found to bind to 17P1 and 17P2, but not to bind to 17P3 and 17P4. Kd for 17P2 was 1.63 \u0026micro;mol/L, and for 17P1 it was 2.78 \u0026micro;mol/L (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The enthalpy, ΔS, showed a value of -31.09 J/mol*K for 17P1 and +\u0026thinsp;71.06 J/mol*K for 17P2, which means that the 17P2 compound showed a stronger interaction, confirming the high affinity between 17P2 and AChE (ΔG -13.28 kJ/mol). In the case of BChE, compounds 17P1, 17P2 and 17P3 showed strong binding to BChE. Only 17P4 showed no binding to the active site of the enzyme. The highest affinity, similarly to AChE, was shown by compound 17P2, with a value of -124.98 kJ/mol (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), with a positive reaction enthalpy ΔH\u0026thinsp;=\u0026thinsp;7.69 kJ/mol. The dissociation constant was comparable for 17P1 and 17P2, being 0.03 \u0026micro;mol/L, while compound 17P3 had a slightly lower value of 0.01 \u0026micro;mol/L. The IC50 for AChE showed the lowest ligand dose inhibiting 50% of the enzyme for 17P1, while for BChE it was the 17P1 compound. Fungi are known to produce a diverse array of bioactive metabolites capable of inhibiting acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The extent of cholinesterase inhibition by fungal extracts varies considerably; some exhibit potent AChE inhibition, while others are more selective for BChE [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This variability indicates the presence of multiple bioactive constituents within fungal species. Notably, out of 43 fungal strains screened, six isolates demonstrated differing levels of inhibitory activity were reported, underscoring the strain-specific nature of these effects [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eThermodynamic parameters of interactions between AChE and the separated fractions from\u003c/b\u003e \u003cb\u003eAspergillus westerdijikae\u003c/b\u003e \u003cb\u003e17P cultures.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSeparated frctions\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK\u003csub\u003ed\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e(\u0026micro;mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eK\u003csub\u003ea\u003c/sub\u003e *10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e(L mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e∆H\u003c/p\u003e\u003cp\u003e(kJ mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e∆G\u003c/p\u003e\u003cp\u003e(kJ mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e∆S\u003c/p\u003e\u003cp\u003e(J mol*K\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eInhibitor activity [%]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (0.25 \u0026micro;L\u003c/p\u003e\u003cp\u003e\u0026micro;M AChE i 10 \u0026micro;MAchE\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (%))\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eK\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e (\u0026micro;mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) K\u003csub\u003e\u003cem\u003eM\u003c/em\u003e\u003c/sub\u003e ACh\u003c/p\u003e\u003cp\u003e35.91\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17P1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e35.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-71.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e86.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17P2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-13.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-31.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e75.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17P3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17P4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003eValues are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD; n\u0026thinsp;=\u0026thinsp;4; different letters in one column correspond to significant differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05); Nb \u0026ndash; no binding.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eThermodynamic parameters of interactions between BChE and the separated fractions from\u003c/b\u003e \u003cb\u003eAspergillus westerdijikae\u003c/b\u003e \u003cb\u003e17P cultures.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"11\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSeparated frctions\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK\u003csub\u003ed\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e(\u0026micro;mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eK\u003csub\u003ea\u003c/sub\u003e *10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e(L mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e∆H\u003c/p\u003e\u003cp\u003e(kJ mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e∆G\u003c/p\u003e\u003cp\u003e(kJ mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e∆S\u003c/p\u003e\u003cp\u003e(J mol*K\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003eInhibitor activity [%]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (0,25 \u0026micro;L \u0026micro;M\u003c/p\u003e\u003cp\u003eBChE i 10 \u0026micro;M\u003c/p\u003e\u003cp\u003eBChE\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (%))\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eKi (\u0026micro;mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003cp\u003eK\u003csub\u003e\u003cem\u003eM\u003c/em\u003e\u003c/sub\u003e BCh\u003c/p\u003e\u003cp\u003e35.91\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17P1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e-25.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e-27.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e46.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e12.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17P2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e-124.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e-40.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e99.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e4.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17P3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e-18.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e-28.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e62.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e7.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17P4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eNb\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"11\"\u003eValues are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD; n\u0026thinsp;=\u0026thinsp;4; different letters in one column correspond to significant differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05); Nb \u0026ndash; no binding\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMAO-A\u003c/b\u003e Obtained fractions bound at the active site. The results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. The dissociation constant ranged from 0.13 to 1 \u0026micro;mol/L for 17P3 and 17P2, respectively. The highest interaction constant and affinity was shown by compound 17P2, which was ΔH\u0026thinsp;=\u0026thinsp;100 kJ/mol and ΔG\u0026thinsp;=\u0026thinsp;153 kJ/mol. On the other hand, the lowest interaction constant and the highest affinity were observed for compound 17P3, which was ΔH\u0026thinsp;=\u0026thinsp;0.01 kJ/mol and ΔG= -47.23 kJ/mol. Fraction 17P3 also showed the lowest value needed to achieve half-maximal inhibitory effect, which was 4.03 \u0026micro;mol/\u0026micro;mol enzyme. Reports on metabolites with monoamine oxidase A (MAO-A) inhibitory activity from endophytic fungi remain scarce in the literature. Nevertheless, a few fungal species have been identified as sources of metabolites with MAO-A inhibitory potential. For instance, \u003cem\u003eDaldinia fissa\u003c/em\u003e was found to produce 5-hydroxy-2-methylchroman-4-one, which exhibited dual inhibition of MAO-A and MAO-B [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In another study, (\u003cem\u003eR\u003c/em\u003e)-5-methylmellein, isolated from the mycelial fermentation of \u003cem\u003eXylaria nigripes\u003c/em\u003e, acted as a selective MAO-A inhibitor and contributed to the understanding of its antidepressant mechanisms [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eThermodynamic parameters of interactions between MAO-A and the separated fractions from\u003c/b\u003e \u003cb\u003eAspergillus westerdijikae\u003c/b\u003e \u003cb\u003e17P cultures.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSeparated frctions\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK\u003csub\u003ed\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e(\u0026micro;mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eK\u003csub\u003ea\u003c/sub\u003e *10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e(L mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e∆H\u003c/p\u003e\u003cp\u003e(kJ mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e∆G\u003c/p\u003e\u003cp\u003e(kJ mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e∆S\u003c/p\u003e\u003cp\u003e(J mol*K\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eInhibitor activity [%]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (1 \u0026micro;mol L inhibitor\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e: 1 \u0026micro;mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eMAO-A)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eK\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e (\u0026micro;mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003cp\u003eK\u003csub\u003e\u003cem\u003eM\u003c/em\u003e\u003c/sub\u003e 5-HT 0.34 mmol\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17P1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.472\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-37.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e77.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e7.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17P2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.001\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e153.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-71.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e81.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17P3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e118.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-47.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-17.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e97.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17P4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-30.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e94.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003eValues are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD; n\u0026thinsp;=\u0026thinsp;4; different letters in one column correspond to significant differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05); Nb \u0026ndash; no binding\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePPAR-γ\u003c/b\u003e The obtained fractions bind to the active site of PPAR-γ. The results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. The dissociation constant is in the range of 7.13\u0026ndash;8.70 \u0026micro;mol/L, for 17P1 and 17P3, respectively. The lowest reaction enthalpy is characterized by the 17P3 fraction, amounting to -19.65 kJ/mol, but it shows a slightly lower affinity for the receptor compared to the other fractions, amounting to -29.97 kJ/mol. The typical ligand, 15-deoxy-Δ12,14-prostaglandin J2, showed a higher affinity by about \u0026minus;\u0026thinsp;18 kJ/mol. Literature on peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists from endophytic fungi is extremely limited. Three studies to date have reported relevant fungal metabolites. The first described the isolation of five novel compounds from \u003cem\u003eCladosporium oxysporum\u003c/em\u003e [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], while the second identified four new bioactive metabolites through bioassay-guided fractionation of an unidentified fungal strain [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The third described six different endophytes with varying agnostic levels [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eThermodynamic parameters of interactions between PPAR-γ and the separated fractions from\u003c/b\u003e \u003cb\u003eAspergillus westerdijikae\u003c/b\u003e \u003cb\u003e17P cultures.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSeparated fractions\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eK\u003csub\u003ed\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e(\u0026micro;mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eK\u003csub\u003ea\u003c/sub\u003e *10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e(L mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e∆H\u003c/p\u003e\u003cp\u003e(kJ mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e∆G\u003c/p\u003e\u003cp\u003e(kJ mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e∆S\u003c/p\u003e\u003cp\u003e(J mol*K\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17P1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-16.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-30.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e44.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17P2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-11.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-30.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e59.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17P3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-19.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-29.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e33.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17P4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-10.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-30.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e63.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLigand\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e189.75\u0026thinsp;\u0026plusmn;\u0026thinsp;2.15\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-98.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-48.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eValues are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD; n\u0026thinsp;=\u0026thinsp;4; different letters in one column correspond to significant differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05); Nb \u0026ndash; no binding\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of gamma irradiation on pigments production.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eData presented in Table\u0026nbsp;\u003cspan refid=\"Tab9\" class=\"InternalRef\"\u003e9\u003c/span\u003e indicate that exposure to low doses of gamma irradiation (250 and 500 Gy) resulted in spore survival rates of 99.48% and 89.48%, respectively. However, a marked decline in spore viability was observed at 2000 Gy, with survival reduced to 79.38%. At the highest applied dose (16,000 Gy), no fungal growth was detected, indicating a complete loss of viability. These findings suggest a dose-dependent inhibitory effect of gamma irradiation on fungal viability and growth. Correspondingly, dry biomass measurements showed that higher doses of gamma irradiation (4000 and 8000 Gy) led to the most pronounced reductions in biomass accumulation, with yields declining to 3.78 g/L and 1.08 g/L, respectively. This further supports the observation of dose-dependent suppression of fungal growth.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab9\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 9\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eEffect of gamma irradiation on survival rate (%), fungal growth (g L\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e), and yellow, orange and red pigments production by\u003c/b\u003e \u003cb\u003eAspergillus westerdijikae\u003c/b\u003e \u003cb\u003e17P.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eDose\u003c/p\u003e\u003cp\u003e(kGy)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSurvival\u003c/p\u003e\u003cp\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eDry biomass\u003c/p\u003e\u003cp\u003e(g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u003cp\u003ePigment yield (AU g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fresh biomass)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRed\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOrange\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eYellow\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e99.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e89.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e80.78\u0026thinsp;\u0026plusmn;\u0026thinsp;2.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e79.38\u0026thinsp;\u0026plusmn;\u0026thinsp;6.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e39.44\u0026thinsp;\u0026plusmn;\u0026thinsp;12.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLSD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.1873\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.8661\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.3121\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.1198\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.2136\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003eThe concentration was measured at 410, 470, and 510 nm that corresponded to the characteristic absorbance of yellow, orange and red pigments, respectively. The calculated mean is for triplicate measurements from two independent experiments\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. LSD means Least Significant Differences\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eRegarding pigment biosynthesis, exposure to 2000 Gy significantly enhanced the production of yellow, orange, and red pigments by \u003cem\u003eA. westerdijkiae\u003c/em\u003e 17P compared to the non-irradiated control. This dose was identified as optimal for stimulating pigment biosynthesis. However, further increases in irradiation dose (4000\u0026ndash;8000 Gy) resulted in a progressive decline in pigment production across all three pigment classes. In the literature, gamma radiation, a potent form of ionizing energy, can induce substantial biological effects, primarily by generating mutations through DNA strand breaks and errors during subsequent repair processes [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Mutagenesis induced by gamma rays has been widely applied in microbial biotechnology to improve strain performance [\u003cspan additionalcitationids=\"CR49 CR50 CR51 CR52\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. This approach typically involves repeated exposure to physical or chemical mutagens to introduce random genetic variability, followed by screening for strains with enhanced biosynthetic traits [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Our findings demonstrate that gamma irradiation at a dose of 2000 Gy (2 kGy) can significantly enhance pigment production in \u003cem\u003eA. westerdijkiae\u003c/em\u003e, potentially due to mutations induced within biosynthetic gene clusters responsible for secondary metabolite synthesis [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eChemical characterization\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eThe whole biomass extract of the fungus was analyzed via LC-MS dereplication using a database of over 700 fungal metabolites. An [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e ion peak of m/z 241.1544 was observed in the positive mode at 3.12 min, matching to the standard peak for neohydroxyaspergillic acid and with a calculated [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e of m/z 241.1546 and an elution time of 2.99 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Further confirmation came from fragmentation analysis of the observed peak where, five out of eight of the most abundant fragments matched that of the neohydroxyaspergillic acid standard.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo gain more insights into the chemical profile of the investigated fractions (17P1-17P4), UHPLC-ELSD and UHPLC-UV-MS analyses were performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). The ELSD chromatograms revealed a similar chemical composition for the fraction pairs 17P1 and 17P2 as well as for 17P3 and 17P4. To determine the constituents contributing to the shiny orange color of fractions 17P1 and 17P2, size exclusion chromatography was conducted which resulted in five fractions (17P2_I-V), with 17P2_II containing the orange-colored pigments. UHPLC-UV-MS analysis of this fraction enabled the tentative annotation of two constituents (\u003cb\u003e3, 4\u003c/b\u003e) as metal complexes, bearing either iron or aluminum as central metal ions each forming coordinate bonds with three units of aspergillic acids or its isomer, neoaspergillic acid. Such pigments have been previously described for other \u003cem\u003eAspergillus\u003c/em\u003e species [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The two main constituents (\u003cb\u003e1\u003c/b\u003e, \u003cb\u003e2\u003c/b\u003e; both \u003cem\u003em/z\u003c/em\u003e 499.32 in neg. mode) of 17P1 and 17P2 were found in the sub-fractions 17P2_III and 17P2_IV in mixture with other constituents (not shown). Compounds \u003cb\u003e5\u003c/b\u003e and \u003cb\u003e6\u003c/b\u003e present in fractions 17P3 and 17P4 were tentatively annotated as penicillic acid (\u003cem\u003em/z\u003c/em\u003e 171.12 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e) and preussin (\u003cem\u003em/z\u003c/em\u003e 318.27 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e) based on UHPLC-MS, respectively. These annotations were confirmed by 1D and 2D NMR analysis of fraction 17P4, with the detected signals aligned well with previously reported NMR data for compound \u003cb\u003e5\u003c/b\u003e [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] and \u003cb\u003e6\u003c/b\u003e [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Since compound \u003cb\u003e6\u003c/b\u003e was in a mixture, its absolute configuration could not be determined.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab10\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 10\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eStructural annotation of detected compounds in fractions 17P1-17P4 and 17P2_II.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePeak no.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRT\u003c/p\u003e\u003cp\u003e[min]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eUV max [nm]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003em/z\u003c/em\u003e\u003c/p\u003e\u003cp\u003e[pos]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003em/z\u003c/em\u003e\u003c/p\u003e\u003cp\u003e[neg]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eStructure annotation\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003en. d.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003en. d.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e499.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eunknown\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003en. d.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003en. d.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e499.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eunknown\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e316\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e697.5 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 719.5 [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e, 735.5 [M\u0026thinsp;+\u0026thinsp;K]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003en. d.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAspergillic acid aluminium complex\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e309\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e726.5 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 748.5 [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e, 764.5 [M\u0026thinsp;+\u0026thinsp;K]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003en. d.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAspergillic acid iron complex\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003en. d.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e371.13 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003en. d.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePenicillic acid*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003en. d.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e318.27 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003en. d.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePreussin*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003en. d., not detected; *, Identified by NMR.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e\u003cem\u003eAspergillus westerdijkiae\u003c/em\u003e P17, isolated from \u003cem\u003eBetula pendula\u003c/em\u003e, demonstrates strong potential as a sustainable source of multifunctional fungal pigments. Fraction 17P2 exhibited notable antimicrobial, antioxidant, anticancer, neuroprotective, and metabolic regulatory activities. Structural analysis revealed key bioactive metabolites, and pigment production was significantly enhanced by gamma irradiation without affecting fungal viability. These findings highlight the strain's promise for pharmaceutical and industrial applications of natural biopigments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAbirami Baskaran(Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing), Andreas Wasilewicz, Judith M. Rollinger\u003csup\u003e,\u003c/sup\u003e Joanna Grzelczyk\u003csup\u003e,\u003c/sup\u003e Ilona Gałązka-Czarnecka, Grażyna Budryn, Tomasz Strzała, Tyler N. Graf, Nicholas H. Oberlies (Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing \u0026ndash; review \u0026amp; editing), Filip Boratyński (Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Software, Supervision, Validation, Visualization, Writing \u0026ndash; review \u0026amp; editing), El-Sayed R. El-Sayed (Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research is part of project No. 2021/43/P/NZ9/02 241 co-funded by the National Science Centre and the European Union Framework Programme for Research and Innovation Horizon 2020 under the Marie Skłodowska-Curie grant agreement no. 945339.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe publication was prepared within the project PROM \u0026ndash; short-term academic exchange (project no. BPI/PRO/2024/00001/U/00001), financed by the National Agency for Academic Exchange (NAWA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdherence to national and international regulations\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNHO and TNJ are members of the Scientific Advisory Board of Clue Genetics, Inc. NHO is also a member of the Scientific Advisory Boards of Mycosynthetix, Inc. and Ionic Pharmaceuticals, LLC.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMeruvu H, dos Santos JC. Colors of Life: A Review on Fungal Pigments. Crit Rev Biotechnol. 2021;41(8):1153\u0026ndash;77. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/07388551.2021.1901647\u003c/span\u003e\u003cspan address=\"10.1080/07388551.2021.1901647\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eElkhateeb W, Daba GF, Pigments. Their Diversity, Chemistry, Food and Non-Food Applications. Appl Microbiol. 2023;3(3):735\u0026ndash;51. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/applmicrobiol3030051\u003c/span\u003e\u003cspan address=\"10.3390/applmicrobiol3030051\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eToma MA, Rahman MH, Rahman MS, Arif M, Hussain N, Dufoss\u0026eacute; L. Fungal Pigments: Carotenoids, Riboflavin, and Polyketides with Diverse Applications. J Fungi. 2023;9(4):454\u0026ndash;454. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/jof9040454\u003c/span\u003e\u003cspan address=\"10.3390/jof9040454\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGmoser R, Ferreira JA, Lennartsson PR, Taherzadeh MJ. Filamentous Ascomycetes Fungi as a Source of Natural Pigments. Fungal Biology Biotechnol. 2017;4(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s40694-017-0033-2\u003c/span\u003e\u003cspan address=\"10.1186/s40694-017-0033-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVenil CK, Velmurugan P, Dufoss\u0026eacute; L, Renuka Devi P, Veera Ravi A. Fungal Pigments: Potential Coloring Compounds for Wide Ranging Applications in Textile Dyeing. J Fungi. 2020;6(2):68. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/jof6020068\u003c/span\u003e\u003cspan address=\"10.3390/jof6020068\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLin L, Xu J. Production of Fungal Pigments: Molecular Processes and Their Applications. J Fungi. 2022;9(1):44. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/jof9010044\u003c/span\u003e\u003cspan address=\"10.3390/jof9010044\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGomes DC. Fungal Pigments: Applications and Their Medicinal Potential. Fungi Bioactive Metabolites: Integration of Pharmaceutical Applications. Springer Nature Singapore; 2024. pp. 651\u0026ndash;81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-981-99-5696-8_21\u003c/span\u003e\u003cspan address=\"10.1007/978-981-99-5696-8_21\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLin L, Xu JF. Pigments and Their Roles Associated with Human Health. J Fungi. 2020;6(4):280. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/jof6040280\u003c/span\u003e\u003cspan address=\"10.3390/jof6040280\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVenugopalan A, Srivastava S. Endophytes as in Vitro Production Platforms of High Value Plant Secondary Metabolites. Biotechnol Adv. 2015;33(6):873\u0026ndash;87. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biotechadv.2015.07.004\u003c/span\u003e\u003cspan address=\"10.1016/j.biotechadv.2015.07.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGakuubi MM, Munusamy M, Liang Z-X, Ng SB. Fungal Endophytes: A Promising Frontier for Discovery of Novel Bioactive Compounds. J Fungi. 2021;7(10):786. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/jof7100786\u003c/span\u003e\u003cspan address=\"10.3390/jof7100786\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRai N, Kumari Keshri P, Verma A, Kamble SC, Mishra P, Barik S, Kumar Singh S, Gautam V. Plant Associated Fungal Endophytes as a Source of Natural Bioactive Compounds. Mycology. 2021;12(3):139\u0026ndash;59. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/21501203.2020.1870579\u003c/span\u003e\u003cspan address=\"10.1080/21501203.2020.1870579\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDevi R, Verma R, Dhalaria R, Kumar A, Kumar D, Puri S, Thakur M, Chauhan S, Chauhan PP, Nepovimova E, Kuca K. A Systematic Review on Endophytic Fungi and Its Role in the Commercial Applications. Planta. 2023;257(4). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00425-023-04087-2\u003c/span\u003e\u003cspan address=\"10.1007/s00425-023-04087-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAdeleke B, Babalola O. Pharmacological Potential of Fungal Endophytes Associated with Medicinal Plants: A Review. J Fungi. 2021;7(2):147. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/jof7020147\u003c/span\u003e\u003cspan address=\"10.3390/jof7020147\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGupta A, Meshram V, Gupta M, Goyal S, Qureshi KA, Jaremko M, Shukla KK. Fungal Endophytes: Microfactories of Novel Bioactive Compounds with Therapeutic Interventions; a Comprehensive Review on the Biotechnological Developments in the Field of Fungal Endophytic Biology over the Last Decade. Biomolecules. 2023;13(7):1038\u0026ndash;1038. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/biom13071038\u003c/span\u003e\u003cspan address=\"10.3390/biom13071038\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShaban A, Zakaria Z, Abdelhamid IA, Elhakim HKA, El-Sayed E-SR. Novel Myco-Fabrication of Copper and Nickel Nanoparticles and Evaluation of Their Effects against Antibiotic Resistance Genes in Different Bacterial Strains and Anticancer Potentials. Process Biochem. 2024;149:192\u0026ndash;203. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.procbio.2024.12.008\u003c/span\u003e\u003cspan address=\"10.1016/j.procbio.2024.12.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbdelkader DH, Negm WA, Elekhnawy E, Eliwa D, Aldosari BN, Almurshedi AS. Zinc Oxide Nanoparticles as Potential Delivery Carrier: Green Synthesis by Aspergillus Niger Endophytic Fungus, Characterization, and in Vitro/in Vivo Antibacterial Activity. Pharmaceuticals. 2022;15(9):1057. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ph15091057\u003c/span\u003e\u003cspan address=\"10.3390/ph15091057\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePanwar A, Manna S, Sahini G, Kaushik V, Kumar M, Govarthanan M. The Legacy of Endophytes for the Formation of Bioactive Agents, Pigments, Biofertilizers, Nanoparticles and Bioremediation of Environment. World J Microbiol Biotechnol. 2025;41(2). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11274-025-04265-2\u003c/span\u003e\u003cspan address=\"10.1007/s11274-025-04265-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEl-Sayed E-SR, Zaki AG, Ahmed AS, Ismaiel AA. Production of the Anticancer Drug Taxol by the Endophytic Fungus Epicoccum Nigrum TXB502: Enhanced Production by Gamma Irradiation Mutagenesis and Immobilization Technique. Appl Microbiol Biotechnol. 2020;104(16):6991\u0026ndash;7003. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00253-020-10712-x\u003c/span\u003e\u003cspan address=\"10.1007/s00253-020-10712-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWouter DC, Rademakers R. NanoPack2: Population Scale Evaluation of Long-Read Sequencing Data. Bioinformatics. 2023;39(5). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/bioinformatics/btad311\u003c/span\u003e\u003cspan address=\"10.1093/bioinformatics/btad311\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSilva GG, Dutilh BE, Matthews T, Elkins K, Schmieder R, Dinsdale EA, Edwards RA. Combining de Novo and Reference-Guided Assembly with Scaffold_builder. Source Code Biol Med. 2013;8(1):23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1751-0473-8-23\u003c/span\u003e\u003cspan address=\"10.1186/1751-0473-8-23\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRonquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, H\u0026ouml;hna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. MrBayes 3.2: Efficient Bayesian Phylogenetic Inference and Model Choice across a Large Model Space. Syst Biol. 2012;61(3):539\u0026ndash;42. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/sysbio/sys029\u003c/span\u003e\u003cspan address=\"10.1093/sysbio/sys029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMagaldi S, Mata-Essayag S, Hartung de Capriles C, Perez C, Colella MT, Olaizola C, Ontiveros Y. Well Diffusion for Antifungal Susceptibility Testing. Int J Infect Dis. 2004;8(1):39\u0026ndash;45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijid.2003.03.002\u003c/span\u003e\u003cspan address=\"10.1016/j.ijid.2003.03.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEbrahimzadeh MA, Nabavi SM, Nabavi SF, Bahramian F, Bekhradnia AR. Antioxidant and Free Radical Scavenging Activity of H. Officinalis L. Var. Angustifolius, v. Odorata, B. Hyrcana and C. Speciosum. Pak J Pharm Sci. 2010;23(1):29\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evan de Loosdrecht AA, Beelen RHJ, Ossenkoppele GJ, Broekhoven MG, Langenhuijsen MMAC. A Tetrazolium-Based Colorimetric MTT Assay to Quantitate Human Monocyte Mediated Cytotoxicity against Leukemic Cells from Cell Lines and Patients with Acute Myeloid Leukemia. J Immunol Methods. 1994;174(1\u0026ndash;2):311\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0022-1759(94)90034-5\u003c/span\u003e\u003cspan address=\"10.1016/0022-1759(94)90034-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGrzelczyk J, Gałązka-Czarnecka I, Oracz J. Potentiality Assessment of the Acetylcholinesterase-Inhibitory Activity of Olive Oil with an Additive Edible Insect Powder. Molecules. 2023;28(14):5535\u0026ndash;5535. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules28145535\u003c/span\u003e\u003cspan address=\"10.3390/molecules28145535\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGach J, Grzelczyk J, Strzała T, Boratyński F, Olejniczak T. Microbial Metabolites of 3-n-Butylphthalide as Monoamine Oxidase a Inhibitors. Int J Mol Sci. 2023;24(13):10605\u0026ndash;10605. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms241310605\u003c/span\u003e\u003cspan address=\"10.3390/ijms241310605\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGrzelczyk J, Budryn G, P\u0026eacute;rez-S\u0026aacute;nchez H. Evaluation of Affinity of Bioactive Isolates from Various Coffee Extracts through Binding with PPAR-γ with the Use of Isothermal Titration Calorimetry and Docking Simulation to Prevent Antidiabetic Effects. J Therm Anal Calorim. 2020;142(2):877\u0026ndash;87. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10973-020-09801-w\u003c/span\u003e\u003cspan address=\"10.1007/s10973-020-09801-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePaguigan ND, El-Elimat T, Kao D, Raja HA, Pearce CJ, Oberlies NH. Enhanced Dereplication of Fungal Cultures via Use of Mass Defect Filtering. J Antibiot 2017, \u003cem\u003e70\u003c/em\u003e (5), 553\u0026ndash;61. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ja.2016.145\u003c/span\u003e\u003cspan address=\"10.1038/ja.2016.145\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEl-Elimat T, Figueroa M, Ehrmann BM, Cech NB, Pearce CJ, Oberlies NH, High-Resolution MS. MS/MS, and UV Database of Fungal Secondary Metabolites as a Dereplication Protocol for Bioactive Natural Products. J Nat Prod. 2013;76(9):1709\u0026ndash;16. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/np4004307\u003c/span\u003e\u003cspan address=\"10.1021/np4004307\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSamson RA, Hoekstra ES, Frisvad JC. \u003cem\u003eIntroduction to Food and Airborne Fungi. 7th Edition, Centraalbureau voor Schimmelcultures, Utrecht. - References - Scientific Research Publishing\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003c/span\u003e\u003cspan address=\"http://www.scirp.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. https://www.scirp.org/reference/ReferencesPapers?ReferenceID=1444676\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVisagie CM, Varga J, Houbraken J, Meijer M, Kocsub\u0026eacute; S, Yilmaz N, Fotedar R, Seifert KA, Frisvad JC, Samson RA. Ochratoxin Production and Taxonomy of the Yellow Aspergilli (Aspergillus Section Circumdati). Stud Mycol. 2014;78:1\u0026ndash;61. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.simyco.2014.07.001\u003c/span\u003e\u003cspan address=\"10.1016/j.simyco.2014.07.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSaha P, Talukdar AD, Choudhury MD, Nath D. Bioprospecting for Fungal-Endophyte-Derived Natural Products for Drug Discovery. In: Singh B, editor. Advances in Endophytic Fungal Research. Springer International Publishing; 2019. pp. 35\u0026ndash;49. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-3-030-03589-1_3\u003c/span\u003e\u003cspan address=\"10.1007/978-3-030-03589-1_3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEl-Sayed ESR, Baskaran A, Pomarańska O, Mykhailova D, Dunal A, Dudek A, Satam S, Strzała T, Łyczko J, Olejniczak T, Boratyński F. Bioprospecting Endophytic Fungi of Forest Plants for Bioactive Metabolites with Antibacterial, Antifungal, and Antioxidant Potentials. Molecules. 2024;29(19):4746\u0026ndash;4746. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules29194746\u003c/span\u003e\u003cspan address=\"10.3390/molecules29194746\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKumar V, Prasher IB. Phytochemical Analysis and Antioxidant Activity of Endophytic Fungi Isolated from Dillenia Indica Linn. Appl Biochem Biotechnol. 2024;196:332\u0026ndash;49. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12010-023-04498-7\u003c/span\u003e\u003cspan address=\"10.1007/s12010-023-04498-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHridoy M, Gorapi MZH, Noor S, Chowdhury NS, Rahman MM, Muscari I, Masia F, Adorisio S, Delfino DV, Mazid. Md. A. Putative Anticancer Compounds from Plant-Derived Endophytic Fungi: A Review. Molecules. 2022;27(1):296. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules27010296\u003c/span\u003e\u003cspan address=\"10.3390/molecules27010296\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBastos M, Abian O, Johnson CM, Ferreira-da-Silva F, Vega S, Jimenez-Alesanco A, Ortega-Alarcon D, Velazquez-Campoy A. Isothermal Titration Calorimetry. Nat Reviews Methods Primers. 2023;3(1):1\u0026ndash;23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s43586-023-00199-x\u003c/span\u003e\u003cspan address=\"10.1038/s43586-023-00199-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang TY, Ji H, Everton D, Le ATH, Krylova SM, Fournier R, Krylov S. N. Fundamental Determinants of the Accuracy of Equilibrium Constants for Affinity Complexes. Anal Chem. 2023;95(42):15826\u0026ndash;32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.analchem.3c03557\u003c/span\u003e\u003cspan address=\"10.1021/acs.analchem.3c03557\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang TY, Latimer J, Jean-Luc R, Kogan I, Krylova SM, Schreiber S, Kohlmann P, Jose J, Krylov S. N. A Practical Approach to Quantitatively Assessing Equilibrium-Constant Accuracy from a Single Binding Isotherm. Precision Chem. 2024;3(2):89\u0026ndash;104. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/prechem.4c00085\u003c/span\u003e\u003cspan address=\"10.1021/prechem.4c00085\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWeinh\u0026auml;upl K, Meuret L, Desrat S, Roussi F, Morellet N, Beaupierre S, Guillou C, van Heijenoort C, Abian O, Vega S, Wolf I, Akopian T, Krandor O, Rubin E, Velazquez-Campoy A, Gauto D, Fraga H. Identification of New ClpC1-NTD Binders for Mycobacterium Tuberculosis Drug Development. Sci Rep. 2025;15(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-025-87535-1\u003c/span\u003e\u003cspan address=\"10.1038/s41598-025-87535-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSingh B, Bhagat J, Chadha B, Kaur A. Cholinesterase Inhibitory Potential of Different Alternaria Spp. And Their Phylogenetic Relationships. \u003cem\u003eBiologia\u003c/em\u003e 2014, \u003cem\u003e69\u003c/em\u003e (1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2478/s11756-013-0294-z\u003c/span\u003e\u003cspan address=\"10.2478/s11756-013-0294-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEl-Sayed ESR, Grzelczyk J, Strzała T, Gałązka-Czarnecka I, Budryn G, Boratyński F. Bioprospecting Endophytic Fungi of Forest Plants for Their Monoamine Oxidase a and Cholinesterases Inhibitors, and Peroxisome Proliferator-Activated Receptor Gamma Agonists. J Appl Microbiol. 2025;136(3). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jambio/lxaf034\u003c/span\u003e\u003cspan address=\"10.1093/jambio/lxaf034\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJeong G-S, Kang M-G, Han S-A, Noh J-I, Park J-E, Nam S-J, Park D, Yee S-T, Kim H. Selective Inhibition of Human Monoamine Oxidase B by 5-Hydroxy-2-Methyl-Chroman-4-One Isolated from an Endogenous Lichen Fungus Daldinia Fissa. J Fungi. 2021;7(2):84\u0026ndash;84. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/jof7020084\u003c/span\u003e\u003cspan address=\"10.3390/jof7020084\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang C, Xiong J, Guan H-D, Wang C-H, Lei X, Hu J-F, Discovery. Synthesis, Biological Evaluation and Molecular Docking Study of (R)-5-Methylmellein and Its Analogs as Selective Monoamine Oxidase a Inhibitors. Bioorg Med Chem. 2019;27(10):2027\u0026ndash;40. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bmc.2019.03.060\u003c/span\u003e\u003cspan address=\"10.1016/j.bmc.2019.03.060\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi D, Luo X, Ying W, La Kim E, Hong J, Lee J, Jung JH. Peroxisome Proliferator Activated Receptor-γ Agonistic Compounds from the Jellyfish‐Derived Fungus \u003cem\u003eCladosporium Oxysporum\u003c/em\u003e. Chem Biodivers. 2023;20(9). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/cbdv.202300851\u003c/span\u003e\u003cspan address=\"10.1002/cbdv.202300851\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee D, Lee JH, Cai XF, Shin JC, Lee K, Hong Y-S, Lee JJ. Fungal Metabolites, Sorbicillinoid Polyketides and Their Effects on the Activation of Peroxisome Proliferator-Activated Receptor γ. J Antibiot. 2005;58(10):615\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ja.2005.84\u003c/span\u003e\u003cspan address=\"10.1038/ja.2005.84\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEl-Sayed ESR, Mousa SA, Strzała T, Boratyński F. Enhancing Bioprocessing of Red Pigment from Immobilized Culture of Gamma Rays Mutant of the Endophytic Fungus Monascus Ruber SRZ112. J Biol Eng. 2024;18(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13036-024-00439-y\u003c/span\u003e\u003cspan address=\"10.1186/s13036-024-00439-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eParekh S, Vinci VA, Strobel RJ. Improvement of Microbial Strains and Fermentation Processes. Appl Microbiol Biotechnol. 2000;54(3):287\u0026ndash;301. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s002530000403\u003c/span\u003e\u003cspan address=\"10.1007/s002530000403\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThacker J. Repair of Ionizing Radiation Damage in Mammalian Cells. Alternative Pathways and Their Fidelity. \u003cem\u003eComptes Rendus de l\u0026rsquo;Acad\u0026eacute;mie des Sciences - Series III - Sciences de la Vie\u003c/em\u003e 2000, \u003cem\u003e322\u003c/em\u003e (2\u0026ndash;3), 103\u0026ndash;108. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0764-4469(99)80030-4\u003c/span\u003e\u003cspan address=\"10.1016/S0764-4469(99)80030-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChopra VL, Mutagenesis. Investigating the Process and Processing the Outcome for Crop Improvement. Curr Sci. 2005;89(2):353\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2307/24110583\u003c/span\u003e\u003cspan address=\"10.2307/24110583\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKonar A, Datta S. Strain Improvement of Microbes. In \u003cem\u003eIndustrial Microbiology and Biotechnology\u003c/em\u003e; Verma, P., Ed.; Springer Singapore, 2022; pp. 169\u0026ndash;193. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-981-16-5214-1_6\u003c/span\u003e\u003cspan address=\"10.1007/978-981-16-5214-1_6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIsmaiel AA, Ahmed AS, El-Sayed ER. Immobilization Technique for Enhanced Production of the Immunosuppressant Mycophenolic Acid by Ultraviolet and Gamma-Irradiated Penicillium Roqueforti. J Appl Microbiol. 2015;119(1):112\u0026ndash;26. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jam.12828\u003c/span\u003e\u003cspan address=\"10.1111/jam.12828\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEl-Sayed ESR, El-Sayyad GS, Abdel-Fatah SS, El-Batal AI, Boratyński F. Novel Nanoconjugates of Metal Oxides and Natural Red Pigment from the Endophyte Monascus Ruber Using Solid-State Fermentation. Microb Cell Fact. 2024;23(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12934-024-02533-8\u003c/span\u003e\u003cspan address=\"10.1186/s12934-024-02533-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZaki AG, El-Sayed E-SR. New and Potent Production Platform of the Acetylcholinesterase Inhibitor Huperzine a by Gamma-Irradiated Alternaria Brassicae under Solid-State Fermentation. Appl Microbiol Biotechnol. 2021;105(23):8869\u0026ndash;80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00253-021-11678-0\u003c/span\u003e\u003cspan address=\"10.1007/s00253-021-11678-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHazaa MA, Shebl MM, El-Sayed E-SR, Mahmoud SR, Khattab AA, Amer MM. Bioprospecting Endophytic Fungi for Antifeedants and Larvicides and Their Enhancement by Gamma Irradiation. AMB Express. 2022;12(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13568-022-01461-3\u003c/span\u003e\u003cspan address=\"10.1186/s13568-022-01461-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBleisch R, Freitag L, Ihadjadene Y, Sprenger U, Steingr\u0026ouml;wer J, Walther T, Krujatz F. Strain Development in Microalgal Biotechnology\u0026mdash;Random Mutagenesis Techniques. Life (2075 \u0026ndash; 1729). 2022;12(7):961\u0026ndash;961. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/life12070961\u003c/span\u003e\u003cspan address=\"10.3390/life12070961\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAssante G, Camarda L, Locci R, Merlini L, Nasini G, Papadopoulos E. Isolation and Structure of Red Pigments from Aspergillus Flavus and Related Species, Grown on a Differential Medium. J Agric Food Chem. 1981;29(4):785\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jf00106a023\u003c/span\u003e\u003cspan address=\"10.1021/jf00106a023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVansteelandt M, Blanchet E, Egorov M, Petit F, Toupet L, Bondon A, Monteau F, Bizec BL, Thomas OP, Pouchus YF, Bot RL, Grovel O. Ligerin, an Antiproliferative Chlorinated Sesquiterpenoid from a Marine-Derived \u003cem\u003ePenicillium\u003c/em\u003e Strain. J Nat Prod. 2013;76(2):297\u0026ndash;301. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/np3007364\u003c/span\u003e\u003cspan address=\"10.1021/np3007364\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePak CS, Lee GH. Total Synthesis of (+)-Preussin, a Novel Antifungal Agent. J Org Chem. 1991;56(3):1128\u0026ndash;33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jo00003a040\u003c/span\u003e\u003cspan address=\"10.1021/jo00003a040\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGu B-B, Jiao F-R, Wu W, Jiao W, Li L, Sun F, Wang S-P, Yang F, Lin H-W. Preussins with Inhibition of IL-6 Expression from \u003cem\u003eAspergillus Flocculosus\u003c/em\u003e 16D-1, a Fungus Isolated from the Marine Sponge \u003cem\u003ePhakellia Fusca\u003c/em\u003e. J Nat Prod. 2018;81(10):2275\u0026ndash;81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.jnatprod.8b00662\u003c/span\u003e\u003cspan address=\"10.1021/acs.jnatprod.8b00662\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Natural Pigments, Monoamine Oxidase, Cholinesterase Inhibitors, PPAR-γ, Fungal Endophytes","lastPublishedDoi":"10.21203/rs.3.rs-7546670/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7546670/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe growing consumer preference for natural and sustainable products has heightened interest in biopigments across pharmaceutical, cosmetic, and food industries. In this study, we investigate endophytic fungi as a viable and eco-friendly source for the production of bioactive natural pigments.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eA promising strain, \u003cem\u003eAspergillus westerdijkiae\u003c/em\u003e P17, was isolated from \u003cem\u003eBetula pendula\u003c/em\u003e and assessed for its pigment-producing potential and associated bioactivities. The cell-free culture extract was fractionated, and the resulting components were evaluated for antimicrobial, antioxidant, anticancer, neuroprotective, and peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist activities. Among the fractions, 17P2 exhibited broad-spectrum antimicrobial effects, notable antioxidant activity (83% DPPH radical scavenging at 1000 mg/mL), and cytotoxicity against MCF-7 and HepG2 cancer cell lines, with IC₅₀ values of 250 mg/mL. Isothermal titration calorimetry (ITC) demonstrated strong binding affinities of 17P2 to acetylcholinesterase (Kd\u0026thinsp;=\u0026thinsp;1.63 \u0026micro;M) and butyrylcholinesterase (Kd\u0026thinsp;=\u0026thinsp;0.03 \u0026micro;M), indicating potential anti-Alzheimer\u0026rsquo;s properties. Additionally, significant interactions with monoamine oxidase A and PPAR-γ suggest possible antidepressant and antidiabetic applications. Four major pigment fractions (17P1\u0026ndash;17P4) were purified and structurally characterized using UHPLC-MS and NMR, revealing key metabolites such as aluminium and iron aspergillic acid complexes, penicillic acid, and preussin. Notably, gamma irradiation at 2000 Gy significantly enhanced pigment yield without compromising fungal viability.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eCollectively, these findings position \u003cem\u003eA. westerdijkiae\u003c/em\u003e P17 as a valuable and versatile biotechnological resource for the sustainable production of multifunctional fungal pigments with potential industrial and therapeutic applications.\u003c/p\u003e","manuscriptTitle":"Natural Pigments from the Endophyte Aspergillus westerdijkiae and Evaluation of their Bioactivities","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-09 10:06:08","doi":"10.21203/rs.3.rs-7546670/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-11T18:29:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-25T11:56:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-23T07:35:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"265687134365932343810748321392407160094","date":"2025-10-20T00:46:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"312512733072333677144668132658156557000","date":"2025-10-15T02:44:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"331015600408453187868215456271631221395","date":"2025-10-14T22:31:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"309851631478936907050070389439479179047","date":"2025-10-14T21:19:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-14T20:26:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-08T01:11:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-08T01:10:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Cell Factories","date":"2025-09-05T18:48:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3625dd0a-8eec-45ac-8577-111012e510a3","owner":[],"postedDate":"September 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-05T16:01:10+00:00","versionOfRecord":{"articleIdentity":"rs-7546670","link":"https://doi.org/10.1186/s12934-025-02905-8","journal":{"identity":"microbial-cell-factories","isVorOnly":false,"title":"Microbial Cell Factories"},"publishedOn":"2025-12-31 15:58:01","publishedOnDateReadable":"December 31st, 2025"},"versionCreatedAt":"2025-09-09 10:06:08","video":"","vorDoi":"10.1186/s12934-025-02905-8","vorDoiUrl":"https://doi.org/10.1186/s12934-025-02905-8","workflowStages":[]},"version":"v1","identity":"rs-7546670","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7546670","identity":"rs-7546670","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}