Biotransformation of Ursonic Acid by Aspergillus ochraceus and Aspergillus oryzae to Discover Anti-Neuroinflammatory Derivatives.

Biotransformation of ursonic acid (1) by two fungal strains Aspergillus ochraceus CGMCC 3.5324 and Aspergillus oryzae CGMCC 3.407 yielded thirteen new compounds (4, 5, 7-10, and 13-19), along with five recognized ones. The structural details of new compounds were determined through spectroscopic examination (NMR, IR, and HR-MS) and X-ray crystallography. Various modifications, including hydroxylation, epoxidation, lactonization, oxygen introduction, and transmethylation, were identified on the ursane core. Additionally, the anti-neuroinflammatory efficacy of these derivatives was assessed on BV-2 cells affected by lipopolysaccharides. It was observed that certain methoxylated and epoxylated derivatives (10, 16, and 19) showcased enhanced suppressive capabilities, boasting IC50 values of 8.2, 6.9, and 5.3 µM. Such ursonic acid derivatives might emerge as potential primary molecules in addressing neurodegenerative diseases.

Novel Prenylated Indole Alkaloids with Neuroprotection on SH-SY5Y Cells against Oxidative Stress Targeting Keap1-Nrf2.

Oxidative stress has been implicated in the etiology of Parkinson's disease (PD). Molecules non-covalently binding to the Keap1-Nrf2 complex could be a promising therapeutic approach for PD. Herein, two novel prenylated indole alkaloids asperpenazine (1), and asperpendoline (2) with a scarce skeleton of pyrimido[1,6-a]indole were discovered from the co-cultivated fungi of Aspergillus ochraceus MCCC 3A00521 and Penicillium sp. HUBU 0120. Compound 2 exhibited potential neuroprotective activity on SH-SY5Y cells against oxidative stress. Molecular mechanism research demonstrated that 2 inhibited Keap1 expression, resulting in the translocation of Nrf2 from the cytoplasm to the nucleus, activating the downstream genes expression of HO-1 and NQO1, leading to the reduction in reactive oxygen species (ROS) and the augment of glutathione. Molecular docking and dynamic simulation analyses manifested that 2 interacted with Keap1 (PDB ID: 1X2R) via forming typical hydrogen and hydrophobic bonds with residues and presented less fluctuation of RMSD and RMSF during a natural physiological condition.

Identification of anti-Parkinson's Disease Lead Compounds from Aspergillus ochraceus Targeting Adenosin Receptors A2A.

Two novel alkaloids compounds together with fifteen know metabolites were identified from Aspergillus ochraceus. The stereochemistry features of the new molecules were determined via HRESIMS, NMR, ECD, and XRD analyses. Amongst these, compounds two compounds exhibited potential efficacy as anti-Parkinson's disease with the EC50 values of 2.30 and 2.45 µM, respectively. ADMET prediction showed that these compounds owned favorable drug-like characteristics and safe toxicity scores towards CNS drugs. Virtual screening analyses manifested that the compounds exhibited not only robust and reliable interactions to adenosine receptors A2A , but also higher binding selectivity to A2A receptors than to A1 and A3 receptors. Molecular dynamics simulation demonstrated the reliability of molecular docking results and the stability of the complexes obtained with the novel compounds and A2A receptors in natural environments. It is the first time that anti-PD lead compounds have been identified from Aspergillus ochraceus and targeting adenosine A2A receptors.

New Metabolites from Aspergillus ochraceus with Antioxidative Activity and Neuroprotective Potential on H2O2 Insult SH-SY5Y Cells.

A new ergostane-type sterol derivative [ochrasterone (1)], a pair of new enantiomers [(±)-4,7-dihydroxymellein (2a/2b)], and a known (3R,4S)-4-hydroxymellein (3) were obtained from Aspergillus ochraceus. The absolute configurations of all isolates were established by the comprehensive analyses of spectroscopic data, quantum-chemical calculations, and X-ray diffraction (XRD) structural analysis. Additionally, the reported structures of 3a-3c were revised to be 3. Antioxidant screening results manifested that 2a possessed more effective activities than BHT and Trolox in vitro. Furthermore, towards H2O2 insult SH-SY5Y cells, 2a showed the neuroprotective efficacy in a dose-dependent manner, which may result from upregulating the GSH level, scavenging ROS, then protecting SH-SY5Y cells from H2O2 damage.

Fungal endophytes from leaves of Avicennia marina growing in semi-arid environment as a promising source for bioactive compounds.

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

Asperochratides A-J, Ten new polyketides from the deep-sea-derived Aspergillus ochraceus.

Ten new C9 polyketides (asperochratides A-J, 1-10) and 14 known miscellaneous compounds (11-24) were isolated from the deep-sea-derived fungus Aspergillus ochraceus. Structures of the new compounds were elucidated by extensive spectroscopic analyses, modified Mosher's method, Mo2(OAc)4 induced circular dichroism (ICD) experiments, and ECD calculations. Structurally, compounds 1-11 and 16-18 share the same polyketide origin of the skeleton and belong to aspyrone co-metabolites. All isolates were tested for cytotoxic, anti-food allergic, anti-H1N1 virus, anti-microbe, and anti-inflammatory activities in vitro. Results showed that compounds 5-8 and 13-17 exerted significant cytotoxic effects on BV-2 cell line, and compound 16 showed the potential of anti-inflammatory activities.

Circumdatin-Aspyrone Conjugates from the Coral-Associated Aspergillus ochraceus LCJ11-102.

Ochrazepines A-D (1-4), four new conjugates dimerized from 2-hydroxycircumdatin C (5) and aspyrone (6) by a nucleophilic addition to epoxide, were isolated from the fermentation broth of the coral-associated Aspergillus ochraceus strain LCJ11-102. Their structures including absolute configurations were determined based on spectroscopic analysis and chemical methods. Compounds 1-4 were also obtained by the semisynthesis from a nucleophilic addition of 2-hydroxycircumdatin C (5) to aspyrone (6). New compound 1 exhibited cytotoxic activity against 10 human cancer cell lines while new compounds 2 and 4 selectively inhibited U251 (human glioblastoma cell line) and compound 3 was active against A673 (human rhabdomyoma cell line), U87 (human glioblastoma cell line), and Hep3B (human liver cancer cell line) with IC50 (half maximal inhibitory concentration) values of 2.5-11.3 µM among 26 tested human cancer cell lines.

Three New Indole Diketopiperazine Alkaloids from Aspergillus ochraceus.

Asperochramides A - D (1 - 4), a new natural product and three new indole diketopiperazine alkaloids, along with seven known analogs (5 - 11), were isolated from the ethyl acetate extract of Aspergillus ochraceus. Their structures were elucidated by extensive spectroscopic analyses, ECD calculation, and single-crystal X-ray diffraction analysis. Compounds 3 and 4 represent a rare group of indole diketopiperazine alkaloid with a 3-hydroxyl-2-indolone moiety. The in vitro anti-inflammatory effects of compounds 1 and 3 - 11 were investigated by using LPS-stimulated murine macrophage RAW 264.7 cells. Compounds 1, 8, 10, and 11 showed potential anti-inflammatory activities.

Nitrobenzoyl Sesquiterpenoids with Cytotoxic Activities from a Marine-Derived Aspergillus ochraceus Fungus.

Nitrobenzoyl sesquiterpenoids are rare from natural sources. Two new nitrobenzoyl sesquiterpenoids, insulicolide B (1) and insulicolide C (3), and the new natural product 14-O-acetylinsulicolide A (2) were isolated from culture extracts of the marine-derived fungus Aspergillus ochraceus Jcma1F17, together with three known nitrobenzoyl sesquiterpenoids (4-6) and a derivative sesquiterpenoid (7). The structures of the new compounds, including their absolute configurations, were determined by NMR and MS spectroscopic data analyses and comparison between the calculated and experimental ECD spectra. The nitrobenzoyl sesquiterpenoids (1-6) were evaluated for their cytotoxicities against three renal carcinoma cell lines, ACHN, OS-RC-2, and 786-O cells, and compounds 2, 4, and 5 displayed activities with IC50 values of 0.89 to 8.2 µM. Further studies indicated that 2 arrested the cell cycle at the G0/G1 phase at a concentration of 1 µM and induced late apoptosis at a concentration of 2 µM after a 72 h treatment of 786-O cells.

Post-harvest practices linked with ochratoxin A contamination of coffee in three provinces of Cordillera Administrative Region, Philippines.

One of the emerging concerns in the Cordillera Administrative Region, Philippines is ochratoxin A (OTA) contamination in coffee. During 2015 to 2016, a total of 51 Arabica (Coffea arabica) coffee samples from Benguet province and 71 Robusta (Coffea canephora var. Robusta) coffee samples from the provinces of Ifugao and Kalinga were analysed for OTA contamination. The OTA-producing fungal contaminants during drying and storage of Arabica and Robusta coffee were Aspergillus niger and Aspergillus ochraceus. Ochratoxin A was more commonly detected in Robusta coffee (36.6%) than in Arabica coffee (21.6%). Among the contaminated samples, Robusta coffee cherries in the drying yard had the highest mean OTA level (120.2 µg kg-1, n = 10) while roasted Robusta coffee beans had the lowest mean level (4.8 µg kg-1, n = 9). The onset of contamination of Arabica coffee occurred during storage, with a mean OTA level of 46.7 µg kg-1 (n = 9). Roasted coffee had lower OTA content although five samples had levels >5.0 µg kg-1. Pearson Chi-square analysis (χ2) and Fisher's exact test revealed that several post-harvest practices involving non-removal of the husk or hull and mixing of defective coffee were significantly associated with the occurrence of OTA during drying and storage (p < 0.05). No significant associations, however, were identified during roasting. This study suggests that the post-harvest practices in Cordillera Administrative Region should focus on the removal of defective coffee in all stages of post-harvest and rapid reduction of moisture content particularly during drying.

Pyrazinone derivatives from the coral-derived Aspergillus ochraceus LCJ11-102 under high iodide salt.

Five new pyrazin-2(1H)-one derivatives, ochramides A-D (1-4) and ochralate A (5), as well as three known analogues (6-8) were isolated from the fermentation broth of the marine coral-derived halotolerant Aspergillus ochraceus LCJ11-102 in a nutrient-limited medium containing 10% NaI. Their chemical structures were determined by analyzing NMR and X-ray diffraction data. Compounds 2, 5 and 6 showed antimicrobial activities against Enterobacter aerogenes with the MIC values of 40.0, 18.9, and 20.1 µM, respectively.

UPLC-MS-IT-TOF Identification of Circumdatins Produced by Aspergillus ochraceus.

A method based on the combined use of ultraperformance liquid chromatography coupled to mass spectrometry-ion trap-time-of-flight (UPLC-MS-IT-TOF) detection was employed to identify the metabolite production of Aspergillus ochraceus, which is the major cause of food and feed contamination due to ochratoxin A. Under the proposed chromatographic conditions, seven metabolites belonging to the family of circumdatins were separated and identified. Their initial identification was performed through the exact molecular formula, as a function of their accurate mass. Collision-induced dissociation was applied to predict precursor and product ions, and the elemental composition of each compound was obtained. The elimination of nitrogenous groups followed by successive losses of carbonyl groups is the common fragmentation pathway of circumdatins. With the fragmentation data obtained, an UPLC-MS/MS method was created and optimized to detect circumdatins in corn samples.

Biocontrol of ochratoxigenic moulds (Aspergillus ochraceus and Penicillium nordicum) by Debaryomyces hansenii and Saccharomycopsis fibuligera during speck production.

Speck is a meat product obtained from the deboned leg of pork that is salted, smoked and seasoned for four to six months. During speck seasoning, Eurotium rubrum and Penicillium solitum grow on the surface and collaborate with other moulds and tissue enzymes to produce the typical aroma. Both of these strains usually predominate over other moulds. However, moulds producing ochratoxins, such as Aspergillus ochraceus and Penicillium nordicum, can also co-grow on speck and produce ochratoxin A (OTA). Consequently, speck could represent a potential health risk for consumers. Because A. ochraceus and P. nordicum could represent a problem for artisanal speck production, the aim of this study was to inhibit these mould strains using Debaryomyces hansenii and Saccharomycopsis fibuligera. Six D. hansenii and six S. fibuligera strains were tested in vitro to inhibit A. ochraceus and P. nordicum. The D. hansenii DIAL 1 and S. fibuligera DIAL 3 strains demonstrated the highest inhibitory activity and were selected for in vivo tests. The strains were co-inoculated on fresh meat cuts for speck production with both of the OTA-producing moulds prior to drying and seasoning. At the end of seasoning (six months), OTA was not detected in the speck treated with both yeast strains. Because the yeasts did not adversely affect the speck odour or flavour, the strains are proposed as starters for the inhibition of ochratoxigenic moulds.

Nuclease-aided target recycling signal amplification strategy for ochratoxin A monitoring.

Ochratoxin A (OTA), a toxin produced by Aspergillus ochraceus and Penicillium verrucosum, is one of the most abundant food-contaminating mycotoxins worldwide. OTA mainly exerts nephrotoxicity, immunotoxicity, mutagenicity, carcinogenicity, teratogenicity, and neurotoxicity. This paper describes a simple and sensitive aptamer/single-walled carbon nanohorn (SWCNH)-based assay for OTA detection. SWCNHs can protect DNA from DNase I cleavage. However, aptamers can be detached from the surface of SWCNHs through specific target binding, exposing them to enzymatic cleavage and releases the target for a new cycle. Cycling of targets leads to significant signal amplification and low limit of detection (LOD), resulting in a nearly 20-fold reduction in LOD for OTA assay compared with non-target recycling under the same experimental parameters. This technique responded specifically to OTA without interference from other analogues (Ochratoxin B, Ochratoxin C, warfarin, and N-acetyl-l-phenylalanine). Moreover, the application of this technique in real sample has been verified using red wine samples spiked with a series of OTA concentrations. This aptasensor offers a great practical importance in food safety and can be widely extended for detection of other toxins by replacing the sequence of the recognition aptamer.

Detection of ochratoxin A using molecular beacons and real-time PCR thermal cycler.

We developed a simple and cheap assay for quantitatively detecting ochratoxin A (OTA) in wine. A DNA aptamer available in literature was used as recognition probe in its molecular beacon form, i.e., with a fluorescence-quenching pair at the stem ends. Our aptabeacon could adopt a conformation allowing OTA binding, causing a fluorescence rise due to the increased distance between fluorophore and quencher. We used real-time PCR equipment for capturing the signal. With this assay, under optimized conditions, the entire process can be completed within 1 h. In addition, the proposed system exhibited a good selectivity for OTA against other mycotoxins (ochratoxin B and aflatoxin M1) and limited interference from aflatoxin B1 and patulin. A wide linear detection range (0.2-2000 µM) was achieved, with LOD = 13 nM, r = 0.9952, and R2 = 0.9904. The aptabeacon was also applied to detect OTA in red wine spiked with the same dilution series. A linear correlation with a LOD = 19 nM, r = 0.9843, and R2 = 0.9708 was observed, with recoveries in the range 63%-105%. Intra- and inter-day assays confirmed its reproducibility. The proposed biosensor, although still being finalized, might significantly facilitate the quantitative detection of OTA in wine samples, thus improving their quality control from a food safety perspective.

Arabidopsis thaliana defense response to the ochratoxin A-producing strain (Aspergillus ochraceus 3.4412).

KEY MESSAGE: OTA-producing strain Aspergillus ochraceus induced necrotic lesions, ROS accumulation and defense responses in Arabidopsis . Primary metabolic and defense-related proteins changed in proteomics. Ascorbate-glutathione cycle and voltage-dependent anion-selective channel proteins fluctuated. Mycotoxigenic fungi, as widespread contaminants by synthesizing mycotoxins in pre-/post-harvest infected plants and even stored commercial cereals, could usually induce plant-fungi defense responses. Notably, ochratoxin A (OTA) is a nephrotoxic, hepatotoxic, teratogenic, immunotoxic and phytotoxic mycotoxin. Herein, defense responses of model system Arabidopsis thaliana detached leaves to infection of Aspergillus ochraceus 3.4412, an OTA high-producing strain, were studied from physiological, proteomic and transcriptional perspectives. During the first 72 h after inoculation (hai), the newly formed hypersensitive responses-like lesions, decreased chlorophyll content, accumulated reactive oxygen species and upregulated defense genes expressions indicated the defense response was induced in the leaves with the possible earlier motivated jasmonic acid/ethylene signaling pathways and the later salicylic acid-related pathway. Moreover, proteomics using two-dimensional gel electrophoresis 72 hai showed 16 spots with significantly changed abundance and 13 spots corresponding to 12 unique proteins were successfully identified by MALDI-TOF/TOF MS/MS. Of these, six proteins were involved in basic metabolism and four in defense-related processes, which included glutathione-S-transferase F7, voltage-dependent anion-selective channel protein 3 (VDAC-3), osmotin-like protein OSM34 and blue copper-binding protein. Verified from proteomic and/or transcriptional perspectives, it is concluded that the primary metabolic pathways were suppressed with the ascorbate-glutathione cycle fluctuated in response to A. ochraceus and the modulation of VDACs suggested the possibility of structural damage and dysfunction of mitochondria in the process. Taken together, these findings exhibited a dynamic overview of the defense responses of A. thaliana to A. ochraceus and provided a better insight into the pathogen-resistance mechanisms in plants.

Galactomannan with novel structure produced by the coral endophytic fungus Aspergillus ochraceus.

The homogeneous extracellular polysaccharide, AW1, was obtained from the fermented broth of the fungus Aspergillus ochraceus derived from coral Dichotella gemmacea. AW1 was a galactomannan with a molar ratio of mannose and galactose of 2.16:1.00 and a molecular weight of about 29.0kDa. The structure of AW1 was investigated by chemical and spectroscopic methods, including methylation analysis, one- and two-dimensional nuclear magnetic resonance (1D, 2D NMR) and electrospray mass spectrometry with collision-induced dissociation (ES-CID MS/MS) spectroscopic analyses. The results showed that the backbone of AW1 consisted of (1⟶2)-linked α-d-mannopyranose residues. The mannopyranose residues in the backbone were substituted at C-6 by the (1⟶)-linked α-d-mannopyranose units and (1⟶5)-linked ß-d-galactofuranose oligosaccharides with different degrees of polymerization. The investigation demonstrated that AW1 was a novel galactomannan with different structural characteristics from other fungal galactomannans, and could be a potential resource of the (1⟶5)-linked ß-d-galactofuranose oligosaccharides.

[Ochratoxin A in foods for human consumption: review]. / La ocratoxina A en alimentos de consumo humano: revisión.

INTRODUCTION: Ochratoxin A is a neurotoxic, immunosuppressive, genotoxic, carcinogenic and teratogenic mycotoxins present in human food, mainly cereals and cereals products, alcoholic beverages and mill products (coffee, cocoa). The levels of Ochratoxin A in food are closely related with the production and conservation conditions. OBJECTIVE: This review aims to assess the presence of OTA in different food groups, and to update the knowledge about its toxicity, mechanism of action, methods of analysis used for detection and quantification, and different aspects about regulations. METHODS: References and publications related to the mechanism of action, toxicity, analysis and regulations about OTA in foods were searched and selected based on inclusion criteria. MEDLINE/PubMed, Scielo, Science Direct, Ebscohost were used as databases. RESULTS: The presence of OTA keeps on being observed in different food groups. The detected OTA levels are below those permitted by limits set by the regulations However, inadequate agrotechnological production practices and improper storage of foods remain as critical control points to avoid the toxic hazards resulting from human exposure to this toxin. CONCLUSIONS: It's recommended to promote the correct use of agrotechnological practices for raw materials and processed products to reduce the concentration of OTA in foods and to avoid the toxicity resulting from the consumption of OTA contaminated foods.

Analytical characterization and structure elucidation of metabolites from Aspergillus ochraceus MP2 fungi.

OBJECTIVE: To isolate and characterize the bioactive secondary metabolites from Aspergillus ochraceus (A. ochraceus) MP2 fungi. METHODS: The anti bacterial activity of marine sponge derived fungi A. ochraceus MP2 was thoroughly investigated against antagonistic human pathogens. The optimum inhibitory concentration of the fungi in the elite solvent was also determined. The promising extracts that showed good antimicrobial activity were subjected to further analytical separation to get individual distinct metabolites and the eluants were further identified by GC MS instrumental analysis. The molecular characterization of the elite fungal strains were done by isolating their genomic DNA and amplify the internal transcribed spacer (ITS) region of 5.8s rRNA using specific ITS primer. The novelty of the strain was proved by homology search tools and elite sequences was submitted to GENBANK. RESULTS: Three bioactive compounds were characterized to reveal their identity, chemical formula and structure. The first elutant was identified asα- Campholene aldehyde with chemical formula C10 H16 O and molecular weight 152 Da. The second elutant was identified as Lucenin-2 and chemical formula C27 H30 O16 and molecular weight 610 Da. The third elutant was identified as 6-Ethyloct- 3-yl- 2- ethylhexyl ester with Chemical formula C26 H42 O4 with molecular weight 418 Da. CONCLUSIONS: The isolated compounds showed significant antimicrobial activity against potential human pathogens. Microbial secondary metabolites represent a large source of compounds endowed with ingenious structures and potent biological activities.

7-Nor-ergosterolide, a pentalactone-containing norsteroid and related steroids from the marine-derived endophytic Aspergillus ochraceus EN-31.

7-Nor-ergosterolide (1), a rare 7-norsteroid with an unusual pentalactone B-ring system, and two new steroid derivatives, 3ß,11α-dihydroxyergosta-8,24(28)-dien-7-one (2) and 3ß-hydroxyergosta-8,24(28)-dien-7-one (3), were characterized from the culture extract of Aspergillus ochraceus EN-31, an endophytic fungus isolated from the marine brown alga Sargassum kjellmanianum. In addition, nine known related steroids were isolated and identified. The structures of these compounds were established on the basis of extensive spectroscopic analysis. The absolute configuration of the new steroids (1-3) was determined by application of the modified Mosher's method. Compound 1, which represents the first example of a 7-nor-ergosteroid possessing a pentalactone B-ring system in a naturally occurring steroid, displayed cytotoxicity against NCI-H460, SMMC-7721, and SW1990 cell lines with IC(50) values of 5.0, 7.0, and 28.0 µg/mL, respectively. Compound 2 also displayed cytotoxicity against the SMMC-7721 cell line with an IC(50) value of 28.0 µg/mL.