Solid Rice Medium Research Articles

MOLECULAR NETWORKING-BASED DEREPLICATION OF AMBUIC ACID DERIVATIVES FROM THE MARINE FUNGUS PESTALOTIOPSIS SP. 4A11

Ambuic acid (AA) is a highly-modified cyclohexenone and known as a promising inhibitor of quorum sensing in methicillinresistant Staphylococcus aureus, and is thus a candidate as an antivirulence drug. This molecule is mainly produced by the species of Pestalotiopsis and, since its discovery twenty years ago, only a restricted amount of AA-derivatives have been described. Despite being a promising subject, methods for the analysis of modified AA-analogues via mass spectrometry remain unexplored. In order to adress this question, the marine fungus Pestalotiopsis sp. 4A11 associated with the ascidian Didemnum perlucidum was grown in a solid rice medium and its crude extract was chemically studied. From this extract, AA and 10-hydroxy ambuic acid (10-HAA) were isolated and identified using NMR spectroscopy with the aim of obtaining model compounds for the MS analysis. These served as reference compounds (seeds) to guide the dereplication of other AA-analogues via LC-MS/MS-based molecular networking. Based on the manual interpretation of the fragmentation pathways of the seeds and related compounds observed in the networks, six AA-derivatives were dereplicated in the extract. Furthermore, three analogues with unprecedented chemical formulas were proposed as putative unprecedented AA-derivatives. The fragmentation annotation proposed represents a fast and feasible method for characterizing AA-derivatives.

Induction of ambuic acid derivatives by the endophytic fungus Pestalotiopsis lespedezae through an OSMAC approach

Ten new ambuic acid derivatives, pestallic acids H–Q including one new iodinated natural product along with two known compounds, ambuic acid and ambuic acid 18-acetate, were obtained through fermentation of the endophytic fungus Pestalotiopsis lespedezae on solid rice medium with 3.5% NaI. Pestallic acids H–Q were undetectable in cultures of the fungus grown on solid rice medium lacking NaI or in those where NaI had been replaced by NaCl or NaBr. The structures of the new metabolites were established on basis of 1D/2D NMR and HRESIMS data. Their absolute configurations were determined by Mosher’s method and TDDFT-ECD calculations. The compounds failed to show antibacterial activity against S. aureus (ATCC29213), drug-resistant Acinetobacter baumannii (BAA1605) or Mycobacterium tuberculosis , as well as cytotoxicity against the mouse lymphoma cell line L5178Y. • Fermentation of Pestalotiopsis lespedezae yielded ten new ambuic acid derivatives. • One new iodinated natural product was induced. • ECD calculations were performed to determine the absolute configuration.

Polyketides from the marine-derived fungus Aspergillus falconensis: In silico and in vitro cytotoxicity studies

Fermentation of the marine-derived fungus Aspergillus falconensis, isolated from sediment collected from the Red Sea, Egypt on solid rice medium containing 3.5% NaCl yielded a new dibenzoxepin derivative (1) and a new natural isocoumarin (2) along with six known compounds (3–8). Changes in the metabolic profile of the fungus were induced by replacing NaCl with 3.5% (NH4)2SO4 that resulted in the accumulation of three further known compounds (9–11), which were not detected when the fungus was cultivated in the presence of NaCl. The structures of the new compounds were elucidated by HRESIMS and 1D/2D NMR as well as by comparison with the literature. Molecular docking was conducted for all isolated compounds on crucial enzymes involved in the formation, progression and metastasis of cancer which included human cyclin-dependent kinase 2 (CDK-2), human DNA topoisomerase II (TOP-2) and matrix metalloproteinase 13 (MMP-13). Diorcinol (7), sulochrin (9) and monochlorosulochrin (10) displayed notable stability within the active pocket of CDK-2 with free binding energy (ΔG) equals to −25.72, −25.03 and −25.37 Kcal/mol, respectively whereas sulochrin (9) exerted the highest fitting score within MMP-13 active center (ΔG = −33.83 Kcal/mol). In vitro cytotoxic assessment using MTT assay showed that sulochrin (9) exhibited cytotoxic activity versus L5178Y mouse lymphoma cells with an IC50 value of 5.1 µM and inhibition of migration of MDA-MB 231 breast cancer cells at a concentration of 70 µM.

Induction of New Lactam Derivatives From the Endophytic Fungus Aplosporella javeedii Through an OSMAC Approach.

Fermentation of the endophytic fungus Aplosporella javeedii on solid rice medium in presence of either 3.5% NaNO3 or 3.5% monosodium glutamate caused a significant change of the fungal metabolite pattern compared to fungal controls grown only on rice. Chemical investigation of the former fungal extracts yielded 11 new lactam derivatives, aplosporellins A–K (2–12), in addition to the known compound, pramanicin A (1). All of these compounds were not detected when the fungus was grown on rice medium without these activators thereby indicating the power of this OSMAC approach. The structures of the new compounds were elucidated by one- and two- dimensional NMR spectroscopy, DFT-NMR calculations and by mass spectrometry as well as by comparison with the literature whereas the absolute configuration of the lactam core was determined by TDDFT-ECD and OR calculations. Pramanicin A (1) showed strong cytotoxicity against human lymphoma (Ramos) and leukemia (Jurkat J16) cells with IC50 values of 4.7 and 4.4 μM, respectively. Mechanistic studies indicated that 1 activates caspase-3 and induces apoptotic cell death.

Allantopyrone E, a rare α-pyrone metabolite from the mangrove derived fungus Aspergillus versicolor

A new α-pyrone derivative, with a rare substitution pattern, was isolated from the solid rice media of the fungal endophyte Aspergillus versicolor associated with the fruit of the mangrove Avicennia marina. It was named allantopyrone E, after allantopyrones C and D. The aforementioned three are the only diaryl-substituted α-pyrones to be reported from nature. The unique structure was elucidated based on one- and two dimensional NMR data as well as high resolution mass spectroscopy. A biosynthetic scheme was proposed for the new compound. Furthermore, allantopyrone E showed cytotoxic effect on HeLa cells with IC50= 50.97 μM.

Cytotoxic Hydroperoxycochliodinol Derivative from Endophytic Chaetomium sp. Isolated from Salvia officinalis

The endophytic fungus Chaetomium sp. was separated from the medicinal plant Salvia officinalis growing in Egypt and cultivated on solid rice medium. Chemical investigation of the EtOAc extract yielded a new hydroperoxycochliodinol derivative (1) in addition to several known compounds, which included cochliodinol (2), isocochliodinol (3), chaetomin (4), and two simple indole derivatives (5, 6). The structure of the new compound was determined by comprehensive NMR studies including DEPT, COSY, HMQC, HMBC, and HR-MS. All compounds were tested for their cytotoxic activity against L5178Y cells. Compounds 1, 2, and 4 showed cytotoxic activity with EC50 values between 3.5–5.0 μg/mL, whereas compounds 3, 5, and 6 were inactive.

Chemical Profiles and Bio-Activities of Different Extracts of Terfezia Species and their Other Associated Fungi

Background:Desert truffles (Terfezia species) are known by their vital nutritional benefits as they are considered as rich sources of vitamins, fatty acid, minerals and proteins.Methods:The chemical constituents of the different solvent extracts of Terfezia species were isolated and identified by column chromatography, spectroscopic and GC-MS analyses. Also, the ethyl acetate and acetone extracts of different fungal isolates, associated Terfezia, after grown on rice medium were screened for their antimicrobial, anticancer and antioxidant activities via disc agar plate, micro culture tetrazolium (MTT) and 2,2-azino-di-[3-ethylbenzo-thiazolin-sulphonate] (ABTS) assays, respectively. The promising fugal strains were molecularly identified by 18SrRNA tool.Results:Bio-guided separation of methylene chloride, ethyl acetate and n-butanol fractions of Terfezia species led to identification of nine compounds namely; (R)-4,8-dihydroxy-7-hydroxymethyl-6- methoxy isochroman-1-one (1), 4-deoxy-4α-phorbal-12-(2,3-dimethyl)butyrate-13-isobutyrate (2), oxyphylline B (3), terfezien A (4), latilagascene D (5), amaiouine (6), senbusine acetate (7), terfezien B (8) and marinoquinoline D (9). Moreover, sixteen compounds were identified in the n-hexane extract via GC-MS analysis, accounting for 93.69% of the total detected components in the extract. While, twenty five components were detected in the methylene chloride extract, representing 43.86% from total detected components in the extract. Eight fungal strains were isolated from Terfezia sp., powder by serial dilution methods and these fungi were cultivated on solid rice medium. Also, their ethyl acetate and acetone extracts were subjected to biological studies including antimicrobial, antioxidant and anticancer activities. The three potent fungal strains (1M, 4M and 8M) were identified by the molecular technique 18SrRNA as Aspergillus niger 1M-EGY-IQ, Penicillium crustosum 4M-EGY-IQ, and Fusarium proliferatum 8M-EGY-IQ for 1M, 4M and 8M, respectively.Conclusion:Terfezia sp., comprise a rich source of bioactive compounds and could be considered as an interesting candidate for the treatment of infectious diseases.

Azaphilones from the Red Sea Fungus Aspergillus falconensis.

The marine-derived fungus Aspergillus falconensis, isolated from sediment collected from the Canyon at Dahab, Red Sea, yielded two new chlorinated azaphilones, falconensins O and P (1 and 2) in addition to four known azaphilone derivatives (3−6) following fermentation of the fungus on solid rice medium containing 3.5% NaCl. Replacing NaCl with 3.5% NaBr induced accumulation of three additional new azaphilones, falconensins Q−S (7−9) including two brominated derivatives (7 and 8) together with three known analogues (10−12). The structures of the new compounds were elucidated by 1D and 2D NMR spectroscopy and HRESIMS data as well as by comparison with the literature. The absolute configuration of the azaphilone derivatives was established based on single-crystal X-ray diffraction analysis of 5, comparison of NMR data and optical rotations as well as on biogenetic considerations. Compounds 1, 3−9, and 11 showed NF-κB inhibitory activity against the triple negative breast cancer cell line MDA-MB-231 with IC50 values ranging from 11.9 to 72.0 µM.

Isolation, identification and antimicrobial activity of secondary metabolites of endophytic fungi from annona leaves (Annona squamosa L.) growing in dry land

Research on isolation, identification, and antimicrobial activity of secondary metabolites of endophytic fungi from leaves of Annona squamosa growing in dryland had been undertaken. The work includes the isolation of endophytic fungi, cultivation, and extraction of fungal extract and identification of chemical metabolites together with the antibacterial test. The pure colony of endophytic fungi was grown on solid rice media using 1 L Erlenmeyer flask. The grown fungi were extracted with ethyl acetate. The ethyl acetate crude extract was then further subjected to chemical analysis and its antibacterial properties. Endophytic fungi species was identified as Aspergillus niger based on macroscopic and microscopic analysis. Analysis LC-MS/MS had revealed the presence of five metabolites including ephedradine A, ergosine, Ia, mudanpioside H, and trichosanic acid. The extract showed strong inhibition against Staphyloccus aureus with diameter of zone inhibition of 16.1 mm and moderate inhibition against Escherichia coli 0175H7 and Salmonella enteritidis ATCC 6939 with the observed diameter of zone inhibition of 9.6 mm and 11.3 mm, respectively.

Polysubstituted Phenyl Glucosides Produced by the Fungus Metarrhizium anisopliae

SummaryMetarhizosides A–G (1–7), seven new polysubstituted phenyl glucosides, were isolated from the extracts of solid rice medium of a marine-derived fungus Metarrhizium anisopliae. Compounds 1–7 all contain a polysubstituted phenyl group and the sugar unit is identified as 4′-O-methyl-β-D-glucopyranose. Their structures were elucidated by NMR spectroscopy and chemical method. These compounds were evaluated for anti-inflammatory activity by using LPS-stimulated murine macrophage RAW 264.7 cells and the cytotoxicities against four human cancer cell lines.

Furoic acid derivatives from the endophytic fungus Coniothyrium sp.

The endophytic fungus Coniothyrium sp. was isolated from leaves of Quercus robur. Fermentation of this fungus on solid rice medium yielded two new furoic acid derivatives (1 and 2) and two additional known compounds. The structures of the new compounds were determined by extensive analysis of 1D and 2D nuclear magnetic resonance spectra as well as high-resolution mass spectrometry data. Compound 1, containing three aromatic chromophores attached by rotatable sigma bonds and a chirality center in benzylic position, was found to be a scalemic mixture with an excess of the (S) enantiomer, the absolute configuration of which was elucidated as by the solution time-dependent density functional theory-electronic circular dichroism approach. The ωB97X/TZVP PCM/MeCN and SOGGA11-X/TZVP SMD/MeCN methods were used for geometry reoptimization to reproduce the solution conformational ensemble. All isolated compounds were tested for their cytotoxicity but proved to be inactive.

Cladosins L-O, new hybrid polyketides from the endophytic fungus Cladosporium sphaerospermum WBS017

The endophytic fungus Cladosporium sphaerospermum WBS017 was obtained from healthy bulbs of Fritillaria unibracteata var. wabuensis. Fermentation of C. sphaerospermum on solid rice medium yielded three new hybrid polyketides, cladosins L−N (1–3), and a known derivative cladodionen (4). Further cultivation of this fungus on white bean medium afforded an additional new hybrid polyketide, cladosin O (5) along with three known analogues (6–8). The structures of the new compounds were elucidated using a combination of NMR and HRESIMS data. The absolute configurations of compounds 2 and 3 were determined by Mosher’s method and TDDFT-ECD calculations. All isolated compounds were evaluated for their cytotoxic and antimicrobial activities. Cladodionen (4) exhibited cytotoxicity against the mouse lymphoma cell line L5178Y with an IC50 value of 3.7 μM, and also exhibited antifungal activity against Ustilago maydis and Saccharomyces cerevisiae, while cladosin L (1) displayed week antibacterial activity against Staphylococcus aureus ATCC 29213 and S. aureus ATCC 700699 with MIC values of 50 and 25 μM, respectively.

R-Hexitronic acid, a new tetronic acid derivative isolated from a soil fungus FG9RK

A new tetronic acid derivative (1) together with terrestric acid (2), a known metabolite of Penicillium species, was isolated from the soil fungus, FG9RK following fermentation on solid rice medium. The structure of 1 was elucidated by one- and two-dimensional NMR and MS measurements. The absolute configuration of the oxygenated carbon in the side chain of 1 was identified as S by converting the compound into its Mosher ester whereas the absolute configuration of the lactone ring was deduced based on biogenetic considerations and comparison with 2.

Biotransformation of Host Plant Flavonoids by the Fungal Endophyte Epicoccum nigrum

AbstractFermentation of the fungus Epicoccum nigrum isolated from leaves of Salix sp. on green lentil solid medium yielded the flavonol kaempferol (3) as well as two kaempferol O‐diglycosides (1 and 2) including the new compound 1. The fungal flavonoids bear strong structural similarities to kaempferol derivatives such as kaempferol O‐glycoside (4) being present in green lentils. Furthermore, feeding experiments were conducted by adding flavonoids (kaempferol and rutin) as precursors to solid rice media followed by HPLC and LC‐MS analyses. Fermentation of the fungus on flavonoid free solid rice medium afforded flavonoid free extracts indicating that the fungal flavonoids originate through hydrolytic cleavage of kaempferol glycosides such as 4 followed by glycosylation and acetylation. This study suggests that E. nigrum is capable of biotransformation reactions of plant derived flavonoids whereas de novo biosynthesis of flavonoids is less likely.

Liposoluble compounds from Ganoderma lipsiense grown on solid red rice medium with antiparasitic and antibacterial properties.

Liposoluble molecules are a group of compounds that display potent biological and therapeutic properties. The present study aimed to identify liposoluble molecules produced by Ganoderma lipsiense grown in red rice medium using solid-state fermentation (SSF) techniques, and to investigate the antigiardial and antibacterial activities potential of extracts in vitro. Eighteen fatty acids and derivatives were identified by gas chromatograph-mass spectrometry (GC-MS) analysis in G. lipsense extract. Qualitative (Fourier transform infrared spectroscopy and nuclear magnetic resonance) characterizations identified the steroid ergosta-6,22-diene-3β,5α,8α-triol in purified hexane subfraction (HEXsf) F19 isolated from hexane fraction (HEXf) of crude extract (CE). Ergosta-6,22-diene-3β,5α,8α-triol exhibited significant inhibitory activity against Giardia duodenalis throphozoites (93.6%) in in vitro assays. CE and HEXf inhibited 95.38% and 92.74% of the G. duodenalis throphozoites in 100µg mL-1 , whereas CE and their fractions dichloromethane (DCMf) and ethyl acetate (EAf) showed antibacterial activities against Pseudomonas aeruginosa and Staphylococcus aureus at 500µg mL-1 . Importantly, some liposoluble compounds produced and identified in G. lipsiense are unpublished for this species. This is first report for the production of ergosta-6,22-diene-3β,5α,8α-triol by G. lipsiense and its antiparasitic activity.

Single Production of Kojic Acid by Aspergillus flavus and the Revision of Flufuran.

Timor Island is very hot and dry due to the high intensity of sunlight experienced throughout the year. The endophytic fungi Aspergillus flavus had been isolated from medicinal plants such as Catharanthus roseus, Annona squamosa and Curcuma xanthorisa. The endophytic fungi A. flavus from each plant was cultivated on solid rice media and then analyzed for its capability for producing kojic acid. The production of kojic acid was analyzed by HPLC; the highest amount of kojic acid was observed from the endophytic fungi A. flavus, isolated from the stem of Catharanthus roseus, followed by A. flavus from Annona squamosa and Curcuma xanthorisa. Simple VLC fractionation of the extract of A. flavus from C.roseus led to the isolation of around 11.1 g of pure kojic acid. The structure of kojic acid (1) was confirmed by NMR and MS spectroscopic data. A comparison of the NMR data with the literature supported the revision of the natural product flufuran to kojic acid. To the best of our knowledge, this is the first report of a strain of endophytic fungi producing only kojic acid without any other toxic metabolites such as alfatoxins. Therefore, this Aspergillus flavus strain can be applied as a potential producer of kojic acid for industrial use.

Eutypellacytosporins A-D, Meroterpenoids from the Arctic Fungus Eutypella sp. D-1.

The Arctic fungus Eutypella sp. D-1, previously found to produce a variety of cytotoxic cyclopropyl-fused and cyclobutyl-fused pimarane diterpenoids when grown in the defined medium, was induced to produce unusual metabolites by growing on solid rice medium. A chemical investigation on the rice medium extract led to the isolation of four new meroterpenoids, eutypellacytosporins A-D (1-4), along with the known biogenetically related compound cytosporin D (5). The structures of the new compounds were elucidated by their detailed spectroscopic analysis and modified Mosher's method. Compounds 1-4 may be formed by the 12,32-ester linkage of two moieties, cytosporin D (5) and decipienolide A or B. All isolated compounds, except 5, showed weak cytotoxicity against DU145, SW1990, Huh7, and PANC-1 cell lines with IC50 values ranging from 4.9 to 17.1 μM.

New quinoline alkaloid and bisabolane-type sesquiterpenoid derivatives from the deep-sea-derived fungus Aspergillus sp. SCSIO06786

One new quinoline alkaloid (1), two new bisabolane-type sesquiterpene derivatives (2 and 3), and a new natural product (4) along with ten known compounds (514) were isolated from the deep sea-derived fungus Aspergillus sp. SCSIO06786 which cultured on solid rice medium. Three new structures were elucidated by analysis of 1D/2D NMR data and HR-ESI-MS. The absolute configurations of 2 and 3 were established by comparison of the experimental and reported ECD values. Compounds 11–13 exhibited moderate selective inhibitory activities against the tested pathogenic bacteria with MIC values among 3.13–12.5 μg/mL.

Secondary metabolites from the Aspergillus sp. in the rhizosphere soil of Phoenix dactylifera (Palm tree)

The soil-derived fungus Aspergillus sp. isolated from the rhizospheric soil of Phoenix dactylifera (Date palm tree) and cultured on the large scale solid rice medium yielded a novel compound 1-(4-hydroxy-2,6-dimethoxy-3,5-dimethylphenyl)-2-methyl-1-butanone (1) and four known compounds; citricin (2), dihydrocitrinone (3), 2, 3, 4-trimethyl-5, 7-dihydroxy-2, 3-dihydrobenzofuran (4) and oricinol (5). The structures of the isolated compounds were elucidated by MS, 1H, 13C and 2D NMR spectra. Compound (1) exhibited potent antimicrobial activities against Staphylococcus aureus with MIC values of 2.3 μg mL−1 and significant growth inhibitions of 82.3 ± 3.3 against Candida albicans and of 79.2 ± 2.6 against Candida parapsilosis. This is the first report to isolate metabolites from the fungus Aspergillus found in temperate region date plant rhizospheres.

New Bioactive Metabolites from the Thermophilic FungusPenicilliumsp. Isolated from Ghamiqa Hot Spring in Saudi Arabia

The thermophilic fungusPenicilliumspecies was isolated from Ghamiqa hot spring sediments in Saudi Arabia. Extract ofPenicilliumspecies cultured on solid rice medium yielded two new compounds 3-(furan 12-carboxylic acid)-6-(methoxycarbonyl)-4-hydroxy-4-methyl-4 and 5-dihydro-2H-pyran13α-methyl-7-hydroxy-5-carboxylic acid methyl ester-1-indanone2. In addition, three known compounds, austinol3, emodin4, and 2-methyl-penicinoline5, were isolated. The structures of the new compounds were unambiguously determined by comprehensive analysis of spectroscopic data, one- and two-dimensional NMR spectroscopy, and high-resolution mass spectrometry. All isolated metabolites were studied for their antibiotic effect against several pathogenic bacteria and for their cytotoxicity against the lymphoma human cancer cell line HTB-176. Austinol3exhibited strong antibacterial activity againstP. aeruginosabacterial strain with an MIC value of 0.13 ± 0.4 µg·mL−1, whereas emodin4demonstrated significant cytotoxicity against the tested HTB-176 cell line with an IC50value of 2 ± 7.6 µM, while the other compounds were moderate to inactive with IC50ranging from 4.3 ± 0.25–22 ± 2.94 µM in this assay.