In Vitro Phytobiological Investigation of Bioactive Secondary Metabolites from the Malus domestica-Derived Endophytic Fungus Aspergillus tubingensis Strain AN103.

Endophytic fungi including black aspergilli have the potential to synthesize multiple bioactive secondary metabolites. Therefore, the search for active metabolites from endophytic fungi against pathogenic microbes has become a necessity for alternative and promising strategies. In this study, 25 endophytic fungal isolates associated with Malus domestica were isolated, grown, and fermented on a solid rice medium. Subsequently, their ethyl acetate crude extracts were pretested for biological activity. One endophytic fungal isolate demonstrated the highest activity and was chosen for further investigation. Based on its phenotypic, ITS ribosomal gene sequences, and phylogenetic characterization, this isolate was identified as Aspergillus tubingensis strain AN103 with the accession number (KR184138). Chemical investigations of its fermented cultures yielded four compounds: Pyranonigrin A (1), Fonsecin (2), TMC 256 A1 (3), and Asperazine (4). Furthermore, 1H-NMR, HPLC, and LC-MS were performed for the identification and structure elucidation of these metabolites. The isolated pure compounds showed moderate-to-potent antibacterial activities against Pseudomonas aeruginosa and Escherichia coli (MIC value ranged from 31 and 121 to 14.5 and 58.3 µg/mL), respectively; in addition, the time−kill kinetics for the highly sensitive bacteria against isolated compounds was also investigated. The antifungal activity results show that (3) and (4) had the maximum effect against Fusarium solani and A. niger with inhibition zones of 16.40 ± 0.55 and 16.20 ± 0.20 mm, respectively, and (2) had the best effect against Candida albicans, with an inhibition zone of 17.8 ± 1.35 mm. Moreover, in a cytotoxicity assay against mouse lymphoma cell line L5178Y, (4) exhibited moderate cytotoxicity (49% inhibition), whereas (1−3) reported weak cytotoxicity (15, 26, and 19% inhibition), respectively. Our results reveal that these compounds might be useful to develop potential cytotoxic and antimicrobial drugs and an alternative source for various medical and pharmaceutical fields.

Cyclic heptapeptides from the soil-derived fungus Clonostachys rosea.

Three new cyclic heptapeptides (1-3) together with three known compounds (4-6) were isolated from a solid rice culture of the soil-derived fungus Clonostachys rosea. Fermentation of the fungus on white beans instead of rice afforded a new γ-lactam (7) and a known γ-lactone (8) that were not detected in the former extracts. The structures of the new compounds were elucidated on the basis of 1D and 2D NMR spectra as well as by HRESIMS data. Compounds 1 and 4 exhibited significant cytotoxicity against the L5178Y mouse lymphoma cell line with IC50 values of 4.1 and 0.1 µM, respectively. Compound 4 also displayed cytotoxicity against the A2780 human ovarian cancer cell line with an IC50 value of 3.5 µM. The preliminary structure-activity relationships are discussed.

Induction of Secondary Metabolites from the Marine-Derived Fungus Aspergillus versicolor through Co-cultivation with Bacillus subtilis.

A new cyclic pentapeptide, cotteslosin C (1: ), a new aflaquinolone, 22-epi-aflaquinolone B (3: ), and two new anthraquinones (9: and 10: ), along with thirty known compounds (2, 4:  - 8, 11:  - 34: ) were isolated from a co-culture of the sponge-associated fungus Aspergillus versicolor with Bacillus subtilis. The new metabolites were only detected in the co-culture extract, but not when the fungus was grown under axenic conditions. Furthermore, the co-culture extract exhibited an enhanced accumulation of the known constituents versicolorin B (14: ), averufin (16: ), and sterigmatocyctin (19: ) by factors of 1.5, 2.0, and 4.7, respectively, compared to the axenic fungal culture. The structures of the isolated compounds were elucidated on the basis of 1D and 2D NMR spectra and mass spectrometry as well as by comparison with literature data. The absolute configuration of compounds 3, 9: , and 10: was determined by ECD (electronic circular dichroism) analysis aided by TDDFT-ECD (time-dependent density functional theory electronic circular dichroism) calculations. Compounds 15, 18:  - 21: , and 26: exhibited strong to moderate cytotoxic activity against the mouse lymphoma cell line L5178Y, with IC50 values ranging from 2.0 to 21.2 µM, while compounds 14, 16, 31, 32: , and 33: displayed moderate inhibitory activities against several gram-positive bacteria, with MIC values ranging from 12.5 to 50 µM.

Germacrane-Type Sesquiterpenoids with Antiproliferative Activities from Eupatorium chinense.

Ten new germacrane-type sesquiterpenoids (1-10) were isolated from a whole plant extract of Eupatorium chinense. The structures were elucidated by analysis of their NMR and MS data as well as by comparison with literature values. The absolute configuration of eupachinsin A (1) was determined by single-crystal X-ray diffraction analysis. Compounds 3 and 4 exhibited cytotoxicity against a human breast cancer cell line (MDA-MB-231), with IC50 values of 0.8 and 3.4 µM, respectively. In addition, compounds 3-5 showed cytotoxicity against the human hepatocellular carcinoma cell line (HepG2), with IC50 values ranging from 3.6 to 7.6 µM.

Antibacterial and Cytotoxic Phenolic Metabolites from the Fruits of Amorpha fruticosa.

Fourteen new natural products, namely, 2-[(Z)-styryl]-5-geranylresorcin-1-carboxylic acid (1), amorfrutin D (2), 4-O-demethylamorfrutin D (3), 8-geranyl-3,5,7-trihydroxyflavanone (4), 8-geranyl-5,7,3'-trihydroxy-4'-methoxyisoflavone (5), 6-geranyl-5,7,3'-trihydroxy-4'-methoxyisoflavone (6), 8-geranyl-7,3'-dihydroxy-4'-methoxyisoflavone (7), 3-O-demethyldalbinol (8), 6a,12a-dehydro-3-O-demethylamorphigenin (9), (6aR,12aR,5'R)-amorphigenin (10), amorphispironones B and C (11 and 12), resokaempferol 3-O-ß-d-glucopyranosyl-(1→2)-ß-d-glucopyranoside-7-O-α-l-rhamnopyranoside (13), and daidzein 7-O-ß-d-glucopyranosyl-(1→2)-ß-d-glucopyranoside (14), together with 40 known compounds, were isolated from the fruits of Amorpha fruticosa. The structures of the new compounds were elucidated by 1D and 2D NMR spectroscopic analysis as well as from the mass spectrometry data. ECD calculations were performed to determine the absolute configurations of 11 and 15. Compounds 1, 4-6, and 16-23 showed potent to moderate antibacterial activities against several Gram-positive bacteria with MIC values ranging from 3.1 to 100 µM. In addition, compounds 11 and 24-33 were significantly cytotoxic against the L5178Y mouse lymphoma cell line and exhibited IC50 values from 0.2 to 10.2 µM.

Lactones from the Sponge-Derived Fungus Talaromyces rugulosus.

The marine-derived fungus Talaromyces rugulosus isolated from the Mediterranean sponge Axinella cannabina and cultured on solid rice medium yielded seventeen lactone derivatives including five butenolides (1-5), seven (3S)-resorcylide derivatives (6-12), two butenolide-resorcylide dimers (13 and 14), and three dihydroisocoumarins (15-17). Among them, fourteen compounds (1-3, 6-16) are new natural products. The structures of the isolated compounds were elucidated by 1D and 2D NMR (Nuclear Magnetic Resonance) spectroscopy as well as by ESI-HRMS (ElectroSpray Ionization-High Resolution Mass Spectrometry). TDDFT-ECD (Time-Dependent Density Functional Theory-Electronic Circular Dichroism) calculations were performed to determine the absolute configurations of chiral compounds. The butenolide-resorcylide dimers talarodilactones A and B (13 and 14) exhibited potent cytotoxicity against the L5178Y murine lymphoma cell line with IC50 values of 3.9 and 1.3 µM, respectively.

Metabolites from the Fungal Endophyte Aspergillus austroafricanus in Axenic Culture and in Fungal-Bacterial Mixed Cultures.

The endophytic fungus Aspergillus austroafricanus isolated from leaves of the aquatic plant Eichhornia crassipes was fermented axenically on solid rice medium as well as in mixed cultures with Bacillus subtilis or with Streptomyces lividans. Chromatographic analysis of EtOAc extract of axenic cultures afforded two new metabolites, namely, the xanthone dimer austradixanthone (1) and the sesquiterpene (+)-austrosene (2), along with five known compounds (3-7). Austradixanthone (1) represents the first highly oxygenated heterodimeric xanthone derivative. When A. austroafricanus was grown in mixed cultures with B. subtilis or with S. lividans, several diphenyl ethers (8-11) including the new austramide (8) were induced up to 29-fold. The structures of new compounds were unambiguously elucidated using 1D- and 2D-NMR spectroscopy, HRESIMS, and chemical derivatization. Compound 7 exhibited weak cytotoxicity against the murine lymphoma L5178Y cell line (EC50 is 12.6 µM). In addition, compounds 9 and 10, which were enhanced in mixed fungal/bacterial cultures, proved to be active against Staphylococcus aureus (ATCC 700699) with minimal inhibitory concentrations (MICs) of 25 µM each (6.6 µg/mL), whereas compound 11 revealed moderate antibacterial activity against B. subtilis 168 trpC2 with an MIC value of 34.8 µM (8 µg/mL).

Investigation of iso-propylchaetominine anticancer activity on apoptosis, cell cycle and Wnt signaling pathway in different cancer models.

Dysregulation of the Wnt signaling pathway contributes to the development of many cancer types. Natural compounds produced with biotechnological systems have been the focus of research for being a new drug candidate both with unlimited resources and cost-effective production. In this study, it was aimed to reveal the effects of isopropylchaetominine on cytotoxic, cytostatic, apoptotic and Wnt signaling pathways in brain, pancreatic and prostate cancer. The IC50 values of isopropylchaetominine in U-87 MG, PANC1, PC3 and LNCaP cells were calculated as 91.94 µM, 41.68 µM, 54.54 µM and 7.86 µM in 72nd h, respectively. The metabolite arrests the cell cycle in G0/G1 phase in each cancer cells. Iso-propylchaetominine induced a 4.3-fold and 1.9-fold increase in apoptosis in PC3 and PANC1 cells, respectively. The toxicity of isopropylchaetominine in healthy fibroblast cells was assessed using the annexin V method, and no significant apoptotic activity was observed between the groups treated with the active substance and untreated. In U-87 MG, PANC1, PC3, and LNCaP cells under treatment with isopropylchaetominin, the expression levels of DKK3, TLE1, AES, DKK1, FRZB, DAB2, AXIN1/2, PPARD, SFRP4, APC and SOX17 tumor suppressor genes increased significantly. Decreases in expression of Wnt1, Wnt2, Wnt3, Wnt4, Wnt5, Wnt6, Wnt10, Wnt11, FRZ2, FRZ3, FRZ7, TCF7L1, BCL9, PYGO, CCND2, c-MYC, WISP1 and CTNNB1 oncogenic genes were detected. All these result shows that isopropylchaetominine can present promising new treatment strategy in different cancer types.

Alkaloids and polyketides from Penicillium citrinum, an endophyte isolated from the Moroccan plant Ceratonia siliqua.

The endophytic fungus Penicillium citrinum was isolated from a fresh stem of the Moroccan plant Ceratonia siliqua. Extracts of P. citrinum grown on rice and white bean media yielded five new compounds, namely, citriquinochroman (1), tanzawaic acids G and H (2 and 3), 6-methylcurvulinic acid (4), 8-methoxy-3,5-dimethylisoquinolin-6-ol (5), and one new natural product, 1,2,3,11b-tetrahydroquinolactacide (6), which had previously been described as a synthetic compound. In addition, 13 known compounds including seven alkaloids and six polyketides were isolated. The structures of the new compounds were unambiguously determined on the basis of one- and two-dimensional NMR spectroscopy as well as by high-resolution mass spectrometry. Citriquinochroman (1) features a new skeleton, consisting of quinolactacide and (3S)-6-hydroxy-8-methoxy-3,5-dimethylisochroman linked by a C-C bond. 1,2,3,11b-Tetrahydroquinolactacide (6) may be a biogenetic precursor of quinolactacide. Citriquinochroman (1) showed cytotoxicity against the murine lymphoma L5178Y cell line with an IC50 value of 6.1 µM, while the other compounds were inactive (IC50 >10 µM) in this assay.

O-heterocyclic embeurekols from Embellisia eureka, an endophyte of Cladanthus arabicus.

Three new polyketides ((-)-1, (+)-1, and 2) were isolated from the EtOAc extract of the fungus Embellisia eureka, an endophyte of the Moroccan plant Cladanthus arabicus (Asteraceae). The structures of these new compounds were determined on the basis of one- and two-dimensional NMR spectroscopy as well as by high-resolution mass spectrometry. The absolute configurations of (-)-1, (+)-1, and 2 were determined by TDDFT ECD calculations of solution conformers, online HPLC-ECD analysis, and the modified Mosher method.

Cytotoxic rocaglamide derivatives from Aglaia duppereana.

Phytochemical investigation of Aglaia duppereana flowers led to the isolation of a new rocaglamide derivative and twelve known congeners. The structure of the new compound was unambiguously elucidated by spectroscopic techniques (1D- and 2D-NMR, HRESIMS). The isolated compounds exhibited a potent cytotoxic activity against mouse lymphoma (L5178Y) cells with EC50 values ranging from 5.1 to 54.8 nM.

Aspergillus Carneus metabolite Averufanin induced cell cycle arrest and apoptotic cell death on cancer cell lines via inducing DNA damage.

Cancer is one of the leading causes of death worldwide, accounting for nearly 10 million deaths in 2020. Current treatment methods include hormone therapy, γ-radiation, immunotherapy, and chemotherapy. Although chemotherapy is the most effective treatment, there are major obstacles posed by resistance mechanisms of cancer cells and side-effects of the drugs, thus the search for novel anti-cancer compounds, especially from natural sources, is crucial for cancer pharmaceutics research. One natural source worthy of investigation is fungal species. In this study, the cytotoxicity of 5 metabolic compounds isolated from filamentous fungus Aspergillus Carneus. Arugosin C, Averufin, Averufanin, Nidurifin and Versicolorin C were analyzed using NCI-SRB assay on 10 different cell lines of breast cancer, ovarian cancer, glioblastoma and non-tumorigenic cell lines. Averufanin showed highest cytotoxicity with lowest IC50 concentrations especially on breast cancer cells. Therefore, Averufanin was further investigated to enlighten cell death and molecular mechanisms of action involved. Cell cycle analysis showed increase in SubG1 phase suggesting apoptosis induction which was further confirmed by Annexin V and Caspase 3/7 Assays. H2A.X staining revealed accumulation of DNA damage in cells treated with Averufanin and finally western blot analysis validated DNA damage response and downstream effects of Averufanin treatment in various signaling pathways. Consequently, this study shows that Averufanin compound induces cell cycle arrest and cell death via apoptosis through causing DNA damage and can be contemplated and further explored as a new therapeutic strategy in breast cancer.

Mycotoxins from Tomato Pathogenic Alternaria alternata and Their Combined Cytotoxic Effects on Human Cell Lines and Male Albino Rats.

The Alternaria species are considered to produce a plethora of several mycotoxins constituting a risk factor for both human and animal health. This work aimed mainly to explore the cytotoxicity of a combined mixture of altenuene (ALT), alternariol (AOH), tenuazonic acid (TeA), and altenuisol (AS) toxins produced by pathogenic A. alternata toward human oral epithelial cells (PCS-200-014), lung fibroblast cells (WI-38), and male albino rats. The sequencing of the multi-locus, RNA polymerase second largest subunit (rpb2), glyceraldehyde-3-phosphate dehydrogenase (gapdh), and Alternaria major allergen gene (Alt a 1) was performed to infer relationships among isolated Alternaria species. The phylogenetic analysis of gapdh, rpb2, and Alt-a 1 sequence data indicated that all isolates resided in A. alternata. The pathogenic potentiality of A. alternata was investigated on tomato plants cv. super strain B under greenhouse conditions, and all isolates were pathogenic to tomato plants, with significant (p < 0.05) variations. The ability of A. alternata isolates to produce mycotoxins was also explored using high-performance liquid chromatography (HPLC). All tested isolates were able to produce at least one of the assessed mycotoxins-ALT, AOH, TeA, and AS-and ALT was reported as the dominant mycotoxin, produced by 80% of A. alternata isolates. The cytotoxic properties of the combined mixture of ALT, AOH, TeA, and AS at concentrations of 31.25, 62.50, 125, 250, and 500 µg/mL were assessed via the MTT assay method after exposure for 24 h versus the control. The treatment of both cell lines with combined mixtures of ALT, AOH, TeA, and AS showed a dose-dependent decrease in cell viability. The highest concentrations tested at 62.50, 125, 250, and 500 µg/mL significantly decreased cell viability and caused cell damage compared to the lowest concentration of 31.25 µg/mL and the control. The cytotoxicity and genotoxicity of the combined mixtures of ALT, AOH, TeA, and AS on male albino rats were also investigated via the gene expression of (TNF-α) and using hematological (CBC), chemical (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and urea and creatinine), and histopathological analyses. A marked increase was observed in the levels of ALT, AST, urea and creatinine, TNF-α gene expression, red blood cells (RBCs), white blood cells (WBCs), hemoglobin (Hb), and packed cell volume % (PCV) after 28 days of exposure relative to the untreated control. Pathological alterations were also observed in the liver and kidney tissues of rats. Conclusively, this work provides a new understanding on the cytotoxicity and genotoxicity of mycotoxins of pathogenic A. alternata from tomatoes.

Pullularins E and F, two new peptides from the endophytic fungus Bionectria ochroleuca isolated from the mangrove plant Sonneratia caseolaris.

Chemical investigation of the EtOAc extract of the endophytic fungus Bionectria ochroleuca, isolated from the inner leaf tissues of the plant Sonneratia caseolaris (Sonneratiaceae) from Hainan island (China), yielded two new peptides, pullularins E and F (1 and 2) together with three known compounds (3-5). The structures of the new compounds were unambiguously determined on the basis of one- and two-dimensional NMR spectroscopy as well as by high-resolution mass spectrometry. The absolute configurations of amino acids were determined by HPLC analysis of acid hydrolysates using Marfey's method. The isolated compounds exhibited pronounced to moderate cytotoxic activity against the mouse lymphoma cells (L5178Y) with EC50 values ranging between 0.1 and 6.7 µg/mL.

A new depsidone derivative from mangrove sediment derived fungus Lasiodiplodia theobromae.

A new depsidone derivative botryorhodine I (1), along with eight known compounds (2-9) were obtained from solid rice cultures of the fungal strain, Lasiodiplodia theobromae M4.2-2 isolated from a mangrove sediment sample. The structures of the isolated compounds were elucidated on the basis of 1 D and 2 D NMR analysis as well as by HRESIMS. All compounds were evaluated for their cytotoxic potential against the mouse lymphoma cell line L5178Y as well as for their antibacterial activities against a panel of Gram-positive and Gram-negative bacterial strains. Compound 3 revealed potent cytotoxic activity with an IC50 of 7.3 µM whereas compound 7 showed selective anti-bacterial activity against different S. aureus and E. faecium bacterial strains with MIC value of 25 µg/ml.

Pyrone derivatives from Helichrysum italicum.

Chemical investigation of aerial parts of Helichrysum italicum yielded two new pyrone derivatives (1 and 2) along with ten known compounds. The structures of the new compounds were established by 1D and 2D NMR spectra as well as by HRESIMS data. Compound 1 represented a rare dimer of substituted α- and γ-pyrone units. DFT-NMR and TDDFT-ECD calculations were carried out to determine the absolute configuration of 1 but failed, representing the limitation of TDDFT-ECD calculation for the configurational assignment. All compounds were measured for their antibacterial and cytotoxic activity but proved to be inactive.

Co-culture of the fungus Fusarium tricinctum with Streptomyces lividans induces production of cryptic naphthoquinone dimers.

Co-cultivation of the endophytic fungus Fusarium tricinctum with Streptomyces lividans on solid rice medium led to the production of four new naphthoquinone dimers, fusatricinones A-D (1-4), and a new lateropyrone derivative, dihydrolateropyrone (5), that were not detected in axenic fungal controls. In addition, four known cryptic compounds, zearalenone (7), (-)-citreoisocoumarin (8), macrocarpon C (9) and 7-hydroxy-2-(2-hydroxypropyl)-5-methylchromone (10), that were likewise undetectable in extracts from fungal controls, were obtained from the co-culture extracts. The known antibiotically active compound lateropyrone (6), the depsipeptides enniatins B (11), B1 (12) and A1 (13), and the lipopeptide fusaristatin A (14), that were present in axenic fungal controls and in co-culture extracts, were upregulated in the latter. The structures of the new compounds were elucidated by 1D and 2D NMR spectra as well as by HRESIMS data. The relative and absolute configuration of dihydrolateropyrone (5) was elucidated by TDDFT-ECD calculations.

New eremophilane-type sesquiterpenes and maleimide-bearing compounds from Carpesium abrotanoides L.

Two new eremophilane-type sesquiterpenes, carperemophilanes A and B (1-2), three new maleimide-bearing compounds, carpesiumaleimides A-C (3-5), along with a known sesquiterpene, carabrol (6), were isolated from the ethanol extract of Carpesium abrotanoides L. Their structures were elucidated by analysis of their NMR and MS data as well as by comparison with the literature. The absolute configuration of carperemophilane A (1) was determined by single-crystal X-ray diffraction analysis. All isolated compounds (1-6) were evaluated in vitro for cytotoxicity against two human cancer cell lines MDA-MB-231 and HGC-27 using the MTT method. Compounds 1, 2 and 6 showed cytotoxic activities with IC50 values ranging from 7.45 to 37.35 µM.

Isolation and X-ray structure analysis of citreohybridonol from marine-derived Penicillium atrovenetum.

The anti-neuroinflammatory meroterpenoid citreohybridonol was isolated for the first time from a sponge-derived fungus Penicillium atrovenetum. In this study, in addition to isolation and structure featuring, its unambiguous absolute configuration was determined exclusively by single crystal X-ray diffraction. The C-17-keto tautomer was clearly observed in X-ray analysis. The substance crystallises in the monoclinic space group P21 with a = 10.7496(5) Å, b = 14.3286(7) Å, c = 17.4909(8) Å, ß = 103.235(2)°, V = 2622.5(2) Å3, Z = 2, Dcalcd = 1.280 g/cm3. The chirality of the asymmetric carbon atoms was as follows: C3 (S), C5 (R), C6 (S), C8 (S), C9 (R), C10 (R), C13 (R), C14 (R).

Natural Products from Deep-Sea-Derived Fungi ̶ A New Source of Novel Bioactive Compounds?

BACKGROUND: Over the last two decades, deep-sea-derived fungi are considered to be a new source of pharmacologically active secondary metabolites for drug discovery mainly based on the underlying assumption that the uniqueness of the deep sea will give rise to equally unprecedented natural products. Indeed, up to now over 200 new metabolites have been identified from deep-sea fungi, which is in support of the statement made above. RESULTS: This review summarizes the new and/or bioactive compounds reported from deepsea- derived fungi in the last six years (2010 - October 2016) and critically evaluates whether the data published so far really support the notion that these fungi are a promising source of new bioactive chemical entities.