Plasmodium-Resistant Indole Diterpenoid Biosynthesis Gene Cluster Derived from Aspergillus oryzae Was Activated by Exogenous P450 Gene Ast B.

Synthetic biology is an effective way to activate silent biosynthetic gene clusters. Five new indole diterpenoids (1, 2, 5, 9, and 10), together with 10 known derivatives (3, 4, 6-8, and 11-15) were activated from Aspergillus oryzae transformants by an exogenous P450 gene Ast B and obtained under the guidance of molecular networking. Their planar structures were determined by NMR and HR-ESI-MS. The absolute configuration of compound 1 was determined by single crystal X-ray diffraction, and those of compounds 2, 5 , 9, and 10 were confirmed by comparing the observed ECD with the calculated ECD. HPLC analysis suggested that the BGCs of indole diterpenoids in A. oryzae were activated by exogenous P450 gene Ast B. Compounds 1-4, 7, 8, and 11 displayed strong activity against chloroquine-sensitive plasmodium strain P.f.3D7 with IC50 values ranging from 0.84 to 2.9 µM. It is the first report that indole diterpenoids have potential antimalarial activity. The structure-activity relationship study showed that the linear indole diterpenoids contribute significantly to the antiparasite activity. Molecular docking studies showed that 1 and positive control chloroquine were at the center of the active pocket of PfHsp90, while 11 was far from the active site.

The Polyketides with Antimicrobial Activities from a Mangrove Endophytic Fungus Trichoderma lentiforme ML-P8-2.

Five new polyketides, including two chromones (1-2), two phenyl derivatives (4-5), and a tandyukusin derivative (6), along with five known polyketides (3 and 7-10) were isolated from mangrove endophytic fungus Trichoderma lentiforme ML-P8-2. The planar structures of compounds were elucidated via detailed 1D, 2D NMR, and HR-ESI-MS analysis. ECD spectra, optical rotation values calculation, and alkali hydrolysis were applied in the determination of the absolute configuration of the new compounds. In bioassays, 6 and 9 exhibited promising antifungal activities against Penicillium italicum, with an MIC value of 6.25 µM for both compounds. Moreover, 3 displayed moderate AChE inhibitory activity with an IC50 value of 20.6 ± 0.3 µM.

Antioxidative Indenone and Benzophenone Derivatives from the Mangrove-Derived Fungus Cytospora heveae NSHSJ-2.

Seven new polyketides, including four indenone derivatives, cytoindenones A-C (1, 3-4), 3'-methoxycytoindenone A (2), a benzophenone derivative, cytorhizophin J (6), and a pair of tetralone enantiomers, (±)-4,6-dihydroxy-5-methoxy-α-tetralone (7), together with a known compound (5) were obtained from the endophytic fungus Cytospora heveae NSHSJ-2 isolated from the fresh stem of the mangrove plant Sonneratia caseolaris. Compound 3 represented the first natural indenone monomer substituted by two benzene moieties at C-2 and C-3. Their structures were determined by the analysis of 1D and 2D NMR, as well as mass spectroscopic data, and the absolute configurations of (±)-7 were determined on the basis of the observed specific rotation value compared with those of the tetralone derivatives previously reported. In bioactivity assays, compounds 1, 4-6 showed potent DPPH· scavenging activities, with EC50 values ranging from 9.5 to 16.6 µM, better than the positive control ascorbic acid (21.9 µM); compounds 2-3 also exhibited DPPH· scavenging activities comparable to ascorbic acid.

Quinolinones Alkaloids with AChE Inhibitory Activity from Mangrove Endophytic Fungus Penicillium citrinum YX-002.

Acetylcholinesterase (AChE) inhibitory activity-guided studies on the mangrove-derived endophytic fungus Penicillium citrinum YX-002 led to the isolation of nine secondary metabolites, including one new quinolinone derivative, quinolactone A (1), a pair of epimers quinolactacin C1 (2) and 3-epi-quinolactacin C1 (3), together with six known analogs (4-9). Their structures were elucidated based on extensive mass spectrometry (MS) and 1D/2D nuclear magnetic resonance (NMR) spectroscopic analyses, and compared with data in the literature. The absolute configurations of compounds 1-3 was determined by combination of electronic circular dichroism (ECD) calculations and X-Ray single crystal diffraction technique using CuKα radiation. In bioassays, compounds 1, 4 and 7 showed moderate AChE inhibitory activities with IC50 values of 27.6, 19.4 and 11.2 µmol/L, respectively. The structure-activity relationships (SARs) analysis suggested that the existence of carbonyl group on C-3 and the oxygen atom on the five-membered ring were beneficial to the activity. Molecular docking results showed that compound 7 had a lower affinity interaction energy (-9.3 kcal/mol) with stronger interactions with different sites in AChE activities, which explained its higher activities.

Antiplasmodial Asperterpenoids from Two Aspergillus oryzae Transformants with Heterologous Expression of Sesterterpene Genes.

The synthetic biology approach enables efficient and directional mining of target compounds during drug discovery. Ten new asperterpenoids (6-15) and five known analogues (1-5), possessing a rare 5/7/3/6/5 skeleton, were obtained from two Aspergillus oryzae transformants with heterologous expression of a terpene cyclase gene AstC with one or two P450 genes AstB/A under the guidance of molecular networking. Their planar structures were determined by 1D and 2D NMR and HR-ESI-MS. The absolute configurations of compounds 6 and 9-13 were determined by single crystal X-ray diffraction, and those of compounds 7-8 and 14-15 were compared with the ECD of known compounds. Seven of all the compounds are the first asperterpenoid oxidation products at C-17 or at C-25. In bioassay, compounds 1-2, 4-5, and 6-8 displayed moderate to strong eliminating activities against chloroquine-sensitive strain (P.f.3D7) with EC50 values ranging from 2.1 to 19.3 µM. The structure-activity relationship (SAR) was discussed, which showed that substituents at C-3, C-11, C-17, C-18, and C-23 of asperterpenoids significantly affected anti-plasma parasite activity.

Cytotoxic Bromine- and Iodine-Containing Cytochalasins Produced by the Mangrove Endophytic Fungus Phomopsis sp. QYM-13 Using the OSMAC Approach.

Twelve new cytochalasins, phomopchalasins D-O (1-3, 5-12, and 14), including one brominated (2) and two iodinated cytochalasins (3 and 6), together with six known analogues (4, 13, and 15-18) were isolated from the mangrove-derived fungus Phomopsis sp. QYM-13 treated with 3% NaBr or 3% KI in potato liquid medium. Their structures and absolute configurations were established by extensive spectroscopic analysis (1D and 2D NMR, HRESIMS), electronic circular dichroism calculations, and a single-crystal X-ray diffraction experiment. Compounds 3 and 6 represent the first iodinated cytochalasins. Compounds 2, 15, 17, and 18 exhibited significant cytotoxicity against human cancer cell line MDA-MB-435 with IC50 values ranging from 0.2 to 8.2 µM.

Azaphilone derivatives with anti-inflammatory activity from the mangrove endophytic fungus Penicillium sclerotiorum ZJHJJ-18.

Nine undescribed azaphilone derivatives, sclerazaphilones A-H (1-9), and three known analogues (10-12), were obtained and identified from the fermented rice cultures of a mangrove endophytic fungus Penicillium sclerotiorum ZJHJJ-18. 1D and 2D NMR, HRESIMS and spectral data indicated the chemical structures of 1-9, and their absolute configurations were assigned by experimental and computational analyses of electronic circular dichroism (ECD) spectra, and application of the chemical transformations. Compounds 1-4 were the first reported N-containing azaphilone derivatives with 5/6 dicyclic core. The bioassay results showed that compounds 3-5 exhibited effective inhibitory effects on the nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells with IC50 values in the range of 6.30-9.45 µM. Moreover, a molecular docking study was conducted to investigate the probable binding interaction of 3-5 with inducible nitric oxide synthase (iNOS).

Hemiacetalmeroterpenoids A-C and Astellolide Q with Antimicrobial Activity from the Marine-Derived Fungus Penicillium sp. N-5.

Four new compounds including three andrastin-type meroterpenoids hemiacetalmeroterpenoids A-C (1-3), and a drimane sesquiterpenoid astellolide Q (15), together with eleven known compounds (4-14) were isolated from the cultures of the marine-derived fungus Penicillium sp. N-5, while compound 14 was first isolated from a natural source. The structures of the new compounds were determined by analysis of detailed spectroscopic data, and the absolute configurations were further decided by a comparison of the experimental and calculated ECD spectra. Hemiacetalmeroterpenoid A (1) possesses a unique and highly congested 6,6,6,6,5,5-hexa-cyclic skeleton. Moreover, the absolute configuration of compound 14 was also reported for the first time. Compounds 1, 5 and 10 exhibited significant antimicrobial activities against Penicillium italicum and Colletrichum gloeosporioides with MIC values ranging from 1.56 to 6.25 µg/mL.

Pyrone Derivatives from a Mangrove Endophytic Fungus Phomopsis†asparagi LSLYZ-87.

Chemical investigation of the endophytic fungus Phomopsis asparagi LSLYZ-87 cultured on PDB medium led to the isolation of two new pyrone derivatives, phomasparapyrone A (1), and phomasparapyrone B (2), together with the known kojic acid (3). Their planar structures were connected through 1D and 2D NMR spectroscopic data. And the stereo structures of 1 and 2 were defined by comparison of the experimental ECD spectra to calculated one. All isolates were evaluated for their anti-neuroinflammatory activities. Among them, compound 2 showed moderate inhibition on NO accumulation induced by LPS on BV-2 cells in a dose dependent manner at 30, 40 and 50 µM, and without cytotoxicity in a concentration of 50.0 µM.

A Pair of Chromone Epimers and an Acetophenone Glucoside from the Mangrove Endophytic Fungus Mycosphaerella sp. L3A1.

Three new compounds, including a pair of chromone derivatives (1-2), and an acetophenone glucoside (3), together with three known compounds (4-6), were isolated from the mangrove endophytic fungus Mycosphaerella sp. L3A1. Their structures were elucidated by HR-ESI-MS analysis and extensive spectroscopic data. The absolute configurations of new compounds were determined using single-crystal X-ray diffraction analysis, electronic circular dichroism (ECD) calculations and chemical hydrolysis. In bioassays, compound 4 showed moderate cytotoxic activity against MDA-MB-435, HCT116, and SNB19 with IC50 values in the range of 18.5-26.29 µM.

Two Antimicrobial Heterodimeric Tetrahydroxanthones with a 7,7'-Linkage from Mangrove Endophytic Fungus Aspergillus flavus QQYZ.

Mangrove endophytic fungi represent significant and sustainable sources of novel metabolites with unique structures and excellent biological activities, attracting extensive chemical investigations. In this research, two novel heterodimeric tetrahydroxanthones, aflaxanthones A (1) and B (2), dimerized via an unprecedented 7,7'-linkage, a sp3-sp3 dimeric manner, were isolated from the mangrove endophytic fungus Aspergillus flavus QQYZ. Their structures were elucidated through high resolution electrospray ionization mass spectroscopy (HRESIMS) and nuclear magnetic resonance (NMR) spectroscopy, the absolute configurations of them were determined by a single-crystal X-ray diffraction combined with calculated electronic circular dichroism (ECD) spectra and a 1D potential energy scan. These compounds were evaluated for antifungal activities in vitro and exhibited broad-spectrum and potential antifungal activities against several pathogenic fungi with minimum inhibitory concentration (MIC) values in the range of 3.13-50 µM. They also performed moderate antibacterial activities against several bacteria with MIC values in the range of 12.5-25 µM. This research enriched the resources of lead compounds and templates for marine-derived antimicrobial drugs.

Bioactive sesquiterpene derivatives from mangrove endophytic fungus Phomopsis sp. SYSU-QYP-23: Structures and nitric oxide inhibitory activities.

Eight new sesquiterpene derivatives (2, 4-6 and 10-13), along with five known analogues were isolated from the mangrove endophytic fungus Phomopsis sp. SYSU-QYP-23. Their structures of new compounds were established by spectroscopic methods, and the absolute configurations were confirmed by single-crystal X-ray diffraction analysis and comparison of the experimental ECD spectra. The absolute configuration of the side chain in 1 was first defined by modified Mosher's method. Compounds 1-7 showed potent inhibitory activities against nitric oxide (NO) production in lipopolysaccharides (LPS) induced RAW 264.7 cells with IC50 values ranging from 8.6 to 14.5 µM. The molecular docking results implied that the bioactive sesquiterpenes may directly bind with targeting residues in the active cavity of iNOS protein.

Metabolites with Anti-Inflammatory Activity from the Mangrove Endophytic Fungus Diaporthe sp. QYM12.

One new diterpenoid, diaporpenoid A (1), two new sesquiterpenoids, diaporpenoids B-C (2,3) and three new α-pyrone derivatives, diaporpyrones A-C (4-6) were isolated from an MeOH extract obtained from cultures of the mangrove endophytic fungus Diaporthe sp. QYM12. Their structures were elucidated by extensive analysis of spectroscopic data. The absolute configurations were determined by electronic circular dichroism (ECD) calculations and a comparison of the specific rotation. Compound 1 had an unusual 5/10/5-fused tricyclic ring system. Compounds 1 and 4 showed potent anti-inflammatory activities by inhibiting the production of nitric oxide (NO) in lipopolysaccharide (LPS)-induced RAW264.7 cells with IC50 values of 21.5 and 12.5 µM, respectively.

Secondary Metabolites with α-Glucosidase Inhibitory Activity from Mangrove Endophytic Fungus Talaromyces sp. CY-3.

Eight new compounds, including two sambutoxin derivatives (1-2), two highly oxygenated cyclopentenones (7-8), four highly oxygenated cyclohexenones (9-12), together with four known sambutoxin derivatives (3-6), were isolated from semimangrove endophytic fungus Talaromyces sp. CY-3, under the guidance of molecular networking. The structures of new isolates were elucidated by analysis of detailed spectroscopic data, ECD spectra, chemical hydrolysis, 13C NMR calculation, and DP4+ analysis. In bioassays, compounds 1-5 displayed better α-glucosidase inhibitory activity than the positive control 1-deoxynojirimycin (IC50 = 80.8 ± 0.3 µM), and the IC50 value was in the range of 12.6 ± 0.9 to 57.3 ± 1.3 µM.

Furobenzotropolones A, B and 3-Hydroxyepicoccone B with Antioxidative Activity from Mangrove Endophytic Fungus Epicoccum nigrum MLY-3.

Three new metabolites, furobenzotropolones A, B (1-2) with unusual benzene and dihydrofuran moieties and 3-hydroxyepicoccone B (3), together with seven known compounds (4-10) were obtained from the endophytic fungus Epicoccum nigrum MLY-3 isolated from the fresh leaf of mangrove plant Bruguiear gymnorrhiza collected from Zhuhai. Their structures were assigned by the analysis of UV, IR, NMR, and mass spectroscopic data. Compound 1 was further confirmed by single-crystal X-ray diffraction experiment using Cu Kα radiation. In antioxidant activities in vitro, compounds 2, 3, 5, and 8 showed promising DPPH· scavenging activity with IC50 values ranging from 14.7 to 29.3 µM. Compounds 2, 3, 5, 7, and 8 exhibited promising potent activity in scavenging ABTS· with IC50 values in the range of 18-29.2 µM, which was stronger than that of the positive control ascorbic acid (IC50 = 33.6 ± 0.8 µM).

Anti-inflammatory activities of alkaloids from the mangrove endophytic fungus Phomopsis sp. SYSUQYP-23.

Five new maleimide derivatives, (+)- and (-)- farinomalein F (1), (+)- and (-)- farinomalein G (2), farinomalein H (3) and one new linearly fused prenylated indole alkaloid phomoamide (8), along with five known compounds 4-7 and 9 were isolated from the mangrove endophytic fungus Phomopsis sp. SYSUQYP-23. Their structures and absolute configurations were determined by HRESIMS, spectroscopic and electronic circular dichroism (ECD) calculations. Bioassay results showed that compounds 3-9 exhibited significant inhibitory activities against nitric oxide (NO) production in lipopolysaccharides (LPS) induced RAW 264.7 cells, with IC50 values ranging from 4.5 to 25 µM. Moreover, the molecular docking study implied the probable binding interaction of compounds 4 and 5 with nitric oxide synthase.

An anti-inflammatory isoflavone from soybean inoculated with a marine fungus Aspergillus terreus C23-3.

A new isoflavone derivative compound 1 (psoralenone) was isolated from soybean inoculated with a marine fungus Aspergillus terreus C23-3, together with seven known compounds including isoflavones 2-6, butyrolactone I (7) and blumenol A (8). Their structures were elucidated by MS, NMR, and ECD. Psoralenone displayed moderate in vitro anti-inflammatory activity in the LPS-induced RAW264.7 cell model. Compound 2 (genistein) showed moderate acetylcholinesterase (AChE) inhibitory activity whereas compounds 2, 5 (biochanin A), 6 (psoralenol), and 7 exhibited potent larvicidal activity against brine shrimp. Compounds 3 (daidzein), 4 (4'-hydroxy-6,7-dimethoxyisoflavone), and 5-7 showed broad-spectrum anti-microbial activity, and compound 7 also showed moderate 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity.

Benzopyran Derivatives and an Aliphatic Compound from a Mangrove Endophytic Fungus Penicillium citrinum QJF-22.

Two new benzopyran derivatives, (2R,4S)-5-methoxy-2-methyl-3,4-dihydro-2H-1-benzopyran-4-ol and (2S,4R,2'S,4'R)-4,4'-oxybis(5-methoxy-2-methyl-3,4-dihydro-2H-1-benzopyran), and a new aliphatic compound, (3E,5Z,8S,10E)-8-hydroxytrideca-3,5,10,12-tetraen-2-one, together with three known benzopyran derivatives, were obtained from a mangrove endophytic fungus Penicillium citrinum QJF-22 collected in Hainan island. Their structures were determined by analysis of spectroscopic data and the relative configuration of (2R,4S)-5-methoxy-2-methyl-3,4-dihydro-2H-1-benzopyran-4-ol was also confirmed by single-crystal X-ray diffraction. The absolute configurations of four compounds were established by comparison of ECD spectra to calculations. The configuration of (3E,5Z,8S,10E)-8-hydroxytrideca-3,5,10,12-tetraen-2-one was confirmed by comparison of optical value to the similar compound. The configurations of the compounds (2S,4S)-5-methoxy-2-methyl-3,4-dihydro-2H-1-benzopyran-4-ol and (2R,4R)-5-methoxy-2-methyl-3,4-dihydro-2H-1-benzopyran-4-ol were first determined. (3R,4S)-3,4,8-Trihydroxy-3,4-dihydronaphthalen-1(2H)-one exhibited moderate inhibitory effects on LPS-induced NO production in RAW264.7 cells with IC50 of 44.7 µM, and without cytotoxicity to RAW264.7 cells within 50 µM.

Chemical Composition and Anti-Alzheimer's Disease-Related Activities of a Functional Oil from the Edible Seaweed Hizikia fusiforme.

Cholinergic disorder, oxidative stress, and neuroinflammation play important roles in the pathology of Alzheimer's disease. To explore the healthy potential of the edible seaweed Hizikia fusiforme on this aspect, a functional oil (HFFO) was extracted from this alga and investigated on its constituents by gas chromatography-mass spectrometry (GC/MS) in this study. Its anti-Alzheimer's related bioactivities including acetylcholinesterase (AChE) inhibition, antioxidation, and anti-neuroinflammation were evaluated, traced, and simulated by in vitro and in silico methods. GC/MS analysis indicated that HFFO mainly contained arachidonic acid (ARA), 11,14,17-eicosatrienoic acid (ETrA), palmitic acid, phytol, etc. HFFO showed moderate AChE inhibition and antioxidant activity. Bioactivity tracing using commercial standards verified that AChE inhibition of HFFO mainly originated from ARA and ETrA, whereas antioxidant activity mainly from ARA. Lineweaver-Burk plots showed that both ARA and ETrA are noncompetitive AChE inhibitors. A molecular docking study demonstrated low CDOCKER interaction energy of -26.33 kcal/mol for ARA and -43.70 kcal/mol for ETrA when interacting with AChE and multiple interactions in the ARA (or ETrA)-AChE complex. In the anti-neuroinflammatory evaluation, HFFO showed no toxicity toward BV-2 cells at 20 µg/mL and effectively inhibited the production of nitroxide and reduced the level of reactive oxygen species in lipopolysaccharide-induced BV-2 cells. The results indicated that HFFO could be used in functional foods for its anti-Alzheimer's disease-related activities.

Depsidone Derivatives and a Cyclopeptide Produced by Marine Fungus Aspergillus unguis under Chemical Induction and by Its Plasma Induced Mutant.

A new depsidone derivative (1), aspergillusidone G, was isolated from a marine fungus Aspergillus unguis, together with eight known depsidones (2‒9) and a cyclic peptide (10): agonodepside A (2), nornidulin (3), nidulin (4), aspergillusidone F (5), unguinol (6), aspergillusidone C (7), 2-chlorounguinol (8), aspergillusidone A (9), and unguisin A (10). Compounds 1‒4 and 7‒9 were obtained from the plasma induced mutant of this fungus, while 5, 6, and 10 were isolated from the original strain under chemical induction. Their structures were identified using spectroscopic analysis, as well as by comparison with literature data. The HPLC fingerprint analysis indicates that chemical induction and plasma mutagenesis effectively influenced the secondary metabolism, which may be due to their regulation in the key steps in depsidone biosynthesis. In bioassays, compound 9 inhibited acetylcholinesterase (AChE) with IC50 in 56.75 µM. Compounds 1, 5, 7, 8, and 9 showed moderate to strong activity towards different microbes. Compounds 3, 4, and 5 exhibited potent larvicidality against brine shrimp. In docking studies, higher negative CDOCKER interaction energy and richer strong interactions between AChE and 9 explained the greater activity of 9 compared to 1. Chemical induction and plasma mutagenesis can be used as tools to expand the chemodiversity of fungi and obtain useful natural products.