The oxygenated products of cryptotanshinone by biotransformation with Cunninghamella elegans exerting anti-neuroinflammatory effects by inhibiting TLR 4-mediated MAPK signaling pathway.

Cryptotanshinone (1), a major bioactive constituent in the traditional Chinese medicinal herb Dan-Shen Salvia miltiorrhiza Bunge, has been reported to possess remarkable pharmacological activities. To improve its bioactivities and physicochemical properties, in the present study, cryptotanshinone (1) was biotransformed with the fungus Cunninghamella elegans AS3.2028. Three oxygenated products (2-4) at C-3 of cryptotanshinone (1) were obtained, among them 2 was a new compound. Their structures were elucidated by comprehensive spectroscopic analysis including HRESIMS, NMR and ECD data. All of the biotransformation products (2-4) were found to inhibit significantly lipopolysaccharide-induced nitric oxide production in BV2 microglia cells with the IC50 values of 0.16-1.16 µM, approximately 2-20 folds stronger than the substrate (1). These biotransformation products also displayed remarkably improved inhibitory effects on the production of inflammatory cytokines (IL-1ß, IL-6, TNF-α, COX-2 and iNOS) in BV-2 cells via targeting TLR4 compared to substrate (1). The underlying mechanism of 2 was elucidated by comparative transcriptome analysis, which suggested that it reduced neuroinflammatory mainly through mitogen-activated protein kinase (MAPK) signaling pathway. Western blotting results revealed that 2 downregulated LPS-induced phosphorylation of JNK, ERK, and p38 in MAPK signaling pathway. These findings provide a basal material for the discovery of candidates in treating Alzheimer's disease.

Annular oxygenation and rearrangement products of cryptotanshinone by biotransformation with marine-derived fungi Cochliobolus lunatus and Aspergillus terreus.

Structural modification of natural products by biotransformation with fungi is an attractive tool to obtain novel bioactive derivatives. In the present study, cryptotanshinone (1), a quinoid abietane diterpene from traditional Chinese medicine Salvia miltiorrhiza (Danshen), was transformed by two marine-derived fungi. By using Cochliobolus lunatus TA26-46, one new oxygenated and rearranged product (2), containing a 5,6-dihydropyrano[4,3-b]chromene moiety, together with one known metabolite (10), were obtained from the converted broth of cryptotanshinone (1) with the isolated yields of 1.0% and 2.1%, respectively. While, under the action of Aspergillus terreus RA2905, seven new transformation products (3-9) as well as 10 with the fragments of 2-methylpropan-1-ol and oxygenated p-benzoquinone were produced and obtained with the isolated yields of 0.1%-1.3%. The structures of the new compounds were elucidated by comprehensive spectroscopic analysis including High Resolution Electrospray Ionization Mass Spectroscopy (HRESIMS), Nuclear Magnetic Resonance (NMR) and Electronic Circular Dichroism (ECD). The metabolic pathways of cryptotanshinone by these two fungi were presumed to be the opening and rearrangement of furan ring, and/or oxygenation of cyclohexane ring. Cryptotanshinone (1) and its metabolites displayed anti-inflammatory activities against NO production in LPS-stimulated BV-2 cells and antibacterial activities towards methicillin-resistant Staphylococcus aureus. These findings revealed the potential of marine fungi to transform the structures of natural products by biotransformation.

New Thiodiketopiperazine and 3,4-Dihydroisocoumarin Derivatives from the Marine-Derived Fungus Aspergillus terreus.

Aspergillus terreus has been reported to produce many secondary metabolites that exhibit potential bioactivities, such as antibiotic, hypoglycemic, and lipid-lowering activities. In the present study, two new thiodiketopiperazines, emestrins L (1) and M (2), together with five known analogues (3-7), and five known dihydroisocoumarins (8-12), were obtained from the marine-derived fungus Aspergillus terreus RA2905. The structures of the new compounds were elucidated by analysis of the comprehensive spectroscopic data, including high-resolution electrospray ionization mass spectrometry (HRESIMS), one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR), and electronic circular dichroism (ECD) data. This is the first time that the spectroscopic data of compounds 3, 8, and 9 have been reported. Compound 3 displayed antibacterial activity against Pseudomonas aeruginosa (minimum inhibitory concentration (MIC) = 32 µg/mL) and antifungal activity against Candida albicans (MIC = 32 µg/mL). In addition, compound 3 exhibited an inhibitory effect on protein tyrosine phosphatase 1 B (PTP1B), an important hypoglycemic target, with an inhibitory concentration (IC)50 value of 12.25 µM.

Benzyl Furanones and Pyrones from the Marine-Derived Fungus Aspergillus terreus Induced by Chemical Epigenetic Modification.

Chemical epigenetic modification on a marine-derived fungus Aspergillus terreus RA2905 using a histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), resulted in a significantly changed metabolic profile. A chemical investigation of its ethyl acetate (EtOAc) extract led to the isolation of a racemate of benzyl furanone racemate (±)-1, which further separated chirally as a pair of new enantiomers, (+)- and (-)-asperfuranone (1), together with two new benzyl pyrones, asperpyranones A (2) and B (3). Their structures were elucidated by analysis of the comprehensive spectroscopic data, including one-dimensional (1D) and two-dimensional (2D) NMR, and HRESIMS. The absolute configurations were determined by electronic circular dichroism (ECD) calculation and single-crystal X-ray crystallographic experiment. The structures with benzyl furanone or benzyl pyrone skeletons were discovered from natural products for the first time. Compounds (±)-1, (+)-1, (-)-1, and 2 displayed the antifungal activities against Candida albicans with MIC values of 32, 16, 64, and 64 µg/mL and PTP1B inhibitory activities with the IC50 values of 45.79, 17.32, 35.50, and 42.32 µM, respectively. Compound 2 exhibited antibacterial activity against Pseudomonas aeruginosa with the MIC value of 32 µg/mL.

Induction of Secondary Metabolite Biosynthesis by Deleting the Histone Deacetylase HdaA in the Marine-Derived Fungus Aspergillus terreus RA2905.

Aspergillus terreus is well-known for its ability to biosynthesize valuable pharmaceuticals as well as structurally unique secondary metabolites. However, numerous promising cryptic secondary metabolites in this strain regulated by silent gene clusters remain unidentified. In this study, to further explore the secondary metabolite potential of A. terreus, the essential histone deacetylase hdaA gene was deleted in the marine-derived A. terreus RA2905. The results showed that HdaA plays a vital and negative regulatory role in both conidiation and secondary metabolism. Loss of HdaA in A. terreus RA2905 not only resulted in the improvement in butyrolactone production, but also activated the biosynthesis of new azaphilone derivatives. After scaled fermentation, two new azaphilones, asperterilones A and B (1 and 2), were isolated from ΔhdaA mutant. The planar structures of compounds 1 and 2 were undoubtedly characterized by NMR spectroscopy and mass spectrometry analysis. Their absolute configurations were assigned by circular dichroism spectra analysis and proposed biosynthesis pathway. Compounds 1 and 2 displayed moderate anti-Candida activities with the MIC values ranging from 18.0 to 47.9 µM, and compound 1 exhibited significant cytotoxic activity against human breast cancer cell line MDA-MB-231. This study provides novel evidence that hdaA plays essential and global roles in repressing secondary metabolite gene expression in fungi, and its deletion represents an efficient strategy to mine new compounds from A. terreus and other available marine-derived fungi.

Sorbicillinoid Derivatives From Sponge-Derived Fungus Trichoderma reesei (HN-2016-018).

Six new sorbicillinoids, trichoreeseione A (1) and B (2), trichodermolide B (3), 13-hydroxy-trichodermolide (4), 24-hydroxy-trichodimerol (5), 15-hydroxy-bisvertinol (7), together with three known analogs, trichodimerol (6), 24-hydroxy-bisvertinol (8), and bisvertinol (9), were isolated from the sponge-derived fungus Trichoderma reesei (HN-2016-018). Their structures including absolute configurations were elucidated by analysis of NMR, MS data, and calculated ECD spectra. Compounds 1 and 2 with a characteristic naphthalene-trione ring were firstly reported in sorbicillinoid family. Compounds 3 and 4 were two rare sorbicillinoids containing a unique bicycle [3.2.1] lactone skeleton, while 3 with a propan-2-one moiety was also recorded first time in this family. Compound 5 displayed cytotoxic activity against A549, MCF-7, and HCT116 cell lines with the IC50 values of 5.1, 9.5, and 13.7 µM, respectively.

Epigenetic Agents Trigger the Production of Bioactive Nucleoside Derivatives and Bisabolane Sesquiterpenes From the Marine-Derived Fungus Aspergillus versicolor.

Epigenetic agents, histone deacetylase inhibitor (SAHA) and DNA methyltransferase inhibitor (5-Aza), were added to Czapek-Dox medium to trigger the chemical diversity of marine-derived fungus Aspergillus versicolor XS-20090066. By HPLC and 1H NMR analysis, the diversity of fungal secondary metabolites was significantly increased compared with the control. With the aid of MS/MS-based molecular networking, two new nucleoside derivatives, kipukasins K (1) and L (2) were obtained. Meanwhile, the yields of four known nucleoside derivatives were significantly enhanced. In addition, one new bisabolane sesquiterpene, aspergillusene E (7), along with ten known derivatives were also isolated. The structures were elucidated by comprehensive spectroscopic methods of NMR and HRESIMS analysis. Compounds 1 and 7 displayed antibacterial activities against Staphylococcus epidermidis and Staphylococcus aureus with the MIC values of 8-16 µg/mL. Our study revealed that the fungus A. versicolor XS-20090066 has been effectively induced by chemical epigenetic manipulation with a combination of SAHA and 5-Aza to produce new metabolites.

Terpenoids From the Coral-Derived Fungus Trichoderma harzianum (XS-20090075) Induced by Chemical Epigenetic Manipulation.

The soft coral-derived fungus Trichoderma harzianum (XS-20090075) was found to be a potential strain to produce substantial new compounds in our previous study. In order to explore its potential to produce more metabolites, chemical epigenetic manipulation was used on this fungus to wake its sleeping genes, leading to the significant changes of its secondary metabolites by using a histone deacetylase (HDAC) inhibitor. The most obvious difference was the original main products harziane diterpenoids were changed into cyclonerane sesquiterpenoids. Three new terpenoids were isolated from the fungal culture treated with 10 µM sodium butyrate, including cleistanthane diterpenoid, harzianolic acid A (1), harziane diterpenoid, harzianone E (2), and cyclonerane sesquiterpenoid, 3,7,11-trihydroxy-cycloneran (3), together with 11 known sesquiterpenoids (4-14). The absolute configurations of 1-3 were determined by single-crystal X-ray diffraction, ECD and OR calculations, and biogenetic considerations. This was the first time to obtain cleistanthane diterpenoid and africane sesquiterpenoid from genus Trichoderma, and this was the first chlorinated cleistanthane diterpenoid. These results demonstrated that the chemical epigenetic manipulation should be an efficient technique for the discovery of new secondary metabolites from marine-derived fungi.

Co-cultivation With 5-Azacytidine Induced New Metabolites From the Zoanthid-Derived Fungus Cochliobolus lunatus.

The zoanthid-derived fungus Cochliobolus lunatus (TA26-46) has been proven to be a source of bioactive 14-membered resorcylic acid lactones (RALs). In the present study, chemical epigenetic manipulation was applied to this fungal strain with a DNA methyltransferase inhibitor resulting in the significant changes of the secondary metabolites. Cultivation of C. lunatus (TA26-46) with 10 µM 5-azacytidine in Czapek-Dox liquid medium led to the isolation of new types of metabolites, including two α-pyrones, cochliobopyrones A (1) and B (2), along with three isocoumarins (3-5) and one chromone (6). The planar structures of the new compounds (1-2) were elucidated by comprehensive analyses of NMR and HRESIMS data. Their challenging relative configurations were established by a combination of acetonide reaction, coupling constants and NOESY correlations analysis, and DP4+ probability calculation. Their absolute configurations were determined by comparing with the ECD calculation data of the fragment molecules, 6-(1,2-dihydroxypropyl)-4-methoxy-2H-pyran-2-ones. It is the first time to obtain α-pyrone compounds with the epoxy ring or bromine atom on the seven-numbered side chain. It could be concluded that chemical epigenetic agents could induce C. lunatus to produce new types of secondary metabolites differing from its original products (RALs).

Biotransfomation of cyperenoic acid by Cunninghamella elegans AS 3.2028 and the potent anti-angiogenic activities of its metabolites.

Cyperenoic acid (1) is one of the major sesquiterpenes isolated from Croton crassifolius, which exhibited potent anti-angiogenic activity. Traditional structural modification of 1 is difficult to perform by chemical technology due to the remarkable stability of the patchoulane skeleton. In order to overcome chemical synthesis difficulties and obtain structurally diverse derivations, microbial transformation of 1 by using Cunninghamella elegans AS 3.2028 was studied for the first time. Five new hydroxylated products 2-6 were obtained. Furthermore, cytotoxicity and anti-angiogenic activities of all the biotransformation products were evaluated by MTT assay and ELISA in HepG2 and MCF-7 cells. These results indicated that hydroxylated modification products 2-4 significantly inhibited VEGF release, which suggest the potential use of hydroxylated modification products for cancer therapy.