New cyathane diterpenoids with neurotrophic and anti-neuroinflammatory activity from the bird's nest fungus Cyathus africanus.

Eleven new cyathane diterpenoids, designated cyafricanins A-K (1-11), were isolated from the culture broth of the baisidiomycete Cyathus africanus (Nidulariaceae, Bird's nest fungi). Their structures were elucidated by comprehensive analysis of their NMR and HRESIMS data. Cyafricanins A (1) was found to possess an unusual 3,4-seco­carbon skeleton. All compounds were evaluated for their neurotrophic activity in PC-12 cells and anti-neuroinflammatory activity in BV2 microglia cells. All of the diterpenoids showed nerve growth factor induced neurite outgrowth-promoting activity at concentration of 20 µM. Among them, cyafricanin B (2) and cyafricanin G (7) exhibited promising neurotrophic activity, and cyafricanin A (1) showed strong inhibitory effects on both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. Furthermore, molecular docking studies revealed that cyafricanin A (1) showed strong interactions with the iNOs protein in the active cavity.

Molecular Diversity and Potential Anti-neuroinflammatory Activities of Cyathane Diterpenoids from the Basidiomycete Cyathus africanus.

Ten new polyoxygenated cyathane diterpenoids, named neocyathins A-J (1-10), together with four known diterpenes (11-14), were isolated from the liquid culture of the medicinal basidiomycete fungus Cyathus africanus. The structures and configurations of these new compounds were elucidated through comprehensive spectroscopic analyses including 1D NMR, 2D NMR (HSQC, HMBC, NOESY) and HRESIMS, and electronic circular dichroism (ECD) data. Neuroinflammation is implicated in the pathogenesis of various neurodegenerative diseases, such as Alzheimers' disease (AD). All isolated compounds were evaluated for the potential anti-neuroinflammatory activities in BV2 microglia cells. Several compounds showed differential effects on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-stimulated and Aß1-42-treated mouse microglia cell line BV-2. Molecular docking revealed that bioactive compounds (e.g., 11) could interact with iNOS protein other than COX-2 protein. Collectively, our results suggested that this class of cyathane diterpenoids might serve as important lead compounds for drug discovery against neuroinflammation in AD.

Pyristriatins A and B: Pyridino-Cyathane Antibiotics from the Basidiomycete Cyathus cf. striatus.

Two novel pyridino-cyathane diterpenoids, pyristriatins A and B (1 and 2), together with striatin C (3) were isolated from cultures of Cyathus cf. striatus, a basidiomycete that was found during a field trip in northern Thailand. The pyristriatins showed antimicrobial effects against Gram-positive bacteria and fungi. The isolation, structure elucidation, relative configuration, and biological and cytotoxic activity are described. Their structures were assigned by HRMS and NMR spectroscopy. We also describe the first 2D NMR assignment of striatin C. Pyristriatins A and B are the first cyathane natural products featuring a pyridine ring.

Three new cyathane diterpenoids from the medicinal fungus Cyathus africanus.

Three new cyathane diterpenoids, cyathin W (1), cyathin V (2), and cyathin T (3), were isolated from the solid culture of Cyathus africanus. The structures and configurations of these new compounds were elucidated on the basis of comprehensive spectroscopic analysis including 1D NMR, 2D NMR (HSQC, HMBC, NOESY), and HR-ESI-MS experiments. Compounds 1 and 3 showed moderate inhibition against nitric oxide production in lipopolysaccaride-activated macrophages with IC50 value of 80.07 and 88.87 µM, respectively. In cytotoxicity assay, compound 1 showed weak cytotoxicity against K562 cell line with IC50 value of 12.1 µM.

Striatoids A-F, Cyathane Diterpenoids with Neurotrophic Activity from Cultures of the Fungus Cyathus striatus.

Six new highly oxygenated polycyclic cyathane-xylosides, named striatoids A-F (1-6), were isolated from the cultures of the basidiomycete Cyathus striatus. Their structures were established by comprehensive spectroscopic analysis including 2D NMR (HMBC, HSQC, ROESY, (1)H-(1)H-COSY) and HRESIMS experiments. Compounds 2 and 3 possess an unusual 15,4'-ether ring system. The isolated compounds dose-dependently enhanced nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma (PC-12) cells.

Development of an enzyme linked immunosorbent assay for detection of cyathane diterpenoids.

BACKGROUND: So-called cyathane type diterpenoids are produced as secondary metabolites by basidiomycetes. Based on their antibacterial, fungicidal, and cytotoxic properties, cyathane type terpenoids represent interesting target compounds in fungal biotechnology. RESULTS: An indirect competitive enzyme linked immunosorbent assay has been developed for detection of cyathane type diterpenoids. Rabbit polyclonal antibodies were raised against a mixture of striatal A and B conjugated to bovine serum albumin. The conditions for direct attachment of the hapten striatal B to a solid phase by passive adsorption were optimized. The cross reactivities of the striatals A, C and D, of the striatins A and B, and of the erinacines C and P to striatal B were determined. The validation study showed that the ELISA was precise and sensitive. The average IC50 of striatal B was 36.0 ng mL-1 with an inter-assay coefficient of variation (CV) of 13.2% (n = 5). Recoveries from striatal B spiked samples in the assay were in the range of 97.3 - 125.9%. A good correlation between the striatal B concentration measured by the ELISA and by HPLC-DAD (y = 1.1122× - 0.1585, R2 = 0.9942) was obtained from linear regression analysis. The suitability of the ELISA for detection of cyathane type diterpenoids in submerged cultures and fruiting bodies of H. erinaceus was studied. It showed cross reactivity with supernatants from submerged cultures and extracts thereof, but did not show cross reactivity with extracts from fruiting bodies. CONCLUSIONS: The developed method is appropriate for qualitative and quantitative detection of cyathane diterpenoids in complex mixtures. Due to its high sensitivity and specificity, it represents an ideal screening method for discovering new cyathane diterpenoids and new potential producers of them.

Structural insights into 2,2'-azino-Bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)-mediated degradation of reactive blue 21 by engineered Cyathus bulleri Laccase and characterization of degradation products.

Advanced oxidation processes are currently used for the treatment of different reactive dyes which involve use of toxic catalysts. Peroxidases are reported to be effective on such dyes and require hydrogen peroxide and/or metal ions. Cyathus bulleri laccase, expressed in Pichia pastoris, catalyzes efficient degradation (78 to 85%) of reactive azo dyes (reactive black 5, reactive orange 16, and reactive red 198) in the presence of synthetic mediator ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)]. This laccase was engineered to degrade effectively reactive blue 21 (RB21), a phthalocyanine dye reported to be decolorized only by peroxidases. The 816-bp segment (toward the C terminus) of the lcc gene was subjected to random mutagenesis and enzyme variants (Lcc35, Lcc61, and Lcc62) were selected based on increased ABTS oxidizing ability. Around 78 to 95% decolorization of RB21 was observed with the ABTS-supplemented Lcc variants in 30 min. Analysis of the degradation products by mass spectrometry indicated the formation of several low-molecular-weight compounds. Mapping the mutations on the modeled structure implicated residues both near and far from the T1 Cu site that affected the catalytic efficiency of the mutant enzymes on ABTS and, in turn, the rate of oxidation of RB21. Several inactive clones were also mapped. The importance of geometry as well as electronic changes on the reactivity of laccases was indicated.

Production of bioactive cyathane diterpenes by a bird's nest fungus Cyathus gansuensis growing on cooked rice.

Cyathane diterpenes are important bioactive substances produced by some edible and medicinal fungi. Seven new cyathane type diterpenes, named as cyathins J-P (1-7), together with two known diterpenes (8 and 9), were isolated from the solid culture of the bird's nest fungus Cyathus gansuensis growing on cooked rice. The structures of the new secondary metabolites were elucidated by NMR experiments. Bioactivity screening indicated that compounds 1, 2, 4, and 8 showed moderate inhibitory activity against NO production in lipopolysaccharide-activated macrophages with an IC50 value of 42, 78, 80, and 16 µM, respectively. The fungus C. gansuensis is a promising source of bioactive secondary metabolites and has application potential in preparing healthy food.

Analysis of cyathane-type diterpenoids from Cyathus striatus and Hericium erinaceus by high-resolution MALDI MS imaging.

Fungal secondary metabolites in both fruiting bodies and pellets from submerged cultures of basidiomycetes were analyzed by atmospheric pressure matrix-assisted laser desorption/ionization-mass spectrometry imaging at a lateral resolution of 15 µm, a mass resolution of 140,000 at m/z 200 and a mass accuracy of better than 2 ppm. The striatals A, B, C, and D, and a number of erinacine type metabolites were detected in the basidiomycetes Cyathus striatus and Hericium erinaceus, respectively. The two fungi were selected as model species, as they are well-known for efficient production of terpenoid secondary metabolites with interesting biological activities, e.g., antibacterial, fungicidal, cytotoxic properties, and stimulating effects on nerve growth factor synthesis. The localization of metabolites revealed a mostly homogeneous distribution of the striatals in the pellets of C. striatus, while a concentration gradient, increasing to the center, was observed in the pellets of H. erinaceus. A mostly homogeneous distribution of metabolites was also found in the fruiting body of H. erinaceus.

Isolation and identification of a new anti-inflammatory cyathane diterpenoid from the medicinal fungus Cyathus hookeri Berk.

A new cyathane diterpene, named as cyathin I (1), as well as two known cyathane diterpenes (12R)-11a,14a-epoxy-13a,14b,15-trihydroxycyath-3-ene (2) and erinacine I (3), were isolated from the liquid culture of Cyathus hookeri Berk. Their structures were elucidated on the basis of extensive spectroscopic analysis. Compounds 1-3 showed inhibition against nitric oxide production in macrophages with an IC50 value of 15.5, 52.3, and 16.8 µM, respectively.

Anti-inflammatory and cytotoxic cyathane diterpenoids from the medicinal fungus Cyathus africanus.

Five novel cyathane diterpenes, cyathins D-H (1-5), as well as three known diterpenes, neosarcodonin O (6), cyathatriol (7),and 11-O-acetylcyathatriol (8), were isolated from the solid culture of Cyathus africanus. The structures of the new compounds were elucidated by spectroscopic methods. The absolute configurations of compounds 2 and 8 were determined by single-crystal X-ray crystallographic analysis, whereas the absolute configuration of C-14 in 1 was determined via the circular dichroism data of the [Rh(2)(OCOCF(3))(4)] complex. Compounds 3, 5, 6, 8, and 9 showed potent inhibition against nitric oxide production in lipopolysaccaride-activated macrophages with an IC(50) value of 2.57, 1.45, 12.0, 10.73, and 9.45µM, respectively. Compounds 6 and 8 showed strong cytotoxicity against Hela and K562 cell lines with the IC(50) value less than 10µM.

Total synthesis of cyathin A(3) and cyathin B(2).

A stereoselective synthesis of cyathin A(3) and cyathin B(2) has been achieved by a Prins-type reaction of a cycloalkenyl cyclopropanol. Particularly noteworthy is the use of a spirocyclobutanone moiety as a convenient scaffold for an efficient ring-closing metathesis to stereoselectively construct a suitably functionalized seven-membered ring (see scheme).