Galewone, an Anti-Fibrotic Polyketide from Daldinia eschscholzii with an Undescribed Carbon Skeleton

A novel polyphenolic natural product, galewone, with undescribed carbon skeleton, was isolated as a racemate from the culture of Daldinia eschscholzii IFB-TL01, a fungus obtained from the mantis (Tenodera aridifolia) gut. The galewone structure was elucidated by a combination of MS and NMR spectra, and substantiated by X-ray crystallographic diffraction. The absolute stereochemistry of each galewone enantiomers was determined by the CD spectrum. In compliance of the structural similarities, galewone might be the shunt products of the dalesconol biosynthetic pathway. Both (−)- and ( + )-galewones were evaluated to be anti-fibrotic against activated hepatic stellate cell line, CFSC-8B, with the IC50 values being 3.73 ± 0.21 and 10.10 ± 0.41 μM, respectively. Thus, galewone may serve as a starting molecule for the discovery of new anti-fibrotic drug.


Results
Owing to its production of a series of bioactive naphthol radical polymerized metabolites with undescribed architectures, the mantis-associated fungus Daldinia eschscholzii IFB-TL01 has attracted remarkable attention [8][9][10][11][12][13] . As part of our continuing research on discovery of anti-fibrotic metabolites, the EtOAc extract of the D. eschscholzii culture was selected for investigation. As a result, the new naphthol derivative, galewone, was characterized and demonstrated to possess a promising potential in anti-fibrotic action. Herein, we present the structure elucidation, bioactivity evaluation, and plausible biogenetic pathway of galewone.
Galewone afforded as red needles, was ascertained to have a molecular formula of C 32 H 20 O 9 from the sodium-liganded molecular ion at m/z 571.09910 (calcd. for C 32 H 20 O 9 Na, 571.09995) in its high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). This observation, along with its 1D and 2D NMR spectra (Table S2 and Figs 6-11 suggested that it was probably hydroxymethylated spirodalesol 12 . The 1 H NMR spectrum of galewone suggested the presence of two groups of ortho-coupled protons (δ H 7.01/H-15 and 7.28/H-16; δ H 6.94/H-27 and 7.48/H-28), two groups of three ortho-protons (δ H 6.97/H-4, 7.68/H-5, and 7.75/H-6; δ H 6.54/H-21, 6.99/H-22, and 6.77/H-23), one hydroxyethyl group (δ H 4.85), one methoxyl group (δ H 3.59), and three phenolic protons (δ H 8.79/HO-3, 12.78/HO-14, and 12.70/HO-24). The 13 C NMR and DEPT experiments evidenced the presence of 32 carbons grouped, including three carbonyl signals (δ C 197.6/C-1, 189.6/C-12, and 189.8/C-26). The most possible structure of galewone was assigned via the HMBC correlations of the H-6 with δ C 124.1/C-2 and 139.8/C-8, of the H-10 with δ C 139.8/C-8, 189.6/C-12 and 132.3/C-18, and of the H-16 with δ C 162.0/C-14, 132.3/C-18 and 47.5/C-19, and of the H-21 with δ C 47.5/C-19, 117.8/C-23 and 115.1/C-25, and of the H-31 with δ C 131.6/C-10 and 189.6/C-12. Subsequently, the novel skeleton and relative configuration of galewone were confirmed by its single crystal X-ray diffraction (Fig. 1). Since galewone was obtained as a racemic mixture, chiral HPLC separation was performed to give two enantiomers ( + )-galewone and (−)-galewone, which were disclosed to possess (19 R,29 S)-and (19 S,29 R)-configurations (Fig. 2), respectively, by comparing their CD curves with the ECD spectra computed for all optional stereoisomers. In the calculated curves, the first Cotton effect centered at 218 nm, arising from the electronic transition from filled C=C bonding molecular orbital (MO, π C=C ) to the anti-bonding C-C orbital (σ −  Table S4), corresponded to the peak at 218 nm in the acquired CD spectra. The next Cotton effect at 321 nm in the recorded CD spectra could be correlated to the shoulder peaks at 298 and 351 nm in the computed counterparts. The electronic transitions from π C=C and the lone pair orbital of oxygen (n O ) to the π = ⁎ C C orbital contributed to these absorption bands (MO 140 → 145 and MO 137 → 143). In addition, the Cotton effects at 251 and 375 nm in the acquired CD spectra were also reproduced by the calculations at 236 and 374 nm, respectively. The electronic transitions from π C=C to the π = ⁎ C C orbitals contributed to these absorption bands (MO 133 → 145 and MO 142 → 145, Table S4).
With the absolute configuration of galewone enantiomers determined, we initiated their biosynthesis through probing into their possible naphthol precursors. The structural similarities between galewone and dalesconols suggested that they might share a similar mechanism to perform their biosynthesis 10 . Galewone embodied two sets of intramolecular hydrogen bonds between phenolic hydroxyl group and adjacent carbonyl group similar to those of dalesconols A-C (Fig. 3), together with that they each consisted of seven rings. The compounds obtained from six-acetyl-CoA 9 provided the possible naphthol radicals, which possessed unique carbon chains and participated in the formation of galewone. For instance, the existence of key intermediate 2-(hydroxymethyl)-naphthalene-1,8-diol ( Fig. 4) was rationalized by such related compound as 3,4,8-trihydroxy-6-(hydroxymethyl)-3,4-dihydronaphthalen-1(2 H)-one 9 . These facts demonstrated that galewone might originate from an similar biogenetic mechanism analogous to that of dalesconols, which initiated by the combination of three naphthol radicals (Fig. 4).
Activated hepatic stellate cells, such as CFSC-8B, have a strong migration capability and express some proteins aggravating liver fibrosis. Inhibition of the proliferation of CFSC-8B could prevent the migration and alleviate liver fibrosis. In our study, wound-healing and invasion chamber assay suggested that (−)-galewone blocked migration of activated hepatic stellate cells. In model group, FBS could induce wound closure 24 h after wound  www.nature.com/scientificreports www.nature.com/scientificreports/ in gowning to CFSC-8B cells migration. However, activated hepatic stellate cells migration induced by FBS was dramatically and dose-dependently inhibited by (−)-galewone as indicated by larger the wounded area (Fig. 5a). Moreover, in BIOCOAT MATRIGEL chamber systems,10% FBS incubation with CFSC-8B cells induced migration of activated hepatic stellate cells was also significantly inhibited by (−)-galewone (Fig. 5b,c). Previous report suggested that activated hepatic stellate cells were responsible for the enhanced expression of α-SMA, Col1A1 and Col3A1 18,19 . As determined by Western bloting, (−)-galewone addition suppressed the expression of the marked proteins of CFSC-8B cells in a dose-dependent manner (Fig. 5d,e).

Discussion
In conclusion, we have discovered a structurally intriguing metabolite from Daldinia eschscholzii IFB-TL01, and proposed a possible pathway for its formation. The naphthol radicals as unique starter units might be coupled with intermolecular logic participating in the construction of the neat sequential compartmentalization of unforeseeable seven ring frameworks, and this finding could set the foundation for further characterization of distinctive radical machineries to produce galewone analogues with more complex biological functionalizations by combinatorial biosynthesis. Specifically, the anti-fibrotic activities of (−)-, (+)-, and (±)-galewone were observed to reduce the cell viability of activated hepatic stellate cells, but to inhibit very weakly that of Lx-2 cells in quiescent phase (Table S1), indicating that galewone may serve as a starting molecule for the discovery of new anti-fibrotic drug. www.nature.com/scientificreports www.nature.com/scientificreports/ experimental Section ethics approval. Specific pathogen-free, 8-10-week-old female BALB/c mice were purchased from Experimental Animal Center of Jiangsu Province (Jiangsu, China). They were maintained with free access to pellet food and water in plastic cages at 25 ± 2 °C and kept on a 12 h light/dark cycle. Animal welfare and experimental procedures were carried out strictly in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, the United States) and the related ethical regulations of our university. All efforts were made to minimize animals' suffering and to reduce the number of animals used.

Statement.
All methods were carried out in accordance with relevant guidelines and regulations, in addition all experimental protocols were approved by Nanjing University and Hohai University.
General instrument and fungal Strain. As described 8,13 . cultivation and metabolites isolation. D. eschscholzii IFB-TL01 was cultured on slants of potato dextrose agar (PDA) at 25 °C for 5 days. The fresh mycelium taken from the fungal colony in the Petri Dish was inoculated into the 1000 mL conical flasks each containing 300 mL of malt-extract medium (20 g/L malt extract, 20 g/L sucrose, 1 g/L peptone), and then the culture was shaken at 28 °C and 200 rpm/min for 3 days. The fermentation was repeated (6 × 300 mL) in order to inoculate into a 50 L fermentor containing 30 L of sterilized malt-extract medium. After a subsequent fermentation for 36 h, these were then seeded into scale-up cultures (350 L malt-extract broth in 500 L fermentor) at 25 °C and 100 rpm for 7 days (antifoam 0.1‰, aeration rate 8000 NL/h). The broth was collected and extracted with EtOAc, and a solid (302.7 g) obtained from in vauo evaporation of solvent of the extract was subjected to column chromatography (CC) over silica gel (1000 g, 300-400 mesh, 50 × 15 cm) eluted with CH 2 Cl 2 /MeOH mixtures (v/v 100:0, 100:1, 100:2, 100:4,100:8, 100:16, 100:32, 0:100). Further purification of the "100:4"-eluted CC fraction was accomplished by gel filtration over Sephadex LH-20 in MeOH, followed by HPLC separation to afford galewone (3 mg).
Single crystal X-ray diffraction. The structure was solved by direct methods (SHELXS-97) and refined to optimize the geometries of the studied systems, taking crystal structures as the original configuration. The solvent effects on the electronic structures of the concerned systems were evaluated by quantum chemistry method through the polarizable continuum model (PCM, dielectric constant ε = 32.64 for CH 3 OH). Then, the corresponding excited-state calculations were performed at the ground-state optimized geometries. Time-dependent DFT in combination with PCM model (TD-DFT/PCM) with the same basis set was carried out to calculate the spin-allowed excitation energy and rotatory strength of the lowest 100 excited states. The UV and ECD spectra were generated using the program SpecDis by applying a Gaussian band shape with the width of 0.20 eV, from oscillator strengths and dipole-velocity rotational strengths, respectively. All the calculations were performed with the Gaussian09 program. cell culture and Mtt proliferation Assay. Lymph node cells isolated from female C57BL/6 mice were maintained in RPMI 1640 medium supplemented with 100 μgmL −1 of streptomycin, 100 UmL −1 of penicillin and 10% fetal calf serum under a humidified 5% (v/v) CO 2 atmosphere at 37 °C. To activate lymph node cells, 5 μgmL −1 concanavalin A (Con A) was used. For the MTT proliferation assay, lymph node cells were cultured in 96-well plates either at a density of 3 × 10 5 cells/well in RPMI 1640 medium (0.2 mL), if stimulated with 5 μgmL −1 of Con-A, or at 5 × 10 5 cells/well in the same medium but without Con-A. After a 48 h treatment with and without the test samples ((−)-, (+)-, and (±)-galewone) at various concentrations, MTT (Sigma; 4 mg mL −1 in PBS; 20 mL) was added to each well for a subsequent 4 h incubation. Subsequently, the supernatant was discarded, DMSO (200 mL) was added, and the OD540 was determined by using an ELISA reader (Sunrise, Tecan, Austria).
Immortalized human hepatic stellate cell line LX-2 and activated rat hepatic stellate cell line CFSC-8B were maintained on glass chamber slides in Dulbecco's modified Eagle's medium supplemented with 100 μgmL −1 of streptomycin, 100 UmL −1 of penicillin and 10% fetal calf serum under a humidified 5% (v/v) CO 2 atmosphere at 37 °C. Cell viability was measured by MTT proliferation assay. LX-2 cells cultured 3 days as quiescent cells 4

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Hepatic stellate cells migration assay. The migratory capacity of CFSC-8B was investigated as described before 7 . Specifically, confluent CFSC-8B at the top of BIOCOAT MATRIGEL invasion chamber was incubated in serum-free medium for 24 h. The lower chamber was filled with 10% fetal calf serum in the presence or absence of (−)-galewone at incremental concentrations. Incubating cells for 24 h, then, CFSC-8B from the upper surface of membranes were completely removed with gentle wiping. The migrated cells on the lower surface of membranes were fixed and stained with crystal violet. Migration rate of CFSC-8B was determined by counting the number of stained cells on membranes in six randomly selected fields at high power.
Hepatic stellate cells wound-healing assay. For determination of cell migration during wound healing, hepatic stellate cells wound-healing assay was measured as described before 5 . CFSC-8B were seeded in 6-wellplates and grown to confluence in Dulbecco's modified Eagle's medium containing 10% FBS. Confluent CFSC-8B were deprived of serum for 24 h, and then disrupted to generate a linear wound, followed by incubation in Dulbecco's modified Eagle's medium containing 10% FBS in the absence or presence of (−)-galewone for 24 h. Cells were subsequently fixed and observed under phase contrast microscopy, five randomly selected points of each well along each wound were measured.