Two previously undescribed phthalides from Talaromyces amestolkiae, a symbiotic fungus of Syngnathus acus

Abstract Amestolkins A (1) and B (2), two previously undescribed phthalides sharing the same planar structure of (1, 5-dihydroxyhexyl)-7-hydroxyisobenzofuran-1(3H)-one were isolated from Talaromyces amestolkiae. Their absolute configurations were elucidated by comprehensive analyses of spectroscopic evidences in high-resolution electrospray mass spectra (HRESIMS) and nuclear magnetic resonance (NMR) combined with electronic circular dichroism (ECD) and NMR calculations. 1 and 2 showed anti-neuroinflammatory activity by inhibiting the gene expressions of proinflammatory factors including C-C motif chemokine ligand 2 (CCL-2), tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6), as well as attenuating the excretion of inducible nitric oxide synthase (iNOS) in BV-2 microglial cells at the concentration of 30 μM. Graphical Abstract

With the purpose of searching secondary metabolites with novel structures from T. amestolkiae, large-scale solid fermentation on T. amestolkiae strain was carried out, and two new phthalides were subsequently obtained ( Figure 1). Their structures and absolute configurations were clarified by comprehensive spectral analysis combined with nuclear magnetic resonance (NMR) as well as electronic circular dichroism (ECD) calculations. Anti-inflammatory assays in BV-2 microglial cells indicated amestolkins A (1) and B (2) can inhibit the gene expressions as well as production of some proinflammatory mediators at the concentration of 30 lM. Herein, the isolation, structure elucidation and anti-inflammatory assays evaluation of these previously undescribed compounds were discussed.
Cell counting Kit-8 (CCK-8) results showed that both 1 and 2 did not inhibit cell viability even at a high concentration, suggesting that the two compounds were nontoxic to BV-2 microglial cells. In the further pharmacological study of the two  compounds, lipopolysaccharide (LPS) was utilized to stimulate the inflammation of BV-2 microglial cells, and the anti-inflammatory effects of 1 and 2 were observed by reverse transcription-polymerase chain reaction (RT-PCR) and western blot experiments. The RT-PCR results ( Figure 4) showed that 1 significantly inhibited gene expressions of C-C motif chemokine ligand 2 (CCL-2), tumor necrosis factor-a (TNF-a) and interleukin 6 (IL-6) ( # P＜0.05, ## P＜0.01), which involved in activated inflammation. However, 2 did not reduce the gene levels of CCL-2, TNF-a and IL-6. Consistent with the RT-PCR results ( Figure 5), the Western blot results displayed that both 1 and 2 significantly downregulated the protein level of inducible nitric oxide synthase (iNOS) ( ## P＜ 0.01), which promotes the production of NO and inflammation. All these results indicated that 1 and 2 possessed the ability of antiinflammation in BV-2 microglial cells. BV-2 microglial cells is the intrinsic immune cell of central nervous system [9], so 1 and 2 were considered to possess the potency of anti-neuroinflammation.   Elmer Lambda 35 UV-VIS spectrophotometer (Perkin Elmer, Inc., Waltham, MA, USA). The IR spectra were recorded on a Cary 600 Series FT-IR (KBr) spectrometer (Agilent Technologies Inc., California, USA). The ECD spectrum was measured on a Chirascan circular dichroism spectrometer (Applied Photophysics Ltd., Leatherhead,UK). The 1D ( 1 H and 13 C) and 2D ( 1 H-1 H COSY, HSQC, HMBC and NOESY) NMR data were obtained using a Bruker-Ascend-700-MHz spectrometer (Bruker Corporation, Billerica, MA, USA), with TMS as internal standard. The HRESIMS data were obtained using a Q Exactive mass spectrometer (Thermo Fisher Scientific, MA, USA). Column chromatographs (CC) were performed with silica gel (200-300 mesh, Qingdao Haiyang Chemical Co., Qingdao, China) and Sephadex LH-20 (GE-Healthcare Bio-Sciences AB, Uppsala, Sweden). Preparative HPLC was performed on a NP7000 serials pump (Hanbon Sci. & Tech., Jiangsu, China) equipped with a YMC ODS column (10 mm Â 250 mm, 5 lm, Akzo Nobel Pulp and Performance Chemicals AB, Bohus, Sweden) using a NU3000 serials UV detector (Hanbon Sci. &Tech., Jiangsu, China). A Chiralpak IC column (4.6 mm Â 250 mm, 5 lm; Chiral Technologies, West Chester, PA, USA) was applied for chiral resolution.

Fermentation and extraction
T. amestolkiae strain was placed on an ultra-clean bench, and the strain was inoculated into M1 liquid medium with a sterilized inoculum needle at 30 C and 120 r/ min. The fermentation broth was obtained by incubating the strain for 7 days. Then large-scale fermentation was carried out in 200 tissue culture flasks of 330 ml, each containing 2 g of peptone, 40 g of brown rice, 25 ml of water and 10 ml of seed culture for 30 days at room temperature.
The fermentation product was extracted by ultrasonic extraction with methanol thrice (10 L each time), and the extract was concentrated under reduced pressure to obtain a flowing infusion, which was dispersed with water and extracted with the same volume of ethyl acetate. The extract was concentrated to obtain 65.35 g of extract.

NMR and ECD calculations
Conformational searches for compounds 1-2 were performed in Conflex 8 software. The preliminary conformers were optimized via Gaussian 16 software to generate the energy-minimized conformers. The ECD curve of each conformer was simulated through the time-dependent density functional theory (TD-DFT) method and drawn by SpecDis 1.71 software. The calculated ECD spectrum of each compound was generated by Boltzmann weighting and magnified or reduced according to the experimental values [11]. Shielding tensor calculations for xB97XD/DGDZVP-optimized conformers (Boltzmann distribution ! 1%) were performed at the PCM/ mPW1PW91/6-311 þ G (d, p) level in methanol using the GIAO method. The isotropic values of TMS were calculated at the same level and used as references. The DP4þ parameters were calculated using an Excel file provided by literature [12]. Computational details are elaborated in the Supporting Information.

Cell culture
BV-2 microglial cells were cultured in dulbecco's modified eagle medium (DMEM) medium containing 10% fetal bovine serum and antibiotics (100 U/ml of penicillin and 100 U/ml of streptomycin) and maintained in a humidified 5%-CO 2 incubator at 37 C. For the experiment, the BV-2 microglial cells were treated with compounds for 1 h and then stimulated with 1 lg/ml LPS for 24 h. Meanwhile, BV-2 microglial cells treated with dimethyl sulfoxide (DMSO) (0.1%) was set as the negative control.

Cell viability assay
The cell viability was investigated using a Cell Counting Kit-8 (MCE, HY-K0301, USA). CCK-8 was performed to estimate the cytotoxic effects of 1-2 on BV-2 microglial cells. The BV-2 microglial cells were seeded into 96-well plates. Compounds 1-2 at 0 lM, 7.5 lM, 15 lM, 30 lM, 60 lM and 120 lM were added to the cultured cells for 24 h. Then, the medium was replaced by 100 lL of fresh media containing 10 lL CCK-8. After 4-h incubation at 37 C, cell viability was examined by measuring the absorption at 450 nm using a microplate reader.

Statistical analysis
Data were presented as the mean ± standard error of the mean (SEM) and analyzed using GraphPad Prism 7.0 software (San Diego, CA, USA). The differences between different groups were evaluated through an unpaired two-tailed Student's t test. Ã p value <0.05 was considered statistically significant.