Isolation and identification of two new compounds from the Penicillium sp. SYPF7381

Abstract Two new compounds, (7R, 2E, 5E)-3,5,7-trimethyl-2,5-octadienedioic-8-methyl ester (1) and neovasipyridone G (3), together with a new natural product compound (7R,2E,5E)-3,5,7-trimethyl-2,5-octadienedioic acid (2), and six known compounds (4–9) were isolated from Penicillium sp. SYPF7381. Their structures were elucidated on the basis of extensive spectroscopic analysis, and the absolute configurations of compounds 1 and 2 were determined by optical rotation. The absolute configuration of compound 3 was determined by means of electronic circular dichroism (ECD) calculation. In addition, the in vitro anti-inflammatory activities of all compounds were assayed in RAW 264.7 cells by assessing LPS-induced NO production. Furthermore, the structure-antiinflammation activity relationships for these isolated compounds were summarized based on the experimental as well as the docking results. Graphical abstract


Introduction
Inflammation is a complex pathophysiological process mediated by a variety of signaling molecules produced by macrophages, leukocytes, mast cells, etc. Macrophages play a central role in host defense against foreign agents by releasing various proinflammatory cytokines and inflammatory mediators such as nitric oxide (NO), tumor necrosis factor-alpha (TNF-a) and cyclooxygenase (COX-2) (Makchuchit et al. 2017;Wu et al. 2018). Immoderate inflammation can be harmful, leading to the pathogenesis of many diseases, such as atheriosclerosis, arthritis, cardiovascular disease and cancer (Park et al. 2018). In the past few decades, it's has been being a hotspot to explore new biologically active compounds from fungi in the rhizosphere soil of officinal plants (Strobel et al. 2004). These metabolites are usually structurally novel and display potent biological or pharmaceutical properties, such as antiacancer, antiviral, antimicrobic and anti-inflammatory activity Wang et al. 2016). In our previous research on the fungus Penicillium sp. SYPF7381, four new hybrid polyketide-terpenoid metabolites were isolated (Feng et al. 2018). It has been reported that hybrid polyketide-terpenoid metabolites exhibited promising anti-inflammatory activity (Chen et al. 2018). The aim of the present study was to identify and characterize more potential therapeutic agents from the metabolites of fungus Penicillium sp. SYPF7381 to prevent and/or mitigate inflammation. Thus, the chemical constituent of Penicillium sp. and anti-inflammatory of the isolates from it were investigated with regard to their in vitro inhibitory activities on NO production using LPS-activated RAW 264.7 macrophage cells.
In addition, the in vitro anti-inflammatory activities of compounds 1-6 were evaluated in RAW 264.7 cells assessing LPS-induced NO production. Cell viability assays showed that compounds 1-6 had no toxic effects on RAW 264.7 cells at concentrations of 50 lM. As a result, all compounds exhibited anti-inflammatory activities at tested concentrations, which were shown in Figure S36. Compounds 2, 3, 5, 6, 7, 8 and 9 can significantly inhibit the production of NO. Compounds 1 exhibited weak anti-inflammatory activities. In a docking analysis, the glide gscore/glide energy of compounds 1 and 3 were À3.512/À37.338 and À4.741/À48.062, respectively. The glide energy of compound 3 showing stronger anti-inflammation activity than 1 was lower than compound 1 ( Figure S37). Compound 6, with stronger anti-inflammation activity than 7, had lower glide energy being compared to 7 (Table S2, Figure S37). Therefore the 8-R absolute configurations of neovasifuranones (compounds 6-7) might play a role in enhancing the anti-inflammatory activity.

General experimental procedures
NMR spectra were performed on Bruker ARX-400 or ARX-600 spectrometer with TMS as an internal standard. Bruker micro TOF-Q mass spectrometer was used to collect HR ESI-TOF MS data in m/z (rel. %). IR spectra were taken on a Bruker IFS-55 infrared spectrophotometer with a KBr disks. Optical rotations were measured on a Peking-Elmer 241 MC Spectropolarimeter at 20 C (MeOH, c in g/100 mL). MPLC was performed on Agela ODS flash column (C-18, 120 g, 20-45 lm), and the MPLC system was equipped with HP UV2040 detector and HP P050 pump. HPLC separation was carried out on an semipreparative YMC-pack ODS-A column (250 Â 10mm) equipped with Shimadzu SPD-10A UV detector and Shimadzu LC-8A series pumping system. Silica gel GF254 for TLC and silica gel (200-300 mesh) for column chromatography were obtained from Qingdao Haiyang Chemical Factory.

Fungal material and fermentation
The fungal strain used in this work was isolated from the rhizosphere soil of Pulsatilla chinensis, which was collected from Huludao, Liaoning province of China in October 2014. The sequence data derived from this strain has been submitted and deposited in GeneBank with the accession number MF568058. A voucher specimen (No. SYPF7381) was deposited at the School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang (Feng et al. 2018). The interrhizospheric fungus Penicillium sp. was fermented on autoclaved rice solid-substrate medium (one hundred and sixty 500 mL Erlenmeyer flasks, each containing 80 g rice, 110 mL water) for 30 days at 26 C.

Anti-inflammatory activity in vitro
Accumulation of nitrite, an indicator of NO synthase activity, in culture medium was measured using the Griess reaction. Cells (2.5 Â 10 5 cells/well) were placed on 96-well microtiter plates and treated with each compound (20, 50 lM) in presence of bacterial lipopolysacchride (LPS; 200 ng/mL) for 24h. Fifty microliter culture medium supernatants were mixed with 50 ll Griess reagent (part I: 1% sulfanilamide; part II: 0.1% naphthylethylene diamide dihydrochloride and 2% phosphoric acid) at 37 C. 10 minutes later, the absorbance was measured at 540 nm.

Molecular docking method
To perform a docking analysis, Schrodinger 2013 package suite (Glide) was used to understand the interactions between the compounds 1-3, 6-7 and this receptor iNOS (PDB ID: 3NQS) ( Figure S37) (Makchuchit et al. 2017;Rosenfeld et al. 2010). Before performing the docking procedures, the protein structure was prepared by removing all water molecules. The interactions between ligand and receptor were evaluated for appropriate orientation with lower binding energy level.

Conclusions
In our study, two new compounds (1 and 3) together with a new natural product compound (2), and six known compounds (4-9) were isolated from Penicillium sp. SYPF7381. The anti-inflammatory activities of all compounds were assayed in RAW 264.7 cells by assessing LPS-induced NO production. And the simple structure-antiinflammation activity relationship for these isolated compounds (6 and 7) were summarized based on the experimental as well as the docking results.

Disclosure statement
No potential conflict of interest was reported by the authors.