Anti-neuroinflammatory constituents from the fungus Penicillium purpurogenum MHZ 111

Abstract A new dihydroisocoumarin (1) and a new coumarin (2), along with eight known metabolites (3–10), were isolated from the solid substrate fermentation cultures of the fungus Penicillium purpurogenum MHZ 111. Their structures were elucidated by extensive spectroscopic analysis and comparison of their spectroscopic and physicochemical data with the literature values. Compounds 2 and 8 showed inhibition of nitric oxide production in lipopolysaccharide-activated BV-2 microglial cells with IC50 values of 26.5 and 52.7 μM, respectively.


Introduction
Neuroinflammation is characterised by the excessive production of inflammatory mediators in the brain, and it is becoming accepted as a critical factor in the damage of brain function, ranging from acute conditions including delirium and postoperative cognitive dysfunction to chronic diseases such as Alzheimer's disease and multiple sclerosis (Collins et al. 2012;Heneka et al. 2014). Microglia are major immune cells of the central nervous system. Increasing evidence indicate that activated microglia are involved in various neuroinflammatory pathology, resulting in progressive neurodegenerative disorders (Walsh et al. 2014;Yuste et al. 2015). Therefore, inhibition of microglial activation and subsequent neuroinflammation may represent an attractive and effective therapeutic strategy for the treatment of neurodegenerative diseases.

General experimental procedures
optical rotations were measured on a Rudolph Autopol IV automatic polarimeter. uV spectra were measured on a Shimadzu uV-2401PC spectrophotometer (Tokyo, Japan). eCD spectrum was obtained on a JASCo J-810 CD spectrometer. IR spectra were recorded on a Thermo Nicolet Nexus 470 FT-IR spectrophotometer with KBr pellets. The HReSIMS data were determined on an LCMS-IT-ToF system (Shimadzu, Kyoto, Japan). The NMR spectra were recorded on Varian INoVA-500 spectrometers operating at 500 MHz for 1 H and 125 MHz for 13 C, respectively. Silica gel (200-300 mesh, Qingdao Marine Chemical Inc., Qingdao, People′s Republic of China), LiChroprep RP-C 18 gel (40-63 μm, Merck, Germany), and Sephadex LH-20 (Pharmacia) were used for open column chromatography. Semipreparative HPLC was performed on a YMC-Pack oDS column (YMC-Pack oDS-A, 250 × 10 mm, 5 μm), equipped with a Shimadzu LC-20AT pump system and an SPD-M20A photodiode array detector (Shimadzu Corporation, Tokyo, Japan).

Fungal material
The fungal strain P. purpurogenum MHZ 111 was isolated from a soil sample collected from Mohe, Heilongjiang Province, P.R. China, in July 2011, by martin medium. The fungus was authenticated according to its morphological characteristics and analyses of its 18S and ITS rDNA sequences (S12 and S13). The strain has been deposited at the China General Microbiological Culture Collection Center with the accession number CGMCC No.11630.

Cell culture and measurement of NO production
BV-2 microglial cells were purchased from Peking union Medical College (PuMC) Cell Bank (Beijing, People's Republic of China). Cell maintenance, experimental procedures, and data determination for the inhibition of No production are the same as previously described (Li et al. 2012a;Huang et al. 2015). The IC 50 values were determined using GraphPad Prism 5 software from experiments performed in triplicate (GraphPad Software, Inc., San Diego, CA, uSA). Indomethacin (IC 50 value of 35.6 ± 1.3 μM) was used as a positive control.

Conclusion
In this study, a new dihydroisocoumarin (1) and a new coumarin (2), along with eight known metabolites (3-10), were isolated from the fungus P. purpurogenum. Among them, compounds 3-5, and 7 were isolated from the genus Penicillium for the first time, while compounds 6 and 8-10 were firstly reported from P. purpurogenum. Compounds 2 and 8 showed inhibitory activity against No production with IC 50 values of 26.5 and 52.7 μM, respectively.

Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1-S13.

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

Funding
The project was financially supported by the Program for New Century excellent Talents