New p-terphenyl and benzoquinone metabolites from the bioluminescent mushroom Neonothopanus nambi

Abstract Two new p-terphenyls, neonambiterphenyls A and B (1–2), a new benzoquinone, neonambiquinone A (3), together with six known sesquiterpenes (4–9), were isolated from the bioluminescent mushroom Neonothopanus nambi PW3. The isolated compounds were identified by mass, IR and spectroscopic analyses (1D and 2D NMR). Compounds 1–3 and 5–7 showed cytotoxicity against cancer cell lines such as KB, NCI-H187 and MCF-7 with IC50 values ranging from 1.45 to 49.31 µg/mL. In addition, compounds 1 and 5 showed cytotoxicity against Vero cells with IC50 values of 38.72 and 32.90 µg/mL, respectively. Graphical Abstract

The molecular formula of 2 was determined as C 20 H 14 O 8 on the basis of HRESITOFMS (m/z 405.0580 [M þ Na] þ ). The IR spectrum showed the presence of hydroxyl (3354 cm À1 ), ester (1730 cm À1 ) and aromatic (1469, 1426 cm À1 ) groups. The 1 H and 13 C NMR spectral data were similar to those of 1, except for position C-3 00 at d 144.2, where H-3 00 in 1 is replaced by a hydroxyl group. The key HMBC correlations between C-4 0 and C-6 00 with H-2 00 and H-6 00 supported this assignment. Therefore, 2 was assigned as a new p-terphenyl derivative, named neonambiterphenyl B.
Since compounds 4 and 8 have already been reported in our previous study (Kanokmedhakul et al. 2012), the newly isolated compounds (1-3, 5-7 and 9) were tested against several targets (Tables S1 and S2). Compounds 1-3 and 5-7 showed cytotoxicity against NCI-H187 cell lines with IC 50 values ranging from 5.03 to 49.31 lg/mL. Among these 2, 5 and 6 showed potent cytotoxicity with IC 50 values of 5.60, 5.03 and 9.40 mg/mL, respectively. Compounds 1, 2 and 5 showed cytotoxicity against KB cell lines with IC 50 values of 9.12, 40.90 and 1.45 lg/mL, respectively. Moreover, compounds 1 and 5 exhibited cytotoxicity against MCF-7 cell lines with IC 50 values of 11.82 and 18.64 mg/mL, respectively. However, they also showed cytotoxicity to Vero cells with IC 50 values of 38.72 and 32.90 mg/mL, respectively. In addition, compounds 1-6 were evaluated for antibacterial activity against three Gram-negative and two Gram-positive bacteria (Table S2). These bacteria are human opportunistic pathogen involved in infections acquired in a hospital setting and resistant to disinfectants as well as antibiotics. All tested compounds exhibited moderate antibacterial activity against Bacillus cereus with MIC values in the range of 64-128 lg/mL. Compounds 1, 2 and 3 exhibited antibacterial activity against Staphylococcus aureus with MIC values of 4, 8 and 64 lg/mL, respectively. Moreover, compounds 1, 4 and 5 also exhibited moderate antibacterial activity against Pseudomonas aeruginosa with the equal MIC value of 128 lg/mL. Only compound 1 showed antibacterial activity against Shigella sonnei with MIC value of 128 mg/mL.

General experimental procedures
Melting points were determined using an Electrothermal IA9200 digital melting point apparatus (Bibby Scientific Limited, Staffordshire, UK). Optical rotations were measured on a JASCO-DIP-1000 digital polarimeter (JASCO Inc., USA). IR spectra were obtained using a Bruker Tenser 27 spectrophotometer (Bruker, Germany). NMR spectra were recorded on a Varian Mercury Plus 400 spectrometer (Varian Inc., USA) using CDCl 3 and CD 3 OD as solvents. The internal standards were referenced from the residue of those solvents. The HR-ESI-TOF-MS were recorded on a Bruker micrOTOF mass spectrometer (Bruker, Germany). Column chromatography was carried out on MERCK silica gel 60 (230-400 mesh) (Merck, Darmstadt, Germany). Thin-layer chromatography was carried out with pre-coated MERCK silica gel 60 PF254 (Merck, Darmstadt, Germany); the spots were visualized under UV light (254 and 365 nm) and further stained by spraying with anisaldehyde and then heated until charred.

Fungus material
The luminous mushroom was collected in 2015 from the Plant Genetic Conservation Project under Royal Initiation by Her Royal Highness Princess Maha Chakri Sirindhorn at Kok Phutaka area, Wiang Kao District, Khon Kaen Province, Thailand and was identified by Prof. Weerasak Saksirirat as N. Nambi PW3. The voucher specimen was deposited at the Department of Plant Science and Agricultural Resources, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand. The mushroom was cultivated on potato dextrose broth without shaking with 2 h of light per day at 25 C for 30 days (Bua-art et al. 2011).

Extraction and isolation
The cultured mycelium of N. nambi PW3 (650 g) was extracted with EtOAc (3 x 4 L). Removal of solvents under reduced pressure gave 95 g of crude EtOAc extact.

Antiplasmodial assay
Antimalarial activity was evaluated against the parasite Plasmodium falciparum (K1, multidrug resistant strain), using the method of Trager and Jensen (Trager and Jensen 1976). Quantitative assessment of malarial activity in vitro was determined by the micro-culture radio isotope technique based on the method described by Desjardins (Desjardins et al. 1979). The inhibitory concentration (IC 50 ) represents the concentration which causes 50% reduction in parasite growth as indicated by the in vitro incorporation of [ 3 H]-hypoxanthine by P. falciparum. The standard compound was dihydroartemisinin.

Cytotoxicity assay
The cytotoxic assays against human epidermoid carcinoma (KB), human small cell lung cancer (NCI-H187) and human breast cancer (MCF-7) cell lines were performed employing the colorimetric method as described by Skehan (Skehan et al. 1990). The reference substances were ellipticine and doxorubicin.

Antibacterial assay
The minimum inhibitory concentrations (MICs) were determined by the dilution method as described in the M07-A9 (Clinical and Laboratory Standards Institute 2012). Resazurin in solution was used as an indicator of microbial growth. MICs were recorded by reading the lowest concentration capable of inhibiting visible growth. The tests were performed in triplicate. Kanamycin, gentamicin and vancomycin were used as positive control drugs. Five microorganism cultures (E. coli ATCC 25922, P. aeruginosa ATCC 27853, S. sonnei ATCC 11060, B. cereus ATCC 11778 and S. aureus ATCC 25923) were used. The experiment was performed at the Department of Microbiology, Faculty of Science, Khon Kaen University, Thailand.

Conclusions
The cultured mycelium of the bioluminescent mushroom Neonothopanus nambi PW3 was investigated and led to the isolation of nine compounds, including two new p-terphenyls (1-2), a new benzoquinone (3), and six known sesquiterpenes (4-9). Besides compounds 1-3, 5 and 7 are reported for the first time in the genus Neonothopanus and compound 9 is reported for the first time in the family Omphalotaceae. Compounds 1-3 and 5-7 showed cytotoxicity against KB, NCI-H187 and MCF-7 cancer cell lines with IC 50 values ranging from 1.45 to 49.31 mg/mL. Compounds 1 and 5 showed cytotoxicity against Vero cells with IC 50 values of 38.72 and 32.90 mg/mL, respectively. In addition, compounds 1 and 2 showed antibacterial activity against S. aureus with MIC values of 4 and 8 mg/mL, respectively.