A new polyoxygenated farnesylcyclohexenone from Fungus Penicillium sp.

A new polyoxygenated farnesylcyclohexenone, peniginsengin A (1), was isolated from the fermentation of Penicillium sp. YIM PH30003, an endophytic fungus associated with Panax notoginseng (Burk.) F. H. Chen. The structure was assigned based on a combination of 1 D and 2 D NMR and mass spectral data. The cytotoxicity and antimicrobial activities of compound 1 were investigated.


Introduction
The genus Penicillium is one of the largest and most intensively investigated mangrove endophytic fungal genera. Diverse structures and interesting biological activities from endophytic Penicillium species have been characterised (Motohashi et al. 2009;Wang et al. 2011;Yang et al. 2013;Rukachaisirikul et al. 2014). As part of our ongoing research on new bioactive compounds from endophytic fungi. The broth culture of the rhizosphere fungus Penicillium sp. YIM PH30003 exhibited antimicrobial activity towards Fusarium solani, the pathogenic fungus of Panax notoginseng. We reported herein the isolation and identification of one new compound from the fungus Penicillium sp., together with its bioactivities. The structure was determined as peniginsengin A (1) (Figure 1) by extensive spectroscopic analyses. The cytotoxicity against human promyelocytic leukemia HL-60, human hepatoma SMMC-7721, non-small-cell lung cancer A-549, breast cancer MCF-7 and human colorectal carcinoma SW4801 cell lines, and antimicrobial activities against F. solani and Staphylococcus aureus of compound 1 were investigated.

Results and discussion
HR-ESIMS analysis of compound 1 revealed quasi-molecular ion peaks at 357.1757 [M þ Na] þ . The 1 H and 13 C NMR spectra, including DEPT, clearly showed three olefinic methyl singlets, two oxygenated methines, one oxygenated quaternary carbon, three olefinic methines, three olefinic quaternary carbons, two carbonyl carbons and five methylenes. The 1 H NMR and 13 C NMR of 1 empressed the skeleton of polyoxygenated farnesylcyclohexenones isolated from Penicillium (Li et al. 2003). The cyclohexanone structure unit in compound 1 was confirmed by HMBC and 1 H-1 H COSY spectra ( Figure S1). The HMBC correlations between H-7 and C-4, C-5 and C-6 suggested a methyl group connected to a double bond at C-5 position. The 1 H-1 H COSY between H-3 and H-4 indicated that C-3 was connected to C-4. The structure of the side chain, comprising two isoprene and one acetic acid fragments, could also be established by the information provided by 1 H-1 H COSY and HMBC spectra ( Figure S1). The HMBC signals between H-1 0 and C-2 indicated that the side chain was connected to C-2 position. The relative stereochemistry of this metabolite was determined by the coupling constant and the NOESY data. The coupling constant of H-3 and H-4 was observed to be 2.2 Hz and a broad singlet when compared with ambuic acid derivatives, suggesting that H-3 and H-4 are situated in a cis relationship (Ding et al. 2009). The orientations of double bonds were determined by the NOESY data from H-2 0 to H-4 0 , H-4 0 to H-6 0 , H-6 0 to H-8 0 and NMR when compared with 7-deacetoxyyanuthone A (Li et al. 2003).

General experimental procedures
Silica gel Qingdao Marine Chemical Group Co.,Shangdong,China) and Sephadex LH-20 (GE Healthcare Co., Buckinghamshire, UK) were employed for column chromatography. All chemicals were purchased from Beijing Greenherbs Science and Technology Development Co. (Beijing, China). Optical rotations were measured with a Jasco P-1020 (Jasco Co., Tokyo, Japan). 1D-and 2D-NMR spectra were obtained on Bruker 500 MHz instruments (Bruker, Karlsruhe, Germany) with tetramethylsilane (TMS) (Sigma-Aldrich Co., Shanghai, China) as internal standard. MS spectrum was recorded using an Agilent G3250AA system (Agilent, Santa Clara, CA, USA).

Biological material and cultivation of fungal strain
Penicillium sp. was isolated on PDA medium (infusion of 200 g fresh potato, dextrose 15 g and 1 L distilled water, agar 15.0 g, pH 7.0) from the P. notoginseng collected from Wenshan, Yunnan Province, China, in March 2012. The stock culture of Penicillium sp. was grown on the slant of PDA medium at 48C. Identification of the strain was performed by rDNA-ITS molecularphylogenetic analysis and morphological characteristics of its different growth stages. A voucher specimen (no. YIM PH30003) was preserved at the Yunnan Institute of Microbiology, Kunming, P.R. China.

Fermentation and isolation
The fungus Penicillium YIM PH 30003 was maintained on the seed medium (PDB, potato infusion of 200 g fresh potato, dextrose 20 g, distilled water 1.0 L, pH 7.0) in a 500-mL Erlenmeyer flask for 30 min at room temperature (rt). The flasks were incubated on a rotary shaker at 288C at 130 rpm for 3 days. The seed culture (10%) was then transferred into a 1000 mL Erlenmeyer flask containing 250 mL of seed medium as the production medium. Fermentation was performed on a rotary shaker at 288C at 130 rpm for 7 days.

The cytotoxicity and antimicrobial assays
The cytotoxicities of compound 1, against HL-60, SMMC-7721, A-549, MCF-7 and SW4801 were determined in vitro by MTS method. Briefly, cells were seeded in 96-well plates at a density of 5.0 £ 10 3 to 1 £ 10 4 cells/well. Cells were treated with different concentrations of compound 1 for 48 h, following incubation with MTS solution for 4 h. The absorbance was measured using a microplate reader (Bio-Rad 680, Bio-Rad, Hercules, USA) at a wavelength of 490 nm. Cisplatin was used as a positive control, generating IC 50 values of 1. 93, 10.21, 6.59, 8.20 and 12.16 mM against HL-60, SMMC-7721, A-549, MCF-7 and SW480 cells, respectively, and taxol was used as a positive control with IC 50 , 0.008 mM.
Antimicrobial assays were performed in 96-well sterilised microplates using a microdilution method. Briefly, 4-day-old spores from F. solani (grown on PDB medium: potato 200 g, glucose 20 g and distilled water 1000 mL), and the test concentration was 1 £ 10 3 spores/mL. The 18-hour-old bacterial cultures from S. aureus (grown on LB medium: yeast extract 5 g, tryptone 10 g, NaCl 10 g and distilled water 1000 mL, pH 7.0) were grown until they reach 1 £ 10 5 colony-forming units/mL. The test samples were dissolved in DMSO, and their final concentrations ranged from 512 to 0.5 mg/mL, which was determined using a 2-fold Natural Product Research serial dilution method. The final concentration of DMSO did not exceed 5%. The wells containing test strains and diluted samples were incubated at 288C (4 days) for fungi and 378C (24 h) for bacteria. The wells containing a culture suspension and DMSO were run as negative controls. As a positive control, nystatin (Taicheng Pharmaceutical Co., Ltd., Guangdong, China) had antifungal activity against F. solani with an MIC of 4 mg/mL, kanamycin (Yunke Biotechnology, Kunming, China) showed antibacterial activity against S. aureus with an MIC of 4 mg/mL. All experiments were repeated three times. The growth of test strains was observed using a CX21BIM-set5 microscope (Olympus Corp., Tokyo, Japan). MICs were determined as the lowest concentrations that produce complete growth inhibition of the tested microorganisms.

Conclusion
Penicillium sp. YIM PH30003 associated with P. notoginseng produced a new small molecule with antifungal activity against F. solani, the pathogenic fungus of P. notoginseng.

Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1 -S8.