New chromone analog and pyrrole alkaloid produced by Penicillium sclerotiorum and their antibacterial activity

Abstract A new chromone analog (1) and a new pyrrole alkaloid (2), together with four known compounds, were isolated from the endophytic fungus Penicillium sclerotiorum MPT-250 obtained from the stems of Taxus wallichiana var. chinensis (Pilger) Florin. The structural elucidation of these metabolites was performed by high-resolution mass spectrometry and NMR spectroscopy. Compounds 1 and 5 exhibited significant antibacterial activity against carbapenems-resistant Pseudomonas aeruginosa and multidrug-resistant Enterococcus faecium with an minimum inhibitory concentration (MIC) value of 3.13 μg/ml respectively. Graphical Abstract


Introduction
Bacterial resistance, a terrible hazard to human health, can directly lead to patient treatment failure, increased medical costs, rising mortality, etc. More seriously, development of drug-resistant bacteria even may re-expose humans to the threat of infectious diseases [1][2][3]. Therefore, the investigation for novel antibiotics against resistant bacteria is urgent and necessary. Discovery of antibacterial drugs such as penicillin, tetracycline, streptomycin, and vancomycin has proved that it is an efficient way to exploit lead compounds from microbial metabolites depending on their structural diversities and extensive bioactivities [4][5][6][7][8]. However, the development and application of microbial active substances make it increasingly difficult to search for novel active structures from conventional microorganisms, and then, the special habitat microorganisms have attracted more attention. Due to their special living environment and long-term mutually beneficial symbiotic relationship with host plants, endophytic fungi can produce secondary metabolites with novel structures and unique activities that may be great potential leads for the development of new pharmaceutical agents [9,10].

Results and discussion
Compound 1 was isolated as a yellowish powder. The molecular formula of C 16    . These data indicated compound 2 to be a pyrrole alkaloid [17]. Exhaustive analyses of the 2D NMR spectra revealed compound 2 was similar with 4-(2-formyl-5-methoxymethylpyrrol-1yl)butyric acid methyl ester, a pyrrole alkaloid isolated from Chinese folk medicine Bolbostemma paniculatum [17]. However, the methyl butyrate fragment connected at   Figure 2) and HR-ESI-MS data. Therefore, compound 2 was characterized as 5-(2-formyl-5-methoxymethylpyrrol-1-yl)pentanoic acid methyl ester. The antibacterial activity against eight clinically important bacteria of compounds 1-6 were assayed by microbroth dilution method [22]. Compound 1 displayed significant antibacterial activity against carbapenems-resistant Pseudomonas aeruginosa with an minimum inhibitory concentration (MIC) value of 3.13 lg/ml and compound 5 was active against multidrug-resistant Enterococcus faecalis with an MIC value of 3.13 lg/ml (Supplemental Table S1).

Fungal material
The fungal strain MPT-250 was isolated from the stems of Taxus wallichiana var. chinensis (Pilger) collected in Tianshui, Gansu Province, China. The internal transcribed spacer (ITS) region was amplified and identified as Penicillium sclerotiorum (GenBank Accession No. OK357287). The voucher sample, MPT-250, has been preserved in the culture collection center of South Central Minzu University. All drugresistant bacteria for biological assays were obtained as gifts from Army Military Medical University.

Fermentation, extraction, and isolation
The fungus MPT-250 was incubated on potato dextrose agar (PDA) medium at 28 C for 5 days. Subsequently, the agar cultures were cut into small pieces (0.5 Â 0.5 cm 2 ) and then inoculated into 100 Â 500 ml culture flasks containing 100 g rice and 80 ml distilled water. After 40 days incubation at room temperature, the fermented material was distilled with MeOH and then extracted three times with EtOAc. The solvent was evaporated to dryness under reduced pressure and the EtOAc extract (64 g) was subjected to silica gel column chromatography (CC, 80-120 mesh) eluting with CH 2 Cl 2 -MeOH

Disclosure statement
Authors declare no conflict of interest.