Paenibacillin A, a new 2(1H)-pyrazinone ring-containing natural product from the endophytic bacterium Paenibacillus sp. Xy-2

A new 2(1H)-pyrazinone ring-containing natural product, paenibacillin A (1), together with five known diketopiperazine derivatives 2–6 and two known isoflavones 7–8, was isolated from the culture of an endophytic bacterium Paenibacillus sp. Xy-2. The structure of compound 1 was elucidated by extensive spectral methods, including UV, IR, HR-ESI-MS, 1D and 2D NMR and ECD experiments. Compound 1 exhibited moderate cytotoxicity against HL-60 cell line with IC50 value of 50.48 μM.


Introduction
Endophytic bacteria are common inhabitants of both the surfaces and the internal tissues of most plants, and do not visibly harm the plants (Hallmann et al. 1997). Actually, they may have diverse effects on the development and physiology of the host plant (Bibi et al. 2012;Venkateswarlu 2013;Jalgaonwala and Mahajan 2014). Endophytic bacteria are important resources of novel and bioactive compounds. Their secondary metabolites including alkaloids, anthraquinones, terpenoids, isocoumarin derivatives, phenols and aliphatic compounds showed antimicrobial, nematicidal activities and cytotoxicities on some tumour cell lines (Piel 2004). Previous research has shown that the ethyl acetate extract of the endophytic bacterium Paenibacillus sp. Xy-2 obtained from the plant of Houttuyniae cordata exhibited relatively strong cytotoxic activity against HL-60 cell line. Up to now, the secondary metabolites of this bacterium have not been studied. Our present investigation on the endophytic bacterium Paenibacillus sp. Xy-2 led to the isolation of eight compounds, including a new 2(1H)pyrazinone ring-containing natural product, paenibacillin A (1), five diketopiperazine derivatives, containing cyclo(Pro-Val) (2), cyclo(Pro-Ile) (3), cyclo(Pro-Leu) (4), cyclo(Pro-Phe) (5) and cyclo(Leu-Hyp) (6), and two isoflavones 4 0 , 5, 7-trihydroxyisoflavone (7), 4 0 , 7dihydroxyisoflavone (8) (Figure 1). Herein, we reported the isolation, structural elucidation and cytotoxic activity of the new compound (1).
The stereochemistry of compound 1 at C-1 0 was proposed according to ECD ( Figure 2). The ECD spectrum of 1 showed a positive cotton effect at 254 nm (D1 þ 3.24) and two negative cotton effects at 224 and 317 nm (D1 2 11.44 and 2 5.16, respectively), which matched well with the experimental one. It allowed us to unambiguously determine the structure of 1 with the absolute configuration of 1 0 R. Consequently, the structure of compound 1 was established as paenibacillin A (1).
It was found that the natural products containing 2(1H)-pyrazinone ring such as compound 1 were rare in the secondary metabolites of bacteria, and almost all of them have been isolated from fungus and actinomycetes (Okada et al. 1996;Motohashi et al. 2011;Wyatt et al. 2012).

General experimental procedures
The optical rotation was measured using a Perkin-Elmer 241 polarimeter (Perkin Elmer, Manhattan, North America) with a 1 cm cell at 258C. UV spectrum was recorded on a Shimadzu UV-1601 (Kyoto, Japan). IR spectrum was obtained on a Bruker IFS-55 infrared spectrophotometer (Bruker TFS-55, Aargau, Switzerland) with KBr pellets. CD spectrum was measured on a JASCO CD-2095 Chiral Detector (Bio-logic Co., Claix, France). The HR-ESI-MS data were detected on a Bruker microTOF-Q mass spectrometer (Bruker Co., Karlsruhe, Germany). The NMR spectral data were recorded on Bruker ARX-400 (400 MHz for 1 H and 100 MHz for 13 C) and Bruker AV-600 (600 MHz for HSQC and HMBC) (Bruker Co., Billerica, MA, USA) with TMS as the internal standard. TLC analyses were carried out using precoated silica gel GF254 plates (Qingdao Marine Chemical Plant, Qingdao, China). Column chromatography was performed on silica gel (200 -300 mesh; Qingdao Marine Chemical Plant, Qingdao, China) and Sephadex LH-20 (Pharmacia, Piscataway, NJ, USA).

Isolation and identification of the bacterial strain
The endophytic bacterium Xy-2 was originally obtained from fresh, healthy plant of H. cordata, which was collected in April 2012 in Shenyang, China. It was obtained using the standard protocol for the isolation of endophytic microbes from plant materials. This strain was identified as Paenibacillus sp. on the basis of in vitro colony growth and micromorphology. The strain was also identified using DNA amplification and sequencing of the ITS. The sequence data has been deposited at GenBank (Accession no. KP715166). A voucher strain has been stored at one of the author's laboratory (Jiao Bai).

Fermentation and extraction
The Paenibacillus sp. Xy-2 growing on LB medium at 288C for 5 days was inoculated in the liquid medium and cultured at 288C for 5 days under shaking conditions at 180 rpm. The liquid medium was composed of starch of 1%, yeast extract of 0.4% and tryptone of 0.2% which were dissolved in water. After 5 days, the fermented broth (130 L) was centrifuged to be separated into the supernatant and the mycelia. Finally, the supernatant was concentrated to 10 L and successively extracted with ethyl acetate, and then the crude extract (19.6 g) was obtained.
The compounds were dissolved in DMSO, and the amount of DMSO was controlled at lower than 0.1% in the final concentration. Cells were incubated with various drug concentrations for 3 days. The number of cells was determined by haemocytometer, and its viability was determined using trypan blue staining. The growth inhibitory ability of the compound was calculated and expressed using the IC 50 value (half-inhibitory concentration). 5-FU (80 mmol/L) and 0.1% DMSO were used as a positive control and a negative control, respectively.

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

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

Funding
This work was supported by the Department of Education of Liaoning Province (L2014376). It was also supported by the Program for Innovative Research Team of the Ministry of Education and the Program for Liaoning Innovative Research Team in University.