A new phenone from the roots of Paeonia suffruticosa Andrews

Abstract Thirteen phenones were obtained from the 70% ethanol extract of Paeonia suffruticosa Andrews. Their structures were determined on the basis of chemical methods and spectral data. Among them, compound 1 was identified as a new compound, and compounds 5 and 13 were obtained from genus Paeonia for the first time. The inhibitory effects of isolated compounds (1–12) on nitric oxide (NO) production in lipopolysaccharide-activated macrophages were evaluated, and NO production was suppressed significantly by compound 7.


Results and discussion
Compound 1 was isolated as brown amorphous powder, which gave a positive reaction with FeCl 3 -K 3 [Fe(CN) 6 ] reagent, suggesting the presence of a phenolic hydroxyl (Shanta & Mathew 1970). Its molecular formula was determined as C 15 H 20 O 9 from its positive-ion HR-ESI-MS (367.1002 [M + Na] + , calcd. for C 15 H 20 O 9 Na + , 367.1005). The 1 H NMR spectrum of 1 displayed a methyl group at δ H 2.54 (3H, s), a methoxy group at δ H 3.89 (3H, s), two para-coupled aromatic protons at δ H 6.50 (1H, s), 7.64 (1H, s) and an anomeric proton at δ H 4.78 (1H, d, J = 7.2 Hz). The 1 H and 13 C NMR data as well as acid hydrolysis and GC comparison with an authentic sample indicated the presence of a d-glucopyranosyl moiety. The β-configuration of the glycosidic linkage was determined from the coupling constant of the anomeric proton. The 13 C NMR spectra of compound 1 were similar to that of paeonol, except for the appearance of a glucosyl group, assignments confirmed with the help of 2D NMR (HSQC, HMBC and NOESY) experiments. The HMBC correlations from the methyl protons to C-1, C-6 and ketone carbonyl carbon, from the methoxy protons to C-4, and from H-1′ to C-5 supported the above deduction. The linked position of the glucosyl group was further established from the NOESY spectrum, in which H-6 (δ 7.64) exhibited a correlation with H-1′ (δ 4.78), indicating that the glucosyl moiety was located at C-5. On the basis of these data, the structure of 1 was elucidated as 2-hydroxy-4-methoxy-5-O-β-d-glucopyranosyl-acetophenone, named as mudanoside C.
Compounds 1-12 were evaluated for their inhibitory effects on NO production induced by LPS in macrophages (see Table 1). In addition, the cell viability in the present experiment was determined by the MTT method to find whether inhibition on NO production was due to the cytotoxicity of the test compounds. As shown in Table 1, hydrocortisone (IC 50 40.64 ± 3.22 μM) was used as positive control. It is pity that only compound 7 significantly suppressed NO production in LPS-activated macrophages.

General experimental procedures
uV spectra were recorded on a Perkin-Elmer 241 polarimeter. IR spectra were recorded on a Bruker IFS55 spectrometer. The 1 H, 13 C and 2D NMR spectra were performed on Bruker ARX-300 and ARX-600 spectrometers, using TMS as an internal standard. HR-ESI-MS spectra were obtained on an Agilent 6210 TOF mass spectrometer. Circular dichroism was measured on a MSO450 spectropolarimeter. Preparative HPLC was carried out on a LC-6AD liquid chromatograph with an YMC Pack ODS-A column (250 × 20 mm, 5 μm, 120 Å) and SPD-10A VP uV/VIS detector. Macroporous resin D101 was purchased from the Chemical Plant of Nankai

Plant material
The roots of P. suffruticosa Andrews from Hebei Province of China were purchased from Liaoning Yaocai Co., Shenyang province, China, and were identified by Prof. Qishi Sun, Department of Pharmaceutical Botany, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical university. A voucher specimen (Y20010062) was deposited in the herbarium of the Department of Natural Products Chemistry, Shenyang Pharmaceutical university.

Acid hydrolysis of compound 1
Compound 1 (2 mg) was dissolved in 2 N HCl (2 mL) heated at 90 °C for 2 h and partitioned with cyclohexane and H 2 O (each 1 mL × 3). The aqueous layer was evaporated to dryness to give residue, which was dissolved in anhydrous pyridine (2 mL), and L-cysteine methyl ester hydrochloride (2 mg) was mixed, and then warmed at 60 °C for 2 h. The trimethylsilylation reagent trimethylsilylimidazole (0.15 mL) was added, followed by warming at 60 °C for another 1 h. After drying the solution, the residue was partitioned between H 2 O and cyclohexane. The cyclohexane layer was concentrated, then dissolved in 200 μL of cyclohexane and analysed by GC. The identification of the resulting glucose was carried out as described in the previous paper (Ding et al. 2012a).

Cell culture and nitrite determination
RAW 264.7 cells were harvested with trypsin and diluted to a suspension in fresh medium, seeded in 96-well plates at 1 × 10 6 cells/mL, 200 μL/well and allowed to adhere for 2 h at 37 °C in 5% CO 2 in air. Then, the cells were treated with 1 μg/mL of LPS for 24 h with or without various concentrations of test compounds. DMSO was used as a solvent for the test compounds, which were applied at a final concentration of 0.2% (v/v) in cell culture supernatants. NO production was determined by measuring the accumulation of nitrite in the culture supernatant using Griess reagent (Dirsch et al. 1998). Briefly, 100 μL of the culture supernatants from incubates was mixed with an equal volume of Griess reagent (a mixture with equal amount of 0.1% N- [1-naphthyl]ethylenediamine and 1% sulphanilamide in 5% H 3 PO 4 ). The concentration of NO 2 − was calculated by using a prepared working line 0, 1, 2, 5, 10, 20, 50 and 100 μM sodium nitrite solutions, and the inhibitory rate on NO production induced by LPS was calculated by the NO 2 − levels as follows: Experiments were performed in triplicate, and data were expressed as the mean ± SD of three independent experiments. After 100 μL of the culture supernatants was taken out for nitrite determination, cytotoxicity was evaluated by the MTT colorimetric assay (Qin et al. 2015). Briefly, an MTT solution was added at the final concentration of 200 μg/mL, and the cells were incubated for 4 h at 37 °C. The supernatant was removed, and 100 μL of DMSO was added to dissolve the formazan. The absorbance at 570 nm was measured by using a microplate reader. The LPS-treated cells were considered as having 100% of viable cells.

Conclusions
Previous phytochemical research on P. suffruticosa has led to the identification of many bioactive compounds; they were confirmed as monoterpenoids and their glycosides with a unique 'cage-like' skeleton, which occurred ubiquitously in all the species examined of the family Paeoniaceae (Yoshikawa et al. 1992;Lin et al. 1996;Okasaka et al. 2008;Duan et al. 2009). However, phenones are also characteristic and major constituents of P. suffruticosa, in which acetophenones are predominant. In this investigation, thirteen phenones were isolated from the roots of P. suffruticosa Andrews. Among them, compound 1 is a new compound, and compounds 5 and 13 were obtained from the genus Paeonia for the first time.
Compounds 1-12 were evaluated for their inhibitory effects on NO production induced by LPS in macrophages. As shown in Table 1, comparing with positive control hydrocortisone (IC 50 40.64 ± 3.22 μM), it is pity that only compound 7 displayed strong inhibitory activities on NO production. Analogs displayed less or no inhibitory activity.

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.