Phytochemical constituents of the pericarps of Illicium difengpi and their anti-inflammatory activity

Abstract Phytochemical investigation on the pericarps of Illicium difengpi lead to the isolation and structure elucidation of a new sesquiterpene, sesquicaranoic acid C (1), a new neolignan, difengpiol C (2), and 10 known compounds. The structures and absolute configurations of two new compounds were determined by a combination of NMR and CD spectroscopic analyses. All isolates were evaluated for their inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells. Graphical Abstract


Introduction
Illicium difengpi K. I. B et K. I. M. (Illiciaceae) is a shrub endemic to China, mainly distributed in the Karst region of Guangxi Province. Its stem barks is listed in Chinese Pharmacopeia as a traditional Chinese medicine to treat rheumatic arthritis (Editorial Committee of Chinese Pharmacopoeia 2015). A series of different types of compounds had been isolated from the stem barks of this plant, including phenylpropanoids, lignans, triterpene acids, sesquiterpenes and others (Huang et al. 1996;Huang et al. 1997;Kouno et al. 1992;Kouno et al. 1993;Fang et al. 2010;Fang et al. 2011;Li et al. 2015aLi et al. , 2015bYang et al. 2016). Our previous paper reported four new and seven known lignan glycosides isolated from the n-BuOH-soluble fraction of an EtOH extract of the stem barks (Pan et al. 2016). However, there are no reports concerning the phytochemistry of the pericarps of I. difengpi so far. In this study, as our continuous search for structurally diverse and biologically interesting natural product from Karst plants, a new sesquiterpene, sesquicaranoic acid C (1), a new neolignan, difengpiol C (2), and 10 known compounds (3-12) (Figure 1) were isolated from the pericarps. Herein, we describe the isolation and structure elucidation of the new compounds, as well as the anti-inflammatory activity of all isolates.

Results and discussion
Compound 1 was obtained as amorphous oil and its molecular formula was determined to be C 16 H 22 O 4 by the negative HR-ESI-MS ion at m/z 277.1426 [M-H] -, requiring six degrees of unsaturation. The 1 H-NMR spectrum showed the presence of two tertiary methyls at d H 0.93 (3H, s) and 1.84 (3H, s), one methoxy group at d H 3.71 (3H, s), and two olefinic protons at d H 6.78 (1H, t, J ¼ 7.5 Hz) and 7.18 (1H, d, J ¼ 5.5 Hz). The 13 C-NMR spectrum displayed 16 carbon signals, with the aid of DEPT and HMBC spectra, were assigned to three methyl, four methylene, four methine, and five quaternary carbons including two carbonyl at d C 170.2 and 171.2. The NMR data of 1 were similar to those of sesquicaranoic acid A (Zhang et al. 2014), a sesquiterpene isolated from the stems of Illicium jiadifengpi. The absence of two oxygenated carbons and the presence of a trisubstituted double bond between C-10 and C-11 in 1 was proven by the 1 HÀ 1 H COSY experiment which showed the presence of the fragment À CH 2 (8)ÀCH 2 (9)ÀCH(10)À, which was further confirmed by the HMBC correlations of H-10 with C-8, C-9, C-12 and C-13 ( Figure S1). Another difference was the absence of one methyl group and the presence of a carbonyl (d C 170.2) and a methoxy group (d C 52.2; d H 3.71, s) in 1. The carbonyl group established at C-12 and the OCH 3 moiety attached to C-12 were determined from the HMBC correlations from OCH 3 /H-10 with C-12. In the ROESY spectrum, the correlations between H-1/H-6 and H-8 ( Figure S8) indicated that H-1, H-6 and C-8 were on the same side. The negative Cotton effects at 213 nm in the CD spectrum ( Figure S10) allowed the assignment of 1S configurations (Weiss and Ziffer 1963;Yogev et al. 1967;Beecham 1971), which further assigned the absolute configurations of 6R and 7S. Therefore, compound 1 was identified as (1S,6R,7S)-7-((Z)-5-methoxy-4-methyl-5-oxopent-3-en-1-yl)-7-methylbicyclo[4.1.0]hept-2-ene-3-carboxylic acid and named sesquicaranoic acid C.
The isolates were evaluated for their effects on the inhibition of nitric oxide production in LPS-activated RAW264.7 cells. All the compounds exhibited no cytotoxicity at their effective concentration based on the MTT assay. As shown in Table 1, compounds 1, 3, 4, 6, 8 and 9 exhibited inhibitory activities of NO production, with IC 50 values of 22.59-46.32 lM, while the other compounds were inactive (IC 50 > 50 lM). Among them, palmarumycin SA1 (3) and matairesinol (8) showed significant inhibitory activity with the IC 50 values of 22.59 and 27.06 lM, respectively, indicating that these two compounds would be identified as an anti-inflammatory lead compound.

General experimental procedures
Optical rotations were measured with a JASCO P-1020 polarimeter. UV spectra were obtained on a Shimadzu UV-2401A spectrophotometer. CD spectra were recorded on a JASCO J-810 CD spectrometer. IR spectroscopy was measured in a Bio-Rad FTS-135 spectrometer with KBr pellets. HRESIMS were recorded on a Shimadu LCMS-IT-TOF spectrometer. The NMR spectra were recorded on Bruker DRX-500 spectrometers with TMS as internal standard, and chemical shifts (d) were expressed in ppm with reference to the solvent signals. Silica gel (200-300 mesh; Qingdao Marine Chemical Inc., Qingdao, China), RP-18 gel (40-63 lm, Merck, Darmstadt, Germany), MCI gel (75-150 lm; Mitsubishi Chemical Corporation, Japan), and Sephadex LH-20 (Amersham Pharmacia biotech, Sweden) were used for column chromatography. Semipreparative HPLC was performed on an Agilent 1200 apparatus equipped with a UV detector and a Zorbax SB-C-18 (9.4 mm Â 25cm, Agilent, USA) column. Fractions were monitored by TLC and spots were visualized by heating silica gel plates sprayed with 10% H 2 SO 4 in EtOH. All solvents were distilled prior to use.

Inhibitory assay of NO production
Inhibitory assays of NO production were carried out as previously described (Yan et al. 2016). Briefly, RAW264.7 cells were harvested and seeded into 96-well plates (3 Â 10 4 cells/well) and preincubated at 37 C for 24 h. After serum starvation for 12 h, cells were pretreated with various concentrations of compounds (0-50 lM) for 30 min, and then stimulated with 1 lg/mL of LPS for 24 h. The nitrite concentration in the culture supernatant was evaluated by using the Griess reagent. MTT assay was used to evaluate the effect of compounds on cell viability.

Conclusion
Two new and 10 known compounds were isolated from the pericarps of Illicium difengpi. All the compounds were isolated from this species for the first time. palmarumycin SA1 (3) and matairesinol (8) exhibited significant inhibitory activity with the IC 50 values of 22.59 and 27.06 lM, respectively, which were worth of further research.

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