Sesquiterpenes from the fruits of Illicium Simonsii maxim

Abstract A new allo-cedrane sesquiterpene glycoside (1) and nine known compounds (2–10) were isolated from the ethanol extract of the fruit of Illicium simonii Maxim. Their structures were elucidated by spectroscopic methods, including 1D-, 2D-NMR, and HRESIMS. The absolute configuration of compound 1 was confirmed by CD experiments. Among them, compounds 1, 4, 5, and 7 displayed moderate anti-inflammatory activities by use of an in vitro bioassay. Graphical Abstract


Introduction
The genus Illicium, belonging to the family Illiciaceae, is a rich source of prenylated C 6 -C 3 compounds (Fukuyama et al. 1994;Kubo et al. 2011). Neolignans and sesquiterpene lactones are major constituents in this genus (Bai et al. 2012;Fang et al. 2013;Liu et al. 2011). Illicium simonsii Maxim., an evergreen shrub or tree of this genus, mainly distributed in India, Myanmar, and the southwestern part of China (Li 2002).
Several parts of this plant have been used as a folk medicine. Its roots, leaves and fruits are commonly used to cure gastro-frigid vomiting, cystic hernia, and gas pains in chest and scabies; its fruits are rich in volatile oil and commonly used as food flavors, possessing anti-cough, expectorant, analgesic, and antibacterial activities. Previous investigations on this plant have proved that it is a rich source of sesquiterpenoids, and some highly oxygenated seco-prezizaane sesquiterpenes have been obtained from the stems, leaves, and fruits of I. simonsii Maxim. (Kouno et al. 1994;Yin et al. 2013;Ma et al. 2017;Ruan et al. 1991;Dong et al. 2013;Liu et al. 2010) In a previous study of a 95% ethanol extract of from the ethanol extract of the fruit of I. simonii Maxim., 3 new compounds were obtained (Zhuang et al. 2018). In a continuing search for bioactive metabolites from the same extract, a new allo-cedrane sesquiterpene glycoside (1) and nine known compounds (2-10) were isolated from the ethanol extract of the fruit of I. simonii Maxim (Figure 1). Their structures were determined by spectroscopic analyses. The anti-inflammatory activities of 1-10 were also evaluated. )]. The 13 C NMR and HSQC spectra of 1 in MeOD indicated 21 carbon resonances including three methyls (d C 17.9, 15.3, and 9.9), five methylenes, eight methines (one methinedioxy at d C 96.3, five oxymethines at d C 78.9, 78.4, 75.4, 74.0, and 71.0, and two sp3 hybridized), and five quaternary carbons (a ketone carbonyl at d C 216.6, a ester carbonyl at d C 175.4, and three sp3 hybridized). The above information coupled with biogenetic considerations and literature references indicated that 1 was a sesquiterpene glycoside and its aglycone displayed the allocedrane skeleton (Nunez et al. 2007).

Results and discussion
In the HMBC experiment, correlation of H-15/d C 75.4 suggested an additional hydroxyl group at C-2. A series of HMBC correlations from H-7, H-10, and H-12 to d C 216.6 and from H-4 and H-14 to d C 175.4 located the ketone group at C-11 and the ester carbonyl at C-13, respectively. The linkage of the sugar unit at C-13 was supported by the HMBC correlation from H-1 0 to C-13. Thus, the planar structure of 1 was established. Its planar structure was the same as (1R,2S,4S,5R,6R,9S)-2-hydroxy-11oxoallo-cedra-13-oic acid 13-O-b-D-glucopyranosyl ester .
The main differences between the two compounds were the stereo configurations. NOESY correlations of H-14 with H-4 and H-10a (b-oriented) and H-15 with H-4 and H-2 suggested that H-4, CH 3 -14, and CH 3 -15 were b-oriented and that 2-OH was a-oriented. The absolute configuration for the aglycone of 1 was determined by analysis of its CD spectrum. The negative Cotton effect at 303 nm indicated that the absolute configuration of the aglycone was 1S, 2S, 4 R, 5 R, 6 R, and 9S based on the octant rule for cyclohexanone (Minkin et al, 1977). The large coupling constant (8.0 Hz) of the glucose anomeric proton suggested that it was in a b-configuration, and its D-configuration was established by GC analysis. Therefore, 1 was determined to be (1S, 2S, 4 R, 5 R, 6 R, 9S)-2-hydroxy-11oxoallo-cedra-13-oic acid 13-O-b-D-glucopyranosyl ester.
In order to evaluate their anti-inflammatory activities, all isolated compounds were evaluated on murine microglial BV2 cells by LPS-stimulated NO production (curcumin as a positive control). 5 showed the strongest inhibitory potency with IC 50 value of 8.9 lM, following 4,7, and 1 with IC 50 values of 15.7, 21.2, and 26.9 lM, respectively, but the others were inactive (IC 50 value >50 lM).

General
Optical rotations were measured with a Perkin-Elmer 241 automatic digital polarimeter. CD spectra were measured on a JASCO J-810 spectro-polarimeter. NMR spectra were obtained on an INOVA-500 spectrometer using the solvent signals (MeOD: d H 3.31 and d C 49.00) as internal standards. GC data were obtained using an Agilent 7890 A instrument. HRESIMS data were recorded using an Agilent 6250 Accurate-Mass Q-TOF LC/MS spectrometer. Medium pressure chromatography system was equipped with two piston pumps, a dual-wavelength UV detector, and a fraction collector (H&E Co., Ltd, Beijing, China). Preparative HPLC was performed on a Shimadzu LC-6AD instrument with an SPD-10A detector using a YMC-Pack ODS-A column (250 Â 20 mm, 5 lm). Column chromatography (CC) was performed with silica gel (200-300 mesh, Qingdao Marine Chemical Inc., Qingdao, China) and ODS (50 mm, YMC, Japan), respectively. TLC was carried out with glass precoated silica gel GF 254 plates. Spots were visualized under UV light or by spraying with 10% H 2 SO 4 in EtOH followed by heating.

Acid hydrolysis of compound 1
Compound 1 (2 mg) was dissolved in 2 M HCl (aq) (5 mL) and heated at 70 C for 8 h under constant stirring. The reaction mixture was diluted with H 2 O and extracted with EtOAc (3 Â 5 mL). The EtOAc layers were combined and evaporated to dryness and then subjected to preparative HPLC using the mobile phase CH 3 CN/H 2 O (17:83) to give the aglycone 1a (0.9 mg). The aqueous layers were combined, evaporated and cryodesiccated. The residue was dissolved in anhydrous pyridine (1 mL), and then Lcysteine methyl ester hydrochloride (2 mg) was added. The mixture was stirred at 60 C for 2 h, and then 0.2 mL of N-trimethylsilylimidazole was added, followed by heating to dryness at 60 C for 2 h. The dried reactant was partitioned between n-hexane and H 2 O (0.2 mL), and the n-hexane fraction was subjected to gas chromatography (GC) (column: DM-5, 0.25 mm Â30 m Â 25 lM; detector: FID; temperature: 280 C; injector temperature: 250 C; carrier: N 2 gas). The sugar was identified by comparison of the retention time with the authentic standard.

Inhibition of nitric oxide production assay
The murine microglial BV2 cells, purchased from the Cell Culture Centre at the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heatinactivated fetal bovine serum (FBS), at 37 C atmosphere, 5% CO 2 . The microglia cells were placed in 96-well cell culture plates (2 Â 10 4 cell/mL) and preincubated for 24 h. Then the cells were treated with various concentrations of isolated compounds in triplicate for 1 h and continuously incubated with LPS (Sigma-Aldrich) (0.3 lg/mL) for 24 h. Curcumin was used as the positive control. After incubation, the supernatants (100 lL) were added to a solution of 100 lL of Griess reagent (a 1:1 mixture of 0.1% naphthyl ethylene diamine and 1% sulfanilamide in 5% H 3 PO 4 ) at room temperature for 20 min. NO concentration was quantified by a microplate reader at 540 nm for the amount of stable nitrite produced in the cell culture supernatants using the Griess assay.

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