Lignan Glucosides from the Stem Barks of Illicium difengpi

In this study, four new lignan glucosides, named difengpiosides A–D (1–4), were isolated from the stem barks of Illicium difengpi, together with seven known compounds 5–11. Their structures were identified on the basis of spectroscopic analyses (1D and 2D NMR, HRESIMS, CD) and a comparison with literature data. All the compounds were evaluated for their inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells.


Introduction
Illicium difengpi K. I. B et K. I. M. (Illiciaceae), native to China, is a toxic shrub that grows in the mountainous areas of Guangxi Province. Its stem bark is listed in the Chinese Pharmacopeia as a traditional Chinese medicine to treat rheumatic arthritis [1]. Previous phytochemical studies on this plant mainly focused on the low and middle polarity components and reported the isolation of over 40 compounds, including phenylpropanoids, lignans, triterpene acids, sesquiterpenes and others [2][3][4][5][6][7]. However, there are few investigations on the polar substances of I. difengpi. In order to provide comprehensive chemistry support for pharmacological studies and quality control of I. difengpi, the present study describes the isolation and structure elucidation of four new lignan glycosides together with seven known compounds from the n-BuOH-soluble fraction of an EtOH extract of the stem barks of this plant, as well as their inhibitory activities against nitric oxide production in lipopolysaccharide-induced RAW264.7 cells.
Molecules 2016, 21, 607 2 of 8 and aromatic ring (1595 and 1501 cm −1 ) groups. The 1 H-NMR spectrum (Table 1) displayed signals at δH 7.07 (1H, d, J = 8.5 Hz), 7.01 (1H, d, J = 2.0 Hz) and 6.89 (1H, dd, J = 8.5, 2.0 Hz) for an AMX aromatic spin system, and four broad singlets at δH 6.92 (2H) and 6.72 (2H) for two tetrasubstituted aromatic rings, as well as three aromatic methoxy groups at δH 3.85 (6H, s) and 3.80 (3H, s). The remaining signals indicated the presence of a sugar moiety, an n-propanol and two −O−CH−CH−CH2O− spin systems, which were also confirmed by the 1 H-1 H COSY spectrum ( Figure 2). The 13 C-NMR spectrum (     These spectral features implied that the aglycone of 1 should be a dimer of dihydrobenzofuran neolignan and was the same as vitrifol A [8]. The rhamnose unit was located at C-4 as elucidated by the HMBC correlation between δ H 5.33 (H-1 111 ) and δ C 146.7 (C-4) ( Figure 2). The α-orientation of the sugar was determined by the small coupling constant (J = 1.5 Hz) of the anomeric proton H-1 111 and its L-configuration was established by HPLC analysis after acid hydrolysis. By comparison with reported data [9], the relative configuration of H-7/H-8 and H-7 1 /H-8 1 was determined as threo according to their coupling constants (J 7,8 = 6.0 Hz and J 7 1 ,8 1 = 6.5 Hz). The absolute configuration of 1 was established as 7R,8S and 7 11 R,8 11 S on the basis of the negative Cotton effects at 237 and 290 nm in the circular dichroism (CD) spectrum as shown in Figure S8, Supplementary Materials [10]. From the above analysis, compound 1 was determined to be vitrifol A 4-α-L-rhamnopyranoside, and named as difengpioside A. To the best of our knowledge, this is the first report of a dihydrobenzofuran sesquilignan glucoside from the family Illiciaceae.
Compound 2 was isolated as an amorphous powder. The HRESIMS spectrum showed a peak at m/z 487.1572 [M + Na] + (calcd. for 487.1575), corresponding to the molecular formula C 23 H 28 O 10 . The IR spectrum displayed the presence of hydroxyl (3412 cm´1) and aromatic ring (1595 and 1501 cm´1) groups. The 1 H-NMR spectrum (Table 2) displayed AMX aromatic spin-system signals at δ H 6.76 (1H, d, J = 8.0 Hz), 6.83 (1H, dd, J = 8.0, 1.5 Hz) and 6.97 (1H, d, J = 1.5 Hz) , two aromatic H-atom signals at δ H 7.02 (1H, s) and 6.56 (1H, s), three aromatic methoxy groups at δ H 3.83 (3H, s), 3.82 (3H, s) and 3.79 (3H, s), and an anomeric proton of xylose at δ H 4.31 (1H, d, J = 7.5 Hz) which indicated a β-configurantion for the xylosyl moiety. The 13 C-NMR spectrum (Table 2) showed the presence of 23 carbons including twelve aromatic carbon signals of two benzene rings, an oxygenated methine carbon at δ C 89.0, a methyleneoxy carbon at δ C 72.8, three methoxy carbons at δ C 57.8, 56.6 and 56.4, and a methine carbon at δ C 52.7. Additionally, five carbon signals (δ C 105.0, 77.9, 74.9, 71.2, and 66.9) were ascribed to a xylosyl moiety. The NMR data were quite identical to those of the known compound 2,3-dihydro-7-methoxy-2-(4 1 -hydroxy-3 1 -methoxyphenyl)-3a-O-β-D-xylopyranosyloxymethyl-5-benzofuranpropanol [5]. The only difference lies in that the propanol moiety at C-1 1 of the known compound was replaced by a methoxyl group in 2, as confirmed by the HBMC correlation from δ H 3.82 (OCH 3 ) to δ C 151.6 (C-1 1 ) (Figure 2) and the molecular formula of 2. The xylose unit was located at C-9 as elucidated by the HMBC correlation between δ H 4.31 (H-1 11 ) and δ C 72.8 (C-9) and its D-configuration was detected by direct comparison with an authentic sample on HPLC after acid hydrolysis. The absolute configurations of C-7 and Molecules 2016, 21, 607 4 of 8 C-8 of 2 were determined as 7R,8S, on the basis of their coupling constant (J 7,8 = 6.5 Hz), indicating H-7 and H-8 to be threo, and the negative Cotton effects at 243 and 287 nm in the CD spectrum [10]. Compound 2 was thus identified as (2R,3S)-2,3-dihydro-5,7-dimethoxy-2-(4 1 -hydroxy-3 1 -methoxyphenyl)-3a-O-β-D-xylopyranosyloxymethylbenzofuran and named difengpioside B.  9) was assigned to a xylose moiety and its β-orientation was determined by the large coupling constant (J = 7.5 Hz) of the anomeric proton at δ H 4.22. The 13 C-NMR (Table 2) and HSQC spectra revealed that the remaining signals of 3 contains twelve aromatic carbons of three benzene rings, three methylene carbons including two oxygenated ones at δ C 73.9 and 61.6, two methoxy carbons at δ C 56.4 and 56.3, three methine carbons at δ C 48.1, 47.8 and 37.4. The above data were very similar to those of (+)-isolariciresinol 9-O-β-D-xylopyranoside (5) [11], except that the difference in the chemical shifts of C-9 and C-9 1 . This indicated the xylosyl moiety was positioned at C-9 1 , which was further supported by HMBC correlation observed between H-1 11 (δ H 4.22) and C-9 1 (δ C 73.9). The configuration of the xylosyl unit was established as D by HPLC analysis after acid hydrolysis. The absolute configuration of the chiral centers was established to be the same as that of (+)-isolariciresinol from the results of CD spectral analysis [12]. Therefore, compound 3 was elucidated as (+)-isolariciresinol-9 1 -O-β-D-xylopyranoside, with the trivial name difengpioside C. It is worth noting that although the literature had been reported a compound with the same name as 3 [13], analysis of NMR spectra revealed that the compound actually was identical with (+)-isolariciresinol 9-O-β-D-xylopyranoside (5 Table 2) showed signals of 1,3,4-trisubstituted aromatic ring at δ H 6.65 (1H, d, J = 8.5 Hz), 6.52 (1H, s) and 6.50 (1H, d, J = 8.5 Hz), a methoxy group at δ H 3.73 (3H, s), and an anomeric proton of rhamnose at δ H 4.63 (1H, d, J = 1.5 Hz) which indicated an α-configuration for the sugar moiety. Sixteen carbon signals, including six aromatic carbons, two methylene carbons (including one oxygenated), one methine carbon, one methoxy carbon and six rhamaopyranosyl signals, were supported by the 13 C-NMR (Table 2) and HSQC spectra. The above spectral data combined with the molecular formula indicated 4 possessed a highly symmetrical skeleton. Acid hydrolysis of 4 liberated the L-rhamnose moiety, which was determined by HPLC analysis. The HMBC correlation between δ H 4.63 (H-1 11 ) and δ C 69.4 (C-9) confirmed that L-rhamnose was linked to C-9. The NMR data of 4 were very similar to those of secoisolariciresinol diglucoside [14], except for the sugar moieties, suggesting that 4 was a diphenylbutane-type lignan dirhamnoside. The symmetrical structure feature of 4 indicated its configuration should be either 8S, 8 1 S or 8R, 8 1 R. Since two negative Cotton effects at 228 and 280 nm were observed in the CD spectrum [15], the absolute configuration of 4 was determined to be 8R,8 1 R. Thus, compound 4 was elucidated as (´)-secoisolariciresinol 9,9 1 -di-O-α-L-rhamnopyranoside, which was named as difengpioside D.
Inhibitors of NO release are considered as potential anti-inflammatory agents [19]. Since the stem barks of Illicium difengpi have been applied for the treatment of rheumatic arthritis in China, the isolated compounds were evaluated for their effects on the inhibition of NO production in LPS-activated RAW264.7 cells. As shown in Table 3, the dihydrobenzofuran-type (1, 2, 9, and 10) and aryltetralin-type (3, 5, 6, and 7) lignan glycosides exhibited weak inhibitory effect against NO with inhibition ratios in the range of 3.29% to 10.53% at a concentration of 25 µM, while the dibenzylbutane-type lignan glycosides 4 and 8 and neolignan glucoside 11 showed no inhibitory effect at the same concentration.

General Information
Melting points were obtained on an X-4 micro melting point apparatus (Shanghai Jingke Scientific Instrument Co., Ltd, Shanghai, China). Optical rotations were measured with a P-1020 polarimeter (JASCO, Tokyo, Japan). UV spectra were obtained on a UV-2401A spectrophotometer (Shimadzu, Kyoto, Japan). CD spectra were recorded on a J-810 CD spectrometer (JASCO, Tokyo, Japan). IR spectra were measured in a FTS-135 spectrometer (Bio-Rad, Richmond, CA, USA) with KBr pellets. HRESIMS were recorded on a LCMS-IT-TOF spectrometer (Shimadzu, Kyoto, Japan). The NMR spectra were recorded on a DRX-500 spectrometer (Bruker Co., Ettlingen, Germany) with TMS as internal standard, and chemical shifts (δ) were expressed in ppm with reference to the solvent signals. Silica gel (200-300 mesh; Qingdao Marine Chemical Inc., Qingdao, China), D101 macroporous resin (Nankai University, Tianjin, China), ODS (40-63 µm; Merck, Darmstadt, Germany), and Sephadex LH-20 (Amersham Pharmacia Biotech, Uppsala, 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 mmˆ25 cm, Agilent Technologies, Santa Clara, CA, USA) column. Analytical HPLC was performed on a Shimadzu HPLC system equipped with a refractive index detector and a CARBOSep COREGEL-87C Ca + (7.8ˆ300 mm, 9 µm, Transgenomic Inc., Omaha, NE, 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. Solvents were distilled before use.

Plant Material
The stem barks of Illicium difengpi were collected from Longzhou County, Guangxi Province, China, in October 2010 and identified by Prof. H. Tang. A voucher specimen (CTM201002) was deposited at the Guangxi Key Laboratory of Functional Phytochemicals Research and Utilization, Guangxi Institute of Botany, China.

Determination of the Absolute Configuration of the Sugars in Compounds 1´4
Each compound (1.5 mg) was dissolved in 0.5 M HCl (0.2 mL) and heated at 80˝C for 2 h. The mixture was desalinated by passing through columns of anion and cation exchange resin and then filtered. The filtrate was directly analyzed by a Shimadzu HPLC system equipped with a refractive index detector and a CARBOSep COREGEL-87C Ca + column (7.8ˆ300 mm, 9 µm, Transgenomic Inc.) at 85˝C with elution of HPLC grade water for 18 min at a flow rate of 0.5 mL/min. The injection volume was 20 µL. The standards L-rhamnose and D-xylose were treated by the same water and chromatographic conditions. The sugars from each compound were identified by comparison of their retention times with those for authentic standards (t R : 13.67 min for L-rhamnose, 13.13 min for D-xylose).

NO Production Inhibition Assay
Assays for NO production were carried out as previously described [20]. Briefly, RAW 264.7 macrophages were harvested and seeded in 96-well plates (3ˆ10 4 cells/well) for measurement of NO production. The plates were pretreated with various samples for 30 min and then incubated with 1 µg/mL LPS for 24 h. The inhibitory effects of the isolated compounds on NO production were evaluated by using the Griess reagent.

Conclusions
Eleven compounds, including four new lignan glucosides 1-4, named difengpiosides A-D, were isolated from the n-BuOH-soluble fraction of an EtOH extract of the stem barks of I. difengpi. All compounds showed weak or no inhibitory activities against NO production at the concentration of 25 µM, indicating that the polar constituents of this plant showed no inflammatory activity in vitro.