The chemical constituents from the roots of Gentiana crassicaulis and their inhibitory effects on inflammatory mediators NO and TNF-α

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Introduction
Rheumatoid arthritis (RA) is characterized as a chronic inflammatory disease, in which the immune system destroys synovial joints and accessory structures. 1 "Qin-Jiao" is a wellknown traditional Chinese medicinal (TCM) herb commonly used for fighting RA since ancient times. 2 In the Chinese Pharmacopoeia, the roots of four plants from the genus Gentiana (Gentianaceae), G. macrophylla, G. crassicaulis, G. straminea, G. duhurica, are used as the original materials of "Qin-Jiao". 3 Among them, G. macrophylla have been chemically and biological investigation by several groups, [4][5][6] whose results suggested that the water extract of G. macrophylla could obviously resist RA. 7 The mechanism might be the inhibition of not only phagocyte to produce and release prostaglandin E2 (PGE2), but also the activity of cyclooxygenase-2 (COX-2). 8 Moreover, gentiopicroside (5), one of the major compounds in G. macrophylla, also showed inhibitory effects on inflammatory mediators NO and COX-2. 9 However, the anti-inflammatory constituents from G. crassicaulis have not been reported to date.
As a part of our research on new bioactive compounds from Gentiana medicinal plants, [10][11][12][13][14][15] the present investigation led to the isolation of two new compounds (1 and 2) from the roots of G. crassicaulis, together with 13 known ones. Their structures were elucidated by detailed 1D and 2D NMR spectroscopic analysis and chemical methods. Most of the isolates (1, 4-6 and 8-11) were evaluated for their antiinflammatory activities by inhibition of inflammatory mediators NO and TNF- and their structure-activity relationships (SARs) were discussed.
Compound 1, a colorless amorphous powder, had a molecular formula C 17 H 24 O 11 as deduced from the HRESIMS (m/z 439.1011 [M + Cl] -). The 13 C NMR and DEPT data established the presence of one methyl, one oxygen-bearing methylene, three methines linked with oxygen, one tetra-and one tri-substituted double bonds, one carbonyl, and one methoxy groups, in addition to a hexosyl moiety. In the 1 H NMR spectrum of 1 (Table 1) , a methoxy proton (δ 3.59, s) and some aliphatic protons were observed. The aforementioned NMR data suggested that 1 was an analogue of compound 9, an iridoid glucoside from the title plant and G. macrophylla. 10 Instead of the terminal double bond between C-10 and C-8 in 9, compound 1 had a trisubstituted double bond (olefinic proton at δ 6.58, q, J = 7.3 Hz) linked with a methyl group (δ C 14.3, δ H 1.99, d, J = 7.3 Hz). In addition, obvious difference at C-8 (δ and 134.5 for 1 and 9, respectively) was observed. The above data revealed that the terminal double bond between C-10 and C-8 in 9 was rearranged to between C-9 and C-8 in 1. The locations of the methyl and trisubstituted double bond were further confirmed by the HMBC correlations of the methyl group (δ 1.99) with C-8 (δ 137.2) and C-9 (δ 129.0), and the trisubstituted olefinic proton (δ 6.58, H-8) with C-1 (δ 91.2) and C-5 (δ 145.5) (Figure 2). In the ROESY spectrum of 1 (Figure 2), correlations of H-3/H-1, and the broad singlet H-6 (δ 4.56, br. s) revealed the equatorial orientations of both H-3 and H-6, respectively, that is, both the C-3 methoxyl and C-6 hydroxyl groups were  oriented. In addition, significant ROESY correlations of the C-10 methyl protons at δ 1.99 with H-1 (δ 6.25) and the olefinic proton (δ 6.58, H-8) with H-6 (δ 4.56) were observed, revealing the configuration of the double bond between C-8 and C-9. Therefore, the structure of qinjiaoside B (1) was assinged as shown in Figure 1.
Compound 2 was obtained as a colorless amorphous powder and its molecular formula C 21 H 32 O 14 was deduced on the basis of HRESIMS (m/z 507.1715 [M -H] -). The 1 H NMR spectrum (Table 1) displayed a doublet methyl signal (δ 1.10, d, J = 6.9 Hz), a singlet olefinic proton (δ 7.04, s), two anomeric protons (δ 4.86, d, J = 8.5 Hz; 4.97, d, J = 3.6 Hz), as well as some aliphatic protons. The 13 C NMR and DEPT data showed the presence of 21 cabon signals, refering to one methyl, one methylene, five methines including two oxygenbearing ones, one trisubstituted double bond, one carbonyl, and 11 sugar carbon signals arising from a hexosyl and a pentosyl units. Acid hydrolysis of 2 afforded D-glucose and D-xylose as sugar residue, which were confirmed by GC analysis of their corresponding trimethylsilated L-cysteine adducts. The above data (Table 1)    correlated with the glucosyl C-6' (δ 65.9), indicating the xylosyl moiety was linked to the glucosyl C-6' in 2. In addition, the chemical shift of the glucosyl C-6' of 2 was down-field shifted by 4.6 ppm in the 13 C NMR spectrum, relative to 4. In the ROESY spectrum of 2 (Figure 2 The isolated iridoid glycosides 1, 4-6 and 8-11 were tested for their anti-inflammatory activities by inhibitory effects on LPS-induced NO and TNF-α production in macrophage RAW264.7 cells ( Table 2). All of them showed inhibitory effects on inflammatory mediators NO at a concentration of 15 μM, while 5 and 9 displayed the most potential inhibitory effects on TNF- with IC 50 of 0.06 and 0.05 μM, respectively. It is noted that the terminal double bond between C-9 and C-10 should be a key to the inhibitory effect on TNF-. For example, compound 1 did not display activity due to the absence of the terminal double bond. In addition, compounds 6 and 10 and 11, possessing longer sugar chain than that of 5, had not inhibitory effects on TNF-, revealing that the number of monosaccharide units in their sugar chains could also affect their anti-inflammatory activities.
In conclusion, 15 compounds including two new iridoid glycosides (1 and 2) were identified. The iridoid glycosides 1, 4-6, and 8-11 showed inhibitory effects on inflammatory mediators NO, while only 4, 5, 8 and 9 displayed potential inhibitory effects on TNF-. This study provided valuable information for "Qin-Jiao" as a TCM herb traditionally used for fighting RA. Further chemical and biological investigations on the iridoid scaffolds from Gentiana are currently underway, in order to explore the therapeutic potential of this important class of natural products as anti-inflammatory leads for drug discovery.

Experimental Section
General Experimental Procedures. Optical rotations were performed on a P-1020 polarimeter (JASCO, Tokyo, Japan). IR spectra were measured on a Bruker Tensor 27 spectrometer with KBr pellets. 1D and 2D NMR spectra were run on Bruker AM-400 and DRX-500 instruments operating at 400 and 500 MHz for 1 H, and 100 and 125 MHz for 13 C, respectively. Coupling constants are expressed in Hertz and chemical shifts are given on a ppm scale with tetramethylsilane as internal standard. The MS data were recorded on a VG Auto Spec-3000 spectrometer (VG, Manchester, U.K.) with glycerol as the matrix. HRESIMS were recorded on an API Qstar Pulsa LC/TOF spectrometer. GC analysis was run on a Shimadzu GC-14C gas chromatograph.
Cell Culture. 26 The RAW264.7 murine macrophage cell line (ATCC TIB-71; American Type Culture Collection, Manassas,VA, USA) was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heatinactivated fetal bovineserum (FBS), 2 mM L-glutamine, 100 IU mL -1 penicillin and 100 μg mL -1 streptomycin in a 37 °C incubator with 5% CO 2 . For all experiments, the cells were grown to 80-90% confluence, with no more than 20 passages. Cells were stimulated by LPS (0.5 μg mL -1 ) in the presence or absence of compounds for the measurement of the production of pro-inflammatory cytokines (TNF-α) and accumulation of nitric oxide (NO).
Measurement of NO release. 26 RAW264.7 cells were pretreated by tested compounds for 1 h and stimulated by LPS (0.5 μg mL -1 ) after 18 h of incubation. NO production was estimated from the amount of stable nitrite produced in the cell culture supernatants measured photometrically by the Griess assay against a standard curve obtained with different concentrations of sodium nitrite. Each experiment was performed three times in duplicate.
Measurement of TNF-α production. 26 RAW264.7 cells were cultured in 96-well plates (1 × 10 4 cell mL -1 ) and preincubated with compounds for 1 h, followed by a further 18 h treatment with LPS for measurement of TNF-α. Contents of TNF-α in the culture medium were measured by ELISA using anti-mouse TNF-α antibodies and a biotinylated secondary antibody, according to the manufacturer's instructions. The optical density of each well was measured at 450 nm with an ELISA reader (Molecular Devices 5, Menlo Park, CA, USA).
Statistical Analysis. 26 Results are expressed as the mean ± SD of three experiments. Statistical significance was evaluated by one-way ANOVA followed by Student's t test for paired populations. P values < 0.05 are considered statistically significant.

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/ 10.1007/s13659-012-0067-3 and is accessible for authorized users.