Two new monoterpene glucosides from Xanthium strumarium subsp. sibiricum with their anti-inflammatory activity

Abstract Two new monoterpene glucosides: xanmonoter A (1) and xanmonoter B (2) were isolated from Xanthium strumarium. Their structures were elucidated on the basis of 1D and 2D NMR, MS and CD analysis. Compounds 1 and 2 were tested for their anti-inflammatory activity with IC50 values of 17.4, 22.1 μM, respectively.


Introduction
Xanthium strumarium subsp. sibiricum (Compositae) is a medicinal plant which is widely distributed in the tropical and subtropical regions of southeast Asia (Qiu et al. 2010). Its fruits which is called 'Fructus Xanthii' in Chinese has been used as Chinese folk medicine for the treatment of anemofrigid cold, rheumatic arthralgia, pruritus, and nasosinusitis. Recent studies showed that 'Fructus Xanthii' contained several classes of compounds, such as sesquiterpene lactones Bui et al. 2012;Favier et al. 2005), ent-kauranoid glycosides (MacLeod et al. 1990;Jiang et al. 2013), thiazines (Lee et al. 2008;Tsankova et al. 1994), and so on (Zhang et al. 2006;Kan et al. 2011;Chen et al. 2015;Olivaro et al. 2016;Taranto et al. 2017) with significant biological activities (Zhao et al. 2008;Song et al. 2009). Based on previous research, we examined the 70% ethanol extract of the fruits of this plant and isolated two new monoterpene glucosides: xanmonoter A (1) and xanmonoter B (2) (Figure 1). In this paper, we report the structure elucidation of the new monoterpene glucosides as well as their anti-inflammatory activity.

Results and discussion
The air-dried and powdered fruits of X. strumarium, were extracted three times with 70% ethanol. Removal of the ethanol under reduced pressure yielded an ethanol extract. The residue was partitioned by EtOAc and n-BuOH. Repeated chromatography on silica gel of the n-BuOH extracts afforded the new monoterpene glucosides 1 and 2.
Compound 1 was isolated as a white amorphous powder with -42.6 (c ¼ 0.1 MeOH). HR-ESI-MS gave a quasi-molecular ion peak at m/z 487.2134 [M þ Na] þ (Calcd 487.2155) in the positive-ion mode. In conjunction with the analysis of 1 H and 13 C NMR spectra, the formula of compound 1 was deduced as C 21 H 36 O 11 . The IR spectrum showed the presence of hydroxyl and an double bond at 3394 and 1635 cm À1 , respectively. The 1 H NMR spectrum showed two methyl signals at d H 1.60, 1.66; four olefinic protons at d H 5.11 (t, J ¼ 6.0 Hz), 5.30 (dd, J ¼ 17.2, 1.6 Hz), 5.16 (dd, J ¼ 10.8, 1.6 Hz), 5.87 (dd, J ¼ 17.2, 10.8 Hz); two anomeric protons at d H 4.25 (d, J ¼ 7.6 Hz), 5.00 (d, J ¼ 2.4 Hz). Except for two sugar carbons, the 13 C NMR spectrum dispayed another ten carbons including two methyls, four methylenes (one olefinic carbon at d C 114.8, one oxygenated carbon at d C 77.4), two olefinic methines, and two quaternary carbons (one olefinic carbon at d C 132.2, one oxygenated carbon at d C 76.2). The information above indicated that the basic structure of compound 1 was monoterpene glucosides (Deliorman et al. 2001;Marino et al. 2014). All carbon-bound protons were assigned from HSQC spectrum. The whole structure was established by analysis of its 1D and 2D NMR spectra. From the 1 H-1 H COSY spectrum, two substructures (drawn with bolds in Figure S11, see Supporting Information) were identified. In the HMBC spectrum, the correlations from d H 1.60 (3H, s), 1.66 (3H, s) to C-7 (d C 132.2) suggested both methyl groups of C-8/9 were attached to C-7. The correlations between d H 3.60, 3.98 (H 2 -10) and d C 76.2 (C-3) together with the molecular formula above indicated that the presence of hydroxymethyl and hydroxyl groups at C-3. Finally, the HMBC correlations from d H 5.00 (d, J ¼ 2.4 Hz) to C-6 0 (d C 68.8) and d H 4.25 (d, J ¼ 7.6 Hz) to C-10 (d C 77.4) exhibited that the two sugars was connected by C-6 0 and the sugar chain was located at C-10. The configuration of the sugars were assigned after hydrolysis of 1 with 3 mol/L CF 3 COOH. The sugars of 1 were determined to be D-glucose and D-apiose on the basis of the TLC method comparison with authentic monosaccharide (CHCl 3 : MeOH: H 2 O ¼ 3:2:0.2, visualization with ethanol-5% H 2 SO 4 spraying), followed by gas chromatography. The absolute configuration of the C-3 in algucone was assigned by using the CD data of the situ formed [Rh 2 (OCOCF 3 ) 4 ] complex, with the inherent contribution subtracted. In this experiment, the Rh-complex of aglucone 1 showed a significant negative Cotton effect at k max 350 nm indicated that the chirality at C-3 was R (Frelek and Szczepek 1999;Gerards and Snatzke 1990). Therefore, the structure of compound 1 was established as shown and named xanmonoter A.
Compund 2 was was isolated as a white amorphous powder with -35.3 (c ¼ 0.1 MeOH). The molecular formular C 22 H 32 O 7 (m/z [M þ Na]þ487.2132, Calcd 487.2155) was established by HR-ESI-MS. The 1 H NMR and 13 C NMR spectra were very similar to those of compound 1 except the 10-hydroxy methylene group at C-3 in compound 1 transferred to C-2 in compound 2. This was fully confirmed by the analysis of the HSQC and HMBC spectrum. The coupled HSQC spectrum showed that the oxymethylene proton at d H 3.75, 3.22 (m, H 2 -10) is directly attached to an oxygenate methylene carbon resonating at d C 74.9 (C-10), the HMBC spectrum showed that the protons at d H 3.22 (m) had the correlations to olefinic carbon at d C 111.4 (C-1) and 149.7 (C-2). Thus, the different substituents were elaborated clearly. The Rh-complex of aglucone 2 showed a significant positive Cotton effect at k max 350 nm indicated that the chirality at C-3 was S. Thus, the structure of compound 2 was elucidated as shown and named xanmonoter B.
Considering this medicinal herb as a therapeutical agent for the treatment of arthritis, the isolated two compounds were studied for their anti-inflammatory activity on lipopoly-saccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7. Results showed that compounds 1 and 2 displayed moderate inhibitory activities with IC 50 values of 17.4, 22.1 lM, respectively, compared with aminoguanidine (IC 50 3.6 lM) as positive control. Further studies are needed to focus on the anti-inflammatory activity of monoterpene glucosides from X. strumarium and clarify their structure-activity relationship.

General experimental procedures
Optical rotations were obtained on a Perkin-Elmer 341 digital polarimeter. UV and IR spectra were recorded on Shimadzu UV2550 and FTIR-8400S spectrometers, respectively. CD spectra were recorded on a JASCO J-815 spectropolarimeter. NMR spectra were obtained with a Bruker AV IN 400 NMR spectrometer (chemical shift values are presented as d values with TMS as the internal standard). HRESIMS spectra were performed on a LTQ-Obitrap XL spectrometer. Preparative HPLC was performed on a Lumtech K-1001 analytic LC equipped with two pumps of K-501, a UV detector of K-2600, and an YMC Pack C 18 column (250 mm Â 10 mm, i.d., 5 lM, YMC Co. Ltd., Japan) eluted with CH 3 OH-H 2 O at a flow rate of 2 mL/min. C 18 reversed-phase silica gel (40 $ 63 lM, Merk, Darmstadt, Germany), Sephadex LH-20 (Pharmacia, Uppsala, Sweden), MCI gel (CHP 20P, 75 $ 150 lM, Mitsubishi Chemical Corporation, Tokyo, Japan) and silica gel (100 $ 200 and 300 $ 400 mesh, Qingdao Marine Chemical plant, Qingdao, People's Republic of China) were used for column chromatography. And pre-coated silica gel GF 254 plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, People's Republic of China) were used for TLC. All solvents used were of analytical grade (Beijing Chemical Works).

Plant material
Fructus Xanthii was collected from Heilongjiang Province, China, in August 2011, and then authenticated by Prof. Wang Zhen-Yue, Heilongjiang University of Chinese Medicine, Harbin, China. A voucher specimen (No. 20111077) was deposited at the herbarium of Heilongjiang University of Chinese Medicine.

Acid hydrolysis of 1-2
Each compound (3 mg) was heated in 3 mol/L CF 3 COOH (4 mL) for 3 h in a water bath. Each mixture was then extracted with EtOAc. The aqueous layer was evaporated to dryness with ethanol in vacuo at 50 C until neutral. The residues were determined in comparison with authentic monosaccharides using TLC (CHCl 3 : MeOH: H 2 O ¼ 3:2:0.2, visualization with etha-nol-5% H 2 SO 4 spraying).The R f of glucose and D-apiose by TLC were 0.26, 0.38, respectively. Furthermore, the absolute configurations of the sugars were determined by gas chromatography. By this method, L-cysteine methyl ester hydrochloride (0.06 mol/L) and hexam-ethyldisilazane -trimethylchlorosilane (HMDS-TMCS, 3:1) were added to the aqueous residue for derivatization. The solution was then centrifuged and the precipitate was removed. After these processes, n-hexane was used to extract derivate and analyzed by GC. D-Glucose (t R ¼ 25.6 min) and D-apiose (t R ¼ 30.4 min) was detected by comparing with authentic monosaccharides.

Assay for anti-inflammatory ability
RAW 264.7 macrophages were seeded in 24-well plates (10 5 cells/well). The cells were coincubated with drugs and LPS 1 lg/mL) for 24 h. The amount of NO was assessed by determining the nitrite concentration in the cultured RAW 264.7 macrophage supernatants with Griess reagent. Aliquots of supernatants (100 lL) were incubated, insequence, with 50 lL of 1% sulfanilamide and 50 lL of 0.1% naphthylethylenediamine in 2.5% phosphoric acid solution. The absorbance was recorded on a microplate reader at a wavelength of 570 nm.

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

Funding
The work was supported financially by the National Natural