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Bioactive sulfur-containing compounds from Xanthium sibiricum, including a revision of the structure of xanthiazinone
Phytochemistry 173 (2020) 112293
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Bioactive sulfur-containing compounds from Xanthium sibiricum, including a revision of the structure of xanthiazinone
T
Zhao Xia, Tian-qi Xu, Hai-xin Zhang, Yi-min Chen, Wei Xu, Guang-xiong Zhou∗ Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou, China
ARTICLE INFO
ABSTRACT
Keywords: Xanthium sibiricum Asteraceae Xanthii Fructus Thiazinedione Anti-inflammatory Cytotoxicity
Seven previously undescribed sulfur-containing compounds, (+)- and (−)-xanthiazinone A, (+)- and (−)-xanthiazinone B, (+)- and (−)-xanthiazinone C and xanthiazinone D, and four known thiazinedione derivatives, together with three thiophene derivatives were isolated from the fruits of Xanthium sibiricum. Racemic xanthiazinones A-C were separated by chiral HPLC columns. Their chemical structures were elucidated based on extensive spectroscopic analyses, ECD calculations, and single crystal X-ray diffractions. The X-ray crystallographic analyses for xanthiazinones A-C represent the first example described for the structure elucidation of the thiazinedione with the five-membered lactone ring attached via an oxygen atom. Accordingly, the previously proposed structure for xanthiazinone was revised. The anti-inflammatory and cytotoxic activities were evaluated for all the isolated compounds. (+)-xanthiazinone B and 2-hydroxy-xanthiazone exhibited potent inhibitory effects against nitric oxide production in lipopolysaccharide-activated RAW 264.7 mouse macrophage cells with IC50 values of 8.75 and 10.90 μM, respectively. All compounds obviously were inactive for three human tumor cell lines (HepG2, MCF-7, and A549) with IC50 values more than 10 μM.
1. Introduction Xanthium (Asteraceae) is a relatively small genus of plants of worldwide distribution, and only four species and two varieties grow in China (Lv et al., 2001). Xanthii Fructus is the dried ripe fruits of Xanthium sibiricum Patrin ex Widder, an annual gregarious herb. These fruits are commonly known as Chinese Materia Medicinal material “Cang-Er-Zi”. In traditional Chinese medicine, Xanthii Fructus has been widely used in the treatment of leukoderma, fever, scrofula, sinusitis, headache, herpes, and cancer (Lin et al., 2014; Fan et al., 2019; Xue et al., 2014; Hsu et al., 2000). Xanthii Fructus contains different types of chemical constituents, including sesquiterpene lactones (Tao et al., 2016; Shi et al., 2015a), monoterpenoids (Shi et al., 2015b; Jiang et al., 2018), glycosides (Jiang et al., 2013, 2016), lignans (Shi et al., 2014, 2015a; Yin et al., 2016; Jiang et al., 2017), thiazinediones (Lee et al., 2008; Cheng et al., 2013; Qin et al., 2006; Ma et al., 1998; Han et al., 2006; Dai et al., 2008; Yin et al., 2016), thiophenes (Shi et al., 2015b), caffeoylquinic acids (Yoon et al., 2013; Chen et al., 2012; Agata et al., 1993), and other constituents. These compounds showed potential pharmacological activities, such as anti-inflammatory (Shi et al., 2015a; An et al., 2004; Huang et al., 2011; Yeom et al., 2015), antioxidant (Ingawale et al., 2018; Huang et al., 2011), antiviral (Shi et al., 2015a), ∗
antiproliferative (Jiang et al., 2016; An et al., 2004), antitumor (Tao et al., 2016), anti-allergic rhinitis (Peng et al., 2014, 2019) effects, which supported its traditional medicinal usage in inflammatory diseases. Our preliminary study showed that an ethyl acetate (EtOAc) extract of Xanthii Fructus exhibited anti-inflammatory activity in LPSinduced RAW 264.7 cells with an IC50 value of 90.09 ± 2.56 μg/mL (Supporting Information Table S2). To identify the anti-inflammatory constituents of Xanthii Fructus, a 95% ethanol (EtOH) extract of this fruits was phytochemically investigated, resulting in the isolation of seven previously undescribed thiazinedione derivatives (1a, 1b, 2a, 2b, 3a, 3b, and 4) and seven known sulfur-containing compounds (5–11) (Fig. 1). The previously reported structure of xanthiazinone was revised based on the spectroscopic analyses and the result of a single crystal Xray diffraction of 1 (Fig. 2). Herein, the separation, structural elucidation, anti-inflammatory and cytotoxic evaluation of these compounds are reported. 2. Results and discussion Compound 1 was obtained as colorless needle crystals. The sodium adduction at m/z 345.0884 [M + Na]+ by HRESIMS demonstrated that the molecular formula of 1 was C15H18N2O4S. IR spectrum showed the
Corresponding author. E-mail address: [email protected] (G.-x. Zhou).
https://doi.org/10.1016/j.phytochem.2020.112293 Received 20 October 2019; Received in revised form 1 February 2020; Accepted 2 February 2020 0031-9422/ © 2020 Elsevier Ltd. All rights reserved.
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Fig. 1. Structures of compounds 1–11.
presence of hydroxyl (3362 cm-1) and amide carbonyl (1620 cm-1) groups. The 1H NMR data of 1 displayed signals for one olefinic proton at δH 6.32 (1H, s), two methyl groups at δH 1.37 (3H, s) and 1.38 (3H, s), four methylene groups at δH 3.48 (2H, s), δH 4.28 (1H, dd, J = 16.0, 1.5 Hz) and 4.39 (1H, dd, J = 16.0, 1.6 Hz), δH 1.98 (1H, m) and 2.25 (1H, m), δH 2.06 (1H, m) and 2.32 (1H, m), and an oxymethine group at δH 5.00 (1H, d, J = 6.1 Hz) (Table 1). The 13C NMR and DEPT data revealed the presence of fifteen carbons (Table 2) for two amide carbonyl groups (δC 162.3 and 177.6), one conjugated carbonyl group (δC
Fig. 2. Structure revision of xanthiazinone. Table 1 1 H NMR data for compounds 1–4. no.
1a
2a
3a
4a
1b
2 6
3.48 s 6.32 s
3.35 d (3.2c) a 2.47 dd (17.3, 10.8) b 2.58 dd (17.3, 4.5) 2.13 m 1.17 s 1.29 s a 3.20 dd (9.5, 8.1) b 3.68 dd (9.5, 4.4) a 1.84 m b 2.18 m a 2.01 m b 2.24 m 4.84 d (6.2) 8.65 s 9.17 s
3.35 d (3.2) a 2.47 dd (17.3, 10.8) b 2.58 dd (17.3, 4.5) 2.13 m 1.17 s 1.29 s a 3.40 dd (9.6, 7.7) b 3.50 dd (9.6, 4.2) a 1.84 m b 2.18 m a 2.01 m b 2.24 m 4.86 d (6.2) 8.67 s 9.16 s
3.34 d (6.3) a 2.45 dd (17.3, 11.4) b 2.60 dd (17.3, 4.3) 1.96 ddd (12.3, 8.4, 4.3) 1.16 s 1.28 s a 3.28 m b 3.65 m
3.47 s 6.46 s
7 9 10 11 3′ 4′ 5′ 1′-NH 4-NH 11-OH
1.37 s 1.38 s a 4.28 dd (16.0, 1.5) b 4.39 dd (16.0, 1.6) a 1.98 m b 2.25 m a 2.06 m b 2.32 m 5.00 d (6.1) 8.75 s 9.36 s
“m” means overlapped or multiplet with other signals. a Data collected at 600 MHz in DMSO‑d6. b Data collected at 600 MHz in CD3OD. c Data are the values of J.
2
9.14 s 4.66 t (4.8)
1.45 s 1.45 s a 4.31 dd (15.5, 1.6) b 4.43 dd (15.5, 1.7) a 2.22 m b 2.50 m a 2.13 m b 2.37 m 5.10 d (5.9)
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an oxygenated carbon (δC 85.8). Comparison of these data with those of xanthiazone (Ma et al., 1998), which was previously isolated from X. sibiricum, suggested that 1 was a thiazinedione derivative. The presence of a substituted pyrrolidinone moiety was suggested by the typical 13C NMR resonances [δC 177.6 (C-2′), 27.5 (C-3′), 28.0 (C-4′), and 85.8 (C4′)] as well as the HMBC correlations from H-5′ to C-3′ and C-2′ and from H2-3′a/b to C-5′ (Fig. 4). The HMBC correlation from H-5′ to C-11 and the chemical shifts of H-5′ (δH 5.00) and C-11 (δC 64.4) showed that C-5′ was connected with C-11 through an oxygen atom. The planar structure of 1 was therefore determined. After careful analyses of NMR data, we found that the data of 1 was similar with the known natural product xanthiazinone also isolated from X. sibiricum (Dai et al., 2008), suggesting that compound 1 and xanthiazinone might be the same compound. The key HMBC correlations from H-1′ to C-3′ and C-4′ as well as 1H–1H COSY correlation of H1′ with H-5′ (Fig. 4), and the active hydrogen chemical shift (δH8.75) determined the thiazinedione moiety to be at oxygen atom. However, the thiazinedione moiety in xanthiazinone was assigned at nitrogen atom, and its NMR spectra were determined in CD3OD, whereas the data for 1 were recorded in DMSO. To clarify this problem, the NMR spectra for 1 were reacquired in CD3OD, and the new data were virtually identical to those of xanthiazinone (Supporting Information Table S1). The aforementioned spectroscopic data suggested that 1 and
Table 2 13 C NMR data for compounds 1–4. no.
1a
2a
3a
4a
1b
2 3 4a 5 6 7 8 8a 9 10 11 2′ 3′ 4′ 5′
28.6 162.3 129.9 174.9 121.3 165.3 41.9 140.9 26.6 26.9 64.4 177.6 27.5 28.0 85.8
28.7 162.1 128.9 186.8 36.3 42.6 38.4 144.8 22.2 25.5 66.5 177.4 27.6 28.0 85.6
28.7 162.1 128.9 186.9 36.3 42.6 38.3 144.9 22.2 25.5 66.2 177.5 27.7 28.0 85.3
28.7 162.1 128.8 187.3 36.0 45.2 38.4 145.3 22.0 25.4 60.7
29.8 164.6 131.0 177.0 123.0 167.3 43.5 143.3 27.5 27.4 66.3 181.6 29.3 29.2 87.9
a b
Data collected at 150 MHz in DMSO‑d6. Data collected at 150 MHz in CD3OD.
174.9), two methyl groups (δC 26.6 and 26.9), four methylene groups (δC 27.5, 28.0, 28.6, and 64.4), and one quaternary carbon (δC 41.9), four olefinic carbons (δC 121.3, 129.9, 140.9, and 165.3) together with
Fig. 3. Perspective drawing of the X-ray structures of 1–3, 5, and 7 (with thermal ellipsoid probability of 30%). 3
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Fig. 4. Key 2D NMR correlations of 1a, 2a and 4.
Fig. 5. Experimental and calculated ECD spectra of 1–4.
xanthiazinone were the same compound and the thiazinedione moiety should be reassigned from nitrogen atom to oxygen atom (Fig. 2). The absolute configuration of xanthiazinone was not determined in the initial report (Dai et al., 2008), so further efforts were to address this issue. Crystals suitable for X-ray diffraction were obtained in methanol with a small amount of dichloromethane. X-ray diffraction analysis (Fig. 3), conducted with Cu Kα radiation, not only confirmed the revision, but also revealed 1 to be racemic, which was further corroborated by the baseline ECD spectrum as well as the optical inactivity. Therefore, a chiral HPLC column was used to separate racemate 1 to yield a pair of optically pure enantiomers, 1a and 1b. The absolute configurations of 1a and 1b were assigned as (5′R)-1 and (5′S)1 by comparing the calculated ECD results with the experimental data (Fig. 5), respectively. Compounds 1a and 1b were thus structurally elucidated as shown in Fig. 1 and named (+)-xanthiazinone A and (−)-xanthiazinone A, respectively. Compound 2 was also obtained as colorless needle crystals and its molecular formula of C15H20N2O4S was deduced from the HRESIMS ion at m/z 347.1042 [M + Na]+, two mass units higher than that of 1. The IR spectrum of 2 also showed the presence of hydroxyl (3384 cm−1) and amide carbonyl (1638 cm−1) groups. The general features of its 1H and 13C NMR data suggested that 2 was also a thiazinedione derivative with a similar structure to 1. However, the olefinic proton and carbon signals for the double bond at δH 6.32/δC 121.3 (CH, C-6) and δC 165.3
(C-7) in 1 disappeared in the spectra of 2. Instead, a methylene signal and an sp3 methine signal were observed at δC 36.3 (CH2, C-6) and 42.6 (CH, C-7) in the 13C NMR spectrum of 2, which were not present in the spectra of 1. Accordingly, the COSY correlations of H-7 with H2-6a/b and H2-11a/b and HMBC correlations from H-7 to C-5/C-8a/C-9 and from H2-6a/b to C-4a/C-8/C-11 suggested that a 6,7-alkene moiety at C-6 and C-7 in 1 was replaced by a –CH2-CH- fragment in 2 (Fig. 4). After measuring the physicochemical data of 2, we found that the specific optical rotation was near zero and the cotton effects in the ECD spectrum were very weak. These results indicated 2 was racemic or scalemic mixture. Subsequent chiral HPLC analysis clearly revealed two peaks in a ratio of approximately 1:1 and then 2a and 2b with identical 1 H NMR spectra but totally contrary optical rotation values and CD curves were obtained by chiral separation. The NOESY spectrum of 2 provided little information for the assignment of the relative configurations, so we resorted to X-ray crystallography for a solution. Suitable crystals of 2 were eventually obtained in methanol containing a small amount of dichloromethane and subjected to X-ray diffraction analysis, which determined the relative configuration and further confirmed the planar structure of 2 (Fig. 3). To determine the absolute configurations of 2a and 2b, the ECD spectra of 2a and 2b were calculated by using the TDDFT method. By comparing the calculated CD curves of 2a and 2b with the experimental CD spectra (Fig. 5), the absolute configurations of 2a and 2b were assigned as (7R, 5′R)-2 and (7S, 5′S)-2, respectively. 4
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Finally, compounds 2a and 2b were identified as (+)-xanthiazinone B and (−)-xanthiazinone B (Fig. 1). Compound 3 had the same molecular formula (C15H20N2O4S) as 2, and its 1H and 13C NMR data (Tables 1 and 2) and physicochemical properties were also very similar to those of 2. The structure of compound 3 was assigned as an epimer of 2 at C-5′, which was supported by the comparison of their different chemical shifts at the 5′ position (δH 4.84 for 2 and δH 4.86 for 3) and 11 position (δH 3.68, 3.20 for 2 and δH 3.50, 3.40 for 3). In our study, crystallization of 3 from methanol resulted in colorless needle crystals. X-ray single crystal diffraction analysis not only unambiguously established the structure and relative configuration (Fig. 3), but also indicated that 3 was a racemic mixture in stereochemical consideration. Similar to 2a and 2b, compounds 3a and 3b were eventually obtained in enantiomerically pure form by chiral HPLC separation, and their absolute configurations were assigned as (7S, 5′R)-3 and (7R, 5′S)-3 by comparison of their measured ECD spectra with the theoretically computed ones (Fig. 5). The structures of compounds 3a and 3b were thus determined as shown in Fig. 1 and they were named (+)-xanthiazinone C and (−)-xanthiazinone C. Compound 4 was obtained as a colorless oil. Its molecular formula was determined to be C11H15NO3S on the basis of the sodiated molecular ion peak observed at m/z 242.0846 [M + Na]+ by HRESIMS analysis, indicating five degrees of unsaturation. IR spectrum showed the presence of hydroxyl (3279 cm−1) and amide carbonyl (1638 cm−1) groups. The 1H NMR spectrum indicated that 4 had two sets of methyl protons, two methylene protons, and one sp3 methine proton (Table 1). The 13C NMR and DEPT spectra of 4 exhibited 11 carbon signals (Table 2), which were classified by chemical shifts and HSQC spectrum as two methyls, three methylenes, one methine, and five quaternary carbons (including two olefinic, one amide carbonyl, and one carbonyl carbons). According to the above features, the NMR spectroscopic data of compound 4 were very similar to those of 5, except that C-6/C-7 double bond in 5 was reduced to an sp3 methine and an sp3 methylene in 4, which was confirmed by HMBC correlations from H-7 to C-5/C-8a/C-9 and the 1H–1H COSY correlations of H-7 with H2-6a/b and H2-11a/b (Fig. 4). The absolute configuration of C-7 was determined as 7S by comparing its calculated ECD curve with the experimental CD spectrum (Fig. 5). Thus, the structure of compound 4 was built as shown in Fig. 1, with the name of xanthiazinone D. The known compounds were identified as xanthiazone (5) (Ma et al., 1998), 2-hydroxy-xanthiazone (6) (Yin et al., 2016), xanthialdehyde (7) (Lee et al., 2008), xanthienopyran (8) (Shi et al., 2015b), dihydro-xanthienopyran (9) (Wang et al., 2017), sibiricumthionol (10) (Shi et al., 2015b), xanthiside (11) (Dai et al., 2008) basing on their NMR and MS data and comparison with the data in these literatures. Nitric oxide (NO) plays an important role in the inflammatory process, and an inhibitor of NO production may be considered as a potential anti-inflammatory agent (Liu et al., 2013). All isolated compounds were tested for their inhibitory activity in the NO production. The results (Table 3) indicated compounds 2a, 3b, 4, 6 and 8 indirectly exhibited anti-inflammatory activity by suppressing LPS-induced NO production in RAW 264.7 cells with IC50 values of 8.75, 19.44, 33.78,
Table 4 Cytotoxicity (IC50, μM) against Human Liver Cancer (HepG2), Human Breast Cancer (MCF-7), and Human Lung Cancer (A549) cellsa.
IC50 (μM)
CC50 (μM)
2a 3b 4 6 8 dexamethasoneb
8.75 ± 0.43 19.44 ± 0.97 33.78 ± 1.20 10.90 ± 1.09 17.40 ± 1.12 10.50 ± 0.40
> 50 > 50 > 50 > 50 > 50 > 50
a b
HepG2
MCF-7
A549
1b 2a 3a 3b cisplatinb
85.80 ± 3.90 82.42 ± 1.61 67.04 ± 2.73 24.23 ± 1.79 3.65 ± 0.17
83.52 ± 3.78 87.22 ± 2.79 70.98 ± 2.80 27.00 ± 1.87 3.58 ± 2.18
80.58 82.61 87.00 26.64 13.53
a b
± ± ± ± ±
3.82 3.65 3.63 1.90 2.21
Other compounds: IC50 > 100 μM. Positive control.
10.90, and 17.40 μM, respectively. The positive control dexamethasone showed activity with an IC50 value of 10.50 μM. The other compounds were inactive (IC50 > 50 μM) for the inhibition of NO production. The isolates were also tested for their cytotoxicity. As shown in Table 4, compounds 1b, 2a, 3a, and 3b exhibited inhibitory effects against three human tumor cell lines (HepG2, MCF-7, and A549), showing IC50 values ranging from 24.23 to 87.22 μM. 3. Conclusions Seven undescribed thiazinedione derivatives (1a, 1b, 2a, 2b, 3a, 3b, and 4), including three pairs of enantiomers, together with seven known sulfur-containing compounds (5–11) were isolated from the fruits of Xanthium sibiricum. The enantiomers were separated by chiral HPLC columns and the previously proposed structure for xanthiazinone was revised to 1 by the spectroscopic data and single crystal X-ray diffraction analysis of 1. Compounds 2a and 6 significantly inhibited the production of NO production in LPS-induced RAW 264.7 cells with IC50 values of 8.75 and 10.90 μM, respectively. None of the isolated compounds showed cytotoxicity against three human cancer cell lines (MCF-7, HepG2, and A549) with IC50 values more than 10 μM. Considering the importance of finding undescribed drug ingredients to treat different types of inflammation, the results have certain practical significance, and constitute a significant contribution to the chemistry and medicinal properties of Xanthium plants. 4. Experimental 4.1. General experimental procedures UV spectra were taken on a Jasco V-550 UV/VIS spectrometer (Jasco Corporation, Tokyo, Japan). The optical rotations were obtained on a Jasco digital polarimeter (Jasco Corporation, Tokyo, Japan). IR spectra were carried out with a Nicolet Impact 410-FTIR spectrometer (Jasco Corporation, Tokyo, Japan). CD spectra were measured with a J810 spectrometer using MeOH as solvent. The NMR spectra were recorded on Bruker AV-300, AV-400 and AV-600 spectrometers (Bruker Instrument, Inc., Zurich, Switzerland) used TMS as an internal standard. HPLC was performed on an Agilent 1200 HPLC system equipped with a diode array detector, using a column A (Ultimate XB-C18, 5 μm, 4.6 × 250 mm, Welch, Potamac, MA, USA) for analysis, a column B (Ultimate XB-C18, 5 μm, 10 × 250 mm, Welch, Potamac, MA, USA), a column C (Chiralpak AD-H, 5 μm, 4.6 × 250 mm) and a column D (ChromegaChiral CC4, 5 μm, 4.6 × 250 mm) for semi-preparative purification. HR-ESI-MS was achieved on an Agilent 6210 LC/MS TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Open column chromatography (CC) was performed on silica gel (200–300 mesh, Haiyang Chemical Group Corporation, Qingdao, China), ODS (50 μm, YMC, Tokyo, Japan) and Sephadex LH-20 (25–100 μm, Pharmacia, Uppsala, Sweden). HSGF254 silica gel TLC plates (0.2 mm thickness, 200 × 200 mm, Qingdao Marine Chemical, Qingdao, China) were used for routine TLC analysis. The spraying reagent used for TLC detection was 10% H2SO4 in EtOH.
Table 3 Inhibition of NO production in LPS-induced RAW 264.7 cells and cytotoxicity against RAW 264.7 cells of some isolatesa. Compounds
Compounds
Inactive compounds are omitted. Positive control. 5
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4.2. Plant material
4.3.3. (+)-Xanthiazinone C (3a) and (−)-xanthiazinone C (3b) Colorless needle crystals; 3a/3b, UV (MeOH) λmax (log ε) 242 (4.03), 324 (4.05) nm; IR (KBr) νmax 3287, 2949, 1649, 1409, 1112, 1062 cm−1; 3a, [ ]D 20 +8.2 (c 0.05, MeOH); CD (MeOH) λmax (Δε): 215 (−12.25), 230 (+4.92), 246 (−9.78), 306 (−7.25), 338 (+8.75) nm; 3b, [ ]D 20 -8.4 (c 0.05, MeOH); CD (MeOH) λmax (Δε): 215 (+14.54), 230 (−6.25), 246 (+11.02), 306 (+8.24), 338 (−11.02) nm; HRESIMS (positive) m/z 347.1042 [M + Na]+ (calcd for C15H20N2O4SNa, 347.1045); 1D NMR data, see Tables 1 and 2
Xanthii Fructus, the ripe fruits of Xanthium sibiricum Patrin ex Widder (Asteraceae), was purchased from Bozhou Market of Chinese Herb Materials, Anhui Province, China, in August 2018. It is said that Xanthii Fructus was collected from Jinzhai County of Anhui Province in July 2017, and authenticated by Prof. G-X Zhou, College of Pharmacy Jinan University at Guangzhou, China. A voucher specimen (No. 201808) was deposited in the Teaching & Research Office of Pharmacognosy, Jinan University.
4.3.4. Xanthiazinone D (4) Colorless oil; [ ]D 20 -5.6 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 234 (3.87), 322 (3.86) nm; IR (KBr) νmax 3279, 1638, 1109, 1014 cm−1; CD (MeOH) λmax (Δε): 215 (−3.85), 230 (+9.95), 244 (−13.75), 301 (−10.01), 334 (+12.02) nm; HRESIMS (positive) m/z 242.0846 [M + H]+ (calcd for C11H16NO3S, 242.0851); 1D NMR data, see Tables 1 and 2
4.3. Extraction and isolation The Xanthii Fructus (20 kg) was milled and extracted with 95% EtOH (40 L × 2 h × 3). The resulting dried extract (3.09 kg) was obtained after concentrated in vacuum, then suspended in H2O (5 L), and partitioned successively with petroleum ether and EtOAc. The EtOAc extract (148 g) was subjected to silica gel CC and eluted with CH2Cl2/CH3OH (100:1, 80:1, 50:1, 20:1, 10:1, 5:1, 4:1, 2:1, 1:1, and 0:1 v/v) to obtain eighteen fractions (EA-ER). Fraction ED (8 g) was chromatographed over silica gel using CH2Cl2/EtOAc (from 80:1 to 0:1 v/v) to produce seven fractions (ED1-ED7). Fraction ED4 (1.2 g) was applied to silica gel and eluted with CH2Cl2/EtOAc (100:1 to 0:1 v/v) to yield subfractions ED4-1 to ED4-6. ED4-3 (150 mg) was chromatographed over a Sephadex LH-20 column (100% MeOH) and was further purified with RP-semipreparative HPLC (solvent system: MeOH/H2O (34:66)) to yield compounds 2 (4.0 mg), 3 (5.2 mg) and 4 (5.4 mg). ED4-4 was subjected to a Sephadex LH-20 column eluted with CH2Cl2/CH3OH (1:1 v/v) and was further purified with RP-semipreparative HPLC (solvent system: MeOH/H2O (48:52)) to yield compounds 1 (4.2 mg), 5 (40 mg) and 6 (12 mg). Fraction ED5 (1.5 g) was subjected to silica gel and eluted with a gradient of increasing CH3OH (0–100%) in CH2Cl2 to provide subfractions ED5-1 to ED5-5. ED5-4 was separated by RPsemipreparative HPLC (solvent system: MeOH/H2O (37:63)) to afford compounds 7 (7.0 mg), 8 (2.4 mg) and 9 (3.8 mg). The separation of EF (4.8 g) using an RP-C18 silica gel column and subsequent elution with MeOH/H2O (30:70, 50:50, 70:30, and 100:0 v/v) provided fractions EF1 to EF7. EF6 (180 mg) was separated by RP-semipreparative HPLC (solvent system: MeOH/H2O (28:72)) to give compounds 10 (6.0 mg) and 11 (75 mg). The chiral separation were conducted on an Agilent 1260 system with a ChromegaChiral CC4 or Chiralpak AD-H column (5 μm, 4.6 × 250 mm) at a flow rate of 1 mL/min. Compounds 1a (1.2 mg) and 1b (1.6 mg) were separated from 1 by a ChromegaChiral CC4 column, eluting with MeOH/H2O (55:45). Compounds 2 and 3 were chromatographed on a Chiralpak AD-H column with n-hexane/ EtOH (85:15 and 82:18, respectively) as a mobile phase, to yield 2a (1.2 mg), 2b (1.5 mg), 3a (1.6 mg) and 3b (1.3 mg).
4.3.5. X-ray crystal data for compound 1 Colorless crystal of C15H18N2O4S, M = 322.37, triclinic, space group P-1, a = 7.5086 (4) Å, b = 7.7367 (4) Å, c = 13.4690 (8) Å, V = 749.02 (7) Å3, Z = 2, T = 293 (2) K, μ(Cu Kα) = 2.107 mm−1, = 1.429 Mg/m3, 4772 reflections measured Dcalc (6.806° ≤ 2θ ≤ 147.276°), 2869 unique (Rint = 0.0295, Rsigma = 0.0432), which were used in all calculations. The final R1 was 0.0827 (I > 2σ(I)) and wR2 was 0.2590 (all data). Crystallographic data for 1 (CCDC, 1952175) can be obtained from the Cambridge Crystallographic Data Centre for free via www.ccdc.cam.ac.uk. 4.3.6. X-ray crystal data for compound 2 Colorless crystal of C15H20N2O4S, M = 324.39, triclinic, space group P-1, a = 7.6241 (6) Å, b = 7.6479 (5) Å, c = 13.8127 (14) Å, V = 746.86 (11) Å3, Z = 2, T = 293 (2) K, μ(Cu Kα) = 2.114 mm−1, Dcalc = 1.442 Mg/m3, 4908 reflections measured (6.714° ≤ 2θ ≤ 147.27°), 2898 unique (Rint = 0.0247, Rsigma = 0.0368), which were used in all calculations. The final R1 was 0.0372 (I > 2σ(I)) and wR2 was 0.1242 (all data). Crystallographic data for 2 (CCDC, 1952178) can be obtained from the Cambridge Crystallographic Data Centre for free via www.ccdc.cam.ac.uk. 4.3.7. X-ray crystal data for compound 3 Colorless crystal of C15H20N2O4S, M = 324.39, monoclinic, space group P21/c, a = 7.7957 (7) Å, b = 8.8535 (9) Å, c = 22.296 (2) Å, V = 1527.4 (3) Å3, Z = 4, T = 293 (2) K, μ(Cu Kα) = 2.067 mm−1, = 1.411 Mg/m3, 5322 reflections measured Dcalc (7.99° ≤ 2θ ≤ 146.614°), 2988 unique (Rint = 0.0476, Rsigma = 0.0718), which were used in all calculations. The final R1 was 0.0533 (I > 2σ(I)) and wR2 was 0.1641 (all data). Crystallographic data for 3 (CCDC, 1952176) can be obtained from the Cambridge Crystallographic Data Centre for free via www.ccdc.cam.ac.uk.
4.3.1. (+)-Xanthiazinone A (1a) and (−)-xanthiazinone A (1b) Colorless needle crystals; 1a and 1b, UV (MeOH) λmax (log ε) 248 (3.60), 340 (3.14) nm; IR (KBr) νmax 3362, 2979, 1619, 1463, 1152, 1107 cm−1; 1a, [ ]D 20 +4.5 (c 0.05, MeOH); CD (MeOH) λmax (Δε): 218 (+2.00), 248 (−1.98), 342 (+0.2) nm; 1b, [ ]D 20 -4.2 (c 0.05, MeOH); CD (MeOH) λmax (Δε): 218 (−2.98), 248 (+2.01), 342 (−1.1) nm; HRESIMS (positive) m/z 345.0884 [M + Na]+ (calcd for C15H18N2O4SNa, 345.0886); 1D NMR data, see Tables 1 and 2
4.3.8. X-ray crystal data for compound 5 Colorless crystal of C11H13NO3S, M = 239.28, monoclinic, space group P21/c, a = 7.4626 (10) Å, b = 7.6479 (10) Å, c = 19.5553 (3) Å, V = 1115.10 (3) Å3, Z = 4, T = 293 (2) K, μ(Cu Kα) = 2.530 mm−1, Dcalc = 1.425 Mg/m3, 6654 reflections measured (9.052° ≤ 2θ ≤ 147.078°), 2201 unique (Rint = 0.0390, Rsigma = 0.0295), which were used in all calculations. The final R1 was 0.0563 (I > 2σ(I)) and wR2 was 0.1620 (all data). Crystallographic data for 5 (CCDC, 1954325) can be obtained from the Cambridge Crystallographic Data Centre for free via www.ccdc.cam.ac.uk.
4.3.2. (+)-Xanthiazinone B (2a) and (−)-xanthiazinone B (2b) Colorless needle crystals; 2a and 2b, UV (MeOH) λmax (log ε) 238 (3.77), 322 (3.80) nm; IR (KBr) νmax 3384, 2922, 1638, 1459, 1091, 1059 cm−1; 2a, [ ]D 20 +9.8 (c 0.05, MeOH); CD (MeOH) λmax (Δε): 230 (+23.02), 246 (−24.25), 304 (−18.25), 338 (+22.50) nm; 2b, [ ]D 20 -10.2 (c 0.05, MeOH); CD (MeOH) λmax (Δε): 230 (−19.04), 246 (+16.24), 304 (+12.25), 338 (−18.72) nm; HRESIMS (positive) m/z 347.1042 [M + Na]+ (calcd for C15H20N2O4SNa, 347.1043); 1D NMR data, see Tables 1 and 2
4.3.9. X-ray crystal data for compound 7 Colorless crystal of C11H11NO3S, M = 237.27, triclinic, space group P-1, a = 6.3633 (15) Å, b = 7.1217 (9) Å, c = 12.2520 (2) Å, V = 534.94 (18) Å3, Z = 2, T = 293 (2) K, μ(Cu Kα) = 2.636 mm−1, 6
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Dcalc = 1.473 Mg/m3, 3257 reflections measured (7.430° ≤ 2θ ≤ 153.596°), 2066 unique (Rint = 0.0598, Rsigma = 0.0812), which were used in all calculations. The final R1 was 0.1690 (I > 2σ(I)) and wR2 was 0.3676 (all data). Crystallographic data for 7 (CCDC, 1954326) can be obtained from the Cambridge Crystallographic Data Centre for free via www.ccdc.cam.ac.uk.
2004. Xanthii Fructus inhibits inflammatory responses in LPS-stimulated mouse peritoneal macrophages. Inflammation 28, 263–270. Chen, B., Ma, L.H., Wang, X.B., Shen, Y.P., Jia, X.B., 2012. Simultaneous determination of 5 phenolic acids in fried Fructus Xanthii from different production sites and its dispensing granules by using ultra-pressure liquid chromatography. Phcog. Mag. 9, 103–108. Cheng, Z., Wang, L., Li, F., Wang, M.K., 2013. A new thiazinedione glycoside from the fruits of Xanthium sibiricum. Chem. Nat. Compd. 49, 977–979. Dai, Y.H., Cui, Z., Li, J.L., Wang, D., 2008. A new thiazinedione from the fruits of Xanthium sibiricum. J. Asian Nat. Prod. Res. 10, 303–305. Fan, W.X., Fan, L.H., Peng, C.Y., Zhang, Q., Wang, L., Li, L., Wang, J.L., Zhang, D.Y., Peng, W., Wu, C.J., 2019. Traditional uses, botany, phytochemistry, pharmacology, pharmacokinetics and toxicology of Xanthium strumarium L.: a review. Molecules 24, 359–399. Hsu, F.L., Chen, Y.C., Cheng, J.T., 2000. Caffeic acid as active principle from the fruit of Xanthium strumarium to lower plasma glucose in diabetic rats. Planta Med. 66, 228–230. Han, T., Li, H.L., Zhang, Q.Y., Zheng, H.C., Qin, L.P., 2006. New thiazinediones and other components from Xanthium strumarium. Chem. Nat. Compd. 42, 567–570. Huang, M.H., Wang, B.S., Chiu, C.S., Amagaya, S., Hsieh, W.H., Huang, S.S., Shie, P.H., Huang, G.J., 2011. Antioxidant, antinociceptive, and anti-inflammatory activities of Xanthii Fructus extract. J. Ethnopharmacol. 135, 545–552. Ingawale, A.S., Sadiq, M.B., Nguyen, L.T., Ngan, T.B., 2018. Optimization of extraction conditions and assessment of antioxidant, α-glucosidase inhibitory and antimicrobial activities of Xanthium strumarium L. fruits. Biocatal. Agric. Biotechnol. 14, 40–47. Jiang, H., Yang, L., Liu, C., Hou, H., Wang, Q.H., Wang, Z.B., Yang, B.Y., Kuang, H.X., 2013. Four new glycosides from the fruits of Xanthium sibiricum Patr. Molecules 18, 12464–12473. 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(–)-Xanthienopyran, a new inhibitor of superoxide anion generation by activated neutrophils, and further constituents of the seeds of Xanthium strumarium. Planta Med. 74, 1276–1279. Liu, Y., Ma, J.H., Zhao, Q., Liao, C.R., Ding, L.Q., Chen, L.X., Zhao, F., Qiu, F., 2013. Guaiane-type sesquiterpenes from curcuma phaeocaulis and their inhibitory effects on nitric oxide production. J. Nat. Prod. 76, 1150–1156. Lin, B., Zhao, Y., Han, P., Yue, W., Ma, X.Q., Rahman, K., Zheng, C.J., Qin, L.P., Han, T., 2014. Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund's adjuvant induced arthritis in rats. J. Ethnopharmacol. 155, 248–255. Ma, Y.T., Huang, M.C., Hsu, F.L., Chang, H.F., 1998. Thiazinedione from Xanthium strumarium. Phytochemistry 48, 1083–1085. Peng, W., Ming, Q.L., Han, P., Zhang, Q.Y., Jiang, Y.P., Zheng, C.J., Han, T., Qin, L.P., 2014. Anti-allergic rhinitis effect of caffeoylxanthiazonoside isolated from fruits of Xanthium strumarium L. in rodent animals. Phytomedicine 21, 824–829. Peng, W., Han, P., Yu, L.Y., Chen, Y., Ye, B.Z., Qin, L.Q., Xin, H.L., Han, T., 2019. Antiallergic rhinitis effects of caffeoylquinic acids from the fruits of Xanthium strumarium in rodent animals via alleviating allergic and inflammatory reactions. Rev. Bras. Farmacogn. 29, 46–53. Qin, L.P., Han, T., Li, H.L., Zhang, Q.Y., Zheng, H.C., 2006. A new thiazinedione from Xanthium strumarium. Fitoterapia 77, 245–246. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty per cent endpoints. Am. J. Epidemiol. 27, 493–497. Shi, Y.S., Liu, Y.B., Li, Y., Li, L., Qu, J., Ma, S.G., Yu, S.S., 2014. Chiral resolution and absolute configuration of a pair of rare racemic spirodienone sesquineolignans from Xanthium sibiricum. Org. Lett. 16, 5406–5409. Shi, Y.S., Liu, Y.B., Ma, S.G., Li, Y., Qu, J., Li, L., Yuan, S.P., Hou, Q., Li, Y.H., Jiang, J.D., Yu, S.S., 2015a. Bioactive sesquiterpenes and lignans from the fruits of Xanthium sibiricum. J. Nat. Prod. 78, 1526–1535. Shi, Y.S., Liu, Y.B., Ma, S.G., Li, Y., Qu, J., Liu, Q., Shen, Z.F., Chen, X.G., Yu, S.S., 2015b. A new thiophene and two new monoterpenoids from Xanthium sibiricum. J. Asian Nat. Prod. Res. 17, 1039–1047. Tao, L., Sheng, X.S., Zhang, L., Li, W.D., Wei, D.H., Zhu, P.T., Zhang, F., Wang, A.Y., James, R.W., Lu, Y., 2016. Xanthatin anti-tumor cytotoxicity is mediated via glycogen synthase kinase-3β and β-catenin. Biochem. Pharmacol. 115, 18–27. Wang, R.B., Deng, S.D., Qu, J., Yu, S.S., 2017. Chemical constituents from fruits of Xanthium chinense Mill. Nat. Prod. Res. Dev. 29, 787–790. Xue, L.M., Zhang, Q.Y., Han, P., Jiang, Y.P., Yan, R.D., Wang, Y., Rahman, K., Jia, M., Han, T., Qin, L.P., 2014. Hepatotoxic constituents and toxicological mechanism of Xanthium strumarium L. fruits. J. Ethnopharmacol. 152, 272–282. Yoon, H.N., Lee, M.Y., Kim, J.K., Suh, H.W., Lim, S.S., 2013. Aldose reductase inhibitory compounds from Xanthium strumarium. Arch Pharm. Res. 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4.4. Anti-inflammatory assay The anti-inflammatory activity was investigated based on the inhibition of NO generation. The NO concentration was detected by the Griess reagent. Briefly, the RAW 264.7 cell line was cultured in DMEM with 10% FBS. The RAW 264.7 cells were seeded at the density of 4 × 104 cells per well in 96-well culture plate (100 μL per well), then with LPS (0.1 μg/mL) in the presence or absence of test compounds. After 24 h, culture supernatant was reacted with Griess reagent for 10 min at room temperature in the dark. The optical density at 540 nm was monitored by using microplate reader. Inhibition (%) = 100 × (ALPS treated − ALPS+sample treated)/(ALPS treated − Auntreated). Dexamethasone was used as the positive control. 4.5. Cytotoxicity assay Three human tumor cell lines, HepG2 (liver cancer), MCF-7 (breast cancer), and A549 (lung cancer) were used in the cytotoxicity assays, which were obtained from ATCC. Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum. Cells (100 μL) were seeded into 96-well culture plate at a concentration of 5 × 103 cells/well. Following a 24 h incubation period, different concentrations of samples dissolved in DMSO were added to each well, respectively. Each experiment was performed in triplicate. Negative controls were treated with DMSO alone, and positive controls with cisplatin. After being incubated for a further 24 h, MTT solution (30 μL) was added to each well and the cells were further incubated at 37 °C for 4 h. The optical density was measured at 570 nm using a MULTISKAN FC, and the IC50 value of each compound was calculated through non-linear regression analysis (Reed and Muench, 1938). Declaration of competing interest The authors have declared that there is no conflict of interest. We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. Acknowledgments This work was supported by the National Natural Science Foundation of China [No. 81573578] and a grant (2017-110108-83-03001423) from the State Bureau of Chinese Medicine. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2020.112293. References Agata, I., Goto, S., Hatano, T., Nishibe, S., Okuda, T., 1993. 1,3,5-tri-O-caffeoylquinic acid from Xanthium strumarium. Phytochemistry 33, 508–509. An, H.J., Jeong, H.J., Lee, E.H., Kim, Y.K., Hwang, W.J., Yoo, S.J., Hong, S.H., Kim, H.M.,
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Bioactive sulfur-containing compounds from Xanthium sibiricum, including a revision of the structure of xanthiazinone
T
Zhao Xia, Tian-qi Xu, Hai-xin Zhang, Yi-min Chen, Wei Xu, Guang-xiong Zhou∗ Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou, China
ARTICLE INFO
ABSTRACT
Keywords: Xanthium sibiricum Asteraceae Xanthii Fructus Thiazinedione Anti-inflammatory Cytotoxicity
Seven previously undescribed sulfur-containing compounds, (+)- and (−)-xanthiazinone A, (+)- and (−)-xanthiazinone B, (+)- and (−)-xanthiazinone C and xanthiazinone D, and four known thiazinedione derivatives, together with three thiophene derivatives were isolated from the fruits of Xanthium sibiricum. Racemic xanthiazinones A-C were separated by chiral HPLC columns. Their chemical structures were elucidated based on extensive spectroscopic analyses, ECD calculations, and single crystal X-ray diffractions. The X-ray crystallographic analyses for xanthiazinones A-C represent the first example described for the structure elucidation of the thiazinedione with the five-membered lactone ring attached via an oxygen atom. Accordingly, the previously proposed structure for xanthiazinone was revised. The anti-inflammatory and cytotoxic activities were evaluated for all the isolated compounds. (+)-xanthiazinone B and 2-hydroxy-xanthiazone exhibited potent inhibitory effects against nitric oxide production in lipopolysaccharide-activated RAW 264.7 mouse macrophage cells with IC50 values of 8.75 and 10.90 μM, respectively. All compounds obviously were inactive for three human tumor cell lines (HepG2, MCF-7, and A549) with IC50 values more than 10 μM.
1. Introduction Xanthium (Asteraceae) is a relatively small genus of plants of worldwide distribution, and only four species and two varieties grow in China (Lv et al., 2001). Xanthii Fructus is the dried ripe fruits of Xanthium sibiricum Patrin ex Widder, an annual gregarious herb. These fruits are commonly known as Chinese Materia Medicinal material “Cang-Er-Zi”. In traditional Chinese medicine, Xanthii Fructus has been widely used in the treatment of leukoderma, fever, scrofula, sinusitis, headache, herpes, and cancer (Lin et al., 2014; Fan et al., 2019; Xue et al., 2014; Hsu et al., 2000). Xanthii Fructus contains different types of chemical constituents, including sesquiterpene lactones (Tao et al., 2016; Shi et al., 2015a), monoterpenoids (Shi et al., 2015b; Jiang et al., 2018), glycosides (Jiang et al., 2013, 2016), lignans (Shi et al., 2014, 2015a; Yin et al., 2016; Jiang et al., 2017), thiazinediones (Lee et al., 2008; Cheng et al., 2013; Qin et al., 2006; Ma et al., 1998; Han et al., 2006; Dai et al., 2008; Yin et al., 2016), thiophenes (Shi et al., 2015b), caffeoylquinic acids (Yoon et al., 2013; Chen et al., 2012; Agata et al., 1993), and other constituents. These compounds showed potential pharmacological activities, such as anti-inflammatory (Shi et al., 2015a; An et al., 2004; Huang et al., 2011; Yeom et al., 2015), antioxidant (Ingawale et al., 2018; Huang et al., 2011), antiviral (Shi et al., 2015a), ∗
antiproliferative (Jiang et al., 2016; An et al., 2004), antitumor (Tao et al., 2016), anti-allergic rhinitis (Peng et al., 2014, 2019) effects, which supported its traditional medicinal usage in inflammatory diseases. Our preliminary study showed that an ethyl acetate (EtOAc) extract of Xanthii Fructus exhibited anti-inflammatory activity in LPSinduced RAW 264.7 cells with an IC50 value of 90.09 ± 2.56 μg/mL (Supporting Information Table S2). To identify the anti-inflammatory constituents of Xanthii Fructus, a 95% ethanol (EtOH) extract of this fruits was phytochemically investigated, resulting in the isolation of seven previously undescribed thiazinedione derivatives (1a, 1b, 2a, 2b, 3a, 3b, and 4) and seven known sulfur-containing compounds (5–11) (Fig. 1). The previously reported structure of xanthiazinone was revised based on the spectroscopic analyses and the result of a single crystal Xray diffraction of 1 (Fig. 2). Herein, the separation, structural elucidation, anti-inflammatory and cytotoxic evaluation of these compounds are reported. 2. Results and discussion Compound 1 was obtained as colorless needle crystals. The sodium adduction at m/z 345.0884 [M + Na]+ by HRESIMS demonstrated that the molecular formula of 1 was C15H18N2O4S. IR spectrum showed the
Corresponding author. E-mail address: [email protected] (G.-x. Zhou).
https://doi.org/10.1016/j.phytochem.2020.112293 Received 20 October 2019; Received in revised form 1 February 2020; Accepted 2 February 2020 0031-9422/ © 2020 Elsevier Ltd. All rights reserved.
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Fig. 1. Structures of compounds 1–11.
presence of hydroxyl (3362 cm-1) and amide carbonyl (1620 cm-1) groups. The 1H NMR data of 1 displayed signals for one olefinic proton at δH 6.32 (1H, s), two methyl groups at δH 1.37 (3H, s) and 1.38 (3H, s), four methylene groups at δH 3.48 (2H, s), δH 4.28 (1H, dd, J = 16.0, 1.5 Hz) and 4.39 (1H, dd, J = 16.0, 1.6 Hz), δH 1.98 (1H, m) and 2.25 (1H, m), δH 2.06 (1H, m) and 2.32 (1H, m), and an oxymethine group at δH 5.00 (1H, d, J = 6.1 Hz) (Table 1). The 13C NMR and DEPT data revealed the presence of fifteen carbons (Table 2) for two amide carbonyl groups (δC 162.3 and 177.6), one conjugated carbonyl group (δC
Fig. 2. Structure revision of xanthiazinone. Table 1 1 H NMR data for compounds 1–4. no.
1a
2a
3a
4a
1b
2 6
3.48 s 6.32 s
3.35 d (3.2c) a 2.47 dd (17.3, 10.8) b 2.58 dd (17.3, 4.5) 2.13 m 1.17 s 1.29 s a 3.20 dd (9.5, 8.1) b 3.68 dd (9.5, 4.4) a 1.84 m b 2.18 m a 2.01 m b 2.24 m 4.84 d (6.2) 8.65 s 9.17 s
3.35 d (3.2) a 2.47 dd (17.3, 10.8) b 2.58 dd (17.3, 4.5) 2.13 m 1.17 s 1.29 s a 3.40 dd (9.6, 7.7) b 3.50 dd (9.6, 4.2) a 1.84 m b 2.18 m a 2.01 m b 2.24 m 4.86 d (6.2) 8.67 s 9.16 s
3.34 d (6.3) a 2.45 dd (17.3, 11.4) b 2.60 dd (17.3, 4.3) 1.96 ddd (12.3, 8.4, 4.3) 1.16 s 1.28 s a 3.28 m b 3.65 m
3.47 s 6.46 s
7 9 10 11 3′ 4′ 5′ 1′-NH 4-NH 11-OH
1.37 s 1.38 s a 4.28 dd (16.0, 1.5) b 4.39 dd (16.0, 1.6) a 1.98 m b 2.25 m a 2.06 m b 2.32 m 5.00 d (6.1) 8.75 s 9.36 s
“m” means overlapped or multiplet with other signals. a Data collected at 600 MHz in DMSO‑d6. b Data collected at 600 MHz in CD3OD. c Data are the values of J.
2
9.14 s 4.66 t (4.8)
1.45 s 1.45 s a 4.31 dd (15.5, 1.6) b 4.43 dd (15.5, 1.7) a 2.22 m b 2.50 m a 2.13 m b 2.37 m 5.10 d (5.9)
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an oxygenated carbon (δC 85.8). Comparison of these data with those of xanthiazone (Ma et al., 1998), which was previously isolated from X. sibiricum, suggested that 1 was a thiazinedione derivative. The presence of a substituted pyrrolidinone moiety was suggested by the typical 13C NMR resonances [δC 177.6 (C-2′), 27.5 (C-3′), 28.0 (C-4′), and 85.8 (C4′)] as well as the HMBC correlations from H-5′ to C-3′ and C-2′ and from H2-3′a/b to C-5′ (Fig. 4). The HMBC correlation from H-5′ to C-11 and the chemical shifts of H-5′ (δH 5.00) and C-11 (δC 64.4) showed that C-5′ was connected with C-11 through an oxygen atom. The planar structure of 1 was therefore determined. After careful analyses of NMR data, we found that the data of 1 was similar with the known natural product xanthiazinone also isolated from X. sibiricum (Dai et al., 2008), suggesting that compound 1 and xanthiazinone might be the same compound. The key HMBC correlations from H-1′ to C-3′ and C-4′ as well as 1H–1H COSY correlation of H1′ with H-5′ (Fig. 4), and the active hydrogen chemical shift (δH8.75) determined the thiazinedione moiety to be at oxygen atom. However, the thiazinedione moiety in xanthiazinone was assigned at nitrogen atom, and its NMR spectra were determined in CD3OD, whereas the data for 1 were recorded in DMSO. To clarify this problem, the NMR spectra for 1 were reacquired in CD3OD, and the new data were virtually identical to those of xanthiazinone (Supporting Information Table S1). The aforementioned spectroscopic data suggested that 1 and
Table 2 13 C NMR data for compounds 1–4. no.
1a
2a
3a
4a
1b
2 3 4a 5 6 7 8 8a 9 10 11 2′ 3′ 4′ 5′
28.6 162.3 129.9 174.9 121.3 165.3 41.9 140.9 26.6 26.9 64.4 177.6 27.5 28.0 85.8
28.7 162.1 128.9 186.8 36.3 42.6 38.4 144.8 22.2 25.5 66.5 177.4 27.6 28.0 85.6
28.7 162.1 128.9 186.9 36.3 42.6 38.3 144.9 22.2 25.5 66.2 177.5 27.7 28.0 85.3
28.7 162.1 128.8 187.3 36.0 45.2 38.4 145.3 22.0 25.4 60.7
29.8 164.6 131.0 177.0 123.0 167.3 43.5 143.3 27.5 27.4 66.3 181.6 29.3 29.2 87.9
a b
Data collected at 150 MHz in DMSO‑d6. Data collected at 150 MHz in CD3OD.
174.9), two methyl groups (δC 26.6 and 26.9), four methylene groups (δC 27.5, 28.0, 28.6, and 64.4), and one quaternary carbon (δC 41.9), four olefinic carbons (δC 121.3, 129.9, 140.9, and 165.3) together with
Fig. 3. Perspective drawing of the X-ray structures of 1–3, 5, and 7 (with thermal ellipsoid probability of 30%). 3
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Fig. 4. Key 2D NMR correlations of 1a, 2a and 4.
Fig. 5. Experimental and calculated ECD spectra of 1–4.
xanthiazinone were the same compound and the thiazinedione moiety should be reassigned from nitrogen atom to oxygen atom (Fig. 2). The absolute configuration of xanthiazinone was not determined in the initial report (Dai et al., 2008), so further efforts were to address this issue. Crystals suitable for X-ray diffraction were obtained in methanol with a small amount of dichloromethane. X-ray diffraction analysis (Fig. 3), conducted with Cu Kα radiation, not only confirmed the revision, but also revealed 1 to be racemic, which was further corroborated by the baseline ECD spectrum as well as the optical inactivity. Therefore, a chiral HPLC column was used to separate racemate 1 to yield a pair of optically pure enantiomers, 1a and 1b. The absolute configurations of 1a and 1b were assigned as (5′R)-1 and (5′S)1 by comparing the calculated ECD results with the experimental data (Fig. 5), respectively. Compounds 1a and 1b were thus structurally elucidated as shown in Fig. 1 and named (+)-xanthiazinone A and (−)-xanthiazinone A, respectively. Compound 2 was also obtained as colorless needle crystals and its molecular formula of C15H20N2O4S was deduced from the HRESIMS ion at m/z 347.1042 [M + Na]+, two mass units higher than that of 1. The IR spectrum of 2 also showed the presence of hydroxyl (3384 cm−1) and amide carbonyl (1638 cm−1) groups. The general features of its 1H and 13C NMR data suggested that 2 was also a thiazinedione derivative with a similar structure to 1. However, the olefinic proton and carbon signals for the double bond at δH 6.32/δC 121.3 (CH, C-6) and δC 165.3
(C-7) in 1 disappeared in the spectra of 2. Instead, a methylene signal and an sp3 methine signal were observed at δC 36.3 (CH2, C-6) and 42.6 (CH, C-7) in the 13C NMR spectrum of 2, which were not present in the spectra of 1. Accordingly, the COSY correlations of H-7 with H2-6a/b and H2-11a/b and HMBC correlations from H-7 to C-5/C-8a/C-9 and from H2-6a/b to C-4a/C-8/C-11 suggested that a 6,7-alkene moiety at C-6 and C-7 in 1 was replaced by a –CH2-CH- fragment in 2 (Fig. 4). After measuring the physicochemical data of 2, we found that the specific optical rotation was near zero and the cotton effects in the ECD spectrum were very weak. These results indicated 2 was racemic or scalemic mixture. Subsequent chiral HPLC analysis clearly revealed two peaks in a ratio of approximately 1:1 and then 2a and 2b with identical 1 H NMR spectra but totally contrary optical rotation values and CD curves were obtained by chiral separation. The NOESY spectrum of 2 provided little information for the assignment of the relative configurations, so we resorted to X-ray crystallography for a solution. Suitable crystals of 2 were eventually obtained in methanol containing a small amount of dichloromethane and subjected to X-ray diffraction analysis, which determined the relative configuration and further confirmed the planar structure of 2 (Fig. 3). To determine the absolute configurations of 2a and 2b, the ECD spectra of 2a and 2b were calculated by using the TDDFT method. By comparing the calculated CD curves of 2a and 2b with the experimental CD spectra (Fig. 5), the absolute configurations of 2a and 2b were assigned as (7R, 5′R)-2 and (7S, 5′S)-2, respectively. 4
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Finally, compounds 2a and 2b were identified as (+)-xanthiazinone B and (−)-xanthiazinone B (Fig. 1). Compound 3 had the same molecular formula (C15H20N2O4S) as 2, and its 1H and 13C NMR data (Tables 1 and 2) and physicochemical properties were also very similar to those of 2. The structure of compound 3 was assigned as an epimer of 2 at C-5′, which was supported by the comparison of their different chemical shifts at the 5′ position (δH 4.84 for 2 and δH 4.86 for 3) and 11 position (δH 3.68, 3.20 for 2 and δH 3.50, 3.40 for 3). In our study, crystallization of 3 from methanol resulted in colorless needle crystals. X-ray single crystal diffraction analysis not only unambiguously established the structure and relative configuration (Fig. 3), but also indicated that 3 was a racemic mixture in stereochemical consideration. Similar to 2a and 2b, compounds 3a and 3b were eventually obtained in enantiomerically pure form by chiral HPLC separation, and their absolute configurations were assigned as (7S, 5′R)-3 and (7R, 5′S)-3 by comparison of their measured ECD spectra with the theoretically computed ones (Fig. 5). The structures of compounds 3a and 3b were thus determined as shown in Fig. 1 and they were named (+)-xanthiazinone C and (−)-xanthiazinone C. Compound 4 was obtained as a colorless oil. Its molecular formula was determined to be C11H15NO3S on the basis of the sodiated molecular ion peak observed at m/z 242.0846 [M + Na]+ by HRESIMS analysis, indicating five degrees of unsaturation. IR spectrum showed the presence of hydroxyl (3279 cm−1) and amide carbonyl (1638 cm−1) groups. The 1H NMR spectrum indicated that 4 had two sets of methyl protons, two methylene protons, and one sp3 methine proton (Table 1). The 13C NMR and DEPT spectra of 4 exhibited 11 carbon signals (Table 2), which were classified by chemical shifts and HSQC spectrum as two methyls, three methylenes, one methine, and five quaternary carbons (including two olefinic, one amide carbonyl, and one carbonyl carbons). According to the above features, the NMR spectroscopic data of compound 4 were very similar to those of 5, except that C-6/C-7 double bond in 5 was reduced to an sp3 methine and an sp3 methylene in 4, which was confirmed by HMBC correlations from H-7 to C-5/C-8a/C-9 and the 1H–1H COSY correlations of H-7 with H2-6a/b and H2-11a/b (Fig. 4). The absolute configuration of C-7 was determined as 7S by comparing its calculated ECD curve with the experimental CD spectrum (Fig. 5). Thus, the structure of compound 4 was built as shown in Fig. 1, with the name of xanthiazinone D. The known compounds were identified as xanthiazone (5) (Ma et al., 1998), 2-hydroxy-xanthiazone (6) (Yin et al., 2016), xanthialdehyde (7) (Lee et al., 2008), xanthienopyran (8) (Shi et al., 2015b), dihydro-xanthienopyran (9) (Wang et al., 2017), sibiricumthionol (10) (Shi et al., 2015b), xanthiside (11) (Dai et al., 2008) basing on their NMR and MS data and comparison with the data in these literatures. Nitric oxide (NO) plays an important role in the inflammatory process, and an inhibitor of NO production may be considered as a potential anti-inflammatory agent (Liu et al., 2013). All isolated compounds were tested for their inhibitory activity in the NO production. The results (Table 3) indicated compounds 2a, 3b, 4, 6 and 8 indirectly exhibited anti-inflammatory activity by suppressing LPS-induced NO production in RAW 264.7 cells with IC50 values of 8.75, 19.44, 33.78,
Table 4 Cytotoxicity (IC50, μM) against Human Liver Cancer (HepG2), Human Breast Cancer (MCF-7), and Human Lung Cancer (A549) cellsa.
IC50 (μM)
CC50 (μM)
2a 3b 4 6 8 dexamethasoneb
8.75 ± 0.43 19.44 ± 0.97 33.78 ± 1.20 10.90 ± 1.09 17.40 ± 1.12 10.50 ± 0.40
> 50 > 50 > 50 > 50 > 50 > 50
a b
HepG2
MCF-7
A549
1b 2a 3a 3b cisplatinb
85.80 ± 3.90 82.42 ± 1.61 67.04 ± 2.73 24.23 ± 1.79 3.65 ± 0.17
83.52 ± 3.78 87.22 ± 2.79 70.98 ± 2.80 27.00 ± 1.87 3.58 ± 2.18
80.58 82.61 87.00 26.64 13.53
a b
± ± ± ± ±
3.82 3.65 3.63 1.90 2.21
Other compounds: IC50 > 100 μM. Positive control.
10.90, and 17.40 μM, respectively. The positive control dexamethasone showed activity with an IC50 value of 10.50 μM. The other compounds were inactive (IC50 > 50 μM) for the inhibition of NO production. The isolates were also tested for their cytotoxicity. As shown in Table 4, compounds 1b, 2a, 3a, and 3b exhibited inhibitory effects against three human tumor cell lines (HepG2, MCF-7, and A549), showing IC50 values ranging from 24.23 to 87.22 μM. 3. Conclusions Seven undescribed thiazinedione derivatives (1a, 1b, 2a, 2b, 3a, 3b, and 4), including three pairs of enantiomers, together with seven known sulfur-containing compounds (5–11) were isolated from the fruits of Xanthium sibiricum. The enantiomers were separated by chiral HPLC columns and the previously proposed structure for xanthiazinone was revised to 1 by the spectroscopic data and single crystal X-ray diffraction analysis of 1. Compounds 2a and 6 significantly inhibited the production of NO production in LPS-induced RAW 264.7 cells with IC50 values of 8.75 and 10.90 μM, respectively. None of the isolated compounds showed cytotoxicity against three human cancer cell lines (MCF-7, HepG2, and A549) with IC50 values more than 10 μM. Considering the importance of finding undescribed drug ingredients to treat different types of inflammation, the results have certain practical significance, and constitute a significant contribution to the chemistry and medicinal properties of Xanthium plants. 4. Experimental 4.1. General experimental procedures UV spectra were taken on a Jasco V-550 UV/VIS spectrometer (Jasco Corporation, Tokyo, Japan). The optical rotations were obtained on a Jasco digital polarimeter (Jasco Corporation, Tokyo, Japan). IR spectra were carried out with a Nicolet Impact 410-FTIR spectrometer (Jasco Corporation, Tokyo, Japan). CD spectra were measured with a J810 spectrometer using MeOH as solvent. The NMR spectra were recorded on Bruker AV-300, AV-400 and AV-600 spectrometers (Bruker Instrument, Inc., Zurich, Switzerland) used TMS as an internal standard. HPLC was performed on an Agilent 1200 HPLC system equipped with a diode array detector, using a column A (Ultimate XB-C18, 5 μm, 4.6 × 250 mm, Welch, Potamac, MA, USA) for analysis, a column B (Ultimate XB-C18, 5 μm, 10 × 250 mm, Welch, Potamac, MA, USA), a column C (Chiralpak AD-H, 5 μm, 4.6 × 250 mm) and a column D (ChromegaChiral CC4, 5 μm, 4.6 × 250 mm) for semi-preparative purification. HR-ESI-MS was achieved on an Agilent 6210 LC/MS TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Open column chromatography (CC) was performed on silica gel (200–300 mesh, Haiyang Chemical Group Corporation, Qingdao, China), ODS (50 μm, YMC, Tokyo, Japan) and Sephadex LH-20 (25–100 μm, Pharmacia, Uppsala, Sweden). HSGF254 silica gel TLC plates (0.2 mm thickness, 200 × 200 mm, Qingdao Marine Chemical, Qingdao, China) were used for routine TLC analysis. The spraying reagent used for TLC detection was 10% H2SO4 in EtOH.
Table 3 Inhibition of NO production in LPS-induced RAW 264.7 cells and cytotoxicity against RAW 264.7 cells of some isolatesa. Compounds
Compounds
Inactive compounds are omitted. Positive control. 5
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4.2. Plant material
4.3.3. (+)-Xanthiazinone C (3a) and (−)-xanthiazinone C (3b) Colorless needle crystals; 3a/3b, UV (MeOH) λmax (log ε) 242 (4.03), 324 (4.05) nm; IR (KBr) νmax 3287, 2949, 1649, 1409, 1112, 1062 cm−1; 3a, [ ]D 20 +8.2 (c 0.05, MeOH); CD (MeOH) λmax (Δε): 215 (−12.25), 230 (+4.92), 246 (−9.78), 306 (−7.25), 338 (+8.75) nm; 3b, [ ]D 20 -8.4 (c 0.05, MeOH); CD (MeOH) λmax (Δε): 215 (+14.54), 230 (−6.25), 246 (+11.02), 306 (+8.24), 338 (−11.02) nm; HRESIMS (positive) m/z 347.1042 [M + Na]+ (calcd for C15H20N2O4SNa, 347.1045); 1D NMR data, see Tables 1 and 2
Xanthii Fructus, the ripe fruits of Xanthium sibiricum Patrin ex Widder (Asteraceae), was purchased from Bozhou Market of Chinese Herb Materials, Anhui Province, China, in August 2018. It is said that Xanthii Fructus was collected from Jinzhai County of Anhui Province in July 2017, and authenticated by Prof. G-X Zhou, College of Pharmacy Jinan University at Guangzhou, China. A voucher specimen (No. 201808) was deposited in the Teaching & Research Office of Pharmacognosy, Jinan University.
4.3.4. Xanthiazinone D (4) Colorless oil; [ ]D 20 -5.6 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 234 (3.87), 322 (3.86) nm; IR (KBr) νmax 3279, 1638, 1109, 1014 cm−1; CD (MeOH) λmax (Δε): 215 (−3.85), 230 (+9.95), 244 (−13.75), 301 (−10.01), 334 (+12.02) nm; HRESIMS (positive) m/z 242.0846 [M + H]+ (calcd for C11H16NO3S, 242.0851); 1D NMR data, see Tables 1 and 2
4.3. Extraction and isolation The Xanthii Fructus (20 kg) was milled and extracted with 95% EtOH (40 L × 2 h × 3). The resulting dried extract (3.09 kg) was obtained after concentrated in vacuum, then suspended in H2O (5 L), and partitioned successively with petroleum ether and EtOAc. The EtOAc extract (148 g) was subjected to silica gel CC and eluted with CH2Cl2/CH3OH (100:1, 80:1, 50:1, 20:1, 10:1, 5:1, 4:1, 2:1, 1:1, and 0:1 v/v) to obtain eighteen fractions (EA-ER). Fraction ED (8 g) was chromatographed over silica gel using CH2Cl2/EtOAc (from 80:1 to 0:1 v/v) to produce seven fractions (ED1-ED7). Fraction ED4 (1.2 g) was applied to silica gel and eluted with CH2Cl2/EtOAc (100:1 to 0:1 v/v) to yield subfractions ED4-1 to ED4-6. ED4-3 (150 mg) was chromatographed over a Sephadex LH-20 column (100% MeOH) and was further purified with RP-semipreparative HPLC (solvent system: MeOH/H2O (34:66)) to yield compounds 2 (4.0 mg), 3 (5.2 mg) and 4 (5.4 mg). ED4-4 was subjected to a Sephadex LH-20 column eluted with CH2Cl2/CH3OH (1:1 v/v) and was further purified with RP-semipreparative HPLC (solvent system: MeOH/H2O (48:52)) to yield compounds 1 (4.2 mg), 5 (40 mg) and 6 (12 mg). Fraction ED5 (1.5 g) was subjected to silica gel and eluted with a gradient of increasing CH3OH (0–100%) in CH2Cl2 to provide subfractions ED5-1 to ED5-5. ED5-4 was separated by RPsemipreparative HPLC (solvent system: MeOH/H2O (37:63)) to afford compounds 7 (7.0 mg), 8 (2.4 mg) and 9 (3.8 mg). The separation of EF (4.8 g) using an RP-C18 silica gel column and subsequent elution with MeOH/H2O (30:70, 50:50, 70:30, and 100:0 v/v) provided fractions EF1 to EF7. EF6 (180 mg) was separated by RP-semipreparative HPLC (solvent system: MeOH/H2O (28:72)) to give compounds 10 (6.0 mg) and 11 (75 mg). The chiral separation were conducted on an Agilent 1260 system with a ChromegaChiral CC4 or Chiralpak AD-H column (5 μm, 4.6 × 250 mm) at a flow rate of 1 mL/min. Compounds 1a (1.2 mg) and 1b (1.6 mg) were separated from 1 by a ChromegaChiral CC4 column, eluting with MeOH/H2O (55:45). Compounds 2 and 3 were chromatographed on a Chiralpak AD-H column with n-hexane/ EtOH (85:15 and 82:18, respectively) as a mobile phase, to yield 2a (1.2 mg), 2b (1.5 mg), 3a (1.6 mg) and 3b (1.3 mg).
4.3.5. X-ray crystal data for compound 1 Colorless crystal of C15H18N2O4S, M = 322.37, triclinic, space group P-1, a = 7.5086 (4) Å, b = 7.7367 (4) Å, c = 13.4690 (8) Å, V = 749.02 (7) Å3, Z = 2, T = 293 (2) K, μ(Cu Kα) = 2.107 mm−1, = 1.429 Mg/m3, 4772 reflections measured Dcalc (6.806° ≤ 2θ ≤ 147.276°), 2869 unique (Rint = 0.0295, Rsigma = 0.0432), which were used in all calculations. The final R1 was 0.0827 (I > 2σ(I)) and wR2 was 0.2590 (all data). Crystallographic data for 1 (CCDC, 1952175) can be obtained from the Cambridge Crystallographic Data Centre for free via www.ccdc.cam.ac.uk. 4.3.6. X-ray crystal data for compound 2 Colorless crystal of C15H20N2O4S, M = 324.39, triclinic, space group P-1, a = 7.6241 (6) Å, b = 7.6479 (5) Å, c = 13.8127 (14) Å, V = 746.86 (11) Å3, Z = 2, T = 293 (2) K, μ(Cu Kα) = 2.114 mm−1, Dcalc = 1.442 Mg/m3, 4908 reflections measured (6.714° ≤ 2θ ≤ 147.27°), 2898 unique (Rint = 0.0247, Rsigma = 0.0368), which were used in all calculations. The final R1 was 0.0372 (I > 2σ(I)) and wR2 was 0.1242 (all data). Crystallographic data for 2 (CCDC, 1952178) can be obtained from the Cambridge Crystallographic Data Centre for free via www.ccdc.cam.ac.uk. 4.3.7. X-ray crystal data for compound 3 Colorless crystal of C15H20N2O4S, M = 324.39, monoclinic, space group P21/c, a = 7.7957 (7) Å, b = 8.8535 (9) Å, c = 22.296 (2) Å, V = 1527.4 (3) Å3, Z = 4, T = 293 (2) K, μ(Cu Kα) = 2.067 mm−1, = 1.411 Mg/m3, 5322 reflections measured Dcalc (7.99° ≤ 2θ ≤ 146.614°), 2988 unique (Rint = 0.0476, Rsigma = 0.0718), which were used in all calculations. The final R1 was 0.0533 (I > 2σ(I)) and wR2 was 0.1641 (all data). Crystallographic data for 3 (CCDC, 1952176) can be obtained from the Cambridge Crystallographic Data Centre for free via www.ccdc.cam.ac.uk.
4.3.1. (+)-Xanthiazinone A (1a) and (−)-xanthiazinone A (1b) Colorless needle crystals; 1a and 1b, UV (MeOH) λmax (log ε) 248 (3.60), 340 (3.14) nm; IR (KBr) νmax 3362, 2979, 1619, 1463, 1152, 1107 cm−1; 1a, [ ]D 20 +4.5 (c 0.05, MeOH); CD (MeOH) λmax (Δε): 218 (+2.00), 248 (−1.98), 342 (+0.2) nm; 1b, [ ]D 20 -4.2 (c 0.05, MeOH); CD (MeOH) λmax (Δε): 218 (−2.98), 248 (+2.01), 342 (−1.1) nm; HRESIMS (positive) m/z 345.0884 [M + Na]+ (calcd for C15H18N2O4SNa, 345.0886); 1D NMR data, see Tables 1 and 2
4.3.8. X-ray crystal data for compound 5 Colorless crystal of C11H13NO3S, M = 239.28, monoclinic, space group P21/c, a = 7.4626 (10) Å, b = 7.6479 (10) Å, c = 19.5553 (3) Å, V = 1115.10 (3) Å3, Z = 4, T = 293 (2) K, μ(Cu Kα) = 2.530 mm−1, Dcalc = 1.425 Mg/m3, 6654 reflections measured (9.052° ≤ 2θ ≤ 147.078°), 2201 unique (Rint = 0.0390, Rsigma = 0.0295), which were used in all calculations. The final R1 was 0.0563 (I > 2σ(I)) and wR2 was 0.1620 (all data). Crystallographic data for 5 (CCDC, 1954325) can be obtained from the Cambridge Crystallographic Data Centre for free via www.ccdc.cam.ac.uk.
4.3.2. (+)-Xanthiazinone B (2a) and (−)-xanthiazinone B (2b) Colorless needle crystals; 2a and 2b, UV (MeOH) λmax (log ε) 238 (3.77), 322 (3.80) nm; IR (KBr) νmax 3384, 2922, 1638, 1459, 1091, 1059 cm−1; 2a, [ ]D 20 +9.8 (c 0.05, MeOH); CD (MeOH) λmax (Δε): 230 (+23.02), 246 (−24.25), 304 (−18.25), 338 (+22.50) nm; 2b, [ ]D 20 -10.2 (c 0.05, MeOH); CD (MeOH) λmax (Δε): 230 (−19.04), 246 (+16.24), 304 (+12.25), 338 (−18.72) nm; HRESIMS (positive) m/z 347.1042 [M + Na]+ (calcd for C15H20N2O4SNa, 347.1043); 1D NMR data, see Tables 1 and 2
4.3.9. X-ray crystal data for compound 7 Colorless crystal of C11H11NO3S, M = 237.27, triclinic, space group P-1, a = 6.3633 (15) Å, b = 7.1217 (9) Å, c = 12.2520 (2) Å, V = 534.94 (18) Å3, Z = 2, T = 293 (2) K, μ(Cu Kα) = 2.636 mm−1, 6
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Dcalc = 1.473 Mg/m3, 3257 reflections measured (7.430° ≤ 2θ ≤ 153.596°), 2066 unique (Rint = 0.0598, Rsigma = 0.0812), which were used in all calculations. The final R1 was 0.1690 (I > 2σ(I)) and wR2 was 0.3676 (all data). Crystallographic data for 7 (CCDC, 1954326) can be obtained from the Cambridge Crystallographic Data Centre for free via www.ccdc.cam.ac.uk.
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4.4. Anti-inflammatory assay The anti-inflammatory activity was investigated based on the inhibition of NO generation. The NO concentration was detected by the Griess reagent. Briefly, the RAW 264.7 cell line was cultured in DMEM with 10% FBS. The RAW 264.7 cells were seeded at the density of 4 × 104 cells per well in 96-well culture plate (100 μL per well), then with LPS (0.1 μg/mL) in the presence or absence of test compounds. After 24 h, culture supernatant was reacted with Griess reagent for 10 min at room temperature in the dark. The optical density at 540 nm was monitored by using microplate reader. Inhibition (%) = 100 × (ALPS treated − ALPS+sample treated)/(ALPS treated − Auntreated). Dexamethasone was used as the positive control. 4.5. Cytotoxicity assay Three human tumor cell lines, HepG2 (liver cancer), MCF-7 (breast cancer), and A549 (lung cancer) were used in the cytotoxicity assays, which were obtained from ATCC. Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum. Cells (100 μL) were seeded into 96-well culture plate at a concentration of 5 × 103 cells/well. Following a 24 h incubation period, different concentrations of samples dissolved in DMSO were added to each well, respectively. Each experiment was performed in triplicate. Negative controls were treated with DMSO alone, and positive controls with cisplatin. After being incubated for a further 24 h, MTT solution (30 μL) was added to each well and the cells were further incubated at 37 °C for 4 h. The optical density was measured at 570 nm using a MULTISKAN FC, and the IC50 value of each compound was calculated through non-linear regression analysis (Reed and Muench, 1938). Declaration of competing interest The authors have declared that there is no conflict of interest. We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. Acknowledgments This work was supported by the National Natural Science Foundation of China [No. 81573578] and a grant (2017-110108-83-03001423) from the State Bureau of Chinese Medicine. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2020.112293. References Agata, I., Goto, S., Hatano, T., Nishibe, S., Okuda, T., 1993. 1,3,5-tri-O-caffeoylquinic acid from Xanthium strumarium. Phytochemistry 33, 508–509. An, H.J., Jeong, H.J., Lee, E.H., Kim, Y.K., Hwang, W.J., Yoo, S.J., Hong, S.H., Kim, H.M.,
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