Article pubs.acs.org/jnp
Carthorquinosides A and B, Quinochalcone C‑Glycosides with Diverse Dimeric Skeletons from Carthamus tinctorius Shi-Jun Yue,† Cheng Qu,† Peng-Xuan Zhang,† Yu-Ping Tang,*,† Yi Jin,† Jian-Shuang Jiang,‡ Ya-Nan Yang,‡ Pei-Cheng Zhang,‡ and Jin-Ao Duan*,† †
Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization and Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, People’s Republic of China ‡ State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100050, People’s Republic of China S Supporting Information *
ABSTRACT: Two novel quinochalcone C-glycosides, carthorquinosides A (1) and B (2), were isolated from the florets of Carthamus tinctorius. Their structures, including the absolute configurations, were established by analysis of NMR and MS data, together with chemical degradation and electronic circular dichroism spectra. Compound 1 has an unprecedented quinochalcone−flavonol structure linked via a methylene bridge, and compound 2 comprises two glucopyranosylquinochalcone moieties linked via the formyl carbon of an acyclic glucosyl unit. A potential biosynthesis pathway is also proposed. Compounds 1 and 2 exhibited anti-inflammatory activities in LPS-stimulated HUVEC cells by regulating IL-1, IL-6, IL-10, and IFN-γ mRNA expression at concentrations as low as 4 μM, and compound 2 also showed inhibitory activity against topoisomerase I at100 μM.
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molecular formula C49H52O28 (24 indices of hydrogen deficiency). Four bands in the UV spectrum (λmax 228, 271, 342, and 402 nm) showed the presence of a highly conjugated system, while the IR spectrum showed absorption bands attributed to hydroxy (3434 cm−1) and carbonyl groups (1641 cm−1) and aromatic functionalities (1539 and 1401 cm−1). The 1 H NMR spectrum (Table 1) in DMSO-d6 revealed signals characteristic of a quinochalcone moiety, i.e., two trans-olefinic protons at δH 7.40 (1H, d, J = 16.0 Hz) and 7.33 (1H, d, J = 16.0 Hz), aromatic protons of a AA′BB′ spin system at δH 7.43 (2H, d, J = 8.5 Hz) and 6.77 (2H, d, J = 8.5 Hz), a phenolic proton at δH 9.83 (1H, s), and a characteristic deshielded proton at δH 18.67 (1H, s). Signals for the protons of a second AA′BB′ system were observed at δH 8.46 (2H, d, J = 8.5 Hz) and 6.89 (2H, d, J = 8.5 Hz) as well as three phenolic protons at δH 13.47 (1H, br s), 12.82 (1H, s), and 10.15 (1H, s). Proton resonances at δH 4.11 (1H, d, J = 14.5 Hz) and 3.32 (1H, d, J = 14.5 Hz) indicated the presence of an isolated methylene group. Additionally, three anomeric protons resonated at δH 5.50 (1H, d, J = 7.2 Hz), 4.98 (1H, d, J = 7.5 Hz), and 3.52 (1H, d, J = 9.5 Hz). The 13C NMR and HSQC spectra of 1 revealed the presence of 49 carbons assignable to an oxygenated tertiary carbon, three carbonyls, four methylenes, eight olefinic carbons (two oxygenated ones at δC 184.6 and 188.9), 15 methines, and 18 aromatic carbons (O-linked, C-
uinochalcones, i.e., quinone-containing chalcones, belong to the flavonoid family and are widely distributed in fruits, vegetables, spices, etc.1 Structurally, quinochalcones may be classified into five main groups, namely, prenylated chalcones, triketone-based chalcones, quinochalcones with Osubstitution, C-glycosylated quinochalcones, and quinochalcone derivatives.1 Interestingly, C-glycosylated quinochalcones are the rarest among all naturally occurring quinochalcones and to date have been discovered only in Carthamus tinctorius. Fourteen quinochalcone C-glycosides (QCGs) and five QCG dimers,2 with anticoagulant, antioxidant, anti-inflammatory, hepatoprotective, and antitumor activities, have been reported.3,4 These QCGs are regarded as the main bioactive constituents of C. tinctorius, in addition to having chemotaxonomic significance.3 Notably, hydroxysafflor yellow A, the most iconic QCG, has been developed into an intravenous fluid in China with good clinical effects for the treatment of cardiaccerebral vascular ailments.3 In the ongoing investigation of bioactive natural products from C. tinctorius,5 two novel QCGs, carthorquinoside A (1) and carthorquinoside B (2), were isolated. The isolation and structural elucidation of compounds 1 and 2 are presented herein, along with their plausible biogenetic pathway and biological evaluation.
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RESULTS AND DISCUSSION Carthorquinoside A (1) was obtained as a yellow, amorphous powder. Its negative HRESIMS molecular ion at m/z 1087.2563 (calcd for C49H51O28, 1087.2572) and 13C NMR spectroscopic data (Table 1) were consistent with the © 2016 American Chemical Society and American Society of Pharmacognosy
Received: June 19, 2016 Published: October 17, 2016 2644
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by the HMBC correlations observed between the anomeric proton H-1⁗ (δH 3.52) and C-3 (δC 194.9) and C-5 (δC 188.9). Additionally, the locations of OH-4′ (δH 9.83), OH-5″ (δH 12.82), OH-7″ (δH 13.47), and OH-4‴ (δH 10.15) were unequivocally assigned on the basis of 13C NMR chemical shifts as well as HMBC correlations of the phenolic protons with nearby carbons. The trans-p-hydroxycinnamoyl group of the QCG moiety was evident from the distinct correlations from H-8 (δH 7.40) to C-7 (δC 177.8), C-9 (δC 137.1), and C-1′ (δC 127.0) and from H-9 (δH 7.33) to C-7, C-8 (δC 121.6), C-1′, and C-2′/6′ (δC 129.6) in the HMBC spectrum, along with the proton spin systems of H-2′/6′ and H-3′/5′ in the 1H−1H COSY spectrum. Noteworthy, the HMBC correlations between the hydrogen-bonded enolic proton at δH 18.67 and the five carbon signals at δC 184.6 (C-1), 177.8 (C-7), 121.6 (C-8), 106.9 (C-2), and 103.3 (C-6) were observed, indicating that the QCG moiety of compound 1 was a tautomeric mixture of the 1-enol-3,7-diketo and 7-enol-1,3-diketo forms in DMSO-d6. According to the relationship between the preferred keto−enol tautomer and the features of the molecular structure proposed by Feng et al.,7 the 1-enol-3,7-diketo form was the predominant tautomer for the QCG moiety of compound 1. The electronic circular dichroism (ECD) spectrum of 1 showed λmax (nm) (Δε) values of 212 (−21.80), 245 (+9.45), 268 (−30.53), and 415 (+16.61) (Figure 3). Thus, the (4S) configuration was determined by comparing the negative Cotton effect at around 270 nm with those reported for saffloquinosides A and B and carthamin.5,9 Thus, the structure of 1, carthorquinoside A, was assigned as depicted. Carthorquinoside B (2) was obtained as a dark orange, amorphous powder. Its molecular formula, C48H52O26 with 23 indices of hydrogen deficiency, was established by the deprotonated molecular ion [M − H]− at m/z 1043.2679 (calcd for C48H51O26, 1043.2674) in the negative HRESIMS in conjunction with the 13C NMR data. The molecular formula is identical to that of anhydrosafflor yellow B, a major compound from the florets of C. tinctorius.10 The IR spectrum showed the presence of hydroxy (3433 cm−1), carbonyl (1640 cm−1), and aromatic (1513 and 1400 cm−1) functions. The UV absorption maxima at 228, 344, and 420 nm were characteristic of a quinochalcone chromophore. The 1H NMR data (Table 2) in DMSO-d6 exhibited signals of a trans-p-hydroxycinnamoyl group at δH 7.51 (1H, d, J = 16.0 Hz), 7.19 (1H, d, J = 16.0 Hz), 7.39 (2H, d, J = 8.5 Hz), 6.76 (2H, d, J = 8.5 Hz), and 9.70 (1H, br s) and a second trans-p-hydroxycinnamoyl group at δH 7.45 (1H, d, J = 16.0 Hz), 7.16 (1H, d, J = 16.0 Hz), 7.37 (2H, d, J = 8.5 Hz), and 6.76 (2H, d, J = 8.5 Hz), together with signals for three glycosyl groups at δH 2.9−4.6 ppm, including two C-glucosyl anomeric protons at δH 3.78 (1H, d, J = 9.0 Hz) and 3.57 (1H, d, J = 9.5 Hz) and a deoxyglucitol anomeric proton at δH 4.56 (1H, d, J = 8.0 Hz). Additionally, a characteristic hydrogen-bonded enolic proton at δH 18.67 (1H, s) and two phenolic protons at δH 4.98 (1H, s) and 4.89 (1H, s) were also present in the 1H NMR spectrum of 2. This, together with the molecular formula and 13C NMR data (Table 2), prompted consideration of an asymmetric dimeric nature of compound 2, isomeric with anhydrosafflor yellow B.10 Likewise, the MS/MS spectrum of compound 2 (Figure S19, Supporting Information) showed the characteristic ions at m/z 1025.25 [M − H − H2O]−, 923.22 [M − H − C8H8O]−, 593.15 [M − H − C21H22O11]−, and 449.11 [M − H − C27H30O15]−, which were consistent with those of anhydrosafflor yellow B.11 However, there is a distinct difference of retention times on a C18
linked, and unsubstituted). Among them, three sets of carbon signals of the saccharide moieties were ascribed to three βglucopyranosyl moieties based on the coupling constants of their anomeric protons.6 Comparison of the 13C NMR data of compound 1 with those of hydroxysafflor yellow A and 6hydroxykaempferol-3,6-di-O-β-D-glucoside,7,8 i.e., the two main constituents of C. tinctorius, highlighted that it was a heterodimer composed of a QCG moiety and a glucosylated flavonoid moiety. The main differences included the absence of one of the C-glucopyranosyl residues and the presence of an additional methylene (δC 17.1) group in 1. The two nonequivalent methylene proton signals at δH 4.11 and 3.32 showing HMBC correlations with C-6 (δC 103.3), C-8″ (δC 107.2), C-7″ (δC 149.3), C-9″ (δC 157.5), C-1 (δC 184.6), and C-5 (δC 188.9) and the absence of 1H−1H COSY correlations with other protons revealed that C-6 is connected to C-8″ by a methylene bridge (Figure 1). The C-6/CH2/C-8″ linkage was further supported by the characteristic fragment ions at m/z 625.14 ([M − H − C22H22O11]−) and 461.11 ([M − H − C27H30O17]−), similar to hydroxysafflor yellow A and 6hydroxykaempferol-3,6-di-O-β-D-glucoside, obtained by a McLafferty rearrangement in the MS/MS spectra of 1 (Figure 2). Two O-β-glucopyranosyl residues were attached at C-3″ and C-6″ as shown by the HMBC cross-peaks between H-1⁗″ (δH 5.50) and C-3″ (δC 132.5) and between H-1⁗′ (δH 4.98) and C-6″ (δC 128.3). The 3″- and 6″-glucopyranosyloxy residues of 1 were confirmed to be D-glucose by acid hydrolysis and the HPLC analysis of their thiazolidine thiocarbamoyl derivatives (Figure S27, Supporting Information). The remaining C-β-glucopyranosyl residue at C-4 was confirmed 2645
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Table 1. NMR Spectroscopic Data (1H 500 MHz and 13C 125 MHz, DMSO-d6) for Compound 1a no.
a
δC
1 2 3 4 5 6
184.6 106.9 194.9 83.7 188.9 103.3
7 8 9 1′ 2′, 6′ 3′, 5′
177.8 121.6 137.1 127.0 129.6 115.6
4′ 1″
158.8 17.1
2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ 1‴ 2‴, 6‴ 3‴, 5‴ 4‴
δH, mult., J in Hz
HMBC (H→C)
no.
δC
1⁗ 2⁗ 3⁗ 4⁗ 5⁗ 6⁗
85.7 69.2 78.2 69.7 79.7 60.5 103.0 74.3 76.5 69.8 77.4 60.7
7.40 d (16) 7.33 d (16)
7, 9, 1′ 7, 8, 1′, 2′/6′
7.43 d (8.5) 6.77 d (8.5)
9, 4′ 1′, 4′
1⁗′ 2⁗′ 3⁗′ 4⁗′ 5⁗′ 6⁗′
4.11 d (14.5) 3.32 d (14.5)
1, 5, 6, 7″, 8″, 9″
1⁗″ 2⁗″
100.9 74.4
156.5 132.5 177.8 150.4
3⁗″ 4⁗″ 5⁗″ 6⁗″
76.4 69.9 77.2 60.8
128.3 149.3 107.2 157.5 103.6 121.4 131.6 114.8 159.8
1-OH 4′-OH 5″-OH 7″-OH 4‴-OH 8.46 d (8.5) 6.89 d (8.5)
δH, mult., J in Hz 3.52 3.31 3.12 3.08 2.87 3.58 3.44 4.98 3.22 3.25 3.09 3.16 3.58 3.44 5.50 3.21 3.23 3.09 3.16 3.58 3.44 18.67 9.83 12.82 13.47 10.15
d (9.5) m m m m m m d (7.5) m m m m m m d (7.2) m m m m m m s s s br s s
HMBC (H→C) 3, 5, 2⁗ 1″ 3⁗, 5⁗ 3⁗
6″ 2⁗′, 4⁗′ 3⁗′, 5⁗′
3″, 2⁗″, 3⁗″ 1⁗″, 3⁗″ 2⁗″, 4⁗″ 3⁗″, 5⁗″
1, 2, 6, 7, 8 3′, 5′ 5″, 6″, 10″ 3‴, 5‴
2′, 4‴ 1‴, 4‴
The assignments were based on 1D 1H and 13C and 2D 1H−1H COSY, HSQC, and HMBC experiments.
Figure 1. Key 1H−1H COSY and HMBC correlations for compounds 1 and 2.
ether bond is present between OH-2⁗″ and the C-1 enolic group based upon the fact that the chemical shifts of C-2⁗″ of deoxyglucitol at δC 90.1 and C-1 of the quinochalcone moiety at δC 177.8 were shifted to lower and higher field, respectively,
reversed-phase column between compound 2 and anhydrosafflor yellow B (Figure S26, Supporting Information). Interpretation of the 1D and 2D NMR spectra of 2 permitted construction of the most likely 2D structure (Figure 1). An 2646
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Figure 2. McLafferty rearrangement for cleavage about the methylene bridge in the MS/MS spectra of 1 (negative mode).
the 2,5-cyclohexadienone and/or cyclohexanedione moieties found in other known QCG dimers. Compound 2 had a large positive specific rotation of +135 and a negative ECD Cotton effect at ca. 272 nm (Figure 3), consistent with a (4S, 4″S) absolute configuration.9,13 Assuming that the deoxyglucitol moiety was biogenetically derived from D-glucose and the coupling constant of its anomeric proton was 8.0 Hz,10,12,13 it may be concluded that the five-membered etherocycle is trans-configured and the deoxyglucitol is 1-deoxyD-glucitol with a (1⁗″S) absolute configuration. Collectively, these data led to the structural assignment of 2 (carthorquinoside B) as depicted. Although bisflavonoids exhibit a wide taxonomic distribution ranging from bryophytes to angiosperms,14 heterodimers comprising quinochalcone and flavonoid moieties are hitherto unknown. Compound 1 represents the first case of compounds comprising a QCG and a flavonol glycoside unit linked by a methylene bridge. From a chemotaxonomic point of view, it is interesting to note that QCGs have only been found in C. tinctorius. Only five QCG dimers have been reported thus far, including safflor yellow B, anhydrosafflor yellow B, precarthamin, carthamin, and hydroxyethylcarthamin, and there have been no reports of new QCG dimers in the literature during the past decade.3 Instead of the 2,5cyclohexadienone and/or cyclohexaendione moieties found in the five known QCG dimers, 2 was the first compound that possessed A and A′ rings in the 2,4-cyclohexadienone form. Therefore, this compound supplements the structural diversity of QCGs and provides a significant clue for the elucidation of the biosynthetic pathway of precarthamin and carthamin. A putative genetic pathway for compounds 1 and 2 is proposed in Figure 4. Both of these originated from a pentahydroxychalcone. The methylene bridge in compound 1 might be formed
Figure 3. ECD spectra of compounds 1 and 2 in MeOH.
than those of the corresponding carbons in safflor yellow B.12 This was supported by the HMBC correlation from H-2⁗″ to C-1. Furthermore, compound 2 did not undergo keto−enol tautomerism and existed in the 1-enol-3,7-diketo form, which was verified by the HMBC cross-peaks between the hydrogenbonded OH proton at δH 18.31 and C-1″ (δC 181.5), C-2″ (δC 106.0), and C-6″ (δC 101.4). Four sets of key HMBC correlations (H-1⁗ with C-3, C-5; H-1⁗′ with C-3″, C-5″; OH-4 with C-3, C-4, C-5; and OH-4″ with C-3″, C-4″, C-5″) demonstrated that two nonequivalent C-β-glucopyranosyl residues and two OH groups were connected at C-4 and C4″, respectively. Thus, both the A and A′ rings in 2 were concluded to be the 2,4-cyclohexadienone moieties, instead of 2647
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Table 2. NMR Spectroscopic Data (1H 500 MHz and 13C 125 MHz, DMSO-d6) for Compound 2a no. 1, 1″
7 8 9 1′ 2′, 6′ 3′, 5′ 4′ 7″ 8″ 9″ 1‴
116.0 158.9 179.8 124.3 134.9 127.8
2‴, 6‴ 3‴, 5‴ 4‴ 1⁗
129.6 115.9 158.5 86.2
2, 2″ 3, 3″ 4, 4″ 5, 5″ 6, 6″
a
δC 177.8 181.5 102.3 106.0 195.8 195.5 85.2 189.5 187.7 103.6 101.4 182.4 128.1 135.2 128.2 129.6
δH, mult., J in Hz
HMBC (H→C)
7.51 d (16.0) 7.19 d (16.0)
7, 9, 1′, 2′/6′ 7, 8, 1′, 2′/6′
7.39 d (8.5)
9, 4′
no.
6.76 d (8.5)
7.45 d (16.0) 7.16 d (16.0)
7″, 9″, 1‴, 2‴/6‴ 7″, 8″, 1‴, 2‴/6‴
7.37 d (8.5) 6.76 d (8.5)
9″, 4‴
3.57 d (9.5)
3, 5
δC
δH, mult., J in Hz
2⁗
69.6
3.28 m
3⁗
78.7
3.08 m
4⁗
69.3
3.06 m
5⁗ 6⁗
78.2 60.5
1⁗′
85.4
2.91 3.50 3.18 3.78
m m m d (9.0)
2⁗′ 3⁗′ 4⁗′ 5⁗′ 6⁗′
69.8 78.9 67.9 79.7 58.0
1⁗″ 2⁗″ 3⁗″ 4⁗″ 5⁗″ 6⁗″
36.0 90.1 71.2 73.5 73.8 64.5
3.49 3.17 3.15 2.94 3.40 3.09 4.56 4.79 3.52 3.53 3.62 3.62 3.42 18.31 9.70 4.89 4.98
m m m m m m d (8.0) m m m m m m s br s s s
1″-OH 4′-OH 4-OH 4″-OH
HMBC (H→C)
3″, 5″
1, 1″, 5, 5″ 6, 6″, 2⁗″ 1, 6, 6″
1″, 2″, 6″ 3, 4, 5 3″, 4″, 5″
The assignments were based on 1D 1H and 13C and 2D 1H−1H COSY, HSQC, and HMBC experiments. spectra were obtained using a Thermo Scientific LTQ Orbitrap XL spectrometer. Preparative HPLC was performed on a Waters auto purification system using an XBridge C18 OBD column (150 × 30 mm, 5 μm) coupled with a UV−vis detector. Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden; 40−70 μm) and macroporous adsorbent resin D101 (Qingdao Hai Yang Chemical Group Co. Ltd., Qingdao, China; 0.3−1.25 mm) were used for column chromatography. Plant Material. The florets of C. tinctorius were purchased from Tongling Traditional Chinese Medicine Co. Ltd. in March 2013 and identified by Prof. C.-G. Wang (College of Pharmacy, Nanjing University of Chinese Medicine). A voucher specimen (No. NJUTCM-20130308) was deposited in the herbarium of Nanjing University of Chinese Medicine, Nanjing, People’s Republic of China. Extraction and Isolation. The dried florets of C. tinctorius (15 kg) were percolated with 95% and 70% EtOH until they were colorless. The brownish solid (1950 g) obtained by evaporating the EtOH extract to dryness below 45 °C was suspended in H2O (5 L) and extracted with petroleum ether (10 L) and EtOAc (10 L). The acqueous phase was concentrated and chromatographed over a D101 column. After eluting with H2O, the D101 column was successively eluted with 5%, 30%, 50%, and 95% EtOH. The 30% EtOH portion was separated into 30 fractions by Sephadex LH-20 using step gradient elution with H2O−MeOH (from 100:0 to 0:100). Fr.3 and Fr.9 were further purified by preparative HPLC to yield compounds 1 (20 mg) and 2 (6 mg). Mobile phases A and B were 0.1% aqueous formic acid and MeOH, respectively. The gradient elution was optimized as follows: 0−5 min, 16−19% B; 5−15 min, 19% B; 15−20 min, 19−44% B; 20−30 min, 44% B; 30−32 min, 44−100% B. The flow rate was 15 mL/min, and the detection wavelength was 407 nm. Carthorquinoside A (1): yellow, amorphous powder; [α]20D +364 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (4.37), 271 (4.20), 342 (4.33), 402 (4.24); ECD (0.46 mM, MeOH) λmax (Δε) 212 (−21.80),
by the nucleophilic addition of C-6 and C-8″ and endogenous formaldehyde, whereas the carbon system of compound 2 may be constructed from the intramolecular dehydration between C-1 and C-2⁗″ of a 1-deoxy-D-glucitol moiety. The aqueous extracts of C. tinctorius and hydroxysafflor yellow A were reported to have significant anti-inflammatory activity.15 In an in vitro anti-inflammatory assay, compound 1 significantly suppressed the overexpressions of IL-6 and IL-1 (pro-inflammatory cytokines) at the mRNA level, while it showed a dose-dependent up-regulation of anti-inflammatory cytokine IL-10 expression after lipopolysaccharide (LPS) stimulation (Figure 5A); compound 2 selectively downregulated the mRNA expressions of IL-6, IL-1, and IFN-γ and increased IL-10 mRNA expression in a dose-dependent manner (Figure 5B). Evidence of the inhibitory activity of compound 2 was also found against topoisomerase I, when assayed for the relaxation of supercoiled plasmid DNA at 100 μM (Figure 6). However, neither 1 or 2, in a range of concentrations between 3.125 and 200 μM, showed significant cytotoxicity toward the HeLa, HepG-2, A549, K562, and HCT116 cell lines.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 digital polarimeter. IR spectra were recorded on a Nicolet-Nexus-470 spectrometer. UV spectra were measured on a UV-2000 UV−visible spectrometer (Beijing LabTech Instruments Co. Ltd., Beijing, China). ECD spectra were recorded on a JASCO J-815-150S spectrometer. NMR spectra were recorded on a Varian 500 MHz NMR spectrometer (500 MHz for 1H and 125 MHz for 13C) with tetramethylsilane as an internal standard. HRESIMS 2648
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Figure 4. Plausible biosynthesis pathway toward the formation of 1 and 2. 245 (+9.45), 268 (−30.53), 415 (+16.61) nm; IR (KBr) νmax 3434, 1641, 1539, 1401 cm−1; NMR (DMSO-d6) data, see Table 1; HRESIMS m/z 1087.2563 [M − H]− (calcd for C49H51O28, 1087.2572); MS/MS (CID 25.0%; [M − H]−) m/z (%) 1069 (7), 1043 (6), 1025 (4), 879 (5), 625 (92), 461 (100). Carthorquinoside B (2): dark orange, amorphous powder; [α]20D +135 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (4.39), 344 (4.35), 420 (4.54); ECD (0.49 mM, MeOH) λmax (Δε) 215 (−57.66), 249 (+10.89), 272 (−60.21), 330 (−2.85), 382 (−38.16), 442 (+78.82) nm; IR (KBr) νmax 3433, 1640, 1513 cm−1; NMR (DMSOd6) data, see Table 2; HRESIMS m/z 1043.2679 [M − H]− (calcd for C48H51O26, 1043.2674); MS/MS (CID 25.0%; [M − H]−) m/z (%) 1025 (18), 923 (6), 593 (26), 449 (100). Determination of the Absolute Configuration of the Sugar. Compound 1 (2 mg) was dissolved in 2 N trifluoroacetic acid (0.1
mL), and the mixture heated at 120 °C for 6 h. The mixture was evaporated to dryness, and the residue was extracted three times with EtOAc (8 mL). The aqueous layer was concentrated to furnish a monosaccharide residue. The residue was dissolved in pyridine (0.5 mL), containing L-cysteine methyl ester hydrochloride (2 mg), and the reaction mixture was heated at 60 °C for 1 h. Phenyl isothiocyanate (2 μL) was added to the mixture, and heating was continued for 1 h at 60 °C. The mixture was concentrated and redissolved in MeOH (0.5 mL). Ten microliters of the solution was analyzed by HPLC with a YMC-Pack ODS-A (250 mm × 4.6 mm, 5 μm) at 30 °C with 25% MeCN−H2O (0.01% formic acid) at a flow rate of 0.8 mL/min. The retention time of the derivative was the same as that of a similar Dglucose derivative. In Vitro Anti-inflammatory Assay. HUVEC cells were purchased from the Nanjing KeyGen Biotech Company (Nanjing, 2649
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Figure 5. Effects of compounds 1 (A) and 2 (B) together with hydroxysafflor yellow A (C, positive control) on LPS-stimulated cytokine mRNA expression in HUVEC cells (each bar represents the mean ± SD of three independent experiments; statistical significance relative to LPS group is indicated: *, p < 0.05; **, p < 0.01). Topoisomerase I-Mediated DNA Cleavage Assay. Relaxation activity of calf-thymus topoisomerase I (Topo I) was performed as described by Bogurcu et al., with minor modifications.17 Hydroxycamptothecine (OPT) was employed as a positive control. Cytotoxicity Assays. The cytotoxicity assays against the HeLa (human epitheloid carcinoma), HepG-2 (human hepatoma), A549 (human lung carcinoma), K562 (human chronic myelogenous leukemia), and HCT-116 (human colon carcinoma) cell lines were assessed using a reported procedure.18 Paclitaxel (IC50: HeLa, 1.7 μM; HepG-2, 6.7 μM; A549, 5.5 μM; K562, 2.7 μM; HCT-116, 3.3 μM) was used as a positive control.
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ASSOCIATED CONTENT
S Supporting Information *
Figure 6. Effects of compounds 1 and 2 on Topo I-mediated supercoiled pBR322 relaxation (negatively supercoiled pBR322 (SC) and relaxed DNA (RLX) are marked).
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00561. 1
China). The HUVEC cells were cultured in DMEM (Gibco, Eggenstein, Germany). All culture media were supplemented with 10% FBS (Gibco, Carlsbad, CA, USA), 100 mg/mL streptomycin (complete medium), and 100 mg/mL penicillin. Cells were divided into the following seven groups (n = 3 in each group): drug groups (0.8, 4, 20, 100 μM drug + 10 μg/mL LPS), a model group (medium + 10 μg/mL LPS), and a control group (medium + medium). The cells were maintained in a humidified 5% CO2, 95% air atmosphere at 37 °C for different periods of time and were then harvested for measurements. Total RNA was isolated and extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and CHCl3. qRT-PCR was performed using a reported procedure.16 The primers are shown in Table S1 (Supporting Information).
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H NMR, 13C NMR, HSQC, COSY, HMBC, ROESY, HRESIMS, and ECD spectra of compounds 1 and 2 (PDF)
AUTHOR INFORMATION
Corresponding Authors
*Tel/Fax: 86-25-85811695. E-mail:
[email protected] (Y.-P. Tang). *Tel/Fax: 86-25-85811916. E-mail:
[email protected] (J.-A. Duan). Notes
The authors declare no competing financial interest. 2650
DOI: 10.1021/acs.jnatprod.6b00561 J. Nat. Prod. 2016, 79, 2644−2651
Journal of Natural Products
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Article
(15) (a) Wu, Y.; Wang, L.; Jin, M.; Zang, B. X. Biol. Pharm. Bull. 2012, 35, 515−522. (b) Song, L. J.; Zhu, Y.; Jin, M.; Zang, B. X. Fitoterapia 2013, 84, 107−114. (16) Lan, Y.; Liu, X. F.; Zhang, R.; Wang, K.; Wang, Y.; Hua, Z. C. BioMetals 2013, 26, 241−254. (17) Bogurcu, N.; Sevimli-Gur, C.; Ozmen, B.; Bedir, E.; Korkmaz, K. S. Biochem. Biophys. Res. Commun. 2011, 409, 738−744. (18) Allard, P. M.; Dau, E. T. H.; Eydoux, C.; Guillemot, J. C.; Dumontet, V.; Poullain, C.; Canard, B.; Guéritte, F.; Litaudon, M. J. Nat. Prod. 2011, 74, 2446−2453.
ACKNOWLEDGMENTS We thank Prof. C.-Y. Wang and Dr. L. Zhang (School of Medicine and Pharmacy, Ocean University of China) for proofreading the manuscript. We also thank Dr. Y.-Y. Liu (Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences) for assisting in the purification of the compounds. This research was supported by National Natural Science Foundation of China (81274058, 81573714), the Open Project Program of Jiangsu Key Laboratory for High Technology Research of TCM Formulae, and Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization (FJGJS-2015-11). This research was also financially supported by a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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