Communication pubs.acs.org/jnp
Anti-inflammatory Flavan-3-ol-dihydroretrochalcones from Daemonorops draco Ping-Chung Kuo,†,# Hsin-Yi Hung,†,# Tsong-Long Hwang,‡ Wen-Ke Du,† Hsiang-Chih Ku,‡ E-Jian Lee,§ Shih-Huang Tai,§ Fu-An Chen,⊥ and Tian-Shung Wu*,†,⊥ †
School of Pharmacy, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan ‡ Graduate Institute of Natural Products, College of Medicine, Chang Gung University; Research Center for Chinese Herbal Medicine, Research Center for Food and Cosmetic Safety, and Graduate Institute of Health Industry Technology, College of Human Ecology, Chang Gung University of Science and Technology; Department of Anesthesiology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan § Department of Surgery and Anesthesiology, and Institute of Biomedical Engineering, National Cheng Kung University, Medical Center and Medical School, Tainan 701, Taiwan ⊥ Department of Pharmacy, College of Pharmacy and Health Care, Tajen University, Pingtung 907, Taiwan S Supporting Information *
ABSTRACT: Four A-type flavan-3-ol-dihydroretrochalcone dimers, dragonins A−D (1−4), were characterized from the traditional Chinese medicine Sanguis Draconis. The structures of 1−4 were elucidated by spectroscopic and spectrometric analyses. Compounds 1 and 2 exhibited significant inhibition of fMLP/CB-induced superoxide anion and elastase. The signaling pathways accounting for the inhibitory effects of compound 2 were also elucidated. These purified A-type flavan-3-ol-dihydroretrochalcones are new potential leads for the development of anti-inflammatory drugs.
D
methylflavan.4 Some purified constituents were identified as the products of the coupling reactions between 7-hydroxy-5methoxyflavan and the quinone methide that resulted from the oxidation of 7-hydroxy-5-methoxy-6-methylflavan.4,23 Therefore, in the present study, dragonins A−D (1−4) (Figure 1), four flavan-3-ol-dihydroretrochalcones with unprecedented carbon skeletons that are produced by the coupling reaction of 7-hydroxy-5-methoxy-6-methylflavan and different quinone methides, were characterized from D. draco by conventional column chromatographic techniques. Their presence in the crude extracts was identified by LC/MS/MS (Figures S34− S39, Supporting Information), and their structures were determined by spectroscopic and spectrometric techniques. The absolute configurations were established by electronic circular dichroism (ECD) analysis. Dragonin A (1) was obtained as a colorless powder, and its molecular formula was established as C 34 H 32 O 8 from HRESIMS data analysis (m/z 591.1992 [M + Na]+, calcd. 591.1989). The UV absorption maxima (λmax) at 280, 242, and 209 nm indicated the presence of a benzene chromophore.24 The IR absorption bands at 3310, 1671, and 1607 cm−1 confirmed the presence of hydroxy, α,β-unsaturated carbonyl,
ragon’s blood, usually produced from several plant genera including Croton, Daemonorops, Dracaena, and Pterocarpus, is a bright red resin that has been in continuous use as a dye, varnish, incense, and medicine since ancient times.1,2 Only the East Asian resin collected from some Daemonorops species (Sanguis Draconis) is currently commercially available.3,4 The characteristic flavonoid constituents from this species are flavan analogues containing A- and C-ring hydroxy groups.4 Currently, Daemonorops draco (Willd.) Blume from Indonesia is the most commonly available species for Chinese medicinal formulas.2 Pharmacological studies indicate that D. dracoderived constituents exhibit anticoagulation,5−8 antiviral,2 antibacterial,9 anticancer,10−15 anti-inflammatory,4 and osteogenic bioactivities.16 Previous investigations of the chemical composition of D. draco indicated the presences of flavans, chalcones, and bisflavans.10,17−22 However, in recent decades, little progress had been made in the characterization of the compounds from Sanguis Draconis, which may be attributed to difficulties and limitations regarding the purification of the constituents. In addition, decomposition of some isolated compounds may be a serious problem, and solving this issue is a challenging task.9 In the previous report, several deoxyproanthocyanidins were characterized and deduced as the oxidation products of the two simple units found in the resin, i.e., 7-hydroxy-5-methoxyflavan and 7-hydroxy-5-methoxy-6© 2017 American Chemical Society and American Society of Pharmacognosy
Received: January 14, 2017 Published: April 11, 2017 783
DOI: 10.1021/acs.jnatprod.7b00039 J. Nat. Prod. 2017, 80, 783−789
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Figure 1. Structures of 1−4.
Figure 2. Significant HMBC correlations of 1−4.
and C−C double bond functionalities. In the 1H NMR spectrum, there were typical signals at δ 8.28 (1H, s, OH-7), 7.92−7.42 (10 H, H-10−H-14, H-2″−H-6″), and 6.23 (1H, s, H-8), which were characteristic of the aromatic moieties of the flavan-3-ol and dihydroretrochalcone constituent units. The mutually coupled oxygenated methines including δ 4.90 (1H, dd, J = 5.6, 3.2 Hz, H-3), 4.45 (1H, d, J = 5.6 Hz, OH-3), and 3.87 (1H, d, J = 3.2 Hz, H-4) constructed the partial structure of the flavan-3-ol moiety. Two methylene groups at δ 2.94 (1H, m, H-3‴), 2.87 (2H, m, H-2‴), and 2.50 (1H, m, H-3‴) and the sp2 carbon singlet of an α,β-unsaturated carbonyl moiety at δ 5.40 (1H, s, H-5′) suggested the presence of a chalcone unit. In addition, there were two methoxy groups at δ 3.81 (3H, s, CH3O-6′) and 3.67 (3H, s, CH3O-5) and two methyls attached to unsaturated functionalities at δ 2.00 (3H, s, CH3-6) and 1.60 (3H, s, CH3-3′). In the 13C NMR spectrum, two carbonyl carbons at δ 199.2 (C-1‴) and 187.2 (C-4′) and one hemiacetal carbon at δ 104.2 (C-2) could be characterized. Thus, compound 1 was composed of a flavan-3-ol and a dihydroretrochalcone unit, and these two units were attached via C-2 and C-4 of the flavan-3-ol moiety. Further HMBC analysis (Figure 2) indicated 2J and 3J correlations from H-4 to C-1′; from H-5′ to C-1′ and C-3′; and from CH3-3′ to C-2′, C3′, and C-4′, confirming the linkage between C-1′ and C-4, the same as reported in the dracoflavans.4 The major differences between 1 and the dracoflavans included replacement of the flavan moiety by a dihydroretrochalcone unit in 1. In addition, the B-ring of the dihydroretrochalcone moiety in 1 was changed to a 4,4-disubstituted 5-methoxy-2-methyl-3-oxycyclohexa-2,5-dien-1-one unit; therefore, an unprecedented carbon skeleton was revealed. The relative configuration of 1 was defined via NOESY data, in which correlations of H-3/H10, H-3/H-2‴, H-3/H-3‴, and H-4/H-3‴ indicated the 2R, 3S, 4S, 1′S or 2S, 3R, 4R, 1′R configurations (Figure 3). The ECD spectrum of 1 exhibited positive Cotton effects at 284, 246, and 217 nm, and according to the time-dependent density functional theory (TDDFT) calculations, the absolute config-
Figure 3. 3D structure of 1.
uration of 1 was determined as 2S, 3R, 4R, 1′R (Figures S8 and S9, Supporting Information). Based on the spectroscopic evidence, the structure of 1 was assigned as shown in Figure 1. The 1H and 13C NMR signals were assigned as listed in Table 1. The molecular formula of compound 2 was determined as identical to that of 1, and its structure should also be similar to 1 according to the spectroscopic data. The difference between the spectra of 1 and 2 included the absence of the sp2 singlet of the α,β-unsaturated carbonyl moiety (δ 5.40 in 1), replacement of the methoxy group (δ 3.81 in 1) by a hydroxy group in 2, and the presence of a methyl group (δ 1.69 in 2). The HMBC analysis revealed the 2J, 3J correlations from H-4 to C-2, C-1′; from CH3-1′ to C-1′, C-2′; from CH3-3′ to C-2′, C-4′; and from OH-6′ to C-1′, C-5′. In the dihydroretrochalcone moiety, the phenylpropanone fragment was attached to C-5′ rather than C-1′, and a methyl group was present at C-1′ to form the new carbon skeleton. The relative configuration of 2 was the same as 1 according to the NOESY correlations from H-3 to H784
DOI: 10.1021/acs.jnatprod.7b00039 J. Nat. Prod. 2017, 80, 783−789
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Table 1. 1H and 13C NMR Spectroscopic Data of Compounds 1−4 1a c
position 2 3 4 4a 5 6 7 8 8a 9 10, 14 11, 13 12 OH-3 OH-7 OH-6′ 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″,6″ 3″,5″ 4″ 1‴ 2‴ 3‴ Me-6 Me-1′ Me-3′ OMe-5 OMe-6′
H
4.90 dd (5.6, 3.2) 3.87 d (3.2)
6.23 s
7.76 7.43 7.43 4.45 8.28
d (7.6) m m d (5.6) s
5.40 s
7.92 d (7.6) 7.50 t (7.6) 7.61 t (7.6) 2.87 m
2b C 104.2 65.3 43.4 104.9 159.6 111.2 156.6 98.0 153.1 139.6 127.5 128.6 129.5
52.2 161.1 124.9 187.2 103.8 173.1 137.7 128.8 129.4 133.8 199.2 34.5
2.94 m 2.50 m 2.00 s
30.2
1.60 s 3.67 s 3.81 s
8.3 60.9 56.1
8.9
3a
H 4.57 dd (5.6, 3.2) 3.73 d (3.2)
6.10 s
7.69 7.47 7.47 5.45 9.33 10.04
d (7.6) m m d (5.6) s s
7.96 d (7.6) 7.55 t (7.6) 7.66 t (7.6) 2.70 m 2.55 m 2.46 m 1.84 1.69 1.51 3.55
C 102.8 63.3 41.3 103.8 158.3 109.2 155.4 96.8 151.5 138.6 126.5 128.7 127.9
46.3 161.6 120.4 186.1 111.8 169.6 136.5 127.9 128.7 133.2 200.4 37.0
s s s s
17.4 8.8 22.5 8.4 60.7
H 5.52 dd (7.4, 5.4) 3.69 d (7.4)
6.32 s
7.31 7.31 7.51 3.86 8.41
m m m d (5.4) s
6.15 s
7.93 d (7.6) 7.46 t (7.6) 7.57 t (7.6) 2.96 ddd (16.8, 11.2, 4.4) 2.70 ddd (16.8, 11.2, 4.4) 2.47 ddd (14.8, 11.2, 4.4) 2.17 m 2.06 s 0.99 s 3.67 s 4.09 s
4a C 85.8 65.9 42.2 103.7 160.1 110.9 157.2 99.1 152.1 137.6 128.1 128.1 127.1
62.3 204.6 72.3 191.8 108.3 176.4 138.0 128.7 129.4 133.6 199.6 34.5 25.1 9.3 11.0 61.0 58.1
H 5.44 dd (5.6, 4.0) 3.88 dd (4.0, 4.0)
6.31 s
7.57 7.55 7.53 4.16 8.50
m m m d (5.6) s
4.00 dd (4.0, 2.4) 3.43 d (2.4)
8.05 d (7.6) 7.30 m 7.64 t (7.6) 3.19 m 3.11 m 2.74 m 2.65 m 2.10 s
3.85 s 4.20 s
C 83.4 62.1 37.7 105.7 158.7 111.5 157.4 99.4 151.9 139.2 127.4 128.5 128.4
56.1 199.1 77.3 188.9 121.9 170.2 137.7 128.9 129.5 133.8 200.2 36.1 19.3 9.2
61.4 57.0
a1
H NMR data measured in acetone-d6 at 400 MHz, and 13C NMR data measured in acetone-d6 at 100 MHz. b1H NMR data measured in DMSO-d6 at 400 MHz, and 13C NMR data measured in DMSO-d6 at 100 MHz. cδH mult (J in Hz).
OMe-6′, H-4/H-3‴, Me-3′/H-14, and H-2‴/OMe-6′. The absolute configuration of 3 was established as 2S, 3R, 4R, 1′S, 3′R according to its ECD spectrum and TDDFT-computed results (Figures S24 and S25, Supporting Information), and the structure of 3 was assigned as shown in Figure 1. The molecular formula of dragonin D (4) was determined as C33H30O8 from its HRESIMS data, which suggested that one methyl group was replaced by a hydrogen compared to 3. Most of its NMR spectroscopic characteristics were similar to those of 3; however, the absence of the sp2 singlet of the α,βunsaturated carbonyl fragment was similar to that of 2. The HMBC data showed that the phenylpropanone moiety was attached to C-5′ (correlation of H-3‴/C-4′), and the replacement of CH3-3′ (correlation of H-3′/C-2′) by a hydrogen in 4 provided evidence for the unprecedented carbon skeleton. Thus, the 2D structure of 4 was determined as shown, and the absolute configuration was further established as 2R, 3R, 4S, 1′S, 3′S (Figure 1) according to its NOESY and ECD spectra (Figures S31 and S33, Supporting Information).
10 and from OH-3 to H-14. The ECD data (Figure S16, Supporting Information) confirmed the absolute configuration as 2R, 3S, 4S, 1′S. Thus, the structure of dragonin B (2) was established as shown in Figure 1. Dragonin C (3) was also an isomer of 1 according to the HRESIMS data. The 1H and 13C NMR spectra displayed one extra carbonyl at δ 204.6 (C-2′) and reduction of one C−C double bond. These suggested that the 4,4-disubstituted 5methoxy-2-methyl-3-oxycyclohexa-2,5-dien-1-one moiety of 1 was changed to a 6-methoxy-3-methyl-5-ene-2,4-dione unit in 3. Correspondingly, the connectivities between the flavan-3-ol and dihydroretrochalcone moieties were from C-1′ to C-4 and from C-3′ to C-2, as was confirmed by the 2J, 3J-HMBC crosspeaks from H-4 to C-2, C-1′, C-2′; from CH3-3′ to C-2, C-2′, C-4′; and from H-5′ to C-1′, C-3′, C-6′. Although the phenylpropanone fragment of the dihydroretrochalcone moiety was attached to C-1′ as in 1, the union between the flavan-3-ol and dihydroretrochalcone moieties was changed from a pyrane ring to a cyclohexanone unit. The relative configuration of 3 was determined by the NOESY cross-peaks of H-3/H-10, H-3/ 785
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Figure 4. Plausible biosynthetic pathway toward 1−4.
The biosynthesis for 1−4 is proposed as shown in Figure 4. Cyclization and tautomerization of the retrochalcone resulted in the flav-3-ene 5. Radical-induced oxidation of the phenolic moiety in the dihydroretrochalcone produced the quinone radical 6. The radical attack of 6 on the double bond of 5 from the re-face produced 7 and, following ring cyclization, afforded the flavan-3-ol-dihydroretrochalcone 1. The si-face radical attack of 6 to 5 produced stereoisomer 8, and phenylpropanoyl rearrangement resulted in quinone radical 9. The methyl group shift from the neighboring methoxy group afforded compound 2. Formation of quinone radical 10 via 7 and subsequent cyclization between the flav-3-ene moiety and the quinone radical would give 3. If cyclization is accompanied by phenylpropanoyl rearrangement to produce radical 11, subsequent demethylation would yield 4.25 The anti-inflammatory activity of the isolates was evaluated by the cellular model in human neutrophils. Compounds 1−4 were tested for their inhibition on the production of superoxide anion and elastase by human neutrophils in response to formylL-methionyl-L-leucyl-L-phenylalanine/cytochalasin B (fMLP/ CB).26,27 Compounds 1 and 2 exhibited inhibition with IC50 values in the range of 1.3 ± 0.5 and 4.5 ± 0.8 μM (Table 2). According to the above data, the purified flavan-3-oldihydroretrochalcones are new potential leads for the development of anti-inflammatory drugs. To test whether fMLFmediated downstream signals, calcium and mitogen-activated protein kinases (MAPKs), are related to the inhibitory effects of compound 2, intracellular calcium ion concentration ([Ca2+]i) and ERK, p38 MAPK, and JNK phosphorylation were assayed. The duration and magnitude of the [Ca2+]i signal response to
Table 2. Inhibitory Effects of Compounds 1−4 on Superoxide Anion Generation and Elastase Release by Human Neutrophils in Response to fMLP/CB IC50 (μM)a compound
superoxide anion generation
elastase release
1 2 3 4 LY294002c
3.1 ± 0.9b 1.3 ± 0.5b >10 >10 0.4 ± 0.02b
4.5 ± 0.8b 3.1 ± 0.5b >10 >10 1.5 ± 0.3b
a Concentration necessary for 50% inhibition. The results are presented as the means ± SEM (n = 3−5). bp < 0.001 compared to the control value. cA phosphatidylinositol-3-kinase inhibitor was used as a positive control.
fMLF are important for neutrophil superoxide anion generation and elastase release.28,29 Compound 2 failed to affect the fMLFinduced peak [Ca2+]i values, but half of the peak value (t1/2) was considerably reduced by compound 2 (Figure 5). In addition, human neutrophils stimulated with fMLF afforded the rapid phosphorylation of ERK, p38 MAPK, and JNK.26 Compound 2 inhibited the phosphorylation of JNK and p38 but not ERK (Figure 6). The results suggested that the inhibitory effects of compound 2 in fMLF-induced activation of human neutrophils are partially mediated by the reduction of JNK and p38 activation as well as calcium mobilization. 786
DOI: 10.1021/acs.jnatprod.7b00039 J. Nat. Prod. 2017, 80, 783−789
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400 spectrometer. HRESIMS spectra were acquired from Varian ProStar LC/Varian 901 FT-ICR and Bruker APEX II FT mass spectrometers. TLC and preparative TLC (pTLC) analyses were carried out with silica gel 60 F254 (Merck KGaA, Darmstadt, Germany). Column chromatography (CC) was performed on Geduran Si 60 (40−63 μm, Merck) and Sephadex LH-20 (25−100 μm, Sigma-Aldrich, St. Louis, MO, USA). All solvents used in column chromatography were of pesticide residue analysis grade (Fluka, Munich, Germany; Sigma-Aldrich), analytical grade (J. T. Baker, Avantor, Center Valley, PA, USA), and chromatographic grade (Merck). Plant Material. The resin of Daemonorops draco was bought from Chuang Song Zong Pharmaceutical Co. Ltd., Taiwan, in 2010 and identified by Prof. Chang-Sheng Kuoh, Department of Life Science, National Cheng Kung University (NCKU), Tainan, Taiwan. A voucher specimen (Wu-2010002) was stored in the School of Pharmacy, NCKU. Extraction and Isolation. The resin of D. draco (3.0 kg) was dissolved in CHCl3 (12.0 L). The insoluble materials were filtered, and the filtrate was partitioned with H2O (6.0 L) to yield a CHCl3-soluble fraction (approximately 3.0 kg) and a water-soluble fraction (70 g). Part of the CHCl3-soluble extract (0.5 kg) was purified on a silica gel column with a step gradient (n-hexane−acetone, 4:1, 2:1, 1:1, and 0:1) to give 10 fractions. Fraction 8 (31.2 g) was chromatographed on Sephadex LH-20 with an eluent mixture of H2O and MeOH to produce nine subfractions (Fr. 8.1−8.9). Further purification of Fr. 8.5 yielded dragonin B (2) (50.6 mg). Fraction 9 (42.3 g) was separated by silica gel CC with a step gradient (CHCl3−acetone, 20:1, 15:1, 10:1, 5:1, 2:1, 1:1, and 0:1) to produce eight subfractions (Fr. 9.1− 9.8). Fr. 9.3 was purified by silica gel CC with a step gradient (CHCl3−MeOH, 80:1, 40:1, 10:1, 5:1, 1:1, and 0:1) to provide four minor fractions (Fr. 9.3.1−9.3.4). Fr. 9.3.4 was chromatographed on Sephadex LH-20 with an eluent mixture of H2O and MeOH, and
Figure 5. Compound 2 inhibited Ca2+ mobilization in fMLF-activated neutrophils. Fluo-3/AM-labeled human neutrophils were incubated with DMSO (as control) or 2 (10 μM) for 5 min before stimulation of fMLF (0.1 μM). Ca2+ mobilization was recorded in a spectrofluorometer in real time. The intracellular calcium concentration ([Ca2+]i) peak and reduction of the time taken to decrease to half of its peak values (t1/2) are shown as the mean ± SEM (n = 3). **p < 0.01.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured with a JASCO P-2000 polarimeter with a 589 nm filter. UV spectra were recorded on a Hitachi U-0080-D spectrophotometer with a 0.1 dm length cell. IR spectra data were collected on a PerkinElmer FT-IR Spectrum RX I spectrometer using KBr pellets. ECD spectra were obtained using a JASCO J-720 spectropolarimeter. 1 H, 13C, and 2D NMR spectra were measured on the Bruker AVIII
Figure 6. Compound 2 inhibited the phosphorylation of JNK and p38 but not ERK in fMLF-activated human neutrophils. Human neutrophils were preincubated with DMSO or 2 (10 μM) before stimulation with fMLF (0.1 μM). The Western blotting was performed under the same condition. After transferring the blots onto nitrocellulose membranes, we immediately cropped the targeted blots according to reference indicating markers, and then the targeted protein was immunoblotted with its specific antibody. Representative images from one of three independent experiments of Western blotting using antiphospho antibodies directed against JNK, p38, and ERK are shown. Bands on the blots were analyzed using a densitometer, and the quantitative ratios for all samples were normalized to the corresponding total protein. Data are shown as the mean ± SEM (n = 3). **p < 0.01; ***p < 0.001. 787
DOI: 10.1021/acs.jnatprod.7b00039 J. Nat. Prod. 2017, 80, 783−789
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Statistical Analysis. Experimental results were illustrated as the mean ± SEM. The half-maximal inhibitory concentration (IC50) was computer-assisted calculated (PHARM/PCS v.4.2). Statistical comparisons between groups were performed through Student’s t test. pValues less than 0.05 were considered to be significant statistically.
further purification was achieved using pTLC to give dragonin A (1) (2.2 mg), C (3) (2.6 mg), and D (4) (1.0 mg). Dragonin A (1): colorless powder (CHCl3); [α]26D +9 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 280 (3.50), 242 (4.14), 209 (4.42) nm; ECD (MeOH) 284 (Δε +0.9), 246 (Δε +1.0), 217 (Δε +2.8) nm; IR (neat) νmax 3310, 2928, 2854, 1671, 1607, 1451, 1231, 1097, 960, 696 cm−1; 1H NMR (400 MHz, acetone-d6) and 13C NMR (100 MHz, acetone-d6), see Table 1; HRESIMS m/z 591.1992 ([M + Na]+, calcd for C34H32O8Na, 591.1989). Dragonin B (2): colorless powder (CHCl3); [α]26D −2 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 283 (3.70), 247 (4.30), 209 (4.58) nm; ECD (MeOH) 281 (Δε −0.5), 234 (Δε −1.5), 209 (Δε −1.9) nm; IR (neat) νmax 3397, 2931, 1668, 1594, 1445, 1365, 1107, 953, 694 cm−1; 1H NMR (400 MHz, DMSO-d6) and 13C NMR (100 MHz, DMSO-d6), see Table 1; HRESIMS m/z 591.1991 ([M + Na]+, calcd for C34H32O8Na, 591.1989). Dragonin C (3): colorless powder (CHCl3); [α]26D +1 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 281 (3.69), 247 (4.07), 210 (4.42) nm; ECD (MeOH) 295 (Δε −0.5), 206 (Δε −1.7) nm; IR (neat) νmax 3410, 2945, 1733, 1708, 1660, 1597, 1355, 1231, 1125, 697 cm−1; 1H NMR (400 MHz, acetone-d6) and 13C NMR (100 MHz, acetone-d6), see Table 1; HRESIMS m/z 591.1991 ([M + Na]+, calcd for C34H32O8Na, 591.1989). Dragonin D (4): colorless powder (CHCl3); [α]26D +3 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 284 (3.72), 249 (3.90), 210 (4.35) nm; ECD (MeOH) 297 (Δε −1.1), 211 (Δε −1.3) nm; IR (neat) νmax 3405, 2926, 2855, 1737, 1663, 1594, 1357, 1224, 1115, 696 cm−1; 1H NMR (400 MHz, acetone-d6) and 13C NMR (100 MHz, acetone-d6), see Table 1; HRESIMS m/z 577.1834 ([M + Na]+, calcd for C33H30O8Na, 577.1833). Quantum Chemical Calculations. The compound structure models were constructed according to the NOE analysis. The ECD calculations were performed with the Gaussian 09 software as described previously.30 Preparation of Human Neutrophils. A study involving human neutrophils was approved by the Institutional Review Board at Chang Gung Memorial Hospital, Taoyuan, Taiwan, and was conducted following the Declaration of Helsinki (2013). Neutrophils were purified as described previously.26,27 Superoxide Anion Generation Measurement. The assay of the generation of superoxide anion was based on the SOD-inhibited reduction of ferricytochrome c.26,27 Elastase Release Measurement. Degranulation of azurophilic granules was determined by elastase release as described previously.26,27 Examination of ([Ca2+]i). Human neutrophils (6 × 105 cells/mL) were treated with Fluo-3/AM (2 μM) for 30 min at 37 °C, followed by centrifugation and resuspension in HBSS solution containing CaCl2 (1 mM). The Fluo-3/AM-labeled human neutrophils were treated with DMSO (0.1%, as control) or compound 2 (10 μM) for 5 min. The [Ca2+]i in response to fMLF (0.1 μM) was measured under continuous stirring using a spectrophotometer with an excitation wavelength of 488 nm and an emission wavelength of 520 nm.26 Western Blot of Whole-Cell Lysates. Human neutrophils were incubated with DMSO (0.1%, as control) or compound 2 (10 μM) for 5 min and then activated by fMLF (0.1 μM) for 30 s in the pretreatment of CB (1 μg/mL). The reaction was stopped by heating at 100 °C for 15 min with sample buffer (62.5 mM pH 6.8 Tris-HCl, 4% sodium dodecyl sulfate, 5% β-mercaptoethanol, 2.5 mM Na3VO4, 0.00125% bromophenol blue, 10 mM p-nitrophenyl phosphate di(tris) salt, and 8.75% glycerol). Samples were centrifuged at 14000g for 20 min, and the supernatant was subjected to the immunoblotting assay. The target protein was characterized by immunoblotting with the corresponding antibody overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated, secondary anti-rabbit antibody (Cell Signaling Technology, Beverly, MA, USA) at room temperature for 1 h. The enhanced chemiluminescence solution was added to the membranes, and protein bands were detected using the BioSpectrum Imaging System (UVP; Upland, CA, USA).
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00039. Supplementary spectroscopic data (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel: 886-6-2747538. Fax: 886-6-2740552. E-mail: tswu@mail. ncku.edu.tw (T.-S. Wu). ORCID
Tian-Shung Wu: 0000-0002-2117-0266 Author Contributions #
P. C. Kuo and H. Y. Hung contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was sponsored by the Ministry of Science and Technology, Taiwan, granted to T.-S.W. and P.-C.K. The authors are also thankful to Chang Gung Memorial Hospital (CMRPD1B0281-3, CMRPF1D0442-3, CMRPF 1F0011-3, CMRPF1F0061-3, and BMRP450 granted to H.-L.H.) for partial financial support of the present research.
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