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Flavonoid dimers from the total phenolic extract of Chinese dragon's blood, the red resin of Dracaena cochinchinensis
Fitoterapia 115 (2016) 135–141
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Flavonoid dimers from the total phenolic extract of Chinese dragon's blood, the red resin of Dracaena cochinchinensis Dao-Ran Pang a,b, Xiao-Qin Su a,b, Zhi-Xiang Zhu a, Jing Sun a,b, Yue-Ting Li a,b, Yue-Lin Song a, Yun-Fang Zhao a, Peng-Fei Tu a, Jiao Zheng a,⁎, Jun Li a,⁎ a b
Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, People's Republic of China School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, People's Republic of China
a r t i c l e
i n f o
Article history: Received 22 August 2016 Received in revised form 12 October 2016 Accepted 15 October 2016 Available online 18 October 2016 Keywords: Longxuetongluo capsule Dracaena cochinchinensis Chinese dragon's blood Flavonoid dimer Electronic circular dichroism
a b s t r a c t Eight new flavonoid dimers, named cochinchinenins I–M (1–5), including three pairs of enantiomers (1a/1b–3a/ 3b) and two optically pure flavonoid dimers (4–5), along with a known analogue (6), were isolated from total phenolic extract of the red resin of Dracaena cochinchinensis (Chinese dragon's blood). The planar structures of 1–5 were elucidated by extensive spectroscopic analysis including HRESIMS and 1D/2D NMR. Their absolute configurations were determined on the basis of experimental and calculated electronic circular dichroism (ECD) data. Compounds 4 and 5 exhibited significant inhibition of nitric oxide production in lipopolysaccharide-stimulated BV-2 microglial cells with IC50 value of 4.9 ± 0.4 and 5.4 ± 0.6 μM, respectively. © 2016 Published by Elsevier B.V.
1. Introduction Ischemic stroke accounts for 70–80% of the stroke incidence, which brings a big burden to the healthcare system and society. Although great advances have been made in understanding the pathophysiologic mechanisms of ischemic stroke, clinically effective therapies are limited [1]. Therefore, it's in urgent need to develop effective agents for prevention and treatment of this disease. There is increasing evidence that post-ischemic inflammation contributes to ischemic brain injury. Post-ischemic inflammation is, therefore, a promising target for therapeutic intervention in ischemic stroke [1]. Chinese dragon's blood, Longxuejie in Chinese, is the red resin of Dracaena cochinchinensis (Lour.) S. C. Chen growing in Yunnan and Guangxi provinces in China, which was firstly discovered by Cai and Xu in 1979 [2]. It was considered to be able to serve as a substitute for Sanguis Draconis (Xuejie in Chinese), a precious crude drug known as “panacea of blood activating” in traditional Chinese medicine, which has a reputation for facilitating blood circulation and dispersing blood stasis [3]. Pharmacological studies showed that the pure compounds and crude extracts of the red resin of D. cochinchinensis possess anti-inflammatory and analgesic, anti-platelet aggregation, antithrombotic, cerebral protective, radioprotective, wound healing, anti-diabetic, anti-microbial, antioxidant, and cytotoxic activities [3,4]. Previous phytochemical investigations on the resin and other parts of D. cochinchinensis have resulted in the isolation of flavonoid and its oligomers, stilbenes, and ⁎ Corresponding authors. E-mail addresses: [email protected] (J. Zheng), [email protected] (J. Li).
http://dx.doi.org/10.1016/j.fitote.2016.10.004 0367-326X/© 2016 Published by Elsevier B.V.
several steroids, and the phenolic compounds were considered to be responsible for its main biological activities [3–8]. In June 2013, Longxuetongluo Capsule (LTC), a new drug consisting of the total phenolic extract of Chinese dragon's blood, was approved for the treatment of ischemic stroke by China Food and Drug Administration. Recently, we demonstrated that LTC could ameliorate erythrocyte deformability and osmotic fragility through the reduction of lipid peroxidation on plasma and erythrocyte membranes in HFD-induced ApoE−/− mice [8]. However, the major phenolic constituents of this material have not yet been fully described because of the difficulties associated with the separation of these compounds, especially for flavonoid oligomers [6]. As an ongoing effort to search for bioactive components of the total phenolic extract of Chinese dragon's blood, an HPLC-MS-guided separation procedure was performed to target the flavonoid oligomers, which led to the isolation of eight new flavonoid dimers (1–5), including three pairs of enantiomers (1a/1b–3a/3b) and two optically pure flavonoid dimers (4–5) along with a known analogue (6). Herein, the isolation and structural elucidation of the new compounds are described as well as their inhibitory effects on NO production in LPS-stimulated BV-2 microglial cells.
2. Experimental 2.1. General experimental procedures Optical rotations were measured on a Rudolph Autopol IV automatic polarimeter (NJ, USA). ECD spectra were recorded on a JASCO J-810 CD
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spectrophotometer (JASCO, Japan). IR spectra were obtained using a Thermo Nicolet Nexus 470 FT-IR spectrophotometer (MA, USA) with KBr pellets. UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer (Tokyo, Japan). NMR spectra were obtained on a Varian INOVA-500 spectrometer (CA, USA) operating at 500 MHz for 1H NMR and 125 MHz for 13C NMR, respectively. The HRESIMS data were recorded on an LCMS-IT-TOF system equipped with a Prominence UFLC system and an ESI interface (Shimadzu, Kyoto, Japan). Silica gel (200– 300 mesh, Qingdao Marine Chemical Inc., Qingdao, People's Republic of China), LiChroprep RP-C18 gel (40–63 μm, Merck, Germany), and Sephadex LH-20 (Pharmacia, LA, USA) were employed for open column chromatography (CC). HPLC was performed on a Shimadzu LC-20AT pump system (Shimadzu Corporation, Tokyo, Japan) equipped with an SPD-M20A photodiode array detector monitoring at 230 and 280 nm. A semi-preparative HPLC SPOLAR C18 column (250 × 10 mm, 5 μm) was employed for the isolation. Chiral HPLC was done with a Daicel CHIRALPAC IC column (150 × 4.6 mm, 5 μm). TLCs were performed using pre-coated GF254 plates. All purified compounds submitted for bioassay were at least 95% pure as judged by HPLC and supported by 1 H NMR analysis. 2.2. Plant material The total phenolic extract of Chinese dragon's blood (LTC140601) was provided by Jiangsu Kanion Pharmaceutical Co. Ltd. A voucher sample is deposited at the Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China. 2.3. Extraction and isolation The air-dried and powdered red resin of D. cochinchinensis (4.5 kg) was extracted with 95% EtOH at room temperature (45 L × 3, 72 h each). The ethanol extracts were combined and evaporated to dryness under reduced pressure. The dry residue (920 g) was defatted with petroleum ether (5 L × 1), and then extracted with EtOAc (5 L × 2) to produce the total phenolic extract of Chinese dragon's blood. The total phenolic extract (600 g) was subjected to silica gel CC and eluted with a stepwise gradient of petroleum ether–EtOAc (10:1 → 1:3) and then CH2Cl2–MeOH (20:1 → 0:1), to afford ten fractions (Fr. A–J). Afterwards, an HPLC-MS-guided separation and purification procedure [0.1% formic acid aqueous solution (v/v, solvent A) and acetonitrile (solvent B): 0– 30 min, 5–95% B; 30–40 min, 95% B, (v/v)] was performed to screen out the targeted fractions containing flavonoid dimers. Fr. G (61.5 g) containing flavonoid dimers was chromatographed on silica gel CC eluting with petroleum ether–EtOAc (3:1 → 0:1) to yield five subfractions (Fr. G1–G5). Fr. G3 (2.2 g) containing the targeted compounds was chromatographed over Sephadex LH-20 CC eluting with MeOH to yield four portions (Fr. G3a–G3d). Fr. G3b (1.3 g) was resolved by RPC18 CC eluting with a stepwise gradient of MeOH–H2O (30:70 → 100:0) to afford sub‐Fr. G3b1–G3b3. Sub-Fr. G3b1 was purified by semi-preparative HPLC using MeOH–H2O (60:40, 2.0 mL/min) to yield 2b (15.4 mg, tR 55.8 min) and 3a (4.1 mg, tR 78.1 min). Sub-Fr. G3b2 was separated on semi-preparative RP-HPLC (MeCN–H2O, 45:55, 3.0 mL/min) to give 6 (9.3 mg, tR 33.4 min), 4 (4.3 mg, tR 34.8 min), and 3b (7.1 mg, tR 36.0 min). Sub-Fr. G3b3 was purified by semi-preparative RP-HPLC (MeCN–H2O, 55:45 → 80:20, 0–30 min, 3.0 mL/min) to give 2a (4.2 mg, tR 13.4 min) and 5 (6.3 mg, tR 14.1 min). Compound 1 (15.0 mg, tR 21.0 min) was obtained from Fr. G3c (500 mg) by semi-preparative RP-HPLC (MeCN–H2O, 35:65, 3.0 mL/min). Furthermore, direct enantiomeric separation of 1 was achieved by chiral HPLC with n-hexane-ethanol (95:5 → 80:20, 0–30 min, 1.0 mL/min), affording 1b (3.1 mg, tR 23.0 min) and 1a (2.6 mg, tR 26.0 min), respectively. 2.3.1. (±)-Cochinchinenin I (1) Yellow amorphous powder; UV λMeOHmax(log ε): 226 (2.94), 281 (2.88) nm; IR (KBr) νmax: 3369, 2954, 2918, 2850, 1654, 1598, 1511,
1455, 1368, 1230, 1169, 1092 cm−1; 1H and 13C NMR data, see Table 1; negative-ion HRESIMS: m/z 499.1767 [M − H]− (calcd for C30H27O7, 499.1762). (−)-Cochinchinenin I (1a): [α]25D: − 6.0 (c 0.10, MeOH); ECD (c 1.0 × 10−3 M, MeOH), λmax (Δε) 208 (−2.33), 222 (− 2.98), 246 (+ 2.72), 292 (+ 0.64) nm. (+)-Cochinchinenin I (1b): [α]25D: + 6.0 (c 0.10, MeOH); ECD (c 1.0 × 10−3 M, MeOH) λmax (Δε) 204 (+ 4.67), 222 (+ 3.41), 240 (− 2.22), 294 (−0.31) nm. 2.3.2. (−)-Cochinchinenin J (2a) Pale brown, amorphous powder; [α]25D: −20.0 (c 0.09, MeOH); UV MeOH λ max(log ε): 215 (3.41), 222 (3.40), 279 (2.80) nm; ECD (c 8.3 × 10−4 M, MeOH), λmax (Δε) 216 (−6.43), 226 (+2.08), 240 (−12.31), 290 (+ 2.28) nm; IR (KBr) νmax: 3431, 2927, 2852, 1614, 1513, 1489, 1459, 1436, 1384, 1229, 1198, 1158, 1115, 1038 cm−1; 1H and 13C NMR data, see Table 2; negative-ion HRESIMS: m/z 541.2215 [M − H]− (calcd for C33H33O7, 541.2232). 2.3.3. (+)-Cochinchinenin J (2b) Pale brown, amorphous powder; [α]25D: +31.0 (c 0.07, MeOH); UV λMeOHmax(log ε): 212 (2.83), 230 (3.48), 280 (2.88) nm; ECD (c 6.5 × 10−4 M, MeOH) λmax (Δε) 216 (+5.94), 226 (− 7.65), 244 (+20.47), 276 (+5.68), 292 (−4.14) nm; IR (KBr) νmax: 3441, 2920, 2851, 1615, 1512, 1489, 1458, 1436, 1198, 1158, 1115, 1038 cm−1; 1H and 13C NMR data, see Table 2; negative-ion HRESIMS: m/z 541.2208 [M − H]− (calcd for C33H33O7, 541.2232). 2.3.4. (−)-Cochinchinenin K (3a) Pale brown, amorphous powder; [α]25D: −20.0 (c 0.12, MeOH); UV MeOH −3 M, λ max(log ε): 217 (3.30), 282 (2.89) nm; ECD (c 1.1 × 10 MeOH) λmax (Δε) 216 (− 5.4), 232 (− 3.83), 244 (+ 1.24), 294 (+0.57) nm; IR (KBr) νmax: 3441, 2924, 2852, 1616, 1510, 1456, 1435, 1399, 1385, 1274, 1235, 1175, 1157, 1116, 1071, 1035 cm−1; 1H and 13 C NMR data, see Table 3; negative-ion HRESIMS: m/z 527.2086 [M − H]− (calcd for C32H31O7, 527.2075). 2.3.5. (+)-Cochinchinenin K (3b) Pale brown, amorphous powder; [α]25D: +22.0 (c 0.09, MeOH); UV λMeOHmax(log ε): 211 (3.50), 230 (3.38), 281 (2.94) nm; ECD (c 8.5 × 10−4 M, MeOH) λmax (Δε) 214 (+ 5.83), 226 (+ 8.96), 240 (− 4.01), 276 (+1.00), 294 (−1.85) nm; IR (KBr) νmax: 3438, 2927, 2852, 1617, 1510, 1438, 1384, 1273, 1234, 1176, 1157, 1117, 1071, 1035 cm−1; 1H and 13C NMR data, see Table 3; negative-ion HRESIMS: m/z 527.2085 [M − H]− (calcd for C32H31O7, 527.2075). 2.3.6. Cochinchinenin L (4) Pale brown, amorphous powder; [α]25D: −36.0 (c 0.10, MeOH); UV MeOH λ max(log ε): 210 (3.51), 230 (3.41), 281 (2.76) nm; ECD (c 9.5 × Table 1 1 H (500 MHz) and 13C (125 MHz) NMR data of compound 1 (δ in ppm, in CD3OD). Position 1
Position 1
δH, multi. (J in Hz) δC, type 1 2 3 4 5 6 α β C =O γ α' β' 1′
6.28, s
6.89, s 3.15, t (7.0) 2.86, t (7.0) 4.14, t (8.0) 2.07, m 2.38, m
119.5, C 154.5, C 103.7, CH 154.9, C 124.5, C 130.2, CH 40.1, CH2 27.1, CH2 202.4, C 43.2. CH 37.3, CH2 29.5, CH2 130.1, C
δH, multi. (J in Hz) δC, type 2′, 6′ 3′, 5′ 4′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴, 6‴ 3‴, 5‴ 4‴
7.88, d (9.0) 6.80, d (9.0)
6.24, d (2.0) 6.18,dd (8.0, 2.0) 6.75, d (8.0) 7.03, d (8.5) 6.63, d (8.5)
132.1, CH 116.2, CH 163.9, C 121.8, C 156.9, C 103.4, CH 157.1, C 107.2, CH 131.4,CH 138.6,C 130.0, CH 115.7, CH 155.9, C
D.-R. Pang et al. / Fitoterapia 115 (2016) 135–141 Table 2 1 H (500 MHz) and 13C (125 MHz) NMR data of compound 2 (δ in ppm, in CD3OD). Position
2aa δH, multi. (J in Hz)
δC, type
δH, multi. (J in Hz)
δC, type
2
4.11, d (10.5) 3.72, m 2.12, m 2.63, dd (16.0, 5.0) 2.37, dd (16.0, 9.0) 6.55, s
70.9, CH2
4.12, d (10.5) 3.71, m 2.12, m 2.62, dd (16.0, 5.0) 2.32, dd (16.0, 9.0) 6.53, s
70.9, CH2
3 4 5 6 7 8 9 10 11 α β γ 1′ 2′, 6′ 3′, 5′ 4′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴, 6‴ 3‴, 5‴ 1‴ 2″-OCH3 8-OCH3 a
2.53, m 2.12, m; 2.05, m 2.42, m 4.16, t (8.0) 6.99, d (8.5) 6.71, d (8.5)
6.36, d (2.0) 6.26, dd (8.0, 2.0) 6.80, d (8.0) 7.06, d (8.5) 6.65, d (8.5) 3.72, s 3.75, s
2ba
35.8, CH 31.6, CH2 123.8, CH 126.1, C 147.0, C 136.3, C 146.6, C 114.1, C 38.0, CH2 37.1, CH2 29.5, CH2 43.6, CH 131.8, C 131.0, CH 116.2, CH 156.7, C 123.2, C 159.7, C 99.8, CH 157.6, C 107.5, CH 131.1, CH 138.1, C 130.0, CH 115.7, CH 156.0, C 55.6, CH3 61.0, CH3
2.50, m 2.16, m; 2.03, m 2.40, m 4.16, t (8.0) 6.98, d (8.5) 6.71, d (8.5)
6.34, d (2.0) 6.25, dd (8.0, 2.0) 6.79, d (8.0) 7.07, d (8.5) 6.67, d (8.5) 3.67, s 3.75, s
35.9, CH 31.7, CH2 123.8, CH 126.0, C 147.0, C 136.3, C 146.6, C 114.2, C 38.0, CH2 37.1, CH2 29.5, CH2 43.6, CH 131.8, C 131.0, CH 116.1, CH 156.7, C 123.1, C 159.6, C 99.7, CH 157.5, C 107.5, CH 131.1, CH 138.1, C 130.0, CH 115.7, CH 156.0, C 55.6, CH3 61.0, CH3
Assignments were carried out based on HSQC and HMBC experiments.
10−4 M, MeOH) λmax (Δε) 212 (− 6.26), 226 (+12.81), 240 (− 4.52), 276 (+ 0.84), 290 (0.42) nm; IR (KBr) νmax: 3441, 2925, 2853, 1616, 1511, 1458, 1436, 1384, 1272, 1234, 1198, 1158, 1117, 1070, 1035 cm−1; 1H and 13C NMR data, see Table 3; negative-ion HRESIMS: m/z 527.2074 [M − H]− (calcd for C32H31O7, 527.2075). 2.3.7. Cochinchinenin M (5) Pale brown, amorphous powder; [α]25D: +27.0 (c 0.11, MeOH); UV λMeOHmax(log ε): 212 (3.43), 230 (3.35), 281 (2.88) nm; ECD (c 1.0 × 10−3 M, MeOH) λmax (Δε) 212 (+ 4.51), 228 (+ 1.68), 242 (− 1.01), 276 (+0.43), 296 (−0.47) nm; IR (KBr) νmax: 3432, 2924, 2852, 1618, 1512, 1444, 1399, 1385, 1273, 1223, 1161, 1112, 1072, 1035 cm−1; 1H and 13C NMR data, see Table 3; negative-ion HRESIMS: m/z 527.2066 [M − H]− (calcd for C32H31O7, 527.2075). 2.4. Biological assays BV-2 microglial cells were purchased from Peking Union Medical College (PUMC) Cell Bank (Beijing, People's Republic of China). Cell culture, viability assay, and assay for inhibition ability against LPS-stimulated NO production were performed according to our previous reports [9–12], with indomethacin (IC50 = 44.1 ± 5.8 μM) as the positive control. The IC50 values were determined using GraphPad Prism 5 software from experiments performed in triplicate (GraphPad Software, Inc., San Diego, CA, USA). All the compounds were prepared as stock solutions in DMSO (final solvent concentration b 0.5% in all assays). 2.5. Computational details Preliminary conformational analysis of compounds 1–4 was performed with the SYBYL-X 2.0 software package using the random search method with the MMFF94s force field. The obtained conformers were
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used for geometry reoptimizations at the B3LYP/6-31G(d) level in the Gaussian 09 package. The ECD spectra for the optimized conformers were calculated by the TDDFT method at the B3LYP/6–31 + G(d) level with the CPCM model in methanol solution. ECD spectra of different conformers were simulated using SpecDis v1.51 with a half-bandwith of 0.16 eV, and the final ECD spectra were obtained according to the Boltzmann-calculated contribution of each conformer after UV correction. The calculated ECD spectra were compared with the experimental data [13]. 3. Results and discussion 3.1. Structural elucidation (±)-Cochinchinenin I (1) was obtained as a yellow amorphous powder. Its molecular formula was determined to be C30H28O7 on the basis of the 13C NMR and negative-ion HRESIMS data (m/z 499.1767 [M − H]−, calcd for C30H27O7, 499.1762), indicating 17 indices of hydrogen deficiency. The IR spectrum indicated the presence of hydroxy functionality (3369 cm−1), carbonyl group (1654 cm−1), and aromatic ring (1598, 1511 cm−1). The 1H NMR spectrum of 1 demonstrated the resonances of two sets of 1,4-disubstituted aromatic rings [δH 7.88 (2H, d, J = 9.0 Hz, H-2′, 6′), 6.80 (2H, d, J = 9.0 Hz, H-3′, 5′); 7.03 (2H, d, J = 8.5 Hz, H-2‴, 6‴), 6.63 (2H, d, J = 8.5 Hz, H-3‴, 5‴)]; one set of ABX coupling system [δH 6.75 (1H, d, J = 8.0 Hz, H-6″), 6.24 (1H, d, J = 2.0 Hz, H3″), 6.18 (1H, dd, J = 8.0, 2.0 Hz, H-5″)]; two aromatic singlets [δH 6.89 (1H, s, H-6), 6.28 (1H, s, H-3)]; eight characteristic aliphatic protons [δH 3.15 (2H, t, J = 7.0 Hz, H2-α), 2.86 (2H, t, J = 7.0 Hz, H2-β), 2.38 (2H, m, H2-β′), 2.07 (2H, m, H2-α′)]; and one methine proton at δH 4.14 (1H, t, J = 8.0 Hz, H-γ). The 13C NMR, together with the HSQC experiment, showed 30 carbon resonances comprising one carbonyl, 11 sp2 quaternary, 13 sp2 methine, one sp3 methine, and four sp3 methylene carbons. The aforementioned data revealed that compound 1 is a flavonoid dimer composing of dihydrochalcone and deoxotetrahydrochalcone moieties [14–16]. The 1H and 13C NMR spectroscopic data (Table 1) of 1 were comparable to those of cochinchinenin [14], except for the replacement of a methoxy group at C-2″ in cochinchinenin by a hydroxy group in 1. The planar structure of 1 was further confirmed by the HMBC correlations between H-3 and C-1/C-5; H-6 and C-2/C-4/C-β/Cγ; H-2′/H-6′ and C-4′/C=O; H-3′/H-5′ and C-1′; H-3″ and C-1″/C-5″; H-5″ and C-1″/C-3″; H-6″ and C-β′/C-2″/C-4″; H-2‴/H-6‴ and C-γ/C4‴; H-3‴/H-5‴ and C-1‴; H2-α and C-1; H2-β and C-2/C-6; H2-α′ and C-5/C-1″/C-1‴; H2-β′ and C-γ/C-2″/C-6″; and H-γ and C-4/C-6/C-2‴/C6‴/C-β′ (Fig. 2). Compound 1 exhibited a specific optical rotation approaching zero and showed no Cotton effect in its electronic circular dichroism (ECD) spectrum, suggesting that 1 was a racemic mixture. Subsequent chiral HPLC resolution of 1 led to the separation of a pair of enantiomers, 1a and 1b in a ratio of ca. 1:1 (Fig. 3). As expected, 1a and 1b exhibited mirror imaged Cotton effects in their ECD spectra (Fig. 4A) and totally opposite specific optical rotations. The absolute configurations of 1a/1b were established by the experimental and quantum chemical calculated ECD spectra. The experimental ECD spectra of 1a/1b matched well with calculated ECD spectra of (γS)-1 and (γR)-1 (Fig. 4A), respectively. Therefore, the structures of 1a and 1b were elucidated as shown in Fig. 1. (−)-Cochinchinenin J (2a) was obtained as a pale brown, amorphous powder. Its molecular formula was established as C33H34O7 with 17 indices of hydrogen deficiency by a deprotonated ion at m/z 541.2215 [M − H]− (calcd for C33H33O7, 541.2232) in the negativeion HRESIMS in conjunction with 13C NMR data. The IR spectrum showed the presence of hydroxy functionality (3431 cm−1) and aromatic ring (1614, 1513 cm−1). The 1H NMR spectrum of 2a showed resonances for a set of ABX-coupled aromatic protons [δH 6.80 (1H, d, J = 8.0 Hz, H-6″), 6.36 (1H, d, J = 2.0 Hz, H-3″), and 6.26 (1H, dd, J = 8.0, 2.0 Hz, H-5″)]; two sets of AA′BB′-coupled aromatic protons [δH 6.99 (2H, d, J = 8.5 Hz, H-2′, 6′), 6.71 (2H, d, J = 8.5 Hz, H-3′, 5′); 7.06
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Table 3 1 H (500 MHz) and 13C (125 MHz) NMR data of compounds 3–5 (δ in ppm, in CD3OD). Position
2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴, 6‴ 3‴, 5‴ 4‴ α β γ 3′-OCH3 4′-OCH3 2″-OCH3 4″-OCH3 a
3aa
3ba
4a
5a
δH, multi. (J in Hz)
δC, type
δH, multi. (J in Hz)
δC, type
δH, multi. (J in Hz)
δC, type
δH, multi. (J in Hz)
δC, type
4.86, dd (10.0, 2.0) 2.09, m 1.96, m 2.85, m 2.61, m 6.81, s
78.8, CH 31.6, CH2
4.84, dd (10.0, 2.0) 2.09, m 1.97, m 2.81, m 2.63, m 6.81, s
78.7, CH 31.5, CH2
4.88, dd (10.0, 2.0) 2.08, m 1.99, m 2.87, m 2.63, m 6.82, s
79.2, CH 31.6, CH2
4.84, overlapped 2.10, m 1.96, m 2.82, m 2.65, m 6.86, s
78.8, CH 31.6, CH2
25.6, CH2
2.11, m 2.43, m 4.18, t (8.0)
129.4, CH 126.4, C 154.9, C 103.8, CH 154.7, C 113.8, C 136.6, C 114.3, CH 147.5, C 148.6, C 112.6, CH 118.5, CH 123.3, C 159.7, C 99.8, CH 157.6, C 107.5, CH 131.1, CH 138.2, C 130.1, CH 115.7, CH 156.0, C 37.3, CH2 29.6, CH2 43.3, CH
3.84, s 3.75, s
56.5, CH3 55.7, CH3
6.24, s
6.86, d (2.0)
6.89, d (8.0) 6.83, dd (8.0, 2.0)
6.36, d (2.0) 6.26, dd (8.0, 2.0) 6.82, d (8.0) 7.08, d (8.5) 6.67, d (8.5)
25.6, CH2
2.11, m 2.43, m 4.18, t (8.0)
129.3, CH 126.4, C 154.8, C 103.8, CH 154.7, C 113.8, C 136.5, C 114.3, CH 147.5, C 148.6, C 112.6, CH 118.5, CH 123.3, C 159.7, C 99.8, CH 157.6, C 107.5, CH 131.1, CH 138.3, C 130.1, CH 115.7, CH 156.0, C 37.2, CH2 29.6, CH2 43.2, CH
3.84, s 3.74, s
56.5, CH3 55.7, CH3
6.25, s
6.87, d (2.0)
6.89, d (8.0) 6.83, dd (8.0, 2.0)
6.36, d (2.0) 6.26, dd (8.0, 2.0) 6.82, d (8.0) 7.08, d (8.5) 6.67, d (8.5)
25.8, CH2
2.11, m 2.44, m 4.18, t (8.0) 3.83, s
129.4, CH 126.4, C 154.9, C 103.8, CH 154.8, C 113.8, C 135.1, C 111.0, CH 148.9, C 147.2, C 116.0, CH 120.0, CH 123.3, C 159.7, C 99.8, CH 157.6, C 107.6, CH 131.1, CH 138.3, C 130.1, CH 115.7, CH 156.0, C 37.3, CH2 29.6, CH2 43.3, CH 56.4, CH3
3.76, s
55.7, CH3
6.24, s
6.97, d (2.0)
6.77, d (8.5) 6.83, dd (8.5, 2.0)
6.36, d (2.0) 6.26, dd (8.0, 2.0) 6.81, d (8.0) 7.08, d (8.5) 6.67, d (8.5)
25.6, CH2
2.15, m 2.46, m 4.21, t (8.0)
129.4, CH 126.3, C 154.9, C 103.9, CH 154.7, C 113.9, C 136.6, C 114.3, CH 147.5, C 148.6, C 112.6, CH 118.5, CH 123.0, C 157.0, C 102.4, CH 160.2, C 105.5, CH 131.3, CH 138.4, C 130.1, CH 115.7, CH 156.0, C 37.0, CH2 29.5, CH2 43.3, CH
3.85, s
56.5, CH3
3.71, s
55.6, CH3
6.25, s
6.87, d (2.0)
6.90, d (8.0) 6.84, dd (8.0, 2.0)
6.33, d (2.0) 6.30, dd (8.0, 2.0) 6.86, d (8.0) 7.10, d (8.5) 6.67, d (8.5)
Assignments were carried out based on HSQC and HMBC experiments.
(2H, d, J = 8.5 Hz, H-2‴, 6‴), and 6.65 (2H, d, J = 8.5 Hz, H-3‴, 5‴)]; one aromatic singlet [δH 6.55 (1H, s, H-5)]; twelve aliphatic protons [δH 4.16 (1H, t, J = 8.0 Hz, H-γ), 4.11 (1H, d, J = 10.5 Hz, H-2a), 3.72 (1H, m, H2b), 2.63 (1H, dd, J = 16.0, 5.0 Hz, H-4a), 2.53 (2H, m, H2-11), 2.42 (2H, m, H2-β), 2.37 (1H, dd, J = 16.0, 9.0 Hz, H-4b), 2.12 (1H, m, H-3), 2.05, 2.12 (each 1H, m, H2-α)], and two methoxy signals [δH 3.72 (3H, s, 2″OCH3), 3.75 (3H, s, 8-OCH3)]. The 13C NMR data of 2a showed 33 carbon resonances comprising 12 sp2 quaternary, 12 sp2 methine, two sp3 methine, five sp3 methylene, and two methoxy carbons. These spectroscopic data indicated 2a to be a flavonoid dimer consisting of homoisoflavan and deoxotetrahydrochalcone moieties [16,17]. The 1H and 13C NMR spectroscopic data (Table 2) of 2a were quite similar to those of homoisosocotrin-4′-ol (6) [17], except for the presence of an additional methoxy group in 2a. The significantly deshielded C-8 resonance (δC 136.3; ΔδC + 36.0) as well as shielded C-7 (δC 147.0; ΔδC − 7.6) and C-8a (δC 146.6; ΔδC − 9.1) resonances indicated that the methoxy group is located at C-8. The proposed structure of 2a was supported by the HMBC correlations between H-5 and C-γ/C-4/C-7/C-8a; H-9 and C-2/C-4/C-2′/C-6′; H-2′/6′ and C-4′/C-9; H-6″ and C-β/C-2″/C4″; H-2‴/6‴ and C-γ/C-4‴; H2-α and C-6/C-1″/C-1‴; H2-β and C-γ/C2″/C-6″; H-γ and C-β/C-5/C-7/C-2‴/C-6‴; 8-OCH3 and C-8; and 2″OCH3 and C-2″ (Fig. 2). NOE correlation between methoxy (OCH3) protons and H-3″ further confirmed the location of 2″-OCH3 (Fig. 2). The absolute configuration of 2a was determined by comparison of experimental and calculated ECD spectra (Fig. 4B). The experimental spectrum of 2a showed negative Cotton effect (CE) at 216, 240 nm and positive CE at 226, 290 nm, which were similar to the calculated spectrum of the (3R,γS) enantiomer [18,19]. Thus, the absolute configuration of 2a was established as (3R,γS). Compound 2b was obtained as a pale brown, amorphous powder. Its molecular formula, C33H34O7, was determined to be the same as
that of 2a on the basis of negative-ion HRESIMS (m/z 541.2208, [M − H]−) and 13C NMR data. Slight differences in 1H and 13C NMR spectroscopic data (Table 2) of 2b in comparison with those of 2a suggested that they could be stereoisomeric compounds. 2D NMR experiments including HSQC, HMBC, and NOESY (Fig. 2) facilitated the assembly of the structure of 2b, the same as that of 2a. However, compounds 2b and 2a showed opposite specific optical rotations ([α]25D: − 20.0 for 2a; [α]25D: + 31.0 for 2b) and mirror-imaged Cotton effects in their ECD spectra (Fig. 4B), which suggested they are enantiomers. The calculated spectrum of the (3S,γR) enantiomer was similar to the experimental spectrum of 2b (Fig. 4B). Therefore, the structure of 2b was defined as shown in Fig. 1, and named (+)cochinchinenin J. Compound 3a was isolated as a pale brown, amorphous powder, [α]25D: − 20.0 (c 0.12, MeOH). Its molecular formula was determined to be C32H32O7 by the negative-ion HRESIMS (m/z 527.2086 [M − H]− ) and the 13C NMR data, indicating 17 indices of hydrogen deficiency. The IR spectrum showed the presence of hydroxy group (3441 cm−1) and aromatic ring (1616, 1510 cm−1). The 1H and 13C NMR spectroscopic data (Table 3) of 3a were highly similar to those of 8-methylsocotrin4′-methoxy-3′-ol [20], which was isolated from the stem of Dracaena cambodiana, except for the absence of a methyl group at C-8 in 3a. These were supported by the significantly shielded C-8 (δC 103.8; ΔδC − 9.7) resonance. The planar structure of 3a was confirmed by the HMBC correlations between H-5 and C-γ/C-4/C-7/C-9; H-8 and C6/C-10; H-2′ and C-2/C-4′; H-6″ and C-2″/C-4″/C-β; H-2‴/6‴ and C-γ/ C-4‴; H2-β and C-2″/C-6″/C-γ; H-γ and C-5/C-7/C-2‴/C-6‴; 4′-OCH3 and C-4′; and 2″-OCH3 and C-2″ (Fig. 2). NOE correlations between methoxy protons at δH 3.84 and H-5′ (δH 6.89) as well as methoxy protons at δH 3.74 and H-3″ (δH 6.36) further confirmed the locations of 4′OCH3 and 2″-OCH3 (Fig. 2). In order to assign the absolute configuration
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Fig. 1. Structures of compounds 1–6 from total phenolic extract of Chinese dragon's blood.
of 3a, ECD spectra were calculated for four possible enantiomers, (2R,γR), (2S,γR), (2S,γS), and (2R,γS), based on TD-DFT calculations of ECD spectra. The calculated spectrum of (2R,γS) enantiomer was close
to the experimental ECD spectrum of 3a (Fig. 4C). Thus, the structure of 3a was characterized as shown in Fig. 1, and named (−)cochinchinenin K.
Fig. 2. Key HMBC and NOE correlations of compounds 1–3.
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Fig. 3. Chiral HPLC analysis of compound 1.
Fig. 4. Experimental and calculated ECD spectra of 1–6 (in MeOH).
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Compound 3b have the same molecular formula (C32H32O7) as that of 3a, as indicated by the negative-ion HRESIMS (m/z 527.2085 [M − H]−) and 13C NMR data. From the IR spectrum, hydroxy group (3438 cm−1) and aromatic ring (1617, 1510 cm−1) were evident. The resemblance of the 1H and 13C NMR data (Table 3) of 3b and 3a suggested the stereoisomeric nature of these compounds. The planar structure of 3b was the same as that of 3a on the basis of 2D–NMR experiments including HSQC, HMBC, and NOESY. The opposite specific optical rotations ([α]25D: −20.0 for 3a; [α]25D: +22.0 for 3b) and mirror-like Cotton effects in their ECD spectra (Fig. 4C) indicated that 3a and 3b are enantiomers. The calculated spectrum of (2S,γR) enantiomer was similar to the experimental spectrum of 3b (Fig. 4C). Therefore, the structure of 3b was defined as shown in Fig. 1, and named (+)-cochinchinenin K. The molecular formula of compound 4 was assigned as C32H32O7 on the basis of its 13C NMR (Table 3) and negative-ion HRESIMS (m/z 527.2078 [M − H]−) data, which was the same as that of cochinchinenin K (3a/3b). The 1H and 13C NMR data of 4 were highly similar to those of cochinchinenin K, except for the presence of 3′OCH3 and 4′-OH in 4 instead of 3′-OH and 4′-OCH3 in cochinchinenin K. This deduction was supported by the HMBC correlations between H-2′ and C-2/C-4′/C-6′; H-5′ and C-1′/C-3′; H-6′ and C-2/C-2′/C-4′; and 3′-OCH3 and C-3′; as well as NOE correlation between methoxy protons at δH 3.83 and H-2′ (δH 6.97). The ECD spectrum of 4 showed positive Cotton effects at 226, 276 nm, and negative Cotton effects at 212, 240 nm, respectively, which was in good agreement with calculated ECD spectrum of (2R,γR)-4 (Fig. 4D). Therefore, the structure of 4 was elucidated as shown in Fig. 1, and named cochinchinenin L. Cochinchinenin M (5) was obtained as a pale brown, amorphous powder, [α]25D: +27.0 (c 0.11, MeOH). It showed a deprotonated ion at m/z 527.2066 [M − H]− (calcd for C32H31O7, 527.2075) in the negativeion HRESIMS corresponding to the molecular formula C33H34O7, which was the same as that of cochinchinenin K (3a/3b). The 1H and 13C NMR data of 5 were very similar to those of cochinchinenin K, except for the presence of 2″-OH and 4″-OCH3 in 5 instead of 2″-OCH3 and 4″-OH in cochinchinenin K. This assignment was supported by the HMBC correlations between H-3″ and C-1″; H-5″ and C-1″/C-3″; H-6″ and C-2″/C-4″/Cβ; H2-β and C-2″/C-6″/C-γ; and 4″-OCH3 and C-4″; as well as NOE correlations between methoxy protons at δH 3.71 and H-3″/H-5″. The absolute configuration of 5 was deduced by comparing the specific optical rotation and ECD data with those of (+)-cochinchinenin K (3b). The similar specific optical rotations ([α]25D: +22.0 for 3b; [α]25D: +27.0 for 5) and Cotton effects in the ECD spectra of 5 and (+)-cochinchinenin K (3b) (Fig. 4E) suggested the (2S,γR) absolute configuration of 5. Thus, the structure of cochinchinenin M (5) was established as shown in Fig. 1. Compound 6 was identified as homoisosocotrin-4′-ol by comparing its spectroscopic data with literature values [17]. Its absolute configuration was defined as (3S,γR) by comparing its ECD spectrum with that of (+)-cochinchinenin J (2b) (Fig. 4F).
3.2. Bioactivity evaluation Compounds 1a, 1b, 2b, 3b, and 4–6 were evaluated for their inhibitory effects on the NO production in LPS-stimulated BV-2 microglial cells using Griess assay [9–12]. Indomethacin was used as positive control (IC50 = 44.1 ± 5.8 μM). Meanwhile, the effects of these compounds on cell proliferation/viability were measured using the MTT method. Compounds 4 and 5 showed significant inhibitory activity against NO production with IC50 values of 4.9 ± 0.4 and 5.4 ± 0.6 μM, respectively, and they (up to 40 μM) did not show any significant cytotoxicity in BV-2 microglial cells with LPS treatment for 24 h. Since compounds 2b, 3b, and 6 showed N 90% inhibition on cell proliferation/viability at the concentration of 20 μM, their NO inhibitory effects were not determined. Compounds 1a and 1b were inactive (b 50% inhibition at 40 μM, the highest concentration tested).
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Conflict of interest All the authors declare that there is no conflict of interest concerning this work.
Acknowledgment The project was financially supported by the National Natural Science Foundation of China (No.81573572), Program for New Century Excellent Talents in University (NCET-13-0693, to J.L.), and graduate students independent subject of Beijing University of Chinese Medicine (No. 2016-JYB-XS085). Appendix A. Supplementary data The 1H and 13C NMR, HSQC, HMBC, NOESY, UV, IR, and HRESIMS spectra for compounds 1–5. Supplementary data associated with this article can be found in the online version, at 10.1016/j.fitote.2016.10. 004.
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Flavonoid dimers from the total phenolic extract of Chinese dragon's blood, the red resin of Dracaena cochinchinensis Dao-Ran Pang a,b, Xiao-Qin Su a,b, Zhi-Xiang Zhu a, Jing Sun a,b, Yue-Ting Li a,b, Yue-Lin Song a, Yun-Fang Zhao a, Peng-Fei Tu a, Jiao Zheng a,⁎, Jun Li a,⁎ a b
Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, People's Republic of China School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, People's Republic of China
a r t i c l e
i n f o
Article history: Received 22 August 2016 Received in revised form 12 October 2016 Accepted 15 October 2016 Available online 18 October 2016 Keywords: Longxuetongluo capsule Dracaena cochinchinensis Chinese dragon's blood Flavonoid dimer Electronic circular dichroism
a b s t r a c t Eight new flavonoid dimers, named cochinchinenins I–M (1–5), including three pairs of enantiomers (1a/1b–3a/ 3b) and two optically pure flavonoid dimers (4–5), along with a known analogue (6), were isolated from total phenolic extract of the red resin of Dracaena cochinchinensis (Chinese dragon's blood). The planar structures of 1–5 were elucidated by extensive spectroscopic analysis including HRESIMS and 1D/2D NMR. Their absolute configurations were determined on the basis of experimental and calculated electronic circular dichroism (ECD) data. Compounds 4 and 5 exhibited significant inhibition of nitric oxide production in lipopolysaccharide-stimulated BV-2 microglial cells with IC50 value of 4.9 ± 0.4 and 5.4 ± 0.6 μM, respectively. © 2016 Published by Elsevier B.V.
1. Introduction Ischemic stroke accounts for 70–80% of the stroke incidence, which brings a big burden to the healthcare system and society. Although great advances have been made in understanding the pathophysiologic mechanisms of ischemic stroke, clinically effective therapies are limited [1]. Therefore, it's in urgent need to develop effective agents for prevention and treatment of this disease. There is increasing evidence that post-ischemic inflammation contributes to ischemic brain injury. Post-ischemic inflammation is, therefore, a promising target for therapeutic intervention in ischemic stroke [1]. Chinese dragon's blood, Longxuejie in Chinese, is the red resin of Dracaena cochinchinensis (Lour.) S. C. Chen growing in Yunnan and Guangxi provinces in China, which was firstly discovered by Cai and Xu in 1979 [2]. It was considered to be able to serve as a substitute for Sanguis Draconis (Xuejie in Chinese), a precious crude drug known as “panacea of blood activating” in traditional Chinese medicine, which has a reputation for facilitating blood circulation and dispersing blood stasis [3]. Pharmacological studies showed that the pure compounds and crude extracts of the red resin of D. cochinchinensis possess anti-inflammatory and analgesic, anti-platelet aggregation, antithrombotic, cerebral protective, radioprotective, wound healing, anti-diabetic, anti-microbial, antioxidant, and cytotoxic activities [3,4]. Previous phytochemical investigations on the resin and other parts of D. cochinchinensis have resulted in the isolation of flavonoid and its oligomers, stilbenes, and ⁎ Corresponding authors. E-mail addresses: [email protected] (J. Zheng), [email protected] (J. Li).
http://dx.doi.org/10.1016/j.fitote.2016.10.004 0367-326X/© 2016 Published by Elsevier B.V.
several steroids, and the phenolic compounds were considered to be responsible for its main biological activities [3–8]. In June 2013, Longxuetongluo Capsule (LTC), a new drug consisting of the total phenolic extract of Chinese dragon's blood, was approved for the treatment of ischemic stroke by China Food and Drug Administration. Recently, we demonstrated that LTC could ameliorate erythrocyte deformability and osmotic fragility through the reduction of lipid peroxidation on plasma and erythrocyte membranes in HFD-induced ApoE−/− mice [8]. However, the major phenolic constituents of this material have not yet been fully described because of the difficulties associated with the separation of these compounds, especially for flavonoid oligomers [6]. As an ongoing effort to search for bioactive components of the total phenolic extract of Chinese dragon's blood, an HPLC-MS-guided separation procedure was performed to target the flavonoid oligomers, which led to the isolation of eight new flavonoid dimers (1–5), including three pairs of enantiomers (1a/1b–3a/3b) and two optically pure flavonoid dimers (4–5) along with a known analogue (6). Herein, the isolation and structural elucidation of the new compounds are described as well as their inhibitory effects on NO production in LPS-stimulated BV-2 microglial cells.
2. Experimental 2.1. General experimental procedures Optical rotations were measured on a Rudolph Autopol IV automatic polarimeter (NJ, USA). ECD spectra were recorded on a JASCO J-810 CD
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spectrophotometer (JASCO, Japan). IR spectra were obtained using a Thermo Nicolet Nexus 470 FT-IR spectrophotometer (MA, USA) with KBr pellets. UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer (Tokyo, Japan). NMR spectra were obtained on a Varian INOVA-500 spectrometer (CA, USA) operating at 500 MHz for 1H NMR and 125 MHz for 13C NMR, respectively. The HRESIMS data were recorded on an LCMS-IT-TOF system equipped with a Prominence UFLC system and an ESI interface (Shimadzu, Kyoto, Japan). Silica gel (200– 300 mesh, Qingdao Marine Chemical Inc., Qingdao, People's Republic of China), LiChroprep RP-C18 gel (40–63 μm, Merck, Germany), and Sephadex LH-20 (Pharmacia, LA, USA) were employed for open column chromatography (CC). HPLC was performed on a Shimadzu LC-20AT pump system (Shimadzu Corporation, Tokyo, Japan) equipped with an SPD-M20A photodiode array detector monitoring at 230 and 280 nm. A semi-preparative HPLC SPOLAR C18 column (250 × 10 mm, 5 μm) was employed for the isolation. Chiral HPLC was done with a Daicel CHIRALPAC IC column (150 × 4.6 mm, 5 μm). TLCs were performed using pre-coated GF254 plates. All purified compounds submitted for bioassay were at least 95% pure as judged by HPLC and supported by 1 H NMR analysis. 2.2. Plant material The total phenolic extract of Chinese dragon's blood (LTC140601) was provided by Jiangsu Kanion Pharmaceutical Co. Ltd. A voucher sample is deposited at the Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China. 2.3. Extraction and isolation The air-dried and powdered red resin of D. cochinchinensis (4.5 kg) was extracted with 95% EtOH at room temperature (45 L × 3, 72 h each). The ethanol extracts were combined and evaporated to dryness under reduced pressure. The dry residue (920 g) was defatted with petroleum ether (5 L × 1), and then extracted with EtOAc (5 L × 2) to produce the total phenolic extract of Chinese dragon's blood. The total phenolic extract (600 g) was subjected to silica gel CC and eluted with a stepwise gradient of petroleum ether–EtOAc (10:1 → 1:3) and then CH2Cl2–MeOH (20:1 → 0:1), to afford ten fractions (Fr. A–J). Afterwards, an HPLC-MS-guided separation and purification procedure [0.1% formic acid aqueous solution (v/v, solvent A) and acetonitrile (solvent B): 0– 30 min, 5–95% B; 30–40 min, 95% B, (v/v)] was performed to screen out the targeted fractions containing flavonoid dimers. Fr. G (61.5 g) containing flavonoid dimers was chromatographed on silica gel CC eluting with petroleum ether–EtOAc (3:1 → 0:1) to yield five subfractions (Fr. G1–G5). Fr. G3 (2.2 g) containing the targeted compounds was chromatographed over Sephadex LH-20 CC eluting with MeOH to yield four portions (Fr. G3a–G3d). Fr. G3b (1.3 g) was resolved by RPC18 CC eluting with a stepwise gradient of MeOH–H2O (30:70 → 100:0) to afford sub‐Fr. G3b1–G3b3. Sub-Fr. G3b1 was purified by semi-preparative HPLC using MeOH–H2O (60:40, 2.0 mL/min) to yield 2b (15.4 mg, tR 55.8 min) and 3a (4.1 mg, tR 78.1 min). Sub-Fr. G3b2 was separated on semi-preparative RP-HPLC (MeCN–H2O, 45:55, 3.0 mL/min) to give 6 (9.3 mg, tR 33.4 min), 4 (4.3 mg, tR 34.8 min), and 3b (7.1 mg, tR 36.0 min). Sub-Fr. G3b3 was purified by semi-preparative RP-HPLC (MeCN–H2O, 55:45 → 80:20, 0–30 min, 3.0 mL/min) to give 2a (4.2 mg, tR 13.4 min) and 5 (6.3 mg, tR 14.1 min). Compound 1 (15.0 mg, tR 21.0 min) was obtained from Fr. G3c (500 mg) by semi-preparative RP-HPLC (MeCN–H2O, 35:65, 3.0 mL/min). Furthermore, direct enantiomeric separation of 1 was achieved by chiral HPLC with n-hexane-ethanol (95:5 → 80:20, 0–30 min, 1.0 mL/min), affording 1b (3.1 mg, tR 23.0 min) and 1a (2.6 mg, tR 26.0 min), respectively. 2.3.1. (±)-Cochinchinenin I (1) Yellow amorphous powder; UV λMeOHmax(log ε): 226 (2.94), 281 (2.88) nm; IR (KBr) νmax: 3369, 2954, 2918, 2850, 1654, 1598, 1511,
1455, 1368, 1230, 1169, 1092 cm−1; 1H and 13C NMR data, see Table 1; negative-ion HRESIMS: m/z 499.1767 [M − H]− (calcd for C30H27O7, 499.1762). (−)-Cochinchinenin I (1a): [α]25D: − 6.0 (c 0.10, MeOH); ECD (c 1.0 × 10−3 M, MeOH), λmax (Δε) 208 (−2.33), 222 (− 2.98), 246 (+ 2.72), 292 (+ 0.64) nm. (+)-Cochinchinenin I (1b): [α]25D: + 6.0 (c 0.10, MeOH); ECD (c 1.0 × 10−3 M, MeOH) λmax (Δε) 204 (+ 4.67), 222 (+ 3.41), 240 (− 2.22), 294 (−0.31) nm. 2.3.2. (−)-Cochinchinenin J (2a) Pale brown, amorphous powder; [α]25D: −20.0 (c 0.09, MeOH); UV MeOH λ max(log ε): 215 (3.41), 222 (3.40), 279 (2.80) nm; ECD (c 8.3 × 10−4 M, MeOH), λmax (Δε) 216 (−6.43), 226 (+2.08), 240 (−12.31), 290 (+ 2.28) nm; IR (KBr) νmax: 3431, 2927, 2852, 1614, 1513, 1489, 1459, 1436, 1384, 1229, 1198, 1158, 1115, 1038 cm−1; 1H and 13C NMR data, see Table 2; negative-ion HRESIMS: m/z 541.2215 [M − H]− (calcd for C33H33O7, 541.2232). 2.3.3. (+)-Cochinchinenin J (2b) Pale brown, amorphous powder; [α]25D: +31.0 (c 0.07, MeOH); UV λMeOHmax(log ε): 212 (2.83), 230 (3.48), 280 (2.88) nm; ECD (c 6.5 × 10−4 M, MeOH) λmax (Δε) 216 (+5.94), 226 (− 7.65), 244 (+20.47), 276 (+5.68), 292 (−4.14) nm; IR (KBr) νmax: 3441, 2920, 2851, 1615, 1512, 1489, 1458, 1436, 1198, 1158, 1115, 1038 cm−1; 1H and 13C NMR data, see Table 2; negative-ion HRESIMS: m/z 541.2208 [M − H]− (calcd for C33H33O7, 541.2232). 2.3.4. (−)-Cochinchinenin K (3a) Pale brown, amorphous powder; [α]25D: −20.0 (c 0.12, MeOH); UV MeOH −3 M, λ max(log ε): 217 (3.30), 282 (2.89) nm; ECD (c 1.1 × 10 MeOH) λmax (Δε) 216 (− 5.4), 232 (− 3.83), 244 (+ 1.24), 294 (+0.57) nm; IR (KBr) νmax: 3441, 2924, 2852, 1616, 1510, 1456, 1435, 1399, 1385, 1274, 1235, 1175, 1157, 1116, 1071, 1035 cm−1; 1H and 13 C NMR data, see Table 3; negative-ion HRESIMS: m/z 527.2086 [M − H]− (calcd for C32H31O7, 527.2075). 2.3.5. (+)-Cochinchinenin K (3b) Pale brown, amorphous powder; [α]25D: +22.0 (c 0.09, MeOH); UV λMeOHmax(log ε): 211 (3.50), 230 (3.38), 281 (2.94) nm; ECD (c 8.5 × 10−4 M, MeOH) λmax (Δε) 214 (+ 5.83), 226 (+ 8.96), 240 (− 4.01), 276 (+1.00), 294 (−1.85) nm; IR (KBr) νmax: 3438, 2927, 2852, 1617, 1510, 1438, 1384, 1273, 1234, 1176, 1157, 1117, 1071, 1035 cm−1; 1H and 13C NMR data, see Table 3; negative-ion HRESIMS: m/z 527.2085 [M − H]− (calcd for C32H31O7, 527.2075). 2.3.6. Cochinchinenin L (4) Pale brown, amorphous powder; [α]25D: −36.0 (c 0.10, MeOH); UV MeOH λ max(log ε): 210 (3.51), 230 (3.41), 281 (2.76) nm; ECD (c 9.5 × Table 1 1 H (500 MHz) and 13C (125 MHz) NMR data of compound 1 (δ in ppm, in CD3OD). Position 1
Position 1
δH, multi. (J in Hz) δC, type 1 2 3 4 5 6 α β C =O γ α' β' 1′
6.28, s
6.89, s 3.15, t (7.0) 2.86, t (7.0) 4.14, t (8.0) 2.07, m 2.38, m
119.5, C 154.5, C 103.7, CH 154.9, C 124.5, C 130.2, CH 40.1, CH2 27.1, CH2 202.4, C 43.2. CH 37.3, CH2 29.5, CH2 130.1, C
δH, multi. (J in Hz) δC, type 2′, 6′ 3′, 5′ 4′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴, 6‴ 3‴, 5‴ 4‴
7.88, d (9.0) 6.80, d (9.0)
6.24, d (2.0) 6.18,dd (8.0, 2.0) 6.75, d (8.0) 7.03, d (8.5) 6.63, d (8.5)
132.1, CH 116.2, CH 163.9, C 121.8, C 156.9, C 103.4, CH 157.1, C 107.2, CH 131.4,CH 138.6,C 130.0, CH 115.7, CH 155.9, C
D.-R. Pang et al. / Fitoterapia 115 (2016) 135–141 Table 2 1 H (500 MHz) and 13C (125 MHz) NMR data of compound 2 (δ in ppm, in CD3OD). Position
2aa δH, multi. (J in Hz)
δC, type
δH, multi. (J in Hz)
δC, type
2
4.11, d (10.5) 3.72, m 2.12, m 2.63, dd (16.0, 5.0) 2.37, dd (16.0, 9.0) 6.55, s
70.9, CH2
4.12, d (10.5) 3.71, m 2.12, m 2.62, dd (16.0, 5.0) 2.32, dd (16.0, 9.0) 6.53, s
70.9, CH2
3 4 5 6 7 8 9 10 11 α β γ 1′ 2′, 6′ 3′, 5′ 4′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴, 6‴ 3‴, 5‴ 1‴ 2″-OCH3 8-OCH3 a
2.53, m 2.12, m; 2.05, m 2.42, m 4.16, t (8.0) 6.99, d (8.5) 6.71, d (8.5)
6.36, d (2.0) 6.26, dd (8.0, 2.0) 6.80, d (8.0) 7.06, d (8.5) 6.65, d (8.5) 3.72, s 3.75, s
2ba
35.8, CH 31.6, CH2 123.8, CH 126.1, C 147.0, C 136.3, C 146.6, C 114.1, C 38.0, CH2 37.1, CH2 29.5, CH2 43.6, CH 131.8, C 131.0, CH 116.2, CH 156.7, C 123.2, C 159.7, C 99.8, CH 157.6, C 107.5, CH 131.1, CH 138.1, C 130.0, CH 115.7, CH 156.0, C 55.6, CH3 61.0, CH3
2.50, m 2.16, m; 2.03, m 2.40, m 4.16, t (8.0) 6.98, d (8.5) 6.71, d (8.5)
6.34, d (2.0) 6.25, dd (8.0, 2.0) 6.79, d (8.0) 7.07, d (8.5) 6.67, d (8.5) 3.67, s 3.75, s
35.9, CH 31.7, CH2 123.8, CH 126.0, C 147.0, C 136.3, C 146.6, C 114.2, C 38.0, CH2 37.1, CH2 29.5, CH2 43.6, CH 131.8, C 131.0, CH 116.1, CH 156.7, C 123.1, C 159.6, C 99.7, CH 157.5, C 107.5, CH 131.1, CH 138.1, C 130.0, CH 115.7, CH 156.0, C 55.6, CH3 61.0, CH3
Assignments were carried out based on HSQC and HMBC experiments.
10−4 M, MeOH) λmax (Δε) 212 (− 6.26), 226 (+12.81), 240 (− 4.52), 276 (+ 0.84), 290 (0.42) nm; IR (KBr) νmax: 3441, 2925, 2853, 1616, 1511, 1458, 1436, 1384, 1272, 1234, 1198, 1158, 1117, 1070, 1035 cm−1; 1H and 13C NMR data, see Table 3; negative-ion HRESIMS: m/z 527.2074 [M − H]− (calcd for C32H31O7, 527.2075). 2.3.7. Cochinchinenin M (5) Pale brown, amorphous powder; [α]25D: +27.0 (c 0.11, MeOH); UV λMeOHmax(log ε): 212 (3.43), 230 (3.35), 281 (2.88) nm; ECD (c 1.0 × 10−3 M, MeOH) λmax (Δε) 212 (+ 4.51), 228 (+ 1.68), 242 (− 1.01), 276 (+0.43), 296 (−0.47) nm; IR (KBr) νmax: 3432, 2924, 2852, 1618, 1512, 1444, 1399, 1385, 1273, 1223, 1161, 1112, 1072, 1035 cm−1; 1H and 13C NMR data, see Table 3; negative-ion HRESIMS: m/z 527.2066 [M − H]− (calcd for C32H31O7, 527.2075). 2.4. Biological assays BV-2 microglial cells were purchased from Peking Union Medical College (PUMC) Cell Bank (Beijing, People's Republic of China). Cell culture, viability assay, and assay for inhibition ability against LPS-stimulated NO production were performed according to our previous reports [9–12], with indomethacin (IC50 = 44.1 ± 5.8 μM) as the positive control. The IC50 values were determined using GraphPad Prism 5 software from experiments performed in triplicate (GraphPad Software, Inc., San Diego, CA, USA). All the compounds were prepared as stock solutions in DMSO (final solvent concentration b 0.5% in all assays). 2.5. Computational details Preliminary conformational analysis of compounds 1–4 was performed with the SYBYL-X 2.0 software package using the random search method with the MMFF94s force field. The obtained conformers were
137
used for geometry reoptimizations at the B3LYP/6-31G(d) level in the Gaussian 09 package. The ECD spectra for the optimized conformers were calculated by the TDDFT method at the B3LYP/6–31 + G(d) level with the CPCM model in methanol solution. ECD spectra of different conformers were simulated using SpecDis v1.51 with a half-bandwith of 0.16 eV, and the final ECD spectra were obtained according to the Boltzmann-calculated contribution of each conformer after UV correction. The calculated ECD spectra were compared with the experimental data [13]. 3. Results and discussion 3.1. Structural elucidation (±)-Cochinchinenin I (1) was obtained as a yellow amorphous powder. Its molecular formula was determined to be C30H28O7 on the basis of the 13C NMR and negative-ion HRESIMS data (m/z 499.1767 [M − H]−, calcd for C30H27O7, 499.1762), indicating 17 indices of hydrogen deficiency. The IR spectrum indicated the presence of hydroxy functionality (3369 cm−1), carbonyl group (1654 cm−1), and aromatic ring (1598, 1511 cm−1). The 1H NMR spectrum of 1 demonstrated the resonances of two sets of 1,4-disubstituted aromatic rings [δH 7.88 (2H, d, J = 9.0 Hz, H-2′, 6′), 6.80 (2H, d, J = 9.0 Hz, H-3′, 5′); 7.03 (2H, d, J = 8.5 Hz, H-2‴, 6‴), 6.63 (2H, d, J = 8.5 Hz, H-3‴, 5‴)]; one set of ABX coupling system [δH 6.75 (1H, d, J = 8.0 Hz, H-6″), 6.24 (1H, d, J = 2.0 Hz, H3″), 6.18 (1H, dd, J = 8.0, 2.0 Hz, H-5″)]; two aromatic singlets [δH 6.89 (1H, s, H-6), 6.28 (1H, s, H-3)]; eight characteristic aliphatic protons [δH 3.15 (2H, t, J = 7.0 Hz, H2-α), 2.86 (2H, t, J = 7.0 Hz, H2-β), 2.38 (2H, m, H2-β′), 2.07 (2H, m, H2-α′)]; and one methine proton at δH 4.14 (1H, t, J = 8.0 Hz, H-γ). The 13C NMR, together with the HSQC experiment, showed 30 carbon resonances comprising one carbonyl, 11 sp2 quaternary, 13 sp2 methine, one sp3 methine, and four sp3 methylene carbons. The aforementioned data revealed that compound 1 is a flavonoid dimer composing of dihydrochalcone and deoxotetrahydrochalcone moieties [14–16]. The 1H and 13C NMR spectroscopic data (Table 1) of 1 were comparable to those of cochinchinenin [14], except for the replacement of a methoxy group at C-2″ in cochinchinenin by a hydroxy group in 1. The planar structure of 1 was further confirmed by the HMBC correlations between H-3 and C-1/C-5; H-6 and C-2/C-4/C-β/Cγ; H-2′/H-6′ and C-4′/C=O; H-3′/H-5′ and C-1′; H-3″ and C-1″/C-5″; H-5″ and C-1″/C-3″; H-6″ and C-β′/C-2″/C-4″; H-2‴/H-6‴ and C-γ/C4‴; H-3‴/H-5‴ and C-1‴; H2-α and C-1; H2-β and C-2/C-6; H2-α′ and C-5/C-1″/C-1‴; H2-β′ and C-γ/C-2″/C-6″; and H-γ and C-4/C-6/C-2‴/C6‴/C-β′ (Fig. 2). Compound 1 exhibited a specific optical rotation approaching zero and showed no Cotton effect in its electronic circular dichroism (ECD) spectrum, suggesting that 1 was a racemic mixture. Subsequent chiral HPLC resolution of 1 led to the separation of a pair of enantiomers, 1a and 1b in a ratio of ca. 1:1 (Fig. 3). As expected, 1a and 1b exhibited mirror imaged Cotton effects in their ECD spectra (Fig. 4A) and totally opposite specific optical rotations. The absolute configurations of 1a/1b were established by the experimental and quantum chemical calculated ECD spectra. The experimental ECD spectra of 1a/1b matched well with calculated ECD spectra of (γS)-1 and (γR)-1 (Fig. 4A), respectively. Therefore, the structures of 1a and 1b were elucidated as shown in Fig. 1. (−)-Cochinchinenin J (2a) was obtained as a pale brown, amorphous powder. Its molecular formula was established as C33H34O7 with 17 indices of hydrogen deficiency by a deprotonated ion at m/z 541.2215 [M − H]− (calcd for C33H33O7, 541.2232) in the negativeion HRESIMS in conjunction with 13C NMR data. The IR spectrum showed the presence of hydroxy functionality (3431 cm−1) and aromatic ring (1614, 1513 cm−1). The 1H NMR spectrum of 2a showed resonances for a set of ABX-coupled aromatic protons [δH 6.80 (1H, d, J = 8.0 Hz, H-6″), 6.36 (1H, d, J = 2.0 Hz, H-3″), and 6.26 (1H, dd, J = 8.0, 2.0 Hz, H-5″)]; two sets of AA′BB′-coupled aromatic protons [δH 6.99 (2H, d, J = 8.5 Hz, H-2′, 6′), 6.71 (2H, d, J = 8.5 Hz, H-3′, 5′); 7.06
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D.-R. Pang et al. / Fitoterapia 115 (2016) 135–141
Table 3 1 H (500 MHz) and 13C (125 MHz) NMR data of compounds 3–5 (δ in ppm, in CD3OD). Position
2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴, 6‴ 3‴, 5‴ 4‴ α β γ 3′-OCH3 4′-OCH3 2″-OCH3 4″-OCH3 a
3aa
3ba
4a
5a
δH, multi. (J in Hz)
δC, type
δH, multi. (J in Hz)
δC, type
δH, multi. (J in Hz)
δC, type
δH, multi. (J in Hz)
δC, type
4.86, dd (10.0, 2.0) 2.09, m 1.96, m 2.85, m 2.61, m 6.81, s
78.8, CH 31.6, CH2
4.84, dd (10.0, 2.0) 2.09, m 1.97, m 2.81, m 2.63, m 6.81, s
78.7, CH 31.5, CH2
4.88, dd (10.0, 2.0) 2.08, m 1.99, m 2.87, m 2.63, m 6.82, s
79.2, CH 31.6, CH2
4.84, overlapped 2.10, m 1.96, m 2.82, m 2.65, m 6.86, s
78.8, CH 31.6, CH2
25.6, CH2
2.11, m 2.43, m 4.18, t (8.0)
129.4, CH 126.4, C 154.9, C 103.8, CH 154.7, C 113.8, C 136.6, C 114.3, CH 147.5, C 148.6, C 112.6, CH 118.5, CH 123.3, C 159.7, C 99.8, CH 157.6, C 107.5, CH 131.1, CH 138.2, C 130.1, CH 115.7, CH 156.0, C 37.3, CH2 29.6, CH2 43.3, CH
3.84, s 3.75, s
56.5, CH3 55.7, CH3
6.24, s
6.86, d (2.0)
6.89, d (8.0) 6.83, dd (8.0, 2.0)
6.36, d (2.0) 6.26, dd (8.0, 2.0) 6.82, d (8.0) 7.08, d (8.5) 6.67, d (8.5)
25.6, CH2
2.11, m 2.43, m 4.18, t (8.0)
129.3, CH 126.4, C 154.8, C 103.8, CH 154.7, C 113.8, C 136.5, C 114.3, CH 147.5, C 148.6, C 112.6, CH 118.5, CH 123.3, C 159.7, C 99.8, CH 157.6, C 107.5, CH 131.1, CH 138.3, C 130.1, CH 115.7, CH 156.0, C 37.2, CH2 29.6, CH2 43.2, CH
3.84, s 3.74, s
56.5, CH3 55.7, CH3
6.25, s
6.87, d (2.0)
6.89, d (8.0) 6.83, dd (8.0, 2.0)
6.36, d (2.0) 6.26, dd (8.0, 2.0) 6.82, d (8.0) 7.08, d (8.5) 6.67, d (8.5)
25.8, CH2
2.11, m 2.44, m 4.18, t (8.0) 3.83, s
129.4, CH 126.4, C 154.9, C 103.8, CH 154.8, C 113.8, C 135.1, C 111.0, CH 148.9, C 147.2, C 116.0, CH 120.0, CH 123.3, C 159.7, C 99.8, CH 157.6, C 107.6, CH 131.1, CH 138.3, C 130.1, CH 115.7, CH 156.0, C 37.3, CH2 29.6, CH2 43.3, CH 56.4, CH3
3.76, s
55.7, CH3
6.24, s
6.97, d (2.0)
6.77, d (8.5) 6.83, dd (8.5, 2.0)
6.36, d (2.0) 6.26, dd (8.0, 2.0) 6.81, d (8.0) 7.08, d (8.5) 6.67, d (8.5)
25.6, CH2
2.15, m 2.46, m 4.21, t (8.0)
129.4, CH 126.3, C 154.9, C 103.9, CH 154.7, C 113.9, C 136.6, C 114.3, CH 147.5, C 148.6, C 112.6, CH 118.5, CH 123.0, C 157.0, C 102.4, CH 160.2, C 105.5, CH 131.3, CH 138.4, C 130.1, CH 115.7, CH 156.0, C 37.0, CH2 29.5, CH2 43.3, CH
3.85, s
56.5, CH3
3.71, s
55.6, CH3
6.25, s
6.87, d (2.0)
6.90, d (8.0) 6.84, dd (8.0, 2.0)
6.33, d (2.0) 6.30, dd (8.0, 2.0) 6.86, d (8.0) 7.10, d (8.5) 6.67, d (8.5)
Assignments were carried out based on HSQC and HMBC experiments.
(2H, d, J = 8.5 Hz, H-2‴, 6‴), and 6.65 (2H, d, J = 8.5 Hz, H-3‴, 5‴)]; one aromatic singlet [δH 6.55 (1H, s, H-5)]; twelve aliphatic protons [δH 4.16 (1H, t, J = 8.0 Hz, H-γ), 4.11 (1H, d, J = 10.5 Hz, H-2a), 3.72 (1H, m, H2b), 2.63 (1H, dd, J = 16.0, 5.0 Hz, H-4a), 2.53 (2H, m, H2-11), 2.42 (2H, m, H2-β), 2.37 (1H, dd, J = 16.0, 9.0 Hz, H-4b), 2.12 (1H, m, H-3), 2.05, 2.12 (each 1H, m, H2-α)], and two methoxy signals [δH 3.72 (3H, s, 2″OCH3), 3.75 (3H, s, 8-OCH3)]. The 13C NMR data of 2a showed 33 carbon resonances comprising 12 sp2 quaternary, 12 sp2 methine, two sp3 methine, five sp3 methylene, and two methoxy carbons. These spectroscopic data indicated 2a to be a flavonoid dimer consisting of homoisoflavan and deoxotetrahydrochalcone moieties [16,17]. The 1H and 13C NMR spectroscopic data (Table 2) of 2a were quite similar to those of homoisosocotrin-4′-ol (6) [17], except for the presence of an additional methoxy group in 2a. The significantly deshielded C-8 resonance (δC 136.3; ΔδC + 36.0) as well as shielded C-7 (δC 147.0; ΔδC − 7.6) and C-8a (δC 146.6; ΔδC − 9.1) resonances indicated that the methoxy group is located at C-8. The proposed structure of 2a was supported by the HMBC correlations between H-5 and C-γ/C-4/C-7/C-8a; H-9 and C-2/C-4/C-2′/C-6′; H-2′/6′ and C-4′/C-9; H-6″ and C-β/C-2″/C4″; H-2‴/6‴ and C-γ/C-4‴; H2-α and C-6/C-1″/C-1‴; H2-β and C-γ/C2″/C-6″; H-γ and C-β/C-5/C-7/C-2‴/C-6‴; 8-OCH3 and C-8; and 2″OCH3 and C-2″ (Fig. 2). NOE correlation between methoxy (OCH3) protons and H-3″ further confirmed the location of 2″-OCH3 (Fig. 2). The absolute configuration of 2a was determined by comparison of experimental and calculated ECD spectra (Fig. 4B). The experimental spectrum of 2a showed negative Cotton effect (CE) at 216, 240 nm and positive CE at 226, 290 nm, which were similar to the calculated spectrum of the (3R,γS) enantiomer [18,19]. Thus, the absolute configuration of 2a was established as (3R,γS). Compound 2b was obtained as a pale brown, amorphous powder. Its molecular formula, C33H34O7, was determined to be the same as
that of 2a on the basis of negative-ion HRESIMS (m/z 541.2208, [M − H]−) and 13C NMR data. Slight differences in 1H and 13C NMR spectroscopic data (Table 2) of 2b in comparison with those of 2a suggested that they could be stereoisomeric compounds. 2D NMR experiments including HSQC, HMBC, and NOESY (Fig. 2) facilitated the assembly of the structure of 2b, the same as that of 2a. However, compounds 2b and 2a showed opposite specific optical rotations ([α]25D: − 20.0 for 2a; [α]25D: + 31.0 for 2b) and mirror-imaged Cotton effects in their ECD spectra (Fig. 4B), which suggested they are enantiomers. The calculated spectrum of the (3S,γR) enantiomer was similar to the experimental spectrum of 2b (Fig. 4B). Therefore, the structure of 2b was defined as shown in Fig. 1, and named (+)cochinchinenin J. Compound 3a was isolated as a pale brown, amorphous powder, [α]25D: − 20.0 (c 0.12, MeOH). Its molecular formula was determined to be C32H32O7 by the negative-ion HRESIMS (m/z 527.2086 [M − H]− ) and the 13C NMR data, indicating 17 indices of hydrogen deficiency. The IR spectrum showed the presence of hydroxy group (3441 cm−1) and aromatic ring (1616, 1510 cm−1). The 1H and 13C NMR spectroscopic data (Table 3) of 3a were highly similar to those of 8-methylsocotrin4′-methoxy-3′-ol [20], which was isolated from the stem of Dracaena cambodiana, except for the absence of a methyl group at C-8 in 3a. These were supported by the significantly shielded C-8 (δC 103.8; ΔδC − 9.7) resonance. The planar structure of 3a was confirmed by the HMBC correlations between H-5 and C-γ/C-4/C-7/C-9; H-8 and C6/C-10; H-2′ and C-2/C-4′; H-6″ and C-2″/C-4″/C-β; H-2‴/6‴ and C-γ/ C-4‴; H2-β and C-2″/C-6″/C-γ; H-γ and C-5/C-7/C-2‴/C-6‴; 4′-OCH3 and C-4′; and 2″-OCH3 and C-2″ (Fig. 2). NOE correlations between methoxy protons at δH 3.84 and H-5′ (δH 6.89) as well as methoxy protons at δH 3.74 and H-3″ (δH 6.36) further confirmed the locations of 4′OCH3 and 2″-OCH3 (Fig. 2). In order to assign the absolute configuration
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Fig. 1. Structures of compounds 1–6 from total phenolic extract of Chinese dragon's blood.
of 3a, ECD spectra were calculated for four possible enantiomers, (2R,γR), (2S,γR), (2S,γS), and (2R,γS), based on TD-DFT calculations of ECD spectra. The calculated spectrum of (2R,γS) enantiomer was close
to the experimental ECD spectrum of 3a (Fig. 4C). Thus, the structure of 3a was characterized as shown in Fig. 1, and named (−)cochinchinenin K.
Fig. 2. Key HMBC and NOE correlations of compounds 1–3.
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Fig. 3. Chiral HPLC analysis of compound 1.
Fig. 4. Experimental and calculated ECD spectra of 1–6 (in MeOH).
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Compound 3b have the same molecular formula (C32H32O7) as that of 3a, as indicated by the negative-ion HRESIMS (m/z 527.2085 [M − H]−) and 13C NMR data. From the IR spectrum, hydroxy group (3438 cm−1) and aromatic ring (1617, 1510 cm−1) were evident. The resemblance of the 1H and 13C NMR data (Table 3) of 3b and 3a suggested the stereoisomeric nature of these compounds. The planar structure of 3b was the same as that of 3a on the basis of 2D–NMR experiments including HSQC, HMBC, and NOESY. The opposite specific optical rotations ([α]25D: −20.0 for 3a; [α]25D: +22.0 for 3b) and mirror-like Cotton effects in their ECD spectra (Fig. 4C) indicated that 3a and 3b are enantiomers. The calculated spectrum of (2S,γR) enantiomer was similar to the experimental spectrum of 3b (Fig. 4C). Therefore, the structure of 3b was defined as shown in Fig. 1, and named (+)-cochinchinenin K. The molecular formula of compound 4 was assigned as C32H32O7 on the basis of its 13C NMR (Table 3) and negative-ion HRESIMS (m/z 527.2078 [M − H]−) data, which was the same as that of cochinchinenin K (3a/3b). The 1H and 13C NMR data of 4 were highly similar to those of cochinchinenin K, except for the presence of 3′OCH3 and 4′-OH in 4 instead of 3′-OH and 4′-OCH3 in cochinchinenin K. This deduction was supported by the HMBC correlations between H-2′ and C-2/C-4′/C-6′; H-5′ and C-1′/C-3′; H-6′ and C-2/C-2′/C-4′; and 3′-OCH3 and C-3′; as well as NOE correlation between methoxy protons at δH 3.83 and H-2′ (δH 6.97). The ECD spectrum of 4 showed positive Cotton effects at 226, 276 nm, and negative Cotton effects at 212, 240 nm, respectively, which was in good agreement with calculated ECD spectrum of (2R,γR)-4 (Fig. 4D). Therefore, the structure of 4 was elucidated as shown in Fig. 1, and named cochinchinenin L. Cochinchinenin M (5) was obtained as a pale brown, amorphous powder, [α]25D: +27.0 (c 0.11, MeOH). It showed a deprotonated ion at m/z 527.2066 [M − H]− (calcd for C32H31O7, 527.2075) in the negativeion HRESIMS corresponding to the molecular formula C33H34O7, which was the same as that of cochinchinenin K (3a/3b). The 1H and 13C NMR data of 5 were very similar to those of cochinchinenin K, except for the presence of 2″-OH and 4″-OCH3 in 5 instead of 2″-OCH3 and 4″-OH in cochinchinenin K. This assignment was supported by the HMBC correlations between H-3″ and C-1″; H-5″ and C-1″/C-3″; H-6″ and C-2″/C-4″/Cβ; H2-β and C-2″/C-6″/C-γ; and 4″-OCH3 and C-4″; as well as NOE correlations between methoxy protons at δH 3.71 and H-3″/H-5″. The absolute configuration of 5 was deduced by comparing the specific optical rotation and ECD data with those of (+)-cochinchinenin K (3b). The similar specific optical rotations ([α]25D: +22.0 for 3b; [α]25D: +27.0 for 5) and Cotton effects in the ECD spectra of 5 and (+)-cochinchinenin K (3b) (Fig. 4E) suggested the (2S,γR) absolute configuration of 5. Thus, the structure of cochinchinenin M (5) was established as shown in Fig. 1. Compound 6 was identified as homoisosocotrin-4′-ol by comparing its spectroscopic data with literature values [17]. Its absolute configuration was defined as (3S,γR) by comparing its ECD spectrum with that of (+)-cochinchinenin J (2b) (Fig. 4F).
3.2. Bioactivity evaluation Compounds 1a, 1b, 2b, 3b, and 4–6 were evaluated for their inhibitory effects on the NO production in LPS-stimulated BV-2 microglial cells using Griess assay [9–12]. Indomethacin was used as positive control (IC50 = 44.1 ± 5.8 μM). Meanwhile, the effects of these compounds on cell proliferation/viability were measured using the MTT method. Compounds 4 and 5 showed significant inhibitory activity against NO production with IC50 values of 4.9 ± 0.4 and 5.4 ± 0.6 μM, respectively, and they (up to 40 μM) did not show any significant cytotoxicity in BV-2 microglial cells with LPS treatment for 24 h. Since compounds 2b, 3b, and 6 showed N 90% inhibition on cell proliferation/viability at the concentration of 20 μM, their NO inhibitory effects were not determined. Compounds 1a and 1b were inactive (b 50% inhibition at 40 μM, the highest concentration tested).
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Conflict of interest All the authors declare that there is no conflict of interest concerning this work.
Acknowledgment The project was financially supported by the National Natural Science Foundation of China (No.81573572), Program for New Century Excellent Talents in University (NCET-13-0693, to J.L.), and graduate students independent subject of Beijing University of Chinese Medicine (No. 2016-JYB-XS085). Appendix A. Supplementary data The 1H and 13C NMR, HSQC, HMBC, NOESY, UV, IR, and HRESIMS spectra for compounds 1–5. Supplementary data associated with this article can be found in the online version, at 10.1016/j.fitote.2016.10. 004.
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