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Flavanoids from the stems of Aquilaria sinensis
Chinese Journal of Natural Medicines 2012, 10(4): 02870291
Chinese Journal of Natural Medicines
Flavanoids from the stems of Aquilaria sinensis CHEN Dong, BI Dan, SONG Yue-Lin, TU Peng-Fei* State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100101, China Available online 20 July 2012
[ABSTRACT] AIM: To study the chemical constituents from the stems of Aquilaria sinensis (Lour.) Gilg. METHODS: Chromatographic separations of silica gel, Sephadex LH-20 and prep-HPLC were used. All the structures were elucidated on the basis of chemical and spectroscopic analysis. RESULTS: Twelve flavonoids were isolated and their structures were identified as aquilarinoside A1 (1), lethedioside A (2), 7, 4-dimethylapigenin-5-O-xylosylglucoside (3), lethedoside A (4), 7-hydroxyl-4-methyl-5-O- glucosideflavonoid (5), 7, 3-dimethyl-4-hydroxyl-5-O-glucosideflavonoide (6), 7, 4-dimethyl-5-O-glucosideflavonoide (7), 5-hydroxyl-7, 4-dimethoxyflavone (8), luteolin-7, 3, 4-trimethyl (9), hydroxylgenkwanin (10), 5, 7-dihydroxyl-4-methoxyflavone (11), and formononetin (12). CONCLUSION: Compound 1 is a new flavone glycoside, and compounds 2, 47 were isolated from genus Aquilaria sinensis for the first time. 5-O-glycosylflavones 1-7 showed inhibitory activity for nitric oxide (NO) production by activated RAW 264.7 cells. [KEY WORDS] Aquilaria sinensis; Chemical constituents; Flavonoids; NO
[CLC Number] R284.1, R965
1
[Document code] A
[Article ID] 1672-3651(2012)04-0287-05
Introduction
Aquilaria sinensis (Lour).Gilg is the only authorized source of Lignum Aquilariae Resinatum in Chinese Pharmacopoeia (2010 Edition). It is distributed in Guangdong, Hainan, Guangxi and Taiwan, China. Lignum Aquilariae Resinatum, the resin-deposited wood of Aquilaria sinensis (Lour.) Gilg, has been used as a traditional incense, sedative, analgesic, and digestive medicine in China and other Asian countries [1]. With increased consumption of Lignum Aquilariae Resinatum in recent years, over-exploitation of A. sinensis in Southeast Asian forest caused depletion of the natural resources. In order to solve this problem, the plant has been large-scale cultivated in Guangdong, and Hainan of China[2-4]. In order to compare the chemical constituents of Lignum Aquilariae Resinatum and the normal stems of A. sinensis, discover bioactive compounds, and provide valuable refer[Received on] 19-Sep.-2011 [Research funding] This project was supported by the Special Program for New Drug Innovation of the Ministry of Science and Technology, China (Nos. 2009ZX311-004, 2009ZX0308-004). [ Corresponding author] TU Peng-fei: Prof., Tel/Fax: 86-1082802750, E-mail: pengfeitu@ vip.163.com These authors have no any conflict of interest to declare. Copyright © 2012, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved
ences for the development of A. sinensis, a systematic phytochemical investigation on the stems of A. sinensis was carried out. As a result, twelve falvonoids were isolated and identified from the plant. Among them, compound 1 was a new flavone glycoside , and compounds 2, 47 were isolated from genus Aquilaria for the first time.
2
Experimental
General experimental procedures The UV spectra were obtained on Shimadzu UV-1650PC spectrophotometer. IR spectra were recorded on an Avater-360 spectrometer. NMR spectra were performed on JEOL JUM-300 and Bruker-400 spectrometer with TMS as an internal standard. HRESI-MS was measured on a Bruker APEX IV FT-MS (7.0 T) mass spectrometer in positive-ion mode. ESI-MS was obtained on an Agilent XCT 6320 IT mass spectrometer. Semi-preparative HPLC was carried on Waters 600 instrument with ODS column (Alltech, 250 mm × 10 mm ID, 5μm). Silica gel (4575 μm, 75150 μm, Qingdao Marine Chemical Factory), Sephadex LH-20 (Amersham Pharmacia Biotech), ODS (50 μm, YMC Co. Ltd) and D101 porous polymer (Tianjin Chem. Ind. Co., Ltd.) were used for column chromatography. Solvents of analytical grade were purchased from Beijing Chemical Factory. The stems of Aquilaria sinensis (Lour.) Gilg were provided by Dianbai Nanyao Pharmaceutical Co., Ltd. in December 2009 from 2.1
CHEN Dong, et al. /Chinese Journal of Natural Medicines 2012, 10(4): 287291
Dianbai County, Guangdong province of China, and identified by Professor TU Peng-Fei (School of Pharmaceutical Sciences, Peking University). A voucher specimen was deposited at the herbarium of Peking University Modern Research Center for Traditional Chinese Medicines (No. 200912011). 2.2 Extraction and isolation The dried powder (40 kg) of the stems of Aquilaria sinensis (Lour.) Gilg was refluxed three times with 70% ethanol for 2 h each time. The extracts were combined and evaporated in vacuum to yield 2 kg of residue. The residue was suspended in water and successively extracted with petroleum ether (PE), EtOAc and n-BuOH successively. The EtOAc fraction (200 g) was subjected to silica gel (75150 μm) column chromatography, eluted with CHCl3MeOH (50 : 15 : 1, V/V) gradiently, to give six fractions (Fr.E1E6). Compounds 8 (500 mg) and 9 (10 mg) were obtained by column chromatography on silica gel (PEEtOAc 5 : 12 : 1, V/V) and Sephadex LH-20 (CHCl3-MeOH 1 : 1, V/V) from Fr. E2. Compound 10 (8 mg) was obtained by column chromatography on ODS (MeOHH2O 4 : 66 : 4, V/V) and Sephadex LH-20 (MeOH) from Fr. E5. A portion of the n-BuOH extract (400 g) was subjected to a D101 resin and successively washed with H2O, 20%, 30%, and 50% EtOH. The 30% EtOH elute (100 g) was subjected to silica gel (4575 μm) and eluted with CHCl3MeOH (20 : 12 : 1, V/V) gradiently to afford six fractions (Fr.AF). Fr.B (4.4 g) was subjected to a silica gel (4575 μm) column chromatography, and eluted with EtOAcMeOH (20 : 110 : 1, V/V) to give 42 subfractions. Sub-Fr.1618 were subjected to ODS column chromatography (MeOHH2O 70 : 30, V/V) and purified by Sephadex LH-20 (MeOH) to give 12 (55 mg); Sub-Fr. 2327 were subjected to ODS column chromatography (MeOHH2O 40 : 60, V/V) and purified by Sephadex LH-20 (MeOH) to give 4 (16 mg). Fr. C (20.0 g) was subjected to a silica gel (4575 μm) column chromatography, and eluted with CHCl3MeOH (15 : 15 : 1, V/V) to give 46 subfractions. Sub-Fr. 39 were further purified with Sephadex LH-20 and eluted with MeOH to yield 11 (40 mg); Sub-Fr.1519 was purified by a prep-HPLC (MeOH-H2O 40 : 60, V/V) to yield 5 (50 mg, tR 28 min); Sub-Fr. 3540 were further purified with Sephadex LH-20 and eluted with MeOH to yield 6 (14 mg). Fr. D (15.0 g) was subjected to silica gel (4575 μm) column chromatography, and eluted with CHCl3-MeOH-H2O (6 : 1 : 0.12 : 1 : 0.1, V/V) to give 34 subfractions. Sub-Fr. 619 were purified by a prep-HPLC (MeOHH2O 45 : 55, V/V) to yield 1 (27 mg, tR 28 min). Sub-Fr. 2329 were purified by a prep-HPLC (MeOHH2O 50 : 50, V/V) to yield 2 (28 mg, tR 24 min). The 50% EtOH eluate (18 g) was subjected to a silica gel H and eluted with CHCl3MeOH (30 : 13 : 1, V/V) to afford fraction 56. Fr. 916 were further purified with a Sephadex LH-20 column and eluted with (MeOHH2O 70 : 30, V/V) to yield 7 (23 mg). Fr. 2123 were purified by prep-HPLC (MeOHH2O 40 : 60,
V/V) to yield 3 (50 mg; tR 29 min). 2.3 Acid hydrolysis of compound 1 The flavonoid glycoside (3 mg) was kept at 90 ºC, 24 h, in 1 molL-1 HCl (1 mL). After neutralization by addition of NaHCO3, the reaction mixture was subjected to co-TLC with D-glucose, xylose and the flavone. 2.4 Assay for inhibitory activities against LPS-induced NO production RAW 264.7 cells grown on a 100 mm culture dish were harvested and seeded in 48-well plates at 6 × 104 cells/well for NO production. The plates were pretreated with various concentrations of samples for 30 min and then incubated for 24 h with or without 1 gmL1 of LPS. The nitrite concentration in the culture supernatant was measured by the Griess reaction. Cell viability was measured by a MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay (Sigma-Aldrich).
3
Results and discussion
Compound 1 was obtained as pale yellow amorphous powder. The HRMS (ESI) of 1 revealed an ion peak at m/z 579.170 8 [M + H]+, corresponding to the molecular formula C28H32O15 (Calcd. 579.171 7). The IR spectrum of 1 indicated the presence of hydroxyl (3 383 cm1), a carbonyl (1 652 cm1) and an aromatic (1 604, 1 504, 1 466 cm1) functionalities. The UV spectrum showed absorption maxima at 330 nm (bandĉ) and 260 nm (bandĊ) in MeOH, a characteristic of flavonoid system. 1 H NMR spectrum (Table 1) of 1 displayed signals for one methoxy group at 3.87; seven aromatic protons: one singlet at 6.67 attributed to H-3; two doublets at 7.01 and 6.85 characteristic of meta-protons on a tetrasubstituted benzene ring; and four protons at 7.89 and 6.89 in an AA'XX' coupling system. HMBC spectrum showed that methoxy protons 3.87 showed long-range heteronuclear correlations with 160.8 (C-4), leading to the location of methoxy group on C-4. In association with 13C NMR data (Table 1), and analysis of the HSQC and HMBC spectra, compound 1 was characteristic of 5, 7-dihydroxyl-4-methoxyflavone skeleton with two sugar moieties.
Fig. 1 Key HMBC correlations of compound 1
The configuration of the glucopyranosyl and xylopyranosyl were assigned to be -D based on the coupling constant of the anomeric proton H-1 (H 4.75, d, J = 7.2 Hz) and H-1 (H 4.17, d, J = 7.6 Hz). The glucosyl residue was located at the 5-O-position of the flavone skeleton according to long-range HMBC correlations between 158.1 (C-5) and
CHEN Dong, et al. /Chinese Journal of Natural Medicines 2012, 10(4): 287291
the glucosy anomeric proton H-1. The H-1of -xylosyl located to C-6 of glucose according to long-rang HMBC correlations between 68.6 (C-6) and the anomeric proton H-1. (Fig.1) Consequently, 1 was determined to be 7-hydroxyl- 4-methoxy-5-O--xylopyranosyl (1ė6)-- glucopyranflavonoside, named as aquilarinoside A1. Table 1 NMR data of compound 1 (400/100 MHz, DMSO-d6), J in Hz No.
1
H NMR
2
13
C NMR No.
4
3.27 (1H, m)
69.5
5
3.30 (1H, m)
75.6
2
4
176.8
5
158.1 103.7
6
C NMR
3
106.5
6.85 (1H, d, J = 2.0)
ǂ H NMR
103.7
1
6.67 (1H, s)
13
4.75 (1H, d, J = 7.2) 2.97 (1H, dd, J = 9.0, 7.0) 3.11 (1H, m)
161.3
3
1
73.3 76.5
4
Identification
Aquilarinoside A1 (1) Yellow powder (MeOH). 50.0 (c 0.30, pyridine). HRESI-MS m/z 579.170 8 [M []20 D + H]+ (Calcd. 579.171 7). IR (KBr): max 3 383, 1 652, 1 604, 1 504, 1 466 cm1. UV (MeOH) max: 214, 260, 330. 1H NMR (400 MHz, DMSO-d6) and 13C NMR (100 MHz, DMSO-d6) data, see Table 1. Lethedioside A (2) Yellow power (MeOH). []20 D 50.6 (c 0.31, pyridine). ESI-MS m/z 623.0 [M + Na]+. 1H NMR (400 MHz, DMSO-d6): 7.65 (1H, d, J = 8.5, 2.0 Hz, H-6), 7.54 (1H, d, J = 2.0 Hz, H-2), 7.10 (1H, d, J = 8.5 Hz, H-5), 7.05 (1H, d, J = 2.4 Hz, H-8), 6.86 (1H, d, J = 2.4 Hz, H-6), 6.68 (1H, s, H-3), 3.88 (3H, s, 4-OCH3), 4.78 (1H, d, J = 7.5 Hz, H-1), 3.283.56 (4H, m, H-2 ,3, 4, 5), 3.97 (1H, dd, J = 11.0, 5.6 Hz, H-6), 3.65 (1H, dd, J = 11.0, 2.0 Hz, H-6), 4.18 (1H, d, J = 7.5 Hz, H-1), 3.37 (1H, dd, J = 9.0, 7.8 Hz, H-2), 3.56 (1H, t, J = 9.0, H-3), 3.24 (1H, m, H-4), 3.68 (1H, m, H-5), 3.02 (1H, m, H-5); 13C NMR (100 MHz, DMSO-d6) data see Table 3. All the data above are in agreement with those of lethedioside A[7]. 7, 4-Dimethylapigenin-5-O-xylosylglucoside (3) Pale
9
158.5
3.95 (1H, dd, J = 11.0, 2.0) 3.65 (1H, dd, J = 11.0, 5.6) 1 4.17 (1H, d, J = 7.6)
10
109.3
2
3.33 (1H, m)
73.4
1'
121.1
3
3.55 (1H, t, J = 9.0)
75.9
yellow powder (MeOH). [ ]20 50.2 (c 0.35, pyridine). D
128.1
4
3.23 (1H, m)
69.7
115.8
5
3.02 (1H, m)
65.6
ESI-MS m/z 593.0 [M + Na]+. 1H NMR (400 MHz, DMSO-d6): 8.04 (2H, d, J = 8.0 Hz, H-2, 6), 7.10 (2H, d, J = 8.0 Hz, H-3, 5), 7.10 (1H, d, J = 2.5 Hz, H-8), 6.88 (1H, d, J = 2.5 Hz, H-6), 6.79 (1H, s, H-3), 3.88 (3H, s, 4-OCH3), 4.80 (1H, d, J = 7.5 Hz, H-1), 3.28-3.56 (4H, m, H-2, 3, 4, 5), 3.99 (1H, dd, J = 11.0, 5.6 Hz, H-6), 3.65 (1H, dd, J = 11.0, 2.0 Hz, H-6), 4.20 (1H, d, J = 7.5 Hz, H-1), 3.37 (1H, dd, J = 9.0, 7.8 Hz, H-2), 3.58 (1H, t, J = 9.0 Hz, H-3), 3.22 (1H, m, H-4), 3.66 (1H, m, H-5), 3.02 (1H, m, H-5); 13C NMR (DMSO-d6, 100 MHz) see Table 3. The NMR data were in good agreement with those of 7, 4'-dimethylapigenin-5-O-xylosylglucoside[7].
7 8
163.6 7.01 (1H, d, J = 2.0)
7.89 (2H, d, 2', 6' J = 8.8) 6.89 (2H, d, 3', 5' J = 8.8) 4'
6
96.6
160.8
OCH3 3.87 (3H, s) 10.34 OH (1H, brs)
68.6
104.0
3.63 (1H,m)
56.0
5-O-glycosylflavones 17 were examined with respect to their inhibitory activities against LPS-induced NO production in RAW 264.7 macrophages. Macrophages play major roles in the immunity and inflammatory responses involved in host defense. Once activated, they initiate the production of cytokines, oxygen and nitrogen species and eicosanoids. The nitric oxide (NO) radical is known to play a central role in inflammatory and immune reactions. However, excessive production of NO may cause tissue damage .In inflammatory diseases such as rheumatoid arthritis, excessive NO production by activated macrophages has been observed[5-6]. The 5-O-glycosylflavones 17 exhibited potent inhibitory activity against NO production compared to that of ibuprofen, a clinical anti-inflammatory agent with an IC50 of 94.12 molL1 (Table 2). Table 2 Inhibitory effects of flavonoids (17) against LPS-induced NO production in RAW 264.7 Compounds
1
2
3
4
5
6
7
IC50/ 13.49 7.91 20.02 8.89 14.56 8.60 9.19 (molL--1)
Ibuprofen 94.12
Lethedoside A (4) Yellow powder (MeOH). []20 D 51.0 (c 0.35, pyridine). ESI-MS m/z 491.0 [M + H]+. 1H NMR (400 MHz, DMSO-d6): 7.68 (1H, dd, J = 8.0, 2.8 Hz, H-6), 7.55 (1H, d, J = 2.8 Hz, H-2), 7.11 (1H, d, J = 8.0 Hz, H-5), 7.09 (1H, d, J = 2.4 Hz, H-8), 6.89 (1H, d, J = 2.4 Hz, H-6), 6.88 (1H, s, H-3), 3.89 (3H, s, 7-OCH3), 3.87 (3H, s, 4-OCH3), 3.84 (3H, s, 3-OCH3), 4.76 (1H, d, J = 6.8 Hz, H-1), 3.28-3.56 (4H, m, H-2, 3, 4, 5), 3.75 (1H, dd, J = 10.0, 5.0 Hz, H-6), 3.48 (1H, m, H-6); 13C NMR (100 MHz, DMSO-d6) data see Table 3. All the data above are in agreement with those of lethedoside A[7]. 7-Hydroxyl-4-methyl-5-O-glucosideflavonoid (5) Yellow powder (MeOH). []20 36.4 (c 0.30, pyridine). D ESI-MS m/z 447.0 [M + Na]+. 1H NMR (400 MHz, DMSO-d6): 7.92 (2H, dd, J = 8.0 Hz, H-2, 6), 6.91 (2H, d, J = 8.0 Hz, H-3, 5), 7.04 (1H, d, J = 2.4 Hz, H-8), 6.89 (1H, d, J = 2.4 Hz, H-6), 6.70 (1H, s, H-3), 3.88 (3H, s, 4-OCH3), 4.74 (1H, d, J = 7.5 Hz, H-1), 3.283.56 (4H, m, H-2, 3,
CHEN Dong, et al. /Chinese Journal of Natural Medicines 2012, 10(4): 287291
4, 5), 3.73 (1H, dd, J = 12.0, 5.0 Hz, H-6), 3.48 (1H, m, H-6); 13C NMR (100 MHz, DMSO-d6) data see Table 3. All the data above are in agreetment with those of 7-hydroxyl-4methyl-5-O-glucosideflavonoid[8]. Table 3 13C NMR data of compounds 27 (100 MHz, DMSO-d6) Carbon
2
3
4
5
6
7
2
160.4
160.9
160.9
160.4
160.7
161.0
3
106.2
106.5
106.7
105.2
105.6
106.5
4
176.3
176.9
176.9
176.3
176.4
177.0
5
157.6
158.1
158.2
157.5
157.6
158.2
6
102.3
102.9
103.4
103.4
103.0
103.7
7
163.1
163.6
163.6
162.9
162.9
163.6
8
96.1
96.6
96.6
96.1
96.1
96.6
9
157.9
158.4
158.5
157.8
157.9
158.5
10
108.6
109.2
109.2
108.7
108.7
109.3
1'
122.2
122.7
122.8
120.4
120.8
122.7
2'
108.6
128.0
109.2
127.6
109.4
128.0
3'
151.3
114.5
151.9
115.4
149.8
114.5
4'
148.4
162.1
149.0
160.8
147.4
162.1
5'
111.1
114.5
111.7
115.4
115.1
114.5
127.6
6'
119.1
128.0
119.7
119.5
128.0
OMe-7
55.6
56.1
56.1
55.5
56.1
OMe-3
55.3
55.9
54.7
OMe-4
55.2
55.5
55.7
55.5
1
103.2
103.7
104.0
103.0
103.7
104.5
2
72.8
73.4
73.5
73.1
73.0
73.6
3
76.0
76.6
77.6
77.1
77.1
77.7
4
69.1
69.5
69.9
69.4
69.4
70.0
5
76.0
75.7
75.7
75.2
75.2
75.8
60.9
60.4
60.4
61.0
6
68.1
68.7
1
103.5
104.1
2
72.9
73.4
3
75.4
76.0
4
69.0
69.8
5
65.1
65.6
7,
55.6
3-Dimethyl-4-hydroxyl-5-O-glucosideflavonoide
(6) Yellow powder (MeOH). []20 40.6 (c 0.35, pyridine). D ESI-MS m/z 477.0 [M + H]+. 1H NMR (DMSO-d6, 400 MHz): 9.88(1H, brs, 4-OH), 7.57 (1H, dd, J = 8.0, 2.5 Hz, H-6), 7.55 (1H, d, J = 2.5 Hz, H-2), 6.93 (1H, d, J = 8.0 Hz, H-5), 7.08 (1H, d, J = 2.5 Hz, H-8), 6.90 (1H, d, J = 2.5 Hz, H-6), 6.82 (1H, s, H-3), 3.89 (3H, s, 7-OCH3), 3.89 (3H, s, 3-OCH3), 4.76 (1H, d, J = 6.8 Hz, H-1), 3.28-3.56 (4H, m, H-2, 3, 4, 5), 3.87 (1H, dd, J = 10.0, 5.0 Hz, H-6), 3.48 (1H, m, H-6); 13C NMR (100 MHz, DMSO-d6) data see Table 3. All above data were in agreement with those of 7,
3-dimethyl-4-hydroxyl-5-O-glucosideflavonoide[9]. 7, 4-Dimethyl-5-O-glucosideflavonoide (7) Yellow powder (MeOH). []20 40.2 (c 0.32, pyridine). ESI-MS D m/z 483.0 [M + Na]+. 1H NMR (400 MHz, DMSO-d6): 8.02 (2H, d, J = 8.0 Hz, H-2, 6), 7.10 (2H, d, J = 8.0 Hz, H-3, 5), 7.05 (1H, d, J = 2.4 Hz, H-8), 6.90 (1H, d, J = 2.4 Hz, H-6), 6.77 (1H, s, H-3), 3.88 (3H, s, 7-OCH3), 3.84 (3H, s, 4-OCH3), 4.75 (1H, d, J = 7.2 Hz, H-1), 3.18-3.56 (4H, m, H-2, 3, 4, 5), 3.73 (1H, dd, J = 12.0, 5.0 Hz, H-6), 3.52(1H, m, H-6); 13C NMR (100 MHz, DMSO-d6) data see Table 3. All above data were in agreement with those of 7, 4-dimethyl-5-O-glucoside flavonoide[9]. 5-Hydroxyl-7, 4-dimethoxyflavone (8) Yellow needles (CHCl3-MeOH). ESI-MS m/z 299.0 [M + H]+. 1H NMR (300 MHz, DMSO-d6): 12.93 (1H, s, 5-OH), 8.07 (2H, d, J = 9.0 Hz, H-2, 6), 7.12 (2H, d, J = 9.0 Hz, H-3, 5), 6.95 (1H, s, H-3), 6.81 (1H, d, J = 2.1 Hz, H-8), 6.39 (1H, d, J = 2.1 Hz, H-6), 3.87 (6H, s, 2 × OCH3). The NMR data were in good agreement with those of 5-hydroxyl-7, 4-dimethoxyflavone [10]. Luteolin-7, 3, 4-trimethyl (9) Yellow needles (MeOH). ESI-MS m/z 285.0 [M + H]+. 1H NMR (300 MHz, DMSO-d6): 12.93 (1H, s, 5-OH), 7.72 (1H, dd, J = 8.7 Hz, 2.1 Hz, H-6), 7.69 (1H, d, J = 2.1 Hz, H-2), 7.13 (1H, d, J = 8.7 Hz, H-5), 7.04 (1H, s, H-3), 6.81 (1H, d, J = 2.1 Hz, H-8), 6.39 (1H, d, J = 2.1 Hz, H-6), 3.87 (3H, s, -OCH3), 3.86 (3H, s, -OCH3), 3.84 (3H, s, -OCH3). These data were in agreement with those of luteolin-7, 3, 4-trimethyl [11]. Hydroxylgenkwanin (10) Yellow powder (CHCl3). ESI-MS m/z 285.0 [M + H]+. 1H NMR (300 MHz, DMSO-d6): 12.93 (1H, s, 5-OH), 9.96 (1H, brs, 4-OH), 9.38 (1H, brs, 3-OH), 7.46 (1H, dd, J = 8.4, 2.5 Hz, H-6), 7.43 (1H, d, J = 2.5 Hz, H-2), 6.90 (1H, d, J = 8.4 Hz, H-5), 6.73 (1H, s, H-3), 6.72 (1H, d, J = 2.1 Hz, H-8), 6.37 (1H, d, J = 2.1 Hz, H-6), 3.86 (3H, s, 7-OCH3). All above data were in agreement with those of hydroxylgenkwanin [12]. 5, 7-Dihydroxyl-4-methoxyflavone (11) Yellow powder (CHCl3). ESI-MS m/z 285.0 [M + H]+ . 1H NMR (400 MHz, DMSO-d6): 12.96 (1H, s, 5-OH), 10.38 (1H, s, 7-OH), 7.92 (2H, d, J = 9.0 Hz, H-2, 6), 6.93 (2H, d, J = 9.0 Hz, H-3, 5), 6.85 (1H, s, H-3), 6.77 (1H, d, J = 2.5 Hz, H-8), 6.37 (1H, d, J = 2.5 Hz, H-6), 3.87 (3H, s, -OCH3); 13C NMR (100 MHz, DMSO-d6): 163.5 (C-2), 102.5 (C-3), 181.4 (C-4), 160.7 (C-5), 97.5 (C-6), 164.5 (C-7), 92.1 (C-8), 156.6 (C-9), 104.2 (C-10), 120.5 (C-1), 128.0 (C-2, 6), 160.6 (C-4), 115.4 (C-3, 5), 55.5 (4-OCH3). 1H and 13C NMR data above were in agreement with those of 5, 7-dihydroxyl-4-methoxyflavone [13]. Formononetin (12) Colorless needles (CHCl3). ESI-MS m/z 269.0 [M + H]+ , 1H NMR (400 MHz, DMSO-d6) : 10.82 (1H, s, 4-OH), 8.33 (1H, s, H-2), 7.96 (1H, d, J = 8.4 Hz, H-5), 7.49 (2H, d, J = 9.0 Hz, H-2, 6), 6.98 (2H, d, J = 9.0 Hz, H-3, 5), 6.94 (1H, dd, J = 8.4, 2.4 Hz, H-6), 6.86 (1H, d, J = 2.4 Hz, H-8), 3.78 (3H, s, -OCH3); 13C NMR (100
CHEN Dong, et al. /Chinese Journal of Natural Medicines 2012, 10(4): 287291
MHz, DMSO-d6): 174.7 (C-3), 162.6 (C-7), 159.0 (C-9), 157.5 (C-4), 153.2 (C-2), 130.1 (C-2, 6), 127.4 (C-5), 124.3 (C-1), 123.2 (C-3), 116.7 (C-10), 115.2 (C-6), 113.6 (C-3, 5), 102.2 (C-8), 55.1 (-OCH3).These data were in agreement with those of formononetin[14].
[2]
[3] [4]
[5] [6]
[8]
[9]
References [1]
[7]
Editorial Committee of Chinese Pharmacopeia. Chinese Pharmacopoeia [M] (2010 ed.). Beijing: China Medical Science Press, 2010, 172-173. Editorial Committee of Flora Reipublicae Popularis Sinicae. Flora Reipublica Popularis Sinicae [M]. Beijing: Science Press, 1999, 52(1): 288-291. Yang JS. Review of the chemical constituents isolated from Chen-Xiang [J]. Nat Prod Res Dev, 1998, 10(1): 99-103. Tian YZ, Mi XY, Piao XL. Studies on chemical constituents, pharmaceutical effects and clinical application of Aquilaria sinensis [J]. Chin J Minzu Univ (Nat Sci Ed), 2010, 19(1): 77-81. Kuo PC, Schroeder RA. The emerging multifaceted roles of nitric oxide [J]. Ann Surg, 1995, 221(3): 220-235. Pacher P, Joseph S, Beckman JS, et al. Nitric oxide and peroxynitrite in health and disease [J]. Physiol Rev, 2007, 87: 315-424.
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Zahir A, Jossang A, Bode B, et al. Five new flavone 5-O-glycosides from Lethedon tannaensis: Lethdosides and lethediosides [J]. J Nat Prod, 1999, 62: 241-243. Hashim OK, Abou-Zaid MM, Saleh NAM. The flavonoids of egyptian Chrozophora species [J]. Bioche System Ecol, 1990, 18(2-3): 151-152. Ulubelen A, Bucker R, Mabry TJ. Flavone 5-O-glucosides from Daphne sericea [J]. Phytochemistry, 1882, 21(3): 801-803. Wang HG, Zhou MH, Lu JJ, et al. Antitumor constituents from the leaves of Aquilaria sinensis (Lour.) Gilg [J]. Chem Ind Forest Prod, 2008, 28(2): 1-5. Peng JY, Fan GR, Wu YT. Studies on chemical constituents of Patrinia villosa [J]. China J Chin Mater Med, 2006, 31(2): 128-130. Wang CF, Hu XJ, Xie SM. Isolation and identification of flavonoids from the leaves of Daphne Genkwa [J]. J Zhengzhou Univ (Med Sci), 2003, 38(1): 107-108. Ding L, Liu GA, He L, et al. Studies on the flavonoid constituents in herb of Eremosparton songoricum [J]. China J Chin Mater Med, 2005, 30(2): 126. Zheng Y, Liu P, Bai YJ, et al. Five flavonoids from Spatholobus suberectus [J]. China J Chin Mater Med, 2008, 33(2): 152-154.
➸㚟㼄䐱⭥㵋㏁⧪ ч
Պ, Θ
ͺ࠸Ӗ༰ྐ༰ၝ
ӥ, ണၥॿ, ෑକ*׆ ඟ௶ྐֿރಓྐޥڳᄷԤಬི, ͺ࠸
100191
ᨬ 㽕Ⳃⱘ˖ཙࡎ͂ઁະ࠴ઁԅܤ༰юדdᮍ⊩˖ၮဈ߫ڡcી߫ᅘౄୖۤغ໒ྡອౄୖԉֺ֥ࠩۦܤԅדशҢ ܤdඹڶसߋܤιۤϏୖࠓࠩࠒߋٲՇd㒧ᵰ˖דशߋՇॴ 12 ّܻරफۦܤ, דιน͂ઁະ፣ A1 (1), lethedioside A (2), 7, 4-֝ުཾོݮஸഭ-5-O-ઁൺൺ፣ (3), lethedoside A (4), 7-ᰥݮ-4-ުཾݮ-5-O-ൺܻර፣ (5), 7, 3-֝ުཾݮ-4-ᰥݮ-5-Oൺܻර፣ (6), 7, 4-֝ުཾݮ-5-O-ൺܻර፣ (7), ོஸഭ-7, 4-֝ުਸ਼ (8), ઁЄഭ-7, 3, 4-సުਸ਼ (9), ᰥݮፌܝഭ (10), ࠡܫۦഭ (11), ਇσܝഭ (12)d㒧䆎˖ۦܤ 1 นܻර፣, ۦܤ 2, 47 นұҶ؞೫ᄑᄯדशԄӾdדशԄӾԅ 5-Oܻර፣ۦܤ 1-7 ပᄥ LPS ယӽೀͦಓю NO ݣd ݇䬂䆡 ͂ઁະ; ܤ༰юܻ ;דර; NO
䞥乍Ⳃ
ޥڳqᄷӖྐҗᄥrࢳޏᅥົ(Nos. 2009ZX311-004, 2009ZX0308-004)ᆇᅙົં
Chinese Journal of Natural Medicines
Flavanoids from the stems of Aquilaria sinensis CHEN Dong, BI Dan, SONG Yue-Lin, TU Peng-Fei* State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100101, China Available online 20 July 2012
[ABSTRACT] AIM: To study the chemical constituents from the stems of Aquilaria sinensis (Lour.) Gilg. METHODS: Chromatographic separations of silica gel, Sephadex LH-20 and prep-HPLC were used. All the structures were elucidated on the basis of chemical and spectroscopic analysis. RESULTS: Twelve flavonoids were isolated and their structures were identified as aquilarinoside A1 (1), lethedioside A (2), 7, 4-dimethylapigenin-5-O-xylosylglucoside (3), lethedoside A (4), 7-hydroxyl-4-methyl-5-O- glucosideflavonoid (5), 7, 3-dimethyl-4-hydroxyl-5-O-glucosideflavonoide (6), 7, 4-dimethyl-5-O-glucosideflavonoide (7), 5-hydroxyl-7, 4-dimethoxyflavone (8), luteolin-7, 3, 4-trimethyl (9), hydroxylgenkwanin (10), 5, 7-dihydroxyl-4-methoxyflavone (11), and formononetin (12). CONCLUSION: Compound 1 is a new flavone glycoside, and compounds 2, 47 were isolated from genus Aquilaria sinensis for the first time. 5-O-glycosylflavones 1-7 showed inhibitory activity for nitric oxide (NO) production by activated RAW 264.7 cells. [KEY WORDS] Aquilaria sinensis; Chemical constituents; Flavonoids; NO
[CLC Number] R284.1, R965
1
[Document code] A
[Article ID] 1672-3651(2012)04-0287-05
Introduction
Aquilaria sinensis (Lour).Gilg is the only authorized source of Lignum Aquilariae Resinatum in Chinese Pharmacopoeia (2010 Edition). It is distributed in Guangdong, Hainan, Guangxi and Taiwan, China. Lignum Aquilariae Resinatum, the resin-deposited wood of Aquilaria sinensis (Lour.) Gilg, has been used as a traditional incense, sedative, analgesic, and digestive medicine in China and other Asian countries [1]. With increased consumption of Lignum Aquilariae Resinatum in recent years, over-exploitation of A. sinensis in Southeast Asian forest caused depletion of the natural resources. In order to solve this problem, the plant has been large-scale cultivated in Guangdong, and Hainan of China[2-4]. In order to compare the chemical constituents of Lignum Aquilariae Resinatum and the normal stems of A. sinensis, discover bioactive compounds, and provide valuable refer[Received on] 19-Sep.-2011 [Research funding] This project was supported by the Special Program for New Drug Innovation of the Ministry of Science and Technology, China (Nos. 2009ZX311-004, 2009ZX0308-004). [ Corresponding author] TU Peng-fei: Prof., Tel/Fax: 86-1082802750, E-mail: pengfeitu@ vip.163.com These authors have no any conflict of interest to declare. Copyright © 2012, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved
ences for the development of A. sinensis, a systematic phytochemical investigation on the stems of A. sinensis was carried out. As a result, twelve falvonoids were isolated and identified from the plant. Among them, compound 1 was a new flavone glycoside , and compounds 2, 47 were isolated from genus Aquilaria for the first time.
2
Experimental
General experimental procedures The UV spectra were obtained on Shimadzu UV-1650PC spectrophotometer. IR spectra were recorded on an Avater-360 spectrometer. NMR spectra were performed on JEOL JUM-300 and Bruker-400 spectrometer with TMS as an internal standard. HRESI-MS was measured on a Bruker APEX IV FT-MS (7.0 T) mass spectrometer in positive-ion mode. ESI-MS was obtained on an Agilent XCT 6320 IT mass spectrometer. Semi-preparative HPLC was carried on Waters 600 instrument with ODS column (Alltech, 250 mm × 10 mm ID, 5μm). Silica gel (4575 μm, 75150 μm, Qingdao Marine Chemical Factory), Sephadex LH-20 (Amersham Pharmacia Biotech), ODS (50 μm, YMC Co. Ltd) and D101 porous polymer (Tianjin Chem. Ind. Co., Ltd.) were used for column chromatography. Solvents of analytical grade were purchased from Beijing Chemical Factory. The stems of Aquilaria sinensis (Lour.) Gilg were provided by Dianbai Nanyao Pharmaceutical Co., Ltd. in December 2009 from 2.1
CHEN Dong, et al. /Chinese Journal of Natural Medicines 2012, 10(4): 287291
Dianbai County, Guangdong province of China, and identified by Professor TU Peng-Fei (School of Pharmaceutical Sciences, Peking University). A voucher specimen was deposited at the herbarium of Peking University Modern Research Center for Traditional Chinese Medicines (No. 200912011). 2.2 Extraction and isolation The dried powder (40 kg) of the stems of Aquilaria sinensis (Lour.) Gilg was refluxed three times with 70% ethanol for 2 h each time. The extracts were combined and evaporated in vacuum to yield 2 kg of residue. The residue was suspended in water and successively extracted with petroleum ether (PE), EtOAc and n-BuOH successively. The EtOAc fraction (200 g) was subjected to silica gel (75150 μm) column chromatography, eluted with CHCl3MeOH (50 : 15 : 1, V/V) gradiently, to give six fractions (Fr.E1E6). Compounds 8 (500 mg) and 9 (10 mg) were obtained by column chromatography on silica gel (PEEtOAc 5 : 12 : 1, V/V) and Sephadex LH-20 (CHCl3-MeOH 1 : 1, V/V) from Fr. E2. Compound 10 (8 mg) was obtained by column chromatography on ODS (MeOHH2O 4 : 66 : 4, V/V) and Sephadex LH-20 (MeOH) from Fr. E5. A portion of the n-BuOH extract (400 g) was subjected to a D101 resin and successively washed with H2O, 20%, 30%, and 50% EtOH. The 30% EtOH elute (100 g) was subjected to silica gel (4575 μm) and eluted with CHCl3MeOH (20 : 12 : 1, V/V) gradiently to afford six fractions (Fr.AF). Fr.B (4.4 g) was subjected to a silica gel (4575 μm) column chromatography, and eluted with EtOAcMeOH (20 : 110 : 1, V/V) to give 42 subfractions. Sub-Fr.1618 were subjected to ODS column chromatography (MeOHH2O 70 : 30, V/V) and purified by Sephadex LH-20 (MeOH) to give 12 (55 mg); Sub-Fr. 2327 were subjected to ODS column chromatography (MeOHH2O 40 : 60, V/V) and purified by Sephadex LH-20 (MeOH) to give 4 (16 mg). Fr. C (20.0 g) was subjected to a silica gel (4575 μm) column chromatography, and eluted with CHCl3MeOH (15 : 15 : 1, V/V) to give 46 subfractions. Sub-Fr. 39 were further purified with Sephadex LH-20 and eluted with MeOH to yield 11 (40 mg); Sub-Fr.1519 was purified by a prep-HPLC (MeOH-H2O 40 : 60, V/V) to yield 5 (50 mg, tR 28 min); Sub-Fr. 3540 were further purified with Sephadex LH-20 and eluted with MeOH to yield 6 (14 mg). Fr. D (15.0 g) was subjected to silica gel (4575 μm) column chromatography, and eluted with CHCl3-MeOH-H2O (6 : 1 : 0.12 : 1 : 0.1, V/V) to give 34 subfractions. Sub-Fr. 619 were purified by a prep-HPLC (MeOHH2O 45 : 55, V/V) to yield 1 (27 mg, tR 28 min). Sub-Fr. 2329 were purified by a prep-HPLC (MeOHH2O 50 : 50, V/V) to yield 2 (28 mg, tR 24 min). The 50% EtOH eluate (18 g) was subjected to a silica gel H and eluted with CHCl3MeOH (30 : 13 : 1, V/V) to afford fraction 56. Fr. 916 were further purified with a Sephadex LH-20 column and eluted with (MeOHH2O 70 : 30, V/V) to yield 7 (23 mg). Fr. 2123 were purified by prep-HPLC (MeOHH2O 40 : 60,
V/V) to yield 3 (50 mg; tR 29 min). 2.3 Acid hydrolysis of compound 1 The flavonoid glycoside (3 mg) was kept at 90 ºC, 24 h, in 1 molL-1 HCl (1 mL). After neutralization by addition of NaHCO3, the reaction mixture was subjected to co-TLC with D-glucose, xylose and the flavone. 2.4 Assay for inhibitory activities against LPS-induced NO production RAW 264.7 cells grown on a 100 mm culture dish were harvested and seeded in 48-well plates at 6 × 104 cells/well for NO production. The plates were pretreated with various concentrations of samples for 30 min and then incubated for 24 h with or without 1 gmL1 of LPS. The nitrite concentration in the culture supernatant was measured by the Griess reaction. Cell viability was measured by a MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay (Sigma-Aldrich).
3
Results and discussion
Compound 1 was obtained as pale yellow amorphous powder. The HRMS (ESI) of 1 revealed an ion peak at m/z 579.170 8 [M + H]+, corresponding to the molecular formula C28H32O15 (Calcd. 579.171 7). The IR spectrum of 1 indicated the presence of hydroxyl (3 383 cm1), a carbonyl (1 652 cm1) and an aromatic (1 604, 1 504, 1 466 cm1) functionalities. The UV spectrum showed absorption maxima at 330 nm (bandĉ) and 260 nm (bandĊ) in MeOH, a characteristic of flavonoid system. 1 H NMR spectrum (Table 1) of 1 displayed signals for one methoxy group at 3.87; seven aromatic protons: one singlet at 6.67 attributed to H-3; two doublets at 7.01 and 6.85 characteristic of meta-protons on a tetrasubstituted benzene ring; and four protons at 7.89 and 6.89 in an AA'XX' coupling system. HMBC spectrum showed that methoxy protons 3.87 showed long-range heteronuclear correlations with 160.8 (C-4), leading to the location of methoxy group on C-4. In association with 13C NMR data (Table 1), and analysis of the HSQC and HMBC spectra, compound 1 was characteristic of 5, 7-dihydroxyl-4-methoxyflavone skeleton with two sugar moieties.
Fig. 1 Key HMBC correlations of compound 1
The configuration of the glucopyranosyl and xylopyranosyl were assigned to be -D based on the coupling constant of the anomeric proton H-1 (H 4.75, d, J = 7.2 Hz) and H-1 (H 4.17, d, J = 7.6 Hz). The glucosyl residue was located at the 5-O-position of the flavone skeleton according to long-range HMBC correlations between 158.1 (C-5) and
CHEN Dong, et al. /Chinese Journal of Natural Medicines 2012, 10(4): 287291
the glucosy anomeric proton H-1. The H-1of -xylosyl located to C-6 of glucose according to long-rang HMBC correlations between 68.6 (C-6) and the anomeric proton H-1. (Fig.1) Consequently, 1 was determined to be 7-hydroxyl- 4-methoxy-5-O--xylopyranosyl (1ė6)-- glucopyranflavonoside, named as aquilarinoside A1. Table 1 NMR data of compound 1 (400/100 MHz, DMSO-d6), J in Hz No.
1
H NMR
2
13
C NMR No.
4
3.27 (1H, m)
69.5
5
3.30 (1H, m)
75.6
2
4
176.8
5
158.1 103.7
6
C NMR
3
106.5
6.85 (1H, d, J = 2.0)
ǂ H NMR
103.7
1
6.67 (1H, s)
13
4.75 (1H, d, J = 7.2) 2.97 (1H, dd, J = 9.0, 7.0) 3.11 (1H, m)
161.3
3
1
73.3 76.5
4
Identification
Aquilarinoside A1 (1) Yellow powder (MeOH). 50.0 (c 0.30, pyridine). HRESI-MS m/z 579.170 8 [M []20 D + H]+ (Calcd. 579.171 7). IR (KBr): max 3 383, 1 652, 1 604, 1 504, 1 466 cm1. UV (MeOH) max: 214, 260, 330. 1H NMR (400 MHz, DMSO-d6) and 13C NMR (100 MHz, DMSO-d6) data, see Table 1. Lethedioside A (2) Yellow power (MeOH). []20 D 50.6 (c 0.31, pyridine). ESI-MS m/z 623.0 [M + Na]+. 1H NMR (400 MHz, DMSO-d6): 7.65 (1H, d, J = 8.5, 2.0 Hz, H-6), 7.54 (1H, d, J = 2.0 Hz, H-2), 7.10 (1H, d, J = 8.5 Hz, H-5), 7.05 (1H, d, J = 2.4 Hz, H-8), 6.86 (1H, d, J = 2.4 Hz, H-6), 6.68 (1H, s, H-3), 3.88 (3H, s, 4-OCH3), 4.78 (1H, d, J = 7.5 Hz, H-1), 3.283.56 (4H, m, H-2 ,3, 4, 5), 3.97 (1H, dd, J = 11.0, 5.6 Hz, H-6), 3.65 (1H, dd, J = 11.0, 2.0 Hz, H-6), 4.18 (1H, d, J = 7.5 Hz, H-1), 3.37 (1H, dd, J = 9.0, 7.8 Hz, H-2), 3.56 (1H, t, J = 9.0, H-3), 3.24 (1H, m, H-4), 3.68 (1H, m, H-5), 3.02 (1H, m, H-5); 13C NMR (100 MHz, DMSO-d6) data see Table 3. All the data above are in agreement with those of lethedioside A[7]. 7, 4-Dimethylapigenin-5-O-xylosylglucoside (3) Pale
9
158.5
3.95 (1H, dd, J = 11.0, 2.0) 3.65 (1H, dd, J = 11.0, 5.6) 1 4.17 (1H, d, J = 7.6)
10
109.3
2
3.33 (1H, m)
73.4
1'
121.1
3
3.55 (1H, t, J = 9.0)
75.9
yellow powder (MeOH). [ ]20 50.2 (c 0.35, pyridine). D
128.1
4
3.23 (1H, m)
69.7
115.8
5
3.02 (1H, m)
65.6
ESI-MS m/z 593.0 [M + Na]+. 1H NMR (400 MHz, DMSO-d6): 8.04 (2H, d, J = 8.0 Hz, H-2, 6), 7.10 (2H, d, J = 8.0 Hz, H-3, 5), 7.10 (1H, d, J = 2.5 Hz, H-8), 6.88 (1H, d, J = 2.5 Hz, H-6), 6.79 (1H, s, H-3), 3.88 (3H, s, 4-OCH3), 4.80 (1H, d, J = 7.5 Hz, H-1), 3.28-3.56 (4H, m, H-2, 3, 4, 5), 3.99 (1H, dd, J = 11.0, 5.6 Hz, H-6), 3.65 (1H, dd, J = 11.0, 2.0 Hz, H-6), 4.20 (1H, d, J = 7.5 Hz, H-1), 3.37 (1H, dd, J = 9.0, 7.8 Hz, H-2), 3.58 (1H, t, J = 9.0 Hz, H-3), 3.22 (1H, m, H-4), 3.66 (1H, m, H-5), 3.02 (1H, m, H-5); 13C NMR (DMSO-d6, 100 MHz) see Table 3. The NMR data were in good agreement with those of 7, 4'-dimethylapigenin-5-O-xylosylglucoside[7].
7 8
163.6 7.01 (1H, d, J = 2.0)
7.89 (2H, d, 2', 6' J = 8.8) 6.89 (2H, d, 3', 5' J = 8.8) 4'
6
96.6
160.8
OCH3 3.87 (3H, s) 10.34 OH (1H, brs)
68.6
104.0
3.63 (1H,m)
56.0
5-O-glycosylflavones 17 were examined with respect to their inhibitory activities against LPS-induced NO production in RAW 264.7 macrophages. Macrophages play major roles in the immunity and inflammatory responses involved in host defense. Once activated, they initiate the production of cytokines, oxygen and nitrogen species and eicosanoids. The nitric oxide (NO) radical is known to play a central role in inflammatory and immune reactions. However, excessive production of NO may cause tissue damage .In inflammatory diseases such as rheumatoid arthritis, excessive NO production by activated macrophages has been observed[5-6]. The 5-O-glycosylflavones 17 exhibited potent inhibitory activity against NO production compared to that of ibuprofen, a clinical anti-inflammatory agent with an IC50 of 94.12 molL1 (Table 2). Table 2 Inhibitory effects of flavonoids (17) against LPS-induced NO production in RAW 264.7 Compounds
1
2
3
4
5
6
7
IC50/ 13.49 7.91 20.02 8.89 14.56 8.60 9.19 (molL--1)
Ibuprofen 94.12
Lethedoside A (4) Yellow powder (MeOH). []20 D 51.0 (c 0.35, pyridine). ESI-MS m/z 491.0 [M + H]+. 1H NMR (400 MHz, DMSO-d6): 7.68 (1H, dd, J = 8.0, 2.8 Hz, H-6), 7.55 (1H, d, J = 2.8 Hz, H-2), 7.11 (1H, d, J = 8.0 Hz, H-5), 7.09 (1H, d, J = 2.4 Hz, H-8), 6.89 (1H, d, J = 2.4 Hz, H-6), 6.88 (1H, s, H-3), 3.89 (3H, s, 7-OCH3), 3.87 (3H, s, 4-OCH3), 3.84 (3H, s, 3-OCH3), 4.76 (1H, d, J = 6.8 Hz, H-1), 3.28-3.56 (4H, m, H-2, 3, 4, 5), 3.75 (1H, dd, J = 10.0, 5.0 Hz, H-6), 3.48 (1H, m, H-6); 13C NMR (100 MHz, DMSO-d6) data see Table 3. All the data above are in agreement with those of lethedoside A[7]. 7-Hydroxyl-4-methyl-5-O-glucosideflavonoid (5) Yellow powder (MeOH). []20 36.4 (c 0.30, pyridine). D ESI-MS m/z 447.0 [M + Na]+. 1H NMR (400 MHz, DMSO-d6): 7.92 (2H, dd, J = 8.0 Hz, H-2, 6), 6.91 (2H, d, J = 8.0 Hz, H-3, 5), 7.04 (1H, d, J = 2.4 Hz, H-8), 6.89 (1H, d, J = 2.4 Hz, H-6), 6.70 (1H, s, H-3), 3.88 (3H, s, 4-OCH3), 4.74 (1H, d, J = 7.5 Hz, H-1), 3.283.56 (4H, m, H-2, 3,
CHEN Dong, et al. /Chinese Journal of Natural Medicines 2012, 10(4): 287291
4, 5), 3.73 (1H, dd, J = 12.0, 5.0 Hz, H-6), 3.48 (1H, m, H-6); 13C NMR (100 MHz, DMSO-d6) data see Table 3. All the data above are in agreetment with those of 7-hydroxyl-4methyl-5-O-glucosideflavonoid[8]. Table 3 13C NMR data of compounds 27 (100 MHz, DMSO-d6) Carbon
2
3
4
5
6
7
2
160.4
160.9
160.9
160.4
160.7
161.0
3
106.2
106.5
106.7
105.2
105.6
106.5
4
176.3
176.9
176.9
176.3
176.4
177.0
5
157.6
158.1
158.2
157.5
157.6
158.2
6
102.3
102.9
103.4
103.4
103.0
103.7
7
163.1
163.6
163.6
162.9
162.9
163.6
8
96.1
96.6
96.6
96.1
96.1
96.6
9
157.9
158.4
158.5
157.8
157.9
158.5
10
108.6
109.2
109.2
108.7
108.7
109.3
1'
122.2
122.7
122.8
120.4
120.8
122.7
2'
108.6
128.0
109.2
127.6
109.4
128.0
3'
151.3
114.5
151.9
115.4
149.8
114.5
4'
148.4
162.1
149.0
160.8
147.4
162.1
5'
111.1
114.5
111.7
115.4
115.1
114.5
127.6
6'
119.1
128.0
119.7
119.5
128.0
OMe-7
55.6
56.1
56.1
55.5
56.1
OMe-3
55.3
55.9
54.7
OMe-4
55.2
55.5
55.7
55.5
1
103.2
103.7
104.0
103.0
103.7
104.5
2
72.8
73.4
73.5
73.1
73.0
73.6
3
76.0
76.6
77.6
77.1
77.1
77.7
4
69.1
69.5
69.9
69.4
69.4
70.0
5
76.0
75.7
75.7
75.2
75.2
75.8
60.9
60.4
60.4
61.0
6
68.1
68.7
1
103.5
104.1
2
72.9
73.4
3
75.4
76.0
4
69.0
69.8
5
65.1
65.6
7,
55.6
3-Dimethyl-4-hydroxyl-5-O-glucosideflavonoide
(6) Yellow powder (MeOH). []20 40.6 (c 0.35, pyridine). D ESI-MS m/z 477.0 [M + H]+. 1H NMR (DMSO-d6, 400 MHz): 9.88(1H, brs, 4-OH), 7.57 (1H, dd, J = 8.0, 2.5 Hz, H-6), 7.55 (1H, d, J = 2.5 Hz, H-2), 6.93 (1H, d, J = 8.0 Hz, H-5), 7.08 (1H, d, J = 2.5 Hz, H-8), 6.90 (1H, d, J = 2.5 Hz, H-6), 6.82 (1H, s, H-3), 3.89 (3H, s, 7-OCH3), 3.89 (3H, s, 3-OCH3), 4.76 (1H, d, J = 6.8 Hz, H-1), 3.28-3.56 (4H, m, H-2, 3, 4, 5), 3.87 (1H, dd, J = 10.0, 5.0 Hz, H-6), 3.48 (1H, m, H-6); 13C NMR (100 MHz, DMSO-d6) data see Table 3. All above data were in agreement with those of 7,
3-dimethyl-4-hydroxyl-5-O-glucosideflavonoide[9]. 7, 4-Dimethyl-5-O-glucosideflavonoide (7) Yellow powder (MeOH). []20 40.2 (c 0.32, pyridine). ESI-MS D m/z 483.0 [M + Na]+. 1H NMR (400 MHz, DMSO-d6): 8.02 (2H, d, J = 8.0 Hz, H-2, 6), 7.10 (2H, d, J = 8.0 Hz, H-3, 5), 7.05 (1H, d, J = 2.4 Hz, H-8), 6.90 (1H, d, J = 2.4 Hz, H-6), 6.77 (1H, s, H-3), 3.88 (3H, s, 7-OCH3), 3.84 (3H, s, 4-OCH3), 4.75 (1H, d, J = 7.2 Hz, H-1), 3.18-3.56 (4H, m, H-2, 3, 4, 5), 3.73 (1H, dd, J = 12.0, 5.0 Hz, H-6), 3.52(1H, m, H-6); 13C NMR (100 MHz, DMSO-d6) data see Table 3. All above data were in agreement with those of 7, 4-dimethyl-5-O-glucoside flavonoide[9]. 5-Hydroxyl-7, 4-dimethoxyflavone (8) Yellow needles (CHCl3-MeOH). ESI-MS m/z 299.0 [M + H]+. 1H NMR (300 MHz, DMSO-d6): 12.93 (1H, s, 5-OH), 8.07 (2H, d, J = 9.0 Hz, H-2, 6), 7.12 (2H, d, J = 9.0 Hz, H-3, 5), 6.95 (1H, s, H-3), 6.81 (1H, d, J = 2.1 Hz, H-8), 6.39 (1H, d, J = 2.1 Hz, H-6), 3.87 (6H, s, 2 × OCH3). The NMR data were in good agreement with those of 5-hydroxyl-7, 4-dimethoxyflavone [10]. Luteolin-7, 3, 4-trimethyl (9) Yellow needles (MeOH). ESI-MS m/z 285.0 [M + H]+. 1H NMR (300 MHz, DMSO-d6): 12.93 (1H, s, 5-OH), 7.72 (1H, dd, J = 8.7 Hz, 2.1 Hz, H-6), 7.69 (1H, d, J = 2.1 Hz, H-2), 7.13 (1H, d, J = 8.7 Hz, H-5), 7.04 (1H, s, H-3), 6.81 (1H, d, J = 2.1 Hz, H-8), 6.39 (1H, d, J = 2.1 Hz, H-6), 3.87 (3H, s, -OCH3), 3.86 (3H, s, -OCH3), 3.84 (3H, s, -OCH3). These data were in agreement with those of luteolin-7, 3, 4-trimethyl [11]. Hydroxylgenkwanin (10) Yellow powder (CHCl3). ESI-MS m/z 285.0 [M + H]+. 1H NMR (300 MHz, DMSO-d6): 12.93 (1H, s, 5-OH), 9.96 (1H, brs, 4-OH), 9.38 (1H, brs, 3-OH), 7.46 (1H, dd, J = 8.4, 2.5 Hz, H-6), 7.43 (1H, d, J = 2.5 Hz, H-2), 6.90 (1H, d, J = 8.4 Hz, H-5), 6.73 (1H, s, H-3), 6.72 (1H, d, J = 2.1 Hz, H-8), 6.37 (1H, d, J = 2.1 Hz, H-6), 3.86 (3H, s, 7-OCH3). All above data were in agreement with those of hydroxylgenkwanin [12]. 5, 7-Dihydroxyl-4-methoxyflavone (11) Yellow powder (CHCl3). ESI-MS m/z 285.0 [M + H]+ . 1H NMR (400 MHz, DMSO-d6): 12.96 (1H, s, 5-OH), 10.38 (1H, s, 7-OH), 7.92 (2H, d, J = 9.0 Hz, H-2, 6), 6.93 (2H, d, J = 9.0 Hz, H-3, 5), 6.85 (1H, s, H-3), 6.77 (1H, d, J = 2.5 Hz, H-8), 6.37 (1H, d, J = 2.5 Hz, H-6), 3.87 (3H, s, -OCH3); 13C NMR (100 MHz, DMSO-d6): 163.5 (C-2), 102.5 (C-3), 181.4 (C-4), 160.7 (C-5), 97.5 (C-6), 164.5 (C-7), 92.1 (C-8), 156.6 (C-9), 104.2 (C-10), 120.5 (C-1), 128.0 (C-2, 6), 160.6 (C-4), 115.4 (C-3, 5), 55.5 (4-OCH3). 1H and 13C NMR data above were in agreement with those of 5, 7-dihydroxyl-4-methoxyflavone [13]. Formononetin (12) Colorless needles (CHCl3). ESI-MS m/z 269.0 [M + H]+ , 1H NMR (400 MHz, DMSO-d6) : 10.82 (1H, s, 4-OH), 8.33 (1H, s, H-2), 7.96 (1H, d, J = 8.4 Hz, H-5), 7.49 (2H, d, J = 9.0 Hz, H-2, 6), 6.98 (2H, d, J = 9.0 Hz, H-3, 5), 6.94 (1H, dd, J = 8.4, 2.4 Hz, H-6), 6.86 (1H, d, J = 2.4 Hz, H-8), 3.78 (3H, s, -OCH3); 13C NMR (100
CHEN Dong, et al. /Chinese Journal of Natural Medicines 2012, 10(4): 287291
MHz, DMSO-d6): 174.7 (C-3), 162.6 (C-7), 159.0 (C-9), 157.5 (C-4), 153.2 (C-2), 130.1 (C-2, 6), 127.4 (C-5), 124.3 (C-1), 123.2 (C-3), 116.7 (C-10), 115.2 (C-6), 113.6 (C-3, 5), 102.2 (C-8), 55.1 (-OCH3).These data were in agreement with those of formononetin[14].
[2]
[3] [4]
[5] [6]
[8]
[9]
References [1]
[7]
Editorial Committee of Chinese Pharmacopeia. Chinese Pharmacopoeia [M] (2010 ed.). Beijing: China Medical Science Press, 2010, 172-173. Editorial Committee of Flora Reipublicae Popularis Sinicae. Flora Reipublica Popularis Sinicae [M]. Beijing: Science Press, 1999, 52(1): 288-291. Yang JS. Review of the chemical constituents isolated from Chen-Xiang [J]. Nat Prod Res Dev, 1998, 10(1): 99-103. Tian YZ, Mi XY, Piao XL. Studies on chemical constituents, pharmaceutical effects and clinical application of Aquilaria sinensis [J]. Chin J Minzu Univ (Nat Sci Ed), 2010, 19(1): 77-81. Kuo PC, Schroeder RA. The emerging multifaceted roles of nitric oxide [J]. Ann Surg, 1995, 221(3): 220-235. Pacher P, Joseph S, Beckman JS, et al. Nitric oxide and peroxynitrite in health and disease [J]. Physiol Rev, 2007, 87: 315-424.
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Zahir A, Jossang A, Bode B, et al. Five new flavone 5-O-glycosides from Lethedon tannaensis: Lethdosides and lethediosides [J]. J Nat Prod, 1999, 62: 241-243. Hashim OK, Abou-Zaid MM, Saleh NAM. The flavonoids of egyptian Chrozophora species [J]. Bioche System Ecol, 1990, 18(2-3): 151-152. Ulubelen A, Bucker R, Mabry TJ. Flavone 5-O-glucosides from Daphne sericea [J]. Phytochemistry, 1882, 21(3): 801-803. Wang HG, Zhou MH, Lu JJ, et al. Antitumor constituents from the leaves of Aquilaria sinensis (Lour.) Gilg [J]. Chem Ind Forest Prod, 2008, 28(2): 1-5. Peng JY, Fan GR, Wu YT. Studies on chemical constituents of Patrinia villosa [J]. China J Chin Mater Med, 2006, 31(2): 128-130. Wang CF, Hu XJ, Xie SM. Isolation and identification of flavonoids from the leaves of Daphne Genkwa [J]. J Zhengzhou Univ (Med Sci), 2003, 38(1): 107-108. Ding L, Liu GA, He L, et al. Studies on the flavonoid constituents in herb of Eremosparton songoricum [J]. China J Chin Mater Med, 2005, 30(2): 126. Zheng Y, Liu P, Bai YJ, et al. Five flavonoids from Spatholobus suberectus [J]. China J Chin Mater Med, 2008, 33(2): 152-154.
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ӥ, ണၥॿ, ෑକ*׆ ඟ௶ྐֿރಓྐޥڳᄷԤಬི, ͺ࠸
100191
ᨬ 㽕Ⳃⱘ˖ཙࡎ͂ઁະ࠴ઁԅܤ༰юדdᮍ⊩˖ၮဈ߫ڡcી߫ᅘౄୖۤغ໒ྡອౄୖԉֺ֥ࠩۦܤԅדशҢ ܤdඹڶसߋܤιۤϏୖࠓࠩࠒߋٲՇd㒧ᵰ˖דशߋՇॴ 12 ّܻරफۦܤ, דιน͂ઁະ፣ A1 (1), lethedioside A (2), 7, 4-֝ުཾོݮஸഭ-5-O-ઁൺൺ፣ (3), lethedoside A (4), 7-ᰥݮ-4-ުཾݮ-5-O-ൺܻර፣ (5), 7, 3-֝ުཾݮ-4-ᰥݮ-5-Oൺܻර፣ (6), 7, 4-֝ުཾݮ-5-O-ൺܻර፣ (7), ོஸഭ-7, 4-֝ުਸ਼ (8), ઁЄഭ-7, 3, 4-సުਸ਼ (9), ᰥݮፌܝഭ (10), ࠡܫۦഭ (11), ਇσܝഭ (12)d㒧䆎˖ۦܤ 1 นܻර፣, ۦܤ 2, 47 นұҶ؞೫ᄑᄯדशԄӾdדशԄӾԅ 5-Oܻර፣ۦܤ 1-7 ပᄥ LPS ယӽೀͦಓю NO ݣd ݇䬂䆡 ͂ઁະ; ܤ༰юܻ ;דර; NO
䞥乍Ⳃ
ޥڳqᄷӖྐҗᄥrࢳޏᅥົ(Nos. 2009ZX311-004, 2009ZX0308-004)ᆇᅙົં