Arch. Pharm. Res. DOI 10.1007/s12272-014-0388-3
RESEARCH ARTICLE
Three new sesquiterpene lactones from Inula britannica Xu-Feng Zhang • Jing-Ling Du • Jie Ren • Feng-Mei Ye • Yang-Guo Xie • Xiang-Rong Cheng Shi-Kai Yan • Hui-Zi Jin
•
Received: 16 January 2014 / Accepted: 2 April 2014 Ó The Pharmaceutical Society of Korea 2014
Keywords Inula britannica 1,10-Secoeudesmanolide Eudesmanolide NO production
Chinese Materia Medica 1977; Bensky et al. 1993). Various bioactive secondary metabolites, such as sesquiterpene lactones, have been isolated from this species (Khan et al. 2010). Our current research of the I. britannica led to the isolation of three new sesquiterpenes (1–3), together with nine known compounds (4–12) (Fig. 1). Compound 4 was obtained from this plant for the first time. In this paper, we described the isolation and structure elucidation of these new sesquiterpenes. Additionally, anti-inflammatory activities of these new isolates against LPS-induced NO production in RAW 264.7 macrophages were also evaluated (Fig. 2).
Introduction
Materials and methods
The Inula genus, which comprises more than 100 species of the Asteraceae family, is found mainly in Asia, Europe and Africa (Flora of China, 1989). Inula britannica L. is mainly distributed in China, Korea and Japan. As one of the most popular traditional Chinese medicine (TCM), I. britannica has been reported to treat bronchitis, digestive, disorders and inflammation (Dictionary of Traditional
General procedures
Abstract One new 1,10-secoeudesmanolide (1), two eudesmanolides (2 and 3), together with nine known compounds (4–12) were isolated from the aerial parts of Inula britannica. The structures of the new compounds were elucidated by detailed spectroscopic analysis, including HRESIMS and 2D-NMR spectroscopic method. In addition, compounds 1–4 were tested for their inhibitory effects against LPS-induced NO production in RAW264.7 macrophages.
Xu-Feng Zhang and Jing-Ling Du have contributed equally to this work. X.-F. Zhang J. Ren F.-M. Ye Y.-G. Xie X.-R. Cheng S.-K. Yan H.-Z. Jin (&) School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China e-mail: [email protected] J.-L. Du No. 117 Hospital, Chinese People’s Liberation Army, Hangzhou 310013, People’s Republic of China
Optical rotations were measured on a Perkin-Elmer 341 digital polarimeter. Circular dichroism (CD) data get on a JASCO J-815 CD spectrometer. The IR spectra were obtained on a Bruker FTIR Vector 22 spectrometer with KBr pellets. 1D and 2D NMR spectra were recorded on Bruker Avance-400 or Avance-500 spectrometers. ESI–MS were recorded on a Varian MAT-212 mass spectrometer. The TOF-ESI spectra were carried out on a Q-Tof micro YA019 mass spectrometer. The normal phase silica gel (100–200, 200–300 mesh, Yantai), MCI gel (CHP20P 75–150 lm, Mitsubishi Chemical Co.), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden) were used for column chromatography, and precoated silica HSGF254 (10–40 lm, Yantai) plates were used for TLC analysis. HPLC and preparative HPLC were performed with SHIMADZU LC 2010AHT, Agilent Technologies 1200 series and SHIMADZU LPD-20A. Optical density was measured using a multifunctional microplate reader
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Fig. 1 Structures of 1–12
Department of Pharmacognosy, School of Pharmacy, Second Military Medical University. A voucher specimen (No. IB201209) was deposited at School of Pharmacy, Shanghai Jiao Tong University.
Fig. 2 Key 1H–1H COSY and HMBC correlations of 1
Extraction and isolation
Spectromax M5 (Molecular Devices, American) in antiinflammatory activities assay.
The air-dried and powdered aerial parts of I. britannica (15 kg) were percolated with 95 % EtOH three times (each for 24 h) at room temperature. The combined extracts were concentrated under reduced pressure to afford a residue extract (1.8 kg), which was further suspended in H2O and partitioned with petroleum ether (PE), CH2Cl2, and ethyl acetate (EA), successively. The CH2Cl2 fraction (113.8 g) was chromatographed on a silica gel column eluting with a
Plant material The aerial parts of I. britannica were collected in Heilongjiang province PR China, in September, 2012, and were authenticated by Professor Hanming Zhang,
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Three new sesquiterpene lactones from I. britannica
CH2Cl2/MeOH (100:0–1:1 v/v) gradient to give 11 fractions A-K. Compounds 5 (10.5 g) and 6 (940.2 mg) were crystallized from Fr. E and Fr. F, respectively. Fr. D was purified with a combination of silica gel, macroporous resin MCI (MeOH–H2O, 9:1 v/v), and Sephadex LH-20 (MeOH) to afford compounds 8 (9.2 mg), and 9 (24.5 mg). Fr. E (22.0 g) was subjected to a silica gel CC with mixtures of CH2Cl2/MeOH (100:0–1:1 v/v) as eluents in a stepwise gradient mode to obtain subfractions E1–E7. Compounds 3 (8.6 mg) and 10 (13.9 mg) were isolated after CC over Sephadex LH-20 followed by preparative HPLC (RP18, 210 nm, 40 % MeOH) from Fr. E2. Fr. E3 was subjected to Sephadex LH-20 and purified by preparative HPLC (45 % MeOH) to obtain compounds 7 (37.0 mg). Using the same procedures, Fr. E4 was subjected to preparative HPLC (65 % MeOH) to afford compounds 4 (18.0 mg). Fr. F (12.0 g) was subjected to CC over macroporous resin MCI and silica gel eluted with CH2Cl2/MeOH (100:0–1:1 v/v) to give subfractions F1–F6. Fr. F3 was subjected to Sephadex LH-20 and purified by preparative HPLC (40 % MeOH) to yield compound 1 (20.3 mg). Fr. G (15.0 g) was subjected to CC over macroporous resin MCI and silica gel eluted with CH2Cl2/MeOH (100:0–1:1 v/v) to yield subfractions G1–G9. Fr. G4 was subjected to Sephadex LH-20 and purified by preparative HPLC (50 % MeOH) to obtain compound 2 (11.8 mg). Compounds 11 (13.9 mg) and 12 (30.6 mg) were isolated after CC over Sephadex LH-20. (4S,6S,7S,8S,9S)-1-acetoxy-6,9-dihydroxy-1,10secoeudesma-5(10),11(13)-dien-12,8-olide (1) Orthorhombic crystals, m.p. 88.1–90.3 °C; [a]20 D ? 66.4 (c 0.1, MeOH); IR (KBr) mmax 3,425, 2,934, 1,775, 1,738, 1,655, 1,384, 1,272, 1,145, 1,034, 819 cm-1; ESIMS m/z 347 [M ? Na]?; HRESIMS (positive) m/z 325.1,650 [M ? H]? (calcd. for C17H25O6, 325.1,646); 1H and 13C NMR data, see Tables 1 and 2.
Table 1
13
C-NMR (100 MHz) data for 1–3 1a
Position
2b
3a
1
65.7 t
73.9 d
2
28.1 t
32.4 t
32.4 t
3
32.6 t
121.7 d
36.2 t
4
34.9 d
132.9 s
145.3 s
5
143.4 s
50.9 d
52.7 d
6
69.3 d
77.3 d
68.0 d
7
46.5 d
54.2 d
49.7 d
8
80.7 d
63.4 d
79.5 d
9
72.3 d
41.7 t
38.0 t
10
135.2 s
40.6 s
42.1 s
11 12
138.3 s 171.4 s
135.9 s 171.1 s
144.8 s 172.6 s
13
125.7 t
118.0 t
122.8 s
14
19.3 q
12.7 q
13.0 q
15
19.7 q
23.2 q
110.3 t
10
173.3 s
0
21.1 q
2 a
Measured in CD3OD;
b
79.9 d
measured in DMSO-d6; d in ppm
Table 2 1H-NMR (400 MHz) data for 1–3 Position
1
2
3
1
3.89 m; 3.91 m
3.42 dt (9.2, 7.2)
3.37 dd (11.6, 4.6)
2
1.40 m; 1.20 m
2.16 m; 1.82 m
1.75 m; 1.50 m
3
1.30 m; 1.03 m
5.30 brs
2.10 m; 2.30 m
4
2.70 m
5
2.37 brd (11.2)
1.91 brd (10.9)
6
4.11 d (2.0)
4.30 t (11.2)
3.60 s
7
3.6 m
2.73 brd (8.2)
2.80 dd (8.8, 5.5)
8
5.00 dd (7.8, 2.4)
4.42 brs
4.70 dt (8.8, 1.5)
9
4.15 d (2.4)
2.04 brd (12.0)
2.52 dd (15.8, 1.5)
1.32 dd (12.0, 2.1)
1.60 m
6.15 s; 5.82 s
Crystallographic data of 1 (copper radiation)
10 11
MeOH, M = 324.36, orthorhombic, space group P2 (1) ˚ , a = 90°; b = 12.3273 2(1) 2(1), a = 7.93460 (10) A ˚ ˚, (2) A, b = 90°; c = 16.5776 (2) A c = 90°; 3 ˚ V = 1,621.49 (4) A , Z = 4, Dcalcd = 1.329 mg m-3, crystal size 0.350 9 0.320 9 0.280 mm3, Cu Ka ˚ ). F (000) = 696, T = 140 (2) K. The final (k = 1.54178 A R values were R1 = 0.0429, wR2 = 0.1206, for 10,801 observed reflections [I [ 2r (I)]. The absolute structure parameter was -0.11 (6).
12 13
6.26 d (2.8);
6.00 d (2.8);
5.88 d (2.4)
5.54 d (2.4)
14
1.85 s
1.85 s
0.78 s
15
1.09 d (7.0)
1.09 d (7.0)
5.00 s; 4.78 s
10 20
2.01 s
a
Measured in CD3OD; b measured in DMSO-d6; d in ppm, J value in Hz within parentheses
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1b,6a-Dihydroxy-5aH-eudesma-3(4),11(13)-dien-12,8bolide (2) White amorphous powder, m.p. 155.7–158.2 °C; [a]20 D ? 21.5 (c 0.1, MeOH); IR (KBr) mmax 3,423, 2,922, 1,757, 1,635, 1,400, 1,130 cm-1; ESIMS m/z 287 [M ? Na]?; 263 [M - H]-; HRESIMS (positive) m/z 287.1,258 [M ? Na]? (calcd. for C15H20O4Na, 287.1,254); 1 H and 13C NMR data, see Tables 1 and 2. 1b,6a-Dihydroxy-5aH-eudesma-4(15),11 (13)-dien-12,8bolide (3) Yellowish oil, [a]20 D ? 84.2 (c 0.1, MeOH); IR (KBr) mmax 3,432, 2,937, 1,755, 1,645, 1,384, 1,270, 1,174, 1,123, 1,051, 1,018, 907 cm-1; ESIMS m/z 287 [M ? Na]?; 263 [M - H]-; HRESIMS (positive) m/z 265.1,455 [M ? H]? (calcd. for C15H21O4, 265.1,434); 1H and 13C NMR data, see Tables 1 and 2. 1b-Hydroxy-8b-acetoxy-isocostic acid methyl ester (4) Colorless gum, ESIMS m/z 345 [M ? Na]?; C18H26O5; 1HNMR (400 MHz, CDCl3, d): 6.27 (1H, s, H-13a), 5.59 (1H, s, H-13b), 5.32 (1H, m, H-3), 5.30 (1H, m, H-8), 3.77 (3H, s, H-30 ), 3.57 (1H, dd, J = 11.0, 5.0 Hz, H-1), 2.87 (1H, brd, J = 12.5 Hz, H-7), 2.30 (1H, m, H-2a), 2.22 (1H, dd, J = 15.0, 2.5 Hz, H-9a), 1.93 (3H, m, H3-20 ), 1.92 (1H, m, H-2b), 1.69 (1H, m, H-6a), 1.68 (3H, s, H3-15), 1.61 (1H, m, H-6b), 1.44 (1H, dd, J = 15.0, 3.0 Hz, H-9b), 0.95 (3H, s, H3-14); 13C-NMR (400 MHz, CDCl3, d): 11.0 (C-14), 20.8 (C-15), 21.2 (C-20 ), 23.3 (C-6), 32.3 (C-2), 37.3 (C-10), 39.4 (C-9), 42.5 (C-7), 46.6 (C-5), 52.0 (C-30 ), 69.4 (C-8), 76.5 (C-1), 120.0 (C-3), 125.3 (C-13), 134.2 (C-4), 141.0 (C-11), 167.1 (C-12), 170.0 (C-10 ) (Yang et al. 2003). 1-Acetoxy-6a-hydroxyeriolanolide (5) Colorless cubes, ESIMS m/z 331 [M ? Na]?; 307 [M H]-; C17H24O5; 1H-NMR (500 MHz, CDCl3, d): 6.30 (1H, d, J = 3.0 Hz, H-13a), 5.70 (1H, d, J = 3.0 Hz, H-13b), 5.00 (1H, ddd, J = 8.0, 4.0, 2.0 Hz, H-8), 4.17 (1H, d, J = 2.0 Hz, H-6), 3.95 (1H, m, H-1a), 3.90 (1H, m, H-1b), 3.52 (1H, m, H-7), 2.82 (1H, dd, J = 16.0, 4.0 Hz, H-9a), 2.69 (1H, m, H-4), 2.45 (1H, dd, J = 16.0, 4.0 Hz, H-9b), 2.03 (3H, s, H3-20 ), 1.75 (3H, s, H3-14), 1.39 (1H, m, H-2a), 1.26 (1H, m, H-3a), 1.23 (1H, m, H-2b), 1.07 (3H, d, J = 7.0 Hz, H3-15), 1.02 (1H, m, H-3b); 13C-NMR (500 MHz, CDCl3, d): 20.2 (C-15), 21.2 (C-14), 22.0 (C20 ), 27.5 (C-2), 32.2 (C-3), 33.9 (C-4), 35.4 (C-9), 46.1 (C7), 65.3 (C-1), 69.3(C-6), 77.0 (C-8), 124.8 (C-13), 132.0 (C-5), 137.6 (C-10), 138.0 (C-11), 171.0 (C-12), 172.2 (C10 ) (Jin et al. 2006).
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1,6a-Dihydroxyeriolanolide (6) Colorless cubic crystals, ESIMS m/z 289 [M ? Na]?; 265 [M - H]-; C15H22O4; 1H-NMR (500 MHz, CDCl3, d): 6.21 (1H, d, J = 2.5 Hz, H-13a), 5.82 (1H, d, J = 2.5 Hz, H-13b), 5.05 (1H, m, H-8), 4.15 (1H, d, J = 2.0 Hz, H-6), 3.48 (1H, m, H-7), 3.47 (1H, m, H-1a), 3.36 (1H, m, H-1b), 2.86 (1H, dd, J = 16.0, 2.0 Hz, H-9a), 2.69 (1H, m, H-4), 2.36 (1H, dd, J = 16.0, 2.0 Hz, H-9b), 1.74 (3H, s, H3-14), 1.34 (1H, m, H-2a), 1.30 (1H, m, H-3a), 1.15 (1H, m, H-2b), 1.06 (3H, d, J = 7.0 Hz, H3-15), 1.00 (1H, m, H-3b); 13C-NMR (500 MHz, CDCl3, d): 19.0 (C-15), 20.0 (C-14), 31.3 (C-3), 32.2 (C-2), 34.0 (C-4), 35.0 (C-9), 46.1 (C-7), 62.6 (C-1), 68.4 (C-6), 78.0 (C-8), 124.2 (C-13), 130.6 (C-5), 137.4 (C-10), 138.5 (C-11), 171.2 (C-12) (Jin et al. 2006). 8,10-Dihydroxy-9-isobutyryloxythymol (7) Colorless oil, ESIMS m/z 291 [M ? Na]?; 267 [M - H]-; C14H20O5; 1H-NMR (400 MHz, CD3OD, d): 7.16 (1H, d, J = 8.0 Hz, H-5), 6.64 (1H, dd, J = 8.0, 1.0 Hz, H-6), 6.60 (1H, d, J = 1.0 Hz, H-2), 4.55 (1H, d, J = 11.0 Hz, H-9a), 4.41 (1H, d, J = 11.0 Hz, H-9b), 3.91 (1H, d, J = 11.5 Hz, H-10a), 3.84 (1H, d, J = 11.5 Hz, H-10b), 2.49 (1H, m, H-20 ), 2.22 (3H, s, H3-7), 1.05 (3H, d, J = 7.0 Hz, H3-30 ), 1.03 (3H, d, J = 7.0 Hz, H3-40 ); 13CNMR (400 MHz, CD3OD, d): 19.2 (C-40 ), 19.3 (C-30 ), 21.1 (C-7), 35.2 (C-20 ), 66.7 (C-10), 68.3 (C-9), 78.7 (C-8), 118.1 (C-2), 121.2 (C-6), 123.9 (C-4), 128.6 (C-5), 140.0 (C-1), 156.7 (C-3), 178.8 (C-10 ) (Zhu et al. 2011). Chrysoeriol (8) Yellow powder, ESIMS m/z 323 [M ? Na]?; 299 [M H]-; C16H12O2; 1H-NMR (500 MHz, DMSO-d6, d): 12.98 (1H, brs, 5-OH), 7.70 (2H, d, J = 6.7 Hz, H-50 , 60 ), 6.94 (1H, d, J = 2.8 Hz, H-20 ), 6.90 (1H, s, H-3), 6.51 (1H, brs, H-8), 6.21 (1H, brs, H-6), 3.89 (3H, s, 30 -OCH3); 13C-NMR (500 MHz, DMSO-d6, d): 182.9(C-4), 165.4 (C-7), 165.0 (C-2), 162.6 (C-9), 158.3 (C-5), 152.1 (C-30 ), 149.5 (C-40 ), 123.1 (C-60 ), 121.8 (C-10 ), 117.2 (C-50 ), 111.6 (C-20 ), 104.9 (C-10), 104.3 (C-3), 100.0 (C-6), 95.6 (C-8), 57.1 (30 OCH3) (Agrawal 1989). 5, 6-Dihydroxy-3, 7, 40 -trimethoxyflavonol (9) Yellow powder, ESIMS m/z 367 [M ? Na]?, 343 [M H]-; C18H16O7; H-NMR (400 MHz, DMSO-d6, d): 12.32 (1H, s, 5-OH), 8.01 (2H, d, J = 9.0 Hz, H-20 , 60 ), 7.10 (2H, d, J = 9.0 Hz, H-30 , 50 ), 6.90 (H, s, H-80 ), 6.90 (H, s, H-80 ), 3.92 (3H, s, –OCH3), 3.87(3H, s, –OCH3), 3.86 (3H, s, –OCH3); 13C-NMR (400 MHz, DMSO-d6, d): 155.3 (C-2),
Three new sesquiterpene lactones from I. britannica
137.8 (C-3), 178.2 (C-4), 145.7 (C-5), 129.7 (C-6), 154.6 (C-7), 91.0 (C-8), 48.9 (C-9), 105.7 (C-10), 122.4 (C-10 ), 130.0 (C-20 , 60 ), 114.2 (C-30 , 50 ), 161.3 (C-40 ), 59.7 (3OCH3), 56.4 (7-OCH3), 55.5 (40 -OCH3) (Horie et al. 1998).
concentrations (50.0, 10.0, 2.0, and 0.4 lM) of samples for 30 min and incubated with LPS (1 lg ml-1) for 24 h. The amount of NO was determined by the nitrite concentration in the cultured RAW264.7 macrophage supernatants with the Griess reagent.
Luteolin (10) Yellow powder, ESIMS m/z 309 [M ? Na]?; m/z 285 [M - H]-; C15H10O6; 1H-NMR (400 MHz, DMSO-d6, d): 12.97 (1H, s, 5-OH), 10.85 (1H, brs, 7-OH), 7.42 (1H, brs, H-60 ), 7.40 (1H, s, H-20 ), 6.89 (1H, d, J = 8.1 Hz, H-50 ), 6.67 (1H, s, H-3), 6.44 (1H, d, J = 1.4 Hz, H-8), 6.19 (1H, d, J = 1.4 Hz, H-6); 13C-NMR (400 MHz, DMSO-d6, d): 93.9 (C-8), 98.9 (C-6), 102.9 (C-3), 103.7 (C-10), 113.4 (C20 ), 116.1 (C-50 ), 119.0 (C-60 ), 121.6 (C-10 ), 145.7 (C-30 ), 157.3 (C-5), 149.7 (C-40 ), 161.5 (C-9), 163.9 (C-2), 164.2 (C-7), 181.7 (C-4) (Liang et al. 2011). Tamarixetin (11) Yellow powder, ESIMS m/z 339 [M ? Na]?, m/z 315 [M - H]-; C15H10O6; 1H-NMR (400 MHz, DMSO-d6, d): 12.46 (1H, s, 5-OH), 7.71 (1H, d, J = 2.0 Hz, H-20 ), 7.68 (1H, dd, J = 8.0, 2.0 Hz, H-60 ), 6.93 (1H, d, J = 8.0 Hz, H-50 ), 6.56 (1H, s, H-8), 6.21 (1H, s, H-6), 3.80 (1H, s, 40 OCH3); 13C-NMR (400 MHz, DMSO-d6, d): 56.1 (40 OCH3), 94.1 (C-8), 98.6 (C-6), 103.4 (C-10), 112.1 (C-50 ), 115.8 (C-20 ), 122.1 (C-60 ), 122.3 (C-10 ), 135.7 (C-3), 147.7 (C-30 ), 149.2 (C-2), 151.8 (C-40 ), 157.7 (C-5), 164.4 (C-9), 167.4 (C-7), 176.2 (C-4) (Yuan et al. 2007). Artemetin (12) Yellow powder, ESIMS m/z 411 [M ? Na]?, m/z 387 [M - H]-; C20H20O8; 1H-NMR (400 MHz, DMSO-d6, d): 12.59 (1H, brs, 5-OH), 7.75 (1H, dd, J = 8.6, 1.8 Hz, H-60 ), 7.67 (1H, d, J = 1.8 Hz, H-20 ), 7.16 (1H, d, J = 8.6 Hz, H-50 ), 6.93 (1H, s, H-8), 3.93, 3.82, 3.74 (each 3H, s, 39 –OCH3), 3.33 (6H, s, 29 –OCH3); 13C-NMR (400 MHz, DMSO-d6, d): 91.5 (C-8), 105.6 (C-10), 111.3 (C-20 ), 111.6 (C-50 ), 122.0 (C-60 ), 122.1 (C-10 ), 131.6 (C6), 138.1 (C-3), 148.5 (C-30 ), 151.3 (C-40 ), 151.6 (C-5), 151.8(C-2), 155.5 (C-9), 158.7 (C-7), 178.3 (C-4), 60.0, 59.8, 56.5, 55.7, 55.6 (59 –OCH3) (Ahmed et al. 1988).
Results and discussion Compound 1 was isolated as orthorhombic crystals. Its molecular formula C17H24O6 was characterized by HRESIMS (positive) m/z 325.1,650 [M ? H]? (calcd. for C17H25O6, 325.1,646), indicating six degrees of unsaturation. The IR spectrum showed bands characteristic of hydroxyl (3,425 cm-1), carbonyl (1,762 and 1,738 cm-1), and olefinic bond (1,655 cm-1). The 1H and 13C NMR spectroscopic data of 1 were very similar to those of compound 5, except that one methylene of 5 was found to be an oxymethyne [dC 72.3 (C-9), dH 4.15 (1H, d, J = 2.4 Hz, H-9)] in compound 1 (Jin et al. 2006). These observations are consistent with a 16 Da surplus in molecular weight, indicating 1 a hydroxylated derivative of 5. Detailed analysis the 1H–1H COSY and HMBC plots suggested the hydroxyl attaching at C-9. The relative configuration of 1 was determined by the following NOESY correlations: H-6/H3-15 and H-7/H-8 (Fig. 3). Furthermore, the absolute stereochemistry of 1 was determined by an X-ray analysis using Cu Ka radiation (Fig. 4). Thus, the structure of 1 was determined as (4S,6S,7S,8S,9S)-1acetoxy-6,9-dihydroxy-1,10-secoeudesma-5(10),11(13)dien-12,8-olide. Compound 2 was obtained as white amorphous powder with a molecular formula C15H20O4 established by HRESIMS (positive) m/z 265.1,364 [M ? H]? (calcd. for C15H21O4, 265.1,373). Its IR spectrum showed absorption bands of hydroxyl (3,423 cm-1), carbonyl (1,757 cm-1), and olefinic bond (1,634 cm-1). The 1H NMR spectrum exhibited one methyl singlet [dH 1.85 (3H, s, H3-14)], one methyl doublet [dH 1.09 (3H, d, J = 7.0 Hz, H3-15)], three oxymethine [dH 3.42 (1H, dt, J = 9.2, 7.2 Hz, H-1); dH 4.30 (1H, t, J = 11.2 Hz, H-6); dH 4.42 (1H, brs, H-8)], and three olefinic protons [dH 5.30 (1H, brs, H-3); dH 6.00 (1H, d, J = 2.8 Hz, H-13a); dH 5.54 (1H, d, J = 2.4 Hz,
Determination of NO production Inhibitory activities against LPS-induced NO production in RAW 264.7 macrophages (Schmidt and Kelm 1996) were selected as the anti-inflammatory screening method. RAW264.7 cells grown on 100 mm culture dish were harvested and seeded in 96-well plates (1 9 105 cells/well) for NO production. The plates were pretreated with various
Fig. 3 Key NOESY correlations of 1
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X.-F. Zhang et al. Fig. 4 Single-crystal X-ray structure (copper radiation) of 1
Fig. 5 Key 1H–1H COSY and HMBC correlations of 2
H-13b)]. The corresponding carbon signals were differentiated by 13C NMR and DEPT spectra (Table 2) as two methyl [dC 12.7 (C-14) and 23.2 (C-15)], three oxymethine [dC 73.9 (C-1), 77.3 (C-6) and 63.4 (C-8)], and two olefinic carbons [dC 121.7 (C-3) and 118.0 (C-15)]. The 13C NMR spectrum also revealed three sp2 disclosed quaternary carbons including two olefinic, and one easter carbonyl carbons. The remaining one degree of unsaturation was due to a tricyclic core. The analysis of 1H–1H COSY spectrum established the key sequences of H-1/H2-2/H-3 and H-5/H6/H-7/H-8/H2-9 (Fig. 5). In addition, the HMBC correlations from H2-2 to C-3 and C-4, H-8 to C-6 and C-10, H213 to C-7, C-11 and C-12, H3-14 to C-1, C-5, C-9 and C-10, H3-15 to C-3, C-4 and C-5, and the chemical shift of C-8 (dH 4.42) indicated the planar structure of 2 was 1,6dihydroxy-eudesma-3(4),11(13)-dien-12,8-olide (Fig. 5). In the NOESY spectrum (Fig. 6), correlations between H-1/H-5, H-5/H-7, H-7/H-8, and H-6/H3-14 were observed. Thus, compound 2 was determined to be 1b,6a-dihydroxy5aH-eudesma-3(4),11 (13)-dien-12,8b-olide. Compound 3 was isolated as yellowish oil and share the same molecular formula of C15H20O4 with 2 deduced from HRESIMS (positive) m/z 265.1,339 [M ? H]? (calcd. for
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Fig. 6 Key NOESY correlations of 2
C15H21O4, 265.1,332). A close comparison of NMR spectra between the two compounds indicated that 3 was also a eudesmane-type sesquiterpene lactone with hydroxyl groups substituted at C-1 and C-6. The presence of a methylene [dC 36.2 (C-3), dH 2.30 (m, H-3a) and 2.10 (m, H-3b)] and olefinic methylene [dC 110.3 (C-15), dH 5.00 (s, H-15a) and 4.78 (s, H-15b)] in compound 3 instead of the corresponding methyl and olefinic methane at C-3 and C-15 in compound 3 suggested that the olefinic group of 3 was located at C-4 and C-15 rather than at C-3 and C-4. This inference was further determined by the 1H–1H COSY and HMBC experiments. The NOESY experiment revealed the relative configuration of 3, which was similar to that of 2. Therefore, 3 was identified as 1b,6a-dihydroxy-5aHeudesma-4(15),11(13)-dien-12,8b-olide. Compounds 1–4 were tested for their inhibitory effects against LPS-induced NO production in RAW264.7
Three new sesquiterpene lactones from I. britannica
macrophages with aminoguanidine as positive control (Schmidt and Kelm 1996). Compounds 1–3 exhibited significant inhibitory activities with IC50 values of 3.99, 5.47, and 3.47 lM, respectively. Whereas, compound 4 showed moderate activity, with IC50 value of 28.65 lM. Acknowledgments The work was supported by program NCET Foundation, NSFC (81230090 and 81102778), partially supported by Global Research Network for Medicinal Plants (GRNMP) and King Saud University, Shanghai Leading Academic Discipline Project (B906), FP7-PEOPLE-IRSES-2008 (TCMCANCER Project 230232), Key laboratory of drug research for special environments, PLA, Shanghai Engineering Research Center for the Preparation of Bioactive Natural Products (10DZ2251300) and the Scientific Foundation of Shanghai China (10DZ1971700, 12401900501). National Major Project of China (2011ZX09307-002-03 and 2011ZX09102-006-02). National Key Technology R&D Program of China (2012BAI29B06).
References Agrawal, P.K. 1989. Carbon-13 NMR of flavonoids, 102. Elsevier Scientific Publishing Company: Amsterdam. Ahmed, D., M.I. Choudhary, S. Turkoz, and B. Sener. 1988. Chemical constituents of Buxus sempervirens. Planta Medica 54(2): 173–174. Bensky, D., A. Gamble, T.J. Kaptchuk, and I.L. Bensky. 1993. Chinese herbal medicine: Materia Medica, 193–194. Seattle: Eastland Press. Horie, T., Y. Ohtsuru, K. Shibata, K. Yamashita, M. Tsukayama, and Y. Kawamura. 1998. 13C NMR spectral assignment of the A-ring of polyoxygenated flavones. Phytochemistry 47: 865–874.
Jiangsu New Medical College. 1977. Dictionary of Traditional Chinese Materia Medica, vol. 2, 2216–2219. Shanghai: Shanghai People’s Press. Jin, H.Z., D.H. Lee, J.H. Lee, K. Lee, Y.S. Hong, D.H. Choung, H.Y. Kim, and J.J. Lee. 2006. New sesquiterpene dimers from Inula britannica inhibit NF-jB activation and NO and TNF-production in LPS-stimulated RAW264.7 cells. Planta Medica 72(1): 40–45. Khan, A.L., J. Hussain, M. Hamayun, S.A. Gilani, S. Ahmad, G. Rehman, Y.-H. Kim, S.-M. Kang, and I.-J. Lee. 2010. Secondary metabolites from Inula britannica L. and their biological activities. Molecules 15: 1562–1577. Lin, R., D.J. Yu, and Z.Y. Wu. 1989. Flora of China, 263–265. Beijing: Science Press. Liang, Y.H., M. Ye, J. Han, B.R. Rong, and D.A. Guo. 2011. Lignans and flavonoids from rhizome of Drynaria fortune. Chinese Traditional and Herbal Drugs 42(1): 25–30. Schmidt, H.H.H.W., and M. Kelm. 1996. Determination of nitrite and nitrate by the Griess reaction. In Methods in Nitric oxide research, Chap. 33, ed. M. Feelisch, and J. Stramler, 491–497. New York: Wiley. Yang, C., C.M. Wang, and Z.J. Jia. 2003. Sesquiterpenes and other constituents from the aerial parts of Inula japonica. Planta Medica 69: 662–666. Yuan, J.Q., Y.N. Yan, Y.F. Liu, and J.H. Miu. 2007. Studies on flavonoids of Eupatorium odoratum L. Journal of Chinese Medicinal Materials 30(6): 657–660. Zhu, J.X., J.J. Qin, H.R. Wang, Y. Zhu, W.D. Zhang, and H.Z. Jin. 2011. Monoterpenes and other chemical constituents from the aerial parts of Inula japonica. Chemistry of Natural Compounds 47(2): 303–305.
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RESEARCH ARTICLE
Three new sesquiterpene lactones from Inula britannica Xu-Feng Zhang • Jing-Ling Du • Jie Ren • Feng-Mei Ye • Yang-Guo Xie • Xiang-Rong Cheng Shi-Kai Yan • Hui-Zi Jin
•
Received: 16 January 2014 / Accepted: 2 April 2014 Ó The Pharmaceutical Society of Korea 2014
Keywords Inula britannica 1,10-Secoeudesmanolide Eudesmanolide NO production
Chinese Materia Medica 1977; Bensky et al. 1993). Various bioactive secondary metabolites, such as sesquiterpene lactones, have been isolated from this species (Khan et al. 2010). Our current research of the I. britannica led to the isolation of three new sesquiterpenes (1–3), together with nine known compounds (4–12) (Fig. 1). Compound 4 was obtained from this plant for the first time. In this paper, we described the isolation and structure elucidation of these new sesquiterpenes. Additionally, anti-inflammatory activities of these new isolates against LPS-induced NO production in RAW 264.7 macrophages were also evaluated (Fig. 2).
Introduction
Materials and methods
The Inula genus, which comprises more than 100 species of the Asteraceae family, is found mainly in Asia, Europe and Africa (Flora of China, 1989). Inula britannica L. is mainly distributed in China, Korea and Japan. As one of the most popular traditional Chinese medicine (TCM), I. britannica has been reported to treat bronchitis, digestive, disorders and inflammation (Dictionary of Traditional
General procedures
Abstract One new 1,10-secoeudesmanolide (1), two eudesmanolides (2 and 3), together with nine known compounds (4–12) were isolated from the aerial parts of Inula britannica. The structures of the new compounds were elucidated by detailed spectroscopic analysis, including HRESIMS and 2D-NMR spectroscopic method. In addition, compounds 1–4 were tested for their inhibitory effects against LPS-induced NO production in RAW264.7 macrophages.
Xu-Feng Zhang and Jing-Ling Du have contributed equally to this work. X.-F. Zhang J. Ren F.-M. Ye Y.-G. Xie X.-R. Cheng S.-K. Yan H.-Z. Jin (&) School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China e-mail: [email protected] J.-L. Du No. 117 Hospital, Chinese People’s Liberation Army, Hangzhou 310013, People’s Republic of China
Optical rotations were measured on a Perkin-Elmer 341 digital polarimeter. Circular dichroism (CD) data get on a JASCO J-815 CD spectrometer. The IR spectra were obtained on a Bruker FTIR Vector 22 spectrometer with KBr pellets. 1D and 2D NMR spectra were recorded on Bruker Avance-400 or Avance-500 spectrometers. ESI–MS were recorded on a Varian MAT-212 mass spectrometer. The TOF-ESI spectra were carried out on a Q-Tof micro YA019 mass spectrometer. The normal phase silica gel (100–200, 200–300 mesh, Yantai), MCI gel (CHP20P 75–150 lm, Mitsubishi Chemical Co.), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden) were used for column chromatography, and precoated silica HSGF254 (10–40 lm, Yantai) plates were used for TLC analysis. HPLC and preparative HPLC were performed with SHIMADZU LC 2010AHT, Agilent Technologies 1200 series and SHIMADZU LPD-20A. Optical density was measured using a multifunctional microplate reader
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Fig. 1 Structures of 1–12
Department of Pharmacognosy, School of Pharmacy, Second Military Medical University. A voucher specimen (No. IB201209) was deposited at School of Pharmacy, Shanghai Jiao Tong University.
Fig. 2 Key 1H–1H COSY and HMBC correlations of 1
Extraction and isolation
Spectromax M5 (Molecular Devices, American) in antiinflammatory activities assay.
The air-dried and powdered aerial parts of I. britannica (15 kg) were percolated with 95 % EtOH three times (each for 24 h) at room temperature. The combined extracts were concentrated under reduced pressure to afford a residue extract (1.8 kg), which was further suspended in H2O and partitioned with petroleum ether (PE), CH2Cl2, and ethyl acetate (EA), successively. The CH2Cl2 fraction (113.8 g) was chromatographed on a silica gel column eluting with a
Plant material The aerial parts of I. britannica were collected in Heilongjiang province PR China, in September, 2012, and were authenticated by Professor Hanming Zhang,
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Three new sesquiterpene lactones from I. britannica
CH2Cl2/MeOH (100:0–1:1 v/v) gradient to give 11 fractions A-K. Compounds 5 (10.5 g) and 6 (940.2 mg) were crystallized from Fr. E and Fr. F, respectively. Fr. D was purified with a combination of silica gel, macroporous resin MCI (MeOH–H2O, 9:1 v/v), and Sephadex LH-20 (MeOH) to afford compounds 8 (9.2 mg), and 9 (24.5 mg). Fr. E (22.0 g) was subjected to a silica gel CC with mixtures of CH2Cl2/MeOH (100:0–1:1 v/v) as eluents in a stepwise gradient mode to obtain subfractions E1–E7. Compounds 3 (8.6 mg) and 10 (13.9 mg) were isolated after CC over Sephadex LH-20 followed by preparative HPLC (RP18, 210 nm, 40 % MeOH) from Fr. E2. Fr. E3 was subjected to Sephadex LH-20 and purified by preparative HPLC (45 % MeOH) to obtain compounds 7 (37.0 mg). Using the same procedures, Fr. E4 was subjected to preparative HPLC (65 % MeOH) to afford compounds 4 (18.0 mg). Fr. F (12.0 g) was subjected to CC over macroporous resin MCI and silica gel eluted with CH2Cl2/MeOH (100:0–1:1 v/v) to give subfractions F1–F6. Fr. F3 was subjected to Sephadex LH-20 and purified by preparative HPLC (40 % MeOH) to yield compound 1 (20.3 mg). Fr. G (15.0 g) was subjected to CC over macroporous resin MCI and silica gel eluted with CH2Cl2/MeOH (100:0–1:1 v/v) to yield subfractions G1–G9. Fr. G4 was subjected to Sephadex LH-20 and purified by preparative HPLC (50 % MeOH) to obtain compound 2 (11.8 mg). Compounds 11 (13.9 mg) and 12 (30.6 mg) were isolated after CC over Sephadex LH-20. (4S,6S,7S,8S,9S)-1-acetoxy-6,9-dihydroxy-1,10secoeudesma-5(10),11(13)-dien-12,8-olide (1) Orthorhombic crystals, m.p. 88.1–90.3 °C; [a]20 D ? 66.4 (c 0.1, MeOH); IR (KBr) mmax 3,425, 2,934, 1,775, 1,738, 1,655, 1,384, 1,272, 1,145, 1,034, 819 cm-1; ESIMS m/z 347 [M ? Na]?; HRESIMS (positive) m/z 325.1,650 [M ? H]? (calcd. for C17H25O6, 325.1,646); 1H and 13C NMR data, see Tables 1 and 2.
Table 1
13
C-NMR (100 MHz) data for 1–3 1a
Position
2b
3a
1
65.7 t
73.9 d
2
28.1 t
32.4 t
32.4 t
3
32.6 t
121.7 d
36.2 t
4
34.9 d
132.9 s
145.3 s
5
143.4 s
50.9 d
52.7 d
6
69.3 d
77.3 d
68.0 d
7
46.5 d
54.2 d
49.7 d
8
80.7 d
63.4 d
79.5 d
9
72.3 d
41.7 t
38.0 t
10
135.2 s
40.6 s
42.1 s
11 12
138.3 s 171.4 s
135.9 s 171.1 s
144.8 s 172.6 s
13
125.7 t
118.0 t
122.8 s
14
19.3 q
12.7 q
13.0 q
15
19.7 q
23.2 q
110.3 t
10
173.3 s
0
21.1 q
2 a
Measured in CD3OD;
b
79.9 d
measured in DMSO-d6; d in ppm
Table 2 1H-NMR (400 MHz) data for 1–3 Position
1
2
3
1
3.89 m; 3.91 m
3.42 dt (9.2, 7.2)
3.37 dd (11.6, 4.6)
2
1.40 m; 1.20 m
2.16 m; 1.82 m
1.75 m; 1.50 m
3
1.30 m; 1.03 m
5.30 brs
2.10 m; 2.30 m
4
2.70 m
5
2.37 brd (11.2)
1.91 brd (10.9)
6
4.11 d (2.0)
4.30 t (11.2)
3.60 s
7
3.6 m
2.73 brd (8.2)
2.80 dd (8.8, 5.5)
8
5.00 dd (7.8, 2.4)
4.42 brs
4.70 dt (8.8, 1.5)
9
4.15 d (2.4)
2.04 brd (12.0)
2.52 dd (15.8, 1.5)
1.32 dd (12.0, 2.1)
1.60 m
6.15 s; 5.82 s
Crystallographic data of 1 (copper radiation)
10 11
MeOH, M = 324.36, orthorhombic, space group P2 (1) ˚ , a = 90°; b = 12.3273 2(1) 2(1), a = 7.93460 (10) A ˚ ˚, (2) A, b = 90°; c = 16.5776 (2) A c = 90°; 3 ˚ V = 1,621.49 (4) A , Z = 4, Dcalcd = 1.329 mg m-3, crystal size 0.350 9 0.320 9 0.280 mm3, Cu Ka ˚ ). F (000) = 696, T = 140 (2) K. The final (k = 1.54178 A R values were R1 = 0.0429, wR2 = 0.1206, for 10,801 observed reflections [I [ 2r (I)]. The absolute structure parameter was -0.11 (6).
12 13
6.26 d (2.8);
6.00 d (2.8);
5.88 d (2.4)
5.54 d (2.4)
14
1.85 s
1.85 s
0.78 s
15
1.09 d (7.0)
1.09 d (7.0)
5.00 s; 4.78 s
10 20
2.01 s
a
Measured in CD3OD; b measured in DMSO-d6; d in ppm, J value in Hz within parentheses
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1b,6a-Dihydroxy-5aH-eudesma-3(4),11(13)-dien-12,8bolide (2) White amorphous powder, m.p. 155.7–158.2 °C; [a]20 D ? 21.5 (c 0.1, MeOH); IR (KBr) mmax 3,423, 2,922, 1,757, 1,635, 1,400, 1,130 cm-1; ESIMS m/z 287 [M ? Na]?; 263 [M - H]-; HRESIMS (positive) m/z 287.1,258 [M ? Na]? (calcd. for C15H20O4Na, 287.1,254); 1 H and 13C NMR data, see Tables 1 and 2. 1b,6a-Dihydroxy-5aH-eudesma-4(15),11 (13)-dien-12,8bolide (3) Yellowish oil, [a]20 D ? 84.2 (c 0.1, MeOH); IR (KBr) mmax 3,432, 2,937, 1,755, 1,645, 1,384, 1,270, 1,174, 1,123, 1,051, 1,018, 907 cm-1; ESIMS m/z 287 [M ? Na]?; 263 [M - H]-; HRESIMS (positive) m/z 265.1,455 [M ? H]? (calcd. for C15H21O4, 265.1,434); 1H and 13C NMR data, see Tables 1 and 2. 1b-Hydroxy-8b-acetoxy-isocostic acid methyl ester (4) Colorless gum, ESIMS m/z 345 [M ? Na]?; C18H26O5; 1HNMR (400 MHz, CDCl3, d): 6.27 (1H, s, H-13a), 5.59 (1H, s, H-13b), 5.32 (1H, m, H-3), 5.30 (1H, m, H-8), 3.77 (3H, s, H-30 ), 3.57 (1H, dd, J = 11.0, 5.0 Hz, H-1), 2.87 (1H, brd, J = 12.5 Hz, H-7), 2.30 (1H, m, H-2a), 2.22 (1H, dd, J = 15.0, 2.5 Hz, H-9a), 1.93 (3H, m, H3-20 ), 1.92 (1H, m, H-2b), 1.69 (1H, m, H-6a), 1.68 (3H, s, H3-15), 1.61 (1H, m, H-6b), 1.44 (1H, dd, J = 15.0, 3.0 Hz, H-9b), 0.95 (3H, s, H3-14); 13C-NMR (400 MHz, CDCl3, d): 11.0 (C-14), 20.8 (C-15), 21.2 (C-20 ), 23.3 (C-6), 32.3 (C-2), 37.3 (C-10), 39.4 (C-9), 42.5 (C-7), 46.6 (C-5), 52.0 (C-30 ), 69.4 (C-8), 76.5 (C-1), 120.0 (C-3), 125.3 (C-13), 134.2 (C-4), 141.0 (C-11), 167.1 (C-12), 170.0 (C-10 ) (Yang et al. 2003). 1-Acetoxy-6a-hydroxyeriolanolide (5) Colorless cubes, ESIMS m/z 331 [M ? Na]?; 307 [M H]-; C17H24O5; 1H-NMR (500 MHz, CDCl3, d): 6.30 (1H, d, J = 3.0 Hz, H-13a), 5.70 (1H, d, J = 3.0 Hz, H-13b), 5.00 (1H, ddd, J = 8.0, 4.0, 2.0 Hz, H-8), 4.17 (1H, d, J = 2.0 Hz, H-6), 3.95 (1H, m, H-1a), 3.90 (1H, m, H-1b), 3.52 (1H, m, H-7), 2.82 (1H, dd, J = 16.0, 4.0 Hz, H-9a), 2.69 (1H, m, H-4), 2.45 (1H, dd, J = 16.0, 4.0 Hz, H-9b), 2.03 (3H, s, H3-20 ), 1.75 (3H, s, H3-14), 1.39 (1H, m, H-2a), 1.26 (1H, m, H-3a), 1.23 (1H, m, H-2b), 1.07 (3H, d, J = 7.0 Hz, H3-15), 1.02 (1H, m, H-3b); 13C-NMR (500 MHz, CDCl3, d): 20.2 (C-15), 21.2 (C-14), 22.0 (C20 ), 27.5 (C-2), 32.2 (C-3), 33.9 (C-4), 35.4 (C-9), 46.1 (C7), 65.3 (C-1), 69.3(C-6), 77.0 (C-8), 124.8 (C-13), 132.0 (C-5), 137.6 (C-10), 138.0 (C-11), 171.0 (C-12), 172.2 (C10 ) (Jin et al. 2006).
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1,6a-Dihydroxyeriolanolide (6) Colorless cubic crystals, ESIMS m/z 289 [M ? Na]?; 265 [M - H]-; C15H22O4; 1H-NMR (500 MHz, CDCl3, d): 6.21 (1H, d, J = 2.5 Hz, H-13a), 5.82 (1H, d, J = 2.5 Hz, H-13b), 5.05 (1H, m, H-8), 4.15 (1H, d, J = 2.0 Hz, H-6), 3.48 (1H, m, H-7), 3.47 (1H, m, H-1a), 3.36 (1H, m, H-1b), 2.86 (1H, dd, J = 16.0, 2.0 Hz, H-9a), 2.69 (1H, m, H-4), 2.36 (1H, dd, J = 16.0, 2.0 Hz, H-9b), 1.74 (3H, s, H3-14), 1.34 (1H, m, H-2a), 1.30 (1H, m, H-3a), 1.15 (1H, m, H-2b), 1.06 (3H, d, J = 7.0 Hz, H3-15), 1.00 (1H, m, H-3b); 13C-NMR (500 MHz, CDCl3, d): 19.0 (C-15), 20.0 (C-14), 31.3 (C-3), 32.2 (C-2), 34.0 (C-4), 35.0 (C-9), 46.1 (C-7), 62.6 (C-1), 68.4 (C-6), 78.0 (C-8), 124.2 (C-13), 130.6 (C-5), 137.4 (C-10), 138.5 (C-11), 171.2 (C-12) (Jin et al. 2006). 8,10-Dihydroxy-9-isobutyryloxythymol (7) Colorless oil, ESIMS m/z 291 [M ? Na]?; 267 [M - H]-; C14H20O5; 1H-NMR (400 MHz, CD3OD, d): 7.16 (1H, d, J = 8.0 Hz, H-5), 6.64 (1H, dd, J = 8.0, 1.0 Hz, H-6), 6.60 (1H, d, J = 1.0 Hz, H-2), 4.55 (1H, d, J = 11.0 Hz, H-9a), 4.41 (1H, d, J = 11.0 Hz, H-9b), 3.91 (1H, d, J = 11.5 Hz, H-10a), 3.84 (1H, d, J = 11.5 Hz, H-10b), 2.49 (1H, m, H-20 ), 2.22 (3H, s, H3-7), 1.05 (3H, d, J = 7.0 Hz, H3-30 ), 1.03 (3H, d, J = 7.0 Hz, H3-40 ); 13CNMR (400 MHz, CD3OD, d): 19.2 (C-40 ), 19.3 (C-30 ), 21.1 (C-7), 35.2 (C-20 ), 66.7 (C-10), 68.3 (C-9), 78.7 (C-8), 118.1 (C-2), 121.2 (C-6), 123.9 (C-4), 128.6 (C-5), 140.0 (C-1), 156.7 (C-3), 178.8 (C-10 ) (Zhu et al. 2011). Chrysoeriol (8) Yellow powder, ESIMS m/z 323 [M ? Na]?; 299 [M H]-; C16H12O2; 1H-NMR (500 MHz, DMSO-d6, d): 12.98 (1H, brs, 5-OH), 7.70 (2H, d, J = 6.7 Hz, H-50 , 60 ), 6.94 (1H, d, J = 2.8 Hz, H-20 ), 6.90 (1H, s, H-3), 6.51 (1H, brs, H-8), 6.21 (1H, brs, H-6), 3.89 (3H, s, 30 -OCH3); 13C-NMR (500 MHz, DMSO-d6, d): 182.9(C-4), 165.4 (C-7), 165.0 (C-2), 162.6 (C-9), 158.3 (C-5), 152.1 (C-30 ), 149.5 (C-40 ), 123.1 (C-60 ), 121.8 (C-10 ), 117.2 (C-50 ), 111.6 (C-20 ), 104.9 (C-10), 104.3 (C-3), 100.0 (C-6), 95.6 (C-8), 57.1 (30 OCH3) (Agrawal 1989). 5, 6-Dihydroxy-3, 7, 40 -trimethoxyflavonol (9) Yellow powder, ESIMS m/z 367 [M ? Na]?, 343 [M H]-; C18H16O7; H-NMR (400 MHz, DMSO-d6, d): 12.32 (1H, s, 5-OH), 8.01 (2H, d, J = 9.0 Hz, H-20 , 60 ), 7.10 (2H, d, J = 9.0 Hz, H-30 , 50 ), 6.90 (H, s, H-80 ), 6.90 (H, s, H-80 ), 3.92 (3H, s, –OCH3), 3.87(3H, s, –OCH3), 3.86 (3H, s, –OCH3); 13C-NMR (400 MHz, DMSO-d6, d): 155.3 (C-2),
Three new sesquiterpene lactones from I. britannica
137.8 (C-3), 178.2 (C-4), 145.7 (C-5), 129.7 (C-6), 154.6 (C-7), 91.0 (C-8), 48.9 (C-9), 105.7 (C-10), 122.4 (C-10 ), 130.0 (C-20 , 60 ), 114.2 (C-30 , 50 ), 161.3 (C-40 ), 59.7 (3OCH3), 56.4 (7-OCH3), 55.5 (40 -OCH3) (Horie et al. 1998).
concentrations (50.0, 10.0, 2.0, and 0.4 lM) of samples for 30 min and incubated with LPS (1 lg ml-1) for 24 h. The amount of NO was determined by the nitrite concentration in the cultured RAW264.7 macrophage supernatants with the Griess reagent.
Luteolin (10) Yellow powder, ESIMS m/z 309 [M ? Na]?; m/z 285 [M - H]-; C15H10O6; 1H-NMR (400 MHz, DMSO-d6, d): 12.97 (1H, s, 5-OH), 10.85 (1H, brs, 7-OH), 7.42 (1H, brs, H-60 ), 7.40 (1H, s, H-20 ), 6.89 (1H, d, J = 8.1 Hz, H-50 ), 6.67 (1H, s, H-3), 6.44 (1H, d, J = 1.4 Hz, H-8), 6.19 (1H, d, J = 1.4 Hz, H-6); 13C-NMR (400 MHz, DMSO-d6, d): 93.9 (C-8), 98.9 (C-6), 102.9 (C-3), 103.7 (C-10), 113.4 (C20 ), 116.1 (C-50 ), 119.0 (C-60 ), 121.6 (C-10 ), 145.7 (C-30 ), 157.3 (C-5), 149.7 (C-40 ), 161.5 (C-9), 163.9 (C-2), 164.2 (C-7), 181.7 (C-4) (Liang et al. 2011). Tamarixetin (11) Yellow powder, ESIMS m/z 339 [M ? Na]?, m/z 315 [M - H]-; C15H10O6; 1H-NMR (400 MHz, DMSO-d6, d): 12.46 (1H, s, 5-OH), 7.71 (1H, d, J = 2.0 Hz, H-20 ), 7.68 (1H, dd, J = 8.0, 2.0 Hz, H-60 ), 6.93 (1H, d, J = 8.0 Hz, H-50 ), 6.56 (1H, s, H-8), 6.21 (1H, s, H-6), 3.80 (1H, s, 40 OCH3); 13C-NMR (400 MHz, DMSO-d6, d): 56.1 (40 OCH3), 94.1 (C-8), 98.6 (C-6), 103.4 (C-10), 112.1 (C-50 ), 115.8 (C-20 ), 122.1 (C-60 ), 122.3 (C-10 ), 135.7 (C-3), 147.7 (C-30 ), 149.2 (C-2), 151.8 (C-40 ), 157.7 (C-5), 164.4 (C-9), 167.4 (C-7), 176.2 (C-4) (Yuan et al. 2007). Artemetin (12) Yellow powder, ESIMS m/z 411 [M ? Na]?, m/z 387 [M - H]-; C20H20O8; 1H-NMR (400 MHz, DMSO-d6, d): 12.59 (1H, brs, 5-OH), 7.75 (1H, dd, J = 8.6, 1.8 Hz, H-60 ), 7.67 (1H, d, J = 1.8 Hz, H-20 ), 7.16 (1H, d, J = 8.6 Hz, H-50 ), 6.93 (1H, s, H-8), 3.93, 3.82, 3.74 (each 3H, s, 39 –OCH3), 3.33 (6H, s, 29 –OCH3); 13C-NMR (400 MHz, DMSO-d6, d): 91.5 (C-8), 105.6 (C-10), 111.3 (C-20 ), 111.6 (C-50 ), 122.0 (C-60 ), 122.1 (C-10 ), 131.6 (C6), 138.1 (C-3), 148.5 (C-30 ), 151.3 (C-40 ), 151.6 (C-5), 151.8(C-2), 155.5 (C-9), 158.7 (C-7), 178.3 (C-4), 60.0, 59.8, 56.5, 55.7, 55.6 (59 –OCH3) (Ahmed et al. 1988).
Results and discussion Compound 1 was isolated as orthorhombic crystals. Its molecular formula C17H24O6 was characterized by HRESIMS (positive) m/z 325.1,650 [M ? H]? (calcd. for C17H25O6, 325.1,646), indicating six degrees of unsaturation. The IR spectrum showed bands characteristic of hydroxyl (3,425 cm-1), carbonyl (1,762 and 1,738 cm-1), and olefinic bond (1,655 cm-1). The 1H and 13C NMR spectroscopic data of 1 were very similar to those of compound 5, except that one methylene of 5 was found to be an oxymethyne [dC 72.3 (C-9), dH 4.15 (1H, d, J = 2.4 Hz, H-9)] in compound 1 (Jin et al. 2006). These observations are consistent with a 16 Da surplus in molecular weight, indicating 1 a hydroxylated derivative of 5. Detailed analysis the 1H–1H COSY and HMBC plots suggested the hydroxyl attaching at C-9. The relative configuration of 1 was determined by the following NOESY correlations: H-6/H3-15 and H-7/H-8 (Fig. 3). Furthermore, the absolute stereochemistry of 1 was determined by an X-ray analysis using Cu Ka radiation (Fig. 4). Thus, the structure of 1 was determined as (4S,6S,7S,8S,9S)-1acetoxy-6,9-dihydroxy-1,10-secoeudesma-5(10),11(13)dien-12,8-olide. Compound 2 was obtained as white amorphous powder with a molecular formula C15H20O4 established by HRESIMS (positive) m/z 265.1,364 [M ? H]? (calcd. for C15H21O4, 265.1,373). Its IR spectrum showed absorption bands of hydroxyl (3,423 cm-1), carbonyl (1,757 cm-1), and olefinic bond (1,634 cm-1). The 1H NMR spectrum exhibited one methyl singlet [dH 1.85 (3H, s, H3-14)], one methyl doublet [dH 1.09 (3H, d, J = 7.0 Hz, H3-15)], three oxymethine [dH 3.42 (1H, dt, J = 9.2, 7.2 Hz, H-1); dH 4.30 (1H, t, J = 11.2 Hz, H-6); dH 4.42 (1H, brs, H-8)], and three olefinic protons [dH 5.30 (1H, brs, H-3); dH 6.00 (1H, d, J = 2.8 Hz, H-13a); dH 5.54 (1H, d, J = 2.4 Hz,
Determination of NO production Inhibitory activities against LPS-induced NO production in RAW 264.7 macrophages (Schmidt and Kelm 1996) were selected as the anti-inflammatory screening method. RAW264.7 cells grown on 100 mm culture dish were harvested and seeded in 96-well plates (1 9 105 cells/well) for NO production. The plates were pretreated with various
Fig. 3 Key NOESY correlations of 1
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X.-F. Zhang et al. Fig. 4 Single-crystal X-ray structure (copper radiation) of 1
Fig. 5 Key 1H–1H COSY and HMBC correlations of 2
H-13b)]. The corresponding carbon signals were differentiated by 13C NMR and DEPT spectra (Table 2) as two methyl [dC 12.7 (C-14) and 23.2 (C-15)], three oxymethine [dC 73.9 (C-1), 77.3 (C-6) and 63.4 (C-8)], and two olefinic carbons [dC 121.7 (C-3) and 118.0 (C-15)]. The 13C NMR spectrum also revealed three sp2 disclosed quaternary carbons including two olefinic, and one easter carbonyl carbons. The remaining one degree of unsaturation was due to a tricyclic core. The analysis of 1H–1H COSY spectrum established the key sequences of H-1/H2-2/H-3 and H-5/H6/H-7/H-8/H2-9 (Fig. 5). In addition, the HMBC correlations from H2-2 to C-3 and C-4, H-8 to C-6 and C-10, H213 to C-7, C-11 and C-12, H3-14 to C-1, C-5, C-9 and C-10, H3-15 to C-3, C-4 and C-5, and the chemical shift of C-8 (dH 4.42) indicated the planar structure of 2 was 1,6dihydroxy-eudesma-3(4),11(13)-dien-12,8-olide (Fig. 5). In the NOESY spectrum (Fig. 6), correlations between H-1/H-5, H-5/H-7, H-7/H-8, and H-6/H3-14 were observed. Thus, compound 2 was determined to be 1b,6a-dihydroxy5aH-eudesma-3(4),11 (13)-dien-12,8b-olide. Compound 3 was isolated as yellowish oil and share the same molecular formula of C15H20O4 with 2 deduced from HRESIMS (positive) m/z 265.1,339 [M ? H]? (calcd. for
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Fig. 6 Key NOESY correlations of 2
C15H21O4, 265.1,332). A close comparison of NMR spectra between the two compounds indicated that 3 was also a eudesmane-type sesquiterpene lactone with hydroxyl groups substituted at C-1 and C-6. The presence of a methylene [dC 36.2 (C-3), dH 2.30 (m, H-3a) and 2.10 (m, H-3b)] and olefinic methylene [dC 110.3 (C-15), dH 5.00 (s, H-15a) and 4.78 (s, H-15b)] in compound 3 instead of the corresponding methyl and olefinic methane at C-3 and C-15 in compound 3 suggested that the olefinic group of 3 was located at C-4 and C-15 rather than at C-3 and C-4. This inference was further determined by the 1H–1H COSY and HMBC experiments. The NOESY experiment revealed the relative configuration of 3, which was similar to that of 2. Therefore, 3 was identified as 1b,6a-dihydroxy-5aHeudesma-4(15),11(13)-dien-12,8b-olide. Compounds 1–4 were tested for their inhibitory effects against LPS-induced NO production in RAW264.7
Three new sesquiterpene lactones from I. britannica
macrophages with aminoguanidine as positive control (Schmidt and Kelm 1996). Compounds 1–3 exhibited significant inhibitory activities with IC50 values of 3.99, 5.47, and 3.47 lM, respectively. Whereas, compound 4 showed moderate activity, with IC50 value of 28.65 lM. Acknowledgments The work was supported by program NCET Foundation, NSFC (81230090 and 81102778), partially supported by Global Research Network for Medicinal Plants (GRNMP) and King Saud University, Shanghai Leading Academic Discipline Project (B906), FP7-PEOPLE-IRSES-2008 (TCMCANCER Project 230232), Key laboratory of drug research for special environments, PLA, Shanghai Engineering Research Center for the Preparation of Bioactive Natural Products (10DZ2251300) and the Scientific Foundation of Shanghai China (10DZ1971700, 12401900501). National Major Project of China (2011ZX09307-002-03 and 2011ZX09102-006-02). National Key Technology R&D Program of China (2012BAI29B06).
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