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Planta Med 2000; 66(2): 189-191
DOI: 10.1055/s-0029-1243131
Letter

© Georg Thieme Verlag Stuttgart · New York

Eremophilenolides from Ligularia fischeri

Wen-Shu Wang1 , Kun Gao1 , Li Yang1 , Zhong-Jian Jia1
  • 1Department of Chemistry, National Laboratory of Applied Organic Chemistry, Lanzhou University, People's Republic of China
Further Information

Prof. Zhong-Jian Jia

Department of Chemistry

Lanzhou University

Lanzhou 730000

People's Republic of China

Email: zhengrl@lzu.edu.cn

Fax: +86-0931-8912582

Publication History

Received: March 22, 1999

Accepted: May 22, 1999

Publication Date:
24 November 2009 (online)

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Table of Contents #

Abstract

The chemical investigation of Ligularia fischeri afforded three new eremophilenolides which were identified as 6β-methoxy-8β-hydroxy-eremophil-7(11)-en-12,8α-olide, ligufischerin, and 6-oxo-8β-hydroxy-eremophil-7(11)- en-12,8α-olide by 1D- and 2D-NMR spectra. In addition, six known eremophilenolides were also obtained.

The roots of Ligularia fischeri (Ledeb.) Turcz. (Compositae) have been used as a traditional Chinese medicine to reduce phlegm, relieve coughing, invigorate the circulation of blood, and alleviate pain [1]. Earlier studies of this plant by Japanese research groups showed its main constituents to be furaneremophilanes [2], [3], [4], [5], eremophilenolides [2], and sesquiterpene oxides of the guaiane series [3]. The photosensitized oxygenation of furaneremophilanes as a biogenic process can produce a series of eremophilenolides [6], [7], [8], [9], which may account for the abundance of eremophilenolides in our study of the plant grown in China. From it we have isolated three new eremophilenolides (7, 8, 9) and six known compounds: 6β-hydroxy-8α-methoxy-eremophil-7(11)-en- 12,8β-olide (1) [6], [8], [9], [10], 6β-hydroxy-eremophil- 7(11)-en-12,8α-olide (2) [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13], 6β,8β-dihydroxy-eremophil-7(11)-en-12,8α-olide (3) [6], [9], [10], [11], [12], 6β-hydroxy-8β-methoxy-eremophil-7(11)-en- 12,8α-olide (4) [6] [9] [11], 6β-hydroxy-eremophil- 7(11)-en-12,8β-olide (5) obtained as a natural product for the first time and 8β-hydroxy-eremophil-7(11)-en- 12,8α-olide (6) [7] [14]. Because of the methanol extraction, compounds 1 and 4 might be artifacts of compound 2 [11]. In this article, we present the isolation and structural determination of the new compounds and give the 13C-NMR data of 5, 6 that have not been reported before.

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Compound 7 was deduced as C16H24O4 from the molecular ion peak m/z = 280 in EI-MS and combination of 13C-NMR and DEPT spectra. Actually, 7, 1, and 4 had not only the same [M]+ and very similar fragment peaks in EI-MS, but also similar features in their 1H- and 13C-NMR spectra [11]. Thus, 7 could be an isomer of 1 and 4. However, the hemi-ketal carbon signal of 7 was at a higher field, while the signal for C-6 was at a lower field than the corresponding carbons in 1 and 4 ([Table 3]). We attributed this to 8-hydroxy, 6-methoxy groups in 7 [15]. For eremophilenolides containing an 8β- lactone, the CH3-15 signal appeared at a lower field than the CH3-14 signal, whereas this phenomenon was reversed in 8α-lactones [6] [10] [11]. Therefore, 7 was assigned as an 8α-lactone by its methyl signals in 1H-NMR spectrum ([Table 1]). The H-6α configuration in 7 came from the absence of a long-range coupling between the olefinic methyl protons (CH3-13) and the allylic proton (H-6α) in this 8α-lactone [10]. Hence 7 was 6β-methoxy-8β-hydroxy-eremophil-7(11)-en-12,8α-olide.

Table 1  1H-NMR spectral data of compounds 5, 7, 8, and 9a (400 MHz, CDCl3, TMS as internal standard).
H 5 7 8 9
6 5.09 d (1.3) 4.11 s 4.60 d (1.1)
8α 4.81 m
9α 2.04–2.11 m 2.57 dd (14.8, 1.5) 2.63 d (16.0)
9β 1.76 dd (14.8, 3.4) 2.30 dd (16.0, 4.0)
13 2.04 dd (1.3, 1.6) 1.91 s 1.95 d (1.1) 2.03 s
14 0.82 s 1.10 s 0.87 s 1.13 s
15 1.02 d (7.2) 0.76 d (5.7) 0.96 d (8.8) 0.85 d (6.8)
OMe 3.37 s
a Chemical shifts (ppm), multiplicity, and coupling constants (Hz in parentheses).

Compound 8 had a molecular ion peak m/z = 248 in EI-MS and fifteen 13C signals (3 × CH3, 4 × CH2, 3 × CH, 5 × C) in the 13C- NMR spectrum. Thus its formula was C15H20O3. Three signals of methyl protons and a proton bearing oxygen in the 1H- NMR spectrum ([Table 1]) showed that 8 was an eremophilenolide with a 8β-lactone unit [6] [10] [11]. However, in the 13C-NMR spectrum the ketal carbon signal of 8 shifted to δ 90.76, and some other carbon signals also shifted when compared with the data of other eremophilenolides ([Table 3]). So in accordance with its unsaturation degree, an ether bond between C-8 and C-6 existed in 8, forming a four-membered ring. 2D-NMR spectra (HMQC, HMBC) strongly supported these assignments (Table2). Based on stereomodels, the oxygen ring had to have an α configuration which increases the angle between H-6β and CH3-13 causing a long range coupling between them [6] [10] [11]. At the same time, the stereochemistry of 8 was supported by the correlation peak between H-6β and CH3-15β in the NOESY spectrum.

The IR spectrum of compound 9 at 1740cm–1 verified the unsaturated γ-lactone, while the absorption at 1700cm–1 showed another carbonyl. The 1H- and 13C-NMR spectra ([Table 1] and [Table 3]) demonstrated it to be another eremophilenolide with the formula C15H20O4 suggested by EI-MS. However, a signal at δ = 186.67 in the 13C-NMR spectrum showed a carbonyl group rather than a hydroxy group in 9. The strong correlation between δ 1.13 (3H, s, CH3-14) and δ 186.67 in HMBC ([Table 2]) indicated that C-6 was a carbonyl carbon. We compared the three methyl proton signals of 9 with those of 7 in 1H-NMR ([Table 1]) and concluded that 9 had a 8β-hydroxy substituent. Finally, 9 was deduced as 6-oxo-8β-hydroxy-eremophil-7(11)-en-12,8α-olide.

Table 2 HMBC correlations of compounds 8 and 9.
8 9
H-6 C-14, C-10, C-11, C-7, C-5 H-9 C-10, C-8
H-9 C-10, C-8, C-5 H-13 C-12, C-11, C-7
H-13 C-12, C-11, C-7 H-14 C-10, C-6, C-5, C-4
H-14 C-6, C-5, C-4 H-15 C-5, C-4, C-3
H-15 C-5, C-4, C-3
Table 3  13C-NMR (DEPT) spectral data of compounds 5, 6, 7, 8, and 9 (100 MHz, CDCl3).
C 5 6 7 8 9 DEPT
1 28.33 26.16 29.18 29.95 29.95 CH2
2 20.17 21.54 25.36 21.48 26.51 CH2
3 28.33 30.51 30.38 30.97 30.85 CH2
4 31.68 29.59 28.91 38.00 35.81 CH
5 44.76 40.36 42.87 47.58 47.01 C
6 71.18 38.99 80.46 76.86 186.67* CH
7 162.95 158.87 154.07 164.19 162.93 C
8 77.55 103.69 103.68 90.76 103.28 C
9 35.88 39.52 39.01 32.94 39.80 CH2
10 35.31 34.99 34.30 38.00 38.78 CH
11 122.08 122.67 127.00 125.75 134.85 C
12 175.15 172.17 171.21 173.79 171.49 C
13 8.93 8.27 8.35 9.48 9.00 CH3
14 18.96 20.51 16.28 17.06 20.01 CH3
15 15.14 16.01 16.09 13.47 16.18 CH3
OMe 58.25 CH3
* quaternary C in DEPT.
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Materials and Methods

All optional rotations were measured on a Perkin-Elmer 241 polarimeter. IR spectra were taken on a Nicolet 170sx FT-IR spectrometer. UV spectra were recorded on a Shimadzu UV-260 UV spectrometer. 1H-NMR (400MHz, CDCl3), 13C-NMR (100MHz, CDCl3) spectra, and 2D-NMR spectra (HMQC, HMBC) were recorded on a Bruker AM 400FT-NMR spectrometer with TMS as internal standard. EIMS data were obtained on an HP-5988 MS spectrometer. Silica gel (200–300 mesh) was used for CC and silica GF254 for TLC. Spots were detected on TLC under UV or by heating after spraying with 5 % H2SO4 in C2H5OH.

The whole plant of L.fischeri was collected in Shennongjia, Hubei Province, China, in 1994, and identified by Prof. Z. N. Zhao of the Wuhan Institute of Botany, Chinese Academy of Sciences. A voucher specimen (No. 949701) is deposited in Chemistry Department of Lanzhou University.

The chopped whole plant material (4.0kg) was extracted repeatedly (3 times, 7 days each time) with petroleum ether (60–90 °C)-Et2O-methanol (1 : 1:1) at room temperature to give a residue (120g) after evaporation. This residue was separated on CC over 1200 g silica gel (200–300 mesh) with a gradient (3000 ml each eluant) of petroleum ether-acetone (30 : 1, 20 : 1, 15 : 1, 10 : 1, 5 : 1, 3 : 1, 1 : 1, 0 : 1). From the fraction of petroleum ether-acetone (15 : 1), crude crystals of 1, 2 and 3 were obtained successively, then recrystallized from petroleum ether to give 1 (40mg), 2 (60mg), 3 (80mg). The mother liquid from the crystals of 1 was separated by repeated (3 times) preparative TLC with CHCl3-CH3COCH3 (20 : 1, 3 × 70ml) to afford 4 (10mg), Rf = 0.45 (CHCl3-CH3COCH3, 10 : 1). The fraction of petroleum ether-acetone (10 : 1, 15g) was separated on CC over 200 g silica gel (200–300 mesh) with benzene-AcOEt (40 : 1, 900 ml, 30 : 1, 900ml) to give two subfractions A and B. Fraction A (7.5g) was placed on CC over 70 g silica gel (200–300 mesh) with petroleum ether-AcOEt (5 : 1, 400ml) to give crude 6, 7, and 8. Subsequently, each of them was purified by preparative TLC with petroleum ether-AcOEt (5 : 1, each with 80ml) to afford 6 (8mg), Rf = 0.50 (petroleum ether-AcOEt, 5 : 1).

Fraction B (5.8g) was separated on CC over 60 g silica gel (200–300 mesh) with petroleum ether-AcOEt (5 : 1, 350ml); 7 (20mg), Rf = 0.47 (petroleum ether-AcOEt, 5 : 1) and 8 (30mg), Rf = 0.36 (petroleum ether-AcOEt, 5 : 1).

The resulting mixture of 5 and 9 was separated by repeated (3 times) preparative TLC with CHCl3-CH3COCH3 (20 : 1, 3 × 80ml) to afford 5 (10mg), Rf = 0.40 (CHCl3-CH3COCH3, 10 : 1); 9 (15mg) Rf = 0.50 (CHCl3-CH3COCH3, 10 : 1).

6α-Methoxy-8β-hydroxy-eremophil-7(11)-en- 12,8α-olide (7): m. p. 148–150 °C; [α]20 D: + 55° (acetone, c 1.0); IR: νKBr max = 3877 (OH), 1736 (γ-unsaturated lactone), 1650 (C = C) cm–1; EI-MS: m/z (%) = 280 [M]+ (3), 262 [M–H2O]+ (8), 180 (14), 174 (32), 156 (480), 154 (95), 140 (10), 124 (15), 109 (100), 59 (5), 43 (16); UV: λMeOH max = 221nm; 1H-NMR data ([Table 1]); 13C-NMR data ([Table 3]).

Ligufischerin (8): m. p. 221–223 °C; [α]20 D: + 20.0° (acetone c 1.0); IR: νKBr max = 1774 (γ-unsaturated lactone), 1670 (C = C) cm–1; EI-MS: m/z (%) = 248 [M]+ (52), 232 [M – O]+ (100), 231 (44), 123 (6), 109 (17), 95 (5), 81 (5), 69 (5), 67 (4); UV: λMeOH max = 233nm; 1H-NMR data ([Table 1]); 13C-NMR data ([Table 3]).

6-Oxo-8β-hydroxy-eremophil-7(11)-en-12,8α-olide (9): m. p. 216–218 °C; [α]20 D: + 27° (acetone c 1.0); IR: νKBr max = 3335 (OH), 1740 (γ-unsaturated lactone), 1700, 1677 (C = C) cm–1; EI-MS: m/z (%) = 264 [M]+ (6), 246 [M –H2O]+ (23), 230 (51), 217 (53), 213 (33), 203 (55), 189 (54), 149 (76), 119 (32), 109 (100), 84 (46) 81 (19), 67 (19), 43 (13); UV: λMeOH max = 261nm; 1H-NMR data ([Table 1]); 13C-NMR data ([Table 3]).

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Acknowledgements

This work was financed by the National Natural Science Foundation of China and the Foundation of the State Education Ministry of China for Doctoral Program.

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References

Prof. Zhong-Jian Jia

Department of Chemistry

Lanzhou University

Lanzhou 730000

People's Republic of China

Email: zhengrl@lzu.edu.cn

Fax: +86-0931-8912582

#

References

Prof. Zhong-Jian Jia

Department of Chemistry

Lanzhou University

Lanzhou 730000

People's Republic of China

Email: zhengrl@lzu.edu.cn

Fax: +86-0931-8912582

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