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Planta Med 2004; 70(5): 479-482
DOI: 10.1055/s-2004-818983
Letter
© Georg Thieme Verlag KG Stuttgart · New York

Eremophilane Sesquiterpene Lactones from Ligularia virgaurea ssp. oligocephala

Quan-Xiang Wu1 , Yan-Ping Shi1 , 2 , Li Yang1
  • 1National Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou, P. R. China
  • 2Key Laboratory of Natural Medicine for Gansu Province, Lanzhou Institute of Chemical Physics (LICP), Chinese Academy of Sciences (CAS), Lanzhou, P. R. China
Further Information

Prof. Yan-Ping Shi

Lanzhou Institute of Chemical Physics

Chinese Academy of Sciences

Lanzhou 730000

Peoples Republic of China

Fax: +86-931-8277088

Email: shiyp@lzu.edu.cn

Email: shiyp@ns.lzb.ac.cn

Publication History

Received: October 17, 2003

Accepted: February 22, 2004

Publication Date:
04 May 2004 (online)

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Table of Contents #

Abstract

From an extract of the whole plant of Ligularia virgaurea ssp. oligocephala, three new eremophilane sesquiterpene lactones, 10α-hydroxy-1-oxoeremophila-7(11),8(9)-dien-12,8-olide (1), 6α,10α-dihydroxy-1-oxoeremophila-7(11),8(9)-dien-12,8-olide (2), 6β,10β-dihydroxyeremophila-7(11),8(9)-dien-12,8-olide (3), as well as four known sesquiterpene lactones with the same carbon skeleton, named toluccanolides A - C (4, 5, 7) and 6β-hydroxy-8α-methoxyeremophila-1(10),7(11)-dien-12,8β-olide (6) were isolated. Their structures were elucidated by spectroscopic methods including intensive 2D NMR techniques (gCOSY, gHMQC, gHMBC and 1H-1H NOESY for 2) and HR-ESI-MS.

We have isolated several new sesquiterpenoids from the genera belonging to Artemisia, Senecio, Carpesium, Halenia, Salvia, and Ligularia [1], [2], [3], [4], [5], [6], [7]. The genus Liguaria was found to be an important source of sesquiterpenes of the eremophilane type. In the course of our search for bioactive sesquiterpenoids, we selected Ligularia virgaurea ssp. oligocephala, which has long been used as a traditional folk medicine for the treatment of stomachache and nausea [8]. In this study we describe the isolation and structure elucidation of eremophilane sesquiterpene lactones 1 - 7 (Fig. [1]), of which compounds 1 - 3 are new constituents obtained from this species.

Three new eremophilanolides 1 - 3 and four known ones 4 - 7 were isolated from the alcoholic extract of the whole plant of Ligularia virgaurea ssp. oligocephala. The known compounds 4 - 7 were identified by direct comparison of their spectral data (1H-NMR and 13C-NMR and DEPT) and melting points with those reported in the literature [9], [10]. To the best of our knowledge compounds 1 - 3 are new constituents isolated from this species.

Compound 1 was obtained as colorless needles. Its molecular formula of C15H18O4 was deduced from HR-ESI-MS (m/z = 263.1277 [M + H]+) and indicated that it has 16 mass units less than 2. Its IR spectrum showed absorptions for hydroxy (3446 cm-1), ketone groups (1717 cm-1) as well as α, β-unsaturated γ-lactone (1772 cm-1) and double bond (1647 cm-1) functions. The 1H- and 13C-NMR spectra (Tables [1] and 2) of 1 were close to those of 2. The oxymethine proton at C-6 in 2 (δH = 4.50 and δC = 68.62) was replaced in 1 by signals at δH = 2.53, 2.75 and δC = 30.47, indicative of a methylene group. Consequently, the structure of compound 1 was determined as 10α-hydroxy-1-oxoeremophila-7(11),8(9)-dien-12,8-olide.

Compound 2 was obtained as a colorless gum. The molecular formula was assigned as C15H18O5 on the basis of the HR-ESI-MS (m/z = 574.2641 [2M + NH4]+). The 13C-NMR spectrum displayed 15 carbons including three methyls, two methylenes, three methines and seven quaternary carbons, assigned by DEPT experiment. Its IR spectrum showed absorption bands for hydroxy (3425 cm-1) and ketone (1722 cm-1) groups, as well as an α,β-unsaturated γ-lactone (1775 cm-1) and double bond (1670 cm-1) functions. In the downfield region of the 13C-NMR spectrum, there were some characteristic signals at δC = 207.10 due to a ketone carbonyl group, and δC = 146.51, 127.31; δC = 150.15, 104.41 and δH = 6.18 ascribed to two double bond functions, and δC = 170.32 to a carbonyl group indicating an α,β-unsaturated γ-lactone, along with δC = 68.62 and δH = 4.50 (1H, s) indicating an oxymethine, and δC = 79.99 an oxygen-bonded quaternary carbon. Based on the above spectral data, compound 2 was considered to be a α,β-unsaturated-γ-lactone sesquiterpene with a ketone and two hydroxy groups. From detailed inspection of the 1H- and 13C-NMR, and comparison of its spectral data with those of known eremophilanes [9], [10], [11], [12], [13], [14], [15], 2 was further confirmed as an eremophilenolide for its methyl group pattern: δH = 2.05 (3H, s) and δC = 8.78 (olefinic methyl group), δH = 0.57 (3H, d), J = 6.8, 1.06 Hz (3H, s) and δC = 13.15, 14.11. To establish the position of the only ketone carbonyl group, compound 2 was subjected to gCOSY, gHMQC and gHMBC experiments, respectively. The location of the carbonyl group was assigned using a gCOSY experiment: H-2αβ (δH = 3.15, 2.32) showing a correlation with H-3αβ (δH = 1.62, 1.93), H-3αβ showing a correlation with H-4α (δH = 3.12), and H-4α showing a correlation with CH3 - 15 (δH = 1.06), and using a gHMBC experiment: H-2α (δH = 3.15), H-3αβ (δH = 1.62, 1.93) and H-9 (δH = 6.18) showing a correlation with C-1 (δC = 207.10) (Fig. [2]). The NOESY experiment on 2 established the relative stereochemistry of the OH group at C-6 as being α-oriented based on correlations of H-6β with H-14, H-15 and H-13, respectively. The hydroxy group attached at C-10 was established as being α-oriented by the larger coupling constant between H-3β (axial bond) and H-4α (axial bond), J 3 β , 4 α = 13.6 Hz, in accordance with 4β,5β-Me. (Table [1]). Assignments of the 13C-NMR data were based on a gHMQC experiment. Hence, compound 2 was elucidated as 6α,10α-dihydroxy-1-oxoeremophila-7(11),8(9)-dien-12,8-olide.

Compound 3 was obtained as a colorless gum. Its molecular formula was assigned as C15H20O4 on the basis of the HR-ESI-MS (m/z = 264.1593 [M]+). The 13C-NMR spectrum displayed 15 carbons including three methyls, three methylenes, three methines and six quaternary carbons, assigned by a DEPT experiment. The IR spectrum showed absorption bands for hydroxy (3444 cm-1), α,β-unsaturated γ-lactone (1763 cm-1) and double bond (1690, 1653 cm-1) functions. The 1H- and 13C-NMR spectra of 3 (Tables [1] and 2) were close to those of 2. Careful inspection of the 1H- and 13C-NMR of compounds 2 and 3 suggested that compound 3 was also an eremophilanolide lacking the ketone group present in 2. The relative stereochemistry of 10-OH was determined as being in a β-orientation based on the following data: firstly, the larger change of the chemical shift of CH3 - 14, i. e., CH3 - 14 downfield shifted from δH = 0.57 ppm in 2 to δH = 1.27 ppm in 3. Secondly, the almost same NMR chemical shifts of CH3 - 13, CH3 - 14 and CH3 - 15, along with C-1, C-2, C-3, C-4 and C-10 of 3 (Tables [1] and 2) when compared to the known compound 8 (Fig. [1]), possessing a cis-fusion of the A/B rings [literature data: δC = 8.8, δH = 1.92 ppm (CH3 - 13), δC = 10.5, δH = 1.23 ppm (CH3 - 14), δC = 16.1, δH = 0.85 ppm (CH3 - 15)] [11], [12]. Hence, the structure of compound 3 was elucidated as 6β,10β-dihydroxyeremophila-7(11),8(9)-dien-12,8-olide.

Zoom Image

Fig. 1 Structures of sesquiterpenes 1 - 8.

Table 1 1H-NMR spectral data of compounds 1, 2 and 3 (400.16 MHz, in CDCl3, δ values, TMS); coupling constants (Hz) are in parentheses
No. 1 2 3
1α
1β		 - - 1.81 - 1.88 m
1.15 - 1.43 m
2β
2α		 2.28 dd (5.2, 13.6)
3.14 ddd (8.4, 13.6, 13.6)		 2.32 dd (4.4, 13.6)
3.15 ddd (7.8, 13.6, 13.6)		 1.58 - 1.66 m
1.15 - 1.43 m
3β
3α		 1.60 dddd
(5.2, 13.6, 13.6, 13.6)
1.88 m		 1.62 dddd
(4.4, 13.6, 13.6, 13.6)
1.93 m		 1.15 - 1.13 m
4α 2.64 m 3.12 dq (13.6, 6.8) 1.15 - 1.43 m
6 2.75 d (16.8)
2.53 d (16.8)		 4.50 s 4.64 s
9 6.12 s 6.18 s 5.52 s
13 1.92 s 2.05 s 2.11 s
14 0.64 s 0.57 s 1.27 s
15 0.93 d (7.6) 1.06 d (6.8) 0.78 d (6.4)
Table 2 13C-NMR spectral data of compounds 1, 2 and 3 (100.32 MHz, in CDCl3, δ values, TMS); multiplication determined by DEPT
No. 1 2 3
1 208.5 s 207.10 s 36.73 t
2 36.35 t 36.47 t 22.43 t
3 29.92 t 29.16 t 30.07 t
4 32.88 d 28.17 d 34.33 d
5 45.72 s 47.37 s 45.33 s
6 30.47 t 68.62 d 68.86 d
7 146.12 s 146.51 s 147.37 s
8 151.97 s 150.15 s 149.07 s
9 103.86 d 104.41 d 111.16 d
10 78.47 s 79.99 s 76.70 s
11 124.21 s 127.31 s 125.07 s
12 170.49 s 170.32 s 170.71 s
13 8.72 q 8.78 q 8.44 q
14 13.50 q 13.15 q 10.54 q
15 14.46 q 14.11 q 15.85 q
Zoom Image

Fig. 2 Key gHMBC correlations (H to C) of 2.

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Materials and Methods

Melting points were determined with an X-4 Digital Display Micro-Melting point apparatus and are uncorrected. Optical rotations recorded in CHCl3 using a Perkin Elmer 241 polarimeter. UV spectra were measured on a Spect 50-UV/Vis instrument (Analytic Jena AG). IR spectra were measured on an FTS165-IR instrument (Bio-Rad, USA). NMR spectra and 2D-NMR were recorded on a Varian INOVA-400 FT-NMR spectrometer (USA) in CDCl3 or acetone-d 6 with TMS as internal standard. HR-ESI-MS were recorded on a Bruker APEX II. Silica gel (200 - 300 mesh) used for column chromatography and silica gel (GF254) for TLC were supplied by the Qingdao Marine Chemical Factory in China. Spots were detected on TLC by visualization under UV light, or by spraying with 98 % H2SO4-EtOH (V:V = 5 : 95) followed by heating at 110 °C.

Ligularia virgaurea ssp. oligocephala Good (Compositae) was collected in Huzhu County, Qinghai province, P. R. China in August 2002 and was identified by adjunct Prof. Ji Ma, Faculty of Pharmacy, First Military Medical University of PLA, Gangzhou, P. R. China. A voucher specimen (No. 2 002 001) has been deposited at Key Laboratory of Natural Medicine for Gansu province.

The air-dried whole plant (4 kg) was powdered and extracted with 95 % EtOH at room temperature (10 L × 4, each extraction lasted 7 days). The combined extracts were evaporated to dryness under reduced pressure. The residue (340 g) was then suspended in H2O (2.2 L), extracted with petroleum ether (60 - 90 °C) (2.5 L), EtOAc (1.0 L) and n-BuOH (1.0 L), respectively. The EtOAc extract (75g) was subjected to column chromatography on silica gel (1000 g) using petroleum ether (60 - 90 °C) with increasing volumes of acetone (V:V = 50 : 1, 30 : 1, 15 : 1, 10 : 1, 7 : 1, 5 : 1, 3 : 1, 1 : 1, each about 3.0 L) as eluent. Fractions were examined by TLC and combined to afford 8 pooled fractions (1A - 1H). Fraction 1C (4.2 g) was further fractionated on a silica gel column (90 g) using petroleum ether-EtOAc (8 : 1, 1100 mL) to give impure compound 7 that was purified, firstly, on a silica gel column (70 g) using petroleum ether-acetone (15 : 1, 800 mL), then by preparative TLC (using petroleum ether-Et2O = 1 : 1 as a mobile phase) to yield pure 7 (50 mg). Fraction 1D (3.4 g) was further fractionated on a silica gel column (35 g) and eluting with petroleum ether-acetone (15 : 1, 1000 mL) to give two fractions (fr. 1D1, 700 mL and fr. 1D2, 300 mL). Fr. 1D1 (2.0 g) was further subjected to column chromatography on silica gel (20 g) and eluting with petroleum ether-EtOAc (8 : 1, 800 mL) to yield impure 6, which was purified on a silica gel column (15 g) eluting with CH2Cl2-EtOAc (100 : 1, 300 mL) to obtain pure 6 (10 mg). Fr. 1D2 (0.8 g) was further purified by preparative TLC using petroleum ether-EtOAc (2 : 1) to give pure 4 (10 mg) and impurity compound 1 that was further purified by preparative TLC using petroleum ether-acetone (2 : 1) to give pure 1 (7 mg). Fraction 1E (6.0 g) was further fractionated on a silica gel column (80 g), eluting with petroleum ether-EtOAc (7 : 1) to give three fractions (fr. 1E1, 200 mL fr. 1E2, 500 mL, and fr. 1E3, 300 mL). Fr. 1E2 (1.5 g) was further subjected to column chromatography on silica gel (70 g) eluting with petroleum ether-acetone (8 : 1, 800 mL) to obtain compounds 2 (5.1 mg) and 3 (6.5 mg). Fraction 1E3 (2.1 g) was purified on a silica gel (30 g) column using CH2Cl2-EtOAc as eluent to give 5 (23 mg).

10α-Hydroxy-1-oxoeremophila-7(11),8(9)-dien-12,8-olide, C15H18O4 (1): Colorless needles from acetone, m. p. 177 - 178 °C, [α]D 26: -87.0° (c 0.17, CHCl3), Rf value: 0.50 (petroleum ether-acetone, 8 : 1). IR (film): νmax = 3446, 2359, 2341, 1772 (α,β-unsaturated γ-lactone), 1717 (ketone carbonyl function), 1647 (double bond), 1558, 1008, 757, 552 cm-1; UV (MeOH): λ (log ε) = 213.4 (4.97), 273.2 (5.04) nm. HR-ESI-MS: m/z = 263.1277 [M + H]+; required: m/z = 263.1278 for C15H19O4; 1H- and 13C-NMR: see Tables [1] and 2, respectively.

6α,10α-Dihydroxy-1-oxoeremophila-7(11),8(9)-dien-12,8-olide, C15H18O5 (2): Colorless gum, [α]D 26: -38.0° (c 0.14, CHCl3); Rf value: 0.30 (petroleum ether-acetone, 8 : 1); IR (film): νmax = 3425, 2955, 2934, 2868, 2360, 1775 (α,β-unsaturated γ-lactone), 1722 (ketone carbonyl function), 1670 (double bond), 1378, 1023, 751, 552 cm-1; UV (MeOH): λ (log ε) = 212.8 (4.87), 274.7 (4.61) nm; HR-ESI-MS: m/z = 574.2641; [2M + NH4]+; required: m/z = 574.2647 for C30H44O10N. 1H- and 13C-NMR: see Tables [1] and 2, respectively.

6β,10β-Dihydroxyeremophila-7(11),8(9)-dien-12,8-olide, C15H20O4 (3): Colorless gum, [α]D 26: -20.0° (c 0.05, CHCl3); Rf value: 0.35 (petroleum ether-acetone, 8 : 1); IR (film): νmax = 3444, 2938, 2879, 2360, 2341, 1763 (α,β-unsaturated γ-lactone), 1690, 1653 (double bond), 1381, 1008, 755, 668 cm-1; UV (MeOH): λ (log ε) = 213.6 (5.30), 278.1 (4.74) nm; HR-ESI-MS: m/z = 264.1593 [M]+; required: m/z = 264.1356; 1H- and 13C-NMR: see Tables [1] and 2, respectively.

Toluccanolides A - C (4, 5, 7) and 6β-hydroxy-8α-methoxyeremophila-1(10),7(11)-dien-12,8β-olide (6): all as colorless needles. For melting point and 1H-NMR data see [9], [10], and for 13C-NMR see Table [3]; [α]D 24: -85.0° (c 0.20, CHCl3) for 4, -77.0° (c 0.20, CHCl3) for 5, -115.0° (c 0.26, CHCl3) for 6, -115.0° (c 0.32, CHCl3) for 7. Additionally, copies of original spectra (1H-NMR, 13C-NMR and DEPT) are obtainable from the author of correspondence.

Table 3 13C-NMR spectral data of compounds 4, 5, 6 and 7 (100.32 MHz, in CDCl3, δ values, TMS); multiplicity determined by DEPT
No 4 5a 6 7
1 128.92 d 128.61 d 129.06 d 128.92 d
2 23.60 t 25.21 t 24.75 t 24.71 t
3 27.46 t 30.37 t 28.60 t 28.57 t
4 35.52 d 38.12 d 37.78 d 37.73 d
5 45.64 s 45.72 s 46.86 s 46.77 s
6 78.58 d 78.13 d 78.67 d 78.63 d
7 161.76 s 160.82 s 157.31 s 158.20 s
8 77.91 d 103.21 s 105.16 s 105.22 s
9 40.06 t 45.66 t 43.42 t 43.48 t
10 134.07 s 136.73 s 134.47 s 134.45 s
11 128.92 s 123.79 s 125.95 s 128.82 s
12 175.20 s 172.54 s 171.80 s 172.12 s
13 8.01 q 8.81 q 8.84 q 8.79 q
14 17.34 q 18.61 q 18.52 q 18.45 q
15 13.91 q 13.02 q 12.51 q 12.53 q
OCH3 - - 50.52 q -
OCH2CH3 - - - 58.64, 15.25
a Run in acetone-d 6.
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Acknowledgements

The authors are greatly indebted to adjunct Professor Ji Ma (Faculty of Pharmacy, First Military Medical University of PLA, Gangzhou, P. R. China) for her help in identification of the plant material.

#

References

Prof. Yan-Ping Shi

Lanzhou Institute of Chemical Physics

Chinese Academy of Sciences

Lanzhou 730000

Peoples Republic of China

Fax: +86-931-8277088

Email: shiyp@lzu.edu.cn

Email: shiyp@ns.lzb.ac.cn

#

References

Prof. Yan-Ping Shi

Lanzhou Institute of Chemical Physics

Chinese Academy of Sciences

Lanzhou 730000

Peoples Republic of China

Fax: +86-931-8277088

Email: shiyp@lzu.edu.cn

Email: shiyp@ns.lzb.ac.cn

   Permissions and Reprints
Zoom Image

Fig. 1 Structures of sesquiterpenes 1 - 8.

Zoom Image

Fig. 2 Key gHMBC correlations (H to C) of 2.