New Weakly Cytotoxic Eremophilane Sesquiterpenes from the Roots of Ligularia virgaurea

• Abstract
• Introduction
• Materials and Methods
• Apparatus
• Plant material
• Extraction and Isolation
• Results and Discussion
• Acknowledgements
• References

Abstract

Six new eremophilane sesquiterpenes, including a novel nortrieremophilane carbon skeleton, were isolated from the roots of Ligularia virgaurea. Their structures were elucidated as 3α,4α-epoxy-6α-(2′-methylacryloyl)oxy-8α-methoxyeremophil-7(11)-en-8β,12-olide (1), 3α,4α-epoxy-6α-(2′-methylacryloyl)oxy-8α-ethoxyeremophil-7(11)-en-8β,12-olide (2), 1β,10β-epoxy-6β-(2′-methylacryloyl)oxy-8β-methoxyeremophil-7(11)-en-8α,12-olide (3), 1β,10β-epoxy-6β-angeloyloxy-8β-methoxyeremophil-7(11)-en-8α,12-olide (4), 6β-methoxyeremophil-7(11)-en-8β,12-olide (5), and 5β-angeloyloxy-3a,4,5,6,7,7a-hexahydro-3aβ-methyl-1H-indene-2,4β-dioic acid methyl ester (6) by spectral methods, including IR, HR-ESI-MS, 1D and 2D NMR techniques. All of compounds were evaluated for their in vitro cytotoxic activities against human hepatoma (SMMC-7721), human promyelocytic leukemia (HL-60), and human hepatocyte (L-02) cells.

Introduction

The genus Ligularia has been taxonomically placed in the Compositae with ca. 110 species distributed within China [1]. More than 27 species have long been used as folk remedies due to their antibiotic, antiphlogistic, and antitumor activities [2]. Based on phytochemical investigations of many Ligularia species by our group [3], [4], [5], [6], [7], [8], sesquiterpenes are found to be the most widespread secondary metabolites. Continuing our research for novel bioactive sesquiterpenes from Ligularia species and the influence of different ecological environments on the chemical constituents of plants, we focused our attention on Ligularia virgaurea (Maxim.) Mattf. Previous chemical work on this plant has led to the isolation of some benzofuranosesquiterpenes and eremophilanes [9], [10], [11], [12], [13]. In this study, we reinvestigated the roots of this species collected from Gannan Tibetan Autonomous Region (S.A. 2200 - 3800 m) in the Gansu Province of People’s Republic of China which represents a different region and altitude from previous studies. Here we describe six new eremophilane sesquiterpenes (1 - 6; Fig. [1]) isolated from this species, and their cytotoxic activities evaluation.

Materials and Methods

Apparatus

Melting points were determined on an X-4 digital display micromelting point apparatus, and are uncorrected. Optical rotations were measured on a Perkin Elmer 341 polarimeter. IR spectra were taken on a Nicolet NEXUS 670 FT-IR spectrometer. NMR spectra were recorded on a Varian Mercury plus-400 NMR spectrometer with TMS as internal standard. HR-ESI-MS data were recorded on a Bruker Daltonics APEX II 47e spectrometer. EI-MS data were recorded on an HP5988A GC/MS spectrometer. Silica gel (200 - 300 mesh) used for column chromatography and silica gel GF₂₅₄ (10 - 40 μ) used for TLC were supplied by the Qingdao Marine Chemical Factory (Qingdao, P. R. China). Spots were detected on TLC under UV light or by heating after spraying with 5 % H₂SO₄ in C₂H₅OH (v/v).

Plant material

The roots of Ligularia virgaurea (Maxim.) Mattf. were collected in the Gannan Tibetan Autonomous Region, Gansu Province, People’s Republic of China, in August 2005. It was identified by Prof. Guo-Liang Zhang, School of Life Sciences, Lanzhou University. A voucher specimen (No. 20 050 801) was deposited in College of Chemistry and Chemical Engineering, Lanzhou University.

Extraction and Isolation

The air-dried roots of L. virgaurea (3.8 kg) were pulverized and extracted with petroleum ether (60 - 90 °C) (PE)-Et₂O-MeOH (1 : 1 : 1) (6 days × 3 times) at room temperature. The extract was concentrated under reduced pressure giving a residue (256 g), which was chromatographed on a silica gel column (200 - 300 mesh, 2000 g, 8.0 × 150 cm) with a gradient of PE-acetone (AC) (30 : 1; 15 : 1; 8 : 1; 5 : 1; 3 : 1; 1 : 1 and 0 : 1). According to TLC analysis, seven crude fractions (fr. A, 30 : 1, 6 L; fr. B, 15 : 1, 10 L; fr. C, 15 : 1 - 8 : 1, 12 L; fr. D, 8 : 1, 10 L; fr. E, 8 : 1 - 5 : 1, 10 L; fr. F, 5 : 1 - 3 : 1, 6 L and fr. G, 1 : 1 - 0 : 1, 6 L) were collected. Fr. C (50.0 g) was chromatographed on a silica gel column (500 g, 6.3 × 110 cm) with a gradient of CHCl₃-EtOAc (80 : 1; 50 : 1; 30 : 1; 10 : 1 and 0 : 1) to give five fractions (fr. C.1, 80 : 1, 2 L; fr. C.2, 80 : 1 - 50 : 1, 3 L; fr. C.3, 50 : 1 - 30 : 1, 3 L; fr. C.4, 30 : 1 - 10 : 1, 3 L and fr. C.5, 10 : 1 - 0 : 1, 2 L). Fr. C.1 (8.0 g) was chromatographed on a silica gel column (100 g, 2.8 × 60 cm) with PE-AC (20 : 1; 10 : 1; 5 : 1 and 0 : 1) to give seven subfractions (fr. C.1.1, 20 : 1, 0.2 L; fr. C.1.2, 20 : 1, 0.3 L; fr. C.1.3, 20 : 1 - 10 : 1, 0.3 L; fr. C.1.4, 10 : 1, 0.3 L; fr. C.1.5, 10 : 1, 0.2 L; fr. C.1.6, 5 : 1, 0.3 L and fr. C.1.7, 5 : 1 - 0 : 1, 0.3 L). Fr. C.1.4 (1.2 g) was chromatographed on a silica gel column (20 g, 2.0 × 30 cm) with PE-AC (10 : 1, 185 mL) and then worked-up by crystallization to afford compound 5 (65 mg). Fr. D (41.0 g) was chromatographed on a silica gel column (460 g, 6.3 × 110 cm) with CHCl₃-EtOAc (60 : 1; 40 : 1; 20 : 1 and 10 : 1) to afford six fractions (fr. D.1, 60 : 1, 2 L; fr. D.2, 60 : 1 - 40 : 1, 3 L; fr. D.3, 40 : 1, 3 L; fr. D.4, 40 : 1 - 20 : 1, 3 L; fr. D.5, 20 : 1, 3 L and fr. D.6, 10 : 1, 2 L). Fr. D.2 (6.5 g) was chromatographed on a silica gel column (80 g, 2.8 × 60 cm) with PE-AC (20 : 1; 10 : 1; 5 : 1 and 3 : 1) to give five fractions (fr. D.2.1, 20 : 1, 0.3 L; fr. D.2.2, 20 : 1 - 10 : 1, 0.4 L; fr. D.2.3, 10 : 1 - 5 : 1, 0.3 L; fr. D.2.4, 5 : 1, 0.3 L and fr. D.2.5, 3 : 1, 0.3 L). Fr. D.2.2 (1.3 g) was chromatographed on a silica gel column (20 g, 2.0 × 30 cm) with PE-EtOAc (8 : 1) to afford three fractions (fr. D.2.2.1, 85 mL; fr. D.2.2.2, 105 mL and fr. D.2.2.3, 98 mL). Fr. D.2.2.1 (106 mg) was purified by preparative TLC (silica gel GF₂₅₄, 10 - 40 μ, 25 × 25 cm) with PE-EtOAc (12 : 1, four developments), to afford three pure compounds: 1 (8 mg, Rf = 0.25, PE-EtOAc, 10 : 1), 3 (28 mg, Rf = 0.29, PE-EtOAc, 10 : 1), and 4 (20 mg, Rf = 0.35, PE-EtOAc, 10 : 1). Fr. D.2.2.2 (60 mg) was chromatographed with PE-AC (15 : 1, 56 mL) to give compound 2 (5 mg). Fr. D.2.3 (1.6 g) was chromatographed on a silica gel column (20 g, 2.0 × 30 cm) with PE-AC (13 : 1) to give four fractions (fr. D.2.3.1, 85 mL; fr. D.2.3.2, 203 mL; fr. D.2.3.3, 108 mL and fr. D.2.3.4, 85 mL). Fr. D.2.3.2 (560 mg) was further chromatographed with PE-EtOAc (8 : 1) to afford three subfractions (fr. D.2.3.2.1, 25 mL; fr. D.2.3.2.2, 38 mL and fr. D.2.3.2.3, 26 mL). Compound 6 (12 mg) was obtained from fr. D.2.3.2.2 (85 mg) by repeated column chromatography with PE-EtOAc (10 : 1, 36 mL). These solvents were recycled many times after careful distillations.

3α,4α-Epoxy-6α-(2′-methylacryloyl)oxy-8α-methoxyeremophil-7(11)-en-8β,12-olide (1): colorless gum, [α]_D ²⁰: + 102 (c 0.7, CHCl₃); IR (KBr): ν _max = 2966, 2935, 1775, 1721, 1636, 1454, 1315, 1158, 996 cm^-1; ¹H-NMR and ¹³C-NMR: see Tables [1] and 2, respectively; EI-MS: m/z (%) = 362 [M]⁺ (11), 330 (3), 293 (14), 276 (11), 261 (25), 244 (28), 224 (85), 192 (68), 69 (100); HR-ESI-MS: m/z = 380.2069 [M + NH₄]⁺ (calcd. for C₂₀H₃₀O₆N: 380.2068).

3α,4α-Epoxy-6α-(2′-methylacryloyl)oxy-8α-ethoxyeremophil-7(11)-en-8β,12-olide (2): colorless gum, [α]_D ²⁰: + 76 (c 0.4, CHCl₃); IR (KBr): ν _max = 2978, 2933, 1774, 1721, 1637, 1454, 1309, 1157, 996 cm^-1; ¹H-NMR and ¹³C-NMR: see Tables [1] and 2, respectively; EI-MS: m/z (%) = 376 [M]⁺ (3), 330 (1), 307 (5), 290 (3), 261 (7), 244 (4), 69 (100); HR-ESI-MS: m/z = 394.2230 [M + NH₄]⁺ (calcd. for C₂₁H₃₂O₆N: 394.2224).

1β,10β-Epoxy-6β-(2′-methylacryloyl)oxy-8β-methoxyeremophil-7(11)-en-8α,12-olide (3): colorless gum, [α]_D ²⁰: -37 (c 1.7, CHCl₃); IR (KBr): ν _max = 2972, 2936, 1774, 1723, 1636, 1452, 1289, 1152, 1015, 923 cm^-1; ¹H-NMR and ¹³C-NMR: see Tables [1] and 2, respectively; EI-MS: m/z (%) = 362 [M]⁺ (1), 330 (1), 293 (1), 276 (1), 261 (1), 244 (2), 224 (4), 192 (4), 69 (100); HR-ESI-MS: m/z = 380.2064 [M + NH₄]⁺ (calcd. for C₂₀H₃₀O₆N: 380.2068).

1β,10β-Epoxy-6β-angeloyloxy-8β-methoxyeremophil-7(11)-en-8α,12-olide (4): colorless gum, [α]_D ²⁰: -90 (c 1.5, CHCl₃); IR (KBr): ν _max = 2972, 2936, 1774, 1722, 1644, 1456, 1290, 1226, 1148, 1014, 924 cm^-1; ¹H-NMR and ¹³C-NMR: see Tables [1] and 2, respectively; EI-MS: m/z (%) = 376 [M]⁺ (1), 344 (1), 293 (1), 276 (1), 261 (1), 244 (1), 83 (100); HR-ESI-MS: m/z = 394.2231 [M + NH₄]⁺ (calcd. for C₂₁H₃₂O₆N: 394.2224).

6β-Methoxyeremophil-7(11)-en-8β,12-olide (5): colorless crystals, m. p. 86 - 88 °C; [α]_D ²⁰: -188 (c 1.1, CHCl₃); IR (KBr): ν _max = 2922, 2863, 1742, 1671, 1452, 1207, 1112, 1094, 1041, 761 cm^-1; ¹H-NMR and ¹³C-NMR: see Tables [1] and 2, respectively; EI-MS: m/z (%) = 264 [M]⁺ (11), 232 (1), 155 (35), 140 (100), 83 (63); HR-ESI-MS: m/z = 265.1799 [M + H]⁺ (calcd. for C₁₆H₂₅O₃: 265.1798).

5β-Angeloyloxy-3a,4,5,6,7,7a-hexahydro-3aβ-methyl-1H-indene-2,4β-dioic acid methyl ester (6): colorless gum, [α]_D ²⁰: + 14 (c 1.2, CHCl₃); IR (KBr): ν _max = 2952, 2881, 1734, 1649, 1623, 1439, 1257, 1214, 1191, 1148, 753 cm^-1; ¹H-NMR and ¹³C-NMR: see Tables [1] and 2, respectively; EI-MS: m/z (%) = 350 [M]⁺ (2), 318 (22), 267 (3), 250 (16), 218 (46), 83 (100); HR-ESI-MS: m/z = 368.2064 [M + NH₄]⁺ (calcd. for C₁₉H₃₀O₆N: 368.2068).

Results and Discussion

Compound 1 was obtained as a colorless gum. The molecular formula was deduced as C₂₀H₂₆O₆ from the HR-ESI-MS (at m/z = 380.2069 [M + NH₄]⁺, calcd.: 380.2068). The IR spectrum showed absorption bands for ester carbonyls (1775, 1721 cm^-1) and a double bond (1636 cm^-1). The ¹H- and ¹³C-NMR data of 1 (Tables [1] and 2) displayed the signals of a methoxy and a 2-methylacryloyloxy group. The EI-MS of 1 showed fragmental peaks at m/z = 276 [M - C₄H₆O₂]⁺ and 69 [C₄H₅O]⁺ which further confirmed the above inferences. Apart from the above groups, the ¹³C-NMR spectrum of 1 showed 15 signals for three methyls, three methylenes, three methines (two were oxygenated), and six quaternary carbons (one carbonyl, one ketal and two olefinic carbons), indicating a sesquiterpene skeleton with an α,β-unsaturated γ-lactone unit and in agreement with an eremophil-7(11)-en-8,12-olide structure [14], [15]. The remaining unsaturation could be an epoxy group, by the characteristic signals at δ _H = 2.94 (1H, brd, J = 2.2 Hz) and δ _C = 61.1 (CH) and 59.6 (C) [16]. The homonuclear coupling correlations from CH₂ (9) through CH (10), CH₂ (1), CH₂ (2) to CH (3) in the ¹H-¹H COSY spectrum, and the long-range correlations between H-3 with C-1, C-2, C-4, and C-5, H-6 with C-4, C-5, C-7, C-8, C-10, C-11, and C-1′, and the methoxy protons with C-8 in the gHMBC spectrum (Fig. [2]), indicated that an epoxy group existed between C-3 and C-4, that a 2-methylacryloyloxy group was attached to C-6, and that a methoxy group was located at C-8. Stereochemically, in the biogenetic consideration of eremophilane derivatives isolated from Compositae species, the methyls at C-4 and C-5 were both assigned the β-orientation [17], [18], thus, the epoxy group between C-3 and C-4 should be α-oriented. The NOE difference spectra of 1 showed that Me-14 had an NOE effect on H-10 (1.07 %), Me-15 had an effect on H-6 (2.33 %), but H-6 had no effect on 8-OMe. These observations implied a cis-fused A/B ring system, in which H-6 was β and 8-OMe was α-oriented. The α-orientation of 8-OMe was also confirmed by the absence of homoallylic coupling between H-6β and Me-13 in the ¹H-NMR spectrum [18]. Thus, the structure of compound 1 was determined as 3α,4α-epoxy-6α-(2′-methylacryloyl)oxy-8α-methoxyeremophil-7(11)-en-8β,12-olide.

The HR-ESI-MS with an [M + NH₄]⁺ peak at m/z = 394.2230 (calcd.: 394.2224) of 2 was in agreement with a molecular formula of C₂₁H₂₈O₆. The ¹H- and ¹³C-NMR spectra of 2 were very similar to those of 1, except for an ethoxy group [δ _H = 1.05 (3H, t, J = 7.2 Hz), 3.23 (1H, dq, J = 7.2, 8.8 Hz), and 3.43 (1H, dq, J = 7.2, 8.8 Hz); δ _C = 58.6 (CH₂) and 14.6 (CH₃)] at C-8 in 2 instead of a methoxy group as in 1. The relative configuration of 2 was supported by NOE difference spectra: there were effects between Me-14 with H-10 (1.09 %), Me-15 with H-6 (2.36 %), but no effect between H-6 with 8-OEt, indicating that H-10 and H-6 were β, and 8-OEt was α-oriented. The absence of homoallylic coupling between H-6β and Me-13 also indicated that 8-OEt was α-oriented [18]. Therefore, 2 was assigned as 3α,4α-epoxy-6α-(2′-methylacryloyl)oxy-8α-ethoxyeremophil-7(11)-en-8β,12-olide.

Compound 3 exhibited a molecular formula C₂₀H₂₆O₆ based on its HR-ESI-MS at m/z = 380.2064 [M + NH₄]⁺ (calcd.: 380.2068). The IR spectrum showed absorption bands for ester carbonyls (1774, 1723 cm^-1) and a double bond (1636 cm^-1). The NMR spectra of 3 (Tables [1] and 2) showed the presence of a methoxy, an epoxy and a 2-methylacryloyloxy groups. Comparison of the NMR spectra of 3 with those of 1 or 2 showed that they contained the same eremophilanolide carbon skeleton. In the gHMBC spectrum of 3, the correlations between the methoxy protons with C-8, H-1 with C-2, C-3, C-5, C-9 and C-10, H-6 with C-4, C-5, C-7, C-11, C-14 and C-1′, indicated the methoxy group at C-8, the epoxy group at C-1 and C-10, as well as the 2-methylacryloyloxy group at C-6, respectively. The stereochemistry of 3 was determined by the rules reported by Naya [19]. In the ¹H-NMR spectrum, the chemical shift of the tertiary methyl group [δ _H = 1.14 (s, Me-14)] was downfield comparing to the secondary methyl group [δ _H = 1.05, (d, J = 7.2 Hz, Me-15)], indicating the presence of an 8α(12)-olide. Furthermore, in the NOE experiment, irradiation of H-6 enhanced the signals of H-1 (1.29 %) and H-4α (5.01 %), but did not enhance the signals of 8-OMe, indicating that H-1 and H-6 were α and 8-OMe was β-oriented. Thus, 3 was identified as 1β,10β-epoxy-6β-(2′-methylacryloyl)oxy-8β-methoxyeremophil-7(11)-en-8α,12-olide.

The HR-ESI-MS of compound 4 gave an [M + NH₄]⁺ peak at m/z = 394.2231 (calcd.: 394.2224), establishing the molecular formula as C₂₁H₂₈O₆. The NMR data of 4 (Tables [1] and 2) showed a close resemblance to those of 3, and the only difference was an angeloyloxy group at C-6 in 4 instead of a 2-methylacryloyloxy group as in 3. Stereochemically, the downfield shift of the tertiary methyl group [δ _H = 1.14 (s, Me-14)], comparing to the secondary methyl group [δ _H = 1.05 (d, J = 7.6 Hz, Me-15)] [19], indicated the presence of an 8α(12)-olide. Furthermore, in the NOE difference spectrum, irradiation of H-6 enhanced the signals of H-1 (1.43 %) and H-4α (5.80 %), but did not enhance the signal of 8-OMe, indicating that H-1 and H-6 were α and 8-OMe was β-oriented. Thus, 4 was assigned as 1β,10β-epoxy-6β-angeloyloxy-8β-methoxyeremophil-7(11)-en-8α,12-olide.

Compound 5 was obtained as colorless crystals. The molecular formula was determined to be C₁₆H₂₄O₃ by its HR-ESI-MS (at m/z =265.1799 [M + H]⁺, calcd.: 265.1798). The IR spectrum showed absorption bands for an α,β-unsaturated γ-lactone (1742, 1671 cm^-1). The NMR spectra (Tables [1] and 2) showed compound 5 to be another eremophil-7(11)-en-8,12-olide with a methoxy substitutent. The location of the methoxy group at C-6 was deduced from the gHMBC correlations between H-6 with C-4, C-5, C-7, C-8, C-10, C-11 and the methoxy carbon. In the NOE difference spectrum, irradiation of Me-14 enhanced the signal of H-10 (1.11 %), indicating that H-10 was β-oriented. In addition, the chemical shift of the tertiary methyl group [δ _H = 0.77 (s, Me-14)] was moved upfield comparing to the secondary methyl group [δ _H = 1.05 (d, J = 7.2 Hz, Me-15)] [19], in combination with the presence of the homoallylic coupling (J = 1.6 Hz) between H-6α and Me-13 [18], indicating that H-8 was α, and the methoxy at C-6 was β-oriented. Thus, compound 5 was proposed as 6β-methoxyeremophil-7(11)-en-8β,12-olide.

The molecular formula of compound 6 was deduced as C₁₉H₂₆O₆ from its HR-ESI-MS (at m/z = 368.2064 [M + NH₄]⁺, calcd.: 368.2068). The IR spectrum displayed absorption bands for ester carbonyl (1734 cm^-1) and double bonds (1649, 1623 cm^-1). The ¹H-NMR spectrum of 6 (Table [1]) showed an angeloyloxy group, as well as two methoxy groups attributed to two carboxylic methyl ester groups, respectively. Apart from the above groups, the ¹³C-NMR spectrum of 6 (Table [2]) showed 12 signals for one methyl, three methylenes, four methines (one sp² hybridized), and four quaternary carbons (one sp² hybridized and two ester carbonyls). To accommodate seven degrees of unsaturation, compound 6 apparently contains two rings besides the above functional groups. The ¹H-¹H COSY spectrum (Fig. [3]) showed the fragment CH(4)-CH(3)-CH₂(2)-CH₂(1)-CH(9)-CH₂(8). The gHMBC spectrum (Fig. [3]) indicated correlations between the methyl at C-5 with C-4, C-5, C-6 and C-9, H-6 with C-5, C-7, C-8, C-9 and C-10, H-3 with C-1′, H-4 with C-12, two methoxy protons with C-10 and C-12, respectively. These results indicated that compound 6 possessed a novel nortrieremophilane carbon skeleton, with a double bond assigned between C-6 and C-7, an angeloyloxy group located at C-3, and two -COOMe groups attached to C-4 and C-7 respectively. Stereochemically, compound 6, apparently as a noreremophilane derivative, should possess 11β-Me and 12β-COOMe, since all the eremophilane derivatives isolated from Compositae plants have β-oriented substituents (normally a Me, or a CH₂OH, or a COOMe) at C-4 and C-5 [20]. In the NOE difference spectra, irradiation of Me-11 enhanced the signal of H-9 (1.11 %), and irradiation of H-3 enhanced the signal of H-4α (2.72 %), indicating that H-9 and the angeloyloxy group at C-3 were both β-oriented. Therefore, the structure of 6 was elucidated as 5β-angeloyloxy-3a,4,5,6,7,7a-hexahydro-3aβ-methyl-1H-indene-2,4β-dioic acid methyl ester.

Compounds 1 - 6 were evaluated for their cytotoxic activities against human hepatoma (SMMC-7721), human promyelocytic leukemia (HL-60), and human hepatocyte (L-02) cells, according to the sulforhodamine B (SRB) method [21]. Vincristine sulfate was used as positive control. The IC₅₀ (half inhibition concentration) values are summarized in Table [3]. These data showed that the investigated compounds possessed weak cytotoxic activities. The likely reason for the weak activity is the lack of an exocyclic methylene group in the lactone ring and, as a consequence, the inability to form adducts with thiol groups.

Acknowledgements

This work was supported by State Key Laboratory of Applied Organic Chemistry, Lanzhou University.

References

• 1 How F C. A dictionary of the families and genera of Chinese seed plants, 2nd edition. Beijing; Science Press 1982.
  Search in Google Scholar
• 2 Jiangsu College of New Medicine. A dictionary of the traditional Chinese medicines. Shanghai; Shanghai Science and Technology Press 1977: 7, 154, 549, 1152, 2349.
  Search in Google Scholar
• 3 Zhao Y, Jia Z J, Peng H R. Eight new eremophilane derivatives from the roots of Ligularia przewalskii .  J Nat Prod. 1995;  58 1358-64.
  CrossrefPubMedSearch in Google Scholar
• 4 Ma B, Gao K, Shi Y P, Jia Z J. Phenol derivatives from Ligularia intermedia .  Phytochemistry. 1997;  46 915-9.
  CrossrefPubMedSearch in Google Scholar
• 5 Gao K, Yang L, Jia Z J. New sesquiterpenes from the roots of Ligularia dentata .  Planta Med. 1997;  63 461-3.
  PubMedSearch in Google Scholar
• 6 Li X Q, Gao K, Jia Z J. Eremophilenolides and other contituents from the roots of Ligularia sagitta .  Planta Med. 2003;  69 356-60.
  Thieme ConnectPubMedSearch in Google Scholar
• 7 Han Y F, Pan J, Gao K, Jia Z J. Sesquiterpenes, nortriterpenes and other constituents from Ligularia tongolensis .  Chem Pharm Bull. 2005;  53 1338-41.
  CrossrefPubMedSearch in Google Scholar
• 8 Gao X, Lin C J, Xie W D, Shen T, Jia Z J. New oplopane-type sesquiterpenes from Ligularia narynensis .  Helv Chim Acta. 2006;  89 1387-94.
  CrossrefPubMedSearch in Google Scholar
• 9 Chen H M, Jia Z J. Benzofuranosesquiterpenes from Ligularia virgaurea .  Phytochemistry. 1991;  30 3132-4.
  CrossrefPubMedSearch in Google Scholar
• 10 Chen H M, Wang B G, Jia Z J. Novel sesquiterpenes from Ligularia virgaurea .  Indian J Chem. 1996;  35B 1304-7.
  PubMedSearch in Google Scholar
• 11 Wang B G, Yang X P, Jia Z J. Two minor benzofunanosesquiterpene dimers from Ligularia virgaurea .  Planta Med. 1997;  63 577-8.
  Thieme ConnectPubMedSearch in Google Scholar
• 12 Wang B G, Yang L, Chen H M, Jia Z J. New sesquiterpenes from the roots of Ligularia virgaurea .  Indian J Chem. 1998;  37B 669-71.
  PubMedSearch in Google Scholar
• 13 Tori M, Honda K, Nakamizo H, Okamoto Y, Sakaoku M, Takaoka S. et al . Chemical constituents of Ligularia virgaurea and its diversity in southwestern Sichuan of China.  Tetrahedron. 2006;  62 4988-95.
  CrossrefPubMedSearch in Google Scholar
• 14 Sugama K, Hayashi K, Mitsuhashi H. Eremophilenolides from Petasites japonicus .  Phytochemistry. 1985;  24 1531-5.
  CrossrefPubMedSearch in Google Scholar
• 15 Tori M, Kawahara M, Sono M. Eremophilane-type sesquiterpenes from fresh rhizomes of Petasites japonicus .  Phytochemistry. 1998;  47 401-9.
  CrossrefPubMedSearch in Google Scholar
• 16 Wang W S, Gao K, Jia Z J. New eremophilenolides from Ligularia shichuana .  J Nat Prod. 2002;  65 714-7.
  CrossrefPubMedSearch in Google Scholar
• 17 Zhao Y, Parsons S, Smart B, Tan R X, Jia Z J, Sun H D. Eremophilane derivatives with a novel carbon skeleton from Ligularia veitchiana .  Tetrahedron. 1997;  53 6195-208.
  CrossrefPubMedSearch in Google Scholar
• 18 Moriyama Y, Takahashi T. New sesquiterpene lactones of eremophilane-type from Ligularia fauriei (FR.) KOIDZ .  Bull Chem Soc Jpn. 1976;  49 3196-9.
  PubMedSearch in Google Scholar
• 19 Naya K, Nogi N, Makiyama Y, Takashina H, Imagawa T. The photosensitized oxygenation of furanoeremophilanes. The preparation and stereochemistry of the tsomeric hydroperoxides and corresponding lactones from furanofukinki and furanoeremophilane.  Bull Chem Soc Jpn. 1977;  50 3002-6.
  PubMedSearch in Google Scholar
• 20 Li Y S, Wang Z T, Zhang M, Chen J J, Luo S D. Two new norsesquiterpenes from Ligularia lapathifolia .  Nat Prod Res. 2004;  18 99-104.
  CrossrefPubMedSearch in Google Scholar
• 21 Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D. et al . New colorimetric cytotoxicity assay for anticancer-drug screening.  J Natl Cancer Inst. 1990;  82 1107-12.
  CrossrefPubMedSearch in Google Scholar

Prof. Zhong-Jian Jia

College of Chemistry and Chemical Engineering

State Key Laboratory of Applied Organic Chemistry

Lanzhou University

Lanzhou 730000

People’s Republic of China

Phone: +86-931-891-2408

Fax: +86-931-891-2582

Email: jiazj@lzu.edu.cn

References

• 1 How F C. A dictionary of the families and genera of Chinese seed plants, 2nd edition. Beijing; Science Press 1982.
  Search in Google Scholar
• 2 Jiangsu College of New Medicine. A dictionary of the traditional Chinese medicines. Shanghai; Shanghai Science and Technology Press 1977: 7, 154, 549, 1152, 2349.
  Search in Google Scholar
• 3 Zhao Y, Jia Z J, Peng H R. Eight new eremophilane derivatives from the roots of Ligularia przewalskii .  J Nat Prod. 1995;  58 1358-64.
  CrossrefPubMedSearch in Google Scholar
• 4 Ma B, Gao K, Shi Y P, Jia Z J. Phenol derivatives from Ligularia intermedia .  Phytochemistry. 1997;  46 915-9.
  CrossrefPubMedSearch in Google Scholar
• 5 Gao K, Yang L, Jia Z J. New sesquiterpenes from the roots of Ligularia dentata .  Planta Med. 1997;  63 461-3.
  PubMedSearch in Google Scholar
• 6 Li X Q, Gao K, Jia Z J. Eremophilenolides and other contituents from the roots of Ligularia sagitta .  Planta Med. 2003;  69 356-60.
  Thieme ConnectPubMedSearch in Google Scholar
• 7 Han Y F, Pan J, Gao K, Jia Z J. Sesquiterpenes, nortriterpenes and other constituents from Ligularia tongolensis .  Chem Pharm Bull. 2005;  53 1338-41.
  CrossrefPubMedSearch in Google Scholar
• 8 Gao X, Lin C J, Xie W D, Shen T, Jia Z J. New oplopane-type sesquiterpenes from Ligularia narynensis .  Helv Chim Acta. 2006;  89 1387-94.
  CrossrefPubMedSearch in Google Scholar
• 9 Chen H M, Jia Z J. Benzofuranosesquiterpenes from Ligularia virgaurea .  Phytochemistry. 1991;  30 3132-4.
  CrossrefPubMedSearch in Google Scholar
• 10 Chen H M, Wang B G, Jia Z J. Novel sesquiterpenes from Ligularia virgaurea .  Indian J Chem. 1996;  35B 1304-7.
  PubMedSearch in Google Scholar
• 11 Wang B G, Yang X P, Jia Z J. Two minor benzofunanosesquiterpene dimers from Ligularia virgaurea .  Planta Med. 1997;  63 577-8.
  Thieme ConnectPubMedSearch in Google Scholar
• 12 Wang B G, Yang L, Chen H M, Jia Z J. New sesquiterpenes from the roots of Ligularia virgaurea .  Indian J Chem. 1998;  37B 669-71.
  PubMedSearch in Google Scholar
• 13 Tori M, Honda K, Nakamizo H, Okamoto Y, Sakaoku M, Takaoka S. et al . Chemical constituents of Ligularia virgaurea and its diversity in southwestern Sichuan of China.  Tetrahedron. 2006;  62 4988-95.
  CrossrefPubMedSearch in Google Scholar
• 14 Sugama K, Hayashi K, Mitsuhashi H. Eremophilenolides from Petasites japonicus .  Phytochemistry. 1985;  24 1531-5.
  CrossrefPubMedSearch in Google Scholar
• 15 Tori M, Kawahara M, Sono M. Eremophilane-type sesquiterpenes from fresh rhizomes of Petasites japonicus .  Phytochemistry. 1998;  47 401-9.
  CrossrefPubMedSearch in Google Scholar
• 16 Wang W S, Gao K, Jia Z J. New eremophilenolides from Ligularia shichuana .  J Nat Prod. 2002;  65 714-7.
  CrossrefPubMedSearch in Google Scholar
• 17 Zhao Y, Parsons S, Smart B, Tan R X, Jia Z J, Sun H D. Eremophilane derivatives with a novel carbon skeleton from Ligularia veitchiana .  Tetrahedron. 1997;  53 6195-208.
  CrossrefPubMedSearch in Google Scholar
• 18 Moriyama Y, Takahashi T. New sesquiterpene lactones of eremophilane-type from Ligularia fauriei (FR.) KOIDZ .  Bull Chem Soc Jpn. 1976;  49 3196-9.
  PubMedSearch in Google Scholar
• 19 Naya K, Nogi N, Makiyama Y, Takashina H, Imagawa T. The photosensitized oxygenation of furanoeremophilanes. The preparation and stereochemistry of the tsomeric hydroperoxides and corresponding lactones from furanofukinki and furanoeremophilane.  Bull Chem Soc Jpn. 1977;  50 3002-6.
  PubMedSearch in Google Scholar
• 20 Li Y S, Wang Z T, Zhang M, Chen J J, Luo S D. Two new norsesquiterpenes from Ligularia lapathifolia .  Nat Prod Res. 2004;  18 99-104.
  CrossrefPubMedSearch in Google Scholar
• 21 Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D. et al . New colorimetric cytotoxicity assay for anticancer-drug screening.  J Natl Cancer Inst. 1990;  82 1107-12.
  CrossrefPubMedSearch in Google Scholar

Prof. Zhong-Jian Jia

College of Chemistry and Chemical Engineering

State Key Laboratory of Applied Organic Chemistry

Lanzhou University

Lanzhou 730000

People’s Republic of China

Phone: +86-931-891-2408

Fax: +86-931-891-2582

Email: jiazj@lzu.edu.cn