Terpenoids from Ligularia kangtingensis.

BACKGROUND: Ligularia kangtingensis, a species from the genus Ligularia (Compositae), is an indigenous plant in Southwest China and more than 20 species in this genus have been used as folk medicines in China.
OBJECTIVE: The chemical constituents of the whole plant of L. kangtingensis were studied.
MATERIALS AND METHODS: The dried whole plants were extracted with ethanol. Its chemical constituents were mainly isolated and purified by silica gel and Sephadex LH-20 column chromatography and their structures were identified on the basis of spectral analysis.
RESULTS: Twelve known terpenoids, including two monoterpenoids, five sesquiterpenoids and five triterpenoids, were isolated and identified from the whole plant of L. kangtingensis.
CONCLUSION: All of the 12 known compounds were isolated for the first time from L. kangtingensis.

INTRODUCTION

The genus Ligularia, a member from the family compositae, comprises approximately 150 species worldwide and nearly 120 species are distributed in China.[1] The roots and rhizomes of many Ligularia plants have long been used as folk medicines for their antibacterial and anti-inflammatory activities in China.[2] The former phytochemical studies on this genus have revealed that it was an important source of terpenoids.[3]

Ligularia kangtingensis S. W. Liu is an indigenous plant in Sichuan province, China, and it well adapted to highlands around 4000 m.[4] A literature survey indicated that no phytochemical research was reported, except a study on volatile oil.[5] As a continuation of our phytochemical studies on medicinal plants, herein we report the isolation of 12 known compounds, including two monoterpenoids, namely (3R, 4R, 6S)-3, 6-dihydroxy-1-menthene (11), 5-p-methane-1, 2-diol (12), five sesquiterpenoids, namely ligudentatin A (2), liguhodgsonal (3), oplopanone (4), 1β, 6α-dihydroxy-4β (15)-epoxyeudesmane (5), 8β-ethoxyeremophil-3, 7 (11)-diene-8α, 12 (6α, 15)-diolide (10) and 5 triterpenoids, namely lupeol (1), oleanolic acid (6), ursolic acid (7), pomolic acid (8), taraxerol (9) from the whole plant of L. kangtingensis.

MATERIALS AND METHODS

General

Nuclear magnetic resonance (NMR) spectra were recorded on Varian Unity 400/54 spectrometer with tetramethylsilane as an internal standard. Column chromatography (CC) was carried out by using silica gel (Qingdao Marine Chemical Industry, 200–300 mesh) and Sephadex LH-20 (GE Healthcare). All the reagents and solvents used for separation and purification were analytical grade and purchased from local firms.

Plant material

The whole plant of L. kangtingensis was collected from Kangding County, Sichuan Province, China, in August, 2010. The plant was identified by Qin-Mao Fang, Institute of TCM Medicinal Resources and Cultivation, Sichuan Academy of Chinese Medicine Sciences. A voucher specimen (No. LK1008) was deposited in the School of Life Science and Technology, University of Electronic Science and Technology of China.

Extraction and isolation

The air-dried whole plant of L. kangtingensis (5 kg) was powdered and extracted three times with 95% EtOH under reflux. The solvents were evaporated in vacuo to yield ethanol extract, which was suspended in H2O and then extracted with petroleum ether and EtOAc, respectively. The petroleum ether extract (185 g) was subjected to CC over silica gel (200–300 mesh, 2 kg) and eluted with a gradient solvent system (CHCl3-MeOH, 90:1–2:1) to give 12 fractions (Fr. 1~12). Fr. 2 (1.4 g) was isolated by silica gel chromatography eluted with solvent systems of cyclohexane-EtOAc (18:1) and CHCl3-acetone (600:1) to afford compound 1 (50 mg). Fr. 5 (2.7 g) was separated by silica gel chromatography (petroleum ether-EtOAc, 10:1–6:1) to give eight subfractions (Fr. 5–1 ~ 5–8). Subfraction 5–2 (35 mg) was separated by preparative thin-layer chromatography (TLC) (CHCl3-acetone, 37:2) and purified by Sephadex LH-20 chromatography (CHCl3-MeOH, 2:1) to give compound 2 (5 mg); Subfraction 5–3 (30 mg) was separated by preparative TLC (CHCl3-acetone, 20:1) to give compound 3 (11 mg); Subfraction 5–4 (60 mg) was separated by silica gel chromatography (CHCl3-acetone, 70:1) and purified by Sephadex LH-20 chromatography (CHCl3-MeOH, 2:1) to give compound 4 (7 mg); Subfraction 5–6 (95 mg) was separated by silica gel chromatography (CHCl3-acetone, 55:1) and purified by Sephadex LH-20 chromatography (CHCl3-MeOH, 2:1) to give compound 5 (6 mg). Fr. 7 (750 mg) was isolated by silica gel chromatography (petroleum ether-EtOAc, 9:1; CHCl3-acetone, 50:1) and purified by Sephadex LH-20 chromatography (CHCl3-MeOH, 2:1) to afford compound 6 (11 mg). Fr. 9 (1 g) was isolated by silica gel chromatography eluted with solvent systems of CHCl3-acetone (60:1) and cyclohexane-EtOAc (6.5:1) to afford compound 7 (10 mg). Fr. 10 (3.5 g) was isolated by silica gel chromatography (petroleum ether-EtOAc, 6:1; CHCl3-acetone, 40:1) and purified by Sephadex LH-20 chromatography (CHCl3-MeOH, 2:1) to afford compound 8 (11 mg). The EtOAc extract (38 g) was subjected to CC over silica gel (200–300 mesh, 500 g) and eluted with a gradient solvent system (cyclohexane-acetone, 30:1–1:1) to give 10 fractions (Fr.A~J). Fr. C (1.1 g) was isolated by silica gel chromatography (cyclohexane-EtOAc, 6:1) and recrystallized from cyclohexane to afford compound 9 (20 mg). Fr. D (180 mg) was isolated by silica gel chromatography (petroleum ether-EtOAc, 12:1) and purified by Sephadex LH-20 chromatography (CHCl3-MeOH, 2:1) to give compound 10 (6 mg). Fr. F (2.1 g) was isolated by silica gel chromatography (cyclohexane-EtOAc, 3:2; petroleum ether-acetone, 4:1) to afford subfraction F-1 and F-2. From subfraction F-1 (30 mg), compound 11 (7 mg) was recrystallized with CHCl3. Subfraction F-2 (240 mg) was isolated by silica gel chromatography (CHCl3-acetone, 35:1) and purified by Sephadex LH-20 chromatography (CHCl3-MeOH, 2:1) to afford compound 12 (6 mg).

RESULTS AND DISCUSSION

Compound 1: White amorphous powder. 1H (400 MHz, CDCl3): dH 4.68, 4.56 (each 1H, br s, H2-29), 3.17 (1H, m, H-3), 1.68, 1.03, 0.96, 0.94, 0.83, 0.79, 0.76 (each 3H, s, 7 × CH3). 13C NMR (100 MHz, CDCl3) [Table 1].

Table 1

13C-NMR spectroscopic data for compound 1, 6, 7, 8 and 9

Compound 2: Colorless gum. 1H (400 MHz, CDCl3): dH 7.17 (1H, d, J = 2.8 Hz, H-3), 6.75 (1H, d, J = 2.8 Hz, H-1), 4.77 (2H, br s, H2-12), 3.86 (3H, s, OCH3), 3.17 (1H, dd, J = 17.6, 4.8 Hz, H-6α), 2.85 (2H, m, H2-9), 2.78 (1H, dd, J = 17.2, 11.2 Hz, H-6β), 2.26 (1H, m, H-7), 1.94 (1H, m, H-8α), 1.80 (3H, s, H3-13), 1.62 (1H, m, H-8β). 13C NMR (100 MHz, CDCl3): dC 168.1 (C-14), 152.5 (C-2), 149.3 (C-11), 139.4 (C-10), 130.4 (C-4), 130.0 (C-5), 119.3 (C-1), 114.9 (C-3), 109.2 (C-12), 51.9 (OMe), 41.7 (C-7), 32.5 (C-6), 30.2 (C-9), 27.2 (C-8), 20.7 (C-13).

Compound 3: Colorless amorphous solid. 1H (400 MHz, CDCl3): dH 10.25 (1H, s, H-14), 7.17 (1H, d, J = 2.8 Hz, H-3), 6.86 (1H, d, J = 2.4 Hz, H-12), 5.63 (1H, br s, OH), 4.79 (2H, br d, J = 9.2 Hz, H2-12),3.40 (1H, dd, J = 17.2, 4.8 Hz, H-6α), 1.81 (3H, s, H3-13). 13C NMR (100 MHz, CDCl3): dC 192.6 (C-14), 153.5 (C-2), 149.0 (C-11), 139.8 (C-10), 134.6 (C-4), 131.7 (C-5), 121.7 (C-1), 115.6 (C-3), 109.6 (C-12), 41.4 (C-7), 30.7 (C-9), 29.9 (C-6), 27.0 (C-8), 20.7 (C-13).

Compound 4: Colorless gum. 1H (400 MHz, CDCl3): dH 2.64 (1H, ddd, J = 14.2, 9.2, 5.2 Hz, H-3), 2.18 (3H, s, H3-15), 1.19 (3H, s, H3-14), 0.88 (3H, d, J = 6.8 Hz, H3-13), 0.68 (3H, d, J = 6.8 Hz, H3-12). 13C NMR (100 MHz, CDCl3): dC 211.5 (C-4), 73.1 (C-10), 56.9 (C-1), 55.6 (C-5), 49.3 (C-7), 46.6 (C-6), 41.9 (C-9), 29.6 (C-15), 29.5 (C-11), 28.5 (C-3), 25.2 (C-2), 22.9 (C-8), 21.9 (C-13), 20.3 (C-14), 15.5 (C-12).

Compound 5: Colorless gum. 1H (400 MHz, CDCl3): dH 3.43 (1H, t, J = 10.0 Hz, H-6), 3.42 (1H, dd, J = 12.4, 4.4 Hz, H-1), 3.21 (1H, dd, J = 3.6, 2.0 Hz, H-15a), 2.77 (1H, d, J = 3.6 Hz, H-15b), 1.65 (1H, d, J = 10.0 Hz, H-5), 0.91 (3H, d, J = 7.2 Hz, H3-12), 0.86 (3H, s, H3-14), 0.80 (3H, d, J = 6.8 Hz, H3-13). 13C NMR (100 MHz, CDCl3): dC 78.0 (C-1), 67.5 (C-6), 61.5 (C-4), 51.5 (C-15), 49.6 (C-5), 49.6 (C-7), 41.8 (C-10), 36.7 (C-9), 33.1 (C-3), 29.2 (C-2), 24.9 (C-11), 20.9 (C-13), 17.8 (C-8), 15.8 (C-12), 12.1 (C-14).

Compound 6: White amorphous powder. 1H (400 MHz, CDCl3): dH 5.27 (1H, br s, H-12), 3.21 (1H, m, H-3), 1.25, 1.13, 1.05, 0.98, 0.92, 0.90, 0.90 (each 3H, s, 7 × CH3). 13C NMR (100 MHz, CDCl3) [Table 1].

Compound 7: White amorphous powder. 1H (400 MHz, CD3OD): dH 5.24 (1H, br s, H-12), 3.19 (1H, t, J = 8.0 Hz, H-3), 1.25, 1.26, 1.09, 0.98, 0.93 (each 3H, s, 5 × CH3), 0.95 (3H, d, J = 6.0 Hz, CH3), 0.86 (3H, d, J = 6.4 Hz, CH3). 13C NMR (100 MHz, CD3OD) [Table 1].

Coumpound 8: White amorphous powder. 1H (400 MHz, CDCl3): dH 5.25 (1H, br s, H-12), 3.28 (1H, m, H-3), 1.17, 1.12, 0.89, 0.81, 0.68, 0.67 (each 3H, s, 6 × CH3), 0.85 (3H, d, J = 6.0 Hz, CH3). 13C NMR (100 MHz, CDCl3) [Table 1].

Compound 9: White needle crystal (cyclohexane). 1H (400 MHz, CDCl3): dH 5.52 (1H, dd, J = 8.0, 2.8 Hz, H-15), 3.18 (1H, dd, J = 10.8, 4.4 Hz, H-3), 1.08, 0.97, 0.94, 0.92, 0.90, 0.81, 0.79 (each 3H, s, 7 × CH3). 13C NMR (100 MHz, CDCl3) [Table 1].

Compound 10: Colorless gum. 1H (400 MHz, CDCl3): dH 6.85 (1H, t, J = 3.0 Hz, H-3), 5.04 (1H, dd, J = 2.8, 1.6 Hz, H-6), 3.53, 3.32 (each 1H, dq, J = 6.0, 4.8 Hz, H2-1′), 2.24 (1H, dd, J = 8.8, 3.2 Hz, H-9a), 2.00 (3H, d, J = 1.2 Hz, H3-13), 1.41 (3H, s, H3-14), 1.21 (3H, t, J = 4.8 Hz, H3-2′). 13C NMR (100 MHz, CDCl3): dC 170.5 (C-12), 168.5 (C-15), 152.7 (C-7), 136.9 (C-3), 129.7 (C-4), 128.3 (C-11), 105.1 (C-8), 82.2 (C-6), 59.2 (C-1′), 44.0 (C-5), 35.6 (C-9), 33.0 (C-10), 26.9 (C-14), 21.7 (C-2), 21.5 (C-1), 15.1 (C-2′), 9.1 (C-13).

Compound 11: Colorless needle crystal (CHCl3). 1H (400 MHz, CD3OD): dH 5.46 (1H, s, H-2), 3.90 (1H, br s, H-3), 3.84 (1H, d, J = 9.2 Hz, H-6), 2.10 (1H, m, H-8), 1.76 (3H, s, H3-7), 1.71 (1H, m, H-4),1.58 (1H, m, H-5a), 1.38 (1H, dt, J = 13.2, 4.0 Hz, H-5b), 0.96 (3H, d, J = 7.2 Hz, H3-10), 0.81 (3H, d, J = 6.8 Hz, H3-9). 13C NMR (100 MHz, CDCl3) dC 136.9 (C-1), 129.6 (C-2), 69.3 (C-3), 67.9 (C-6), 42.4 (C-4), 29.9 (C-5), 26.3 (C-8), 20.9 (C-9), 20.3 (C-10), 17.1 (C-7).

Compound 12: Colorless gum. 1H (400 MHz, CDCl3): dH 5.71 (1H, dd, J = 10, 2.8 Hz, H-5), 5.61 (1H, dd, J = 10.4, 1.2 Hz, H-6), 3.79 (1H, dd, J = 7.6, 3.2 Hz, H-2), 2.14 (1H, m, H-4), 1.80 (1H, m, H-8), 1.71 (2H, m, H2-3), 1.30 (3H, s, H3-7), 0.93 (3H, d, J = 6.4 Hz, H3-10), 0.92 (3H, d, J = 6.8 Hz, H3-9). 13C NMR (100 MHz, CDCl3): dC 133.1 (C-5), 132.5 (C-6), 74.0 (C-2), 71.2 (C-1), 39.5 (C-4), 33.1 (C-8), 30.1 (C-3), 24.5 (C-7), 20.4 (C-10), 20.2 (C-9).

Identification of the 12 compounds isolated [Figure 1] was based on comparison of 1H-NMR and 13C-NMR data with those reported in literature, and their structures were elucidated as lupeol (1),[6] ligudentatin A (2),[7] liguhodgsonal (3),[8] oplopanone (4),[9] 1β, 6α-dihydroxy-4β (15)-epoxyeudesmane (5),[10] oleanolic acid (6),[11] ursolic acid (7),[11] pomolic acid (8),[11] taraxerol (9),[12] 8β-ethoxyeremophil-3, 7 (11)-diene-8α, 12 (6α, 15)-diolide (10),[13] (3R, 4R, 6S)-3, 6-dihydroxy-1-menthene (11),[14] 5-p-methene-1, 2-diol (12).[15]

Figure 1

Structure of compounds 1–12 isolated from Ligularia kangtingensis

CONCLUSIONS

Terpenoids, especially sesquiterpenoids, are the important secondary metabolites from plants for their diverse bioactivities, and this type of compounds is the major chemical constituent in the genus Ligularia. Here, our phytochemical investigation on the whole plant of L. kangtingensis has led to the isolation of 12 known terpenoids, including two monoterpenoids, five sesquiterpenoids and five triterpenoids, from this plant for the first time.