A New Eremophilanolide Sesquiterpene From Ligularia macrophylla and Diversity of the Species

Abstract

A new eremophilanolide sesquiterpene, 1α,6β,10β-trihydroxyeremophila-7(11),8-dien-12,8-olide (macrophyllalarin A, 1), and along with 5 known eremophilanolide sesquiterpenes, 6α,10β-dihydroxyeremophila-7(11)-en-12,8-olide (2), 8β-hydroxyeremophila-7(11)-en-12,8α(15,6α)-diolide (3), 6α,15β-epoxy-8β-hydroxyeremophila-7(11)-en-8α,12-olide (4), 6β,10α-dihydroxy-1-oxo-eremophila-7(11),8-dien-8,12-olide (5), and 1β,10-epoxy-6β,8β-dihydroxyeremophila-7(11)-en-8α,12-olide (6), were isolated from the roots and rhizomes of Ligularia macrophylla. The structure of the new compound was identified by comprehensive spectral analysis.

There are more than 110 species of Ligularia (Compositae) in China, mainly distributed in southwest, northwest, and north China. ^1,2 So far, about 40 species of Ligularia have been studied for their chemical constituents. Many research groups have isolated and identified various chemical constituents (sesquiterpenoids, monoterpenes, diterpenoids, triterpenoids, lignans, sterols, benzofurans, phenylpropanes, pyrrolizidine alkaloids [PA], phenylpropanoids, and flavonoids) from the genus Ligularia. ^{3 -5} Some previous study reported that the chemical constituents of Ligularia macrophylla collected from different places showed large differences, then the diversity of chemical substances in this species is present. ^{6 -9} In order to enrich the chemical composition diversity of the same plant species at different collection sites, the chemical constituents of L. macrophylla which grows in the Yili river valley of Xinjiang were systematically studied. This paper reports the isolation and characterization of 1 new eremophilanolide sesquiterpenes, macrophyllalarin A (1), together with 5 known analogs, 6α,10β-dihydroxyeremophila-7(11)-en-12,8-olide (2), ¹⁰ 8β-hydroxyeremophila-7(11)-en-12,8α(15,6α)-diolide (3), ¹¹ 6α,15β-epoxy-8β-hydroxyeremophila-7(11)-en-8α,12-olide (4), ¹² 6β,10α-dihydroxy-1-oxo-eremophila-7(11),8-dien-8,12-olide (5), ¹³ and 1β,10-epoxy-6β,8β-dihydroxyeremophila-7(11)-en-8α,12-olide (6) ¹⁴ (Figure 1).

Figure 1

The structures of compounds 1 to 6.

Compound 1 was obtained as colorless crystal. The HR-ESI-MS gave m/z 303.1207 [M+Na]⁺ (calcd. 303.1203), and molecular formula C₁₅H₂₀O₅. The IR spectrum indicated absorption bands for hydroxy (3444 cm⁻¹), ester carbonyl (1766 cm⁻¹), and double bond (1625 cm⁻¹) moieties. The ¹H and ¹³C NMR spectra (Table 1) of 1 showed resonances for 3 methyl groups [δ _H 0.81 (3H, d, J = 5.3 Hz), δ _C 15.9 (CH₃); δ _H 1.27 (3H, s), δ _C 11.5 (CH₃); and δ _H 1.99 (3H, s), δ _C 8.3 (CH₃)], 2 double bond signals [δ _H 6.01 (1H, s), δ _C 108.1 (CH), 150.3 (C) and δ _C 149.0 (C), 125.5 (C)], 3 oxygen-bearing carbons include 2 oxygenated methine carbon signals [δ _H 3.85 (1H, dd, J = 4.4, 12.1 Hz), δ _C 72.8 (CH) and δ _H 4.64 (1H, s), δ _C 70.0 (CH)] and 1 oxygenated quaternary carbon [δ _C 79.0 (C)], and 1 carbonyl group [δ _C 172.6 (C=O)]. In addition to the above signals, 2 methylene signals [δ _H 1.32 (1H, m), 1.78 m (1H, m), δ _C 31.8 (CH₂) and δ _H 1.45 (2H, m), δ _C 30.0 (CH₂)], 1 methine signal [δ _H 1.47 (1H, m), δ _C 35.3 (CH)], and 1 quaternary carbon signal [δ _C 46.7 (C)] were also observed. Combined with the above 1D NMR spectra signals, the correlations of H-1/H-2/H-3/H-4/H-15 which were found in the spectrum of ¹H-¹H COSY (Figure 2) indicated that the compound contains (-CH-CH₂-CH₂-CH-CH₃-) fragment. The HMBC spectra exhibited the long-range correlations of the compound: H-1/C-2, H-6/C-7; H-9/C-8; H-13/C-7, C-11, C-12; H-14/C-5, C-6, C-10; and H-15/C-3, C-4 (Figure 2). It is preliminarily speculated that the compound is a lactone eremophilane type sesquiterpene with 2 double bonds between C-8/C-9 and C-7/C-11, and a lactone ring linked between C-8 and C-12. ¹⁵ Combined with the molecular formula C₁₅H₂₀O₅ and the HMBC correlations of H-9 with C-10, C-8; H-6 with C-5, C-7; and H-1 with C-5, C-2, and C-10, 3 hydroxy groups should be located at C-1, C-6, and C-10, respectively.

Table 1

NMR Spectral Data of Compounds 1 (100 and 400 MHz, CD₃OD, δ in ppm, J in Hz).

Position	δ H	δ C
1	3.85 (1H, dd,12.1, 4.4 Hz)	72.8
2	1.32 (1H, m), 1.78 m (1H, m)	31.8
3	1.45 (2H, m)	30.0
4	1.47 (1H, m)	35.3
5	-	46.7
6	4.64 (1H, s)	70.0
7	-	149.0
8	-	150.3
9	6.01 (1H, s)	108.1
10	-	79.0
11	-	125.5
12	-	172.6
13	1.99 (3H, s)	8.3
14	1.27 (3H, s)	11.5
15	0.81 (3H, d, 5.3 Hz)	15.9

Figure 2

Key HMBC, ¹H-¹H COSY, and NOESY correlations of 1.

The stereochemistry of the compound 1 was determined by the NOESY experiment and analysis of ¹H-NMR characteristic signals. β-Orientation was assigned to the C-15 and C-14 methyl groups on the basis of biogenetic precedents. ¹⁶ The NOESY correlation between H-1/H₃-14 and the coupling pattern of H-1 [δ _H 3.85 (1H, dd, J = 4.4, 12.1 Hz)] determined the same β-axial orientation of both CH₃-14 and H-1, thus the OH at C-1 should be α-configuration. In the like manner, the NOESY correlation between H-6/H-4 indicated that the OH at C-6 should be β-configuration. Considering relative low-field chemical shift of CH₃-14 (δ _H 1.27) in relation to CH₃-15 (δ _H 0.81), the type of A/B condensed ring is cis-fused, so the OH at C-10 was determined to be β-orientated. ^{17 -19} Thus, compound 1 was determined as 1α,6β,10β-trihydroxyeremophila-7(11),8-dien-12,8-olide and given the name macrophyllalarin A.

Compound 1 was the major eremophilane component of the present sample. A related compound, 1α,6β,10β-trihydroxyfurano-eremophilane (euryopsol), has been isolated from other Senecioneae species, an Euryops species, ²⁰ and Senecio nemorensis. ²¹ Because furanoeremophilanes and eremophilane-8,12-olides are strongly correlated, ²² the present results indicate some correlation with these genera.

Ligularia macrophylla is mainly distributed in China. ¹ It is reported that the characteristic chemical constituents of L. macrophylla collected from Lijiang in Yunnan province are lignans, ²³ unlike this the characteristic chemical constituents of L. macrophylla collected from Xinjiang regions are eremophilane sesquiterpenes. Focusing on eremophilanes in L. macrophylla, most of them are furanoeremophilanes or eremophilan-12,8-olides with a variety of oxygen functionalities. In a sample from Kashikar region of south Xinjiang, the major characteristic chemical constituents were 9-oxygenated furanoeremophilanes. ^6,12 These compounds were not obtained from a sample in Manas River of north Xinjiang, but benzofuran type of rearranged eremophilanes was isolated together with 1(10)-epoxyfuranoeremophilanes. ⁷ In contrast, less-oxidized eremophilanes, such as furanoeremophilan-15,6a-olide, were isolated from a sample of Kaskelen canyon of Kazakhstan. ²⁴ Interestingly, the samples from Yili River of north Xinjiang (this study, 600 km east from Manas River), both 15,6α-olide (3), as in the Kazakhstan sample, and 1(10)-oxidized compounds, as in the Manas River sample, were obtained. The results indicate that L. macrophylla is diverse in chemical components caused by geographical separation, but the composition of the eremophilane compounds is not clearly separated but continuous.

Experimental

General

Melting points were determined by YRT-3 Melting Point Instrument (Henan Mingjun Instrument Co., Ltd. Henan, China) and are uncorrected. The IR spectra were recorded as KBr pellets on an Avatar 360 FT-IR ESP spectrophotometer (Nicolet, United States). The 1D and 2D NMR spectra also were measured by a Varian MR 400 NMR and a Bruker AV400 NMR (Bruker Corp, Germany). AR 2140 one part per million electronic balance (Mettler Corp, Ltd., Switzerland). HR-ESI-TOF-MS spectra were acquired using an Agilent 6210 ESI/TOF (Agilent Technologies, Santa Clara, CA, United States). UV-2401 PC UV-visible spectrophotometer (Shimadzu, Japan). FTS165 infrared spectrum analyzer (Bole company, United States). Spots of compounds were observed under UV light and visualized by spraying with vanillin and 10% H₂SO₄, followed by heating. Column chromatography (CC) was performed on silica gel (200-300 and 100-200 mesh; Qingdao Marine Chemical Factory, Qingdao, China).

Plant Material

The roots and rhizomes of L. macrophylla DC. were collected in July 2009 in Yili Nileke County, Xinjiang Province, P.R. China. The plant material was identified by Prof. Ping Yan, College of Life Sciences, Shihezi University, and a voucher specimen (WQ-LM-09-01) has been stored at the Herbarium, Department of Pharmacognosy, School of Pharmacy, Shihezi University, Xinjiang, China.

Extraction and Isolation

The air-dried roots and rhizomes parts (10.6 kg) of L. macrophylla were soaked and extracted with 95% EtOH at room temperature. The EtOH extract (3 × 15 L, 7 days each time) was concentrated under reduced pressure to give a semisolid (1900 g), a portion of which (1900 g) was suspended in H₂O (2 L) and partitioned successively with petroleum ether (PE), AcOEt, and BuOH (each 3 × 2 L). The PE extracts (334 g) were chromatographed on silica gel column (5 kg of SiO₂) by the use of a gradient of PE/acetone (from 100:1 to 0:100) to afford 11 fractions (Fr.1-Fr.11). After appropriate fractions were assembled, 3 major components were selected for further isolations. Fr.8 was subjected to repeated CC (SiO₂; PE/acetone 8:1) to give subfractions Fr.8 A-D, Fr.8-A was subjected to repeated CC (SiO₂; PE/AcOEt 8:1) to give 1 (22 mg); Fr.8-D was subjected to repeated CC (SiO₂; PE/AcOEt 8:1) and followed by PTLC with CHCl₃/MeOH (30:1) to afford 6 (10 mg). Fr.7 was subjected to repeated CC (SiO₂; PE/acetone 10:1) and purified by PTLC with benzene/acetone (4:1) to afford 2 (2 mg). Fr.6 was subjected to repeated CC (SiO₂; PE/acetone 20:1) to give subfractions Fr.6 A-G; silica gel CC was used for elution separation of the flow fraction Fr.6- C, and 4 flow fractions were obtained (Fr.6-C-1-4), Fr.6-C-1 was subjected to repeated CC (SiO₂; PE/acetone 15:1) to give 4 (8 mg), The impure crystal was found in the fluid fraction (Fr.6-C-2) and compound 3 (5 mg) was obtained by multiple recrystallization. Fr.6-D was subjected to repeated CC (SiO₂; PE/acetone 15:1) and 3 flow fractions (Fr.6-D-1-3) were obtained. Crystallization was found in the first flow fraction and compound 5 (18 mg) was obtained by multiple recrystallization.

1α,6β,10β-Trihydroxy-Eremophil-7(11),8(9)-Dien-8(12)-Olide (Macrophyllalarin A, 1)

Colorless Crystal, M.p. 176°C-178°C (CH₃OH);

${\left[\alpha \right]}_{\mathrm{D}}^{25}$ : +53 (c 0.15, CH₃OH);

UV (MeOH) λ _max (log ε) 227 (3.256) nm;

IR (KBr): 3444, 2924, 1766, 1625, 1375, 1163, 1078, 1056 cm⁻¹.

¹H and ¹³C NMR (CD₃OD): Table 1.

HRMS-ESI: m/z [M+Na]+calcd for C₁₅H₂₀O₅Na⁺ 303.1203; found: 303.1207.

Supplemental Material

Supporting information - Supplemental material for A New Eremophilanolide Sesquiterpene From Ligularia macrophylla and Diversity of the Species

Supplemental material, Supporting information, for A New Eremophilanolide Sesquiterpene From Ligularia macrophylla and Diversity of the Species by Chuanqi Lu, Wei Dai, Wei Zhou, Lin Zhang and Qi Wang in Natural Product Communications