A pair epimers of eremophilane sesquiterpene from Ligularia macrophylla

Abstract

Two oxygenated eremophilane-type sesquiterpenoids, possessing C₂₁ skeleton, a pair epimers, have been isolated from the whole plants of Ligularia macrophylla (Compositae). Their structures were determined by analysis of infrared, ultraviolet, nuclear magnetic resonance, high-resolution electrospray ionization mass spectrometry data, and electronic circular dichroism as well as comparison with literature data. Of the two compounds, the compound 1 is unknown, and the other is known. These compounds are evaluated for antibacterial activity of Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Bacillus cereus.

A pair epimers of eremophilane sesquiterpene from Ligularia macrophylla, collected from Xinjiang Province, China

Introduction

Ligularia macrophylla (Ledeb.) DC. belongs to Senecioneae genus (Compositae) ligularia blue-gray group (Sect. Thrynoides); the distribution of this species is extensive throughout the Tianshan Mountains and Altay region of Xinjiang. Altay was the source of type specimens. It grows on the banks of river valleys, grasslands, reed swamps, shady slopes, and forest at an altitude of 700–2900 m. ¹ The rhizomes and roots of L. macrophylla are utilized as “Aster” ² in traditional Chinese medicine ³ in numerous regions of China, which has the effects of warming the lungs, lowering qi, eliminating phlegm, and relieving cough.^4–6 It is used by folks to treat diseases such as tuberculosis, ⁷ pharyngitis, bronchitis, urethritis, hepatitis, and rheumatism. It has been reported that the chemical components in Ligularia macrophylla mainly contain fatty acids, ⁸ polyolefins, ⁹ erimofenane sesquiterpenes,^10–13 tetrahydrofuran, ¹⁴ and benzofurans. ¹⁵ Some of which demonstrate in vitro cytotoxic effects on human tumor cell lines MCF-7 (breast cancer) and A549 (lung cancer). ¹⁶ In this study, two erimofenane sesquiterpenes epimers (compounds 1 and 2 ¹⁷ ) were discovered in the L. macrophylla. The same planar structure of compounds 1 and 2 were ascertained by the ¹H-¹HCOSY and HMBC, HSQC. The relative configurations of compounds 1 and 2 are identified as NOESY correlations, and the absolute configurations are confirmed by the electronic circular dichroism (ECD) spectra of compounds 1 and 2. So, we reported the determination of the chemical structure and separation of compounds 1 and 2 (Figure 1) and their antibacterial activity.

Figure 1.

Structures of compounds 1 and 2.

Result and discussion

Compound 1 is isolated in the form of a uncolored gum, the molecular formula was identified as C₂₁H₂₈O₇ based on the high-resolution electrospray ionization mass spectrometry (HRESIMS) sodium adduct peak at m/z 415.1727 [M + Na]⁺ (calcd for C₂₁H₂₈O₇Na, 415.1727), displaying seven degrees of unsaturation. The infrared (IR) spectrum revealed that the compound 1 has alkenyl (1455 cm⁻¹), carbonyl (1774 cm⁻¹), and hydroxy (3522 cm⁻¹). The signals of δ_H [6.55 (q, J = 7.3, 6.9 Hz, H), 2.11 (m, 3H), 4.27 (d, J = 13.4 Hz, H), 4.32 (d, J = 13.4 Hz, H), 3.08 (s, 3H)] in the ¹H NMR spectra displayed that the compound 1 have a methoxy and (Z)-2’-hydroxymethylbut-2’-enoyloxy, which was confirmed by the signals of ¹³C NMR at δ_C [167.1, 132.4, 142.6, 15.9, 63.6, 50.7]. Besides the above signals, the ¹³C NMR also showed 15 carbon signals, comprising three methyl (δ_C 9.8, 12.8, 15.8), three methylene (δ_C 23.7, 26.6, 41.7), three methines (two oxygenated) (δ_C 36.6, 62.5, 72.0), and six quaternary carbons (including two sp² hybrid (δ_C 154.7, 128.1), two oxygenated (δ_C 61.0, 107.6), and one acyl group (δ_C 172.2)) (Table 1), indicates that 1 has a sesquiterpene skeleton. The characteristic signals of the ¹H NMR at δ_H (0.95 (d, J = 6.7 Hz, 3H), 1.06 (s, 3H), 2.03 (s, 3H)) and the signals of ¹³C NMR at δ107.6 displayed an eremophil-7(11)-en-8(12)-olide skeleton. The δ_C 62.5 and 61.0 signal in ¹³C NMR showed that the compound 1 has an epoxy moiety. Cross-peaks from H-6 to C-4, C-5, C-7, C-8 and C-10, and from H-9 to C-1, C-5, C-7, C-8 and C-10 in the HMBC spectrum, shows that the location of an epoxy moiety is between C-10 and C-1. The positioning of the (Z)-2’-hydroxymethylbut-2’-enoyloxy group at C-6 was determined by analyzing the correlation from H-6 to C-1’ in the HMBC spectrum. Similarly, the correlation from OCH₃ to C-8 proved the OCH₃ at C-8. Combining the spin-coupling system H-1/H₂-2/H₂-3/H-4/H₃-15, as observed in the ¹H-¹H COSY spectrum, the planar configuration structure of 1 was determined (Figure 2).

Figure 2.

¹H-¹H COSY (blue bold), key HMBC (red arrows) of compounds 1 and 2.

Table 1.

The ¹H (600 MHz) and ¹³C NMR (150 MHz) data for compounds 1 and 2 in CD₃OD.

Position	1		2
	δ C	δH (J Hz)	δ C	δH (J Hz)	HMBC
1	62.5	2.91, br s	63.7	3.15, d (4.4)	H-1/C-2,3
2	26.6	1.97, m; 1.52, m	20.8	1.98, m; 1.98, m
3	23.7	1.27, m; 1.17, m	24.7	1.65, m; 1.36, m
4	36.6	1.02, m	33.0	1.82, m
5	47.6		44.4
6	72.0	5.64, s	74.2	5.82, d (1.6)	H-6/C-4, 5, 7, 8, 10, 11, 1′
7	154.7		154.7
8	107.6		105.6
9	41.7	2.95, d (14.5);1.80, d (14.5)	43.9	2.35, d (13.4)1.73, d (13.4)	H-9/ C-1,5,7,8,10
10	61.0		62.0
11	128.4		127.9
12	172.4		172.2
13	9.80	2.02, s	8.3	1.85, d (1.6)	H-13/C-7,11,12
14	12.8	1.06, s	15.6	1.17, s	H-14/C-4,5,6,10
15	15.8	0.95, d (6.7)	16.1	1.05, d (7.2)	H-15/C-3,4,5
OCH3	50.7	3.08, s	50.9	3.27, s	CH3/C-8
1′	167.1		166.9
2′	132.4		132.4
3′	142.6	6.55, q (7.3)	143.3	6.59, q (7.2)
4′	15.9	2.11, br d (7.3)	16.1	2.12, br d (7.2)	H-4′/C-2′,3′
5′	63.6	4.32, d (13.4);4.27, d (13.4)	63.9	4.34, d (13.1);4.26, d (13.1)	H-5′/C-1′,2′,3′

The relative stereochemistry of compound 1 was ascertained by the NOESY experiment and ³J coupling constants. According to the biosynthetic pathway of eremophilane-type sesquiterpenoids derived from Compositae species, the CH₃-14 and CH₃-15 are determined as β-oriented. ¹⁸ The chemical shift of CH₃-14 and CH₃-15 indicate that the 1,10-epoxy group is in α-orientation and the A/B ring system is transfused. ¹⁹ The correlations of H-1/H-6, H-1/H₃-14, H-1/ H₃-15, and H₃-14/H₃-15 in NOESY (Figure 3) further support the assignment of β-orientation for H-1, H-6, H-14, and H-15. Similarly, the H-4 and C-8 OCH₃ are α-oriented. The ¹H and ¹³C NMR signals (Table 1) of 1 were almost identical to those of 2. The discriminating coupling constants of H-6, H-1, and H-10 indicated the relative stereochemistry of 1 was different from 2; thus, 1 was suggested to be the epimer of 2. The ECD spectra (Figure 4) confirmed that the Cotton effect at 210 nm exhibited by compound 1 was exactly opposite to that of compound 2. Therefore, the absolute stereochemistry of compound 1 was confirmed to be 1S, 4S, 5S, 6R, 8R, and 10S.

Figure 3.

NOESY (black double arrow) correlations of compounds 1 and 2.

Figure 4.

The ECD spectra of compounds 1 and 2.

Compound 2 was obtained as a colorless gum. Compounds 1 and 2 have the same molecular formula based on the HRESIMS at m/z 415.1731 [M + Na]⁺ (calcd for C₂₁H₂₈O₇ Na, 415.1727). The IR spectrum showed that the compound 2 have hydroxy (3504 cm⁻¹), carbonyl (1772 cm⁻¹), and alkenyl (1456 cm⁻¹). The ¹H NMR and ¹³C NMR spectra (Table 1) also showed a high degree of agreement between compounds 1 and 2. Similarly, the plane configuration of compound 2 is identical to 1, which is supported by the data of the ¹H-¹H COSY, HSQC, and HMBC spectral of compound 2. The value for ³J_H-1 and the cross-peaks of H-1/H₃-14 in NOESY spectrum indicated compound 2 was suggested to be the epimer of 1 at H-1 and H-6. Furthermore, the known compound 2 was determined as sagittacin C through a comparison of the NMR data with literature report. ¹⁷

The antibacterial activities of compounds 1 and 2 against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Bacillus cereus were tested with a microbroth dilution method by using ampicillin as a positive control. The results demonstrated that compounds 1 and 2 exhibited equivalent antibacterial activity; the minimum inhibitory concentration (MIC) values were between 60.0 and 120.0 μg/m (Table 2).

Table 2.

Antibacterial activity (MIC) of compounds 1 and 2 (μg/mL).

	S. aureus	E. coli	B. subtilis	B. cereus
1	116.0	61.0	109.5	57.0
2	120.0	63.5	118.0	60.5
ampicillin	23.5	94.5	11.5	22.5

MIC (μg/mL): minimum inhibitory concentration.

Conclusion

Two oxygenated eremophilane-type sesquiterpenoids have been isolated from the whole plants of L. macrophylla, including a new compound 1 along with the reported sagittacin C (2). The antibacterial activity compounds 1 and 2 have been evaluated by a microbroth dilution method against four bacteria. Compounds 1 and 2 did played inhibitory activity against to four bacteria and displayed moderate activity (MIC = 60.0–120.0).

Experimental section

General experimental procedures

The RUDOLPH AUTOPOL IV polarimeter (Rudolph Research Analytical, Madison, WI, USA) was utilized to measure the optical rotations. A Shimadzu UV-260 spectrophotometer (Shimadzu Corporation, Tokyo., Japan) was employed for the measurement of UV spectra. IR spectra were obtained from a BRUKER TENSOR27 spectrometer (Rudolph Research Analytical, Karlsruhe, Baden-Württemberg, Germany). A BRUKER 500 MHz AVANCE NEO (Rudolph Research Analytical, Madison, WI, USA) was used to acquire the NMR spectra. The ECD spectra were obtained using a JASCO J1500 spectrometer (JASCO Corporation, Hachioji-shi, Tokyo, Japan). HRESIMS data were acquired using a Bruker APEX-II mass spectrometer (Rudolph Research Analytical, Madison, WI, USA). A Shimadzu High-Performance Liquid Chromatograph (Shimadzu LC-2030C series) was used for semi-preparation. Normal phase column chromatography (CC) is conducted by silica gel (300−400 mesh, 200−300 mesh, 100−200 mesh, Qingdao Marine Chemical Company, China) and Sephadex LH-20 gel (20–150 µm, Pharmacia, China). Macroporous resin (DIAION HP20, Mitsubishi Group, Japan), MCI GEL CHP20P (75–150 µm, Mitsubishi Group, Japan), reversed-phase silica gel (LiChroprep RP-18 (25–40 µm), Merck, Germany). The thin-layer chromatography (TLC) was performed using the silica gel plate GF254 (Merck).

Plant material

The L. macrophylla were gathered from Ili city (30°45’00.00’’N, 105°58’00.00’’E), Xinjiang Province in September 2020 and were identified by Professor Sheng Que of Normal School for Nationalities, Qinghai Normal University. The voucher specimen (No: DYTW-20200920) has been submitted to the Institute for the Development of Natural Medicines, Lanzhou Jiaotong University.

Extraction and isolation

The entire plants of L. macrophylla (6.0 kg) were subjected to air-drying and subsequently extracted using industrial-grade methanol (3 × 4 L, 7 days each time) at room temperature (RT) to yield a crude extract weighing 290 g. The extract was pinched and dissolved in warm water, then extracted with petroleum ether, ethyl acetate, and n-butanol to obtain extracts 100, 90, and 20 g, respectively. The ethyl acetate extract (90 g) was separated by macroporous resin column and eluted with methanol/water (30:70, 50:50, 80:20, 90:10, 100:0) afforded fractions F1-F5. The F3 (26.52 g) was separated by normal phase column chromatography and elution with petroleum ether (PE)/ethyl acetate (EtOAc) (100:1 to 1:1) to give 10 fractions A-J. Fraction D was separated by the preparative with gradients (flow rate, 2.0 mL/min, C₂H₃N/H₂O, v/v, 6/4) to afforded compounds 1 (8 mg) and 2 (10 mg).

Antibacterial assay

Four bacteria, B. cereus, B. subtilis, E. coli, and S. aureus, which have lyophilized and preserved, were streaked and inoculated on TSA solid medium and subjected to incubation at a temperature of 37°C for a duration of 24 h. Next, the bacteria were suspended in the Mueller−Hinton broth until reaching a turbidity equivalent to a 0.5 McFarland standard (approximately 1.5 × 10⁸ CFU/mL). Stock solutions containing compounds 1 and 2 and ampicillin, which were used at once, were prepared with sterile water at a concentration of 1 g/L. Then, these samples were diluted in the Mueller−Hinton broth and reallocated into 96-well microtiter plates with a capacity of 1 mL for inoculation. The final inoculation concentration is controlled between 2 × 10⁵ and 7 × 10⁵ CFU/mL. The minimum inhibitory concentration (MIC) is defined as the lowest amount of a substance that hinders observable proliferation. Triplicate experiments were conducted for each test.

Compound characterization

Compound 1

Colorless gelatinous, ${[\alpha ]}_{\mathrm{D}+93}^{21}$ (C 1.0. MeOH); IR (KBr) ν_max 3521, 2963, 2933, 1774, 1721, 1455, 1433 cm⁻¹; UV (MeOH) λ_max (log ε) 220 nm; ¹H NMR (500 MHz, CD₃OD) and ¹³C NMR data (125 MHz, CD₃OD), see Table 1; HRESIMS: m/z 415.1727 [M + Na]⁺ (calcd for C₂₁H₂₈O₇ Na, 415.1727).

Compound 2

Colorless gelatinous, ${[\alpha ]}_{\mathrm{D}-81}^{21}$ (C 1.0. MeOH); IR (KBr) ν_max 3504, 2973, 2940, 1772, 1723, 1456, 1440 cm⁻¹; UV (MeOH) λ_max (log ε) 210 nm; ¹H NMR (500 MHz, CD₃OD) and ¹³C NMR data (125 MHz, CD₃OD), see Table 1; HRESIMS: m/z 415.1731 [M + H]⁺ (calcd for C₂₁H₂₈O₇ Na, 415.1727).

Supplemental Material

sj-pdf-1-chl-10.1177_17475198241255788 – Supplemental material for A pair epimers of eremophilane sesquiterpene from Ligularia macrophylla

Supplemental material, sj-pdf-1-chl-10.1177_17475198241255788 for A pair epimers of eremophilane sesquiterpene from Ligularia macrophylla by Tong Shen, Xiang-Tao Zha, Mei Yu, Guo-Li Li and Yi-Lin He in Journal of Chemical Research