New Sesquiterpenoids from Ambrosia artemisiifolia L.

Abstract

A new pseudoguaianolide 1 and two new guaiane-type sesquiterpene glucosides 2 and 3, were isolated from the aerial parts of Ambrosia artemisiifolia L together with two known sesquiterpene dilactones 4 and 5. The new compounds were determined on the basis of spectroscopic and chemical methods to be 3β-acetoxy-4β-hydroxy-1α,7α, 10β,11αH-pseudoguaia-12,8β-olide (1), 1β,7β,9β,10β,13αH-guaia-4(5)-en-12,6β-olide 9-O-β-d-glucoside (2) and 4β-hydroxy-1α,5α,7α,9αH-guaia-10(14),11(13)-dien-12-acid 9-O-β-d-glucoside (3). The isolated compounds were evaluated for cytotoxicity against human promyelocytic leukemia HL-60 cell lines in vitro, but were all inactive.

1. Introduction

Ambrosia artemisiifolia L. (Asteraceae), an invasive alien plant species in China, is nowadays widespread in an area ranging from Guangdong Province in the south to Heilongjiang Province in the north [1]. This species is considered as a harmful weed capable of quickly colonizing both agricultural and urban areas with competitive seeds, and its pollen can induce serious allergic disorders during a certain period of its development [2]. However, many investigations into the composition and properties of A. artemisiifolia indicate that this species can serve a valuable source of biologically active substances. The extracts of this plant showed various bioactivities, such as molluscicidal [3], plant growth inhibitory [4], anti-inflammatory [5], antithrombin [6], antibacterial [7], insecticidal [8], hepatoprotective and hypolipemic activities [9]. On the other hand, the main chemical components of this species were identified as sesquiterpene lactones, which account for the antihelminthic, cardiotonic, antiinflammatory, analgesic, sedative (calming), antimalarial, anti-tumor, and other types of pharmacological activity [10,11]. As part of an ongoing search for novel bioactive compounds, we studied Ambrosia artemisiifolia L. growing in Hunan Province of China. Herein, this paper reports the isolation and identification of three new sesquiterpenoids from the aerial parts of the plant.

2. Results and Discussion

The dried aerial parts of A. artemisiifolia were extracted three times with MeOH at room temperature. The MeOH extract residue was suspended in water and then partitioned successively with petroleum ether and EtOAc. Column chromatography of the EtOAc-soluble fraction yielded three new compounds 1–3 and two known compounds, psilostachyin B (4) [12] and psilostachyin (5) [13] (Figure 1).

Figure 1. Structures of compounds 1–5 isolated from A. artemisiifolia.

Figure 1. Structures of compounds 1–5 isolated from A. artemisiifolia.

Compound 1 was obtained as white amorphous powder, and the molecular formula was assigned as C₁₇H₂₆O₅ from its HRESIMS (m/z 311.1852 [M+H]⁺) and NMR data. The ¹H-NMR spectrum displayed readily recognizable signals for four methyl groups [δ_H 1.18 (3H, s, H-15), 2.04 (3H, s, H-17), 0.90 (3H, d, J = 7.0 Hz, H-14) and 1.02 (3H, d, J = 7.4 Hz, H-13)] (Table 1). The ¹³C-NMR and DEPT spectra of 1 exhibited 17 carbons corresponding to four methyls, three methylenes, seven methines (including three oxy-methine carbons), a quaternary carbon, and two quaternary carbonyl groups (Table 1). The structure of 1 was deduced based on ¹H-¹H COSY, HSQC, and HMBC techniques using those methyl groups as starting points. The HMBC correlations from the H-atoms of the four methyl groups to corresponding C-atoms [H₃-17 (δ_H 2.04) to C-16; H₃-14 (δ_H 0.90) to C-1, C-9 and C-10; H₃-13 (δ_H 1.02) to C-7, C-11 and C-12; H₃-15 (δ_H 1.18) to C-1, C-4, C-5 and C-6) (Table 1)] established a typical acetyl moiety (δ_H 2.04, δ_C 169.8 and 20.3) and three fragments as ¹⁴CH₃-¹⁰CH(¹CH)-⁹CH-, ¹³CH₃-¹¹CH (⁷CH)-¹²COOH, and ¹⁵CH₃-⁵C(⁴CH/¹CH)-⁶CH₂- (Figure 2). Those fragments were further linked to form a 5/7-membered fused-ring structure based on the analysis of the ¹H-¹H-COSY correlations (Figure 2). Considering the molecular formula C₁₇H₂₆O₅ and the presence of two C=O bonds, 1 was suggested to be an acetylated derivative of pseudoguaianolide [14]. The lactone ring was formed between C-12 and C-8, which deduced from the low-field chemical shift of C-12 (δ_C 177.8) and C-8 (δ_H 4.58 and δ_C 79.7) [15]. While the acetyl moiety was found to be attached to C-3 via an ester linkage from long-range correlation of H-3 [δ 5.45 (ddd, J = 8.1, 7.5, 5.0 Hz)] with C-16 (δ_C 169.8) observed in the HMBC spectrum (Figure 2). Thus the planar structure of 1 was deduced as 3-acetoxy-4-hydroxy-pseudoguaia-12,8-olide. The relative stereochemistry of 1 was assigned by analyses of the NOESY spectrum and the proton coupling patterns (Table 1). The key NOE correlation observed between H-7 and H-8, as well as the large coupling constant measured for H-8 (8.6 Hz), indicating the presence of a cis-fused lactone ring [16]. While the NOE correlation was absent between H-1 and H₃-15 suggesting a typical trans-fused 5/7-membered ring, a model of this molecule indicated that H-1 was α-orientation [14]. Meanwhile, the correlations of H-1 with H-3, H-4 and H-7, and H-7 with H-8 and H-11 revealed that all of these protons (H-1, H-3, H-4, H-7 and H-11) were cofacial and were arbitrarily assigned α-oriented. The β-orientation of H-10 was revealed by the coupling constant measured H-1 (J_H-1/H-10 = 13.6 Hz), and corroborated by an NOE between H-10 and H₃-15. Finally, compound 1 was established as 3β-acetoxy-4β-hydroxy-1α,7α,10β,11αH-pseudoguaia-12,8β-olide.

Figure 2. Key ¹H-¹H COSY, HMBC and NOESY correlations of 1.

Figure 2. Key ¹H-¹H COSY, HMBC and NOESY correlations of 1.

Table 1. NMR assignments of 1 by DEPT, HSQC, HMBC, and NOESY experiments in C₅D₅N ^a.

Position	δH (J in Hz)	δC, Mult.	HMBC	NOESY
1	1.51, ddd (13.6, 5.6, 5.6)	39.0 CH	C-2, C-4, C-5, C-10, C-14,C-15	H-3, H-4, H-7
2	Hα: 1.82, m	31.5 CH2	C-1, C-3
	Hβ: 2.01, m		C-1, C-3
3	5.45, ddd (8.1, 7.5, 5.0)	71.0 CH	C-1, C-4, C-5, C-16	Hα-2, Hβ-2, H-4
4	3.64, d (7.5)	75.9 CH	C-3, C-5, C-6, C-15	H-1, H-3, H-7
5	–	43.3 qC
6	Hα: 1.94, br d (14.3)	30.5 CH2	C-5, C-7, C-15	H-4, H-7
	Hβ: 1.16, overlapped			Hβ-9
7	2.51, m	36.7 CH	C-5, C-6, C-11, C12	H-1, H-4, H-8, H-11
8	4.58, ddd (13.2, 8.6, 2.8)	79.7 CH	C-7	H-7, Hα-9, Hβ-9, H-11
9	Hα: 2.07, m	36.4 CH2	C-1, C-7, C-8, C-10, C-14
	Hβ: 1.66, br d (12.7)		C-1, C-7, C-8, C-10	Hβ-6, H-14, H-15,
10	1.80, m	28.5 CH	C-1, C-5, C-14
11	2.94, m	37.5 CH	C-6, C-7, C-12, C-13	H-7, H-8, H-13
12	–	177.8 qC
13	1.02, d (7.4)	9.7 CH3	C-7, C-11, C-12	Hβ-6, H-7, H-11
14	0.90, d (7.0)	16.4 CH3	C-1, C9, C-10	Hα-9
15	1.18, s	17.5 CH3	C-1, C-4, C-5, C-6	Hβ-9, H-10
16	–	169.8 qC
17	2.04, s	20.3 CH3	C-16	H-3

Note: ^{a 1}H (400 MHz) and ¹³C (100 MHz) NMR; δ in ppm.

Table 1. NMR assignments of 1 by DEPT, HSQC, HMBC, and NOESY experiments in C₅D₅N ^a.

Position	δH (J in Hz)	δC, Mult.	HMBC	NOESY
1	1.51, ddd (13.6, 5.6, 5.6)	39.0 CH	C-2, C-4, C-5, C-10, C-14,C-15	H-3, H-4, H-7
2	Hα: 1.82, m	31.5 CH2	C-1, C-3
	Hβ: 2.01, m		C-1, C-3
3	5.45, ddd (8.1, 7.5, 5.0)	71.0 CH	C-1, C-4, C-5, C-16	Hα-2, Hβ-2, H-4
4	3.64, d (7.5)	75.9 CH	C-3, C-5, C-6, C-15	H-1, H-3, H-7
5	–	43.3 qC
6	Hα: 1.94, br d (14.3)	30.5 CH2	C-5, C-7, C-15	H-4, H-7
	Hβ: 1.16, overlapped			Hβ-9
7	2.51, m	36.7 CH	C-5, C-6, C-11, C12	H-1, H-4, H-8, H-11
8	4.58, ddd (13.2, 8.6, 2.8)	79.7 CH	C-7	H-7, Hα-9, Hβ-9, H-11
9	Hα: 2.07, m	36.4 CH2	C-1, C-7, C-8, C-10, C-14
	Hβ: 1.66, br d (12.7)		C-1, C-7, C-8, C-10	Hβ-6, H-14, H-15,
10	1.80, m	28.5 CH	C-1, C-5, C-14
11	2.94, m	37.5 CH	C-6, C-7, C-12, C-13	H-7, H-8, H-13
12	–	177.8 qC
13	1.02, d (7.4)	9.7 CH3	C-7, C-11, C-12	Hβ-6, H-7, H-11
14	0.90, d (7.0)	16.4 CH3	C-1, C9, C-10	Hα-9
15	1.18, s	17.5 CH3	C-1, C-4, C-5, C-6	Hβ-9, H-10
16	–	169.8 qC
17	2.04, s	20.3 CH3	C-16	H-3

Note: ^{a 1}H (400 MHz) and ¹³C (100 MHz) NMR; δ in ppm.

Compound 2 has the molecular formula C₂₁H₃₂O₈ according to its HRESIMS and ¹³C-NMR data. ¹H-, ¹³C-NMR and DEPT spectra revealed that 2 contained a typical β-d-glucopyranosyl [δ_H 4.38 (d, J = 7.8, H-1'); δ_C 102.5, 75.2, 77.9, 71.8, 78.2, 62.8], which was confirmed by acid hydrolysis and then co-chromatography with an authentic sample. Besides, the remaining 15 carbon signals, which belong to the aglycone, were attributable to three methyls, three methylenes, six methines (including two oxy-methines), two olefinic quaternary carbons, and a quaternary carbonyl group (Table 2). To deduce the structure of the aglycone and the glycosidic connection, a complete ¹H and ¹³C-NMR spectral assignment was carried out using a combination of DEPT, HSQC, ¹H-¹H COSY, HMBC and NOESY experiments. The ¹H-¹H COSY spectrum of 2 revealed a fragment with eleven carbons [-³CH₂-²CH₂-¹CH-¹⁰CH(¹⁴CH₃)-⁹CH(OR)-⁸CH₂-⁷CH(⁶CHOR)-¹¹CH-¹³CH₃] which was corroborated by HMBC correlations (Figure 3). The fragment was further connected by HMBC spectrum, the key HMBC correlations from H-1 to C-4, C-5 and C-6, from H-6 to C-1, C-4 and C-5, from H-13 to C-11, C-12 and C-7, and from H-15 to C-3, C-4 and C-5 indicated a sesquiterpene skeleton with a 5/7-membered fused-ring, which belong to that kind of guaiane-type sesquiterpenoid (Figure 3) [17,18]. In addition, the oxy-methine carbon δ_C 82.6 (C-9) showed a long-range correlation with the anomeric proton of glucose at δ_H 4.38 (d, J = 7.8 Hz, H-1'), indicating that the hydroxyl group at C-9 is glucosylated. The low-field chemical shift of δ_C 181.2 (C-12) and δ_C 83.3 (C-6), as well as the molecular formula C₂₁H₃₂O₈ revealed that a lactone ring was formed between C-12 and C-6 [18,19]. Therefore, the planar structure of compound 2 was determined as guaia-4(5)-en-12,6-olide 9-O-β-d-glucoside. In the NOESY spectrum, there were no NOE correlations between H-6 and H-7, as well as the large values of the constants of J_6,7 and J_7,11 (10–11 Hz), suggesting the presence of a trans-fused lactone ring [19]. Moreover, the correlations of H-7 with H-1 and H-9, and H-6 with H-11 and H₃-14 revealed that protons H-7, H-1 and H-9 were cofacial, while H-6, H-11 and H₃-14 were on the opposite side. Assume the usual α-configuration for the isopropyl at C-7 [19], the stereochemistry of 2 was established as 1β,7β,9β,10β,13αH-guaia-4(5)-en-12,6β-olide 9-O-β-d-glucoside.

Table 2. ¹H- (400 MHz) and ¹³C- (100 MHz) NMR spectral data of compounds 2 and 3.

Position	2 a		3 b
	δH (J in Hz)	δC, Mult.	δH (J in Hz)	δC, Mult.
1	3.09, m	49.9 CH	3.24, m	45.4 CH
2	Hα: 1.56, m; Hβ: 2.10, overlapped	29.4 CH2	1.96, m	27.0 CH2
3	2.36, m	39.4 CH2	Hβ: 1.77, m; Hα: 1.98, m	40.9 CH2
4	–	131.6 qC	–	80.7 qC
5	–	142.5 qC	2.38, br t (10.8)	54.2 CH
6	4.80, d (11.5)	83.3 CH	Hβ: 1.39, ddd (12.2, 11.0, 10.8); Hα: 2.01, br d (12.2)	33.4 CH2
7	1.89, ddd (11.5, 11.6, 11.6)	46.8 CH	2.92, br t (11.0)	39.8 CH
8	Hα: 1.67, m; Hβ: 2.14, overlapped	32.2 CH	Hα: 1.88, m; Hβ: 2.62, br d (11.8)	45.3 CH2
9	3.88, m	82.6 CH	4.79, dd (11.0, 3.0)	79.5 CH
10	2.11, overlapped	41.3 CH	–	152.5 qC
11	2.34, m	42.5 CH	–	149.0 qC
12	–	181.2 qC	–	169.8 qC
13	1.18, d (6.9)	12.5 CH3	5.55, br s; 6.42, br s	121.9 CH2
14	0.80, d (7.0)	8.1 CH3	5.27, br s; 6.18, br s	110.1 CH2
15	1.81, s	15.5 CH3	1.32, s	24.9 CH3
Glc-1'	4.38, d (7.8)	102.5 CH	5.06, d (7.7)	101.1 CH
2'	3.15, dd (8.0, 7.8)	75.2 CH	4.11, dd (8.2, 7.7)	75.8 CH
3'	3.35, m	77.9 CH	4.23, m	78.7 CH
4'	3.31, m	71.8 CH	4.26, m	71.9 CH
5'	3.27, m	78.2 CH	3.88, m	78.8 CH
6'	3.66, dd (11.6, 5.2); 3.88, br d (11.6)	62.8 CH2	4.36, dd (11.9, 5.2); 4.51, dd (11.9, 2.2)	62.9 CH2

Notes: ^a measured in CD₃OD, ^b measured in C₅D₅N; δ in ppm.

Table 2. ¹H- (400 MHz) and ¹³C- (100 MHz) NMR spectral data of compounds 2 and 3.

Position	2 a		3 b
	δH (J in Hz)	δC, Mult.	δH (J in Hz)	δC, Mult.
1	3.09, m	49.9 CH	3.24, m	45.4 CH
2	Hα: 1.56, m; Hβ: 2.10, overlapped	29.4 CH2	1.96, m	27.0 CH2
3	2.36, m	39.4 CH2	Hβ: 1.77, m; Hα: 1.98, m	40.9 CH2
4	–	131.6 qC	–	80.7 qC
5	–	142.5 qC	2.38, br t (10.8)	54.2 CH
6	4.80, d (11.5)	83.3 CH	Hβ: 1.39, ddd (12.2, 11.0, 10.8); Hα: 2.01, br d (12.2)	33.4 CH2
7	1.89, ddd (11.5, 11.6, 11.6)	46.8 CH	2.92, br t (11.0)	39.8 CH
8	Hα: 1.67, m; Hβ: 2.14, overlapped	32.2 CH	Hα: 1.88, m; Hβ: 2.62, br d (11.8)	45.3 CH2
9	3.88, m	82.6 CH	4.79, dd (11.0, 3.0)	79.5 CH
10	2.11, overlapped	41.3 CH	–	152.5 qC
11	2.34, m	42.5 CH	–	149.0 qC
12	–	181.2 qC	–	169.8 qC
13	1.18, d (6.9)	12.5 CH3	5.55, br s; 6.42, br s	121.9 CH2
14	0.80, d (7.0)	8.1 CH3	5.27, br s; 6.18, br s	110.1 CH2
15	1.81, s	15.5 CH3	1.32, s	24.9 CH3
Glc-1'	4.38, d (7.8)	102.5 CH	5.06, d (7.7)	101.1 CH
2'	3.15, dd (8.0, 7.8)	75.2 CH	4.11, dd (8.2, 7.7)	75.8 CH
3'	3.35, m	77.9 CH	4.23, m	78.7 CH
4'	3.31, m	71.8 CH	4.26, m	71.9 CH
5'	3.27, m	78.2 CH	3.88, m	78.8 CH
6'	3.66, dd (11.6, 5.2); 3.88, br d (11.6)	62.8 CH2	4.36, dd (11.9, 5.2); 4.51, dd (11.9, 2.2)	62.9 CH2

Notes: ^a measured in CD₃OD, ^b measured in C₅D₅N; δ in ppm.

Figure 3. Key ¹H-¹H COSY, HMBC and NOESY correlations of 2.

Figure 3. Key ¹H-¹H COSY, HMBC and NOESY correlations of 2.

Compound 3 was obtained as a colorless gum. The molecular formula was shown to be C₂₁H₃₂O₉ by its HRESIMS and ¹³C-NMR data. The structure of 3 contained a β-d-glucopyranosyl, which was confirmed by methods previously described in compound 2. Moreover, two pair of exomethylene singlets at δ_H 5.55 /6.42 (br s, H₂-13) and 5.27 /6.18 (br s, H₂-14), along with a methyl singlets δ_H 1.32 (s, H₃-15) was clearly observed in ¹H-NMR spectrum. The ¹³C-NMR and DEPT spectra (Table 2) showed a total of 21 carbon signals, among which, 15 were ascribable to the aglycone. The structure of the aglycone and the glycosidic connection were carried out by 2D-NMR methods (HSQC, ¹H-¹H COSY, HMBC and NOESY experiments). The HMBC correlations from the H-atoms of the exomethylenes and methyl groups to corresponding C-atoms revealed three C₄ units as ¹³CH₂=¹¹C (⁷CH)-¹²COOH, ¹⁴CH₂=¹⁰C(¹CH)-⁹CH-, and ¹⁵CH₃-⁴C(³CH₂)-⁵CH-, respectively. Those C₄ units in combination with a C₈ unit -³CH₂-²CH₂-¹CH-⁵CH-⁶CH₂-⁷CH-⁸CH₂-⁹CHOR deduced from ¹H-¹H COSY correlations (Figure 4), established the structure of the aglycone as 4,9-dihydroxyguaia-10(14),11(13)-dien-12-acid, which was good agree with the formula C₂₁H₃₂O₉. Moreover, an informative correlation was also observed between the anomeric proton signal at δ_H 5.06 (d, J = 7.7 Hz, H-1') and a methine carbon signal at δ_C 79.5 (C-9) in the HMBC spectrum (Figure 4), implying that the sugar moiety was linked at the C-9 position. Therefore, the planar structure of 3 was established as 4-hydroxyguaia-10(14),11(13)-dien-12-acid 9-O-β-d-glucoside. The key NOESY correlations of H-1 with H-5 and H-9, and H-5 with H-7, H-9 and H₃-15 showed that all of those protons (H-1, H-5, H-7, H-9 and H₃-15) were cofacial. Meanwhile, the coupling constants of H-9 (δ_H 4.79, dd, J = 11.0, 3.0 Hz) revealed its α-oriented position. Thus compound 3 was determined to be 4β-hydroxy-1α,5α,7α,9αH-guaia-10(14),11(13)-dien-12-acid 9-O-β-d-glucoside.

Figure 4. Key ¹H-¹H COSY, HMBC and NOESY correlations of 3.

Figure 4. Key ¹H-¹H COSY, HMBC and NOESY correlations of 3.

The cytotoxicity of compounds 1–5 were tested against human promyelocytic leukemia HL-60 cell by the MTT method in vitro. The results revealed that all compounds were inactive (LC₅₀ > 100 μM).

3. Experimental Section

3.1. General

Optical rotations were measured on WZZ2B automatic polarimeter (Precision Instrument Co., Shanghai, China); melting points were obtained on a SGW X-4 micromelting point apparatus (INESA Physico Optical Instrument Co., Ltd, Shanghai, China). The 1D- and 2D-NMR spectra were measured with a Bruker DRX-400 instrument (Bruker BioS-pin GmbH Company, Rheinstetten, Germany) with TMS as internal standard. ESIMS data were recorded on an API QSTAR mass spectrometer (Applied Biosystem/MSD Sciex, Concord, ON, Canada). Column chromatography was performed on silica gel 60 (200–300 mesh, Qingdao Marine Chemical Ltd, Qingdao, China), Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) and Develosil ODS (50 μm, Nomura Chemical Co. Ltd., Osaka, Japan). Preparative HPLC was performed on a Waters 1525 Binary HPLC pump and a Waters 2414 refractive index detector (Waters Corp, Millipore, Milford, MA, USA) using a YMC-Pack ODS-A column (250 mm × 10 mm I.D.; S-5 μm, 12 nm).

3.2. Plant Material

The aerial parts of A. artemisiifolia were collected from Miluo, Hunan Province, P. R. China, in August 2012, and identified by Prof. Zhongshi Zhou, Institute of Plant Protection, Chinese Academy of Agricultural Sciences. The voucher specimen (No. 20120821) was deposited in Hunan Agricultural University.

3.3. Extraction and Isolation

Dried aerial parts of A. artemisiifolia (10.0 kg) were powdered and extracted three times with MeOH (95% v/v) at room temperature, then concentrated under reduced pressure to obtain a crude residue (0.5 kg). The residue was further suspended in H₂O (2 L) and extracted with petroleum ether (PE) and EtOAc successively, to yield a PE-soluble fraction (90.0 g), an EtOAc-soluble fraction (90.0 g). The EtOAc-soluble fraction was subjected to silica gel column chromatography (CC) (100–200 mesh) with elution of CHCl₃–MeOH (100:0 → 60:40, v/v) to give six fractions (Fr. C₁–C₆). The Fr. C₂ was further separated by ODS-C₁₈ CC (MeOH–H₂O 30:70 → 70:30, v/v) to produce seven sub-fractions (C₂-1–C₂-7), then sub-fraction C₂-3 was further purified by Sephadex LH-20 column (MeOH) and normal silica gel CC (petroleum ether–EtOAc, 9:1) to yield colourless crystal 4 (100.0 mg) and 5 (30.0 mg). Similarly, Fractions C₃ (6.0 g) and C₅ (8.3 g) were fractionated by an ODS-C₁₈ column with elution of MeOH–H₂O (30:70 → 70:30, v/v), respectively. Compound 1 (60.0 mg) was obtained from sub-fraction C₃-5 by Sephadex LH-20 column (MeOH) and semi-preparative HPLC chromatography (MeOH-H₂O 48%, v/v, flow rate 3 mL/min, t_R = 23.6 min). Compounds 2 (8.0 mg) and 3 (6.0 mg) were obtained from sub-fraction C₅-2 which purified successively on Sephadex LH-20 column (MeOH) and semi-preparative HPLC chromatography (MeOH-H₂O 38%, v/v, flow rate 3 mL/min; t_R-3 = 37.3 min, t_R-2 = 50 min).

3.4. Characterization of Compounds 1–3

Compound 1: white powder, mp 290–293 °C, ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{25}$ −44.8 (c 0.9, MeOH); IR (KBr) υ_max 3342, 2951, 1769, 1740, 1727, 1704, 1660, 1176, 1035 cm⁻¹; ¹H-NMR (400 MHz, C₅D₅N) and ¹³C-NMR (100 MHz, C₅D₅N) spectroscopic data, see Table 1; positive ion ESIMS m/z: 311 [M+H]⁺, 333 [M+Na]⁺; negative ESIMS m/z: 655 [2M+Cl]⁻; HRESIMS m/z: 311.1852 [M+H]⁺ (calcd for C₁₇H₂₇O₅, 311.1853).

Compound 2: white powder, mp 305–307 °C, ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{25}$ +33.2 (c 0.5, MeOH); IR (KBr) υ_max 3392, 2921, 2850, 1754, 1688, 1458, 1384, 1229, 1176 cm⁻¹; ¹H-NMR (400 MHz, MeOD) and ¹³C-NMR (100 MHz, MeOD) spectroscopic data, see Table 2; positive ion ESIMS m/z: 413 [M+H]⁺, 435 [M+Na]⁺; negative ESIMS m/z: 411 [M−H]⁻, 859 [2M+Cl]⁻; HRESIMS m/z: 435.1971 [M+Na]⁺ (calcd for C₂₁H₃₂O₈Na, 435.1989).

Compound 3: yellowish syrup, ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{25}$ −37.7 (c 1.1, MeOH); IR (KBr) υ_max 3400, 2923, 2870, 1702, 1458, 1380, 1221 cm⁻¹; ¹H-NMR (400 MHz, C₅D₅N) and ¹³C-NMR (100 MHz, C₅D₅N) spectroscopic data, see Table 2; positive ion ESIMS m/z: 451 [M+Na]⁺, 879 [2M+Na]⁺; negative ESIMS m/z: 427 [M−H]⁻, 463 [M+Cl]⁻; HRESIMS m/z: 451.1935 [M+Na] ⁺ (calcd for C₂₁H₃₂O₉Na, 451.1939).

3.5. Determination of the Configurations of Sugar Unit in 2 and 3

Compounds 2 (2.0 mg) in 1 N HCl (5 mL, 1,4-dioxane–H₂O, 1:1) was heated under reflux for 8 h. After removal of the solvent, the residue was partitioned between Et₂O and H₂O. The water layer was neutralized with 5% NaOH and desalted (Sephadex LH-20, MeOH) to afford the sugar residue (1.0 mg). The sugar residue was derivatized with Sigma Sil-A for 35 min at 70 °C and analyzed by GC-MS [HP-5MS column (30 m × 0.25 mm, 0.25 mm); injection temperature: 250.0 °C; column flow: 1.33 mL/min; ion source temperature: 200.0 °C; interface temperature: 220.0 °C]. In the acid hydrolysate of 2, d-Glucose was confirmed by comparison of the retention times of the aforementioned derivative with the authentic d-glucose derivative prepared in a similar way, both of which showed identical retention time of 12.17 min. By the same method, the sugar moiety of 3 was identified as d-glucose.

3.6. Cytotoxicity Assays

The cytotoxic activity of each compound against human promyelocytic leukemia HL-60 cell was examined in vitro at the Second Xiangya Hospital of Central South University, and was determined by the MTT assay [20,21].

4. Conclusions

The phytochemical investigation of the aerial parts of A. artemisiifolia afforded a new pseudoguaianolide (1), two new guaiane-type sesquiterpene glucosides (2 and 3), as well as two known sesquiterpene dilactones psilostachyin B (4) and psilostachyin (5). Although all the isolated compounds were inactive against human promyelocytic leukemia HL-60 cell, the discovery of these new compounds further expands our knowledge of the structural diversity of the sesquiterpenoids produced by the plant A. artemisiifolia.