Artemisiaside A: A new Lignan Glycoside and its Analogues From the Aerial Parts of Artemisia argyi

Abstract

Chemical analysis of the aerial parts of Artemisia argyi H. Lév. & Vaniot led to the isolation of 6 lignans, including a new lignan glycoside, artemisiaside A, using various chromatographic techniques. Detailed spectroscopic (including 1D, 2D- nuclear magnetic resonance) and high resolution mass spectroscopy procedures, and electronic circular dichroism were used to ascertain the structural orientations of these compounds. The anti-inflammatory activities of compounds 1 to 6 were evaluated by measuring their inhibitory effects on lipopolysaccharide (LPS) -induced nitric oxide (NO) production in RAW264.7 LPS-activated macrophages. At 50 μM, compound 1 showed moderate anti-inflammatory activity with an inhibition rate of 61.2%.

Keywords

compositae lignan glycosides anti-inflammatory activity

Introduction

Compositae, the largest family of flowering plants, encompasses more than 1600 genera and 22,000 species, with a worldwide distribution in all continents except Antarctica.¹ This family has enormous economic benefits by providing food, cosmetics, and pharmaceuticals. Artemisia is a large genus in the Compositae family, with more than 350 species distributed across parts of the northern hemisphere temperate regions.² Artemisia species have been used to treat a variety of diseases, from simple fevers to malaria.^3,4 Secondary metabolites of Artemisia species reported in the literature include phenolic compounds, alkaloids, terpenoids, sterols, and coumarins.⁵ Especially, the discovery of artemisinin from Artemisia annua has brought more attention to this genus. Artemisia argyi, a perennial plant, known as wormwood in traditional Chinese medicines, is mainly found across parts of eastern Asia and Europe.⁶ The stems and leaves of A argyi have a particularly strong fragrance. The whole plant was generally used as a Chinese traditional herb to treat warming meridians to dissipate cold, to stop bleeding, as an anti-inflammatory, to relieve cough-melt sputum, and so on. In addition, it can also be used as a dietary source for treating gastric mucosal injury or unhealthy eating habits.⁷ Previous study has shown that the leaves and stems of A argyi mainly contain volatile oil, sesquiterpenoids, flavonoids, phenols, alkaloids, and sugars.⁸ In our phytochemical investigation of the methanol extract of the aerial parts of A argyi, we report the isolation and structure determination of 3 lignans and 3 lignan glycosides, including 1 previously undescribed compound (1), and 5 known compounds (2-6). Their anti-inflammatory activities were assessed by measuring the inhibitory effects on lipopolysaccharide (LPS)-induced NO production in macrophages activated by RAW264.7 LPS.

Result and Discussion

Compound 1 was obtained as a colorless amorphous powder. Based on the high resolution electrospray ionization mass spectroscopy (HRESIMS) ion peak at m/z 623.2341 [M-H]⁻ (calcd 623.2340), its molecular formula was determined as C₃₀H₄₀O₁₄, suggesting eleven degrees of unsaturation. Analysis of the ¹³C nuclear magnetic resonance (NMR) data (Table 1) with the aid of distortionless enhancement by polarization transfer experiment uncovered the presence of 1 methyl, 4 methylenes (including 3 oxygenated), 11 methines (including 3 aromatics, 5 oxygenated), 10 quaternary carbons (including 9 aromatics, 1 acetyl), and 4 methoxyl groups. The ¹H and ¹³C NMR

Table 1.

¹H and ¹³C NMR Data of Compounds 1 and 1b.

	Compound 1^a		Compound 1b^b
No.	δ_H (J in HZ)	δ_C, type	δ_H (J in Hz)	δ_C, type
1		129.9, s		129.5,s
2		125.9, s		125.6, s
3		147.4, s		146.8, s
4		138.9, s		138.3, s
5		148.8, s		147.8, s
6	6.58 (s)	107.6, d	6.57 (s)	107.0, d
7	2.84 (dd, 15.3, 4.3)	34.1, t	2.70	33.4, t
	2.67 (dd, 15.3, 10.6)
8	1.86 (m)	38.6, d	1.67	40.2, d
9	3.98 (dd, 9.6, 4.5)	73.8, t	3.64	65.4, t
	3.48 (dd, 9.6, 6.6)		3.54
3-OMe	3.28 (s)	59.9, q	3.33 (s)	59.3, q
5-OMe	3.85 (s)	56.5, q	3.82 (s)	56.2, q
1′		138.9, s		138.4, s
2′	6.32 (s)	106.7, d	6.40 (s)	106.7, d
3′		149.0, s		148.3, s
4′		134.7, s		134.9, s
5′		149.0, s		148.3, s
6′	6.32 (s)	106.7, d	6.40 (s)	106.7, d
7′	4.14 (d,6.6)	43.4, d	4.20 (d,6.2)	42.6, d
8′	2.15 (m)	46.2, d	2.21	44.9, d
9′	4.21 (overlap)	65.8, t	4.13	65.3, t
	4.02 (dd, 9.6, 6.6)		3.97
9′-carbonyl		173.3, s		171.2, s
9′-acetonyl	2.08 (s)	20.9, q		20.8, q
3′-OMe	3.73 (s)	56.7, q	3.71 (s)	56.6, q
5′-OMe	3.73 (s)	56.7, q	3.71 (s)	56.6, q
1″	4.20 (d, 7.8)	104.7, d
2″	3.16 (dd, 8.0, 7.9)	75.1, d
3″	3.32 (overlap)	78.1, d
4″	3.26 (overlap)	71.6, d
5″	3.23 (overlap)	77.9, d
6″	3.83 (overlap)	62.7, t
	3.65 (dd, 11.9, 5.4)

The ¹H and ¹³C NMR data of compound 1 were recorded at 600 MHz and 150 MHz, with CD₃OD as solvent, respectively.

The ¹H and ¹³C NMR data of the compound reported in the literature were recorded with a Bruker DRX-360, with (CD₃)₂CO as solvent^.⁹.

data (Table 1) were reminiscent of 2-acetoxymethyl-1,2,3,4-tetrahydro-7-hydroxy-1-(4-hydroxy-3,5 -dimethoxyphenyl)-3-hydroxymethyl-6,8-dimethoxynaphthalene (compound 1b).⁹ However, it is noteworthy that there is a slight difference in the carbon chemical shift at C-7 and C-7′, which probably resulted from the introduction of a sugar moiety at C-9. The key 1H detected heteronuclear multiple bond correlation (HMBC) correlations (Figure 2) of H-9 with C-1″ confirmed that the glycosyl moiety was attached to C-9. The planar structure of 1 was further determined by a comprehensive interpretation of the ¹H-¹H COSY, HSQC, and HMBC spectra. To determine the absolute configuration of 1, it was acid hydrolyzed to obtain an aglycon and sugar. For the aglycone (1c), due to the low amount, the ¹H NMR and ¹³C NMR data could not be completed, but CD (Figure 3) and specific rotation data (Supplementary Figure S22) were provided; its electronic circular dichroism (ECD) spectrum was almost identical to that of urinatetralin,¹⁰ phyltetralin,¹⁰ and strychnovanosides B-C,¹¹ and the absolute configuration of the sugar was confirmed as D-glucose by comparison with standard glucose using a chiral HPLC column analysis (Supplementary Figure S20). Thus, the absolute configuration of 1 was established as 7′R, 8S, 8′S. Accordingly, compound 1 was established as shown in Figure 1 and was named artemisiaside A.

Figure 1.

Chemical structures of compounds 1 to 6 and compound 1b.

Figure 2.

¹H-¹H COSY and key HMBC correlations of compound 1.

Figure 3.

Experimental ECD spectra of compound 1.

By comparing their spectroscopic data with those reported in the literature, the other 5 known compounds 2-6 were identified as (-)-lyoniresinol 3α-O-β- D-glucopyranoside,¹² (6R,7S,8S)-7α-[(β-D-glucopyranosyl)oxy]lyoniresinol,¹³ ( + )-lyonire sinol,¹² 5-methoxy-( + )-isolariciresinol,¹⁴ and burselignan,¹⁴ respectively.

As a traditional (folk) medicine, A argyi exhibits multifarious bioactivities and is used to control dysmenorrhea, abdominal pain, and inflammation.¹⁵ For instance, the essential oil from A argyi (AAEO) dose-dependently suppressed the release of pro-inflammatory mediators (nitric oxide [NO], PGE2, and ROS) and cytokines (TNF-α, IL-6, IFN-β, and MCP-1) in LPS-induced RAW264.7 macrophages.¹⁶ As well as the crude extract, dehydromatricarin A was also identified as an active component of this plant against inflammation of the lung.¹⁷ Moreover, several sesquiterpenoids obtained from A argyi have shown an enormous potential for inhibitory activity of NO production in vitro.^18–20 Therefore, in order to explore and enrich the variety of compounds with anti-inflammatory activity from A argyi, the anti-inflammatory activities of compounds 1 to 6 were evaluated by measuring their inhibitory effects on LPS-induced NO production in RAW264.7 LPS-activated macrophages. At 50 µM, compound 1 displayed moderate activity with an inhibition rate of 61.2% ( Table 2 ). At the same concentration, the inhibition rate of the positive control (L-NMMA) was 55.5%.

Table 2.

Inhibitory Rate of Lipopolysaccharide (LPS)-Induced Nitric Oxide (NO) Production in RAW246.7 Cells Treated With Compounds 1 to 6.

Compound	Concentration（μM）	NO Inhibitory Rate（%）
1	50	61.2 ± 0.5
2	50	25.5 ± 2.5
3	50	21.6 ± 2.0
4	50	18.7 ± 2.4
5	50	22.6 ± 1.8
6	50	19.5 ± 2.5
L-NMMA	50	55.5 ± 2.3

Conclusion

Six lignan analogues, including 1 new lignan glycoside, were obtained from the aerial parts of A argyi. Their structures were determined from extensive spectroscopic and mass spectrometric data and ECD. Compounds 1 to 6 were assessed by measuring the inhibitory effects on LPS-induced NO production in RAW264.7 LPS-activated macrophages. The results indicated that compound 1 has moderate anti-inflammatory activity.

Experimental

General Experimental Procedures

Column chromatography (CC) was performed on silica gel (200-300 mesh, Qingdao Marine Chemical Factory), Sephadex LH-20 (20-100 μm, Amersham Pharmacia Biotech), and MCI gel CHP-20P (70-150 μm, Mitsubishi Chemical Corp.). Standard thin-layer chromatography (TLC) was carried out on silica gel (GF254, 10-40 μm, Qingdao Marine Chemical Factory). Compounds were visualized on the TLC plates under UV light and by spraying with 5% H₂SO₄ in EtOH (v/v) followed by heating. HPLC analyses were performed on an Agilent 1260 HPLC system using an ODS column (C18, 250 × 4.6 mm, YMC Pak, 5 μm; detector: UV) and DAICEL chiral column (AGP 100.4 100 × 4.0) with a flow rate of 1.0 mL/min. Preparative HPLC was performed on an Agilent 1100 series with a Zorbax SB-C18 column (5 μm, 9.4 × 150 mm, Agilent) with a flow rate of 8.0 mL/min. NMR experiments were carried out using a cryogenic Bruker AV-600 spectrometer with TMS as internal standard. Mass spectra were measured on an Agilent G6230 spectrometer. UV spectra were obtained on either a Shimadzu 2700 or 2401 PC double-beam spectrophotometer. Optical rotations were measured with a Horiba SEPA-300 polarimeter. ECD experiment was conducted with an Agilent Applied Photophysics circular dichroism spectrometer.

Plant Material

The aerial parts of A argyi (Compositae) were collected from Xichang (GPS, 30^o52′N/104^o44′E) of Sichuan province, China, in July 2021 (wet season). The plant was identified by Prof. Qing-Shan Yang (Anhui University of Chinese Medicine), and an authentic sample (A20210708) is kept in the Laboratory of Research and Development of Medicinal Plants in the Panxi Region.

Extraction and Isolation

Air-dried and powdered aerial parts of A argyi (2 kg) were milled and soaked in methanol (3L) at room temperature for 48 h, and the extraction was conducted 3 times under the same conditions. The combined extract was evaporated to dryness under reduced pressure to yield an oily residue (106 g), which was further subjected to silica gel CC eluting with CH₂Cl₂ and then with CH₂Cl₂/MeOH stepwise-gradient (50:0, 25:1, 20:1, 15:1, 10:1, 5:1, 2:1, 0:1, v/v) to give 3 fractions (Frs. I-III).

Fr. I (15 g) was fractionated by silica gel CC with CH₂Cl₂/MeOH (from 50:1 to 20:1, v/v) as the eluent to give 3 subfractions (Frs. IA-IC). Fr. IC (500 mg) was applied to a silica gel column and eluted with CH₂Cl₂/MeOH (30:1, v/v) to afford 4 additional subfractions (Fr. IC-1-Fr. IC-4). Fr. IC-2 (108 mg) was passed through a Sephadex LH-20 column eluting with CH₂Cl₂/MeOH (1:1, v/v) to obtain 3 additional subfractions (Fr. IC-2a-Fr. IC-2C). Fr. IC-2b (30 mg) was separated on a Sephadex LH-20 column eluting with CH₂Cl₂/MeOH (1:1, v/v) and then subjected to semi-preparative RP-HPLC using MeCN-H₂O (15:85-25:75, v/v) as the mobile phase to yield 4 (t_R 30 min, 3.2 mg), 5 (t_R 34 min, 4.8 mg), and 6 (t_R 40 min, 5.2 mg).

Fr. III (12 g) was separated on a silica gel column eluting with CH₂Cl₂/MeOH (from 15:1 to 1:1, v/v) to afford 3 subfractions (Frs. IIIA-IIIC). Fr. IIIA (3 g) was subjected to silica gel CC with CH₂Cl₂/MeOH (5:1, v/v) as the eluent and then was chromatographed over a Sephadex LH-20 column eluting with CH₂Cl₂/MeOH (1:1, v/v) to obtain 3 additional subfractions (Fr. IIIA-1-Fr. IIIA-3). Fr. IIIA-2 (76 mg) was separated on a Sephadex LH-20 column eluting with CH₂Cl₂/MeOH (1:1, v/v) and then was applied to semi-preparative RP-HPLC using MeCN-H₂O (5:95-20:80, v/v) as the mobile phase to yield 1 (t_R 23 min, 2.1 mg), 2 (t_R 25 min, 4.8 mg), and 3 (t_R 30 min, 5.6 mg).

Artemisiaside A (1)

Colorless amorphous powder, [α] D²⁵ −54 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 275 (3.54) nm; ECD (MeOH) λ_max (Δε) 216 ( + 8.31), 244 (−3.68), 273 (−0.92), 287 ( + 0.86) nm; HRESIMS m/z 623.2341[M-H]⁻ (calc. for C₃₀H₃₉O₁₄⁻, m/z 623.2340); ¹H and ¹³C NMR data; see Table 1 .

Aglycone (1c) of Compound 1

Colorless amorphous powder, [α] D²⁵ −4 (c 0.02, MeOH); ESIMS m/z 463 [M + H]⁺ (calc. for C₂₄H₃₁O₉⁺, m/z 463), CD (c = 0.0667mg/mL, CH₃OH) (Δε) 312 ( + 0.33), 299 ( + 0.33), 274 (−0.91), 265 (−0.36), 244 (−3.68), 225 ( + 2.92), 213 ( + 4.96).

Determination of Sugar Components

Compound 1 (2 mg) was refluxed in 10% HCl/dioxane 1:1 (2 mL) for 2 h. The reaction mixture was extracted with ethyl acetate (5 mL × 3) and further purified by silica gel CC using light petroleum–acetone (5:1) to give compound 1c (0.4 mg). The sugar component in the aqueous layer was evaporated under reduced pressure, dissolved in water (1 mL) and subjected to HPLC analysis (Column: ChromCore Suger-10Ca, 6 μm; Mobile Phase: 0.5 mL/min). Standard D-glucose (Sigma) was subjected to the same reaction and HPLC analysis under the same conditions.

Measurement of Nitric Oxide Production

NO production was assayed in supernatants of cultured RAW264.7 cells using an NO assay kit (Beyotime Institute of Biotechnology). Cells were seeded in 96 well culture plates, and pretreated with the tested compounds (50 µM) and stimulated with LPS (1 μg/mL) for 24 h. The supernatant was mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2.5% phosphoric acid) and incubated at room temperature for 24 h. The concentration of nitrite was measured by reading the absorbance at 570 nm.^21,22 L-NMMA was used as a positive control.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221118552 - Supplemental material for Artemisiaside A: A new Lignan Glycoside and its Analogues From the Aerial Parts of Artemisia argyi

Supplemental material, sj-docx-1-npx-10.1177_1934578X221118552 for Artemisiaside A: A new Lignan Glycoside and its Analogues From the Aerial Parts of Artemisia argyi by Chuan-Wang Tong, Ming Tao, En-Bo Zhang, Yi Huang, Hao Geng and Yang Yu in Natural Product Communications

Footnotes

Acknowledgements

The authors would like to thank the members of the analytical center of Kunming Institute of Botany, Chinese Academy of Science, for the measurement of all spectra. This study was financially supported by the Natural Science Key Research Program of Anhui Province University (KJ2020A0913, KJ2020A0915), Foundation of Anhui Province Key Laboratory of Research & Development of Chinese Medicine (AKLPDCM202004), the Doctoral Scientific Research and Double High Talent Project of Xichang University (YBZ202114, LGLZ201811), and the Key Research and Development Program of Science and Technology Bureau of Liangshan Yi Autonomous Prefecture (21ZDYF0019). Computational resources used in this work were supported in part by SciGrid, Chinese Academy of Sciences.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Doctoral Scientific Research and Double High Talent Project of Xichang University, Key Research and Development Program of Science and Technology Bureau of Liangshan Yi Autonomous Prefecture, Natural Science Key Research Program of Anhui Province University, Foundation of Anhui Province Key Laboratory of Research & Development of Chinese Medicine (grant numbers YBZ202114, LGLZ201811, 21ZDYF0019, KJ2020A0913, KJ2020A0915, gxgnfx2022133 and AKLPDCM202004).

ORCID iD

Hao Geng

Supplemental Material

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