Two new eudesmane sesquiterpene glucosides from the aerial parts of Artemisia vulgaris

Abstract Using combined chromatographic methods, two new sesquiterpene glucosides, vulgarosides A (1) and B (2), and two known analogs ainsliaside E (3) and pumilaside A (4) were isolated from the aerial parts of Artemisia vulgaris. Their chemical structures were established by spectroscopic methods, including one and two-dimensional nuclear magnetic resonance (1 D and 2 D-NMR) spectroscopy and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). In addition, their cytotoxicity on five human cancer cell lines, including KB (epidermoid carcinoma), HepG2 (hepatocarcinoma), MCF7 (breast carcinoma), SK-Mel-2 (melanoma), and LNCaP (prostate cancer) was also evaluated by the SRB assay. However, none of the tested eudesmane sesquiterpene glycosides showed significant cytotoxicity (IC50>100 µM). Graphical Abstract


Introduction
The genus Artemisia is one of the largest and most widely distributed genera of the family Asteraceae (Compositae) consisting of over 500 diverse species (Bora and Sharma 2011;Giang et al. 2014;Liu et al. 2020;Cao et al. 2021;Zhang et al. 2021). Artemisia vulgaris L. is one of the best-known species of this genus, which has a widespread distribution in the natural habitats worldwide (Europe, Asia, North and South America, and Africa) (Judzentiene and Budiene 2018). This plant has been used as a medicinal herb in Vietnamese traditional medicine for the treatment of metrorrhagia, leucorrhea, and irregular menstruation (Dũng et al. 1992;Thao et al. 2004). Several chemical constituents have been reported from A. vulgaris, including essential oils, flavonoids, sesquiterpene lactones, phenolics, flavonoids, coumarin derivatives, and other groups of metabolites (Bora and Sharma 2011). A. vulgaris has been reported to have diverse pharmacological activities, including anti-inflammatory (Afsar et al. 2013), antioxidant (Melguizo-Melguizo et al. 2014, antifertility (Shaik et al. 2014), hepatoprotective (Gilani et al. 2005), and cytotoxic activities (Saleh et al. 2014). Indeed, the 70% ethanolic extract of A. vulgaris has been shown to have preferential cytotoxicity against PANC-1 human pancreatic cancer cells under a nutrient-deprived condition with PC 50 12.5 lg/mL (Omar et al. 2019). In our ongoing study on the chemical of A. vulgaris (Hanh et al. 2018); we report herein the isolation, structural identification, and cytotoxicity of four eudesmane sesquiterpene glucosides.

Results and discussion
The MeOH residue (150 g) was suspended in water (W) and successively extracted with nhexane (H), dichloromethane (D), and ethyl acetate (E) to yield the H extract (30 g), D extract (12 g), E extract (10 g) and water layer (W), respectively. Repeated purification via column chromatography of the water layer extract of A. vulgaris led to the isolation of four eudesmane-type sesquiterpenoid glucosides (1-4), including two new compounds (1 and 2). The known compounds, ainsliaside E (3) (Miyase et al. 1991) and pumilaside A (4) (Xu et al. 2010) were identified by interpretation of their spectroscopic data in comparison with those reported in literatures. To the best of our knowledge, this is the first report of compound 3 from Artemisia and compound 4 from A. vulgaris.
Compound 1 was purified as a colorless oil. The HR-ESI-MS showed a protonated molecular ion [M þ H]  .7 (C-10), as well as two methines and four methylene groups. The remaining signals at d C 100.1 (CH, C-1 0 )/d H 4.61 (1H, d, J ¼ 8.0 Hz, H-1 0 ), 75.6 (CH, C-2 0 ), 78.2 (CH, C-3 0 ), 71.8 (CH, C-4 0 ), 78.3 (CH, C-5 0 ), and 63.0 (CH 2 , C-6 0 ) were assignable to a b-glucopyranosyl unit (Kitajima et al. 2000). The D configuration of the glucose moiety was assigned on the basis of biosynthetic reasons, analogy with ainsliaside E (3) and pumilaside A (4), coexistence in the plant A. vulgaris. The 1 H and 13 C NMR data of 1 exhibited the characteristic resonance for an eudesmane-type sesquiterpene glycoside, one of main components reported from Artemisia species (Ivanescu et al. 2015 Figure S2, Supplementary data). The heteronuclear multiple bond correlations (HMBC) correlation between the anomeric Glc H-1 0 (d H 4.61) and C-6 (d C 78.5) confirmed that the glucose unit was linked at C-6 ( Figure S2, Supplementary data). The relative configuration of 1 was determined by comparison of the 1 H and 13 C NMR data of 1 with those pumilaside A (Kitajima et al. 2000) and further substantiated by the nuclear overhauser effect spectroscopy (NOESY) experiment.
In particular, NOESY correlations observed between H-6/H 3 -14 and H 3 -15 and between H 3 -14/H 3 -15 indicated that H-6, H 3 -14, and H 3 -15 have the same orientation ( Figure S2). Furthermore, the NOESY correlations of H-5 with H-1 and H 2 -12, the large coupling constant (J ¼ 12.0 Hz) between H-5/H ax -6, the small coupling constant (J ¼ 5.0 Hz) between H ax -6/H-7, and the lack of NOE between H-5/H-7 suggested that the orientation of H-1, H-5, and the isopropyl group at C-7 should be oriented to the opposite face of H-6, H 3 -14, and H 3 -15 ( Figure S2). On the basis of the spectroscopic analysis, the structure of 1 was elucidated as a new compound and named vulgaroside A.
Compound 2 was also afforded as a colorless oil. The HR-ESI-MS showed a sodium molecular ion [M þ Na] þ at m/z 457.2412 (calcd. for C 21 H 38 NaO 9 þ , 457.2408), suggesting that the molecular formula of 2 is C 21 H 38 O 9 . Comparative analysis of the 1 H and 13 C NMR data of 2 with those of 1 resulted in a close similarity, except for the replacement of the terminal disubstituted double bond in 1 by a CH 3 -CH-fragment in 2. This was revealed by the presence of a carbon signal of a methine at d C 34.8 (C-11) and a methyl at d C 17.5 (C-12) in the 13 C NMR spectrum of 2, along with HMBC cross-peaks observed from H-12 (3H, d, J ¼ 6.5 Hz) to C-7 (d C 38.4), C-11 (d C 34.8), and C-13 (d C 67.7). The relative configuration of 2 was suggested to be the same as that of 1 by NOESY spectroscopic analysis as well as the large coupling constant (J ¼ 12.0 Hz) between H-5/H ax -6 and the small coupling constant (J ¼ 4.0 Hz) between H ax -6/H-7. Consequently, the structure of 2 was established as shown in Figure 1, namely vulgaroside B. The cytotoxic activity of the compounds against human cancer cell lines, KB (epidermoid carcinoma), HepG2 (hepatocarcinoma), MCF7 (breast carcinoma), SK-Mel-2 (melanoma), and LNCaP (prostate cancer) was examined by the SRB assay. However, none of the tested eudesmane sesquiterpene glycosides showed significant cytotoxicity (IC 50 >100 mM). The positive control, ellipticine showed cytotoxicity toward the KB, HepG2, MCF7, SK-Mel2, and LNCap cell lines, with IC 50 values of 1.83 ± 0.20, 1.14 ± 0.04, 1.79 ± 0.12, 1.71 ± 0.08, 1.54 ± 0.16 mM, respectively.

Plant material
The aerial parts of Artemisia vulgaris were harvested at Bac Tu Liem, Hanoi, Vietnam (March 2016). The plant material was identified by Dr. Nguyen The Cuong, a botanist from the Institute of Ecology and Biological Resources, VAST, Vietnam. A voucher specimen (TPCN-07) is deposited at the herbarium of Institute of Ecology and Biological Resources and Institute of Marine Biochemistry, VAST, Vietnam.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was financially supported by L'Oreal-UNESCO for Women in Science National Fellowship, Vietnam 2019 and a grant from Vietnam Academy of Science and Technology (grant number: T -DNDTP.05/19-21).