A new benzofuran from Artemisia halodendron Turcz. ex Bess.

Abstract A new benzofuran, methyl (2S,2″S,3′E)-[2-(1″-acetoxypropan-2-yl)-2,3-dihydrobenzofuran-5-yl]acrylate (1), and 13 known compounds (2–14) were isolated from an ethanol extract of Artemisia halodendron Turcz. ex Bess. The chemical structures of these compounds were determined by 1D and 2D NMR (1H-1H COSY, HMBC, HMQC and NOESY) and HR-ESI-MS spectra, and results were compared with data from the literature. The effects of compounds 1–14 were measured on NF-κB activation, with compounds 2 and 3 exhibiting inhibitory activities against TNF-α-induced NF-κB reporter gene expression in HeLa cells from 10 to 100 μM.


Introduction
Artemisia, comprising up to 350 species, is the largest genus in the plant family Asteraceae. The use of Artemisia species as a penetrant is of interest in the field of natural products research. Recently, irregular monoterpene ester with potential antifungal activity were isolated from Artemisia incisa (Rashid et al. 2017), the extract of A. quettensis had significant antibacterial and anticancer effects (Baqheri Farahani et al. 2017), two new amorphane derivatives and one modified seco-eudesmane were purified from A. alba growing in the North-East of Italy (Peron et al. 2017), the aerial parts ethanolic extract of A. marschalliana was observed to be an effective antioxidant and anticancer agent (Salehi et al. 2017). In addition, artemisinin, a sesquiterpene lactone with antimalarial properties, was isolated from A. annua Linn in 1971 (Klayman 1985). Another interesting species is A. halodendron Turcz. ex Bess., a typical sandy shrub widely distributed in Chinese deserts and mainly used for windbreaks and dune fixation. The aerial parts of A. halodendron have been traditionally used to treat chronic bronchitis, asthma, rheumatoid arthritis and inflammation (Wang et al. 2004(Wang et al. , 2017. Despite these medicinal applications, however, A. halodendron is mostly used as animal fodder, and its chemical composition has not been well characterised. In this study, further phytochemical investigation of A. halodendron was therefore performed to search for additional compounds with novel structures and potent bioactivities. Nuclear factor-κB (NF-κB) transcription factors are involved in many physiological processes, including apoptosis, immunity, and inflammation. NF-κB regulates the transcription of various inflammatory cytokines, including interleukin 8 (IL-8), tumour necrosis factor alpha (TNF-α), and monocyte chemotactic protein 1 (Ma et al. 2012). NF-κB is thus primarily an inducer of inflammatory cytokines; consequently, its inhibitors may be useful as anti-inflammatory agents.
The present study aimed to analyse the chemical structures of one new benzofuran (1), one known phenoxychromone (2), nine known flavonoids (3-11), and three known benzofurans (12-14) isolated from the aerial parts of A. halodendron. The NF-κB inhibitory activities of compounds 1-14 on TNF-α-induced NF-κB reporter gene expression in HeLa cells were investigated.

Plant material
The aerial parts of A. halodendron were collected from the prairie of Tongliao, Inner Mongolia, China in August 2015, and identified taxonomically by Prof. Gao Li (College of Pharmacy, Yanbian University). The voucher specimen (voucher number: YB-SHZ-1508) has been deposited at the Pharmacognosy Laboratory of the College of Pharmacy, Yanbian University.

NF-κB luciferase reporter assay
NF-κB-dependent luciferase activity was measured using the Dual Luciferase Reporter Assay system (Ma et al. 2016). Briefly, HeLa cells (1 × 10 5 cells/well) were seeded in a 96-well plate for 24 h. The cells were then transfected with plasmids for each well and then incubated a transfection period of 48 h. After that, the cell culture medium was removed and replaced with fresh medium containing different concentrations (10, 30 and 100 μM) of the compounds for 8 h, followed by treatment with 10 ng/mL of TNF-α for 8 h. Luciferase activity was determined in MicroLumat Plus Luminometer by injecting 100 μL of assay buffer containing luciferin and measuring light emission for 10 s. Co-transfection with pRL-CMV, which expresses renilla luciferase, was performed to enable normalisation of data for transfection efficiency. The data are presented as means ± SD of three independent experiments.

Conclusions
A. halodendron is widely distributed in Chinese deserts, but very few of its chemical components have been identified thus far. In this continuation of our studies on A. halodendron, one new benzofuran (1), one phenoxychromone (2), nine known flavonoids (3-11), and three known benzofurans (12-14) were isolated from this plant. Among them, compounds 4 and 8 are reported for the first time from the genus Artemisia, and compounds 2, 3, 5, 6, 9, 11, 12-14 have been isolated from A. halodendron for the first time. Compounds 2 and 3 at concentrations from 10 to 100 μM exhibited obvious inhibitory activity against TNF-αinduced NF-κB reporter gene expression in HeLa cells. Our results may provide a scientific basis for the completion of the chemical characterisation of A. halodendron and for the development of compounds with novel pharmacological activity.