Artemyrianins A–G from Artemisia myriantha and Their Cytotoxicity Against HepG2 Cells

Abstract Four new sesquiterpenoids, artemyrianins A–D (1–4), and three new norlignans, artemyrianins E–G (5–7), together with five known compounds (8–12), were isolated from the aerial parts of Artemisia myriantha (Asteraceae). The new compounds were established by spectroscopic data analyses (HRMS, IR, 1D and 2D NMR), and their absolute configurations were confirmed by the single-crystal X-ray diffraction or ECD calculations. The isolates showed cytotoxicity against HepG2 cells with IC50 values ranging from 33.3 to 145.2 μM. Graphic Abstract Electronic supplementary material The online version of this article (10.1007/s13659-020-00255-z) contains supplementary material, which is available to authorized users.


Introduction
Hepatocellular carcinoma (HCC) resulting in 780000 deaths every year is the fourth cause of cancer mortality [1]. The treatment for advanced HCC has long been unsatisfied until the authorization of sorafenib in 2007. Afterwards, six drugs involving nivolumab, regorafenib, lenvatinib, Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1365 9-020-00255 -z) contains supplementary material, which is available to authorized users. pembrolizumab, ramucirumab and cabozantinib have been approved by the Food and Drug Administration (FDA). Whereas, the drug resistance and side effects are inevitable [2]. Natural products, especially sesquiterpenoids, such as artemisinin, thapsigargin, parthenolide [3] and arglabin [4] are leading compounds for the development of potent therapeutic agents for neoplastic diseases, which inspired us to search for anti-HCC active compounds with high efficiency and low toxicity from natural sources. Artemisia myriantha Wall. ex Bess., a folk medicine herb, has been used to treat inflammation and menorrhagia diseases [5]. According to previous investigation, sesquiterpenoids [5][6][7][8][9], dimeric sesquiterpenoids [5], flavones [7] and volatile oils [10][11][12] with antibacterial, anticancer and immunomodulating activity have been reported from this plant. In order to investigate the bioactive constituents of A. myriantha, our phytochemical study led to the isolation of four new sesquiterpenoids, artemyrianins A-D (1-4), three new norlignans, artemyrianins E-G (5-7), as well as five known compounds (Fig. 1). Herein, we reported their isolation, structural identification and cytotoxicity on HepG2 cells.
Artemyrianin E (5) was isolated as a yellowish oil and had a chemical composition of C 19 . The absolute stereochemistry of 5 was elucidated by the high agreement between the experimental and calculated ECD spectra. Hence, compound 5 was assigned as 7′E-(8S)-artemisarin.
Artemyrianin F (6) gave a molecular formula of C 19 H 18 O 5 from the HREIMS at m/z 326.1152 ([M] + , calcd for 326.1154). Its IR spectrum presented the characteristic absorption bands of hydroxy (3404 cm −1 ) and phenyl ring (1607, 1503 and 1442 cm −1 ) functionalities. The high resemblance between the 1 H and 13 C NMR (DEPT) data of 6 and (+)-artemisarin [16] manifested their same planar structure. The geometry of Δ 7 � was Z-form as deduced from the small J 7 ′ ,8 ′ value of 11.6 Hz. The ECD spectrum of 6 was superimposable to the calculated one, compound 6 was thus elucidated as 7′Z-(8R)-artemisarin.
Artemyrianin G (7) had a molecular formula of C 19 H 18 O 6 based on the HREIMS at m/z 342.1104 ([M] + , calcd for 342.1103). The existence of hydroxy (3406 cm −1 ) and phenyl ring (1608, 1503 and 1443 cm −1 ) groups was recognized in the IR spectrum. Its 1 H and 13 C NMR (DEPT) spectroscopic data were close to those of mandshuricol A [17], but the main difference was that C-8′ in 7 was a methine attached to a hydroxy with chemical shift of δ C 79.6 and C-8′ in mandshuricol A was a quaternary carbon attached to a hydroxy and a hydroxymethyl group with chemical shift of δ C 83.4, which suggested that 7 was de-hydroxymethyl derivative at C-8′ of mandshuricol A. The above deduction was supported by the 1   Accordingly, compound 7 was established as (8R,7′R,8′R)-8′-dehydroxymethylmandshuricol A.
In addition to four sesquiterpenoids (1-4), five lignans (5-9), two coumarins (10 and 11) and a benzofuran (12) were isolated from A. myriantha for the first time. The isolates were assayed for their cytotoxicity against HepG2 cells with sorafenib as the positive control. As a result (Table 3), all compounds showed cytotoxic activity with IC 50 values ranging from 33.3 to 145.2 μM.

Extraction and Isolation
The air-dried aerial parts of A. myriantha (24.7 kg) were smashed and extracted with 90% EtOH for two times at room temperature (each 100 L, four days). The combined extracts were concentrated and suspended in H 2 O, which was extracted with EtOAc. The EtOAc portion (1.

Spectroscopy Data of Compounds
The 1 H NMR spectra of artemyrianins A-G (1-7) were recorded in CDCl 3 at 600 MHz, and their 13 C NMR (DEPT) spectra were recorded in CDCl 3 at 150 MHz.

X-ray Crystallographic Analysis of Artemyrianin A (1)
Single crystals of compound 1 were obtained from MeOH. X-ray crystallographic data were collected on a Bruker D8 Quest apparatus employing graphite-monochromated Cu Kα radiation at 100.(2) K. Cell refinement and data reduction were carried out by the aid of Bruker SAINT. The structure was solved by direct methods with SHELXS-97. All non-hydrogen atoms were refined anisotropically by means of the least-squares method, and all hydrogen atoms were positioned using difference Fourier overlapping calculation and geometric calculations. Flack parameter = 0.18 (14).

Computational Details
The ECD calculations for compounds 2-7 were achieved by Gaussian 09 program. The configurations of compounds 2-4 and 7 were initially established on the basis of their  ROESY data, and further optimized by means of the DFT calculation at b3lyp/6-31G(d,p) level in the gas phase. The imaginary frequencies were excluded by the aid of frequency calculations at the same level. Taking the solvent effects into consideration, ECD calculations were carried out using the TDDFT methodology at b3lyp/6-311+g(d,p) level. The ECD curves were plotted by the Origin Pro 9 program (OriginLab Corporation, Northampton, USA).

Cytotoxicity Assay
MTT assays were conducted to evaluate the cytotoxic activity of the isolates on HepG2 cells [23]. HepG2 cells were seeded onto 96-well plates at a density of 1 × 10 4 cells per well. Following 24 h incubation at 37 °C with 5% CO 2 , cells were treated with 100 μL of culture medium containing tested samples at gradient concentrations for 48 h. Next, the medium was removed and replaced by 100 μL MTT solution (1 mg/mL), and the plates were further incubated in dark for four h. After removing the medium, 100 μL DMSO was added into each well to dissolve the MTT formazan salt. Then the plates were measured at 490 nm on a microplate reader (Bio-Rad, USA). The cytotoxicity of compounds was expressed as IC 50 values which calculated by Graph-Pad prism 5 (GraphPad Software, San Diego, California, USA). The HepG2 cells (Jining Biotechnology Co., Ltd.,   (Table 3).