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Sesquiterpene lactones from Artemisia vulgaris L. as potential NO inhibitors in LPS-induced RAW264.7 macrophage cells

Twelve new guaianolide sesquiterpene lactones (1–12), along with ten known analogs (13–22) were isolated from an EtOH extract of the dried aerial parts of Artemisia vulgaris L. The new structures were elucidated via abundant spectroscopic data analyses (HRESIMS, IR, 1D, and 2D NMR), and the absolute configurations of these compounds were determined by X-ray crystallography and ECD calculations. The compounds (1−22) were identified as guaiane-type sesquiterpenes with characteristic α-methylene-γ-lactone and α,β-unsaturated carbonyl moieties. All compounds were tested for their inhibitory activity against NO production in lipopolysaccharide-stimulated RAW264.7 macrophages. The isolated sesquiterpenoids dose-dependently exhibited an NO production inhibitory activity by inhibiting the expression of inducible NO oxidase (iNOS) and cyclooxygenase-2 (COX-2) with IC50 values ranging from 1.0 to 3.6 μM. The inhibitory effect on the NO production of the compounds (1–4 and 6–22) is better than that of the positive control (dexamethasone). The different substitutions of compounds on C-8 influence anti-inflammatory effects, as evidenced by the in silico analysis of related binding interactions of new compounds (1–12) with iNOS.


Introduction
Artemisia vulgaris L. (mugwort), belonging to the family of Asteraceae, is widespread throughout Asia, North America, and Europe (Weston et al., 2005). A. vulgaris, as a kind of traditional medicinal plant, has been extensively used for relieving pain and treating gynecological symptoms in folks (Hershoff, 2001;Pires et al., 2009). The chemical constituents of A. vulgaris contain mainly polysaccharides, flavonoids, terpenoids, and sterols, showing anti-tumor, anti-inflammatory, hepatoprotective, anti-oxidant, immunomodulatory, anti-allergic, and antibacterial activities (Schmid-Grendelmeier et al., 2003;Blagojević et al., 2006;Saleh et al., 2014;Abiri et al., 2018;Soon et al., 2019;Ekiert et al., 2020). Previous phytochemical investigations reported that sesquiterpenoids are among the most critical ingredients of secondary metabolites in the genus Artemisia and have strong application values in contemporary medicine, food, and the perfume industry (Duke and Bogenschutz, 1994;Bora and Sharma, 2011;Rasheed et al., 2017;Sundararajan and Kumari, 2017). It is widely known that artemisinin and arglabin are promising potent remedies in the treatment of malaria and cancer (Lone et al., 2015;Kumari et al., 2019). A. vulgaris and Artemisia annua L. are very similar in many aspects (Funk, 2009). Therefore, A. vulgaris is regarded as an abundant producer of biologically effective sesquiterpene lactones, prompting us to select it for a detailed study.
Inflammation is an essential immune response to pathogens, toxins, and local injuries. The beneficial effects in the physiological or acute inflammation might turn deleterious in a persistent or over-inflammatory response. Macrophage is a primary cell type in directing the host's inflammatory and immune processes, and the excessive release of nitric oxide is an essential sign of an inflammatory response. Testing the inhibitory effect of compounds on NO's release in macrophages is an integral approach for revealing novel small molecules with anti-inflammatory activities. Our investigation for architecturally unique and effective chemical constituents from A. vulgaris led to the isolation and structural elucidation of 22 compounds, including 12 new sesquiterpenoids. And, all the compounds were assessed for anti-inflammatory effects by LPSinduced RAW264.7 macrophage cells.
2 Materials and methods 2.1 General experimental procedures UV spectra were attained on a JASCO V-730 spectrophotometer. IR data were obtained on a Nicolet iS50 spectrometer (Thermo Fisher Scientific, United States) using KBr pellets. Optical rotations were measured using a JASCO P-2000 polarimeter at room temperature. HRESIMS spectra were recorded with an AB Sciex Triple-TOF 5600+ apparatus. ECD measurements were conducted on a Chirascan Plus spectrometer (Applied Photophysics, United Kingdom). 1D and 2D NMR spectra were obtained on a Bruker AVANCE III 500 or 600 MHz instruments. Chemical shifts were reported in ppm (δ) with coupling constants (J) in hertz. The residual signals of CDCl 3 were used as references. Single crystal X-ray diffraction (Rigaku Oxford Diffraction Supernova Dual Source) was used to measure the crystal structures. Silica gel Qingdao Marine Chemical Co.,Ltd.,China), Sephadex LH-20 gel (GE Healthcare, Sweden), YMC ODS-A-HG gel (50 μm, YMC, Japan), and MCI gel (SaiPuRuiSi. Beijing, China) were used to perform column chromatography (CC). Semi-Preparative HPLC was performed on a Wufeng LC-100 apparatus (Shanghai Wufeng Co., Ltd., China) using photodiode array (PDA) UV analysis at 210 nm with a YMC-Pack ODS-A column (250 × 10 mm, 5 μm, 3 ml/min).

X-ray crystallographic analyses
All crystals were obtained by recrystallization from MeOH. The X-ray diffraction data of compounds 6, 8, 10, and 12 were collected on an Agilent SuperNova four-circle instrument by means of Cu Kα radiation. The structures were solved by direct methods and refined by the full-matrix leastsquares process on F 2 using the SHELXTL or the Olex2 software package. X-ray data can be obtained free from the Cambridge Crystallographic Data Centre via https://www.ccdc.cam.ac.uk/structures/.

ECD calculations
Conformational analyses for new compounds were carried out using MOE software with MMFF94s. The obtained stable conformers were optimized at the b3lyp/6-31+g(d) level in the gas phase and further subjected to ECD calculations at cam-b3lyp/6-31+g(d) level in the PCM model of methanol using the Gaussian 09 program. The ECD spectra were weighted according to the Boltzmann distributions.

Cytotoxicity
For the cytotoxicity assay, RAW 264.7 cells were grown in DMEM containing 10% FBS and cultured at 37°C. Then, the cells were seeded in a 96-well plate (1 × 10 4 cells/well) before the cells were incubated for 24 h in various concentrations of compounds (3.125-200 μM). Cell viability was examined using a CCK8-kit. The CCK8 solution (5 µl) was added to each well and incubated for 1-2 h at 37°C. The OD value at 450 nm was quantified by Graphpad software, and the corresponding 50% cytotoxic concentration (CC 50 ) of the compounds was subsequently obtained.

Measurement of NO production
The level of accumulated nitrite in the culture media reflected the level of NO using the classic Griess reagent (Beyotime, Jiangsu, China). The RAW264.7 cells were seeded in a 96-well plate with a 6 ×10 4 cells/ml density for the indicated time. Then, the cells were pretreated for 1 h with test compounds (0.25-4 μM), followed by stimulation with LPS (1 μg/ml) for 18 h. Finally, 50 ul of the culture supernatant was mixed with an equal amount of the Griess reagent, and the optical densities at 570 nm were read using a microplate reader. The nitric oxide concentration of the samples was calculated according to the standard curve.

Western blotting
RAW264.7 cells with the indicated treatment were harvested and treated in a RIPA lysis buffer (Beyotime, Shanghai, China) containing a protease and phosphatase inhibitor cocktail (Beyotime, Shanghai, China) to obtain the lysates. Then, the total proteins were measured using the bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China) and regulated by a loading buffer and the RIPA lysis buffer. The samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride membranes (Millipore), followed by blocking with 5% non-fat milk. The membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with specific secondary antibodies. Finally, the protein bands were detected using the ImageJ software (Bio-Rad, Hercules, CA, United States).

Molecular docking
In order to explore the possibility of compounds 1-6 binding to the INOS target, Autodock vina and AutoDockTools-1.5.6 (Vina, 2010;Forli et al., 2016) were used to predict the free binding energy. Autodock vina used the Broyden-Fletcher-Goldfarb-Shanno (BFGS) 19 method to obtain the optimal conformation (Nguyen et al., 2020). First, the three-dimensional structure of INOS (PDB ID:3E6T) was obtained from the Protein Data Bank (http://www.rcsb.org) (Burley et al., 2021), whose resolution was 2.5 Å. The three-dimensional structure of compounds 1-6 was built and optimized by ChemDraw Ultra 8.0. Pymol molecular graphics software and AutodockTools-1.5. 6 were used to remove ligand, dehydrate, hydrogenate, and charge the target (Liang, 2003;Seeliger and de Groot, 2010). The cubic grid box was calculated by AutoDockTools-1.5.6 positioned at the center of (122.65, 114.0, 36.63) with a spacing of 0.375 Å. All docking parameters were set to default values, but the modes and exhaustiveness were set to 10. The docking results were further analyzed and presented using pymol.
The relative configuration was determined by the correlations between H-7/H 2 -13, H-7/H-5, and H-6/H-8 in the NOESY spectrum (Figure 2). The absolute configuration of 1 was assigned according to the ECD calculations on the arbitrarily chosen enantiomers. Based on the evidence mentioned previously, the stereochemistry of 1 was eventually assigned as 1S,5R,6S,7R,8S.
Artemvulactone M (6) was determined as C 19 H 24 O 6 according to the HRESIMS ([M + Na] + , m/z 371.1450, calcd for 371.1465). The IR absorption bands at 3,495, 1,772, and 1,731 cm −1 were marks of hydroxy and carbonyl groups. The 1 H and 13 C NMR spectra of 6 were comparable to 4, but the Frontiers in Chemistry frontiersin.org isovaleryloxy group is absent. It could be aided by the HMBC correlation between the proton signal at δ H 4.96 (1H, td, 9.9, 2.1 Hz, H-8) and the carbonyl carbon signal at δ C 176.4 (C-1′). In addition, the main difference was ascribed to the absence of a cyclic olefinic bond at C-3 and C-4 in 6, but an additional ring connected via an oxygen atom. The aforementioned reasoning was based on the correlation of H-2′/H 3 -3′ (H 3 -4′) in the 1 H− 1 H COSY spectrum and the cross peak from H-2′/H 3 -3′ (H 3 -4′) to C-1′ in the HMBC spectrum (Figure 1). The single-crystal X-ray diffraction experiment with Cu Kα radiation [CCDC,2164101] confirmed the 2D structure of 6 and defined its absolute configuration as 1S,3R,4S,5R,6S,7R,8S. Therefore, the absolute configuration of compound 6 was established as shown ( Figure 4). Artemvulactone N (7) was obtained as a colorless oil. The molecular formula C 20 H 24 O 5 was deduced from the HRESIMS at m/z 345.1703 [M + H] + (calcd for 345.1697). The IR spectra revealed that 7 possessed hydroxy, carbonyl, and olefinic groups. In the NMR spectra of 7 (Tables 1, 3), the signals at δ H 5.16 (1H, s, H-14a), 5.44 (1H, s, H-14b), δ C 144.1 (C-10), and 117.6 (C-14) Frontiers in Chemistry frontiersin.org revealed that a terminal double bond was located at C-10, which was supported by the HMBC correlations from H 2 -14 to C-1 and C-8. The NMR spectra of 7 (Tables 1, 3) were very similar to compound 4, except that the isovaleryloxy substituent at C-8 in 4 was replaced by an angeloyloxy group in 7. The absolute configuration of 1S,5R,6S,7R,8S was confirmed by the comparison of experimental and calculated ECD spectra (Figure 3). Artemvulactone O (8) gave a molecular formula of C 19 H 24 O 6 as defined by the HRESIMS ion at m/z 371.1456 [M + Na] + (calcd for 371.1465). The presence of hydroxy (3,476 cm −1 ), carbonyl (1,769 and 1,730 cm −1 ), and olefinic (1,658 cm −1 ) functionalities was evident from the spectroscopic data. The 1 H and 13 C NMR spectra of 8 manifested that it was structurally similar to 6. The main difference was the position of a double bond, which was verified by the HMBC correlations from H 3 -14 to C-9 ( Figure 1). The absolute configuration of 1S,3R,4S,5R,6S,7R,8S was determined by means of a single crystal X-ray crystallographic diffraction experiment with Cu Kα radiation (CCDC, 2164105, Figure 4). Artemvulactone P (9) was isolated as a colorless oil. According to the HRESIMS ([M + H] + , m/z 317.1370, calcd for 317.1384) and the 1D NMR spectrum, the molecular formula of 9 was assigned as C 18 H 20 O 5 . The IR absorption bands at 1,773, 1,737, and 1,673 cm −1 confirmed the existence of carbonyl and olefinic groups, with one less hydroxyl group than compound 1. Additionally, the propionyl group was attached to oxygen-linked carbon at C-8, which could be demonstrated by the signals at δ C 173.3 (C-1′), 27.8 (C-2′), and 9.1 (C-3′). In the 1D NMR spectrum, the characteristic signals of methylene [δ H 2.65 (2H, m, H-2); δ C 46.3 (C-2)] in 1 were replaced by a carbonyl in 9 at C-2 (δ C 195.1). Based on the application of the TD-SCF ECD calculation method, the similarity of the calculated ECD spectrum with its experimental spectrum indicated the 5R,6R,7S,8R configuration of 9 (Figure 3).
Artemvulactone Q (10) showed the same molecular formula as that of compound 8. The carbonyl and olefinic groups also existed in the structure of 10, which were attributable to the IR absorptions at 1,770, 1,736, and 1,629 cm −1 . The compound 10 differed from 9 by one more methoxy group and one less cyclic olefinic bond in its chemical structure at C-10. The aforementioned analysis was confirmed by the 1 H-1 H COSY correlations of H-1/H-9/H 3 -14/H 3 -16 and HMBC correlations from δ H 3.24 (-OCH 3 ) to δ C 76.5 (C-10). The absolute configuration of compound 10 was established based on a single-crystal X-ray crystallographic diffraction experiment with Cu Kα radiation (Figure 4).
Artemvulactone R (11) was obtained as a white powder and assigned a molecular formula of C 18 H 22 O 6 by HRESIMS and 13 C NMR data. The IR absorption bands of 11 implied hydroxy, carbonyl, and olefinic groups. The 1D NMR data (Tables 2, 3) revealed a definite structural variation between 11 and 8. The main difference was that, compound 11 possessed one less methyl group at C-2′ [δ H 2.42 (2H, dd, 15.0, 7.5 Hz, H-2′); δ C 27.9 (C-2′)], which was defined based on the 1 H− 1 H COSY

FIGURE 8
Compound 1 on the downregulation of LPS-induced iNOS and COX-2 mRNA expression in RAW264.7 cells. *p < 0.05, **p < 0.01, ***p < 0.001, compared to the LPS-treated groups. 3.2 Anti-inflammatory effects for the intervention of NO production in LPSinduced RAW264.7 cells Inhibition of NO overexpression is an essential indicator for evaluating small molecules with anti-inflammatory activities. Thus, the inhibitory effects of compounds 1-22 on LPSmediated NO production were tested, and it was found that isolated sesquiterpenoids except compound 5 exhibited a concentration-dependent NO production inhibitory activity with IC 50 values ranging from 1.0 to 3.6 μM (Table 4). It was preliminarily concluded that the isolated compounds had the potential to be developed into anti-inflammatory drugs.
NO production is directly related to the expressions of iNOS and COX-2. We selected the most active compound 1 for gene and protein level verification. The results of experiments demonstrated that compound 1 at 4 μM reduced more expressions of iNOS and COX-2 than the positive drug (Dexamethasone) in LPS-induced macrophages by quantitative real-time PCR and Western blotting (Figures 6, 7). By comparing the structure and activity of the compounds, we preliminarily summarize the structure-activity relationship of guaiane-type sesquiterpene lactones in inhibiting inflammation: α-methyleneγ-lactone, α,β-unsaturated carbonyl moieties, and the ester side chain at C-8 are critical for cytotoxic activity.
To better compare the binding ability of docking compounds, dexamethasone and INOS were used for redocking. Molecular docking results showed that 1-12 and dexamethasone had a good interaction with the INOS targets by targeting residues in pockets. Figure 8 revealed the predicted geometry of 1, 7, 4 and 5 bound to the INOS protein. It was conformable to the results obtained through biological experiments. The free-binding energy and the number of binding residues are shown in Table 5, and that compounds 7, 5, and 4 were predicted to possess a stronger association with the protein. Previous research has shown that the type of substituent at C-8 also affects the compound activity: isovaleryloxy > acetyl > angeloyloxy > methylbutyryloxy (Sun et al., 2020). Combined with biological experiments, the hydroxyl group at C-1 was inferred to influence compound activity, and the following trend in activity for the side chain was summarized: angeloyloxy > methylbutyryloxy > isovaleryloxy. The aforementioned information rationalizes the results of molecular docking and biological experiments.

Conclusion
In conclusion, in our endeavor to search for antiinflammatory compounds from Artemisia vulgaris L., 12 new and 10 known guaiane-type sesquiterpenoids were isolated and identified, of which compound 1 was the most potent inhibitor on NO release in LPS-stimulated RAW264.7 cells. The biological data confirmed that the expression of inflammatory enzymes of iNOS and COX-2 was suppressed by 1 in LPS-stimulated RAW264.7 cells. The binding interactions of compound 1 with iNOS also confirmed the conclusion. Further mechanisms need to be further explored. Accordingly, 1 displayed the therapeutic potential for modulating inflammation.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.