The First Phytochemical Investigation of Artemisia divaricate: Sesquiterpenes and Their Anti-Inflammatory Activity

Artemisia divaricate belongs to the Artemisia genus of the family of Compositae, a sort of perennial herb endemic in most regions of China. For the first time, a phytochemical investigation was carried out on the whole plant of Artemisia divaricate, resulting in the identification of 39 sesquiterpenes, with 9 of them being new (1–9). The structures of the new compounds were fully established using extensive analysis of MS and 1D and 2D NMR spectroscopic data and density functional theory (DFT) NMR calculations. Their structures involve germacrane, eudesmane, and bisabolane types. All the new isolates were evaluated for their anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated murine macrophages of RAW 264.7 cells. Compounds 2 and 8 showed a significant inhibition effect on NO production, with IC50 values of 5.35 ± 0.75 and 7.68 ± 0.54 µM, respectively.


Introduction
Natural products, defined as components or metabolites of animals, plants, or microorganisms, play a highly important role in drug discovery and development due to their diversified structures and bioactivities [1]. Sesquiterpenes, which are an essential class of secondary metabolites, have a wide distribution in plants, especially those of the Asteraceae family [2]. The Artemisia genus, one of the largest genera of the Asteraceae family, possesses a unique position in traditional Chinese medicine, as a variety of Artemisia plants has a long history of being used as a medicine [3]. Extensively, phytochemical investigations on Artemisia plants have been launched for decades, yielding numerous sesquiterpenoid compounds, including mainly eudesmanolides, guaianolides, and germacranolides [4][5][6][7]. It is noteworthy that germacranolides exist as the largest group, which act as biogenetic precursors for the other types of sesquiterpene lactones. Previous investigations revealed the diversity of the pharmacological properties of germacranolides, such as antitumor [8], anti-inflammatory [9], antibacterial [10], cytotoxic [11], and immune [12] effects.
Artemisia divaricate (Pamp.) Pamp., a species endemic in China, is a perennial herbaceous plant mainly distributed in western Hubei, western Sichuan, and northern Yunnan [13]. So far, no phytochemical investigations have been reported on this plant.
In our continuous effort to search for bioactive constituents from natural sources, a systematic investigation of A. divaricate was performed, resulting in the isolation of 39 sesquiterpenes from the title plant for the first time. Herein, we describe the isolation and structural elucidation of nine undescribed sesquiterpenes of eudesmane or germacrane types (Figure 1; structures of known compounds, see Figure S1 in Supporting Information). Their structures were elucidated using extensive analysis of the spectroscopic data and
Based on the HR-ESIMS data, compound 2 (white powder) was designated a molecular formula of C 19 H 28 O 6 , the same as that of 1. The IR showed absorption bands for hydroxy (3440 cm −1 ) and carbonyl groups (1715 and 1626 cm −1 ). A detailed NMR analysis of compound 2 (Tables 1 and 2) showed high similarities between these two compounds, suggesting they might share the same planar structure. Detailed analysis of its 1 H-1 H COSY and HMBC data further supported such elucidation ( Figure 2).  Given the acetoxyl group is likely to be attached to C-8 or C-6, there were two possible structures for compounds 1 and 2, namely the 8-OAc and 6-OAc isomers ( Figure S18). DFT NMR calculation was performed on the two isomers to figure out the most possible structure. A conformational search was conducted using Conflex in a 5.0 kcal/mol energy window [14]. All conformers were reoptimized at the B3LYP/6-31G(d) in vacuo, and their 1 H and 13 C NMR chemical shifts were calculated at the level of mPW1PW91/6-311G (d,p) with the PCM solvent mode for chloroform [15]. The improved statistical method DP4+ Molecules 2023, 28, 4254 4 of 14 was used to analyze the calculated data of two possible structures and the experimental data [16]. The results of compound 1 gave a 100.00% (all data) possibility for the 8-OAc isomer, and compound 2 gave 97.20% (all data) for the same isomer (Figures S19 and S20), suggesting both are 8-OAc derivatives. Based on the HR-ESIMS data, compound 2 (white powder) was designated a molecular formula of C19H28O6, the same as that of 1. The IR showed absorption bands for hydroxy (3440 cm −1 ) and carbonyl groups (1715 and 1626 cm −1 ). A detailed NMR analysis of compound 2 (Tables 1 and 2) showed high similarities between these two compounds, suggesting they might share the same planar structure. Detailed analysis of its 1 H-1 H COSY and HMBC data further supported such elucidation ( Figure 2).
Given the acetoxyl group is likely to be attached to C-8 or C-6, there were two possible structures for compounds 1 and 2, namely the 8-OAc and 6-OAc isomers ( Figure S18). DFT NMR calculation was performed on the two isomers to figure out the most possible structure. A conformational search was conducted using Conflex in a 5.0 kcal/mol energy window [14]. All conformers were reoptimized at the B3LYP/6-31G(d) in vacuo, and their 1 H and 13 C NMR chemical shifts were calculated at the level of mPW1PW91/6-311G (d,p) with the PCM solvent mode for chloroform [15]. The improved statistical method DP4+ was used to analyze the calculated data of two possible structures and the experimental data [16]. The results of compound 1 gave a 100.00% (all data) possibility for the 8-OAc isomer, and compound 2 gave 97.20% (all data) for the same isomer (Figures S19 and S20), suggesting both are 8-OAc derivatives.
The relative configurations of 1 and 2 were determined using the coupling constant and NOESY cross-peaks. The Z-form of C-3 and C-4 was revealed using the NOESY correlation of H-3/H3-15 in both 1 and 2. The correlations of H3-14/H-8/H-6 were observed in the NOESY spectrum of 1 and 2, which supported that H3-14, H-8, and H-6 were on the same face and in β-orientation. The coupling constant of J6-7 (10.4 Hz for 1, 10.5 Hz for 2) indicated that these two protons in both compounds were in a trans form. The correlation of H-1/H-5 and H-5/H-7 was observed for 1, suggesting that H-1, H-5, and H-7 were on the same face and in α-orientation. The correlations of H-5/H-7 and H3-14/H-1 were observed for 2, suggesting that H-5 and H-7 were α-orientated, while H-1 was β-orientated ( Figure 2). Thus, compound 2 was proposed as a C-1 epimer of 1. Therefore, the full structures of compounds 1 and 2 were established as shown and named divaricanolides A and B.
Compound 3 was obtained as a colorless oil. Its molecular formula C19H28O6 was deduced from the HR-ESIMS data, indicative of six indices of hydrogen deficiency. However, in the 13 C NMR and DEPT spectra, only 18 carbon resonances were well resolved. After a detailed analysis of its HSQC and HMBC data, two methine carbon signals (δC 54.80; δH 2.62, 1.83) were found to have overlapped.
A comparison of its NMR data (Tables 1 and 2) with those of 1 showed that they possessed the same skeleton, having an acetoxyl (δH 1.95; δC 170.3, 21.2) located at C-8, with an ethyl ester group located at C-13. The diagnostic signals for a terminal double bond (δH 5.03, 4.76; δC 109.1, 144.6) were observed for 3, which were located at C-4 and C-15 using HMBC correlation ( Figure 3  The relative configurations of 1 and 2 were determined using the coupling constant and NOESY cross-peaks. The Z-form of C-3 and C-4 was revealed using the NOESY correlation of H-3/H 3 -15 in both 1 and 2. The correlations of H 3 -14/H-8/H-6 were observed in the NOESY spectrum of 1 and 2, which supported that H 3 -14, H-8, and H-6 were on the same face and in β-orientation. The coupling constant of J 6-7 (10.4 Hz for 1, 10.5 Hz for 2) indicated that these two protons in both compounds were in a trans form. The correlation of H-1/H-5 and H-5/H-7 was observed for 1, suggesting that H-1, H-5, and H-7 were on the same face and in α-orientation. The correlations of H-5/H-7 and H 3 -14/H-1 were observed for 2, suggesting that H-5 and H-7 were α-orientated, while H-1 was β-orientated ( Figure 2). Thus, compound 2 was proposed as a C-1 epimer of 1. Therefore, the full structures of compounds 1 and 2 were established as shown and named divaricanolides A and B.
Compound 3 was obtained as a colorless oil. Its molecular formula C 19 H 28 O 6 was deduced from the HR-ESIMS data, indicative of six indices of hydrogen deficiency. However, in the 13 C NMR and DEPT spectra, only 18 carbon resonances were well resolved. After a detailed analysis of its HSQC and HMBC data, two methine carbon signals (δ C 54.80; δ H 2.62, 1.83) were found to have overlapped.
A comparison of its NMR data (Tables 1 and 2) with those of 1 showed that they possessed the same skeleton, having an acetoxyl (δ H 1.95; δ C 170.3, 21.2) located at C-8, with an ethyl ester group located at C-13. The diagnostic signals for a terminal double bond (δ H 5.03, 4.76; δ C 109.1, 144.6) were observed for 3, which were located at C-4 and C-15 using HMBC correlation (  Figure 3). Therefore, the structure of 3 was proposed and named divaricanolide C.   Table 2) exhibited two methyl groups at δH 1.39 (s) and 1.50 (overlapped) and five olefinic protons at δH 6.17 (d), 5.71 (d), 5.46 (d), 5.18 (dd), and 5.15 (dd). The above spectroscopic data accounted for seven degrees of unsaturation, and the remaining two degrees of unsaturation were represented by a bicyclic carbon skeleton in compound 4.
In  The NOESY correlations between H-3/H-5 and the coupling constant of J 8-9 (16.1 Hz) inferred E-geometry for both C-3/C-4 and C-8/C-9 double bonds ( Figure 5). Due to the fact that the α-orientation of H-7 was reported for the majority of the naturally occurring germacrane-type sesquiterpenes, H-7 in compound 4 was tentatively defined as α-orientation. The coupling constant of J 6-7 (9.8 Hz) indicated that these two protons were in a trans form; namely, H-6 was β-orientated. The relative configuration of the OH-10 remained unclear. To further elucidate the relative configuration, the same DFT NMR calculation method used to describe compounds 1 and 2 was performed on two possible isomers with rel-(6R,7S,10R) and rel-(6R,7S,10S) configurations ( Figure S37). The DP4+ statistical analysis showed a 100% DP4+ probability (all data) for the rel-(6R,7S,10S) isomer ( Figure S38). Therefore, the structure of 4 was proposed as shown and named divaricanolide D.
form; namely, H-6 was β-orientated. The relative configuration of the OH-10 remai unclear. To further elucidate the relative configuration, the same DFT NMR calcula method used to describe compounds 1 and 2 was performed on two possible isomers w rel- (6R,7S,10R) and rel-(6R,7S,10S) configurations ( Figure S37). The DP4+ statistical an sis showed a 100% DP4+ probability (all data) for the rel-(6R,7S,10S) isomer (Figure S Therefore, the structure of 4 was proposed as shown and named divaricanolide D. Compound 5, a colorless oil, had a molecular formula of C15H22O3 established us HR-ESIMS. Its NMR data (Tables 1 and 2) were similar to those of compound 4, sugges that 5 might be an analogue of 4. A comparison of their 1 H and 13 C NMR data revea that an additional methyl signal (δC 12.6 and δH 1.19) was observed in 5, accompanied the absence of an exocyclic double bond when compared with 4. The position of the ad tional methyl group was designated as Me-13 using the 1 H-1 H COSY correlations (Fig  4) of H-7 (δH 2.62)/H-11 (δH 2.43)/H3-13 (δH 1. 19).
Compound 8 had a molecular formula of C 15 H 22 O established using HR-ESIMS, corresponding to five indices of hydrogen deficiency. The IR absorption at 3450 was assigned to the hydroxyl group. The 13 C NMR spectrum of 8 (Table 1) was very similar to that of 7, suggesting they shared the same germacrane skeleton with similar functional groups. As compound 8 had one more degree of unsaturation than 7, compound 8 might possess one more ring in the molecule. Given the existence of one nonproton-bearing oxygenated carbon at δ C 73.7 in 8 and less H 2 O in the molecular formula when compared with 7, an oxygen bridge between C-1 and C-4 was constructed, which was consistent with the low-field resonance of H-1 (δ H 4.11, dd). Thus, the planar structure of 8 was established.
The C-5/C-6 double bond was given E-geometry using the NOESY correlations of H-5/H-7 and the coupling constant of J 5-6 (16.0 Hz) ( Figure 5 and Table 3). The presence of the NOESY correlations of H-1/H a -3 and H a -3/H 3 -15 implied that H-1 and H 3 -15 were on the same face. However, the relationship between H-1/H 3 -15 and H-7 was uncertain. Similarly, DFT NMR calculation was performed on two possible isomers with relative configurations of rel-(1R,4S,7R) and rel-(1S,4R,7R) ( Figure S77). The rel-(1R,4S,7R) configuration was finally designated for 8 using DP4+ probability with a 100% possibility for all data ( Figure S78). Thus, the structure of 8 was proposed and named divaricanolide H.

Anti-Inflammatory Activity Assay
Macrophage inflammation plays a vital role in metabolic diseases, neurodegenerative diseases, and cancers [39]. The current research suggests that inflammation involves a lot of pro-inflammatory cytokines. NO is an important inflammatory mediator in inflammation. Natural products have been considered important sources to identify antiinflammatory agents [40]. Herein, eight new compounds (1−8) were tested for their inhibitory effects on NO production in LPS-stimulated RAW 264.7 macrophages for a preliminary evaluation of their anti-inflammatory activity ( Figure 6). Firstly, the noncytotoxic concentrations of compounds 1-8 were evaluated using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The MTT results showed that compounds 2, 5-6, and 8 did not show evident cytotoxicity up to 10 µM ( Figure 6B). Among these four compounds, only compounds 2 and 8 showed a significant inhibitory effect on the release of nitric oxide (NO) from RAW 264.7 cells ( Figure 6A). Next, the IC 50 values of compounds 2 and 8 in inhibiting NO production were evaluated, which were 5.35 ± 0.75 and 7.68 ± 0.54 µM, respectively.

Computational Section
DFT NMR was performed using the Gaussian 16 program [14]. Conformational searching was conducted using Conflex 8.0 software with the MMFF force field within an energy window of 5.0 kcal/mol [14]. Conformers with the Boltzmann population above 0.1% were reoptimized at the B3LYP/6-311G(d) level in vacuo, and then their NMR data were calculated at the level of mPW1PW91/6-311G(d, p) with the PCM solvent mode for chloroform [15]. A possible configuration was specified using DP4+ probability [16] 3.5. Cell Culture RAW 264.7 macrophages were purchased from American Type Cell Collection (Manassas, VA, USA) and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and 1% penicillin-streptomycin (Gibco, Carlsbad, CA, USA) in a humidified incubator with 5% CO 2 at 37 • C.

Cell Viability
The cell viability was evaluated using an MTT colorimetric assay [41]. RAW 264.7 macrophages were inoculated onto 96-well plates (at a concentration of 1 × 10 4 cells per well) and allowed to adhere to the bottom of the plates and incubated in an incubator for 24 h. Then, the cells were treated with or without compounds at 10 µM and incubated for 18 h, and then the cells with DMEM medium containing 1 mg/mL MTT (Sigma-Aldrich, St. Louis, MO, USA) were incubated for 4 h. After that, DMSO was added to solubilize formazan precipitates. The optical density (OD) at 540 nm was measured using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The calculation equation for relative cell viability is as follows: cell viability (%) = (As − A0)/(Ac − A0) × 100%, where As, A0, and Ac are the absorptions of test sample, blank control, and negative control (DMSO).

Measurement of Nitric Oxide (NO) Production
RAW 264.7 macrophages were inoculated onto 96-well plates (at a concentration of 1 × 10 4 cells per well) and allowed to adhere to the bottom of the plates and incubated in an incubator for 24 h. The cells were then treated with different concentrations of compounds or vehicle (DMSO) for 1 h, followed by stimulation with 1 µg/mL LPS. DMSO was used as vehicle, with the final concentration of DMSO being maintained at 0.1% of all cultures. After 18 h incubation, the supernatant was collected to determine NO content using Griess reagent (Sigma, St. Louis, MO, USA), as described previously. The absorbance at 490 nm was measured using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA) [42].

Statistical Analysis
All data were expressed as mean ± SEM based on at least three independent experiments and analyzed using GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). One-way ANOVA was used for statistical comparison, and p-values less than 0.05 were considered statistically significant.

Conclusions
In summary, for the first time, a phytochemical investigation of A. divaricate was carried out, leading to the characterization of 39 sesquiterpenes, including 9 new ones. The structures of the isolated compounds involve germacrane-, eudesmane-, and bisabolanetype sesquiterpenes, which is consistent with the chemical constituents isolated from other Artemisia plants. The structure elucidation of the new compounds, especially the stereochemistry, were greatly supported by DFT NMR calculations. As a matter of fact, due to the existence of the flexible 10-membered ring of the germacrane-type sesquiterpenes, the NOESY correlations could not be used to convince the relative configurations. DFT NMR calculation could provide a powerful tool to figure out the relative configuration. Given the biogenetic relationship, absolute configuration could be further proposed. In the anti-inflammatory activity assay, compounds 2 and 8 showed a significant inhibitory effect on NO production in LPS-stimulated RAW 264.7 macrophages, with IC 50 values of 5.35 ± 0.75 and 7.68 ± 0.54 µM, respectively. Our findings provide the first understanding of the chemical constituents of the medicinal plant A. divaricate and enrich the structural diversity of the sesquiterpenes of the Artemisia plants.