Secondary Metabolites and Their Cytotoxic Activity of Artemisia nitrosa Weber. and Artemisia marschalliana Spreng.

As a promising source of biologically active substances, the Artemisia species from Kazakhstan have not been investigated efficiently. Considering the rich history, medicinal values, and availability of the Artemisia plants, systematic investigations of two Artemisia species growing in the East Kazakhstan region were conducted. In this study, one new germacrane-type sesquiterpene lactone (11), together with 10 known sesquiterpenes and its dimer, were characterized from A. nitrosa Weber. Additionally, one new chromene derivative (1’) with another 12 known compounds, including coumarins, sesquiterpene diketones, phenyl propanoids, polyacetylenics, dihydroxycinnamic acid derivatives, fatty acids, naphthalene derivatives, flavones, and caffeic acid derivatives were isolated from A. marschalliana Spreng. All compounds were isolated and identified for the first time from these two Artemisia species. The structures of new compounds (11, 1’) were established by using UV, TOFMS, LC–MS, 1D and 2D NMR spectroscopic analyses. The cytotoxicity of all isolated compounds was evaluated. As a result, all compounds did not show significant inhibition against HL-60 and A-549 cell lines. The sesquiterpenoids isolated from A. nitrosa were tested for their inhibitory activity against the LPS-induced NO release from the RAW624.7 cells, and neither of them exhibited significant activity.

Artemisia is one of the largest genera in the Asteraceae family, encompassing more than 400 species, and is widely distributed all over the world [18,19]. The most significant number of species are found in Russia and China and, in Kazakhstan, 81 species were documented, with 19 being endemic, and 34 growing in the territory of Central

General Experimental Procedures
To distinguish a certain substance, the combination of NMR (1D and 2D) analytical techniques with other experimental methods, such as LC-MS, UV, IR, preparative HPLC, and semi-preparative HPLC were used. A Shimadzu UV-2550 UV-vis spectrophotometer is used for the measurement of UV spectra. The IR spectra are registered on a Thermo Nicolet FTIR IS 5 spectrophotometer. The HR-ESIMS spectra were measured on a Waters Synapt G2-Si Q-TOF instrument with a Waters BEH C18 column (1.7 µm, 2.1 mm× 50 mm, CH 3 CN:H 2 O with 0.1% formic acid, from 5% to 95%, 0-9 min, flow rate 0.4 mL/min, 45 • C). Analytical HPLC was performed on a Waters e2695 system equipped with a Waters 2998 photodiode array detector (PDA), a Waters 2424 evaporative light-scattering detector (ELSD), and a Waters 3100 MS detector, using a Waters Sunfire RP C18 column (5 µm, 4.6 mm × 150 mm, CH 3 CN:H 2 O with 0.1% formic acid, from 5% to 95%, 0-25 min, flow rate 1.0 mL/min, 30 • C). Preparative HPLC was run on a Waters system equipped with a Waters 2767 autosampler, a Waters 2545 pump, a Waters 2489 PDA and an Acuity ELSD using a Waters Sunfire RP C18 column (5 µm, 30 mm × 150 mm, flow rate 30 mL/min).
The NMR spectra were recorded on a Bruker Avance III (Bruker, Zurich, Switzerland) using a 500 M NMR spectrometer with TMS as the internal standard. The chemical shift (δ) values were given in ppm and coupling constants (J) in Hz. All solvents used for CC were of at least analytical grade (Shanghai Chemical Reagents Co., Ltd., Shanghai, China), and solvents used for HPLC were of HPLC grade (Merck KGaA, Darmstadt, Germany).

Plant Materials
Here, A. nitrosa and A. marschalliana were gathered from East Kazakhstan at the end of July 2020 and identified by experts of the Republican State Enterprise on the subject of economic management at the "Institute of Botany and Phytointroduction" of the Committee of Forestry and Wildlife of the Ministry of Ecology, Geology, and Natural Resources of the Republic of Kazakhstan. A sample of A. nitrosa (No. ANI-07) and a sample of A. marschalliana (AMA-07) were deposited in the herbarium of the Research Center for Medicinal Plants, Faculty of Chemistry and Chemical Technology, Al-Farabi Kazakh National University, Almaty, Kazakhstan ( Figures S31 and S32). The air-dried whole plants of A. nitrosa (14 kg) and A. marschalliana (13 Kg) were cut into small pieces and stored at room temperature.

Cytotoxicity Assay
The cytotoxic effects of A. nitrosa and A. marschalliana were determined using the colorimetric (CCK8) method [28] and the sulforhodamine B (SRB) protein staining method [29]. The CCK8 method was used to detect the growth inhibition of HL-60 cell lines. Cells with a logarithmic growth phase were seeded into a 96-well culture plate at a specific density (90 µL per well); after culturing overnight, different concentrations of drugs were added for 72 h. Three replicate wells were set up for each concentration, which corresponds to concentrations of vehicle control and cell-free zero adjustment wells. Then, 10 µL of CCK-8 was added to each well. After incubating for 2~3 h in the incubator, the

Cytotoxicity Assay
The cytotoxic effects of A. nitrosa and A. marschalliana were determined using the colorimetric (CCK8) method [28] and the sulforhodamine B (SRB) protein staining method [29]. The CCK8 method was used to detect the growth inhibition of HL-60 cell lines. Cells with a logarithmic growth phase were seeded into a 96-well culture plate at a specific density (90 µL per well); after culturing overnight, different concentrations of drugs were added for 72 h. Three replicate wells were set up for each concentration, which corresponds to concentrations of vehicle control and cell-free zero adjustment wells. Then, 10 µL of CCK-8 was added to each well. After incubating for 2~3 h in the incubator, the Spectra-Max 190 microplate reader was used to measure the optical density (OD value) at the 450 nm wavelength.
The compound's inhibitory effect on the proliferation of A549 cells was detected by the sulforhodamine B (SRB) protein staining method. The specific steps are as fol-lows: A549 cells in the logarithmic growth phase are seeded into a 96-well culture plate at an appropriate density, 90 µL per well; after overnight culture, different con-centrations of compounds (DMSO concentration less than 0.5%) are added for 72 h, each set has three wells for each concentration, and a solvent control group (negative control) is set. After the effect is over, the culture medium is discarded, and 10% (w/v) trichloroacetic acid (100 µL/well) is added; the solution is fixed at 4 • C for 1 h, then washed with distilled water five times, before being dried at room temperature. Then, we added 100µL of SRB solution (4 mg/mL, dissolved in 1% glacial acetic acid), incu-bated it for 15 min at room temperature, rinsed with 1% glacial acetic acid five times to wash away unbound SRB, and added 10 mM Tris solution 100 µL to each well after drying at room temperature, before using a full-wavelength microplate reader Spec-traMax 190 at the 515 nm wavelength to determine the OD value.
The inhibitory rate of the compound on cell proliferation is calculated by the following formula:

Cell Viability Evaluation
Here, RAW264.7 cells were seeded into 96-well plates at a concentration of 1 × 10 4 cells per well and allowed to adhere to the bottom of the plate overnight. Then, the cells were treated with different concentrations of compounds for 18 h. The cell viability was determined by MTT assay, as described previously [30]. Then, cell viability was determined by incubation with DMEM containing MTT (1 mg·mL −1 ) for 4 h, followed by dissolving the formazan crystals with 150 µL DMSO. The absorbance at 540 nm was measured by a SpectraMax M5 microplate reader (Molecular Devices, San Jose, CA, USA).

Measurement of Nitric Oxide (NO) Production
Here, RAW264.7 cells were seeded into 96-well plates (1 × 10 4 cells per well) and allowed to adhere for 24 h. The cells were then treated with different concentrations of compounds or vehicles (DMSO) followed by stimulation with 1 µg·mL −1 lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, MO, USA). The DMSO was used as the vehicle, with the final concentration of DMSO being maintained at 0.1% of all cultures. After 18 h of incubation, the supernatant was collected to determine the NO content using the Griess reagent (Sigma-Aldrich, St. Louis, MO, USA) as described previously [31]. The absorbance at 490 nm was measured by a SpectraMax M5 microplate reader (Molecular Devices, San Jose, CA, USA).
Earlier phytochemical studies on A. marschalliana harvested in the Iranian prov-ince of East Azerbaijan led to the isolation and identification of a high concentration of oxygenated sesquiterpenes [26,27], which is surprising due to fewer plants growing in Kazakhstan containing sesquiterpenoid compounds.

Cytotoxicity Activity
The separated compounds of A. nitrosa and A. marschalliana were examined for their cytotoxicity against human myeloid leukemia HL-60 cells and A-549 human lung cancer cell lines by the CCK8 and the sulforhodamine B (SRB) protein staining methods, respectively. The results (Tables 3 and 4) showed that monomeric sesquiterpene lactones from A. nitrosa showed weak cytotoxic activities against both A-549 and HL-60 cell lines, while the compounds from A. marschalliana did not show any effect on the growth of A-549 and HL-60 cell lines (Tables 3 and 4).

Anti-Inflammatory Activity
The sesquiterpenoids isolated from A. nitrosa were tested for their inhibitory effects against NO production on LPS-stimulated RAW264.7 macrophages. Firstly, the cytotoxicity of compounds 1-11 was evaluated using the MTT assay to determine the toxicity. Most compounds did not show obvious cytotoxicity towards RAW264.7 cells up to 10 µM ( Figure S30). Among the isolates, compounds 2, 9, and 11 showed weak NO inhibitory effects at a concentration of 2.5 µM ( Figure S30). Dexamethasone (Dex) was used as the positive control.

Conclusions
In this work, a phytochemical study of the whole plants of A. nitrosa and A. marschalliana growing in Kazakhstan was carried out for the first time. Twelve compounds were purified from A. nitrosa, including eight germacranolides, two eudesmanolides, one guaianolide, and one sesquiterpene dimer. Among them, compound 11 is a new germacrene-type sesquiterpene lactone. Moreover, a total of 13 compounds were isolated and identified from A. marschalliana, including 1 new chromene derivative (1'), and other known coumarins, sesquiterpene diketone, phenyl propanoid, polyacetylene compounds, fatty acids, naphthalene derivative, flavone, and caffeic acid derivative, respectively. The results revealed the chemical constituents of these two Artemisia plants of Kazakhstan for the first time. Their chemical constituents differed a lot from each other. The characteristic sesquiterpenoids were disclosed from A. nitrosa, while A. marschalliana was rich in other types of structures rather than sesquiterpenoids. It should be pointed out that the previous investigation of A. marschalliana led to the isolation of rich content of oxygenated sesquiterpenes, which suggested a more in-depth investigation for this species. All the known sesquiterpenes (1-10, 12) have been already reported from the Artemisia species, such as A. herba-alba, A. barrelieri, and A. gypsacea. Artebarrolide (12) is the first dimeric germacranolide reported from A. barrelieri, and it was found for the second time in this study. The biological assay of these compounds is rare in previous investigations.
In this study, the cytotoxicity assay of all isolated compounds and the anti-inflammatory assay of the sesquiterpenoids were performed. The results of the cytotoxicity assay showed that none of these compounds showed significant inhibition against A-549 and HL-60 cell lines. The sesquiterpenoids isolated from A. nitrosa did not show significant inhibition on the LPS-induced NO release from RAW-264.7 cells at the concentrations of 10 and 2.5 µM, which closely correlates to the anti-inflammatory activity. Compared with the compounds isolated from A. heptapotamica in the previous study [24], we found that the sesquiterpenoids obtained from A. nitrosa lack the α,β-unsaturated ketone moiety in their structures, which might be pivotal to the anti-inflammatory activity. It is obvious that more in-depth investigations are needed to discover bioactive compounds from the Artemisia species in Kazakhstan.