UPLC-MS Analysis, Quantification of Compounds, and Comparison of Bioactivity of Methanol Extract and Its Fractions from Qiai (Artemisia argyi Lévl. et Van.)

The Artemisia argyi Lévl. et Van. growing in the surrounding areas of Qichun County in China are called Qiai (QA). Qiai is a crop that can be used both as food and in traditional folk medicine. However, detailed qualitative and quantitative analyses of its compounds remain scarce. The process of identifying chemical structures in complex natural products can be streamlined by combining UPLC-Q-TOF/MS data with the UNIFI information management platform and its embedded Traditional Medicine Library. For the first time, 68 compounds in QA were reported by the method in this study. The method of simultaneous quantification of 14 active components in QA using UPLC-TQ-MS/MS was reported for the first time. Following a screening of the activity of QA 70% methanol total extract and its three fractions (petroleum ether, ethyl acetate, and water), it was discovered that the ethyl acetate fraction enriched with flavonoids such as eupatilin and jaceosidin had the strongest anti-inflammatory activity, while the water fraction enriched with chlorogenic acid derivatives such as 3,5-di-O-caffeoylquinic acid had the strongest antioxidant and antibacterial activity. The results provided the theoretical basis for the use of QA in the food and pharmaceutical industries.


Introduction
Artemisia argyi Lévl. et Van. is widely distributed in East Asian countries, especially in China. Artemisia argyi is a common flavoring and colorant in the food industry, and also a traditional medicine used to manage dysmenorrhea and inflammation [1]. Another use is in moxibustion, a form of traditional Chinese medicine that involves burning the plant materials over acupuncture points [2]. The mugwort grown in Qichun County, Hubei Province, China, is called "Qiai". According to Li Shizhen's "Compendium of Materia Medica", a classical Chinese medicine work, the quality of Qiai is superior to other regions [3]. Modern studies suggest that Qiai contains a wide range of active ingredients, including phenolic acids, terpenes, polysaccharides, and essential oils [4][5][6]. Furthermore, the essential oil, tannins, and flavonoid concentration in Qiai are higher than in other production areas [7][8][9]. Although the prices of Qiai are higher than in other production areas, its demand remains robust. As research progresses, the pharmacological effects of Artemisia argyi, such as anti-inflammatory [10], anti-tumor [11], and obesity improvement [12], become clearer, and more and more Artemisia argyi products are developed and utilized [13]. By 2021, the planting area in Qiai reached 20,000 hectares, with an industrial output value of 1.16 billion dollars.
The QA extract solution was detected using UPLC-Q-TOF/MS technology under chromatographic and mass spectrometry conditions. The rapid, efficient and validated UPLC-Q-TOF/MS analytical method was established for the identification of the main chemical components in QA. The base peak ion chromatograms (Figure 1) provide the metabolomic analysis, also known as the analytical fingerprint for plant identification and authentication, a fairly integrated frame.
The collected MS data were imported into the UNIFI information management platform. In the UNIFI information software, the theoretical database of QA leaf compounds and the physical database of reference substances were established. As shown in Tables 1 and 2, a total of 68 compounds were identified in QA leaves, with 47 compounds identified by positive ion mode collection and 43 compounds identified by negative ion mode (22 compounds were collected by both positive and negative ions). This is the first time that the combination of UPLC-Q-TOF/MS and the UNIFI platform has been applied to characterize the compounds in QA, and the established method has successfully identified the largest number of compounds. Among these are well-known phytochemicals, such as The collected MS data were imported into the UNIFI information man form. In the UNIFI information software, the theoretical database of QA le and the physical database of reference substances were established. As show and 2, a total of 68 compounds were identified in QA leaves, with 47 comp fied by positive ion mode collection and 43 compounds identified by nega (22 compounds were collected by both positive and negative ions). This is that the combination of UPLC-Q-TOF/MS and the UNIFI platform has be characterize the compounds in QA, and the established method has succe fied the largest number of compounds. Among these are well-known ph such as chlorogenic acid, jaceosidin, eupatilin, quercetin, and 3,5-di-O-caffeo which possess antioxidant, anti-inflammatory, cancer chemopreventive, pression, and food additive properties.     Figure 2 depicts the representative UPLC-TQ-MS/MS total ion chromatogram of standards, QA-TE, QA-FEA, and QA-FWT. Figure 3 depicts the ion chromatograms of 14 standards under the optimal UPLC-TQ-MS/MS conditions. The method's linearity, sensitivity, precision, and accuracy satisfy international standards. The linearity of the standard solution was assessed by analyzing the standard solution over a concentration range satisfactory for the quantification of the relevant analytes in the sample. All analytes' regression equations had excellent linearities, with the determination coefficient R 2 ≥ 0.9967 ( Table 3). All analyte detection limits ranged from 0.48 to 5.32 ng/mL (Table 3), while all analyte quantitation limits ranged from 1.45 to 15.89 ng/mL (Table 3). To the best of our knowledge, this is the lowest limit of the quantification method for the simultaneous quantification of compounds in QA. Additionally, for the peak region of all analytes, the intra-day and inter-day RSDs were less than 2.31% and 2.16%, respectively (   Figure 3 depicts the ion chromatogram standards under the optimal UPLC-TQ-MS/MS conditions. The method's linearity, tivity, precision, and accuracy satisfy international standards. The linearity of the ard solution was assessed by analyzing the standard solution over a concentration satisfactory for the quantification of the relevant analytes in the sample. All analyt gression equations had excellent linearities, with the determination coefficient R 2 ≥ (Table 3). All analyte detection limits ranged from 0.48 to 5.32 ng/mL (Table 3), wh analyte quantitation limits ranged from 1.45 to 15.89 ng/mL (Table 3). To the best knowledge, this is the lowest limit of the quantification method for the simulta quantification of compounds in QA. Additionally, for the peak region of all analyt intra-day and inter-day RSDs were less than 2.31% and 2.16%, respectively (Table 3   These findings demonstrate that the approach has good precision whether used to measure on an intra-day or day-to-day basis. Additionally, the range of spiking recoveries for all analytes was 99.79% to 104.37% (Table 3), demonstrating that the method has adequate accuracy. Furthermore, the analyte recovery range was measured to be 97.56% to 101.74% (Table 3). The findings indicate that the adopted methodology has good linearity, sensitivity, precision, accuracy, and stability, and can be used to quantify fourteen characteristic compounds from QA leaves.

Quantitative Analysis
The developed UPLC-TQ-MS/MS method was subsequently applied to quantify 14 bioactive compounds in leaves of A. argyi. Table 4 shows the quantification results for extracts and fractions. The p-values for all compounds measured were less than 0.05. Figure 4 depicts the structures of quantified compounds in Qiai. The quantified compounds belonged to two classes, eight flavonoids (chrysoeriol 7-O-glucoside, chrysoeriol, schaftoside, isoschaftoside, hyperoside, hispidulin, eupatilin, and jaceosidin) and six chlorogenic acid derivatives (3,5-di-O-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, chlorogenic acid, neochlorogenic acid, and 4-Dicaffeoylquinic acid). Among them, hyperoside (Rt = 8.25 min), chrysoeriol 7-O-glucoside (Rt = 11.10 min), and chrysoeriol (Rt = 13.68 min) displayed deprotonated molecules at the m/z ratio of 463.03, 461.10, and 299.03, respectively. This is the first report of the quantification of these three flavonoids in QA that we are aware of. In addition, for the first time, the method of simultaneous quantification of 14 active components in QA using UPLC-TQ-MS/MS was reported.  According to the data in Table 4, chrysoeriol, hispidulin, eupatilin, and jaceosidin in the total extract were enriched in QA-FEA. Hyperoside, schaftoside, isoschaftoside, and six chlorogenic acid derivatives were enriched in QA-FWT after fractionation. This proves that these compounds were mostly extracted using ethyl acetate and methanol. The 14 bioactive compounds include analgesic, anti-inflammatory, and antipyretic properties that can be used to treat a variety of disorders [24][25][26]. Therefore, we can infer that the pharmacological activity of fractions depends on the content of active compounds in them. According to the data in Table 4, chrysoeriol, hispidulin, eupatilin, and jaceosidin in the total extract were enriched in QA-FEA. Hyperoside, schaftoside, isoschaftoside, and six chlorogenic acid derivatives were enriched in QA-FWT after fractionation. This proves that these compounds were mostly extracted using ethyl acetate and methanol. The 14 bioactive compounds include analgesic, anti-inflammatory, and antipyretic properties that can be used to treat a variety of disorders [24][25][26]. Therefore, we can infer that the pharmacological activity of fractions depends on the content of active compounds in them.

Evaluation of Antioxidant Potential
The antioxidant potential of the total extract and fractions was analyzed using the DPPH colorimetric and ABTS colorimetric assays. The details are shown in Table S2. The radical scavenging activities of the total extract, fractions, and trolox were expressed as IC50. Except for QA-FPE, all tested total extracts and fractions had a significant DPPH and ABTS scavenging potential. This may be due to the presence of phenolic compounds in QA. Hydroxyl groups in phenolic compounds react with various kinds of free radicals [27]. In the radical scavenging assay, it was understood that QA-FWT, with IC50 58.34 µg/mL (DPPH) and IC50 270.00 µg/mL (ABTS), was the most active of all the tested samples, which was lower than trolox. The antioxidant activity is closely related to the content of phenolic compounds [28]. It is known that phenolic compounds, particularly chlorogenic acids derivatives, and flavonoids are predominant in QA. Different phenolic components have different solubility in the extraction solvent (petroleum ether, ethyl acetate, and water). The antioxidant activity might be related to the majority quantities of chlorogenic acids derivatives in QA-FWT and flavonoids in QA-FEA.

Inhibition of the NO Release Capacity
NO release inhibition by LPS-stimulated RAW 264.7 cells was performed using five different concentrations of the total extract and fractions at 5, 10, 15, 20, and 25 µg/mL. Details are provided in the Table S3. First, to ensure that the effects on NO release were not caused by reduced cell viability, the potential toxicity of the test materials was evaluated against RAW 264.7 cells. Samples showed cell viability of over 90%, indicating that none of the samples were harmful to the cells. Interestingly, among the samples capable of scavenging radicals, QA-FEA and QA-FWT inhibited NO significantly. Furthermore, QA-FEA showed higher activities than QA-FWT. This is because the main components in QA-FEA were flavonoids, whereas the main components in QA-FWT were chlorogenic acids. Moreover, studies have confirmed that the anti-inflammatory activities of eupatilin and jaceosidin [29] were significantly higher than chlorogenic acids. Eupatilin and jaceosidin are the main components of flavonoids enriched in QA-FEA. Inflammatory mediators are important factors to promote the occurrence of inflammation. Eupatilin and jaceosidin can effectively regulate the expression of related enzymes to inhibit the production of inflammatory mediators and prevent future inflammation. This confirms that flavonoids are more responsible for the anti-inflammatory activity of QA than chlorogenic acid derivatives.

Antibacterial Activities
We assessed the diameters of the inhibition zone of the total extract and three fractions against different bacteria ( Figure S1). The findings are detailed in Table S4 indicating that QA has a wide antibacterial spectrum. QA-FWT had better anti-bacterial activity against different bacteria as evidenced by the diameters of the inhibition zone. This is due to the chlorogenic acid derivatives in QA that can destroy the cell wall and cell membrane structure of bacteria and certainly have an inhibitory effect on bacteria [30,31]. Beyond that, the hydroxylation at C5 and C7 of flavonoid compounds can increase the inhibition of bacterial growth [32]. The C5 and C7 of jaceosidin, eupatilin, and hispidulin riched in QA-FEA are replaced by hydroxyl groups, and the antimicrobial activity of QA-FEA is increased. This provides a theoretical basis for the application of QA as a natural antibacterial agent in food and agriculture.
Dulbecco's modified Eagle's medium (DMEM) was purchased from Servicebio (Wuhan, China), dimethyl sulfoxide (DMSO) was purchased from Aladdin (Shanghai, China), fetal bovine serum was purchased from Newzerun (Wuhan, China), phosphate buffered saline was purchased from Hyclone (Shanghai, China), mueller hinton agar (MHA) and mueller hinton broth (MHB) were purchased from Hopebio (Qingdao, China). The DPPH Free radical Scavenging Ability assay kit and the ABTS Free radical Scavenging Ability assay kit were purchased from Jiancheng Bioengineering Institute (Nanjing, China), and the Nitric Oxide assay kit was purchased from Beyotime (Shanghai, China).

Plant Material
The plant samples ( Figure 5 methanol, ethanol) were acquired from Chron Chemicals (Chengdu, China). A Milli-Q purification system (Millipore, France) was used to create the ultra-pure water. Dulbecco's modified Eagle's medium (DMEM) was purchased from Servicebio (Wuhan, China), dimethyl sulfoxide (DMSO) was purchased from Aladdin (Shanghai, China), fetal bovine serum was purchased from Newzerun (Wuhan, China), phosphate buffered saline was purchased from Hyclone (Shanghai, China), mueller hinton agar (MHA) and mueller hinton broth (MHB) were purchased from Hopebio (Qingdao, China). The DPPH Free radical Scavenging Ability assay kit and the ABTS Free radical Scavenging Ability assay kit were purchased from Jiancheng Bioengineering Institute (Nanjing, China), and the Nitric Oxide assay kit was purchased from Beyotime (Shanghai, China).

Plant Material
The plant samples ( Figure 5) were collected from Zhulin Lake in Qichun County, Huanggang City, Hubei Province, China. The plant was collected in June 2021 and verified by Prof. Dr. Dingrong Wan, South-Central Minzu University (SCMU). Voucher specimens of Qiai plants were deposited in SCMU with the number QA2021060403. The majority of the collected plant leaves was shade dried for 7 days and then pulverized with an electric grinder to give Mugwort leaf powder.

Preparation of Extract and Fractions
Mugwort leaf powder (50.0 g) was extracted with 70% methanol. Extraction (1:20, w/v) was performed by maceration for 3 h at room temperature, heated for reflux three times in a water bath (2.5 h each time), combined with filtrate, and concentrated under

Preparation of Extract and Fractions
Mugwort leaf powder (50.0 g) was extracted with 70% methanol. Extraction (1:20, w/v) was performed by maceration for 3 h at room temperature, heated for reflux three times in a water bath (2.5 h each time), combined with filtrate, and concentrated under vacuum to 7.6 g of the total crude extract (QA-TE). Warm water was used to dissolve 6 g of QA-TE before it was progressively partitioned with 500 mL petroleum ether (PE) and 500 mL ethyl acetate (EtOAc) to produce the PE fraction (QA-FPE, 2.0 g), EtOAc fraction (QA-FEA, 1.2 g), and water fraction (QA-FWT, 2.4 g), respectively. The extract and fractions were stored at −20 • C until use.

UPLC-Q-TOF/MS Analysis
Chromatographic analysis was performed on an ultra-performance liquid chromatography system equipped with a four-element pump, an online degassing machine, an automatic sampler, and a thermostatically controlled column chamber. The separation was performed on an ACQUITY UPLC HSS T3 column (100 × 2.1 mm, 1.8 µm). The mobile phase was composed of solvent A (0.1% Formic acid in H 2 O) and solvent B (0.1% Formic acid in acetonitrile: methanol, 9:1), and the elution gradient system was optimized on this basis. Elution gradient technology was used for the study, with a constant flow rate of 0.4 mL/min. The injection volume was 2 µL. The gradient proceeded as follows: 0-1. The column and autosampler were kept at 45 and 4 • C, respectively. MS detection was carried out on Synapt-G2-SI MS system. The high collision energy ranged from 15 to 25 eV, whereas the low collision energy was fixed at 6 eV, and the ionization mode was set as ESI + and ESI − . The mass ranged from 50 to 1200 Da. The cone voltage was 40 V, the capillary voltage was 3.00 kV in the negative mode and 2.59 kV in the positive mode. The desolvation temperature was fixed at 500 • C, while the ion source temperature remained at 150 • C. Desolvation gas (N2) flowed at 800 L/h while cone gas (N2) flowed at 50 L/h.

Construction of UNIFI Theoretical Library on Chemical Constituents of QA
SciFinder, PubMed, PubChem, and Reaxys are a few of the internet databases that were used to compile a list of the compounds mentioned in the literature on QA. Search terms "Artemisia argyi" were employed to search published literature up to April 2022. The process of identifying chemical structures in complex natural products can be streamlined by combining UPLC-Q-TOF/MS data with the UNIFI information management platform and its embedded Traditional Medicine Library. Finally, the structure of 208 compounds reported from A. argyi species was collected and saved in a .sdf file as a theoretical library. The MS data of the QA-TE was imported into the UNIFI platform for rapid matching screening with the theoretical library data of A. argyi compounds.

Preparation of Standard Solution and Sample Solution
Flavonoids and chlorogenic acids are important components in QA, which are closely related to the pharmacological action of QA. Therefore, it is significant to quantify the main flavonoids and chlorogenic acids in QA.
A total of 2-3 mg samples were taken, QA-TE was dissolved in methanol:water (1:1), and QA-FEA and QA-FWT were dissolved in methanol. The sample solution was centrifuged with a centrifuge (Eppendorf 5810R) at 10,000 r/min, and the supernatant was used for the test. The QA-TE and QA-FWT were diluted to 50 µg/mL and the QA-FEA to 10 µg/mL.

Instrumentation and Analytical Conditions
Chromatographic analysis was the same as 2.4. The Xevo TQ-S MS/MS system was used to perform the mass spectrometry detection. The ionization mode for was set to ESI + and ESI − mode for the determination of the main chemical constituents of QA by the UNIFI theoretical library. The quantitative data acquisition mode was set to multiple reaction monitoring (MRM), the ionization mode was set to ESI − , and the other analysis conditions of mass spectrometry were consistent with 2.4. Each analyte's collision energy and particular fragmentor voltage were tuned in order to produce the strongest quantitative change. Table S1 in the supplementary document includes the optimum values for these critical parameters for the fourteen target compounds.

DPPH Assay
The scavenging activities of the total extract and three fractions were evaluated using a 2.2-dy-phenyl-1-picrylhydrazyl (DPPH) Free Radical Scavenging Ability Assay kit with slight modifications [33]. DPPH (600 µL) was admixed with 400 µL of fractions and standard (4.0-426.0 µg/mL), respectively. After being vortexed, the reaction mixture was left at room temperature in the dark for 30 min. After incubation, absorbance was assessed at 517 nm using a spectrophotometer. Methanol was employed as a blank, and trolox served as the positive control (standard). Each blank, samples, and standards' absorbance were measured in triplicate. The ability to scavenge the DPPH radical was measured by the following equation: A i : absorbance of DPPH radical + fraction/standard; A j : absorbance of fraction/standard + methanol; A 0 : absorbance of DPPH radical + methanol. By graphing the sample concentration vs. the scavenging capacity using a logarithm function, the IC50 (Half-maximal Inhibitory Concentration) value was determined.

ABTS Assay
The scavenging activity of the total extract and three fractions was evaluated using a 2,2 -Azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) Free Radical Scavenging Ability Assay kit. The detection buffer, ABTS solution, and hydrogen peroxide solution (76:5:4) were mixed to prepare the ABTS working solution. Trolox was used as a positive control (standard). ABTS (170 µL), and peroxidase solution (20 µL) were admixed with 10 µL of fractions and standard (51.8-837.0 µg/mL), respectively. The reaction mixture was vortexed and left at room temperature in the dark for 6 min. After incubation, absorbance was measured by an enzyme standard instrument at 405 nm. The ability to scavenge the ABTS radical was measured by the following equation: A i : absorbance of ABTS radical + peroxidase solution+ fraction/standard; A 0 : absorbance of ABTS radical + peroxidase solution+ H 2 O.

Determination of Anti-Inflammatory Activity by Inhibition of NO
The inhibiting effect on nitric oxide (NO) production in LPS-stimulated RAW 264.7 (Wuhan, China) macrophage cells served as a metric for the anti-inflammatory action. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum and 0.5% penicillin/streptomycin. The cells were cultivated in a humidified incubator at 37 • C with 5% CO 2 and 95% air. Measurements were made of the samples' ability to inhibit NO generation. In 96-well culture plates filled with 100 L of DMEM media, RAW 264.7 cells (6 × 10 4 ) were planted. After 2 h of cell adhesion, the cells were starved for 12 h. LPS (1 µg/mL) and different concentrations of sample solution (25, 20, 15, 10, 5 µg/mL) were added simultaneously. The cells were incubated at 37 • C with 5% CO 2 for 24 h. After 24 h of incubation, 50 µL of the supernatant was collected for nitrite assay with a NO assay kit by using the Griess reaction [34]. The remaining medium was taken out, and the CCK-8 technique was used to assess the cell viability. The absorbance was measured at 450 nm.

Disc Diffusion Assay
The agar plates' preparation was performed for the disc diffusion technique to examine the antibacterial activity of the extract and fractions. Two Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis) and three Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, P. vulgaris) were chosen for antibacterial activities of the total extract and fractions. Each strain was cultivated for 24 h, and the bacterial culture was diluted to a concentration of about 10 6 CFU/mL. A total of 0.2 mL of the diluted solution was then evenly dispersed over the agar plates. Samples were diluted with methanol at 50 mg/mL. Then, 0.2 mL of the sample solution was injected into a 6 mm diameter hole placed in the agar plates. The plates were cultured at 37 • C for 16 h. To assess the antibacterial activity of the strains, the widths of their inhibition zones were evaluated. Methanol (ME) was used as a negative control, and 5 µg of ciprofloxacin hydrochloride (CH) was used as a positive control.

Conclusions
In conclusion, this study established a rapid identification method for compounds in QA by combining UPLC-Q-TOF/MS with the UNIFI information management platform. Meanwhile, the study provided an effective method for the quantitative analysis of 14 compounds in QA by UPLC-TQ-MS/MS. This method could quantify 14 compounds simultaneously and be verified by LODs, LOQs, precision, repeatability, stability, and recovery range. The QA-FEA obtained from the QA-TE significantly reduced the NO release by LPS-stimulated RAW 264.7 cells. Meanwhile, QA-FWT has the highest DPPH and ABTS free radical scavenging ability and antibacterial ability. This is because QA-FEA has the highest flavonoid content and QA-FWT has the highest phenolic acid content. The results showed that Artemisia argyi Lévl. et Van., as dietary and traditional Chinese medicine, was an excellent source of natural antioxidants, anti-inflammatory drugs, and antibacterial agents. The results provided the theoretical basis for the use of QA in the food and pharmaceutical industries. The plant material selected for this study was from one production area, so there are some limitations. Factors such as geographical location, variety, and climate can have significant effects on the chemical composition of Artemisia argyi Lévl. et Van. In the future, we will work to improve the information on the chemical composition of Artemisia argyi in terms of different cultivars and origins to provide more comprehensive and reliable information for the research and application of Artemisia argyi.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules28052022/s1, Table S1. The optimized MRM parameters of the fourteen target analytes. Table S2. Antioxidant potential of extract and fractions of QA evaluated by DPPH and ABTS method. Table S3. Inhibition of NO release by LPS-stimulated Raw264.7 cells of extract and fractions of QA. Table S4. Diameter of inhibition zone of the extract and fractions of QA. Figure S1. Bacterial inhibition of P. vulgaris (A), B.subtilis (B), S.aureus (C), E.coli (D), P.aeruginosa (E) by samples in Disc diffusion assay.
Funding: This research was funded by Fundamental Research Funds for the Central Universities, South-Central Minzu University grant number CZZ22002. And The APC was funded by Xinzhou Yang.