New Evidence for Artemisia absinthium as an Alternative to Classical Antibiotics: Chemical Analysis of Phenolic Compounds, Screening for Antimicrobial Activity

Artemisia absinthium, an important herb of the Artemisia genus, was evaluated in this study for its potential as an alternative to classical antibiotics. The antimicrobial activity of methanol extracts of A. absinthium (MEAA) was evaluated using the broth microdilution method, revealing that A. absinthium exhibited broad-spectrum antibacterial and antifungal activity. Ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS) was used to analyze the chemical profile of the MEAA, with a focus on flavonoids, quinic acids, and glucaric acids. A total of 90 compounds were identified, 69 of which were described for the first time in A. absinthium. Additionally, a new class of caffeoyl methyl glucaric acids was identified. The main active compounds were quantified and screened for antimicrobial activity. A. absinthium was found to be rich in quinic acids and flavonoids. The screening for antimicrobial activity also revealed that salicylic acid, caffeic acid, casticin, and 3,4-dicaffeoylquinic acid had varying degrees of antimicrobial activity. The acute toxicity of MEAA was examined following OECD guidelines. The administration of 5000 mg/kg bw of MEAA did not result in mortality in male and female mice. Furthermore, there were no observed effects on the visceral organs or general behavior of the mice, demonstrating the good safety of MEAA. This study provides new evidence for the use of A. absinthium as an alternative to classical antibiotics in addressing the problem of bacterial resistance.


Introduction
The emergence and spread of drug-resistant strains pose a serious threat to global public health [1]. To address the declining susceptibility of strains to antibiotics, researchers have proposed alternative therapies, such as traditional herbal medicines, probiotics, vaccines, immunoglobulins, and bacteriophages. These therapies can help reduce selection pressure and minimize resistance to classical antibiotics [2][3][4][5][6]. Traditional herbal medicines have been used for thousands of years to treat bacterial infections, with Artemisia species in particular excelling in traditional antimicrobial herbal medicine [7,8]. Extracts from Artemisia species, such as Artemisia annua, Artemisia argyi, and Artemisia indica, have demonstrated varying degrees of antimicrobial activity against different strains [9][10][11]. Thus, it is worth examining the antimicrobial activity of another important species in the Artemisia genus, A. absinthium.
A. absinthium, a perennial herb in the Asteraceae family, is widely distributed in Iran, India, Pakistan, and European countries. This herb has a long history of medicinal use, particularly in Europe and Central Asia, where it is used to treat fever, stomachache, indigestion, anorexia, and hepatitis. A. absinthium is rich in essential oils, flavonoids, phenolic acids and terpenoids [12] (Figure 1), and modern pharmacological studies have shown that it possesses antimicrobial, antioxidant and neuroprotective pharmacological activities. 2 of 20 Notably, A. absinthium is widely used not only in medicine but also in food, cosmetics, and animal husbandry. In the food sector, A. absinthium is an important compound in absinthe, which has been consumed in Europe for more than 300 years and is believed to have appetizing, stomachic, and tonic properties. In cosmetics, A. absinthium is used in a range of products based on its excellent antimicrobial and antioxidant activity. In the livestock industry, A. absinthium is used as a special feed additive to reduce the risk of intestinal diseases in livestock [13,14]. Given the numerous practical applications of A. absinthium, it is suggested that it possesses superior antimicrobial activity. Although there are many reports analyzing the antimicrobial activity of the essential oil of A. absinthium, there is still a lack of research on the composition analysis and evaluation of the antimicrobial activity of the methanol extracts of A. absinthium (MEAA). phenolic acids and terpenoids [12] (Figure 1), and modern pharmacological studies have shown that it possesses antimicrobial, antioxidant and neuroprotective pharmacological activities. Notably, A. absinthium is widely used not only in medicine but also in food, cosmetics, and animal husbandry. In the food sector, A. absinthium is an important compound in absinthe, which has been consumed in Europe for more than 300 years and is believed to have appetizing, stomachic, and tonic properties. In cosmetics, A. absinthium is used in a range of products based on its excellent antimicrobial and antioxidant activity.
In the livestock industry, A. absinthium is used as a special feed additive to reduce the risk of intestinal diseases in livestock [13,14]. Given the numerous practical applications of A. absinthium, it is suggested that it possesses superior antimicrobial activity. Although there are many reports analyzing the antimicrobial activity of the essential oil of A. absinthium, there is still a lack of research on the composition analysis and evaluation of the antimicrobial activity of the methanol extracts of A. absinthium (MEAA). In recent years, advanced technologies such as high-resolution mass spectrometry (HRMS) [15,16], ion mobility spectrometry [17], diagnostic product ions (DPIs) filters [18], and liquid chromatograph mass spectrometer (LC-MS) data acquisition [19] have been continuously developed and improved, enabling metabolomics studies based on highperformance liquid chromatography-tandem mass spectrometry to be widely used in the metabolite analysis and structural characterization of traditional herbal medicines. However, the identification of positional and geometric isomers among a large number of metabolites, as well as the identification of new chemical structures, is often hindered by the lack of standards and reference data [20,21]. To address this issue, previous studies have analyzed and summarized various metabolite identification strategies based on limited representative standards, such as MS/MS fragmentation laws [22], ion mobility spectra [17], and quantitative structure-retaining relationships [23]. In addition, chromatographic retention time prediction based on molecular hydrogen bonding analysis and ClogP calculation for isomers has also been used for isomer identification [24]. In this study, we analyzed and summarized the identification strategy based on past studies to characterize the chemical profile of A. absinthium.
In this study, we aimed to investigate the potential of A. absinthium as an alternative to classical antibiotics by evaluating the antimicrobial activity of its methanol extracts. Additionally, we conducted an analysis of the MEAA using ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS) and employed structure analysis and isomer identification strategies to focus on flavonoids, In recent years, advanced technologies such as high-resolution mass spectrometry (HRMS) [15,16], ion mobility spectrometry [17], diagnostic product ions (DPIs) filters [18], and liquid chromatograph mass spectrometer (LC-MS) data acquisition [19] have been continuously developed and improved, enabling metabolomics studies based on highperformance liquid chromatography-tandem mass spectrometry to be widely used in the metabolite analysis and structural characterization of traditional herbal medicines. However, the identification of positional and geometric isomers among a large number of metabolites, as well as the identification of new chemical structures, is often hindered by the lack of standards and reference data [20,21]. To address this issue, previous studies have analyzed and summarized various metabolite identification strategies based on limited representative standards, such as MS/MS fragmentation laws [22], ion mobility spectra [17], and quantitative structure-retaining relationships [23]. In addition, chromatographic retention time prediction based on molecular hydrogen bonding analysis and ClogP calculation for isomers has also been used for isomer identification [24]. In this study, we analyzed and summarized the identification strategy based on past studies to characterize the chemical profile of A. absinthium.
In this study, we aimed to investigate the potential of A. absinthium as an alternative to classical antibiotics by evaluating the antimicrobial activity of its methanol extracts. Additionally, we conducted an analysis of the MEAA using ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS) and employed structure analysis and isomer identification strategies to focus on flavonoids, quinic acid, and glucaric acid compounds. We also quantified the main active compounds present in A. absinthium using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and evaluated their antimicrobial activity through the broth microdilution method. By identifying the compounds and conducting quantitative analysis, antimicrobial activity screening, and evaluation of acute toxicity, we aimed to provide more evidence for A. absinthium as a viable alternative to classical antibiotics.

Antimicrobial Activity of MEAA
This study investigated the antimicrobial activity of MEAA against nine clinically common strains, including four Gram-positive and four Gram-negative bacteria, and one fungal strain. The broth microdilution method was used to determine the MIC and MBC values of the methanol extracts. The results, presented in Table 1, demonstrate that the MEAA exhibits broad-spectrum antibacterial activity with different degrees of potency against the tested strains. Gram-positive bacteria were found to be more susceptible to the methanol extracts compared to Gram-negative bacteria, as indicated by lower MIC/MBC values. Notably, the methanol extracts demonstrated superior activity against S. aureus (MIC = 1.25 mg/mL, MBC = 1.25 mg/mL), S. epidermidis (MIC = 0.625 mg/mL, MBC = 1.25 mg/mL), P. aeruginosa, and B. cereus (MIC = 1.25 mg/mL, MBC = 1.25 mg/mL), with S. epidermidis being the most sensitive among the tested bacteria. The fungus C. albicans was also inhibited by the methanol extracts (MIC = 2.5 mg/mL, MBC = 5.0 mg/mL). Similar to some other Artemisia species, such as A. annua, A. argyi, and A. indica, A. absinthium also exhibited antimicrobial activity, particularly against Gram-positive bacteria. This is consistent with what has been reported so far. This may be due to differences in the structure of cell membranes between Gram-positive and Gram-negative bacteria. Hydrophilic compounds, which are present in the methanol extracts, require permeation across the membrane to be effective. However, the presence of lipopolysaccharides in the outer membrane of Gram-negative bacteria can limit the penetration of hydrophobic antibacterial compounds, resulting in the need for higher doses of these compounds [25,26].

Chemical Profile of A. absinthium
The chemical profile of A. absinthium was investigated using Ultra Performance Liquid Chromatography-Quadrupole-Time of Flight Mass Spectrometry (UPLC-Q-TOF-MS) ( Figure 2). The identified compounds were compared with reference standards and literature data, and a total of 90 compounds were tentatively identified, including 31 flavonoids, 20 quinic acids, 17 glucaric acids, 8 other organic acids, and 14 other polyphenols. Among these, 69 compounds were identified for the first time in A. absinthium. Detailed information about these identified compounds is presented in Table 2. This study used a comprehensive information about these identified compounds is presented in Table 2. This study used a comprehensive and relatively complete identification strategy, integrating the identification strategies of previous researchers, to effectively identify many positional and geometric isomers, thereby solving the problem of compound identification in A. absinthium.  Table 1.

Quinic Acids
The diagnostic ion for quinic acids is usually m/z 191.06. In this study, 20 quinic acids were identified ( Figure 4A), which are formed by the combination of quinic acid with caffeoyl-, coumaroyl-, and feruloyl- [30]. Among them, eight compounds (#19, #21, #27,  #30, #32, Figure 4C). The eight isomers are formed by the combination of cis-caffeoyland trans-caffeoylon the OH groups at the 1,2,3,4 positions of quinic acid [31]. The elution order of positional isomers is determined by ClogP, with smaller ClogP indicating a smaller retention time. After calculating the ClogP of positional isomers and combining it with the literature data, the elution order of 4 positional isomers is 3-/5-/4-/1-caffeoylquinic acids ( Figure 4D) [20,32]. To further identify the geometric isomers, molecular simulation results summarized the formation rules of intramolecular hydrogen bonds of these isomers. It showed that 5-cis-caffeoylquinic acids were more hydrophobic and eluted later than 5-trans-caffeoylquinic acids, while 3-cis-and 4-cis-caffeoylquinic acids were the opposite. The possible reason for this phenomenon is that 5-cis-are able to form at least one hydrogen bond that is not present in 5-trans-caffeoylquinic acids, and that hydrogen bonds cannot be formed in 3-cis-and 4-cis-caffeoylquinic acids [32]. Therefore, the structures of 4 pairs of caffeoylquinic acids isomers were tentatively identified, with the elution order of isomers being 3-cis, 3-trans, 5-trans, 5-cis, 4-cis, 4-trans, 1-cis, and 1-trans [20]. The PR of DPI at m/z 191.06 to m/z 179.03 showed remarkably consistent change among these isomers. The trans-isomers had higher PR values than their corresponding cis-isomers. Among the eight caffeoylquinic acid isomers, 4-trans-caffeoylquinic acid had the highest PR value, reaching 113.70. Additionally, the PR values of 1-cis-caffeoylquinic acid were similar to those of 3-cis-caffeoylquinic acid, while the PR values of 4-cis-caffeoylquinic acid were similar to those of 5-cis-caffeoylquinic acid. Similarly, the PR values of 1-trans-caffeoylquinic acid were similar to those of 5-trans-caffeoylquinic acid. The DPI ratios and elution order based on ClogP can accurately identify positional and geometric caffeoylquinic acids or other acylated quinic acids. Based on the PRs of DPI at m/z 191.06 to m/z 163.04, 3 pcoumaroylquinic acids were identified [31]. Additionally, four dicaffeoylquinic acids (#67, #70, #73, #79), one tricaffeoylquinic acid (#83), and three feruloylquinic acids (#48, #54, #58) were identified, referring to the literature data [33][34][35].

Glucaric Acids
Glucaric acids are a group of compounds found widely in plants, formed by the combination of glucaric acid and one to four acylated or combined residues [29]. These compounds have a unique structure of positional and geometric isomers that remain the focus of ongoing research [36]. In this study, we identified eight compounds (#8, #9 Figure 5B) [30]. To further analyze the positional and geometric isomers, we compared the intra-molecular hydrogen bonds of these isomers. The intra-molecular hydrogen bonds formed by 1(6)-OH and 2(5)-CO in 2-trans-and 5-trans-caffeoylglucaric acids were found to be more stable than in 3-trans and 4-trans caffeoylglucaric acids [37]. Therefore, the 3-trans and 4-trans caffeoylglucaric acids eluted with a shorter retention time than the 2-trans-and 5-trans-caffeoylglucaric acids. Additionally, because the intra-molecular hydrogen bond on the same side is more stable than the intra-molecular hydrogen bond on the reverse side, the total intra-molecular hydrogen bonds of 3-trans-caffeoylglucaric acid formed at 3-/4-and 3-/2-were weaker than that of 4-trans-caffeoylglucaric acid. Likewise, the 5-transcaffeoylglucaric acid was stronger than its 2-trans isomer (2-trans-caffeoylglucaric acid). Therefore, the elution order of 4 trans-isomers was 3-trans, 4-trans, 2-trans, and 5-trans [32]. Based on the DPIs ratio rules of previous studies [37,38], the elution order of 4 pairs of caffeoylglucaric acid isomers was 3-trans, 4-trans, 3-cis, 4-cis, 2-trans, 5-trans, 2-cis, and 5-cis. After calculating the PRs of DPI at m/z 209.03 to m/z 191.02, the developed PR rules were supplemented ( Figure 5C). The PR values of cis-caffeoylglucaric acid were similar to that of the corresponding trans-caffeoylglucaric acid, which was consistent with previous studies [39]. Furthermore, the PRs of 3-cis, 4-cis-and 5-cis-caffeoylglucaric acid were higher than that of corresponding 3-trans, 4-trans-and 5-trans-caffeoylglucaric acid, and the PR values of 2-trans-caffeoylglucaric acid were higher to that of the corresponding 2-cis caffeoylglucaric acid. Therefore, the PR rules supplemented on the basis of previous studies could be used to accurately identify the caffeoylglucaric acid isomers and other acylated caffeoylglucaric acids.

Glucaric Acids
Glucaric acids are a group of compounds found widely in plants, formed by the combination of glucaric acid and one to four acylated or combined residues [29]. These compounds have a unique structure of positional and geometric isomers that remain the focus acid were similar to that of the corresponding trans-caffeoylglucaric acid, which was consistent with previous studies [39]. Furthermore, the PRs of 3-cis, 4-cis-and 5-ciscaffeoylglucaric acid were higher than that of corresponding 3-trans, 4-trans-and 5-transcaffeoylglucaric acid, and the PR values of 2-trans-caffeoylglucaric acid were higher to that of the corresponding 2-cis caffeoylglucaric acid. Therefore, the PR rules supplemented on the basis of previous studies could be used to accurately identify the caffeoylglucaric acid isomers and other acylated caffeoylglucaric acids.     Note: *, unknown compounds; +, compounds identified using chemical reference compounds.

A New Class of Caffeoyl Methyl Glucaric Acid Isomers
During the process of identifying feruloylglucaric acids, we identified eight compounds (#23, #28, #33, #38, #39, #41, #45, #49) with an m/z of 385.0711 [29]. However, the diagnostic product ions of these compounds (385.0711, 223.0454, 205.0348) were significantly different from those of feruloylglucaric acids (385.0711, 209.0297, 191.0192) ( Figure 6A). In the case of feruloylglucaric acids, the DPI at 209.0297 was produced by the removal of a feruloyl-from feruloylglucaric acids, while the DPI at 223.0454 was produced by the removal of a caffeoyl-from the newly discovered compounds. Feruloyl-has one more methyl-than caffeoyl-. In the case of [M-H] − ions consistency, those compounds that we identified might be formed by the combination of caffeoyl-and a methylated glucaric acid. After checking databases such as PubChem (https://pubchem.ncbi.nlm.nih.gov/, accessed on 30 January 2023) and ChemSpider (http://www.chemspider.com/, accessed on 30 January 2023), the DPI at 223.0454 might be derived from methyl glucaric acid. Therefore, we tentatively identified these compounds as a new class of caffeoyl methyl glucaric acid isomers ( Figure 6B). To identify positional and geometric isomers, we followed the identification rules of glucaric acid isomers. Hence, the elution order of caffeoyl methyl glucaric acids was tentatively identified as 3-cis, 4-cis, 3-trans, 2-cis, 4-trans, 2-trans, 5-trans, and 5-cis.

Content of 14 Active Compounds in A. absinthium
The MEAA was found to be rich in phenolic acids and flavonoids, which are known to have antimicrobial properties and are commonly used in traditional herbal medicine [43,44]. For instance, the casticin, chlorogenic acid, 3,5-dicaffeoylquinic acid, 4,5dicaffeoylquinic acid, and cynaroside are the quality control compounds of the traditional

Content of 14 Active Compounds in A. absinthium
The MEAA was found to be rich in phenolic acids and flavonoids, which are known to have antimicrobial properties and are commonly used in traditional herbal medicine [43,44]. For instance, the casticin, chlorogenic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, and cynaroside are the quality control compounds of the traditional antimicrobial Chinese medicine Vitex trifolia fruit and Lonicera japonica flower in the Chinese Pharmacopoeia [45]. To more accurately determine the content of antimicrobial compounds in A. absinthium and to establish its quality standards, we selected eight phenolic acids and six flavonoids and determined their content. Among these compounds, chlorogenic acid, rutin, kaempferide, and casticin had higher content than others, with values up to 25.5950 ± 0.544 µg/g, 11.3730 ± 0.368 µg/g, 8.8500 ± 0.258 µg/g, and 3.5470 ± 0.148 µg/g, respectively. The results of the content determination of the 14 compounds are shown in Table 3.

Antimicrobial Activity of 14 Active Compounds in A. absinthium
To further investigate the antimicrobial effect of MEAA, we examined the antimicrobial activity of 14 active compounds against nine strains. After the determination of MIC and MBC values of different compounds against different strains, the results revealed that salicylic acid, caffeic acid, casticin, and 3,4-dicaffeoylquinic acid had strong antimicrobial effects and inhibited all nine strains to varying degrees. However, the remaining 10 compounds had higher MBC values than 1 mg/mL for different strains and did not exhibit significant sensitivity. Salicylic acid displayed a good inhibitory effect on all nine strains, as indicated by its low MIC and MBC values. The sensitivity of Gram-positive bacteria to the four compounds was higher than that of Gram-negative bacteria, consistent with the results of the methanol extracts. Among the fungal strains, only salicylic acid and caffeic acid exhibited potent antimicrobial activity against C. albicans. Detailed data are shown in Table 4. The four compounds screened were more effective in antibacterial activity compared to the other compounds, which seems to explain the antibacterial activity of the methanol extracts. Salicylic acid, caffeic acid, and 3,4-dicaffeoylquinic acid belong to the class of phenolic acids, which are known for their proven antimicrobial activity [45]. Salicylic acid and caffeic acid have been extensively studied for their antimicrobial effects, which involve reducing the hardness of bacterial cell walls and breaking down bacterial cell membranes [46]. Among the quinic acids, 3,4-dicaffeoylquinic acid is more sensitive to nine strains, and further investigation is required to understand the differences in the antimicrobial activity of dicaffeoylquinic acid isomers due to their structural specificity. Fiamegos et al. [33] reported that dicaffeoylquinic acid isomers act as a pump inhibitor that targets the efflux pump system of Gram-positive bacteria, revealing a potential mechanism for bacterial inhibition by dicaffeoylquinic acid isomers. The sensitivity of dicaffeoylquinic acid isomers to Gram-positive bacteria is consistent with the finding of the current study. The study also highlights the potential antimicrobial activity of casticin, the primary active compound in Vitex trifolia fruit, which is known for its antitumor and anti-inflammatory activity [46]. Additionally, further trials are needed to confirm whether the newly identified caffeoyl methyl glucaric acids have antimicrobial activity.

Acute Toxicity
During the observation period, mice in both the control and treated groups exhibited no mortality. Activity levels, hair condition, and defecation did not reveal any significant abnormalities in any of the groups. After oral administration of MEAA, body weight changes remained within the normal physiological range for all groups of mice for 14 days (Figure 7). No significant differences were found in body weight between male and female mice in the treated group when compared to the control group ( Table 5). The major organs, including the heart, spleen, kidney, lung, liver, and thymus, were examined in each group of mice. No significant lesions were observed in any of the major organs in all groups of mice ( Figure A1). The organ coefficients of male and female mice in both control and treated groups are presented in Table 6. There were no significant differences in the organ coefficients of mice in the treated group compared to those in the control group. The above results indicate that the LD50 of MEAA is greater than 5000 mg/kg bw, demonstrating that MEAA possesses a good safety profile. This is consistent with the reported claims of better safety of other Artemisia plant extracts. Mekonen et al. reported on the acute toxicity of the aqueous extracts of Artemisia afra, which did not cause mortality in mice at a dose of 5000 mg/kg bw, indicating a good safety profile [47]. Similarly, Dib s study showed that after a single dose of 6000 mg/kg bw of crude extract of Artemisia campestris L., no significant abnormalities were observed in mice after 14 days [48]. In addition, there are still many reports that support the better safety of Artemisia plant extracts. Many pieces of evidence show that a multitude of Artemisia plant extracts exert therapeutic effects with better safety, which also applies to MEAA.   Note: Values expressed as mean ± SEM (n = 5). "ns": no significant differences.

Plant Materials and Chemicals
The above results indicate that the LD 50 of MEAA is greater than 5000 mg/kg bw, demonstrating that MEAA possesses a good safety profile. This is consistent with the reported claims of better safety of other Artemisia plant extracts. Mekonen et al. reported on the acute toxicity of the aqueous extracts of Artemisia afra, which did not cause mortality in mice at a dose of 5000 mg/kg bw, indicating a good safety profile [47]. Similarly, Dib's study showed that after a single dose of 6000 mg/kg bw of crude extract of Artemisia campestris L., no significant abnormalities were observed in mice after 14 days [48]. In addition, there are still many reports that support the better safety of Artemisia plant extracts. Many pieces of evidence show that a multitude of Artemisia plant extracts exert therapeutic effects with better safety, which also applies to MEAA.

Plant Materials and Chemicals
The dried plants of A. absinthium were obtained from Xinjiang herbal medicine market in China and were confirmed as genuine using a molecular identification method that we had previously established [49]. The HPLC-grade solvents (methanol and acetonitrile) were purchased from Merck KGaA (Darmstadt, Germany).

Preparation of Plant Extracts
The Dried plants were pulverized into a fine powder using a tissue crusher. A sample of 100 mg of the powder was placed into a centrifuge tube, to which 2 mL of 70% methanol was added. The mixture was extracted using sonication at 25 • C and 40 KHz for 30 min. Following centrifugation at 13,000 rpm for 10 min, the supernatant was filtered through a 0.2 µm membrane filter and utilized for chemical analysis. The analytical concentration was 50 mg/mL (w/v). Another 100 g of the sample powder was extracted using the same extraction method as described above to obtain 2 L of extract, which was allowed to settle and subsequently filtered. The extract was then filtered, concentrated to a paste using a rotary evaporator, and dried in a freeze dryer for use in the screening of antimicrobial activity and the evaluation of acute toxicity.

Experimental Animals
For the acute toxicity study, 20 SPF-graded ICR mice, consisting of 10 females and 10 males with an average weight of 18-22 g, were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), with an animal license no. SYXK (Beijing, China) 2021-0006. The mice were housed in the SPF-grade animal facility at the In-stitute of Traditional Chinese Medicine, Chinese Academy of Traditional Chinese Medicine, under standard laboratory conditions (12 h light/dark cycle, 23 ± 2 • C). They were fed on a standard rodent diet and could drink freely. These experiments were conducted following internationally accepted guidelines for evaluating the safety of MEAA [50] and were approved by the Animal Welfare Ethics Committee of the Institute of Traditional Chinese Medicine, Chinese Academy of Traditional Chinese Medicine. The mobile phase flow rate was 0.5 mL/min, and the injection volume was 1 µL.
The mass spectrometry acquisition was conducted in negative ion mode with an acquisition mode of MS E . The parameters were set as follows: capillary voltage, 2000 V; cone voltage, 40 V; desolvent gas, nitrogen; gas flow rate, 900 L/h; desolvation gas temperature, 450 • C; ion source temperature, 100 • C; mass range, m/z 50-1200; collision gas, argon gas; low energy scanning trap voltage, 6 eV; high energy scanning trap voltage, 50-70 eV. To correct the mass, Leucine enkephalin was used as the mass correction solution.
Mass spectrometry acquisition was performed in negative ion mode using an electrospray ion source (EIS) with an ion source temperature of 550 • C. Quantitative analysis was performed using a Multi-response monitoring (MRM) model, and the mass spectrometry conditions, and their optimized condition parameters are provided in Table A1.

Microbial Strains and Culture Media
The tested microbial strains included Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella aerogenes, Klebsiella pneumoniae, and Candida albicans, all of which were purchased from BeNa Culture Collection (Beijing, China). Bacterial strains were cultured in Luria-Bertani medium at 37 • C after activation, whereas the fungal strains were cultured in Sabouraud medium at 30 • C. Single colonies from the plate were cultured in a liquid medium, and the turbidity of the bacterial suspension was diluted to 0.5 McFarland when the concentration of the bacterial suspension was 10 6 cfu/mL.

Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
The optimized broth microdilution method and 2,3,5-triphenyltetrazolium chloride (TTC) staining was used to determine the MIC and MBC of the methanol extracts and their main active compounds [11,52]. The alcohol extract (100 mg/mL) and 14 active compounds (20 mg/mL) were dissolved in dimethyl sulfoxide (DMSO) to prepare the stock solutions, which were then added to a 96-well microplate and diluted with the medium in the range of 0.156-5.00 mg/mL using the two-fold serial dilution method. The medium was inoculated with 0.5 × 10 6 cfu/mL of the strain. The positive control was tetracycline or fluconazole (stock solutions concentration, 2 mg/mL) co-cultured with the bacterial suspension, while the negative control was the sample solvent co-cultured with the bacterial suspension. All wells contained no more than 2.5% DMSO and had a final volume of 200 µL. The MIC was determined as the lowest concentration at which the microorganisms did not demonstrate any visible growth after 24 h of incubation at 37 • C or 30 • C, and the MBC was defined as the lowest concentration at which the medium with 20 µL of 0.5% TTC solution added did not turn red.

Acute Toxicity Study
The mice were acclimatized and fed for 7 days prior to the experiments. Before administration, mice were fasted for 4 h, although they were allowed to drink freely. The MEAA dissolving in a 0.5% CMC-Na solution and administered as a single dose by gavage at 5000 mg/kg bw to one male and one female mouse. During the subsequent 24 h period, the activity level, hair condition, defecation, and death of these two mice were observed. Under normal conditions for the initial two mice, four male and four female mice were administered the same dose, totaling five treated mice in each group. Simultaneously, five male and five female mice were given 0.5% CMC-Na solution by gavage to establish the control group. The animals were observed individually for the first 30 min after treatment, mainly to observe the level of activity, hair condition, defecation, and general states. Special attention was given during the first 4 h, with regular observations during the first 24 h and daily thereafter for a total of 14 days. During this period, the body weight of each group of mice was measured and recorded daily. At the end of the observation period, the mice were weighed, humanely killed, and subjected to gross necropsy. The lesions of the major organs, including the heart, spleen, kidney, lung, liver, and thymus, were observed and weighed for the calculation of organ coefficients [51]. The data were analyzed using GraphPad Prism software (version 7.0, San Diego, CA, USA) and are presented as mean ± standard error of the mean (SEM). Significant differences among groups were assessed using one-way ANOVA. Statistical significance was determined at p < 0.05.

Conclusions
In this study, we evaluated the antimicrobial activity of MEAA, which has received limited attention in previous years. The extracts exhibited broad-spectrum antibacterial and antifungal activity. To understand the active compounds responsible for this activity, we summarized previous identification strategies and analyzed the phenolic compounds and their isomers in A. absinthium. We identified a total of 90 compounds, including flavonoids, quinic acids, and glucaric acids, with most of them being reported for the first time in A. absinthium. Notably, we identified a new class of caffeoyl methyl glucaric acids. We quantitatively analyzed and screened 14 primary active compounds for antimicrobial activity. Salicylic acid, caffeic acid, casticin, and 3,4-dicaffeoylquinic acid showed promising antimicrobial activity. In the acute toxicity study, the 5000 mg/kg bw dose of MEAA had no significant effect on the general status, body weight, and organ condition of the mice. This indicates that MEAA has a good safety profile. Our findings suggest that A. absinthium can be used as an antibiotic alternative to combat bacterial resistance, and the identification and screening of phenolic compounds provide a basis for further exploration of its antimicrobial properties.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.  Figure A1. The lesion status of the heart, spleen, kidney, lung, liver, and thymus in each group of mice. The scale is 1 cm. Figure A1. The lesion status of the heart, spleen, kidney, lung, liver, and thymus in each group of mice. The scale is 1 cm.