The antimicrobial effect and mechanism of the Artemisia argyi essential oil against bacteria and fungus

Abstract

Artemisia argyi is a traditional Chinese herb with antibacterial, antifungal, and antitumor activities. The essential oil of Artemisia argyi was extracted using the steam distillation method in this study. The chemical composition of the essential oil was analyzed using the gas chromatography–mass spectrometry method. Agar disc diffusion and double-broth dilution assays were used to detect the antimicrobial activity of the essential oil. Subsequently, the antimicrobial mechanisms were explored through cytomembrane permeability assay and electron microscopy. Based on gas chromatography–mass spectrometry analysis, 25 compounds were detected, including 13.76% cineole, 6.77% terpinen-4-ol, 6.68% 3-dione, 1,7,7-trimethyl-, 4.07% 3-cyclohexen-1-ol, 4-methyl-1-(1-methylethyl)-acetate, 3.58% 1-isopropyl-2-methylbenzene, and 1.58% g-terpinene. The essential oil was tested for antimicrobial activity, and the IC₅₀ values for Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Listeria monocytogenes, Pseudomonas aeruginosa, Streptococcus pneumoniae, and Candida albicans were determined to be 25.51 ± 2.29, 49.53 ± 0.86, 52.40 ± 1.49, 52.76 ± 1.60, 73.99 ± 1.38, 65.52 ± 0.95, and 214.98 ± 3.27 μg mL⁻¹, respectively. For essential oil interaction with cytoderm, the microorganisms treated by 1 × IC₅₀ and 2 × IC₅₀ concentration of essential oil both represented positive test results. Additionally, the alkaline phosphatase levels showed a direct correlation with concentration and treatment duration (range from 0 to 8 h). The interaction between essential oils and the cytomembrane was investigated by examining samples containing one of three test strains (Staphylococcus aureus, Escherichia coli, and Candida albicans), essential oil, and voltage-sensitive fluorescent dye disc35. The results demonstrated a significant increase in fluorescence levels within the solution upon introduction of the essential oil-treated strains. The findings of our research suggest that the essential oil disrupts the cytoderm and cytomembrane, thereby exhibiting antimicrobial activity.

Introduction

Artemisia argyi is a traditional Chinese medicine utilized for the treatment of asthma, inflammation, hepatitis, pathogen infections, and menstrual symptoms [1]. The essential oil extracted from A. argyi has been found to possess anti-inflammatory, antifungal, disinfectant, and anthelmintic properties [2]. Studies on the A. argyi compound have shown that its essential oil and extraction exhibit the main pharmacological activities [2, 3]. Some studies indicated that A. argyi essential oil had antibacterial activities against S. aureus, E. coli, and S. enteritidis [3, 4].

The treatment of pathogenic infections has gained public attention. According to clinical research, pathogenic bacteria such as E. coli, P. aeruginosa, and P. vulgaris can cause urinary tract infections, food poisoning, diarrhea, and bromatoxism [5]. However, due to drug resistance, the therapy of pathogenic bacterial infections using antibiotic are confronted with difficulty [6]. Pathogenic bacteria have the ability to form a biofilm during the process of developing resistance to antibiotics as a way to evade the attack of these drugs [6, 7]. On the other hand, the essential oil of A. argyi has been reported to exhibit anti-inflammatory activity, albeit with slight side effects [8]. In addition, there have been limited reports on the antimicrobial effects and mechanisms of A. argyi essential oil.

In this paper, essential oil was extracted from A. argyi using steam distillation. Gas chromatography–mass spectrometry was used to identify the components of the essential oil. The essential oil’s antimicrobial effects were detected using an agar disc diffusion assay, which was represented by the half-maximal inhibitory concentration (IC₅₀) [9]. The antimicrobial mechanism was studied by conducting cell permeability and electron microscopy assays.

Materials and methods

Chemical reagents and microorganisms

The conventional chemical reagents utilized in the experiment and positive drugs such as ampicillin sodium and streptomycin sulfate were procured from Aladdin Reagent Co., Ltd. (Shanghai, China). The positive control drugs used in the antibacterial Gram-positive bacteria, Gram-negative bacteria, and fungi were ampicillin sodium, streptomycin sulfate, and fluconazole, respectively. HPLC-grade derivatives of cineole (CAS NO. 470826), terpinen-4-ol (CAS NO. 562743), and 3-dione, 1,7,7-trimethyl- (CAS NO. 2767842) were purchased from Sigma-Aldrich Co. (Shanghai, China), to authenticate antimicrobial activities of the main chemical components in essential oil. The microorganisms used in this study were divided into bacteria and fungi. The detailed information of the microorganisms is listed in Table 1. Here are the cultivation conditions for different microorganisms: Enterococcus faecalis, Streptococcus agalactiae, Proteus mirabilis, and Streptococcus pneumoniae cultivated at 30 °C using blood agar medium [10–13]; Candida albicans cultivated at 30 °C using Martin medium [14]; for the other six bacteria, Luria–Bertani medium is the preferred medium at 30 °C. All mediums were procured from Aladdin Reagent Co., Ltd. (Shanghai, China). The microorganisms that were added to the test tube slants were stored at − 4 °C.

Table 1.

The microorganism’s detailed information

NO	Strain	Strain type	Source
1	Staphylococcus aureus	Gram-positive bacteria	CGMCC
2	Streptococcus pneumoniae	Gram-positive bacteria	CGMCC
3	Bacillus subtilis	Gram-positive bacteria	CGMCC
4	Listeria monocytogenes	Gram-positive bacteria	CGMCC
5	Enterococcus faecalis	Gram-positive bacteria	Hospital
6	Streptococcus agalactiae	Gram-positive bacteria	Hospital
7	Escherichia coli	Gram-negative bacteria	CGMCC
8	Pseudomonas aeruginosa	Gram-negative bacteria	CGMCC
9	Proteus mirabilis	Gram-negative bacteria	Hospital
10	Candida albicans	Fungus	CGMCC

CGMCC, China General Microbiological Culture Collection Center

Hospital: Laboratory Department of the First People’s Hospital of Lu’an City, Anhui Province

Preparation of the essential oil

Fresh Artemisia argyi Levl. et Van. var. argyi cv. was harvested from Anhui Yeji plantation base and identified by Prof. Naifu Chen of West Anhui University’s College of Biotechnology and Pharmaceutical Engineering. The plant samples were air-dried at room temperature (~ 28 °C) and screened for impurities using a 50 mesh. The leaves and stems were then crushed using a plant powder mill, and the resulting powder was washed with sterile water to prepare the oil–water mixture. Two hundred fifty grams of the powder samples were added to a 1-L round flask and soaked in 3 L of distilled water for 24 h. The essential oil was extracted using a volatile oil extractor for 8 h, and the extracted oil was dried with anhydrous sodium sulfate. The quantity of essential oil was then measured using an analytical balance.

Component analysis of the essential oil

To analyze the components of the essential oil, we performed GC–MS analysis using modified conditions from our previous research [15]. For GC–MS analysis, the equipment used was Trace 1300 gas chromatograph coupled to ISQ mass spectrometer (Thermo Fisher Scientific, West Palm Beach, FL, USA). Separation of essential oil was carried out using a TG-5 MS capillary column of 30 m × 0.25 mm with 0.25 mm film thickness. The total program time for GC–MS was 42 min and helium chosen as the carrier gas. The column oven temperature program was set as follows: 70 °C maintained for 5 min, increased to 85 °C at 1.4 °C min⁻¹, then increased to 95 °C at 1.4 °C min⁻¹ and maintained for 3 min, further increased to 125 °C at 1.3 °C min⁻¹, then to 155 °C at 1.4 °C min⁻¹, to 171 °C at 0.8 °C min⁻¹, and finally, to 181 °C at 10 °C min⁻¹, and maintained for 1 min. The injection speed was set to 1.0 mL min⁻¹ with a split ratio of 5:1. The mass spectrum conditions were set as follows: electron ionization (EI) was used as an ion source, with a 2.5 mL injection volume, 280 °C MS transfer line, 250 °C ion source, electron impact as MS mode, and a mass range scan of 50–350 atomic mass units.

To identify the chemical compounds of essential oil, the retention indices, retention times, and mass spectra were compared to the National Institute of Standards and Technology’s mass spectra libraries. All peaks detected in at least two-thirds of the total ion chromatograms were considered when calculating the total peak area (100%) and the relative areas of the essential oil ingredients.

Antimicrobial activities of the essential oil

To evaluate the antimicrobial effect of the essential oil, ten different kinds of microorganisms, including six kinds of Gram-positive bacteria, three kinds of Gram-negative bacteria, and one kind of fungus, were used as test strains. According to the filter disc diffusion plate method which described by Kil et al. [16], we carried out the antimicrobial activity experiment. When each strain was cultivated to the logarithmic growth stage, the culture concentration was diluted to 10⁶–10⁸ colony forming units per mL (CFU mL⁻¹). The antimicrobial activity tests were performed on 96-well plates using corresponding positive drug as a control group. The initial concentration of essential oil was prepared to 2048 μg mL⁻¹mixing with 2048 μg essential oil, 980 μL LB liquid medium, and 20 μL DMSO. The initial concentration of the positive control drug was 100 μg mL⁻¹ including ampicillin sodium, streptomycin sulfate, and fluconazole. Subsequently, the essential oil was dissolved with LB media at twofold serial dilutions (2048, 1024, 512, 256, 128, 64, 32, and 16 μg mL⁻¹), while positive control drug dissolving with LB media at twofold serial dilutions (100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0.78125 μg mL⁻¹) for the antimicrobial tests. Thereafter, 200 μL essential oil with 2048, 1024, 512, 256, 128, 64, 32, and16 μg mL⁻¹ were added into 8 wells in the longitudinal direction of the plate orderly. Then, 50 μL microorganism solution to be measured was added into each well. The ninth well was designated as the blank control, filled with 200 μL of LB liquid medium and 50 μL of microorganism solution. Three replicate wells were prepared for each experiment. The bacterial samples were incubated in a rotary shaker at 180 rpm min⁻¹ and 37 °C for 24 h. In contrast, the mixed fungus samples were incubated at 180 rpm min⁻¹ and 25 °C for the same duration. Subsequently, 0.1 mL of the mixture solution from the 96-well plates was aseptically inoculated onto agar plates. Finally, the agar plates were incubated at a constant temperature of 37 °C for 24 h. The formula for calculating the inhibition ratio is as follows: inhibition ratio = $\left(\mathrm{The}\mathrm{counts}\mathrm{of}\mathrm{blank}\mathrm{strain},-,\left(\mathrm{The}\mathrm{counts}\mathrm{of}\mathrm{tested}\mathrm{strain}\right)\right)/\mathrm{The}\mathrm{counts}\mathrm{of}\mathrm{blank}\mathrm{strain}\times 100\%$.

Antimicrobial mechanism of the essential oil

To investigate the antimicrobial mechanism of the essential oil, Staphylococcus aureus, Escherichia coli, and Candida albicans were selected as test strains to assess cell cytomembrane permeability, cell constituent leakage, and electron microscopy following treatment with the essential oil.

Determination of cytomembrane permeability

To assess the integrity of the cytomembrane following exposure to essential oil, a cytomembrane permeability assay was conducted. After incubation at 37 °C for 24 h, the strains were harvested and resuspended in a buffered solution (5 mM Hepes, pH 7.2, 5 mM glucose) once the optical density at 600 nm reached 0.6. The strains suspension obtained above was diluted to an OD₆₀₀ value of 0.05 using an ultraviolet spectrophotometer. Subsequently, the mixture containing 3600 μL of strain suspension and 400 μL of diSC35 with a concentration of 0.4 μM was incubated at 37 °C for 2 h. Following this, the mixture was treated with essential oil at a concentration equal to 1 × IC₅₀ for 24 h. Fluorescence levels were measured using a fluorescence spectrophotometer with excitation and emission wavelengths of 627 nm and 670 nm, respectively [17].

The essential oil was added to a suspension of Staphylococcus aureus, resulting in the final concentration of the essential oil being equivalent to 1 × IC₅₀ value and 2 × IC₅₀ value. The mixture was cultured under conditions of 37 °C and 100 rpm min⁻¹ shaking cultivation. Distilled water was used as a control group instead of the essential oil. Time sampling was conducted at 0, 2, 6, and 8 h. The collected samples were centrifuged at 3000 rpm min⁻¹ for 10 min. Subsequently, following the manufacturer’s protocol, the alkaline phosphatase (AKP) content in the liquid supernatant collection was measured using an alkaline phosphatase kit. Finally, the relationship between essential oil concentration and AKP content was observed.

Electron microscopy

The essential oil-treated strains were analyzed using environmental scanning electron microscopy (ESEM) to elucidate the potential functional antimicrobial mechanism of the essential oil [17]. The mixture containing the strain and essential oil was incubated at 37 ℃ and 150 rpm min⁻¹ in a constant temperature oscillation incubator for approximately 8 h until reaching the logarithmic growth phase, with DMSO serving as the blank control. The concentration of the essential oil was adjusted to attain a value equal 1 × IC₅₀. The sample preparation centrifuge 10 mL of the strain solution at a speed of 6000 rpm min⁻¹ for 10 min, discard the liquid supernatant, immobilize the cells using 5 mL of 2.5% glutaraldehyde for a duration of 12 h, rinse the cells with six different concentrations of ethyl alcohol (30%, 50%, 70%, 80%, 90%, and pure) sequentially for a duration of 15 min each, perform freeze-drying on the samples, and finally, apply gold coating by spraying. Subsequently, the samples were subjected to examination using environmental scanning electron microscopy.

Statistical analysis

The preparation of essential oil, inhibitory microbial activity, and cell permeability were conducted in triplicates, with the data recorded as mean ± SD. Statistical analysis was performed using SPSS23.0 software, and regression analysis was employed to determine significant differences.

Results and discussion

Components of the essential oil

The GC–MS assay was conducted to identify the constituents of essential oil extracted from Artemisia argyi. The results are presented in Fig. 1 and Table 2, which provide a comprehensive list of 25 chemical components. The essential oil primarily consisted of 13.76% cineole, 6.77% terpinen-4-ol, 6.68% 3-dione, 1,7,7-trimethyl-, 4.07% 3-cyclohexen-1-ol,4-methyl-1-(1-methylethyl)-, acetate, 3.58% 1-isopropyl-2-methylbenzene, and 1.58% g-terpinene. According to the research conducted by Dongyun Guo et al., GC–MS analysis detected relative contents of 0.45%, 0.31%, 0.31%, 0.45%, and 0.43% for 2-carene in A. argyi essential oil from Zhejiang Quzhou, Shanxi Changzhi, Anhui Chizhou, and Hunan Shaoyang in China, respectively [18]. Our findings revealed that the relative content of 2-carene determined in Anhui Yeji, China, remained consistent at 0.48% when compared to other research results. Previous studies have reported the anti-inflammatory activity of 2-carene in Zingiber anamalayanum rhizome oil [19]. While the cineole relative content was 16.82%, 11.39%, 22.93%, 3.51%, 18.08%, 33.34%, 12.27%, 21.69%, 3.78%, and 11.34% in A. argyi essential oil from Zhejiang Quzhou, Shanxi Changzhi, Hebei Quyang, Chongqing Linjiang, Shandong Heze, Henan Tangyin, Anhui Chizhou, Hubei Qichun, Hunan Shaoyang, and Jiangxi Poyang of China, respectively [18]. The relative content of cineole in Anhui Yeji, China, was found to be 13.76% in our study. It suggested that the contents of A. argyi essential oil presented difference during different regions.

Fig. 1.

Mass spectra of Artemisia argyi essential oil

Table 2.

Component of the essential oil. ^aRI: Kováts retention indices calculated on column HP-5 according to Bajer et al. (2017) [20]

Compound name	CAS NO	RIa	RT	Area%
Isophorone	78591	989.26	11.15	0.09
1,3, 5-Trimethylbenzene	526738	996.63	11.5	0.11
A-terpene	99865	1015.72	12.94	0.92
Benzene,1,2,4-trimethyl	95636	1019.41	13.24	0.07
1-Isopropyl-2-methylbenzene	527844	1022.97	13.53	3.58
2-Carene	554610	1026.9	13.85	0.48
Cineole	470826	1032.8	14.33	13.76
g-Terpinene	99854	1055.03	16.14	1.58
Linalool	78,706	1068.8	17.26	0.06
4-Isopropylidene-1-methylcyclo	586629	1097.67	19.61	0.05
Benzene,4-ethyl-1,2-dimethyl-	934805	1111.95	21.07	0.03
2,6-Xylenol	576261	1120.6	21.99	0.18
1,2,4,5-Tetramethylbenzene	95932	1131.42	23.14	0.06
3-Dione, 1,7,7-trimethyl-	2767842	1173.1	27.57	6.68
Terpinen-4-ol	562743	1182.13	28.53	6.77
Phenyl thiocyanate	5285870	1187.21	29.07	0.47
Methyl salicylate	119368	1190.78	29.45	0.02
3-Cyclohexen-1-ol,4-methyl-1-(1-methylethyl)-, acetate	4821049	1196.61	30.07	4.07
3-Cyclohexene-1-acetaldehyda, a,4-dimethyl-	29548149	1204.51	30.93	0.06
Phenol, o-tert-butyl- (8CI)	88186	1207.48	31.26	0.38
cis-Anethol	104461	1241.75	35.06	0.09
Nonanoic acid	112050	1280.79	39.39	0.17
Thymol	89838	1291.7	40.6	0.09
5-Isopropyl-2-methylphenol	499752	1298.74	41.38	0.25
Eugenol	97530	1349.86	46.73	0.81

Antimicrobial activities of the essential oil

The experiment on antimicrobial activities revealed that the essential oil inhibited Gram-positive and Gram-negative bacteria as well as fungus to varying degrees. As shown in Table 3, the essential oil exhibited antimicrobial activities against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Listeria monocytogenes, Pseudomonas aeruginosa, Streptococcus pneumoniae, Proteus mirabilis, Enterococcus faecalis, Streptococcus agalactiae, and Candida albicans with half maximal inhibitory concentrations of 25.51 ± 2.29 μg mL⁻¹, 49.53 ± 0.86 μg mL⁻¹, 52.40 ± 1.49 μg mL⁻¹, 52.76 ± 1.60 μg mL⁻¹, 73.99 ± 1.38 μg mL⁻¹, 65.52 ± 0.95 μg mL⁻¹, 1215.65 ± 3.40 μg mL⁻¹, 1535.50 ± 3.28 μg mL⁻¹, 1847.23 ± 2.66 μg mL⁻¹, and 214.98 ± 3.27 μg mL⁻¹, respectively. The corresponding positive drugs exhibited antimicrobial activities against the aforementioned microorganisms, with half maximal inhibitory concentrations of 1.89 ± 0.09 μg mL⁻¹, 16.39 ± 0.88 μg mL⁻¹, 13.72 ± 0.85 μg mL⁻¹, 2.94 ± 0.29 μg mL⁻¹, 12.86 ± 0.38 μg mL⁻¹, 1.59 ± 0.16 μg mL⁻¹, 8.54 ± 0.66 μg mL⁻¹, 2.74 ± 0.35 μg mL⁻¹, 2.17 ± 0.14 μg mL⁻¹, and 58.61 ± 0.82 μg mL⁻¹, respectively.

Table 3.

Antimicrobial activities of the essential oil and positive drugs

Microorganisms	IC50 of essential oil (μg mL−1)	Positive drug	IC50 of positive drugs (μg mL−1)
Staphylococcus aureus	25.51 ± 2.29	Ampicillin sodium	1.89 ± 0.09
Escherichia coli	49.53 ± 0.86	Streptomycin sulfate	16.39 ± 0.88
Bacillus subtilis	52.40 ± 1.49	Ampicillin sodium	13.72 ± 0.85
Listeria monocytogenes	52.76 ± 1.60	Ampicillin sodium	2.94 ± 0.29
Pseudomonas aeruginosa	73.99 ± 1.38	Streptomycin sulfate	12.86 ± 0.38
Streptococcus pneumoniae	65.52 ± 0.95	Ampicillin sodium	1.59 ± 0.16
Proteus mirabilis	1215.65 ± 3.40	Streptomycin sulfate	8.54 ± 0.66
Enterococcus faecalis	1535.50 ± 3.28	Ampicillin sodium	2.74 ± 0.35
Streptococcus agalactiae	1847.23 ± 2.66	Ampicillin sodium	2.17 ± 0.14
Candida albicans	214.98 ± 3.27	Fluconazole	58.61 ± 0.82

The results indicated that four kinds of Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, Listeria monocytogenes, Streptococcus pneumoniae) and two kinds of Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) were susceptible to essential oil. However, three kinds of bacteria obtained from Clinical patients including Enterococcus faecalis, Streptococcus agalactiae, and Proteus mirabilis displayed insensitivity to essential oil. Interestingly, the essential oil could inhibit half of the Candida albicans which belonged to fungus at 214.98 ± 3.27 μg mL⁻¹. To our knowledge, there had been no study detected that Artemisia argyi could inhibit Candida albicans.

The main components in essential oil were identified as cineole (CAS NO. 470826), terpinen-4-ol (CAS NO. 562743), and 3-dione, 1,7,7-trimethyl- (CAS NO. 2767842) through GC–MS analysis. Their standard reagents’ antimicrobial activities against Staphylococcus aureus, Listeria monocytogenes, and Candida albicans were subsequently detected. The antimicrobial activities of standard reagents against Staphylococcus aureus, Listeria monocytogenes, and Candida albicans are presented in Table 4 with their respective IC₅₀ values. Among the three standards, terpinen-4-ol exhibited the highest inhibitory activity against Staphylococcus aureus, Listeria monocytogenes, and Candida albicans with IC₅₀ values of 14.75 ± 3.51, 33.47 ± 3.46, and 196.72 ± 4.81 μg mL⁻¹, respectively. Following with terpinen-4-ol, cineole exhibited the inhibitory activity against Staphylococcus aureus, Listeria monocytogenes, and Candida albicans with IC₅₀ values of 32.37 ± 1.86, 66.25 ± 3.91, and 293.14 ± 4.52 μg mL⁻¹, respectively. The antimicrobial activity of 3-dione, 1,7,7-trimethyl- was not detected in the experimental analysis.

Table 4.

Antimicrobial activities of the standard reagents’ antimicrobial activities

Microorganisms	IC50 of cineole (μg mL−1)	IC50 of terpinen-4-ol (μg mL−1)	IC50 of 3-dione, 1,7,7-trimethyl- (μg mL−1)
Staphylococcus aureus	32.37 ± 1.86	14.75 ± 3.51	-
Listeria monocytogenes	66.25 ± 3.91	33.47 ± 3.46	-
Candida albicans	293.14 ± 4.52	196.72 ± 4.81	-

Several studies have demonstrated that terpinen-4-ol and cineole exhibit significant antimicrobial properties among the plant essential oil components [21–25]. The antimicrobial activities of terpinen-4-ol in tea tree oil were found to be superior to those of other components, as reported by Badr et al. [22], and these findings are consistent with the earlier observations made by R. Loughlin et al. [24]. The findings of our study align with the outcomes reported in previous research [22, 24, 25].

Effect of Artemisia argyi essential oil on the cytomembrane of microorganisms

The voltage-sensitive fluorescent dye disc35 can effectively bind to lipid molecules present on the cellular cytomembrane [26]. Due to the dye disc35 binding with lipid molecules could be released into the solution, it could be used as a fluorescent marker for the investigation of the cell cytomembrane destruction [27]. Based on the results of antimicrobial activity, Staphylococcus aureus, Escherichia coli, and Candida albicans were selected for determination of cytomembrane permeability. The concentration of essential oil treated with the strains was set at 1 × IC₅₀ value. Treatment with essential oil resulted in a nearly 15-fold increase in fluorescence levels in the Staphylococcus aureus cell solution, as shown in Fig. 2A. In Fig. 2B, which represents the change in fluorescence levels of the Escherichia coli solution, the fold was 11. Similarly, Fig. 2C demonstrated a fold change of 10 in fluorescence levels of the Candida albicans solution. Combined with the analysis of antimicrobial activities, the results of cytomembrane permeability were consistent with the trends observed in the antimicrobial results. It is noteworthy that although Candida albicans exhibited the lowest susceptibility to essential oil, with an IC₅₀ value of 214.98 ± 3.27 μg mL⁻¹, it only showed a slightly lower fluorescence level compared to the other two strains in the cytomembrane permeability assay. The discrepancy between the changes in fluorescence values and IC₅₀ values among these three groups of strains warrants further investigation. The experiment’s fluorescence levels revealed that essential oil permeabilized the cytomembrane, resulting in diSC35 release into solution. This finding suggests that the cytomembrane may be a target for essential oil.

Fig. 2.

Cytoplasmic membrane permeability of Staphylococcus aureus (A), Escherichia coli (B), and Candida albicans (C) treatment with the essential oil. Red curves indicate the fluorescence levels of stimulated strains treatment with the essential oil at a concentration equivalent to 1 × IC₅₀ value, respectively, while the black curve represents the untreated strains

Effect of Artemisia argyi essential oil on the cytoderm of microorganisms

The alkaline phosphatase, which is typically found in the interstitial space between the cytoderm and cytomembrane, cannot be detected in the solution of microorganisms [28]. When the cytoderm suffered damage, the leakage of alkaline phosphatase occurred [28]. To investigate the impact of the essential oil on cytoderm, we detected the leakage of alkaline phosphatase and measured the AKP content in the liquid supernatant using an alkaline phosphatase kit following the manufacturer’s protocol. The effects of essential oil permeabilization on cytoderm were observed in Staphylococcus aureus, Escherichia coli, and Candida albicans (Fig. 3). Upon treatment with 1 × IC₅₀ and 2 × IC₅₀ concentrations of essential oil, there was an increase in alkaline phosphatase levels from 0 to 8 h for all strains. As time progressed, higher AKP contents were detected in strains treated with 2 × IC₅₀ compared to those treated with 1 × IC₅₀. The analysis of cytomembrane permeabilization showed consistent results with the portion related to cytoderm permeabilization. This indicates that Artemisia argyi essential oil has the ability to damage both cytoderm and cytomembrane in microorganisms including Gram-positive bacteria, Gram-negative bacteria, and fungi.

Fig. 3.

Effect of essential oil treatment on the AKP leakage of Staphylococcus aureus (B), Escherichia coli (C), and Candida albicans (D) treatment with the essential oil at 1 × IC₅₀ and 2 × IC₅₀ concentrations. A The p-nitrophenol standard curve of the alkaline phosphatase kit

Electron microscopy of strains treated by the essential oil

Environmental scanning electron microscopy was employed to elucidate the mechanism of antimicrobial action. The ESEM micrographs in Fig. 4 depict the effects of essential oil treatment on strains. As depicted in Fig. 4a, Staphylococcus aureus treated with distilled water exhibited a normal cell morphology, while Fig. 4d revealed significant cellular damage resulting from treatment with essential oil at a concentration equivalent to 1 × IC₅₀. Similar observations were made for Escherichia coli in comparison groups, as shown in Fig. 4b vs. Fig. 4e and Fig. 4c vs. Fig. 4f, respectively. Interestingly, despite Staphylococcus aureus displaying higher susceptibility in both antimicrobial and cell permeability tests among all tested strains, Escherichia coli demonstrated the most pronounced morphological disruption under electron microscopy (Fig. 4b vs. Fig. 4e). In summary, these findings strongly indicate that the essential oil exerts a profound impact on cytomembrane integrity.

Fig. 4.

ESEM photographs of Staphylococcus aureus (a, d), Escherichia coli (b, e), and Candida albicans (c, f) treated with essential oil

Conclusions

The A. argyi essential oil was obtained through steam distillation method. In terms of antimicrobial activity, the oil exhibited potent effects against Gram-positive bacteria, Gram-negative bacteria, and fungi. Regarding the mechanism of action, the essential oil disrupted the cytoderm and cytomembrane of the test strain, resulting in lipid molecule leakage and AKP release. These findings suggest that A. argyi essential oil holds promising potential as a natural source for anti-inflammatory medicine.

Funding

This research was supported by the Anhui Scientific Research and Innovation Team of Quality Evaluation and Improvement of Traditional Chinese Medicine (2022AH010090), the Provincial Level Nature Science Foundation of Anhui Education Department (KJ2019A0628, KJ2019A0617, KJ2019A0626, KJ2021A0957, KJ2020A0634, and GXYQ2020127), Postdoctoral Science Foundation of West Anhui University (WXBSH2021001), and The High-level Talent Project of West Anhui University (WGKQ2021021).

Declarations

Conflict of interest

The authors declare no competing interests.