Artemisia Extracts Suppress H1N1 Influenza A Virus Infection by Targeting Viral HA/NA Proteins and Modulating the TLR4/MyD88/NF-κB Signaling Axis

Abstract

Background: Influenza A virus is an acute respiratory virus that spreads quickly, affects a broad range of populations, and can lead to many complications and mortality. Artemisia L. species are widely used in traditional medicine, but their antiviral potential against H1N1 remains uncertain. Methodology: Network pharmacology and molecular docking were used to computationally explore their potential function in this domain, and to investigate how their invasion mechanisms and adsorption occur. UPLC-MS/MS analysis identified the main components of the extracts. The anti-H1N1 mechanism of Artemisia L. extracts was studied in vitro. Results: Network pharmacology identified 95 key targets between Artemisia L. and IAV, with quercetin and luteolin as core active compounds. Molecular docking predicted strong binding affinities between these compounds and influenza virus proteins. UPLC-MS/MS analysis identified 75, 100, and 64 chemical components in ACBE, AALE, and ACTE, respectively, mainly flavonoids and terpenoids. Artemisia L. extracts exhibited both preventive and therapeutic effects against H1N1, reducing progeny virus NP mRNA and protein levels. In vitro experiments showed that higher concentrations of the extracts prevent virus attachment to MDCK cells by denaturing the HA protein. NA plays an essential role in progeny virus release. We found that a high concentration of ACTE can inhibit NA up to 85%, and ACBE showed a low inhibitory effect on NA. Conclusions: In terms of therapeutic effects, Artemisia L. extracts can regulate intracellular inflammatory factors via the TLR4/NF-κB/MyD88 signaling pathways and reduce the expression of IL-1β, IL-6, TNF-α, TLR4, NF-κB, p65, and MyD88 at the mRNA level, thereby inhibiting H1N1 virus replication. These results suggest that bioactive components in Artemisia L. extracts may inhibit H1N1, potentially leading to the development of natural-product-based anti-influenza agents.

1. Introduction

The influenza virus poses a serious threat to public health. Approximately 10% of the world’s population contracts it each year. Those at higher risk of severe illness are mainly people aged 65 and older and children under 2 years old. The number of influenza-related deaths annually can range from 290,000 to 640,000 [1]. The influenza virus is part of the Orthomyxoviridae family. It is a negative-sense, single-stranded RNA virus, categorized into types A, B, C, and D [2]. There have been four global influenza pandemics in history; the 1918 “Spanish flu (H1N1)” can be called the “largest plague” in human history. About 1 billion people were infected worldwide, and between 50 million and 100 million people died. In 2009, the “swine flu” caused by the new H1N1 virus swept across the world again [3].

Influenza A virus is the main seasonal strain responsible for causing mild to severe respiratory infections and other complications in humans. Currently, anti-influenza drugs on the market primarily target neuraminidase (NA), the viral RNA polymerase, and the M2 ion channel. Commonly used therapeutic drugs include bavirin, peramivir, rimantadine, and others, but resistance to these medications has been widely reported. The spread of the influenza virus depends on factors such as virus type, environmental conditions, and other factors. Transmission of the virus can be reduced through natural, non-pharmaceutical, and pharmaceutical barriers [4]. Therefore, the search for new, safe, and promising drugs is essential.

The genus Artemisia L. belongs to the dicotyledonous family Asteraceae. It can be sustained by diverse resources and is widely found in the northern temperate regions of Asia, Europe, and North America. It includes over 500 species, such as Artemisia caruifolia Buch.-Ham. ex Roxb, Artemisia argyi Levl. et Vant, and Artemisia capillaris Thunb [5]. Polysaccharides, coumarins, terpenoids, flavonoids, and volatile oils are secondary metabolites with significant pharmacological value [6]. Artemisia L. and some of its active ingredients are commonly used as dietary supplements, functional foods, and medicines [7] for the treatment of malaria, jaundice, viral infections, tuberculosis, and for their antibacterial, anti-inflammatory, and immunomodulatory properties [8]. These three Artemisia L. species share similar compounds, so quantifying these compounds affects H1N1 treatment in a dose-dependent manner; this is why these three plants were chosen.

Several Artemisia species have demonstrated notable preclinical antiviral activity against influenza viruses. Extracts of Artemisia annua have shown direct inhibition of influenza A virus by targeting the viral nucleoprotein and reducing virus-induced apoptosis in cell and mouse models [9]. Flavonoids from Artemisia scoparia, particularly cirsimaritin, significantly reduce viral RNA and protein synthesis while suppressing the NF-κB and MAPK signaling pathways involved in viral replication and inflammation [10]. Artemisia cina (santonica flower) extracts and their major constituent santonin exhibit strong virucidal activity against H5N1 and H1N1, attributed partly to neuraminidase interaction [11]. Collectively, these studies suggest that Artemisia species contain bioactive compounds that can interfere with influenza virus replication through diverse mechanisms, although clinical efficacy in humans has not yet been established.

Artemisia L. is also used in Chinese patent and herbal medicines to prevent and treat various diseases. However, whether Artemisia L. can inhibit the influenza virus and its mechanism of action has not yet been reported. Therefore, this paper employs network pharmacology to explore, from a theoretical perspective, the potential of Artemisia L. plants to inhibit H1N1; additionally, it utilizes Artemisia caruifolia Buch.-Ham. ex Roxb extract (ACBE), Artemisia argyi Levl. et Vant extract (AALE), and Artemisia capillaris Thunb. extract (ACTE). This study mainly focuses on blocking the influenza virus from entering cells and examining its treatment mechanism.

2. Results

2.1. Network Pharmacology Explores the Multi-Target Effects of Artemisia L. Plants Against Influenza Viruses

2.1.1. Screening of Active Ingredient Targets from Artemisia L. Plants

According to the TCMSP database, the number of active ingredients in Qinghao, Aiye, and Yinchen was 126, 135, and 53, respectively. The targets corresponding to each drug’s active ingredients were then screened. According to OB > 30% and DL > 0.18, 44 active ingredients were identified (Table S1), as well as 157 drug targets.

Using the GeneCards database, 3281 disease-related targets were obtained. The intersection of the active ingredients and the influenza virus targets mentioned above was identified as comprising 95 key targets. The results are shown in Figure 1.

2.1.2. Construction of the PPI Network Between Artemisia Plants and the Influenza Virus

The PPI network of 95 key targets was constructed by STRING (https://string-db.org) with a confidence level of 0.4. After removing isolated proteins, an interaction network of 140 proteins was obtained, comprising 95 nodes and 586 edges, which highlights the main interactions between Artemisia L. plant compounds and influenza viruses. Cytoscape software was used to visualize the protein network diagram, as shown in Figure 2.

Combined with the Analyze Network, the topological features of the PPI network were examined to determine each node gene’s degree and rank the degrees. The top five genes were TNF, AKT1, TP53, IL-6, and IL-1β. These five targets are the key nodes in the PPI network (

Table 1. PPI network topology analysis of common targets.

Name	Degree	Betweenness Centrality	Clustering Coefficient
TNF	158	0.039099664	0.503407984
AKT1	158	0.067001705	0.489126907
TP53	150	0.048932214	0.504864865
IL-6	150	0.031887528	0.540900901
IL-1β	144	0.025259772	0.563380282
MMP9	142	0.017747865	0.585110664
CASP3	140	0.021139784	0.579710145
PTGS2	138	0.019117295	0.588661552
EGFR	134	0.018423848	0.582089552
HIF1A	132	0.015809889	0.618648019

and Figure 2).

2.1.3. Enrichment Analysis of Target Genes of Artemisia L. Plants and Influenza Virus

GO functional annotations were performed for the 95 key target genes to identify active ingredient targets and respiratory infection disease targets, and the GO enrichment results were visualized using the R package (V 4.4.2) “enrichplot”. The results are shown in the following figure. In terms of biological processes (BP), a total of 506 terms were obtained that were significantly related to signal transduction, apoptosis, inflammatory response, and other processes. In terms of molecular functions (MF), 103 terms were identified, and key target genes were significantly associated with protein binding, cytokine activity, transcription factor activity, etc. In terms of cell composition (CC), 49 terms were identified, and key target genes were significantly associated with the cytoplasm, nucleus, chromatin, and other cellular components.

KEGG functional enrichment analysis of 95 key target genes identified 165 related pathways. The R package “enrichplot” was used to create a bar chart showing the top 15 pathways. From the figure, we can see that the KEGG enrichment results include lipid metabolism and atherosclerosis, as well as the AGE-RAGE signaling pathway in diabetic complications, the TNF signaling pathway, and the IL-17 signaling pathway, among others. This suggests that the key target genes are significantly associated with pathways such as the IL-17 and TNF signaling pathways (Figure 3).

2.1.4. Visualization of Key Components and Core Proteins

TP53, IL-6, AKT1, TNF, IL-1β, and their active ingredients were selected for molecular docking. Figure 4 is a visualization of receptor and ligand docking, and the molecular docking energy parameters are shown in

Table 2. Target and active ingredient molecular docking table.

Target	PDB ID	Active Ingredients	Docking Energy (kcal/mol)
TP53	1a1u	Quercetin	−5.90
IL-6	5zo6	Luteolin	−5.15
TNF	1pk6	Luteolin	−4.35
AKT1	1unr	Quercetin	−6.70
IL-1β	1l1b	Quercetin	−8.58

.

The docking energy of TP53 and quercetin is −5.90 kcal/mol, as shown in Figure 4C; the docking energy of IL-6 and luteolin is −5.15 kcal/mol, as shown in Figure 4A; the docking energy of TNF and luteolin is −4.35 kcal/mol, as shown in Figure 4E; the docking energy of AKT1 and quercetin is −6.70 kcal/mol, as shown in Figure 4B; the docking energy of IL-1β and quercetin is −8.58 kcal/mol, as shown in Figure 4D. When the docking energy is less than −4.25 kcal/mol, it indicates some binding activity between two components; a value below −5.0 kcal/mol indicates vigorous binding activity. It is important to note that molecular docking only predicts binding potential and does not confirm biological activity; these results provide supportive evidence for subsequent experimental validation of key interactions. These active ingredients are expected to play a key role in inhibiting the influenza virus (Figures S2–S6).

2.2. UPLC-MS/MS Analysis of Artemisia L. Extracts

Compared with traditional chemical reagent-based reaction determination, UPLC-MS/MS offers high sensitivity and good specificity. It can quickly separate and quantitatively detect samples, reducing operational time and errors. It can detect highly sensitive trace molecules and determine the molecular weight, structure, and composition of target compounds. It has been widely used to analyze the chemical components of various Chinese herbal medicines. A total of 74 chemical components were detected in ACBE, 100 chemical components were detected in AALE, and 65 chemical components were detected in ACTE, mainly flavonoids and terpenes. Seven active ingredients were detected in all three extracts (Figure 5). These primarily consist of flavonoids and terpenoids. The identification results were confirmed by comparison with PubChem and ChemSpider databases and were consistent with previous studies.

2.3. Artemisia L. Extracts Have In Vitro Resistance to H1N1

2.3.1. Cytotoxicity of Artemisia L. Extracts on MDCK Cells

A cytotoxicity assay was conducted to determine the toxic concentration range of ACBE, AALE, and ACTE against MDCK cells. Drugs at concentrations ranging from 0 to 27.0 mg/mL were incubated with MDCK cells for 48 h. The relative cell survival rate was then measured using the CCK-8 method. The IC50 values were 20.0 mg/mL for ACBE, 4.7 mg/mL for AALE, and 3.9 mg/mL for ACTE. These results indicate that all IC50 values are above 2.0 mg/mL, suggesting that their effects are very low and the compounds are generally safe. Statistical analysis by one-way ANOVA showed no significant cytotoxicity at the concentrations used in subsequent antiviral assays (p > 0.05) (Figure 6).

2.3.2. The TCID₅₀ Determination of H1N1

The baseline dose of the H1N1 virus on MDCK cells was determined using a CPE assay. The TCID₅₀ of H1N1 on MDCK cells was calculated using the Reed–Muench method and determined to be 10⁻⁶/mL. Subsequent experiments were performed using the H1N1 virus at an MOI of 0.01 (

Table 3. The infectivity of H1N1 on MDCK cells.

Viral Dilution	Number of Holes	Number of CPE Holes	No Number of CPE Holes	Cumulative Number of CPE Holes	Cumulative Number of No CPE Holes	Total	CPE Ratio	CPE Percentage
10−4	4	4	0	10	0	10	10/10	100%
10−5	4	4	0	6	0	6	6/6	100%
10−6	4	2	2	2	2	4	2/4	50%
10−7	4	0	4	0	4	4	0/4	0%

).

2.3.3. Artemisia L. Extracts Prevent and Treat H1N1 Infection

During the direct action, Artemisia L. extracts were mixed with the H1N1 virus and incubated for 5 h. The mixture was then inoculated into MDCK cells for 1 h. After 48 h, ACBE and ACTE did not affect H1N1. The EC₅₀ of AALE for H1N1 was 2.3 mg/mL, and the SI was 2, indicating that AALE also had no effect. Hence, Artemisia L. extracts had no direct inactivation effect on H1N1. Statistical analysis by one-way ANOVA showed no significant difference compared to the virus control group (p > 0.05) (Figure 7A).

To evaluate the activity of Artemisia L. extracts against H1N1, CPE and cell viability assays were used. In the prevention phase, MDCK cells were incubated with Artemisia L. extracts for 5 h, followed by 1.5 h of H1N1 virus infection (Figure 7A). After 48h, the cell survival rate of MDCK cells treated with Artemisia L. extracts showed a gradient-dependent increase, and the EC₅₀ of ACTE was 70.22 μg/mL and SI was 56.63; the EC₅₀ of AALE was 15.10 μg/mL and SI was 313.8; The EC₅₀ of ACBE was 81.94 μg/mL, SI was 244.48, and both SI > 2 (Figure 7B), indicating that Artemisia L. extracts may interact with host cells and be able to block the viral invasion of cells. Statistical analysis by one-way ANOVA showed no significant difference compared to the virus control group (p > 0.05). For treatment, MDCK cells were infected with the H1N1 virus for 1 h and then incubated with Artemisia L. extracts. After 48 h, the survival rate of MDCK cells treated with Artemisia L. extracts increased in a gradient-dependent manner. The EC₅₀ of ACBE was 669 μg/mL, and the SI was 29.9. The EC₅₀ of AALE was 110.2 μg/mL, and SI was 42.7; the EC₅₀ of ACTE was 188.1 μg/mL, and SI was 21.14; both SI > 2. Therefore, Artemisia L. extracts can be used to treat H1N1. Statistical analysis by one-way ANOVA showed no significant difference compared to the virus control group (p > 0.05) (Figure 7C).

2.3.4. Artemisia L. Extracts Inhibited the Virus Content in Progeny

To investigate whether Artemisia L. extracts inhibit viral replication in H1N1-infected MDCK cells, we investigated whether they inhibited NP gene content, protein expression, and immunofluorescence. Compared with the H1N1 control group, Artemisia L. extracts significantly reduced progeny virus expression during prophylaxis (Figure 8 and Figure 9).

2.4. The Effect of Artemisia L. Extracts on the Reproduction Cycle of H1N1

2.4.1. Artemisia L. Extracts Inhibit the Attachment Phase of H1N1

To explore which stage of viral infection is associated with the antiviral effects of Artemisia L. extracts, viral infection at attachment during H1N1 infection was evaluated. Artemisia L. extracts and H1N1 virus liquid were mixed and incubated for 1 h at 4 °C. Then, a virus maintenance solution was added, and the mixture was cultured at 37 °C for 24 h to observe CPE and cell viability. Statistical analysis by one-way ANOVA confirmed significant differences compared to the virus control group (p < 0.001) (Figure 10). The results showed that different concentrations of Artemisia L. extracts can inhibit H1N1 virus attachment.

In the virus-only control group (no drug), guinea pig blood agglutinated. When Artemisia L. extracts were added, blood agglutination was inhibited to varying degrees. The higher the virus titer, the greater the virulence. An ACBE concentration of 12.0 mg/mL could destroy the HA protein, and AALE and ACTE concentrations of 12.0 mg/mL could still destroy the HA protein of the virus stock solution (Figure 11A). A virus titer was chosen to explore an ACTE concentration of more than 0.75 mg/mL, and an AALE concentration of more than 0.187 mg/mL; an ACBE concentration of more than 3.0 mg/mL can destroy HA (Figure 11B). These results demonstrate that high concentrations of Artemisia L. extracts can effectively inhibit HA, suggesting that Artemisia L. extracts may have a target site that interacts with HA, thereby blocking influenza virus adsorption to cells.

2.4.2. ACBE and AALE Reduce Calcium Ion Content in H1N1-Infected MDCK Cells

The HA of H1N1 attaches virions to cells to initiate the infection cycle by binding to terminal sialic acid residues on glycoproteins, and HA binds to the voltage-dependent Ca²⁺ channel Cav1.2 to trigger intracellular Ca²⁺ oscillations and subsequent H1N1 entry and replication. Cell calcium levels were measured using Fluo-4 dye to determine whether Artemisia L. extracts inhibited Ca²⁺ content in H1N1-infected MDCK cells. Figure 12 shows that AALE significantly affects intracellular Ca²⁺ content. In contrast, ACBE has a significant effect on intracellular calcium ion content only at high concentrations, whereas ACTE shows no significant change in intracellular Ca²⁺ content. Statistical analysis by one-way ANOVA confirmed significant differences compared to the virus control group (p < 0.05). In contrast, ACBE has a significant effect on intracellular calcium ion content only at high concentrations (p < 0.05) **, whereas ACTE shows no significant change in intracellular Ca²⁺ content ** (p > 0.05).

2.4.3. ACBT and ACTE Reduce NA Activity and Inhibit H1N1

Artemisia L. extracts can inhibit viral release by targeting the NA protein. We used the NA kit to investigate whether Artemisia L. extracts affected NA. Peramivir is a classic NA inhibitor and served as a positive control. It was found that ACBE and ACTE could inhibit NA, and as their concentration decreased, the inhibition rate also decreased. AALE had no significant inhibitory effect on NA. Statistical analysis by one-way ANOVA confirmed significant differences compared to the virus control group (p < 0.05, p < 0.01, p < 0.001) **. AALE had no significant inhibitory effect on NA ** (p > 0.05) (Figure 13).

2.4.4. Effect of Artemisia L. Extracts on mRNA Expression of Related Genes in MDCK Cells in the Treatment Group

Artemisia L. extracts decreased the gene expression of intracellular inflammatory factors. Under their therapeutic effect, the differences in the expression of IL-1β, IL-6, and TNF-α at the mRNA level in the Artemisia L. extract treatment group compared with the virus control group are shown in Figure 14. IL-1β, IL-6, and TNF-α were reduced at the mRNA level. Statistical analysis by one-way ANOVA confirmed significant differences compared to the virus control group (p < 0.05, p < 0.01, p < 0.001).

2.4.5. Effect of Artemisia L. Extracts on the Expression of Proteins Related to TLR4/NF-κB/MyD88 Signaling Pathway in MDCK Cells in the Treatment Group

Artemisia L. extracts treat H1N1 through the TLR4/NF-κB/MyD88 signaling pathway. The differences in the expression levels of the TLR4, NF-κB, and MyD88 proteins in the Artemisia L. extract-treated cell group compared to the virus control group are shown in Figure 15. After the virus invades cells, the TLR4/NF-κB/MyD88 signaling pathway is activated, leading to an inflammatory response upon ACBE intervention. The protein content of this pathway is downregulated to varying degrees, including the content of TLR4, NF-κB, and MyD88. Statistical analysis by one-way ANOVA confirmed significant differences compared to the virus control group (p < 0.05).

2.4.6. Effect of Artemisia L. Extracts on the Expression of Proteins Related to TGF-β/MAPK/P-JNK Signaling Pathway in MDCK Cells in the Treatment Group

Compared with the virus control group, the expression of the P-JNK gene in MDCK cells pre-treated with Artemisia plant extracts was significantly decreased. Statistical analysis by one-way ANOVA confirmed significant differences compared to the virus control group (p < 0.05). AALE can increase MAPK gene expression—statistical analysis by one-way ANOVA confirmed significant differences compared to the virus control group (p < 0.05) **—whereas ACBE and ACTE have no regulatory effect on it ** (p > 0.05). AALE can increase MAPK gene expression, whereas ACBE and ACTE have no regulatory effect on it. The above results indicate that ACBE can resist the influenza A virus by regulating the content of P-JNK genes in cells. AALE can resist the influenza A virus by regulating the content of MAPK genes. ACTE can resist the influenza A virus infection by regulating p-JNK expression (Figure 16).

3. Discussion

IAV spreads quickly and affects a wide range of people. Currently, the most commonly used biological means of preventing the virus is vaccination, which is highly effective at reducing its spread. The current vaccine primarily consists of specific antibodies targeting HA and NA; however, the HA protein accumulates mutations, necessitating updates [12]. Some people have also expressed concerns and doubts about the safety and necessity of influenza vaccines [13]. Therefore, enhancing the body’s immune system with natural products, functional foods, and nutritional supplements is the safest and most cost-effective strategy for preventing viral infections [14]. Therefore, it is crucial to prevent influenza using natural products.

Artemisia L. species are widely distributed worldwide, rich in diverse chemical components, and exhibit a range of pharmacological effects. They are common Chinese medicinal materials. We first utilized network pharmacology and molecular docking to investigate whether Artemisia L. affects influenza and explore its potential applications [15].

The advantages of Artemisia L. species are primarily reflected in the following: (1) Multi-target synergistic potential: Their rich content of flavonoids, lignans, and volatile oils may simultaneously inhibit viral invasion and replication as well as regulate host immune/inflammatory responses. (2) Comprehensive regulation of the “virus–host-immunity” chain: Unlike many single-target synthetic antiviral drugs, Artemisia L. extracts often exhibit anti-inflammatory and immunomodulatory effects while inhibiting viruses, helping to alleviate excessive inflammatory damage in the later stages of infection. (3) Combination of traditional wisdom and modern validation: Their traditional efficacy in “clearing heat and detoxifying” is backed by the clinical efficacy of classic formulas such as Haoqin Qingdan Decoction and Retoxin Injection, as well as modern preparations, providing a solid foundation for the applications and research motivations of this study. Disadvantages of Artemisia L. species are primarily reflected in the following: (1) Complex composition: The crude extract has a complex composition, making it difficult to identify the specific active components clearly. (2) Variety and regional differences: The active ingredient profiles and contents of Artemisia annua vary significantly depending on the species and origin, directly affecting the stability and reproducibility of their effects [16,17].

The active ingredients of Qinghao, Aiye, and Yinchen were retrieved through the TCMSP database, and a total of 44 active ingredients corresponding to 157 drug targets were obtained; among them, there were 95 key targets that exerted effects on both the active ingredient targets of Artemisia L. and influenza virus targets. In virtual screening, a threshold of OB ≥ 30% and DL ≥ 0.18 is usually established because, through multiple rounds of filtering with these OB and DL values, a list is obtained of candidate molecules that not only have strong binding capabilities but also have a higher likelihood of becoming successful drugs, thereby significantly enhancing the efficiency of subsequent experimental validation. This paper is the first to simulate the interactions of luteolin and quercetin with IL-6 (5ZO6), TNF (1PK6), AKT1 (1UNR), and IL-1B (1L1B). Our research aims to predict their potential mechanisms of action from a computational chemistry perspective, providing target presuppositions for subsequent experimental verification. The docking targets of the most effective active ingredients and hub genes were obtained through molecular docking. Figure 4 shows that quercetin exhibits strong binding to AKT1 and IL-1β, suggesting that it may play a key role in influenza virus [18].

Artemisia L. has been widely reported to be rich in flavonoids (such as quercetin) and flavonoid lignans in previous studies. Multiple independent studies have confirmed that these components exhibit inhibitory activity against various viral models (including influenza virus, herpesvirus, and coronavirus). Therefore, they were selected based on a “known active ingredient tracking” strategy [19]. These components are characteristic secondary metabolites of this genus, present in relatively high amounts, and easily isolated and identified, forming an important material basis for their pharmacological effects.

This study selected Artemisia annua, Artemisia argyi, and Artemisia capillaris, all previously reported in the Artemisia genus, and identified their main flavonoids and flavonoid lignans, including quercetin and flavonoid lignans, in extracts taken by UPLC-MS/MS to explore their potential interactions with the viral targets of interest in this study.

UPLC-MS/MS combines the separation capability of liquid chromatography with the high sensitivity and high resolution of mass spectrometry. It possesses fast analysis, high sensitivity, and strong anti-interference capabilities, thereby enhancing the accuracy of multi-component analysis [20]. A total of 75 chemical components were identified in ACBE: 10 flavonoids, 12 organic compounds, 5 alkaloids, and 6 terpenes; 100 in AALE: 19 flavonoids, 6 terpenoids, 10 organic compounds, and 5 amino acid compounds; and 64 in ACTE: 11 flavonoids, 10 organic compounds, 4 organic bases, and 3 alkaloids. The types of flavonoids account for a high proportion, among which quercetin, luteolin, schaftoside, baicalin, etc., are common flavonoids; the terpenoids detected include scutellaria lactone, dechamozulene, parthenolide, hydrogenated costus lactone, glycyrrhetinic acid, valeric acid, artemisinin, etc. In vitro cell experiments were then used to demonstrate that Artemisia L. can inhibit the H1N1 virus at both preventive and therapeutic stages within a non-toxic range. The NP mRNA and protein expression of influenza virus progeny were significantly reduced under the preventive effect. AALE, ACBE, and ACTE can inhibit the proliferation of progeny viruses by inhibiting the attachment of H1N1 to MDCK cells. In this study, we used three different approaches to assess the inhibitory effects of Artemisia L. extracts on the H1N1 virus. Prevention: Cells were cultured first with an extract, then infected with viral fluid. Direct: The extract solution was directly mixed with the virus to infect cells. Therapeutic: Cells were first infected with viral fluid, then treated with an extract after viral infection (Figure S1). But no study has combined to evaluate the dose-dependent effects of these compounds on N1H1 in this way.

The normal viral replication cycle comprises six stages: adsorption, invasion, uncoating, biosynthesis, assembly, and release [21]. The attachment of viral particles to cells via HA binding to terminal sialic acid residues on glycoproteins is crucial for viral binding, fusion, and entry, thereby initiating the infection cycle [22]. Figure 10 shows that Artemisia L. extracts inhibit virus attachment to the cell surface. Figure 11 shows that some components of Artemisia L. extracts may interact with the HA protein at the target site, thereby blocking the attachment of the influenza virus to cells. Glycyrrhetinic acid was detected by UPLC-MS/MS, and previous studies have demonstrated that glycyrrhetinic acid exhibits inhibitory activity against sialase [23]. In previous studies, pentacyclic triterpenes in Artemisia L. plants have been shown to prevent viral entry into cells [24]. Peroxymethylated pentacyclic triterpenes can also disrupt the interaction between influenza HA and the host receptor, preventing the virus from entering host cells [25].

Extracellular Ca²⁺ influx is crucial for influenza virus entry into host cells via endocytosis. Extracellular Ca²⁺ influx can inhibit the internalization of the influenza virus in host cells; however, it does not affect virus adsorption to the cell surface. Inhibiting extracellular Ca²⁺ influx can effectively prevent viral infection [26]. Figure 12 shows that AALE significantly affects intracellular Ca²⁺ content. In contrast, ACBE has a significant effect on intracellular Ca²⁺ content only at high concentrations, whereas ACTE has no significant effect. Previous studies show that coumarins and flavonoids can reduce intracellular Ca²⁺ levels. Twelve coumarins and flavonoids were detected in ACBE and ACTE, respectively, while twenty-one coumarins and flavonoids were detected in AALE.

Progeny virus particles must be released into the extracellular space, where they infect new host cells after the NA cleaves the connection between sialic acid and HA [27]. Figure 13 shows that ACBE and ACTE can effectively inhibit NA. In addition, UPLC-MS/MS identified chlorogenic acid, and both chlorogenic acid and its protein conjugates showed higher inhibitory activity against NA targets in the late-release phase. A diet containing chlorogenic acid and its derivatives can help prevent influenza A virus infection [28].

Under therapeutic conditions, Artemisia L. extracts regulate intracellular inflammatory factors via the TLR4/NF-κB/MyD88 signaling pathway, reducing the expression of IL-1β, IL-6, TNF-α, TLR4, NF-κB p65, and MyD88 at the mRNA level, thereby inhibiting viral infection. In the innate immune mechanism, pattern recognition receptors (PRRs) and their downstream signaling pathways play a key role in the body’s clearance of viruses and the induction of severe pneumonia. TLR3, a member of the TLR family, recognizes viral double-stranded RNA and regulates downstream signaling through the TRIF pathway. In contrast, TLR7/8 can recognize single-stranded RNA and stimulate the production of numerous inflammatory factors and chemokines through the MyD88-NF-κB pathway. TLR4 is located on the cell membrane and can recognize pathogen-associated molecular patterns (PAMPs), such as bacterial lipopolysaccharides and viral envelope proteins, thereby triggering the recruitment of MyD88 and subsequently activating downstream NF-κB and other inflammatory factors, including IL-6 and TNF-α, through a series of cascades. The results showed that chlorogenic acid, quercetin, luteolin, genistein, baicalin, isoliquiritigenin, diosmin, berberine, montanolide, calycosin, glycyrrhizic acid, parthenolide, and sinomenine can inhibit TLR4 activity.

4. Materials and Methods

4.1. Chemicals and Reagents

Peramivir (P129200), acetonitrile (A298777), and formic acid (F112034) were purchased from Aladdin Biotechnology Co., Ltd. (Shanghai, China); cell culture medium (DMEM), fetal bovine serum (FBS), and 0.25% EDTA-trypsin (25200-056) were purchased from Gibco (Waltham, MA, USA); Trizol (B511311-0100) and CCK-8 (C0038) were purchased from Bio-Time (Xiamen, China). The One Step TB Green PrimeScript RT-PCR Kit II (RR086A) was from Takara (Kusatsu, Japan). The NA kit, intracellular calcium ion content assay kit, and RIPA lysis buffer were purchased from Biotech (Winooski, VT, USA). Abcam (Cambridge, UK) supplied NP, GAPDH, Goat anti-mouse IgG, and Goat anti-rabbit IgG. Proteintech (Wuhan, China) provided NF-κB p65 Polyclonal antibody, MyD88 Polyclonal antibody, and TLR4 Monoclonal antibody.

4.2. Preparation of Artemisia L. Extracts

The Artemisia caruifolia Buch.-Ham. ex Roxb. (Qinghao) used in this study was collected from the Wuling Mountain Area of Chongqing (29.5° N, 118.0° E). The above-ground parts of the plants were harvested during the peak bud stage from July to September. Artemisia capillaris Thunb. (Yinchen) was collected from the Yimeng Mountain area of Shandong (35.5° N, 118.0° E). The above-ground parts of these plants were harvested from March to April. The collected plant samples were identified as belonging to the genus Artemisia L. of the Asteraceae family, named Qinghao (20230713-3), Aiye (202305115-4), and Yinchen (20230329-2). Then, 1 kg of Artemisia L. (Qinghao, Aiye, and Yinchen) was crushed and passed through a 10-mesh sieve to obtain Artemisia L. powder. The powder was placed into a 5 L extraction vessel, 200 mL of 50% alcohol solution was added to the bottom, and supercritical carbon dioxide extraction was performed. The extraction conditions were as follows: pressure of 25 MPa and temperature of 50 °C for 2 h. For separation, extract 1 was obtained in separation kettle 1 (pressure 8 MPa, temperature 60 °C), and extract 2 in separation kettle 2 (pressure 5 MPa, temperature 30 °C), yielding approximately 4%. Next, ethyl acetate extraction was performed on extract 1. Ultrasonic extraction lasted 30 min at a 1:10 (w/v) extract-to-ethyl acetate ratio, followed by suction filtration. The residue on the filter was extracted three more times, and the combined filtrates were concentrated to remove ethyl acetate by rotary evaporation, yielding extracts of Artemisia caruifolia Buch.-Ham. ex Roxb. (ACBE), Artemisia argyi Levl.et Vant. (AALE), and Artemisia capillaris Thunb. (ACTE), which were used in subsequent tests.

4.3. The Components of Artemisia L. Share Targets with the Influenza Virus

Target information for compounds in Qinghao, Aiye, and Yinchen was obtained from the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP, https://tcmsp-e.com/tcmsp.php) and the Swiss Target Prediction Database (http://www.swisstargetprediction.ch/). At the same time, the keyword “influenza virus” was searched in GeneCards (https://www.genecards.org/) and OMIM (https://www.omim.org/) databases to identify disease targets. These targets were then cross-analyzed using the Draw Venn Diagram (https://bioinfogp.cnb.csic.es/tools/venny/index.html). Subsequently, the shared genes were uploaded to the STRING (https://cn.string-db.org/) online software, which was used to obtain protein interaction information, and the data were imported into Cytoscape 3.9.1 to build a protein–protein interaction (PPI) network [29]. DAVID Functional Annotation Tools (https://davidbioinformatics.nih.gov/) conducted bioinformatics analysis on Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Finally, bar charts and bubble charts displayed the top ten enriched items [30].

4.4. Molecular Docking of Therapeutic Targets for the Influenza Virus in Artemisia L.

The PDB format files of core target proteins were obtained from the PubChem database (http://pubchem.ncbi.nlm.nih.gov) and the PDB database (https://www.rcsb.org/). PyMOL viewer software (V3.1) was used to open the structure and remove water molecules and ligands. AutoDockTools (V 4.2.6) was used to perform hydrogen-bond treatment on the target, with the grid box set to cover the entire structure, and Autogrid run for the initial docking. After using the Genetic algorithm for calculation, the AutoDock program was used for the second docking process. A statistical binding energy of ≤−5 kJ/mol indicates successful docking. The selected binding sites were saved as PDBQT files, and then the PyMOL viewer was used for visualization [25].

4.5. Identification of the Active Components of Artemisia L. Extracts by UPLC-MS/MS

Analysis was conducted using Shimadzu Nexera Series and X500 QTOF liquid chromatography-mass spectrometry (Tokyo, Japan). Chromatographic separation was employed on a Dikmatech Leapsil 2.7 μm C18 (2.1 mm × 150 mm) column. In positive-ion mode, the mobile phase consisted of 0.1% formic acid (A) and acetonitrile (B), delivered at 0.3 mL/min. The injection volume was 3 µL, with the column maintained at 40 °C. In negative-ion mode, the mobile phase consisted of a 5 mM aqueous ammonium acetate solution (A) and acetonitrile (B), both at 0.3 mL/min. The injection volume remained 3 µL, with the column temperature held at 40 °C. Gradient elution conditions: 0–5 min, 5% B; 5–18 min, 5–55% B; 18–25 min, 55–80% B; 25–31 min, 80–90% B; 31–38 min, 90–95% B; 38–42 min, 95–5% B. Both modes were scanned using information-dependent acquisition (IDA), with a positive-ion mass range of 80 to 1300 Da. Parameters included the following: spray voltage of 4200 V, collision energy of 30 V, and ion source temperature of 550 °C. For negative ion mode, the spray voltage was 4200 V, with the same collision energy and temperature.

4.6. Cell and Virus Culture

Madin–Darby canine kidney (MDCK) cells and H1N1 (A/Victoria/4897/2022) were acquired from the Shenzhen Center for Disease Control and Prevention (Shenzhen, China). MDCK cells were cultured in a constant temperature incubator at 37 °C with 5% CO₂, while H1N1 was grown at 35 °C with 5% CO₂ for 72 h. The virus was divided into small aliquots and stored at −80 °C until further use.

4.7. Determination of Median Tissue Culture Infective Dose (TCID₅₀) of H1N1

MDCK cells were inoculated into 96-well plates (100 µL per well) and cultured at 37 °C with 5% CO₂ for 24 h. The supernatant from the MDCK cells was discarded, and the cells were washed twice with PBS. The virus was diluted tenfold with the virus maintenance solution, and seven dilution gradients were prepared for analysis. The MDCK cells were then treated with different concentrations of diluted H1N1 virus (100 µL per well). Cells without virus served as the negative control, with four replicates per group. All cells were cultured at 37 °C with 5% CO₂ for 1.5 h. The H1N1 virus suspension was then discarded, and the cells were washed twice with PBS. Next, 150 μL of virus growth medium was added. After three days of incubation, 1% guinea pig blood was used to evaluate the results. The TCID₅₀ and virus titer (MOI) were calculated using the Reed–Muench method [31]. We conducted experiments to explore the range of non-toxic drug concentrations for cells. Subsequent experiments were carried out assuming that the drug concentration does not affect normal cell growth.

$$\mathrm{Distance} \mathrm{Ratio}= {\displaystyle \frac{\mathrm{More} \mathrm{than} 50\% \mathrm{lesion} \mathrm{rate} -50\%}{\mathrm{More} \mathrm{than} 50\% \mathrm{lesion} \mathrm{rate}-\mathrm{Less} \mathrm{than} 50\% \mathrm{lesion} \mathrm{rate}}}$$

$$\mathrm{T}\mathrm{C}\mathrm{I}\mathrm{D}50=\mathrm{l}\mathrm{o}\mathrm{g}\mathrm{a}\mathrm{r}\mathrm{i}\mathrm{t}\mathrm{h}\mathrm{m} \mathrm{o}\mathrm{f} \mathrm{v}\mathrm{i}\mathrm{r}\mathrm{u}\mathrm{s} \mathrm{d}\mathrm{i}\mathrm{l}\mathrm{u}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{w}\mathrm{i}\mathrm{t}\mathrm{h} \mathrm{a} \mathrm{l}\mathrm{e}\mathrm{s}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e} \mathrm{h}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{e}\mathrm{r} \mathrm{t}\mathrm{h}\mathrm{a}\mathrm{n} 50\%+ \mathrm{d}\mathrm{i}\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}$$

$$\mathrm{M}\mathrm{O}\mathrm{I}=0.7\times \mathrm{T}\mathrm{C}\mathrm{I}\mathrm{D}50\times \mathrm{V}\mathrm{i}\mathrm{r}\mathrm{u}\mathrm{s} \mathrm{v}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e}/\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}$$

4.8. Using CCK-8 to Measure Cell Viability

The cytopathic effect (CPE) was observed under a microscope to determine whether it was rounded or demural. CCK-8 was performed when the lesion reached 75% in the viral control group. A total of 10% CCK-8 solution was added to the sample and incubated at 37 °C in the dark for 40 min. The optical density (OD) was measured at 450 nm using a microplate reader. The cell survival rate was calculated [32]. The cell survival rate formula is as follows:

$$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{s}\mathrm{u}\mathrm{r}\mathrm{v}\mathrm{i}\mathrm{v}\mathrm{a}\mathrm{l} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e} (\%)={\displaystyle \frac{\mathrm{O}\mathrm{D} \left(\mathrm{e}\mathrm{x}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}\right)-\mathrm{O}\mathrm{D} \left(\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}\right)}{\mathrm{O}\mathrm{D} \left(\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)-\mathrm{O}\mathrm{D} \left(\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}\right)}}\times 100\%$$

4.9. In Vitro Methods of Action of Artemisia L. Extracts Against H1N1

The Artemisia L. extracts were diluted in a serum-free culture medium, and three different dosing regimens were used to assess their inhibition of H1N1. MDCK cells were cultured in 96-well plates for 24 h. In the prevention mode, the drug solution was added to the wells and incubated for 5 h, after which the MDCK cells were infected with H1N1, washed with PBS, and then cultured in the medium. The prevention phase refers to the application of the extract before virus exposure to reduce viral attachment or replication.

In the direct mode, the virus and drug solution were mixed and incubated at 37 °C for 5 h; the mixture was added to the wells for 1 h to infect the cells, washed with PBS, and then cultured in serum-free DMEM. The direct mode refers to applying the extract during the viral exposure to inactivate the virus. In the treatment mode, H1N1 was incubated with MDCK cells for 1 h, washed with PBS, and then the drug solution was added for continuous culture. The treatment mode refers to applying the extract after the virus has infected the cells. The experiments also included a virus control group (only virus, no drug) and a cell control group (no virus, no drug). The effects were assessed by CPE expression and cell viability [33]. The half cytotoxic concentration (IC₅₀) and half effective concentration (EC₅₀) of the Artemisia L. extracts were calculated, and the selection index (SI) was used as the evaluation index (SI = IC50/EC50) to measure the inhibitory efficacy of the drug against the virus.

$$\mathrm{P}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e} \mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}={\displaystyle \frac{\mathrm{O}\mathrm{D} \left(\mathrm{e}\mathrm{x}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{l}\right)-\mathrm{O}\mathrm{D} \left(\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}\right)}{\mathrm{O}\mathrm{D} \left(\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)-\mathrm{O}\mathrm{D} \left(\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}\right)}}\times 100\%$$

4.10. The Effect of Artemisia L. Extracts on Cells Invaded by H1N1

The virus pretreatment protocol was divided into an attachment step, which is relevant to how it is blocked during host infection. For the attachment assay, cells were infected with a mixture of Artemisia L. extracts and H1N1 at 4 °C for 1 h. This period was defined as the time for the virus to attach to the cell. The drug–disease venom was abandoned, and the PBS was washed twice. The incubation was then performed at 37 °C for 24 h, after which CPE was detected, and cell viability was determined [34].

4.11. The Effect of Artemisia L. Extracts on Intracellular Ca²⁺ Content

A flat-bottom 96-well plate was filled with 15,000 MDCK cells per well and cultured for 24 h. The MDCK cells were incubated with Artemisia L. extracts for 5 h. After 1.5 h of infection with the H1N1 virus, the cells were cultured in the virus proliferation solution, and intracellular Ca²⁺ levels were measured 48 h later. Fluo-4 AM (500×) 0.2 μL was added to each well, along with 0.2 μL of Solubility Enhancer (500×), and 99.6 μL of Assay Buffer. The plates were then incubated at 37 °C for 30 min to evaluate intracellular Ca²⁺ content, and the fluorescence (RFU) was measured using a microplate reader [34]. The excitation wavelength (Ex) was set at 490 nm, and the emission wavelength (Em) was set at 525 nm.

4.12. Determination of Target Gene Expression in MDCK Cells Using Real-Time Fluorescence Quantitative PCR (qPCR)

Total RNA was extracted using Trizol reagent. The One Step TB Green PrimeScript RT-PCR Kit II was employed. The qPCR instrument (Real-time PCR instrument; Manufacturer: Applied Biosystems (Foster City, CA, USA); Model: 7500), and its software (7500 V 2.3) were powered on to configure the reaction program. Stage 1 and 2: Reverse transcription: 42 °C for 5 min, then 95 °C for 10 s; Stage 3: PCR: 95 °C for 5 s, 60 °C for 34 s, for 40 cycles; Stage 4: Dissociation protocol: 95 °C for 15 s, 60 °C for 1 min, then 95 °C for 15 s [35] (

Table 4. Primer details.

Primer Name	Primer Sequences
GAPDH-F	5-GCACCGTCAAGGCTGAGAAC-3
GAPDH-R	5-TGGTGAAGACGCCAGTGGA-3
NP-F	5-TTCATCAGAGGGACAAGAGTGG-3
NP-R	5-TCAGTTCAAGAGTGTTGGAGTC-3
IL-1β-F	5-TGAAGTCACCATAGCTCCAAAAA-3
IL-1β-R	5-GCATGTCGCATCTGTAGCTC-3
IL-6-F	5-TGACCCAACCACAGACGCCAG-3
IL-6-R	5-AGGAATGCCCATGAACTACAGC-3
TNF-α-F	5-CGAACCCCAAGTGACAAGCC-3
TNF-α-R	5-TCTGTCAGCTCCACGCCGTTG-3
p65 NF-κB-F	5-GCACAGACACCACCAAGACCCAC-3
p65 NF-κB-R	5-CGGCAGTCTTTCCCCACAAGCTC-3
MyD88-F	3-CCTGAGCGTTTTGATGCCTT-3
MyD88-R	5-ACTTCAGCCGATAGTTTGTCT-3
TLR4-F	5-TGCCAGAATGATGTCTCCTACCC-3
TLR4-R	5-CTCAGGTCCAGTTTCTCGGTT-3

).

4.13. Western Blot Analysis of Related Protein Levels in MDCK Cells

Cells were lysed with RIPA lysis buffer to obtain a total protein lysate. Protein samples were separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked in blocking solution for 10 min, incubated with the primary antibody at 4 °C overnight, and then incubated with the secondary antibody at room temperature for 1 h. A developer was added and observed under a developer [35].

4.14. Immunofluorescence Was Used to Investigate the Expression of the NP Protein in Different Treatment Groups of MDCK Cells

Fix with 4% paraformaldehyde at room temperature for 15–20 min, then wash twice with PBS. Permeabilize the membrane with 0.3% Triton solution, let stand at room temperature for 10 min, and wash twice with PBS. Fix with 5% BSA solution at room temperature for 1 h, then wash twice with PBS. Add the primary antibody (anti-NP mouse monoclonal antibody diluted 1:200 in 5% BSA solution) and incubate at 4 °C overnight. Add secondary antibody (Alexa Fluor 488-labeled goat anti-mouse IgG) and incubate at room temperature for 1 h in the dark. Add DAPI and incubate at room temperature in the dark for 5 min. Observe with a fluorescent inverted microscope and take pictures to record the results [34].

4.15. The Effect of Artemisia L. Extracts on Hemagglutinin

Take a 96-well hemagglutination plate, dilute the virus with a stock titer of 1:32 using a two-fold series, and add 25 μL of the virus solution and 25 μL of PBS to each well. Set up a drug control group by adding 25 μL of the virus solution and 25 μL of the drug to each well. Then, add 50 μL of prepared 1% guinea pig blood to each well, mix well, and incubate at 37 °C for 30 min. Observe the red blood cell agglutination. Select the next virus dilution and multiply according to the hemagglutination test results.

To assess the effect of Artemisia L. extracts on H1N1 adsorption to host cells, the drug solution was diluted twofold from 12.0 mg/mL to create 8 concentrations (25 μL/well) with PBS. Then, 25 μL of virus solution was added to each well, along with a virus control group (25 μL virus solution + 25 μL PBS) and a blank control group (50 μL PBS). Three replicate wells were prepared, and 50 μL of 1% guinea pig red blood cells was added to each well. The plates were incubated at 37 °C for 15 min, and hemagglutination was observed.

4.16. The Effect of Artemisia L. Extracts on Neuraminidase

NA was measured using a neuraminidase (NA) assay kit. The concentrations of the 70 μL reaction buffer, 10 μL NA, 10 μL NA substrate, and 10 μL inhibitor were 3 mg/mL, 1 mg/mL, and 0.33 mg/mL, respectively, containing Artemisia L. extracts. Peramivir at 5 μg/mL served as the positive control. Then, the mixture was incubated at 37 °C for 30 min, and its RFU was measured with Ex/Em = 322/450 nm [34]. The inhibition rate of NA was calculated using the following formula.

$$\mathrm{D}\mathrm{r}\mathrm{u}\mathrm{g} \mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{N}\mathrm{A} \left(\%\right)={\displaystyle \frac{\mathrm{f}\mathrm{l}\mathrm{u}\mathrm{o}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{p}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l} \mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}-\mathrm{f}\mathrm{l}\mathrm{u}\mathrm{o}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g} \mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}}{\mathrm{f}\mathrm{l}\mathrm{u}\mathrm{o}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{p}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l} \mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}}}\times 100$$

4.17. Statistical Analysis

All results from this study are presented as the mean ± standard deviation (SD) of three independent experiments. The 2^(−ΔΔCt) method was used to analyze the viral gene mRNA level. GraphPad Prism 8.0 software was utilized for statistical analysis. The grayscale values of Western blot bands were analyzed using ImageJ (V 1.54r). A p-value of less than 0.05 was considered statistically significant.

5. Conclusions

This study employed network pharmacology and molecular docking technology to investigate the key components, core targets, and signaling pathways of Artemisia L. extracts in the treatment of influenza virus, providing a theoretical basis and reference value for further research on the therapeutic mechanism of the influenza virus. Artemisia L. extracts can exert an inhibitory effect on H1N1 from two perspectives: early prevention and later treatment. ACBE inhibits H1N1 infection mainly by affecting the intracellular Ca²⁺ level and the release of progeny viruses in MDCK cells; AALE has no inhibitory effect on NA; ACTE has no regulatory effect on intracellular Ca²⁺. These results show that Artemisia L. extracts can inhibit H1N1 and provide a scientific basis for further development of natural product-based anti-influenza strategies.