Based on Network Pharmacology and Molecular Mechanism of Action of Ganlu Yin in the Treatment of COVID-19

Luotong JING Yingcai WEI Jiao HUANG Ning LIANG

Abstract ［Objectives］This study was conducted to explore the mechanism of Ganlu Yin in the treatment of COVID-19 based on network pharmacology and molecular docking technology.

［Methods］ TCMSP, ETCM and TCMID databases were used to dig out the chemical components in the compound ingredients of Ganlu Yin. Swiss ADME platform was used for ADME screening and Swiss Target Prediction server was used to predict potential targets of chemical components. The COVID-19 related genes were obtained from GeneCards database and Drugbank website, and the intersection targets between Ganlu Yin and COVID-19 were obtained by Venn analysis. The targets were imported into String analysis platform to construct a protein interaction network, and Cytoscape 3.9.0 software was used for visualization processing. GO enrichment and KEGG pathway analysis were performed on the intersection targets through David database. The core components, clinical antiviral drugs, core targets and novel Coronavirus (SARS-COV-2) hydrolase 3CLpro (Mpro), omicron B.1.1.529 spike protein and angiotensin converting enzyme 2(ACE2) were tested by molecular docking.

［Results］ A total of 213 effective chemical components and 1 023 drug targets in Ganlu Yin were screened out, including 4 620 COVID-19 related genes and 328 intersection targets. The core components mainly included β-sitosterol, quercetin, and kaherol. The key core targets involved GAPDH, AKT1, TNF, ALB, and EGFR, etc. A total of 241 items were screened by GO functional enrichment, including inflammatory response, protein phosphorylation, positive regulation of gene expression and other biological processes. A total of 85 KEGG pathways were screened out, which were mainly enriched in influenza A, HIF-1 signaling pathway, hepatitis C and other pathways. The results of molecular docking showed that the core chemical components in Ganlu Yin had good binding ability with core targets and disease targets ACE2, 3CLpro and spike protein, especially kahenol with ACE2, β-sitosterol with 3CLpro, and quercetin with spike protein.

［Conclusions］ Prescription Ganlu Yin has the characteristics of multi-component and multi-target action, and can treat COVID-19 through multiple signaling pathways.

Key words COVID-19; Ganlu Yin; Network pharmacology; Molecular docking; Omicron

Received: June 23, 2022  Accepted: August 27, 2022

Luotong JING (1997-), female, P. R. China, master candidate, devoted to research about pharmacodynamic evaluation and application research of traditional Chinese medicine.

*Corresponding author. Ning LIANG (1974-), female, P. R. China, master’s supervisor, devoted to research about  new immunomodulatory drugs of traditional Chinese medicine.  E-mail: molycn@163.com.

Ganlu Yin comes from Taipinghuiminhejiju Fang·Volume VI［1］. It is composed of Rehmanniae Radix, Rehmanniae Radix Praeparata, Radix Ophiopogonis, Asparagi Radix, Dendrobii Caulis, Herba Artemisiae Scopariae, Radix Scutellariae, Fructus Aurantii, Folium Eriobotryae, and Radix Rhizoma Glycyrrhizae. The complementary and synergistic effects of various components in Ganlu Yin is an important basis for its pharmacological action of nourishing yin and promoting the secretion of saliva, clearing away heat and toxic materials, removing dampness and regulating the middle warmer［2］. At present, it is widely used in keratoconjunctiva xerosis, yin deficiency and damp-heat due to diabetic nephropathy, periodontitis, herpetic angina, etc.［3-6］. Traditional Chinese medicine believes that most viral pneumonias are exogenous fever caused by pathogenic cold. Syndrome differentiation and treatment according to the pathogenesis is the main method of traditional Chinese medicine in the treatment of new coronary pneumonia at this stage, that is, the holistic approach to clear away heat is an important means. Studies have shown that Ganlu Yin is effective in treating patients with asymptomatic infection of 2019-nCoV and has no obvious adverse reactions［7-8］. Traditional Chinese medicine compounds have the characteristics of multi-component, multi-target and multi-pathway. An effective method to study the mechanism of action of compound prescriptions is network pharmacology, and the active ingredients, potential targets and mechanisms of action of the classic prescription Ganlu Yin on COVID-19 can be analyzed based on network pharmacology.

The novel coronavirus pneumonia (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is highly contagious and has had a serious impact on the lives of people all over the world and on global public health. According to research reports［9-10］, the new coronavirus (SARS-CoV-2) is an enveloped single-stranded RNA beta coronavirus that can  enters host cells through the binding between the viral structural spike (S) protein and the angiotensin-converting enzyme 2 (ACE2) receptor and causes the disease. The major coronavirus hydrolase 3CLpro (Mpro) plays a crucial role in the life cycle of the virus. After the SARS-CoV-2 virus enters host cells, the positive-strand RNA genome is translated, and processed by the 3CLpro virus protease into a protein, which forms an active polymerase complex after correct folding and assembling. Therefore, preventing 3CLpro from functioning when viral transcription or replication occurs can effectively shut down the life cycle of the virus［11］. Omicron［12］, which is currently infecting globally, is an emerging SARS-CoV-2 variant with stronger immune evasion ability than other identified mutants, and WHO defines it as the fifth VOC (virus of concern)［13］. The Omicron spike protein has more mutations than other variants. Through the analysis of the gene sequence of the Omicron variant, it was found that there are about 50 nucleotide mutation sites in the Omicron variant, of which more than 30 mutation sites are located on the spike protein of the virus. The spike protein is the key for the virus to infect human cells, and mutation can increase the binding ability of the virus to human cells, thereby increasing the spread of the virus. It may also cause more immune evasion, resulting in secondary infections and decreased vaccine effectiveness［14］. China has adopted conventional treatment combined with traditional Chinese medicine for COVID-19 patients, which have proved that traditional Chinese medicine has a satisfactory effect in improving clinical symptoms, delaying disease progression, and reducing mortality and recurrence rate, and relevant active components of traditional Chinese medicine are potential candidates for development［15-16］. Traditional Chinese medicine treatment of new coronary pneumonia is still an important choice for all mankind to fight the epidemic.

In this study, the chemical components in Ganlu Yin compound ingredients were explored by network pharmacology to screen potential targets of chemical components. The intersection targets of Ganlu Yin and COVID-19 were imported into the String analysis platform to construct a protein interaction network, and the intersection targets were subjected to GO function enrichment and KEGG pathway analysis through the David database, and the key active components were docked molecularly to core targets, Mpro, ACE2, and the Omicron B.1.1.529 spike protein. The molecular docking method was used to verify the correlation between Ganlu Yin and the new coronavirus, aiming to provide a theoretical reference for the treatment of COVID-19 with traditional Chinese medicine Ganlu Yin.

Materials and Methods

Materials

Databases and software involved in this study: Traditional Chinese Medicine System Pharmacology Database and Analysis Platform TCMSP database (TCMSP, http://ibts.hkbu.edu.hk/LSP/tcmsp.php); TCMID database (TCMID, http://www .megabionet.org/tcmid/); ETCM database (ETCM, http://www.tcmip.cn/ETCM/index.php/Home/Index/index.html); Venny2.1 server (http://bioinfogp. cnb.csic.es/tools/venny/index.html); Pubchem Organic Small Molecule Bioactivity Database (https://pubchem.ncbi.nlm.nih.gov/); Swiss ADME (www.SwissADME.ch); Swiss Target Prediction server (http://new.swisstargetprediction.ch/); GeneCards database (https://www.genecards.org/); Drugbank (https://go.Drugbank.com/); DAVID database (https: //david.ncifcrf.gov/); STRING database (https://string-db.org/); PDB database (https://www.rcsb.org/); Cytoscape 3.9.0 software; Bioinformatics website (http://www.bioinformatics.com.cn/); AutoDockTools 1.5.6 Software; Pymol software.

Collection of active chemical components of Ganlu Yin

The chemical components of Rehmanniae Radix Praeparata, Asparagi Radix, Herba Artemisiae Scopariae, Radix Scutellariae, Fructus Aurantii, Folium Eriobotryae, and Radix Rhizoma Glycyrrhizae in Ganlu Yin compound were searched using the TCMSP database with OB (oral bioavailability)≥30 and DL (drug-like properties)≥ 0.18  as the screening criteria. The chemical components of Radix Ophiopogonis and Dendrobii Caulis were retrieved from the ETCM platform, and the chemical components of Rehmanniae Radix, which were not included in the above platforms, were searched from the TCMID database. The chemical composition information, compound structures and CAS numbers of Radix Ophiopogonis, Dendrobii Caulis and Rehmanniae Radix were supplemented and screened through the chemical professional database and Huayuan website; Pubchem database was used to determine their component structure and SMILES numbers; and Swiss ADME (www.SwissADME.ch) was used for drug activity screening［17］. Taking the compounds conforming to two or more drug-like principles of Lipinski, Ghose, Veber, Egan and Muegge and the gastrointestinal absorption (GI absorption) as "High" as the ADME inclusion criteria, the active chemical components of Radix Ophiopogonis, Dendrobii Caulis and Rehmanniae Radix with good drug-forming potential and good oral bioavailability were screened out.

Prediction of targets of active components in Ganlu Yin

The targets of the active components screened from ten traditional Chinese medicines in Ganlu Yin were predicted using Swiss Target Prediction (www.SwissTargetPrediction.ch). The targets of each active ingredient with a "probability" value greater than 0 were selected, and summarized and integrated into an EXCEL file. The above-obtained active components and targets were imported into Cytoscape 3.9.0 software to construct a network map of "Ganlu Yin-active components-targets".

Acquisition of COVID-19 targets

COVID-19-related targets were collected using the GeneCards database (https://www.genecards.org/) and Drugbank (https://go.Drugbank.com/), with "COVID-19" as the keyword.

Identification of potential targets of action

The Venny2.1 server (http://bioinfogp.cnb.csic.es/tools/venny/index.html) was used to perform Venn analysis on the main chemical components in Ganlu Yin and the COVID-19 target data set, to obtain a common target set of Ganlu Yin. The intersection targets of medicines and the disease were shown in the form of Venn diagrams to initially explore the targets of Ganlu Yin in the treatment of the disease.

Construction of intersection target PPI network and screening of key targets

The intersection genes were uploaded to the STRING database (https://string-db.org/) for protein interaction (PPI) analysis with the species selected as "Homosa-piens". Episomal genes were eliminated, and the intersecting genes were screened with the lowest interaction score > 0.4 to obtain key genes, which were imported into the Cytoscape software to construct a visual network diagram. The cytoHubba plug-in was used to screen the core genes of Ganlu Yin in the treatment of new coronary pneumonia.

Network analysis of GO enrichment and KEGG pathway enrichment

The intersection targets were imported into the DAVID database (https://david.ncifcrf.gov/) for gene function GO enrichment and KEGG pathway enrichment analysis based on the Kyoto Encyclopedia of Genes and Genomes. The GO analysis results were drawn as histograms and the KEGG pathway enrichment results were drawn as bubble heat maps using the Bioinformatics online website.

Molecular docking

The molecular structures of Mpro (PDB ID: 6LU7), omicron B.1.1.529 spike protein (PDB ID: 7T9J), ACE2 (PDB ID: 1R42) and 5 key targets were downloaded from the PDB database (https://www.rcsb.org/), and the protein receptors were pretreated by dehydration and ligand removal using Pymol software. The 2D structures of key active components in Ganlu Yin were downloaded from the Pubchem database (https://pubchem.ncbi.nlm.nih.gov/) as small molecule ligand structures, which were optimized into 3D structures using ChemBioOffice software and saved in mol2 format. The receptors and ligands were hydrogenated by the software AutoDockTools 1.5.6, and the molecules and proteins were docked by the software Autodock Vina 1.1.2. Molecular docking visualization analysis was performed using Pymol software.

Results

Active components of Ganlu Yin

After searching and screening in TCMSP, ETCM and TCMID databases, a total of 246 chemical components were obtained. The SMILES numbers of the chemical components were imported into the Swiss Target Prediction (www.SwissTargetPrediction.ch) to predict the targets using the Pubchem database, and 23 chemical components predicted with no targets were eliminated, and finally 213 active compounds in the Ganlu Yin prescription were obtained, including 86 of Radix Rhizoma Glycyrrhizae, 33 of Radix Scutellariae, 14 of Folium Eriobotryae, 8 of Asparagi Radix, 4 of Fructus Aurantii, 13 of Herba Artemisiae Scopariae, 16 of Rehmanniae Radix, 2 of Rehmanniae Radix Praeparata, 25 of Radix Ophiopogonis, and 22 of Dendrobii Caulis. Among them, nine compounds were shared by multiple ingredients in the prescription (Table 1).

Prediction of Ganlu Yin targets

The targets of chemical components in Ganlu Yin were predicted using Swiss Target Prediction (www.SwissTargetPrediction.ch), and a total of 1 023 drug target genes were obtained after sorting and deduplication. The 213 active compounds and gene targets were imported into Cytoscape software to construct a visual map of "Ganlu Yin-active component-target" (Fig. 1). The network consists of 1 237 nodes and 14 470 edges, and the yellow nodes are the compound ingredients in Ganlu Yin: Rehmanniae Radix Praeparata, Asparagi Radix, Herba Artemisiae Scopariae, Radix Scutellariae, Fructus Aurantii, Folium Eriobotryae, Radix Rhizoma Glycyrrhizae, Rehmanniae Radix, Radix Ophiopogonis, and Dendrobii Caulis. The circular nodes represent the chemical substances of traditional Chinese medicine ingredients, and the numbers on the nodes are the PubChem CIDs of corresponding compounds. The light yellow rectangles are the predicted target genes in Ganlu Yin. Among the chemical components, quercetin (A2), isorhamnetin (A4), naringenin (A7), β-sitosterol (A1), 7-methoxy-2-methyl isoflavone (A6), and kaempferol (A9) had higher degree values.

Identification of COVID-19 targets and potential targets

In the GeneCards database, 4 600 disease targets were retrieved with the keyword "COVID-19". Twenty five disease-related targets were retrieved from the Drugbank website. After integration and deduplication, a total of 4 620 disease targets were obtained. When using the Venny2.1 server to intersect the targets predicted by Compound Ganlu Yin with the targets of COVID-19, 328 common potential targets of Ganlu Yin and COVID-19 were obtained (Fig. 2).

The ingredients of Ganlu Yin and the intersection targets were imported into the Cytoscape software to construct a "Ganlu Yin-potential target-COVID-19" interaction network (Fig. 3). There are 349 nodes and 5 339 edges in the network. In the figure, the red rectangles represent the traditional Chinese medicine compound and the disease; the orange circles represent ten traditional Chinese medicines in Ganlu Yin; the blue triangles are nine common compounds in the compound; and the green diamonds represent the 328 potential targets shared by Ganlu Yin and COVID-19.

Construction of protein-protein interaction (PPI) network of potential targets

The 328 intersection targets were imported into the String database to obtain potential target-protein interaction relationship, and TSV data files were exported. The Cytoscape software was employed to draw a PPI visualization network graph (Fig. 4), which consists of 326 nodes and 4 953 edges. The CytoHubba plug-in was used to arrange the nodes according to degree values of the nodes. The color of the nodes in the figure changed from dark red to light pink, and the darker the color, the greater the node degree.

The top 10 gene targets in the PPI network graph are GAPDH, AKT1, TNF, ALB, EGFR, HSP90AA1, VEGFA, SRC, IL1B, and STAT3 (Fig. 4) according to their degree values. The degree values of these nodes were all greater than 120 (Table 2), indicating that they played a key role in the entire network and might be core targets of Ganlu Yin in the treatment of new coronary pneumonia. The GAPDH gene could be predicted from coniferin in Rehmanniae Radix. AKT1 was mainly from compounds such as quercetin, isorhamnetin, 7-methoxy-2-methyl isoflavone, and kaempferol. TNF was mainly predicted from compounds in Radix Rhizoma Glycyrrhizae such as iso-licoflavonol.

GO enrichment analysis

Through the analysis of the 328 intersection gene targets in the Metascape database, a total of 241 GO functional items were obtained (P<0.01, FDR<0.01), including 152 biological processes (BPs), 45 cellular components (CCs), and 44 molecular functions (MFs). The enrichment results were sorted from small to large, and the top ten items were selected to draw a histogram (Fig. 5). As shown in the figure, BPs mainly involved protein phosphorylation, response to drug, peptidyl-tyrosine phosphorylation, inflammatory response, and positive regulation of gene expression and other processes. CCs mainly concentrated on plasma membrane, cytoplasm, and cell surface. MFs mainly involved ATP binding, enzyme binding, protein kinase activity, and protein serine/threonine kinase activity.

KEGG pathway enrichment analysis

The 328 core targets were subjected to KEGG pathway enrichment. A total of 85 pathways were screened (P<0.01, FDR<0.01), and 20 pathways with smaller P values were selected for visualization (Fig. 6). The results showed that the core genes were mainly enriched in Influenza A, Hepatitis C, PI3K-AKT signaling pathway, HIF-1 signaling pathway, Hepatitis B, Viral carcinogenesis and other pathways. The KEGG pathway enrichment results showed that Ganlu Yin could play a role in the treatment of COVID-19 by regulating the coordination of multiple biological processes and pathways.

Molecular docking results of core compounds in Ganlu Yin

The clinical drug remdesivir is a broad-spectrum antiviral nucleotide prodrug［18］, which is used to treat patients with new coronary pneumonia. Arbidol is used in the treatment of COVID-19 with a rate as high as 98%［19］, which is the most commonly used antiviral drug in clinic. The core components in Ganlu Yin with degree values ranking the top six and clinical drugs (remdesivir, arbidol) were molecularly docked to the core targets with degree values ranking the top five, as well as Mpro, omicron B.1.1.529 spike protein, and ACE2, and the results are shown in Table 3.

Smaller binding energy during molecular docking suggests greater affinity between protein receptors and small ligand molecules and more stable binding conformation. If the binding energy is less than 0, it indicates that the two can spontaneously bind; and the binding energy ≤-5.0 kcal/mol indicates that the two have good binding ability, and the binding energy≤-7.0 kcal/mol indicates that they have strong binding ability［20］. The molecular docking results showed that all the core chemical components in Ganlu Yin and the clinical drugs all docked to the targets with ability less than -5.0 kcal/mol, indicating that they had good binding ability with receptors.

The docking results of the compounds in Ganlu Yin and clinical drugs with the core targets showed that remdesivir (Fig.7-A) had the lowest binding energy to GAPDH. The schematic diagram of molecular docking showed that remdesivir had multiple hydrogen bonds with GAPDH, so the binding was more stable. The docking energy of quercetin with AKT1 molecules was lower than those of other compounds, indicating that the binding of AKT1 to quercetin (Fig. 7-B) was easier than that with other compounds. Analyzing from the binding energy of molecular docking, it could be seen that the affinity of TNF with the compounds and clinical drugs was weaker than that with other core targets, but the binding energies were still all ≤-5.0 kcal/mol, and the binding energy of TNF and quercetin (Fig. 7-C) was the lowest at -7.0 kcal/mol, suggesting that TNF and quercetin had strong binding ability.

The absolute values of the binding energies of the core compounds in Ganlu Yin and the clinical drugs with ACE2 were generally higher than the binding energies with 3CLpro, suggesting that the core compounds could bind better with ACE2 and the structures were more stable. β-Sitosterol, quercetin, and kaempferol (Fig. 8-A) all had greater binding energies to ACE2 than arbidol and remdesivir. Except for 7-methoxy-2-methyl isoflavone, the binding energies of other compounds to 3CLpro were greater than that of remdesivir. Among them, the binding energy of β-sitosterol (Fig. 8-B) to 3CLpro was -7.2 kcal/mol, which was lower than other compounds, so their binding performance was better. The binding energies of the compounds and clinical drugs to the Omicron B.1.1.529 spike protein were all ≤-7.0 kcal/mol, indicating that the ligand molecules had a strong binding ability to the receptor proteins. The binding energy of quercetin (Fig. 8-C) to the spike protein of Omicron B.1.1.529 was lower than that of remdesivir (Fig. 8-D) to the spike protein receptor molecule, suggesting that quercetin had a stronger affinity with the receptor protein and the conformation was more stable.

Discussion

The 2019 novel coronavirus pneumonia (COVID-19) rapidly swept across the globe, severely impacting public health systems and causing social and economic crises, making it one of the most devastating outbreaks in human history. In a global pandemic environment, many countries continue to endure the detrimental effects of multiple outbreaks of this viral disease. In the past two years, a large number of mutations have led to the evolution of the virus, so SARS-CoV-2 has produced different variant strains［21］. There are five "variants of concern" (VOC) defined by WHO (Alpha, Beta, Gamma, Delta, Omicron). Among them, the Omicron variant exhibits more than 30 amino acid mutations in the spike protein, which can enhance transmissibility and immune evasion. The emergence of SARS-CoV-2 variant strains exacerbates the spread of the disease and increases the difficulty of epidemic prevention and control.

Through network pharmacology, it was found that there were 213 active components and 1 023 drug targets in Ganlu Yin. The top six compounds according to the interaction degree values were: quercetin, isorhamnetin, naringenin, β-sitosterol, 7-methoxy-2-methyl isoflavone, and kaempferol. Except for β-sitosterol, the rest belong to flavonoids. Flavonoids have biological effects such as anti-inflammatory, antiviral, antioxidant, and analgesic effects［22］, and can exert immunomodulatory effects through NF-κB, Wnt/β-catenin, and Toll-like receptor signaling pathways. They are natural immune modulators. Quercetin has anti-inflammatory, anti-cancer, and antioxidant effects. Xu et al.［23］ found that quercetin promoted LPS-induced chondrocyte proliferation and inhibited inflammation by regulating autophagy. Kaempferol has antioxidant, anti-anxiety, anti-inflammatory, anti-allergic and anti-apoptotic effects. Kaempferol can reduce the mRNA expression of IL-6, TNF-α and IL-β in the CFA model mice and alleviate the inflammatory pain in the model mice, and has a significant anti-inflammatory and analgesic effect［24］. β-Sitosterol is a phytosterol, widely present in nuts and vegetable oils, and is an important compound in many traditional Chinese medicines. It can exert anti-apoptotic, antibacterial, anti-inflammatory, and anti-atherosclerotic effects through multiple pathways［25］. Zhao et al.［26］ found that β-sitosterol could affect the proliferation and apoptosis of granulosa cells through the PI3K/AKT signaling pathway.

In this study, a protein interaction PPI network for the intersection targets was constructed by string database analysis and Cytoscape software visualization processing, and it was found that the core targets of Ganlu Yin in the treatment of COVID-19 mainly included GAPDH, AKT1, TNF, ALB, EGFR, HSP90AA1, VEGFA, SRC, IL1B, and STAT3. Among them, tumor necrosis factor (TNF) and interleukin 1B (IL1B) are all factors related to inflammation. TNF can also be used as an important predictor for judging the severity of COVID-19［27］. According to existing reports, SARS-CoV-2 can manipulate the host defense system, resulting in the overexpression of pro-inflammatory factors IL-6, IL1B, and TNF［28］, which further leads to the generation of cytokine storm phenomenon. The main central mediator of the cytokine storm phenomenon may be TNF［29-30］, which is closely related to the function of tumor necrosis factor in blood coagulation, insulin resistance, heart failure and other related diseases. In fact, in severe cases of COVID-19, most patients with new coronary pneumonia die from acute respiratory distress syndrome (ARDS), heart failure, renal failure, severe multiple organ dysfunction, and uncontrolled immune function. These uncontrolled immune expression and the outbreak of pro-inflammatory genes lead to the deterioration of the condition of the illness, and usually, immunosuppressive drugs can be used for reducing the occurrence of cytokine storm in the treatment of severe acute respiratory failure in COVID-19, with an obvious therapeutic effect［31］. Protein kinase (AKT1) is a key factor regulating cell growth, metabolism, autophagy and other functions and controlling the PI3K/Akt1 signaling pathway cascade. It has been reported［32］ that cells infected with SARS-CoV-2 show the activation of autophagy inhibitors (AKT1, SKP2) and the reduction of phagocytic cell formation (BECN1), and SARS-CoV-2 leads to the down-regulation of cell autophagy, thereby enabling virus transmission. Effective compounds can be used in virus treatment to induce autophagy and limit viruses’ ability to spread.

Through the GO function and KEGG pathway enrichment analysis, it was found that the treatment of new coronary pneumonia with Ganlu Yin mainly involved biological functions such as inflammatory response, protein phosphorylation, and positive regulation of gene expression. The 85 pathways screened by KEGG pathway enrichment included influenza A, hepatitis C, hepatitis B and other pathways related to this outbreak. Secondly, Ganlu Yin also played a role through immune and inflammation-related signaling pathways such as tumor necrosis factor signaling pathway, cell apoptosis, Toll-like receptor signaling pathway, and T cell receptor signaling pathway, in reducing the occurrence of cytokine storm, and regulating the immune system and improving inflammation symptoms.

Angiotensin-converting enzyme 2 (ACE2), a transmembrane glycoprotein, is an important component of the renin-angiotensin system (RAS), and ACE2 has been identified as the SARS-CoV-2 receptor that mediates viral entry into host cells, endangering human health［33］.  The coronavirus hydrolase 3CLpro is the core protease for the hydrolysis of single-stranded RNA viral precursor polyprotein and plays an important role in the viral replication process. Less than a month after the discovery of the Omicron variant, it was on the rise in countries such as South Africa, the United Kingdom, and Denmark［34］. It was found that the omicron B.1.1.529 spike protein has a higher affinity to the human ACE2 receptor and proliferates approximately 70 times faster than the delta variant in human airway tissue［35］. Its spike protein has about 32 mutations, which is associated with antibody binding sites, and also means that it can evade hosts’ immunity and the immunity conferred by vaccination, and its transmission is more insidious. Through molecular docking, it was found that the compounds in Ganlu Yin showed strong binding ability to the core targets and disease-acting protein targets, and the binding energies of its core components to ACE2, 3CLpro, and the Omicron B.1.1.529 spike protein were all <-5.0 kcal/mol, indicating that the ligand molecules had good binding ability to receptor proteins. Among them, quercetin, naringenin, and kaempferol showed the binding ability to the Omicron B.1.1.529 spike protein higher than that of arbidol and remdesivir, suggesting that these three compounds had good binding performance to receptor proteins, and the binding conformation was more stable.

In summary, the potential mechanism of Ganlu Yin in the treatment of new coronary pneumonia (COVID-19) based on network pharmacology and molecular docking technology was studied, suggesting that Ganlu Yin exerts its effects on new coronary pneumonia through multiple components, multiple targets, and multiple pathways, which also provides a theoretical reference for the traditional Chinese medicine treatment of COVID-19. However, there are certain limitations in the study. In the future, relevant pharmacological research and clinical experiments can be carried out, so as to more effectively guide the clinical use of Ganlu Yin to prevent and treat new coronary pneumonia.

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