Exploring the role of Xingren on COVID‐19 based on network pharmacology and molecular docking

Abstract Since the outbreak of novel Coronavirus Pneumonia 2019 (COVID‐19), the role of Almonds (Xingren) in the protection and treatment of COVID‐19 is not clear. Network pharmacology and molecular docking were used to explore the potential mechanism and potential key targets of Xingren on COVID‐19. A total of nine common targets between them were obtained, and these targets were involved in multiple related processes of GO and KEGG pathway enrichment analysis. Molecular docking showed that licochalcone B has the best binding energy (−9.33 kJ·mol−1) to PTGS2. They are maybe the important ingredient and key potential target. Its possible mechanism is to intervene anxiety disorder in the process of disease development, such as regulation of blood pressure, reactive oxygen species metabolic process, leishmaniasis peroxisome, and IL‐17 signaling pathway. Practical applications Xingren is a traditional Chinese medicine that has been used and developed in China for many years. It contains a variety of active ingredients and also has the functions of relieving cough, relieving asthma, enhancing human immunity, delaying aging, regulating blood lipids, nourishing brain, and improving intelligence. In this article, the possible mechanisms of action and important targets of Xingren in the prevention and treatment of COVID‐19 were discussed through network pharmacology and molecular docking. We also found that active ingredient licochalcone B and the potential target PTGS2 are worthy of further research and analysis. At the same time, the study also provides a theoretical basis and reference for the prevention and treatment of COVID‐19 and the development of new drugs.

relatively closed environments, the incubation period of the virus is 1-14 days, and patients can be of all ages, and the main clinical manifestations are fever, dry cough, and malaise (Kang et al., 2022;Ni et al., 2020). Since China has a long history of Chinese medicine research and rich experience in epidemic prevention and control, Chinese medicine will play a great role in the fight against epidemics in the absence of specific drugs and vaccines. Currently, the National Health Commission of the People's Republic of China has published the "New Coronavirus Pneumonia Treatment Protocol," which also includes Chinese medicine, and continues from the "Third Edition" to the "Ninth Edition," and has certain efficacy in patients with mild, normal or severe COVID-19 infection (Ding & Bian, 2020;Fang et al., 2022;Lin & Li, 2020). According to their researches, it was found that among the various ingredient preparations of traditional Chinese medicine, Almond (Xingren) is used most frequently in the therapeutic period and can be used in combination with some Chinese medicines for prescriptions (Yue et al., 2020;Zhou et al., 2020). In the treatment of COVID-19, Xingren is also the most frequently used Chinese medicine in combination, and it is not clear whether it is used as a "ruler" medicine.
Xingren is the seeds of the deciduous tree plant apricot (Prunus armeniaca L.) or bilberry, family Rosaceae, it has the effect of relieving cough and asthma and laxative and is useful for the respiratory system, digestive system, inflammatory reaction, and tumor treatment (Zheng & Shen, 2020). There are few studies on this drug in China and abroad, none of which involve studies on COVID-19 treatment. In view of the new findings and researches for Xingren on COVID-19, Xingren has an effect on COVID-19, but its mechanism and core active ingredients on it are still unclear (Yue et al., 2020;Zeng et al., 2020;Zhou et al., 2020). Consequently, our team used the current emerging network pharmacology and molecular docking to investigate the material basis and potential mechanism of action of Xingren in interfering with COVID-19 through a comprehensive analysis of the effective active ingredients, action targets and related pathways of Xingren by biological systems (Yu et al., 2022).
Meanwhile, to provide a certain theoretical basis and reference for the screening of active ingredients, prevention and treatment of COVID-19 and new drug research of Xingren by preliminary validation of molecular docking methods. The study flow is shown in

| Screening of active ingredients and action targets of Xingren
The TCMSP platform (http://tcmspw.com/tcmsp.php) was used to search for "Xingren" as the keyword, and the retrieved ingredients were screened for active ingredients with oral bioavailability (OB) > 30% and drug-likeness (DL) > 0.18, and finally the ingredient s that met the criteria were The active ingredients were finally obtained. The active ingredients obtained from the screening were predicted using Related Targets in the TCMSP platform for the active ingredient-related targets.

| Collection of disease targets
The GeneCards (https://www.genec ards.org/) and OMIM (https:// omim.org/) platforms was searched by the keyword "novel coronavirus pneumonia", and the relevant targets of COVID-19 were obtained. To exclude targets with low relevance to COVID-19, we scored according to the relevant scores of GeneCards, and we included targets with a score of ≥2. After that, we merged the two databases and deduplicated to obtain the relevant targets of COVID-19. By taking the intersection of Xingren active ingredientrelated targets and COVID-19-related targets, the target that was re-summed was the common action target of Xingren active ingredient and COVID-19.

| Construction of protein interaction network and active ingredient-disease-target network
To explore the interaction between target proteins, the common target of Xingren and COVID-19 was uploaded to STRING (https://strin g-db.org/) platform, and the species screening was set to "Homo sapiens," and no combined score screening was performed to obtain the protein interaction PPI network. The related targets' information of Xingren and COVID-19 was imported into Cytoscape 3.7.2 software to construct the active ingredient-disease-target network.

| GO function and KEGG pathway enrichment analysis
We used R 3.6.3 software to import the coding genes of Xingren core targets into it, and executed the Cludterirofiler and pathview commands of the Bioconductor bioinformatics software package.

Gene Ontology (GO) functional enrichment analysis and Kyoto
Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed at p < .05, and the related histograms, pathway cnetplot, and bubble diagrams were obtained.

| Preparation of small molecule structures
The molecular structures of major four targets for this docking were obtained from the PubChem database (https://pubch em.ncbi.nlm.nih.gov/), and Chem3D was used for format conversion as well as energy minimization, after which all structures were imported into Schrodinger software to create a database, which was saved as a database of ligand molecules for molecular docking through hydrogenation, structure optimization, and energy minimization.

| Preparation of target protein structures
The structures of major target proteins were obtained from the RCSB database (https://www.rcsb.org/). The protein structures were processed on the Maestro 11.9 platform, and the proteins were treated with Schrodinger's Protein Preparation Wizard to remove crystalline water, fill in missing hydrogen atoms, and repair missing bond information, repair missing peptides, and finally optimize the protein for energy minimization as well as geometric structure (Fazi et al., 2015; Rajeswari et al., 2014).

| Docking results screening and analysis
The mode of interaction between the compound and the target protein was analyzed to obtain the interaction of the compound with the protein residues, such as the resulting hydrogen bonding interaction, π-π interaction, hydrophobic interaction, etc. The docking score of the compound was then referred to infer whether the compound to be screened has certain active effects. Besides, the R software was used to draw a heat map based on the molecular docking results to analyze which active ingredients were more relevant to the target.

| Screening results of active ingredients and action targets of Xingren
A total of 113 ingredients were retrieved from Xingren on the TCMSP platform, and 19 active ingredients and 74 drug action targets were obtained after screening, and the results are shown in Table 1.

| Results of disease target collection
A search of the GeneCards and OMIM database for relevant targets of COVID-19 yielded 698 targets, which were filtered and deduplicated. The regulatory targets of the active ingredients of Xingren were intersected with the relevant targets of COVID-19 using R 3.6.3 software to obtain nine common targets and the Venn diagrams of the relevant targets, and the results are shown in Figure 2.

| Construction of protein interaction network and active ingredient-disease-target network
By uploading the co-interaction target of Xingren and COVID-19 to the STRING (https://strin g-db.org/) platform, and setting the species filter to "Homo sapiens" without the combined score filter, we obtained the protein Interacting PPI networks and related information, and the results are shown in Figure 3. This included 9 protein nodes, 21 interaction links, and a PPI-enriched p-value of 1.39e −07 (p < .05). The construction of active ingredient-disease-target network by using Cytoscape 3.7.2 software ( Figure 4). In the network diagram, purple represents Xingren, red represents COVID-19, yellow represents the active ingredient of Xingren, and green represents their common genes. From the diagram, it can be seen that the action targets of different active ingredients can be the same or different, which reflects the multicomponent and multi-target action mechanism of Xingren on COVID-19, and the results are shown in Figure 4. More importantly, according to the degree values of active ingredients and key targets, the top four active ingredients were glycyrol, licochalcone B, glabridin, and phaseol, respectively.

| GO functional enrichment analysis
Importing nine relevant key targets of Xingren into R 3.6.3 software and performing GO functional enrichment analysis by executing DOSE, clusterProfiler, and pathview in R 3.6.3 software and obtaining a total of 30 biological processes, molecular functions, and cellular components at p < .05, the results are shown in Figure 5. Among it, the common action targets are mainly enriched in regulation of blood pressure, reactive oxygen species metabolic process, cellular response to chemical stress, peroxisome, microbody, peroxisomal matrix, antioxidant activity, heme binding, tetrapyrrole binding, etc.

| KEEG pathway enrichment analysis
The nine related targets of almond were imported into R 3.6.3 software, and KEEG pathway enrichment analysis was performed by executing DOSE, clusterProfiler, and pathview in R 3.6.3 software, and

| Molecular docking
In this experiment, compounds glabridin, phaseol, licochalcone B, and glycyrol were molecularly docked with MAKP14, CCNA2, PPARG, and PTGS2 target proteins. The molecular docking results showed that the four compounds bind well to the four target proteins and have a good match, and the results are shown in Table 2.
The complexes formed by the compound and the protein after docking were visualized using Pymol 2.1 software (the compound with the most negative docking score was selected for each target) to obtain the binding pattern of the compound to the protein, and the amino acid residues that bind the compound to the protein pocket can be clearly seen based on the binding pattern.

| DISCUSS ION
In this study, the active ingredients of Xingren were first screened from the TCMSP platform. The GeneGards database was used to screen out the targets of COVID-19, and the targets related to the active ingredient of Xingren were intersected with the targets related to COVID-19, then construction of PPI networks and Xingren active ingredient-disease-target networks. We also performed GO function and KEGG pathway enrichment analysis of the active ingredient targets of Xingren using R 3.6.3 software, so as to discover Viral and bacterial infections cause inflammatory storms that accelerate disease progression, and this is also true for the COVID-19

TA B L E 1 The active ingredients and action targets of Xingren
virus, which is a major factor in accelerating disease progression (Bird, 2018;Liu et al., 2016;Ye et al., 2020). According to the "Novel Coronavirus Pneumonia Treatment Protocol (Trial Version 9)," inflammatory exudates and mucus are seen in the lungs of patients with neocoronavirus infection, and inflammatory cells mainly include monocytes and lymphocytes (Fang et al., 2022). Neutrophil and lymphocyte infiltration is also seen in the heart and blood vessels, so that neocoronavirus can cause the production and release of inflammatory factors, while in a study by Xu it was also confirmed that the appearance of respiratory symptoms in patients with neocoronavirus infection is associated with the activation and release of inflammatory factors and endotoxins . Licochalcone B, a derivative of licochalcone, it can inhibit the inflammatory response in macrophages and to protect mice from endotoxic shock (Park et al., 2014). In an in vitro experimental study, licochalcone B inhibited NO and pro-inflammatory cytokine production by suppressing the activation of nuclear factor-kB and activator protein 1 in lipopolysaccharide in RAW264.7 cells. In animal models, licochalcone B may protect BALB/c mice from lipopolysaccharide-induced endotoxic shock by inhibiting the production of inflammatory cytokines. Other researchers also found that licochalcone B has antiinflammatory, antibacterial, antioxidant and anticancer activities (Kang et al., 2017).
When Jiang and colleagues extracted active compounds from rhododendron clover root, its compound (phaseol) showed moderate inhibition and no cytotoxic effect when the anti-inflammatory activity of phaseol was evaluated by inhibiting NO production in lipopolysaccharide-activated murine macrophages RAW 264.7 cells (Jiang et al., 2019). Furthermore, in the p-coumarin-extracted phaseol by Li et al. was found to reduce LP26-induced production of inflammatory mediators (e.g., NO, PGE2 and ROS) in RAW 264.7 macrophages . The results of both suggest that phaseol may achieve anti-inflammatory effects by inhibiting the production of inflammatory mediators.
In a study, Glabridin reduced inflammation and injury perception in rodents by activating BKCa channels and decreasing NO levels (Parlar et al., 2020). This compound shows an anti-injury sensing response mainly by activating BKCa channels and downregulating NO levels and part of the transient receptor potential vanilloid-1 pathway, it also shows anti-inflammatory effects by inhibiting COX activity without cytotoxicity. Glabridin also reduces LDH activity and decreases LD concentration, thereby inhibiting glycolytic metabolism and regulating energy metabolism in breast cancer cells, and may be used as a potential anticancer agent or anticancer adjuvant . One study has shown that Glabridin may also achieve therapeutic effects in atherosclerosis by regulating the expression and downregulation of the activity of MLCK (Wang et al., 2019). In addition, Glabridin has been suggested to have a regulatory effect on MAPK pathway signaling, but further studies are needed. In the opinions from Fu showed that Glycyrol inhibited IL-2 expression by decreasing NF-κB and NFAT transcriptional activity (Fu et al., 2014). It also has a therapeutic effect on autoimmune and inflammatory reactions, especially in rheumatoid arthritis diseases. Another study from Shin confirmed the anti-inflammatory effect of Glycyrol, which was attributed to the inhibition of phosphorylation of I-κBα (Shin et al., 2008).
In the active ingredient disease-target network, a total of nine targets were obtained, namely PTGS2, CAT, NOS2, PPARG, MAPK14,

F I G U R E 4
The active ingredient-disease-target network of Xingren on COVID-19 GSK3B, CCNA2, AR, and SOD1. The top four of the degree rankings are PTGS2, PPARG, MAPK14, and CCNA2. PTGS2 are inflammatory response genes, and it is also closely related to the development of tumors. It has been found that PTGS2 play a key role in the development of ovarian and intestinal tumors (Cabrera et al., 2006;Chulada et al., 2000;Habermann et al., 2013;Yucesoy et al., 2016). At present, a number of studies have also confirmed that PTGS2 is a potential target for the treatment of COVID-19, and it has great potential as a target for the development of new drugs Li, Qiu, et al., 2021;Passos et al., 2022). Two studies found that there are different re- tion and other processes. It has also been shown that when MAKP14 is suppressed, the chance of viral infection is reduced (Wang et al., 2017).
Currently, no research report between MAKP14 and COVID-19 has been found. CCNA2 is a regulator of cyclin-dependent kinases, which exhibits the characteristics of changes in protein abundance with cell cycle during the cell cycle, and is a highly conserved member of the cyclin family (Gan et al., 2018). It has been found that CCNA2 expression is increased in many types of cancer . Additionally, CCNA2 is also a potential therapeutic target for COVID-19 infection caused by SARS-CoV-2 (Chen et al., 2022). Notes: A is the 3D structure of the active ingredient in complex with the core target protein, B is the 2D binding mode of the complex, and C is the 3D binding mode of the complex.

| CONCLUSION
In summary, Xingren have potential preventive and curative effects against COVID-19. Although there are few common target proteins between the two, Xingren is active in many formulations targeting COVID-19, and it is assumed that it is most likely to be used as a "minister" drug rather than a "ruler" drug, and its efficacy is good, which is of great value for research. In addition, we found that licochalcone B in Xingren binds well to PTGS2, the target protein of COVID-19, and licochalcone B deserves further study and analysis. Finally, this study also provides some theoretical basis and reference for the excavation of the active ingredients of Xingren, licochalcone B to prevent and control COVID-19 and the development of new drugs for COVID-19.

AUTH O R CO NTR I B UTI O N S
BY and MW designed the study; JW supervised the study; MW, YW, and LL performed the research, analyzed the data and wrote the manuscript; JW and BY revised the manuscript. All authors reviewed the manuscript. And all authors read and approved the final version of the manuscript.

CO N FLI C T O F I NTE R E S T
All authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.