Research on active compounds regarding SGMD for the treatment of COVID-19 based on network pharmacology and molecular docking

Background: Retrieve Curative effect of Six Gentlemen Modied Decoction (SGMD) in treating with coronavirus disease ( COVID-19 ) by network pharmacology and verify its authenticity by molecular docking. Methods: The chemical constituents, effective components, and action targets were screened using TCMSP. COVID-19 related targets were retrieved by the GeneCards and NCBI databases, and drug targets and disease targets were mapped by Venny to obtain potential targets for treatment. The regulatory network of traditional Chinese medicine (TCM) compounds was established with Cytoscape to obtain the key components, and the PPI network and its network topology were established with the Bisogenet and CytoNCA plug-ins to obtain the core targets. Bioconductor was used for GO function analysis and KEGG pathway analysis to obtain the relevant functions and pathways. Results: 173 effective components, 253 targets, and 348 targets related to COVID-19 were obtained after screening, 50 cross targets were shown, and the key components of the top 15 are avonoids such as quercetin, luteolin, kaempferol, naringenin, licochalcone A, etc. The top 28 core targets include TP53, EGFR, SRC, AR, ABL1, and others. Biological processes such as the responses to metal ions, molecules of bacterial origin, lipopolysaccharide, toxic substances, and oxidative stress were involved. The main pathway involved the AGE−RAGE signaling pathway in diabetic complications as well as the TNF and IL-17 signaling pathways. The average binding energies of the rst three core components connected with 6LU7 and 1R42 were -4.16 kJ/mol and -4.12 kJ/mol, respectively. Conclusion: The core compounds of SGMD can spontaneously combine with SARS-CoV-2 3CL hydrolase and ACE2 to treat COVID-19. C/EBPα-binding

6 g of Amomi Fructus and 6 g of Glycyrrhizae Radix et Rhizoma. SGD was recorded in the book entitled, "Ancient and Modern Famous Medical Prescriptions".The decoction strengthens the spleen, supplements the lung, and bene ts the qi for resolving dampness. It is often used for the treatment of spleen stomach qi de ciency and retention of dampness and stagnation of qi. Astragali Radix and Pogostemonis Herba were added into this formula. Astragali Radix is the essential medicine for elevating yangqi. It can tonify the qi of lung and spleen as well as cure internal heat and diabetes. Pogostemonis Herba is a kind of aromatic damp-resolving drug, and it is recorded in the book "Medicinal meaning". It is used along with fragrant qi, which is good for regulating the spleen and stomach of the middle jiao, and it has the effect of activating the spleen and enhancing appetite. It can also reduce distension syndrome of the middle jiao and then cure vomiti and cholera. Modern studies [3][4][5][6] have shown that the decoction warms the spleen and stomach, invigorating and regulating qi. Thus, it can be used to clear the qi of the middle jiao, regulate the gastrointestinal tract, enhance the appetite of patients, and regulate metabolism, endocrine and immune functions.
This study is based on network pharmacology and sought to screen the active ingredients of SGMD in order to determine its effective components and targets. The COVID-19 screening found the intersection of drug targets and disease targets. Furthermore, the mechanism of action of this prescription was elucidated to provide a scienti c basis for the development and clinical application of COVID-19 drugs.

Retrieval of effective ingredients and targets
In the above retrieval results, the bioavailability (OB) > 30% and drug-like property (DL) > 0.18 were selected as the screening conditions for the secondary retrieval to collect the effective ingredients and targets of various herbs in this prescription, and the collected target proteins were inputted into the UniProt database (https://www.uniprot.org/) for gene name correction.

Prediction of potential targets of COVID-19 in drug therapy
The intersection of drug targets and disease targets was obtained using the Venny mapping software, and the cross targets were the potential targets of SGMD relevant to COVID-19.

The establishment of regulatory network of TCM compounds
The targets of SGMD relevant to the treatment of COVID-19 were uploaded to the Cytoscape software 3.7.1 http://www. cytoscape. Org/). Then, we obtained an interaction diagram of the TCM -componenttarget -disease network, which means the regulation network of TCM compounds was constructed.

The establishment of an PPI network
The names of the cross targets were imported into the Bisogenet plug-in in cytoscape-3.7.1 software to create a new network, and the species option was selected as "Homo sapiens". Then, the construct protein interaction network button was selected. At last, we generated the PPI network, which was used to identify the functional partnerships and interactions between proteins.

Network topology analysis
Traditional Chinese medicine, effective ingredients, targets and disease are the nodes in the network of TCM compounds regulation. When there is an association among these nodes, the Networkanalysis plugin in cytoscape-3.7.1 software can be used to analyze the topological structure. In addition, the core targets of SGMD relevant to COVID-19 can be screened out.

GO function analysis and KEGG pathway analysis
The Bioconductor database (http://www.bioconductor.org/) was used for the GO function analysis and the enrichment of KEGG. The targets of drugs treatment related to COVID-19 were entered, and the column diagram of the molecular function, cell composition, biological process and pathway process were determined. Finally, we speculated the most probable mechanism of action in the treatment of COVID-19.

Components-targets molecular docking
The structures of SARS-CoV-2 3 CL hydrolase (Mpro) and angiotensin converting enzyme (ACE2) were found using the RCSB database (http://www.rcsb.org), and the result showed that the protein codes (PDB IDS) were 6 lu7 and 1 r42, respectively. Mgl tools 1.5.6 (ttp://mgltools.scripps.edu/) were used for protein processing, and the results were saved in the PDBQT format after water removal and hydro-charging. The ligands (active compounds) in the form of Mol2 were treated in the same way and stored in the same format for later use. The dock key components and the above two targets were used with AutoDock 4.2.6 (http://autodock.scripps.edu/).Then,the docking les were converted to PDB format with OpenBabel2.4.1 software (http://openbabel.org/wiki/Main_ Page). Finally, the results of the docking between the components and the targets were observed and analyzed in Pymol 2.3.3 (hts:// pymol.org/2/).

Retrieval of chemical compositions, effective components and targets
There were a large number of pharmaceutical chemical components in SGMD that were retrieved by TCMSP. A total of 194 effective ingredients were obtained after the second retrieval based on the screening conditions of bioavailability (OB) > 30% and drug-like property (DL) > 0. 18

Retrieval of COVID-19 related targets
The GeneCards and NCBI databases were used to retrieve the keyword "novel coronavirus". In total, 346 related targets were found in the GeneCards database, and 48 related targets were found in the NCBI database, among which 46 were common targets. After removing common targets, 348 targets related to COVID-19 were obtained.

Potential targets of COVID-19 in drug therapy
The Venny mapping software was used to perform intersection alignment of 253 effective ingredients regulated targets retrieved from the TCMSP database, and a total of 348 targets related to COVID-19 identi ed from the GeneCards and NCBI databases. 50 cross-targets were obtained, which were potential targets for the treatment of COVID-19 by SGMD, as shown in g.1.

Analysis of regulatory network of TCM compounds
After the cross-targets were imported into Cytoscape 3.7.1 software (http://www. cytoscape.org/), the TCM compound regulatory network was obtained, with 168 nodes and 588 edges, as shown in g. 2. The 118 effective ingredients in SGMD acted on 50 cross-targets of drugs and diseases to treat COVID-19. In g. 2, the ginger yellow nodes represent the effective ingredients of Pinelliae Rhizoma, the grey nodes represent the effective ingredients of Citri Reticulatae Pericarpium, and the purple nodes represent the effective ingredients of Codonopsis Radix. The light-yellow nodes represent the effective ingredients of Astragali Radix, and the yellow node represents the effective ingredients of Atractylodis Macrocephalae Rhizoma, while the brown node represents the effective ingredients of Poria, and the pink node represents the effective ingredients of Pogostemonis Herba. The blue node represents the effective ingredients of Amomi Fructus, and the green node represents the effective ingredients of Glycyrrhizae Radix et Rhizoma, while the red nodes represent the chemical components that exist in multi-herb Chinese medicine and act on COVID-19. The sky-blue nodes represent the potential targets of drug treatment, and each edge represents the interaction between the effective ingredients and the potential therapeutic targets. There

PPI network
Fifty drug and disease cross-targets were imported into the Bisogenet plug-in, and the protein-protein interaction (PPI) network was obtained after the network analysis, as shown in g. 3. A total of 2,133 target proteins were loaded.

Network topology analysis
The CytoNCA plug-in was used to carry out the topological analysis of target proteins in the PPI network.

GO function analysis and KEGG pathway analysis
SGMD treatment targets related to COVID-19 were entered into Bioconductor for the GO functional analysis and KEGG enrichment analysis. P value <0.05 was set as the default option, and the rst 20 lines of the screening results were plotted into a histogram. The GO functional analysis includes the biological process (BP), molecular function (MF) and cell composition (CC). According to panels A, B and C in g. 5, the relevant targets for the treatment of COVID-19 were mainly concentrated in biological processes such as responses to metal ions, molecules of bacterial origin, lipopolysaccharides, toxic substances and oxidative stress. The main molecular functions were concentrated in cytokine receptor binding, phosphatase binding, BH domain binding, cytokine activity and protein phosphatase binding. The main cellular components were concentrated in membrane raft, membrane microdomain and the membrane region. According to g. 5d, the relevant targets for the treatment of COVID-19 were produced by treating the effect mainly through the AGE−RAGE signaling pathway in diabetic complications as well as the TNF and IL-17 signaling pathways.

Target -KEGG pathway network
Cytoscape 3.7.1 software was used to map the 20 pathways and path-related targets obtained from the KEGG enrichment analysis in order to understand the pathway mechanism. The hsa04933-AGE−RAGE signaling pathway in diabetic complications as well as the hsa04668-TNF and hsa04657-IL−17 signaling pathways are of great importance in the enriched pathways. MAPK1, MAPK3, RELA, IL6, MAPK14 and other targets also play important roles in this network ( g. 6).

Discussion
In the investigation and analysis of TCM symptoms of COVID-19 patients in convalescence, it was found that about 26.1% of the patients presented with de ciency of lung and spleen [7]. According to the diagnosis and treatment protocol for COVID-19 issued by the national health commission, the trial version 7 describes the symptoms of COVID-19 convalescent patients with de ciency of lung and spleen, and the main clinical manifestations are shortness of breath, fatigue and weakness, anorexia and nausea, fullness, and uncomfortable loose stool. The tongue is light and fat, and the coating is white and greasy. In traditional Chinese medicine, the spleen is the mother of the lung, and the lung is the main source of qi, while the spleen nourishes the qi. The lung is the master of qi, and the shortness of breath is the symptom of de ciency of lung qi. The spleen is the foundation of acquired constitution. Once the human body is in the state of spleen de ciency, production of qi and blood would be de cient, leading to water and body uid metabolism disorders, which would then lead to the viscera loss of nutrition, water and grain transport disorders, internal generation of phlegm and dampness, which can cause the symptoms of fatigue, weakness, anorexia, nausea, fullness, and uncomfortable loose stool. Therefore, clinical treatment should follow the principle of strengthening the spleen, supplementing the lungs and bene ting the qi for resolving dampness. Multiple Chinese medicines of SGMD used in combination can restore the function of all vessels converging in the lung and promote digestion and absorption. In this study, network pharmacology and molecular docking technology were used to further explore the therapeutic mechanism of the recommended prescription of SGMD.
The network pharmacology results are as follows: there are 173 active ingredients and 253 targets that re ect the characteristics of multiple components and targets of traditional Chinese medicine. From the regulatory network of TCM compounds, the key components in the treatment of COVID-19 include avonoids such as quercetin, luteolin, kaempferol, naringin, licochalcone A, nobiletin, irisolidone, baicalein, formononetin, isorhamnetin, 7-O-methylisomucronulatol, Odoratin, Vestitol, 7-Acetoxy-2methyliso avone, Quercetin der., etc. The PPI network and its topology suggest that the core targets of this decoction are TP53, EGFR, SRC, AR, ABL1, GRB2, AKT1, MAPK1, and others, totalling 28 targets. The GO function analysis suggests that the biological processes of this decoction include responses to metal ions, molecules of bacterial origin, lipopolysaccharides, toxic substances and oxidative stress. The KEGG pathway analysis suggests that pathways play a therapeutic role of this decoction, including the AGE−RAGE signaling pathway in diabetic complications as well as the TNF and IL-17 signaling pathways. In addition, the targets with high frequencies involved in the above pathways are MAPK1, MAPK3, RELA, IL6, MAPK14.
The molecular docking technique was used to verify the components and targets. The results were as follows: formononetin, Quercetin der. and 7-Acetoxy-2-methyliso avone blocked the replication of the virus by binding to the 6LU7 receptor protein through SER-10,ALA-7, LYS-5 and other cytokines, and the binding energies were -4.28, -4.12, -4.08 (unit kJ/mol), with an average of -4.16.7-Acetoxy-2methyliso avone, irisolidone and formononetin bind to the human receptor protein 1R42 to prevent virus invasion through the cytokines such as IL-256, MET-249, SER-254 and ASN-250, and the binding energies were -4.26, -4.16 and -3.93 kJ/mol, with an average of -4.12 kJ/mol. Previous studies have suggested that binding energies less than 0 indicate that the ligand and receptor can spontaneously bind [8]. The above results indicated that the core components of SGMD exhibited good binding properties with COVID-19related target proteins, which veri ed the therapeutic effect of this decoction.
Pharmacological studies have shown that formononetin may inhibit the in ammatory response of rats by controlling the expression of the Sph k1-s1p signaling pathway [9]. It can inhibit spleen cell apoptosis and improve its conversion rate, improve the innate immune function of mice [9], suppress the proliferation of leukemia HL-60 cells and increase the apoptosis rate [10] and protect myocardial cells [11]. Quercetin der. is a derivative of Quercetin. Quercetin can reduce the apoptosis rate of rat alveolar cells and inhibit in ammation by suppressing the expression of TGF-β1, TNF-α and so on [12]. In addition, studies have shown that quercetin also has a great diversity of pharmacological activities like antioxidation [13][14][15], antitumor [16][17][18][19], antiviral [20][21][22][23] and immunoregulation [24][25][26]. The pharmacological effects of irisolidone include anti-in ammatory, antioxidation, antibacterial and antitumor impacts [27]. MAPK1, MAPK3 and MAPK14 are all belong to the mitogen-activated protein kinase (MAPK). They are mainly engaged in cell activation, growth, proliferation, apoptosis and immunoregulation, which can inhibit the expression of in ammatory factors and reduce lung injury. RELA can promote the apoptosis of neutrophils [28] and reduce the in ammatory response. IL6 is an in ammatory mediator, which is not only related to the occurrence and spread of in ammation [29][30][31], but it is also involved in the suppression of in ammation [32][33][34]. The AGE-RAGE signaling pathway reacts on cell activation and tissue damage and has the activity of promoting the expression and release of in ammatory factors [35]. The TNF and IL-17 signaling pathways also play a crucial part in the occurrence and development of in ammation [26,36,37].

Conclusions
In summary, through network pharmacology, this paper collated the effective ingredients, action targets, potential action targets, key components and core targets related to treating COVID-19 as well as the biological functions and pathways of potential action targets in SGMD. The molecular docking technology was used to verify the reliability of the therapeutic effect of this decoction.
This study is only based on the pharmacology theory of TCM and does not consider other in uential factors such as the compatibility dosage of prescription and processed products, so studies on the mechanism of action of SGMD to restore symptoms of COVID-19 patients still needs to be further improved.

Declarations
Ethics approval and consent to participate: Not applicable. Consent for publication: I understand that the text and any pictures or videos published in the article will be freely available on the internet and may be seen by the general public. The pictures, videos and text may also appear on other websites or in print, may be translated into other languages or used for commercial purposes.
Availability of data and materials: All data generated or analysed during this study are included in this published article.
Competing interests: The authors declare no con ict of interest.   TCM compound regulatory network. The triangle represents the chemical composition, the square represents the target, and the side represents the relationship between the two Figure 2 TCM compound regulatory network. The triangle represents the chemical composition, the square represents the target, and the side represents the relationship between the two Figure 3 Protein-protein interaction network.  The PPI topology analysis Figure 4 The PPI topology analysis Analysis of the GO function (a, b and c) and the KEGG pathway (d) The abscissa represents the number of enrichments, the ordinate represents the name, and the color represents the signi cance. Redder the column corresponds to more signi cant in the enrichment. Analysis of the GO function (a, b and c) and the KEGG pathway (d) The abscissa represents the number of enrichments, the ordinate represents the name, and the color represents the signi cance. Redder the column corresponds to more signi cant in the enrichment.

Figure 6
Target-pathway network. The yellow square nodes represent the targets, and the red v-shaped nodes represent the pathways. Larger squares correspond to more important targets in the pathway.
Page 23/25 Figure 6 Target-pathway network. The yellow square nodes represent the targets, and the red v-shaped nodes represent the pathways. Larger squares correspond to more important targets in the pathway.