key: cord-0938070-em4arah8
authors: Simayi, Jimilihan; Nuermaimaiti, Maimaitiming; Wumaier, Ainiwaer; Khan, Nawaz; Yusufu, Maierdan; Nuer, Muhadaisi; Maihemuti, Nulibiya; Bayinsang,; Adurusul, Kaysar; Zhou, Wenting
title: Analysis of the active components and mechanism of Shufeng Jiedu capsule against COVID-19 based on network pharmacology and molecular docking
date: 2022-01-07
journal: Medicine (Baltimore)
DOI: 10.1097/md.0000000000028286
sha: 3dfeeb05b3d158ea9b8b9117491fe9db9b66138d
doc_id: 938070
cord_uid: em4arah8

This study investigates the active components and mechanism of Shufeng Jiedu Capsules (SFJDC) against novel coronavirus through network pharmacology and molecular docking. The TCMSP, TCMID, and BATMAN-TCM databases were used to retrieve the components of SFJDC. The active components were screened by ADME (absorption, distribution, metabolism, and excretion) parameters, and identified by Pubchem, Chemical Book, and ChemDraw softwares. The molecular docking ligands were constructed. SARS Coronavirus-2 Major Protease (SARS-CoV-2-M(pro)) and angiotension converting enzyme 2 (ACE2) were used as molecular docking receptors. AutoDock software was used for molecular docking. Cytoscape 3.7.1 software was used to generate an herbs-active components-targets network. Gene Ontology gene function and Kyoto Encyclopedia of Genes and Genomes signal pathway analysis were performed by DAVID data. A total of 1244 components were identified from SFJDC, and 210 active components were obtained. Among them, 97 active components were used as docking ligands to dock with SARS-CoV-2-M(pro) and ACE2. There were 48 components with good binding activity to SARS-CoV-2-M(pro). Ten active components (including 7-Acetoxy-2-methylisoflavone, Kaempferol, Quercetin, Baicalein, Glabrene, Glucobrassicin, Isoglycyrol, Wogonin, Petunidin, and Luteolin) combined with SARS-CoV-2-M(pro) and ACE2 simultaneously. Among them, Kaempferol, Wogonin, and Baicalein showed higher binding activity. The herbs-active components-targets network contained 7 herbs, 10 active components, and 225 targets. The 225 target targets were involved in 653 biological processes of Gene Ontology analysis and 130 signal pathways (false discovery rate ≤ 0.01) of Kyoto Encyclopedia of Genes and Genomes analysis. The active components of SFJDC (such as Kaempferol, Wogonin, and Baicalein) may combine with ACE2 and act on multiple signaling pathways and targets to exert therapeutic effect on novel coronavirus.

In December 2019, an outbreak of pneumonia caused by the SARS-CoV-2 occurred in Wuhan, China, which is later named as COVID-19 and has been spreading since. The novel coronavirus (COVID-19) is highly contagious. As of April 30, 2020, more than 80,000 cases have been diagnosed in China, and more than 3 million have been diagnosed worldwide. Patients have severe acute respiratory symptoms (SARS), including fever, dyspnea, fatigue, cough, and pneumonia. [1] Recent research results showed that SARS-CoV-2 was most similar to a group of SARS-like corona viruses in pathogenesis and clinical manifestations. [2] SARS Coronavirus-2 Major Protease (SARS-CoV-2-M pro ), the main proteolytic enzyme of SARS coronavirus, can cut the replicase of SARS coronavirus into functional proteins, thus playing an important role in the life cycle of SARS-coronavirus. [3] Because the hydrolysis specificity of the M pro is similar to that of the 3C protease (3C pro ) of picornaviruses, M pro is also called 3Clike protease (3CL pro ). [3] It has been reported that the SARS-CoV-2 may have the same receptor as that of SARS coronavirus (i.e., angiotension converting enzyme 2 [ACE2]), although its binding ability to ACE2 may be weaker than that of SARS coronavirus. [4] ACE2 was further confirmed to be indeed necessary for SARS-CoV-2 to infect cells. [5] Therefore, a deep understanding of the distribution and expression of ACE2 is of great significance for the prevention and control of COVID-19. [5] Shufeng Jiedu Capsules (SFJDC) is a pure Chinese medicine preparation made from 8 Chinese herbs, including Polygonum cuspidatum (Chinese name Huzhang), Forsythia suspensa (Chinese name Lianqiao), Isatis tinctoria L. (Chinese name Banlangen), Bupleurum chinense DC. (Chinese name Chaihu), Patrinia Scabiosaefolia Fisch (Chinese name Baijiangcao), Verbena officinalis L. (Chinese name Mabiancao), Phragmites communis (Chinese name Lugen), and Glycyrrhiza uralensis Fisch (Chinese name Gancao). Among them, Polygonum cuspidatum (Chinese name Huzhang) has the effect of dispelling wind and eliminating dampness and is the monarch drug in SFJDC. Studies have shown that the Polygonum cuspidatum extract or its purified active ingredients (such as resveratrol) can inhibit HIV-1 virus replication. [6, 7] Forsythia suspensa (Chinese name Lianqiao) has the effect of promoting blood circulation and removing blood stasis, and is the ministerial drug in SFJDC. Forsythia suspensa and its main active ingredient quercetin have anti-human cytomegalovirus effects and cytotoxicity in vitro. [8] The effective ingredient of Forsythia suspensa also has anti-respiratory syncytial virus effects in vitro. [9] Isatis tinctoria L. (Chinese name Banlangen) has heatclearing and detoxicating effects and is the ministerial drug in SFJDC. It can significantly improve the inflammatory response caused by influenza virus FM1, and can repair the pathological damage of trachea and lung tissue. [10] Bupleurum chinense DC. (Chinese name Chaihu) has reconciling superficies and interior effects and is the adjuvant drug in SFJDC. It is reported that Bupleurum chinense DC. (Chinese name Chaihu) could alleviate acute lung injury in mice induced by lipopolysaccharide. [11] As an adjuvant drug in SFJDC, Patrinia Scabiosaefolia Fisch (Chinese name Baijiangcao) can exert heat-clearing and detoxicating effects. Cho et al [12] reported that the methanol extract of Patrinia scabiosaefolia played an anti-inflammatory role in mice with ulcerative colitis. Verbena officinalis L. (Chinese name Mabiancao), another adjuvant drug of SFJDC, can promote blood circulation, remove blood stasis, and has heat-clearing and detoxicating effects. Its extracts have anti-inflammatory activity. [13] Phragmites communis (Chinese name Lugen) can help produce saliva and slake thirst and serves as an adjuvant drug of SFJDC. Glycyrrhiza uralensis Fisch (Chinese name Gancao) is a conductant drug of SFJDC. In traditional Chinese medicine, Glycyrrhiza uralensis Fisch (Chinese name Gancao) is used to treat respiratory diseases such as cough, bronchitis, and pneumonia. It also has anti-viral effects. [14] SFJDC has functions of anti-viral and anti-bacterial infection, and can enhance immunity. [15] It is often used clinically for the treatment of acute viral upper respiratory infection with wind-heat syndrome. [16] It is also used for treating acute exacerbations of chronic obstructive pulmonary disease. [17] After years of clinical observation, its effect is definite, and it is an ideal drug for anti-viral infection. [18, 19] Since the outbreak, SFJDC has been included in the diagnosis and treatment guidelines for COVID-19. [20] The traditional Chinese medicine plays an important role in the treatment of various diseases and has achieved significant clinical efficacy. [21] However, the potential components, targets and mechanism of the traditional Chinese medicine have not yet been clarified. Network pharmacology is an advanced approach to identify drug components, which can systematically explain the relationship between drugs and diseases. [22] Molecule docking can use chemometric methods to simulate the geometry and intermolecular forces of molecules, and to study the interactions between molecules, [23] which allows us to identify the active sites of small molecules (or ligands) and large molecules (or receptors) of known structure at low energy. [24] In this paper, active components of SFJDC and its mechanisms against SARS-CoV-2 were investigated. The structure of SARS-CoV-2-M pro was used as a template for network pharmacology and molecular docking. The active components of SFJDC were used as the matching library. SARS-CoV-2-M pro and ACE2 were used as molecular docking receptors. Gene functions and metabolic pathways of components-targets were then analyzed. Our findings may provide experimental evidence for developing new drugs for the treatment of COVID-19.

Ethical approval was not necessary because this study did not involve animals or human subjects (tissues).

The active components of SFJDC were collected through the TCMSP platform (http://lsp.nwu.edu.cn/tcmsp.php), TCMID (http://bionet.ncpid.org/), and BATMAN-TCM (http://bionet. ncpsb.org/batman-tcm/). These active components were further screened by the oral bioavailability [25] and drug-likeness. [26] As previously described, [27] oral bioavailability ≥ 30 and druglikeness ≥ 0.18 were set as the criteria for screening active components in this study. The structures of these components were confirmed by Pubchem and Chemical Book databases (https://www.chemicalbook.com/, https://www.ncbi.nlm.nih. gov/). For components with no available structure, Chemdraw (Version: 16.0, https://www.chemdraw.com.cn) was used to draw the component structure.

AutoTools was used to pre-treat high-resolution crystal structure of SARS-CoV-2-M pro (PDB ID: 6LU7) and ACE2 proteins (PDB ID: 1R42). The excess protein chains and ligands were removed. The water molecules were also removed by hydrogenation. The Gasteiger charge was calculated and saved as a pdbqt file for molecular docking. Then Autodock Vina (version: 1.2, http:// vina.scripps.edu/index.html) was used for small molecule and 

TCMSP (http://lsp.nwu.edu.cn/tcmsp.php) was used to predict and screen targets corresponding to the active components with better binding energy to SARS-CoV-2-M pro and ACE2 in molecular docking. Protein names of the targets were converted into gene names based on the Uniprot database (https://www. uniprot.org/) using the keyword "Homo sapiens" (human genera). The herbs-active components-targets network (HB-C-T Network) of SFJDC was constructed using Cytoscape 3.7.1 software (version: 3.7.1, https://cytoscape.org).

The targets of SFJDC active components were analyzed by DAVID6.8 database (https://david.ncifcrf.gov/) using Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway analysis.

We collected a total of 1244 components from 8 herbs of SFJDC by TCMSP, TCMID, and BATMAN-TCM analysis platform (Table 1) . Among them, 210 active components were obtained by screening the components through ADME (absorption, distribution, metabolism, and excretion) parameters. The basic information of some active components of SFJDC is shown in Table 2 .

Molecular docking was performed using 97 active components of SFJDC as ligands, and proteins of SARS-CoV-2-M pro and ACE2 as receptors. The binding between the active component and the target was evaluated by the binding energy. The larger the binding energy, the more stable the ligand is bound to the receptor. [28] The docking results showed that 10 of the 97 active components of SFJDC exhibited good binding activity with SARS-CoV-2-M pro and ACE2 ( Table 3 ).

The binding energy of the current clinically recommended chemical drugs (Lopinavir, Ritonavir, and Remdesivir) was also analyzed. The results showed that the optimal binding energy of Remdesivir was the lowest among the 3 drugs (-4.9 kcal mol À1 ) ( Table 3 ). The binding energy of Kaempferol, Baicalein, and Wogonin with SARS-CoV-2-M pro was lower than -4.9 kcal mol À1 , indicating good binding activity. Kaempferol, Baicalein, and Wogonin also had optimal binding energy with ACE2. As shown in Figure 1 , Kaempferol, Baicalein, and Wogonin bound to the active site of the ACE2 protein, respectively. They formed hydrogen bonding interactions with 2 amino acids of UNK910 and ALA614 of ACE2 protein. They also bound to the active sites of SARS-CoV-2-M pro protein and formed hydrogen bonding interactions with 5 amino acids (THR26, ASN140, ASN142, GLU166, and PHE140). They also formed pi-pi interactions with VAL3 amino acids of SARS-CoV-2-M pro protein. The results indicate that hydrogen bonding plays a key role in the recognition and binding stability of active components of SFJDC with ACE2 and SARS-CoV-2-M pro protein. This also suggests that the main active components of SFJDC exert therapeutic effects in the treatment of COVID-19 by interacting with related proteins.

The active targets of these 10 active components were screened through the TCMSP analysis platform. The gene name-protein name conversion of targets was conducted through the Uniprot database. Totally, 384 targets were identified. After deleting the duplicate target names, 225 target targets were obtained. An HB-C-T network was constructed through network analysis to clarify the relationship between herbs, active components, and target targets (Fig. 2) . The average Degree of the entire constructed network was 3.18. For the components, there were 9 components with Degree greater than 3.18. About 70% of the components had more than 20 targets on average, indicating that there may be a few key components in the network that can act on most of the SFJDC targets. Among them, Quercetin (Degree = 153), Kaempferol (Degree = 61), Luteolin (Degree = 58), Baicalein (Degree = 27), 7-Acetoxy-2-methylisoflavone (C1, Degree = 26), and Glabrene (Degree = 20), Liquiritigenin (Degree = 20) had the most targets. For the targets, the more components a single target is affected by, the more likely SFJDC can act on this target. There were 29 targets with Degree greater than 3.18. Among them, AR (Degree = 7), PRSS1 (Degree = 7), NCOA2 (Degree = 7), PPARG (Degree = 6), PTGS2 (Degree = 6), and HSP90AA1 (Degree = 6) were affected by more than 6 components. Therefore, multiple Table 1 The number of components each herb of SFJDC.

Herb 

To further understand the effect of SFJDC, we used GO gene biological processes and KEGG signal pathway analysis to analyze the 225 targets through the DAVID database. Our results showed that the 225 targets were related to 653 biological processes and 130 signal pathways (Fig. 3) . The biological processes were mainly focused on the RNA polymerase II promoter and apoptosis, as well as positive regulation of gene expression, signal transduction, protein phosphorylation, proteolysis, immune response, inflammatory response, drug response, and response to viruses. The main Table 2 Active components from SFJDC screened by ADME. H1 to H8 = as indicated in Table 1 . ADME = absorption, distribution, metabolism, and excretion, DL = drug-likeness, OB = oral bioavailability, SFJDC = Shufeng Jiedu Capsules. 

This study investigated the active components and mechanism of SFJDC against COVID-19 by network pharmacology and molecular docking. When performing molecular docking, it is generally believed that lower binding energy indicates higher binding ability. [28] In this paper, Redoxivir had a binding energy of -4.9 kcal mol À1 with SARS-CoV-2-M pro , which was the lowest among the 3 chemical drugs. [29] [30] [31] The HB-C-T network analysis showed that, Kaempferol, Wogonin, and Baicalein had the highest node degrees, indicating Figure 2 . Herbal-active component-action target network. The network consists of 242 nodes (7 herbs, 10 active components, and 225 active targets) and 385 edges. The edges between HB (yellow octagon), C (red quadrilateral), and T (green circle) represent interactions. A degree of node (Degree) represents the number of nodes that directly interact with the node in the protein interaction network. The size of the node is proportional to the degree. The greater the degree of a node, the more biological functions it participates in, and the stronger its biological significance.

Simayi et al. Medicine (2022) 101:1 Medicine that they may participate in many biological functions. It has been reported that Kaempferol inhibits the NF-kB signaling pathway by reducing oxidative stress and TNF-a, IL-6, and IL-1b inflammatory factors in bronchoalveolar lavage fluid. [32] It can inhibit the excessive activation of the complement system in the body and improve the acute lung injury induced by influenza A virus. [33, 34] Wogonin reduces inflammatory pathological damage of lung tissue by inhibiting the expression of TNF-a and IL1-1b. [35] Baicalein can inhibit systemic allergic reactions by inhibiting the release of inflammatory mediators and mast cell degranulation. [36] Baicalein can also inhibit vascular remodeling and improve rat pulmonary arterial hypertension induced by crocin and the mechanism may be related to its inhibition of mitogen-activated protein kinase and NF-kB signaling pathway. [37] The recent studies on COVID-19 also reported the anti-COVID-19 potential of flavonoids such as Kaempferol and Baicalein, [38, 39] which is consistent with our results. Therefore, Kaempferol, Wogonin, and Baicalein are main active components of SFJDC in treating COVID-19. We further performed GO and KEGG analysis on the identified targets of SFJDC. The GO biological processes mainly included the RNA polymerase II promoter and apoptosis, as well as positive regulation of gene expression, signal transduction, protein phosphorylation, proteolysis, immune response, inflammatory response, drug response, and response to viruses. The main virus-relevant pathways obtained by KEGG analysis were the PI3K-Akt signaling pathway, as well as the signaling pathways related to the virus' natural immune response, such as the NOD-like receptor signaling pathway and the Toll-like receptor signaling pathway. Many viruses can regulate the host cell PI3K-Akt signaling pathway during infection to complete virus replication. [40] Targets that are involved in these 3 signaling pathways included MAPK1, RELA, IL6, and IKBKB. Chen et al [41] reported that RELA, MAPK1, and IL6 were important targets of SFJDC in treating COVID-19. Zhuang et al [42] also found that RELA and CASP9 were key targets of SFJDC in treating COVID-19. These findings further confirm the accuracy of the prediction results of this study. Among them, RELA was the target of Kaempferol, Baicalein, and Wogonin. However, whether the main active components in SFJDC regulate the PI3K-Akt signaling pathway, the NOD-like receptor signaling pathway, and the Toll-like receptor signaling pathway by acting on MAPK1, RELA, IL6, and IKBKB targets needs further study.

In conclusion, our results show that SFJDC may exert therapeutic effects on COVID-19 through the synergistic effect of multiple components and multiple targets. However, due to the limitations of network pharmacology and molecular docking, more experiments are needed to provide theoretical and experimental basis for SFJDC treatment of COVID-19 and later drug development. 

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