Research on Active Compounds of Huanglian Jiedu Decoction for the Treatment of Corona Virus Disease 2019 Based on Network Pharmacology and Molecular Docking

Background Huanglian Jiedu Decoction (HLJDD) is a traditional Chinese prescription for the treatment of inuenza, inammation and other ailments related to heat-syndrome, a typical pathological symptom in Traditional Chinese Medicine. It was recommended as one of the basic prescriptions among the Proposed Diagnoses and Treatment issued by China’s National Health Commission. In this work we investigated the molecular mechanism of action of Huanglian Jiedu Decoction in the treatment of Corona Virus Disease 2019 (COVID-19) through network pharmacology and molecular docking approaches. Methods The chemical constituents and action targets of Coptis chinensis, Scutellaria baicalensis, Phellodendron amurense, Gardenia jasminoides in HLJDD were retrieved on Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). The database of UniProt and GeneCards were used to query the target genes that corresponding to the active compounds, and then a compound-target network was constructed using Cytoscape 3.7.2. GO database was used to annotate GO functions. Reactome was used to analyze KEGG enrichment pathway, predicting the possible mechanisms of active compounds. DAVID database was used to analysis the tissue enrichment. The main active ingredient is molecularly docked with the SARS-CoV-2, ACE2 and TMPRSS2. Results We screened 84 compounds and obtained 341 corresponding target genes in the network. Gene annotation showed that the targets were involved mainly in 457 biological functions. 306 signaling pathways was enriched, involving chemokine and cytokine signaling pathways that mediate inammation, interferon-γ signaling pathway, p53 pathway. And the targets mainly distributed in the lung liver and placenta, involving a variety of immune cells, such as T cells, B cells. The molecular docking results showed that core compounds such as beta-sitosterol, stigmasterol and quercetin had high anity with SARS-CoV-2, ACE2 and TMPRSS2, which was comparable with drugs like abidol used to COVID-19 treatment by.

bloating and constipation. This prescription is a well-known prescription for treating heat-syndrome in China and was rst mentioned in the "WaiTaiMiYao" by a medical scientist who named Wang Tao in the Tang Dynasty. It is an aqueous extract of 4 herbal materials, Coptis chinensis (Huanglian, the rhizomes of Coptis chinensis Franch, Ranunculaceae), Scutellaria baicalensis (Huangqin, the dried roots of S. baicalensis Georgi, Lamiaceae), Phellodendron amurense (Huangbo, the dried bark of Phellodendron amurense Rupr., Rutaceae ), and Gardenia jasminoides(Zhizi, dried ripe fruit of Gardenia jasminoides J.Ellis, Rubiaceae) with the ratio of 3:2:2:3. This formula has been used since ancient times, and has been widely used in clinical practice, and is o cially included in the Chinese Pharmacopoeia [4]. In gastrointestinal diseases, in ammation, cardiovascular diseases and Alzheimer's disease, HLJDD has good clinical effects [5][6][7]. Modern pharmacological studies have also elucidated the pharmacokinetics and pharmacodynamics of HLJDD, and found that the main compounds of iridoids, avonoids and alkaloids in HLJDD can play an anti-in ammatory effect [8]. Baicalein and coptisine, the active ingredients in Huanglian Jiedu Decoction, interfere with the arachidonic acid cascade by inhibiting the lipoxygenase pathway and exert anti-in ammatory effects [9]. Liu et al. studied the effect of HLJDD on the urine metabolomics of healthy people and found seven potential biomarkers, including 2-(formylamino) -benzoic acid, whicn has proved the mechanism of in treating heat syndrome HLJDD from pharmacology [1,10]. In the in vitro study, the modi ed HLJDD also activated the plasma bactericidal activities [10]. However, the role and mechanism of HLJDD's in COVID-19 are unclear and needs further investigation.
Network pharmacology is a new discipline that combines the functions of drug compounds, disease targets, and biological signaling pathways based on computer network analysis [11,12], which is suitable to analyze the traditional Chinese medicine because of the multiple targets affected by the multi-components. Network pharmacology is able to visualize, systematize, and informatize the principles of the process of treating diseases by traditional Chinese medicine. The molecular docking technology predicts the binding mode and a nity between two molecules by analyzing the physical and chemical properties of the molecules and by computer simulation [13]. Molecular docking plays an important role in the mechanism of active ingredients and target proteins of traditional Chinese medicine. It can clarify the principle of the e cacy of traditional Chinese medicine at the molecular level, making the research of traditional Chinese medicine microscopic and scienti c. SARS-CoV-2 crystal structure has been determined by Shanghai Tech University (PDB 6LU7). SARS-CoV-2 invades cells by binding the ACE2 receptor on the surface of human cells with the S protein of its spinous. Recently, German scientist Markus Hoffmann discovered that SARS-CoV-2 needs the help of TMPRSS2(Transmembrane Protease Serine 2) protein to enter cells. This article intends to analyze the active ingredients and target genes of HLJDD through network pharmacology, and dock the main active ingredients with SARS-CoV-2 hydrolase, ACE2 and TMPRSS2 to provide a theoretical basis for its clinical application.

Methods
The work ow is demostrated in Fig. 1.
Bioavailability refers to the relative amount of drugs that is absorbed into the systemic blood circulation and metabolized after being administered via an extravascular route. Drug-like properties are usually used to evaluate the possible failure characteristics of a compound. The signi cance of this standard lies in bioavailability. The higher the degree and drug-like properties, the more research signi cance the human body has. Through the TCMSP platform, the target components are collected in the corresponding target database to obtain the target of the component [16].

Establishing the ingredient-target network
The collected ingredients and targets are sorted and imported into Cytoscape 3.7.2 software (http://www.Cytoscape.org/) [17] to generate a network of active ingredients-target interactions of HLJDD. Visualize the pharmacological action mechanism of HLJDD.

Target pathway analysis
In order to further understand the function of the selected target protein genes and the role in the signaling pathway, the target protein obtained after weight reduction of the predicted target point is imported into the GO database (http://geneontology.org/) and set threshold FDR < 0.05. After annotating GO function, make analysis of KEGG enrichment pathway through reactome platform (https://reactome.org/Pathway Browser/), and the pathways related to HLJDD for the treatment of new coronavirus pneumonia were obtained by consulting the literature and the KEGG database. Use Omicshare (http://www.omicshare.com/tools/index.php/) to visualize the enrichment analysis results. And further carry out tissue enrichment analysis on target protein through David database (https://david.ncifcrf.gov/ ).

Component-target molecular docking
Using the PDB database [18] (https://www.rcsb.org/) to load SARS-COV-2 3CL hydrolase, ACE2 3d structure * PDB format and we predicted the structure of the TMPRSS2, and using ZINC (http://ZINC.docking.org ) to collect and analyze the rst ve core ligands * MOL2 format. The target proteins were dewatered and hydrogenated by PyMOL software, and the compounds and the target proteins were converted to pdbqt format by Auto Dock Vina software [19]. The docking results were visualized in Pymol.

Component-target network of HLJDD
The corresponding targets of the main compounds of HLJDD were collected in the TCMSP database, and the results were imported into Cytoscape 3.7.2. The active ingredient-prediction target network was constructed, and 425 nodes (84 active ingredient nodes and 341 predicted target nodes) and 1801 interaction relationships were obtained, as shown in Figure 2. Among them, yellow represents the compound of Scutellaria baicalensis, green represents the compound of Coptis chinensis , red represents the compound of Phellodendron amurense, and blue represents the compound of Gardenia jasminoides.
Intersecting compounds in HLJDD Further carry out tissue enrichment analysis on targets. As shown in Figure 7, tissue enrichment revealed that targets expression sites are mainly distributed in lung tissue, liver, and placenta, and involve a variety of immune cells, such as T cells and B cells. It shows that the key targets of the active ingredients of HLJDD are mainly expressed in lung tissues and immune cells.

Component-target molecular docking
It is generally believed that the lower the energy is, the more stable conformation of the ligand-receptor binding is, the more likely the interaction is. The results of molecular docking showed that the a nity of the core active compounds in HLJDD to SARS-COV-2 3CL hydrolase, ACE2 and TMPRSS2 was less than zero, which indicated that the core active compounds in HLJDD could spontaneously bind to SARS-COV-2 3CL hydrolase, ACE2, TMPRSS2. The results are shown in table 2.The result of molecular docking showed that the binding energy of ACE2 with stigmasterol was -9.1KJ/Mol.And the binding energy of SARS-COV-2 3CL hydrolase and TMPRSS2 with beta sitosterol was -7.5KJ/Mol and 8.4KJ/Mol respectively. The results show that the conformation of the Ligand and its receptor is the most stable. The binding ability of the major components in HLJDD to the SARS-COV-2 3CL hydrolase, ACE2, TMPRSS2 is lower than that of the effective drugs reported so far, which indicates that the major components in HLJDD bind to the SARS-COV-2 3CL hydrolase, ACE2, TMPRSS2 more stable and have more possibility to play a role. The docking results are shown in Figure 8-1,2,3.  [20]. Since ancient times, traditional Chinese medicine has played a very good role in treating complex diseases and new diseases. From the outbreak of the epidemic situation, traditional Chinese medicine has also had relatively obvious curative effects in this new coronary pneumonia. This prescription is mainly used for the treatment of patients with moderate to severe illness. Therefore, studying the active ingredients, therapeutic targets and molecular docking mechanisms of this prescription can provide a theoretical basis for the treatment of a large number of patients with moderate and severe illness. Interventional treatment in the middle and severe stages of the disease, preventing the development of the critically ill patients in the critical direction, greatly saving medical resources, reducing the burden on the country, society and individuals, is of great signi cance to ending the epidemic as soon as possible.
In this study, after analyzing the active ingredients through the network pharmacology method, 341 potential targets, 307 biological processes, 107 molecular functions, and 306 KEGG pathways were obtained. After preliminary clinical observation, the common clinical symptoms of the new coronavirus infection are dyspnea, and severely ill patients will have a signi cant increase in proin ammatory cytokines such as IL-6, TNF-a, IFN-γ, which has the characteristics of cytokine storm [21]. Studies have found that serum matrix metalloproteinase 9 (MMP9) and transforming growth factor beta (TGF-β) are common airway remodeling related proteins [22,23]. They can mediate the in ammatory response and damage the airway. In severe cases, the air ow is restricted, and the restricted air ow can lead to a lack of effective ventilation. The main manifestations in clinical symptoms are dyspnea and decreased lung function [24,25]. We speculate that Huanglian Jiedu Decoction may inhibit airway remodeling and improve dyspnea by inhibiting the expression of MMP9 and TGF-β. The cytokine storm, also known as the "in ammatory storm", is actually an important node in the transition from mild patients to critically ill patients, and it is also a cause of death of severe patients now [26][27][28][29]. Once an in ammatory storm is formed, while the immune system kills the virus, it will also kill a large number of normal cells in the lung, severely destroying the lung's ventilation function, leading to respiratory failure until hypoxia and death. It has also been found in recent years that the alveolar in ammatory response caused by the release of in ammatory cytokines is the main cause of lung injury [28]. IL6 is a pro-in ammatory factor, and its main function is to accelerate the alveolar in ammation in the early stage of pulmonary brosis through chemotactic in ammatory cell aggregation and promote in ammatory cell in ltration, and then mediate the occurrence of idiopathic pulmonary brosis [27,30]. IL10 is a class of anti-in ammatory factors that play an important role in the occurrence of various organ brotic diseases. At the same time, it has immunomodulatory effects such as inhibiting monocytes / macrophages, Th1 cell functions, and enhancing B cell functions [31,32]. And the latest research found that interleukin-6 (IL-6) is an important in ammatory factor that induces the in ammatory storm of neocorona virus pneumonia [33,34].
Based on the preliminary understanding of the mechanism and the KEGG analysis results, it is speculated that the core active compounds in HLJDD may regulate cytokine signaling and interleukin signaling in the immune system by acting on genes such as IL6 and IL10 (show as Fig. 9). From the perspective of immune regulation and suppression of in ammation, inhibit excessive immune cell activation and cytokine production to block the occurrence of in ammatory storms; Pulmonary microangiogenesis is also an important part of pulmonary brosis. VEGF is an important factor that promotes angiogenesis. It mainly exerts its physiological function by binding with receptors (VEGFR1, VEGFR2, etc.) [35]. Under pathological conditions, their combination can inhibit the apoptosis of vascular endothelial cells, promote their proliferation, migration and differentiation, increase vascular permeability, and stimulate neovascularization in the body [36]. Moreover, some studies have found that NO / NOS (nitrogen oxide synthase) pathway may be involved in its role in promoting angiogenesis [37]. Therefore, it is speculated that Huanglian Jiedu Decoction may inhibit VEGF signal transduction by acting on VEGFA, NOS2, NOS3 and other genes, reduce pulmonary brosis, and play a role in treating COVID-19.
The results of molecular docking showed that the conformations of ACE2 hydrolase and Stigmasterol was the most stable and the possibility of their action was the greatest. This indicates that Stigmasterol in Scutellaria baicalensis and Phellodendron amurense plays a more important role in the treatment of new coronary pneumonia. Studies have shown that Stigmasterol has potent anti-tumor, anti-in ammatory and anti-oxidative effects [38,39]. SARS-CoV-2 3CL hydrolase and TMPRSS2 also showed good binding activity to beta sitosterol, which was mainly derived from Scutellaria baicalensis, Phellodendron amurense and Gardenia jasminoides. Some studies have shown that beta sitosterol has certain anti-in ammatory effects [40,41], and can be combined with PLY cholesterol binding site to prevent the toxin from binding to membrane, playing a competitive antagonistic role, thus to have an anti-infective effect [42]. Therefore, it is speculated that HLJDD may play a better role in the treatment of COVID-19 through its competitive antagonism, anti-in ammation and anti-oxidation.

Conclusion
Overall, this study applied network pharmacology and molecular docking methods to explore the chemical constituents, action targets and its core active compounds in HLJDD, as well as the binding energy with SARS-CoV-2 3CL hydrolase, ACE2 and TMPRSS2. The active compounds such as stigmasterol, beta-  Figure 1 The target network diagram of the compound of Huanglian Jiedu Decoction. Inputting the active ingredients and targets of HLJDD from TCSMP into Cytoscape 3.7.2 software and producting the network upside. Among the network gure, yellow represents the compound of Scutellaria baicalensis, green represents the compound of Coptis chinensis , red represents the compound of Phellodendron amurense, and blue represents the compound of Gardenia jasminoides; the nodes` size represents the degree, the edges between the nodes represents the interrelations of the active compounds and targets.    KEGG enrichment pathway annotation A total of 306 signal pathways were obtained through Pathway analysis by reactome (P <0.05). The core targets in the network mainly involved the immune system, signal transduction, gene expression, programmed cell apoptosis, cell cycle, and cell response to external stimuli and so on. The rightmost color band indicates the main enrichment path, and the P value gradually increases as the color deepens. The center of each part is the root of a top-level path. One step away from the center represents the next lower hierarchy. Yellow indicates the obvious expression of the path, and gray indicates the path without obvious expression.

Figure 6
Tissue enrichment analysis of Huanglian Jiedu Decoction target As shown in Figure 7, tissue enrichment revealed that targets expression sites are mainly distributed in lung tissue, liver, and placenta, kidney, blood and involve a variety of immune cells, such as T cells and B cells.  KEGG pathway predicts the mechanism of HLJDD intervention in interleukin signaling in the immune system The core targets are mainly interferon signaling, interleukin signaling, growth hormone receptor signaling, prolactin receptor signaling, TNFR2 non-canonical NF-κB pathway, and FLT3 signaling in cytokine signaling in the immune system. The interleukin signaling pathway mainly involves IL-1 family signaling, IL-2 family signaling, IL-4 and IL-13 signaling, IL-3,IL-5 and GM-CSF signaling, IL-6 family signaling, IL-7 signaling , IL-10 signaling, IL-12 family signaling, IL-17 signaling, and IL-20 family signaling. The far right color band indicates that the P value gradually increases as the color deepens.