Preliminary Exploration Of The Mechanism Of Compound Qinlan Oral Liquid Against New Coronavirus Pneumonia Based On Network Pharmacology And Molecular Docking Technology

For more information:ali.ma@wecistanche.com

Abstract

Objective: Applying Traditional Chinese Medicine (TCM) network pharmacology and molecular docking technology to explore the mechanism of anti-coronary virus pneumonia (Corona Virus Disease 2019, COVID-19) of Compound Qinlan oral liquid. Methods: Traditional Chinese Medicines Integrated Database (TCMID), Traditional Chinese Medicine Systems Pharmacology (TCMSP), OMIM, GeneCards, String, and other online databases were used for building a series of networks and selecting the core targets, and analyzing the signal pathways. Finally, Discovery Studio 2016 software was used to conduct molecular docking of the main compounds (Chinese Medicine Legal Quality Control Compound) of Compound Qinlan oral liquid with key targets ACE2, 3CLpro, etc. Results: The results showed that Compound Qinlan oral liquid has specific effects on lung, heart, and stomach diseases. The Compound Qinlan oral liquid compound-pneumonia target network contained 98 compounds and 184 corresponding targets, and the core targets involved INS, TP53, IL6, VEGFA, ALB, and JUN. GO (GeneOntology) function enrichment analysis yielded 653 GO entries, and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment screening yielded 112 related pathways, including hypoxia-inducible factor-1 (HIF-1) and Toll-like receptor (TLRs) signaling pathway related to pneumonia, as well as Influenza A signaling pathway and Hepatitis B signaling pathway related to microbial infection. The results of molecular docking show that Isochlorogenic acid C, Baicalein, etc have a good binding capacity with ACE2, 3CLpro, AKT1, and other proteins. Conclusion: In this paper, we preliminarily explored the potential therapeutic mechanism for Compound Qinlan oral liquid against coronavirus pneumonia (COVID-19) and predicted the active ingredients. We hope that the results will help to further study the active ingredients and mechanism of Compound Qinlan oral liquid for anti-COVID-19.

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Keywords: COVID-19, Compound Qinlan oral liquid, Network Pharmacology, Molecular mechanism

Background

The new coronavirus(SARS-CoV-2)outbreak has been going on for seven months, leading to an examination of the role of traditional Chinese medicine (TCM) in the prevention and control of this new coronavirus pneumonia (COVID-19)epidemic. For example, the ability of TCM to reduce symptoms and control the progress of the disease has been recognized. In multiple editions of the 'New Coronavirus Infected Pneumonia Diagnosis and Treatment Program (Trial)' issued by the National Health Commission of the People's Republic of China, some Chinese patent medicines and decoctions have been proposed for the treatment of COVID-19, such as Huoxiang Zhengqi capsules (pills, water, oral solution), Jinhua Qinggan granules and Lianhua Qingwen capsules(particles). It can be seen from the above that TCM is widely used for blocking COVID-19, with significant effects and a considerable impact. It is highly popular amongst people in Asian countries. There is no doubt that TCM will undergo a development period as a result of this epidemic.

Compound Oinlan oral liquid is prepared from Jinyinhua(Lonicerae Japomicae Flos, Emperor drug), Huangqin (Scutellariae Radix, Minister drug), Lianqiao(Forsythiae Fructus, Assistant drug), and Banlangen (Isatidis Radix, Assistant drug), which has the functions of relieving surfaces, clearing away heat and detoxification. Traditionally, compound Qinlan oral liquid is mainly used for fever, cough, and sore throat, and has a clinically reported therapeutic effect on acute upper respiratory tract infection as well [1-3], with good safety features [4]. Based on the above advantages, Compound Qinlan oral liquid was selected from the list of recommended Chinese patent medicine preparations in the"Prevention and Treatment Program for Pneumonia with New Coronavirus Infection in Heilongjiang Province (Second Edition)". At present, compound Qinlan oral liquid has been applied to patients with new coronavirus pneumonia in the medical observation period and clinical treatment period in Hubei and other provinces. Clinical feedback showed that compound Qinlan oral liquid can effectively ameliorate clinical symptoms in diagnosed patients [5]. Recently, the Cui laboratory constructed a model of human coronavirus pneumonia and acute respiratory distress syndrome in BALB/c mice. Some mice were given compound Qinlan oral liquid 11 mL/kg/d and 22 mL/kg/d for 3 days, with a good effect on inhibiting virus replication, improving gastrointestinal function, enhancing immunity, and reducing lung cell inflammatory factors. This treatment thus had a good therapeutic effect in mouse coronavirus pneumonia. The results provide laboratory data support for the clinical application of compound Qinlan oral liquid, which can be used for clinical reference [5]. Therefore, it can be seen that the compound Qinlan oral liquid has shown good anti-COVID-19 efficacy in clinical applications and basic research.

Compound TCM is a pharmaceutical system based on the meticulous composition of medicinal materials, abundant active ingredients, as well as intricate targets and signaling pathways. These characteristics are consistent with the overall characteristics of TCM. Due to the complexity of the system, modern research and interpretation of TCM pose great difficulties and challenges. Network pharmacology [6] integrates disciplines such as systems biology, multi-directional pharmacology, computational biology, and network analysis to explore the correlation between Chinese medicine and disease from a holistic perspective, providing a system-level search for potential Chinese medicine prescriptions. The new strategy of active ingredients and targets of action is consistent with the multicomponent-multitarget interaction relationship of TCM, which is consistent with the viewpoint that TCM regulates the body at an overall level to exert a therapeutic effect. Therefore, network pharmacology has been widely used in the field of TCM for a preliminary prediction of the mechanism of action. Although Compound Qinlan oral liquid has significant clinical activity against COVID-19, there is still no literature report on its related mechanism of action. This study explored the biological pathways of COVID-19 clinical symptoms through network pharmacology to carry out a preliminary analysis of active substances and perform a preliminary exploration of the mechanism. These results represent an in-depth study of the anti-COVID-19 mechanism of compound Qinlan oral liquid.

Materials and methods

The establishment of a database of potential targets for compound Qinlan oral liquid

The obtained active ingredients were respectively searched for regarding their relevant targets in the TCMSP database, and after removing duplicates, the target of compound Qinlan oral liquid was obtained. Pneumonia and coronavirus were also used as keywords in the search, and after removing duplicates, the targets related to pneumonia and coronavirus were obtained. The two targets were combined to obtain a database of new coronavirus pneumonia-related targets. The targets of compound Qinlan oral liquid and new coronavirus pneumonia-related targets were taken to obtain the potential targets of compound Qinlan oral liquid for treating pneumonia caused by the new coronavirus.

Results

Meridian information network

The network of compound Qinlan oral liquid was established, as shown in Figure 1. The blue nodes represent the four herbal medicines, and the orange nodes represent various sutras. Three of the four herbs are attributed to the heart meridian, followed by the lung meridian, small intestine meridian, and stomach meridian.

Scree ning of active compounds

After searching through the three databases, there were 136 non-repetitive compounds that met the conditions OB≥30% and DL≥0.18. Among them, Lonicerae. Japonicae Flos contained 41, Scutellariae Radix contained 45, Forsythia Fructus contained 23, and Isatidis Radix contained 42.

Potential targets of compound Oinlan oral liquid In this study, we first retrieved 5,135 targets related to pneumonia and 351 targets related to coronavirus through three disease target databases. After deleting duplicates, 5,226 targets related to the target disease were obtained. In addition, a total of 248 targets related to drugs were retrieved. By taking the intersection of the two types of targets, we obtained 184 potential targets of compound Qinlan oral liquid.

Pharmacological network of compound Qinlan oral liquid

The compound Qinlan oral liquid - medicine -the compound-target-disease pharmacological network was established, as shown in Figure 2. The blue nodes are compound nodes, the red nodes are potential target nodes, the yellow nodes are disease nodes and the green nodes are medicinal material nodes. The network consists of 289 nodes(98 compound nodes and 184 target nodes) and 1,285 edges. In this network, we found that, on average, each compound was connected to 9.56 targets, and each target was linked to 5.09compounds so that multiple compounds act on the same target at the same time and a single compound acts on multiple targets. The phenomenon also reflects the therapeutic characteristics of multiple components of TCM acting on multiple targets. From the point of view of the target, the more targets a single target is affected, the more the compound from compound Qinlan oral liquid may be to this target. It was found that 10.33% of the targets had no less than ten compounds. Interactions occurred where 12 targets were linked to more than 20 compounds.

PPI network of potential targets

Using the STRING website to analyze the obtained potential targets, a PPI network diagram was obtained, as shown in Figure 3.

The size of each node represents the grade value (degree), the color from light to dark represents the centrality value (betweenness centrality), and the thickness and color of the line represent edge betweenness and combined score, respectively. Topological analysis of the PPI network can determine hub proteins in this network. After comprehensively examining the three indicators of degree, betweenness centrality, and closeness centrality, the top six hubs were INS, TP53, IL6, VEGFA, ALB, and JUN. The level values were all over 110.

Using the MCODE function in Cytoscape to conduct a module analysis of the PPI network, there were a total of six modules that met the conditions. Such modules indicate that the proteins in them are closely related. Furthermore, they may jointly perform a biological process, as shown in Figure 4. The quadrilateral label in the figure is the original node, and MCODE expands from this node to find the node that meets the requirements among its neighboring nodes. Qualified nodes are indicated by round labels. The shade of each node’s color indicates its MCODE score. When using MCODE for module analysis, the score of module 1 was 45.62, the score of module 2 was 8.00, the score of module 3 was 4.00, the score of module 4 was 4.00, the score of module 5 was 3.33 and the score of module 6 was 3.2.

Results of GO enrichment analysis and KEGG pathway analysis 

Using the DAVID website to perform GO enrichment analysis on potential targets, a total of 653 GO entries with a P-value < 0.05 were obtained, including 482 biological processes (BP) entries, 54 cell composition (CC) entries, and molecules. There were 117 molecular functions (MF) entries; the top 20 channels of each category are shown in Figure 5. A total of 112 channels with a P-value < 0.05 were obtained through KEGG pathway analysis, with the top 30 pathways shown in Figure 5. In addition to some cancer pathways, these mainly included pathways related to inflammation and microbial infections.

Molecular docking 

A total of six proteins and 17 compounds were selected for molecular docking. The relevant information of every single protein and compound is shown in Table 1 and Table 2.

The results of molecular docking are shown in Table 3. Except for the 7BW4 protein that had no relevant ligands in the original structure, other proteins use the original ligand for docking and to calculate the RMSD values. These values were less than 2, indicating a small difference from the real situation with the docking mode obtained using molecular docking. The theoretical calculation reproduced the experimental results, and the current docking method was feasible. In the docking results, the index used was the opposite of the binding energy. Some small molecules fail to meet the relevant thresholds during docking, so there is no docking result. This is considered to indicate an extremely unmatched receptor and ligand and docking cannot be performed. Among the docking results of 1R4L and 6WX4 proteins, the main chemical components of compound Qinlan oral liquid were not as good as the original ligand. The other four proteins were better than the original ligand. The two compounds with the best docking for each protein were selected for mapping, as shown in Figure 6.

Discussion

According to a recent report, the Institute of Chinese Materia Medica of the Chinese Academy of Chinese Medical Sciences has successfully constructed an animal model suitable for the evaluation of the efficacy of TCM in the treatment of new coronavirus pneumonia. This model was used to verify the recommended series of prescriptions, such as Lianhua Qingwen capsules, Jinhua Qinggan granules, and Shufeng Jiedu capsules. At the same time, it verified a variety of Chinese medicines that had been recommended in Hubei and other provinces, such as Kesuzhi syrup and compound Oinlan oral liquid, which have been used clinically for the treatment of new coronavirus pneumonia. The results show that these drugs can significantly reduce inflammatory injury to the lungs in mice, reduce the viral load and the level of inflammatory factors in the lung tissue, and increase the proportion of immune cells in the peripheral blood. Drug efficacy is consistent with this clinical effect. Clinical feedback shows that the clinical symptoms of patients with new coronavirus pneumonia are mostly fever, cough, and nasal congestion, and rhinorrhea [7. Therefore, it is scientifically reasonable to carry out a TCM intervention for such symptoms. According to reports in the literature, a multi-center clinical evaluation of compound Qinlan oral liquid for treating acute upper respiratory tract infection with the exogenous wind-heat syndrome has been confirmed [1l, and compound Oinlan oral liquid has a significant effect on acute upper respiratory tract infections, as it can target nasal congestion, runny nose, fever, dry or sore throat, and cough with a good therapeutic effect and good safety [2].

This study is the first to analyze the potential anti-COVID-19 mechanism of compound Qinlan oral liquid by network pharmacology. By querying various online databases to obtain the meridian, ingredients, and target information of the four Chinese medicine, we then constructed and analyzed the Chinese medicine-meridian network, target network, disease pathways, and basic biological pathways for important target interventions to explore the anti-COVID-19 effects of compound Qinlan oral liquid using TCM syndrome differentiation and a multi-component and multi-target molecular mechanism.

Based on a theoretical analysis of TCM, we can see that the cause of the new coronavirus pneumonia is mainly 'wet'[8], and the heat is deep. It should disperse cold and dampness, aromatize turbidity, penetrate the surface to disperse evil, and lift the spleen and stomach. Therefore, the related organs are the lung, large intestine, spleen and stomach (humidification), and skin (relief). Aromatisation is related to multiple organs, i.e.the lung, spleen and stomach, liver, kidney, and skin. According to clinical feedback, the new coronavirus pneumonia not only induces lung damage but also involves organs such as the heart and kidneys. "Therefore, Chinese medicine theory and Western medicine diagnosis have good practical consistency. According to the meridian analysis of compound Qinlan oral liquid, according to the theory of Chinese medicine, three of the four Chinese herbal medicines are all attributed to the heart channel, followed by the lung channel, the small intestine channel, and the stomach channel. It can protect the heart, stomach, intestines, and other organs.

Moreover, by searching the database and the Chinese Pharmacopoeia (2015 version) and literature, a total of 184 chemical components were obtained, and 5,226 viral pneumonia targets were obtained. After intersecting with drug targets,184 targets were obtained. The anti-COVID-19 effect is achieved by a variety of active ingredients through a wide range of targets. PPI network topology analysis was used to demonstrate the compound protein Qinlan oral liquid - medicinal herbs -active ingredients - targets - hub proteins in pneumonia network, of which the top six are INS, TP53, IL6, VEGFA.ALB and JUN.GO enrichment analysis of potential targets revealed that the cell composition was mainly enriched in the outer space of the cell, and the molecular function was mainly enriched in the items related to protein binding. In terms of biological processes, it was mainly enriched in terms related to the cell cycle, inflammation, and responses to some substances. 

Inflammation is an important symptom of pneumonia. At the same time, we also obtained an entry related to responses to lipopolysaccharide. As a component of the outer cell wall of Gram-negative bacteria, lipopolysaccharide is also a component of the new coronavirus. These results suggest that compound Qinlan oral liquid has a certain effect in the treatment of pneumonia caused by the new coronavirus. It is worth noting that, in these three GO analyses, items related to RNA polymerase transcription factors were enriched; current treatment with the new drug ribavirin impairs RNA-guided RNA polymerase in the treatment of related diseases. It has been speculated that compound Qinlan oral liquid has a similar effect in the treatment of pneumonia caused by the new coronavirus. In the KEGG pathway analysis, in addition to cancer-related pathways, we obtained three types of pathways. The first was inflammation-related pathways, including the PI3K-Akt, HF-1, and TNF signaling pathways.

The second was pathways related to microbial infections, including hepatitis B, influenza A, hepatitis CTuberculasis and other pathways. The third was pathways related to immunity, including the NF-KB, T cell receptor, and NOD-like receptor signaling pathways. These three aspects are closely related to pneumonia caused by the new coronavirus. It is speculated that the compound Qinlan oral liquid may play a therapeutic role by inhibiting inflammation, eliminating pathogens, and enhancing immune function. This was confirmed by GO analysis and KEGG analysis.

When screening the proteins used for docking, we first searched for proteins related to the new coronavirus in the RCSB database. At present, there are three kinds of proteins that are resolved, which are the main protein, papain-like protein, and RNA-dependent RNA polymerase. Among them, RNA-dependent RNA polymerase is likely to be a target for the drug ribavirin, which has been used in the treatment of new coronavirus pneumonia. At the same time, we found the carboxypeptidase (ACE2)associated with the human angiotensin-converting enzyme, which is considered as the target of the new coronavirus, and used these four proteins as virus-related targets. Secondly, we also selected two existing therapeutic targets, i.e. transmembrane serine protease and cathepsin B. According to the verification of the RMSD value, the docking conditions were suitable, but in the docking results of the 1R4L and 6WX4 proteins, the docking results of the main chemical components of compound Qinlan oral liquid are not as good as those of the original ligand. The reason why the selected compound docking results are worse than the original ligand is probably that the original ligand is extremely tightly bound. We can see that the reverse number of the original ligand's binding energy is much larger than that of several other proteins. Therefore, for these two proteins, even if the binding energy of the selected ligand is less than the original ligand, it should still have a certain significance. In the docking process, is chlorogenic acid C, quercetin, kaempferol, and caffeic acid all performed well? It has been reported [9,10] that the main active ingredients of compound Qinlan oral liquid are chlorogenic acid 1.05 mg/mL,caffeic acid 0.29 mg/mL, baicalin 13.78 mg/mL, baicalin 0.48 mg/mL，（R，S）-gaovichun 8.88 μg/mL and forsythin 0.5 mg/mL. The Chinese Pharmacopoeia (2015 edition)stipulates that baicalin, chlorogenic acid, and forsythin should be used as quality control indicators for compound Qinlan oral liquid. Chlorogenic acid has broad-spectrum antibacterial activity and has a certain inhibitory effect on E. col. It has good resistance to Staphylococcus aureus, yeast, Aspergillus niger, and Bacillus subtilis, as well as Staphylococcus aureus and E. cli, the effect on fungi is stronger than that on bacteria [1]. Chlorogenic acid has a significant inhibitory effect on respiratory syncytial virus, a synergistic effect when combined with licorice, and enhances the anti-respiratory syncytial virus effect in vitro[12]. Baicalin also has an anti-RSV role in vivo and in vitro, can inhibit multiple stages of viral infection, and improves lung damage caused by RSV infection. Baicalin has better anti-inflammatory and antioxidant effects than ribavirin(Tavivir)and can significantly improve the symptoms of severe influenza patients and shorten the course of disease [131. Clinically, Su et al.[14] found that the main ingredients of the compound Qinlan oral liquid can directly inhibit respiratory viruses. Forsythia suspensa extracts have good anti-influenza A effects in vivo and in vitro [15.16]. so this chemical may be the main active ingredient of compound Qinlan oral liquid against COVID-19.

In summary, this study used network pharmacology to predict the biological mechanism of compound Qinlan oral liquid against new coronavirus pneumonia (COVID-19). We initially explored the compound Qinlan oral liquid against 2019-nCoV-induced pneumonia biological pathways and the main pharmacodynamic substances of the prescription, which provides a certain reference for the clinical of follow-up Chinese medicine. Without a doubt, the results require further clinical verification.

Conclusion

Based on the previous reports of its clinical efficacy and laboratory evidence, we further predicted that compound Qinlan oral liquid mainly acts on the lungs and heart Meridian. The network established under the indicator components and other main components from CQOL showed that the mechanism involved in its function is hypoxia-inducible factor-1 (HIF-1)and Toll-like receptor (TLRs) signaling pathways related to pneumonia, as well as Influenza A and Hepatitis B signaling pathways related to microbial infection. Characteristic active ingredients such as is chlorogenic acid C, quercetin, kaempferol, and caffeic acid showed well binding energy with the selected representative protein target, which consolidated clinical efficacy and mechanism of action. Collectively, these results suggest that COOL might be a reliable modem herbal medicine for relieving COVID-19 symptoms.

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