Screening method of application of mysorethorn cold mixture in treating respiratory syncytial virus

Abstract

The invention discloses a screening method of application of a mysorethorn cold mixture to treatment of respiratory syncytial virus. According to the method, network pharmacology and a molecular docking technology are combined, and active ingredients, action targets and action mechanisms of the mysorethorn cold mixture for treating respiratory syncytial virus diseases are explored; an experimental basis is provided for treating the respiratory syncytial virus disease by the mysorethorn cold mixture, and the mysorethorn cold mixture is of great significance in promoting clinical application of traditional Chinese medicines in treating the respiratory syncytial virus disease.

Description

[0001] This application is a divisional application of Patent Application No. CN202311488234.0

[0002] Original filing date: November 29, 2023

[0003] Original application number: CN202311488234.0

[0004] Original invention title: A new use of Yunshi Ganmao Mixture and its screening method Technical field

[0005] The present invention relates to the technical field of traditional Chinese medicine preparations, and specifically relates to a screening method for the use of Yunshi Ganmao Mixture in the treatment of respiratory syncytial virus Background art

[0006] Human Respiratory Syncytial Virus (HRSV) is the most common pathogen causing acute lower respiratory tract infections in infants and young children. The main pathological changes involve bronchioles, trachea and alveoli, leading to HRSV pneumonia. Clinical manifestations include fever, cough, dyspnea, wheezing, etc [1] A retrospective study showed that there were 33.1 million newly diagnosed cases of acute lower respiratory tract infections caused by HRSV in children under 5 years old globally, among which 2.9 million children needed hospitalization and 59,600 children died; among children under 6 months old, 1.4 million children needed hospitalization, among which 27,300 children died [2] ; According to the report of the National Respiratory and Enteric Virus Surveillance System (NREVSS) in the United States, HRSV is also one of the main causes of upper respiratory tract symptoms in adults and causes a relatively high mortality rate in infections of immunocompromised patients [3] Currently, there are no specific drugs and vaccines against HRSV infection, and clinical treatment mainly focuses on symptomatic treatment and supportive treatment. When patients use ribavirin, interferon, bronchodilators, hormonal drugs, etc., they often experience drug adverse reactions such as fever and allergy [4-6] Traditional Chinese medicine, guided by the overall concept and syndrome differentiation and treatment, has proposed a series of treatment methods for anti-HRSV infection, such as clearing the lung, resolving phlegm, and relieving cough, and has achieved good clinical efficacy. Zhao Xia et al [7-8] The study confirmed that Jinxin Koufuye may antagonize its inhibitory effect on apoptosis by regulating apoptosis-related genes in the early stage of HRSV infection, thereby affecting the virus replication and proliferation process entering cells and preventing the spread of the virus; Zheng Yan et al [9] The study confirmed that Shanggan Keli can inhibit the expression of TLR3 and PKR mRNA in the lung tissue of mice with viral upper respiratory tract infection; Lan Dan et al

[10] The experimental results showed that HRSV can induce bronchial epithelial cells (NHBE) to secrete TSLP, and Retoxin can inhibit the increase in TSLP secretion caused by HRSV, and the effect is more obvious when administered as a preventive measure; Wang Xue

[11] et al. screened the anti-respiratory syncytial virus targets of Jinchan oral liquid based on network pharmacology, and the results showed that there were 11 compounds involved in the target network of the main active ingredients, and there were 22 corresponding anti-HRSV infection target proteins. It can be seen that traditional Chinese medicine treatment of HRSV shows a multi-target therapeutic effect.

[0007] Yunshi Cold Mixture is an exclusive product of Guizhou Liangji Pharmaceutical Co., Ltd., and obtained a new approval number in 2016: National Medicine Standard Z20163021. Yunshi Cold Mixture is a folk prescription used by the Miao people in Guizhou to prevent and treat colds for a long time. This prescription is included in the monograph "Miao Pharmacy". The prescription is composed of four Chinese medicinal materials: Yunshi peel, Lanbuzheng, verbena, and ginger. Modern pharmacological analysis shows that the chemical components contained in the above medicinal materials have obvious effects of enhancing immunity, antiviral, antibacterial, anti-inflammatory, and relieving cold symptoms such as aversion to cold and fever, nasal congestion and runny nose, body aches, cough and sputum. It is safe and has no toxic side effects. It is a cold medicine that can be taken by the elderly, children and pregnant women. Yunshi Cold Mixture is specially used to treat colds caused by wind and cold (and wind and heat colds), has clear efficacy, has the triple effects of rapid sweating, improving dizziness and aches, and relieving cough and reducing phlegm, and is fast and effective, and can quickly improve the four major cold symptoms such as nasal congestion, aversion to cold, aches, and coughs. It treats both the symptoms and the root cause, and has unique ingredients that enhance immunity, preventing and treating colds from the root.

[0008] The present invention takes Yunshi cold mixture as the research object. In exploring the mechanism of action of Yunshi cold mixture in treating colds caused by wind and cold, it is found that Yunshi cold mixture can be used for the new use of treating respiratory syncytial virus. Then, the present invention studies the immune mechanism of Yunshi cold mixture in treating respiratory syncytial virus, and constructs the Yunshi cold mixture-active ingredient-key target-disease network through network pharmacology and molecular docking technology, providing pharmacological evidence for the new use of Yunshi cold mixture against respiratory syncytial virus. It also provides an experimental basis for Yunshi cold mixture in treating respiratory syncytial virus, which is of great significance for promoting the clinical application of traditional Chinese medicine in treating respiratory syncytial virus. Summary of the invention

[0009] The purpose of the invention is to provide a new use of Yunshi cold mixture.

[0010] Another object of the present invention is to provide a method for screening new uses of Yunshi cold mixture.

[0011] The new use specifically includes use in preparing drugs for treating respiratory syncytial virus diseases, and use of the active ingredients in the Yunshi cold mixture screened by the method in preparing drugs for treating respiratory syncytial virus diseases.

[0012] The present invention is achieved through the following technical solutions:

[0013] Regarding the new use of Yunshi Ganmao Mixture of the present invention, the active ingredients of the Yunshi Ganmao Mixture are prepared from the following Chinese herbal medicine raw materials in parts by weight: 200 parts of Yunshi bark, 300 parts of Geum japonicum Thunb., 300 parts of Verbena officinalis L., and 200 parts of ginger. The application of the Yunshi Ganmao Mixture in the preparation of drugs for treating respiratory syncytial virus diseases.

[0014] Regarding the screening method for the new use of Yunshi Ganmao Mixture of the present invention, the method is to analyze its mechanism of action by combining network pharmacology and molecular docking technology, and specifically includes the following steps:

[0015] (1) Prediction of active ingredients and key core targets of Yunshi Ganmao Mixture:

[0016] Input the single herbs in the Yunshi Ganmao Mixture into the Traditional Chinese Medicine Systems Pharmacology Analysis Platform respectively. Taking the oral bioavailability of the biological ≥ 30% and the drug-likeness (DL) ≥ 0.18 as the conditions for screening active ingredients, and record their targets. Use the Swiss TargetPrediction online website and TargetNet online website to supplement the targets to obtain the action targets of the Yunshi Ganmao Mixture.

[0017] (2) Obtaining potential targets of respiratory syncytial virus:

[0018] Search for "Respiratory Syncytial Virus" in the GeneCards database, the Online Mendelian Inheritance in Man (OMIM) database, and the DisGeNet database respectively, and convert the protein targets to Gene Symbol through the UniProt database; after screening out duplicate values, obtain the anti-HRSV related targets.

[0019] (3) Obtaining common targets of Yunshi Ganmao Mixture and respiratory syncytial virus:

[0020] Input the action targets of the Yunshi Ganmao Mixture obtained in step (1) and the anti-HRSV related targets obtained in step (2) into the Venny 2.1.0 online platform to obtain the Venn diagram of the intersection of the two target proteins.

[0021] (4) Construction of the traditional Chinese medicine-ingredient-target network diagram:

[0022] Use Cytoscape 3.2.1 software to construct the traditional Chinese medicine-ingredient-target network diagram in the Yunshi Ganmao Mixture, and use the "Analyze network" function of this software to perform topological analysis of the Yunshi Ganmao Mixture.

[0023] (5) Construction of protein - protein interaction (PPI) network:

[0024] Input the 71 targets obtained in step (3) into the STRING database, analyze and predict the interaction relationships between proteins through the STRING database, including direct and indirect relationships between protein interactions, construct a PPI network, and analyze to obtain core acting targets;

[0025] (6) Gene Ontology (GO) biological function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis:

[0026] Input the mapped targets obtained in step (4) into the Metascape database, perform GO analysis and KEGG related pathway analysis using the Metascape database, and visualize them with the online drawing website Microbial Informatics.

[0027] (7) Verification of "component - key core target" molecular docking analysis:

[0028] Perform molecular docking on the two top - ranked active components of Yunshi Ganmao Mixture obtained in step (1) and the top - ranked core acting targets of Yunshi Ganmao Mixture obtained in step (5); download the high - resolution three - dimensional crystal structure of the target protein from the PBD database, and perform molecular pre - processing using PyMol and Autodock Tools; run Autodock Vina for batch semi - flexible molecular docking, and visualize the output results of small molecules with the PyMol software.

[0029] The visualization analysis of the present invention shows that the anti - viral effect of Yunshi Ganmao Mixture mainly involves TNF signaling pathway, IL - 17 signaling pathway, Toll - like signaling pathway, Th17 cell pathway, C - type lectin receptor signaling pathway, PD - 1 / PD - L1 pathway, PI3K / AKT pathway, etc.

[0030] The core targets of the present invention are JUN, IL6, and PTGS2.

[0031] The active components of the present invention are quercetin and 3 - deoxysapnaretin.

[0032] The docking analysis of the present invention shows that quercetin binds to the active sites of JUN and IL6 proteins; 3 - deoxysapnaretin binds to the active site of PTGS2 protein.

[0033] The application of quercetin and 3 - deoxysapnaretin of the present invention in the preparation of a medicament for treating respiratory syncytial virus diseases by adding pharmaceutically acceptable excipients to prepare a pharmaceutically acceptable preparation.

[0034] The preparation of the present invention is a solid preparation or a liquid preparation.

[0035] The solid preparations of the present invention are tablets, capsules, granules, pills, powders, and lyophilized powder preparations. The liquid preparations of the present invention are injections and oral liquids.

[0036] The beneficial effects of the present invention are as follows:

[0037] 1. The Yunshi Ganmao Mixture of the present invention can be used to treat respiratory syncytial virus diseases. By applying the Yunshi Ganmao Mixture to respiratory syncytial virus model mice and calculating the inhibition rates of lung indices in each treatment group, it was found that the inhibition rate of the lung index in the ribavirin group was 2.99%, and the treatment groups of the Yunshi Ganmao Mixture at low dose of 1.56 g / kg, medium dose of 7.8 g / kg, and high dose of 39 g / kg reached 1.43%, 3.40%, and 6.86% respectively. That is, after treatment with the Yunshi Ganmao Mixture, the lung index in the high-concentration group of the Yunshi Ganmao Mixture decreased significantly (P < 0.05), and there were also obvious downward adjustments in the ribavirin group, the low-concentration group, and the medium-concentration group of the Yunshi Ganmao Mixture, indicating that the Yunshi Ganmao Mixture can exert an anti-HRSV effect.

[0038] 2. Through network pharmacology prediction of the Yunshi Ganmao Mixture of the present invention, 637 potential active ingredients and 329 anti-HRSV targets of the Yunshi Ganmao Mixture were obtained, and 71 targets were the same between the Yunshi Ganmao Mixture and HRSV. By analyzing the Yunshi Ganmao Mixture and HRSV, the results showed that the Yunshi Ganmao Mixture can exert an anti-HRSV effect through biological processes such as positive regulation of lipopolysaccharide, bacterium-derived molecules, and cytokines by cells.

[0039] 3. The active ingredients quercetin and 3-deoxysarpanchalcone screened from the Yunshi Ganmao Mixture of the present invention have strong binding abilities to the key targets (JUN, IL6, PTGS2); quercetin binds to the active sites of JUN and IL6 proteins; 3-deoxysarpanchalcone binds to the active site of PTGS2 protein. The active ingredients have good binding abilities to the core targets, indicating that the Yunshi Ganmao Mixture has the potential to treat HRSV. It provides pharmacological evidence for the new use of the Yunshi Ganmao Mixture against respiratory syncytial virus. It is of great significance for promoting the clinical application of traditional Chinese medicine in treating respiratory syncytial virus.

[0040] 4. By applying the Yunshi Ganmao Mixture to respiratory syncytial virus model mice, the results showed that after infection with HRSV, both WBC and LYM would decrease, and after treatment with the Yunshi Ganmao Mixture, both WBC and LYM would increase; the Yunshi Ganmao Mixture can reduce the contents of IgM, TNF-a, IL-6, and IL-1β in the serum of mice after HRSV infection; it can also down-regulate the expression levels of TLR4 / NF-κB mRNA. It is proved that the Yunshi Ganmao Mixture can exert an anti-HRSV effect. Description of the Drawings

[0041] Figure 1:Venn diagram of genes of Yunshi Ganmao Mixture and HRSV

[0042] Figure 2 :Network diagram of active ingredients of Yunshi Ganmao Mixture - HRSV targets - pathways

[0043] Figure 3 :PPI network diagram of Yunshi Ganmao Mixture and HRSV targets

[0044] Figure 4 :Bar chart of GO analysis

[0045] Figure 5 :KEGG pathway enrichment analysis

[0046] Figure 6 :Molecular docking of some key targets and active ingredients of Yunshi Ganmao Mixture (where A is quercetin and JUN; B is quercetin and IL6; C is 3 - deoxysarpanchalcone and PTGS2)

[0047] Figure 7 :Morphology of Hep - 2 cells under microscope (where A: control group; B: treated with HRSV for 48h)

[0048] Figure 8 :Respiratory syncytial virus - induced mouse model and body weight changes (where (a): schematic diagram of animal modeling and drug administration; (b): body weight changes of mice in each group;)

[0049] Figure 9 :Comparison of organ indices of mice in each group (Note: compared with the blank group, # P < 0.05; ## P < 0.01, compared with the HRSV group, *P < 0.05; **P < 0.01;)

[0050] Figure 10 :Pathological changes of lung tissues of mice in each group (HE)

[0051] Figure 11 :Flow cytometry diagram of T cell subsets

[0052] Figure 12 :CD3 + Types and numbers of peripheral blood T cell subsets in group

[0053] Figure 13 :CD3 + CD4 + Types and numbers of peripheral blood T cell subsets in group

[0054] Figure 14 :CD3 + CD8 + Types and numbers of peripheral blood T cell subsets in group

[0055] Figure 15 : T cell CD4 + / CD8 + Ratio

[0056] Figure 16 : Relative expression levels of HRSV mRNA in the lung tissues of mice in each group (where, compared with the blank group, #P < 0.05; ##P < 0.01, compared with the HRSV group, *P < 0.05; **P < 0.01.)

[0057] Figure 17 : IgM levels in the sera of mice in each group (where, A: Expression level of IgM in the serum; B, C, D: Cytokines TNF-a, IL-6, IL-1β; compared with the blank group, #P < 0.05; ##P < 0.01, compared with the HRSV group, *P < 0.05; **P < 0.01.)

[0058] Figure 18 : Relative expression levels of TLR4 mRNA in the lung tissues of mice in each group (where, compared with the blank group, #P < 0.05; ##P < 0.01, compared with the HRSV group, *P < 0.05; **P < 0.01.)

[0059] Figure 19 : Relative expression levels of NF-kB mRNA in the lung tissues of mice in each group (where, compared with the blank group, #P < 0.05; ##P < 0.01, compared with the HRSV group, *P < 0.05; **P < 0.01.) Detailed implementation manners

[0060] The technical solutions of the present invention will be further specifically described below through specific embodiments.

[0061] Example 1 Screening method for new uses of Yunshi Ganmao Mixture

[0062] (1) Prediction of active ingredients and key core targets of Yunshi Ganmao Mixture:

[0063] Input the single herbs in Yunshi Ganmao Mixture into the Traditional Chinese Medicine Systems Pharmacology Analysis Platform respectively. With the conditions of bio-oral availability ≥ 30% and drug-likeness (DL) ≥ 0.18 for screening active ingredients, and record their targets. Supplement the targets with the online websites of Swiss TargetPrediction and TargetNet to obtain a total of 637 action targets of Yunshi Ganmao Mixture;

[0064] (2) Obtaining potential targets of respiratory syncytial virus:

[0065] Search for "Respiratory Syncytial Virus" in the GeneCards database, the Online Mendelian Inheritance in Man (OMIM) database, and the DisGeNet database respectively. The search keyword is "Respiratory Syncytial Virus", and the protein targets are converted into Gene Symbols through the UniProt database; after screening out duplicate values, 329 anti-HRSV related target genes are obtained;

[0066] (3) Obtaining the common targets of Yunshi Ganmao Mixture and respiratory syncytial virus:

[0067] Input the action targets of Yunshi Ganmao Mixture obtained in step (1) and the anti-HRSV related targets obtained in step (2) into the online platform Venny 2.1.0 to obtain a Venn diagram of the intersection of the target proteins of the two, and 71 common targets of the two can be obtained;

[0068] (4) Construction of the traditional Chinese medicine-ingredient-target network diagram:

[0069] Use Cytoscape 3.2.1 software to construct the traditional Chinese medicine-ingredient-target network diagram of Yunshi Ganmao Mixture, and use the "Analyze network" function of this software to perform topological analysis of Yunshi Ganmao Mixture;

[0070] (5) Construction of the protein-protein interaction (PPI) network:

[0071] Input the 71 targets obtained in step (3) into the STRING database, analyze and predict the interaction relationships between proteins through the STRING database, including the direct and indirect relationships between protein interactions, construct a PPI network, and the core action targets can be obtained through analysis;

[0072] (6) Gene Ontology (GO) biological function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis:

[0073] Input the mapped targets obtained in step (4) into the Metascape database, use the Metascape database to perform GO analysis and KEGG related pathway analysis, and visualize them using the online drawing website Microbial Informatics;

[0074] Results: 2395 GO analysis entries were obtained, including 2236 in Biological Processes (BP), 61 in Cellular Components (CC), and 98 in Molecular Functions (MF); 164 pathways related to Yunshi Ganmao Mixture against HRSV were enriched by KEGG. The top 6 pathways related to antiviral immunity were selected for result visualization, as shown in Figure 5 . It can be seen that the antiviral effect of Yunshi Ganmao Mixture mainly involves TNF signaling pathway, IL-17 signaling pathway, Toll-like signaling pathway, Th17 cell pathway, C-type lectin receptor signaling pathway, and PD-1 / PD-L1 pathway.

[0075] (7) Verification of "ingredient-key core target" molecular docking analysis:

[0076] Perform molecular docking of the top two active ingredients of Yunshi Ganmao Mixture obtained in step (1) and the top-ranked core action targets of Yunshi Ganmao Mixture obtained in step (5); download the high-resolution three-dimensional crystal structure of the target protein from the PBD database, and perform molecular preprocessing using PyMol and Autodock Tools; run Autodock Vina for batch semi-flexible molecular docking, and visualize the small molecule output results using PyMol software.

[0077] The results showed that quercetin binds to the active sites of JUN and IL6 proteins (see Figure 6 -A, B); the active sites of JUN and IL6 proteins ( Figure 6 -A, B); 3-deoxysapanchalcone binds to the active site of PTGS2 protein ( Figure 6 -C).

[0078] Example 2

[0079] Use of the granule prepared by taking Yunshi Ganmao Mixture as the raw material drug, concentrating, adding 1 / 9 of starch or dextrin, granulating, in the preparation of a drug for treating respiratory syncytial virus diseases.

[0080] Example 3

[0081] Use of the pill prepared by taking Yunshi Ganmao Mixture as the raw material drug, concentrating, adding and mixing evenly with 1 / 11 of starch or dextrin, drying, in the preparation of a drug for treating respiratory syncytial virus diseases.

[0082] Example 4

[0083] Use of the tablet prepared by taking Yunshi Ganmao Mixture as the raw material drug, concentrating, adding 1 / 10 of starch, granulating, and tabletting, in the preparation of a drug for treating respiratory syncytial virus diseases.

[0084] Example 5

[0085] Taking Yunshi Ganmao Mixture as the raw material drug, concentrating it, adding 12 times the amount of injection water, soaking for 1 hour, filtering, and sterilizing to obtain the application of the injection in the preparation of drugs for treating respiratory syncytial virus diseases.

[0086] Example 6

[0087] Taking any one of quercetin and 3-deoxysapachalcone as the raw material drug, or taking quercetin and 3-deoxysapachalcone as the raw material drug in a ratio of 1:1, adding 1 / 9 of starch or dextrin, granulating to obtain the application of the granule in the preparation of drugs for treating respiratory syncytial virus diseases.

[0088] Example 7

[0089] Taking any one of quercetin and 3-deoxysapachalcone as the raw material drug, or taking quercetin and 3-deoxysapachalcone as the raw material drug in a ratio of 1:1, adding and mixing evenly with 1 / 11 of starch or dextrin, drying to make pills to obtain the application of the pills in the preparation of drugs for treating respiratory syncytial virus diseases.

[0090] Example 8

[0091] Taking any one of quercetin and 3-deoxysapachalcone as the raw material drug, or taking quercetin and 3-deoxysapachalcone as the raw material drug in a ratio of 1:1, adding 1 / 10 of starch, granulating, tabletting to make tablets to obtain the application of the tablets in the preparation of drugs for treating respiratory syncytial virus diseases.

[0092] To further verify the feasibility of the present invention, the inventors conducted a series of experiments, and the steps are as follows:

[0093] I. Experimental method: Prediction of key core targets of active components of Yunshi Ganmao Mixture against respiratory syncytial virus

[0094] 1 Prediction of key core targets of active components of Yunshi Ganmao Mixture against respiratory syncytial virus

[0095] 1.1 Prediction of active components of Yunshi Ganmao Mixture and their targets

[0096] Input the single herbs in Yunshi Ganmao Mixture into the Traditional Chinese Medicine Systems Pharmacology Analysis Platform respectively, with the conditions of biological oral bioavailability ≥ 30% and drug-likeness (DL) ≥ 0.18 as the conditions for screening effective components, and record their targets. Use the Swiss TargetPrediction online website and TargetNet online website to supplement the targets to obtain the action targets of Yunshi Ganmao Mixture.

[0097] 1.2 Obtaining potential targets of respiratory syncytial virus

[0098] Search for "Respiratory Syncytial Virus" in the GeneCards database, the Online Mendelian Inheritance in Man (OMIM) database, and the DisGeNet database respectively. The search keywords are "RespiratorySyncytial Virus", etc., and the protein targets are converted into Gene Symbol through the UniProt database; after screening out duplicate values, the anti-HRSV related targets are obtained.

[0099] 1.3 Results of obtaining the common targets of Yunshi Ganmao Mixture and respiratory syncytial virus

[0100] Input the relevant targets under "1.1" and "1.2" into the online platform Venny 2.1.0 to obtain the Venn diagram of the intersection of the two target proteins.

[0101] 1.4 Construction of the traditional Chinese medicine-ingredient-target network diagram

[0102] Use Cytoscape 3.2.1 software to construct the traditional Chinese medicine-ingredient-target network diagram in Yunshi Ganmao Mixture, and use the "Analyze network" function of this software to perform topological analysis on Yunshi Ganmao Mixture.

[0103] 1.5 Construction of the protein-protein interaction (PPI) network

[0104] Analyze and predict the interaction relationship between proteins through the STRING database, including the direct and indirect relationships between protein interactions.

[0105] 1.6 Gene Ontology (GO) biological function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis:

[0106] Input the mapped targets under "1.4" into the Metascape database, use the Metascape database to perform GO analysis and KEGG related pathway analysis, and visualize them with the online drawing website Microbial Informatics.

[0107] 1.7 Molecular docking analysis and verification of "ingredient-key core target":

[0108] To further verify the screened active ingredients and core targets, perform molecular docking on the top two active ingredients of Yunshi Ganmao Mixture obtained under "1.1" and the core action targets of Yunshi Ganmao Mixture under "1.5". Download the high-resolution three-dimensional crystal structure of the target protein from the PBD database, and use PyMol and Autodock Tools for molecular preprocessing. Run Autodock Vina for batch semi-flexible molecular docking, and visualize the small molecule output results with PyMol software.

[0109] Using the top three targets in Yunshi Ganmao Mixture under item "1.1" as small molecule ligands, download the protein ligands of the corresponding targets from the PDB database; use Autodock vina software for docking verification.

[0110] 2 Results

[0111] 2.1 Active Ingredients and Target Prediction of Yunshi Ganmao Mixture

[0112] Through the Traditional Chinese Medicine Systems Pharmacology Analysis Platform, the Swiss Target Prediction online website, and the TargetNet online website, a total of 637 action targets of the effective active ingredients of Yunshi Ganmao Mixture were finally obtained.

[0113] 2.2 Obtaining Potential Targets of Respiratory Syncytial Virus

[0114] Search the GeneCards database, the Online Mendelian Inheritance in Man (OMIM) database, and the DisGeNet database for the English name of respiratory syncytial virus, and convert the protein targets into GeneSymbol through the UniProt database; after screening out duplicate values, 329 anti-HRSV related target genes were obtained.

[0115] 2.3 Results of Obtaining Common Targets of Yunshi Ganmao Mixture and Respiratory Syncytial Virus

[0116] Taking the intersection of the obtained action targets of Yunshi Ganmao Mixture and the anti-HRSV related targets, the common targets of the two can be obtained.

[0117] There are 71 common targets. The results are shown in Figure 1 .

[0118] 2.4 Yunshi Ganmao Mixture Active Ingredient - HRSV Target - Pathway Network Diagram

[0119] Use Cytoscape 3.2.1 software to construct the Yunshi Ganmao Mixture active ingredient - HRSV

[0120] target - pathway network diagram. The results are shown in Figure 2 . (Blue represents the drug, yellow represents the active ingredient, red represents the disease, and pink represents the target. The denser the connection lines, the more important the target.)

[0121] 2.5 Construction of Protein - Protein Interaction (PPI) Network

[0122] Input the obtained 71 targets into the STRING database to construct a PPI network, as shown in Figure 3 . (Nodes represent genes, and connection lines represent protein interaction relationships between two genes)

[0123] 2.6 Gene Ontology (GO) Biological Function Analysis

[0124] A total of 2,395 GO analysis entries were obtained, including 2,236 in Biological Processes (BP), 61 in Cellular Components (CC), and 98 in Molecular Functions (MF). See

[0125] . (The abscissa represents the enrichment number, and the ordinate represents the functions involved. The more enriched, the redder the color; the less enriched, the bluer the color) Figure 4

[0126] 2.7 Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Enrichment Analysis

[0127] A total of 164 pathways of Yunshi Ganmao Mixture against HRSV were obtained by KEGG enrichment. The results of the top 6 pathways related to antiviral immunity were visualized. See Figure 5 (The more target points on the pathway, the redder the color; the fewer target points, the bluer the color). It can be seen that the antiviral effect of Yunshi Ganmao Mixture mainly involves the TNF signaling pathway, IL-17 signaling pathway, Toll-like signaling pathway, Th17 cell pathway, C-type lectin receptor signaling pathway, and PD-1 / PD-L1 pathway.

[0128] 2.8 "Component - Key Core Target" Molecular Docking Analysis

[0129] Molecular docking was performed on the 2 active ingredients (quercetin and 3-deoxysarpanchalcone) and 3 key targets (JUN, IL6, PTGS2) screened. The binding energies after docking are shown in Table 1. It can be seen that the active ingredients of Yunshi Ganmao Mixture, such as quercetin and 3-deoxysarpanchalcone, have strong binding abilities with several key targets (such as JUN, IL6, PTGS2). The molecular docking results show that quercetin binds to the active sites of JUN and IL6 proteins ( Figure 6 -A, B); 3-deoxysarpanchalcone binds to the active site of PTGS2 protein ( Figure 6 -C).

[0130] Table 1 Binding Energies of Key Targets and Active Ingredients in Docking

[0131] Molecular ID Name Target PDB ID Minimum Docking Binding Energy MOL000098 Quercetin JUN 4Y5H -6.5 MOL000098 Quercetin IL6 5SFK -5.7 3-Deoxysapachalcone PTGS2 5F19 -8.7

[0132] 2.9 Summary

[0133] ​A total of 637 action targets of the effective active ingredients of Yunshi Ganmao Mixture were finally obtained through prediction, 329 anti-HRSV related target genes, and 71 common targets between the two. This shows that the same target of the drug can correspond to different active ingredients, and different active ingredients also correspond to multiple targets, reflecting the characteristics of the comprehensive regulation of multiple components and multiple targets of traditional Chinese medicine compound. In this analysis of Yunshi Ganmao Mixture and HRSV, the research results show that the core genes in the PPI network are related to RELA, IL6, TLR4, TNF, JUN, AKT1, ACTB, and PTGS2

[12] It has been confirmed that most of them are involved in processes such as cell proliferation, apoptosis, inflammatory response, and regulation of ion channels. The results of its GO analysis and KEGG analysis show that Yunshi Ganmao Mixture can play an anti-HRSV role through biological processes such as the cell's response to lipopolysaccharide, bacterial-derived molecules, biological stimuli, and positive regulation of cytokines.

[0134] II. Exploration of the mechanism of Yunshi Ganmao Mixture against respiratory syncytial virus model mice in vivo

[0135] 1 Materials and methods

[0136] 1.1 Materials

[0137] 1.1.1 Main instruments and equipment:

[0138] Automatic blood cell analyzer (model: BC-5130 Shenzhen Mindray Bio-Medical Electronics Co., Ltd.), carbon dioxide incubator (model: PBN-80CH Shanghai Yiheng Scientific Instrument Co., Ltd.), ultra-clean workbench (model: cj-1d), ordinary optical microscope (Motic M200), paraffin slicer purchased from Leica Company in Germany, microplate reader, flow cytometer purchased from BD Company in the United States, real-time fluorescence quantitative PCR instrument CFX96 purchased from Bio-Rad Company in the United States, high-throughput tissue grinder purchased from Ningbo Xinyi Ultrasonic Equipment Co., Ltd.

[0139] 1.1.2 Drugs and main reagents:

[0140] Yunshi Ganmao Mixture 60ml / bottle (batch number: 20220303 Guizhou Liangji Pharmaceutical Co., Ltd.), ribavirin granules 50mg / bag (batch number: 201063 Sichuan Baili Pharmaceutical Co., Ltd.), phosphate PBS buffer solution, fetal bovine serum (batch number: 20010401 Zhejiang Tianhang Biotechnology Co., Ltd.), DMEM / F12-GlutaMAX-I (product of Gibco company, cat. no.: 1056), isoflurane anesthetic (model: R510-22 Shenzhen Rewo Life Technology Co., Ltd.), IgM ELISA kit purchased from Hangzhou Lianke Biotechnology Co., Ltd., IL-1 / IL-6 / TNF-α ELISA kit and T cells (CD3, CD4, CD8)

[0141] The subgroup flow antibodies were purchased from Wuhan Elabscience Co., Ltd.

[0142] 1.1.3 Experimental cell lines and virus strains:

[0143] The human laryngeal carcinoma epithelial cell line HEp-2 (ATCC, CCL-23) and the HRSV strain HRSV-A-GZ08-0 were kindly provided by Teacher Zhang Ke of the Department of Pathogen Biology, Guizhou Medical University.

[0144] 1.1.4 Experimental animals:

[0145] 80 female BALB / c mice, 5 - 6 weeks old, with a body weight of (18 ± 2) g, specific pathogen free grade, were purchased from Beijing Huafukang Biotechnology Co., Ltd., license number: SCXK (Jing) 2019 - 0008. The breeding temperature was 22 - 25 °C, the humidity was 50% - 80%, and they were bred with a 12-hour day-night cycle. This experiment was reviewed and approved by the Ethics Committee of Guizhou Medical University, number 2303209.

[0146] 1.2 Methods

[0147] 1.2.1 Amplification of respiratory syncytial virus and determination of TCID50

[0148] Take the subcultured and amplified Hep-2 cells. After inoculating HRSV, harvest the virus suspension after 3 - 7 days of culture. When the Hep-2 cells grow to a monolayer, change to a new culture medium, dilute the virus solution at a ratio of 1:10 for spotting on plates, infect the cells, and set up a cell control group. Calculate TCID50 after treatment.

[0149] 1.2.2 Establishment of a mouse model of respiratory syncytial virus and grouping for drug administration

[0150] Randomly divide 60 SPF-grade female BALB / c mice into 6 groups: normal control group, virus infection group, Yunshi Ganmao Mixture groups (low dose 1.56 g / kg, medium dose 7.8 g / kg, high dose 39 g / kg), ribavirin group (50 mg / kg),

[0151] 10 mice in each group. Number the mice in each group with picric acid. Establish an animal virus infection model on the fourth day of the mice's adaptive growth. Use the gaseous anesthetic isoflurane. After the mice inhale isoflurane and become completely limp, aspirate 100 μL of TCID50 of 1×10 6.67The HRSV virus solution was slowly dripped into the nasal cavities of mice, and the blank control group was dripped with the same volume of normal saline. Three days after HRSV infection, the mice were administered drugs by gavage. The drug administration dose was calculated according to the body weight of the mice at a rate of 0.1 mL / 10 g, and the drugs were administered at the same time every day for 4 consecutive days. The normal control group (normal saline), negative control group (normal saline), experimental group (low concentration of 1.56 g / kg, medium concentration of 7.8 g / kg, high concentration of 39 g / kg of Yunshi Ganmao Mixture), and positive drug group (ribavirin 50 mg / kg).

[0152] 1.2.3 Physiological characteristic detection

[0153] During the experiment, the body temperature and body weight of the mice were measured at the same time every day, and the status of the mice, such as food and water intake, mental state and activity status, was observed and recorded.

[0154] 1.2.4 Blood routine detection of mice

[0155] Blood was taken from the eyeballs and placed in an EP tube containing anticoagulant. The orbital blood of the mice was detected for white blood cell count (WBC), lymphocyte count (LYM),

[0156] neutrophil (NEU) using an automatic blood cell analyzer.

[0157] 1.2.5 Pulmonary function and pathological determination of lung tissue

[0158] Lung index = (mouse lung weight / mouse body weight) × 100%; Lung index inhibition rate = (average lung index of virus control group - average lung index of drug administration group) / average lung index of virus control group × 100%;

[0159] The pathological changes of the lungs were observed by HE staining. Fresh lung tissues of each group of mice were taken, fixed with 4% paraformaldehyde for 24 h, embedded in paraffin, sectioned, dewaxed, stained with HE, and the inflammatory conditions of the lung tissues were observed under an optical microscope, and inflammatory scores were performed.

[0160] 1.2.6 Detection of peripheral blood T cell subsets

[0161] Peripheral blood of each group of mice was aspirated and added on lymphocyte separation medium, centrifuged at 900×g for 30 min using a horizontal rotor. After centrifugation, the second-layer annular milky white cells were aspirated into another clean centrifuge tube, and 10 mL

[0162] Wash the cells with cell washing solution, centrifuge at 250×g for 10 min, discard the supernatant, wash the cells with PBS 3 times, centrifuge at 250×g for 10 min, discard the supernatant, then add 10 μL of each of the CD3+, CD4+, and CD8+ fluorescently labeled antibodies, mix well, place in the dark at 4°C for 30 min, add cell staining buffer to resuspend the cells, centrifuge at 300×g for 5 min, discard the supernatant, add 200 μL of cell staining buffer to resuspend the cells, and detect and analyze using a BD CantoⅡpuls flow cytometer in the United States.

[0163] Detection and analysis.

[0164] 1.2.7 Detection of HRSV viral load in lung tissue

[0165] Take 100 mg of lung tissue and place it in an EP tube to crush it. Add TRIzol reagent to extract total RNA, and measure the RNA concentration and purity. Use qRT-PCR to detect the expression of HRSV mRNA in lung tissue and calculate the viral load.

[0166] 1.2.8 Detection of IgM level and cytokine TNF-a, IL-6, IL-1β levels in mouse serum

[0167] Using the ELISA method, add the serum to the enzyme-linked immunosorbent assay (ELISA) plate, add 100 μl of the sample to each well, then add the detection antibody to each well and cover with a film, incubate at 37°C for 1.5 hours, and then follow the instructions for each step. Add the stop solution and immediately measure the optical density value OD450 nm of each well using an enzyme-linked immunosorbent assay reader. Obtain the concentration of each group of samples according to the standard curve.

[0168] 1.2.9 Detection of mRNA levels of TLR4 / NF-κB in mouse lung tissue

[0169] Take 100 mg of lung tissue and place it in an EP tube to crush it. Add TRIzol reagent to extract total RNA, and measure the RNA concentration and purity. Use qRT-PCR to detect the expression level of TLR4 / NF-κB mRNA (Real-time PCR).

[0170] 1.2.10 Data analysis

[0171] Analyze using SPSS 24.0 statistical software. One-way analysis of variance is used for differences among multiple groups, and the t-test is used for analysis between the experimental group and the control group. A difference with P < 0.05 is considered statistically significant.

[0172] 2 Results

[0173] 2.1 Amplification of respiratory syncytial virus and determination of TCID50

[0174] Normal Hep-2 cells are monolayer adherent cells that are spindle-shaped or polygonal after subculture. After treatment with HRSV, Hep-2 cells are induced to fuse to form multinucleated giant cells, syncytia and other CPE phenomena (see Figure 7 ). The virus TCID50 is approximately 1×10 6.67 (see Table 2) Table 2 Calculation of HRSV TCID50 by Reed-Muench method

[0175]

[0176] 2.2 Establishment of a mouse model of respiratory syncytial virus and detection of physiological characteristics

[0177] An BALB / c mouse model infected with respiratory syncytial virus was established. After inhalation anesthesia, 100 μl of respiratory syncytial virus suspension (1×10 6.67 TCID50) was instilled intranasally. Administration intervention started on the third day after infection until the seventh day, see Figure 8 (a). The body weight percentages of the low, medium, and high dose groups in the Yunshi Ganmao Mixture group were significantly higher than those in the HRSV-infected control group, and the results were significantly different (P<0.05), see Figure 8 (b); it shows that Yunshi Ganmao Mixture can effectively control the body weight changes of mice after HRSV infection.

[0178] 2.3 Blood routine examination of mice

[0179] The blood routine results of mice showed that compared with the normal group, the counts of WBC and LYM in the model group were both down-regulated, and the results were significantly different (P<0.05); compared with the model group, the counts of WBC and LYM in the ribavirin group and the high-concentration group of Yunshi Ganmao Mixture were both significantly up-regulated (P<0.05), and the counts of WBC and LYM in the low-concentration and medium-concentration groups of Yunshi Ganmao Mixture were also significantly up-regulated. The results are shown in Table 2.

[0180] Table 2 Blood routine examination of mice in different treatment groups

[0181]

[0182] Note: Compared with the blank group, #P<0.05; ##P<0.01, compared with the HRSV group, *P<0.05; **P<0.01.

[0183] 2.4 Pulmonary function and pathological determination of lung tissue

[0184] After mice were infected with HRSV, compared with the normal group, the lung index of the HRSV group increased significantly, and the result was significantly different (P<0.05); compared with the model group, after drug treatment, the lung index of the high-concentration group of Yunshi Ganmao Mixture decreased significantly (P<0.05), and the ribavirin group, the low-concentration and medium-concentration groups of Yunshi Ganmao Mixture also decreased significantly, as shown in Figure 9 .

[0185] By calculating the inhibition rate of the lung index in each treatment group, it was found that the inhibition rate of the lung index in the ribavirin group was 2.99%, and the low, medium, and high groups of Yunshi Ganmao Mixture reached 1.43%, 3.40%, and 6.86% respectively, as shown in Table 3.

[0186] In the normal group of mice, the size of the alveolar lumen was normal, the alveolar wall was full, the thickness of the alveolar wall was uniform, and there was no obvious infiltration of inflammatory cells in the alveoli, and there was no secretion in the lumen (see Figure 10 A); compared with the normal group of mice, in the HRSV group of mice, the alveoli were significantly smaller, the alveolar septum was narrower, the alveolar wall was thicker, and there was infiltration of inflammatory cells in the interstitium (see Figure 10 B); in the ribavirin group and the groups of Yunshi Ganmao Mixture (1.56 g / kg, 7.8 g / kg, 39 g / kg), the number of alveoli in the lung tissue of mice increased, the septum became larger, the size of the alveoli tended to be uniform, and the thickness of the alveolar wall was uniform (the results are shown in Figure 10 C, D, E, F).

[0187] Table 3 Comparison of the results of the inhibition rate of the lung index in mice in different drug treatment groups

[0188]

[0189] 2.5 Detection of peripheral blood T lymphocyte subsets

[0190] Compared with the control group, in the HRSV group of mice after respiratory syncytial virus infection, the numbers of CD3 + , CD3 + CD4 + , CD3 + CD8 + , and T lymphocytes decreased significantly (P<0.05), as shown in Figures 11 - 14 , and the ratio of the number of CD4 + / CD8 + T cells increased significantly (P<0.05), as shown in Figure 11 , Figure 15 . Compared with the HRSV group, after drug treatment, the CD3 + , CD3 + CD4 + , CD3 + CD8+ The number of T lymphocytes was significantly higher than that in the HRSV group (P < 0.05), as shown in Figures 11 - 14 , CD4 + / CD8 + The ratio of T cell numbers was significantly decreased (P < 0.05), as shown in Figure 11 , Figure 15 .

[0191] 2.6 Detection of HRSV viral load in lung tissue

[0192] The qPCR results of lung tissue showed that after HRSV-infected mice, compared with the HRSV group, the relative expression levels of HRSV mRNA in the ribavirin group and the Yunshi Ganmao Heji (1.56 g / kg, 7.8 g / kg, 39 g / kg) groups were significantly decreased after drug treatment (P < 0.05), and the results are shown in Figure 16 .

[0193] 2.7 Detection of IgM level and cytokine TNF-a, IL-6, IL-1β levels in mouse serum

[0194] Compared with the normal group, the IgM content in the serum of mice in the HRSV group was significantly higher than that in the normal control group (r < 0.01). Compared with the HRSV group, the IgM content in the ribavirin group and the Yunshi Ganmao Heji (1.56 g / kg, 7.8 g / kg, 39 g / kg) groups was significantly down-regulated after drug treatment (r < 0.01), as shown in Figure 17 A. Compared with the normal group, the contents of cytokines TNF-α, IL-6, and IL-1β in the serum of mice in the HRSV group were significantly up-regulated (P < 0.05), and the contents in the ribavirin group and the Yunshi Ganmao Heji (1.56 g / kg, 7.8 g / kg, 39 g / kg) groups were significantly down-regulated after drug treatment

[0195] (P < 0.05), as shown in Figure 17 B, C, D.

[0196] 2.8 Detection of mRNA levels of TLR4 / NF-κB in mouse lung tissue

[0197] The qPCR results of lung tissue showed that after HRSV-infected mice, compared with the normal group, the relative expression level of TLR4 mRNA in the HRSV group was significantly higher than that in the normal control group (P < 0.01). Compared with the HRSV group, the relative expression levels of TLR4 mRNA in the ribavirin group and the Yunshi Ganmao Heji (1.56 g / kg, 7.8 g / kg, 39 g / kg) groups were significantly decreased (P < 0.05), and the results are shown in Figure 18After infecting mice with HRSV, compared with the normal group, the relative expression level of NF-κB mRNA in the HRSV group was significantly higher than that in the normal control group (P<0.01). Compared with the HRSV group, after drug treatment, the relative expression levels of NF-κB mRNA in the ribavirin group and the Yunshi Ganmao Mixture (1.56 g / kg, 7.8 g / kg, 39 g / kg)

[0198] groups decreased significantly (P<0.05), and the results are shown in Figure 19 ..

[0199] 3. Summary

[0200] By treating mice with respiratory syncytial virus model with various doses of Yunshi Ganmao Mixture (1.56 g / kg, 7.8 g / kg, 39 g / kg), it was found that: 1) Yunshi Ganmao Mixture can effectively control the weight loss of mice after HRSV infection. 2) Yunshi Ganmao Mixture can regulate the pulmonary infection of HRSV by down-regulating the lung index, up-regulating the inhibition rate of the lung index and effectively improving the pathological changes of lung tissue; at the same time, it can effectively down-regulate the viral load in the lung tissue after HRSV infection, indicating that Yunshi Ganmao Mixture can improve pulmonary infection and reduce the viral load by regulating relevant mechanisms. 3) By detecting blood routine, it was found that both WBC and LYM would decrease after HRSV infection, and both WBC and LYM would increase after treatment with Yunshi Ganmao Mixture. Using flow cytometry, it was found that Yunshi Ganmao Mixture can up-regulate T lymphocyte subsets: CD3 + 、CD4 + 、CD8 + immune cell numbers to improve the damage of HRSV to the body's immune system, and can effectively down-regulate the CD4 + / CD8 + ratio to maintain the balance of the body's immune system. 4) Yunshi Ganmao Mixture can reduce the contents of IgM, TNF-a, IL-6, and IL-1β in the serum of mice after HRSV infection. 5) By detecting the expression levels of TLR4 / NF-κBmRNA in the lung tissue of mice by qRT-PCR, it was found that Yunshi Ganmao Mixture can down-regulate the expression level of TLR4 / NF-κB mRNA. Through this part of the mechanism verification experiment, it is suggested that the Miao medicine Yunshi Ganmao Mixture can play a protective role against HRSV on the host through anti-inflammatory and immunomodulatory pathways, and may play its anti-HRSV role by down-regulating TLR4 / NF-κB.

[0201] Although the present invention has been described in detail above with general descriptions, specific embodiments and experiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection of the present invention.

[0202] References:

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Claims

1. A screening method for the use of Yunshi Ganmao Mixture in the treatment of respiratory syncytial virus, characterized in that, The screening method is to analyze its mechanism of action by combining network pharmacology and molecular docking technology, and specifically includes the following steps: (1) Prediction of active ingredients and key core targets of Yunshi Ganmao Mixture: Input the single herbs in Yunshi Ganmao Mixture into the Traditional Chinese Medicine Systems Pharmacology Analysis Platform respectively. With the conditions of biological oral bioavailability ≥ 30% and drug-likeness (DL) ≥ 0.18 as the criteria for screening active ingredients, record their targets, and supplement the targets with the online websites of Swiss TargetPrediction and TargetNet to obtain the action targets of Yunshi Ganmao Mixture; (2) Obtaining potential targets of respiratory syncytial virus: Search for "Respiratory Syncytial Virus" in the GeneCards database, the Online Mendelian Inheritance in Man (OMIM) database, and the DisGeNet database respectively, and convert the protein targets into Gene Symbol through the UniProt database; after screening out duplicate values, obtain the anti-HRSV related targets; (3) Obtaining common targets of Yunshi Ganmao Mixture and respiratory syncytial virus: Input the action targets of Yunshi Ganmao Mixture obtained in step (1) and the anti-HRSV related targets obtained in step (2) into the online platform of Venny 2.1.0 to obtain the Venn diagram of the intersection of the two target proteins; (4) Construction of the traditional Chinese medicine-ingredient-target network diagram: Use Cytoscape 3.2.1 software to construct the traditional Chinese medicine-ingredient-target network diagram of Yunshi Ganmao Mixture, and use the "Analyze network" function of this software to conduct topological analysis of Yunshi Ganmao Mixture; (5) Construction of the protein-protein interaction (PPI) network: Input the 71 targets obtained in step (3) into the STRING database, analyze and predict the interaction relationship between proteins through the STRING database, including the direct and indirect relationships between protein interactions, construct the PPI network, and analyze to obtain the core action targets; (6) Gene Ontology (GO) biological function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis: Input the mapped targets obtained in step (4) into the Metascape database, use the Metascape database to conduct GO analysis and KEGG related pathway analysis, and visualize them with the online drawing website of Microbial Informatics; (7) Verification of "ingredient-key core target" molecular docking analysis: Conduct molecular docking of the top two active ingredients of Yunshi Ganmao Mixture obtained in step (1) and the core action targets of Yunshi Ganmao Mixture obtained in step (5); download the high-resolution three-dimensional crystal structure of the target protein from the PBD database, and use PyMol and Autodock Tools for molecular preprocessing; run Autodock Vina for batch semi-flexible molecular docking, and visualize the output results of small molecules with PyMol software.

2. The screening method according to claim 1, characterized in that, The visualization analysis described in step (6) shows that the anti-viral effects of Yunshi Ganmao Mixture mainly involve the TNF signaling pathway, IL-17 signaling pathway, Toll-like signaling pathway, Th17 cell pathway, C-type lectin receptor signaling pathway, and PD-1 / PD-L1 pathway.

3. The screening method according to claim 1, characterized in that, The core targets described in step (7) are JUN, IL6, and PTGS2.

4. The screening method according to claim 1, characterized in that, The active ingredients described in step (7) are quercetin and 3-deoxysapanchalcone.

5. The screening method according to claim 1, characterized in that, The docking analysis described in step (7) shows that quercetin binds to the active sites of JUN and IL6 proteins; 3-deoxysapanchalcone binds to the active site of PTGS2 protein.

Images