Analysis method of medicinal components of Reiyanning mixture for treating bacterial pneumonia

Abstract

The invention provides a method for analyzing medicinal components of a Reiyanning mixture for treating bacterial pneumonia, which comprises the following steps of: analyzing medicinal materials of the Reiyanning mixture on the basis of ultra-high performance liquid chromatography-quadrupole time-of-flight combined mass spectrometry (UPLC-Q / TOF-MS), and predicting an anti-infection mechanism of the Reiyanning mixture in combination with network pharmacology to obtain GO enrichment analysis and KEGG enrichment analysis results; and a novel method is provided for analysis of medicinal components of the Reiyanning mixture and research on an action mechanism of the Reiyanning mixture.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to an analysis method for the active ingredients of Reyanning mixture for treating bacterial pneumonia. Background Art

[0002] Bacterial pneumonia is the infectious disease with the highest proportion of pneumonia. In terms of clinical manifestations, patients mainly present with symptoms such as fever, cough, sputum congestion, dyspnea and lung rales. Common pathogens include Streptococcus pneumoniae, Staphylococcus aureus and Klebsiella pneumoniae. According to its pathogenesis and symptoms, Chinese medicine classifies it as "pneumonia wheezing". Professor Wang Lie, a master of Chinese medicine, proposed the "heat toxin" theory, pointing out that "heat toxin" is the main cause of bacterial pneumonia, providing a new idea for the treatment of bacterial pneumonia. Reyanning mixture is a traditional Chinese medicine compound formula with the effect of clearing heat and detoxification. It combines the essence of multiple Chinese medicinal materials such as dandelion, Polygonum cuspidatum, Patrinia scabra and Scutellaria barbata. It is an ideal choice for the treatment of exogenous wind-heat, internal depression and fire, and cough with yellow sputum. Modern medical research has found that Reyanning mixture combined with antibiotics has an anti-methicillin-resistant Staphylococcus aureus effect, and main ingredients such as dandelion also have anti-infection effects. However, the material basis of the compound is unclear, and the active ingredients used to treat bacterial pneumonia are not clear, which is not conducive to the quality control of Reyanning Mixture and the development of new indications. Summary of the invention

[0003] In view of the problems existing in the prior art, the present invention provides a method for analyzing the active ingredients of Reyanning mixture for treating bacterial pneumonia.

[0004] The objective of the present invention is achieved through the following technical solutions: The invention provides an analysis method for the active ingredients of Reyanning mixture for treating bacterial pneumonia, characterized in that the analysis method comprises: screening out the chemical ingredients of Reyanning mixture based on UPLC-Q / TOF-MS technology; using a network pharmacology-related database, screening out the effective ingredients of Reyanning mixture and collecting corresponding action targets, and constructing a Reyanning mixture target library; constructing a bacterial pneumonia target library; taking the intersection of the bacterial pneumonia target library and the Reyanning mixture target library to obtain a Reyanning mixture acting on a bacterial pneumonia target database, and obtaining 49 active targets; visualizing the collected active targets and the corresponding effective ingredients using software, constructing a compound network, performing network topology analysis, and obtaining 42 important chemical components and 23 key targets; constructing a protein interaction network; and performing GO enrichment analysis and KEGG enrichment analysis on the active targets obtained by screening to obtain pathways with high enrichment significance.

[0005] Preferably, the method for screening the chemical components of Reyanning mixture includes: Prepare the test solution: take 10 μL of the original solution of Reyanning mixture and add it to 10 mL of acetonitrile solvent (acetonitrile: water = 1:1), mix well, centrifuge, and take the supernatant for liquid chromatography analysis and mass spectrometry analysis; Prepare mixed standard solution: accurately weigh 1 mg each of the reference substances polydatin, rhamnolide, chicoric acid, and baicalin, place them in different centrifuge tubes, add 1 mL of acetonitrile and shake to dissolve; pipette 125 μL of each and add 500 μL of acetonitrile to dilute, centrifuge at 10,000 rpm for 10 min; take 20 μL of the supernatant, add it to 1 mL of pure water, centrifuge at 10,000 rpm for 10 min, take 900 μL and add it to a liquid phase vial to prepare a mixed standard solution, and perform liquid chromatography and mass spectrometry analysis on the machine; Establishment of UNIFI analysis platform for Reyanning mixture: The chemical components of dandelion, Polygonum cuspidatum, Patrinia serrata and Scutellaria barbata were collected and sorted in databases such as TCMSP, BATMAN, symmap, ETCM and PubChem, and the compound information that may be related to Reyanning mixture was obtained, including chemical component name, structural formula, molecular formula, etc. and chemical structure. The compound structural formula was stored in .mol ​​mode, sorted into an Excel table and put into a folder, that is, the chemical component database of Reyanning mixture was constructed, uploaded to the local UNIFI analysis platform, and the compounds were analyzed.

[0006] Data processing and analysis: Manually check the detailed information of compound test samples and standard reference substances to ensure accurate analysis.

[0007] Preferably, the liquid chromatography conditions are as follows: the chromatographic column used in this experiment is Waters ACQUITY UP-LC BEH C 18 (100 mm × 4.6 mm, 1.7 μm); 1 mL / L formic acid aqueous solution (A) - acetonitrile (B) as the mobile phase, gradient elution; the HPLC elution gradient is as follows:

[0008] Preferably, the mass spectrometry conditions are: detection in positive ion mode (ESi+) and negative ion mode (ESi-), respectively, with a scan time of 0.2 s and a scan range of 50m / z -2000 Da; positive ion mode: capillary voltage 2.00 kV, ion source temperature 120°C, desolvation temperature 500°C, desolvation gas nitrogen (N2) flow rate of 1000 L / h, cone gas flow rate 50L / h, and leucine enkephalin is used as a calibration liquid; negative ion mode: capillary voltage 2.50 kV, ion source temperature 100°C, desolvation temperature 450°C, desolvation gas nitrogen (N2) flow rate of 600 L / h, cone gas flow rate 50 L / h, and reserpine is used as a calibration liquid.

[0009] Preferably, the network pharmacology related databases are TCMSP database, BTAMAN-TCM database, ETCM database and SymMAP database.

[0010] Preferably, the method for constructing a bacterial pneumonia target library is as follows: using "pneumonia" and "bacterialpneumonia" as keywords, accessing the DrugBank database, TTD database, GEO database, GeneCards database, OMIM database and PharmGkb database, and inputting the information target names obtained from the database into the UniProt database to obtain the target gene name; searching for direct data of "pneumonia" and "bacterial pneumonia" in the GEO database to obtain transcriptomics data, using the R tool for analysis, and obtaining the intersection of public database targets as the bacterial pneumonia target database.

[0011] Preferably, the method for constructing a protein interaction network is as follows: prepare an input file based on the intersection genes obtained from the bacterial pneumonia target library and the Reyanning mixture target library, and copy the gene names in the intersection file to the corresponding boxes in the string database; select the "Homo Sapiens" species to obtain the constructed protein interaction network; set filtering conditions, delete free nodes, and obtain a clearer protein interaction network diagram.

[0012] Preferably, the key targets are ACE, HMOX1, SERPINE1, MPO, CCL2, IL1β, CYP3A4, ADRB2, PTGS2, ADRB1, MMP9, IL6, CFTR, IL4, BTK, CD40LG, RELA, NOS2, IL13, CYP19A1, CYP1A2, CYP1A1 and TERT.

[0013] Compared with the prior art, the beneficial effects of the present invention are: Based on UPLC-Q / TOF-MS technology and network pharmacology methods, the present invention analyzed the pharmacological material basis of Reyanning mixture, screened 42 compounds, and used network pharmacology methods to construct a drug target database and a disease target database, and obtained 49 targets by taking the intersection. GO and KEGG enrichment analysis were used. The results showed that Reyanning mixture can affect bacterial invasion and regulate inflammation, and the PI3K / AKT pathway is the core pathway of Reyanning mixture in treating bacterial pneumonia. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The chemical structure, extraction flow chart and mass spectrum of apigenin; a Structure and extracted ion flow chart of apigenin; b Primary mass spectrum of apigenin; c Secondary mass spectrum of apigenin; Figure 2 The chemical structure, extraction flow chart and mass spectrum of wogonin; a The structure and extracted ion flow chart of wogonin; b The primary mass spectrum of wogonin; c The secondary mass spectrum of wogonin; Figure 3 The chemical structure, extraction flow chart and mass spectrum of rhein; a The structure and extracted ion flow chart of rhein; b The primary mass spectrum of rhein; c The secondary mass spectrum of rhein; Figure 4 The chemical structure, extraction flow chart and mass spectrum of luteolin; a The structure and extracted ion flow chart of luteolin; b The primary mass spectrum of luteolin; c The secondary mass spectrum of luteolin; Figure 5 Total ion current of Reyanning mixture sample; (A) Total ion current of Reyanning mixture under positive ion mode; (B) Total ion current of Reyanning mixture under negative ion mode.

[0015] Figure 6 Compound-ingredient-target network diagram; Figure 7 Construction of target library of Reyanning Decoction in the treatment of bacterial pneumonia; (A) Volcano plot of transcriptomic analysis of GEO database; (B) Target database of Reyanning Decoction in bacterial pneumonia: (C) Intersection Venn diagram of targets of Reyanning Decoction in bacterial pneumonia.

[0016] Figure 8 Protein interaction network of Reyanning mixture in the treatment of bacterial pneumonia; Fig. 9 GO enrichment analysis of targets of Reyanning mixture in the treatment of bacterial pneumonia; Fig.10 KEGG enrichment analysis of targets of Reyanning mixture in the treatment of bacterial pneumonia; DETAILED DESCRIPTION

[0017] The concept of the present invention will be illustrated below in conjunction with specific implementation methods.

[0018] The effective ingredients of traditional Chinese medicine are the material basis for preventing and curing diseases. This application adopts UPLC-Q / TOF-MS technology and UNIFI analysis platform to separate and screen the chemical components of Reyanning mixture, and screen out the components with possible pharmacological activity to lay its material basis.

[0019] As a perfect combination of modern technology and traditional Chinese medicine theory, network pharmacology of Chinese medicine compound has the same purpose as the "multi-component-multi-target" concept in traditional Chinese medicine theory. This concept emphasizes that multiple components in Chinese medicine can act on multiple targets at the same time, thereby achieving a synergistic therapeutic effect. Network pharmacology technology is based on this concept. By constructing a complex interaction network, it systematically studies the potential components in Chinese medicine and their interactions with biological molecules, and deeply understands the material basis of the efficacy of Chinese medicine. This application will conduct a preliminary discussion on the mechanism of Reyanning mixture in the treatment of bacterial pneumonia by combining the results of in vivo and in vitro experimental studies.

[0020] 1.1 Materials 1.1.1 Experimental instruments Table 1 Instruments Instrument Name Production Company UPLC / Q-TOF-MS system (Vion IMS Qtof) Waters Corporation Electronic balance (YP20001) Shanghai Hengji Company Pipette (Research® plus) Eppendorf Spiral mixer (VORTEX-5) Haimen Qilin Bell Ultrasonic oscillator (KQ-500DE) Kunshan Ultrasonic Instrument Company 1.1.2 Experimental consumables The consumables used in the experiment are shown in the following table. Table 2 Experimental consumables Consumables Name Production Company ACQUITY UPLC BEH C18 Waters Corporation EP tube (10 / 50 mL) Beijing Lanjieke Technology Co., Ltd. Injection vial (1.5 mL) Thermo Fisher Scientific Pipette tips (10 / 200 / 1000 / 5000 μL) Thermo Fisher Scientific 1.1.3 Drug and compound standards Table 3 Experimental compounds Drug Name Production Company Reyanning mixture (RYNM) Tsinghua Deren Xi'an Xingfu Pharmaceutical Co., Ltd. Scutellarin Chengdu Aifa Biological Company Emodin Chengdu Aifa Biological Company Chicoric acid Chengdu Aifa Biological Company Polydatin Chengdu Aifa Biological Company

[0021] (1) Preparation of test solution: Measure 10 μL of the original solution of Reyanning mixture and add it to 10 mL of acetonitrile solvent (acetonitrile: water = 1:1), mix well, centrifuge, and aspirate 900 μL of the supernatant for sample loading.

[0022] (2) Preparation of mixed standard solution: accurately weigh 1 mg each of the reference substances polydatin, rhamnolide, chicoric acid, and baicalin, place them in different centrifuge tubes, add 1 mL of acetonitrile and shake to dissolve; take 125 μL of each and add 500 μL of acetonitrile to dilute, centrifuge at 10,000 rpm for 10 min; take 20 μL of the supernatant and add it to 1 mL of pure water, centrifuge at 10,000 rpm for 10 min, take 900 μL and add it to a liquid phase vial to prepare a mixed standard solution, and load it onto the machine.

[0023] (3) Liquid chromatography conditions: The chromatographic column used in this experiment was Waters ACQUITY UP-LC BEH C 18 (100 mm×4.6 mm, 1.7 μm); 1 mL / L formic acid aqueous solution (A)-acetonitrile (B) was used as the mobile phase for gradient elution. The HPLC elution gradient is shown in Table 4.

[0024] Table 4

[0025] (4) Mass spectrometry conditions: positive ion mode (ESi+) and negative ion mode (ESi-) were used for detection. The scanning time was 0.2s and the scanning range was 50m / z-2000Da. Positive ion mode: capillary voltage 2.00kV, ion source temperature 120℃, desolvation temperature 500℃, desolvation gas nitrogen (N2) flow rate 1000L / h, cone gas flow rate 50L / h, and leucine enkephalin was used as the calibration liquid. Negative ion mode: capillary voltage 2.50kV, ion source temperature 100℃, desolvation temperature 450℃, desolvation gas nitrogen (N2) flow rate 600L / h, cone gas flow rate 50L / h, and reserpine was used as the calibration liquid.

[0026] (5) Establishment of the UNIFI analysis platform for Reyanning mixture: The chemical components of Taraxacum mongolicum, Polygonum cuspidatum, Patrinia serrata, and Scutellaria barbata were collected and sorted in databases such as TCMSP, BATMAN, symmap, ETCM, and PubChem, and information on compounds that may be related to Reyanning mixture was obtained, including chemical component names, structural formulas, molecular formulas, and chemical structures. The compound structural formulas were stored in .mol ​​mode, sorted into Excel tables and put into folders, and the chemical component database of Reyanning mixture was constructed. The chemical components were uploaded to the local UNIFI analysis platform and the compounds were analyzed.

[0027] (6) Optimization of ultra-high performance liquid chromatography and mass spectrometry conditions: UNIFI platform automatically matched and screened. After optimizing the parameters, the screening settings were: Mass error (ppm) ±5, Response ≥ 2000, and [M+H] was selected for the positive ion mode. + and 2×[M+H] + , negative ion mode selects [MH] – and 2×[MH] - .

[0028] (7) Data processing and analysis: Manually check the detailed information of compound test samples and standard reference substances to ensure accurate analysis.

[0029] 1.2.2 Chemical component screening and target collection of Reyanning mixture based on material basis research The literature was combined with the chemical components screened based on UPLC-Q / TOF-MS technology in the TCMSP database, BTAMAN-TCM database, ETCM database, and SymMAP database to obtain the active ingredients of Reyanning mixture, and the corresponding targets were searched in the database. The databases were sorted and summarized to build a drug target library.

[0030] Construction of bacterial pneumonia target library: Using "pneumonia" and "bacterial pneumonia" as keywords, access the DrugBank database, TTD database, GEO database, GeneCards database, OMIM database and PharmGkb database, and input the target names obtained from the database into the UniProt database to obtain the target gene name. Search the GEO database for direct data of "pneumonia" and "bacterial pneumonia" to obtain transcriptomics data, which are analyzed using the R tool to obtain the intersection of public database targets as the bacterial pneumonia target database.

[0031] R software processing: R software was used to intersect the bacterial pneumonia target library with the Reyanning mixture target library, draw a Venn diagram, and obtain active targets that may be effective.

[0032] The collected herbal targets and corresponding active ingredients were visualized using software, compound networks were constructed, and network topology analysis was performed.

[0033] Prepare the input file based on the intersection genes obtained from the bacterial pneumonia target library and the Reyanning mixture target library, and copy the gene names in the intersection file to the corresponding boxes in the string database. Select the "Homo Sapiens" species to obtain the constructed protein interaction network. Set the filtering conditions, delete the free nodes, and obtain a clearer protein interaction network diagram.

[0034] GO enrichment analysis: The intersection target genes obtained by screening were summarized and GO enrichment analysis was performed using the R software toolkit (P ≤ 0.01) to generate results for analysis.

[0035] KEGG enrichment analysis: The intersection target genes obtained by screening were summarized and KEGG enrichment analysis was performed using the R software toolkit (P ≤ 0.01), and the top 20 results were selected for analysis.

[0036] The optimization of ultra-high performance liquid chromatography and mass spectrometry conditions was explored, and the mobile phase was determined to be formic acid aqueous solution and acetonitrile, and the column temperature was 35°C. The addition of formic acid effectively improved the tailing of the chromatographic peak. Considering the complex composition of Reyanning mixture, positive and negative ion modes were used for scanning analysis and screening.

[0037] After high-throughput screening using the UNIFI data analysis platform, 87 chemical components were detected in Reyanning mixture, of which 8 were derived from Patrinia odoratum, 24 from Taraxacum officinale, 32 from Scutellaria barbata, and 23 from Polygonum cuspidatum. Details of the identified components are shown in Table 5, and the identification and ion flow diagram of the standard are shown in Table 6. Four of the compounds were identified by comparing the detailed information with the standard, and the compound structural formula and extracted ion flow diagram mass spectrum are shown in Figure 1-Figure 4 The total ion current diagram of Reyanning mixture is shown in Figure 5 .

[0038] Table 5 Identification table of the active ingredients of Reyanning mixture

[0039] Table 6 Identification results of standard substance of Reyanning mixture

[0040] 1.3.3 Screening of chemical components and target collection of Reyanning mixture based on material basis The chemical components contained in Reyanning mixture were identified and screened under the screening conditions of Mass error (ppm) ±5, Response ≥ 2000. A total of 42 compounds were screened out, and the detailed information is shown in Table 7. Figure 6 As shown in the figure, 244 targets were collected from 42 compound components in various databases. The chemical components of Reyanning mixture and the compound target database were used to construct a "compound-component-target" network system using Cytoscape 3.8.0 software. The network has a total of 416 nodes and 921 edges. Among them, 244 nodes represent the targets received by Reyanning mixture, 34 nodes represent the unique component targets of the four herbs, and the blue in the middle is a total of 244 drug targets. The darker the color and the larger the area of ​​the graph, the more it is associated with the drug. The periphery is the drug composition and active ingredients; different colors represent different categories of herbs.

[0041] Table 7 Screened active ingredients of Reyanning mixture

[0042] 1.3.4 Construction of the target database of Reyanning mixture on bacterial pneumonia The keywords were searched in the PharmGkb database for 46 targets, in the Drugbank database for 10 targets, in the Genecards database for 2543 targets, in the TTD database for 6 targets, and in the OMIM database for 2 targets. The transcriptomic data of GSE35716, GSE40012, and GSE20346 were analyzed in the GEO database, and the control group and the experimental group were selected for analysis and comparison, resulting in 21655 targets. The intersection of the public database and the GEO database targets was used as the target database for bacterial pneumonia, such as Figure 7 (B).

[0043] The intersection of 244 targets acted by the components of the Reyanning mixture and 914 targets related to bacterial pneumonia was taken to obtain the target database of Reyanning mixture on bacterial pneumonia, and a total of 49 targets were obtained. The detailed information of the targets is shown in Table 8, and the specific information of the collected targets is shown in Figure 7 (C).

[0044] Table 8 Targets of Reyanning mixture in the treatment of bacterial pneumonia

[0045] 1.3.5 Construction of protein interaction network and screening of core targets In order to further understand the interaction relationship of Reyanning mixture with disease target proteins, the 49 intersection targets obtained were imported into the database for PPI network construction and analysis, and the discrete proteins were removed to obtain the PPI network diagram (see Figure 8 .

[0046] The PPI results were imported into Cytoscape for topological analysis, and a total of 23 key targets were obtained. After analysis, they were found to be ACE, HMOX1, SERPINE1, MPO, CCL2, IL1β, CYP3A4, ADRB2, PTGS2, ADRB1, MMP9, IL6, CFTR, IL4, BTK, CD40LG, RELA, NOS2, IL13, CYP19A1, CYP1A2, CYP1A1, and TERT (see Figure 8).

[0047] The 49 intersection targets obtained were subjected to GO enrichment analysis and KEGG enrichment analysis.

[0048] GO analysis consists of three parts: BP, CC, and MF. The biological processes that affect the biological function (BP) according to the size of the P value include: regulation of apoptosis signaling pathways, regulation of inflammatory responses, response to bacterial-derived molecules, etc. Cellular components include: cell membrane caveolae, cytoplasmic vesicle cavity, etc. Molecular functions (MF) include: cytokine activity, transcriptional coactivator binding, etc., see Fig. 9 .

[0049] KEGG analysis found that it was related to IL-17 signaling pathway, HIF-1 signaling pathway, PI3K-Akt signaling pathway and other signaling pathways. Fig.10 .

[0050] This application uses UPLC-Q / TOF-MS combined with UNIFI analysis platform to analyze and high-throughput screen the active ingredients of Reyanning mixture. UNIFI analysis has the characteristics of being targeted and can perform high-throughput screening of Chinese medicine ingredients without the need for manual spectrum interpretation. It is an indispensable technical platform for the study of Chinese medicine ingredients. Through this technical study, it was found that 87 chemical components of Reyanning mixture can be separated and identified, of which 8 chemical components are derived from Patrinia serrata, 24 chemical components are derived from Taraxacum mongolicum, 32 chemical components are derived from Scutellaria barbata, and 23 chemical components are derived from Polygonum cuspidatum.

[0051] In order to further determine the mechanism of action of Reyanning mixture in the treatment of bacterial pneumonia, we predicted its anti-infection mechanism by joint network pharmacology. It is known that the main ingredients of Reyanning mixture are dandelion, Polygonum cuspidatum, Patrinia scabra and Scutellaria barbata. The above Chinese medicine ingredients were collected from 6 databases, and 42 effective ingredients were obtained, as shown in Table 7. At the same time, the corresponding disease target genes were obtained from the database based on "bacterial pneumonia" as the keyword. The target genes corresponding to the effective ingredients and the target genes corresponding to the disease were intersected to obtain 49 targets, as shown in Table 8. The compound network was constructed to analyze the relationship between the active ingredients and the targets. Then, the protein interaction network between the target genes was constructed to understand the relationship between proteins, and the obtained network was analyzed. The mechanism of action of Reyanning mixture in the treatment of bacterial pneumonia was deeply explored through GO enrichment analysis. The analysis results showed that Reyanning mixture can regulate a series of important biological processes, including the regulation of apoptosis signaling pathways and the regulation of inflammatory responses. These regulatory effects not only reveal the pharmacological activity of Reyanning mixture in the treatment of bacterial pneumonia, but also provide strong theoretical support for our understanding of its efficacy. In the process of dealing with bacterial invasion, Reyanning mixture can effectively regulate reactions related to bacteria-derived molecules, thereby blocking further bacterial invasion. At the same time, it can also regulate inflammatory responses, alleviate lung inflammation symptoms, and promote recovery from the disease. When performing KEGG enrichment analysis, the pathway results found that the PI3K / AKT pathway plays a key role in the treatment of inflammation.

[0052] The PI3K / AKT pathway works through protein phosphorylation. AKT ensures cell survival by mediating cell growth factors and inactivating pro-apoptotic proteins. Under certain conditions, IKK kinase plays an indirect role in the activation of transcription factor NF-κB after being phosphorylated by AKT. The relationship between AKT and NF-κB is actually complicated. Studies have shown that AKT can promote the phosphorylation of NF-κB subunits by inhibiting the degradation of IκB protein. At the same time, when AKT function in cells is enhanced, the transcriptional activity of NF-κB is upregulated, and the use of AKT inhibitors will interfere with the upregulation of NF-κB. These studies show that the activation of the NF-κB pathway requires the participation of upstream pathway genes. Recent studies have also found that NF-κB can phosphorylate the IKKα complex, and the IKK complex can act on IκB inhibitory proteins and phosphorylate NF-κB / P65 proteins, playing a role in signal transmission between AKT and NF-κB. In this study, Reyanning mixture activated and inhibited the phosphorylation of AKT in the pathway, preventing the pathway from being activated. It also reduced the level of IκB phosphorylation and promoted the reduction of NF-κB activation level, thereby achieving anti-inflammatory effects.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. The method for analyzing the active ingredients of Reyanning mixture for treating bacterial pneumonia is characterized by: The analysis method includes: screening out the chemical components of Reyanning mixture based on UPLC-Q / TOF-MS technology; using network pharmacology related databases, screening out the effective components of Reyanning mixture and collecting corresponding action targets, and constructing a target library of Reyanning mixture; constructing a bacterial pneumonia target library; taking the intersection of the bacterial pneumonia target library and the Reyanning mixture target library to obtain a target database of Reyanning mixture acting on bacterial pneumonia, and obtaining 49 active targets; using software to visualize the collected active targets and corresponding effective components, constructing a compound network, performing network topology analysis, and obtaining important chemical components and key targets; constructing a protein interaction network; performing GO enrichment analysis and KEGG enrichment analysis on the active targets obtained by screening, and obtaining pathways with high enrichment significance.

2. The method according to claim 1, characterized in that The screening method of the chemical components of Reyanning mixture includes: (1) Prepare the test solution: take 10 μL of the original solution of Reyanning mixture and add it to 10 mL of acetonitrile solvent (acetonitrile: water = 1:1), mix well, centrifuge, and take the supernatant for liquid chromatography analysis and mass spectrometry analysis; (2) Preparation of mixed standard solution: accurately weigh 1 mg each of the reference substances polydatin, rhamnolide, chicoric acid, and baicalin, place them in different centrifuge tubes, add 1 mL of acetonitrile and shake to dissolve; take 125 μL of each and add 500 μL of acetonitrile to dilute, centrifuge at 10,000 rpm for 10 min; take 20 μL of the supernatant and add it to 1 mL of pure water, centrifuge at 10,000 rpm for 10 min, take 900 μL and add it to a liquid phase vial to prepare a mixed standard solution, and perform liquid chromatography and mass spectrometry analysis on the machine; (3) Establishment of the UNIFI analysis platform for Reyanning mixture: The chemical components of Taraxacum mongolicum, Polygonum cuspidatum, Patrinia serrata and Scutellaria barbata were collected and sorted in databases such as TCMSP, BATMAN, symmap, ETCM, and PubChem, and information on compounds that may be related to Reyanning mixture was obtained, including chemical component names, structural formulas, molecular formulas, and chemical structures. The compound structural formulas were stored in .mol ​​mode, sorted into Excel tables and put into folders, thus constructing a chemical component database for Reyanning mixture, which was uploaded to the local UNIFI analysis platform for compound analysis.

3. (4) Data processing and analysis: Manually check the detailed information of compound test samples and standard reference substances to ensure accurate analysis.

4. The method according to claim 2, characterized in that: Liquid chromatography conditions: The chromatographic column used in this experiment is Waters ACQUITY UP-LCBEHC 18 (100mm×4.6mm, 1.7μm); 1mL / L formic acid aqueous solution (A)-acetonitrile (B) as the mobile phase, gradient elution; the HPLC elution gradient is as follows:

5. The method according to claim 2, characterized in that: The mass spectrometry conditions were as follows: positive ion mode (ESi+) and negative ion mode (ESi-) were used for detection, with a scan time of 0.2s and a scan range of 50m / z-2000Da; positive ion mode: capillary voltage 2.00kV, ion source temperature 120℃, desolvation temperature 500℃, desolvation gas nitrogen (N2) flow rate 1000L / h, cone gas flow rate 50L / h, and leucine enkephalin was used as the calibration liquid; negative ion mode: capillary voltage 2.50kV, ion source temperature 100℃, desolvation temperature 450℃, desolvation gas nitrogen (N2) flow rate 600L / h, cone gas flow rate 50L / h, and reserpine was used as the calibration liquid.

6. The method according to claim 1, characterized in that The network pharmacology related databases are TCMSP database, BTAMAN-TCM database, ETCM database and SymMAP database.

7. The method according to claim 1, characterized in that The method for constructing a bacterial pneumonia target library is as follows: using "pneumonia" and "bacterialpneumonia" as keywords, accessing the DrugBank database, TTD database, GEO database, GeneCards database, OMIM database and PharmGkb database, and inputting the information target names obtained from the database into the UniProt database to obtain the target gene names; searching for direct data of "pneumonia" and "bacterialpneumonia" in the GEO database to obtain transcriptomics data, using the R tool for analysis, and obtaining the intersection of public database targets as the bacterial pneumonia target database.

8. The method according to claim 1, characterized in that The method for constructing a protein interaction network is as follows: prepare an input file based on the intersection genes obtained from the bacterial pneumonia target library and the Reyanning mixture target library, copy the gene names in the intersection file to the corresponding boxes in the string database; select the "HomoSapiens" species to obtain the constructed protein interaction network; set filtering conditions, delete free nodes, and obtain a clearer protein interaction network diagram.

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