A method for analyzing the active ingredients of Qingjiang Hewei granules in the treatment of non-erosive gastroesophageal reflux disease based on network pharmacology.
Through network pharmacology analysis and molecular docking technology, the active ingredients and targets of Qingjiang Hewei granules in non-erosive gastroesophageal reflux disease were identified, solving the problem of unclear mechanism of action and achieving preliminary verification of its therapeutic mechanism.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HEILONGJIANG ZBD PHARMA
- Filing Date
- 2026-03-31
- Publication Date
- 2026-06-30
AI Technical Summary
In the existing technology, the mechanism of action of Qingjiang Hewei granules in treating non-erosive gastroesophageal reflux disease is unclear, and there is a lack of systematic research on pharmacodynamic components and target verification.
Using network pharmacology methods, combined with HPLC analysis and database retrieval, a component-disease-target network diagram was constructed to screen key components and core targets. GO function and KEGG pathway enrichment analysis were performed, and molecular docking verification was conducted to clarify the interaction between pharmacodynamic components and targets.
For the first time, the pharmacodynamic material basis of Qingjiang Hewei granules in the treatment of non-erosive gastroesophageal reflux disease was clarified, its potential mechanism of action was explored, and a theoretical basis was provided, laying the foundation for further research.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for analyzing the active ingredients of Qingjianghewei granules in the treatment of non-erosive gastroesophageal reflux disease based on network pharmacology. Background Technology
[0002] For understanding the technical content of this invention:
[0003] Non-erosive reflux disease (NERD), also known as endoscopic negative reflux disease, typically presents with symptoms such as acid reflux and heartburn. This disease accounts for approximately 70% of all gastroesophageal reflux disease (GERD).
[0004] Clinically, the treatment principles for gastroesophageal reflux disease (GERD) are: to control symptoms, cure esophagitis, improve quality of life, prevent complications, and prevent recurrence. Western medicine clinical treatment methods include general treatment, drug therapy, and endoscopic or surgical treatment. The purpose of drug therapy is to control reflux, relieve symptoms, further consolidate the therapeutic effect, and prevent recurrence. Commonly used Western medicine drugs mainly include acid-suppressing drugs (proton pump inhibitors such as omeprazole, esomeprazole, and lansoprazole; histamine H2 receptor antagonists such as cimetidine, ranitidine, and famotidine), prokinetic drugs (domperidone, mosapride, and itopride), and mucosal protectants (sucralfate and bismuth tripotassium dicitrate). These drugs primarily treat symptoms and, although effective, can cause adverse reactions such as headache, diarrhea, nausea, or constipation with long-term use.
[0005] The principles of Traditional Chinese Medicine (TCM) treatment are based on syndrome differentiation and treatment. Clinical treatment focuses on promoting the smooth flow of Qi, employing methods such as soothing the liver and clearing heat, harmonizing the stomach and suppressing rebellious Qi, regulating Qi and resolving phlegm, promoting blood circulation and removing blood stasis, and strengthening the spleen and resolving dampness, depending on the condition. If deficiency syndromes are present, the differentiation of Qi, blood, Yin, and Yang should be applied, supplementing without causing stagnation. TCM has been widely used in the treatment of gastroesophageal reflux disease (GERD), with significant efficacy and high safety, making it worthy of widespread clinical application. Qingjiang Hewei Granules, composed of Scutellaria baicalensis, Coptis chinensis, Pinellia ternata, dried ginger, Fritillaria thunbergii, Taraxacum mongolicum, Gentiana scabra, Citrus aurantium, Trichosanthes kirilowii, and Glycyrrhiza uralensis, has the effects of balancing cold and heat, harmonizing the stomach and suppressing rebellious Qi. It is suitable for non-erosive GERD with a TCM syndrome of mixed cold and heat, characterized by heartburn, acid reflux, belching, epigastric pain, preference for warmth and pressure, dry mouth, bitter taste, burning pain behind the sternum, loose stools, red tongue with a yellow and greasy or thin yellow coating, and a weak pulse. This formula is a modified version of Banxia Xiexin Decoction, which has the effects of balancing cold and heat, harmonizing the stomach, and relieving nausea and vomiting. However, its mechanism of action in treating non-erosive gastroesophageal reflux disease is still unclear.
[0006] Therefore, this study aims to use network pharmacology to explore the potential mechanism of action of Qingjiang Hewei granules in treating non-erosive gastroesophageal reflux disease. At the same time, it will use molecular docking technology based on computer simulation to preliminarily verify the predicted key components and core targets, so as to provide a theoretical basis for subsequent research on the mechanism of action of Qingjiang Hewei granules in treating non-erosive gastroesophageal disease. Summary of the Invention
[0007] The purpose of this invention is to provide: A method based on network pharmacology analysis of the active ingredients of Qingjiang Hewei granules in the treatment of non-erosive gastroesophageal reflux disease, and related technologies, to solve technical problems such as exploring the active ingredients of Qingjiang Hewei granules in the treatment of non-erosive gastroesophageal reflux disease, or a combination thereof.
[0008] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.
[0009] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.
[0010] The definition of the standard chemical term can be found in the reference "Network Pharmacology" (Tsinghua University Press, Li Shao, April 2022).
[0011] Unless otherwise stated, conventional methods within the scope of the art, such as HPLC, shall be used. Unless specifically defined, the use of all commercially available products used herein shall employ standard techniques. For example, they may be performed using the manufacturer's instructions for use with the kits, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein are generally performed according to conventional methods well known in the art, based on the descriptions in the various general and more specific documents cited and discussed in this specification.
[0012] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.
[0013] In a first aspect, the present invention provides a method for analyzing the pharmacodynamic components of Qingjiang Hewei granules in the treatment of non-erosive gastroesophageal reflux disease based on network pharmacology. The analytical method employs HPLC, generates a molecular network of chemical components of Qingjiang Hewei granules through standard comparison and database, obtains relevant targets for non-erosive gastroesophageal reflux disease using network pharmacology databases, constructs a "component-disease-target" network diagram and screens key components, constructs a protein interaction network, performs GO function and KEGG pathway enrichment analysis, and conducts preliminary verification of molecular docking between key components and core targets.
[0014] In some embodiments, the non-erosive gastroesophageal reflux disease is specifically caused by lower esophageal sphincter dysfunction.
[0015] In some embodiments, the method includes the following steps: (1) Preparation of test sample: Weigh Qingjiang Hewei granules, dissolve in methanol aqueous solution, filter, take the filtrate to obtain Qingjiang Hewei granules test sample solution; weigh an appropriate amount of reference standard, dissolve, and prepare reference standard solution; (2) HPLC analysis: The retention times of the chromatographic peaks of the test solution and the reference solution of Qingjiang Hewei Granules were compared to obtain the structural information of the chemical components of Qingjiang Hewei Granules; (3) Screening of active chemical components of Qingjiang Hewei Granules: a. Using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), we searched for the active components of Scutellaria baicalensis, Coptis chinensis, Zingiber officinale, Pinellia ternata, Fritillaria thunbergii, Gentiana scabra, Citrus aurantium, Trichosanthes kirilowii, and Glycyrrhiza uralensis in Qingjiang Hewei Granules and their corresponding targets; b. Using the PubChem database, we searched for the active components of Taraxacum mongolicum and copied them to the Swiss Target Prediction database to predict the targets of the active components of Taraxacum mongolicum; c. Based on the components obtained by HPLC analysis, we determined the corresponding targets. (4) Target prediction for non-erosive gastroesophageal reflux disease: Targets for non-erosive gastroesophageal reflux disease were obtained by using the GeneCards and MalaCards databases with the keyword "Gastroesophageal reflux disease". The disease targets obtained from the two databases were merged and duplicate values were removed to obtain the disease targets for gastroesophageal reflux disease. (5) Construction of compound-component-target network diagram and screening of key components: The effective components and targets obtained from the screening of the clearing and gastric granules in steps (2) and (3) are imported into Cytoscape software to construct the compound-component-target network diagram, and 20 key components are screened according to the Degree (DC) value. (6) Intersection target and protein-protein interaction (PPI) analysis of Qingjiang Hewei Granules in the treatment of non-erosive gastroesophageal reflux disease: The disease targets of Qingjiang Hewei Granules and non-erosive gastroesophageal reflux disease were uploaded to the Draw Venn Diagram online platform to obtain the target of Qingjiang Hewei Granules in the treatment of non-erosive gastroesophageal reflux disease. The intersection targets were imported into the STRING online platform, Multiple proteins were selected, and Organisms were selected as Homosapiens to obtain the protein interaction relationship. After exporting the data, it was imported into Cytoscape software to calculate the Degree value. The top 20 core targets were selected based on the Degree, Betweenness centrality and Closeness centrality values. (7) GO function and KEGG pathway enrichment analysis: The intersection targets obtained above were uploaded to the DAVID online database to perform GO and KEGG pathway enrichment analysis. The enrichment results were downloaded and the top 20 results of Biological process, Cellular Components and Molecular Function and KEGG were selected by Degree value respectively. They were then uploaded to the MicroBioinformatics online platform for visualization to obtain pathways with high enrichment significance. (8) Preliminary verification of molecular docking between key components and core targets: Search the PubChem database and download the corresponding SDF structure file of the component. Based on PPI screening, the top 20 targets are obtained and uploaded to the PDB database. Select Homo sapiens and download the corresponding PDB format file of the target. Upload the SDF structure file and PDB format file to the online docking tool CB-Dock 2 for molecular docking and visualize the results.
[0016] In some embodiments, the preparation of the Qingjiang Hewei Granules test solution in step (1) is as follows: take an appropriate amount of Qingjiang Hewei Granules, grind them finely, take 0.25g, accurately weigh them, place them in a 50mL volumetric flask, add 70% methanol, shake well, sonicate for 30min, cool, make up to volume, shake well, filter, and take the filtrate to obtain the Qingjiang Hewei Granules test solution.
[0017] In some embodiments, the preparation of the reference solution in step (1) is specifically as follows: take appropriate amounts of reference standards, accurately weigh them, dissolve them in 70% methanol, and prepare a reference solution with a concentration of 100 μg / mL, thereby obtaining the corresponding reference solution. The reference standards are: baicalin, wogonin, wogonin, sennain A, berberine hydrochloride, epiberberine, berberine hydrochloride, palmatine hydrochloride, berberine hydrochloride, chicoric acid, caffeic acid, gentiopicrin, glycyrrhizin, ammonium glycyrrhizate, naringin, hesperidin, neohesperidin, 6-gingerol, ferulic acid, and vanillic acid.
[0018] In some embodiments, the HPLC chromatographic conditions in step (2) are as follows: acetonitrile is used as mobile phase A, and 20 mmol / L potassium dihydrogen phosphate aqueous solution (pH adjusted to 2.0 with phosphoric acid) is used as mobile phase B for gradient elution; the flow rate is 0.8 mL / min; the detection wavelength is 237 nm; the column temperature is 25 °C; and the elution gradient is: 0-10 min, 19% A; 10-16 min, 19% → 20% A; 16-18 min, 20% → 21%; 18-28 min, 21% → 22%; 28-38 min, 22%; 38-42 min, 22% → 25%; 42-60 min, 25% → 40%; 60-90 min, 40% → 52%; 90-91 min, 52% → 71%; 91-100 min, 71% → 19%; 100-110 min, 19%.
[0019] In some embodiments, the screening criteria for the active ingredient in step (3) are: screening with oral bioavailability ≥30% and drug-likeness ≥0.18.
[0020] In some embodiments, the target in step (3) is predicted based on the TCMSP database and the PharmMapper database respectively, and is selected as a potential target of the component based on the Norm Fit value ≥ 0.5.
[0021] In some embodiments, the target screening criteria in step (4) are: disease targets are screened in the GeneCards database with a median Relevance score ≥ 7.29.
[0022] In some embodiments, the 20 key components in step (5) are: baicalin, wogonin, wogonin, sennain A, berberine hydrochloride, epiberberine, berberine hydrochloride, palmatine hydrochloride, berberine hydrochloride, chicoric acid, caffeic acid, gentiopicrin, glycyrrhizin, ammonium glycyrrhizate, naringin, hesperidin, neohesperidin, 6-gingerol, ferulic acid, and vanillic acid.
[0023] In some embodiments, the first 20 core targets in step (6) are TNF, IL6, STAT3, CASP3, TP53, AKT1, PTGS2, JUN, BCL2, HIF1A, INS, TGFB1, IL1B, MMP9, EGFR, IFNG, CXCL8, ANXA5, SRC, and MYC.
[0024] In some embodiments, the pathways with high enrichment significance in step (7) are PI3K-Akt, MAPK and Ras signaling pathways.
[0025] The present invention has at least the following beneficial effects: Compared with the prior art, this invention clarifies for the first time the pharmacodynamic material basis of Qingjiang Hewei granules in treating non-erosive gastroesophageal reflux disease, and explores for the first time the potential mechanism of action of Qingjiang Hewei granules in treating non-erosive gastroesophageal reflux disease. Attached Figure Description
[0026] Figure 1 Figure 1 shows the component analysis and identification of Qingjiang and Wei granules based on HPLC. Figure 2 Venn diagram of disease targets for non-erosive gastroesophageal reflux disease; Figure 3 Venn diagram of disease intersection targets for Qingjiang Hewei granules in the treatment of non-erosive gastroesophageal reflux disease; Figure 4 : Compound-component-target network diagram of Qingjiang Hewei Granules; Figure 5 PPI analysis of the intersection targets of Qingjiang Hewei Granules in the treatment of non-erosive gastroesophageal disease; Figure 6 Figure: GO-Biological process (BP) enrichment analysis results of Qingjiang Hewei Granules for NERD intersection targets; Figure 7 Figure: GO-Cellular component (CC) enrichment analysis results of Qingjiang Hewei Granules for NERD treatment; Figure 8 Figure: GO-Molecular function (MF) enrichment analysis results of Qingjiang Hewei Granules for NERD intersection targets; Figure 9 Results of KEGG pathway enrichment analysis of Qingjiang Hewei Granules as the intersection target of NERD treatment; Figure 10 Partial results of visualization of the docking between key components and core targets. Detailed Implementation
[0027] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.
[0028] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.
[0029] Example 1: Analysis of components in septic tank and gastric granules based on high performance liquid chromatography (HPLC) The reagents and drugs used in this embodiment are shown in Table 1, and the experimental instruments are shown in Table 2.
[0030] Table 1: Main Reagents and Drugs
[0031] Table 2: Experimental Instruments
[0032] Sample preparation method: (1) Preparation of reference solution: Take appropriate amounts of 20 reference standards of baicalin-vanillic acid in Table 1, weigh them accurately, dissolve them completely, and prepare a reference solution with a concentration of about 100 μg / mL to obtain the corresponding reference solution; (2) Preparation of test solution: Take an appropriate amount of Qingjiang Hewei Granules (batch number: SB20602), grind it finely, take about 0.25 g, weigh it accurately, put it in a 50 mL volumetric flask, add an appropriate amount of 70% methanol, shake it thoroughly, sonicate it for 30 min, cool it, make up to volume, shake it well, filter it, and take the filtrate to obtain the Qingjiang Hewei Granules test solution.
[0033] Chromatographic conditions: Octadecylsilane-bonded silica gel (YMC PACK ODS-AQ, 4.6 × 250 mm, 5 μm) was used as the stationary phase; acetonitrile was used as mobile phase A, and 20 mmol / L potassium dihydrogen phosphate aqueous solution (pH adjusted to 2.0 with phosphoric acid) was used as mobile phase B. Gradient elution was performed according to the elution conditions in Table 3; the flow rate was 0.8 mL / min; the detection wavelength was 237 nm; and the column temperature was 25 °C.
[0034] Table 3: Chromatographic elution conditions
[0035] Based on the above chromatographic conditions, 20 component peaks (peaks 1-20) were obtained in the test sample solution by comparison with the standard solution. These peaks are: gentiopicrin, caffeic acid, vanillic acid, glycyrrhizin, ferulic acid, chicoric acid, naringin, hesperidin, neohesperidin, epiberberine, berberine hydrochloride, baicalin, palmatine hydrochloride, berberine hydrochloride, scutellarin A, wogonin, ammonium glycyrrhizate, 6-gingerol, and wogonin. For detailed results, please refer to [link to results]. Figure 1 Table 4.
[0036] Table 4: Identification of components in sclerosing and gastric granules based on HPLC
[0037] Note: Mol ID refers to the component name in the standardized database after being retrieved from the TCMSP database.
[0038] Example 2: Network pharmacology-based predictive analysis of the effective components and targets of Qingjiang Hewei granules Using the following keywords from the Qingjiang Hewei Granules formula—"Huangqin (HQ)," "Huanglian (HL)," "Gangjiang (GJ)," "Qingbanxia (BX)," "Zhebeimu (BM)," "Pugongying (PGY)," "Longdancao (LD)," "Zhishi (ZS)," "Quangualou (GL)," and "Zhigancao (GC)"—as keywords, the corresponding active ingredients of each herb were retrieved from the Traditional Chinese Medicine Systems Pharmacology (TCMSP, Version 2.3, https: / / www.tcmsp-e.com / index.php). According to literature reports, the effective components of each single herb were screened based on oral bioavailability (OB) ≥30% and drug likeness (DL) ≥0.18. At the same time, the corresponding targets of each effective component were retrieved from the TCMSP database.
[0039] For active ingredients for which no corresponding targets were found in the TCMSP database, they were imported into the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ), and their SMILES were copied to the Swiss Target Prediction database (https: / / www.swisstargetprediction.ch / index.php, version 2019). Homosapiens was selected as the prediction condition to predict possible targets, and then the corresponding targets were saved. During the search, it was found that "dandelion" in the formula could not be found in the TCMSP database. Therefore, the active ingredients of dandelion were further supplemented through literature search. The target prediction and normalization methods for its active ingredients were the same as before. Finally, the normalized targets were merged, and duplicate values were removed to obtain the complete target of the Qingjiang and Wei granules formula.
[0040] In addition, components in the Qingjiang and Wei granules were detected using high-performance liquid chromatography (HPLC). Figure 1 (Table 4) A total of 20 components were directly included in the effective component cohort of the Qingjiang and Wei Granules formula. The corresponding targets of each component were predicted based on the TCMSP database and the PharmMapper database (http: / / www.lilab-ecust.cn / pharmmapper / , Version 2017). Potential targets for each component were selected based on a Norm Fit value ≥ 0.5.
[0041] Based on the screening criteria and the elimination of components whose corresponding targets could not be predicted, a total of 34 effective components were obtained from Scutellaria baicalensis (HQ), 14 from Coptis chinensis (HL), 5 from Zingiber officinale (GJ), 13 from Pinellia ternata (BX), 7 from Fritillaria thunbergii (BM), 10 from Gentiana scabra (LD), 23 from Citrus aurantium (ZS), 11 from Trichosanthes kirilowii (GL), 93 from Glycyrrhiza uralensis (GC), and 40 from Taraxacum mongolicum (PGY). After removing duplicates, the corresponding targets were determined as follows: 146 from Scutellaria baicalensis, 207 from Coptis chinensis, 114 from Pinellia ternata, 45 from Zingiber officinale, 69 from Fritillaria cirrhosa, 112 from Gentiana scabra, 162 from Citrus aurantium, 15 from Trichosanthes kirilowii, 272 from Glycyrrhiza uralensis, and 286 from Taraxacum mongolicum.
[0042] Among them, MOL000358 (beta-sitosterol, defined as A1) is a component common to BM, BC, GJ, and HQ; MOL000359 (sitosterol, defined as A2) is a component common to GC, GJ, HQ, and LD; MOL000098 (quercetin, defined as B1) is a component common to GC, HL, and PGY; MOL002897 (epiberberine, defined as A3) and MOL001458 (coptisine, defined as A4) are components common to HL and HQ; MOL002714 (baicalein, defined as A5) and MOL000449 (stigmasterol, defined as A5) are components common to BM, BC, GJ, and HQ. A6) is a component shared by BX and HQ; MOL002914 (Eriodyctiol (flavanone), defined as A7) is a component shared by HQ and ZS; MOL013352 (obacunone, defined as C1) is a component shared by HL and ZS; MOL000422 (kaempferol, defined as B2) is a component shared by GC and LD; MOL004328 (naringenin, defined as B3) is a component shared by GC and ZS; MOL000006 (luteolin, defined as C2) is a component shared by PGY and ZS; MOL000354 (isorhamnetin, defined as B4) is a component shared by GC and PGY.
[0043] Based on HPLC analysis, a total of 20 effective components of Qingjiang Hewei Granules were identified, and a total of 445 target sites were predicted.
[0044] After merging the target points corresponding to each component, removing unnormalized target points, and eliminating duplicate values, a total of 751 target points for Qingjiang Hewei Granules were obtained.
[0045] Example 3: Target prediction for non-erosive gastroesophageal reflux disease Using "Gastroesophageal reflux disease" as the keyword, data was retrieved from the GeneCards: The Human Gene Database (https: / / www.genecards.org / , Version 5.23, Updated - Dec 23, 2024) and the MalaCards: The Human Disease Database (https: / / www.malacards.org / , Version 5.23 - Updated December 20, 2024) to obtain disease targets for non-erosive gastroesophageal diseases. In the GeneCards database, disease targets were filtered based on a median Relevance score ≥7.29. Subsequently, the disease targets retrieved from the two databases were merged, and duplicate values were removed to obtain the disease targets for gastroesophageal reflux disease.
[0046] Results: A total of 7399 disease targets for gastroesophageal reflux disease (GERD) were obtained from the Genecards database. After filtering based on a median relevance score ≥ 7.29, 3700 targets were identified. A search of the Malacards database yielded 103 disease targets for GERD. Venn diagrams were plotted from both databases, and after removing duplicates, a total of 3712 disease targets for non-erosive GERD were obtained. The results are shown below. Figure 2 .
[0047] To further identify the target sites of Qingjiang Hewei Granules (QJHWG) in the treatment of NERD, Venn diagrams were constructed, yielding 372 intersecting target sites, including MMP2, KCNMA1, XDH, HSPB1, DECR1, PRKCG, NOS2, and CACNA2D1. These are considered potential targets for Qingjiang Hewei Granules in treating NERD. The results are shown in […]. Figure 3 Table 5.
[0048] Table 5: Potential targets of Qingjiang Hewei Granules in the treatment of NERD
[0049] Example 4: Construction of the compound-component-target network diagram and screening of key components The effective components and targets obtained from the screening of Qingjiang and Wei granules were imported into Cytoscape software to construct a compound-component-target network diagram, and key components were screened based on the Degree (DC) value.
[0050] A compound-component-target network diagram was constructed using Cytoscape software to identify potential key components of Qingjiang Hewei granules for treating non-erosive gastroesophageal reflux disease. The top 30 effective components were screened based on Degree (DC) values, including Quercetin (B1), Glycyrrhizinate, Baicalin, Chicoric Acid, Wogonoside, Naringin, Liquiritin, Neohesperidin, Hesperidin, Wogonin, and Oroxylin. A (Phyllostachys A), Beta-sitosterol (A1), 6-Gingerol, Palmatine, Kaempferol (B2), epiberberine, Berberine, Jatrorrhizine, Luteolin (C2), coptisine (A4), Ferulic Acid, Apigenin, Baicalein (A5), Naringenin (B3), Isorhamnetin (B4), Caffeic acid, Stigmasterol (A6), Vanillic acid, 7-Methoxy-2-methyl isoflavone, and formononetin.
[0051] Furthermore, further analysis revealed that 20 targets in Qingjiang Hewei Granules, including PTGS2, AR, HSP90AB1, PTGS1, ESR1, CALM1, NOS2, NCOA2, PRSS1, PRKACA, and PPARG, may be targets for its treatment of non-erosive esophageal reflux disease.
[0052] See results Figure 4 Tables 6 and 7. Figure 4In Chinese: A1 (MOL000358, beta-sitosterol) is a common component of BM, BX, GJ, and HQ; A2 (MOL000359, sitosterol) is a common component of GC, GJ, HQ, and LD; A3 (MOL002897, epiberberine) is a common component of HL and HQ; A4 (MOL001458, coptisine) is a common component of HL and HQ; A5 (MOL002714, baicalein) is a common component of BX and HQ; A6 (MOL000449, stigmasterol) is a common component of BX and HQ; A7 (MOL002914, eriodyne) is a common component of BM, BX, GJ, and HQ. Ctiol (flavanone) is a common component of HQ and ZS; B1 (MOL000098, quercetin) is a common component of GC, HL and PGY; B2 (MOL000422, kaempferol) is a common component of GC and LD; B3 (MOL004328, naringenin) is a common component of GC and ZS; B4 (MOL000354, isorhamnetin) is a common component of GC and PGY; C1 (MOL013352, obacunone) is a common component of HL and ZS; C2 (MOL000006, luteolin) is a common component of PGY and ZS.
[0053] Table 6: Top 30 Main Ingredients of Qingjiang and Wei Granules Ranked by Degree Value
[0054] Note: DC is Degree value; GC is licorice; HL is Coptis chinensis; PGY is dandelion; QJHWG is Qingjiang Hewei Granules; HQ is Scutellaria baicalensis; ZS is Citrus aurantium; BM is Fritillaria cirrhosa; BX is Pinellia ternata; GL is Trichosanthes kirilowii; GJ is dried ginger; LD is gentian.
[0055] Table 7: Top 20 targets of Qingjiang and Wei Granules identified based on compound-component-target screening
[0056] Example 5: Intersecting target and protein-protein interaction (PPI) analysis of Qingjiang Hewei granules in the treatment of non-erosive gastroesophageal reflux disease The disease targets of Qingjiang Hewei Granules and non-erosive gastroesophageal reflux disease (GERD) were uploaded to the DrawVenn Diagram online platform (http: / / bioinformatics.psb.ugent.be / webtools / Venn / ) to obtain the target of Qingjiang Hewei Granules for the treatment of GERD. Then, the intersection targets were imported into the STRING online platform (https: / / cn.string-db.org / , version 2024), where Multiple proteins were selected and Homo sapiens were chosen as the Organisms for protein-protein interaction (PPI) analysis. The PPI analysis results were then exported and imported into Cytoscape software (Version 3.8.2, https: / / cytoscape.org / ) to calculate the Degree value. The top 20 core targets were then selected based on the Degree (DC), BC (Betweenness centrality), and CC (Closeness centrality) values.
[0057] PPI analysis was performed on 372 targets that overlap with the therapeutic effects of Qingjiang and Wei granules. The results showed that the top 20 targets, after screening based on DC, BC, and CC, included TNF (Tumor necrosis factor), IL6 (Interleukin-6), STAT3 (Signal transducer and activator of transcription 3), CASP3 (Caspase-3), TP53 (Cellular tumor antigen p53), AKT1 (RAC-alpha serine / threonine-protein kinase), PTGS2 (Prostaglandin G / H synthase 2), JUN (Transcription factor AP-1), BCL2 (Apoptosis regulator Bcl-2), HIF1A (Hypoxia-inducible factor 1-alpha), INS (Insulin receptor), TGFB1 (Transforming growth factor beta-1), IL1B (Interleukin-1 beta), MMP9 (Matrix metalloproteinase-9), and EGFR (Epidermal growth factor). Targets included factor receptor, IFNG (Interferon gamma), CXCL8 (Interleukin-8), ANXA5 (Annexin A5), SRC (Proto-oncogene tyrosine-protein kinase Src), and MYC (Myc proto-oncogene protein). Results are shown in […]. Figure 5 Table 8.
[0058] Table 8: Top 20 PPI Targets of Qingjiang Hewei Granules in the Treatment of Non-Erosive Gastroesophageal Disease
[0059] Note: DC is the Degree value; BC is the Betweenness centrality; CC is the Closeness centrality.
[0060] Example 6: Enrichment analysis of GO and KEGG pathways The aforementioned intersection targets were uploaded to the DAVID online database (Database for Annotation, Visualization, and Integrated Discovery, https: / / davidbioinformatics.nih.gov / home.jsp, version 2024q4, Released by Nov. 11, 2024) for Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The enrichment results were downloaded and the top 20 results for Biological Process (BP), Cellular Components (CC), Molecular Function (MF), and KEGG were selected by Degree value, and then uploaded to the Microbioinformatics online platform (https: / / www.bioinformatics.com.cn / ) for visualization.
[0061] Enrichment analyses of the GO and KEGG pathways were performed on the intersecting targets, respectively. The results of the GO enrichment analysis are as follows: Figures 6-8 As shown. Figure 6The results showed that the BP process of Qingjiang and Wei granules is mainly enriched in biological processes such as positive regulation of transcription by RNA polymerase II, signal transduction involved in the regulation of gene expression, positive regulation of gene expression, positive regulation of cell population proliferation, negative regulation of apoptotic process, response to xenobiotic stimulus, inflammatory response, and apoptotic process. The CC process focuses on processes involving cellular components such as the cytosol, plasma membrane, cytoplasm, nucleus, nucleoplasm, membrane structures, extracellular region, extracellular space, extracellular exosomes, and mitochondria. Results are shown in […]. Figure 7MF primarily affects protein binding, identical protein binding, metal ion binding, enzyme binding, ATP binding, protein homodimerization activity, DNA binding, protein kinase binding, RNA polymerase II cis-regulatory region sequence-specific DNA binding, DNA-binding transcription factor activity, and RNA polymerase II-specific processes. The results are shown in […]. Figure 8 .
[0062] KEGG pathway enrichment analysis revealed that the intersection targets were mainly concentrated in cancer pathways, metabolic pathways, the PI3K-Akt signaling pathway, the MAPK signaling pathway, the Ras signaling pathway, lipid and atherosclerosis, the AGE-RAGE signaling pathway in diabetic complications, and chemical carcinogenesis-reactive oxygen species pathways. The results are shown in [Figure number missing]. Figure 9 .
[0063] Example 7: Preliminary verification of docking between key components and core target molecules Based on the key components of the Qingjiang and Wei granules identified through component-target network diagram screening, a selection of representative components were used for molecular docking validation. First, the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ) was searched, and the corresponding SDF structure files for each component were downloaded. Then, the core targets were selected based on the top 20 targets identified through PPI screening, uploaded to the Protein Data Bank (PDB) database (https: / / www.rcsb.org / ), and Homo sapiens were chosen. The corresponding PDB format files for each target were then downloaded. Finally, the prepared files were uploaded to the online docking tool CB-Dock 2 (https: / / cadd.labshare.cn / cb-dock2 / php / index.php) for molecular docking (blind docking), and the results were visualized. Generally, a docking binding energy less than -5.0 kcal / mol indicates good docking between the receptor and ligand.
[0064] To preliminarily explore the binding between key components and core targets of Qingjiang Hewei Granules in the treatment of non-erosive gastroesophageal reflux disease, molecular docking was performed on key components that ranked high in the predicted results of Qingjiang Hewei Granules, namely Berberine, Epiberberine, Glycyrrhizinate, Liquiditin, Quercetin, Beta-sitosterol, Kaempferol, Luteolin, Baicalin, Naringin, Neohesperidin, Wogonin, and Wogonoside, as well as some core targets STAT3, AKT1, and PGTS2.
[0065] Docking results showed that the binding energies of the key components Berberine, Epiberberine, Glycyrrhizinate, Liquiditin, Quercetin, Beta-sitosterol, Kaempferol, Luteolin, Baicalin, Naringin, Neohesperidin, Wogonin, and Wogonoside to the core targets STAT3 (PDB ID: 6tlc), AKT1 (PDB ID: 7wm2), and PTGS2 (PDB ID: 6ofy), respectively, were all less than -5.0 kcal / mol, indicating that the key components in Qingjiang and Wei granules have good binding to the core targets. (See attached figures.) Figure 10 Table 9.
[0066] Table 9: Molecular docking results between key components and core targets (kcal / mol)
[0067] In summary, network pharmacology analysis revealed that the following components are present in Qingjiang Hewei Granules: Quercetin (B1), Glycyrrhizinate, Baicalin, Chicoric Acid, Wogonoside, Naringin, Liquiritin, Neohesperidin, Hesperidin, Wogonin (HQ2), and Oroxylin. The key components responsible for its therapeutic effect on NERD include A (skin-layering agent A), Beta-sitosterol (A1), 6-Gingerol, Palmatine, Kaempferol (B2), epiberberine, Berberine, Jatrorrhizine, Luteolin (C2), coptisine (A4), Ferulic Acid, Apigenin (PGY21), Baicalein (A5), Naringenin (B3), Isorhamnetin (B4), Caffeic acid, Stigmasterol (A6), and Vanillic acid. PPI analysis revealed that the core targets of Qingjiang Hewei Granules in treating NERD include TNF, IL6, STAT3, CASP3, TP53, AKT1, PTGS2, JUN, BCL2, HIF1A, INS, TGFB1, IL1B, MMP9, EGFR, IFNG, CXCL8, ANXA5, SRC, and MYC. Further molecular docking results showed good docking between the key components of Qingjiang Hewei Granules and the core targets, indicating that the mechanism by which Qingjiang Hewei Granules improve NERD may be through acting on multiple targets such as AKT1, PTGS2, STAT3, and HIF1A, thereby regulating the expression of proteins related to pathways such as the PI3K-Akt signaling pathway, MAPK signaling pathway, and Ras signaling pathway.
[0068] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.
Claims
1. A method for analyzing the pharmacodynamic components of Qingjiang Hewei granules in the treatment of non-erosive gastroesophageal reflux disease based on network pharmacology. The analytical method uses HPLC, generates a molecular network of chemical components of Qingjiang Hewei granules by comparing with standards and databases, obtains relevant targets for non-erosive gastroesophageal reflux disease using network pharmacology databases, constructs a "component-disease-target" network diagram and screens key components, constructs a protein interaction network, performs GO function and KEGG pathway enrichment analysis, and conducts preliminary verification of molecular docking between key components and core targets.
2. The method according to claim 1, characterized in that, The non-erosive gastroesophageal reflux disease is specifically caused by dysfunction of the lower esophageal sphincter.
3. The method according to claim 1, characterized in that, The method is performed according to the following steps: (1) Preparation of test sample: Weigh Qingjiang Hewei granules, dissolve in methanol aqueous solution, filter, take the filtrate to obtain Qingjiang Hewei granules test sample solution; weigh an appropriate amount of reference standard, dissolve, and prepare reference standard solution; (2) HPLC analysis: The retention times of the chromatographic peaks of the test solution and the reference solution of Qingjiang Hewei Granules were compared to obtain the structural information of the chemical components of Qingjiang Hewei Granules; (3) Screening of active chemical components of Qingjiang Hewei Granules: a. Using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), we searched for the active components of Scutellaria baicalensis, Coptis chinensis, dried ginger, Pinellia ternata, Fritillaria thunbergii, Gentiana scabra, Citrus aurantium, Trichosanthes kirilowii and Glycyrrhiza uralensis in Qingjiang Hewei Granules and their corresponding targets. b. Use the PubChem database to retrieve the active ingredients of dandelion and copy them to the Swiss Target Prediction database to predict the targets of the active ingredients of dandelion; c. Determine the corresponding target points based on the components obtained from HPLC analysis; (4) Target prediction for non-erosive gastroesophageal reflux disease: Targets for non-erosive gastroesophageal reflux disease were obtained by using the GeneCards and MalaCards databases with the keyword "Gastroesophageal reflux disease". The disease targets obtained from the two databases were merged and duplicate values were removed to obtain the disease targets for gastroesophageal reflux disease. (5) Construction of compound-component-target network diagram and screening of key components: The effective components and targets obtained from the screening of the clearing and gastric granules in steps (2) and (3) are imported into Cytoscape software to construct the compound-component-target network diagram, and 20 key components are screened according to the Degree (DC) value. (6) Intersection target and protein-protein interaction (PPI) analysis of Qingjiang Hewei Granules in the treatment of non-erosive gastroesophageal reflux disease: The disease targets of Qingjiang Hewei Granules and non-erosive gastroesophageal reflux disease were uploaded to the DrawVenn Diagram online platform to obtain the target of Qingjiang Hewei Granules in the treatment of non-erosive gastroesophageal reflux disease. The intersection targets were imported into the STRING online platform, Multiple proteins were selected, and Organizations were selected as Homo sapiens to obtain the protein interaction relationship. After exporting the data, it was imported into Cytoscape software to calculate the Degree value. The top 20 core targets were selected based on the Degree, Betweenness centrality and Closeness centrality values. (7) GO function and KEGG pathway enrichment analysis: The intersection targets obtained above were uploaded to the DAVID online database to perform GO and KEGG pathway enrichment analysis. The enrichment results were downloaded and the top 20 results of Biological process, Cellular Components and Molecular Function and KEGG were selected by Degree value respectively. They were then uploaded to the MicroBioinformatics online platform for visualization to obtain pathways with high enrichment significance. (8) Preliminary verification of molecular docking between key components and core targets: Search the PubChem database and download the corresponding SDF structure file of the component. Based on PPI screening, the top 20 targets are obtained and uploaded to the PDB database. Select Homosapiens and download the corresponding PDB format file of the target. Upload the SDF structure file and PDB format file to the online docking tool CB-Dock 2 for molecular docking and visualize the results.
4. The method according to claim 3, characterized in that, The preparation of the Qingjiang Hewei Granules test solution in step (1) is as follows: take an appropriate amount of Qingjiang Hewei Granules, grind them into a fine powder, take 0.25g, accurately weigh it, place it in a 50mL volumetric flask, add 70% methanol, shake it thoroughly, sonicate it for 30min, cool it, make up to volume, shake it thoroughly, filter it, and take the filtrate to obtain the Qingjiang Hewei Granules test solution.
5. The method according to claim 3, characterized in that, The preparation of the reference solution in step (1) is as follows: take an appropriate amount of reference, weigh it accurately, dissolve it in 70% methanol, and prepare a reference solution with a concentration of 100 μg / mL. The reference solutions are: baicalin, wogonin, wogonin, scutellarin A, berberine hydrochloride, epiberberine, berberine hydrochloride, palmatine hydrochloride, berberine hydrochloride, chicoric acid, caffeic acid, gentiopicrin, glycyrrhizin, ammonium glycyrrhizate, naringin, hesperidin, neohesperidin, 6-gingerol, ferulic acid, and vanillic acid.
6. The method according to claim 3, characterized in that, The HPLC chromatographic conditions in step (2) are as follows: acetonitrile is used as mobile phase A, and 20 mmol / L potassium dihydrogen phosphate aqueous solution (pH adjusted to 2.0 with phosphoric acid) is used as mobile phase B for gradient elution; the flow rate is 0.8 mL / min; the detection wavelength is 237 nm; the column temperature is 25 ℃; the elution gradient is: 0-10 min, 19% A; 10-16 min, 19%→20% A; 16-18 min, 20%→21%; 18-28 min, 21%→22%; 28-38 min, 22%; 38-42 min, 22%→25%; 42-60 min, 25%→40%; 60-90 min, 40%→52%; 90-91 min, 52%→71%; 91-100 min, 71%→19%; 100-110 min, 19%.
7. The method according to claim 3, characterized in that, The screening criteria for the active ingredients in step (3) are as follows: oral bioavailability ≥30% and drug-likeness ≥0.18 are used as conditions for screening, and the corresponding targets of each ingredient are predicted based on the TCMSP database and the PharmMapper database respectively. At the same time, the potential targets of the ingredient are screened according to the Norm Fit value ≥0.
5. The screening conditions for the targets in step (4) are as follows: disease targets are screened in the GeneCards database with the median Relevancescore ≥7.29 as conditions.
8. The method according to claim 3, characterized in that, The 20 key components mentioned in step (5) are: baicalin, wogonin, wogonin, sennain A, berberine hydrochloride, epiberberine, berberine hydrochloride, palmatine hydrochloride, berberine hydrochloride, chicoric acid, caffeic acid, gentiopicrin, glycyrrhizin, ammonium glycyrrhizate, naringin, hesperidin, neohesperidin, 6-gingerol, ferulic acid, and vanillic acid.
9. The method according to claim 3, characterized in that, The first 20 core targets mentioned in step (6) are TNF, IL6, STAT3, CASP3, TP53, AKT1, PTGS2, JUN, BCL2, HIF1A, INS, TGFB1, IL1B, MMP9, EGFR, IFNG, CXCL8, ANXA5, SRC, and MYC.
10. The method according to claim 3, characterized in that, The pathways with high enrichment significance in step (7) are the PI3K-Akt, MAPK and Ras signaling pathways.