Application of throat-clearing dropping pill in preparation of medicine for treating cough caused by ACEI

Abstract

The invention provides application of a throat-clearing dropping pill in preparation of a medicine for treating cough caused by ACEI. According to the present invention, the throat heat clearing dropping pill can alleviate ACEI-induced cough and accompanied pharyngeal discomfort symptoms by inhibiting bradykinin accumulation, inhibiting arachidonic acid metabolism, reducing cough high sensitivity, reducing airway hyperresponsiveness and other potential action mechanisms, such that the throat heat clearing dropping pill can be used for preparing drugs for preventing and / or treating ACEI-induced cough and accompanied pharyngeal discomfort symptoms. In addition, the invention further successfully establishes a guinea pig cough inducing experimental model of the ACEI compound cough inducing agent for the first time.

Description

[0001] This application claims the priority of Chinese patent application filed with the Patent Office of the State Intellectual Property Office of China on December 31, 2024, with application number 202411987452.3 and invention name "Use of Qingyan Drops in the Preparation of Medicines for Treating ACEI-Induced Cough", the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application relates to the technical field of traditional Chinese medicine, and in particular to the use of Qingyan drops in the preparation of a medicine for treating ACEI-induced cough. Background Art

[0003] Angiotensin-converting enzyme inhibitors (ACEI) antihypertensive drugs are mainly used to treat hypertension, kidney disease, myocardial infarction, congestive heart failure and other diseases. Cough is the most common adverse reaction of ACEI drugs. The specific mechanism of ACEI-induced dry cough is not very clear, and there are currently no clinical guidelines recommending the use of drugs to relieve ACEI-induced dry cough. Only a small number of clinical studies have reported on some throat and cough and expectorant drugs. No relevant information has been retrieved in the basic research field of ACEI-induced dry cough, and there is a lack of reference materials for model establishment and systematic research.

[0004] Qingyan Drop Pills are a traditional Chinese medicine drop pill preparation developed exclusively by Jinyao Darentang Sixth Traditional Chinese Medicine Factory. The formula is a modified version of Qingyin Pills in Lantai Guifan and Liushen Pills in Houke Xinfa. Although it is derived from an ancient prescription, it is the essence of the formula. It has no toxic ingredients and is highly safe. It can be used for wind-heat throat paralysis, sore throat, dry throat and red and swollen throat. It has significant effects on the treatment of respiratory diseases, especially throat symptoms. The potential therapeutic effect of Qingyan Drop Pills on cough caused by ACEI has not been reported. Summary of the invention

[0005] The inventors of the present application have found through research that Qingyan Drops can relieve ACEI-induced cough and the accompanying throat discomfort symptoms, and thus can be used to prepare a drug for preventing and / or treating ACEI-induced cough and the accompanying throat discomfort symptoms.

[0006] The first aspect of the present application provides the use of Qingyan Drops in preparing a medicament for preventing and / or treating ACEI-induced cough and its accompanying pharyngeal discomfort symptoms.

[0007] In one embodiment of the present application, the pharyngeal discomfort symptoms include at least one of dry throat, itchy throat and sore throat.

[0008] In one embodiment of the present application, the Qingyan Drops prevent and / or treat ACEI-induced cough and its accompanying pharyngeal discomfort symptoms by inhibiting bradykinin accumulation, inhibiting arachidonic acid metabolism, reducing cough hypersensitivity, and reducing airway hyperresponsiveness.

[0009] In one embodiment of the present application, the ACEI includes at least one of captopril, enalapril, benazepril, lisinopril, ramipril, fosinopril, cilazapril, perindopril and imidapril; preferably, the ACEI includes at least one of captopril, enalapril, benazepril, fosinopril and ramipril; more preferably, the ACEI includes captopril.

[0010] In one embodiment of the present application, the Qingyan dropping pills include 80-120 parts of Indigo Naturalis, 80-120 parts of Licorice, 60-100 parts of Terminalia chebula, 60-100 parts of Menthol, 5-30 parts of Borneol and 5-30 parts of Artificial Bezoar; preferably, the Qingyan dropping pills include 90-110 parts of Indigo Naturalis, 90-110 parts of Licorice, 70-90 parts of Terminalia chebula, 70-90 parts of Menthol, 15-25 parts of Borneol and 10-15 parts of Artificial Bezoar; more preferably, the Qingyan dropping pills include 100 parts of Indigo Naturalis, 100 parts of Licorice, 80 parts of Terminalia chebula, 80 parts of Menthol, 20 parts of Borneol and 12 parts of Artificial Bezoar.

[0011] The second aspect of the present application provides the use of a pharmaceutical composition in the preparation of a drug for preventing and / or treating cough caused by ACEI and its accompanying pharyngeal discomfort symptoms; wherein the pharmaceutical composition comprises 80-120 parts of Indigo Naturalis, 80-120 parts of Licorice, 60-100 parts of Terminalia Chebula, 60-100 parts of Menthol, 5-30 parts of Borneol and 5-30 parts of artificial Bezoar; preferably, the pharmaceutical composition comprises 90-110 parts of Indigo Naturalis, 90-110 parts of Licorice, 70-90 parts of Terminalia Chebula, 70-90 parts of Menthol, 15-25 parts of Borneol and 10-15 parts of artificial Bezoar; more preferably, the pharmaceutical composition comprises 100 parts of Indigo Naturalis, 100 parts of Licorice, 80 parts of Terminalia Chebula, 80 parts of Menthol, 20 parts of Borneol and 12 parts of artificial Bezoar.

[0012] In one embodiment of the present application, the pharyngeal discomfort symptoms include at least one of dry throat, itchy throat and sore throat.

[0013] In one embodiment of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0014] In one embodiment of the present application, the pharmaceutically acceptable carrier or excipient includes at least one of a solvent, a diluent, a dispersant, a suspending agent, a surfactant, an isotonic agent, a thickener, an emulsifier, a preservative, a binder, a lubricant, a stabilizer, a hydrating agent, an emulsification accelerator, a buffer, an absorbent, a colorant, a flavoring agent, a sweetener, an ion exchanger, a release agent, a coating agent, a flavoring agent and an antioxidant.

[0015] In one embodiment of the present application, the pharmaceutical composition is formulated into any one dosage form of powder, tablet, capsule, pill, emulsion, suspension or tincture.

[0016] Beneficial effects of this application:

[0017] The present application provides the use of Qingyan Drop Pills in the preparation of drugs for treating ACEI-induced cough. Qingyan Drop Pills can achieve the effect of treating ACEI-induced cough through potential mechanisms such as inhibiting bradykinin accumulation, inhibiting arachidonic acid metabolism, reducing cough hypersensitivity, and reducing airway hyperresponsiveness; at the same time, Qingyan Drop Pills have a relieving effect on pharyngeal discomfort symptoms such as dry throat, itchy throat, and sore throat that may be associated with cough. In short, Qingyan Drop Pills can relieve ACEI-induced cough and its accompanying pharyngeal discomfort symptoms, so it can be used to prepare drugs for preventing and / or treating ACEI-induced cough and its accompanying pharyngeal discomfort symptoms. Further, the pharmaceutical composition of the present application can also be used to prepare drugs for preventing and / or treating ACEI-induced cough and its accompanying pharyngeal discomfort symptoms. In addition, the present application also successfully established a guinea pig cough-inducing experimental model of ACEI compound cough inducer for the first time.

[0018] Of course, implementing any product or method of the present application does not necessarily require achieving all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application, and a person skilled in the art can also obtain other embodiments based on these drawings.

[0020] Figures 1A to 1F The results of network pharmacology analysis; Venn diagram of potential targets of Qingyan Drops compounds and targets of ACEI-induced cough diseases ( Figure 1A ), target protein PPI network analysis diagram ( Figure 1B ), target protein KEGG pathway enrichment analysis diagram (top 20 signal pathways) ( Figure 1C ), target protein GO function enrichment analysis ( Figure 1D ), “Qingyan Drops-Medicinal Materials-Compounds-Targets-Pathways” Network Diagram ( Figure 1E ), "Qingyan Drop Pills-Medicinal Materials-Compounds-Targets-Pharmacological Actions" Network Diagram ( Figure 1F );

[0021] FIG. 2A to FIG. 2F This is a molecular docking demonstration diagram; TRPV1 and terminalia carbolic acid ( Figure 2A ), BDKRB2 and licorice ( Figure 2B ), PTGS2 and indirubin ( Figure 2C), PLA2G7 and hyodeoxycholic acid ( Figure 2D ), PTGER3 and licorice ( Figure 2E ), PTGER2 and glycocholic acid ( Figure 2F )Dock result diagram;

[0022] FIG. 3A to FIG. 3F is the binding curve between the drug component and the protein target (SPR sensor image); the binding curve between hyodeoxycholic acid and PLA2G7 ( Figure 3A ); Binding curve between liquiritigenin and PLA2G7 ( Figure 3B ); Binding curve between licorice and COX-2 ( Figure 3C ); Binding curve between licorice and TRPV1 ( Figure 3D ); Binding curve between hyodeoxycholic acid and TRPA1 ( Figure 3E ); Binding curve between terminalia chebula acid and TRPV1 ( Figure 3F );

[0023] Figure 4A is the inhibition rate of representative ingredients in Qingyan Drops on COX-2 (A) (compared with the negative control group, , , ); Figure 4B is the inhibition rate of the representative ingredients in Qingyan Drops on 5-LOX (B) (compared with the negative control group, , , );

[0024] Figure 5A To investigate the effect of Qingyan Dropping Pills on the protein expression of TRPV1 (A) in guinea pig lung tissue within a certain temperature range (compared with the blank control group, ); Figure 5B The effect of Qingyan Dropping Pills on the protein expression of BDKRB2 (B) in guinea pig lung tissue within a certain temperature range (compared with the blank control group, );

[0025] Figure 6 The effect of Qingyan Drops on the expression of TRPV1, BDKRB2, and PTGER3 proteins in lung tissue in the capsaicin-induced cough animal experiment;

[0026] Figure 7 In the animal experiment of bradykinin-induced cough, Qingyan Drops affected the expression of TRPV1, BDKRB2 and PTGER3 proteins in lung tissue. DETAILED DESCRIPTION

[0027] The following will be combined with the embodiments of the present application and the accompanying drawings to clearly and completely describe the technical solutions in the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those skilled in the art based on the present application belong to the scope of protection of the present application.

[0028] The first aspect of the present application provides the use of Qingyan Drop Pills in the preparation of drugs for preventing and / or treating cough caused by ACEI and its accompanying pharyngeal discomfort symptoms. The inventor found in the study that through network pharmacology analysis, it was shown that Qingyan Drop Pills can achieve the effect of treating ACEI-induced cough by inhibiting bradykinin accumulation, inhibiting arachidonic acid metabolism, reducing cough hypersensitivity, and reducing airway hyperresponsiveness. At the same time, Qingyan Drop Pills have a relieving effect on pharyngeal discomfort symptoms such as dry throat, itchy throat, and sore throat that may be associated with cough. Through in vitro validation experiments, it was shown that Qingyan Drop Pills may relieve ACEI-induced cough by regulating arachidonic acid metabolism, reducing cough hypersensitivity, and other pathways. Through in vivo efficacy evaluation experiments, it was shown that Qingyan Drop Pills can significantly prolong the cough latency of ACEI-induced cough, reduce the number of coughs, and improve the antitussive rate. In short, Qingyan Drop Pills can relieve ACEI-induced cough and its accompanying pharyngeal discomfort symptoms, so that it can be used to prepare drugs for preventing and / or treating ACEI-induced cough and its accompanying pharyngeal discomfort symptoms.

[0029] In the present application, the term "treatment" has its general meaning, and in the present application specifically refers to treating a mammalian individual (preferably a human) who has suffered from ACEI-induced cough and its accompanying pharyngeal discomfort symptoms with the drug of the present application, in the hope of treating, curing, alleviating, alleviating, etc. the disease. Similarly, the term "prevention" as used in the present application has its general meaning, and in the present application specifically refers to treating a mammalian individual who may be at risk of suffering from ACEI-induced cough and its accompanying pharyngeal discomfort symptoms with the drug of the present application, in the hope of preventing, preventing, blocking, isolating, etc. the disease.

[0030] In one embodiment of the present application, the pharyngeal discomfort symptoms include at least one of dry throat, itchy throat and sore throat. In the study, the inventor selected 33 representative chemical components of Qingyan Drop Pills as the research object, obtained the potential action targets of the compound, and used "ACE inhibitor-induced cough" as the keyword to retrieve the disease-related targets of ACEI-induced cough; at the same time, "pharyngeal dryness", "itchy throat" and "sore throat" were used as keywords to retrieve the targets of dry throat, itchy throat and sore throat for cough-related symptoms, and obtained the intersection targets of the potential action targets of the compound with dry throat, itchy throat and sore throat as the target protein of Qingyan Drop Pills for treating cough-related symptoms. Network pharmacology prediction analysis found that Qingyan Drop Pills have a relieving effect on the pharyngeal discomfort symptoms that may be associated with cough, such as dry throat, itchy throat and sore throat.

[0031] In one embodiment of the present application, the Qingyan Drops prevent and / or treat ACEI-induced cough and its accompanying pharyngeal discomfort symptoms by inhibiting bradykinin accumulation, inhibiting arachidonic acid metabolism, reducing cough hypersensitivity, and reducing airway hyperresponsiveness pathways. In the study, the inventors selected 33 representative chemical components of Qingyan Dropping Pills as research objects, obtained potential targets of the compounds, used "ACE inhibitor-induced cough" as keywords, retrieved disease-related targets of ACEI-induced cough, and used Venny analysis to obtain the intersection targets of the potential targets of the compounds and ACEI-induced cough as the target proteins of Qingyan Dropping Pills for treating ACEI-induced cough. Network pharmacology prediction analysis found that Qingyan Dropping Pills can act on protein targets such as MME, BDKRB2, TRPV1, etc. to regulate arachidonic acid metabolism, the regulation of TRP channels by inflammatory mediators, calcium ion signaling pathways, TNF signaling pathways, cholinergic synapses and other signaling pathways, inhibit the accumulation of BK and the arachidonic acid metabolic process, reduce neurogenic inflammation and reduce cough hypersensitivity, relieve airway inflammation and relax tracheal smooth muscles to reduce airway hyperresponsiveness, thereby achieving the effect of treating ACEI-induced cough, and can be used to prepare drugs for preventing and / or treating ACEI-induced cough and its accompanying pharyngeal discomfort symptoms.

[0032] In the present application, the type of ACEI is not particularly limited. For example, the ACEI includes but is not limited to at least one of captopril, enalapril, benazepril, lisinopril, ramipril, fosinopril, cilazapril, perindopril and imidapril.

[0033] In one embodiment of the present application, the ACEI includes at least one of captopril, enalapril, benazepril, lisinopril, ramipril, fosinopril, cilazapril, perindopril and imidapril; preferably, the ACEI includes at least one of captopril, enalapril, benazepril, fosinopril and ramipril; more preferably, the ACEI includes captopril.

[0034] In one embodiment of the present application, the Qingyan dropping pills include 80-120 parts of Indigo Naturalis, 80-120 parts of Licorice, 60-100 parts of Terminalia chebula, 60-100 parts of Menthol, 5-30 parts of Borneol and 5-30 parts of Artificial Bezoar; preferably, the Qingyan dropping pills include 90-110 parts of Indigo Naturalis, 90-110 parts of Licorice, 70-90 parts of Terminalia chebula, 70-90 parts of Menthol, 15-25 parts of Borneol and 10-15 parts of Artificial Bezoar; more preferably, the Qingyan dropping pills include 100 parts of Indigo Naturalis, 100 parts of Licorice, 80 parts of Terminalia chebula, 80 parts of Menthol, 20 parts of Borneol and 12 parts of Artificial Bezoar. For example, the amount of Indigo Naturalis can be 80 parts, 90 parts, 100 parts, 110 parts, 120 parts, or a range consisting of any two of the values ​​therein; the amount of Licorice can be 80 parts, 90 parts, 100 parts, 110 parts, 120 parts, or a range consisting of any two of the values ​​therein; the amount of Terminalia Chebula can be 60 parts, 70 parts, 80 parts, 90 parts, 100 parts, or a range consisting of any two of the values ​​therein; the amount of menthol can be 60 parts, 70 parts, 80 parts, 90 parts, 100 parts, or a range consisting of any two of the values ​​therein; the amount of Borneol can be 5 parts, 10 parts, 15 parts, 20 parts, 25 parts, 30 parts, or a range consisting of any two of the values ​​therein; and the amount of Artificial Bezoar can be 5 parts, 8 parts, 10 parts, 12 parts, 15 parts, 18 parts, 20 parts, 25 parts, 30 parts, or a range consisting of any two of the values ​​therein.

[0035] The second aspect of the present application provides the use of a pharmaceutical composition in the preparation of a drug for preventing and / or treating cough caused by ACEI and its accompanying pharyngeal discomfort symptoms; the pharmaceutical composition comprises 80-120 parts of Indigo Naturalis, 80-120 parts of Licorice, 60-100 parts of Terminalia Chebula, 60-100 parts of Menthol, 5-30 parts of Borneol and 5-30 parts of artificial Bezoar; preferably, the pharmaceutical composition comprises 90-110 parts of Indigo Naturalis, 90-110 parts of Licorice, 70-90 parts of Terminalia Chebula, 70-90 parts of Menthol, 15-25 parts of Borneol and 10-15 parts of artificial Bezoar; more preferably, the pharmaceutical composition comprises 100 parts of Indigo Naturalis, 100 parts of Licorice, 80 parts of Terminalia Chebula, 80 parts of Menthol, 20 parts of Borneol and 12 parts of artificial Bezoar. For example, the amount of Indigo Naturalis can be 80 parts, 90 parts, 100 parts, 110 parts, 120 parts, or a range consisting of any two of the values ​​therein; the amount of Licorice can be 80 parts, 90 parts, 100 parts, 110 parts, 120 parts, or a range consisting of any two of the values ​​therein; the amount of Terminalia Chebula can be 60 parts, 70 parts, 80 parts, 90 parts, 100 parts, or a range consisting of any two of the values ​​therein; the amount of menthol can be 60 parts, 70 parts, 80 parts, 90 parts, 100 parts, or a range consisting of any two of the values ​​therein; the amount of Borneol can be 5 parts, 10 parts, 15 parts, 20 parts, 25 parts, 30 parts, or a range consisting of any two of the values ​​therein; and the amount of Artificial Bezoar can be 5 parts, 8 parts, 10 parts, 12 parts, 15 parts, 18 parts, 20 parts, 25 parts, 30 parts, or a range consisting of any two of the values ​​therein.

[0036] The pharmaceutical composition of the present application can also be used to prepare drugs for preventing and / or treating cough caused by ACEI and its accompanying pharyngeal discomfort symptoms due to the pharmaceutical prescription containing Qingyan Drop Pills. 33 representative chemical components of 6 medicinal materials in the pharmaceutical composition were selected as research objects. Through network pharmacology analysis, it was shown that the pharmaceutical composition can achieve the effect of treating ACEI-induced cough by inhibiting bradykinin accumulation, inhibiting arachidonic acid metabolism, reducing cough hypersensitivity, and reducing airway hyperresponsiveness. At the same time, the pharmaceutical composition has a relieving effect on pharyngeal discomfort symptoms such as dry throat, itchy throat, and sore throat that may be accompanied by cough. Through in vitro validation experiments, it is shown that the pharmaceutical composition may relieve ACEI-induced cough by regulating arachidonic acid metabolism, reducing cough hypersensitivity, and other pathways. Through in vivo efficacy evaluation experiments, it is shown that the pharmaceutical composition can significantly prolong the cough latency of ACEI-induced cough, reduce the number of coughs, and improve the antitussive rate. In short, the pharmaceutical composition can relieve ACEI-induced cough and its accompanying pharyngeal discomfort symptoms, so that it can be used to prepare drugs for preventing and / or treating ACEI-induced cough and its accompanying pharyngeal discomfort symptoms.

[0037] In one embodiment of the present application, the pharyngeal discomfort symptoms include at least one of dry throat, itchy throat and sore throat. 33 representative chemical components of 6 medicinal materials in the pharmaceutical composition were selected as research objects to obtain potential action targets of the compound, and "ACE inhibitor-induced cough" was used as the keyword to retrieve disease-related targets of ACEI-induced cough; at the same time, "pharyngeal dryness", "itchy throat" and "sore throat" were used as keywords to retrieve targets of dry throat, itchy throat and sore throat for cough-related symptoms, and the intersection targets of the potential action targets of the compound and dry throat, itchy throat and sore throat were obtained as target proteins for the pharmaceutical composition to treat cough-related symptoms. Network pharmacology prediction analysis found that the pharmaceutical composition has a relieving effect on pharyngeal discomfort symptoms such as dry throat, itchy throat and sore throat that may be associated with cough.

[0038] In one embodiment of the present application, the pharmaceutical composition prevents and / or treats ACEI-induced cough and its accompanying pharyngeal discomfort symptoms by inhibiting bradykinin accumulation, inhibiting arachidonic acid metabolism, reducing cough hypersensitivity, and reducing airway hyperresponsiveness pathways. In the study, the inventors selected 33 representative chemical components of 6 medicinal materials in the pharmaceutical composition as research objects, obtained potential targets of the compounds, used "ACE inhibitor-induced cough" as keywords, retrieved disease-related targets of ACEI-induced cough, and used Venny analysis to obtain the intersection targets of the potential targets of the compounds and ACEI-induced cough as the target proteins of the pharmaceutical composition for treating ACEI-induced cough. Network pharmacology prediction analysis found that the pharmaceutical composition can act on protein targets such as MME, BDKRB2, TRPV1, etc. to regulate arachidonic acid metabolism, the regulation of TRP channels by inflammatory mediators, calcium ion signaling pathways, TNF signaling pathways, cholinergic synapses and other signaling pathways, inhibit the accumulation of BK and arachidonic acid metabolism processes, reduce neurogenic inflammation and reduce cough hypersensitivity, relieve airway inflammation and relax tracheal smooth muscles to reduce airway hyperresponsiveness, thereby achieving the effect of treating ACEI-induced cough, and can be used to prepare drugs for preventing and / or treating ACEI-induced cough and its accompanying pharyngeal discomfort symptoms.

[0039] In one embodiment of the present application, the ACEI includes at least one of captopril, enalapril, benazepril, lisinopril, ramipril, fosinopril, cilazapril, perindopril and imidapril; preferably, the ACEI includes at least one of captopril, enalapril, benazepril, fosinopril and ramipril; more preferably, the ACEI includes captopril. In one embodiment of the present application, the pharmaceutical composition further includes a pharmaceutically acceptable carrier or excipient. In the present application, "pharmaceutically acceptable" means that there is no substantial toxic effect when used at a usual dosage, so that it can be approved by a government or an international organization equivalent thereto or has been approved for use in animals, more particularly for humans, or is registered in the pharmacopoeia.

[0040] In one embodiment of the present application, the pharmaceutically acceptable carrier or excipient includes at least one of a solvent, a diluent, a dispersant, a suspending agent, a surfactant, an isotonic agent, a thickener, an emulsifier, a preservative, an adhesive, a lubricant, a stabilizer, a hydrating agent, an emulsification accelerator, a buffer, an absorbent, a colorant, a flavoring agent, a sweetener, an ion exchanger, a release agent, a coating agent, a flavoring agent, and an antioxidant. In the present application, the "pharmaceutically acceptable carrier or excipient" available in the pharmaceutical composition of the present application can be any conventional carrier in the field of pharmaceutical preparations, and the selection of a specific carrier will depend on the mode of administration or the type and state of disease used to treat a specific patient. The preparation method of a suitable pharmaceutical composition for a specific mode of administration is completely within the knowledge of a technician in the pharmaceutical field.

[0041] The term "pharmaceutical composition" as used in the present application has its general meaning. In addition, the "pharmaceutical composition" of the present application can also exist or provide in the form of health products, functional foods, foods, food additives, etc. The conventional techniques in the pharmaceutical field, especially the field of preparations, can be used to obtain the active ingredients of the raw materials of the pharmaceutical composition of the present application by the extraction, separation and purification means commonly used in drug production, optionally mixed with one or more pharmaceutically acceptable carriers or excipients, and then formed into the required dosage form to prepare the pharmaceutical composition of the present application. According to the pharmaceutical composition of the present application, it is a pharmaceutical preparation that can be applicable to oral administration, a pharmaceutical preparation (such as solution) applicable to parenteral injection (such as intravenous injection, subcutaneous injection), a pharmaceutical preparation (such as ointment, patch or cream) applicable to surface administration, or a pharmaceutical preparation (such as suppository) applicable to rectal administration, etc. The dosage form for oral administration may include, for example, tablets, pills, hard capsules or soft capsules, solutions, suspensions, emulsions, tinctures, syrups, powders, powders, fine particles, granules, pellets, elixirs, etc., and is not limited thereto. In addition to the active ingredients, these preparations may also contain diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine), lubricants (e.g., silicon dioxide, talc, stearic acid or its magnesium salt, calcium salt, and polyethylene glycol). Tablets may also contain binders such as aluminum magnesium silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidine. If necessary, they may also contain pharmaceutical additives such as disintegrants (e.g., starch, agar, alginic acid or its sodium salt), absorbents, colorants, flavoring agents, sweeteners, and the like. Tablets may be prepared according to commonly used mixing, granulation, or coating methods.

[0042] In one embodiment of the present application, the pharmaceutical composition is formulated into any one dosage form of powder, tablet, capsule, pill, emulsion, suspension or tincture.

[0043] The dosage of the Qingyan dripping pills or pharmaceutical composition of the present application is not particularly limited and can be changed according to the age, sex and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration and the judgment of the diagnostician. Considering these factors, the dosage to be administered is within the level of those skilled in the art.

[0044] The present application is described in detail below in conjunction with specific embodiments. The experimental materials and methods used below are conventional materials and methods unless otherwise specified.

[0045] 1. Qingyan Drop Pills Information

[0046] Qingyan Drops are provided by the Sixth Traditional Chinese Medicine Factory of Tianjin Pharmaceutical Darentang Group Co., Ltd. and its ingredients include 100 parts of Indigo, 100 parts of Licorice, 80 parts of Terminalia chebula, 80 parts of Menthol, 20 parts of Borneol and 12 parts of Artificial Bezoar.

[0047] 2. Network Pharmacology Analysis

[0048] Thirty-three representative chemical components from the six medicinal materials of Qingyan Drop Pills were selected as the research objects, including indigo carmine and indigo in Indigo Naturalis, gallic acid, chebulic acid, digalloyl glucose, terimasin I, corilagin, 1,3,6-tri-O-galloyl-β-D-glucoside, 1,2,3,4-tetra-O-galloyl-β-D-glucose in Terminalia Chebula, 1-palmitoyl-lysophosphatidylcholine and taurocholic acid in artificial bezoar. , glycocholic acid, tauroursodeoxycholic acid, cholic acid, hyodeoxycholic acid, glycoursodeoxycholic acid, chenodeoxycholic acid, bilirubin, menthol in menthol, borneol (borneol) in borneol, liquiritin isomers in licorice, liquiritin, isoliquiritin, formononetin, liquiritigenin, glycyrrhizin, glycyrrhizic acid, glycyrrhizic acid, glycyrrhizic coumarin, linolenic acid, compounds common to terminalia chebula and licorice, linoleic acid, punicic acid, oleic acid; potential targets of compounds were obtained through databases such as TCMSP, Swiss TargetPrediction, and BATMAN-TCM. Using TTD, DrugBank, DisGeNET, and GeneCards databases, with "ACE inhibitor-induced cough" as the keyword, disease-related targets of ACEI-induced cough were retrieved; at the same time, with "pharyngeal dryness", "itchy throat", and "sore throat" as the keywords, targets of cough-related symptoms such as dry throat, itchy throat, and sore throat were retrieved. Venny analysis was used to obtain the intersection of the potential target of the compound and the target of ACEI-induced cough as the target protein of Qingyan Dropping Pills for treating ACEI-induced cough; at the same time, the intersection of the potential target of the compound and dry throat, itchy throat, and sore throat were obtained as the target protein of Qingyan Dropping Pills for treating cough-related symptoms. Further, PPI network analysis, Cluster module analysis, GO analysis, and KEGG analysis were performed on the two groups of target proteins to construct a pharmacological action network relationship diagram of Qingyan Dropping Pills.

[0049] The results of network pharmacology analysis were as follows Figures 1A to 1F Through the prediction of various databases, 544 related targets of 33 compounds of Qingyan Drops were obtained, and 644 disease-related targets were obtained. The intersection was taken to obtain 160 target proteins of Qingyan Drops for the treatment of ACEI-induced cough, see Figure 1A .from Figure 1BIt can be seen that the PPI network has a total of 160 nodes and 2920 edges. By screening nodes with a degree value greater than or equal to 30, 83 protein targets and 1887 edges were obtained. Further screening nodes with a degree value greater than or equal to 2 times the median, i.e. 51, a total of 42 key target proteins were obtained, including prostaglandin-endoperoxide synthase 2 (PTGS2), interleukin 6 (IL6), albumin (ALB), tumor necrosis factor (TNF), Caspase-3 (CASP3), etc. KEGG and GO analysis were performed on 160 targets, and there were 173 signaling pathways with P < 0.05, including: Inflammatory mediator regulation of TRP channels, PI3K-Akt signaling pathway, tumor necrosis factor signaling pathway, interleukin 17 signaling pathway, T cell receptor signaling pathway, Th17 cell differentiation, Ras signaling pathway, vascular endothelial growth factor signaling pathway, MAPK signaling pathway, etc. (see Figure 1C GO analysis see Figure 1D , P < 0.05 of 70 cell components, 130 molecular functions, 553 biological processes, in terms of cell components, they are mainly involved in plasma membrane, membrane rafts, extracellular space, etc.; in terms of molecular functions, they are mainly involved in ATP binding, enzyme binding, protein tyrosine kinase activity, etc.; in terms of biological processes, they are mainly involved in the response to exogenous stimuli, the regulation of apoptosis, inflammatory response, cell proliferation and gene expression, etc. The network relationship diagram of "Qingyan Drop Pills-Medicinal Materials-Compounds-Targets-Pathways" is shown in Figure 1EThe network has 261 nodes and 1294 edges. The average number of adjacent nodes in the network is 9.916, the network density is 0.038, the network heterogeneity is 0.954, and the network centrality is 0.233. The network results show that there is an interaction between one compound and multiple target proteins, and there are also different compounds acting on the same target protein. It also shows that one target can enrich multiple signal pathways, indicating that Qingyan Drops treats ACEI-induced cough through multiple components, multiple targets, and multiple pathways. The network relationship diagram of "Qingyan Drops-Medicinal Materials-Compounds-Targets-Pharmacological Actions" can be found in Figure 1F , network target analysis found that the mechanism of Qingyan Drops in treating ACEI-induced cough is related to pharmacological processes such as inhibiting bradykinin accumulation, inhibiting arachidonic acid metabolism, reducing cough reflex sensitivity, and reducing airway hyperresponsiveness.

[0050] The results showed that Qingyan drops can regulate arachidonic acid metabolism, the regulation of TRP channels by inflammatory mediators, calcium ion signaling pathways, TNF signaling pathways, cholinergic synapses and other signaling pathways by acting on protein targets such as MME, BDKRB2, and TRPV1, inhibit the accumulation of BK and the arachidonic acid metabolism process, reduce neurogenic inflammation and reduce cough hypersensitivity, relieve airway inflammation and relax tracheal smooth muscles to reduce airway hyperresponsiveness, thereby achieving the effect of treating ACEI-induced cough; at the same time, Qingyan drops can relieve the symptoms of pharyngeal discomfort that may be associated with cough, such as dry throat, itchy throat, and sore throat.

[0051] 3. In vitro validation experiments

[0052] (1) Molecular docking

[0053] Molecular docking is a commonly used method in drug discovery because it can accurately predict the conformation of small molecule ligands in suitable target binding sites and evaluate their binding affinity. This application selects targets with strong correlation with four potential mechanisms of action: inhibition of bradykinin accumulation, inhibition of arachidonic acid metabolism, reduction of cough hypersensitivity, and reduction of airway hyperresponsiveness as verification objects, including targets MME and F12 for inhibiting bradykinin accumulation, targets PLA2G7, PTGS2, ALOX5, and PTGES for inhibiting arachidonic acid metabolism, targets TRPV1, TRPA1, BDKRB2, PTGER2, and PTGER3 for reducing cough hypersensitivity, and targets NOS2, CHRM2, and ACHE for reducing airway hyperresponsiveness. The above target protein crystal structures were downloaded from the RCSB PDB database. After downloading, Schrödinger software was used to correct the chemical bond order, add hydrogen, process disulfide bonds and metal ions, remove water molecules and heteroatoms in the crystal structure, and perform other pretreatments and energy optimization. The target protein was used as a rigid receptor, and each ligand molecule was used as a flexible ligand with changeable conformation, and semi-flexible docking was performed through the Ligand Docking module.

[0054] The molecular docking results are shown in Table 1. FIG. 2A to FIG. 2F As shown in Table 1, it can be seen that TRPV1 has good docking values ​​with terminal acid, indirubin, and gallic acid; BDKRB2 has good docking values ​​with glycocholic acid, licorice, and terminal acid; PTGS2 and PLA2G7 have good docking values ​​with indirubin, licorice, and menthol; and PTGER3 has good docking values ​​with licorice, hyodeoxycholic acid, indirubin, and gallic acid. FIG. 2A to FIG. 2F It can be seen that the binding between the drug components and the target protein is mainly hydrogen bonding, and the drug components of Qingyan Drops show low free binding energy with the target protein. It can be preliminarily believed that the mechanism of action of Qingyan Drops in treating ACEI-induced cough is related to targets such as TRPV1, BDKRB2, PTGS2, PLA2G7, and PTGER3.

[0055] Table 1 Molecular docking results

[0056] (2) Surface plasmon resonance (SPR) experiment

[0057] Eight representative ingredients in Qingyan Drops, including hyodeoxycholic acid, glycyrrhizin, chebulic acid, gallic acid, glycyrrhetinic acid, menthol, indirubin and glycocholic acid, were selected as small molecule compounds. PLA2G7 and COX-2, key targets in arachidonic acid metabolism, and TRPV1 and TRPA1, important targets associated with cough hypersensitivity, were selected as target proteins. Small molecule samples and 1% (v / v) DMSO in PBS buffer were added according to the plate position and volume specified by the instrument. The operation was carried out according to the Biacore 8K Molecular Interaction Analysis System Operation Guide to complete the component-target interaction analysis.

[0058] The binding curve between the drug component and the protein target (SPR sensor image) is shown in FIG. 3A to FIG. 3F The equilibrium dissociation constants between the analyte and the ligand are shown in Table 2. FIG. 3A to FIG. 3F As can be seen from Table 2, there is a certain affinity between licorice and COX-2, TRPV1, and PLA2G7 protein, and between hyodeoxycholic acid and PLA2G7 protein. This indicates that Qingyan Drops may play a role in treating ACEI-induced cough by acting on PLA2G7, COX-2 and TRPV1, a target related to cough hypersensitivity, in the AA metabolic pathway.

[0059] Table 2 Equilibrium dissociation constants between analytes and ligands

[0060] Note: “KD” is the equilibrium dissociation constant.

[0061] (3) Enzyme inhibitor screening experiment

[0062] Eight representative ingredients in Qingyan Dropping Pills, including terminalic acid, menthol, glycyrrhizin, gallic acid, glycocholic acid, glycyrrhetinic acid, hyodeoxycholic acid and indirubin, were selected for inhibitor screening reaction with two key enzymes COX-2 and 5-LOX in the arachidonic acid metabolic pathway. The COX-2 inhibitor screening kit (Beyotime, batch number Z984240806) was used to explore the effects of the key ingredients of Qingyan Dropping Pills with the key enzyme COX-2. After the reaction, the excitation wavelength was set to 560 nm and the emission wavelength was set to 590 nm for fluorescence measurement. The fluorescence values ​​of each sample well and blank control well were measured and recorded as RFU, respectively. 空白对照 、RFU 100%酶活性对照 and RFU 样品 RFU, Relative Fluorescence Unit. Calculate the inhibition percentage of each sample using the following formula: Inhibition rate (%) = (RFU 100%酶活性对照 -RFU 样品 ) / (RFU 100%酶活性对照 -RFU空白对照 ) × 100%. The 5-LOX inhibitor screening kit (Cayman, batch number 760700) was used to explore the interaction between the key components of Qingyan Dropping Pills and the key enzyme 5-LOX. After the reaction was completed, the absorbance was detected at a wavelength of 490-500 nm. The absorbance of each sample well and blank control well was measured and recorded as OD 空白对照 OD 100%酶活性对照 and OD 样品 Calculate the inhibition percentage of each sample using the following formula: Inhibition rate (%) = (OD 100%酶活性对照 -OD 样品 ) / (OD 100%酶活性对照 -OD 空白对照 )×100%.

[0063] The results of the inhibition rate of representative ingredients in Qingyan Drops on COX-2 and 5-LOX are as follows FIG. 4A to FIG. 4B As shown, from FIG. 4A to FIG. 4B It can be seen that the representative ingredients in Qingyan Drops, Chebulic Acid, Menthol, Gallic Acid, Glycocholic Acid, Glycyrrhetnic Acid, Hyodeoxycholic Acid, and Indirubin, can inhibit the enzyme activity of COX-2 at the concentrations of 25 μmol / L, 50 μmol / L, and 100 μmol / L; Liquiritigenin, Menthol, Gallic Acid, Glycocholic Acid, Glycyrrhetnic Acid, Hyodeoxycholic Acid, and Indirubin can inhibit the enzyme activity of 5-LOX at the concentrations of 50 μmol / L and 100 μmol / L. The inventors speculate that Qingyan Drops can inhibit the activity of COX-2 and 5-LOX, reduce the production of inflammatory and cough-causing factors in the AA metabolic pathway, and thus play a role in treating ACEI-induced cough.

[0064] (4) Cell thermal migration (CETSA) analysis experiment

[0065] Take an appropriate amount of Qingyan dripping pills, grind them into powder, weigh 1 g of powder, place it in a 100 mL round-bottom flask, add 25 mL of 70% anhydrous ethanol, and weigh it. Reflux extraction for 2 h, weigh it, make up with 70% ethanol, filter it, and concentrate it to obtain the Qingyan dripping pill extract. Take 80 mg of lung tissue and place it in an EP tube. Add non-denaturing tissue / cell lysis buffer at a ratio of 1:9, fully disrupt it with a tissue disruptor, ice bath for 30 min, shake it every 5 min to ensure complete tissue lysis, centrifuge it at 4°C, 12000 r for 10 min, collect the supernatant, and obtain the guinea pig lung protein solution.

[0066] The Qingyan dripping pill extract was prepared into a 200 μg / mL solution with ddH2O, and 44.4 μL of Qingyan dripping pill extract solution and 44.4 μL of ddH2O were incubated with 400 μL of guinea pig lung protein solution at 37°C for 2 h. The solution was then divided and heated at four temperature gradients of 37°C, 42°C, 51°C, and 59°C for 3 minutes. After cooling, the samples were prepared and tested by protein immunoblotting (WB) to explore the interaction between the Qingyan dripping pill extract and the ACEI cough-related targets TRPV1 and BDKRB2. The WB experimental steps are as follows:

[0067] Gel preparation: Prepare 10% gel according to the instructions of the SDS-PAGE gel preparation kit.

[0068] Sample loading: Use a pipette to add 8 μL of the prepared protein sample into each well.

[0069] Electrophoresis: First, perform electrophoresis at 80 V for 30 min. When the indicator reaches the boundary of the gel, adjust the voltage to 120 V until bromophenol blue reaches 1 cm from the bottom of the gel, and then turn off the power.

[0070] Prepare PVDF membrane, filter paper and sponge, place the membrane and gel with strips into the transfer solution, use the sandwich method to arrange the filter paper, sponge and gel in the corresponding order, and pay attention to prevent the generation of bubbles, connect the gel surface to the negative electrode, connect the PVDF membrane to the positive electrode, place it in an ice water bath with a constant current of 300 mA, and transfer the membrane for 90 min.

[0071] After removing the PVDF membrane from the transfer clip, place it in the rapid blocking solution for 1 h. Cut the required protein bands and add TRPV1 and BDKRB2 primary antibodies respectively, and incubate overnight at 4°C. The next day, collect the primary antibodies and store them at -20°C, and wash the membrane with TBST 3-5 times on a shaker. Then add the secondary antibody prepared in the rapid blocking solution and incubate for 1 h, and wash the membrane again. Finally, use ECL luminescent solution to expose in a chemiluminescent imager, save the bands, and further use ImageJ to quantitatively analyze the levels of TRPV1 and BDKRB2.

[0072] In a certain temperature range, the effect of Qingyan Drops on the protein expression of TRPV1 and BDKRB2 in guinea pig lung tissue is as follows FIG. 5A to FIG. 5B As shown, from FIG. 5A to FIG. 5B It can be seen that the extract of Qingyan pills has a certain binding effect with TRPV1 and BDKRB2 targets. Qingyan pills may play a role in treating ACEI-induced cough by acting on TRPV1 and BDKRB2 targets and regulating the high sensitivity of cough.

[0073] In summary, Qingyan drops may relieve ACEI-induced cough by regulating arachidonic acid metabolism and cough hypersensitivity, which preliminarily verifies the prediction results of network pharmacology.

[0074] IV. In vivo efficacy evaluation and mechanism of action study

[0075] 1. Evaluation of the efficacy and mechanism of Qingyan Drops in relieving ACEI-induced cough (capsaicin-induced cough)

[0076] SPF-grade Hartley guinea pigs (male, weighing 300-350 g) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., license number SCXK (Beijing) 2021-0011, and randomly divided into 6 groups, namely, blank group, model group (captopril, dose of 11.6 mg / kg, Changzhou Pharmaceutical Factory Co., Ltd., batch number 22081613z), SB705498 (TRPV1 receptor antagonist, dose of 5 mg / kg, Picasso, batch number 501951-42-4) group, Jinsuozi lozenges (dose of 0.93 g / kg, Guangxi Jinsuozi Co., Ltd., batch number 24031203) group, Qingyan Dropping Pills low-dose group (dose of 27.87 mg / kg), Qingyan Dropping Pills high-dose group (dose of 83.61 mg / kg), Qingyan Dropping Pills low- and high-dose administration suspensions were administered at 0.1% CMC-Na solution was fully suspended and prepared, with concentrations of 0.0149 g / mL and 0.0418 g / mL, respectively, with 10 animals in each group. The blank group was gavaged with 0.1% CMC-Na, and the other groups were gavaged with captopril to establish the ACEI-induced cough animal model. After 4 hours, each group was given corresponding drug treatment (blank group and model group were gavaged with 0.1% CMC-Na) for 14 days, once a day, with a gavage volume of 10 mL / kg.

[0077] On day 0 (one day before administration), blood was collected from the dorsal mid-foot vein of guinea pigs in each group. One hour after the last administration on day 14, blood was collected from the dorsal mid-foot vein of guinea pigs in each group. After blood collection, the guinea pigs were placed in the nebulizer chamber, and 250 μmol / L capsaicin (MCE, batch number 239533) was used as a cough inducer to start timed nebulization. After 5 minutes, they were taken out and placed in an empty cage. They were observed for another 5 minutes, and the coughing situation of each group was recorded. Then, blood was collected from the abdominal aorta, and the guinea pig blood was collected and lung tissue was dissected for biochemical index detection. RT-qPCR and WB were used to detect the expression of cough hypersensitivity-related proteins and genes in lung tissue. The therapeutic effect of Qingyan Drops was evaluated and its possible mechanism of action was preliminarily analyzed.

[0078] Determination of substance P (SP), calcitonin gene-related peptide (CGRP), bradykinin (BK) and neurokinin A (NKA) contents: According to the instructions of the kits of SP (Jianglai Biotechnology, batch number 062730003172620627), CGRP (Jianglai Biotechnology, batch number 062730003120040627), BK (Jianglai Biotechnology, batch number 062844003215320802) and NKA (Shanghai ELISA Biotechnology Co., Ltd., batch number Sep 2024), after the reaction, the wavelength was set to 450 nm, the OD value of each well was determined, and the contents of SP, CGRP, BK and NKA in serum and lung tissue of each group were calculated according to the instructions.

[0079] Determination of the activity of aminopeptidase (APP), neutral endopeptidase (NEP) and dipeptidyl peptidase-4 (DPP IV): According to the instructions of the kits for APP (Jianglai Biotechnology, batch number 062844003546730802), DPP IV (Shanghai ELISA Biotechnology Co., Ltd., batch number Jul 2024) and NEP (Shanghai ELISA Biotechnology Co., Ltd., batch number Jul 2024), after the reaction, set the wavelength to 450 nm, determine the OD value of each well, and calculate the content of APP, NEP and DPP IV in the lung tissue of each group according to the instructions.

[0080] RT-qPCR detection of gene expression levels of TRPV1, BDKRB2, and PTGER3 in lung tissues:

[0081] (1) The PCR primers used are listed below (all purchased from Sangon Biotech (Shanghai) Co., Ltd.):

[0082] (2) RNA extraction:

[0083] Weigh 80 mg of lung tissue and put it into an enzyme-free 1.5 mL EP tube. Add 1 mL Trizol and 2 steel beads and grind the tissue in a tissue crusher. Let it stand for 15 min.

[0084] Add 200 μL of chloroform to the lysate, shake vigorously, let stand at room temperature for 15 min, and then centrifuge at 4°C, 12,000 rpm for 15 min.

[0085] Transfer the supernatant (400 μL) to a new enzyme-free EP tube, add 400 μL of isopropanol, gently shake up and down 3-5 times, let stand at room temperature for 10 min, and then centrifuge at 4°C, 12,000 rpm for 15 min.

[0086] Discard the supernatant, place on ice, add 1 mL of 75% ice ethanol prepared with enzyme-free water, gently blow off the precipitate, and centrifuge at 4°C, 12,000 rpm for 5 min.

[0087] Discard the supernatant and avoid ethanol residue, and place at room temperature for about 5 minutes to allow the precipitate to completely dry. Add 90-150 μL of enzyme-free water to dissolve the precipitate according to the amount of precipitate.

[0088] The RNA concentration was measured using an ultra-micro-nucleic acid protein analyzer.

[0089] (3) Synthesis of cDNA

[0090] Insert the 0.2 mL Eppendorf tube on ice and incubate for 5 min, add template RNA, add RNase free ddH2O to 12 μL, then add 4 μL of 4×gDNA wiper Mix, gently blow and mix with a pipette, incubate at 42℃ for 2 min, then directly add 4 μL of 5×HIScript Ⅱ qRT SuperMix Ⅱ, gently blow and mix with a pipette, incubate at 50℃ for 15 min, and then change the temperature to 85℃ and incubate for 2 min. After the reaction, store the cDNA at -20℃ for later use.

[0091] (4) Real-time fluorescence quantitative PCR:

[0092] Add the following reagents to a 96-well PCR plate:

[0093] After brief centrifugation, the mixed system was placed in a qPCR system and the following amplification program was set:

[0094] Step 1 95℃ 10 min

[0095] Step 2 95℃ 10 s

[0096] Step 3 60℃ 30 s

[0097] Step 2→Step 3 Set the number of cycles to 50 and export the data after the reaction is completed.

[0098] WB detection of protein expression levels of TRPV1, BDKRB2, and PTGER3 in lung tissue:

[0099] (1) Extraction and quantification of total tissue protein

[0100] 50-100 mg of lung tissue from each group was placed in an EP tube, and 9 times the amount of non-denaturing tissue / cell lysis buffer was added. The tube was fully disrupted with a tissue disruptor, placed in an ice bath for 30 min, and shaken once every 5 min to ensure complete tissue lysis. The tube was centrifuged at 12000 r for 10 min, and the supernatant was collected to obtain total protein.

[0101] (2) Protein quantification by BCA method

[0102] According to the instructions of the BCA kit (Solaibao; batch number: 240005014), after the reaction, set the wavelength to 562 nm, measure the sample absorbance, and calculate the protein concentration according to the instructions. Add the protein solution to 4× protein loading buffer at a ratio of 3:1, denature at 100°C for 5 min, cool and store in a -20°C refrigerator for later use.

[0103] (3) SDS-PAGE gel electrophoresis

[0104] Gel preparation: Prepare 10% gel according to the instructions of the SDS-PAGE gel preparation kit.

[0105] Sample loading: Use a pipette to add the sample with the protein concentration to be measured into the sample wells at a protein amount of 70 μg / well.

[0106] Electrophoresis: First, perform electrophoresis at 80 V for 30 min. When the indicator reaches the boundary of the gel, adjust the voltage to 120 V until bromophenol blue reaches 1 cm from the bottom of the gel, and then turn off the power.

[0107] (4) Transfer

[0108] Prepare PVDF membrane, filter paper and sponge, place the membrane and gel with strips into the transfer solution, use the sandwich method to arrange the filter paper, sponge and gel in the corresponding order, and pay attention to prevent the generation of bubbles, connect the gel surface to the negative electrode, connect the PVDF membrane to the positive electrode, place it in an ice water bath with a constant current of 300 mA, and transfer the membrane for 90 min.

[0109] (5) Immunoblotting and protein quantification

[0110] After removing the PVDF membrane from the transfer clip, place it in the rapid blocking solution for 1 h. Cut the required protein bands and add the primary antibody separately, incubate at 4°C overnight. The next day, collect the primary antibody and store it at -20°C, and wash the membrane 3-5 times with TBST on a shaker. Then add the secondary antibody prepared in the rapid blocking solution and incubate for 1 h, and wash the membrane again. Finally, use ECL luminescent solution to expose in a chemiluminescent imager, save the bands, and further perform quantitative analysis using ImageJ.

[0111] The effects of Qingyan drops on the cough latency, number of coughs within 10 minutes and antitussive rate after capsaicin-induced cough are shown in Table 3. As can be seen from Table 3, Qingyan drops can significantly prolong the cough latency of ACEI-induced cough guinea pigs, reduce the number of coughs, and increase the antitussive rate (to more than 60%). The three efficacy indicators are better than those of Yaojinshengzi lozenges.

[0112] Table 3 Effects of Qingyan Drops on cough latency, cough frequency within 10 minutes and antitussive rate after capsaicin-induced cough ;

[0113] Note: Compared with the blank group, ###P<0.001; compared with the model group, .

[0114] In the capsaicin-induced cough animal experiment, the effect of Qingyan Drops on the expression of TRPV1, BDKRB2, and PTGER3 proteins in lung tissue was as follows: Figure 6 Table 4-1 to Table 4-4 show the effects of Qingyan drops on SP, CGRP, BK, and NKA in plasma, Table 5 shows the effects of Qingyan drops on SP, CGRP, BK, and NKA in lung tissue, Table 6 shows the effects of Qingyan drops on DPP IV, NEP, and APP in lung tissue, Table 7 shows the effects of Qingyan drops on the gene expression of TRPV1, BDKRB2, and PTGER3 in guinea pig lung tissue, and Table 8 shows the effects of Qingyan drops on the protein expression of TRPV1, BDKRB2, and PTGER3 in lung tissue.

[0115] Table 4-1 Effects of Qingyan Drops on SP in Plasma ;

[0116] Note: Compared with the blank group, ###P<0.001; compared with the model group, .

[0117] Table 4-2 Effects of Qingyan Drops on CGRP in Plasma ;

[0118] Note: Compared with the blank group, ###P<0.001; compared with the model group, .

[0119] Table 4-3 Effect of Qingyan Drops on BK in Plasma ;

[0120] Note: Compared with the blank group, ###P<0.001; compared with the model group, .

[0121] Table 4-4 Effect of Qingyan Drops on NKA in Plasma ;

[0122] Note: Compared with the blank group, ##P<0.01; compared with the model group, .

[0123] Table 5 Effects of Qingyan Dropping Pills on SP, CGRP, BK and NKA in lung tissue ;

[0124] Note: Compared with the blank group, ##P<0.01, ###P<0.001; compared with the model group, .

[0125] Table 6 Effects of Qingyan Dropping Pills on DPP Ⅳ, NEP and APP in lung tissue ;

[0126] Note: Compared with the blank group, #P<0.05, ###P<0.001; compared with the model group, .

[0127] Table 7 Effect of Qingyan Drops on the expression of TRPV1, BDKRB2 and PTGER3 genes in guinea pig lung tissue ;

[0128] Note: Compared with the blank group, ###P<0.001; compared with the model group, .

[0129] Table 8 Effects of Qingyan Drops on the expression of TRPV1, BDKRB2, and PTGER3 proteins in lung tissue ;

[0130] Note: Compared with the blank group, #P<0.05, ##P<0.01; compared with the model group, .

[0131] from Figure 6 As can be seen from Tables 4-1 to 4-4 and Tables 5 to 8, the possible mechanisms of action of Qingyan Drops in relieving ACEI-induced cough include: inhibiting the accumulation of inflammatory mediator BK by promoting the expression of BK metabolic enzymes; reducing neurogenic inflammatory responses by reducing the release of sensory neuropeptides such as SP, CGRP, and NKA; and inhibiting the phosphorylation level of cough conduction TRPV1 by inhibiting the gene and protein expression of PGE2 receptor PTGER3 and BK receptor BDKRB2, thereby improving ACEI-induced cough.

[0132] (II) Evaluation of the efficacy and mechanism of Qingyan Drops in relieving ACEI-induced cough (bradykinin-induced cough)

[0133] Ordinary Hartley guinea pigs, male, weighing 300-400 g, Tianjin Yuda Experimental Animal Breeding Co., Ltd., license number SCXK (Tianjin) 2021-0001, were selected and randomly divided into 6 groups, namely, blank group, model group (captopril, dose of 11.6 mg / kg, Changzhou Pharmaceutical Factory Co., Ltd., batch number 22081613z), icatibant acetate (bradykinin receptor antagonist, dose of 500 μg / kg, Shanghai Aladdin Biotechnology Co., Ltd., batch number F2303485), Jinsuozi throat lozenges group (dose of 0.93 g / kg, Guangxi Jinsuozi Co., Ltd., batch number 24031203), Qingyan drops low-dose group (dose of 27.87 mg / kg), Qingyan drops high-dose group (dose of 83.61 mg / kg), Qingyan Dropping Pills low- and high-dose suspensions were prepared by fully suspending 0.1% CMC-Na solution, with concentrations of 0.0149 g / mL and 0.0418 g / mL, respectively, with 10 animals in each group. Each group was gavaged with captopril (the blank group was gavaged with 0.1% CMC-Na) to establish an ACEI-induced cough animal model. Four hours later, the Jinsuozi throat tablets and Qingyan Dropping Pills groups were gavaged with corresponding drug treatments (the blank group and the model group were gavaged with 0.1% CMC-Na), and the icatibant acetate group was intracavitary injection once a day for 14 consecutive days, with a gavage volume of 10 mL / kg.

[0134] One hour after the last administration on the 14th day, 5 mg / mL bradykinin (Shanghai McLean Biochemical Technology Co., Ltd., batch number C14596670) was used as a cough inducer, and the guinea pig was placed in the cabin. The timer started when the cabin was filled with gas, and the cabin was atomized for 10 minutes. The time of the guinea pig's first cough and the number of coughs within 10 minutes were recorded. Then, blood was collected from the abdominal aorta, and the guinea pig blood and lung tissue were dissected for biochemical index detection. RT-qPCR and WB were used to detect the expression of cough hypersensitivity-related proteins and genes in lung tissue. The therapeutic effect of Qingyan Drops was evaluated and its possible mechanism of action was preliminarily analyzed.

[0135] Determination of substance P (SP), calcitonin gene-related peptide (CGRP), bradykinin (BK) and nerve growth factor (NGF) contents: According to the instructions of the kits of SP (Jianglai Biotechnology, batch number 062730003172620627), CGRP (Jianglai Biotechnology, batch number 062730003120040627), BK (Jianglai Biotechnology, batch number 062844003215320802) and NGF (Shanghai ELISA Biotechnology Co., Ltd., batch number Sep 2024), after the reaction, the wavelength was set to 450 nm, the OD value of each well was determined, and the contents of SP, CGRP, BK and NGF in serum and lung tissue of each group were calculated according to the instructions.

[0136] Determination of phospholipase A2 (PLA2), cyclooxygenase-2 (COX-2), thromboxane A2 (TXA2) and prostaglandin E2 (PGE2) contents: According to the instructions of the kits for PLA2 (Dogesce, batch number 202409), COX-2 (Jianglai Biotechnology, batch number 062844003507240904), PGE2 (REED BIOTECH, batch number KD071640901001) and TXA2 (REED BIOTECH, batch number KD081340906101), after the reaction, the wavelength was set to 450 nm, the OD value of each well was determined, and the contents of PLA2, COX-2, PGE2 and TXA2 in the lung tissue of each group were calculated according to the instructions.

[0137] RT-qPCR detection of gene expression levels of TRPV1, BDKRB2, and PTGER3 in lung tissues:

[0138] (1) The PCR primers used are listed below (all purchased from Sangon Biotech (Shanghai) Co., Ltd.):

[0139] (2) RNA extraction:

[0140] Weigh 80 mg of lung tissue and put it into an enzyme-free 1.5 mL EP tube. Add 1 mL Trizol and 2 steel beads and grind the tissue in a tissue crusher. Let it stand for 15 min.

[0141] Add 200 μL of chloroform to the lysate, shake vigorously, let stand at room temperature for 15 min, and then centrifuge at 4°C, 12,000 rpm for 15 min.

[0142] Transfer the supernatant (400 μL) to a new enzyme-free EP tube, add 400 μL of isopropanol, gently shake up and down 3-5 times, let stand at room temperature for 10 min, and then centrifuge at 4°C, 12,000 rpm for 15 min.

[0143] Discard the supernatant, place on ice, add 1 mL of 75% ice ethanol prepared with enzyme-free water, gently blow off the precipitate, and centrifuge at 4°C, 12,000 rpm for 5 min.

[0144] Discard the supernatant and avoid ethanol residue, and place at room temperature for about 5 minutes to allow the precipitate to completely dry. Add 90-150 μL of enzyme-free water to dissolve the precipitate according to the amount of precipitate.

[0145] The RNA concentration was measured using an ultra-micro-nucleic acid protein analyzer.

[0146] (3) Synthesis of cDNA

[0147] Insert the 0.2 mL Eppendorf tube on ice and incubate for 5 min, add template RNA, add RNase free ddH2O to 12 μL, then add 4 μL of 4×gDNA wiper Mix, gently blow and mix with a pipette, incubate at 42℃ for 2 min, then directly add 4 μL of 5×HIScript Ⅱ qRT SuperMix Ⅱ, gently blow and mix with a pipette, incubate at 50℃ for 15 min, and then change the temperature to 85℃ and incubate for 2 min. After the reaction, store the cDNA at -20℃ for later use.

[0148] (4) Real-time fluorescence quantitative PCR:

[0149] Add the following reagents to a 96-well PCR plate:

[0150] After brief centrifugation, the mixed system was placed in a qPCR system and the following amplification program was set:

[0151] Step 1 95℃ 10 min

[0152] Step 2 95℃ 10 s

[0153] Step 3 60℃ 30 s

[0154] Step 2→Step 3 Set the number of cycles to 50 and export the data after the reaction is completed.

[0155] WB detection of protein expression levels of TRPV1, BDKRB2, and PTGER3 in lung tissue:

[0156] (1) Extraction and quantification of total tissue protein

[0157] 50-100 mg of lung tissue from each group was placed in an EP tube, and 9 times the amount of non-denaturing tissue / cell lysis buffer was added. The tube was fully disrupted with a tissue disruptor, placed in an ice bath for 30 min, and shaken once every 5 min to ensure complete tissue lysis. The tube was centrifuged at 12000 r for 10 min, and the supernatant was collected to obtain total protein.

[0158] (2) Protein quantification by BCA method

[0159] According to the instructions of the BCA kit (Solaibao; batch number: 240005014), after the reaction, set the wavelength to 562 nm, measure the sample absorbance, and calculate the protein concentration according to the instructions. Add the protein solution to 4× protein loading buffer at a ratio of 3:1, denature at 100°C for 5 min, cool and store in a -20°C refrigerator for later use.

[0160] (3) SDS-PAGE gel electrophoresis

[0161] Gel preparation: Prepare 10% gel according to the instructions of the SDS-PAGE gel preparation kit.

[0162] Sample loading: Use a pipette to add the sample with the protein concentration to be measured into the sample wells at a protein amount of 70 μg / well.

[0163] Electrophoresis: First, perform electrophoresis at 80 V for 30 min. When the indicator reaches the boundary of the gel, adjust the voltage to 120 V until bromophenol blue reaches 1 cm from the bottom of the gel, and then turn off the power.

[0164] (4) Transfer

[0165] Prepare PVDF membrane, filter paper and sponge, place the membrane and gel with strips into the transfer solution, use the sandwich method to arrange the filter paper, sponge and gel in the corresponding order, and pay attention to prevent the generation of bubbles, connect the gel surface to the negative electrode, connect the PVDF membrane to the positive electrode, place it in an ice water bath with a constant current of 300 mA, and transfer the membrane for 90 min.

[0166] (5) Immunoblotting and protein quantification

[0167] After removing the PVDF membrane from the transfer clip, place it in the rapid blocking solution for 1 h. Cut the required protein bands and add the primary antibody separately, incubate at 4°C overnight. The next day, collect the primary antibody and store it at -20°C, and wash the membrane 3-5 times with TBST on a shaker. Then add the secondary antibody prepared in the rapid blocking solution and incubate for 1 h, and wash the membrane again. Finally, use ECL luminescent solution to expose in a chemiluminescent imager, save the bands, and further perform quantitative analysis using ImageJ.

[0168] The effects of Qingyan Drops on the cough latency, the number of coughs within ten minutes, and the antitussive rate after bradykinin-induced cough are shown in Table 9. As can be seen from Table 9, Qingyan Drops can significantly prolong the cough latency of ACEI-induced cough guinea pigs, reduce the number of coughs, and increase the antitussive rate (to more than 60%). The three efficacy indicators are better than Yaojinsuozi lozenges.

[0169] Table 9 Effects of Qingyan Drops on the cough latency, number of coughs within 10 minutes, and antitussive rate after bradykinin-induced cough ;

[0170] Note: Compared with the blank group, ###P<0.001; compared with the model group, .

[0171] In the bradykinin-induced cough animal experiment, the effect of Qingyan Drops on the expression of TRPV1, BDKRB2, and PTGER3 proteins in lung tissue was as follows: Figure 7 Table 10 shows the effects of Qingyan Dropping Pills on SP, CGRP, NGF, and BK in plasma, Table 11 shows the effects of Qingyan Dropping Pills on SP, CGRP, NGF, and BK in lung tissue, Table 12 shows the effects of Qingyan Dropping Pills on PLA2, PGE2, TXA2, and COX-2 in lung tissue, Table 13 shows the effects of Qingyan Dropping Pills on the gene expression of TRPV1, BDKRB2, and PTGER3 in lung tissue, and Table 14 shows the effects of Qingyan Dropping Pills on the protein expression of TRPV1, BDKRB2, and PTGER3 in lung tissue.

[0172] Table 10 Effects of Qingyan Dropping Pills on SP, CGRP, NGF, and BK in plasma ;

[0173] Note: Compared with the blank group, #P<0.05, ###P<0.001; compared with the model group, .

[0174] Table 11 Effects of Qingyan Dropping Pills on SP, CGRP, NGF, and BK in lung tissue ;

[0175] Note: Compared with the blank group, ##P<0.01, ###P<0.001; compared with the model group, .

[0176] Table 12 Effects of Qingyan Drops on PLA2, PGE2, TXA2, and COX-2 in lung tissue ;

[0177] Note: Compared with the blank group, #P<0.05, ##P<0.01, ###P<0.001; compared with the model group, .

[0178] Table 13 Effects of Qingyan Drops on the expression of TRPV1, BDKRB2, and PTGER3 genes in lung tissue ;

[0179] Note: Compared with the blank group, ###P<0.001; compared with the model group, .

[0180] Table 14 Effects of Qingyan Drops on the expression of TRPV1, BDKRB2, and PTGER3 proteins in lung tissue ;

[0181] Note: Compared with the blank group, #P<0.05; compared with the model group, .

[0182] from Figure 7 As can be seen from Tables 10 to 14, the possible mechanisms of Qingyan Drops in relieving ACEI-induced cough include: inhibiting the expression of key enzymes PLA2 and COX-2 in the AA metabolic pathway, reducing the levels of its inflammatory and cough-inducing metabolites PGE2 and TXA2; reducing the release of sensory neuropeptides such as SP, CGRP, and NGF, thereby reducing neurogenic inflammatory responses; and inhibiting the phosphorylation level of cough conduction TRPV1 by inhibiting the gene and protein expression of PGE2 receptor PTGER3 and BK receptor BDKRB2, thereby improving ACEI-induced cough.

[0183] In summary, network pharmacology analysis shows that Qingyan dripping pills can achieve the effect of treating ACEI-induced cough by inhibiting bradykinin accumulation, inhibiting arachidonic acid metabolism, reducing cough hypersensitivity, and reducing airway hyperresponsiveness. At the same time, Qingyan dripping pills have a relieving effect on the pharyngeal discomfort symptoms that may be associated with cough, such as dry throat, itchy throat, and sore throat. Through in vitro validation experiments, it is shown that Qingyan dripping pills may relieve ACEI-induced coughs by regulating arachidonic acid metabolism and reducing cough hypersensitivity. Through in vivo efficacy evaluation experiments, it is shown that Qingyan dripping pills can significantly prolong the cough latency of ACEI-induced cough, reduce the number of coughs, improve the antitussive rate, and evaluate the possible mechanism of action of Qingyan dripping pills to relieve ACEI-induced coughs. In short, Qingyan dripping pills can relieve ACEI-induced coughs and their accompanying pharyngeal discomfort symptoms, so that they can be used to prepare drugs for preventing and / or treating ACEI-induced coughs and their accompanying pharyngeal discomfort symptoms. In addition, the present application has also successfully established a guinea pig cough-inducing experimental model of ACEI compound cough inducers for the first time in in vivo efficacy evaluation and mechanism of action research.

[0184] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. Use of Qingyan Drops in the preparation of a drug for preventing and / or treating ACEI-induced cough and its accompanying pharyngeal discomfort symptoms.

2. The use according to claim 1, wherein The pharyngeal discomfort symptoms include at least one of dry throat, itchy throat and sore throat.

3. The use according to claim 1, wherein The Qingyan Drop Pills prevent and / or treat cough caused by ACEI and the accompanying pharyngeal discomfort symptoms by inhibiting bradykinin accumulation, inhibiting arachidonic acid metabolism, reducing cough hypersensitivity, and reducing airway hyperresponsiveness.

4. The use according to claim 1, wherein The ACEI includes at least one of captopril, enalapril, benazepril, lisinopril, ramipril, fosinopril, cilazapril, perindopril and imidapril.

5. The use according to claim 1, wherein The ACEI includes at least one of captopril, enalapril, benazepril, fosinopril and ramipril.

6. The use according to claim 1, wherein The ACEIs include captopril.

7. The use according to claim 1, wherein The Qingyan dripping pills comprise 80-120 parts of indigo, 80-120 parts of liquorice, 60-100 parts of terminalia chebula, 60-100 parts of menthol, 5-30 parts of borneol and 5-30 parts of artificial bezoar.

8. The use according to claim 1, wherein The Qingyan dripping pills comprise 90-110 parts of indigo, 90-110 parts of liquorice, 70-90 parts of terminalia chebula, 70-90 parts of menthol, 15-25 parts of borneol and 10-15 parts of artificial bezoar.

9. The use according to claim 1, wherein The Qingyan dripping pills comprise 100 parts of indigo, 100 parts of liquorice, 80 parts of terminalia chebula, 80 parts of menthol, 20 parts of borneol and 12 parts of artificial bezoar.

10. Use of the pharmaceutical composition in the preparation of a drug for preventing and / or treating cough caused by ACEI and its associated pharyngeal discomfort symptoms; wherein, The pharmaceutical composition comprises 80-120 parts of indigo naturalis, 80-120 parts of liquorice, 60-100 parts of terminalia chebula, 60-100 parts of menthol, 5-30 parts of borneol and 5-30 parts of artificial bezoar.

11. The use according to claim 10, wherein The pharmaceutical composition comprises 90-110 parts of indigo naturalis, 90-110 parts of liquorice, 70-90 parts of terminalia chebula, 70-90 parts of menthol, 15-25 parts of borneol and 10-15 parts of artificial bezoar.

12. The use according to claim 10, wherein The medicine composition comprises 100 parts of indigo naturalis, 100 parts of liquorice, 80 parts of terminalia chebula, 80 parts of menthol, 20 parts of borneol and 12 parts of artificial bezoar.

13. The use according to claim 10, wherein The pharyngeal discomfort symptoms include at least one of dry throat, itchy throat and sore throat.

14. The use according to claim 10, wherein The pharmaceutical composition further includes a pharmaceutically acceptable carrier or excipient.

15. The use according to claim 14, wherein The pharmaceutically acceptable carrier or excipient includes at least one of a solvent, a diluent, a dispersant, a suspending agent, a surfactant, an isotonic agent, a thickener, an emulsifier, a preservative, a binder, a lubricant, a stabilizer, a hydrating agent, an emulsification accelerator, a buffer, an absorbent, a colorant, a flavoring agent, a sweetener, an ion exchanger, a release agent, a coating agent, a flavoring agent and an antioxidant.

16. The use according to any one of claims 10 to 15, wherein The pharmaceutical composition is prepared in any dosage form of powder, tablet, capsule, pill, emulsion, suspension or tincture.

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