Use of Qingyan Dropping Pills in the preparation of a medicament for treating ACEI-induced cough

Abstract

The present application provides the use of Qingyan Dropping Pills in the preparation of a medicament for treating cough caused by ACEI. Qingyan Dropping Pills can relieve cough caused by ACEI and its accompanying pharyngeal discomfort symptoms through potential action mechanisms such as inhibiting bradykinin accumulation, inhibiting arachidonic acid metabolism, reducing cough hypersensitivity, and reducing airway hyperreactivity, and thus can be used to prepare a medicament for preventing and / or treating cough caused by ACEI and its accompanying pharyngeal discomfort symptoms. In addition, the present application has also successfully established a guinea pig cough induction experimental model with an ACEI compound cough inducer for the first time.

Description

[0001] This application claims the priority of a Chinese patent application titled "Use of Qingyan Dropping Pills in the Preparation of Drugs for Treating Cough Caused by ACEI" with the application number 202411987452.3 filed with the Patent Office of the State Intellectual Property Office of China on December 31, 2024. The entire content thereof is incorporated herein by reference. Technical Field

[0002] This application relates to the field of traditional Chinese medicine technology, and particularly to the use of Qingyan Dropping Pills in the preparation of drugs for treating cough caused by ACEI. Background Art

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

[0004] Qingyan Dropping Pills is a traditional Chinese medicine dropping pill preparation independently developed by the Sixth Traditional Chinese Medicine Factory of Tianjin Pharmaceutical Group. Its formula is modified from Qingyin Pills recorded in "Lantai Guifan" and Liushen Pills recorded in "Laryngeal Medicine Heart Method". Although it is derived from an ancient formula, it extracts the essence, has no toxic components, has high safety, and can be used for wind-heat throat obstruction, sore throat, dry throat, and pharyngeal swelling and redness. It has a significant effect on the treatment of respiratory diseases, especially throat symptoms. The potential therapeutic effect of Qingyan Dropping Pills on cough caused by ACEI has not been reported. Summary of the Invention

[0005] The inventors of this application found through research that Qingyan Dropping Pills can relieve cough caused by ACEI and its accompanying pharyngeal discomfort symptoms, and thus can be used to prepare drugs for preventing and / or treating cough caused by ACEI and its accompanying pharyngeal discomfort symptoms.

[0006] The first aspect of this application provides the use of Qingyan Dropping Pills in the preparation of drugs for preventing and / or treating cough caused by ACEI and its accompanying pharyngeal discomfort symptoms.

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

[0008] In one embodiment of this application, the Qingyan Dropping Pills prevent and / or treat cough caused by ACEI 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 liquorice root, 60 - 100 parts of chebula fruit, 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 liquorice root, 70 - 90 parts of chebula fruit, 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 liquorice root, 80 parts of chebula fruit, 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 includes 80 - 120 parts of indigo naturalis, 80 - 120 parts of liquorice root, 60 - 100 parts of chebula fruit, 60 - 100 parts of menthol, 5 - 30 parts of borneol, and 5 - 30 parts of artificial bezoar; preferably, the pharmaceutical composition includes 90 - 110 parts of indigo naturalis, 90 - 110 parts of liquorice root, 70 - 90 parts of chebula fruit, 70 - 90 parts of menthol, 15 - 25 parts of borneol, and 10 - 15 parts of artificial bezoar; more preferably, the pharmaceutical composition includes 100 parts of indigo naturalis, 100 parts of liquorice root, 80 parts of chebula fruit, 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 includes 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 thickening agent, an emulsifier, a preservative, a binder, a lubricant, a stabilizer, a moisturizer, an emulsification accelerator, a buffer, an absorbent, a colorant, a flavoring agent, a sweetening agent, an ion exchanger, a demolding agent, a coating agent, a flavor correcting agent, and an antioxidant.

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

[0016] Advantages of the present application:

[0017] The present application provides the use of Qingyan Dropping Pills in the preparation of a drug for treating cough caused by ACEI. Qingyan Dropping Pills can achieve the effect of treating ACEI-induced cough through potential action mechanisms such as inhibiting bradykinin accumulation, inhibiting arachidonic acid metabolism, reducing cough hypersensitivity, and reducing airway hyperreactivity; at the same time, Qingyan Dropping Pills can relieve the pharyngeal discomfort symptoms such as dry throat, itchy throat, and sore throat that may accompany cough. In short, Qingyan Dropping Pills can relieve the cough caused by ACEI and its accompanying pharyngeal discomfort symptoms, and thus can be used to prepare a drug for preventing and / or treating the cough caused by ACEI and its accompanying pharyngeal discomfort symptoms. Further, the pharmaceutical composition of the present application can also be used to prepare a drug for preventing and / or treating the cough caused by ACEI and its accompanying pharyngeal discomfort symptoms. In addition, the present application has also successfully established a guinea pig cough induction experimental model of ACEI compound cough inducer for the first time.

[0018] Of course, it is not necessary for any product or method implementing the present application to simultaneously achieve all the above-mentioned advantages. Description of the Drawings

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present application, and those of ordinary skill in the art can also obtain other embodiments based on these drawings.

[0020] Figures 1A to 1F For the results of network pharmacology analysis; Venn diagram of potential action targets of Qingyan Dropping Pills compounds and disease targets of ACEI-induced cough ( Figure 1A ), target protein PPI network analysis diagram ( Figure 1B ), target protein KEGG pathway enrichment analysis diagram (the top 20 signaling pathways) ( Figure 1C ), target protein GO function enrichment analysis ( Figure 1D ), "Qingyan Dropping Pills - Chinese herbal medicine - compound - target - pathway" network diagram ( Figure 1E ), "Qingyan Dropping Pills - Chinese herbal medicine - compound - target - pharmacological action" network diagram ( Figure 1F );

[0021] Figures 2A to 2F For the molecular docking display diagram; TRPV1 and chebulinic acid ( Figure 2A ), BDKRB2 and liquiritin ( Figure 2B ), PTGS2 and indirubin ( Figure 2C), PLA2G7 and hyodeoxycholic acid ( Figure 2D ), PTGER3 and liquiritigenin ( Figure 2E ), PTGER2 and glycocholic acid ( Figure 2F ); docking result diagrams;

[0022] Figures 3A to 3F are the binding curves (SPR sensor images) between drug components and protein targets; the binding curve between hyodeoxycholic acid and PLA2G7 ( Figure 3A ); the binding curve between liquiritigenin and PLA2G7 ( Figure 3B ); the binding curve between liquiritigenin and COX-2 ( Figure 3C ); the binding curve between liquiritigenin and TRPV1 ( Figure 3D ); the binding curve between hyodeoxycholic acid and TRPA1 ( Figure 3E ); the binding curve between chebulinic acid and TRPV1 ( Figure 3F );

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

[0024] Figure 5A is 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 is 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 is the effect of Qingyan dropping pills on the protein expression of TRPV1, BDKRB2, and PTGER3 in lung tissue in the capsaicin-induced cough animal experiment;

[0026] Figure 7 is the effect of Qingyan dropping pills on the protein expression of TRPV1, BDKRB2, and PTGER3 in lung tissue in the bradykinin-induced cough animal experiment. Specific implementation methods

[0027] The technical solutions in the present application will be clearly and completely described below in conjunction with the embodiments and the accompanying drawings of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all of 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 dropping pills in the preparation of a drug for preventing and / or treating cough caused by ACEI and its accompanying pharyngeal discomfort symptoms. The inventors found in the research that through network pharmacology analysis, it was shown that Qingyan dropping pills could achieve the effect of treating ACEI-induced cough through potential action mechanisms such as inhibiting bradykinin accumulation, inhibiting arachidonic acid metabolism, reducing cough hypersensitivity, and reducing airway hyperresponsiveness; at the same time, Qingyan dropping pills had a relieving effect on pharyngeal discomfort symptoms such as dry throat, itchy throat, and sore throat that may accompany cough. Through in vitro verification experiments, it was shown that Qingyan dropping pills might relieve cough caused by ACEI through pathways such as regulating arachidonic acid metabolism and reducing cough hypersensitivity. Through in vivo pharmacodynamic evaluation experiments, it was shown that Qingyan dropping pills could significantly prolong the cough latency of ACEI-induced cough, reduce the number of coughs, and increase the antitussive rate. In summary, Qingyan dropping pills can relieve cough caused by ACEI and its accompanying pharyngeal discomfort symptoms, and thus can be used to prepare a drug for preventing and / or treating cough caused by ACEI and its accompanying pharyngeal discomfort symptoms.

[0029] In the present application, the term "treatment" has its general meaning, and in the present application, it particularly refers to treating a mammalian individual (preferably a human) suffering from cough caused by ACEI and its accompanying pharyngeal discomfort symptoms with the drug of the present application, in the hope of having therapeutic, curative, relieving, alleviating and other effects on the disease. Similarly, as used in the present application, the term "prevention" has its general meaning, and in the present application, it particularly refers to treating a mammalian individual at risk of suffering from cough caused by ACEI and its accompanying pharyngeal discomfort symptoms with the drug of the present application, in the hope of having preventive, prophylactic, blocking, isolating and other effects on 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 inventors' research, 33 representative chemical components of Qingyan dropping pills were selected as the research objects to obtain the potential action targets of the compounds. Using "ACE inhibitor-induced cough" as the keyword, the disease-related targets of ACEI-induced cough were retrieved; at the same time, using "pharyngeal dryness", "itchy throat", and "sore throat" as keywords, the targets of cough-related symptoms of dry throat, itchy throat, and sore throat were retrieved. The intersection targets of the potential action targets of the compounds with dry throat, itchy throat, and sore throat were the target proteins for Qingyan dropping pills to treat cough-related symptoms. Network pharmacology prediction analysis found that Qingyan dropping pills may have a relieving effect on pharyngeal discomfort symptoms such as dry throat, itchy throat, and sore throat that may accompany cough.

[0031] In one embodiment of the present application, the Qingyan dropping pills 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. In the inventors' research, 33 representative chemical components of Qingyan dropping pills were selected as the research objects to obtain the potential action targets of the compounds. Using "ACE inhibitor-induced cough" as the keyword, the disease-related targets of ACEI-induced cough were retrieved. By using Venny analysis, the intersection targets of the potential action targets of the compounds and ACEI-induced cough were the target proteins for Qingyan dropping pills to treat ACEI-induced cough. Network pharmacology prediction analysis found that Qingyan dropping pills can act on protein targets such as MME, BDKRB2, and TRPV1, regulate signal pathways such as arachidonic acid metabolism, regulation of TRP channels by inflammatory mediators, calcium ion signaling pathway, TNF signaling pathway, and cholinergic synapse, inhibit the accumulation of BK and the process of arachidonic acid metabolism, reduce neurogenic inflammation and cough hypersensitivity, relieve airway inflammation and relax tracheal smooth muscle to reduce airway hyperresponsiveness, so as to achieve the effect of treating ACEI-induced cough, and thus 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 types of ACEIs are not particularly limited. For example, the ACEIs include but are 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 liquorice root, 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 liquorice root, 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 liquorice root, 80 parts of terminalia chebula, 80 parts of menthol, 20 parts of borneol, and 12 parts of artificial bezoar. For example, indigo naturalis can be 80 parts, 90 parts, 100 parts, 110 parts, 120 parts, or a range composed of any two of these values; liquorice root can be 80 parts, 90 parts, 100 parts, 110 parts, 120 parts, or a range composed of any two of these values; terminalia chebula can be 60 parts, 70 parts, 80 parts, 90 parts, 100 parts, or a range composed of any two of these values; menthol can be 60 parts, 70 parts, 80 parts, 90 parts, 100 parts, or a range composed of any two of these values; borneol can be 5 parts, 10 parts, 15 parts, 20 parts, 25 parts, 30 parts, or a range composed of any two of these values; 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 composed of any two of these values.

[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 includes 80-120 parts of Indigo Naturalis, 80-120 parts of Liquorice Root, 60-100 parts of Chebula Fruit, 60-100 parts of Menthol, 5-30 parts of Borneol, and 5-30 parts of Calculus Bovis Artifactus; preferably, the pharmaceutical composition includes 90-110 parts of Indigo Naturalis, 90-110 parts of Liquorice Root, 70-90 parts of Chebula Fruit, 70-90 parts of Menthol, 15-25 parts of Borneol, and 10-15 parts of Calculus Bovis Artifactus; more preferably, the pharmaceutical composition includes 100 parts of Indigo Naturalis, 100 parts of Liquorice Root, 80 parts of Chebula Fruit, 80 parts of Menthol, 20 parts of Borneol, and 12 parts of Calculus Bovis Artifactus. For example, Indigo Naturalis can be 80 parts, 90 parts, 100 parts, 110 parts, 120 parts or a range composed of any two of these values, Liquorice Root can be 80 parts, 90 parts, 100 parts, 110 parts, 120 parts or a range composed of any two of these values, Chebula Fruit can be 60 parts, 70 parts, 80 parts, 90 parts, 100 parts or a range composed of any two of these values, Menthol can be 60 parts, 70 parts, 80 parts, 90 parts, 100 parts or a range composed of any two of these values, Borneol can be 5 parts, 10 parts, 15 parts, 20 parts, 25 parts, 30 parts or a range composed of any two of these values, and Calculus Bovis Artifactus can be 5 parts, 8 parts, 10 parts, 12 parts, 15 parts, 18 parts, 20 parts, 25 parts, 30 parts or a range composed of any two of these values.

[0036] Due to containing the drug formula of Qingyan Dropping Pills, the pharmaceutical composition of the present application can also be used in the preparation of a drug for preventing and / or treating cough caused by ACEI and its accompanying pharyngeal discomfort symptoms. Taking 33 representative chemical components of 6 medicinal materials in the pharmaceutical composition as the research objects, through network pharmacology analysis, it is shown that the pharmaceutical composition can achieve the effect of treating ACEI-induced cough through potential action mechanisms such as inhibiting bradykinin accumulation, inhibiting arachidonic acid metabolism, reducing cough hypersensitivity, and reducing airway hyperreactivity; 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 accompany cough. Through in vitro verification experiments, it is shown that the pharmaceutical composition may relieve cough caused by ACEI by regulating arachidonic acid metabolism and reducing cough hypersensitivity. Through in vivo pharmacodynamic 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 increase the antitussive rate. In short, the pharmaceutical composition can relieve cough caused by ACEI and its accompanying pharyngeal discomfort symptoms, and thus can be used to prepare a drug for preventing and / or treating cough caused by ACEI 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. Thirty-three representative chemical components of six medicinal materials in the pharmaceutical composition were selected as the research objects to obtain the potential action targets of the compounds. Using "ACE inhibitor-induced cough" as the keyword, the disease-related targets of ACEI-induced cough were retrieved; at the same time, using "pharyngeal dryness", "itchy throat", and "sore throat" as keywords, the targets of cough-related symptoms of dry throat, itchy throat, and sore throat were retrieved. The intersection targets of the potential action targets of the compounds with dry throat, itchy throat, and sore throat were obtained as the target proteins for the pharmaceutical composition to treat cough-related symptoms. Network pharmacology prediction analysis found that the pharmaceutical composition may have a relieving effect on pharyngeal discomfort symptoms such as dry throat, itchy throat, and sore throat that may accompany 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. In the research, the inventors selected thirty-three representative chemical components of six medicinal materials in the pharmaceutical composition as the research objects to obtain the potential action targets of the compounds. Using "ACE inhibitor-induced cough" as the keyword, the disease-related targets of ACEI-induced cough were retrieved. By using Venny analysis, the intersection targets of the potential action targets of the compounds and ACEI-induced cough were obtained as the target proteins for the pharmaceutical composition to treat ACEI-induced cough. Network pharmacology prediction analysis found that the pharmaceutical composition can act on protein targets such as MME, BDKRB2, and TRPV1, regulate signal pathways such as arachidonic acid metabolism, regulation of TRP channels by inflammatory mediators, calcium ion signaling pathway, TNF signaling pathway, and cholinergic synapse, inhibit the accumulation of BK and the arachidonic acid metabolism process, reduce neurogenic inflammation and cough hypersensitivity, relieve airway inflammation and relax tracheal smooth muscle to reduce airway hyperresponsiveness, so as to achieve the effect of treating ACEI-induced cough, and thus can be used to prepare a drug 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 having no substantial toxic effects when used at normal therapeutic doses, and thus can be approved by a government or an international organization equivalent thereto, or has been approved for use in animals, more particularly in humans, or is listed in a 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 thickening agent, an emulsifying agent, a preservative, a binder, a lubricant, a stabilizer, a hydrating agent, an emulsification accelerator, a buffer, an absorbent, a coloring agent, a flavoring agent, a sweetening agent, an ion exchanger, a release agent, a coating agent, a taste-correcting agent, and an antioxidant. In the present application, the "pharmaceutically acceptable carrier or excipient" usable in the pharmaceutical composition of the present application can be any conventional carrier in the field of pharmaceutical preparations, and the choice of a specific carrier will depend on the mode of administration or the type and condition of the disease for treating a specific patient. The preparation method of a suitable pharmaceutical composition for a specific mode of administration is completely within the knowledge of those skilled in the pharmaceutical art.

[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 Drops 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] II. Network Pharmacology Analysis

[0048] Thirty-three representative chemical components from the six herbs of Qingyan Pills were selected as the research objects, including indirubin and indigo in Indigo Naturalis, gallic acid, chebulinic acid, digalloyl glucose, terchebin, corilagin, 1,3,6-tri-O-galloyl-β-D-glucopyranoside, 1,2,3,4-tetra-O-galloyl-β-D-glucopyranose in Terminalia chebula Retz., 1-palmitoyl-lysophosphatidylcholine, taurocholic acid, glycocholic acid, taurochenodeoxycholic acid, cholic acid, hyodeoxycholic acid, glycochenodeoxycholic acid, chenodeoxycholic acid, bilirubin in Calculus Bovis Artifactus, menthol in Mentha haplocalyx Briq., borneol (borneol) in Borneolum Syntheticum, glycyrrhizin isomers, liquiritin, isoliquiritin, ononin, liquiritigenin, glycyrrhizin, glycyrrhizic acid, glycyrrhetinic acid, glycyrrhisoflavone, linolenic acid in Glycyrrhiza uralensis Fisch., linoleic acid, punica granate acid, oleic acid, the compounds shared by Terminalia chebula Retz. and Glycyrrhiza uralensis Fisch.; through databases such as TCMSP, Swiss TargetPrediction, and BATMAN-TCM, the potential action targets of the compounds were obtained. Using the TTD, DrugBank, DisGeNET, and GeneCards databases, with "ACE inhibitor-induced cough" as the keyword, the disease-related targets of ACEI-induced cough were retrieved; at the same time, with "pharyngeal dryness", "itchy throat", and "sore throat" as the keywords, the targets of dryness of the throat, itchy throat, and sore throat, the symptoms related to cough, were retrieved. Using Venny analysis, the intersection targets of the potential action targets of the compounds and ACEI-induced cough were obtained as the target proteins for Qingyan Pills to treat ACEI-induced cough; at the same time, the intersection targets of the potential action targets of the compounds and dryness of the throat, itchy throat, and sore throat were obtained as the target proteins for Qingyan Pills to treat cough-related symptoms. Further, PPI network analysis, Cluster module analysis, GO analysis, and KEGG analysis were respectively performed on the two groups of target proteins to construct the pharmacological action network relationship diagram of Qingyan Pills.

[0049] The results of network pharmacology analysis are as Figures 1A to 1F shown. Through prediction by each database, 544 related targets of 33 compounds of Qingyan Pills were obtained, 644 disease-related targets were obtained, and 160 target proteins for Qingyan Pills to treat ACEI-induced cough were obtained by taking the intersection, as shown in Figure 1A . From Figure 1BIt can be seen that the PPI network has a total of 160 nodes and 2,920 edges. Nodes with a degree value (i.e., degree value) greater than or equal to 30 are screened, and 83 protein targets and 1,887 edges are obtained. Further screening of nodes with a degree value greater than or equal to twice the median, i.e., 51, a total of 42 key target proteins are obtained, including prostaglandin-endoperoxide synthase 2 (PTGS2), interleukin 6 (IL6), albumin (ALB), tumor necrosis factor (TNF), Caspase-3 (CASP3), etc. KEGG and GO analyses are performed on 160 targets, and there are a total of 173 signaling pathways with P < 0.05. The related pathways include: Inflammatory mediator regulation of TRP channels, PI3K-Akt signaling pathway, TNF signaling pathway, IL-17 signaling pathway, T cell receptor signaling pathway, Th17 cell differentiation, Ras signaling pathway, VEGF signaling pathway, MAPK signaling pathway, etc., as shown in Figure 1C . The GO analysis is shown in Figure 1D . There are 70 cellular components, 130 molecular functions, and 553 biological processes with P < 0.05. In terms of cellular components, it is mainly involved in the plasma membrane, membrane rafts, extracellular space, etc.; in terms of molecular functions, it is mainly involved in ATP binding, enzyme binding, protein tyrosine kinase activity, etc.; in terms of biological processes, it is mainly involved in the response to external stimuli, regulation of apoptosis, inflammatory response, cell proliferation, and regulation of gene expression, etc. The network relationship diagram of "Qingyan Dropping Pills - Medicinal Materials - Compounds - Targets - Pathways" is shown in Figure 1E, The 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 the interaction of one compound with multiple target proteins, and there are also phenomena where different compounds act on the same target protein. At the same time, it also shows that one target can be enriched in multiple signaling pathways, indicating the characteristics of Qingyan dropping pills in treating ACEI-induced cough through multiple components, multiple targets, and multiple pathways. The network relationship diagram of "Qingyan dropping pills - medicinal materials - compounds - targets - pharmacological effects" is shown in Figure 1F , Analysis of the network targets found that the mechanism of Qingyan dropping pills 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 dropping pills can act on protein targets such as MME, BDKRB2, and TRPV1, regulate signaling pathways such as arachidonic acid metabolism, regulation of TRP channels by inflammatory mediators, calcium ion signaling pathway, TNF signaling pathway, and cholinergic synapse, inhibit the accumulation of BK and the process of arachidonic acid metabolism, reduce neurogenic inflammation and cough reflex sensitivity, relieve airway inflammation and relax tracheal smooth muscle to reduce airway hyperresponsiveness, so as to achieve the effect of treating ACEI-induced cough; at the same time, Qingyan dropping pills may relieve symptoms of discomfort in the pharynx accompanying cough, such as dry throat, itchy throat, and sore throat.

[0051] III. In vitro verification experiment

[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 within suitable target binding sites and evaluate their binding affinity. In this application, targets with strong correlations to four potential mechanisms of action, namely inhibiting bradykinin accumulation, inhibiting arachidonic acid metabolism, reducing cough hypersensitivity, and reducing airway hyperresponsiveness, were selected for verification. These targets include MME and F12 for inhibiting bradykinin accumulation, PLA2G7, PTGS2, ALOX5, and PTGES for inhibiting arachidonic acid metabolism, TRPV1, TRPA1, BDKRB2, PTGER2, and PTGER3 for reducing cough hypersensitivity, and NOS2, CHRM2, and ACHE for reducing airway hyperresponsiveness. The crystal structures of these target proteins were downloaded from the RCSB PDB database. After downloading, the Schrödinger software was used to correct the bond order, add hydrogen, process disulfide bonds and metal ions, and remove water molecules and heteroatoms in the crystal structure for preprocessing and energy optimization. The target protein was used as a rigid receptor, and each ligand molecule was used as a flexible ligand with variable conformation. Semi-flexible docking was performed through the Ligand Docking module.

[0054] The results of molecular docking are shown in Table 1, Figures 2A to 2F as follows. It can be seen from Table 1 that TRPV1 has good docking values with chebulinic acid, indirubin, and gallic acid; BDKRB2 has good docking values with glycocholic acid, liquiritin, and chebulinic acid; PTGS2 and PLA2G7 have good docking values with indirubin, liquiritin, and menthol; PTGER3 has good docking values with liquiritin, hyodeoxycholic acid, indirubin, and gallic acid. From Figures 2A to 2F it can be seen that the binding between the drug components and the target protein is mainly through hydrogen bonds. The drug components of Qingyan dropping pills and the target protein all show relatively low free binding energies, and it can be preliminarily considered that the mechanism of action of Qingyan dropping pills in treating ACEI-induced cough is related to targets such as TRPV1, BDKRB2, PTGS2, PLA2G7, and PTGER3.

[0055] Table 1 Results of Molecular Docking

[0056]

[0057] (2) Surface Plasmon Resonance (SPR) Experiment

[0058] Eight representative components in Qingyan dropping pills, namely hyodeoxycholic acid, liquiritin, chebulinic acid, gallic acid, glycyrrhetinic acid, menthol, indirubin, and glycocholic acid, were selected as small molecule compounds. The key targets PLA2G7 and COX-2 in arachidonic acid metabolism and the important targets TRPV1 and TRPA1 related to cough hypersensitivity were used as target proteins. According to the Plate position and volume specified by the instrument, small molecule samples and PBS buffer containing 1% (v / v) DMSO were added, and the interaction analysis between components and targets was completed according to the "Operation Guide of Biacore 8K Molecular Interaction Analysis System".

[0059] The binding curves (SPR sensor images) between drug components and protein targets are shown as Figures 3A to 3F follows. The equilibrium dissociation constants between analytes and ligands are shown in Table 2. As can be seen from Figures 3A to 3F and Table 2, liquiritin has a certain affinity with COX-2, TRPV1, and PLA2G7 proteins, and hyodeoxycholic acid has a certain affinity with PLA2G7 protein. It indicates that Qingyan dropping pills may play a role in treating ACEI-induced cough by acting on PLA2G7, COX-2, and the cough hypersensitivity-related target TRPV1 in the AA metabolic pathway.

[0060] Table 2 Equilibrium dissociation constants between analytes and ligands

[0061]

[0062] Note: "KD" is the value of the equilibrium dissociation constant.

[0063] (3)Enzyme inhibitor screening experiment

[0064] Eight representative components in Qingyan dropping pills, namely chebulinic acid, menthol, liquiritin, gallic acid, glycocholic acid, glycyrrhetinic acid, hyodeoxycholic acid, and indirubin, were selected to conduct inhibitor screening reactions with two key enzymes, COX-2 and 5-LOX, in the arachidonic acid metabolic pathway. According to the instructions of the COX-2 inhibitor screening kit (Beyotime, batch number Z984240806), the interaction of the key components of Qingyan dropping pills with the key enzyme COX-2 was explored. 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 the blank control well were measured and recorded as RFU 空白对照 , RFU 100%酶活性对照 and RFU 样品 . RFU, Relative Fluorescence Unit. The inhibition percentage of each sample was calculated, and the calculation formula was: 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%. Operate according to the instructions of the 5-LOX inhibitor screening kit (Cayman, batch number 760700) to explore the interaction between the key components of Qingyan Dropping Pills and the key enzyme 5-LOX. After the reaction, measure the absorbance at a wavelength of 490 - 500 nm, and measure the absorbance of each sample well and the blank control well, which can be 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. The calculation formula is: 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%.

[0065] The inhibition rate results of the representative components in Qingyan Dropping Pills on COX-2 and 5-LOX are as shown in Figures 4A to 4B . It can be seen from Figures 4A to 4B that the representative components in Qingyan Dropping Pills, such as Chebulic Acid, Menthol, Gallic acid, Glycocholic acid, glycyrrhetnic acid, Hyodeoxycholic acid, and Indirubin, can inhibit the enzyme activity of COX-2 at 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 concentrations of 50 μmol / L and 100 μmol / L. The inventor speculated that Qingyan Dropping Pills can reduce the generation of pro-inflammatory and cough-inducing factors in the AA metabolic pathway by inhibiting the activities of COX-2 and 5-LOX, and thus play a role in treating ACEI-induced cough.

[0066] (4) Cellular thermal shift assay (CETSA) experiment

[0067] Take an appropriate amount of Qingyan Dropping Pills, grind them into powder, weigh 1 g of the powder, place it in a 100 mL round-bottom flask, add 25 mL of 70% anhydrous ethanol, and weigh. Reflux and extract for 2 h, weigh, make up with 70% ethanol, filter, and concentrate to obtain the extract of Qingyan Dropping Pills. Take 80 mg of lung tissue and place it in an EP tube, add non-denaturing tissue / cell lysate according to a ratio of 1:9, fully break it with a tissue homogenizer, ice-bath for 30 min, shake it once every 5 min to ensure complete lysis of the tissue, centrifuge at 12000 r at 4℃ for 10 min, collect the supernatant to obtain the guinea pig lung protein solution.

[0068] Prepare a 200 μg / mL solution of the extract of Qingyan Dropping Pills with ddH2O. Incubate 44.4 μL of the extract solution of Qingyan Dropping Pills, 44.4 μL of ddH2O and 400 μL of the guinea pig lung protein solution at 37℃ for 2 h. Subsequently, divide the solution into aliquots and heat it at four temperature gradients of 37℃, 42℃, 51℃, and 59℃ for 3 minutes. After cooling, prepare the samples and perform Western blot (WB) experiments to detect the interaction between the extract of Qingyan Dropping Pills and the cough-related targets TRPV1 and BDKRB2 of ACEI. The steps of the WB experiment are as follows:

[0069] Gel preparation: Prepare a 10% gel according to the instructions of the SDS-PAGE gel preparation kit.

[0070] Loading: Use a pipette to add the prepared protein sample to the loading well at 8 μL per well.

[0071] 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 the bromophenol blue runs to 1 cm from the bottom of the gel, then turn off the power supply.

[0072] Prepare the PVDF membrane, filter paper and sponge. Put the membrane and the gel with bands into the transfer buffer. Use the sandwich method to arrange the filter paper, sponge and gel in the corresponding order, and pay attention to preventing the generation of bubbles. Connect the gel surface to the negative electrode and the PVDF membrane to the positive electrode, place it in an ice-water bath and transfer at a constant current of 300 mA for 90 min.

[0073] After taking out the PVDF membrane from the transfer clip, put it into the rapid blocking solution and block for 1 h. Cut the required protein bands and add the primary antibodies of TRPV1 and BDKRB2 respectively, and incubate overnight at 4℃. The next day, collect the primary antibodies and store them at -20℃. Wash the membrane 3 - 5 times on a shaker with TBST. Then add the secondary antibody prepared with the rapid blocking solution and incubate for 1 h, and wash the membrane again. Finally, use the ECL luminescent solution to expose in a chemiluminescence imager, save the bands, and further perform quantitative analysis of the levels of TRPV1 and BDKRB2 with ImageJ.

[0074] In a certain temperature range, the effects of Qingyan Dropping Pills on the protein expression of TRPV1 and BDKRB2 in the lung tissue of guinea pigs are as follows Figures 5A to 5B shown. It can be seen from Figures 5A to 5B that the extract of Qingyan Dropping Pills has a certain binding effect with TRPV1 and BDKRB2 targets. Qingyan Dropping Pills may play a role in treating cough caused by ACEI by acting on TRPV1 and BDKRB2 targets and regulating cough hypersensitivity.

[0075] To sum up, Qingyan Dropping Pills may play a role in relieving cough caused by ACEI by regulating arachidonic acid metabolism and cough hypersensitivity, etc., and initially verifying the prediction results of network pharmacology.

[0076] IV. In Vivo Pharmacodynamic Evaluation and Mechanism of Action Research

[0077] (1) Pharmacodynamic Evaluation and Mechanism of Action Research on Qingyan Dropping Pills in Relieving Cough Caused by ACEI (Capsaicin-Induced Cough)

[0078] SPF-grade Hartley guinea pigs, male, weighing 300 - 350 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with the license number SCXK (Beijing) 2021 - 0011. They were randomly divided into 6 groups, namely the blank group, the model group (captopril, dose 11.6 mg / kg, Changzhou Pharmaceutical Factory Co., Ltd., batch number 22081613z), the SB705498 (TRPV1 receptor antagonist, dose 5 mg / kg, Biyaso, batch number 501951 - 42 - 4) group, the Golden Throat Lozenge (dose 0.93 g / kg, Guangxi Golden Throat Co., Ltd., batch number 24031203) group, the low-dose Qingyan Dropping Pills group (dose 27.87 mg / kg), and the high-dose Qingyan Dropping Pills group (dose 83.61 mg / kg). The low- and high-dose administration suspensions of Qingyan Dropping Pills were prepared by fully suspending them in 0.1% CMC-Na solution, with concentrations of 0.0149 g / mL and 0.0418 g / mL respectively, and there were 10 animals in each group. The blank group was given 0.1% CMC-Na by gavage, and the other groups were given captopril by gavage to establish an animal model of cough caused by ACEI. After 4 h, each group was given the corresponding drug treatment (the blank group and the model group were given 0.1% CMC-Na by gavage), which lasted for 14 days, once a day, and the gavage volume was 10 mL / kg.

[0079] On the 0th day (one day before drug administration), blood was collected from the dorsal mid-foot vein of guinea pigs in each group. One hour after the last drug administration on the 14th day, blood was collected from the dorsal mid-foot vein of guinea pigs in each group. After blood collection, the guinea pigs were placed in an atomization chamber, and atomization was started using 250 μmol / L capsaicin (MCE, batch number 239533) as the cough-inducing agent and timing was started. After 5 minutes, they were taken out and placed in an empty cage, and observed for another 5 minutes. The cough conditions of each group were recorded. Subsequently, blood was collected from the abdominal aorta, and the blood of the guinea pigs was collected and the lung tissue was dissected for biochemical index detection. RT-qPCR and WB were used to detect the expression levels of cough hypersensitivity-related proteins and genes in the lung tissue respectively. The therapeutic effect of Qingyan Dropping Pills was evaluated and its possible mechanism of action was preliminarily analyzed.

[0080] Determination of substance P (SP), calcitonin gene-related peptide (CGRP), bradykinin (BK) and neurokinin A (NKA) contents: According to the kit instructions of SP (Jianglai Biotech, batch number 062730003172620627), CGRP (Jianglai Biotech, batch number 062730003120040627), BK (Jianglai Biotech, batch number 062844003215320802) and NKA (Shanghai Enzyme-linked Biotechnology Co., Ltd., batch number Sep 2024), after the reaction ended, the wavelength was set at 450 nm, the OD value of each well was measured, and the contents of SP, CGRP, BK and NKA in the serum and lung tissue of each group were calculated according to the instructions.

[0081] Determination of the activities of aminopeptidase (APP), neutral endopeptidase (NEP) and dipeptidyl peptidase-4 (DPP Ⅳ): According to the kit instructions of APP (Jianglai Biotech, batch number 062844003546730802), DPP Ⅳ (Shanghai Enzyme-linked Biotechnology Co., Ltd., batch number Jul 2024) and NEP (Shanghai Enzyme-linked Biotechnology Co., Ltd., batch number Jul 2024), after the reaction ended, the wavelength was set at 450 nm, the OD value of each well was measured, and the contents of APP, NEP and DPP Ⅳ in the lung tissue of each group were calculated according to the instructions.

[0082] RT-qPCR detection of the gene expression levels of TRPV1, BDKRB2 and PTGER3 in the lung tissue:

[0083] (1) The list of PCR primers used is as follows (all purchased from Sangon Biotech (Shanghai) Co., Ltd.):

[0084]

[0085] (2) RNA extraction:

[0086] Weigh 80 mg of lung tissue and place it in a 1.5 mL enzyme-free EP tube. Add 1 mL of Trizol and 2 steel beads, and grind the tissue in a tissue homogenizer. Let it stand for 15 min.

[0087] Add 200 μL of chloroform to the lysate, shake vigorously, and let it stand at room temperature for 15 min. Then centrifuge at 4°C, 12000 rmp for 15 min.

[0088] 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 it stand at room temperature for 10 min, and then centrifuge at 4°C, 12000 rmp for 15 min.

[0089] Discard the supernatant, operate on ice, add 1 mL of 75% ice ethanol prepared with enzyme-free water, gently disperse the precipitate, and centrifuge at 4°C, 12000 rmp for 5 min.

[0090] Aspirate and discard the supernatant, avoiding ethanol residue, and let it stand at room temperature for about 5 min to completely dry the precipitate. Dissolve it with 90 - 150 μL of enzyme-free water according to the amount of precipitate.

[0091] Use a ultra-micro nucleic acid and protein analyzer to measure the RNA concentration.

[0092] (3)Synthesis of cDNA

[0093] Insert a 0.2 mL Ep tube into ice and incubate for 5 min. Add template RNA, supplement with RNase free ddH2O to make up to 12 μL, then add 4 μL of 4×gDNA wiper Mix, gently pipette and mix well, incubate at 42°C for 2 min, then directly add 4 μL of 5×HIScript Ⅱ qRT SuperMix Ⅱ, gently pipette and mix well, incubate at 50°C for 15 min, and then change the temperature to 85°C for 2 min. After the reaction, store the cDNA at -20°C for later use.

[0094] (4)Real-time fluorescence quantitative PCR:

[0095] Add the following reagents to the PCR 96-well plate respectively:

[0096]

[0097] After briefly centrifuging the mixed system, place it in a qPCR system and set the following amplification program:

[0098] Step 1 95°C 10 min

[0099] Step 2 95°C 10 s

[0100] Step 3: 60 °C for 30 s

[0101] Step 2 → Step 3: Set the number of cycles to 50 times. After the reaction is completed, export the data.

[0102] WB was used to detect the protein expression levels of TRPV1, BDKRB2, and PTGER3 in lung tissues:

[0103] (1) Extraction and quantification of total tissue protein

[0104] Take 50 - 100 mg of lung tissue from each group and place it in an EP tube. Add 9 times the volume of non - denaturing tissue / cell lysate. Use a tissue homogenizer to fully break it up, ice - bath for 30 min, shake it once every 5 min to ensure complete lysis of the tissue, centrifuge at 12000 r for 10 min, and collect the supernatant to obtain the total protein.

[0105] (2) Protein quantification by BCA method

[0106] Operate according to the instructions of the BCA kit (Solarbio; batch number: 240005014). After the reaction is completed, set the wavelength to 562 nm, measure the absorbance value of the sample, and calculate the protein concentration according to the instructions. Add the protein solution and 4× protein loading buffer in a ratio of 3:1, denature at 100 °C for 5 min, cool it, and store it in a - 20 °C refrigerator for later use.

[0107] (3) SDS - PAGE gel electrophoresis

[0108] Gel preparation: Prepare a 10% gel according to the instructions of the SDS - PAGE gel preparation kit.

[0109] Loading: Use a pipette to add the sample with the measured protein concentration to the loading wells at a protein amount of 70 μg per well.

[0110] Electrophoresis: First, perform electrophoresis at 80 V for 30 min. When the indicator reaches the gel boundary line, adjust the voltage to 120 V until the bromophenol blue runs to 1 cm from the bottom of the gel, then turn off the power.

[0111] (4) Transfer

[0112] Prepare the PVDF membrane, filter paper, and sponge. Place the membrane and the gel with bands in the transfer buffer. Use the sandwich method to arrange the filter paper, sponge, and gel in the corresponding order, and pay attention to preventing the generation of bubbles. Connect the gel side to the negative electrode and the PVDF membrane to the positive electrode, place it in an ice - water bath, and transfer at a constant current of 300 mA for 90 min.

[0113] (5) Immunoblotting reaction and protein quantification

[0114] After removing the PVDF membrane from the transfer clip, place it in the rapid blocking solution and block for 1 h. Cut out the required protein bands and add the primary antibody respectively, and incubate overnight at 4 °C. The next day, collect the primary antibody and store it at -20 °C. Wash the membrane 3 - 5 times with TBST on a shaker. Then add the secondary antibody prepared with the rapid blocking solution and incubate for 1 h, and wash the membrane again. Finally, expose it with the ECL luminescent solution in a chemiluminescent imager, save the band, and further perform quantitative analysis with ImageJ.

[0115] The results of the effects of Qingyan dropping pills on the cough latency, the number of coughs within 10 min, and the cough suppression rate after capsaicin-induced cough are shown in Table 3. It can be seen from Table 3 that Qingyan dropping pills can significantly prolong the cough latency of guinea pigs induced by ACEI, reduce the number of coughs, and increase the cough suppression rate (up to more than 60%). All three pharmacodynamic indexes are better than those of Yaojinshangzi throat tablets.

[0116] Table 3 Effects of Qingyan dropping pills on cough latency, number of coughs within 10 min, and cough suppression rate after capsaicin-induced cough ;

[0117]

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

[0119] In the animal experiment of capsaicin-induced cough, the effects of Qingyan dropping pills on the protein expressions of TRPV1, BDKRB2, and PTGER3 in lung tissue are as Figure 6 shown. Tables 4-1 to 4-4 show the effects of Qingyan dropping pills on SP, CGRP, BK, and NKA in plasma, Table 5 shows the effects of Qingyan dropping pills on SP, CGRP, BK, and NKA in lung tissue, Table 6 shows the effects of Qingyan dropping pills on DPP IV, NEP, and APP in lung tissue, Table 7 shows the effects of Qingyan dropping pills on the gene expressions of TRPV1, BDKRB2, and PTGER3 in guinea pig lung tissue, and Table 8 shows the effects of Qingyan dropping pills on the protein expressions of TRPV1, BDKRB2, and PTGER3 in lung tissue.

[0120] Table 4-1 Effects of Qingyan dropping pills on SP in plasma ;

[0121]

[0122] Note: Compared with the blank group, P<0.001; compared with the model group, .

[0123] Table 4-2 Effects of Qingyan dropping pills on CGRP in plasma ;

[0124]

[0125] Note: Compared with the blank group, P < 0.001; compared with the model group, 。

[0126] Table 4-3 Effects of Qingyan Dropping Pills on BK in plasma ;

[0127]

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

[0129] Table 4-4 Effects of Qingyan Dropping Pills on NKA in plasma ;

[0130]

[0131] Note: Compared with the blank group, ##P < 0.01; compared with the model group, 。

[0132] Table 5 Effects of Qingyan Dropping Pills on SP, CGRP, BK, and NKA in lung tissue ;

[0133]

[0134] Note: Compared with the blank group, ##P < 0.01, P < 0.001; compared with the model group, 。

[0135] Table 6 Effects of Qingyan Dropping Pills on DPP Ⅳ, NEP, and APP in lung tissue ;

[0136]

[0137] Note: Compared with the blank group, #P < 0.05, P < 0.001; compared with the model group, 。

[0138] Table 7 Effects of Qingyan Dropping Pills on the gene expression of TRPV1, BDKRB2, and PTGER3 in guinea pig lung tissue ;

[0139]

[0140] Note: Compared with the blank group, P < 0.001; compared with the model group, 。

[0141] Table 8 Effects of Qingyan Drops on the expression of TRPV1, BDKRB2, and PTGER3 proteins in lung tissue ;

[0142]

[0143] Note: Compared with the blank group, #P<0.05, ##P<0.01; compared with the model group, .

[0144] 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.

[0145] (II) Evaluation of the efficacy and mechanism of Qingyan Drops in relieving ACEI-induced cough (bradykinin-induced cough)

[0146] 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.

[0147] At 1 h after the last administration on the 14th day, bradykinin (Shanghai Macklin Biochemical Co., Ltd., batch number C14596670) at a concentration of 5 mg / mL was used as the cough inducer. The guinea pigs were placed in the chamber, and timing started when the chamber was filled with gas. Nebulization was carried out in the chamber for 10 minutes, and the time of the first cough of the guinea pigs and the number of coughs within 10 minutes were recorded. Subsequently, blood was collected from the abdominal aorta, and the blood of the guinea pigs was collected and the lung tissue was dissected for biochemical index detection. RT-qPCR and WB were used to detect the expression levels of cough hypersensitivity-related proteins and genes in the lung tissue respectively. The therapeutic effect of Qingyan dropping pills was evaluated and its possible mechanism of action was preliminarily analyzed.

[0148] Determination of the contents of substance P (SP), calcitonin gene-related peptide (CGRP), bradykinin (BK) and nerve growth factor (NGF): According to the kit instructions of SP (Jianglai Biotech, batch number 062730003172620627), CGRP (Jianglai Biotech, batch number 062730003120040627), BK (Jianglai Biotech, batch number 062844003215320802) and NGF (Shanghai Enzyme-linked Biotechnology Co., Ltd., batch number Sep 2024), after the reaction ended, the wavelength was set at 450 nm, the OD value of each well was measured, and the contents of SP, CGRP, BK and NGF in the serum and lung tissue of each group were calculated according to the instructions.

[0149] Determination of the contents of phospholipase A2 (PLA2), cyclooxygenase-2 (COX-2), thromboxane A2 (TXA2) and prostaglandin E2 (PGE2): According to the kit instructions of PLA2 (Dogesce, batch number 202409), COX-2 (Jianglai Biotech, batch number 062844003507240904), PGE2 (REED BIOTECH, batch number KD071640901001) and TXA2 (REED BIOTECH, batch number KD081340906101), after the reaction ended, the wavelength was set at 450 nm, the OD value of each well was measured, and the contents of PLA2, COX-2, PGE2 and TXA2 in the lung tissue of each group were calculated according to the instructions.

[0150] RT-qPCR detection of the gene expression levels of TRPV1, BDKRB2, and PTGER3 in the lung tissue:

[0151] (1) The list of PCR primers used is as follows (all purchased from Sangon Biotech (Shanghai) Co., Ltd.):

[0152]

[0153] (2) RNA extraction:

[0154] Weigh 80 mg of lung tissue, place it in a 1.5 mL enzyme-free EP tube, add 1 mL of Trizol and 2 steel beads, and grind the tissue in a tissue homogenizer. Let it stand for 15 min.

[0155] Add 200 μL of chloroform to the lysate, shake vigorously, let it stand at room temperature for 15 min, then centrifuge at 4°C, 12000 rmp for 15 min.

[0156] Take the supernatant (400 μL) and transfer it to a new enzyme-free EP tube. Add 400 μL of isopropanol, gently shake it up and down 3 - 5 times, let it stand at room temperature for 10 min, then centrifuge at 4°C, 12000 rmp for 15 min.

[0157] Discard the supernatant, operate on ice, add 1 mL of 75% ice ethanol prepared with enzyme-free water, gently disperse the precipitate, and centrifuge at 4°C, 12000 rmp for 5 min.

[0158] Aspirate and discard the supernatant, avoiding ethanol residue, and let it stand at room temperature for about 5 min to completely dry the precipitate. Dissolve it with 90 - 150 μL of enzyme-free water according to the amount of precipitate.

[0159] Use a ultra-micro nucleic acid and protein analyzer to measure the RNA concentration.

[0160] (3)Synthesis of cDNA

[0161] Insert a 0.2 mL Ep tube into ice for incubation for 5 min, add template RNA, add RNase free ddH2O to make up to 12 μL, then add 4 μL of 4×gDNA wiper Mix, gently pipette and mix well, incubate at 42°C for 2 min, then directly add 4 μL of 5×HIScript Ⅱ qRT SuperMix Ⅱ, gently pipette and mix well, incubate at 50°C for 15 min, and then change the temperature to 85°C for incubation for 2 min. After the reaction, store the cDNA at -20°C for later use.

[0162] (4)Real-time fluorescence quantitative PCR:

[0163] Add the following reagents to the PCR 96-well plate respectively:

[0164]

[0165] After briefly centrifuging the mixed system, place it in a qPCR system and set the following amplification program:

[0166] Step 1 95°C 10 min

[0167] Step 2 95°C 10 s

[0168] Step 3 60℃ 30 s

[0169] Step 2→Step 3 Set the number of cycles to 50 and export the data after the reaction is completed.

[0170] WB detection of protein expression levels of TRPV1, BDKRB2, and PTGER3 in lung tissue:

[0171] (1) Extraction and quantification of total tissue protein

[0172] 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.

[0173] (2) Protein quantification by BCA method

[0174] 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.

[0175] (3) SDS-PAGE gel electrophoresis

[0176] Gel preparation: Prepare 10% gel according to the instructions of the SDS-PAGE gel preparation kit.

[0177] 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.

[0178] 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.

[0179] (4) Transfer

[0180] 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.

[0181] (5) Immunoblotting and protein quantification

[0182] After taking out the PVDF membrane from the membrane transfer clamp, it was placed in a rapid blocking solution for 1 h. The required protein bands were cut out and primary antibodies were added respectively, and incubated overnight at 4°C. The next day, the primary antibodies were collected and stored at -20°C, and the membrane was washed 3-5 times with TBST on a shaker. Then, a secondary antibody prepared with a rapid blocking solution was added and incubated for 1 h, and the membrane was washed again. Finally, it was exposed with an ECL luminescent solution in a chemiluminescent imager, the bands were saved, and further quantitative analysis was performed using ImageJ.

[0183] The effects of Qingyan Dropping Pills on the cough latency, the number of coughs within ten minutes, and the cough suppression rate after cough induction by bradykinin are shown in Table 9. It can be seen from Table 9 that Qingyan Dropping Pills can significantly prolong the cough latency of guinea pigs induced by ACEI, reduce the number of coughs, and increase the cough suppression rate (up to more than 60%). All three pharmacodynamic indexes are better than those of Yaojinshangzi Houpian.

[0184] Table 9 Effects of Qingyan Dropping Pills on cough latency, number of coughs within 10 min, and cough suppression rate after cough induction by bradykinin ;

[0185]

[0186] Note: Compared with the blank group, P<0.001; compared with the model group, .

[0187] In the animal experiment of cough induction by bradykinin, the effects of Qingyan Dropping Pills on the protein expressions of TRPV1, BDKRB2, and PTGER3 in lung tissue are as Figure 7 shown. 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 expressions of TRPV1, BDKRB2, and PTGER3 in lung tissue, and Table 14 shows the effects of Qingyan Dropping Pills on the protein expressions of TRPV1, BDKRB2, and PTGER3 in lung tissue.

[0188] Table 10 Effects of Qingyan Dropping Pills on SP, CGRP, NGF, and BK in plasma ;

[0189]

[0190] Note: Compared with the blank group, #P<0.05, P<0.001; compared with the model group, .

[0191] Table 11 Effects of Qingyan Dropping Pills on SP, CGRP, NGF, and BK in lung tissue ;

[0192]

[0193] Note: Compared with the blank group, ##P < 0.01, P < 0.001; compared with the model group, 。

[0194] Table 12 Effects of Qingyan Dropping Pills on PLA2, PGE2, TXA2, and COX-2 in lung tissue ;

[0195]

[0196] Note: Compared with the blank group, #P < 0.05, ##P < 0.01, P < 0.001; compared with the model group, 。

[0197] Table 13 Effects of Qingyan Dropping Pills on the gene expression of TRPV1, BDKRB2, and PTGER3 in lung tissue ;

[0198]

[0199] Note: Compared with the blank group, P < 0.001; compared with the model group, 。

[0200] Table 14 Effects of Qingyan Dropping Pills on the protein expression of TRPV1, BDKRB2, and PTGER3 in lung tissue ;

[0201]

[0202] Note: Compared with the blank group, #P < 0.05; compared with the model group, 。

[0203] From Figure 7 , it can be seen from Tables 10 to 14 that the possible mechanisms by which Qingyan Dropping Pills relieve cough caused by ACEI are as follows: by inhibiting the expression of key enzymes PLA2 and COX-2 in the AA metabolic pathway, reducing the levels of their pro-inflammatory and cough-inducing metabolites PGE2 and TXA2; by reducing the release of sensory neuropeptides such as SP, CGRP, and NGF, reducing the neurogenic inflammatory response; by inhibiting the gene and protein expression of PGE2 receptor PTGER3 and BK receptor BDKRB2, and inhibiting the phosphorylation level of cough-conducting TRPV1, thereby playing a role in improving cough caused by ACEI.

[0204] In summary, network pharmacology analysis indicates that Qingyan dropping 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 hyperreactivity. At the same time, Qingyan dropping pills can relieve the pharyngeal discomfort symptoms that may accompany cough, such as dry throat, itchy throat, and sore throat. In vitro verification experiments show that Qingyan dropping pills may relieve ACEI-induced cough by regulating arachidonic acid metabolism and reducing cough hypersensitivity. In vivo pharmacodynamic evaluation experiments show that Qingyan dropping 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 dropping pills in relieving ACEI-induced cough. In short, Qingyan dropping pills can relieve ACEI-induced cough and its accompanying pharyngeal discomfort symptoms, and thus can be used to prepare drugs for preventing and / or treating ACEI-induced cough and its accompanying pharyngeal discomfort symptoms. In addition, this application has successfully established a guinea pig cough induction experimental model with an ACEI compound cough inducer for the first time in in vivo pharmacodynamic evaluation and mechanism of action research.

[0205] The above are only the preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of this application shall be included within the scope of protection of this application.

Claims

1. Use of Qingyan Dropping Pills in the preparation of a medicament for preventing and / or treating cough caused by ACEI and its accompanying pharyngeal discomfort symptoms; wherein, The Qingyan dropping pills are prepared from the following traditional Chinese medicine raw materials: 80 - 120 parts of indigo naturalis, 80 - 120 parts of licorice root, 60 - 100 parts of chebula fruit, 60 - 100 parts of menthol, 5 - 30 parts of borneol, and 5 - 30 parts of artificial bezoar.

2. The use according to claim 1, wherein, The described 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 dropping pills 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 hyperreactivity.

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 ACEI includes captopril.

7. The use according to claim 1, wherein The Qingyan dropping pills are prepared from the following traditional Chinese medicine raw materials: 90 - 110 parts of indigo naturalis, 90 - 110 parts of licorice root, 70 - 90 parts of chebula fruit, 70 - 90 parts of menthol, 15 - 25 parts of borneol, and 10 - 15 parts of artificial bezoar.

8. According to the use described in claim 1, wherein The Qingyan dropping pills are prepared from the following traditional Chinese medicine raw materials: 100 parts of indigo naturalis, 100 parts of licorice root, 80 parts of chebula fruit, 80 parts of menthol, 20 parts of borneol, and 12 parts of artificial bezoar.

9. Use of a pharmaceutical composition in the preparation of a medicament for preventing and / or treating cough caused by ACEI and its accompanying pharyngeal discomfort symptoms; wherein, The pharmaceutical composition is prepared from the following traditional Chinese medicine raw materials: 80 - 120 parts of indigo naturalis, 80 - 120 parts of licorice root, 60 - 100 parts of chebula fruit, 60 - 100 parts of menthol, 5 - 30 parts of borneol, and 5 - 30 parts of artificial bezoar.

10. The use according to claim 9, wherein, The pharmaceutical composition is prepared from the following traditional Chinese medicine raw materials: 90 - 110 parts of indigo naturalis, 90 - 110 parts of licorice root, 70 - 90 parts of chebula fruit, 70 - 90 parts of menthol, 15 - 25 parts of borneol, and 10 - 15 parts of artificial bezoar.

11. The use according to claim 9, wherein, The pharmaceutical composition is prepared from the following traditional Chinese medicine raw materials: 100 parts of indigo naturalis, 100 parts of licorice root, 80 parts of chebula fruit, 80 parts of menthol, 20 parts of borneol, and 12 parts of artificial bezoar.

12. The use according to claim 9, wherein, The described pharyngeal discomfort symptoms include at least one of dry throat, itchy throat, and sore throat.

13. The use according to claim 9, wherein, The pharmaceutical composition further includes a pharmaceutically acceptable carrier or excipient.

14. The use according to claim 13, wherein The pharmaceutically acceptable carrier or excipient includes at least one of a solvent, diluent, dispersant, suspending agent, surfactant, isotonic agent, thickening agent, preservative, binder, lubricant, stabilizer, hydrating agent, emulsification accelerator, buffer, absorbent, coloring agent, ion exchanger, demolding agent, coating agent, flavoring agent, and antioxidant.

15. Use according to any one of claims 9 to 14, wherein, The pharmaceutical composition is formulated into any one of the dosage forms of powder, tablet, capsule, pill, emulsion, suspension, or tincture.

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