A method for discovering and the quality marker of a cough-relieving and expectorant granule.

Abstract

This invention relates to the field of traditional Chinese medicine research technology, specifically a method for discovering and identifying quality markers for the antitussive and expectorant effects of Ke Ning granules. This application evaluates the antitussive and expectorant effects of Ke Ning granules using concentrated ammonia-induced cough and phenol red excretion assays. Plasma pharmacochemistry studies of Ke Ning granules were conducted using UHPLC-Q-Exactive Plus Orbitrap HRMS technology, identifying 26 prototype blood-entering components. Through network pharmacological analysis of these blood-entering components, 11 potential active ingredients for antitussive and expectorant effects of Ke Ning granules were screened, involving 40 common targets including IL6, TLR4, and STAT3, and mainly participating in signaling pathways such as PI3K-Akt, HIF-1, and EGFR. Seven prototype blood-entering components were quantitatively analyzed using pharmacokinetic methods. Azelaic acid, caffeic acid, and vanillin were selected as quality markers for the antitussive and expectorant effects of Ke Ning granules based on efficacy, transmissibility, and measurability. The results of this study are of great significance for further clarifying the pharmacodynamic material basis of Ke Ning granules, optimizing quality standards, and promoting its clinical application.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine research technology, specifically to a method for discovering and a quality marker for cough-relieving and expectorant granules. Background Technology

[0002] Ke Ning Granules are a major traditional Chinese medicine product of Guizhou Dalong Pharmaceutical Co., Ltd. Composed of pine cones, loquat leaves, and cotton root, this pure traditional Chinese medicine OTC preparation contains no centrally acting antitussives and has the effects of relieving cough and phlegm, calming asthma, and strengthening the body's resistance. It is listed in the National Drug Standard [WS3-B-1581-93]. It is a classic formula combining warming and cooling properties, and has been used clinically for many years for coughs that worsen with cold, cough with wheezing and chest tightness, as well as coughs from colds and chronic bronchitis, with significant efficacy. In folk medicine, there is the Dong medicine "Lingzhi Pine Cone Cotton Root Soup," which has the effects of invigorating qi and blood, relieving cough and asthma, and is used to treat chronic bronchitis with a clinical efficacy rate as high as 96.7%. Using pine cone decoction to treat chronic bronchitis has been found to have the effects of tonifying the kidneys and strengthening yang, strengthening the body's resistance and eliminating pathogens, expelling cold and dampness from the lungs, and improving the body's immune function. Cotton roots have excellent expectorant, antitussive, and qi-tonifying effects, while loquat leaves have the effects of clearing the lungs and resolving phlegm, relieving cough and asthma, and harmonizing the stomach and relieving nausea. Both have a long history of use in treating coughs. The above research indicates that Ke Ning granules have a promising market prospect and development potential in the treatment of cough and phlegm.

[0003] In November 2020, the Center for Drug Evaluation of the National Medical Products Administration issued the "Technical Guidelines for Research on Quality Standards of New Traditional Chinese Medicines (Trial Implementation)," which emphasized that quality standards should focus on the medicinal material basis as an important research content, centering on the safety and efficacy of drugs, and combining the transmission and traceability of the quantity and quality of traditional Chinese medicines to comprehensively control the quality of medicinal materials and ensure the stability of preparation quality. The guidelines also pointed out that the selection of quality control methods for traditional Chinese medicines should be tailored to the specific medicine, encouraging the integration of multiple methods, and establishing multiple testing and detection items in the quality standards of compound traditional Chinese medicine preparations. Currently, the pharmacodynamic material basis of Ke Ning granules is unclear, and the existing quality standards only include thin-layer chromatography identification of pine cones and cotton root, lacking effective quality control standards and making it difficult to ensure the stability of preparation quality.

[0004] Since Academician Liu Changxiao proposed the new concept of Q-Markers for Traditional Chinese Medicine (TCM), he has elaborated on the research methods of Q-Markers from five aspects: efficacy, specificity, translocation and traceability, measurability, and compatibility environment. These methods have been widely applied in the quality control research of TCM materials and their preparations. Based on the theoretical premise that drug components are absorbed into the bloodstream to exert their efficacy, plasma pharmacochemistry research methods are used to systematically analyze the translocated components from medicinal materials to preparations and then to the blood, thus clarifying the "translocation and traceability" of quality. Network pharmacology research methods are employed to construct a "TCM-blood-disease-target" network based on translocated components, identifying disease-related chemical components and screening for candidate Q-Markers with "efficacy." Only components that enter the body and reach effective concentrations are more likely to be key Q-Markers. Pharmacokinetic studies can clarify the dynamic changes in the absorption degree, concentration changes, and residence time of efficacy-related components in the body. Using pharmacokinetic markers with "measurability" as candidate Q-Markers has certain guiding significance. Based on this, and building upon the previous chemical composition analysis and attribution of Ke Ning granules, this study will conduct ammonia-induced cough and phenol red excretion experiments to verify its antitussive and expectorant effects. Furthermore, by combining plasma pharmacology, network pharmacology, and pharmacokinetic results, the quality markers for the antitussive and expectorant effects of Ke Ning granules will be determined, providing a basis for further exploration of the pharmacodynamic material basis and quality control of Ke Ning granules. Summary of the Invention

[0005] To address the aforementioned technical problems in the prior art, this invention provides a method for discovering a quality marker for cough-relieving and expectorant granules, and the quality marker itself, comprising the following:

[0006] A method for discovering a quality marker for cough-relieving and expectorant granules includes the following steps:

[0007] (1) Analyze and identify the original blood-entering components of Ke Ning granules;

[0008] (2) Using the original blood-entry components as the "drug component set", the potential active ingredients and common targets of Ke Ning Granules for relieving cough and expectoration were screened out by network pharmacology.

[0009] (3) Based on the analysis of blood-entering components, pharmacokinetic quantitative analysis of the in vivo processes of the original blood-entering components is used;

[0010] (4) The quality markers for cough relief and expectoration of Ke Ning Granules were finally determined through effectiveness, transmissibility and measurability.

[0011] Furthermore, the Ke Ning granules are composed of three medicinal materials: pine cone, loquat leaf, and cotton root. Ke Ning granules are a major traditional Chinese medicine product of Guizhou Dalong Pharmaceutical Co., Ltd. It is a pure traditional Chinese medicine OTC preparation composed of pine cone, loquat leaf, and cotton root, and does not contain centrally acting antitussive drugs. It has the effects of relieving cough and phlegm, relieving asthma, and strengthening the body's resistance. It is listed in the National Drug Standard [WS3-B-1581-93].

[0012] Furthermore, the step (1) of analyzing and identifying the prototype blood-entering components of Ke Ning granules is performed using the UHPLC-Q-Exactive Plus Orbitrap HRMS method.

[0013] The specific chromatographic conditions are as follows:

[0014] Chromatographic column: CORTECS UPLC T3 (2.1 mm × 100 mm, 1.6 μm), column temperature: 40 ℃, flow rate: 0.3 mL / min, mobile phase: 0.1% formic acid aqueous solution (A) - acetonitrile methanol (2:1) solution (B), injection volume: 2 μL, gradient elution program: 0–3 min, 1% → 2% B; 3–5 min, 2% → 5% B; 5–10 min, 5% → 30% B; 10–15 min, 30% → 60% B; 15–20 min, 60% → 70% B; 20–22 min, 70% → 95% B; 22–24 min, 95% B; 24–25 min, 95% → 1% B; 25–28 min, 1% B;

[0015] The specific mass spectrometry conditions are as follows:

[0016] The ion source was electrospray ionization (ESI) with a spray voltage of 3500V and an ion lens voltage frequency of 50.0. The sheath gas pressure was 35arb, the auxiliary gas pressure was 10arb, the nebulization temperature was 350℃, the capillary temperature was 320℃, the probe heater temperature was 350℃, and the scanning mode was a full scan + data-dependent secondary scan (ddMS2) positive and negative ion exchange mode with a scanning range of m / z 80~1200, a primary resolution of 70000, and a secondary resolution of 17500.

[0017] Furthermore, in the UHPLC-Q-Exactive Plus Orbitrap HRMS method, the plasma to be tested is processed as follows: 500 μL of plasma is taken, 100 μL of 1% formic acid aqueous solution is added, vortexed for 1 min, 2 mL of methanol is added to precipitate proteins, vortexed for 5 min, sonicated for 10 min, and centrifuged at 8000 r / min at 4℃ for 10 min in a refrigerated high-speed centrifuge. The supernatant is placed in a centrifuge tube and dried with a nitrogen blower at 37℃. The residue is reconstituted with 200 μL of 50% methanol, vortexed for 2 min, sonicated for 10 min, and centrifuged at 12000 r / min at 4℃ for 10 min. The supernatant is collected as the sample solution and analyzed by the UHPLC-Q-Exactive Plus Orbitrap HRMS instrument.

[0018] Furthermore, step (2) specifically includes the following steps:

[0019] ① Screening of potential active ingredients and collection of targets for Ke Ning granules:

[0020] The blood-entering components were imported into the SwissADME database to screen for potential active ingredients. Then, the Swiss TargetPrediction database was used to limit the species to "Homo sapiens" to predict the target of potential active ingredients and remove invalid and duplicate targets.

[0021] ② Collection of targets related to cough suppression and expectoration:

[0022] By searching the GeneCards database using the keywords “Cough” and “Sputum” respectively, disease-related targets were obtained. After removing duplicate genes and genes that do not have Uniprot ID, cough-relieving and expectorant-related targets were obtained.

[0023] ③ Protein-protein interaction network analysis and screening of core targets:

[0024] The Venny online software was used to find the intersection of potential drug active ingredient targets and disease targets and draw a Venn diagram to obtain the common targets of potential active ingredients for treating cough and phlegm. The intersection targets were imported into the String database to obtain the interaction relationships between target proteins. The PPI network diagram was drawn and the topology analysis was performed using the Analyze network tool and CytoNCA plugin in Cytoscape 3.10.0 software to obtain the degree, betweenness, and closeness topology parameters of each node. The top 20 targets were calculated for each node. The intersection targets obtained by the three algorithms are the core targets.

[0025] ④ Construction and analysis of the "drug-component-disease-common target" network:

[0026] After processing the gene data of potential active ingredients, diseases, and common targets of drugs, network and type files are generated, and then imported into Cytoscape 3.10.0 software to draw a network diagram of "drug-ingredient-disease-common target".

[0027] ⑤ Enrichment analysis:

[0028] Intersecting targets were imported into the DAVID database (https: / / david.ncifcrf.gov / ) and enriched in the gene ontology database "Gene ontology, abbr.GO" and the Kyoto Encyclopedia of Genes and Genomes "Kyoto Encyclopedia of Genes and Genomes, abbr.KEGG". Bubble charts and rectangle charts were then created using the online graphing platform MicroBio, and biological interpretations were performed.

[0029] Furthermore, in step (3), the in vivo process of quantitative analysis of the parent compound entering the bloodstream is carried out using pharmacokinetic methods, under the following specific conditions:

[0030] Chromatographic conditions:

[0031] Column: ACQUITY BEH C18 (2.1×50mm, 1.7μm); Guard column: Waters Van Guard BEH C18 (2.1×5mm, 1.7μm); Mobile phase: A: 0.2% formic acid aqueous solution, B: 0.2% formic acid acetonitrile solution; Flow rate: 0.3mL / min; Column temperature: 40℃; Injection volume: 1μL; Gradient elution conditions as follows:

[0032] Table 1 Gradient Elution of Mobile Phase

[0033]

[0034] Mass spectrometry conditions:

[0035] Electrospray ionization (ESI) source, capillary voltage 3kV, ion source temperature 120℃, desolvation gas (nitrogen) 1000L / H, desolvation gas temperature 600℃, collision gas (argon) 0.15mL / min; multiple reaction monitoring (MRM) mode for simultaneous monitoring of positive and negative ions, the monitored ions for each component are as follows:

[0036] Table 2 Multiple Reaction Ion Monitoring (MRM) Mass Spectrometry Conditions

[0037]

[0038] Further, in step (3), the sample to be tested is prepared by the following method: 200 μL of plasma is placed in a 1.5 mL plastic centrifuge tube, 40 μL of 40 ng / mL puerarin (internal standard) solution is added sequentially, followed by 20 μL of 1% formic acid water and 800 μL of acetonitrile. The mixture is vortexed for 3 min, sonicated for 10 min, and centrifuged at 12000 r / min for 10 min at 4 °C. The supernatant is separated and dried under nitrogen at 37 °C. The residue is dissolved in 200 μL of 50% acetonitrile aqueous solution, vortexed for 3 min, sonicated for 10 min, and centrifuged at 14000 r / min for 10 min at 4 °C. The supernatant is then analyzed using an ACQUITY UPLC I-Class / Xevo TQ-S ultra-high performance liquid chromatography triple quadrupole system.

[0039] A quality marker for cough-relieving and expectorant granules is one or more of caffeic acid, azelaic acid, and vanillin.

[0040] A method for detecting multiple components in plasma, using UHPLC-Q-Exactive Plus Orbitrap HRMS to analyze and identify the following components in plasma: nicotinamide, DL-leucine, adenine, adenosine, 2,5-dihydroxybenzoic acid, maltol, 8-hydroxyquinoline, homovanillic acid, caffeic acid, riboflavin, vanillin, 4-hydroxycinnamic acid, benzodihydrofuran-2-carboxylic acid, scopolamine, 4-indolecarboxaldehyde, azelaic acid, 3-tert-butyladipic acid, andrographolide, caffeoyl alcohol, 6-gingerol.

[0041] 2-Hydroxy-4,5',8a'-trimethyl-1'-oxo-4-vinyloctahydro-1'H-spiro[cyclopentane-1,2'-na phthalene]-5'-carboxylic acid, ethyl p-ethoxybenzoate, 3,5-di-tert-butyl-4-hydroxybenzaldehyde, 1,4a-dimethyl-9-oxo-7-(propan-2-yl)-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxylic acid, caffeoyl alcohol, α-linolenic acid;

[0042] The specific chromatographic conditions are as follows:

[0043] Chromatographic column: CORTECS UPLC T3 (2.1 mm × 100 mm, 1.6 μm), column temperature: 40 ℃, flow rate: 0.3 mL / min, mobile phase: 0.1% formic acid aqueous solution (A) - acetonitrile methanol (2:1) solution (B), injection volume: 2 μL, gradient elution program: 0–3 min, 1% → 2% B; 3–5 min, 2% → 5% B; 5–10 min, 5% → 30% B; 10–15 min, 30% → 60% B; 15–20 min, 60% → 70% B; 20–22 min, 70% → 95% B; 22–24 min, 95% B; 24–25 min, 95% → 1% B; 25–28 min, 1% B;

[0044] The specific mass spectrometry conditions are as follows:

[0045] The ion source was electrospray ionization (ESI) with a spray voltage of 3500V and an ion lens voltage frequency of 50.0. The sheath gas pressure was 35arb, the auxiliary gas pressure was 10arb, the nebulization temperature was 350℃, the capillary temperature was 320℃, the probe heater temperature was 350℃, and the scanning mode was a full scan + data-dependent secondary scan (ddMS2) positive and negative ion exchange mode with a scanning range of m / z 80~1200, a primary resolution of 70000, and a secondary resolution of 17500.

[0046] A method for detecting multiple components in plasma, simultaneously determining the content of azelaic acid, nicotinamide, vanillin, caffeic acid, 2,5-dihydroxybenzoic acid, α-linolenic acid and riboflavin in plasma;

[0047] The specific conditions are as follows:

[0048] Chromatographic conditions:

[0049] Column: ACQUITY BEH C18 (2.1×50mm, 1.7μm); Guard column: Waters Van Guard BEH C18 (2.1×5mm, 1.7μm); Mobile phase: A: 0.2% formic acid aqueous solution, B: 0.2% formic acid acetonitrile solution; Flow rate: 0.3mL / min; Column temperature: 40℃; Injection volume: 1μL; Gradient elution conditions as follows:

[0050] Table 1 Gradient Elution of Mobile Phase

[0051]

[0052] Mass spectrometry conditions:

[0053] Electrospray ionization (ESI) source, capillary voltage 3kV, ion source temperature 120℃, desolvation gas (nitrogen) 1000L / H, desolvation gas temperature 600℃, collision gas (argon) 0.15mL / min; multiple reaction monitoring (MRM) mode for simultaneous monitoring of positive and negative ions, the monitored ions for each component are as follows:

[0054] Table 2 Multiple Reaction Ion Monitoring (MRM) Mass Spectrometry Conditions

[0055]

[0056] Compared with the prior art, the technical effects of this invention are reflected in:

[0057] (1) This application evaluates the antitussive and expectorant effects of Ke Ning granules by cough induction with concentrated ammonia and phenol red excretion test, and identifies 26 original blood-entry components of Ke Ning granules by UHPLC-Q-Exactive Plus Orbitrap HRMS method.

[0058] (2) This application uses network pharmacology to screen 11 potential active ingredients of Ke Ning Granules for cough relief and expectoration. They exert their cough relief and expectoration effects mainly by acting on 40 common targets, including IL6, TLR4, and STAT3, and participating in the regulation of signaling pathways such as PI3K-Akt, HIF-1, and EGFR.

[0059] (3) This application used pharmacokinetic quantitative analysis to study the in vivo processes of seven prototype components entering the bloodstream. Azelaic acid, caffeic acid, and vanillin were selected as quality markers for the antitussive and expectorant effects of Ke Ning granules based on efficacy, transmissibility, and measurability. The results of this study are of great significance for further clarifying the pharmacodynamic material basis of Ke Ning granules, optimizing quality standards, and promoting its clinical application. Attached Figure Description

[0060] Figure 1 This study investigated the effect of Ke Ning granules on cough in mice. Note: A: Comparison of cough latency in each group of mice; B: Comparison of the number of coughs within 5 minutes in each group of mice; M: Model group; DEX: Dextromethorphan hydrobromide group; KNKL-L, KNKL-M, and KNKL-H were the low, medium, and high concentrations of Ke Ning granules, respectively. Compared with the model group, # P<0.05, ## P<0.01, ### P<0.001.

[0061] Figure 2This represents the amount of phenol red excreted from the trachea of ​​mice in each group. Note: NC: normal control group; AMB: ambroxol hydrochloride group; KNKL-L, KNKL-M, and KNKL-H are the low, medium, and high dose groups of Ke Ning granules, respectively. Compared with the control group, *P<0.05, **P<0.01.

[0062] Figure 3 This is a chromatogram of plasma containing Ke Ning granules and blank plasma. Note: A. Blank plasma - ESI+; B. Blank plasma - ESI-; C. Plasma containing granules - ESI+; D. Plasma containing granules - ESI-.

[0063] Figure 4 It is a Venn diagram of the intersection of the active ingredients of Ke Ning granules with cough and phlegm genes.

[0064] Figure 5 It is a protein-protein interaction network.

[0065] Figure 6 This is a visualization of the active ingredients of Ke Ning granules and the intersection of cough and target points.

[0066] Figure 7 This is a network diagram of "drug-ingredient-common target-disease" for Ke Ning granules.

[0067] Figure 8 This is a bubble chart of GO biological function enrichment analysis.

[0068] Figure 9 This is a rectangle plot of GO biological function enrichment analysis.

[0069] Figure 10 This is a bubble chart of KEGG pathway enrichment.

[0070] Figure 11 These are UPLC-MS / MS chromatograms of each component.

[0071] Figure 12 This is the average concentration-time curve of seven compounds in rat plasma after oral administration of Ke Ning granules. Detailed Implementation

[0072] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description made.

[0073] Example:

[0074] A Study on Quality Markers for Cough-Relieving and Expectorant Properties of Ke Ning Granules Based on Plasma Pharmacochemical Binding Network Pharmacology and Pharmacokinetics

[0075] 1. Materials

[0076] 1.1 Animals

[0077] Kunming mice (20±2g) were purchased from Changsha Tianqin Biotechnology Co., Ltd., with the production license number SCXK(Xiang)2022-0011; SD rats (250±20g) were purchased from Changsha Tianqin Biotechnology Co., Ltd., with the production license number SCXK(Xiang)2019-0014. Both were SPF-grade animals. They were placed in a light / dark cycle environment at 20-24°C and a relative humidity of 50-70% and raised for one week before being used in experimental studies. During this period, they had free access to water and food. All experimental studies were approved by the Animal Ethics Committee of Guizhou Medical University.

[0078] 1.2 Reagents and Instruments

[0079] Phenol red (Tianjin Kemiou Chemical Reagent Co., Ltd., batch number: HG / T4100-2009); Sodium hydroxide (Chengdu Jinshan Chemical Reagent Co., Ltd., batch number: 20230102); Sodium bicarbonate (Shanghai Aladdin Biochemical Technology Co., Ltd., batch number: C2316287); Ambroxol hydrochloride (Shandong Yuxin Pharmaceutical Co., Ltd., batch number: 622122014); Kening granules (Guizhou Dalong Pharmaceutical Co., Ltd., batch number: 20221102); Ammonia water (National Pharmaceutical Group Chemical Reagent Co., Ltd., batch number: 20230329); Dextromethorphan hydrobromide oral solution (Zhongshan Branch of Zhuhai Federal Pharmaceutical Co., Ltd., batch number: 31044111); Heparin sodium (Beijing Solarbio Science & Technology Co., Ltd., batch number: NO.1031R021); Puerarin (National Institutes for Food and Drug Control, batch number: 110752-202217); Methanol, formic acid, acetonitrile (Merck KGaA, Germany, chromatographic grade); α-Linolenic acid, azelaic acid, caffeic acid (Sichuan维克奇Biotechnology Co., Ltd., batch number: wkq23021006, HPLC≥98%; wkq22091403, HPLC≥98%; wkq22101809, HPLC≥98%); Vanillin, riboflavin, 2,5-dihydroxybenzoic acid (TANMO Quality Inspection, batch number: 22021152, 99.9%; 230503093, 99.1%; 23110122, 99.9%); Nicotinamide (Shanghai Yuanye Bio-Technology Co., Ltd., batch number: KJ0625BA14, HPLC≥99.5%).

[0080] UHPLC-Q-Exactive Plus Orbitrap HRMS (Thermo Fisher Scientific); ACQUITY UPLC I-Class / Xevo TQ-S ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer including a binary gradient pump, autosampler, column oven, triple quadrupole mass analyzer, and Masslynx 4.1 mass spectrometer workstation (Waters Corporation, USA); EL204 0.001 g electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); KQ500DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); THZ-82 water bath constant temperature shaker; Allegra X-30R low temperature high speed centrifuge (Beckman Coulter, USA); Model 680 microplate reader (Bio-Ray Biomedical Products Co., Ltd.); YLS-8A multi-functional cough and asthma induction device (Jinan Yiyan Technology Development Co., Ltd.); nitrogen blowing device (Beijing Zhongxing Huili Technology Development Co., Ltd.).

[0081] 1.3 Databases and Software

[0082] Swiss ADME (http: / / www.swissadme.ch), Swiss Target Prediction(http: / / swisstargetprediction.ch / ), GeneCards(https: / / www.genecards.org / ), Venny2.1.0(htp: / / ioinfogp.cnb.csic.es / tools / venny / ), String(https: / / string-db.org / ), Metascape(https: / / www.meta scape.org / gp / index.html), Cytoscape3.10.0 software, DAVID (0https: / / david.ncifcrf.gov / ), Uniprot (https: / / ww.uniprot.org / ), Pubchem (https: / / pubchem.ncbi.nlm.nih.gov / ), Weisheng Xinping (http: / / www.bioinformatics.com.cn).

[0083] 2 methods

[0084] 2.1 Ammonia-induced coughing test in mice

[0085] 2.1.1 Modeling and Drug Administration

[0086] Thirty-six mice, half male and half female, were randomly divided into six groups: normal control group (NC), model group (M), dextromethorphan hydrobromide group (clinically equivalent dose 18.2 mL / kg, DEX), and low (clinically equivalent dose 22.75 g / kg, KNKL-L), medium (45.5 g / kg, KNKL-M), and high (91 g / kg, KNKL-H) dose groups of Ke Ning granules, with six mice in each group. Mice were fasted for 12 hours before administration but allowed free access to water. The normal control group and model group received the same volume of physiological saline, while the other groups received the medication by gavage at 20 mL / (kg·bw) twice daily for 7 consecutive days. One hour after the last administration, the mice were placed in a YLS-8A cough and asthma induction device. The normal control group received 0.9% physiological saline via nebulization, while the other groups received 28% ammonia via nebulization (5 mL of fresh ammonia was replaced before each animal was placed in the device) for 45 seconds.

[0087] 2.1.2 Detection Indicators

[0088] Record the cough latency (s) and the number of coughs within 5 minutes in mice (defined as severe abdominal muscle contraction, shoulder and back shrugging accompanied by mouth opening and coughing sounds), and calculate the cough suppression rate.

[0089] 2.2 Phenol Red Excretion Experiment in Mice

[0090] 2.2.1 Modeling and Drug Administration

[0091] Thirty mice, half male and half female, were randomly divided into five groups: normal control group (NC), ambroxol hydrochloride group (163.8 mg / kg, AMB), and low (clinically equivalent dose 22.75 g / kg, KNKL-L), medium (45.5 g / kg, KNKL-M), and high (91 g / kg, KNKL-H) dose groups of Ke Ning granules, with six mice in each group. The blank control group was given an equal volume of physiological saline, while the mice in the other groups were administered the drug by gavage twice a day for 7 consecutive days. 30 minutes after the last administration, 5% phenol red solution was injected intraperitoneally at a dose of 10 mL / (kg·bw). 30 minutes later, the mice were euthanized by cervical dislocation, fixed in a supine position on a surgical board, the neck skin was cut open, the trachea was separated, and the tracheal segment from the thyroid cartilage to the tracheal bifurcation was taken. The tracheal segment was placed in 2 mL of physiological saline containing 0.1 mL of 1 mol / L sodium hydroxide (NaOH), shaken well, sonicated for 5 min, shaken for 15 min, and centrifuged at 4000 r / min for 5 min at 4℃. The supernatant was transferred to a centrifuge tube for later use.

[0092] 2.2.2 Detection Indicators

[0093] 200 μL of plasma supernatant was placed in a 96-well plate, and the OD value was measured at 546 nm using a microplate reader. The results were then analyzed based on the return...

[0094] The phenol red content was calculated using a regression equation. The standard phenol red curve was constructed as follows: 0.01 g of phenol red was accurately weighed using an analytical balance and dissolved in 5% sodium bicarbonate (NaHCO3) to prepare a 100 μg / mL phenol red solution. This solution was then serially diluted to 0.1, 0.3, 0.5, 0.7, 1, 3, 5, and 10 μg / mL phenol red solutions. The absorbance was measured at 546 nm using a microplate reader. A standard curve was plotted with phenol red concentration on the x-axis and OD value on the y-axis. The amount of phenol red excreted was calculated based on the regression equation.

[0095] 2.3 Statistical Analysis

[0096] Quantitative data are expressed as mean ± standard deviation. One-way ANOVA was performed using SPSS 23 statistical software. If the variances were homogeneous, LSD multiple comparisons were used for comparisons between groups; if the variances were unequal, Dunnett's T3 multiple comparisons were used for comparisons between groups. P < 0.05 was considered statistically significant.

[0097] 2.4 Blood Component Analysis of Ke Ning Granules

[0098] 2.4.1 Chromatographic conditions

[0099] Chromatographic column: CORTECS UPLC T3 (2.1 mm × 100 mm, 1.6 μm), column temperature: 40 ℃, flow rate: 0.3 mL / min, mobile phase: 0.1% formic acid aqueous solution (A) - acetonitrile methanol (2:1) solution (B), injection volume: 2 μL, gradient elution program: 0–3 min, 1% → 2% B; 3–5 min, 2% → 5% B; 5–10 min, 5% → 30% B; 10–15 min, 30% → 60% B; 15–20 min, 60% → 70% B; 20–22 min, 70% → 95% B; 22–24 min, 95% B; 24–25 min, 95% → 1% B; 25–28 min, 1% B.

[0100] 2.4.2 Mass Spectrometry Conditions

[0101] The ion source was electrospray ionization (ESI) with a spray voltage of 3500V and an ion lens voltage frequency of 50.0. The sheath gas pressure was 35arb, the auxiliary gas pressure was 10arb, the nebulization temperature was 350℃, the capillary temperature was 320℃, the probe heater temperature was 350℃, and the scanning mode was a full scan + data-dependent secondary scan (ddMS2) positive and negative ion exchange mode with a scanning range of m / z 80~1200, a primary resolution of 70000, and a secondary resolution of 17500.

[0102] 2.4.3 Collection and Processing of Biological Samples

[0103] Fourteen mice were randomly divided into a blank control group (n=2) and a cough-relieving granule administration group (n=12, clinically equivalent dose 91 g / kg). The cough-relieving granule administration group was further divided into groups at 15 min, 30 min, 45 min, 60 min, 90 min, and 120 min after administration, with two mice in each group. Mice were fasted for 12 hours before the experiment but had free access to water. Cough-relieving granules were administered by gavage twice daily for 7 consecutive days. The blank control group was administered physiological saline by gavage. After the last administration, blood samples were collected from the eyeballs at 15, 30, 45, 60, 90, and 120 min after administration. The samples were placed in 1.5 mL centrifuge tubes coated with 1% heparin sodium and centrifuged at 4000 r / min and 4℃ for 10 min. The supernatant was collected to obtain blank plasma and drug-containing plasma. The drug-containing plasma from each time point was mixed and stored at -20℃ for later use.

[0104] Take 500 μL of blank plasma and 500 μL of drug-containing plasma, add 100 μL of 1% formic acid aqueous solution, vortex for 1 min, add 2 mL of methanol to precipitate proteins, vortex for 5 min, sonicate for 10 min, centrifuge at 8000 r / min at 4℃ for 10 min in a refrigerated high-speed centrifuge, collect the supernatant and place it in a centrifuge tube, blow dry with nitrogen at 37℃, obtain the residue, add 200 μL of 50% methanol to reconstitute, vortex for 2 min, sonicate for 10 min, centrifuge at 12000 r / min at 4℃ for 10 min, collect the supernatant as the sample solution, and analyze it in a UHPLC-Q-Exactive Plus Orbitrap HRMS instrument.

[0105] 2.5 Network Pharmacological Analysis

[0106] 2.5.1 Screening of potential active ingredients and collection of targets for Ke Ning granules

[0107] The components entering the bloodstream are imported into the SwissADME database to screen for potential active ingredients. Then, the Swiss TargetPrediction database is used to limit the species to "Homo sapiens" to predict the target of potential active ingredients and remove invalid and duplicate targets.

[0108] 2.5.2 Collection of targets related to cough suppression and expectoration

[0109] By searching the GeneCards database using the keywords “Cough” and “Sputum”, disease-related targets were obtained. After removing duplicate genes and genes that do not have Uniprot ID, cough-relieving and expectorant-related targets were identified.

[0110] 2.5.3 Protein-protein interaction network analysis and screening of core targets

[0111] The Venny online software was used to find the intersection of potential drug active ingredient targets and disease targets and draw a Venn diagram to obtain the common targets of potential active ingredients for treating cough and phlegm. The intersection targets were imported into the String database to obtain the interaction relationships between target proteins. The PPI network diagram was drawn and topological analysis was performed using the Analyze network tool and CytoNCA plugin in Cytoscape 3.10.0 software to obtain the degree, betweenness, and closeness topological parameters of each node. The top 20 targets were calculated for each node. The intersection targets obtained by the three algorithms are the core targets.

[0112] 2.5.4 Construction and Analysis of the "Drug-Component-Disease-Common Target" Network

[0113] After processing the gene data of potential active ingredients, diseases, and common targets of drugs, network and type files are generated, and then imported into Cytoscape 3.10.0 software to draw a network diagram of "drug-ingredient-disease-common target".

[0114] 2.5.5 Enrichment Analysis

[0115] Intersecting targets were imported into the DAVID database (https: / / david.ncifcrf.gov / ) and enriched in the gene ontology database "Gene ontology, abbr.GO" and the Kyoto Encyclopedia of Genes and Genomes "Kyoto Encyclopedia of Genes and Genomes, abbr.KEGG". Bubble charts and rectangle charts were then created using the online graphing platform MicroBio, and biological interpretations were performed.

[0116] 2.6 Pharmacokinetic Studies

[0117] 2.6.1 Solution Preparation

[0118] Accurately weigh an appropriate amount of puerarin, place it in a 2 mL volumetric flask, dissolve it in methanol and dilute it to the mark to obtain a puerarin stock solution with a concentration of 1 mg / mL. Accurately pipette an appropriate amount of the stock solution and dilute it to a puerarin internal standard solution with a concentration of 40 ng / mL. Store at -20℃ for later use.

[0119] Accurately weigh appropriate amounts of nicotinamide, 2,5-dihydroxybenzoic acid, α-linolenic acid, azelaic acid, caffeic acid, vanillin, and riboflavin reference standards, respectively, and dissolve them in methanol to prepare single reference standard stock solutions with a mass concentration of 1 mg / mL. Accurately pipette appropriate amounts of the above single reference standard solutions of different volumes, dilute with 50% methanol and make up to volume to prepare mixed reference standard solutions containing the following mass concentrations of seven components: nicotinamide 1 μg / mL, 2,5-dihydroxybenzoic acid 0.3 μg / mL, α-linolenic acid 0.3 μg / mL, azelaic acid 0.1 μg / mL, caffeic acid 0.3 μg / mL, vanillin 1 μg / mL, and riboflavin 1 μg / mL. Store at -20℃ for later use.

[0120] 2.6.2 Sample Collection and Processing

[0121] Eight healthy male SD rats were used. Before administration, the rats were fasted for 12 hours but allowed free access to water. They were given Ke Ning granules (91 g / kg) by a single oral gavage. At 0.083, 0.167, 0.25, 0.33, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 24, and 36 hours after administration, about 0.5 ml of blood was collected from the fundus venous plexus and placed in heparin-coated centrifuge tubes. The tubes were centrifuged at 12,000 r / min for 10 min at 4°C. The supernatant was collected and stored at -80°C for later use.

[0122] Take 200 μL of rat plasma and place it in a 1.5 mL plastic centrifuge tube. Add 40 μL of 40 ng / mL puerarin (internal standard) solution, 20 μL of 1% formic acid solution, and 800 μL of acetonitrile. Vortex for 3 min, sonicate for 10 min, and centrifuge at 12000 r / min for 10 min at 4 °C. Separate the supernatant and dry it under nitrogen at 37 °C. Dissolve the residue in 200 μL of 50% acetonitrile aqueous solution, vortex for 3 min, sonicate for 10 min, and centrifuge at 14000 r / min for 10 min at 4 °C. Take the supernatant and analyze it in an ACQUITY UPLC I-Class / Xevo TQ-S ultra-high performance liquid chromatography triple quadrupole system.

[0123] 2.6.3 Chromatographic conditions

[0124] Column: ACQUITY BEH C 18 (2.1×50mm, 1.7μm); Guard post: Waters Van GuardBEH C 18 (2.1×5mm, 1.7μm); Mobile phase: A: 0.2% formic acid aqueous solution, B: 0.2% formic acid acetonitrile solution; Flow rate: 0.3mL / min; Column temperature: 40℃; Injection volume: 1μL. Gradient elution conditions are shown in Table 1.

[0125] Table 1 Gradient Elution of Mobile Phase

[0126]

[0127]

[0128] 2.6.4 Mass Spectrometry Conditions

[0129] Electrospray ionization (ESI) source, capillary voltage 3kV, ion source temperature 120℃, desolvation gas (nitrogen) 1000L / H, desolvation gas temperature 600℃, collision gas (argon) 0.15mL / min; multiple reaction ion monitoring (MRM) mode for simultaneous monitoring of positive and negative ions, the monitored ions of each component are shown in Table 2.

[0130] Table 2 Multiple Reaction Ion Monitoring (MRM) Mass Spectrometry Conditions

[0131]

[0132] 2.6.5 Methodological Examination

[0133] The specificity was evaluated by analyzing the chromatograms of blank plasma, blank plasma with 7 mixed reference standards and IS solution, and plasma with oral cough granules and IS solution.

[0134] To investigate the linear range and lower limit of quantitation, accurately pipette the reference stock solution, mix it, and dilute it stepwise with methanol to prepare a mixed reference solution. Take 200 μL of blank blood, add 10 μL of each gradient of mixed reference solution, process according to the plasma sample method, and evaluate the linearity.

[0135] Precision and accuracy were assessed using four concentration control samples (n=5), with intraday and interday precision and method accuracy evaluated through testing on the same day and over three consecutive days.

[0136] The matrix effect and extraction recovery were investigated by comparing the peak areas of blank plasma with added quality control samples before and after treatment, and by examining the extraction recovery. The matrix effect was also investigated by comparing the mixed control standards in plasma with those in pure solvent.

[0137] Stability studies assessed the changes in the chemical properties of quality control samples in plasma under different storage conditions. These included short-term stability at 4°C for 12 hours, freeze-thaw stability after three freeze-thaw cycles, and stability at -20°C for 24 hours.

[0138] 2.6.6 Statistical Data Processing

[0139] The non-compartmental model (NCA) analysis method in WinNonLin 8.2.2 (Phoenix, Phasight, USA) data processing software was used to fit the pharmacokinetic parameters of each component.

[0140] 3 Results

[0141] 3.1 Effects of Ke Ning Granules on Cough Latency and Cough Frequency in Ammonia-Induced Cough Mice

[0142] Mice exhibited a significant cough response after ammonia stimulation. Compared with the model group, the cough latency was significantly prolonged in the positive control group and the low, medium, and high dose groups of Ke Ning granules (P<0.001), and the number of coughs within 5 minutes was significantly reduced (P<0.05, P<0.01, P<0.001). The cough suppression rate of Ke Ning granules did not show a significant dose-dependent effect. These results suggest that Ke Ning granules have a good inhibitory effect on ammonia-induced cough (see Table 3). Figure 1 .

[0143] Table 3. Cough suppression rate in mice of each group (n=6)

[0144]

[0145] 3.2 Effects of Ke Ning Granules on Phenol Red Excretion in the Trachea of ​​Mice

[0146] In this experiment, the phenol red standard curve was y = 0.0768x + 0.0388, R0 2 =0.9999. The phenol red excretion rate of each group was calculated using the regression equation. The results showed that, compared with the control group, the low- and medium-dose Ke Ning granule groups had increased phenol red excretion (P < 0.05), and the ambroxol hydrochloride group and the high-dose Ke Ning granule group had increased phenol red excretion compared with the control group, but the differences were not statistically significant, as shown in Table 4. Figure 2 As shown.

[0147] Table 4. Effects of Ke Ning granules on phenol red excretion in the trachea of ​​mice (mean ± SD, n = 6)

[0148]

[0149]

[0150] Note: Compared with the control group, * P<0.05, ** P<0.01.

[0151] 3.3 Blood Component Analysis of Cough-Relieving Granules

[0152] Based on the qualitative analysis of the chemical components of Ke Ning granules, FreeStyle was used. TM1.3 The software analyzes the data, extracting chromatographic peaks from the samples through preprocessing such as peak extraction, peak matching, and background subtraction. By comparing the mass spectrometry characteristics of the treated mice, the blank control group, and the extract of Ke Ning granules, 26 original blood-entering components were detected in the drug-containing plasma of Ke Ning granules. The results are shown in Table 5, and the chromatograms are as follows. Figure 3 As shown.

[0153] Table 5. Chromatographic peak assignments of the original plasma components of Ke Ning granules.

[0154]

[0155]

[0156]

[0157]

[0158] 3.4 Network Pharmacological Analysis of Blood-Entering Components

[0159] 3.4.1 Obtaining the intersection targets of potential active ingredients of Ke Ning granules with antitussive and expectorant targets

[0160] After importing the blood-entering components into the Swiss ADME database for screening, 13 potential active ingredients were obtained. Using a probability greater than 0 as the criterion for inclusion in the Swiss Target Prediction, a total of 479 relevant targets were obtained after deduplication. Disease-related targets were retrieved from the GeneCards database, and 2336 cough targets and 790 phlegm-producing targets with a "Relevance Scores" greater than the median were identified. After deduplication and gene standardization, 1897 cough targets and 698 phlegm-producing targets were obtained. A total of 40 targets were obtained using Venny 2.1.0 online software, and a Venn diagram was plotted. (See figure). Figure 4 .

[0161] 3.4.2 Protein-protein interaction network analysis and screening of core targets

[0162] Forty common targets were imported into the String database, with the protein species defined as "Homo sapiens", the interaction score capped at >0.4, and single targets with no interaction (CPA3) removed. All other parameters remained at their default values ​​to obtain protein-protein interactions. Figure 5 A visualization of protein-protein interactions was created using Cytoscape 3.10.0 software. (See attached image.) Figure 6The core targets were obtained using the Dgree, Betweenness, and Closeness algorithms. It is speculated that the cough-relieving and expectorant effects of Ke Ning granules mainly affect the following 17 targets: IL6, TLR4, STAT3, PTGS2, EGFR, ERBB2, MMP9, PTPRC, MAPK1, MTOR, BRAF, ACE, MPO, CCND1, MMP2, PIK3CG, and NR3C1.

[0163] 3.4.3 Construction of the "Drug-Component-Disease-Common Target" Network and Acquisition of Effective Substance Groups

[0164] like Figure 7 As shown, a network diagram of "drug-component-disease-common target" for the treatment of cough and phlegm by Ke Ning granules is presented. Eleven components in Ke Ning granules, including 8-Hydroxyquinoline, Homovanillic acid, Vanillin, 2,3-Dihydro-1-benzofuran-2-carboxylic acid, 4-Coumaric acid, Scopolettein, Caffeic acid, Azelaic acid, Andrographolide, Kahweol, and (+ / -)-Gingerol, participate in the regulation of cough and phlegm relief. The remaining two components, Nicotinamide and Adenine, do not participate in the regulation. These eleven components constitute the effective substance group for the treatment of cough and phlegm by Ke Ning granules.

[0165] 3.4.5 Discovery of the pathway of cough-relieving and expectorant effects of Ke Ning granules

[0166] Intersecting targets were imported into the DAVID database for GO and KEGG enrichment analysis to interpret the biological processes (abbr.BP), cellular components (abbr.CC), and molecular functions (abbr.MF) involved in the targets. Figure 8 , 9 ), and obtain the main participating signaling pathways ( Figure 10The results yielded 224 entries related to antitussive and expectorant drugs, mainly related to the positive regulation of protein modification and phosphorylation, the metabolism of phosphorus and phosphate, and hormone regulation; 28 entries related to cytokines, mainly involving lysosomes, nuclear membrane, endoplasmic reticulum lumen, and cell basement membrane; and 36 entries related to protein kinase binding and activity, and phosphotransferase activity. These entries primarily involved the following signaling pathways: PI3K-Akt signaling pathway, HIF-1 signaling pathway, COVID-19, Kaposi's sarcoma-associated herpesvirus infection, endocrine resistance, EGFR tyrosine kinase inhibitor resistance, relaxin signaling pathway, and ErbB signaling pathway.

[0167] 3.5 Pharmacokinetic Analysis of Cough-Relieving Granules in Blood

[0168] 3.5.1 Specificity

[0169] Under the selected chromatographic and mass spectrometric conditions, endogenous substances in plasma did not interfere with the determination of the analyte and internal standard. The components showed good separation and peak shapes, indicating good specificity of the method. (See [link to article]). Figure 11 .

[0170] 3.5.2 Standard Curve, Linear Range, and Sensitivity

[0171] Seven components, including nicotinamide, showed good linearity within their linear range in rat plasma, and the correlation coefficients (R2) of the standard curves for each component were all greater than 0.99 (see Table 6).

[0172] Table 6. Linear regression curve equations for 7 components including niacinamide.

[0173]

[0174] 3.5.3 Accuracy and Precision

[0175] The intra-day and inter-day precision RSDs of seven components, including nicotinamide, in plasma were all less than 15%, and the accuracy RE values ​​were in the range of -15% to 15%, indicating that the method has good accuracy and precision and meets the requirements of biological sample analysis methods.

[0176] 3.5.4 Extraction Recovery and Matrix Effect

[0177] The extraction recoveries of seven components, including nicotinamide, in plasma samples at three concentrations ranged from 91.15% to 102.16%, and the matrix effect ranged from 99.49% to 109.74%. The results indicate that the extraction recoveries were good and there was no obvious matrix effect, which met the requirements of biological sample analysis methods.

[0178] 3.5.5 Stability

[0179] The RE values ​​of the seven components in the plasma samples after storage at -20℃ for 24 hours, placement at 4℃ for 12 hours, and three freeze-thaw cycles (at -20℃ room temperature) were all in the range of -15% to 15%, and the RSD was less than 15%, indicating that the plasma biological samples containing these seven components had good stability after storage at -20℃ for 24 hours, placement at 4℃ for 12 hours, and three freeze-thaw cycles.

[0180] 3.5.6 Pharmacokinetic parameters

[0181] The mean plasma concentration-time curves of Ke Ning granules at various time points after oral administration to normal rats were determined using the established UPLC-MS / MS method described above. Figure 12 Pharmacokinetic parameters were calculated using WinNonLin 8.2 software, and a non-compartmental model was used to fit the drug's kinetic process in rats. The relevant pharmacokinetic parameters are shown in Table 8. In normal rats, after oral administration of Ke Ning granules, nicotinamide, riboflavin, caffeic acid, and azelaic acid reached peak concentrations before 3 hours, indicating rapid absorption. The half-lives (T1 / 2) of riboflavin and caffeic acid were less than 3 hours, indicating a short duration of action in vivo. The half-lives (T1 / 2) of nicotinamide and azelaic acid were greater than 10 hours, indicating a longer duration of action in vivo. Vanillin was rapidly absorbed in vivo with a short duration of action. 2,5-Dihydroxybenzoic acid and α-linolenic acid reached peak concentrations around 12 hours, with half-lives (T1 / 2) greater than 10 hours, indicating slower absorption and a longer duration of action in vivo.

[0182] Table 7. Pharmacokinetic parameters of the seven components after oral administration of Ke Ning granules.

[0183]

[0184]

[0185] 4 Discussion

[0186] Quality control of traditional Chinese medicine compound preparations is a crucial prerequisite for ensuring clinical efficacy. Therefore, this study, based on in vivo pharmacodynamics, plasma pharmacochemistry, network pharmacology, and pharmacokinetics, explores the quality markers of Ke Ning granules for relieving cough and expectoration, which is of great significance. The efficacy of Ke Ning granules in relieving cough and expectoration was verified for the first time using classic models of ammonia-induced cough and phenol red excretion experiments, confirming its effectiveness. Using UHPLC-Q-Exactive Plus Orbitrap HRMS technology, 26 components of the original Ke Ning granules were analyzed and entered into the bloodstream, identifying substances that enter the bloodstream and may exert their effects, demonstrating the traceability and transmissibility of the pharmacodynamic material basis. Network pharmacology screening identified 13 potential active ingredients targeting the original blood-entry components. Among them, 11 components, including 8-hydroxyquinoline, homovanillic acid, vanillin, benzodihydrofuran-2-carboxylic acid, p-hydroxycinnamic acid, scopolamine lactone, caffeic acid, azelaic acid, andrographolide, caffeoyl alcohol, and 6-gingerol, are involved in regulating and constituting the effective substance group for cough relief and expectoration of Ke Ning granules. Through "component-target-pathway" visualization network analysis, it was found that the main targets involved are IL-6, STAT3, and EGFR, and KEGG enrichment revealed PI3K-Akt and HIF-1 signaling pathways. This suggests that the cough relief and expectoration effects of Ke Ning granules are achieved through the synergistic action of multiple components and multiple targets. Based on the blood-entry component analysis, pharmacokinetic quantitative detection was performed on the changes of seven components in vivo: azelaic acid, nicotinamide, vanillin, caffeic acid, 2,5-dihydroxybenzoic acid, α-linolenic acid, and riboflavin, demonstrating the measurability of the substances. Studies have found that caffeic acid has anti-influenza virus activity and is a potential active substance for the antitussive, antiasthmatic, anti-inflammatory, and hypoglycemic effects of Huanyangshen (a traditional Chinese medicine formula). Azelaic acid has good anti-inflammatory, antioxidant, antimicrobial, and anti-hemolytic activities. Fang Wei discovered azelaic acid in the blood migration components of rats with chronic obstructive pulmonary disease (COPD) treated with Huatan Jiangqi Fang (a traditional Chinese medicine formula for resolving phlegm and lowering qi), suggesting it may be a potential active substance for this formula. Vanillin has anti-inflammatory and antitussive effects, providing pre-protection against acute lung injury. It also reduces inflammation by regulating ERK, p38, AKT, and NF-κB, thereby lowering the protein expression levels of IL-6 and TNF-α inflammatory factors. These studies corroborate the effectiveness of caffeic acid, azelaic acid, and vanillin.

[0187] In summary, the results of this study indicate that Ke Ning granules have significant antitussive and expectorant effects. Based on the definition of quality markers, caffeic acid, azelaic acid, and vanillin were selected as quality markers for the antitussive and expectorant effects of Ke Ning granules through efficacy, transmissibility, and measurability. These results are of great significance for further clarifying the pharmacodynamic material basis of Ke Ning granules, optimizing quality standards, and promoting its clinical application.

[0188] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. A method for discovering a quality marker for cough-relieving and expectorant granules, characterized in that, Includes the following steps: (1) Analyze and identify the original blood-entering components of Ke Ning granules; (2) Using the original blood-entry components as the "drug component set", the potential active ingredients and common targets of Ke Ning granules for relieving cough and expectoration were screened by network pharmacology method; (3) Based on the analysis of blood-entering components, pharmacokinetic quantitative analysis of the in vivo processes of the original blood-entering components is used; (4) The quality markers for cough relief and expectoration of Ke Ning granules were finally determined through effectiveness, transmissibility and measurability; The step (1) of analyzing and identifying the original blood-entering components of Ke Ning granules is performed using the UHPLC-Q-Exactive PlusOrbitrap HRMS method. The specific chromatographic conditions are as follows: Chromatographic column: CORTECS UPLC T3, 2.1 mm × 100 mm, 1.6 μm; column temperature: 40 ℃; flow rate: 0.3 mL / min; mobile phase: mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was acetonitrile methanol solution, with a volume ratio of 2:1; injection volume: 2 µL; gradient elution program: 0–3 min, 1% → 2% B; 3–5 min, 2% → 5% B; 5–10 min, 5% → 30% B; 10–15 min, 30% → 60% B; 15–20 min, 60% → 70% B; 20–22 min, 70% → 95% B; 22–24 min, 95% B; 24–25 min, 95% → 1% B; 25–28 min, 1% B. The specific mass spectrometry conditions are as follows: The ion source is electrospray, with a spray voltage of 3500V and an ion lens voltage frequency of 50.

0. The sheath gas pressure is 35arb, the auxiliary gas pressure is 10arb, the nebulization temperature is 350℃, the capillary temperature is 320℃, the probe heater temperature is 350℃, the scanning mode is a positive and negative ion exchange mode of full scan + data-dependent secondary scan, the scanning range is 80~1200 m / z, the primary resolution is 70000, and the secondary resolution is 17500. The UHPLC-Q-Exactive Plus Orbitrap HRMS method involves processing the plasma as follows: Take 500 μL of plasma, add 100 μL of 1% formic acid aqueous solution, vortex for 1 min, add 2 mL of methanol to precipitate proteins, vortex for 5 min, sonicate for 10 min, centrifuge at 8000 r / min at 4℃ for 10 min in a refrigerated high-speed centrifuge, collect the supernatant in a centrifuge tube, dry it with nitrogen blowdown at 37℃, obtain the residue, add 200 μL of 50% methanol to redissolve, vortex for 2 min, sonicate for 10 min, centrifuge at 12000 r / min at 4℃ for 10 min, collect the supernatant as the sample solution, and analyze it using the UHPLC-Q-Exactive Plus Orbitrap HRMS instrument; Step (3) involves using pharmacokinetic quantitative analysis to determine the in vivo process of the parent compound entering the bloodstream, under the following specific conditions: Chromatographic conditions: Chromatographic column: ACQUITY UPLC® BEH C18, 2.1×50mm, 1.7μm; Guard column: Waters VanGuard BEH C18, 2.1×5mm, 1.7μm; Mobile phase: A: 0.2% formic acid aqueous solution, B: 0.2% formic acid acetonitrile solution; Flow rate: 0.3 mL / min; Column temperature: 40℃; Injection volume: 1 μL; Gradient elution conditions as follows: mobile phase gradient elution table Mass spectrometry conditions: Electrospray ionization source, capillary voltage 3kV, ion source temperature 120℃, solvent gas flow rate 1000 L / H, solvent gas temperature 600℃, collision gas flow rate 0.15 mL / min; multi-reaction ion monitoring mode for simultaneous monitoring of positive and negative ions, the monitored ions for each component are as follows: Multiple reaction ion monitoring mass spectrometry conditions In step (3), the sample to be tested is prepared as follows: 200 µL of plasma is placed in a 1.5 mL plastic centrifuge tube, 40 µL of 40 ng / mL puerarin internal standard solution is added sequentially, followed by 20 µL of 1% formic acid water and 800 µL of acetonitrile. The mixture is vortexed for 3 min, sonicated for 10 min, and centrifuged at 12000 r / min for 10 min at 4 °C. The supernatant is separated and dried under nitrogen at 37 °C. The residue is dissolved in 200 µL of 50% acetonitrile aqueous solution, vortexed for 3 min, sonicated for 10 min, and centrifuged at 14000 r / min for 10 min at 4 °C. The supernatant is then analyzed using an ACQUITY UPLCI-Class / XevoTQ-S ultra-high performance liquid chromatography-tandem triple quadrupole system.

2. The method for discovering the quality marker for cough-relieving and expectorant granules according to claim 1, characterized in that, The cough-relieving granules are composed of three medicinal materials: pine cone, loquat leaf, and cotton root.

3. The method for discovering the quality marker for cough-relieving and expectorant granules according to claim 1, characterized in that, Step (2) specifically includes the following steps: ① Screening of potential active ingredients and collection of targets for Ke Ning granules: The blood-entering components were imported into the SwissADME database to screen for potential active ingredients. Then, the Swiss TargetPrediction database was used to limit the species to "Homo sapiens" to predict the target of potential active ingredients and remove invalid and duplicate targets. ② Collection of targets related to cough suppression and expectoration: By searching the GeneCards database using the keywords "Cough" and "Sputum" respectively, disease-related targets were obtained. After removing duplicate genes and genes that do not have Uniprot ID, cough-relieving and expectorant-related targets were obtained. ③ Protein-protein interaction network analysis and screening of core targets: The Venny online software was used to find the intersection of potential drug active ingredient targets and disease targets and draw a Venn diagram to obtain the common targets of potential active ingredients for treating cough and expectoration. The intersection targets were imported into the String database to obtain the interaction relationships between target proteins. The PPI network diagram was drawn and the topology analysis was performed using the Analyze network tool and CytoNCA plugin in Cytoscape 3.10.0 software to obtain the degree value, betweenness centrality, and proximity centrality topology parameters of each node. The top 20 targets were calculated respectively. The intersection targets obtained by the three algorithms are the core targets. ④ Construction and analysis of the "drug-component-disease-common target" network: After processing the gene data of potential active ingredients, diseases, and common targets of drugs, network and type files are generated, and then imported into Cytoscape 3.10.0 software to draw a network diagram of "drug-ingredient-disease-common target". ⑤ Enrichment analysis: Intersecting targets were imported into the DAVID database and enriched in the Gene ontology database (abbr.GO) and the Kyoto Encyclopedia of Genes and Genomes (abbr.KEGG). Bubble charts and rectangle charts were then created using the online graphing platform MicroBio, and biological interpretations were performed.

4. A method for detecting multiple components in blood plasma, characterized in that, The following components in plasma were analyzed and identified using UHPLC-Q-Exactive Plus Orbitrap HRMS: nicotinamide, DL-leucine, adenine, adenosine, 2,5-dihydroxybenzoic acid, maltol, 8-hydroxyquinoline, homovanillic acid, caffeic acid, riboflavin, vanillin, 4-hydroxycinnamic acid, benzodihydrofuran-2-carboxylic acid, scopolamine, 4-indolecarboxaldehyde, azelaic acid, 3-tert-butyladipic acid, andrographolide, caffeoyl alcohol, 6-gingerol, and 2-Hydroxy-4,5',8a'-trimethyl-1'-oxo-4-vinyloctahydro-1'H-spiro[cyclopentane-1,2'-naphthalene]-5'-carboxylic acid. acid, ethyl p-ethoxybenzoate, 3,5-di-tert-butyl-4-hydroxybenzaldehyde, 1,4a-dimethyl-9-oxo-7-(propan-2-yl)-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxylic acid, caffeoyl alcohol, α-linolenic acid; The specific chromatographic conditions are as follows: Chromatographic column: CORTECS UPLC T3, 2.1 mm × 100 mm, 1.6 μm; column temperature: 40 ℃; flow rate: 0.3 mL / min; mobile phase: mobile phase A is 0.1% formic acid aqueous solution - mobile phase B is acetonitrile methanol solution, with a volume ratio of 2:1; injection volume: 2 µL; gradient elution program: 0–3 min, 1% → 2% B; 3–5 min, 2% → 5% B; 5–10 min, 5% → 30% B; 10–15 min, 30% → 60% B; 15–20 min, 60% → 70% B; 20–22 min, 70% → 95% B; 22–24 min, 95% B; 24–25 min, 95% → 1% B; 25–28 min, 1% B. The specific mass spectrometry conditions are as follows: The ion source is electrospray, with a spray voltage of 3500V and an ion lens voltage frequency of 50.

0. The sheath gas pressure is 35arb, the auxiliary gas pressure is 10arb, the nebulization temperature is 350℃, the capillary temperature is 320℃, the probe heater temperature is 350℃, the scanning mode is a positive and negative ion exchange mode with full scan and data-dependent secondary scan, the scanning range is 80~1200 m / z, the primary resolution is 70000, and the secondary resolution is 17500. The specific plasma processing method is as follows: Take 500 μL of plasma, add 100 μL of 1% formic acid aqueous solution, vortex for 1 min, add 2 mL of methanol to precipitate proteins, vortex for 5 min, sonicate for 10 min, centrifuge at 8000 r / min at 4℃ for 10 min in a refrigerated high-speed centrifuge, collect the supernatant and place it in a centrifuge tube, blow it dry with nitrogen at 37℃, obtain the residue, add 200 μL of 50% methanol to reconstitute, vortex for 2 min, sonicate for 10 min, centrifuge at 12000 r / min at 4℃ for 10 min, collect the supernatant as the sample solution for detection.

5. A method for detecting multiple components in plasma, characterized in that, Simultaneously, the contents of azelaic acid, nicotinamide, vanillin, caffeic acid, 2,5-dihydroxybenzoic acid, α-linolenic acid and riboflavin in plasma were measured. The specific conditions are as follows: Chromatographic conditions: Chromatographic column: ACQUITY UPLC® BEH C18, 2.1×50mm, 1.7μm; Guard column: Waters VanGuard BEH C18, 2.1×5mm, 1.7μm; Mobile phase: A: 0.2% formic acid aqueous solution, B: 0.2% formic acid acetonitrile solution; Flow rate: 0.3 mL / min; Column temperature: 40℃; Injection volume: 1 μL; Gradient elution conditions as follows: mobile phase gradient elution table Mass spectrometry conditions: Electrospray ionization source, capillary voltage 3kV, ion source temperature 120℃, solvent gas flow rate 1000 L / H, solvent gas temperature 600℃, collision gas flow rate 0.15 mL / min; multi-reaction ion monitoring mode for simultaneous monitoring of positive and negative ions, the monitored ions for each component are as follows: Multiple Reaction Ion Monitoring (MRM) Mass Spectrometry Conditions The specific plasma processing method is as follows: Take 500 μL of plasma, add 100 μL of 1% formic acid aqueous solution, vortex for 1 min, add 2 mL of methanol to precipitate proteins, vortex for 5 min, sonicate for 10 min, centrifuge at 8000 r / min at 4℃ for 10 min in a refrigerated high-speed centrifuge, collect the supernatant and place it in a centrifuge tube, blow it dry with nitrogen at 37℃, obtain the residue, add 200 μL of 50% methanol to reconstitute, vortex for 2 min, sonicate for 10 min, centrifuge at 12000 r / min at 4℃ for 10 min, collect the supernatant as the sample solution for detection.

Images